Glycobiology of Innate Immunology [1st ed. 2022] 9789811690808, 9789811690815, 9811690804

Table of contents :
Contents
Abbreviations
Chapter 1: Repertoire in Innate Immunity
1.1 Historical Expansion of Defense System
1.2 Columbus Era to Modern Revolution in Immunological Defense System
1.3 Historical Profile of Defense Constituents and Progress in Innate Immune Repertoire
1.3.1 Phagocytosis
1.3.2 Leukocytes
1.3.3 Neutrophils
1.3.4 Granulocytes
1.3.5 Monocytes and Macrophages
1.3.6 Dendritic Cells
1.3.7 Complement System
1.3.7.1 Alternative Complement System
1.3.7.2 Lectin Pathway
1.3.8 Opsonization
1.3.9 Lysozyme and Salvarsan
1.3.10 Progress in Innate Immune Response Since Historic Spanish Flu
1.4 The Outline of Innate Immunity
1.4.1 Concept of Immune Receptors
1.4.2 Host Protection from Microbial Invaders of Innate Immunity
1.5 Autophagy from Microbial Invaders and Self-Associated Molecular Patterns (SAMPs) of Innate Immune Cells
References
Chapter 2: Dendritic Cells (DCs) in Innate Immunity
2.1 General Biology of DCs
2.2 Classification and Different Function of DCs
2.2.1 pDC, Lymphoid Organ CD8α+ DC, and Tissue CD103+ DC Interaction with Tregs
2.2.2 DCs Induce Tolerance State
2.2.3 DC co-Stimulatory Receptors
2.2.3.1 CD80/CD86
2.2.3.2 CD70
2.2.3.3 ICOS-L, PD Ligands (PD-L), IL-10, IOL-27, TGFβ, Retinoic Acid, and β-Catenin
2.2.3.4 Indoleamine-2,3-Dioxygenase
2.2.4 Application of DCs to Human Diseases
References
Chapter 3: Glycan Biosynthesis in Eukaryotes
3.1 General Glycosylation Events
3.2 Sugar Nucleotide Transporters Deliver Donor Saccharides to ER-Golgi Network
3.3 Golgi Traffic
3.4 N-glycan Synthesis
3.5 O-glycosylation and Multiple O-Glycan Structures
3.5.1 7 Core O-glycan Structures
3.5.2 Modification of 7 Core O-Glycan Structures
3.6 O-GlcNAcylation, O-Mannosylation, O-β-Glucosylation, O-α-Fucosylation, O-β-Glucosylation, O-β-Galactosylation, C-Glycosyla...
3.6.1 O-GlcNAcylation
3.6.2 O-Mannosylation
3.6.3 O-β-Glucosylation
3.6.4 O-α-Fucosylation
3.6.5 O-β-Glucosylation
3.6.6 O-β-Galactosylation
3.6.7 C-Mannosylation and C-Glycosylation
3.7 Function of O-Glycosylation and O-Glycans
3.8 Glycosaminoglycans (GAGs)
3.8.1 Classification and Biosynthesis of GAGs
3.8.2 Chondroitin Sulfate (CS)
3.8.2.1 Sulfotransferases Sulfate CS/DS Chains
3.8.2.2 Chain Termination in CS Chains
3.8.2.3 Sulfated CS Function as Pathogen Receptor and Co-receptor as Well as CS Binds to Advanced Glycation End-Product Recept...
3.8.2.4 Roles of CS in Embryogenesis and Development
3.8.3 Dermatan Sulfate (DS)
3.8.3.1 Biosynthesis of DS
3.8.3.2 Deficiency Syndrome of DS
3.8.4 Keratan Sulfate (KS)
3.8.5 Heparin and Heparan Sulfate
3.8.6 Hyaluronic Acid (HA) or Hyaluronan
3.8.7 Proteoglycans (PGs)
3.8.7.1 Intracellular PG Type of Mast Cell Granule Serglycin
3.8.7.2 PGs of Syndecans and Glypicans on Cell Surfaces
3.8.8 Extracellular PGs
3.8.8.1 Aggrecan
3.8.8.2 Perlecan
3.8.8.3 Small Leu-rich PGs (SLRPs) of Decorin, Lumican, and Biglycan
3.9 Glycosylphosphatidylinositols (GPIs) Anchor Glycosylation
3.9.1 General Structure of GPI Anchors
3.9.2 Function of GPI-Anchored Protein
3.9.3 Biosynthesis, Structural Assembly, and Transportation of GPI-Anchored Protein
3.9.4 GPIs in Parasites
3.9.5 GPI Interaction with TLRs in Malaria P. falciparum
3.9.6 GPI-Defected Disorders of Paroxysmal Nocturnal Hemoglobinuria (PNH) and Prion Disease
References
Chapter 4: Glycans in Glycoimmunology
4.1 Glycans in Cell Recognition and Evolutionary Adaptation in Organisms
4.2 Changes in Glycan Structure Involved in Coregulated Expression of Glycan-Binding Lectin Counterparts
4.3 Evolution of Lectin: Alternative Splicing Contributes to Variation for Glycan-Binding Receptors
4.4 E-Selectin-Binding Ligand sLex (CD15s) on Neutrophil CD44 N-glycan and Alternatively Spliced Exon 6 Contains Core 2 O-Glyc...
4.5 Glycans Regulate T Cells
4.5.1 Glycans Regulate Development and Differentiation in T Cells
4.5.2 Glycosylation of Notch Receptor Signaling for Thymocyte β Selection and T Cell Function Regulation
4.5.3 Alternatively Spliced Variants Produce Different Glycan Structures of CD43 and CD45 Isoforms in T Cells
4.5.4 T Cells CD43 and CD45 Interaction with Their Counter-Receptor or Lectins to Determine T Cell Fates
4.5.5 TCR Glycosylation Governs Hyper-response and Autoimmune Responses in T Cells and Tregs
4.5.6 SAMP and N-Glycan-Dependent Modulation of Inhibitory T Cell Receptors to Suppress T Cell Functions
4.5.7 Galectins in Suppression of T Cell Functions
4.5.8 Glycans Regulate T Cell-Mediated Immune Suppression and Tolerance in Tumor Progression
4.6 Abnormal N-Glycosylation in Autoimmunity
4.7 Glycan Regulation of NK Cell Receptors
4.7.1 NCRs on NK Cells
4.7.2 NCR Ligands
4.7.3 Interaction of NCRs Ligands with Pathogens
4.7.4 Interaction of NCRs Ligands with Self-Ligands
4.7.5 NK Cells MHC-I-Independent Inhibitory Receptors Siglec-7 and Siglec-9
4.8 Carbohydrate Recognition of Target Antigens by DCs During Infection and Inflammation
4.8.1 Lewis Ligand Recognition by DCs
4.8.2 VIM Ceramide Dodecasaccharide
4.9 Glycan-Specific Trafficking Receptors in DC Maturation
4.10 Glycan Ligands in Trafficking of DC Migration
4.10.1 sLex-PSGL-1 Glycans in DC Trafficking
4.10.2 Ganglioside Recognition by DC Receptors in Trafficking
4.11 Chemokine Receptors in DC Trafficking
4.11.1 Chemokine
4.11.2 Chemokine Receptor
4.11.3 Chemokine-GAG Interaction as a Type of Protein-Glycan Interactions
4.11.4 Molecular Motifs in Chemokine for GAG Recognition
4.11.5 C-C Type Chemokine Receptor 4 (CCR4) and Specific Ligand 17 (CCL17) and Specific Ligand 22 (CCL22)
4.12 Glycan Structure-Recognizing Selectins in DC-Endothelium Interaction During Infection and Inflammation
4.12.1 3 Species of Selectins: E-, L-, and P-selectins
4.12.2 Representative Selectin Ligand PSGL-1 and Role of PSGL-1 O-Glycan
4.12.3 Glycosyltransferases for Biosynthesis of PSGL-1 O-Glycan
4.12.4 Designation of Carbohydrate Glycomimetic Drugs and Natural Inhibitors of Selectins
4.12.5 Glycomimetic Drug Candidates
4.12.6 GAG-Glycomimetic Drugs
Chapter 5: Pathogen-Host Infection Via Glycan Recognition and Interaction
5.1 Lectin Recognition of Glycans on Cell Surface and Soluble Glycans
5.2 Innate Immune-Specific and Host Defensing Lectins of Fungal, Protozoa, Invertebrate, and Lower Vertebrates
5.3 How Do Hosts Interact with Pathogens?
5.3.1 Lectin-Carbohydrate Interaction
5.3.2 Bacterial Glycoconjugates Interact with Host Lectins
5.3.2.1 Glycoproteins
5.3.2.2 Bacterial Capsule of Capsular Polysaccharide (CPS)
5.3.2.3 Function of Bacterial CPS
5.4 Pathogen-Producing Lectins as Receptors to Bind to the Host Carbohydrates
5.4.1 Uropathogenic E. coli (UPEC), Enterohemorrhagic E. coli (EHEC), and Enterotoxigenic E. coli (ETEC)
5.4.1.1 Uropathogenic E. coli (UPEC)
5.4.1.2 Enterotoxigenic E. coli (ETEC)
5.4.1.3 Enterohemorrhagic E. coli (EHEC)
5.4.1.4 EHEC O157:H7 or EPEC Recognition of Core 2 O-Glycans of Mucin Type on Cell Membrane of Host
5.4.2 Lectins and Glycans of Other Pathogenic Bacteria
5.4.2.1 Legionella pneumophila, K. pneumoniae, S. pneumoniae, and B. cepacia Complex
5.4.2.2 Non-typeable H. influenzae and Acinetobacter baumannii
5.4.2.3 H. pylori
5.4.2.4 P. aeruginosa
5.4.2.5 N. gonorrhoeae, N. meningitidis, S. aureus, Chlamydia pneumoniae and Vibrio parahaemolyticus
5.4.3 Viral Lectins or Host Lectin-Binding Glycans
5.5 Host Lectin Defense Mechanisms in Lectin-Carbohydrate Interactions
5.6 Pathogenic Glycans to Trigger Innate Immune Enhancement
5.6.1 Example 1: Polysaccharides with Immune Enhancement of Cyrtomium macrophyllum
5.6.2 Example 2: Activation of Macrophage by Polysaccharide from Paecilomyces cicadae
5.6.3 Example 3: NK Cell-Mediated Cytotoxicity Increased by Arabinogalactan from Anoectochilus formosanus
5.6.4 Example 4: Streptococcus pneumonia Polysaccharides Activate NK Cells, NK-Like T Cells, and Monocytes
5.6.5 Example 5: C. macrophyllum Polysaccharides (CMP) Enhance Lymphocyte Proliferation and Macrophage Function
5.7 TLR4 Receptor-Activating Glycans Activate NO Production in Macrophage
5.8 CBPs or GBPs in Antigen Recognition
References
Chapter 6: Innate Immunity Via Glycan-Binding Lectin Receptors
6.1 Glycosylation Effect on Autoimmunity and Inflammation
6.1.1 Glycosylation in Immunological Recognition and Inflammation
6.1.2 Glycosylation Effect on Autoimmunity
6.2 Glycosylation Effect on Tumor Immunity of Immune Cells
6.3 Immune Tolerance and Defense Mechanisms of Innate Immune DCs During Infection
6.4 How Are Pathogenic Bacteria Recognized by Receptors of DCs of the Host Immune System?
6.4.1 DC Lectins for Glycan Recognition of Invasive Agents
6.4.2 Toll-Like Receptors
6.4.2.1 DC-Receptor-Specific Pathogenic Ligands
6.4.2.2 Signaling upon Receptor-Ligand Interaction
6.4.3 Innate Immune Receptors in Malaria Infection
6.4.3.1 Pathogenic Process in Malaria Plasmodium falciparum Infection
6.4.3.2 P. falciparum GPI Anchor Glycosylation
6.4.3.3 GPI Anchor on the Merozoite Surface in Inflammatory and TLR2, TLR4, and TLR9
6.4.3.4 Inflammasome and Sialic Acid Tropism in Malaria Recognition
6.4.4 Innate Immunity Receptors in Protozoan Parasite Toxoplasma gondii
6.4.4.1 Host Carbohydrate-Binding Domain of Toxoplasma gondii-Secreted Proteins
6.4.4.2 T. gondii GPI-Anchored Protein Recognition of TLR2, TLR4, and Galectins of Host APCs
6.5 Pathogen Recognition and Adaptive Immune Responses in Acquired Immunity
6.6 Galactose-Specific C-Type Lectin: Two Major ASGPR and Macrophage Galactose Lectin (MGL) in the Human
References
Chapter 7: Sialic Acid-Binding Ig-Like Lectins (Siglecs)
7.1 PolySia and Host Sialic Acids Modulate Host Immune Responses as Pathogenic Decoys
7.2 Sialic Acid Recognition by Siglecs for Self- or Nonself-Antigens
7.3 Classification of Siglecs
7.4 Evolution of Siglecs, Sialic Acids, and Sialic Acid O-Acetylation as Host Ligands (Receptors) for Microbes and Innate Immu...
7.5 Microbial Sialic Acid-like Molecules Synthesis and Recognition of Microbial Sialic Acids by DCs and Bacteriophages
7.6 Hematopoietic System in Siglecs
7.7 Structure of Siglecs
7.7.1 Cytoplasmic ITIM and ITAM Domains of Siglecs
7.7.2 Adaptor Proteins Associated with Siglecs
7.7.3 SA-Recognition Tropism of Siglecs
7.8 Inhibitory Signaling of DCs
7.9 Siglec-1 (CD169, Sialoadhesin/Sn)
7.9.1 General SAbinding Specificity of Siglec-1
7.9.2 Siglec-1 Is a Pathogen-Binding Receptor
7.9.3 Siglec-1 Recognizes HIV and Is a Transinfection Receptor Expressed on mDCs
7.10 CD22/Siglec-2
7.10.1 General and Structural Aspects of CD22/Siglec-2
7.10.2 CD22 I Associated with Development of Autoimmune Diseases
7.10.3 CD22 Function in Immune Tolerance Events
7.10.4 Role of CD22 (Siglec-2, Mice Siglec-G) in Immune Responses
7.10.5 Model Ligands for Recognition of CD22 on B Cells
7.10.6 B Cell-Targeted Immunotherapy Through CD22-Positive Targeting of B-Cell Lymphomas
7.10.7 Immune Tolerance Capacity of Neu5Ac-α2,6-Gal Ligands in DCs by ST6Gal-1 of Tumor Cells for Immunesurveillance
7.10.8 CD22 Vs. Pathogens
7.10.9 CD22 Application with CAR-T on Acute Lymphoblastic Leukemia (ALL)
7.10.10 CD22/Siglec-2 Coreceptor, CD45 on T Cells
7.10.10.1 Existence of CD45 Isoforms on T Cells
7.10.10.2 CD45 Function Is Displayed in a Glycan-Dependent Manner on T Cells
7.10.10.3 Two Glycoproteins of CD43 and CD45 in Glycan-Dependent Functions on T Cells
7.11 Siglec-4/Myelin-Associated Glycoprotein (MAG)
7.11.1 General Aspects of MAG/Siglec-4
7.11.2 Siglec-4/MAG in the CNS and Brain Development
7.11.3 Siglec-4/MAG in Hippocampal Long-Term Potentiation
7.12 Siglec-15, Non-CD33-Related Siglecs in Humans
7.12.1 The Structure and Expression of Siglec-15, Called Misnomer ``CD33L3´´ in Humans
7.12.2 DAP12-Syk Pathway in Siglec-15-Mediated Remodeling of the Tumor Microenvironment
7.12.3 Siglec-15 Functions in Osteoclastogenesis
7.13 Siglec-3 (CD33)-Related Siglecs on DCs
7.13.1 Siglec-3 (CD33)
7.13.2 Structure, Natural Ligand, and Cellular Signaling with SHP-1/-2 of Siglec-3/CD33
7.13.3 Pathogen Ligand for CD33
7.13.4 Siglec-3/CD33 Is Related to SOCS3 and Internalization of CD33
7.13.5 Putative Functions of Siglec-3/CD33 in Alzheimer´s Disease (AD)
7.13.6 Siglec-3-/CD33-Based Immunotherapy for AML
7.13.7 Siglec-5/CD170 as a CD33-Related Siglec
7.13.7.1 Paired Siglec-5/Siglec-14 Receptors
7.13.7.2 Role of Siglec-5/CD170 in DCs and Modulation of Immune Responses
7.13.8 Siglec-6 as a CD33-Related Siglec
7.13.9 Siglec-7 (CD328) as a CD33-Related Siglec
7.13.9.1 Basic Properties of Siglec-7 in DCs
7.13.9.2 Siglec-7 Recognition of Pathogenic C. jejuni Sialyl LOSs and Ganglioside Mimics
7.13.9.3 Function of Inhibitory Siglec-7 on NK Cells
7.13.10 Siglec-8 as a CD33-Related Siglec and Siglec-F as a Mouse Paralog
7.13.10.1 Siglec-8-Binding Glycans on Basophils, Eosinophils, and Mast Cells of Humans
7.13.10.2 Siglec-8 Is an Eosinophil-Specific CD33-Related Siglec and Functions in Mast Cells
7.13.11 Siglec-9 as a CD33-Related Siglec and Murine Functional Counterpart, Siglec-E
7.13.11.1 Siglec-9 Function in Immune Surveillance and Survival
7.13.11.2 Immune Suppression of Tumor Cells by Siglec-9-SA Interaction
7.13.11.3 Siglec-9 and its Ligand Mucin in Tumor Biology
7.13.11.4 Siglec-9 Linkage to the CD200-CD200R Axis for Inflammation Inhibition in M1- and M2-Type Macrophages
7.13.11.5 Inhibitory Receptor Engagement in Autoimmune Diseases Linked to the Siglec-9-CD200R-CD200 Axis
7.13.11.6 Cannabinoid (CB) Receptors Also Upregulate CD200-CD200R Interaction and Expressions
7.13.11.7 Siglec-9 in Neutrophil Function and T Cells
7.13.11.8 Siglec-9 Functions as a Leukocyte Ligand for Vascular Adhesion Protein-1 (VAP-1)
7.13.11.9 Siglec-9 Recognition with the Pathogenic Group B Streptococcus (GBS) Sialic Acid Glycan Capsule and Group a Streptoc...
7.13.11.10 Siglec-9 Recognition with Pathogenic Pseudomonas aeruginosa Attenuates Innate Immune Responses
7.13.12 Siglec-10 (Mouse Ortholog Siglec-G) in Humans as a CD33-Related Siglec
7.13.12.1 General Aspects of Siglec-10 (Mouse Ortholog Siglec-G)
7.13.12.2 Human Siglec-10 and Siglec-G as a Mouse Ortholog in Immune Responses and Inflammatory Process
7.13.12.3 Siglec-10 in T Cells during Autoimmune Inflammation
7.13.12.4 Comparison Between the Mouse Siglec-G Ortholog and Human Siglec-10
7.13.13 Human Siglec-11 as a CD33-Related Siglec
7.13.13.1 Siglec-11 Is a Paired Receptor with Siglec-16, Which Is Caused by Gene Conversion in Primates
7.13.13.2 Human Siglec-11 and Mouse Siglec-E Function in Microglial Cells
7.13.13.3 Human Siglec-11 and Mouse Siglec-E Function in Microglial Cells
7.13.14 Siglec-14 in Humans as a CD33-Related Siglec
7.13.14.1 The Structure and Expression of Siglec-14 in Humans, Paired with Siglec-5
7.13.14.2 Role of Siglec-14 in Pathogenic Invasion
7.13.14.3 Expression and SA Specificity of Siglec-14
7.13.15 Siglec-16 as a CD33-Related Siglec Is a Paired Receptor with Siglec-11
7.14 Mouse CD33-Related Siglecs with ITIM-Like Domains
7.14.1 mSiglec-E that Belongs to CD33-Related Siglecs
7.14.2 Siglec-F (Human Paralog Siglec-8) as a CD33-Related Siglec
7.14.3 Human Siglec-10 and Mouse Ortholog Siglec-G as CD33-Related Siglecs
7.14.3.1 Mouse Siglec-G Functions in B-Cell Tolerance
7.14.3.2 Mouse Siglec-G Functions in T-Cell Downregulation
7.14.4 Siglec-H as a CD33-Related Siglec
References
Chapter 8: C-Type Lectin (C-Type Lectin Receptor)
8.1 Evolutionary Diversity of C-Type Lectins
8.2 Ca2+-Dependent Glycan-Binding CTLs
8.3 Myeloid CTL-Like Receptor or Myeloid-Suppressive or Inhibitory CLR (MICL), CLEC 12A
8.4 Macrophage Inducible CTLR (Mincle, Clec4e, ClecSf9)/Macrophage CTL (MCL, CLEC4d, ClecSf8)
8.4.1 Expression and Ligand-Binding Specificity of Mincle, Clec4e, ClecSf9, and MCL
8.4.2 Pathogenic PAMPs-Recognition of Mincle and MCL
8.4.3 Th1/Th17 Activation and T Cell Development in Mincle or MCL Interaction with Host
8.5 Mannose Receptor (MR) as CLR and Macrophage Mannose Receptor
8.5.1 Structural Basis and Functions of MR
8.5.2 MR Expression in Immune Systems and Interaction with Helminth Flatworm Trematodes
8.5.3 Recognition of Pathogenic Microbes by MR
8.6 Mannose (or Mannan)-Binding Protein (MBP) and Mannose-Binding Lectin (MBL)
8.6.1 Structural Basis and Glycan Ligand Binding Specificity of MBL
8.6.2 Immunoprotective Activity of MBL
8.6.3 MBL Function in Diseases
8.7 Fucose-Binding Lectin (FBL) and Ficolin
8.7.1 Fucose-Binding Lectin (FBL) Diversity of F-Lectin Repertoires
8.7.2 Specificity of Ficolins or FBL
8.7.3 Ficolin Functions in the Immune Response
8.7.4 Ficolin Interaction with Microorganisms
8.8 Dectin 1 (CLEC-7A in Human)
8.8.1 Basic Function and Structure of Dectin 1
8.8.2 Dectin-1 Recognizes β1,3/β1,6-glycans in Fungi, Plants, Bacteria, and House Dust Mite
8.8.3 Dectin-1 Cluster Includes CTL-Like Receptor 2 (CLEC-2)
8.8.4 CLEC Structures and Ligand Recognition
8.9 DC-Associated CTL-2 (Dectin-2) Family or CLEC4n
8.9.1 Structural Basis and Function of Dectin-2
8.9.2 Langerhans Cell-Specific Expression of Dectin-2 and Interaction with Fungal High-Man Glycans
8.10 Dectin-3 (Clec4D, Clecsf8, MCL, Macrophage CTL)
References
Chapter 9: Galectins
9.1 General and Structural Aspects of Galectins
9.1.1 Biological Roles of Galectins
9.1.2 Immunological Roles of Galectins
9.1.3 Classification of Galectins
9.1.4 Galectin Ligands in Proteins and Gangliosides
9.1.5 Galectins in Lower Organisms such as Zebrafish or Marine Oyster
9.2 Galectin-2
9.3 Galectin-3 and -8 Recognize GM3, But Not Galectin-4
9.3.1 Galectin-3
9.3.2 Galectin-8
9.4 Galectin-1 and -4 Bind to GM1, But Not GM3
9.4.1 Galectin-4
9.4.2 Galectin-1
9.5 Galactine-9 and Galelctin-10
References
Chapter 10: DC-SIGNs
10.1 DC-Specific ICAM-3-Grabbing Non-integrin, DC-SIGNB (CD209)
10.1.1 Molecular Characteristics of DC-SIGN
10.1.2 General Signaling of DC-SIGN
10.1.3 α2,6 Sialyl IgG Fc Function by DC-SIGN Receptor
10.1.4 DC-SIGN Binds to Pathogens, Antigen, and Glycans
10.1.5 DC-SIGN Role in DC-Mediated Viral Transmission by HIV-1
10.1.6 DC-Mediated Immunosuppression by Mycobacteria
10.1.7 DC-SIGN Recognizes Lewis Antigens Expressed in PMN
10.2 Other DCs-Derived Receptors
10.2.1 Dendritic Cell NK Lectin Group Receptor (DNGR-1; CLEC9A)
10.2.2 CTL-Like Receptor-1 (CLEC-1)
10.2.3 CTL-Like Receptor, CLEC12A, Known as Myeloid Inhibitory CTL-Like Receptor (MICL), CTL-Like Molecule-1 (CLL-1), DC-Assoc...
10.2.4 CD161 (NKR-P1A)
References
Chapter 11: Toll-Like Receptors (TLRs)
11.1 TLR Molecular Structure, Subtypes, and Recognition Ligand
11.2 Signal Initiation and Transduction of TLRs
11.3 Glycosylation of TLRs
11.4 General TLR Functions as Pathogen and Antigen Receptors on DCs
11.5 TLR-9 as a CpG DNA Receptor
11.6 TLR-3 as a dsRNA Receptor
11.7 TLR-4 as the LPS Receptor
11.7.1 Ligands of TRL4 Recognition
11.7.2 MyD88-Dependent Pathway of TLR4
11.7.3 MyD88-Independent Pathway of TLR4
11.8 TLR11
11.8.1 Three Major Domains and Binding Ligand of TLR11
11.8.2 TLR11 Intracellular Signal Transduction
11.9 Inhibition of TLRs by Gangliosides
References
Chapter 12: CD33 and CD33-Related Siglecs in Pathogen Recognition and Endocytosis of DC in the Innate Immune System
12.1 CD33 (Siglec-3)
12.1.1 General Biology of CD33
12.1.2 CD33 (Siglec-3)-Targeting of Acute Myeloid Leukemia (AML)
12.1.3 CD33 (Siglec-3)-Targeting Treatment of Alzheimer´s Disease (AD)
12.2 CD33-Related Siglecs (CD33rSiglecs)
12.2.1 Inhibitory CD33rSiglecs in Escape from Tumor and Bacterial Immunosurveillance
12.2.2 Activating CD33rSiglecs
12.3 Pathogenic Suppression of the Pathogen-Specific Host Immune Response
12.3.1 Inhibitory Receptor CD200R and CD200:CD200R1 Signaling
12.3.2 Pathogenic Decoy Ligands Neutralize Host Immunity Through Eliciting Host CD200-CD200R1 Inhibitory Signaling
12.3.2.1 Murine Cytomegalovirus (MCMV)
12.3.2.2 Human Cytomegalovirus (HCMV)
12.3.2.3 Rat Cytomegalovirus (RCMV)
12.3.2.4 Poxvirus
12.3.2.5 Coronaviruses
12.3.2.6 Influenza Virus
12.3.2.7 Herpesviruses
12.3.2.8 Bacterial Decoy Ligands
12.3.2.9 Neisseria Meningitidis
12.3.2.10 Toxoplasma gondii
12.3.2.11 Leishmania
12.3.2.12 Schistosomes and Salmonella
12.4 DCs Tumor Immunotherapy Through Sialyl Binding of DCs to T Cells
References

Citation preview

Cheorl-Ho Kim

Glycobiology of Innate Immunology

Glycobiology of Innate Immunology

Cheorl-Ho Kim

Glycobiology of Innate Immunology

Cheorl-Ho Kim Molecular and Cellular Glycobiology Lab Sungkyunkwan University, Department of Biological Sciences Suwon, Korea (Republic of)

ISBN 978-981-16-9080-8 ISBN 978-981-16-9081-5 https://doi.org/10.1007/978-981-16-9081-5

(eBook)

© The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2022 This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Singapore Pte Ltd. The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore 189721, Singapore

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Repertoire in Innate Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1 Historical Expansion of Defense System . . . . . . . . . . . . . . . . 1.2 Columbus Era to Modern Revolution in Immunological Defense System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3 Historical Profile of Defense Constituents and Progress in Innate Immune Repertoire . . . . . . . . . . . . . . . . . . . . . . . . . 1.3.1 Phagocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3.2 Leukocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3.3 Neutrophils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3.4 Granulocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3.5 Monocytes and Macrophages . . . . . . . . . . . . . . . . . 1.3.6 Dendritic Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3.7 Complement System . . . . . . . . . . . . . . . . . . . . . . . 1.3.8 Opsonization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3.9 Lysozyme and Salvarsan . . . . . . . . . . . . . . . . . . . . 1.3.10 Progress in Innate Immune Response Since Historic Spanish Flu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4 The Outline of Innate Immunity . . . . . . . . . . . . . . . . . . . . . . 1.4.1 Concept of Immune Receptors . . . . . . . . . . . . . . . . 1.4.2 Host Protection from Microbial Invaders of Innate Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 Autophagy from Microbial Invaders and Self-Associated Molecular Patterns (SAMPs) of Innate Immune Cells . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Dendritic Cells (DCs) in Innate Immunity . . . . . . . . . . . . . . . . . . 2.1 General Biology of DCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Classification and Different Function of DCs . . . . . . . . . . . . . 2.2.1 pDC, Lymphoid Organ CD8α+ DC, and Tissue CD103+ DC Interaction with Tregs . . . . . . . . . . . . .

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2.2.2 2.2.3 2.2.4 References . . . 3

DCs Induce Tolerance State . . . . . . . . . . . . . . . . . . DC co-Stimulatory Receptors . . . . . . . . . . . . . . . . . Application of DCs to Human Diseases . . . . . . . . . . ........................................

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Glycan Biosynthesis in Eukaryotes . . . . . . . . . . . . . . . . . . . . . . . . 3.1 General Glycosylation Events . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Sugar Nucleotide Transporters Deliver Donor Saccharides to ER-Golgi Network . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 Golgi Traffic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4 N-glycan Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5 O-glycosylation and Multiple O-Glycan Structures . . . . . . . . . 3.5.1 7 Core O-glycan Structures . . . . . . . . . . . . . . . . . . 3.5.2 Modification of 7 Core O-Glycan Structures . . . . . . 3.6 O-GlcNAcylation, O-Mannosylation, O-β-Glucosylation, O-α-Fucosylation, O-β-Glucosylation, O-β-Galactosylation, C-Glycosylation, and C-Mannosylation . . . . . . . . . . . . . . . . . 3.6.1 O-GlcNAcylation . . . . . . . . . . . . . . . . . . . . . . . . . 3.6.2 O-Mannosylation . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6.3 O-β-Glucosylation . . . . . . . . . . . . . . . . . . . . . . . . . 3.6.4 O-α-Fucosylation . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6.5 O-β-Glucosylation . . . . . . . . . . . . . . . . . . . . . . . . . 3.6.6 O-β-Galactosylation . . . . . . . . . . . . . . . . . . . . . . . . 3.6.7 C-Mannosylation and C-Glycosylation . . . . . . . . . . 3.7 Function of O-Glycosylation and O-Glycans . . . . . . . . . . . . . 3.8 Glycosaminoglycans (GAGs) . . . . . . . . . . . . . . . . . . . . . . . . 3.8.1 Classification and Biosynthesis of GAGs . . . . . . . . . 3.8.2 Chondroitin Sulfate (CS) . . . . . . . . . . . . . . . . . . . . 3.8.3 Dermatan Sulfate (DS) . . . . . . . . . . . . . . . . . . . . . . 3.8.4 Keratan Sulfate (KS) . . . . . . . . . . . . . . . . . . . . . . . 3.8.5 Heparin and Heparan Sulfate . . . . . . . . . . . . . . . . . 3.8.6 Hyaluronic Acid (HA) or Hyaluronan . . . . . . . . . . . 3.8.7 Proteoglycans (PGs) . . . . . . . . . . . . . . . . . . . . . . . 3.8.8 Extracellular PGs . . . . . . . . . . . . . . . . . . . . . . . . . . 3.9 Glycosylphosphatidylinositols (GPIs) Anchor Glycosylation . . 3.9.1 General Structure of GPI Anchors . . . . . . . . . . . . . . 3.9.2 Function of GPI-Anchored Protein . . . . . . . . . . . . . 3.9.3 Biosynthesis, Structural Assembly, and Transportation of GPI-Anchored Protein . . . . . . 3.9.4 GPIs in Parasites . . . . . . . . . . . . . . . . . . . . . . . . . . 3.9.5 GPI Interaction with TLRs in Malaria P. falciparum . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.9.6 GPI-Defected Disorders of Paroxysmal Nocturnal Hemoglobinuria (PNH) and Prion Disease . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Glycans in Glycoimmunology . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1 Glycans in Cell Recognition and Evolutionary Adaptation in Organisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2 Changes in Glycan Structure Involved in Coregulated Expression of Glycan-Binding Lectin Counterparts . . . . . . . . . 4.3 Evolution of Lectin: Alternative Splicing Contributes to Variation for Glycan-Binding Receptors . . . . . . . . . . . . . . 4.4 E-Selectin-Binding Ligand sLex (CD15s) on Neutrophil CD44 N-glycan and Alternatively Spliced Exon 6 Contains Core 2 O-Glycan sLea (CD44v6) Epitope . . . . . . . . . . . . . . . 4.5 Glycans Regulate T Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5.1 Glycans Regulate Development and Differentiation in T Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5.2 Glycosylation of Notch Receptor Signaling for Thymocyte β Selection and T Cell Function Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5.3 Alternatively Spliced Variants Produce Different Glycan Structures of CD43 and CD45 Isoforms in T Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5.4 T Cells CD43 and CD45 Interaction with Their Counter-Receptor or Lectins to Determine T Cell Fates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5.5 TCR Glycosylation Governs Hyper-response and Autoimmune Responses in T Cells and Tregs . . 4.5.6 SAMP and N-Glycan-Dependent Modulation of Inhibitory T Cell Receptors to Suppress T Cell Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5.7 Galectins in Suppression of T Cell Functions . . . . . . 4.5.8 Glycans Regulate T Cell-Mediated Immune Suppression and Tolerance in Tumor Progression . . 4.6 Abnormal N-Glycosylation in Autoimmunity . . . . . . . . . . . . . 4.7 Glycan Regulation of NK Cell Receptors . . . . . . . . . . . . . . . . 4.7.1 NCRs on NK Cells . . . . . . . . . . . . . . . . . . . . . . . . 4.7.2 NCR Ligands . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.7.3 Interaction of NCRs Ligands with Pathogens . . . . . . 4.7.4 Interaction of NCRs Ligands with Self-Ligands . . . . 4.7.5 NK Cells MHC-I-Independent Inhibitory Receptors Siglec-7 and Siglec-9 . . . . . . . . . . . . . . . . . . . . . . . 4.8 Carbohydrate Recognition of Target Antigens by DCs During Infection and Inflammation . . . . . . . . . . . . . . . . . . . . . . . . . . 4.8.1 Lewis Ligand Recognition by DCs . . . . . . . . . . . . . 4.8.2 VIM Ceramide Dodecasaccharide . . . . . . . . . . . . . . 4.9 Glycan-Specific Trafficking Receptors in DC Maturation . . . . 4.10 Glycan Ligands in Trafficking of DC Migration . . . . . . . . . . .

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sLex-PSGL-1 Glycans in DC Trafficking . . . . . . . . . . Ganglioside Recognition by DC Receptors in Trafficking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.11 Chemokine Receptors in DC Trafficking . . . . . . . . . . . . . . . . . 4.11.1 Chemokine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.11.2 Chemokine Receptor . . . . . . . . . . . . . . . . . . . . . . . . 4.11.3 Chemokine-GAG Interaction as a Type of Protein-Glycan Interactions . . . . . . . . . . . . . . . . . 4.11.4 Molecular Motifs in Chemokine for GAG Recognition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.11.5 C-C Type Chemokine Receptor 4 (CCR4) and Specific Ligand 17 (CCL17) and Specific Ligand 22 (CCL22) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.12 Glycan Structure-Recognizing Selectins in DC-Endothelium Interaction During Infection and Inflammation . . . . . . . . . . . . . 4.12.1 3 Species of Selectins: E-, L-, and P-selectins . . . . . . 4.12.2 Representative Selectin Ligand PSGL-1 and Role of PSGL-1 O-Glycan . . . . . . . . . . . . . . . . . . . . . . . . 4.12.3 Glycosyltransferases for Biosynthesis of PSGL-1 O-Glycan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.12.4 Designation of Carbohydrate Glycomimetic Drugs and Natural Inhibitors of Selectins . . . . . . . . . . . . . . 4.12.5 Glycomimetic Drug Candidates . . . . . . . . . . . . . . . . 4.12.6 GAG-Glycomimetic Drugs . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.10.1 4.10.2

5

Pathogen-Host Infection Via Glycan Recognition and Interaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1 Lectin Recognition of Glycans on Cell Surface and Soluble Glycans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2 Innate Immune-Specific and Host Defensing Lectins of Fungal, Protozoa, Invertebrate, and Lower Vertebrates . . . . . . . . . . . . . 5.3 How Do Hosts Interact with Pathogens? . . . . . . . . . . . . . . . . . 5.3.1 Lectin-Carbohydrate Interaction . . . . . . . . . . . . . . . . 5.3.2 Bacterial Glycoconjugates Interact with Host Lectins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4 Pathogen-Producing Lectins as Receptors to Bind to the Host Carbohydrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4.1 Uropathogenic E. coli (UPEC), Enterohemorrhagic E. coli (EHEC), and Enterotoxigenic E. coli (ETEC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4.2 Lectins and Glycans of Other Pathogenic Bacteria . . . 5.4.3 Viral Lectins or Host Lectin-Binding Glycans . . . . . . 5.5 Host Lectin Defense Mechanisms in Lectin-Carbohydrate Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Pathogenic Glycans to Trigger Innate Immune Enhancement . . . 5.6.1 Example 1: Polysaccharides with Immune Enhancement of Cyrtomium macrophyllum . . . . . . . . 5.6.2 Example 2: Activation of Macrophage by Polysaccharide from Paecilomyces cicadae . . . . . . . . 5.6.3 Example 3: NK Cell-Mediated Cytotoxicity Increased by Arabinogalactan from Anoectochilus formosanus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.6.4 Example 4: Streptococcus pneumonia Polysaccharides Activate NK Cells, NK-Like T Cells, and Monocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.6.5 Example 5: C. macrophyllum Polysaccharides (CMP) Enhance Lymphocyte Proliferation and Macrophage Function . . . . . . . . . . . . . . . . . . . . 5.7 TLR4 Receptor-Activating Glycans Activate NO Production in Macrophage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.8 CBPs or GBPs in Antigen Recognition . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

7

Innate Immunity Via Glycan-Binding Lectin Receptors . . . . . . . . 6.1 Glycosylation Effect on Autoimmunity and Inflammation . . . . 6.1.1 Glycosylation in Immunological Recognition and Inflammation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1.2 Glycosylation Effect on Autoimmunity . . . . . . . . . . 6.2 Glycosylation Effect on Tumor Immunity of Immune Cells . . . 6.3 Immune Tolerance and Defense Mechanisms of Innate Immune DCs During Infection . . . . . . . . . . . . . . . . . . . . . . . 6.4 How Are Pathogenic Bacteria Recognized by Receptors of DCs of the Host Immune System? . . . . . . . . . . . . . . . . . . . 6.4.1 DC Lectins for Glycan Recognition of Invasive Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4.2 Toll-Like Receptors . . . . . . . . . . . . . . . . . . . . . . . . 6.4.3 Innate Immune Receptors in Malaria Infection . . . . . 6.4.4 Innate Immunity Receptors in Protozoan Parasite Toxoplasma gondii . . . . . . . . . . . . . . . . . . . . . . . . 6.5 Pathogen Recognition and Adaptive Immune Responses in Acquired Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.6 Galactose-Specific C-Type Lectin: Two Major ASGPR and Macrophage Galactose Lectin (MGL) in the Human . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Sialic Acid-Binding Ig-Like Lectins (Siglecs) . . . . . . . . . . . . . . . . . 7.1 PolySia and Host Sialic Acids Modulate Host Immune Responses as Pathogenic Decoys . . . . . . . . . . . . . . . . . . . . . . .

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7.2 7.3 7.4

7.5

7.6 7.7

7.8 7.9

7.10

7.11

Sialic Acid Recognition by Siglecs for Selfor Nonself-Antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Classification of Siglecs . . . . . . . . . . . . . . . . . . . . . . . . . . . . Evolution of Siglecs, Sialic Acids, and Sialic Acid O-Acetylation as Host Ligands (Receptors) for Microbes and Innate Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Microbial Sialic Acid-like Molecules Synthesis and Recognition of Microbial Sialic Acids by DCs and Bacteriophages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hematopoietic System in Siglecs . . . . . . . . . . . . . . . . . . . . . . Structure of Siglecs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.7.1 Cytoplasmic ITIM and ITAM Domains of Siglecs . . 7.7.2 Adaptor Proteins Associated with Siglecs . . . . . . . . 7.7.3 SA-Recognition Tropism of Siglecs . . . . . . . . . . . . Inhibitory Signaling of DCs . . . . . . . . . . . . . . . . . . . . . . . . . Siglec-1 (CD169, Sialoadhesin/Sn) . . . . . . . . . . . . . . . . . . . . 7.9.1 General SAbinding Specificity of Siglec-1 . . . . . . . . 7.9.2 Siglec-1 Is a Pathogen-Binding Receptor . . . . . . . . . 7.9.3 Siglec-1 Recognizes HIV and Is a Transinfection Receptor Expressed on mDCs . . . . . . . . . . . . . . . . CD22/Siglec-2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.10.1 General and Structural Aspects of CD22/Siglec-2 . . 7.10.2 CD22 I Associated with Development of Autoimmune Diseases . . . . . . . . . . . . . . . . . . . . . . 7.10.3 CD22 Function in Immune Tolerance Events . . . . . . 7.10.4 Role of CD22 (Siglec-2, Mice Siglec-G) in Immune Responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.10.5 Model Ligands for Recognition of CD22 on B Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.10.6 B Cell-Targeted Immunotherapy Through CD22-Positive Targeting of B-Cell Lymphomas . . . 7.10.7 Immune Tolerance Capacity of Neu5Ac-α2,6-Gal Ligands in DCs by ST6Gal-1 of Tumor Cells for Immunesurveillance . . . . . . . . . . . . . . . . . . . . . . . . 7.10.8 CD22 Vs. Pathogens . . . . . . . . . . . . . . . . . . . . . . . 7.10.9 CD22 Application with CAR-T on Acute Lymphoblastic Leukemia (ALL) . . . . . . . . . . . . . . . 7.10.10 CD22/Siglec-2 Coreceptor, CD45 on T Cells . . . . . . Siglec-4/Myelin-Associated Glycoprotein (MAG) . . . . . . . . . 7.11.1 General Aspects of MAG/Siglec-4 . . . . . . . . . . . . . 7.11.2 Siglec-4/MAG in the CNS and Brain Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.11.3 Siglec-4/MAG in Hippocampal Long-Term Potentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Siglec-15, Non-CD33-Related Siglecs in Humans . . . . . . . . . 7.12.1 The Structure and Expression of Siglec-15, Called Misnomer “CD33L3” in Humans . . . . . . . . . . . . . . 7.12.2 DAP12-Syk Pathway in Siglec-15-Mediated Remodeling of the Tumor Microenvironment . . . . . 7.12.3 Siglec-15 Functions in Osteoclastogenesis . . . . . . . . 7.13 Siglec-3 (CD33)-Related Siglecs on DCs . . . . . . . . . . . . . . . . 7.13.1 Siglec-3 (CD33) . . . . . . . . . . . . . . . . . . . . . . . . . . 7.13.2 Structure, Natural Ligand, and Cellular Signaling with SHP-1/-2 of Siglec-3/CD33 . . . . . . . . . . . . . . 7.13.3 Pathogen Ligand for CD33 . . . . . . . . . . . . . . . . . . . 7.13.4 Siglec-3/CD33 Is Related to SOCS3 and Internalization of CD33 . . . . . . . . . . . . . . . . . . 7.13.5 Putative Functions of Siglec-3/CD33 in Alzheimer’s Disease (AD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.13.6 Siglec-3-/CD33-Based Immunotherapy for AML . . . 7.13.7 Siglec-5/CD170 as a CD33-Related Siglec . . . . . . . 7.13.8 Siglec-6 as a CD33-Related Siglec . . . . . . . . . . . . . 7.13.9 Siglec-7 (CD328) as a CD33-Related Siglec . . . . . . 7.13.10 Siglec-8 as a CD33-Related Siglec and Siglec-F as a Mouse Paralog . . . . . . . . . . . . . . . . . . . . . . . . 7.13.11 Siglec-9 as a CD33-Related Siglec and Murine Functional Counterpart, Siglec-E . . . . . . . . . . . . . . 7.13.12 Siglec-10 (Mouse Ortholog Siglec-G) in Humans as a CD33-Related Siglec . . . . . . . . . . . . . . . . . . . . 7.13.13 Human Siglec-11 as a CD33-Related Siglec . . . . . . 7.13.14 Siglec-14 in Humans as a CD33-Related Siglec . . . . 7.13.15 Siglec-16 as a CD33-Related Siglec Is a Paired Receptor with Siglec-11 . . . . . . . . . . . . . . . . . . . . . 7.14 Mouse CD33-Related Siglecs with ITIM-Like Domains . . . . . 7.14.1 mSiglec-E that Belongs to CD33-Related Siglecs . . . 7.14.2 Siglec-F (Human Paralog Siglec-8) as a CD33-Related Siglec . . . . . . . . . . . . . . . . . . . . . . 7.14.3 Human Siglec-10 and Mouse Ortholog Siglec-G as CD33-Related Siglecs . . . . . . . . . . . . . . . . . . . . 7.14.4 Siglec-H as a CD33-Related Siglec . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

C-Type Lectin (C-Type Lectin Receptor) . . . . . . . . . . . . . . . . . . 8.1 Evolutionary Diversity of C-Type Lectins . . . . . . . . . . . . . . 8.2 Ca2+-Dependent Glycan-Binding CTLs . . . . . . . . . . . . . . . . 8.3 Myeloid CTL-Like Receptor or Myeloid-Suppressive or Inhibitory CLR (MICL), CLEC 12A . . . . . . . . . . . . . . . . 8.4 Macrophage Inducible CTLR (Mincle, Clec4e, ClecSf9)/ Macrophage CTL (MCL, CLEC4d, ClecSf8) . . . . . . . . . . . .

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8.4.1

Expression and Ligand-Binding Specificity of Mincle, Clec4e, ClecSf9, and MCL . . . . . . . . . . 8.4.2 Pathogenic PAMPs-Recognition of Mincle and MCL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.4.3 Th1/Th17 Activation and T Cell Development in Mincle or MCL Interaction with Host . . . . . . . . . 8.5 Mannose Receptor (MR) as CLR and Macrophage Mannose Receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.5.1 Structural Basis and Functions of MR . . . . . . . . . . . 8.5.2 MR Expression in Immune Systems and Interaction with Helminth Flatworm Trematodes . . . . . . . . . . . 8.5.3 Recognition of Pathogenic Microbes by MR . . . . . . 8.6 Mannose (or Mannan)-Binding Protein (MBP) and MannoseBinding Lectin (MBL) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.6.1 Structural Basis and Glycan Ligand Binding Specificity of MBL . . . . . . . . . . . . . . . . . . . . . . . . 8.6.2 Immunoprotective Activity of MBL . . . . . . . . . . . . 8.6.3 MBL Function in Diseases . . . . . . . . . . . . . . . . . . . 8.7 Fucose-Binding Lectin (FBL) and Ficolin . . . . . . . . . . . . . . . 8.7.1 Fucose-Binding Lectin (FBL) Diversity of F-Lectin Repertoires . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.7.2 Specificity of Ficolins or FBL . . . . . . . . . . . . . . . . 8.7.3 Ficolin Functions in the Immune Response . . . . . . . 8.7.4 Ficolin Interaction with Microorganisms . . . . . . . . . 8.8 Dectin 1 (CLEC-7A in Human) . . . . . . . . . . . . . . . . . . . . . . . 8.8.1 Basic Function and Structure of Dectin 1 . . . . . . . . 8.8.2 Dectin-1 Recognizes β1,3/β1,6-glycans in Fungi, Plants, Bacteria, and House Dust Mite . . . . . . . . . . 8.8.3 Dectin-1 Cluster Includes CTL-Like Receptor 2 (CLEC-2) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.8.4 CLEC Structures and Ligand Recognition . . . . . . . . 8.9 DC-Associated CTL-2 (Dectin-2) Family or CLEC4n . . . . . . . 8.9.1 Structural Basis and Function of Dectin-2 . . . . . . . . 8.9.2 Langerhans Cell-Specific Expression of Dectin-2 and Interaction with Fungal High-Man Glycans . . . . 8.10 Dectin-3 (Clec4D, Clecsf8, MCL, Macrophage CTL) . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Galectins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.1 General and Structural Aspects of Galectins . . . . . . . . . . . . . . 9.1.1 Biological Roles of Galectins . . . . . . . . . . . . . . . . . 9.1.2 Immunological Roles of Galectins . . . . . . . . . . . . . 9.1.3 Classification of Galectins . . . . . . . . . . . . . . . . . . . 9.1.4 Galectin Ligands in Proteins and Gangliosides . . . . .

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9.1.5

Galectins in Lower Organisms such as Zebrafish or Marine Oyster . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2 Galectin-2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.3 Galectin-3 and -8 Recognize GM3, But Not Galectin-4 . . . . . . 9.3.1 Galectin-3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.3.2 Galectin-8 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.4 Galectin-1 and -4 Bind to GM1, But Not GM3 . . . . . . . . . . . 9.4.1 Galectin-4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.4.2 Galectin-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.5 Galactine-9 and Galelctin-10 . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

11

DC-SIGNs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.1 DC-Specific ICAM-3-Grabbing Non-integrin, DC-SIGNB (CD209) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.1.1 Molecular Characteristics of DC-SIGN . . . . . . . . . . 10.1.2 General Signaling of DC-SIGN . . . . . . . . . . . . . . . 10.1.3 α2,6 Sialyl IgG Fc Function by DC-SIGN Receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.1.4 DC-SIGN Binds to Pathogens, Antigen, and Glycans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.1.5 DC-SIGN Role in DC-Mediated Viral Transmission by HIV-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.1.6 DC-Mediated Immunosuppression by Mycobacteria . . . . . . . . . . . . . . . . . . . . . . . . . . 10.1.7 DC-SIGN Recognizes Lewis Antigens Expressed in PMN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.2 Other DCs-Derived Receptors . . . . . . . . . . . . . . . . . . . . . . . . 10.2.1 Dendritic Cell NK Lectin Group Receptor (DNGR-1; CLEC9A) . . . . . . . . . . . . . . . . . . . . . . . 10.2.2 CTL-Like Receptor-1 (CLEC-1) . . . . . . . . . . . . . . . 10.2.3 CTL-Like Receptor, CLEC12A, Known as Myeloid Inhibitory CTL-Like Receptor (MICL), CTL-Like Molecule-1 (CLL-1), DC-Associated CTL 2 (DCAL-2), and CD371 . . . . . . . . . . . . . . . . . . . . . 10.2.4 CD161 (NKR-P1A) . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Toll-Like Receptors (TLRs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.1 TLR Molecular Structure, Subtypes, and Recognition Ligand . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.2 Signal Initiation and Transduction of TLRs . . . . . . . . . . . . . . 11.3 Glycosylation of TLRs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.4 General TLR Functions as Pathogen and Antigen Receptors on DCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Contents

11.5 11.6 11.7

TLR-9 as a CpG DNA Receptor . . . . . . . . . . . . . . . . . . . . . . TLR-3 as a dsRNA Receptor . . . . . . . . . . . . . . . . . . . . . . . . TLR-4 as the LPS Receptor . . . . . . . . . . . . . . . . . . . . . . . . . 11.7.1 Ligands of TRL4 Recognition . . . . . . . . . . . . . . . . 11.7.2 MyD88-Dependent Pathway of TLR4 . . . . . . . . . . . 11.7.3 MyD88-Independent Pathway of TLR4 . . . . . . . . . . 11.8 TLR11 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.8.1 Three Major Domains and Binding Ligand of TLR11 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.8.2 TLR11 Intracellular Signal Transduction . . . . . . . . . 11.9 Inhibition of TLRs by Gangliosides . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

CD33 and CD33-Related Siglecs in Pathogen Recognition and Endocytosis of DC in the Innate Immune System . . . . . . . . . 12.1 CD33 (Siglec-3) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.1.1 General Biology of CD33 . . . . . . . . . . . . . . . . . . . 12.1.2 CD33 (Siglec-3)-Targeting of Acute Myeloid Leukemia (AML) . . . . . . . . . . . . . . . . . . . . . . . . . 12.1.3 CD33 (Siglec-3)-Targeting Treatment of Alzheimer’s Disease (AD) . . . . . . . . . . . . . . . . . 12.2 CD33-Related Siglecs (CD33rSiglecs) . . . . . . . . . . . . . . . . . . 12.2.1 Inhibitory CD33rSiglecs in Escape from Tumor and Bacterial Immunosurveillance . . . . . . . . . . . . . 12.2.2 Activating CD33rSiglecs . . . . . . . . . . . . . . . . . . . . 12.3 Pathogenic Suppression of the Pathogen-Specific Host Immune Response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.3.1 Inhibitory Receptor CD200R and CD200:CD200R1 Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.3.2 Pathogenic Decoy Ligands Neutralize Host Immunity Through Eliciting Host CD200-CD200R1 Inhibitory Signaling . . . . . . . . . . . . . . . . . . . . . . . . 12.4 DCs Tumor Immunotherapy Through Sialyl Binding of DCs to T Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abbreviations

AhR AICD ALIS APC AR ASGPR ATCS BAG6 BAT3 Batf3 BGN C2GnT C4ST C6ST CAM CD CEA CFG ChABC ChGn ChPF ChSy-1 CIA CLD CLR CNS CNTN-1 CNX Core 1 β3Gal-T COSMC CR

Aryl hydrocarbon receptor Activation-induced cell death Aggresome-like structure Antigen-presenting cell Autosomal recessive Asialoglycoprotein receptor (ASGPR) Adducted thumb-clubfoot syndrome Bcl2-associated anthogene 6 HLA-B-associated transcript 3 Basic leucine zipper transcription factor ATF-like3 Biglycan Core 2 β1,6N-GlcNAc-transferase Chondroitin 4-O-sulfo-transferase Chondroitin 6-O-sulfo-transferase Cell adhesion molecule Clustered differentiation Carcinoembryonic antigen Consortium for functional glycomics Chondroitinase-A, -B, and -C Chondroitin GalNAcT Chondroitin polymerizing factor Chondroitin synthase Collagen type II-induced arthritic disease Collagen-like domain C-type lectin receptor Central nervous system Contactin-1 Calnexin Core 1 β3-Gal-Transferase Core 1 β3GalT-specific molecular chaperone Complement receptor xv

xvi

CRD CRP CRP CRT CS CSPG CTL CTL-4 CTLs D4ST DALIS DAMP DBL DCN DC-SIGN DLL Dol-P DS DSE DSE-L DTH EDS EDSKT eIF2 EMT ERAD ERGIC ESAM EtN-P Flt3L FUT3 GalNAc-4S-6ST GalNAcT-I GalNAcT-II Gal-T1 GBP GBS GCN2 GIT GITR GlcA GlcAT-II GlcNAcT-II

Abbreviations

Carbohydrate recognition domain Complement regulatory protein C-type receptor protein Calreticulin Chondroitin sulfate CS attached PG C-type lectin Human CTL antigen-4 Cytotoxic T lymphocytes Dermatan 4-O-sulfo-transferase DC Aggresome-like induced structures Damage-associated molecular pattern Duffy binding-like Decorin DC-specific intracellular adhesion molecule-3 non-integrin Delta-like ligands Dolichol phosphate Dermatan sulfate DS epimerase DSE-like gene Delay-type hypersensitivity Ehlers-Danlos syndrome EDS Kosho type Eukaryotic initiation factor 2 Epithelial-mesenchymal transition ER-associated degradation ER-Golgi intermediate compartment Endothelial cell-specific adhesion molecule Phosphoethanolamine FMS-like Tyr kinase 3 ligand α1,3/4-Fuc-Transferase GalNAc-4-sulfate 6-O-sulfo-transferase N-acetylglucosaminyltransferase I N-acetylgalactosaminyltransferase β1,4-galactosyltransferase Glycan-binding protein Group B streptococcus General control non-derepressible 2 Gastrointestinal track Glucocorticoid-induced TNFR-related protein Glucuronic acid Glucuronosyltransferase II N-acetylgalactosaminyltransferase I

grabbing

Abbreviations

GNE GPCR GSK3β GSL GT GvHd HA HBP hIBM HIV hLys HNK HNK-1ST Hp HPMR hPro HS IBD ICOS ICOS-L ID2 IDDM IDO IdoA2S IdoUA IFN IIM IRF8 ITIM KS LacNAc Lag-3 LAP LLC LPS MAL MAP MASP MBL MBP MCEDS MHC MK MLL-5

xvii

UDP-GlcNAc-2-epimerase G-protein coupled receptor Glycogen synthase kinase 3β Glycophospholipid Glycosyltransferase Graft-versus-host disease Hemagglutinin Hexosamine biosynthetic pathway ITM-like hereditary inclusion-body myositis Human immunodeficiency virus Hydroxyl Lys Human natural killer HNK-1 sulfo-transferase Heparan phosphate Hyperphosphatasia mental retardation Hydroxy-proline Heparan sulfate Inflammatory bowel disease Inducible co-stimulator ICOS-ligand DNA-binding protein 2 Insulin-dependent diabetes mellitus Indoleamine-2,3-dioxygenase IdoA 2-sulfated residue Iduronic acid Interferon Idiopathic inflammatory myopathies IFN-regulatory factor 8 Immune receptor tyrosine-based inhibitory motif Keratan sulfate Lactosamine Lymphocytic activation gene-3 Latency-associated protein Lewis lung carcinoma Lipopolysaccharide MYD88 adaptor-like protein MBL-associated protein MBL-associated serine protease Mannose-binding lectin Mannan-binding protein Musculocontractural EDS Histocompatibility complex Midkine Mixed-lineage leukemia-5

xviii

MMP MNK moDC MR MS NA NGS NLR NOD NST OST PAMP PAPS PAPST PARP PC PCNA pDC PD-L1 PE PG PGAP PGI PGRP PMN PNH POM-T PrPc PrPres PRR PSGL-1 PTN QC RA RAG RAGE rER RLR SED SIGNR1 SLC35D1 SLE SLRP SP

Abbreviations

Matrix metalloproteinase ManNAc kinase Monocyte-derived DC Mannose receptor Multiple sclerosis Neuraminidase Next-generation genome sequencing NOD-like receptor Nucleotide oligomerization domain Nucleotide-sugar transporter Oligosaccharyltransferase Pathogen-associated molecular pattern 30 -Phospho-adenosine 50 -phospho-sulfate PAPS transporter Pathogen-associated recognition Phosphatidylcholine Proliferating cell nuclear antigen Plasmacytoid DC PD ligand-1 Phosphatidylethanolamine Proteoglycan Post-GPI-attachment to protein Protein–glycan interaction Peptidoglycan recognition protein Polymorphonuclear neutrophils Paroxysmal nocturnal hemoglobinuria O-mannosyl-Transferase Proteinase-sensitive cellular prion protein Proteinase-resistant prion protein Pattern recognition receptor P-Selectin glycoprotein ligand-1 Pleiotrophin Quality control Retinoic acid Recombination-activating gene Advanced glycation end products Rough ER RA-inducible gene I-like receptor Spondyloepiphyseal dysplasia SIGN receptor-1 Solute carrier 35D1 Systemic lupus erythematosus Small leucine-rich PG Surfactant protein

Abbreviations

ST3Gal-1 T helper-3 Teffs TEP TF TGN TIGIT Tim-3 TIR TLR TM TOR TPA Tr1 TRAM Treg TRIF TRIF TS TSR UA UST uTPA VNTR VSP Xyl-T β3GnT

xix

β-Gal α2,3-SA-transferase 1 TGF-β-expressing Th3 Effector T cells Thioester-containing protein Thomsen-Friedenreich antigen Trans-Golgi network T cell immunoreceptor with Ig and ITIM domains (TIGIT) mucin-domain-containing molecule-3 Toll/IL-1R-homology domain Toll-like receptor Transmembrane Target of rapamycin Tissue plasminogen activator T regulatory-1 TRIF-related adaptor molecule Regulatory T cell TIR-bearing adaptor-inducing IFN-β TIR-domain-containing adaptor protein inducing IFN Thrombospondin Type 1 repeat Uronic acid Uronyl 2-O-sulfo-transferase Urinary type plasminogen activator Variable number tandem repeats Variant surface glycoprotein β-xylosyltransferase β1,3-N-GlcNAc-transferase

Chapter 1

Repertoire in Innate Immunity

1.1

Historical Expansion of Defense System

The first historical depiction of infectious diseases was from the Epic of Gilgamesh, an Akkadian poem, which is the oldest literature that belongs to early Sumerian poems. The Epic of Gilgamesh is a Babylonian poem written around 2000 B.C. that depicts the adventures of Gilgamesh, the king of Uruk. Gilgamesh is speculated to reign sometimes between 2800 and 2500 B.C. and was deified posthumously [1]. There are a few versions of the poem, and the oldest version of it dates back to the seventh century B.C. In this poem, the presence of pestilences and diseases is well recorded. In addition, the Greek mythology also illustrates infectious diseases in the story of the Apollo and Artemis’s murder of Niobe’s sons. In the story, Apollo and Artemis kill Niobe’s sons with the arrows of plague. Apollo, once again, sends plague to the Greek camps during the Trojan War (from 1194 to 1184 B.C.), thereby causing the death of many lives. Infectious diseases have always been with human and, thus naturally, recorded since the very beginning of the written history. Infectious diseases at the beginning of written history were considered as curses of God or somethings mysterious. However, it was Hippocrates, the Greek physician, who has changed the course of medical practices entirely. Hippocrates, famed to be known as the father of modern medicine, ingenuously observed the relationship between hygiene, immunity, and infections. His clever observations made him possible to forecast infectious diseases, and he even mentioned tuberculous spondylitis, malaria, and tetanus [2]. His insights still have bearing on the understanding of innate immunity. Hippocrates’s brilliant clinical observations are well documented in the Corpus Hippocraticum (Hippocratic Collection), and the contents in it are still recognized as useful “lessons” for current and future medical philosophy since they provide archeological portrayals of infectious diseases. The knowledge of infectious diseases was accumulated through the course of history and had influenced the relevant consciousness and awareness of the modern medical practices and education. For instance, the fact that the symptoms © The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2022 C.-H. Kim, Glycobiology of Innate Immunology, https://doi.org/10.1007/978-981-16-9081-5_1

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Repertoire in Innate Immunity

of diseases are associated with the disequilibrium of bodily fluids has been widely accepted from the ancient to the modern eras. Hippocrates was the first to recognize magical miasma was not the cause of diseases and, rather precisely, recorded influenza pandemic [2]. Let’s take a look at how the knowledge of fever was produced to understand how the knowledge of infectious diseases has been developed. During the time of Hippocrates, the tertian paroxysmal and quartan fevers were recognized to be caused by malaria [3]. The tenth-century C.E. Persian physician Akhawayni then created fever curves for tertian and quartan fevers and recorded them in Hidayat al-Mutaʽallemin fi al-Tibb [3]. Likewise, many ancient Greek, Roman, and medieval savants and physicians increasingly gained knowledges about fevers and body temperature contributing to the basic understanding of the role of fever and thermometry. Even fevers as a symptom and as a disease have been distinguished during this time. In the same ear, scholars were able to define the cause of fever and discover methods for measuring fevers. This progression of the understanding of fever enabled the sixteenth-century scientist Galileo to produce thermometric instruments. For understanding fever, clinical thermometry was continuously being useful even until the end of the eighteenth century providing useful tools to measure fever and interpret the role of fever in human health [4]. Many more scientists kept discovering more and more, thanks to pioneers in history. Aulus Cornelius Celsus (ca 25 B.C.–50 A.D.) defined, for the first time, inflammation as calor (warmth), dolor (pain), tumor (swelling), and rubor (redness) [5–8]. In 1443, Thomas of Sarzana, later known as Pope Nicholas V, found the copy of De Medicina which describes the Greco-Roman medicine by Celsus at the library in Milan. This book was published in 1478 and quickly gained its reputation as a standard text of medicine. The descriptions of diseases and rational therapeutic approaches recorded in the book had influenced practices of physicians for many following years. For instance, in the sixteenth century, Ambroise Paré cited Celsus for his use of vessel ligation in order to stop hemorrhage in wounds [9]. Two centuries later, Morgagni utilized De Medicina as a standard reference to understand diseases and develop therapeutic and surgical methods. He also attempted to correlate case studies done in the past with relative pathologies with the help of De Medicina [10]. Muhammad ibn Zakariya al-Razi, who was called Rhazes (ca 850–930), was a Persian physician who first described measles authentically in literature, and he distinguished smallpox from measles for the first time in medical history [11– 13]. Razi wrote medical books such as Kitab al-Mansur’t (Book for al-Mansur) and Kitab al-Hawi (Comprehensive Book on Medicine) while working at hospitals in his hometown of Rayy, near Tehran and Baghdad. These books were known in Latin tradition as Liber Almansoris and Continens and had influenced many European physicians and universities [11]. The concept of diseases founded by medieval Islamic physicians was described in the Greek philosophy and Greco-Roman medicine as “Arabized Galenism.” Many brilliant descriptions of diagnoses are included in Razi’s case studies like the terms “headache caused by a yellow bile vapor” or “illness.” Besides Razi, many other Arabic physicians like Abū al-Qāsim al-Zahrāwi

1.2 Columbus Era to Modern Revolution in Immunological Defense System

3

(known as Albucasus among Europeans), Ibn Sina (Avicenna), and Ali ibn al-'Abbas al-Majusi (Haly Abbas) all have greatly contributed to the knowledge of infectious diseases and inflammation. During 1104–1110 C.E., the plague killed more than 90% of the European population, and it was when the term Black Death was first introduced. During 1346–1353, a new plague broke out over Western Asia, the Middle East, North Africa, and Europe which killed an estimated 75 million people. Like the Latin pestis (curse), plagues were dreaded by the humankind throughout history. Plagues were considered as the Apocalypse, a divine curse, which possess “the power to kill over a fourth of the earth.” It is one of the rare epidemics that have been recurred with highly transmissible nature resulting in brutality, high pathogenicity, strong lethality, and great swiftness, and, moreover, there were virtually no options for preventions and treatments until the twentieth century. Particularly, in the Western world, epidemics have influenced the evolution of societies at both biological and cultural levels [14]. King Philip VI in 1348 supported the study group in the medical faculty of the University of Paris to study the relationship between the constellation of Saturn, Jupiter, and Mars on March 20, 1345, and the dissemination of pestilence in the air [15]. The Swiss-German physician Philippus Aureolus Theophrastus Bombastus von Hohenheim, known as Paracelsus, introduced mercury salt to treat syphilis. The rationale behind mercury salt that Paracelsus thought arsenic was one of the major components of the first effective treatment for syphilis [16]. Paracelsus, the sixteenth-century alchemist and physician, had acquired his skill from barber surgeons, alchemists, and gypsies, and, at the same time, he was also appointed as the professor of medicine in Basel. He was famous for his use of mercury, arsenic, antimony, and tin salts for syphilis, intestinal worms, and endemic diseases during medieval Europe. In assumption, he killed more patients than he cured using such toxic metals. For hundred years later, Paul Ehrlich discovered the arsenic-containing drug “606” (later called Salvarsan) which was the first drug for syphilis and considered to be the “magic bullet.” The drug was utilized until the introduction of another powerful antibiotic in the twentieth century, penicillin.

1.2

Columbus Era to Modern Revolution in Immunological Defense System

The Columbian Exchange includes the multiple influences through exchanges of crops, tropical diseases, religion, ideas, philosophy, and populations between the New World and the Old World. The exchange was initiated by the voyage of Christopher Columbus to America in 1492. Infectious diseases, such as smallpox, measles, chicken pox-varicella zoster, Bordetella pertussis whooping, plague, typhus, and malaria, were entered into the new lands [17]. The New World (Mundus Novus) had gained many things described above as a byproduct of the Columbian Exchange or interchange, but the most important and best-known gains were the

4

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Repertoire in Innate Immunity

supplies of new metals. The Old World had also gained new staple crops like cassavas, maize, potatoes, and sweet potatoes. The exchange of many stuffs like currency was drastically increased throughout many parts of the world. The exchange had introduced many crops of the Old World including coffee and sugar that were well available for the soils of the New World. Hereafter, Martin Luther (1483–1546) initiated the religious reformation after the church sold indulgence to citizens. The first microscope was designed by Antonie van Leeuwenhoek (1632–1723), a Dutch tradesman and scientist from Delft. Using the microscope, he discovered the world of previously invisible creatures which he called animalcules (small animals). van Leeuwenhoek communicated his discoveries in 1674 to the Royal Society of London with detailed drawings of his findings. Elie Metchnikoff, a Russian biologist, zoologist, and protozoologist, was the first to introduce the concept of phagocytosis into broader perspectives. He is the father of natural immunity. Together with Paul Ehrlich, Elie Metchnikoff was a recipient of the 1908 Nobel Prize in Physiology and Medicine from their discoveries and achievements on the conceptional antibody and the important immune aspects. Elie Metchnikoff and Paul Ehrlich shared Nobel Prize to their pioneer works in humoral and adaptation immunology. Metchnikoff achieved the phenomena including leukocyte recruitment and microbe phagocytosis in the defense system of hosts during pathogenic infections, infectious agent-induced inflammation, and systemic immune response of hosts. His work pioneered the contemporary research on innate immunity. During his research era, he stayed in Odessa region of Russia and the Pasteur Institute in Paris, France. At that time, he was enriched with his complex personality, creative ingenuity, imagination, and insight which made him as “the father of natural immunity” although his observation that a thorn of rose caused phagocyte recruitment in a starfish might be a myth [18, 19]. Louis Pasteur (1822–1895), a French chemist, is one of the founders of microbiology. In Louis Pasteur’s scientific career, he laid the foundation for the future study on stereochemistry. He also pointed out the importance of epidemiology and public health. He fought against the idea of spontaneous life generation. He established the concept of immunity and generalized the principle of vaccination. He developed the concept of microorganism-induced infectious diseases and the concept of vaccination [20]. Robert Koch (1843–1910), a German physician, was the first researcher to isolate bacterial pathogen of Bacillus anthracis in 1877. He formulated the Koch postulations that the microorganism is present and discoverable in every case of the disease. He also pointed out that microorganisms need to be cultivated in a pure culture. Inoculations from cultures must reproduce the diseases in susceptible animals. Then, microorganisms must be re-obtained from infected animals and grown again in a pure culture.

1.3 Historical Profile of Defense Constituents and Progress in Innate Immune. . .

1.3 1.3.1

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Historical Profile of Defense Constituents and Progress in Innate Immune Repertoire Phagocytosis

Johann August Ephraim Goeze (1731–1793) is recognized to be the first researcher ever to study phagocytic phenomenon. He first described tardigrades in 1773 and, in 1784, observed pathological outcomes of neurocysticercosis during pathogenic larval stages [21]. He did figure many aquatic invertebrates, particularly insects and worms. Goeze perceived the similarities of the two different sources of tapeworm heads present in the intestinal tract of humans and Cysticercus cellulosaeinvaginated heads in pigs [22] (Fig. 1.1).

1.3.2

Leukocytes

In 1843, Gabriel Andral (1797–1878) and William Addison (1802–1881) described for the first time the leukocytes [23], as it was almost 200 hundred years after the first identification of the red cells by Jan Swammerdam using his designed microscope in 1658. Thereafter, Max Schultze performed functional studies on finely and coarsely granular cells. For the leukocytes, a German anatomist Max Johann Sigismund Schultze (1825–1874) also described for the first time four different types of leukocytes and the existence of intracellular granules. Max Johann Sigismund Fig. 1.1 Johann August Ephraim Goeze (1731–1793), a German zoologist’s portrait estimated to be painted by some painter in the eighteenth century. The current portrait image has been copied from the public domain from Wikipedia in the web address of https://it. wikipedia.org/wiki/Johann_ August_Ephraim_Goeze#/ media/File:Johann_August_ Ephraim_Goeze1.jpg

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Schultze studied on coarse granular cells to observe by a warm stage technique using his microscope at 38
C. The finely and coarsely granular cells of Max Schultze were shown in his article reported in 1865 [24]. He observed the amoeboid behaviors including interacting movement and phagocytosis phenomena. These cells were a type of granular cells like eosinophils and other blood leukocytes. Schultze joined the medical research groups of Greifswald and Berlin. In 1854, after his appointment of associate professorship of anatomy at Halle, he was successfully obtained his full professor in anatomy and histology. Later, he was the director of the Anatomical Institute, the University of Bonn. His recognition was particularly marked for his historical study on the cellular theory. His prominent achievement is attributed to the uniting animal sarcode conception, which was raised by Felix Dujardin, with vegetable protoplasma, which was raised by Hugo von Mohl. His suggestion was the terminology of protoplasm ad its identity. His achievement is his first definition of the cells to nucleated mass of protoplasms with presence or absence of cell walls (this is written through the description of Das Protoplasma der Rhizopoden und der Pflanzenzellen, ein Beitrag zur Theorie der Zelle, 1863). Max Schultze also investigated medicine field with the naturalist Fritz Müller, who was a German biologist and doctor, to follow the debate in Europe about evolution logics of Darwin’s theory. Max exchanged research literatures related to the Darwin’s monograph book of On the Origin of Species, and a simple microscope manufactured with Friedrich Wilhelm Schiek in Berlin in 1857. Using the microscope, Müller insisted on his hypothesis of “all higher Crustacea probably will be traceable to a Zoea ancestor,” which is basically originated from his own investigations. In addition, Müller insisted on his criticism of Darwin’s theory through his book of Für Darwin, which is basically written in defense of Darwin’s theories. He corroborated the Darwin’s theory of natural selection [23]. He established in 1865 the book series of serial articles in the Archiv für mikroskopische Anatomie, which contained several his articles. He highly investigated into depth on his study subject via fine refining (Fig. 1.2). Continuously, Florence Rena Sabin (1871–1953), an American medical scientist and one of the first women as a full professorship at Johns Hopkins School of Medicine, elucidated the differentiation events of white blood cells in the 1920s (Fig. 1.3). In 1924, the origins of blood, blood vessels, and blood cells have been reported by Florence R. Sabin. Thereafter, she further examined the brain histology, tuberculosis pathology, and immunology [26]. In 1925, after movement to the Rockefeller Institute for Medical Research in New York City, she concentrated and focused on the lymphatic organ system and cells. From her achievement of tuberculosis pathology for the functional discovery of monocytes to form tubercles known for granuloma, in 1926, she was invited to the committee of the National Tuberculosis Association.

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Fig. 1.2 Max Schultze’s granular white cells described in his article reported in 1865. The figure has been copied from the review article of Dr. Kay AB (2015) [25], as the captions are copied from Douglas Brewster’s translation [24]. The photo image has been copied from the public domain from Wikipedia in the web address of https://en.wikipedia.org/wiki/Max_Schultze Fig. 1.3 Florence Rena Sabin. The photo image has been copied from the public domain from Wikipedia in the web address of https:// en.wikipedia.org/wiki/ Florence_R._Sabin

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Neutrophils

In the concept of neutrophils, James Gerald Hirsch (1922–1987), an American physician and scientist, for the first time, reported the degranulation and phagocytosis of blood cells (Fig. 1.4) [27]. Using electron microscope, he observed and elucidated degranulation of neutrophils and eosinophils, as such events are specific for phagocytosis. Thus, phagocytic granules are observed because they are released into the phagocytic vacuole. Apart from the works by James Hirsch, Seymour Klebanoff independently discovered the similar granulating events with specific enzymes in the late 1960s. For example, some white blood cells express a myeloperoxidase enzyme that oxidizes the cell membrane components of targets, most well-known for killing of infected microbial pathogens [28, 29]. Therefore, Hirsch is discriminated for his achievement of phagocyte where the white blood cells engulf harmful microbes [26].

1.3.4

Granulocytes

For granulocytes, Niels Borregaard, a Danish physician and scientist, found several intracellular proteins in granulocytes with the synthetic origins, intracellular granule stores, and extracellular release (Fig. 1.5) [31]. From the electron microscopic observation, degranulation of neutrophils and eosinophils has been observed during phagocytosis. Granulation is a crucial process to release components from the Fig. 1.4 James Gerald Hirsch. The photo image has been copied from the public domain from Wikipedia in the web address of https:// en.wikipedia.org/wiki/ James_G._Hirsch

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Fig. 1.5 Niels Borregaard. The photo image has been copied from the public in the web address of https://jlb.onlinelibrary.wiley.com/doi/full/10.1189/jlb.4LT0217-049R, as shown in Fig. 1.4, reading with grandchildren. He was one of the editors of Journal of Leukocyte Biology, Society for Leukocyte Biology [30]

neutrophils in innate immune responses at the infection sites and initiate generation of bactericidal oxygen species for degranulation of granule subsets [32]. The granules are released into the phagocytic vacuole [33–35]. Niels graduated in 1978 from Aarhus University with his medical doctor degree in 1981. He was especially interested in the neutrophils among human phagocytes, as pursued. Niels isolates neutrophil subcellular organelles toward granulopoiesis. In the 1980s, in his work on neutrophil granules and component proteins, he isolated several neutrophil granule proteins and antimicrobial proteins such as α-defensin [30].

1.3.5

Monocytes and Macrophages

Macrophage was initially identified as immune cells to phagocytosize infectious agents [36] in the late nineteenth century. Since its discovery, macrophage received largely attracted attention in its defender roles against infectious pathogens. Later it received a spotlight for its role in tissue homeostasis in organism. The tissue macrophages are characteristically ontogenic and diverse subpopulations with various phenotypes. In current knowledge, macrophages are present in three distinct tissues including yolk sac, fetal liver, and hematopoiesis-lineage stem cells (SCs) in bone marrow (BM). Most macrophage populations are present in the epidermis,

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Fig. 1.6 Zanvil Alexander Cohn. The photo image has been copied from the public in the web address of http://centennial.rucares.org/index.php?page¼Innate_Immunity, as shown in the Rockefeller University Hospital Centennial, Monday, January 21, 2019. Because Cohn joined the Rockefeller University of René Dubos group in 1957 and with James G. Hirsch, he studied on leukocyte ingestion and microbial killing

lung, liver, brain, and spleen. The macrophages are produced before host birth through self-renewal. However, macrophages resident in the gut, dermis, and heart are established from blood-existing monocytes [37, 38]. Macrophage phenotypes are associated with macrophage-associated disorders. Macrophages engulf and digest pathogens, as well as toxins and dead cells. For monocytes and macrophages, Zanvil Alexander Cohn (1926–1993), an American cell biologist and immunologist, is the founder of modern macrophage biology (Fig. 1.6). Dr. Siamon Gordon, born in South Africa in Zanvil A. Cohn lab, conducted pioneer studies on the differentiation of mature macrophages (Fig. 1.7) [39, 40]. His achievement on defense mechanism against infectious pathogens is a spot. He claimed the endocytosis as a general cellular function to regulate the quantal uptake of exogenous molecules via plasma membrane-derived vesicles and vacuoles. Soluble substances are internalized by a type of pinocytosis and particulate substances by phagocytosis to the final destination of the vacuoles. Eukaryotic cells are house-kept and essential in leukocytes, macrophages, capillary endothelial and thyroid epithelial cells, yolk sac, and oocytes. They are involved in host defense, immunological responses, molecular transport, hormone transformations, and the metabolic pathways.

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Fig. 1.7 Siamon Gordon. His professorship in cellular pathology department, the University of Oxford is associated with his prominent outcomes until retirement at the end of 2008. Currently, he is an emeritus professor of the University of Oxford. Siamon Gordon has studied on macrophage heterogeneity and differentiation. https:// royalsociety.org/people/ siamon-gordon-11519/

Fig. 1.8 Ralph Steinman. The photo image has been copied from the public in the web address of https://en. wikipedia.org/wiki/Ralph_ M._Steinman

1.3.6

Dendritic Cells

For DCs, Ralph Steinman (1943–2011), born in Canada, launched the concept of yet another cell-recognizing antigens and the DCs (Fig. 1.8) [41]. He, in 1973, discovered DCs during his joining as a postdoctoral fellow in Zanvil A. Cohn group [41]. Steinman is a recipient of three co-receivers of the 2011 Nobel Prize in Physiology and Medicine, with other two scholars as recipients. The DCs are

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Fig. 1.9 Bernard Babior. The photo image has been copied from the public in the web address of https://en. wikipedia.org/wiki/ Bernard_Babior

functionally bridging the innate to adaptive immunity because DCs are sentinels, capable of presenting antigens through processing of captured antigens to T helper cells. The DCs migrate to lymph nodes, lymph tissues, or antigen-reactive T-cell clones. DCs are sensing responders due to differentiation or maturation capacity, influencing the differentiation of Thl vs. Th2 T cells. The DCs allocate the innate defenses to enhance cytokines and innate lymphocyte behaviors. Three innate features of DCs influence peripheral tolerant pathway. DCs target antigens to differentiate to matured DCs, contributing to actively stimulation of both B cells and T cells [42]. FcR death receptors activate or inhibit DC function because most DCs are immature and microbial stimuli mature DCs to control helper, cytotoxic, and regulatory T cells. Reactive oxygen species (ROS) of DCs are actual parameters to capture death cells or debris. Manfred L. Karnovsky (1918–1998), a South African biochemist, showed the clues how phagocytes convert oxygen to reactive species to kill bacteria or pathogens [43]. Studies on how white blood cells covert oxygen to protect the host by means of defenses against pathogens are his pioneering achievement. Bernard Babior (1935–2004), an American physician and scientist, pioneered research on the oxidase system of neutrophils (Fig. 1.9) [44]. Thus, Babior found that free radicals are crucial for defensing mechanisms of white blood cells. High toxic derivative, superoxide as a ROS is synthesized by a specific enzyme NAPDH oxidase. This kills pathogenic microbes. Babior attempted to expand the NAPDH oxidase property to genetically fatal diseases including immunodeficiency and chronic granulomatous diseases.

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Fig. 1.10 David Lambeth. The photo image has been copied from the public in the web address of http://www. emoryhealthsciblog.com/ tag/david-lambeth/ in Emory University

David Lambeth, MD, PhD, Emory pathologist, discovered the functional explanation of reactive oxygen-producing enzymes (Fig. 1.10). In 1999, he discovered the Duox family of ROS-generating enzymes. NADPH oxidases (Nox) are known for plants to fight off pathogens and for fungi to induce sexual development. In flies, NADPH oxidases also lead for egg laying, and in humans, they could sense gravity. NADPH oxidase catalyzes the superoxide genesis from oxygen and NADPH. The genetic disorder such as chronic granulomatous disease is an oxidase enzymelacking inherited immunodeficiency. Nox (NADPH oxidase) and the related Duox (dual oxidases) are involved in diverse responses via ROS. The Nox/Duox families are widely identified in various organisms including fungi, green plants, fruit flies, green plants, slime molds, nematodes, and mammals [45].

1.3.7

Complement System

Complement is a thioester-containing protein (TEPs including C3 and C4), which is also found in the protease inhibitor a2-macroglobulin. Complement cascade consists of collagen-tulip family molecules, α2-macroglobulin family molecules, and serine protease family molecules. Components of complement are found widely in metazoans. Compared to other defense systems in each organism, the complement belongs to an ancient molecular machinery and innate immunity. The complement system complements the additional functions of immunoglobulin antibodies and phagocytic myeloid cells to make a clearance of invaded pathogenic microbes and damaged self-cells. Eventually, the system stimulates inflammatory response. The complement system can also be acted together with antibodies generated from the adaptive immune cells. The complement system is composed of 40 more proteins in

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Fig. 1.11 Jules Bordet. The photo image has been copied from the public in the web address of https://en. wikipedia.org/wiki/Jules_ Bordet

mammals with soluble forms in blood and some membrane bound on cells. Complements act as an immune surveillance on host cells and nonself-invaders. Upon accounting to damaged cells and microbes, they keep homeostatic status with functionally and structurally multiple roles. The complement is crucial as PRR to detect nonself, PAMPs, and DAMPs and as effector system in both primary innate and adaptive immunities. It influences adaptive immunity, particularly B cells and Ab. It affects many other biological systems as part of homeostasis [46]. Jules Bordet (1870–1961), a Belgian immunologist and microbiologist, described for the first time during the end of the nineteenth century the bacteria-killing agents as he found the bactericidal and bacteriolytic effects (Fig. 1.11). Such bacterialkilling activity has been considered to be the cooperative results between the specific serum antibody and another thermolabile substance, called alexin. Thus, Jules Bordet is a pioneering immunologist in the era of the dawn of molecular immunology. His works are made at l’Institut Pasteur in Paris from 1894 to 1901 and the Pasteur Institute of Brabant in Brussels, when such works are before World War I. His observation was that complements bind to antibody-antigen complexes, designing the assay system of the complement fixation. For example, he identified anaphylatoxin, conglutinin, and whooping cough-causing bacterium of Bordetella pertussis. He identified thrombin formation, platelet clot formation, human milk lysozyme, and bacteriophage biology. From the outcomes of his complement, Jules Bordet was a recipient of the 1919 Nobel Prize for Physiology and Medicine

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[47, 48]. Although he could not attend the award ceremony, the award was from his contribution to the complement. However, complement seemed not to be his research goal but for bacterial killing. Actually, he contributed to bacteriophage life cycle and lysozyme studies. His scientific supervisor Metchnikoff also received a Nobel Prize in 1908 in Medicine from phagocytosis discovery. Interestingly, in 1903, Almroth Wright in London rather discovered the “opsonin” reality and serum opsonic activity [49]. Later, in 1935, Gordon and Thompson demonstrated that complement is the same identity as “serum opsonin” [50].

1.3.7.1

Alternative Complement System

In the same era, alternative complement pathway has been appeared by Louis Pillemer (1908–1957), who was born in South Africa. He isolated properdin from serum. Louis Pillemer traced his ideas about the antibacterial serum component as a complement, originated as early back as the 1790s, when John Hunter in London, UK, observed the blood resistance against putrefaction. A century later, many immunologists including Jozsef Fodor (Hungary), Carl Flugge (Germany), George Nuttall (USA), and Hans Buchner (Germany) studied on antibacterial serum constituents. Nuttall and Buchner reported that the blood constituents having bactericidal activity are heat-sensitive over heating at 55
C or 60
C [51, 52]. Thereafter, Elie Metchnikoff (Ilya Ilyich Mechnikov), the Institut Pasteur, Paris, France, co-worked on the same phenomenon with a visiting researcher Jules Bordet [53]. In 1895, Jules Bordet discovered a heat-stable serum molecule at 56
C for 30 min from immune-injected animals with agglutinated Vibrio cholerae debris. The isolated serum constituent is named sensibilatrices, currently named antibodies. In contrast, a heat-labile agent from nonimmunized animals is named “alexine” by Buchner. Alexine is currently the complement and means the Greek “to defend,” which lyses the bacterium Vibrio cholerae. Bordet evidenced that antibody-foreign erythrocyte complex binds to alexine which leads to lysis of the erythrocytes [54, 55], indicating that the hemolysis indicates the bacteriolysis. Then, the complement fixation reaction enables to develop complement fixation technology to detect the antibodies against bacterial infection including typhoid, plague, and anthrax [56]. Louis Pillemer’s laboratory was in the Institute of Pathology, Case Western Reserve University School of Medicine. His pioneering works largely affected to open the alternative complement pathway, an antibody-nondependent defense system, as he is evaluated as an early researcher of the alternative complement pathway. He described for the first time properdin as a key component of an antibodyindependent complement activation pathway. That was his pioneering work and allowing the discovery of the alternative complement pathway. In the 1970s, properdin was reconfirmed to be a stabilizing agent, required for the alternative pathway convertases, which are used for the complement cascades, where properdin recognizes target cells and infectious microbes to lead to phagocytic clearance. It also recognizes ligands, phagocyte receptors, and serum-regulating proteins [57]. Later, by his laboratory in Case Western Reserve, Irwin H. Lepow purified the C1qrs

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complex and characterized its enzyme, and with the natural serum C1 esterase inhibitor.

1.3.7.2

Lectin Pathway

The lectin pathway was first found in the 1980s, although the lectin pathway was not solidly confirmed till the 1990s. Discovery of the mannose-binding lectin (MBL) from the mammalian sera initiates the reality of lectin pathway in 1987 by Kawasaki group [58]. Toshisuke Kawasaki, a Japanese glycobiologist in Kyoto, isolated and characterized a named mannan-binding protein (MBP). The MBP was isolated as a soluble protein from liver tissues of rabbits in 1978 (Fig. 1.12). The isolated MBP activates complement system via the classical lectin pathway [59]. MBL activation of complement system contributes to the diverse defense mechanism in vertebrates. Hence, because MBL is structurally similar to Ciq in its quaternary structure, the MBL has been considered to be one of the classical pathways. MBL binds to C1r and C1s to activate. MBL was also isolated from human serum in 1983. The membrane protein binds mannan. ManNAc, GlcNAc, and Man inhibit the mannan binding, while GalNAC and M-6-P are inert. His proposed theory was that the MBP is the

Fig. 1.12 Toshisuke Kawasaki. The center is Prof. Kawasaki, and his left are Mrs. Dr. Kawasaki and Dr. SJ Kim (SCM Biotechnology, Director, Korea) and YJ Kang. His right are Prof. Cheorl-Ho Kim (Sungkyunkwan University, Korea), Dr. Tae-Wook Chung (GeneBioCell, Ltd., Korea, Director), and YJ Kang in Glycoconjugate Symposium, Lubeck, Germany. The author Cheorl-ho Kim presented his snap photo due to his personal respect

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hepatic uptake protein of glycoproteins with terminally glycosylated glycoconjugates having GlcNAc and/or Man residues. As mentioned above, the complement also influences adaptive immune responses operated by B cells and antibodies, affecting many other biological systems as part of homeostasis [60]. Components of complement are found widely in metazoans and particularly important for human resistance to bacteria [61]. Human populations frequently exhibit specific resistant phenotypes to infection and manifest of malaria parasites through gene polymorphisms in the antiparasitic thioester-containing protein 1 (TEP1) gene. The polymorphism is related with the variability in parasite killing [62]. Complement is activated on the nonself cell surfaces by the classical, lectin, or alternative pathways. Evasion of complement function is a hallmark of invasive pathogens and hematophagous organisms. The complement system is particularly important for human resistance to bacteria [63] and for mosquito resistance to malaria parasites [64]. For example, the complement is also important for mosquito resistance to malaria parasites. The transmission capacity of carrier, the mosquito Anopheles gambiae species to protozoan parasites of Plasmodium family, is varied in each individual. Because Plasmodium falciparum malaria is an intracellular parasite by Anopheles mosquitoes, most deaths occur as a result of complications of anemia or cerebral malaria named coma. The complement receptor-1 (CR1/CD35) is a regulator present on the red cell surfaces and most leukocytes, in the malaria pathogenesis upon P. falciparum infection. CR1 mediates the interaction between the red blood cells, which are infected by parasites, and normal red blood cells (noninfected cells), forming rosettes and disturbing microcapillaries. CR1 also controls complement activation and immune complex formation during malaria infection. CR1 is a receptor for the invasion of red cells by the parasite. Complement activation on RBCs normally occurs via C3 cleavage and immune complexed with C3b. Complement activation contributes to the loss of RBCs during P. falciparum malaria anemia [65], which occurs via the classical pathway by antibody binding to C1q, MBP binding to pathogenic cells, and basal cleavage of C3 on cell surfaces [66]. RBC-surfaced complement regulatory proteins (CRPs) including CR1/CD35, DAF/CD55, and protectin/CD59 [67] regulate the complementation. CR1 mediates the clearance of immune complexes, and C3b-opsonized immune complexes bind to CR1 on RBCs and endocytosed by liver or spleen macrophages [65]. The CRP clearance reduces the RBC’s capacity to regulate complement deposition [68]. Then, RBCs become susceptible to complement-mediated destruction by phagocytosis. Erythrophagocytosis by macrophages is such a complement-dependent anemia during Plasmodium infection. This process is also enhanced by CD47, a self-marker expressed on RBC. Plasmodium parasite preferentially infects CD47-expressed RBCs to avoid phagocytic clearance [69].

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Opsonization

For opsonic phenomenon, Almroth Wright (1861–1947), Sir Almroth Edward Wright, a British bacteriologist and immunologist, described that the number of organisms ingested increased if incubated in the presence of fresh normal serum (Fig. 1.13) [70]. He developed an antityphoid fever inoculation system in World War I, but his name and work are not well recognized today. After born in 1861 in Yorkshire, where his father was an Irish protestant minister and Hebrew scholar. Almroth’s mother was a Swedish as the daughter of NW Almroth, governor of the mint in Stockholm, Sweden [71]. His two clinical tests of the erythrocyte sedimentation rate and the opsonic index are nonspecific detection parameters for pathophysiologic serum proteins. Although infectious serum contains an increased level of specific antibodies, in healthy subjects, Wright found that antibodies contribute minimally to opsonic activity. That is because the opsonic activity. The activity is present in newborn serum and increased in the acute immune phase, with less specific. His demonstrations of complement-mediated lysis both of normal cells by lectins and of foreign cells by animal lectins are caused by Wright’s serum opsonic activities. His findings influenced the mechanistic understanding of complement activation through lectin pathway by recognition of surface sugars as predictable pathogen patterns that are quite different with the less predictable targets of the adaptive immunity. For healthy subjects, Wright suggested that antibodies contributed to opsonic activity that is the complement-enhanced phagocytosis of

Fig. 1.13 Almroth Wright. The photo image has been copied from the public in the web address of https://en. wikipedia.org/wiki/ Almroth_Wright

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microorganisms. In 1906, he introduced the term “opsonic” (Greek “to cater for”) [70, 72]. In the third Cold Spring Harbor Symposium on immunology in 1989, as Charles Janeway mentioned that the immune system has evolved specifically to interact with infectious microbes and recognizes both specific antigenic determinants and certain characteristic patterns, this is indeed appropriate to Almroth Wright consistence. He proposed and used the “opsonic index,” and this would be meetable too much of Janeway’s address. Among many mechanisms in which immune system fights pathogens, one is opsonization. Opsonization is the immune process of recognizing and targeting invading agents for phagocytosis [73]. Opsonization uses opsonins to tag infectious pathogens to eliminate them by phagocytes. Opsonization of a pathogen occurs by antibodies or the complement system, fighting off foreign pathogens like bacteria and viruses. Opsonization supports self-tolerance and inhibits autoimmunity. Opsonins are used to overcome the repellent force between the negative cell walls and promote uptake of the pathogen by the macrophage. Opsonization is an antimicrobial technique to kill and stop the spread of disease. For acting opsonins, C3b, C4b, and C1q are complement molecules and serve as opsonins. In the alternative complement pathway, the spontaneous complement activation converts C3 to an opsonin C3b upon binding to antigen. Antibodies can also activate complement via the classical pathway, generating C3b and C4b onto the antigen surface. C3b-bound antigen is phagocytosed. CR1 expressed on all phagocytes recognizes complement opsonins, including C3b and C4b which are both parts of C3-convertase. C1q as a C1 complex binds to the Fc region of antibodies. As circulating proteins, pentraxins, collectins, and ficolins are all opsonins and secreted PRRs. These molecules coat the microbes as opsonins to enhance neutrophil response. Pentraxins bind to phosphatidylcholine (PC) and phosphatidylethanolamine (PE) on apoptotic cell membrane. IgM also binds to PC. Currently, the known collectins are MBL, surfactant protein (SP)-A, and SP-D. They recognize unknown ligands on apoptotic cell membranes to interact with phagocyte receptors for phagocytosis.

1.3.9

Lysozyme and Salvarsan

For lysozyme, Alexander Fleming (1881–1955), later respected to Sir Alexander Fleming, who is a Scottish biologist and pharmacologist, discovered in 1922 substance in nasal mucus molecules that causes bacterial cells to disintegrate. The substance was lysozyme (from the Greek lysis), as he named the substance in 1923 (Fig. 1.14). In 1928 he isolated the antibiotic penicillin from the mold Penicillium notatum. It was the world’s first antibiotic substance benzylpenicillin (penicillin G) from the mold. From the discovery, he became the 1945 Nobel Prize recipient in Physiology and Medicine, as the prize was shared with Howard Florey and Ernst Chain [74]. Fleming was knighted for his scientific achievements in 1944 [75]. In 1999, he was nominated in Time magazine’s the 100 Most Important People of the twentieth century. In 2002, he was chosen in the BBC television for the

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Fig. 1.14 Alexander Fleming. The photo image has been copied from the public in the web address of https://en.wikipedia.org/ wiki/Alexander_Fleming

Fig. 1.15 Sahachiro Hata. The photo image has been copied from the public in the web address of https://en. wikipedia.org/wiki/ Sahachiro_Hata

100 Greatest Britons. Again in 2009, he was selected as the third “greatest Scot” in STV, behind the great Robert Burns and William Wallace. For Salvarsan, Sahachiro Hata (1873–1938) (Fig. 1.15), a Japanese bacteriologist, developed Salvarsan in the lab of Paul Ehrlich in 1909. Salvarsan also known as “Präparat 606” or the “magic bullet,” was used to treat syphilis and one of the first

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modern chemotherapeutic agents [76]. He was a prominent Japanese young scientist and bacteriologist who developed the arsphenamine in 1909 under Paul Ehrlich (1854–1915) group. He was the 1908 Nobel Prize recipient in Physiology or Medicine for his achievement of the antibody production theory. Historically, he designated and discovered the antisyphilitic arsenic agent, arsphenamine, Salvarsan, or 606, which was the most valuable chemotherapy in the twentieth century by Paul Ehrlich group with his young members, chemist, Alfred Bertheim, and bacteriologist, Sahachiro Hata. Sahachiro Hata stayed in the Institute for Infectious Diseases (Densenbyogenkyuzo), currently renamed Ikkaken or the Institute for Medical Sciences (Ikkagakugwnkyuzo, IMS), Tokyo, Japan. The magic bullet he established was prescribed to cure or eradicate syphilis for a half and more century until “penicillin” discovery to WWII by Howard Florey (1898–1968). Although Ehrlich is known as the father of chemotherapy followed by Brian Druker, Alexander Fleming (1881–1955), and Hamao Umezawa (1914–1986), his contribution was a real trying to application. Syphilis was used until penicillin and other antibiotic introduction in the 1940s. However, the precise mechanism how the Salvarsan acts to eliminate the syphilis spirochete in vivo is unclear, although other arsenic agents such as melarsoprol and roxarsone are used to treat parasites. Interestingly, arsenic trioxide has recently been reported to cure patients with acute promyelocytic leukemia. Although Sahachiro Hata received a public nomination of the 1911 Nobel Prize in Chemistry and of the 1912/1913 Nobel Prize in Physiology or Medicine [77], unfortunately, it was not.

1.3.10 Progress in Innate Immune Response Since Historic Spanish Flu In influenza virus, Spanish flu (1918–1919) was pandemic, and an influenza pandemic killed European peoples ranged from 20 and 40 million in 1918 and 1919 [78]. Normally, virus subtypes with HA and NA combinations are rarely identified and isolated from mammal species; virus subtypes with all 15 HA subtypes and all 9 NA subtypes were identified and isolated from avian birds. Since entrance to the twentieth century, the emergence of different antigenic strains, which can be transmitted to in human populations, though shifts in antigen genes were identified in 1918 (type of H1N1), 1957 (H2N2), 1968 (H3N2), and 1977 (H1N1) to date with a pandemic propahatopns. The strains emerged after gene assortment of viruses of avian and human origins. During 1996, an H7N7 influenza virus subtype, which was an avian origin, was identified. In addition, during 1997 in Hong Kong, an H5N1 subtype of avian influenza virus was identified, and the H5N1 subtype caused death of humans. During 1999, influenza virus H9N2 subtype was identified as forms of two independent isolations of influenza virus from humans. The gene reassortment could be emerged from virus to infectious cases in the human population. The outbreak was caused by H1N1 influenza virus and the most devastating epidemic

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in modern history. Hong Kong flu (1968–1969) caused by H3N2 influenza virus caused the death of one million people. The pandemic began in 1968 in China and spread rapidly to the Southeast Asia and Australia region. The H3N2 subtype was again transmitted to the USA in September through military personnel returning back from Vietnam to hometown unite states [79]. Acquired immunodeficiency syndrome was discovered in 1981 to now. AIDS was first reported on June 5, 1981, and is caused by the human immunodeficiency virus (HIV), a lentivirus. AIDS killed approximately 25 million people within three decades. Five million people received HIV treatment in 2009. It has been known that 33.3 million people were exposed to HIV in 2009 of which 2.6 million peoples were newly infected and 1.8 million peoples with AIDS-associated lethal deaths were counted [80]. During the immune receptor-ligand interaction, a provisional immunologist, Dr. Charles Alderson Janeway Jr. (1943–2003) claimed the immunological recognition through antigen binding. This largely contributed to the TLR discoveries and prospects toward TLR-carbohydrate recognition in innate immune response. Innate immune recognition relies on germline-encoded receptors. The immune receptors evolve to recognize conserved molecules by pathogenic microbes. Well-defined molecular structures or patterns are bound by the immune defense receptors, and the structural recognition can distinguish invaded or pathogenic nonself from self. The innate immunity molecularly discriminates the self- and nonself-antigens. Immune system evolves basic conceptional strategies of nonself and selfdistinguishment. The innate immune discrimination of nonself and self is possible through occasional recognition of molecular patterns appeared on the invaders and hosts. In sides of endogenous protection of self, the innate immune recognition is defined as discrimination of normal and abnormal self. The abnormal self frequently define damaged self or transformed self. These patterns are deciphered by host receptors, depending on the meaning of these signals. His description of “I believe that immunological recognition extends beyond antigen binding by the clonally distributed receptors ---- and there is still much to learn------I believe that the immune system predates the development of specific immunological recognition mediated by clonally distributed receptors ----” clearly provoked the importance in the immune receptor-ligand interaction. This is really innovative, as implied from “the most likely possibility is that primitive immune effector cells possess receptors recognize certain pathogen-associated molecular pattern (PAMP) not found in the host” and also from “What kind of ligands or patterns should such non-clonally distributed receptors recognise? I think it is likely that such patterns in molecules are found in many microorganisms ---. -- Complex cell wall carbohydrates or lipopolysaccharides are likely ligands . . .” [81–83]. On the other hand, depending on the information derived from the immune responses, studies on the invertebrate immunity have been greatly contributed to the mammal host immunity. For example, Clay G. Huff (1900–1982), an American parasitologist, mentioned the Immunity in Invertebrates. Christiane NüssleinVolhard, a German biologist and geneticist, published in 1985 the first article on the Toll gene product (Fig. 1.16). Later, she received the 1995 Nobel Prize in Physiology or Medicine for her achievements. As the light gradually shed to the

1.3 Historical Profile of Defense Constituents and Progress in Innate Immune. . .

23

Fig. 1.16 Christiane Nüsslein-Volhard. The photo image has been copied from the public in the web address of https://en. wikipedia.org/wiki/ Christiane_N%C3% BCsslein-Volhard

Fig. 1.17 Jules Hoffmann. The photo image has been copied from the public in the web address of https://en. wikipedia.org/wiki/Jules_A. _Hoffmann

appearance of the receptors, Jules Hoffmann, born in Echternach, Luxemburg, as a chemist and biologist together with Bruno Lemaitre, made outbreaking contributions to the function of Toll receptors in antifungal immunity (Fig. 1.17). His finding revolutionized the Toll functions where the dorsoventral modulating gene loci of spaetzle-Toll-cactus modulates the antifungal immunity in the experimental eukaryotic fly, Drosophila [84, 85]. His colleague, Ruslan Medzhitov, a biochemist from Uzbekistan, received the Shaw Prize in Life Science and Medicine 2011 together

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with Bruce Beutler and Jules Hoffmann. He depicted that the innate immunity should be the virtues of non-clonal immune recognition with CA Jr. Janeway [83, 86]. From the continuous progress in the immune receptors to discriminate their ligands even including glycans, lipids, proteins, or nucleic acids, the welldefined word of “PRR” has been come on stage. Richard Ulevitch, a biochemist from the USA, has also focused on the signaling pathways of PRRs. He has organized and defined the TLRs in the innate immune response [87–89]. Later, Shizuo Akira, a Japanese scientist, conducted his research on microbial ligands of PRRs, such as the identification of TLR9 as a CpG DNA receptor, suggesting innate recognition of and regulation by DNA [90, 91]. Currently, the PRRs include human TLRs 1–10, the mannose receptor (MR), Dectin 1, Dectin 2, nucleotide oligomerization domain (NOD)-like receptors (NLRs), retinoic acid (RA)-inducible gene I-like receptors (RLRs), etc.

1.4

The Outline of Innate Immunity

The concept of neutrophils, leukocytes, dendrites, monocytes, and macrophages has been born, and their effects on innate immunity have been fairly established to these days. The infectious pathogen-derived pressure is a powerful driving kinetic and force, contributing to the evolution of mammals. Innate immunity is the vital non-clonal immune response in discrimination of nonself via molecular binding. The responses of innate immunity are rapid and nonspecific to pathogens [83]. The immune system utilizes immune recognition to constantly select against the targets. The target recognition is basic in innate immunity. The recognized targets then interact not only with innate immune cell populations such as cells of innate lymphoid, myeloid, and NK but also with nonimmune cells in specific circumstances and the ancient humoral systems like complements. Innate immune cells capture the invasive targets via the two known ways of encapsulation and phagocytosis. Encapsulation event is restricted to invertebrates in defense against foreign targets too large for phagocytosis by their hemocytes. The encapsulation process in invertebrate immune response requires coordinated action of cellular and humoral factors. Another event named phagocytosis needs phagosome process. When a macrophage ingests a pathogenic microbe, the pathogen is trapped in a phagosome and fused to a cellular lysosome to incorporate a specific intermediate vesicle formation, named phagolysosome. Within the phagolysosome, the incorporated pathogens are digested by phagolysosomal enzymes and toxic peroxides. Humoral antibodies produced by innate immune cells of vertebrates or mammals are expressed in the four modes including neutralization, complement activation, opsonization, and agglutination (Fig. 1.18). Neutralization indicates that antibodies bind to viruses and toxins and then blocks the pathogens’ proliferation and replication. Complement activation indicates the classical pathway that antigen-antibody complexes activate the complement system and dissolves the pathogens. Opsonization suggests that phagocytic cells grab the antibody-bound surface of

1.4 The Outline of Innate Immunity

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A) Neutralization

Virus-infected cell

Virus

Inactivated Virus

B) Complement Activation C 1

Bacterial cell

C C C 4 2 3

C) Opsonization

Bacterial cell

Fc receptor

Phagocytic cell

Phagocytic cell

Fig. 1.18 Antibody-mediated events of innate immune cells in mammals

foreign substances for efficient phagocytosis and then eat them. Similarly, agglutination event indicates the action of an antibody when it cross-links multiple antigens, producing clumps of antigens.

1.4.1

Concept of Immune Receptors

Innate immune responses are displayed by distinct receptors, frequently defined pattern recognition receptors (PRRs). PPRs recognize PAMPs directly (Fig. 1.19). Several PRRs include peptidoglycan recognition protein (PGRP), Toll-like receptor (TLR), lipopolysaccharide (LPS) and β1,3glucan-binding protein, galectin, C-type lectin (CTL), and thioester-containing protein (TEP). For PAMPs, examples of Gram-negative and Gram-positive bacterial PAMPs have been illustrated in Fig. 1.20. Each E. coli and S. aureus strain has been shown with several components found in virus, bacteria, and fungi. Other complement receptors and Toll receptors bind to PAMP recognition products. Among the PRRs, specific interests have been functionally related in multiple forms of CTL, C-type lectin in myelocytes. Currently known and representative CTLs are illustrated (Fig. 1.21). Innate immune responses are common features, which occurred in all the metazoan organisms to protect hosts against pathogenic invaders and opportunistic infectious pathogens because invertebrates lack an adaptive immunity.

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PAMPs

PRRs

Example of PRRs Pattern recognition receptors Cellular signaling

PGRP

Immune response

Peptidoglycan Recognition Protein

TLR

Toll-Like Receptor

LGBP

Lipopolysaccharide, β-1,3-glucan Binding Protein

CTL

C-Type Lectin

GALE

Galectin

TEP

Thioester-containing Protein

Fig. 1.19 Some pattern recognition receptor (PRR) forms in myeloid lineage innate immune cells PAMPs Gram-negative bacteria E. coli

Example of PAMPs Lipopolysaccaride Outer membrane peptidoglycan Inner membrane

Pathogen-associated molecular patterns (PAMPs)

Nucleic Acids

Microbe type

ssRNA

Virus

dsRNA

Virus

CpG

Virus, Bacteria

Dilin Proteins

Bacteria Flagellin

Gram-positive bacteria S. aureus

Glycolipid peptidoglycan

LPS

Gram-negative bacteria

Lipoteichoic-acid

Gram-positive bacteria

Mannan

Fungi, Bacteria

Glucans

Fungi

Cell wall lipids

membrane

Carbohydrates

Fig. 1.20 PAMPs produced by Gram-negative and Gram-positive bacteria and components known in virus, bacteria, and fungi

As a first step, metazoan organisms recognize microbial PAMP structures. CTLs are involved in pathogen recognition, cellular adhesion, antigen uptake, and complement activation. The CTLs as a superfamily of Ca2+-dependent C-type receptor protein (CRP) recognize nonself and invader for clearance through binding to terminal sugars on the microorganisms. For example, mollusk CTLs (CfLec-1 to CfLec-4) identified from Chlamys farreri are involved in the immune response against Vibrio anguillarum PAMPs via the opsonization, encapsulation, and phagocytosis. CfLec-1 serves as a PRR in the PAMP recognition and opsonizes it to clear invaders. Encapsulation, different from opsonization event, is specific for invertebrates against large foreign agents for phagocytosis by individual hemocytes [92]. Like phagocytosis, the cooperation of CfLec-1 and hemocytes activates

1.4 The Outline of Innate Immunity

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C-type lectin Type Ⅰ Type Ⅱ

DEC-205 S S

MMR

DC-SIGN

Langerin DCIR

S S

CLEC-1

Dectin-1

CRD

Dectin-2

DLEC

P P P

Carbohydrate recognition domains (CRD) Tyrosine-based motif for targeting to coated pits and internalization

I T I M

I T A M

Triad of acidic amino acids Fibronectin type Ⅱ repeat Di-leucine motif Tandem repeat

Fig 1.21 The representative C-type lectins (CTLs) in humans

hemocyte encapsulation. CfLec-1 from C. farreri opsonizes with hemocytes against invaders (Fig. 1.22). Receptor engagement can transduce signals to alarm the presence of pathogens from their innate ligands. The innate or primary immune responses are, therefore, the first line of defense system upon infectious invasion. While innate immunity eliminates majority of pathogens quickly, certain infections are not cleared initially due to the virulence factors produced by pathogens. Innate immune responses are not specific originally and have potential to adapt. The nonspecific property of innate immune responses is alternatively complemented and overcame by the PRR concept. The PRR receptors are expressed on the innate immune cells to recognize specific microbial components. PRRs are classified into three types of humoral proteins circulating in the plasma, endocytic receptors, and signaling receptors. Cellular PRRs, which are associated with the innate immune responses, are expressed on antigen-presenting cells (APCs) in adaptive immune response and on epithelial cells that are the first to encounter pathogens during infection. TLRs are well defined in the innate immune responses [88]. Because the innate immune-related myeloid cells respond to foreign invaders as the first defense line during infectious diseases, the elementary mission for the host is to bind to the pathogens. Subsequently, it exerts a rapid and fast response to defend. TLRs or Toll-like receptor families display the primary roles in both vertebrates and invertebrates, reflecting a functional role in host defense. Ruslan Medzhitov, a biochemist from Uzbekistan, received the Shaw Prize in Life Science and Medicine 2011 together with Bruce Beutler and Jules Hoffmann. Richard

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Pathogen (Vibrio anguillarum) PAMPs

C-type lectin (CfLec)

Encapsulation

Opsonization

Immune response Fig. 1.22 Mollusk CTLs (CfLec) of Chlamys farreri are involved in the immune response against Vibrio anguillarum PAMPs via the opsonization, encapsulation, and phagocytosis

Ulevitch is a biochemist from the USA, and he focused on the signaling pathways of pattern recognition receptors. Shizuo Akira, a Japanese scientist, conducted a research on microbial ligands of PRRs, such as the identification of TLR9 as a CpG DNA receptor. Innate recognition of and regulation by DNA has been carried out by Akira Shuzo [93].

1.4.2

Host Protection from Microbial Invaders of Innate Immunity

During an infection, the innate immunity is the first line to be bordered, within a short time, no longer than minutes to hours for full activation. This is because of the host defense in the first infection phase. While innate immunity eliminates the most pathogens quickly, certain infection is initially not cleared just due to the virulence factors produced by pathogens. Innate immune responses are traditionally not specific and potential to adapt. The property that innate immunity is not specific is alternatively complemented and overcome by the PRR concept. The PRRs are expressed on the innate immune cells to recognize specific microbial components. Innate immune cells recognize the difference between Gram-positive and Gramnegative bacteria but not closely related strains [94].

1.4 The Outline of Innate Immunity

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As a model of bacterial pathogens to evade phagocytosis/phagocytic killing, the following mechanistic examples are raised: 1. Evasion of complement-mediated phagocytosis by expression of a polysaccharide capsule common among bacterial pathogens, group B Streptococcus (GBS), Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae: neonatal infectious events are the most common causes of life-threatening pathogen infections in neonates and newborns. Meningitis, pneumonia, and septicemia are infections in adults as a growing clinical problem and skin infections, urinary tract infections, and meningitis in adults with underlying illness. They are often important virulence factors with antiphagocytic properties affecting complement deposition/activation at the bacterial surface. 2. Evasion of non-opsonic phagocytosis. This is a bacterial mechanism to evade phagocytosis/phagocytic killing and evasion of non-opsonic phagocytosis. PRRs exhibit signaling such as TLR that triggers an intracellular signaling which culminates in induced production of multiple mediators including chemokines, proinflammatory cytokines, type I interferon (IFN), and subsequent activation of phagocytes. In phagocytosis, SR-A recognizes and binds with non-opsonic phagocytosis of pathogenic microbes. They are expressed by most macrophage populations. Endocytosis of mLDL contributes to foam cell formation in atherosclerosis. They act as a PRR. Phagocytic receptor mediates direct non-opsonic phagocytosis of several bacterial species and contributes to resistance to experimental infection with Gram-positive bacteria (L. monocytogenes, S. aureus, S. pneumoniae). Bacterial mechanisms to evade phagocytosis/phagocytic killing include targeting of inhibitory receptors in human Siglec-5, where they are expressed by neutrophils, monocytes, macrophages, and basophils. It consists of the extracellular four Ig-like domains and two cytosolic immune receptor Tyr-based inhibitory motifs (ITIMs) to act in cell-cell interactions and inhibition of inflammatory signals. The binding protein of GBS binds to Human Siglec-5 and inhibits phagocytosis. 3. Targeting of inhibitory receptors. 4. Evasion via T3SS. 5. Increased intracellular survival. How can we determine the evolutionary history of innate immunity? Innate immunity displays the rapid evolution. This rapid evolution is frequently caused by interaction between hosts and pathogens as well as pathogen-driven direct selection [95, 96]. Many genes involved in innate immunity undergo evolution along the mammal lineage [97]. Other characteristics of the innate immunity are heterogeneous in immune-responding cells in the different responses against pathogen infection [98, 99] and diversity of PAMPs [100]. The innate immune response against pathogens is controlled to avoid self-damage. The innate immune response is transcriptionally and adaptationally diverse in expression of cells between species and variability. Transcriptionally diverging genes include chemokines and cytokines produced in myeloid cells. The gene expressions are also transcriptionally regulated by each distinct promoter [101]. Thus, each gene expression distinctly differs among

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species and conditions to evolve for adaptive and relevant response [102]. The immune-responding genes change continuously during the evolution because of their repeated exposure to the surrounded antigens. Diverse environmental pathogens influence the changes of genetic markers in each environment [103, 104]. Prolonged adaptation to different ecological niches led to shape the evolution of PRR such as TLRs differently even within some species [105]. The allele frequency of sickle cell β-hemoglobin is increased in humans due to malariaendemic interaction. This process is well described as a commonly accepted example of a deleterious gene polymorphism occurred via selective pressure by a certain harmful parasite [106]. Recent next-generation genome sequencing (NGS) has elucidated that certain immunity-associated human genes such as the viral RNA-editing gene are continuously, but positively, being selecting [107–109] to adapt between pathogens and their hosts. This concept is so-called Red Queen hypothesis [94], but outlined cases between infectious pathogens and co-selection in humans are just dawn stage. Complement components are very ancient. The barriers include tight junctions in epithelial cells, mucins and mucus, stomach acid, and nutrient sequestration (factors which strongly bind iron, biotin, etc.). Cells include phagocytes and other blood cells (macrophages, granulocytes), NK cells, αβ-NKT and γδ T cells (invariant or semiinvariant T-cell receptors). Molecules include antimicrobial peptides (AMP, like defensins, magainins, cecropins), complement (C, like thioester-containing proteins including C4, C3, and C5, TEPs), lectins, ficolins and collectins (MBL), scavenger domain-containing proteins, TLRs, NLRs, cytokines, and chemokines as well as intracellular defenses such as PKR, interferon, lectins, TRIMs, RNAi, etc. NK cells are also crucial for the innate immunity since NK cells as innate lymphocytes bear many receptors with crucial roles in infectious disease, cancer, autoimmunity, transplantation, and reproduction [110]. NK receptors have a function of ligand recognition by lectin or immunoglobulin domains. They have a signaling either activating or inhibitory, exhibiting highly diverse between individuals.

1.5

Autophagy from Microbial Invaders and Self-Associated Molecular Patterns (SAMPs) of Innate Immune Cells

PRRs such as TLR4 also induce autophagy in APCs (Fig. 1.23). Autophagy in immune cells functions as a surveillance way of the intracellular pathogens and SAMPs [111]. A main player of autophagy is the target of rapamycin (TOR) kinase for growth factor receptor signaling, hypoxia, ATP levels, and insulin signaling. TLR signaling induction elicits autophagy in APCs. In fact, autophagy is frequently observed in macrophages treated with certain TLR agonists such as TLR4-specific LPS [111, 112]. For the adaptive immune responses driven by T cells, the CD4+ helper T-cell subsets are normally regulated by major histocompatibility complex

References

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(A) lassical pathway of autophagosome formaon Nutrient depleon or stress mTOR pathway

LC3 fusion

MHC-II compartment

Phagophore

MHC-II compartment /Autolysosome

(B) Endosome mediated autophagy in DCs Bacteria LPS binding to TLR receptor Selecve autophagy MHC-II compartment DC aggresome-like induced structures Early endocyc pathway

Fig. 1.23 Autophagy induction of PRRs such as TLR in APCs

(MHC) class-II (MHC-II)-presented peptidyl antigens on the APCs such as DCs. Endocytosed antigens are delivered to MHC-II compartments, endocytic organelles [113]. Cytosolic and nuclear proteins, including mucin-1 (MUC1) and complement component, are also loaded on MHC-II. Cytoplasmic proteins are delivered to lysosomes through three autophagy pathways including microautophagy, chaperone-mediated autophagy, and macroautophagy [114]. Autophagosome is formed with a nucleation membrane, phagophore, and fuses with an endosomelysosome. Importantly, autophagy in APCs is formed via TLR activation to detect microbes, although classical autophagy is induced by rapamycin or nutrient deprivation. LC3, autophagosome marker, colocalizes with MHC-II compartments. LPS treatment in DCs induces the transient protein aggregates named DC aggresome-like induced structures (DALIS) or ubiquitin-positive aggresome-like structures (ALIS) [114]. In LPS-induced DCs, autophagosomes formed via MHC-II compartments bear the autophagosome markers LC3 and ATG16L [113], calling endosomemediated autophagy. MHC-II compartment-driven autophagosomes preferentially engulf the LPS-induced DALIS, which become later degraded in autolysosomes. The TLR signaling is initiated via two adaptor proteins, MyD88/Toll/IL-1R-homology domain (TIR)-bearing adaptor-inducing IFN-β (TRIF), where they activate protein kinases, ubiquitin ligases, and transcriptional factors.

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59. Kawasaki T, Etoh R, Yamashina I. Isolation and characterization of a mannan-binding protein from rabbit liver. Biochem Biophys Res Commun. 1978;81(3):1018–24. 60. Ricklin D, Hajishengallis G, Yang K, Lambris JD. Complement: a key system for immune surveillance and homeostasis. Nat Rev Immunol. 2010;11:785–97. 61. Garcia BL, Zwarthoff SA, Rooijakkers SH, Geisbrecht BV. Novel evasion mechanisms of the classical complement pathway. J Immunol. 2016;197(6):2051–60. 62. Kamareddine L, Nakhleh J, Osta MA. Functional interaction between Apolipophorins and complement regulate the mosquito immune response to systemic infections. J Innate Immun. 2016;8(3):314–26. 63. Serruto D, Rappuoli R, Scarselli M, Gros P, van Strijp JA. Molecular mechanisms of complement evasion: learning from staphylococci and meningococci. Nat Rev Microbiol. 2010;8(6):393–9. 64. Blandin SA, Wang-Sattler R, Lamacchia M, Gagneur J, Lycett G, Ning Y, Levashina EA, Steinmetz LM. Dissecting the genetic basis of resistance to malaria parasites in Anopheles gambiae. Science. 2009;326(5949):147–50. 65. Biryukov S, Stoute JA. Complement activation in malaria: friend or foe? Trends Mol Med. 2014;20(5):293–301. 66. Walport MJ. Complement. First of two parts. N Engl J Med. 2001;344(14):1058–66. 67. Kim DD, Song WC. Membrane complement regulatory proteins. Clin Immunol. 2006;118 (2–3):127–36. 68. Odhiambo CO, Otieno W, Adhiambo C, Odera MM, Stoute JA. Increased deposition of C3b on red cells with low CR1 and CD55 in a malaria-endemic region of western Kenya: implications for the development of severe anemia. BMC Med. 2008;6:23. 69. Banerjee R, Khandelwal S, Kozakai Y, Sahu B, Kumar S. CD47 regulates the phagocytic clearance and replication of the plasmodium yoelii malaria parasite. Proc Natl Acad Sci U S A. 2015;112(10):3062–7. 70. Mabry DS, Wallace JH, Dodd MC, WrighT CS. Opsonic factors in normal and immune sera in the differential phagocytosis of normal, trypsinized and virus-treated human and rabbit erythrocytes by macrophages in tissue culture. J Immunol. 1956;76(1):62–8. 71. Ellis H. Sir Almroth Wright: pioneer immunologist. Br J Hosp Med (Lond). 2011;72(3):169. 72. Forsdyke DR. Almroth Wright, opsonins, innate immunity and the lectin pathway of complement activation: a historical perspective. Microbes Infect. 2016;18(7–8):450–9. 73. Mevorach D. Opsonization of apoptotic cells. Implications for uptake and autoimmunity. Ann N Y Acad Sci. 2000;926:226–35. 74. Clutterbuck PW, Lovell R, Raistrick H. Studies in the biochemistry of micro-organisms: the formation from glucose by members of the Penicillium chrysogenum series of a pigment, an alkali-soluble protein and penicillin-the antibacterial substance of Fleming. Biochem J. 1932;26(6):1907–18. 75. McIntyre N. Sir Alexander Fleming. J Med Biogr. 2007;15(4):234. 76. Gelpi A, Gilbertson A, Tucker JD. Magic bullet: Paul Ehrlich, Salvarsan and the birth of venereology. Sex Transm Infect. 2015;91(1):68–9. 77. Sachachiro Hata - Nomination Database. https://www.nobelprize.org/nomination/redirector/? redir¼archive/show_people.php&id¼3941 78. Capua I, Alexander DJ. Avian influenza and human health. Acta Trop. 2002;83(1):1–6. 79. Nguyen-Van-Tama HAW. Annual report of the national influenza surveillance scheme. Vaccine. 2003;21(16):1762–8. 80. Richardson ET, Collins SE, Kung T, Jones JH, Hoan Tram K, Boggiano VL, Bekker LG, Zolopa AR. Gender inequality and HIV transmission: a global analysis. J Int AIDS Soc. 2014;17:19035. 81. Janeway CA Jr. Approaching the asymptote? Evolution and revolution in immunology. Cold Spring Harb Symp Quant Biol. 1989;54(Pt 1):1–13. 82. Medzhitov R, Janeway CA Jr. Innate immune induction of the adaptive immune response. Cold Spring Harb Symp Quant Biol. 1999;64(429–35):Review.

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83. Medzhitov R, Janeway CA Jr. Innate immunity: the virtues of a nonclonal system of recognition. Cell. 1997;91(3):295–8. 84. Lemaitre B, Nicolas E, Michaut L, Reichhart JM, Hoffmann JA. The dorsoventral regulatory gene cassette spätzle/toll/cactus controls the potent antifungal response in drosophila adults. Cell. 1996;86(6):973–83. 85. Lemaitre B, Nicolas E, Michaut L, Reichhart JM, Hoffmann JA. Pillars article: the dorsoventral regulatory gene cassette spätzle/Toll/cactus controls the potent antifungal response in Drosophila adults. Cell. 1996. 86: 973-983. J Immunol. 2012;188(11):5210–20. 86. Medzhitov R, Janeway CA Jr. Decoding the patterns of self and nonself by the innate immune system. Science. 2002;296(5566):298–300. 87. Ulevitch RJ. Toll gates for pathogen selection. Nature. 1999;401(6755):755–6. 88. Aderem A, Ulevitch RJ. Toll-like receptors in the induction of the innate immune response. Nature. 2000;406(6797):782–7. Review 89. Beutler B, Hoebe K, Du X, Ulevitch RJ. How we detect microbes and respond to them: the toll-like receptors and their transducers. J Leukoc Biol. 2003;74(4):479–85. 90. Kumagai Y, Takeuchi O, Akira S. TLR9 as a key receptor for the recognition of DNA. Adv Drug Deliv Rev. 2008;60(7):795–804. 91. Hemmi H, Kaisho T, Takeda K, Akira S. The roles of toll-like receptor 9, MyD88, and DNA-dependent protein kinase catalytic subunit in the effects of two distinct CpG DNAs on dendritic cell subsets. J Immunol. 2003;170(6):3059–64. 92. Yang J, Wang L, Zhang H, Qiu L, Wang H, Song L. C-type lectin in Chlamys farreri (CfLec-1) mediating immune recognition and opsonization. PLoS One. 2011;6(2):e17089. 93. Ishii KJ, Akira S. Innate immune recognition of, and regulation by, DNA. Trends Immunol. 2006;27(11):525–32. Review 94. Netea MG, Schlitzer A, Placek K, Joosten LAB, Schultze JL. Innate and adaptive immune memory: an evolutionary continuum in the host’s response to pathogens. Cell Host Microbe. 2019;25(1):13–26. Review 95. Fumagalli M, Sironi M, Pozzoli U, Ferrer-Admetlla A, Pattini L, Nielsen R. Signatures of environmental genetic adaptation pinpoint pathogens as the main selective pressure through human evolution. PLoS Genet. 2011;7:e1002355. 96. Enard D, Cai L, Gwennap C, Petrov DA. Viruses are a dominant driver of protein adaptation in mammals. eLife. 2016;5:e12469. 97. Haygood R, Babbitt CC, Fedrigo O, Wray GA. Contrasts between adaptive coding and noncoding changes during human evolution. Proc Natl Acad Sci U S A. 2010;107:7853–7. 98. Barreiro LB, Quintana-Murci L. From evolutionary genetics to human immunology: how selection shapes host defence genes. Nat Rev Genet. 2010;11:17–30. 99. Avraham R, Haseley N, Brown D, Penaranda C, Jijon HB, Trombetta JJ, Satija R, Shalek AK, Xavier RJ, Regev A, Hung DT. Pathogen cell-to-cell variability drives heterogeneity in host immune responses. Cell. 2015;162:1309–21. 100. Hwang SY, Hur KY, Kim JR, Cho KH, Kim SH, Yoo JY. Biphasic RLR-IFN-β response controls the balance between antiviral immunity and cell damage. J Immunol. 2013;190:1192– 200. 101. Bagheri M, Zahmatkesh A. Evolution and species-specific conservation of toll-like receptors in terrestrial vertebrates. Int Rev Immunol. 2018;37(5):217–28. 102. Hagai T, Chen X, Miragaia RJ, Rostom R, Gomes T, Kunowska N, Henriksson J, Park JE, Proserpio V, Donati G, Bossini-Castillo L, Vieira Braga FA, Naamati G, Fletcher J, Stephenson E, Vegh P, Trynka G, Kondova I, Dennis M, Haniffa M, Nourmohammad A, Lässig M, Teichmann SA. Gene expression variability across cells and species shapes innate immunity. Nature. 2018;563(7730):197–202. 103. Ferrer-Admetlla A, Bosch E, Sikora M, Marquès-Bonet T, Ramírez-Soriano A, Muntasell A, Navarro A, Lazarus R, Calafell F, Bertranpetit J, Casals F. Balancing selection is the main force shaping the evolution of innate immunity genes. J Immunol. 2008;181(2):1315–22.

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104. Netea MG, Wijmenga C, O’Neill LAJ. Genetic variation in toll-like receptors and disease susceptibility. Nat Immunol. 2012;13(6):535. 105. Das A, Guha P, Chaudhuri TK. Environmental selection influences the diversity of TLR genes in ethnic Rajbanshi population of North Bengal Region of India. J Genet Eng Biotechnol. 2016;14(2):241–5. 106. Allison AC. Notes on sickle-cell polymorphism. Ann Hum Genet. 1954;19:39–51. 107. Nakano Y, Aso H, Soper A, Yamada E, Moriwaki M, Juarez-Fernandez G, Koyanagi Y, Sato K. A conflict of interest: the evolutionary arms race between mammalian APOBEC3 and lentiviral Vif. Retrovirology. 2017;14:31. 108. van Valen L. A new evolutionary law. Evol Theory. 1973;1:1–30. 109. Adrian J, Bonsignore P, Hammer S, Frickey T, Hauck CR. Adaptation to host-specific bacterial pathogens drives rapid evolution of a human innate immune receptor. Curr Biol. 2019;29(4):616–30. 110. Parham P. MHC class I molecules and KIRs in human history, health and survival. Nat Rev Immunol. 2005;5:201–14. 111. Deretic V. Autophagy in immunity and cell-autonomous defense against intracellular microbes. Immunol Rev. 2011;240:92–104. 112. Delgado MA, Elmaoued RA, Davis AS, Kyei G, Deretic V. Toll-like receptors control autophagy. EMBO J. 2008;27:1110–21. 113. Kondylis V, van Nispen Tot Pannerden HE, van Dijk S, Ten Broeke T, Wubbolts R, Geerts WJ, Seinen C, Mutis T, Heijnen HF. Endosome-mediated autophagy: an unconventional MIIC-driven autophagic pathway operational in dendritic cells. Autophagy. 2013;9(6): 861–80. 114. Münz C. Enhancing immunity through autophagy. Annu Rev Immunol. 2009;27:423–49.

Chapter 2

Dendritic Cells (DCs) in Innate Immunity

2.1

General Biology of DCs

A representative innate immune cell, dendritic cells (DCs) belong to hematopoietic cells and bridge immune responses between innate immune and adaptive immunities. DCs are the immune cells resided on the first line of interaction with antigens. In addition, the DCs are the primary defense guards for host immune response against pathogens and invaders. Because immune system of mammals consisted of two fundamental immunities of (1) primary or innate and (2) acquired or adaptive ones, DCs belonged to the primary or innate immunity groups. When foreign and invaded antigens are encountered or exposed to the mammal immune system, initial receptors regarding innate immunity system are broadly and systematically selected by molecular and physical recognition. Then, molecular and biochemical reactions are activated. The activation reflects the downstream signaling that has extensively been subjected for biological signal transduction. During the past 3 decades since 1985, the huge clarification of such primary defense system has been made. Once activation of the innate immunity is performed, adaptive immune system is operated by highly compromised and specific T-cell and B-cell receptors. Therefore, the response style and pattern of the two immune systems are quite different. The innate immunity is rapidly processed in short time intervals within hours to days, while adaptive case exhibits slow reaction period such as days to weeks for longer lasting time than innate one. Due to the specific signal transduction-based proposition, clonal amplification of the adaptive immune-related cells is highly outstanding and indeed explosive, but the case of innate immunity is almost constant without amplification. At the stage of completion of the reaction, the adaptive immunity is potentially forwarded to the memory state of the next generation of responses, but not observed in the case of the innate immunity. DCs fall into a heterogeneous group of myeloid lineage APC. They occupy, throughout the entire body, 2–4% of leukocytes. DCs are defined by their surface proteins, localization, and function. In initial defense system of the body, indeed © The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2022 C.-H. Kim, Glycobiology of Innate Immunology, https://doi.org/10.1007/978-981-16-9081-5_2

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38

2 Dendritic Cells (DCs) in Innate Immunity

likely to the innate immunity, the first defense line in cell level is DCs in body fluids. DCs have a diverse and big group of antigen-presenting cells (APCs), which translate the innate immunity-derived information of invaded antigens to adaptive immunity. The basic concept and function of DCs in transmitting the information to the B and T cells are originally described for the initial immune response by Steinman and Cohn in 1973 [1]. At the molecular level, DCs have a unique ability to transmit the collected and processed information to B and T cells of the adaptive immunity [2]. Although there are many different bone marrow-differentiated cells, DCs are a small group of innate immune cells derived from the bone marrow through the mammalian body system. DCs in body fluids are distinct APCs that function to translate the foreign antigens to recognizing cells, specifically through connection of the innate immunity to adaptive immunity. The translation process is operated by initiating T-cell responses in a direct fashion. DCs are therefore one of the most basic actors and the general mediators in the initial immune responses of mammals. Besides DCs, monocyte-translated macrophages play the same roles as DCs in the flamed tissues. In other words, DCs as an essential regulator of innate immune response and docking to trigger the active immune responses are the major mediators and players [2]. More specifically, antigen-captured and eventually antigen-loaded DCs induce a stepwise antigen-specific T-cell immunity that is called adaptive immunity. The DC’s loaded information-receiving receptor is called major histocompatibility complex (MHC), where MHC-II is specific for the B-cell activation, whereas MHC-I is oriented for the tumor- or virus-dependent receptor style in mammals. The MHC-II is expressed on DCs, macrophages, or cervical cells, while the MHC-I is normally expressed on the somatic and nonimmune cell surfaces. On the other hand, in organ tissues but not in body fluids, macrophages are the major actors of the innate immune defense. Initially the macrophage’s precursors, named monocytes, are circulating in the body fluids. Upon tissue damages by pathogen infection and related agents, the macrophage derived from the monocytes acts as a local mediator of the innate immunity through modulation of inflammatory response. Through the continued innate immune responses, macrophages activate the next step of responses, specialized for adaptive immune responses via internalization of antigenic peptides, process, and present the captured foreign antigens to the T cells. Macrophagic differentiation of monocytes or partial DCs involves in differentially expressed phenotypic changes in cell surface antigens of clustered differentiation (CD) markers and the production of mediators responsible for pro- or anti-inflammatory response [3]. Apart from pathogenic invasion, DCs are applicable to the tumor immunity because the versatile capacity of DCs can trigger antigen-specific immune responses to adaptive immune cells. Therefore, application of DC biology has recently received some great interests to cancer immunotherapies. The positive aspect and merit of DCs are attributed to target cancer-associated antigens expressed on tumor cell surfaces. This allows many researchers to the current research trend to use DCs. DCs can be directly isolated from the body or often derived from monocytes and in vitro cultured in order to utilize them to the cancer immunotherapy [4]. In organisms, DCs can be found in many different types of cells, and they reasonably

2.1 General Biology of DCs

39

protect the peripheral tissues. The most general DCs can be seen on the skin, a dermal barrier as peripheral tissues. The skin DCs are indeed non-clonal type with diverse receptors of DCs, and they recognize specific invasive agents, antigens, pathogen-associated recognition patterns (PARPs), or PAMPs. The peripheral DCs ingest and digest such foreign antigens in order to present the processed antigen fragments to adaptive immune cells such as T helper cells, as above mentioned. When DCs present the processed antigens and carry them on MHC-II, they are ready to migrate from the original phagocytic region, lymphatics, to the final destination in near lymph nodes. The antigen-processed fragments are typically oligopeptides to date, and they are subjected to load onto each MHC-II amino acid region. This process is a classical behavior for antigenic presentation after processing to helper T cells resident in lymphoid nodes or tissue [5]. Among the APCs in mammals, therefore, DCs are typical and professional APCs having the co-stimulatory molecules and MHC-II. DCs are consequently regarded as positive stimulators of primary immunity. In addition, DCs are an essential regulator in initiating organism’s innate immune responses, giving a term of “acquired immunity or secondary immunity.” Thereafter, binding of antigen peptides to T-cell receptor (TCR), MHC-antigen fragments, and co-stimulatory proteins on the DCs’ surface eventually activates T cells. The T-cell activation is a multi-complexed process including phosphorylationbased signal transduction and subsequently induction of T-cell differentiation, giving a description of “the antigen-specific response.” On the other hand, apart from DC activation, nonactivated and immature DCs contribute to the constitutive presentation of self-antigen. The status indicates T-cell deletion and also differentiation status of regulatory T cells (Tregs) or suppressor T cells by the interaction with the nonactivated and immature DCs. This process eventually triggers the T-cell behavior including the deletion of T cells and Tregs, and also differentiation of suppressor T cells. Therefore, this status of immune suppression is called “selftolerance.” Thus, the mammalian immunity is established with the appropriate and relevant response to the nonself threats from external environment. Thus, a wellorganized and target-oriented immunity is consequently ensured in the immunology, homeostatic limiting to only foreign pathogens, invaders, or agents [6]. Apart from APC function, DCs induce effector T cells or Treg responses through co-stimulatory signals and cytokine expression. As the most positive APC, DCs bridge between T-cell response and immune tolerance. The consequent case is DC-derived tolerance as a result of rare and distinct aspect of DC-regulated T cells. DCs also influence multiple phenotypes of T cells, including development, differentiation and function, to maintain tolerogenic state. The DC-T-cell interaction is not regularly observed but observed by certain conditions including DC maturation state or tissue microenvironments. The DC and Treg recognition is indispensable in induction of central and peripheral tolerance. DCs are indispensable for Treg differentiation and homeostasis [7].

40

2.2

2 Dendritic Cells (DCs) in Innate Immunity

Classification and Different Function of DCs

Basically, DCs are differentiated to each specific cell type, depending on their functions for the innate immune system. DCs develop from a common BM-derived macrophage or progenitor DC cells, which further progress to the differentiated cell types of monocyte and macrophage lineage or the common DCs [8]. DCs in the BM exist as both forms of the named plasmacytoid DC (pDC) and pre-DC progenitors [9]. Completely matured pDC in the BM moves to the bloodstream. Prematured DCs migrate to the peripheral tissues or lymphatic organs via the vascular vessel. The migrated prematured DCs to peripheral tissues or lymphatic nodes are ready to differentiate into conventional DC subsets of CD8α+/CD103+ DCs or CD11b + DCs [10]. The DCs can be classified depending on their maturation to antigen-specific cell types, presentation to T cells, or migration to lymphatic nodes. Typically, DCs are classified into two distinct classes of (1) pDCs, which produce type I IFN upon viral infection and (2) conventional DCs as APCs, which activate naive T cells. Among conventional DCs, CD8α+ DCs orchestrate immune responses upon infection of intracellular pathogens. In contrast, CD11b+ DCs fight extracellular pathogens. Conventional DCs are classified into two basic categories of (1) migratory DCs such as dermal DCs and Langerhans cells and (2) nonmigratory DCs such as spleen DCs, which are resident in secondary lymphoid organs, depending on their migratory potentials. Also, if foreign pathogens infect the body tissues, DCs are classified into other two different categories of (i) monocyte-derived DCs (moDCs) and (ii) pDCs as a first defense line against pathogenic invasion or type I interferon-releasing DCs. In the meaning of diverse functional phenotypes of DCs, DCs are defined as multifunctional-shaped and phenotyped defense cells against pathogenic infection or agent invasion. In order to accomplish the translational behavior which transmits the information of foreign antigens to T cells as adaptive immune cells and eventually B cells, moDCs are highly migrative to their directed locations to account their counterpart cells such as T cells [11]. For migrative potentials, DCs or their precursors acquire and require two different migration steps, i.e. (i) as a first step, they leave the vascular blood to peripheral tissues through the leukocyte homing process, and (ii) as a next second step, matured DCs leave the peripheral tissues through the homing process to the draining lymphoid nodes. The process is well defined through the leukocyte homing process. DCs and DC precursors include undifferentiated monocytic cells and extravasate to peripheral dermis, tonsils, epidermis, and gastrointestinal tract (GIT) mucosal region across the endothelial lines from the blood. When DCs are translocated to inflammatory tissues, DCs are activated by an uptake of foreign antigens or by proinflammatory cytokines released by diverse inflammatory cells. On the infection or injured site, leukocytes under circulation attach to endothelial cells and platelets as well as the adhered leukocytes. When inflammatory agent or invaded pathogens are encountered to DCs, DCs undergo their maturation process to express their surface antigens such as CD11 series. When the immature DCs are in stages of antigen uptake and processing, they are differentiated to active and mature

2.2 Classification and Different Function of DCs

41 Immunity

Tolerance

CD83+ Immature DC

Stimuli

Mature DC

CD83+ Antigen uptake and capturing,

Antigen presentation, to be able to costimulate and activate T-cell

antigen processing

Fig. 2.1 Maturation of dendritic cell

Pathogens

DC precursor (CD11c+, Mac1+, CCR5+)

Inflammatory monocyte (Mac1+, CCR2+)

Blood

CD83+ Lymph node

Tissue T cells

Fig. 2.2 Maturation of DCs and migration to lymph node and T-cell activation, and chemokinedriven migration of monocytes during pathogenic invasion

DCs. The differentiated DCs exhibit the classical APC roles including antigen presentation, costimulation, and T-cell activation (Fig. 2.1). Thus, DCs are important for initial stage of the immune responses, where immature DCs positively internalize foreign antigens and differentiate to trigger T-cell responses. To accomplish such mission, therefore, DCs serially step down into two essential migration stages. (i) Migration into peripheral tissues is to acquire antigens on the inflamed sites, and (ii) migration to lymph nodes is its final destination to collaborate with the information-receiving cells like B cells and T cells (Fig. 2.2). Cell maturation, positional migration to lymph node, and T-cell activation of DCs are all their

42

2 Dendritic Cells (DCs) in Innate Immunity

Complement-binding protein like domain

w“ˆš”ˆG”Œ”‰™ˆ•Œ

EGF domain

j ›–š–“Š

jG

sTzŒ“ŒŠ›•G

lŸ›™ˆŠŒ““œ“ˆ™G

Lectin domain

uG

lTzŒ“ŒŠ›•G

wTzŒ“ŒŠ›•G

Fig. 2.3 Structure of selectins

pathways collaborated during pathogenic invasion or occurring of inflammatory agents. During inflammatory status, the leukocytes such as DCs are highly activated to express the cell adhesion molecules (CAMs) of selectin molecules, which recognize and tightly bind to the sugar components on the endothelial cells. Selectins expressed on DC surface act as rolling mediator, whereas cell surface β2 integrins act to potentiate arrest, intraluminal crawling, and slow rolling. The adhesion cascade events are comprised of a series of various responses including basic interaction of arrest, crawling deceleration, slow rolling, tethering, and transendothelial migration [12]. In leukocytes, to accomplish the adhesion event, lipid rafts in membranes are highly ordered through assembly with membrane lipid components of cholesterol and sphingolipids, and membrane-selected proteins. Particular membrane proteins participate in lipid rafts via hydrophobic residues in transmembrane domains. Consequently, they are ready to interact with membranelocated GSLs or cholesterol. N- and O-glycans on protruding proteins are also associated with lipid rafts. The dominantly expressed leukocyte ligands are recognized by their receptors of P /L-selectins. The specific P-selectin glycoprotein ligand-1 (PSGL-1) known as the selectin ligand is recognized. Apart from the PSGL-1, other important leukocytic ligands to recognize E-selectin are well defined to date, and several representative candidates of CD44 and CD43 are reported [13]. Among selectins, myeloid lineage leukocyte cells produce L-selectin form, while activated platelets produce P-selectin form on surfaces. In addition, stimulated endothelial cells express E- and P-selectins on their cell surfaces (Fig. 2.3). Through such selectin-ligand interactions, the leukocytes are also ready to bind to the

2.2 Classification and Different Function of DCs

sŒœ’–Š ›Œ

43

i“––‹ ŒššŒ“ w“ˆ›Œ“Œ›

h‹Œš–•

l•‹–›Œ“œ” hŠ›ˆ›–•

p•“›™ˆ›–•

hŠ›ˆ›–•

p•“ˆ””ˆ›–™  Š ›–’•Œš

p•“ˆ””ˆ›–™  ›ššœŒ

a zŒ“ŒŠ›• a zœŽˆ™ ”–“ŒŠœ“Œš

Fig. 2.4 Leukocyte trafficking to inflammatory endothelium and infiltration

peripheral platelets and infiltrate into the inflammatory tissues, where the immune cells resident in the area are also stimulated to activate by various inflammatory mediators including cytokines of IL-1β and TNF-α as well as arachidonic prostanoid metabolites (Fig. 2.4). Among the DC subpopulations, only matured DCs can activate and proliferate T cells after migration to the lymph node, while immature DCs rather induce anergy status, apoptosis, or deletion of T cells, providing the immune tolerance status, and then allowing the immunological homeostasis.

2.2.1

pDC, Lymphoid Organ CD8α+ DC, and Tissue CD103+ DC Interaction with Tregs

The phenotype of pDCs is a characteristic of B220+ PDCA-1+ Siglec-H+ CD11clow MHC-IIlow cells. The pDC subset development depends on E2–2 and IRF8 [14]. pDCs secrete type I IFN, and normally, pDCs are a poor type of APC, although pathogen-stimulated pDCs can prime naive T cells. Normal mouse antigen-pulsed splenic pDCs and human pDCs induce anergy states of antigen-specific mouse T cells and human CD4+ T cells [15], respectively. A subset of naive T cells is activated by CpG-induced pDCs, and activated T cells generate immunosuppressive Treg populations [16]. Human pDCs stimulate differentiation of T cells into IL-10expressing Tregs, and pDCs activate CD4+CD25+ FOXP3+ Tregs. CD8α+ DCs are lymphoid organ-specific subsets. CD103+ DCs are tissue-specific CD103+ DCs. The cells are developed through a fms-like Tyr-kinase 3 ligand (Flt3L), which is known as a distinct ligand of Tyr-kinase receptor [17]. In fact,

44

2 Dendritic Cells (DCs) in Innate Immunity

Flt3L is also known as a specific inhibitor of basic Leu zipper transcription factor ATF-like3 (Batf3) [18], transcription factor IFN-regulatory factor 8 (IRF8), and DNA-binding protein 2 (ID2) [2]. The CD8α+ DCs are involved in peripheral and central tolerances through TGFβ production. This tolerance is nonresponsive to peripheral tissue-associated SAMPs by direct recognition to self-responsive T cells that induce apoptosis. The tolerance of CD8α+DCs is mostly strong when antigens recognize CD205 known as DEC205. CD205 promotes clonal deletion and Treg differentiation [19]. DEC205 + DC depletion induces thymic Treg level and maintenance in mucous [20]. In conventional DC subset, CD11b + DCs are resident in the spleen, lymphoid organ, and peripheral tissues, after development with the help of GM-CSF, Flt3L, IRF2, IRF4, LTβ, Notch2, and RelB [21]. However, CD11b+ DCs are minorly resident in the thymus. CD11b+ DCs express endothelial cell-specific adhesion molecule (ESAM). In spleen-resident CD11b+ DCs [12], ESAM-high-expressing DCs are generated through differentiation from DC progenitors, whereas the ESAMpoorly expressing DCs are derived from circulating monocytes. CD8α CD11b+ DCs exhibit cross-tolerance against intestinal antigens, while CD8α+ DCs do not. For example, antigen-tolerogenic CD11b+ DC subpopulation is specially enriched in the specialized organs such as Peyer’s patches when type II collagen-specific oral tolerance responses are appeared, and therefore, they inhibit development of collagen-induced arthritis [22].

2.2.2

DCs Induce Tolerance State

Apart from APC function, DCs can also induce immune tolerance. DCs induce central and peripheral tolerance via T-cell interaction. Tolerance induction of DCs indicates that the loss of DCs breaks peripheral tolerance. However, constitutive DC depletion does not induce spontaneous autoimmunity but a myeloproliferative disease. In normal condition, DCs maintain immunological homeostasis in organism and also promote peripheral tolerance state of T cells when T cells are presented with foreign non-harmful antigens or self-antigens. DCs assist T-cell development to maintain the T-cell homeostasis in the thymus, as confirmed by two-photon imaging and live intravital microscopic analysis via DC-T-cell binding. DC-derived T-cell homeostasis is based on signals through MHC-TCR complex recognition [23]. DCs are also capable of presenting self-antigens to peripherally resident T cells in the lymphoid node drained. Peripheral tolerance is essential due to the limited central tolerance. Peripheral tolerance potentiates to avoid “horror autotoxicus” [24]. DC-induced T-cell tolerance is acquired by immune checkpoints of CTLA-4 and PD-1 specifically expressed on CD8+ T cells, and also by Treg induction. DC self-antigens induce CD4+ T-cell tolerance, as derived from the interaction between DC PD-L1 and T-cell PD-1 as well as antigen-specific peripheral iTregs. Constitutive DC ablation enhances autoimmunity upon self-antigen immunization [25]. Enhanced production

2.2 Classification and Different Function of DCs

45

level of Flt3L in blood plasma is essential for development of the myeloid proliferative disease because the DCs are absent. The similar examples are observed in patients defected with hereditary monocytes or DC deficiency in humans [26]. However, constitutively depleted DCs in lupus-prone MRL/lpr mice exhibit the improved level of autoimmune response. DCs expand and differentiate T cells. DCs maintain homeostasis of peripheral-resident T-cell populations via prevention of unwanted activation of T cells. DCs rather induce induced type Tregs known as iTregs [8]. Treg proliferation depends on the DCs [27]. Treg depletion accelerates DC maturation and expansion, depending on Flt3. Lamina propria CD103+ DC subset expands when the cells are interacted with Flt3L [27]. CD103+ DCs are the DC subtype resident in peripheral tissues and also corresponded to CD8+ DC type present in the lymphatic nodes or splenic tissues. Binding of Flt3 to Flt3L influences the functions of Treg cells and CD8α+-CD103+ DCs. DCs differentiate and maintain various types of Tregs. For example, IL-10-expressing T regulatory-1 (Tr1), T helper-3 (TGFβ-expressing Th3), thymic-generated Tregs named nTregs and periphery-differentiated Tregs named iTregs, and Foxp3+ T-cell subsets are affected by DCs toward their differentiation. DCs can also induce tolerance of peripheral T cells, and DCs generate antigen-specific peripheral iTregs by PD-L1-PD-1 binding.

2.2.3

DC co-Stimulatory Receptors

2.2.3.1

CD80/CD86

DCs express surfaced CD80/CD86. All T cells including Tregs express CD28. T-cell CD28 binds to DCs CD80/CD86 to develop and maintain thymic and peripheral Tregs. DC CD80/CD86 enhances Treg proliferation [28]. The DC CD80/86 signaling does not affect the development of nTregs, which are derived from the thymus. However, DC CD80/86 induces development of iTreg subsets in peripheral tissues.

2.2.3.2

CD70

DCs and mTECs express CD70, a TNF family, whereas CD70’s receptor is CD27 on developing thymocytes. CD70-CD27 binding develops thymic-derived nTregs. In the thymus, CD70-expressing CD8α+ DCs contribute to development of nTreg cell population. In addition, CD70-CD27 binding positively transduces the nTreg selection and leads to prevention of apoptotic cell death [29]. Peripheral CD70 contributes to Th1 differentiation, while it suppresses Th17 differentiation, reducing autoimmunity [30]. In contrast, CD70 overexpression does not differentiate Th17 without any effect on Treg development.

46

2.2.3.3

2 Dendritic Cells (DCs) in Innate Immunity

ICOS-L, PD Ligands (PD-L), IL-10, IOL-27, TGFβ, Retinoic Acid, and β-Catenin

The cell surface protein ligands include co-stimulatory B7 family members and bind to receptors on lymphocytes toward immune response regulation. Among them, the inducible co-stimulator (ICOS) ligand (ICOS-L) is mainly present in APCs like B cells, DCs, and macrophages. Moreover, the ICOS-L is also expressed from nonimmune cells including lung epithelium, endothelium, and tumor microenvironment cells. ICOS as a co-regulatory receptor of T cells provides a co-stimulatory signal to T cells during antigen-mediated activation. ICOS is a rapidly induced co-stimulator upon T-cell receptor cross-linking. Follicular lymphoma cells generate Treg cells via ICOS/ICOS-L pathway, applicable to treatment by anti-ICOS/ICOS-L therapy [31]. ICOS molecule is found in the T-cell subsets activated including CD8+ and CD4+ T cells and, also, effector T cells including CD4+ T follicular helper cells (Tfh). ICOS-targeting therapy improves antitumor immunity. ICOS signaling activates the effector T cells of CD4+Foxp3 T cells upon tumor immune responses. ICOS-L transfection of tumor cells enhances antitumor immunity when cells are vaccinated with anti-CTLA-4 treatment [32]. ICOS-L also promotes antitumor immune responses [33]. ICOS promotion of immunosuppressive Tregs may impair tumor immunity [34]. Tregs specifically express the transcription factor Foxp3 [35]. Natural and inducible Tregs express CD25, glucocorticoid-induced TNFR-related protein (GITR), CD45RO, and CTLA-4, but lack CD127 [35]. Tregs suppress effector T cells (Teffs) to prevent autoimmune diseases, allergies, infection-induced organ damage, as well as transplant rejection. ICOS-L activates memory and effector T cells upon humoral immune reaction. ICOS expression is increased in rejected allografts [36]. In a negative viewpoint, Tregs are harmful in cancer due to its suppression of antitumor immunity. Tregs actively accumulate in tumor microenvironments with poor antitumor immune response and poor survival. The tumor-associated microenvironment (TAM) favors the phenotype conversion from CD4 + CD25-T-cell subsets to inducible subsets of Tregs. ICOS protein belongs to a CD28 class, which is a co-stimulatory protein, and maintains durable immune reactions upon binding to ICOS-L. ICOS/ICOS-L axis promotes Treg differentiation. Normal tissues express ICOS-L and regulate CD4+ T-cell activation and cytokine production [37]. ICOS+ Tregs dampen T-cell responses via impairing APC with IL-10. ICOS blockade upregulates activated and pathogenic T cells. Certain cancers stimulate ICOS-L to develop immunosuppressive CD4+ T-cell population like Tregs. Thus, tumor progression and survival require ICOS-L expression. During anticancer vaccination or anti-CTLA-4 treatment, ICOS+ T cells exhibit the enhanced CD4+ and CD8+ subset levels, increasing the Teffs/Tregs ratio in tumor microenvironment. Hence, ICOS/ ICOS-L binding improves cancer therapy effect. In airway asthma, ICOS-L-expressing semi-mature DCs induce TGFβ-expressing and antigen-specific iTregs [38]. pDC induces iTreg, an ICOS-L-dependent. In mice, ICOS-L-deficient pDC cannot protect them against asthma. PD-L1-KO APCs

2.2 Classification and Different Function of DCs

47

generate iTregs in vitro. PD-L1-KO APCs stimulate to differentiate the naive CD4+ T-cell subsets to iTreg cell subsets, even to a lesser extent [39]. PD-1 binding stabilizes and strengthens DCs and T-cell recognition [40]. PD-1 and PD-L1 binding inhibits TCR-mediated signaling [40]. DC treatment with soluble PD-1 blocks DC maturation with IL-10 secretion [41]. PD-L1-expressing DCs induce antigenspecific iTreg generation with dampened disease severity. DCs and T cells express IL-10, a regulatory cytokine [42]. IL-10 regulates Treg cells and inhibits APC function with anti-inflammatory activity. IL-10 inhibits maturation of DCs. Moreover, IL-10 suppresses the levels of co-stimulatory proteins, MHC-II, and chemokines of CXC and CC. In addition, IL-10 inhibits expression of proinflammatory cytokines in DCs [43]. IL-10 in human DCs increases the levels of T-cell tolerance and T-cell anergy [44]. IL-10-stimulated DCs inhibit the response level of effector T cells [45], protecting EAE symptoms and inhibiting transplanted graft rejection in hosts [46]. IL-10 controls DCs to inhibit contact hypersensitivity and anti-Leishmania immune response [47]. IL-10 modulation of CD11c+ APCs maintains intestinal immune homeostasis [48]. DC IL-10 expression is important for T-cell anergy and suppression. IL-10-expressing matured pulmonary DCs induce tolerance event through Tr1 cells. BM-derived DCs are transmitted to semi-matured types of DCs when GM-CSF, TNF-α, and IL-10 are present. Those DCs trigger to differentiate suppressive T cells, which express IL-10. Dermatic DCs known as Langerhans cells inhibit IL-10-mediated contact hypersensitivity event and Tr1 cell differentiation [49]. DCs co-cultured with Tregs secrete TGFβ, IL-10, and IL-27, and also generate Tr1 cells. DC IL-27 inhibits the IL-23 and IL-1β expression but activates IL-10 expression. Hence, differentiation into more immunogenic Th17 cell type and the resulting autoimmune potentials are terminated [50]. IL-27 induces c-Maf, ICOS, and IL-21 expression in naive T cells, to collaboratively concert to Tr1 cells [51]. Human DC stimulation with IL-27 increases the level of PD-L1 surface expression, without DC maturation [52]. Stimulation of DCs with IL-27 increases CD39 and suppresses the inflammasome pathway [53]. A regulatory and pleiotropic cytokine TGFβ is effective on T cells and on APCs. TGFβ stimulates conversion of naive T cells, which are peripherally resident to CD4 + CD25+ Treg cell type through Foxp3 gene expression. During treatment with LPS, splenic DCs secrete highly TGFβ to differentiate Tr1 cells. DCs induce extrathymic iTreg differentiation by TGFβ assistance [8]. T-cell-specific TGFβ signaling inhibition using a dominant-negative TGFβRII terminates differentiation of iTreg cells [18]. The integrin-α4β8 activates TGFβ by metalloproteinase degradation of latency-associated protein (LAP) and extracellular TGFβ release [54]. DC-produced retinoic acid (RA) induces oral tolerance by iTregs. Mucosal DCs guide T-cell homing to the gut by DC-derived RA. DC-produced RA inhibits TGFβ-dependent Th17 cell production and also activates Foxp3+ Treg cell differentiation. RA activates iTreg differentiation by inhibition of effector memory T-cell generation, which expresses IFN-γ or IL-21 [55]. RA-forming enzymes depended on β-catenin known as the key canonical protein in the Wnt signaling (Wingless Int) [56], constitutively expressed in DCs. β-Catenin regulates BM-DC maturation. Blocking of β-catenin interaction with E-cadherin of BM-DCs increases the

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expression levels of two major surfaced co-stimulatory molecules and MHC-II but not proinflammatory cytokines, providing tolerant DCs with IL-10-expressing Tregs. CD11c-specific deficiency of β-catenin is sensitive to colitis and Th1-/ Th17-associated EAE and prevents Foxp3+ Treg responses [57]. Wnt/β-catenin signaling suppresses tumor-raised immunity through inhibition of CD8 T-cell-mediated DC priming with IL-10 [58].

2.2.3.4

Indoleamine-2,3-Dioxygenase

A specific catabolic enzyme of a Trp catabolism, known as indoleamine-2,3dioxygenase (IDO), catabolizes Trp residue to its metabolite kynurenine, which induces cell starvation caused by tryptophan loss. In addition, kynurenine activates the general control non-derepressible 2 (GCN2) kinase. The GCN2 kinase specifically phosphorylates the eukaryotic initiation factor 2 (eIF2), and the activated pathway generates Tregs and expands to IDO-rich condition. Kynurenine binding to aryl hydrocarbon receptor (AhR) present in CD4+ T-cell populations inactivates the AhR receptor. This induces immunosuppressive T cells and allows Tregs [59]. AhR also increases IDO expression and RA-synthesizing enzymes directly on the DCs. DC-produced IDO induces tolerance by iTreg generation. iTregs can induce the IDO synthesis in pDC [60]. IDO expression differs between DC subsets. CD8α+ DCs produce highly IDO, although CD8α DCs do not. CD103+ DCs present in intestines also produce highly IDO enzyme and maintain gut homeostasis and oral tolerance [61]. DC CD80/86 binding to CTLA-4 induces IDO expression.

2.2.4

Application of DCs to Human Diseases

DCs are candidate cells to treat human diseases including antitumor immunity and tolerance in transplantation and autoimmunity. Tolerogenic DCs can be obtained by deletion of co-stimulatory receptors. B7-H1 (PD-L1) deletion generates tolerogenic DCs [62], although it is not possible in humans. Cytokine cocktails containing IL-10 or TNFα are alternative to overcome. Another overcoming strategy is generating DCs that induce tolerance in several types of human autoimmune disorders like graft-versus-host disease (GvHd) and collagen-type II-induced arthritic diseases (CIA). Blocking of co-stimulatory protein expression or IDO activation or TGFβ expression in DCs is suggested. Tolerance DCs from the patients can be acquired using cytokine cocktails [63]. The clinical trials with DCs are limited, as boost immunity in cancer therapy is performed currently. However, tolerogenic DCs are not used to treat autoimmunity [64].

References

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Chapter 3

Glycan Biosynthesis in Eukaryotes

For three billion years, terrestrial life has been evolved to adapt to environmental changes with energy production, reproduction, and cellular signal transduction. Each organism has mainly used each DNA genetic code and RNA diversity, structurebased functional proteins, lipid-based membranes, and metabolites. Using such nucleic acid-protein-lipid axis, to some extent, they have acquired their survivals to share with diversity, allowing terminology of evolution. However, the axis is too limited to cover all the diversity in organisms, in addition toward the future evolution probabilities. For organisms to protect themselves from pathogenic infections, selfhyperreactivity, and intellectual differentiation, each organism has acquired diverse cell surface glycans which are essential. The diversity or escape process from pathogens is distinct from genetic code due to importance in organism survival. Hence, any concept to explain the future unidirectional evolution is required in the biotic and abiotic environments. Then, the appropriate field has been raised from the dawn to create a link between water and hydrophilic environments, terming of “glycans” as molecules and “glycobiology” as subject. For the basic background of the steady-state investigation on the glycans, the functional importance and structural diversity are the most well-recognized facts. Glycans such as N-, O-glycans, GSLs, GAGs, GPI anchors, sialic acids, and cytoplasmic and nuclear glycans are particularly characteristics of eukaryotes. In the biosynthesis of glycans, template is not needed because such equivalent is not utilized for the design of glycans, as this is contrast to the DNA biosynthesis. It is reminded that DNA generates the template for the protein. The biosynthesis of glycans consisted of three distinct steps. In the first step, sugar nucleotides are generated in the cytoplasm and supplied. In the next second step, the sugar nucleotides are trafficking to organelle ER or Golgi apparatus by each specific transporter located in the membranes. In the third step, each specific glycosyltransferase attaches the sugars from each sugar nucleotide to an acceptor protein substrate or glycan substrate in the ER and Golgi apparatus, toward Golgi trafficking. In eukaryotes, subcellular organelle ER-Golgi networks create evolutionary glycan diversity and cell surface glycans. In addition, glycan structures are diverse in different organisms. © The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2022 C.-H. Kim, Glycobiology of Innate Immunology, https://doi.org/10.1007/978-981-16-9081-5_3

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Glycan Biosynthesis in Eukaryotes

For example, various N-glycans are structurally defined in different eukaryotes including yeast, insect, plant, and animal (Fig. 3.1).

3.1

General Glycosylation Events

Glycosylation is a post-transcriptional modification with over 50% of total proteins and ubiquitous process in plasma membrane glycolipids. Most cellular interactions are mediated by glycosylation, and disease development involves changes in glycan biosynthesis. All the N-glycans carry an identical and common saccharide linkage generated from the same biosynthesis pathway that normally terminates in the late Golgi step. O-glycosylation also provides diverse linkages with diverse structures in protein glycans. N-glycosylation and O-glycosylation of glycoproteins as well as lipid glycosylation of sphingolipid are responsive to environmental changes and are changed in various diseases. Alterations in O-glycosylation ad N-glycosylation are caused by changed expression levels of biosynthetic enzymes or dysfunctional biosynthetic pathway of the ER-Golgi network. Glycoproteins only carry glycans through three categories of N-linked (Asn), O-linked (Ser, Thr, hydroxy Lys), and C-linked (carboxyl group of Trp). The glycosylation of O-glycans, N-glycans, and glycosphingolipids (GSLs) is performed by the multiple machineries such as endosomal ER and Golgi complexes. Thereafter, the synthesized glycans are localized on their locations in plasma membrane or each destination (Fig. 3.2). Glycosylation event occurs only to proteins in the ER-Golgi, lysosome, plasma membrane, and extracellular protein-trafficking pathway. However, the exceptional cases are for the cytosolic and nuclear proteins which a single O-linked GlcNAc residue is attached. Ribosome-mediated translated proteins are sorted through the N-glycosylation secretion way in rough ER (rER) with the N-terminal ER-specific signal region. ER-processed proteins which are normally folded are trafficking to the Golgi complex through transport vesicles. The defect in innate immune functions

Glucose(Glc)

N-Acetyl glucosamine (GlcNAc)

Xylose (Xyl)

Galactose (Gal)

Fucose (Fuc)

N-Acetyl galactosamine (GalNAc)

Mannose (Man)

Glucuronic acid (GlcA)

Sialic (Sia) or N-Acetyl neuraminic acid (NeuAc)

A) Monosaccahrides

Yeast

Insect

Plant

B) Different N-glycan structures in different organisms

Fig. 3.1 Monosaccharides and N-glycan structures in various eukaryotic organisms

Animal

3.1 General Glycosylation Events

55

A) Scheme of N- and O-glycan biosynthesis

Processing enzymes

B) Core structures of glycans

Golgi

-Ser/Thr

/CP

-Ser/Thr

)CN

)NE

Cer

)CN

LacCer

Flip )NE0#E

GalNAc-T

ER

Ceramide Ser/-Thr protein

)CN0#E

-Asn-

-Ser/Thr

N-glycans

-Ser/Thr

O-glycans

GSLs

Asn protein Flip : flippase

C) ER/Golgi pathway of glycan synthesis

N-Glycans

O-Glycans

GSLs

Rough ER

Golgi

Plasma membrane

Secretory Granule

Fig. 3.2 Mammalian glycan biosynthetic pathways. (a) Scheme of N- and O-glycan biosynthesis. (b) Core glycan structures. (c) ER-Golgi pathway of glycan synthesis. Synthesis of three major glycans in the ER-Golgi, and subsequent modification in the lumen. Genes or glycosylation enzymes are well defined

does not primarily come from one of the glycantransferases or processing enzymes such as hydrolases but is likewise originated from the sugar nucleotides formation, transports to the ER-Golgi trafficking. Each sugar residue has at least three or four recognition sites for glycosidic linkage with adjacent monosaccharide residues. In addition, the resulting anomeric α- or β-configuration is generated in each glycosidic linkage to yield each specific

56

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Glycan Biosynthesis in Eukaryotes

branch in glycan structures. The synthesized glycans consequently exhibit structural diversity compared to other cellular constituents. These linear chains of polymers are only types of the simple linkages formed by peptide or nucleotide bonds. Theoretically, the naturally occurring 9 monosaccharides in humans can link into more than 15 million possible tetrasaccharides. Even simple glycans can have many varieties of forms [1]. Evolution process, therefore, exhibits inter-species and intra-species differences, molecular similarity, structural mimicry, glycosylation adaptations of invasive agents, each specificity of glycosyltransferase, environmental acquisition, and natural coevolution. Differences in glycosylation pattern are found in all eukaryota including arthropoda, deuterostomia, fungi, nematoda, and viridiplantae [2]. Currently, the eukaryotic biosynthesis of glycans is well established, as the representative synthetic pathways of glycans are described.

3.2

Sugar Nucleotide Transporters Deliver Donor Saccharides to ER-Golgi Network

For supplying of sugar nucleotides required for donor substrates, monosaccharides used for the biosynthesis are supplied from the so-called high-energy form of nucleotide sugars. The sugar nucleotides are derived from dietary sugar sources and salvage pathways for the formation. Monosaccharides of Fru and Glc residues are present as the main sugar sources, and therefore, all other monosaccharides can be converted from the two basic hexoses. Continuous enzymatic pathway including phosphorylation, acetylation, and epimerization generates various nucleotide sugar donors through conversion reaction. Nucleotide sugar is biosynthesized in the cytoplasmic space. However, only CMP-SA or CMP-NeuAc is generated in the nuclear region, indicating that synthesis of sugar nucleotides is tightly controlled [3]. For instance, UDP-GlcNAc, an abundant form in cytosol, inhibits the enzyme activity of Gln-Fru-6-phosphate transaminase required for the first step of synthesis of UDP-GlcNAc in the enzyme reaction [3]. In addition, CMP-SA or CMP-NeuAc blocks enzyme activity of the GNE/MNK known as the UDP-GlcNAc-2-epimerase (GNE)/ManNAc kinase (MNK). The enzyme GNE/MNK is the basic two enzymes for the CMP-SA or CMP-NeuAc biosynthesis [4]. Biosynthesis of nucleotide sugars requires ATP, and tightly regulated synthesis of sugar nucleotides indicates that the altered nucleotide sugar impairs cellular pathway for glycosylation. The sugar nucleotides are generated in the cytosolic region, after monosaccharides are delivered to the ER lumen side and/or Golgi apparatus. From the fact that sugar nucleotides cannot transmigrate across the lipid bilayer of membrane, it is suggested that Man and Glc residues transmigrate across the membrane by interaction with the dolichol phosphate (Dol-P) as its specific lipid carrier. The cytoplasmic Dol-P-Man/Dol-P-Glc synthase enzymes recognize each substrate GDP-Man or UDP-Glc for linking the Dol-P present in the cytosolic site. The “flippase” enzyme catalyzes the transport of the Dol-P-monosaccharides from the cytosolic side to ER

3.2 Sugar Nucleotide Transporters Deliver Donor Saccharides to ER-Golgi Network

57

luminal leaflet side. Then, the monosaccharides are ready to be utilized by various ER-resident glycosyltransferases (GTs) [5]. The nucleotide sugar transport is specifically mediated by nucleotide sugar transporters. The nucleotide sugars can cross the ER membranes and also Golgi membrane by means of the nucleotide sugar transporters (NSTs) embedded on the ER and Golgi membranes. They are indeed antiporters. Hence, nucleotide sugars enter into the ER and/or Golgi system from cytosol, and this entry event is coupled to exit of each nucleoside monophosphate to cytosols at the level of equimolecular level from the lumen of ER and Golgi complex [6]. Upon the ER-Golgi lumen transportation of sugar nucleotides, each glycosyltransferase transfers the monosaccharide residue to each glycan substrate. Then, to be equilibrium status in the lumen, the nucleoside diphosphates are again subjected to the molecular conversion to di-anionic nucleoside monophosphates that are utilized by a nucleoside diphosphatase for the antiporter and inorganic phosphate. In the topology, several transporters including UDP-Gal transporter, UDP-GalNAc transporter [7], UDP-glucuronic acid (GlcA) transporter, UDP-GalNAc transporter, UDP-GlcNAc transporter [8], and UDP-Xyl transporter [9] are multiple enzymes with two more substrate specificities. In contrast, the CMP-NeuAc transporter [10] and GDP-Fuc transporter [3] are a mono-type enzyme with a single specificity. Interestingly, many NST transporters such as UDP-GlcNAc, UDP-Fuc, GDP-Xyl, and CMP-SA/NeuAc transporters are reported to be strictly localized on Golgi membrane [6, 9, 11], while the specific transporter for UDP-GlcA is localized in the membrane of ER [8]. UDP-Glc and UDP-Xyl are resident in the ER region, but UDP-Glc and UDP-GlcA are detected in the Golgi apparatus [6]. A special GSL, galactosylceramide (Gal-Cer) is synthesized by UDP-Gal-Cer Gal-transferase present in the ER region of certain cells of myelinating cells, spermatogenic cells, and epithelial cells. However, UDP-Gal transporter as an NST is located in Golgi apparatus. The different localization of the two Cer-Galtransferase and UDP-Gal transporter is surely unmatched issue to fully agree with the substrate and glycosyltransferase relationship. For this inconsistency, several studies have clearly concluded to settle down the discrepancy [11, 12]. First, ER-resident galactosyltransferase is associated with the UDP-Gal transporter (UGT) during ER-Golgi networks, and the ER-Golgi boundary is suggested to be flexible to fuse together [11]. Second, ER and Golgi colocalization of the UDP-Gal transporter is also explained by RNA variants produced via its alternative spicing with two splice forms of UGT1 and UGT2. UGT1 is suggested to be a Golgi type and UGT2 for dual ER and Golgi type operated by a C-terminal di-lysine motif (KVKGS) [12]. GlcNAc-phosphotransferase selectively catalyzes phosphorylation reaction of the N-glycoproteins to move to the lysosome region [13]. Golgi-localized transferases recognize only one monosaccharide residue, a saccharide sequence and target peptides. For example, α2,6-sialyltransferase, ST6Gal I, binds to the terminal LacNAc to form a linkage of the SAα2,6-LacNAc structures present in N-glycan/ O-glycan of glycoproteins and GSLs; however, the β1,4-galactosyltransferase

58

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Glycan Biosynthesis in Eukaryotes

A) Major classes of N/O-glycans on different membrane proteins N-Glycans

O-Glycans

GSLs

High mannose type

Hybrid

Complex

N/O

Core 1

Core 3

Core 2

B) N/O glycans drawn on a membrane protein and GSLs

GM3

Core 4

GM2

GM1

GD3

GD2

1)N[ECPU %QTG

0)N[ECPU 56 %QORNGZ V[RG

*KIJ /CPPQUGV[RG

%QTG 56

*[DTKF V[RG

%QTG

56 0*

)CN )NE /CP

#UP 0*

#UP

56

%QTG 

)5.U )/

)/

)CN0#E )NE0#E

0*

#UP

)&C

)6D

)/ )N[EQRTQVGKP

0GW#E (WE

Fig. 3.3 Structures of representative O-/N-glycoproteins and glycolipids. Representative protein N-glycosylation (complex, high-mannose, and hybrid type), mucin-type O-glycosylation, and gangliosides on mammals are described. (a) Each specific structure drawn on each carrier. (b) N-/O-glycans drawn on a membrane protein

(Gal-T1) catalyzes the galactosylation reaction to any terminal GlcNAc residue. Structures of cell surface glycoproteins and glycolipids are illustrated in Fig. 3.3.

3.3

Golgi Traffic

The Golgi traffic models are not unified and differently explained by Golgi researchers. Thus, the consensus model indicates the complexity. The Golgi compartments include cis, medial, and trans cisternae plus the trans-Golgi network (TGN). For more details, the Golgi apparatus contains cisternae structures, ranged between the nucleus and cis-Golgi system, including cis-compartment, medialcompartment, and trans-compartment of Golgi network. All the glycosylation events

3.3 Golgi Traffic

59

are ended with the trans-Golgi system. The Golgi apparatus is kept by the cytoskeletal matrix with microtubules of actin-spectrin network and intermediate filaments. They are different from their structures, Golgi-localizing enzymes, and COPI- or clathrin-mediated vesicle-forming capacity. The filaments and Golgi membranes are linked through membrane proteins or mechanochemical proteins including dynamin, dynein, kinesin, and myosin [14]. The protein transport from ER to each different Golgi compartment is started from the hundreds of releasing sites located in the ER vesicles coated with COPII. In order to incorporate COPII vesicles, coatamer proteins, termed COPs, binds to transporter molecule domain of the cytosolic tail region in the cargo membranes. The ER export signaling proteins belong to type I membrane protein family, which contains the peptide motifs containing diacidic amino acids or di-hydrophobic amino acids. However, GTs are type II transmembrane (TM) proteins and contain proximal amino acids of RK(X)RK sequence near in the TM domain [15]. Soluble proteins are passively or actively exported from the ER [16]. Golgi glycosyltransferases are polarly distributed. In species of mammals to plants, glycosylation-beginning transferases are accumulated in cis-cisternae, whereas late-acting glycosyltransferases are accumulated in trans-cisternae [17]. Golgi structure varies between organisms. COPI vesicles are associated with Golgi structures in eukaryotes. COPI vesicle is an intra-Golgi carrier. Mammalian Golgi is surrounded by thousand more COPI vesicles. COPI is conserved. COPIIcoated vesicles are fused to the specific vesicle, named ER-Golgi intermediate compartment (ERGIC) complex. COPI vesicles are particularly important for retrograde traffic of the vesicles, in way of COPI vesicles in protein recycling from Golgi complex to ER side. The ER proteins escaped or proteins misfolded are reversely retrotransported to the ER region through vesicles coated with COPI. The cis-Golgi system is again fused with ERGICs. Several proteins enhance smooth transports of vesicles. ARF and Sar1 GTPases stimulate the formation of COPI and COPII vesicles, where Sar1-GTP and ARF-GTP proteins associate with the recruited vesicle coat proteins. Consequently, the GTPases of Rab family selectively target vesicles. The remaining protein, SNARE, leads to fused vesicle formation, where vesicles hold vesicle-SNARE, termed v-SNARE, for specific recognition of tethering-SNARE form, termed t-SNARE, and the fused cargo vesicles are moved to specific compartments [18, 19]. From the sequential glycosyltransferase catalysis, the Golgi enzymes are particularly interested in their roles. Glycosyltransferases are sequentially arranged in the Golgi apparatus for their complete synthetic pathway, while early catalyzing enzymes are present in the cis-Golgi complex and intermediately catalyzing enzyme groups are found in medial Golgi complex. The end-catalyzing enzymes are present in the trans-Golgi complex. In addition, the glycosyltransferases present in a certain Golgi compartment are structurally complexed to allow the steady-state location of the Golgi-resident enzymes [20].

60

3.4

3

Glycan Biosynthesis in Eukaryotes

N-glycan Synthesis

The N-glycosylation event is conserved through eukaryotes. N-glycan structures are heterogeneously generated in the pathway and classified for their backbones to three major types of (A) high-Man, (B) hybrid, and (C) complex. N-glycan attachment to candidate Asn residues is carried out through the commonly observed sequence, triamino acid sequon that is the Asn-X-Ser/Thr. “X” means any other amino acid, barring Pro residue. The N-glycans are commonly featured with the core of Manα1,6 (Manα1,3)Manβ1,4GlcNAcβ1,4GlcNAcβ-1-Asn-X-Ser/Thr- with two independent antennae. The Asn-linked core structure is further processed and trimmed to three distinct groups of N-glycans. The three groups consist of oligomannose type, which contains only Man residues, complex type having sialyl LacNAc on each antenna, and a Fuc residue in the Asn-linked GlcNAc. Hybrid type has a combined oligo-Man type and complex type has Manα1,6 and Manα1,3 antennae. The precursor glycan is serially attached to a dolichol pyrophosphate known as lipid carrier in the ER cytoplasmic face. The first monosaccharide of GlcNAc residue is attached to dolichol phosphate, where a phosphate group is transferred and the GlcNAc residue is linked to yield dolichyl pyrophosphate-GlcNAc. The formed dolichyl pyrophosphate-GlcNAc is then further used to generate dolichyl pyrophosphateGlcNAc2Man5. This substrate is translocated to cross the ER lumenal membrane surface by a specific enzyme flippase. Thereafter, Man residues are serially attached, and the Manα1,3 antenna is attached by a triglucosyl Glcα1–2Glcα1–3Glcα1–3cluster. In this stage, dolichol-P-Man and dolichol-P-Glc are used to be used as donor substrates. The final dolichol-linked glycan is Glc3Man9GlcNAc2-. Using this dolichol-linked glycan, oligosaccharyltransferase (OST) in the ER lumen attaches it to Asn residues on target proteins, and the resulting β-N-glycosidic-linked proteins are yielded [21]. This glycan is further subjected to trimming and processing to mature N-glycans. All fungi, metazoans, and plants use the common biosynthetic pathway in the ER lumen, including the trimming and processing of the sugar structures. The ER chaperon in the pathway contributes to precise folding and quality control of glycoproteins, processing by calnexin (CNX) and calreticulin (CRT). During the quality control (QC) of glycoproteins, the triglucosyl residues monitor the level by α-glucosidases I and II. Therefore, the incomplete structure of folded proteins is α-glucosylated by a specific α-glucotransferase to transfer glucose residue to the terminal α-1-2 Man on the α-1,3 mannosyl antenna and further cycled via the calnexin/calreticulin interaction. If still unfolded, glycoproteins fall into the ER-associated degradation (ERAD) machinery to eliminate them through secretory proteasome to ubiquitination in the ER cytosols. Indeed, the N-glycan-bearing glycoproteins disappeared during the ubiquitination, and the remained protein part is digested in proteasomes [22, 23]. Precisely folded glycoproteins are further glycosylated with trimming and finally sialylation. Finally, the three groups of N-glycans are produced in Golgi apparatus via ER-Golgi network. Among them, the Man-6-phosphate (Man-6-P)-carrying N-glycans recognize the Man-6-P receptor that are localized on membranes of endosomes and lysosomes. Then, the

3.5 O-glycosylation and Multiple O-Glycan Structures

61

Man-6-P-glycoproteins are translocated to lysosomes. Each form of peripheral N-glycan is different from each phylum in plants and invertebrates to mammals [22, 23].

3.5

O-glycosylation and Multiple O-Glycan Structures

Monosaccharides of GalNAc, GlcNAc, glucose, galactose, mannose, xylose, fucose, and arabinose are subjected to the O-glycosidic attachment to Ser/Thr of proteins. The well-known reaction is the monosaccharide GalNAc transfer from the UDP-GalNAc to Ser/Thr through an α-glycosidic linkage, and this is catalyzed by GalNAc transferase. The O-glycan synthesis commences with the protein folding process and subunit oligomerization of proteins between the ER and Golgi compartments. O-glycosylation process is sequentially progressed with the pathway assembled by membrane-bound glycosyl-, O-acetyl-, and sulfotransferases. The O-glycosylation pathway is distinct for GTs, sulfotransferases, and O-acetyltransferase, and O-glycan biosynthetic transferases are mainly localized in the Golgi. Although they similarly catalyze, sequence homology is not high between them. All Golgi GTs belong to the type II TM proteins and consist of a membranespanning domain region, a large catalytic domain in C-terminal region and a short cytosolic domain region in N-terminal region of the Golgi lumen. The β-Gal α2,3SA-transferase 1 (ST3Gal-1) catalyzes the sialylation reaction to the core 1 O-glycosylation structures. Core 1 of O-glycosylation structures is further converted to core 2 mucin type of O-glycan structures through addition of GlcNAc residue by a specific enzyme, core 2 β1,6 N-GlcNAc-transferase (C2GnT). The C2GnT enzyme transfers a β1,6-GlcNAc residue using the donor substrate UDP-GlcNAc to the acceptor substrate, core 1 structure. These core 2 O-glycans have merits for further glycosylation and extension as the substrate, which has a poly-lactosamine (LacNAc) sequence. In addition, core 1 β3-Gal-transferase (core 1 β3Gal-T) enzyme synthesizes core 1 mucin-type (and also core 2 type) O-glycan structures, by means of a distinct chaperone named core 1 β3GalT-specific molecular chaperone (COSMC). The COSMC as an ER-resident enzyme, which is also a specific type II transmembrane protein, is required for the core 1 β3Gal-T enzyme folding. Therefore, if the COSMC is not present in the ER, the core 1 β3Gal-T enzyme is proteolyzed through the proteasome [24].

3.5.1

7 Core O-glycan Structures

The seven distinct O-linked glycans are described (Table 3.1), as subclassified through the first monosaccharide residue linked to Ser/Thr residues or hydroxyl Lys (hLys) residue of O-glycoproteins. O-glycosylation is in other word termed “mucin-type glycosylation” through GalNAc α-linkage formation attached to the

62

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Glycan Biosynthesis in Eukaryotes

Table 3.1 O-glycan types found in humans and different mucin-type O-glycans Types (A) O-glycan types Mucin-type GAG O-GlcNAc O-Gal O-Man O-Glc O-Fuc (B) Mucin-type O-glycans Core-1 Core-2 Core-3 Core-4 Core-5 Core-6 Core-7 Core-8

Glycan linkage GalNAcα1-Ser/Thr GlcAβ1,3Galβ1,3Galβ1,4Xylβ-1-Ser on proteoglycans GlcNAcβ-1-Ser/Thr on nucleus and cytosol proteins Glcα1,2
Galβ-1-O-Lys on collagens NeuAcα2,3Galβ1,4GlcNAcβ1,2Manα-1-Ser/Thr on muscular α-Dystroglycan Xylα1,3Xylα1,3
Glcβ-1-Ser on EGF protein domains NeuAcα2,6Galα1,4GlcNAcβ1,3
Fucα-1-Ser/Thr on EGF domains Glcβ1,3Fucα-1-Ser/Thr on thrombospondin (TSR) repeats

Galβ1,3GalNAcα1-Ser/Thr Galβ1,3(GlcNAcβ1,6)GalNAcα1-Ser/Thr in blood cells types GlcNAcβ1,3GalNAcα1-Ser/Thr in colon and salivary tissue GlcNAcβ1,3(GlcNAcβ1,6)GalNAcα1-Ser/Thr GalNAcα1,3GalNAcα1-Ser/Thr GlcNAcβ1,6GalNAcα1-Ser/Thr in ovarian tissue GlcNAcα1,6GalNAcα1-Ser/Thr Galα1,3GalNAcα1-Ser/Thr in bronchial tissue

hydroxyl (OH)-group attached to Ser/Thr residues. The first GalNAc residue is the first transferred sugar catalyzed by GalNAc-transferase and is thus an initiating sugar of mucin-type O-glycans [24]. The mucins belonged to O-glycoproteins with variable number of tandem repeats (VNTR), which are rich in multiple Ser-Thr-Procontaining domains as the O-glycan substitution sites. The O-glycosylation as a mucin-type occurs at mucosal surfaces and protects the mucosal surfaces from infection [2]. The mucin-type O-glycan is the most common type having a GalNAc residue at the reducing end. Currently, eight mucin-type core structures are found with the different saccharide at the second position and saccharide links. In humans, the core 1 to core 6 and core 8 O-glycans are known (Table 3.1) [25, 26]. For O-glycan consensus sites, there is the consensus amino acid region, which is the recognition and attachment sites of the first saccharide in most O-glycoproteins, but not known for O-GlcNAcylation and mucin-type O-glycosylation. However, specific glycosylation sites for O-Glc glycosylation and O-Fuc glycosylation have been suggested for putative consensus sites [27, 28]. In addition, statistic analysis of the known O-GlcNAcylated proteins and mucin-type O-glycans indicated each specific role for each type of glycosylation. The prediction algorithms for the O-GlcNAcylations and mucin-type O-glycans suggest a ruling mechanism. For example, the NetOglyc 3.1 prediction program exhibits possible prediction of 76% of the glycosyl-targeting amino acid sequences and 93% of the nonglycosylated amino acid sequences from the known proteins [29].

3.5 O-glycosylation and Multiple O-Glycan Structures

63

Apart from mucins, some glycoproteins have only mucin-like domains, but not the VNTR domain. The glycosylation of the mucin-like domain is absolutely the same as the mucin-type O-glycoprotein. The mucins contain the linear core structure, named core 1 (Galβ1,3GalNAc-) and core 3 (GlcNAcβ1,3GalNAc-) structures and also branched core 2 structure of the Galβ1,3(GlcNAcβ1,6)GalNAc- and core 4 structure of the GlcNAcβ1,3(GlcNAcβ1,6)GalNAc- sequences. Backbone units of LacNAc types 1 and 2 (Galβ1,3GlcNAc- and Galβ1,4GlcNAc-) are further added for length extension, respectively. The branched I and linear i antigens of Galβ1,4GlcNAcβ1,3Galβ1,4- are also extended. Complex oligosaccharides such as ABO and Lewis blood group structures are such extended O-glycans, and the glycans can be further subjected to sialylation, fucosylation, and sulfation [24, 30]. The detailed mucin-type O-glycans exhibit their diverse structures, as described previously.

3.5.2

Modification of 7 Core O-Glycan Structures

In the seven core carbohydrates, the GalNAcα-1-Ser- or GalNAcα1-Thr-, termed Tn-antigen, and NeuAcα2,6GalNAcα-1-Ser- or NeuAcα2,6GalNAcα-1-Thr-, termed sialyl Tn (STn) antigenic epitopes, are known. The core carbohydrates can be further modified by the LacNAc unit (Galβ1,4GlcNAc). The LacNAc unit is also by a GlcNAcβ1–6 residue or repeated for the same LacNAc units, called polyLacNAcs. These are also linked to the AB(O)H blood group antigenic epitopes and type 2 Lewis antigenic epitopes of LeX, sialyl LeX (SLeX), and LeY. Poly-LacNAcs are found predominantly on core 2 structures of O-glycans. Nonreducing terminal saccharides are GlcNAc, GalNAc, Fuc, and NeuAc (Neu-SA). Gal residue and GlcNAc residue are also frequently sulfated at the carbon C-6 and at the C-3 or C-6, respectively [31]. NeuAc or SA residues are often O-acetyl esterified at the carbons of C-4, C-7, C-8, and C-9 [32]. The UDP-GalNAc to polypeptide GalNActransferases, termed pp-GalNAc-transferases (EC 2.4.1.41), transfer the GalNAc residue using the donor UDP-GalNAc through an GalNAc-α-OH-Ser/Thr residue to produce the mucin-type O-glycosylations. Currently, the pp-GalNAc-Ts comprises 15 members [33, 34]. Currently, approximately 24 pp-GalNAc-Ts enzymes are known in humans [35]. Each pp-GalNAc-Ts overlaps with different specificity and tissue specificity [31, 34]. No consensus sequence is present in pp-GalNAc-T enzymes, and they have their own linking sites. Ser residue and Thr residue only in protein sequences are targeted to glycosylate, because O-glycosylation is indeed a process after folding process. Thus, O-glycosyl attachment sites are mainly oriented to Ser and Thr, and in certain case to Pro residues.

64

3.6

3.6.1

3

Glycan Biosynthesis in Eukaryotes

O-GlcNAcylation, O-Mannosylation, O-β-Glucosylation, O-α-Fucosylation, O-β-Glucosylation, O-β-Galactosylation, C-Glycosylation, and C-Mannosylation O-GlcNAcylation

The other five O-glycan types have one conformation in each structure. For another type of O-glycosylation, O-β-GlcNAc as a single reside type is also linked to hydroxyl group attached to Ser/Thr residue [35], and this type is frequently found in cytoplasmic and nuclear proteins. Hence, it is regarded as a single GlcNAc modification named GlcNAcylation via a β-glycosidic linkage. The frequently occurring O-glycan synthesis is to attach a GlcNAc residue to proteins which are resident in cytoplasm or nucleus. This posttranslational modification replaces phosphorylation because the process is a reversible process by O-GlcNAc transferase and O-GlcNAcase [36]. The common Ser or Thr site for O-β-GlcNAcylation is possibly competitive to phosphorylation event specific for the same OH-groups. The O-phosphorylation and O-β-GlcNAc glycosylation are frequently known for nuclear proteins. The enzymes of O-GlcNAc-transferase (OGT) and N-acetyl-Dglucosaminidase specific for O-linkage cleavage (O-GlcNAcase) transfer and remove the O-GlcNAc residues, which are linked to the O-GlcNAc-bound proteins, respectively. They are conserved in all metazoans. On the other hand, O-GlcNAcylation on the targeted amino acids linked to EGF repeats is completed through enzymatic catalysis of an extracellularly resident O-GlcNAc transferase enzyme [37].

3.6.2

O-Mannosylation

For minor O-glycan forms, O-mannosylation is known, and this type of glycans has Man-αlinked Ser or Thr on proteins in the brain and muscle of metazoans [38]. O-mannosylated glycans are minorly found in glycoproteins in the brains and nerves as well as skeletal muscles. One representative case of the O-mannosylated glycoproteins is α-dystroglycan in dystroproteins in extracellular matrix (ECM) protein in the skeletal muscle [39]. Skeletal muscle glycoprotein, α-dystroglycan, has mainly the O-mannosyl glycans. Most structures are Neu5Acα2,3Galβ1,4GlcNAcβ1,2Manα-Ser/Thr-. This, only the NeuAcα2,3Galβ1,4GlcNAcβ1,2Man- glycan, is reported in humans. Interestingly, in sheep brain, the α-dystroglycan, which contains the Galβ1,4(Fucα1,3) GlcNAcβ1,2Man-glycan, was known [40, 41]. Also, in rat brain, the O-mannosylated glycan of HSO3-3GlcAβ1,3Galβ1,4GlcNAcβ1,2Man form was found [41, 42]. Mammalian GlcNAc-transferase IX functions specifically to the GlcNAcβ1,2Manα1-Ser/Thr substrates, and thus consequently the O-mannosylated

3.6 O-GlcNAcylation, O-Mannosylation, O-β-Glucosylation, O-α-Fucosylation,. . .

65

glycans with 2,6-branches are found in the brain [43], giving the diverse O-mannosylated glycan forms in humans. However, other fucosylated chain and GlcA-3-sulfated chain and branched chain are also related. The fly Drosophila melanogaster O-glycan-synthetic genes contain at least two genes encoding for Omannosyl-transferase (POM-T1 and POM-T2). The O-mannosyl-transferase enzymes are catalytically active when the POM-T1 and POM-T2 genes, which encode mannosyltransferases, are co-expressed [27].

3.6.3

O-β-Glucosylation

In addition, for the rare types, two different glycan types of O-glucosylation and O-fucosylation are present in the EGF-like homology domains (EGF domains). The EGF-repeated domains are attached by O-Fuc, O-Glc, or O-GlcNAc. Extracellular domain glycosylation contains up to 36 tandem EGF repeats, and they regulate Notch signaling pathway. An EGF domain is often a motif found to involve in interaction between protein and protein. The EGF domain repeats contains approximately 30–40 amino acids in length with the conserved 6 Cys residues and 3 S-S bonds. Glc residue is linked to the OH-group in Ser residue of the Cys1-Xaa-SerXaa-P-Cys2, which is the common consensus sequence [27]. O-Glc residue is further linked to one or two α1,3 Xyl linkages, as found in human coagulation factor VII, coagulation actor IX, and protein Z [28, 44]. Currently known O-fucosyl glycoproteins contain a single Fuc-O-linkage glycan. The O-fucosyl glycoproteins include blood coagulation factors VII and XII, tissue plasminogen activator (TPA), and urinary-type plasminogen activator. One exceptional case, coagulation factor IX, consists of Fuc-O linkage to Ser/Thr, and the Fucα1-Ser/Thr is further modified to the longer tetrasaccharide structure of NeuAcα2,6Galβ1,4GlcNAcβ1,3Fucα1-Ser/ Thr-. Currently known O-fucosyl glycosylation observed in EGF domain repeats utilizes the common motif of Cys2-Xaa3-5Ser/The-Cys3 sequence [28]. Another specific type of O-fucosyl glycans is reported. The thrombospondin (TS) type 1 repeats (TSRs) in the human ECM contain disaccharide-bearing O-fucosylated glycans of the Glcβ1,3Fucα1-Ser/Thr- structure [45].

3.6.4

O-α-Fucosylation

The TSR proteins are expressed in the ECM of cells. A TSR protein has about 60 amino acids in length with the conserved Arg, Cys, Ser, and Trp amino acid residues with the putative sequence of WX5CX2/3S/TCX2G [28]. O-β-glucose and O-α-fucose attached to Ser or Thr are also known in the EGF-repeated domains of Notch and Cripto/FRL/Critic proteins. Notch regulator Fringe protein is a β3GlcNAc-transferase that O-GlcNAcylates O-Fuc residue to regulate Notch ligand recognition. The EGF-repeated domain is O-fucosylated by a specific enzyme of

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protein O-fucosyltransferase 1 (Pofut1) in mammals [46]. O-fucosylation seems to need a consensus Cys-Xaa-Ser/Thr-Cys motif present in the domains with the EGF-like repeats. Currently, two different protein-O-fucosyl-transferases, named POFU-TI and POFU-TII enzymes, catalyze the transferring reaction of Fuc residue from the GDP-Fuc as a donor substrate to acceptor substrate [47]. POFU-TI specifically transfers Fuc residue to the EGF-like repeat domains, whereas the POFU-TII is specific for the O-fucosylation reaction of TSRs. As a product, the tetrasaccharides of Neu5Acα2,3/2,6Galβ1,4GlcNAcβ1,3Fucα-O-Ser and Neu5Acα2,3/ 2,6Galβ1,4GlcNAcβ1,3Fucα-O-Thr are known in the examples of some substrates including blood coagulation factor XII, Cripto factor IX, Delta, Notch, Serrate, and thrombospondin type 1 repeats and urokinase, which are all known as carriers of the EGF-like repeat domains. These proteins also contain single O-fucose residue with the tetrasaccharide [47].

3.6.5

O-β-Glucosylation

O-β-glucosylation is also found at the hydroxyl Ser residue or Thr residue attached to the EGF-like repeated regions with the amino acid sequence of Cys-Xaa-Ser-XaaPro-Cys as the conserved consensus sequence but different from the O-fucosylation sites, and this contains a trisaccharide with two Xyl residues (Xylα1,3Xylα1,3Glcβ-O-). Apart from O-Glc, the EGF repeats are also recipient to receive O-fucose or O-GlcNAc. Several EGF-like repeated domains also have single O-β-Glc attachment in only specialized glycoproteins including factor VII, factor IX, and Notch proteins [48]. Protein O-glucosyltransferase (Poglut), which is a CAP10-like protein, catalyzes the O-glucosylation reaction to the Ser residue present in the common conserved sequences of EGF-repeated region [49]. Deficiency in the mice Poglut gene exhibits embryonic lethal effect, which exhibits the Notch-like phenotype [50]. O-glucose residue is attached to the terminal Xyl residue in the trisaccharyl Xylα1,3Xylα1,3Glcβ1-O-EGF [51]. To catalyze the xylosylation reaction in humans, two UDP-xylose to glucoside α3-xylosyltransferase genes of GXYLT1 and GXYLT2 [52], as well as an ER-localized UDP-xylose to xyloside α3xylosyltransferase gene of XXYLT1, are identified [53]. Unfortunately, the function of O-glucosylation in Notch signaling is not yet elucidated, although the Notch signaling determines cell fate during development and uncontrolled regulation of the Notch signaling is associated with various human diseases. EGF repeats, small domains composed of at least 40 amino acids, are present on cell surfaces and extracellularly secreted in metazoans. It has six Cys residues with three S-S disulfide bonds to form a characteristic three-dimensional structure. Fringe uses folded EGF repeats as a substrate for GlcNAc attachment [54], indicating the glycosyltransferase recognition of the three-dimensional confirmation of EGF-repeated domains. Fringe has O-fucose specificity on some EGF repeats. Mouse Notch1 has the specific 16 amino acid sites modified with O-Glc trisaccharide [55]. The known 17th

3.6 O-GlcNAcylation, O-Mannosylation, O-β-Glucosylation, O-α-Fucosylation,. . .

67

O-glucosylation site present in the EGF9 is the consensus amino acid motif with CASAAC sequence, indicating Ala residue replacement of Pro residue in the consensus sequence. However, bacteria do not transfer the O-glucose glycans, concluding their restricted role in eukaryotes [56].

3.6.6

O-β-Galactosylation

O-β-galactosylation is also known for collagen, forming the hydroxy-Lys and hydroxy-Pro in the collagen with the disaccharide Glcα1,2Galβ-O-Hydroxyl-Lys/ Hydroxyl-Pro attachment [57]. Because collagen protein structure is a trimeric form, which is composed of three left-handed alpha chains with the Gly-X-Y repeats. Hydroxy-proline (hPro) and hLys residues are major amino acids at X and Y positions. Certain hLys is attached by galactose and glucose-galactose units. Collagen glycosylation occurs in the ER before triple-helix formation by β1-O-galactosyland α1,2glucosyltransferase enzymes. O-Galactosylation is present only on collagen proteins. Gal residue or Glcα1,2Gal disaccharide binds to the modified amino acid hLys residue in collagen proteins with covalent linkages [58, 59].

3.6.7

C-Mannosylation and C-Glycosylation

Finally, a very unique type of C-mannosylation is known, and this attaches a single Man residue to the Trp indole ring via a C-linkage in most of eukaryotes, except for yeasts. A specific glycan linkage is formed between a carbohydrate and a protein, and the linkage has been known as C-glycosylation that occurs at a specific Try residue in human RNase Us type [60]. The reason why it has been termed C-glycosylation is the saccharide linkage with the protein via a carbon-carbon bond. Apart from regular protein glycosylation, C-mannosylation of tryptophan residues is unique between mannose and protein. Not for the classical O-glycan type or N-glycan type, a C-C bond is the characteristic site of a single Man attachment. C-mannosylation event is therefore very specific among various glycosylation events of proteins, which differs from types of glycosylation. It involves in a covalent linkage for addition of an α-Man residue to the Trp indole C-2 carbon position via a C-C link. C-mannosylation of tryptophan residues is also an ER-localized catalysis reaction using the donor substrate, Dol-P-Man. The Dol-P sugars are general substrates for several ER mannosyl- and glucosyltransferases for N- and O-glycosylation and mannosyltransferases involved in GPI-anchored biosynthesis. The dolichol-diphosphate (Dol-P-P) oligosaccharide is also a substrate of OSTs during N-glycan synthesis [61]. A conserved amino acid W-X-X-W motif bears a Man residue linked to the initial Trp residue [62, 63], in the MUC5AC and MUC5B CYS domains. The dolichol-phosphate-Man is used as the donor substrate [62]. C-mannosylation contributes to the protein folding process. C-Man linkage is

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particularly identified in thrombospondin (TSN) type 1 repeats and the WSXWS motif present in cytokine receptors of type I. The C-mannosylase gene is not sure, although Caenorhabditis elegans DPY-19 has been suggested to be a C-mannosyltransferase [61]. The DPY-19-coding gene has sequential and topological homologies with the N-glycan OST. This indicates some evolution-based C-glycosylation creation from the house-keeping N-glycosylation. C. elegans receptor proteins of MIG-21 and UNC-5 have been suggested to be acceptor substrates of DPY-19 enzyme to secret soluble MIG-21.

3.7

Function of O-Glycosylation and O-Glycans

Regarding the roles of O-glycoproteins in cells, O-glycan-containing glycoproteins contribute to, like N-glycan-containing glycoproteins, protein structure and protein stability, immunity, nonspecific protein recognitions, receptor signaling, enzyme activity, protease resistance, and cell survival [31]. The functional roles of glycans in proteins or lipids are known to broadly involve in the bigger spectra including development, differentiation, growth, cell function, or survival of organisms. Additionally, O-glycans of mucin-type easily interact with water molecules compared to other types of glycans. Mucins with a heavy glycosylation in proteins, called mucintype O-glycan structure, are present in cell surfaces located on the genital, digestive, and respiratory tracts. The mucins also carry clustered sialylglycans with a strong negative charge affordable to hold water and form mucus. The gel-like sticky and hydrophilic molecules present in nasal secretions are basically composed of secreted MUC2 polypeptides. For the fundamental role of the mucin-type O-glycosylations, protective function of the cells is suggested from bacterial invasions, allowing protection of the cells from bacterial attacks [64]. Similar to O-glycans of mucin type, another polymeric GAGs also show such negative charges from the sulfate group. Another function of O-glycans is to recognize and interact between each protein. Carbohydrate structures are used as substrates for nonenzymatic carbohydrate-binding proteins, named lectins. Upon lectin binding, glycans influence the function and fate of the target proteins. Most of glycan recognition of glycoproteins are ubiquitously taken place in the cellular system. Representatively, selectins and galectins are well characterized to bind to carbohydrate epitopes. Consequently, their recognition to carbohydrate ligands induces cellular events such as apoptosis, signaling, endocytosis, proliferation, cell-to-cell recognition, ECM-cell recognition, ECM assembly, fertilization, and differentiation [65]. In addition, O-mannosylated glycans with sialylation play crucial roles in interaction with ligands for complex of laminin and dystroglycan. The interaction leads to functionality in development of the brain and muscle [41]. Furthermore, O-glycans specifically recognize immunologic antigens in mammals. The O-linked glycosylation therefore affects the signaling molecules such as hormones and cytokines, and certain enzymes, although the influence is not very strong but rather finely regulates them. O-glycans of mucin type decrease the biological activity of cytokine IL-5 [66],

3.8 Glycosaminoglycans (GAGs)

69

but they activate lactase phlorizin hydrolase enzyme of humans [67]. The change in glycan structures also specifically influences the signaling molecules. O-fucosylation in urinary-type plasminogen activator (uTPA) activates the uTPA receptor. Additionally, the O-fucosylations are needed for proper Notch function, as described previously [68]. Also, the O-GlcNAc turnover modification is important for signaling pathways of many different biological phenomena including transcriptional regulation of gene expression, protein degradation via proteasome, and insulin-receptor signaling by competing with phosphorylation. O-GlcNAc modulates neutrophil motility in DCs and macrophages [69]. O-glycans are also required for the protein expression, as found in glycophorin A with a heavy glycosylation. The O-glycans are present on the surfaces of human erythrocytes [70]. The O-glycans influence protein modification and proteolytic processing. Representatively, insulin-like GF-II protein is degraded into the matured IGF-II as a functional form, when amino acid Thr-75 residue is specifically O-glycosylated [71].

3.8

Glycosaminoglycans (GAGs)

Proteoglycans are highly glycosylated proteins, which contain core protein part and GAG sequences with one or more repeats. GAGs and proteoglycans (PGs) constitute the ECM at the cell surfaces. The attached GAG chain number is diverse. GAGs are a different type distinct from common O-glycosylation types and structurally diverse. GAGs are linear, unbranched, and heterogeneous sulfated glycans with negatively charged heteropolysaccharides found in every mammalian tissue [72]. Because sulfotransferases synthesize sulfated GAGs’ side chains present in proteoglycans, GAGs have monosaccharide type and sulfation specificity. The GAG backbones consist of repeated disaccharide units with alternated uronic acid (UA) residue and hexosamine residue units. GAGs have diversity through sulfated reaction and GlcA residue epimerization to iduronic acids. The UA-repeated units are composed of β-D-GlcA or the epimerized form of β-D-GlcA C-5, termed α-liduronic acid (IdoA). In the GAGs, the amino sugar residues are forms of (i) Glc-derived α-D-glucosamine (GlcN)or β-D-GlcN and also (ii) Gal-derived GalNAc residue. The permutation events of the monosaccharides, which are characteristically found in the GAG-repeating units, generate distinct GAG structures. The generated GAG structures include the glucosamine-carrying heparan sulfate (HS) and heparan phosphate (Hp), and the GalNAc-containing dermatan sulfate (DS) and chondroitin sulfate (CS). Meanwhile, another type of GAGs, keratan sulfate (KS), alternates GlcNAc with Gal but does not bear UA. Hyaluronan known as HA alternates GlcNAc with GlcA but does not bear a core protein. GAGs consist of alternated UAs and N-acetylated hexosamine with GlcNAc or GalNAc residue, which is combined with GlcA residue or Gal residue. The GAG linkage tetrasaccharide is the GluAβ1,3Galβ1,3Galβ1,4Xylβ-O-serine, where the repeated disaccharide unit of GAG is generated through the common structure

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with tetrasaccharide of GlcAβ1,3Galβ1,3Galβ1,4Xylβ1-O-Ser sequence and affected by the GTs for the linkage synthesis (Fig. 3.1). The sqv gene products of GAG synthesis (e.g.,, HS or CS) are resident in Golgi apparatus. SQV-1 gene encodes the UDP-GlcA decarboxylase for the UDP-xylose genesis functioning as a first nucleotide sugar donor in GAG synthesis. SQV-6 encodes for xylosyltransferase, SQV-3 for galactosyltransferase I, SQV-2 for galactosyltransferase II, and SQV-8 for glucuronosyltransferase I. SQV-4 gene encodes for the enzyme UDP-Glc dehydrogenase to synthesize UDP-GlcA in the cytoplasm. SQV-7 gene encodes for nucleotide sugar transporter protein that transports UDP-GlcA in Golgi apparatus into the lumen and also translocates UDP-Gal residue and UDP-GalNAc residue. Glucuronosyltransferase (GlcAT-II) and N-acetylgalactosaminyltransferase (GalNAcT-II) act as the acting enzymes for CS chain elongation. N-acetylgalactosaminyltransferase I (GlcNAcT-II) acts in HS synthesis. N-acetylglucosaminyltransferase I (GalNAcT-1) functions in CS chain initiation. Mutations in CS GAG synthesis lead to human diseases such as connective tissue disorder, Ehlers-Danlos syndrome, and hereditary multiple exostoses, which causes inappropriate chondrocyte proliferation and bone growth. Also, defects in HS synthesis cause disorders related to intracellular signaling, Wingless, Hedgehog, and fibroblast grow factor pathways. Defects in GAG synthesis caused animal development abnormality and human disease. For example, mutation in Drosophila HS GAG-synthetic enzymes causes developmental defect. GAGs carry sulfated or non-sulfated monosaccharides. Deacetylated and N-sulfated GlcNAc residues are also found. Most hexosamine units are acetylated and present as GlcA-containing disaccharide repeats. Deacetyl form of hexosamine units are regularly sulfated by sulfonylation enzymes, and they are present as iduronate-containing disaccharide repeats. The ionic carboxylate and sulfate groups in GAGs attract water. GAGs covalently bound to a core protein are PGs or in some case of free chains, HA, or hyaluronan.

3.8.1

Classification and Biosynthesis of GAGs

GAGs are categorized into six classes with hyaluronan (or hyaluronic acid) (HA; GlcA and GlcNAc), DS (iduronic acid or GlcA and GalNAc) and CS (GlcA and GalNAc), heparin, HS (iduronic acid or GlcA and GlcNAc), and keratan sulfate (KS; Gal and GlcNAc) [73]. From structural basis of the disaccharide repeats, GAGs are further classified into three types of (i) DS and CS (GlcA+GalNAc), (ii) heparin-HS (GlcA+GlcNAc), and (iii) KS (Gal+GlcNAc). The epimerization form of GlcA in DS and heparin-HS is called “iduronate.” GAG structures are heterogeneous due to O-sulfation [74]. Heparin is a highly sulfated GAG, whereas HS is sulfated only in a certain region [75]. Sulfated GAG forms are generated at the region of Golgi apparatus with modification by O-sulfotransferases [76]. The biosynthesis of GAGs is quite different from those of other O-glycans, because all the transferases are specific for GAGs,

3.8 Glycosaminoglycans (GAGs)

71

only except for the chondroitin 6-O-sulfotransferase, KS Gal-6-O-sulfotransferase, and GlcNAc 6-O-sulfotransferase, which transfers sulfate group to other LacNAc extensions [77]. CS and DS as well as heparin and HS chains are generated from the known common linker tetrasaccharide of the GlcAβ1,3Galβ1,3Galβ1,4Xyl-structure. GAG chains are assembled in ER-Golgi apparatus. GAG-protein-linked sequence is the GlcAβ1,3Galβ1,3Galβ1,4Xylβ1-O-Ser structure as an initiating unit. The tetrasaccharide in the linkage site is assembled by transfer of a Xyl, 2 Gal, and a GlcA residues by Xyl-T, β1,4GalT-I, β1,3Gal-T II, and β1,3glucuronyl-T I [78], respectively. CS and DS GAGs are generated through the GalNAc linkage, while heparin and HS are generated if GlcNAc residue is added at the first reaction [74]. Chondroitin GalNAc transferase-I, GalNAc transferase-II, and chondroitin synthetase initiate the CS/DS GAG chain synthesis with the first few GalNAc residues. However, chondroitin synthetase functions as a co-polymerase to elongate the CS/DS forms, having the multiple (GalNAcβ1,4GlcAβ1,3)n structure [78, 79]. HA has no core protein and is synthesized at the extracellular surface functioning in tissue freshness and cancer growth. KS-type GAG belongs to the sole GAG species that lacks uronic acid but bears Gal residue in the KS disaccharide units. On the other hand, HA species belongs to non-sulfated KS forms, which are attached to substrate proteins in a N-glycosylation form or core 1 O-glycosylation form. GAG species have a different chain number specificity. One chained GAG, decorin, is known as a small Leu-rich proteoglycan (SLRP). GAG having 100 or more saccharide chains is known for aggrecan [76].

3.8.2

Chondroitin Sulfate (CS)

All the CS-synthetic enzymes are well explained to generate diverse structural formation of CS chains. CS-associated developmental and pathophysiological processes include CS-recognizing molecules with CS receptors. CS is a sulfated GAG as a linear polysaccharide with disaccharide unit repeats of uronic acid and HexNAc. CS-attached PGs (CSPGs) have at least one side chain. CSPGs are involved in cytokinesis, morphogenesis, and neuronal plasticity, skeletal diseases, formation of glial scars, and pathogenic invasions such as bacteria and viruses [74, 80]. Disaccharide unit is [(–4GlcAβ1–3GalNAcβ1–)n] as galactosaminoglycan (Fig. 3.4). In C. elegans, ChSy gene is the orthologue of human ChSy. ChSy family is a group of glycosyltransferases that conduct polymerization of chondroitin sulfate chain through GalNAc transferase-II and GlcA transferase-II activities. Unlike human type, ChSy of C. elegans has not only GalNAc transferase activity but also GalNAc transferase-I activity. Normally, the GalNAc transferase-I activity can be found in ChGn genes, which produce CS chain by addition of GalNAc to tetrasaccharide linker of core protein. That means C. elegans ChSy is indispensable for production of chondroitin proteoglycans. For the roles of chondroitin proteoglycans in C. elegans, knockdown of ChSy was established by soaking in a

72

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Glycan Biosynthesis in Eukaryotes

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Fig. 3.4 GAG structures and glycosyltransferases involved in GAG synthesis. Synthesis of GAG linkage tetrasaccharide, GluAβ1,3Galβ1,3Galβ1,4Xylβ-O-serine, and different GAGs in C. elegans is described. HS, KS, DS, and HA GAG chains as the typical disaccharide units are described. CS-repeated units are GalNAc residue and GlcA residue. DS is a CS stereoisomer having an IdoA residue instead of GlcA. HS repeat units are GlcNAc residue and GlcA residue. The saccharide residues are esterified by sulfate, while HA belongs to a linear polymer with the repeated disaccharides of 4GlcAβ1,3GlcNAcβ1-units. Glycosyltransferases involved in GAG synthesis include (i) GlcAT-II, which is a glucuronosyltransferase, and GalNAcT-II, which is a N-acetylgalactosaminyltransferase for chondroitin chain elongation, (ii) GlcNAcT-II (N-acetylgalactosaminyltransferase I) for heparan sulfate synthesis, and (iii) GalNAcT-1 (N-acetylglucosaminyltransferase II) for chondroitin chain initiation

double-stranded siRNA solution or feeding with E. coli, which produce a doublestranded form of siRNA. As expected, these ChSy knockdown C. elegans show a decrease of chondroitin. Interestingly, ChSy-null strains exhibit abnormal embryogenesis in C. elegans with incomplete mitosis and reversion of cytokinesis. Although zygotes undergo mitosis, cytoplasms are not perfectly separated due to mal-cytokinesis. Nonetheless, nucleus is divided to produce multinucleated cells, leading to embryo death and abnormal reproductivity. In terms of CS or HS, similar phenotypes are observed in blocking tetrasaccharide biosynthesis due to incomplete chondroitin sulfate chain synthesis. But blocking of heparan sulfate chain synthesis does not display any morphological change [81]. Therefore, chondroitin proteoglycans are responsible for cytokinesis of C. elegans embryos, but without detailed mechanism of chondroitin proteoglycans in cytokinesis. Using tetrasaccharide-O-Ser/protein, GalNAcT-I enzyme catalyzes the attachment of GalNAc residue to the GlcA residue terminally present in nonreducing end to form chondroitin stem [74]. The disaccharide GlcA-GalNAc repeats of CS are formed by the alternative catalysis of GlcA residue and GalNAc residue through GlcAT-II and GalNAcT-II enzymatic transfers, respectively. The tetrasaccharide unit is also used for another sulfated GAG, HS which has disaccharide repeats of (–4GlcAβ1,4GlcNAcα1–)n as glucosaminoglycan. Therefore, the alternate GlcNAc

3.8 Glycosaminoglycans (GAGs)

73

forms the HS. Hence, CS and HS chain synthesis needs the first HexNAc addition [74]. To sulfate the chondroitin backbone, sulfotransferases add sulfate group at the Glc-A C-2/GalNAc C-4/GalNAc C-6, forming the disaccharide A, C, D, E, and O units. In addition, the GlcA epimerization to IdoA residue is catalyzed by 2 GlcA C-5 epimerases called DS-epi-1 and DS-epi-2 enzymes [82, 83]. The epimerases convert CS into its stereoisomer, DS, which carries disaccharide repeats of iA, iB, and iE. To date, six glycosyltransferase genes for chondroitin biosynthesis of the disaccharide repeats [(–4GlcAβ1–3GalNAcβ1–)n] were isolated for chondroitin synthase (ChSy)-1, ChSy-2, ChSy-3, chondroitin GalNAcT (ChGn)-1, ChGn-2, and chondroitin-polymerizing factor (ChPF). The three ChSy-1, ChSy-2, and ChSy-3 enzymes bear bifunctional glycosyltransferase enzymes known as GlcAT-II and GalNAcT-II. Of interests, co-expression of two proteins among the four ChPF, ChSy-1, ChSy-2, and ChSy-3 proteins upregulates the activity levels of GlcAT-II and GalNAcT-II enzymes, increasing chain lengths. Thus, chondroitin length is formed by the synthesizing enzyme complexes of chondroitin polymerases as well as their combinations of four proteins including ChPF, ChSy-1, ChSy-2, and ChSy-3. However, ChGn-1 and ChGn-2 bear both enzyme activities of GalNAcTI and GalNAcT-II, catalyzing initiation and elongation of chain synthesis [74].

3.8.2.1

Sulfotransferases Sulfate CS/DS Chains

Seven sulfotransferases sulfate CS and DS chains [84] utilizing the donor substrate 30 -phosphoadenosine-50 -phosphosulfate (PAPS) to supply GlcA, GalNAc, or IdoA in CS and DS GAGs as acceptors. In sulfation of CS units, the common acceptor substrate is an O group saccharide with non-sulfation. The monosulfated A and C units are generated through sulfation reaction at GalNAc 4-O/6-O positions, respectively. Further additional sulfation reaction of A and C saccharides yields disulfated D and E saccharides, respectively. Hence, the sulfo-transferring reaction of chondroitin unit has the two different “4-O/6-O-sulfation” reactions at the initial step. GalNAc-4-O-sylation in CS and DS sugar chains is performed by specific enzymes of three forms, named chondroitin 4-O-sulfotransferase (C4ST)-1, C4ST-2, and C4ST-3, for the CS, while dermatan 4-O-sulfotransferase (D4ST)-1 yields the GalNAc 4-O-sulfation next to IdoA in DS. Therefore, it is clear that the specific enzymes of C4STs and D4ST-1 yield the specific products, A and iA saccharide units, respectively [74]. Chondroitin 6-O-sulfotransferase-1 (C6ST-1) sulfates to the carbon C-6 of GalNAc residue present in C and D units in CS, but not DS. Two UST and GalNAc-4-sulfate 6-O-sulfotransferase (GalNAc-4S-6ST) make the specific disulfate-containing disaccharides linked to CS/DS chains. Uronyl 2-Osulfotransferase (UST) enzyme performs the GlcA 2-O-sulfation reaction in the IdoA and C unit present in the iA unit, reducing the levels of iB chain and D chain, respectively. During E/iE chain synthesis, a specific enzyme, GalNAc-4S6ST, catalyzes the sulfation reaction to the 4-O-sulfated 6-O position of GalNAc in

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the A/iA units, which are generated by enzymes of C4-ST and D4-ST. Human C6ST-1 (CHST3) mutation causes a genetic disorder named spondyloepiphyseal dysplasia (SED) known as an Omani-type disease, as a chondrodysplasia disease. CS, which is 6-O-sulfated by sulfotransferase, C6ST-1 catalysis, is important for development and formation of skeletal bones in humans. In mice-deficient model, mice C4ST-1 (Chst11) mutation also causes chondrodysplasia with neonatal lethality. Human D4ST-1 (CHST14) mutation causes genetic disorders called adducted thumb-clubfoot syndrome (ATCS) and Ehlers-Danlos syndrome (EDS), which are the EDS Kosho-type (EDSKT) and kyphoscoliosis-type EDS as musculocontractural EDS (MCEDS) [74]. The D4ST-1 mutation yields DS deficiency and a hypersynthesis of CS chains. Chondroitin polymerization is performed by the constituent chondroitin polymerases. In the type 1 herpes simplex virus-resistant cells, which were originated from mouse fibroblasts, two mutant cells named gro2C and sog9 were established. The gro2C is HS-deficient due to lack of Ext1 for HS synthesis [84], whereas the sog9 is CS-deficient due to C4ST-1 mutation. CS chain is important in pathogenic infection. For example, deficiency in ER nucleotide sugar transporter gene SLC35D1 (solute carrier 35D1) shortens CS chains with skeletal dysplasia. CS elongation increases binding potential to atherogenic lipids and arteriosclerosis development [85]. In fact, the abnormal CS moieties with long chains generated by C4ST-1 and ChGn-2 are found in the atherosclerosis. TGF-β, EGF, and PDGF induce CS chain elongation. ChGn-1 initiates CS synthesis with an increased chain number. ChGn-1 enzyme aberrantly expressed in chondrosarcoma cells synthesizes a CSPG named aggrecan with multiple CS chains. ChGn-1 enzyme initially synthesizes the CS chains through enzymatic catalysis of the first GalNAc transfer to the acceptor chain of tetrasaccharide. The GalNAc (4-O-sulfate)-linked pentasaccharide carbohydrate located in the nonreducing end is preferably used as the substrate for chondroitin polymerases. Then, the CS chain number is gradually increased. ChGn1 also cooperatively acts with the C4ST-2 enzyme and consequently increases the number of CS chains. Xyl residues are frequently 2-O-phosphorylated in both HS and CS chains. GlcAT-I enzyme catalyzes the transfer reaction of GlcA residue to the phospho-trisaccharide-Ser of the Galβ1,3Galβ1,4Xyl-2-O-P-β1-O-Ser. In proteoglycan decorin, transferring event of GlcA residue is coupled to enzymatic dephosphorylation reaction of the 2-O-phosphorylated Xyl residue by phosphatase. The Xyl phosphorylation reaction is important for complete linkage in the tetrasaccharide-bearing CS and HS chains. Unlike the 2-O-phosphorylation, the Gal sulfation of the linkage is detected only in CS and DS. The sulfate groups on the Gal help the CS-selective assembly on the tetrasaccharide linkage region [86]. Gal C-4 and C-6 sulfations influence GalNAcT-I enzyme activity of ChGn-1. Gal sulfation regulates the CS chain initiation, as C6ST-1 catalyzes the Gal C-6 sulfation.

3.8 Glycosaminoglycans (GAGs)

3.8.2.2

75

Chain Termination in CS Chains

In chain termination of CS, the nonreducing GalNAc residue is 4,6-O-disulfated, and this disulfation reaction is a terminator in CS chain elongation. In rat CSPG aggrecan, CS biosynthesis terminates with sulfated GalNAc in the structure of GalNAc residue with 4,6-O-disulfation. In human aggrecan CS, the level of 4,6-Odisulfated GalNAc is increased. Because the mammalian GalNAc4S-6ST enzyme generates disaccharide E/iE chain units and the non-reduced 4,6-O-disulfonyl GalNAc residue end present in the CS/DS [87], it involves in the chain termination. In GalNAc4S-6ST-deficine mice, the nonreducing terminal sugar structures are absent. Moreover, mast cells derived from the bone marrow, which was isolated from the deficient mice, synthesize longer chains of CS than those produced from mast cells derived from wild-type mice. EXTL2 acts as an inhibitor of GAG biosynthesis where Xyl kinase (FAM20B) enhances the GAG synthesis. The EXTL2 regulation of GAG biosynthesis is a type of “quality control” process appeared in HSPGs and CSPGs. EXTL2-type enzyme is an enzyme produced by the three different EXT-like genes. EXTL2 is an α1,4-HexNAc-T enzyme possessing both α-GalNAc-T and GlcNAc-T-I activities, which transfer α-GalNAc/α-GlcNAc residues to the acceptor of tetrasaccharide chain part, respectively. A product of pentasaccharide-Ser, which is generated by α-GalNAc-T enzyme, has a structure of GalNAcα1,4GlcAβ1,3Galβ1,3Galβ1,4Xylβ1-O-Ser, and this pentasaccharide is not used for an acceptor substrate in CS chain synthesis, because the binding α-GalNAc in the site of β-GalNAc inhibits the continued elongation of CS chains. The GalNAcα capping is not found in natural GAGs. EXTL2 transfers the GlcNAc residue to a phosphoryl tetrasaccharide chain. The phospho-pentasaccharide produced cannot be used for an acceptor substrate in HS or CS polymerization. For O-sulfate-transferring reaction to nonreducing GlcA end of the chain, cellsurfaced thrombomodulin (TM) is detected as both forms of CSPG (βTM) and non-PG form (αTM). αTM is not substituted by CS, hence named “part-time” PG [88]. Interestingly, two differently prepared αTM forms of the recombinant αTM expressed by αTM gene-transfected CHO cells and the human urine-purified αTM exhibit the tetrasaccharide unmodified linked to the GlcAβ1,3Galβ1,3Galβ1,4Xylstructure with GlcA 3-O-sulfation present in the nonreducing end. Human natural killer-1 (HNK-1)-specific MAb binds to the αTM. HNK-1 sulfotransferase (HNK-1ST) catalyzes the transfer of the sulfate group via the 3-O-sulfation reaction to GlcA residue of the nonreducing end in the HNK-1 glycan epitope, and thus, a unique HNK-1 epitope is generated. HNK-ST suppresses CS substitution on TM, and the CS constituents reduce anticoagulant activity of αTM. HNK-1-posessing TM is not used for chondroitin polymerases of ChSy-1 and ChPF. Thus, the terminal GlcA 3-O-sulfation of the chain structure catalyzed by the specific enzyme of HNK-first is used as a blocking mark of the CS substitution in TM. Among several GAG species, a representative chondroitin synthesis has been in part studied in human and C. elegans with regard to its role in developments.

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%J5[

%JQPFTQKVKP RTQVGQIN[ECPU %J5[%JQPFTQKVKPU[PVJCUG )CN0#E )NE# )CN :[N

C. elegans 2RF\WHV

9KNFV[RG

2RF\WHV

'ODT[QU %JQPFTQKVKPFGRNGVKQP

Y%J5[40#K

,QFRPSOHWH F\WRNLQHVLV

&HOOGHDWK

0XOWLQXFOHDWHGFHOOV

Fig. 3.5 C. elegans chondroitin synthase disruption by RNAi knockdown technology blocks the oocyte growth and the developmental stage of 2-cell to 4-cell stage. Adopted from reference No [89]. Chondroitin glycans are key component of embryo development

Chondroitin species are enzymatically synthesized by multiple enzyme genes encoding for chondroitin synthase enzymes. Especially, the detailed enzymatic characterization has been studied in C. elegans. C. elegans chondroitin synthases have been disrupted by a RNAi technique of knockdown. The resultant strains exhibited the defectively reduced oocyte proliferation mainly caused by blocking the developmental stages during two cells to four cells [89]. Hence, it has been concluded that chondroitin glycans are essential for embryo development. However, the current status of the functional roles of the chondroitin carbohydrates is still not well elucidated in the molecular level. Several experiments to demonstrate the synthetic pathway of the chondroitins are partially performed in several laboratories. In C. elegans, chondroitin-synthetic enzyme genes reported include the genes of sqv-1 to sqv-8. These genes seem to be crucial for early embryonic development process and postembryonic vulval morphogenesis, as adopted from the recent study [89] (Fig. 3.5). From eight sqv genes, SQV-4 gene encodes a UDP-Glc dehydrogenase enzyme, which synthesizes UDP-GlcA in the cytoplasm. SQV-7 encodes a sugar nucleotide transporter, which transports UDP-GlcA synthesized at the cytoplasm to the lumen of Golgi apparatus. This also transports the UDP-Gal and UDP-GalNAc at the cytoplasm to Golgi lumen. For the roles in Golgi apparatus,

3.8 Glycosaminoglycans (GAGs)

77

Cytosol

SQV-/-

Golgi lumen SQV-6

Heparan Sulphate SQV-4

SQV-1

SQV-3 GlcAT GlcNAcT-II

SQV-7

…

SQV-2

GlcAT-I SQV-8 Identity 37% human CS synthase

SQV-5 GalNAcT 37% Drosophila CG9220

20% human GalNAcT-I

GlcAT-II

SQV-4 : UDP-glucose dehydrogenase SQV-7 : nucleotide-sugar transporter SQV-1 : decarboxylase for UDP-GlcA SQV-6 : xylosyltransferase SQV-3 : galactosyltransferase I SQV-2 : galactosyltransferase II SQV-8 : glucuronosyltransferase I

Chondroitin …

Glucose

Glucuronic acid

Galactose

UDP

GalNAc

GlcNAc

Xylose

Forming fluid-filled extracellular space

Protein core

Fig. 3.6 C. elegans chondroitin glycan is essential for the oocyte growth and the developmental stage. Adopted from reference No [81, 89]. In C. elegans, chondroitin-synthetic enzyme genes are known for the sqv-1 to sqv-8 and crucial for embryo development with postembryo vulval morphogenesis. From 8 sqv genes, SQV-4 is a cytoplasmic UDP-Glc dehydrogenase. SQV-7 encodes a sugar nucleotide transporter protein. SQV-1 encodes a UDP-GlcA decarboxylase enzyme to generate UDP-Xyl in Golgi apparatus. SQV-3, SQV-6, SqV-2, and SqV-8 denote for Gal-transferase, Xyl-transferase, Gal-transferase II, and glucuronosyltransferase I enzymes

SQV-1 is a UDP-GlcA decarboxylase, and it synthesizes UDP-Xyl that is the first nucleotide sugar donor for GAG synthesis (Fig. 3.6). Next, SQV-6 is a Xyl-transferase and SQV-3 is a Gal-transferase. SQV-2 is Gal-transferase II and SQV-8 glucuronosyltransferase I (Table 3.2).

3.8.2.3

Sulfated CS Function as Pathogen Receptor and Co-receptor as Well as CS Binds to Advanced Glycation End-Product Receptor in Metastasis

Various CS chains can directly recognize a variety of molecules. Among CS chains, HS chains, and sulfated heparin, the sulfated CS chains are particularly important for their binding capacity [74]. Heparin pentasaccharide binds to antithrombin [72]. Spatial distribution of negative charges attached to repeated saccharide units present in CS chains is important, as known for HS/heparin recognition to FGFR and FGFR [72]. CS oligosaccharides interact with a pleiotrophin (PTN) known as heparinbinding growth factor [90]. Therefore, certain pathogens including bacteria, parasites, and viruses utilize CS saccharide chains for attachment and infection. For

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Table 3.2 CS tetrasaccharide chain linkage- and repeated disaccharide chain-synthesizing enzymes and catabolizing enzymes in humans. Sqv genes biosynthesize GAG of HS and chondroitin in Golgi apparatus and GAG linkage tetrasaccharide, GluAβ1,3Galβ1,3Galβ1,4Xylβ-O-serine. SQV-1, UDP-GlcA decarboxylase; SQV-6, xylosyltransferase; SQV-3, galactosyltransferase I; SQV-2, galactosyltransferase II; SQV-8, glucuronosyltransferase I; SQV-4, UDP-glucose dehydrogenase for UDP-GlcA formation; SQV-7, nucleotide sugar transporter for UDP-GlcA transport to Golgi apparatus lumen as well as UDP-Gal and UDP-GalNAc to Golgi. GlcAT-II (glucuronosyltransferase) and GalNAcT-II (N-acetylgalactosaminyltransferase) are used for chondroitin chain elongation. GlcNAcT-II (N-acetylgalactosaminyltransferase I) is used for HS synthesis. GalNAcT-1(N-acetylglucosaminyltransferase II) involves in chondroitin chain initiation Enzymes In tetrasaccharide linkage Xylosyl-T β1,4-Galactosyl-T-I β1,3-Galactosyl-T-II β1,3-Glucuronyl-T-I In repeated disaccharide Chondroitin synthase

Chondroitin-polymerizing factor Chondroitin GalNAc-T

Abbreviation

Gene symbols

Chromosomal loci

XylT GalT-I GalT-II GlcAT-I

XYLT XYLT2 B4GALT7 B3GALT6 B3GAT3

16p12.3 17q21.33 5q35.2–q35.3 1p36.33 11q12.3

ChSy-1

CHSY1

ChSy-2 ChSy-3 ChPF

CHSY2(CSS3) CHSY3(CHPF2) CHPF(CSS2)

ChGn-1

CSGALNACT1

15q26.3(GalNAcT-II, GlcAT-II) 5q23.3 7q36.1 (CSGLCA-T) 2q35(GalNAcT-II, GlcAT-II) 8p21.3 (GalNAcT-I, GalNAcT-II) 10q11.21

ChGn-2 Sulfate-transferases and epimerases Chondroitin 4-O-sulfo-T C4ST-1 C4ST-2 C4ST-3 Dermatan 4-O-sulfo-T D4ST-1 Chondroitin 6-O-sulfo-T C6ST-1 Uronyl 2-O-sulfo-T UST GalNAc 4-S-6-O-sulfo-T GalNAc4S6ST Glucuronyl C-5 epimerase DS-epi1 DS-epi2 Sulfate-transferases and epimerases Glucuronyl C-5 epimerase DS-epi1 DS-epi2 Tetrasaccharide linkage-modifying enzymes Xylose 2-O-kinase XylK Galactose 6-O-sulfo-T C6ST-1 Exostosin-like glycosylEXTL2 T2 Uronyl 3-O-sulfo-T HNK1-ST

CSGALNACT2 C4ST-1 (CHST11) C4ST-2 (CHST12) C4ST-3(CHST13) D4ST1 (CHST14) C6ST-1 (CHST3) UST GALNAC4S-6ST (CHST15) DSE (SART2) DSEL

12q 7p22 3q21.3 15q15.1 10q22.1 6q25.1 10q26 (BRAG)

18q22.1

DSE (SART2) DSEL

6q22 18q22.1

FAM20B (gxk1) (CHST3) EXTL2

1p25 10q22.1 1p21 (GlcNAcT-I)

(CHST10)

2q11.2 (continued)

3.8 Glycosaminoglycans (GAGs)

79

Table 3.2 (continued) Enzymes Abbreviation Chondroitin sulfate hydrolases Endo-β-NHYAL-1 acetylgalactosaminidase HYAL-4 SPAM1

Gene symbols

Chromosomal loci

HYAL-1

3p21.3

HYAL-4 (CSHY) SPAM1

7q31.3 7q31.3

example, the malaria (Plasmodium falciparum)-infected erythrocytes adhere to endothelial cells by recognition of CS saccharide units carrying a low sulfated CS-A structure like monosulfated A unit [91]. HSV recognizes and binds to E unit-rich CS chains as its infection receptor [92, 93]. Hence, C4ST-1 and E unitdeficient sog9 cells cannot be infected by HSV-1. C4ST-1 gene transfection in sog9 cells produces E disaccharide and renders HSV-1 infection [93]. CS chains with D or E units bind to PTN, midkine (MK), FGF, HGF, and BDNF. CS stimulates neurite outgrowth as well as neural stem cell and neuronal progenitor cell growth [74], because CS saccharide chains are co-receptors or holding reservoirs for associated molecules. CS binds to neuronal receptor, named neurite outgrowth identified contactin-1 (CNTN-1), which is necessary for extension and regeneration of neurons. Interestingly, CSPGs as essential CNS components inhibit axon outgrowth during CNS injury. If CS parts of CSPGs are removed in lesion sites, axon regeneration event is induced. However, CS action in neurite outgrowth inhibition is controversial because CS-E stimulates neurite outgrowth in the controlled experiment using cultured primary neurons. Such apparent contradiction is attributed to the structure difference in CS saccharide chains. The dominant CS forms present in mammal tissues include the two monosulfated A and C units. The A unit-enriched CS form (CS-A) inhibits axon guidance event and cerebellar granule neuron growth [94]. However, C chain-enriched CS form (CS-C) does not inhibit and induce axon regeneration [95], and their synthetic enzyme C6ST-1 expression is increased in injury conditions. Hence, neuronal cells have distinct CS-recognizing CS receptors [96]. In two different cell lines such as L cells of mouse fibroblast cells and mutant sog9 cells, the CS-E is generated by C4ST-1 enzyme, and it easily recognizes Wnt-3a. Consequently, it controls β-catenin-dependent canonical Wnt downstream signaling [97]. However, C4ST-1-lacking sog9 cells do not recognize Wnt-3a. C4ST-1 also involves in tumorigenesis, because C4ST-1 genes in colon adenocarcinoma cell and hepatocarcinoma cells of humans are not expressed, although those cells exhibit the activated Wnt/β-catenin signaling [97]. C4ST-1 expression is also inhibited in certain human patients with B-cell lymphoma [98]. In addition, the C4ST-1 expression is correlated with malignant colorectal cancer [99]. A protooncogene HRAS is known to diminish the C4ST-1 gene expression in the Costello syndrome pathogenesis, which germline mutations in the HRAS generate the pathogenesis [100]. Therefore, C4ST-1 can be applied for therapeutic treatment of cancer progressions associated with RAS signaling or canonical Wnt signaling in humans.

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Apart from endogenous CS-E, exogenous CS-E treatment prevents accumulation of β-catenin [97]. CS-E can be used to treat aberrant canonical Wnt-caused diseases. A CS-E binds to its specific neuronal receptor for a CNTN-1 known as a GPI-anchored protein of cell adhesion molecule (CAM) as the Ig superfamily [101]. Only CS-E recognizes the CNTN-1. However, others of CS-A, CS-C, or HS do not bind to the CNTN-1. Certain CS chains, which are resident in extracellular region, intracellularly transduce their signaling. A transmembrane receptor protein phosphatase known as PTPσ is also known as a CS receptor to inhibit axon regeneration [102]. PTPσ and leukocyte-related antigen (LAR) subfamily are also receptors for HSPGs, and they exert to potentiate synapse formation and axon guidance at the early developmental stage [103, 104]. PTPσ regulates bimodally extension of neurons through double actions such as inhibition of CSPG and stimulation of HSPG. In fact, HS induces oligomerization of PTPσ ectodomains. In contrast, CS inhibits the HS-induced oligomerization [104]. Thus, differential action of PTPσ is mediated by their oligomerization modulation of PTPσ, which is used for the common receptor for CSPGs and HSPGs. Currently, TM leukocyte common antigen-related phosphatase (LAR) receptor as a subclass of LAR subfamily, and two family members of Nogo receptors including NgR1 and NgR3, are also known for the CS receptors to inhibit CSPG function [105]. CE-E unit mediates pulmonary metastasis, as confirmed by Lewis lung carcinoma (LLC) cells. A silencing of GalNAc4S-6ST reduces the CS E units and suppresses pulmonary metastasis in LLC cells. CS-E binds to advanced glycation end-product (RAGE) receptor, an Ig superfamily, largely present in lung tissue [106]. RAGE binds to sulfated GAG saccharide chains such as CS-E and HS. Anti-RAGE antibody inhibits CS-E-involved lung metastasis of LLC cells (Fig. 3.4c), allowing RAGE and sulfated GAG for drug targets for pulmonary metastasis.

3.8.2.4

Roles of CS in Embryogenesis and Development

Caenorhabditis elegans generates HS and chondroitin, which is not sulfated in CS. The chondroitin and CS chains are indispensable, as confirmed in the nematode ortholog of ChSy for sqv-5 in the vulva formation [89]. GlcAT-I (B3gat3) KO mice are lethal at embryonic stage of development before the 8-cell stage because of cytokinetic defaults. The two-cell embryos of wild type were treated with chondroitin-digesting enzymes such as chondroitinase-A, chondroitinase-B, and chondroitinase-C (ChABC) from bacterial sources also which show embryonic lethality. Vertebrate bones are formed through the known process of endochondral and intramembranous ossification events. Sulfation of CS/DS chains is important for skeletal bone formation with endochondral ossification. Mouse MC3T3-E1 cells as an osteoblastic line show intramembranous ossification. Cadherin mediates cell-cell interaction during osteogenic differentiation because MC3T3-E1 cells produce cadherin-11 and N-cadherin [81]. In differentiating MC3T3-E1 cells, CS-E unit is increased, where CS-E recognizes N-cadherin or cadherin-11 and consequently enhances osteogenic differentiation [107]. However, CS-A does not bind to the

3.8 Glycosaminoglycans (GAGs)

81

molecules. Interaction between CS-E ligand and the cadherin-11 and N-cadherin-11 as CS receptors influences osteogenic bone formation of MC3T3-E1 cells, which is potentially applicable for patients with osteoporosis. The enforced GalNAc4S-6ST expression for binding ligand CS-E units may enhance adhesion to N-cadherin/ cadherin-11 in MC3T3-E1 cells. Sulfation and CS chains synthesis are also crucial for embryonic development. Incomplete CS synthesis at early stage of embryonic development causes cell death due to reversed cytokinesis. Enforced reduction of CS levels induces both of myogenic differentiation and myofiber regeneration. Therefore, a dystrophin deficiency in mice, which is a typical model animal of Duchenne muscular dystrophy, can be compensated through the intramuscular ChABC injections. This potentially improves the dystrophy pathogenic progress of myofibers. In other words, the abundantly synthesized CS levels are an essential factor for muscular dystrophic protection, regeneration of skeletal muscles, disease cell differentiation, and disease improvement.

3.8.3

Dermatan Sulfate (DS)

In the structure aspect, DS is a linear oligo- or polysaccharide, as a sulfated GAG, with a covalent linkage to the PG core proteins [108, 109]. The DS saccharide units have GalNAc and l-iduronic acid (IdoUA), although CS is indeed a DS stereoisomer, consisting of D-GlcUA instead of IdoUA. Sugars are the general subjects of esterification reaction by sulfate at multiple positions. DS chains are assembled through cooperative action of various GTs and modifying enzymes such as epimerases and sulfotransferases. For the biosynthesis of DS, first, acceptor core proteins are generated. Second, the GAG linker chain of GlcUAβ1,3Galβ1,3Galβ1,4Xylβ1sequence is synthesized by specific transferases of β-xylosyltransferase (Xyl-T), β1,4-Gal-transferase-I (Gal-T-I), β1,3-Gal-transferase-II (Gal-T-II), β1,3-GlcAtransferase-I (GlcAT-I) using the target core proteins for linking to Ser residue. The four different Xyl-T, GlcAT-I, GalT-I, and GalT-II enzymes commonly act to form all GAGs including CS, DS, and HS. Upon formation of the saccharide linker chains, chondroitin synthase enzymes make the assembly with the chondroitin units. Then, the GlcUA epimerization as well as each sugar sulfation occur through enzymatic catalysis of DSE, D4ST, and UST. Xyl-T, Gal-T-I, Gal-T-II, GlcAT-I, GalNAcT-I, GlcAT-II, GalNAcT-II, DS epimerase (DSE), dermatan 4-Osulfotransferase (D4-ST), and UST are involved in the multiple reactions. DS-PGs are abundant in the cartilage, skin, and aortic endothelium in humans. DS-PGs exhibit ubiquitous expression patterns in many organs and tissues including the brain, heart, kidney, liver, and lung. DS and DS-PG synthesis defections are associated with development of skin and skeletal diseases. DS chain units contain disaccharides of GalNAc and IdoUA residues, having 50–200 more repeated units (Fig. 3.4). DS chain units are frequently targeted to sulfate at the carbon C-4 and C-2 sites both on GalNAc and IdoUA residues, respectively. Their localization in ECMs

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exerts to play crucial roles in various biological pathways including growth factormediated signal transduction, anticoagulation, and wound healing [110]. CS consists of another sugar residues GlcUA and GalNAc. After the chondroitin unit formation, the GlcUA residue is targeted to epimerization reaction to form the IdoUA residue by a specific DSE enzyme. Then, the new type chains of CS/DS hybrid can be also synthesized. The small Leu-rich DS-PGs include biglycan, fibromodulin, and decorin, which carry the Leu-rich sequences with small protein core sequences [72]. The PG-deficient KO mice are particularly featured with osteoporosis, skin fragility, collagen fibrils, and abnormal Achilles tendon. Core protein of decorin modulates the collagen fibrogenesis. EDS is classified to a heterogeneous diseases and also heritable connective tissue disorders with the multiple expressions of joint hypermobility, skin hyperextensibility, and tissue fragility.

3.8.3.1

Biosynthesis of DS

CS disaccharide contains GlcA and GalNAc, namely, 4GlcAβ1–3-GalNAcβ1-. While in DS, β-D-GlcA is transformed to α-L-IdoA via the C5 epimerization, which is catalyzed by an epimerase. Then, the CS/DS is frequently present as a single chain hybrid form of the CS/DS hybrids [111]. DS is generated from CS, firstly by GlcA epimerization to IdoA, followed by sulfo-transfer reaction in distinct carbons [112]. The DS-epimerase (DSE) and DSEl genes encode the DS epimerase-1 and DS epimerase-2, respectively. The DS epimerases modify CS to CS/DS hybrid units. The produced CS/DS hybrid units are richer than CS in structure and conformation aspects. The CS/DS hybrid units preferentially interact with ECM proteins and related growth factors. More specifically, after chondroitin backbone synthesis, two DSE enzymes convert GlcA residue to IdoA residue form via the C5-carboxyl group epimerization of GlcA residue [83], forming CS/DS hybrid units with different IdoA contents. C5-epimerized residue is O-sulfated, forming CS/DS chains. The IdoA residue is formed by DS-epi-1 and DS-epi-2 enzymes, genetically coded by the DSE and DSE-like genes (DSE-L), respectively [83]. Therefore, CS/DS consists of alternated units of a hexuronic acid of GlcA or IdoA, and the amino sugar GalNAc. The IdoA-containing units yield long blocks and often are interspersed among unmodified GlcA saccharide units. Missense mutations of homozygous forms in DSE gene are the causing factors of the musculocontractural type of Ehlers-Danlos syndrome (MC-EDS), a connection tissue disease showing fragility complications [113]. Human DSEL (C18orf4) is related to bipolar disorder [114] and depression disorder [115]. Dse KO mice show easy skin fragility caused by the reduced IdoA content in the CS/DS of the small Leu-rich PGs biglycan and decorin, known to assemble collagen fibrils [116]. CS/DS IdoA-null double KO mice die upon birth [117]. The amino sugar of the CS/DS is N-acetylgalactosamine (GalNAc), small leucine-rich PGs that mostly carry DS chains. CS and DS GAG chains may exist as separate entities but can also form hybrid structures along the same GAG chain. The distribution of CS and DS domains has been shown in their tissue and regional differences like in the brain.

3.8 Glycosaminoglycans (GAGs)

83

The repeated disaccharide units of DS, linking to Ser residue of core proteins, exert their functions. The common GAG-protein linker region is the tetrasaccharide with the carbohydrate structure of GlcUA-Gal-Gal-Xyl-O-Ser- [108]. β-Xyl-T (Xyl-T) encoded by XYLT-1 or XYLT-2 catalyzes the transferring reaction of a Xyl residue using the donor substrate UDP-Xyl to a certain Ser residue present in the PG core proteins, which previously is biosynthesized through ER/cis-Golgi complex network, which commences the DS, CS, and HS chain biosynthesis [118]. Two Gal saccharides attached to Xyl-O-Ser-core proteins are formed from the donor UDP-Gal by Gal-T-I and Gal-T-II enzymes that are expressed from their genes of B4GALT7 and B3GALT6, respectively [119]. Thereafter, β1,3-glucuronosyl-T-I (GlcAT-I) expressed by its gene B3GAT3 adds a GlcUA residue using the donor UDP-GlcUA to the acceptor substrate Gal-Gal-Xyl-O-Ser. B4GALT7 (GalT-I) deficiency in GalT-I-encoding B4GALT7 mutation causes EDS-progeroid type 1 [120]. Phenotype includes an appearance aged, craniofacial dysmorphism, delayed development, elastic skin, short stature, osteopenia, hypermobile joints, hypotonic muscles, and wound healing dysfunction. Furthermore, homozygous mutations found in B4GALT7 gene exhibit the similar EDS form and reduce DS side chain lengths of decorin. The mutated fibroblasts showed the reduced sulfation level of HS chains with retarded wound closure [120]. Thus, EDS-progeroid type 1 is caused by defection of HS as well as DS. Homozygous mutation in B4GALT7 generates a certain type of disease such as Larsen syndrome found in regional Reunion Island in France with symptoms including dwarfism, facial features, hyperlaxity, and multiple dislocations [108]. The known Larsen syndrome clinically displays congenital joint dislocations and craniofacial abnormal dysfunctions including dislocated hip, elbow, foot, and knee deformities. Therefore, genetic syndromes expressed as the Larsen in Reunion Island and EDS-progeroid type 1 show common joint dislocations, but the reason why the B4GALT7 mutation makes the two disorders is not known. Another GalT-I-encoding B4GALT6 synthesizes the common linker region tetrasaccharide, GlcUA-Gal-Gal-Xyl- in CS/DS and HS saccharides (Fig. 3.5). Patient-derived fibroblast cells, which have the heterozygous mutations in GalT-I, synthesize low-glycosylated decorin and biglycan PG core proteins with shorter DS chains [121]. B3GALT6 (GalT-II) deficiency shows defects in DS, HS, and CS through mutations and influences the development of the skeleton and skin with different symptoms.

3.8.3.2

Deficiency Syndrome of DS

The repeat of disaccharide units of the chondroitin repeat (-4GlcUAβ1–3GalNAcβ1)n chain is formed by enzymatic reaction of ChSy family [122]. DSE encoded by the specific genes of DSE or DSE2 epimerizes the GlcUA residue to IdoUA residue through the GlcUA C-5 hydroxyl group epimerization [83]. Dermatan chains are sulfated by specific enzymes of dermatan 4-O-sulfotransferase-1 (D4ST1) and UST, encoded by different genes of CHST14 and UST, respectively. Among them, the enzyme D4ST1 transfers the sulfate group using the substrate PAPS to the substrate

84

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Glycan Biosynthesis in Eukaryotes

GalNAc C-4 in DS [123], while UST transfers a sulfate group using PAPS substrate to the substrate IdoUA C-2 of DS [124]. UST deficiency by lack of the UST gene causes EDS-related symptoms. DSE deficiency patients are featured of collagen bundles with collagen fibrillar proteins, the intermittent small flowered fibrils, and granulofilamentous deposits [125]. DSE-lacking patients show a mild symptom of the EDS musculocontractural type, compared to the CHST14-negative patients. The DSE deficiencies influence the DS biosynthesis. Dse/ mice mutants synthesize a less amount of IdoUA residues in the skin tissue and consequently weaken the collagen fibril strength. In addition, DSE enzyme is efficient in forming IdoUA blocks of DS, while DSE2 is more effective to form a CS/DS hybrid chain compared to IdoUA units [83]. CHST14 (D4ST1) deficiency leads to a retrograded epimerization reverseconverting IdoUA residue to GlcUA residue, and this contributed to the DSE-generated chondroitin production, which is 4-O-sulfated by C4ST enzyme to GalNAc residues present in chondroitin, because CS and DS 4-O-sulfation acts as an inhibitor of DSE [126]. Lack of CHST14 causes an autosomal recessive disorder. Chst14/ mutant mice exhibit lower body weights, fragile skin, kinked tails, and low fertility. Moreover, Chst14/ mutant mice are featured with the impaired growth of neural stem cells, defected neurogenesis, and change in glial cell populations [127]. These phenotypes are similar to the D4ST1-deficient EDS patients.

3.8.4

Keratan Sulfate (KS)

Keratan sulfate (KS) disaccharides consist of β-D-GlcNAc and β-D-Gal units. KS is a linear polysaccharide form of LacNAc, Galβ1-4GlcNAcβ1–3, which is Gal and GlcNAc C-6 sulfated [128]. The KS disaccharides can have the six-positional sulfation at most units, although sulfation at GlcNAc is much more frequent. The KS carbohydrate backbone is elongated by two distinct GTs of β1,3-N-GlcNActransferase (β3GnT) and β1,4-Gal-Transferase (β4GalT) [129]. Sulfation of the chain is performed by two sulfotransferases like KS Gal-6-sulfotransferase (KSGal6ST) to Gal. GlcNAc6ST-1 and GlcNAc6ST-5 (CGn6ST) sulfate GlcNAc in the brain tissue and cornea. In humans, β3GnT has eight genes and β4GalT has seven genes. Among these, specific β3GnT7 and β4GalT4 are known for KS chain elongation, because β3GnT7 and β4GalT4 have higher activity for sulfated than non-sulfated substrates [129, 130]. However, there is no direct evidence to support of this possibility. KS is synthesized by two stages. The first stage is the GlcNAc-sulfated poly-Nacetyllactosamine chain synthesis by β3GnT7, β4GalT4, and GlcNAc6ST-5. The second stage is highly sulfated KS synthesis via Gal sulfation by KSG6ST. Most cells do not express KS-specific sulfotransferases in culture [131]. KS is generated by elongating the N-/O-glycans linked to scaffold core proteins [128]. KSPG is mainly expressed in the ECM or extracellular surfaces in the cornea, cartilage, and brain. The approximate MW of KS has been estimated around 20 kDa; therefore,

3.8 Glycosaminoglycans (GAGs)

85

4CFKQTGUKUVCPEGKPFWEVKQP

-GTCVCP UWNHCVGRTQVGQIN[ECP -52)



)

)CN

)NE0#E

5WNHCVG

&+67 &+67



)

&+67 &+67 &+67

3$36

3$367 &+67VXOIRWUDQVIHUDVH

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approximately 45 disaccharide units are assumed as the commonest structured MW. KS is used as an active component present in protective eye drops used for dysfunctional vision. KS is enriched in human cornea tissue. Human central nervous system (CNS) and peripheral nervous system also consist of the KS with low amounts [132]. KS-PG and KS display cellular regulatory roles in epithelial cells and mesenchymal cells as well as bone and cancers. KS contains sulfated chain with non-sulfated poly-LacNAc, monosulfated and disulfated disaccharides. Historically, KS was initially discovered in the corneal region for the first time by Suzuki in 1939, as a mucoid with Gal and Glc as well as acetyl and sulfate groups. Karl Meyer determined the mucinous mucopolysaccharides designating keratosulfate [133]. Does KS-PG protect radiation-mediated apoptosis? In human Burkitt’s lymphoma cells, expression of PAPS transporters (PAPSTs)-1 and -2 (PAPST1 or PAPST2) reduced radiation-induced apoptosis. Cleavage of KS chains by keratanase increases radiation-mediated apoptosis. The KS-mediated apoptosis depends on 6-O-sulfation of GlcNAc residues. GlcNAc-6-O-sulfotransferases (CHST2, CHST6, and CHST7) inhibit apoptotic cell death. PAPST1 increases in the phosphorylation level of p38 MAPK and Akt. Therefore, GlcNAc 6-O-sulfation reaction in KS decreases radiation-induced apoptotic cell death [134]. The sulfation in KS has been summarized in protection from apoptosis (Fig. 3.7).

3.8.5

Heparin and Heparan Sulfate

The heparin (Hp)/heparan sulfate (HS) disaccharides consist of four-linked UA and four-linked α-GlcN unit. HS and Hp carbohydrate structures are different from only in their proportions of the composed monosugar and dissugar. HS bears β-D-GlcA as a key UA form, whereas Hp bears α-l-IdoA residue. GlcA residues in HS are replaced by GlcNAc units, but low level of GlcN-N-sulfated residue and limited level of unsubstituted GlcN residue are also present. Hp predominantly bears IdoA 2-sulfated residue (IdoA2S) with N,6-disulfated GlcN units. The GAGs can be utilized as potentially therapeutic drugs in the forms of the potent antithrombotic and anticoagulant Hp [135]. In fact, mast cell-enriched tissues including swine and cattle intestinal mucosa are the sources of such pharmacological Hp. Hp is currently the widely used GAG substance as the most common therapeutic glycans through

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the world to prevent and treat the thromboembolic prophylaxis. The Hp is simply prepared [86], and currently 20 more mammalian HS-PG core proteins are known [136]. Among GAGs, HS mediates intracellular signaling, Wingless, Hedgehog, and FGF pathway. Therefore, the damaged HS synthesis causes for developmental defect, as observed in Drosophila. During the studies, Hp has been the first GAG having biological function. Hp is the anticoagulant. The FGF recognizes the HS chain of the syndecan proteoglycan and consequently activates the FGFR of cells [137]. Heparanase digestion of cell-surfaced HS and matrixed HSPGs promotes invasion and metastasis [138].

3.8.6

Hyaluronic Acid (HA) or Hyaluronan

HA as a sole GAG form is not sulfated and consists of repeated disaccharide units of β-D-GlcA residue and β-D-GlcNAc residue. HA has the longest chain among all the known GAG types. The MW of HA is approximately above 100 kDa, and the polymerization degree of HA is, therefore, in the range in about 255 disaccharide units per chain up to several million of MW [139]. This extremely high MW polysaccharide has high viscosity even at lower concentrations. Hyaluronan synthase 2 overexpression promotes ErbB2 signaling and progression of breast cancer [140], while its suppression inhibits tumorigenesis and progression [141]. HA influences the metastasis of mouse mammary carcinoma cells [142].

3.8.7

Proteoglycans (PGs)

PGs are GAG-linked proteins. GAGs, except for keratan 6 sulfate, are linked to a Ser in PG protein through the tetrasaccharide GlcAβ1,3Galβ1,3Galβ1,4Xyl-liker. The GAG’s protein core simply acts as a scaffold for GAG activity. PGs are often large in size with heavy glycosylation and membrane attachment. There are about 50 distinct PG genes except for alternative spliced proteins [143]. Although most PGs are Nand O-glycoproteins, however, the only PG definition is based on O-linked GAG chains. Most PGs function mainly at the extracellular area and act for the cytokines, chemokines, growth factors, and morphogens. They also modulate embryonic development, pathogen-infectious inflammation, and cell-cell communication [144]. PGs also influence growth factor action, collagen fibril formation, tumor cell behavior, and corneal transparency for vision. GAGs nonspecifically interact with proteins, and they are located on cell surfaces as proteoglycan forms and adhere to soluble forms of polypeptides such as growth factors via electrostatic interactions. For the most important cellular function, GAGs stabilize growth factors [108]. Some GAGs act as co-receptor for the growth factors or directly bind to cellular receptors or via growth factor sequestration. The GAGs can be used for diagnostic markers and also for targets of potential therapy in

3.8 Glycosaminoglycans (GAGs)

87

cancers. For the application, infrared and Raman spectroscope analysis and bioimaging analysis have been developed to distinguish GAG class. Defect of GAG synthesis leads to diseases such as connective tissue disorder and EhlersDanlos syndrome that displays hereditary multiple exostoses. This syndrome involves in inappropriate chondrocyte proliferation and bone growth. GAGs increase cell adhesion and cancer cell invasion. GAGs regulate cell functions through transforming growth factor (TGF) and FGF signaling [72]. Certain corneal dysfunction is caused by sulfated GAGs. Mutation of CS and DS biosynthetic enzyme genes generates connective tissue-defected diseases [145]. GAGs and proteoglycans regulate cancer progression. CSPGs activate the melanoma growth [146]. CS inhibits the migration of transendothelial monocytes and consequent angiogenesis [108]. In stem cells, GAGs and PGs are biomarkers of progenitor cells [147]. GAG and PGs give “stemness” of stem cells, as evidenced by CSPG role in neural stem cells [148]. HSPG and CSPG also give stemness in hematopoietic precursor cells [149].

3.8.7.1

Intracellular PG Type of Mast Cell Granule Serglycin

The Hp-containing PG serglycin [150] is an unusual PG type because of its existence only in the mast cell granules and mast cell-related cells but not in the ECM. Mast cell-secretory granules contain Hp chains attached to the small peptide of serglycin [151]; consequently, Hp is partially fragmented during mast cell degranulation. Extremely small Hp fragments are the LMWH products such as enoxaparin. Mast cells contain granules packed with secretory proteins. The PG serglycin carries GAG side chains including mainly Hp, but sometimes CS or DS. The granules are closely packed by the help of the PG serglycin, and its GAG side chains [152]. The intracellular GAGs are stored in mast cell granules. In rare cell type, basophils also have granules. Serglycin is the mast cell granule PG and has a small protein core. The human sequence is 158 amino acids long with a signal peptide in the N-terminal region. Serglycin contains a central domain with Ser and Gly alternate. The human sequence has 8 Ser [153]. Ser residues carry galactosaminoglycan side chains of chondroitin-4-sulphate (CS-A) or DS (CS-B) or GAG chains of the HS/Hp family. Rat peritoneal mast cell Hp PG, serglycin, carries about 750 kDa with 75 kDa Hp chains attached to a protein core [154]. Serglycin has a different GAG substitution.

3.8.7.2

PGs of Syndecans and Glypicans on Cell Surfaces

There are two distinct PGs of syndecan and glypican expressed on cell surfaces. Several ECM PGs include small Leu-rich PGs (SLRPs) like decorin, biglycan, and lumican as well as aggrecan. GAG chains are less dense, and the extracellular surfaced GAGs linked to glypicans or syndecans likely act as signaling molecules, or in tissue remodeling. The cell surface glypican PGs function as modulators or

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morphogens of functional action of bone morphogenetic protein, FGF, Sonic Hedgehog, and Wnt. A total of six mammalian glypican genes are known for the core proteins of glypican-1 to glypican-6. They are linked to the outer membrane region through a C-terminal region of GPI anchor [155]. Another cell surface PG, syndecans, includes four mammalian syndecans. Syndecans are composed of a TM region and a cytosolic domain, which recognizes cytoskeleton proteins and protein kinases [156]. Syndecans collaboratively with integrins and hyaluronan signaling via CD44 increase cancer cell motility [157]. GAGs increase metastasis and angiogenesis. Syndecan-1 increases the cancer cell adhesion to lymphatic endothelium [158]. Decorin regulates EGFR signaling and proliferation in melanoma [159]. Decorin binding to VEGFR-2 indicates its antagonistic role [160], because decorin acts as an antagonist to VEGFR-2. In fact, 12-amino acid oligopeptide present in the decorin Leu-rich-repeated 5 domain is the binding site for the VEGFR-2. Consequently, VEGF-VEGFR-2 binding is antagonized [157]. Lumican glycoprotein inhibits melanoma cell migration [161]. GAGs linked to PGs on cell surfaces help viral invasion. Enveloped proteins of yellow fever and dengue viruses, which belonged to flavivirus family, recognize GAGs on surface [162]. Viral carbohydrates also bind to the cell surface receptor such as DC-SIGN [163].

3.8.8

Extracellular PGs

3.8.8.1

Aggrecan

Aggrecan is mostly abundant as PG in tissue ECMs like the cartilage, where aggrecan associates with big HA-aggregated complexes [164]. Thus, this is a target for regenerative medicine. For example, the CS-linked core aggrecans are abundantly present.

3.8.8.2

Perlecan

Multiple domain-containing PG perlecan is present at the region of cellular basement membrane or at the interspaces between pericellular region. Perlecan as a strong HS-consisting PG function as growth factor stores like FGF and in angiogenesis event upon binding to VEGF [164]. Perlecan loss inhibits the cancer growth such as colon carcinoma cells and tumor angiogenic progression [165].

3.8.8.3

Small Leu-rich PGs (SLRPs) of Decorin, Lumican, and Biglycan

The SLRPs include decorin that acts to wrap tendons adjacent to D-band of collagen fibrils [166]. SLRPs form the structure of the cornea and transparency.

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89

The SLRP lumican bearing three N-linked KS chains leads to ordered collagen fibrils of the cornea [167]. Decorin (DCN) deficiency KO mice show the EDS-like phenotype [168]. However, decorin core protein-encoding DCN gene mutations do not cause EDS. But DCN mutation yields only the truncated decorin core protein with limited functions of decorin. Decorin is a prototype SLRP with a DS side chain responsible for autophagy, collagen fibrillogenesis, tumor growth, and wound repair [169]. C-terminal domain of decorin functions for maintenance of the cornea fibrillar reorganization. Dcn/ mutant mice are featured with impaired collagen morphology and skin weakness and fragility [168], resembling the EDS-like symptoms. Dcn/Bgn double deficient mutant mice also exhibit skin fragility and osteopenia similar to the EDS-progeroid-like form [170]. The decorin KO mice exhibit abnormal collagen morphology and human EDS-like pattern [168]. In addition, the double decorin and biglycan KO mice show the human EDS-like progeroid type [170]. Lumican has four sites for KS. The corneal lumican is a KSPG with a molecular weight range of 70–300 kDa KS, giving corneal transparency. In contrast, skin dermal lumican is a glycoprotein with a 57 kDa [161]. Biglycan (BGN) two missense mutations cause the spondyloepimetaphyseal dysplasia as an X-linked inheritance disease in tropical families including Korean, Indian, and Italian with a short stature and joint osteoarthritis [171]. BGN-deficient mice reduce growth and bone mass, promoting myofibroblast differentiation and proliferation through TGFβ and SMAD2 signaling [172]. The KO mice are used as models for human spondyloepimetaphyseal dysplasia or Meester-Loeys syndrome for therapeutic agents for these disorders.

3.9 3.9.1

Glycosylphosphatidylinositols (GPIs) Anchor Glycosylation General Structure of GPI Anchors

GPIs, ubiquitous surface-anchored molecules in eukaryotes, have a basic role, simply to anchor to cell membranes. The main question of “what is GPI membrane anchor? is the conceptional long subject in the membrane protein diversity in organisms. In a single word, GPI stands for glycosylphosphatidylinositol. In other words, a phosphatidylinositol (PI) phospholipid linked via a glycosyl, or sugar chain, component to the protein C-terminal region. What are GPI membrane anchors? GPI stands for glycosylphosphatidylinositol. In other words, a PI phospholipid linked via a glycosyl, or sugar chain, is a component of the protein in the C-terminal region. During the transglycosylation reaction, GPI species is linked to cell wall proteins. Cell wall β1,6-glucan is cross-linked to proteins via its GPI glycan. How are proteins bound to membranes? Chemical and enzymatic reactions of GPI anchors are different from humans and parasites. Differences are present in parasite and host GPI pathways, and therefore, selective inhibitors of the T. brucei

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GPI pathway can be designed. Before understanding of the GPI structures, several basic components are figured out. For example, phosphocholine, phosphoethanolamine, and ethanolamine are basic components. Among them, ethanolamine is a functional group of phospholipids and this is abundant (Fig. 3.8). GPI anchors are holders of surface proteins anchored in eukaryotic membranes. GPI anchors are used for tethering candidate molecules to the exposed extracellular leaf of the lipid bilayered plasma membrane (PM) via their carboxyl termini. GPI anchors are frequently synthesized in most eukaryotes including fungi, invertebrates, protozoa, plants, and mammals [173]. GPI anchors are biosynthesized via the

3.9 Glycosylphosphatidylinositols (GPIs) Anchor Glycosylation

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membrane-associated enzyme complex. Lastly, the inositol-linked palmitic acid is cleaved off. Phospholipase C specific for phosphatidylinositol (PI-PLC) cleaves and releases the peptide part from the cellular PM [174]. The known GPIs consist of a common component of an ethanolamine-phosphate (EtN-P), three Man residues, a glucosamine (GlcN) as a non-acetylated form, and inositol phospholipid (IP or PI). The GPI anchors contain an ethanolamine-phosphate6Manα1,2Manα1,6Manα1,4GlcNα1,6myo-inositol-1-phosphate-lipid. Peptides are linked to amino group in ethanolamine through the NH-CO linkage with C-terminal carboxyl groups. GPI-tethered proteins are removed from the membrane by PI-PLC enzyme. The substitution of palmitic acid on the myoinositol C-2 position inhibits the PI-PLC activity. GPI-anchored proteins (GPI-Aps) are the only forms of posttranslational modification by glycolipid. Fatty acid chains, which are linked to inositol phospholipids, are embedded into the PM outer leaflets of cells. However, protein parts are not directly embedded into the PM of cells. More than approximately 150 proteins of human membrane proteins are reported to be such GPI-APs [175], having their distinct functions. There are many GPI-anchored proteins known from mammals, protozoa, plants, and lower eukaryotes including yeast, fungi, and slime molds, as shown in Table 3.3. 40 more number enzymes are GPI-anchored and include alkaline phosphatases; RECK protease inhibitors; transcytotic transporters; 50 -nucleotidase; dipeptidase; cellular adhesion molecules (CAMs) including CD48, contactins, and glypicans; receptor proteins including GDNF receptor-α series, folate receptors, and FcγR-IIIb; and complement regulatory proteins including CD55 and CD59 as decay-accelerating proteins. GPI-Aps are often associated with lipid raft-microdomains of cellular PM. They are transiently homodimerized or released by enzymatic cleavage from the GPIs [167, 176, 177] and, also, sorted apical directions in certain cells upon polarization stimulation [178]. GPI-APs are required for fertilization, embryo formation, development, neural formation, and immunity [84, 179–181].

3.9.2

Function of GPI-Anchored Protein

For example, CD24 is the cell’s surface antigen consisted of protein and sugar residues. It is also localized at cell’s surface by GPI-anchored link. CD24 GPI-anchored antigen is a neural surface molecule known as a heat-stable antigen. The CD24 has many broad roles in the cells, as many scientists work with the CD24 to identify its interaction molecules and functions. CD24 is also related to cell proliferation, neuronal development, lymphocyte activation, and other cellular processes. It is named a nectadrin and small cell lung cancer antigen cluster-4 [182, 183]. The roles of CD24 in neuronal development and neuronal diseases have been explained at the level of the intracellular signaling. CD24-mediated signaling uncovered its systemic involvement in neural migration, neurite extension, and neurogenesis (Fig. 3.9) [183]. Overexpressed CD24 also inhibits DAMPs with Siglec-10 cis-interaction. The multiple roles of the CD24 are caused by its glycan

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Table 3.3 Examples of GPI-anchored proteins GPI-anchored protein Protozoa T. brucei variant surface glycoprotein (VSG) Leishmania major promastigote surface protease (PSP) T. cruzi GPI-anchored mucins P. falciparum merozoite surface protein 1 (MSP-1) Toxoplasma gondii surface antigen 1 (SAG-1) Entamoeba histolytica GPI proteophosphoglycans Yeast, fungi, and slime mold S. cerevisiae α-agglutinin S. cerevisiae GAS1p Aspergillus fumigatus GEL 1p Candida albicans HWP1 Dictyostelium discoideum prespore antigen (PsA) Dictyostelium discoideum contact-site A (CsA) Plants Pyrus arabinogalactan proteins (AGP) Arabidopsis thaliana metallo- and aspartyl proteases Arabidopsis thaliana β1–3 glucanase Mammals Erythrocyte CD59 and CD55 DAF Alkaline phosphatase 50 -Nucleotidase Renal dipeptidase Trehalase NCAM-120 NCAM TAG-1 CD58 FcgIII receptor Ciliary neurotrophic factor receptor (CNTFR) a subunit Glial cell-derived neurotrophic factor receptor (GDNFR) CD14 Prion protein, PrP Glypican family of GPI-anchored proteoglycans CD24 (heat-stable antigen, nectadrin)

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chains. The CD24 molecular weight is a 20–70 kDa protein, while its predicted molecular weight from its DNA and amino acid sequences is 3 kDa, which is smaller than the cellular protein forms. The difference is derived from CD24 sugar chains. CD24 protein core is glycosylated at different sites with different sugar sequences. Murine CD24 contains seven distinctly predicted O-glycan sites, and three N-glycan sites. Because it has many different glycan structures, the CD24 interacts with many different cellular receptors and shows diverse functions. The CD24 has a variety of

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roles, where one of the cells that function is neuronal cells. CD24 is known to involve neuronal migration, neurite outgrowth, and neurogenesis. CD24 is highly expressed in developing brain stage in the neuroepithelial migratory zone, but lowly in the ventricular zone. CD24 expression is activated in postmitotic neurons during the period of migration. Also, in humans CD24 is expressed highly in neural stem cells in order to differentiate to neuroblasts and neurons. CD24 has multiple potentials to activate or suppress outgrowth of neurites. CD24 expressed in cerebellar, hippocampal, spinal, and cortical non-motor neurons promotes neurite outgrowth, while in retinal, dorsal root ganglia, or motor neurons, it’s expression is inhibited. Three CD24 receptors, L1, contactin, and TAG-1, are known. L1 binds to α-2,3-SA and others bind to Lewis X antigen. TAG-1- and contactin-deficient mice do not exhibit neurite outgrowth at cerebellar neurons. On the other hand, in dorsal root ganglia, CD24 inhibits neurite outgrowth, and CD24 associates with clusters of L1 and contactin or L1 and TAG-1. These parts are characteristics for the inhibited neural outgrowth and the destined nodes of Ranvier. Therefore, promotion and inhibition of outgrowth are determined, depending on the neurons which are myelinated or not. CD24 also interacts with many signaling molecules such as MAPK, NF-kB, Notch and Hedgehog, and other networks. With many signaling pathways, CD24 potentiates cell proliferation, growth, and differentiation and sometimes induces cancerous behaviors. Nonetheless, the relationship between neuronal behavior and CD24 signaling is still not revealed. Further works will allow to know us. Neuronal diseases such as multiple sclerosis (MS) are also related to CD24. MS is a chronic inflammatory disease with widespread loss of myelination and axons, as depicted to CNS and autoreactive lymphocytes. CD24 regulates T cells and lymphocytes, as CD24 expression on CNS-resident lymphocytes increases

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autoreactive T cells. In addition, in neural cancer, CD24 importantly regulates development, invasion, and metastatic progression. In glioblastoma CD24 activates Src kinase to upregulate the growth and invasiveness of glioma. Another CD52 resembles to CD24. CD24 with cooperation of Siglec-10 specifically suppresses damage-caused immune responses in injured tissues. CD52 is a classical GPI-anchored glycoprotein present in hematopoietic lineage cells including monocytes, DCs, B cells, and T cells [184–186]. In T cells, they release the soluble form of CD52 after absolutely activation. Soluble CD52 can bind to SA-binding immunoglobulin-like lectin (Siglec-10) with its N-linked glycan motif, and this downstream signal attenuates the TCR signaling pathway resulting in T-cell suppression. Siglec-10 has an ITIM motif and thus plays an inhibitory role due to its ITIM motif in most of immune cells like B cells, NK cells, DCs, or monocytes. In addition, it has a homology to a murine Siglec-G with a similar structure and function. Siglec-10 alerts the pathogen and regulates p38 and MAPK pathway to exert anti-inflammatory function like IL-10. Siglec-10 is a key molecule during C. jejuni infection in humans. Moreover, Siglec-G expressed on DCs inhibits CD8 T-cell proliferation. For example, when the pathogens break into the host cells, Siglec-G recruits the SHP-1 increasing degradation in phagosome. This pathway inhibits T-cell proliferation. A DAMP protein, HMGB1, mediates the formation of CD52-Siglec-10 complex. In contrast, other types of DAMPs such as HSP70 and HSP90 cannot form the CD52-Siglec-10 complex. As the HMGB1, DAMP can initiate the noninfectious inflammation to express proinflammatory cytokines through MyD88 and NF-kB signaling pathway. Especially, HMGB1 interacts with several immune-related molecules to upregulate the expression of cytokine genes. HMGB1 has two motifs of BOX A and BOX B with each distinct function. BOX A has a p53 transactivation-binding domain, and BOX B has a TLR4-binding cytokine region. Interestingly, Box B is related to the Siglecc-10 functions. If T-cell immune response is overactivated, soluble 52 in T cells binds to Siglec-10 through HMGB1 as a DAMP. In the Siglec-G-deficient mice, inflammatory cytokine expressions are increased. Siglec-10 suppresses soluble CD52 in hyperactivated immune response. The authors of this paper showed one of the DAMP proteins, HMGB1; no other type of DAMPs such as HSP70 and HSP90 is required to make CD52-Siglec-10 complex. The inhibitory receptor Siglec-10 recognizes the soluble CD52 and regulates activation of T cells. In fact, T cells release the soluble form of CD52 upon activation. The N-linked glycans present in soluble form of CD52 released from T cells are recognized by Siglec-10 and attenuate the TCR signaling for T-cell suppression. The Box B domain, not Box A, in HMGB1 having proinflammatory function specifically binds to CD52 and consequently elicits Siglec-10 binding to N-glycans attached to CD52, recognizing only α-2,3 SA-Gal linkage, but not O-linked or N-linked α-2,6 SA linkage. CD52-HMGB1-Siglec-10 trimolecular complex recruits SHP1 and interacts with the T-cell receptor. CD52-bound Siglec10 is then phosphorylated in ITIM motif and associates with SHP1. The phosphorylated SHP1 inhibits the TCR signaling by decreasing phosphorylation level of TCR-associated Tyr kinases such as Lck and ZAP-70. This is the mechanism of T-cell suppression [187]. The CD52-HMGB1-Siglec-10 interacts with the TCR and

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attenuates the hyperactivated T-cell response. For the mechanism with CD52HMGB1-Siglec-10 complex, HMGB1 protein is essential for suppression by CD52. Soluble CD52 can be used as a therapeutic agent. Soluble CD52 requires HMGB1 Box B to bind to Siglec-10, and α-2,3 sialylated CD52 is crucial for HMGB1 interaction and suppressor function. SA is required for the complex formation. The schematic illustration of the CD52 glycan recognition to the proinflammatory B box of HMGB1 has been expressed for engagement of the Siglec-10 toward downregulation of human T-cell function (Fig. 3.10). This effect is significantly decreased when HMGB1 antibody was treated. Altogether, this mechanism underlying the T-cell suppression provides homeostasis of inflammatory responses as a possibility as therapeutic target in inflammation-associated diseases.

3.9.3

Biosynthesis, Structural Assembly, and Transportation of GPI-Anchored Protein

The overall GPI-AP biosynthetic pathway has been established with their structural remodeling and transporting mechanism(s). GPI species is generated through the ER synthetic pathway through amination by specific GPI-transamidase using en bloc transferred target proteins. Regarding biochemical synthesis, GPI formation in the ER is started from free phospho inositol species and transamidated en bloc to proteins through enzymes produced by PIG genes. The initial two synthetic steps are found in the ER cytoplasm region, and the synthesis event is continued on the ER lumen side. For GPI attachment, precursor proteins bear their GPI-attachment signals with three distinct regions of a ω site, a hydrophilic spacer, and a

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hydrophobic region. The protein precursor candidates are independently prepared and catalyzed by the complex of GPI-transamidase enzyme. Therefore, the precursor proteins are posttranslationally modified for attachment to GPIs by the complex enzyme. Later, the glycan and lipid parts are further matured through multiple genes called post-GPI-attachment to protein (PGAP) genes. Once proteins are posttranslationally modified with GPI, structural shift between the GPI glycan and lipid occurs by PGAP genes. The acyl chains are generally the palmitic acid in GPI-AP, indicating most acyl chains are palmitic acid in GPI-AP and the palmitic acid is cleaved by a deacylase enzyme, called PGAP1. The deacylase named PGAP1 cleaves off the acyl chain from the inositol backbone. Similarly, PGAP5 cleaves a phosphoethanolamine (EtN-P) from Man-2 residue, which is responsible for recognition and delivery of p24 family protein to the ER. When GlcN-PIs are flipped across the ER lumen side, GlcN-PIs are additionally modified with an inositol acyl chain. PGAP5 cleaves off an EtN-P from Man-2 to afford the sorting to the ER. During trafficking via secretory vesicles, which is initiated at the ER sites, to the Golgi lumens and cell surfaces for fatty acid modification, COPII vesicles are cooperated with GPI-Aps, where the COPII vesicles are associated with Sec24C and Sec24D proteins. Therefore, the GPI-APs are fused with COPII vesicles with cooperation with Sec24C and Sec24D isoforms, for transportation to the cell surfaces via the Golgi network which further remodels structures such as fatty acid remodeling. GPI-APs delivered to the Golgi network are further remodeled through the process of fatty acid remodeling for the GPI-AP-lipid rafts. PGAP3 removes the unsaturated lipid chain at the sn-2, and PGAP2 adds a saturated lipid chain like stearic acid chain. Sequential modification of the GPI lipid composition reorganizes the membrane lipid raft-competent GPI-APs. The PI in the GPI has the sn-2 diacyl and unsaturated lipids including 1-stearic acid and 2-arachidonic PI. For example, 1-stearic and 2-arachidonic PI are the diacyl PIs. When they are flipped into the ER lumen side, GlcN-PIs are linked to an acylated fatty acid species, representatively like palmitic acid, and the inositol, and thus, the lipid part is exchanged. For synthesis of GPIs in the ER lumen, alkyl lipids such as alkyl-acyl donors come from peroxisomes where alkyl lipids synthesized are used for the 1-alkyl-2acyl formation. Alkyl-acyl donors come from peroxisomes. PGAP1 cleaves an acyl group from the GPI-AP inositol in order to recognize p24 family proteins and GPI-AP delivery to the ERES and remodel glycans by PGAP5. GPI-APs in the Golgi are further remodeled for fatty acid, which are associated with lipid rafts. PGAP3 removes the unsaturated lipid linked to the sn-2 and PGAP2 adds the saturated lipid chain. Biosynthesis of GPI is started on the ER cytoplasmic side by GlcNAc transfer using the donor UDP-GlcNAc to the PI as the acceptor substrate. Thus, the formed GlcNAc-PI is produced by GPI-GlcNAc transferase, a complex monoglycosyltransferase [188]. GlcNAc-PI species is further subjected to the deacetylation reaction to form the product glucosamine-PI by a deacetylase. Flippase is essential for cell survival. During the GPI-AP biosynthesis, GPI is generated from free PI resided in the ER via ten more reactions. 20 PIG genes involve in the synthesis pathway. The initial two steps are operated on the ER cytoplasmic outlets,

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and other continuing reactions occurred at the ER lumen side. Alkyl lipids are supplied from peroxisomal organelle and converted to the 1-alkyl-2-acyl-lipid component of GPIs anchored in the ER luminal side. Paroxysmal nocturnal hemoglobinuria (PNH) occurs at this step, caused by PIG-A defect. Current-reported GPI-deficient disorder is raised by enzymatically defected PIG-M or PIG-V gene mutations at gene levels. Among PIG genes, genetic defected deficiency in PIG-A, PIG-M, or PIG-V gene causes for PNH and some GPI deficiency diseases. For the GPI-synthetic gene-defected diseases, defected GPI genesis may block cell-surfaced GPI-AP location and consequently cause early lethality in embryonic development. However, inherited diseases also occur due to the restricted synthesis of GPI-APs. The disease-causing factor in the patients is a point mutation of the transcriptional factor Sp1-recognition site in the PIG-M gene 5-flanking region. The PIG-M gene encodes the Man-transferase for the attachment of Man-1. The point mutation of Sp1-binding cis-element on the 50 -flanking promoter region of PIG-M gene diminishes the PIG-M protein synthesis. The PIG-M promoter mutation-derived disease phenotype expresses the seizures and venous thrombosis. Administration with a histone deacetylase inhibitor, sodium butyrate, prevents and improves the PIG-M phenotype with GPI-Ap distribution in cells. Another defected GPI-Ap derived from the PIG-V gene mutation, where Man-2 is produced by the PIG-V gene for the Man-transferase enzyme, has been known. The PIG-V gene mutation raises hyperphosphatasia mental retardation (HPMR) disorder, termed Mabry syndrome. The HPMR belongs to an autosomal recessive (AR) form of the inherited diseases. The disease exhibits an abnormal retardation in mental behavior and facially abnormal features. It shows extremely high alkaline phosphatase activity [189]. Just four point mutations are known in the transmembrane domain gene and cause the reduced PIG-V gene expression with the reduced location of surfaced GPI-Aps [190].

3.9.4

GPIs in Parasites

GPI anchors function for stable association of proteins with the plasma membrane, but with measurable “off-rates” from the membrane and the potential to be shed by phospholipases. The very high lateral mobility is potentially obtained on the plane of the lipid bilayer. They insulate the protein domain from the cell interior because this is important in protozoa. They participate in signaling through association with other membrane-spanning components in lipid rafts. In human African sleeping sickness of Trypanosoma brucei, Trypanosoma forms in blood smear from patient with African trypanosomiasis. The surface coat is made of a dense monolayer of VSG. Many protein variants are common GPI anchors. GPI anchors from T. cruzi act as TLR2 ligands and TLR4 ligands. With regard to innate immunity and malaria caused by Haemosporida, the life cycle is complex. They undergo schizogony in the infected body of vertebrates and gametogony in the intermediate hosts. Plasmodium in RBC does not interact with cytosols but forms a called parasitophorous vacuole and proliferates in a way of

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Fig. 3.11 Camillo Golgi (1843–1926), an Italian biologist and pathologist. He studied on the central nervous system at the University of Pavia during 1860 and 1868. His discovered a staining a black reaction called Golgi’s method or Golgi’s staining in 1873. Several findings including Golgi apparatus/ tendon organ/tendon reflex were made. Golgi and Santiago Ramón y Cajal was a recipient of the 1906 Nobel Prize in Physiology or Medicine for his discoveries on the nervous system structure. Adapted from https://en.wikipedia. org/wiki/Camillo_Golgi

schizogony. Periodically, they destroy erythrocytes or RBCs. Currently, only cultivable plasmodium strain is the Plasmodium falciparum, which is resistant to chloroquine. In the liver, forms of sporozoites are produced, and one sporozoite produces thousands of merozoite and they destroy the liver organ. They are released to blood circulation and enter to RBC. This status is called malaria. Schizogony reproduces through its multiple asexual fission. Innate immune receptors of host immune cells rather mediate the systemic inflammation during malaria progression. Like most infectious diseases, the pathology of malaria is also driven by inflammatory cytokines. Consequently, cytokine production results in the symptoms of malaria with fever, chills, rigors, headaches, myalgias, lethargy, and more relatedness. Malaria pathology involves in the following three steps of the excessive production of inflammatory cytokines with septic shock-like syndrome, severe anemia with RBC destruction and destruction of infected red blood cells with erythropoiesis inhibition, and adhesion of infected RBC on capillary blood vessels. For the malaria toxin hypothesis, in 1889, Camillo Golgi (1843–1926), an Italian biologist and pathologist, reported the malarial fever paroxysms. The paroxysms are observed when the protozoan forms of schizonts rupture from erythrocytes of host and release newly produced merozoites (Fig. 3.11). Innate immune receptors are mediators of systemic inflammation and pathogenesis of malaria. Innate immune receptors for malaria parasites define parasite targets for innate immune receptors, identify relevant innate immune receptors, define their role on host-parasite interaction and disease outcome, and elaborate prophylactic and therapeutic interventions employing TLR agonists or antagonists. Plasmodium parasite components activate innate immune receptors toward release of inflammatory mediators. Plasmodium

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membrane GPI anchor functions as LPS-like roles and this is thus the malaria toxin. Merozoite GPI anchor induces inflammation and TLR2 activation but to less extent of TLR4. Malaria PAMP is the hemozoin that is the hemin known as protoporphyrin IX’s crystalline polymer and as hemoglobin component. During the initial malaria study, there were numerous questions on the malaria toxification. For example, how does malaria detoxify the protoporphyrin IX (hemin)? What are the innate immune receptors involved on hemozoin recognition?

3.9.5

GPI Interaction with TLRs in Malaria P. falciparum

What is hemozoin? It is a malaria pigment. Hemozoin is the crystalline breakdown product of hemoglobin. It is how malaria detoxifies protoporphyrin IX, also known as hemin. Hemozoin is present in parasitophorous vacuole. Hemozoin chemistry in solution exhibits that hemin chloride can form β-hematin dimer by bonding between carboxylic groups and heme synthetic hemozoin. Therefore, highly purified hemin lacks cytokine-inducing activity. Hemozoin level in circulating phagocytes correlates with disease severity. For the hypothesis, hemozoin has been regarded as a potential candidate for a proinflammatory substance made by malaria. Toll receptors act in the pathogenesis of malaria through the recognition. For example, the purple sea urchin has 222 TLRs [188, 191]. Shizuo Akira discovered that TLR9 elicits innate immune response upon binding to the malaria pigment hemozoin [192]. When malaria parasites enter to red blood cells (RBCs), the malaria degrade heme components of hemoglobins present in host RBCs and form a hemozoin (HZ), which is a heme polymer with hydrophobicity. The HZ is consequently liberated to the plasma blood and circulated through the blood. The circulating HZ species are consequently captured by the reticuloendothelial system and concentrated in the system. The HZ species strongly induce the host immune responses, although the acting mechanism(s) of the HZ regulation of the innate immunity is(are) not molecularly clearly studied yet. However, the precisely studies evidences indicate that HZ species isolated from the malaria parasitic P. falciparum act as a specific ligand for TLR9, although the HZ is not a DNA-based ligand. The HZ activity to induce the host innate immune responses was demonstrated. The HZ induces the expression of cytokine/chemokines responsible for inflammation response. In addition, the HZ upregulates co-stimulating receptors. In the TLR9-deficient, TLR9/, and MyD88/ animals, the inflammatory induction of innate immune responses is severely impaired. However, the other receptor-deficient animals including Toll/IL1R domain-containing adaptor-inducing IFN-β/, TLR2/, TLR4/, or TLR7/ mouse are not impaired for induction of the inflammatory responses. For the reverse demonstration, the experimental results obtained from the synthetic HZ, which is purely synthesized without any contaminant, TLR9-dependently exhibit in vitro induction of the innate immune responses. Interestingly, the prescribed anti-malaria drug, chloroquine (CQ), abrogated the HZ-induced expression of the related cytokines. Therefore, HZ is concluded to induce the TLR9-driven and

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MyD88-associated innate immune responses, which is highly sensitive to CQ drug. In addition, HZ is a key regulator of interactions between malaria and hosts. Natural hemozoin activates cytokine production via TLR9/MyD88. The stimulatory activity of hemozoin was destroyed by Dnase. Bacterial DNA is non-methylated and rich in CpG, and immunologically active CpG-rich DNA is recognized by TLR9. Shizuo Akira discovered for the first time that hemozoin constitutes the first non-nucleotide ligand for TLR9. Thus, the cytokine-inducing component of hemozoin is DNA. PCR analysis of hemozoin shows that most of its DNA is malarial. Is the DNA on hemozoin human or malarial? Can malaria DNA actually activate cells through its specific receptor TLR9? The biological property of TLR9 is complex. Although TLR9 resides in the ER, TLR9 translocates to the endosomal compartment to bind its ligand known as CpG-rich DNA. Then, does malaria DNAs activate innate immune responses? In fact, malaria DNA is stimulatory for DCs only when introduced into endosomal compartment of cells. Malaria DNA is stimulatory for pDCs only when introduced into cells. The malaria genome contains 269 CpG repeats. Hemozoin functions to traffic DNA into an intracellular compartment to which TLR9 can be recruited. However, most malaria DNA is AT-rich. The malaria genome contains the motif ATTTTTAC over 6000 times. Microarray analysis of 14 patients with febrile P. falciparum showed an IFN signature. Hemozoin-DNA rapidly activates IFN-β production in PBMC, but most malaria DNA is AT-rich. In addition, AT-rich DNA mimics malarial DNA, and transfection of AT-rich DNA drives a variety of promoter constructs like native DNA and activates cytokines such as TNF-α, IL-1β, IL-6 and IFN-β. When six AT-rich motifs of AT1–AT6 have been studied, AT-2 is the GCACACATTTTTACTAAAAC. Human PBMC produces IFN-β in response to AT-rich DNA. AT-rich DNA activates type I IFNs independently of TLRs. AT-rich DNA activates type I IFNs independently of TLRs but dependently on IRF1. Infection with Plasmodium leads to a proinflammatory priming and hyperresponsiveness of TLRs. In mice, TLR9 has an important role in promoting this proinflammatory priming, which is mediated by IL-12 release by dendritic cells and IFN-γ release by T and NK cells. Treatment with an antagonist of nucleic acid sensing TLRs prevents cytokinemia and lethality in a rodent model of cerebral malaria. Can we interfere with pathogenesis of malaria by blocking MyD88 activation? Phagocytized hemozoin colocalizes with TLR9 in the lysosomes. The inflammatory component of hemozoin is DNA. CpG and AT-rich DNA activate different innate immune pathways [193, 194]. In step 1, internalization of hemozoin by phagocytes leads to activation of MyD88 via TLR9 and leads to IFN-g production, inflammasome formation, and caspase-1 activation. In step 2, IFN-γ priming of phagocytes enhances expression of TLRs and pro-IL-1β, TNF-α production, caspase-1 fragmentation, and maturation for inflammation. Therapeutic modulation of nucleic acid sensing TLRs can be the potential target to prevent cerebral and blood malaria symptoms [195]. For malaria hypothesis, hemozoin is internalized into the phagolysosome. TLR9 can be recruited to the phagosome, thus resulting in the proinflammatory mediator genesis such as TNF-α, which causes fever. Later, its DNA fragments are liberated and released from the cell surfaces via the crystal forms by the enzyme activities of

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Modulaon of innate immunity by African Trypanosomes Host • • • •

Infecon progress

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(glycophosphadylinositol)

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$3&

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② Unable to present African Trypanosome

Fig. 3.12 A proposed modulation of innate immunity by African trypanosomes. Deduced from Ref [196]

proteases and other enzymes. Then, why do humans get fever during malaria? The search for the malaria toxin has been started through the world. What innate immune receptors are activated during disease and what microbial products such as malarial toxins activate these receptors? Is the source of inflammation a component of the parasite outer membrane? Is malaria like LPS and Gram-negative bacteria? Is the source of inflammation a component of the merozoite outer membrane? In other words, is the malaria toxin just like LPS? The malarial parasite is coated with a GPI, which activates TLR2. Malaria GPI structures trigger innate immune responses in hosts. GPI anchors from T. cruzi are also TLR2 ligand and, to a lesser extent, TLR4 ligands. GPI-anchored proteins of P. falciparum have the TLR-inducing activity. In the protozoan infectious diseases such as Brucei group African trypanosomes or malaria, the host innate immunity recognizes trypanosome PAMPs expressed as a type of GPI-anchored and shed membrane-bound variant surface glycoprotein (VSG) molecules. GPI substituents induce proinflammatory macrophages and DC responses [196]. Next, the polarized VSG-restricted Th cells express type 1 cytokines. GPI activates the host innate immune system (Fig. 3.12). In malaria, there has been a big question of “Is there any source of inflammation as a component of the merozoite outer membrane? In other words, is the malaria toxin just like Gramnegative bacterial LPS? Currently, it has been accepted that GPI anchors activate TLR2, as the malaria is coated with a GPI anchor. For example, GPI anchors from T. cruzi are TLR2 ligand and, to a lesser extent, TLR4 ligands. GPI anchors from P. falciparum have the similar TLR-inducing activity. For structure of malaria GPIs, as they trigger innate immune responses in hosts, GPIs are frequently expressed in protozoa rather than animals with immunostimulation of the host [197]. Such known

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pathogenic protozoa include Trypanosomes, Leishmania, Toxoplasma, and Plasmodium species. GPIs are the malaria pathogenic factor or malaria parasite-associated molecular patterns (malaria PAMPs). The patterns are malaria molecular signatures on outmost membranes. The primary structural units of GPIs are composed of an ethanolamine-phosphate-substituted sugar moiety, EtN-P6Manα1,2Manα1,6Manα1,4GlcN, which is attached to α1–6-glycosidic PtdIns [198] with structural diversity. GPI in each different organism differs in the structure and composition of the lipids and sugars and/or ethanolamine-phosphates. GPIs vary in the lipid components including diacylglycerol part, 1-alkyl part, 2-acylglycerol part and ceramides), inositol acylation, and ethanolamine-phosphate groups apart from Man C-6 [199]. Malaria GPIs are anchored with several proteins such as MSP-1, MSP-2, MSP-4, MSP-5, and MSP-10 as well as rhoptry membrane proteins, which are required for merozoite invasion to erythrocytes. The malaria GPI structure is different from GPI species produced by humans. Human GPIs contain alkyl moieties at ethanolamine-phosphate substituent on GlcN and β-GalNAc-Man. The GPI acyl groups of human sources are large size with unsaturation at the carbon sites of C22: 4, C22:5, and C22:6 [198]. The variations are related with structure diversity with different GPI roles. During infection, the schizont burst and merozoites are released. Some merozoites are recognized by the host innate immune cells. The merozoites invade RBC erythrocytes to survive [200]. Schizont components induce the innate immune responses. Malaria GPIs activate the host innate immune system [198] during malaria infection. Malaria-infected individuals have rarely anti-GPI antibodies [201], providing protective effects against malaria illness. For example, malaria, P. falciparum-bearing GPIs are regarded as malaria-producing toxins. Host proinflammatory responses are induced by the host upon malaria GPI recognition through TLR2 interaction. GPIs also interact with NKT and B cells toward establishment of adaptive immune responses, thereby producing GPI-binding antibodies of class M or G [198]. GPIs are released during malaria schizont burst. GPI lipid moieties are extracted with hydrophobic solvents. GPIs are cleaved to sugar part and PtdIns lipid part [202]. The question is: how GPIs are bound by TLRs because GPI lipid moieties are not bound by TLRs? In the aspects of GPIs and TLR-mediated signaling, GPIs activate PTK, PKC, and three MAPK species such as ERK, JNK, and p38 and also NF-κB/c-Rel factor toward proinflammatory responses [202]. GPI-anchored proteins also bind to host lectin-like receptors to activate protein tyrosine kinase (PTK). GPI phospholipase D releases diacylglycerol and it activates PKC. Malaria GPI-induced signaling has been known in macrophage and TLR-/MyD88-knockout mice and GPIs are bound by TLR2 or TLR4. In human HEK cells engineered, malaria GPIs bind to the TLR2/ 1 heterodimer, while the sn-2 lipid-deficient GPIs switch binding capacity to TLR2/6 from TLT2/1 [202]. The scavenger receptor CD36 is a co-receptor for diacyl peptides, where diacyl peptides are recognized by TLR2/6 [203]. Hemozoin different from GPIs also presents parasite DNA to TLR9. TLR9 also recognizes parasite factors. Parasite hemozoin activates plasmacytoid DCs through TLR9 [192] in such a way that hemozoin is inactive but presents parasitic DNA to DC TLR9 for

References

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proinflammatory cytokine expression. Parasite hemozoin binds DNA and the complex recognizes TLR9.

3.9.6

GPI-Defected Disorders of Paroxysmal Nocturnal Hemoglobinuria (PNH) and Prion Disease

PNH as a hematopoietic stem cell disorder is originated by a defected GPI-anchored synthesis. PIG-A is defective in PNH patients, causing that PIG-A is the causing gene for PNH. The major PNH symptom is anemia due to hemolysis caused by intravascular erythrocytic destruction. The pathologic cause is derived from the failed and incomplete surface location of complement regulators or GPI-APs CD59 and CD55 (named DAF). The incomplete or failed expression of CD55 or CD59 raises erythrocytes destroyed due to high susception to self-complement attack. The defected expression of the surfaced GPI-APs of CD55 and CD59 is caused by defects of the GPI-anchored synthesis. For example, the PIG-A as the GPI biosynthetic gene is defected in PNH patients, as found in 1993. PIG-A is a causing factor for PNH. PIG-A gene in the X chromosome causes the PNH, among the 20 more genes thought to be associated with the GPI biosynthesis. Genetic mutation in the PIG-A gene raises the defected GPI formation. In contrast, other related genes encoded on autosomes exhibit two site mutations for defected GPI synthesis. For the therapeutic agent, a humanized MAb form of termed eculizumab was generated to bind the complement protein C5, as the anti-hemolytic agent of PNH anemia. On the other hand, prion disease forms insoluble plaques in neuronal cells in the brain. This is involved in neurodegenerative spongiform encephalopathy. Proteinase-sensitive cellular prion protein (PrPc) is shifted to an insoluble and proteinase-resistant prion protein (PrPres) as the deposited plaques. PrPres is also the plaque-causing agent. PrPc and PrPres are GPI-Aps crucial for the pathogenesis. PrPres is generated at the cell surface through the endocytosis. Prion endocytosis through caveolin domain/ lipid rafts requires GPI. GPI anchors propagate and spread out misfolded Sup35, known as an amyloidogenic yeast protein. This induces prion-like protease-resistant aggregates. GPI anchor on PrPc is crucial for the infection of cells.

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Chapter 4

Glycans in Glycoimmunology

4.1

Glycans in Cell Recognition and Evolutionary Adaptation in Organisms

Carbohydrates (or glycans) are ubiquitous and display a broad range of biological functions and disease expressions. Without glycan-mediated events, any biological aspect is not possible in living organisms. For example, protein folding, cell adhesion, trafficking, signaling, fertilization, embryogenesis, pathogen recognition, and immune responses require such glycan-mediated events. The structure of glycans is complex, which is propagated and amplified by the stereoisomers, anomeric configurations, branched chains, and modifications by sulfation, methylation, and phosphorylation. This complexity is distinguished from genomics and proteomics. The face molecules of organisms are glucans, and the functions are dependent from glycan-protein and glycan-lipid interactions. Glycan carbohydrate residues are especially well fit to form a wide range of distinct sequences, due to the specific rings and chains with axial and equatorial presence of the hydroxyl groups. Moreover, the anomeric positioned hydroxyl group favorably forms α- and β-glycosidic linkages. This is one of the evolutionary selections. Then, a question how diverse glycan structures are shared with various eukaryotes is answered by the evolution of glycan-recognizing and processing proteins including glycosidases, glycosyltransferases, sugar transporters, sugarnucleotide transporters, and glycan-binding lectins. Such processed glycans known in eukaryotes are N-/O-glycans, C-mannose, glycolipids, and GAGs. Synthesis of O-/N-glycan carbohydrates is also found in the microbes. The most well-evolved microbe with the N- and O-linked carbohydrate synthesis is the Campylobacter species, because they generate both O- and N-linked glycans to their proteins such as flagellin [1]. Each specific glycan exhibits a key core unit with extended core, differentiating into strain- and phylum-depending structures. Glycans as the key molecules of organisms face the extracellular outer world, allowing a terminology of glycan © The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2022 C.-H. Kim, Glycobiology of Innate Immunology, https://doi.org/10.1007/978-981-16-9081-5_4

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diversity created in each individual environment. Therefore, diverse glycan structure influences phenotype differences [2–7]. The glycobiological process enables possibilities to acquire natural selections in exclusively allowed conditions. Evolutionlinked glycosylation creates each specific glycan to unicellular organisms with monosaccharides. Among them, the eukaryotes utilize restricted numbers of monosaccharide residues without detailed modifications. Glycoforms in each eukaryotic organism are restricted and limited due to the distinct glycosyltransferases and glycosidases, leading to specific glycan structures in individuals through the expression pattern of the related enzymes cooperated with the spatial and temporal expression behavior. Glycans are produced through multiple harmonists such as glycosyltransferases and glycosidases and have various characteristics of sugar component composition, linked sequence, and stereo-conformation depending on cell or tissue dependency [8]. Consequently, the generated glycan structures and patterns are the molecular bases for the non-self- and self-recognition.

4.2

Changes in Glycan Structure Involved in Coregulated Expression of Glycan-Binding Lectin Counterparts

Glycans and lectins or glycan-binding proteins (GBP) reciprocally control innate and adaptive immunity. The glycome is a regulator of the immunity. The development, manifest, display, and function of the immunity rely on glycan structures and GBP as well as their interactions. Lectins recognize glycans for diverse roles of cell sociology. Carbohydrate recognition domain (CRD) is known in the animal or human lectins. The examples are the C-type (referred from Ca2+-dependent glycan recognition), I-type (referred from Ig-like domain specificity), and P-type (referred from Man-6-P recognition specificity) domains. During long historic evolution, structural variations within their genes and protein sequences have been made to the scaffold homologous proteins, consequently generating a wide range of the C-type lectin families [9]. In inflammatory responses, cellular glycosylation event and substitutions with sulfate groups as well as selectin expression are coregulated to recognize modified glycoprotein glycans, as known for the examples of CD34, CD43, and CD44 (Hutch-I, Hermes antigen). In tumor metastasis, glycans of proteins in CD11/ CD18, CD24, or CD66 are coregulated to be recognized by selectins [10]. Such example is also reported in a variant myeloperoxidase that has sialyl and fucosyl glycans as a CD62E (E-selectin, ELAM-1, LECAM-2) counter-receptor on human myeloid cells which are also expressed upon exposure on granulocyte colonystimulating factor [11]. In tumor suppression of tumor biology, the defected tumor suppressor activity causes growth. Tumor suppressor p16INK4a is known to trigger anoikis in pancreatic cancer cells through the galectin-1 and desialylated glycan in α2,6-sialylation with the reduced level of the antiapoptotic galectin-3 [12–14]. In detail, the reengineered pancreatic carcinoma cells having a tumor suppressor p16INK4a gene expression exhibited the coregulation of glycan biosynthesis

4.3 Evolution of Lectin: Alternative Splicing Contributes to Variation for. . .

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genes such as glycosyltransferases and sialyltransferases [15] with expression of two lectins, thereby enabling for the tumor cells to induce lectin-dependent anoikis [12– 14]. More specifically, the reduced α2,6-sialylglycans present in the tumor cell surface are more susceptible for the recognition of fibronectin receptor (FR; α5β1integrin) and galectin-1 recognition, and this interaction consequently activates apoptotic downstream signaling [12, 14]. In addition, expression of galectin-3 as an antagonist to galectin-1 is simultaneously decreased in order to physiologically respond to galectin-1. The glycosylated fibronectin receptors known as α5β1integrin and galectin complexes activate caspase-8. Glycosylation enforced by microenvironmental agents of NO [16] or proteins is not directly related to glycosylation caused by the Rho GTPase Rac1 [17]. In those cases, surfaced α2,6sialylation event is regulated. Desialylation by neuraminidases regulates the hyaluronic acid-CD44 binding [18]. For the same action, neuraminidase activity converts GD1a ganglioside to GM1 form, which is bound by galectin-1 [19, 20]. Considering that lectin is differentially expressed between human and mouse, neither α1,3-gal residues nor NeuGc residue sialylation of N-glycans is found in humans. In addition, CD15 (X-hapten, LeuM1, SSEA-1, Lex), CD75 (LacNAc), and branched N-glycans are highly present in immune cells of humans than mouse [21]. Galectin-1 can bind to GM1 ganglioside in some neuroblastoma cells and the activated effector T cells by the microenvironmental assists of neuraminidase or sialidase that cleaves the terminal SA residue from abundant GD1a moiety into the active counter-receptor GM1 [22–25]. The sialic acid removal facilitates the galectin-1 binding to GM1. Thus, dynamic reconstituting of the cell surface glycans is an effective means for the responsiveness to distinct lectins, without any additional glycan synthesis. The counter-receptor recognition by cellular lectins correctly translates cellular signals.

4.3

Evolution of Lectin: Alternative Splicing Contributes to Variation for Glycan-Binding Receptors

In the side of glycans, glycan structures are timely changed, although glycan structures created from organisms are not genetically encoded. Extracellularly, organisms have acquired glycosylation events as a form of their adaptation to extracellular environments and intracellularly to reflect cellular activation or differentiation. In the side of glycan-binding lectin, during environment-based adaptation and evolution, glycan affinity of lectins has been modulated by sugar structures of monosaccharides and oligosaccharides as well as the three-dimensional recognition of the glycan code. Glycans affect the lectin reactivities. Alternative splicing-derived protein variations alter the acceptor specificity of the glycoprotein and explains the glycan roles of glycoproteins [26].

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E-Selectin-Binding Ligand sLex (CD15s) on Neutrophil CD44 N-glycan and Alternatively Spliced Exon 6 Contains Core 2 O-Glycan sLea (CD44v6) Epitope

As an evolution strategy, eukaryotes have obtained alternative splicing of protein genes. In glycan structural aspects, glycan sequence and structure can be altered by the alternative splicing of protein genes. Thus, alternative splicing event is a regulation type. Glycan affinity of lectins is therefore regulated by the lectin-binding carbohydrate epitope density or number obtained by the glycan core or by branched multiantennary epitope. For example, desialylation unmasks contact sites for a lectin. Leukocyte transendothelial migration is a key step in their recruitment to sites of inflammation by synergic regulation of endothelium-expressed selectins. Selectin and immunoreceptor engagement activate leukocytic integrin affinity, while ligand-induced integrin clustering likewise stabilizes adhesion and initiates transmigration. Homing of leukocytes and their migrative extravasation are forced by selectin-glycan bindings in the endothelial cells on vascular vessels closed to inflammation region and infected tissues. Another case to blocks in immune response is obtained from glycan-synthesizing enzyme-deficient cells. For example, deficiency of GCnT-1, a core 2 β1,6-GlcNAc-transferase, exhibit decreased DCs homing on L-, P- and E-selectin-expressing cells and blocking DCs homing to inflammation area. The glycan-decorating epithelial barrier is a starting line of defense from pathogenic invaders. However, GCnT-2-deficient animals, which encodes the core 2 β1,6-GlcNAc-transferase, are susceptible to infection and inflammation. In addition, the distinct and dynamic contribution of P- and E-selectins mediates β2-integrin-induced PMN transmigration [27]. E-selectin-binding leukocyte ligands are known for CD43, CD44, and PSGL-1. For example, glycan determinants such as CD15s (sialyl Lex) (Fig. 4.1) or CD176 [Thomsen-Friedenreich antigen (TF), Galβ1,3GalNAcα1-O-R, mucin-type O-glycan to protein] have rather become counter-receptors for lectins. E-selectin interacts with PSGL-1 or CD44 ligand towards Src family kinase-integrin αLβ2-mediated leukocyte rolling. P-selectinPSGL-1 binding induces αMβ2-ICAM-1 interaction to potentiate adhesion [27]. CD44 is not a mucin-type protein but modified only with N- and O-glycans [28]. CD44’s N-glycans recognize E-selectin [29]. CD44 and PSGL-1 form membrane lipid rafts microdomain on leukocytic cells. Interaction between selectin and Fig. 4.1 Sialyl-LeX

4.5 Glycans Regulate T Cells

119

CD44 or PSGL-1 expressed on myocytes or any equivalent cells contributes to SKF phosphorylation and signaling mediators, which transduce β2-integrins into active status with extended, intermediate affinity, slowed rolling, and contributed arrest levels [30]. In the leukocytic rolling status, selectin recognition with CD44 or PSGL1 activates a signaling pathway, like the T cell receptor signaling cascade [10]. For example, ligand clustering with non-rafted T cell receptors and with rafted coreceptors initiates downstream signaling. In fact, the PSGL-1-/CD44-involved signaling is mediated through the β2-integrin transduction event and rolling to ICAM-1 [10]. P-selectin-PSGL-1 binding activates β2-integrins and initiate PMN transmigration, whereas E-selectin engages CD44 to influence PMN transmigration, indicating the interaction of P-/E-selectins and their ligands to promote PMN transmigration. The glycoprotein CD44 (Hutch-I, Hermes antigen) has its globular domain in the extracellular region and transmembrane (TM) domain, as spaced by a stem. The conventional stem region is the site responsible for structural variation through alternative splicing to produce variant isoforms. A single gene primary transcript is alternatively spliced between exon No. 5 and No. 16. The CD44 isoform is the removed form for stem extensions. CD44 N-glycans of blood polymorphonuclear neutrophils (PMN) contain sialyl Lex antigen known as CD15s epitope Neu5Acα2,3Gal β1,4(Fucα1,3)GlcNAc that is a counter-receptor for CD62E (E-selectin, ELAM-1, LECAM-2) [31], and the additional exons facilitates the CD44 to have binding sites for O-glycans/mucin types. The exon 6 variant known as CD44v6 is expressed in neutrophils in the pathologic lesion of ulcerative colitis, as SLeA epitopes on core 2 O-glycans modified by α1,3/4-Fuc-transferase (FUT3). By help of the biantennary chains, CD44 can induce mucosal inflammation during cell detachment into the blood lumen and transepithelial migration events [32]. Thus, alternatively spliced forms are responsible for their distinct roles.

4.5

Glycans Regulate T Cells

Glycosylation regulates and controls host immune responses through T cell regulations such as thymocyte precursor development and T helper subset differentiation [33]. N-glycan branching is associated with the immune system. Glycosyltransferases involve in N-glycan branching and their target proteins with various biological functions. N-glycan biosynthesis is distinctly specialized for transfer en bloc, conserved mechanism during evolution, transmembrane GTs, and monogenic substrate specificity. For the folding and quality control of glycoproteins, the synthesis is also well conserved for the step-by-step transfer, interspecies variability, and type II membrane GTs with stage-specific and tissue-specific expression. GTs biosynthesize N-glycans in rough ER and Golgi apparatus. N-glycan branching enzymes exist in Golgi. During thymus development, the antennae N-glycans are branched five-fold more from SP or DN thymocytes. Thereafter, during the transition stage from SP to peripheral T cell types, the N-glycan branching level is declined two-fold more [34]. Hence, the N-glycan branching changes result in the

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level of TCR clustering during thymocyte development. Autoimmune diseases are caused by self-antigen-specific abnormal immune responses. The N-glycans are associated in T cell-driven autoimmune responses. In fact, development and progression of inflammatory bowel disease (IBD), systemic lupus erythematosus (SLE), multiple sclerosis (MS), and insulin-dependent diabetes mellitus (IDDM) are related to the N-glycans. Additionally, complex type of N-glycans essentially recognize the MHC peptide and TCR interaction towards regular T cell development [33]. Changes in glycosylation are frequently observed during activation, development, and differentiation into effector T cells. Ultimately, glycosylation and glycosylated target molecules control a stage-specific pathway of T cell fate and life. Naïve T cells are metabolically quiescent using consuming ATP produced by oxidative phosphorylation. During early development of T cells, variously different T cell subsets are made by distinct glycosylation patterns. Functionally, T cells are developed, learned, educated, and matured in the thymus via final selection pathway through TCRs to phenotypically express an antigen recognition repertoire [35]. The stage-specifically expressed glycans influence the T cell development, and glycosylation also controls thymocyte development, where T cells display alterations in glycosylation for thymus development as well as peripheral activation and survival. T cells alter glycosylation process during thymus development and peripheral activation. Alteration in T cell glycosylation regulates T cell functions. For example, glycans present in two abundant glycoproteins CD43/CD45 expressed in the T cells are dramatically regulated during development, activation, and survival of T cells. N-glycosylation is involved in the thymus seeding and speciation of T cell lineages. N-glycans expressed in T cells elaborately control the T cell roles in activation, development, differentiation, and signaling. Peripherally circulating naive T cells and thymus-resident T cells also produce sialylated core 1 O-glycoproteins to regulate their fates. O-glycans also influence T cell development, for example, upon O-glycan-galectin binding. Among galectins, galectin-1 triggers apoptotic event of immature thymocytes via galectin-1 recognition by core 2 O-glycan structures present in T cell coreceptors of CD43 and CD45. In the special condition that CD45 bears α2,6-sialyl core 1 O-glycans and α2,6-sialyl N-glycans, CD45 on mature thymocytes is resistant to galectin-1 binding [26]. Upon binding to the altered glycosylate antigens, several immune events including antigen capture, antigen presentation to Th cells, cross-presentation to CTL, and anticancer response are progressed. GBPs recognize self by cell adhesion via DC-SIGN and ICAM-1/ ICAM-3 binding and by T cell signaling via GalNAc-bearing CD45 glycoform and MGL binding as well as by discrimination of danger patterns via HMGB1 and Siglec-G/CD24 binding and APC immune or tolerance regulation via galectins binding to APC glycoproteins of Tim-3 and CD43.

4.5 Glycans Regulate T Cells

4.5.1

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Glycans Regulate Development and Differentiation in T Cells

After born in BM, initiation of T cell development and thymic progenitors trafficking to the thymus depend on the P-selectin expression in the thymus epithelial cells or PSGL-1 present in circulating thymus progenitors [36]. α1,3Fucosyl PSGL-1 is a binding site of P-selectin. When thymus progenitors are homing into the thymus, the progenitors bind to α1,3Fucosyl PSGL-1, too [37]. In autoimmunity or cancer, the question of how glycans modulate multiple steps of thymocytic development is raised to answer. The elucidation of the glycan-encoded immune responses is therefore a goal of this chapter. Glycosylation of surfaced membrane receptors on T cells directs T cell functions. In addition, glycans are involved in tolerogenic and immune suppressive responses in cancer progression and autoimmune responses. Alteration of the glycan structures in T cell receptors and tumor cells as glycan code of tumor can modulate the immune response to suppress immune pathways. Such immune suppressions are frequently occurred in the tumor-associated microenvironment with tumor immune escape [38, 39]. Glycans generate the lost immunological tolerance in autoimmune response, giving tolerance in cancer progression. Glycans may exhibit dual roles of immune inhibiting checkpoints or stimulating signals. Elucidation of the glycan roles responsible for autoimmune response and cancer progression will create insights into new concept of potential targets or markers for the immunomodulatory drugs. Immune cells are step-wisely developed from the primitive progenitor cells and further differentiated into each functional CD-expressing cell type. For example, the T cell subpopulation in development is mainly early thymocyte progenitors (ETP). The ETP include various CD-specific subpopulations. Representatively, double negative (DN)-1/DN-2/DN-3/DN-4, double positive (DP), and single positive (SP) populations are classified. The glycosylation of SP subpopulation gives the functional T cell function that can discriminate counterparts via its distinct CD. However, T cell-surfaced glycans act as players in many immunological behaviors including immune unbalance, autoimmunity incidence, and cancer progression. The causing reasons are basically based on that changes in T cell glycosylation pattern often induce reprogrammed and reconstituted immune stimulation as well as immune tolerogenic responses. The interplay of T cell glycans confers both autoreactivity and self-tolerance of T cells. For glycoenzymological synthesis of the T cell interplay, several glycan synthetic GTs are known to form glycans required for T cell function. Eukaryotic proteins are matured by Asn glycans or Ser/Thr glycans at the ER/Golgi networks by means of posttranslational modification. N-glycans are formed at protein N-X-S/T region and are processed by α-mannosidases and GlcNAc-transferases (Mgat)1, Mgat2, Mgat4, and Mgat5 using donor UDP-GlcNAc substrate via the hexosamine biosynthetic pathway (HBP). Glc, glutamine, or GlcNAc residue is used for the UDP-GlcNAc synthesis of the HBP. The product UDP-GlcNAc is then served for the N-glycan synthesis in the ER/Golgi

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apparatus. Mgat1 transfers GlcNAc to Man5GlcNAc2-Asn to start the N-glycan synthesis. Therefore, Mgat1 yields the first antennary. Mgat1 KO mouse embryos lack branching of protein N-glycan with developmental lethal and retardation effects [40]. Some Mgat1 polymorphisms upregulate the mRNA level and enzymatic activity. Consequently, the Mgat4- and Mgat5-mediated UDP-GlcNAc usage is reduced and decreases in N-glycan branching level [41]. Mgat2 yields β1,2 GlcNAc branch with bi-antennary N-glycans as the second antenna. Lack of Mgat2 yields incomplete complex N-glycans with bisecting N-glycan structure in mice [42] with gastrointestinal, hematologic, and osteogenic dysfunctions, like human Mgat2 deficiency phenotype of CDG-II [43]. The Mgat2KO mice to target T cells (Mgat2f/f/Lck-Cre) show severe EAE [44]. Mgat2 lacking remarkably reduces N-glycan branching, compared with Mgat5 deletion. In the Mgat1 deletion [34] or Mgat2 deletion [34, 42] mice, the mice show N-glycan branching-deficient phenotypes. In addition, the total T cell number present in the thymus and spleen tissues is reduced. Mgat4a further branches the β1,4-GlcNAc branches in N-glycan core Man and yields tri-antennary N-glycans. Mgat4a-formed tri-antennary N-glycans bind to galectin-9 present in β island cells of the pancreas. Lack of Mgat4a displays disease phenotypes such as hyperglycemia, insulin resistance, and obesity in mice [45]. The β cells isolated from Mgat4a-transgenic mouse exhibit the insulin sensitivity and ameliorates non-insulin-dependent diabetes [46]. Hence, Mgat4a-catalized branching in Glut2 N-glycans may bind to galectin9 giving prolonged retention time. Mgat5 enzyme forms tetra-antenna N-glycan structures. MGAT5 gene encodes the β1,6-GlcNAc-transferase-V (GnT-V) enzyme, which generates a β1,6-branching enzyme on the complex N-glycan type [47]. The GnT-V branches the antennary structures of N-glycans and is known to control T cell functions. Mgat5-KO mice are hypoglycemic and hypersensitive to fasting time [48, 49]; however, the insulin responses are unchanged. The increased glucagon level indicates their lean phenotype in Mgat5-KO mice [49]. Mgat5-KO mice lead to inflammatory demyelination and neurodegeneration. In addition, Mgat5-KO mice show Tim-3+ Th1 phenotyped cells with high frequency compared to that of PL/J mouse [50]. Mgat5 expression is increased in cancer with the enhanced cell motility, epithelial-mesenchymal transition (EMT), and invasive potentials [48]. Experimental results obtained from mice models have revealed the T cell activation by N-glycans. T cells derived from tissues of the Mgat5-deficient mouse exhibit the lowered activation level threshold rather than T cells isolated from normal mice of wild type. Mgat5 deletion does not affect the T cell number and population resident in the spleen and thymus. The N-glycans with tetra-antennary branches contain poly-LacNAc glycan chains, which are used as the naturally found endogenous ligands for binding to galectin-3. The mice with Mgat5 deficiency showed increased signs of proliferative glomerulonephritis and developed autoimmune responses. In immune synapses, Mgat5 loss reduces the lattice generated from T cell-associated galectin-3 and increases TCR clustering level. Therefore, EAE and delay-type hypersensitivity (DTH) are easily developed. Mgat5-branched N-glycans further attenuate differentiation of Th1 [50] and Th17 cells [51].

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In addition, in human CTL antigen (CTLA)-4, its glycans are the N-glycosylated types at the two Asn sites. One N-glycan site-lost protein is produced from the wild type having two Asn sites in CTLA-4 protein via the Thr17Ala mutagenic polymorphism which is produced in human CTLA-4. The one Asn site-restricted isoform increases MS incidence through the suppression of its surface retention time on T cells [41, 52, 53]. However, the branched N-glycans increased the surface retention time of CTLA-4 [54], where CTLA-4 functions mainly in T cell arrests of hosts. CTLA-4 exhibits a high binding affinity for the counterparts of CD80/CD86 coreceptors present in APCs and suppresses the T cell induction [55]. GnT-V enzyme acts to many T cell receptor proteins like TCR, CD25 (IL-2Rα), and CD4. IL-2Rα known as CD25 is highly glycosylated in N-glycans and O-glycans/mucin type [56]. IL-2Rα (CD25) consists of three distinct N-glycosylation sites. IL-2Rα (CD25) response is activated by N-glycans. The N-glycan number influences T cell growth and differentiation. N-glycosylation inhibition suppresses the surface retention time of IL-2Rα (CD25) present in T cells and IL-2 signaling. The inhibited CD25 suppresses Th-1, Th-2, and Treg cell differentiation but activates development of Th17 cells [56, 57]. Glucosamine as the UDP-GlcNAc substrate in HBP [58], unlike GlcNAc, interferes with N-glycosylation [59]. Glucosamine is known to inhibit inflammatory immune responses and autoimmune diseases [60]. For example, glucosamine administration attenuates differentiation of T cell subsets of Th1, Th2, and Tregs but remarkably induces Th17 cell polarization by blocking the CD25 N-glycosylation and signaling. The attenuation effect of glucosamine is almost the same as those effects obtained from the N-glycosylation blocker tunicamycin treatment. As expected, the restricted glucosamine dose exacerbates the EAE level by enforcing Th17 cell differentiation [56]. This inhibitory effect is also similar to that of non-branched N-glycan formation in EAE incidence. In addition, Glc and glutamine prevent Th17 cell differentiation and also lead to switching to iTreg cells by branched N-glycan and subsequent elevated CD25 retention on cell surface [57]. Such Glc and glutamine supplementation activates N-glycan synthesis and hence increases the surface retention time of CD25 [57]. Tregs or activated T cells produce CD25, and the CD25 recognizes cytokine IL-2 associated with IL-2Rβ and γ chains, activating PI3K/Akt/ mTOR, MAPK, and STAT5 for growth, survival, activation-induced cell death (AICD), and differentiation. Therefore, glycolysis and glutaminolysis events can collaboratively regulate T cells to develop and differentiate as well as enable selftolerance via limited N-glycosylation. Such catalyzed GNT-V-branched products as N-glycan branches influence to T cell phenotypes including proliferation, differentiation, intracellular signaling, and inflammatory cytokine expression. In TCR signaling, glycosylated surface receptor proteins are resistant to proteolysis.

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Glycosylation of Notch Receptor Signaling for Thymocyte β Selection and T Cell Function Regulation

When thymus progenitors enter the thymus, thymus progenitors differentiate into ETPs that are the CD4
CD8
DN1 subsets [26]. The Notch signaling commits CD4
CD8
DN1 thymocytes linking to the T cell populations [61]. The Notch receptors/ligands are glycosylated to transduce Notch signaling. The manic, radical, and lunatic Fringe is the GlcNAc-transferases that catalyzes the GlcNAc transfer from UDP-GlcNAc to fucosyl O-glycans in EGF-like repeated domains of the extracellular region of Notch receptor [62, 63]. Loss of the 3 manic, radical, and lunatic Fringe glycosyltransferases diminishes Notch binding to Delta-like ligands (DLL) [64]. The Fringe-catalyzed Notch glycosylation develops T cells. The lunatic Fringe gene known as Lfng is wrongly expressed by a lck promoter [65]. The defected Notch glycosylation by missed GlcNAc residue of the EGF-like repeated region makes a B cell subset differentiated from thymus lymphatic progenitors. Lfng is weakly regulated in CD4 + CD8 + DP subset thymocytes. Lfng ectopic expression increases Notch recognition with ligands present in stromal cells, inhibiting DN development but potentiating differentiation of B cells [66]. Thus, alteration in the Notch glycosylation affects T cell development. At DN stages, Notch binds to DLLs. The Lfng presence on DNs increases Notch binding to DLLs, and the Lfng defect in DPs leads to Notch-independent development of T cells. The T cell subset lineages develop at the DN3 step, and recombination-activating genes (RAG) rearrange the Tcrb and induce the TCR-β chain (TCR-β) expression and consequently yield a pre-TCR complex [61, 67]. Next, in the presence of IL-7 and Notch, the pre-TCR signaling allows β-selection through the suppressed expression of RAG complex of quiescent DN3 (DN3a) Rag1/2. The DN3a subset differentiates into cycling DN3 thymocytes (DN3b), and this is also differentiated into DN4 cell type. Loss of pre-TCR signaling can be rescued in lck-null cells by artificial Lfng expression. O-GlcNAcylation also regulates the T cell development [68]. After β-selection in DN4 thymocytes, ST6Gal-I (β-galactoside α2,6-sialyltransferase 1) expression is significantly increased up to 10 times more, compared to the DN3 thymocytes with α2,6-sialylglycans [69]. In ST6Gal-I KO mice, the DN subpopulations are decreased. In the same model of ST6Gal-I KO mice, expression of CD96, a receptor of nectin-1 in cellular migration, is decreased in the DN2 and DN3 subpopulations. In addition, ST3Gal-I expression level is reduced in DN or DP, while ST3Gal-I expression is increased in SP thymocytes [70]. In ST3Gal-1 KO mice, the TCR repertoire is altered, and thymocyte selection requires sialylation [71]. The β-selected DN4 cells exhibit rapid self-renewal and differentiation into DP CD4 + CD8+ thymocytes with the expression of TCRαß receptors [72]. TCRαβ carries at least 7 N-glycosylation sites, and TCR-CD3 complex carries 12 N-glycosylation sites responsible for TCR folding and function [73]. In addition, decreased sialylation level in DP CD4 + CD8+ thymocytes increases binding capacity to MHC-I, and the increased sialylation on differentiating SP CD8+ CTLs in the

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thymus decreases the level of MHC-I-CD8 recognition [74]. Furthermore, lack of the GnT-V-encoding Mgat5 gene significantly increases TCR clustering and, consequently, reduces T cell activation and increases the level of autoimmune disease development. The cytosolic regions of the CD4 and CD8 receptors recognize Lck in the molecular level, strengthening TCR signaling to MHC antigen complex during CD4 and CD8 coupling to the TCR [75]. N-glycan branching enhances the DN cell maturation and TCR selection levels. Protein O-glycan and N-glycan structures regulate T cell responses [76]. In the N-glycans on T cell receptors, N-glycans regulate the T cell responses. MHC-I expressed in nucleated cells recognizes TCRs present in CD8+ T cells. However, MHC-II present in APCs including B cells, DCs, macrophages, and TECs recognizes CD4+ T cells [77]. Defection of MHC1a N-glycan synthesis caused by Asn site mutation or conformational shift frequently yields unfolded protein and accumulates proteins in cytosol [78]. MHC-II consists of two α and β subunits. The α subunit carries N-glycans attached to highmannose and complex types. However, the β subunit contains only complex-type N-glycans [79]. Among two different MHC-I and MHC-II, MHC-II glycosylation is necessary in antigen recognition and microbial carbohydrate antigen presentation. The perfect MHC-II glycosylation enables to respond downstream T cell signals.

4.5.3

Alternatively Spliced Variants Produce Different Glycan Structures of CD43 and CD45 Isoforms in T Cells

Glycosylation of coreceptors activates the T cells, as known in the case of TCR-CD45 complex. TCR-CD45 complex is formed by galectin-3, because galectin-3 recognition to poly-LacNAc sequences in branched N-glycans forms molecular lattice. CD45 phosphatase enzyme activity prevents T cell activation and suppresses T cell downstream activation [80]. CD45 gene is also spliced with alternative five variant isoforms including CD45ABC, CD45AB, CD45BC, CD45B, and CD45RO on human leukocytes [81–83]. The alternatively spliced variant protein forms have up to 11 different site glycosylation sites. However, the 11 Nglycosylation forms are different [84, 85] during T cell differentiation and activation [86]. For functional regulation, alternatively spliced transcript of CD45 gene likewise creates differential glycosylation events. CD45 is a predominant transmembrane glycoprotein expressed on T cells, and CD45 contains the cytoplasmic Tyr phosphatase domains. Three exon (4,5,6)-produced protein regions differentiate the common (RO) polypeptide formed by a Cys-rich region and three extracellular fibronectin type III repeats. The exons produce the A, B, and C domains, and the isoforms of RA translated from exon 4 form, RB from exon 5 form, RBC from exon 5 and exon 6 forms, and RABC from exons 4 to 6 forms are generated. The series of CD44 variants target mucin-type O-glycans [26]. The CD45 forms have MWs

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80–230 kDa, larger than the predicted MW of nascent protein not glycosylated (123–141 kDa). Sialylation regulates the CD45 clustering on oligomerization and TCR signaling [26, 87, 88]. During CD8+ T cell regulation, CD45 RO isoform is expressed for higher dimerization. Because of the glycosylation, switching from sialyl CD176 (Thomsen-Friedenreich antigen, TF) to non-sialyl CD176 decreases electrostatic tension when CD45 is dimerized [89]. The α2,6-sialylation on N-glycoproteins, not on core 2 O-glycoproteins, decreases the mature thymocyte capacity of CD45-mediated apoptosis by a galectin [26, 88]. T cells regulate their CD45 glycosylation by alternatively spliced CD45 isoforms having different glycosylation. Consequently, the T cell CD43 and CD45 glycophenotype controls interaction between T cells and endogenous lectins. Two major glycoproteins CD43 and CD45 expressed in the T cells are differentially expressed for the T cell lifespan. Glycans control T cell behaviors. Core 1 sialyl O-glycan is produced on DN thymocytes which are unmatured and matured CD4 or CD8 CTLs in the thymus, although non-sialylated core 1/core 2 O-glycosylations are found in immatured forms of DP thymocytes [90]. Activated CD4+ T cells and CD8+ CTLs exhibit the prevention of sialylation with increase in core 2 O-glycosylation level because of de novo hyposialylated CD43 and CD45 synthesis [89, 91]. Additionally, expression of ST3Gal-1 gene is differentially modulated in CD4+ T cell differentiation to Th-1 and Th-2 subsets [92]. For example, Th2 cells express ST3Gal-1 for core 1 sialyl O-glycans, whereas Th1 cells are negative for ST3Gal-1 expression with non-sialylated core 1 O-glycans [92]. Th-1 and Th-2 cells exhibit C2GnT expression and synthesize core 2 O-glycoproteins [93]. CD43 and CD45 glycosylation in T cells are controlled in thymocyte development and differentiation from DN thymocytic precursors to memory T cells, TCR signaling, apoptosis, migration, and T cell activation [91, 94–96]. CD43 and CD45 glycans control migration, TCR signaling, and apoptosis, at the T cell level. T cell life fate is thus controlled by CD43 and CD45 glycosylation. How does the glycosylated extracellular domain of CD43 influence the CD43-mediated T cell fates? Thymocytes and T cells express tri- or tetra-antennary types of N-glycans due to GnT-V that adds β1,6-GlcNAc to the N-glycan Man core, and then polyLacNAc unit is further added and terminally sialylated in SAα2,3- or SAα2,6glycans. The SAα2,3- and SAα2,6-linked N-glycans are generated by ST enzymes of ST3Gal-4 and ST6Gal-1, respectively. Sialylation reaction is influenced by development of thymic T cells. For example, medullary and cortical thymocytes contain α2,3-sialyl N-glycans, whereas mature medullary thymocytes possess α2,6sialyl N-glycans [96]. Similar to mature thymocytes, the thymus-left naïve T cells express α2,6-sialyl N-glycans. Activated peripheral T cells increase the level of surface complex N-glycans, decreasing α2,6-sialylation [97, 98]. ST6Gal1 increases complex N-glycan levels on CD45. How are T cells modulated by the CD43s 80 O-glycans and 11 N-glycans as well as CD45s 8 ~ 47 O-glycans during the life fate? How do CD43 and CD45 glycans on T cells differ from such glycans expressed by other leukocytes of B cells or DCs? CD43 and PSGL-1 have various O-glycan forms. Core 1-O-glycans and PSGL-1 recognize three selectin types [29, 99]. The core 1 β1,3-Gal-transferase enzyme

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generates the O-glycan core 1 sugar structure of Galβ1,3GalNAcα1-Ser/Thr- and more distal sLex are attached. Neutrophils isolated from core 1 β1,3-Gal-transferasedeficient mice exhibit significantly defected leukocytic rolling driven on P-selectin or E-selectin expressed on the endothelium [29]. PSGL-1 and CD43 associate to form lipid rafts on leukocytes. E-selectin also engages in CD43 on rolling of effector T cells [100] to activate signaling. The CD43 gene has only one exon, and CD43 has about 80 O-glycans on T cells with core 1 O-glycan and core 2 O-glycan structures [101]. The human CD43 extracellular region has about 80 O-glycan structures and has one N-glycan structure. T cells express two CD43 membrane proteins, which are sialylated in core 1 O-glycan structure (115 kDa) and core 2 O-glycan structure (130 kDa) [76], while the predicted weight is about 44 kDa. Naive and activated types of T cells equally produce CD43 protein with peripheral tissue core 1 O-glycosylation structure, whereas the activated type of T cells also synthesizes CD43 protein, which has a core 2 O-glycan structure [100, 102]. The cytoplasmic domain of CD43 as a mucin-type glycoprotein interacts with the cytoskeleton linker proteins (ezrin–radixin–moesin family) to activate T cell activation, migration, and survival [103, 104]. T cells and thymocytes have two different CD43 glycan structures. The CD43 protein extends about 45 nm length from the surface of T cells. The CD43 115 kDa glycoform is primarily expressed by mature CD4 or CD8 SP thymocytes, native T cells, and immature DN thymocytes. The CD43 130 kDa expression is increased in the activated peripheral T cells as well as immatured DP thymocytes. CD45 has five isoforms on human T cell surface due to alternative splicing, and the CD45 isoforms differ in the O-glycosylation level. How does CD45 glycosylation change in development and activation of T cells? The CD45 intracellular domain is commonly present in all CD45 isoforms with tandem phosphatase domains for TCR signaling [105]. Intracellularly, all CD45 isoforms have the enzymatic active domains of phosphatase, which are named phosphatase P1 and phosphatase P2. Among them, the phosphatase P1 has a strong enzyme activity. The alternatively spliced form with extracellular domains of CD45 bears two different O-glycosylation structures of core 1 O-glycan and core 2 O-glycan forms. All CD45 isoforms have commonly three regions of a membrane proximal F region, a CR for N-glycosylation, and a terminal end region [87]. The three regions of the F region, CR region, and terminal end region of CD45 protein have Asn sites for N-glycosylation. The CD45 extracellular region contains three A-domain, B-domain, and C-domain, which are formed by the alternatively spliced variants of exon 4, exon 5, and exon 6, as O-glycosylation sites. From total five isoforms, RO form does not contain alternatively spliced variant domains. Other forms are RB form produced from the exon 5, and the RA form is produced from the exon 4. Also, the RBC form is produced from the exon 5 and exon 6, while the RABC form is produced from the exon 4, exon 5, and exon 6. T cell development and activation regulate alternative forms of CD45. DP thymocytes express predominantly RO, while mature SP thymocytes express mainly RB and RBC. In contrast, naive T cells reside in peripheral region and generate mainly the RB form. The activated T cells and memory T cells generate mainly the RO form [88]. Therefore, immature

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DN T cells in the thymus generate the CD45RA/RBC/RB form, having core 1 sialyl O-glycans. The matured CD4 SP or CD8 SP T cell subsets in the thymus generate the CD45RB/RBC form, having core 1 sialyl O-glycans. In contrast, DP thymocytes express the CD45RO form with both O-glycan types of core 1 O-glycan/core 2 O-glycosylation structures, which are not sialylated. Naive T cell types express the CD45RB isoform, having a core 1 sialyl O-glycan structures in the peripheral organs, whereas activated T cell populations like DP T cells in the thymus generate the CD45RO form with non-sialyl forms of core 1 O-glycan and core 2 O-glycans [106].

4.5.4

T Cells CD43 and CD45 Interaction with Their Counter-Receptor or Lectins to Determine T Cell Fates

CD43 and CD45 function with their counter-receptors or lectin proteins expressed from neighboring cells including immune cells, endothelial cells, and cancer cells [107]. Glycosylation of thymocytes and T cells during development and activation is important in a fashion that T cells interact with the lectins through CD43 and CD45 glycans. Interaction between CD43 and CD45 glucans and endogenous lectins regulates the T cell functions. During T cell migration event, T cells normally transmigrate to inflammation tissue regions and sites. E-selectin as an adhesion molecule is synthesized in activated types of endothelial cells, recruitment of T cells to inflammation sites. The CD43 form is an endogenously produced coreceptor responsible for E-selectin recognition [108], because E-selectin binds to the T cell CD43 130 kDa glycoform bearing sLex tetrasaccharide on core 2 O-glycans [100, 102–104]. Activated T cells increase the synthesized carbohydrates of core 2 O-glycan linked to CD43 protein, which have the SLeX epitopes as the E-selectin ligand. The T cells interact with E-selectin and migrate to inflammation regions. In TCR signaling, TCR signaling is involved in negative and positive selection of thymocytes and peripheral T cell activation. CD45 intracellular phosphatase regulates TCR signaling thresholds [105]. Intensive N- and O-glycans and sialyl residues on the CD45 extracellular domain keeps CD45 molecule separated on the plasma membrane, increasing TCR signaling via the CD45 intracellular phosphatase activation [87]. Reduction in SA content and/or multivalent lectin interaction with the CD45 extracellular domain induces molecular clustering or oligomerization of CD45 molecule, causing TCR signaling dysfunction [87, 88, 106, 107]. CD45 oligomerization also decreases the CD45 intracellular phosphatase activity [88]. Thus, CD45 binding with lectins like placental protein 14 (PP-14) [106, 107], macrophage galactose-type lectin (MGL) [108], or galectin-1 [88] suppresses TCR signaling because such clustered CD45 losses phosphatase activity (Table 4.1). However, the role of T cell CD43 in TCR signaling is not certain compared to that of CD45.

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Table 4.1 Coreceptors of CD43 and CD45 expressed in thymic T cell subsets and peripheral T cells CD43 coreceptor E-selectin Galectin-1 Galectin-3 Macrophage galactose-type lectin Mannose receptor CD45 Coreceptor Galectin-1 Galectin-3 Placental protein 14 CD22/Siglec-2 Macrophage galactose-type lectin Serum mannan-binding protein Mannose receptor

Glycan SLeX on core 2 O-glycan LacNAc LacNAc Terminal GalNAc Man Glycan LacNAc LacNAc LacNAc SAα2,6 Terminal GalNAc Man, GlcNAc Man

Function T cell migration T cell apoptosis Unknown Unknown Unknown Function T cell apoptosis T cell apoptosis T cell apoptosis T cell signaling T cell apoptosis Unknown Unknown

In T cell apoptosis, β-Gal recognition lectin, termed galectin-1, leads to apoptotic cell death of immature thymocytes residing in the thymus. Apoptotic cell death of T cells is elicited by galectin-1 action, and this is a type of negative selection [109]. Both activated peripheral T cells and thymocytes in the thymus are subjected to apoptosis by galectin-1 [110]. Three known counter-receptors including CD43, CD45, and CD7 are directly associated with the regulation of the galectin-1-driven apoptosis of T cells [111–113]. Human T cell CD7 is crucial for galectin-1-mediated apoptosis [112]. CD43 and CD45 are originally targeted by galectin-1 for apoptosis. However, they enhance galectin-1-induced apoptosis [114], because galectin-1 recognizes CD43 and CD45 dependently on their glycan density and type. Originally, galectin-1 preferentially binds to asialo-LacNAc unit in non-α2,6-siyalylated complex N-glycan forms or core 2 O-glycan forms [115]. However, galectin-1 is also capable to recognize the core 1 O-glycan structures such as mucin or CD43 [88, 114], if they are abundantly present. In contrast, core 2 O-glycan types linked to CD45 protein induces the galectin-1-caused apoptosis of T cells [116]. CD43 and CD45 on immature thymocytes contain galectin-1-recognizing core 2 O-glycan forms. However, CD45 present in matured T cells in the thymus have only core 1 O-glycan types and α2,6-sialyl N-glycan type. Thus, the so-called glycophenotype blocks galectin-1 binding, because of the absence of α2,6-sialylation and core 2 O-glycosylation. In addition, alteration of CD43 and CD45 glycosylation renders resistant survival potentials of mature T cells in the thymus against the galectin-1caused apoptotic cell death of the T cells [88, 115]. In the peripheral naive T cells, SAα2,6- and CD45 core 1 O-glycosylation are present, rendering likewise resistance to galectin-1-driven apoptotic cell death. The activated types of CD4+ T cells and CD8+ CTLs also express receptors for CD43 and CD45, which carry the complex types of N-glycan and core 2 O-glycan types with decreased α2,6-SA levels, potentiating the activated T cells to galectin-1-induced apoptotic death

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[88]. Nevertheless, because CD4+ effector Th-2 cells synthesize α2,6-sialyl N-glycan structures and sialyl core 1 O-glycan structures, galectin-1 binding capacity is reduced, and galectin-1-caused apoptotic Th-2 cells death is also reduced [117]. Therefore, even though T cells and thymocytes in the thymus can be exposed to galectin-1 even in the peripheral tissues, certain T cell populations exhibit resistance to galectin-1-caused cell deaths by different glycosylation in CD43 and CD45. As a secondary surface glycoprotein of T cells, CD28 N-glycans occupy about 50% of total molecular mass. N-glycosylated human CD28 suppresses regular CD28-drived adhesion and co-stimulation of T cells during CD28-CD80 recognition. Complete disruption or mutation of the CD28 N-glycosylating sites or treatment of N-glycan synthesis inhibitors enhances the binding to CD80 on APCs in Jurkat T cells [118]. The branched CD25 receptor N-glycans increase its cell surface retention time and consequently confers immune tolerance by T cell differentiation. Experimental restriction of UDP-GlcNAc and lack of branched N-glycans prevent CD25’s cell surface retention and inhibit downstream IL-2 signaling. This consequently activates a Th-17 over-induced differentiation of activated Treg cells (called iTreg cells) [119].

4.5.5

TCR Glycosylation Governs Hyper-response and Autoimmune Responses in T Cells and Tregs

Autoimmunity develops upon the self-tolerance absence or autoimmune responses. Glycans determine fate of self-/non-self-antigens. Certain pathogenic glycans primarily activate the innate immune system; however, the question of how glycans discriminate self/non-self is unclear. Abnormally controlled N-glycan synthesis induces autoimmune and exacerbated immune responses [120]. Lack of β1,6GlcNAc branch synthesis in Mgat5-deficient KO mouse exhibits development of delayed-type hypersensitivity and EAE [121] and colitis [122]. The lack of β1,6 branches in N-glycans breaks normal lattice of T cell surface by abnormal TCR clustering in Mgat5 KO mice. The TCR clustering reduces the TCR threshold and activates T cells in the hyperimmune response in Mgat5 KO mice. Abnormal lattice formation indicates a broken lattice between TCR-β1,6-branched N-glycans and galectins, leading to abnormal TCR downstream signaling [44, 123]. Similarly, lack of β3GnT2 enzyme induces T cell hypersensitivity, as shown in β3GnT2-KO mice. β3GnT2 enzyme attaches GlcNAc residues to the N-glycan, and the GlcNAc residues act as a galectin ligand. However, β3GnT2-KO mice, lacking the GlcNAc residue in the N-glycans, lost galectin ligand, poly-LacNAc on the N-glycans. This is similar to the Mgat5 KO mice [123]. Most galectins bind to cell-surfaced N-glycans to form lattices [124] and increase glycoprotein surface retention time [54]. The galectin-glycan interaction generates the cell surface lattice as the receptor modulator that can regulate cell proliferation

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and response. Galectin-3 binds to Mgat5-synthesized N-glycans with the β1,6GlcNAc branch in TCR proteins of T cells. In CD8+ T cells, the GnT-V activity catalyzes the branched N-glycan formation, and the branched N-glycans again increased the galectin-3-mediated membrane lattice, and this downregulates the glycoprotein recognition, ultimately increasing the T cell activation [125]. Lack of Mgat2 reduces N-glycan branches compared to Mgat5 deficiency. Galectin-3 binding capacities to the total LacNAcs in Mgat2-deficient KO T cells and Mgat5deficient KO T cells are not different for each other. Lack of N-glycan branches yields poly-LacNAc-structured linear N-glycans. Galectin-3 prefers to bind to the poly-LacNAc carbohydrate present ion the Mgat5-driven N-glycan branches on EGFR and TGFβRII, delaying endocytosis [126]. The GnT-V-produced β1,6GlcNAc branches on N-glycans activate CD4+ T cells by inhibition of self-growth and self-signaling levels of T cells [127]. In addition to GnT-V, enzyme activities of α-mannosidase II (α-MII), MGAT1 gene-encoded GlcNAc-transferase I (GnT-I), and MGAT2 gene-encoded GlcNAc-transferase-II (GnT-II) compromise the T cell functions, which initiate autoimmune responses including EAE, IBD, SLE, and IDDM. On the other hand, the T cell self-renewal and development are regulated by the O-GlcNAc-transferase enzyme and GnT-I enzyme, respectively. In addition, core-fucosyl GlcNAc-Asn catalyzed by α1-6 Fuc-transferase (FUT8) affects T cell responses in immune diseases [127, 128]. The FUT8-catalyzed core fucosylation of TCR proteins hyper-activates CD4+ Th cells to display auto-responses of T cells, while FUT-8 core fucosylation to the CTLA-4/PD-1 co-inhibitory receptors induces immune tolerance status. Likely, in the α-mannosidase-II-lacking mice, glomerulonephritis is increased. In addition, glomerular IgM immunocomplex deposits, complement factor 3, and anti-nuclear antibodies are accumulated [129]. These pathologic and biochemical parameters are almost like to those occurred in lupuslike syndrome. Thus, it is considered that N-glycosylation gives a clue in manifest of T cell immunology. Regarding Tregs, SIGN receptor-1 (SIGNR1/CD209b), the murine homolog of DC-SIGN, was discovered as the secretory IgA (SIgA) receptor, where SIgA inhibits DC maturation in signr1 KO BMDCs. IgA is the most abundant antibody isotype in mammals and maintains homeostasis at mucosal surfaces and immune protection. IgA interacts with various receptors such as IgA Fc receptor I (FcαRI), transferrin receptor 1 (CD71), asialoglycoprotein receptor (ASGPR), Fcα/μR, FcRL4, and DC-SIGN/SIGNR1. Upon IgA binding to two receptors of the FcαRI and DC-SIGN/SIGNR1, anti-inflammatory immune responses occurred. SIgA prevents autoimmune responses through the glycan-dependent interaction with SIGNR1 on DCs which induces an immune tolerance via Treg expansion. This sugar interaction prevents tissue damage in multiple autoimmune and inflammatory diseases during SIgA-DC interaction, exhibiting a tolerogenic phenotype with IL-10 expression. These phenotype cells stimulate the IL-10-secreting Treg expansion. Therefore, in vivo trials using the SIgA-DC administration diminish the autoimmune disease development in EAE and IDDM [130]. SIGN1R-mediated signaling activates and expands IL-10-secreting Treg cells. SIGNR1 also regulates colonic immune response and peripheral immunity against systemic pathogenic infection.

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Consequently, immune suppressive SIGNR1 in murine macrophage differentiation and cancer progression is anticipated. In a recent report [131], when SIGNR1positive RAW264.7 macrophages were co-cultured with Lewis lung cancer cells (LLC) with IL-4 induction, LLC-stimulated macrophages expressed IL-10. However, inhibition of SIGNR1 by SIGNR1 (CD209b)-specific antibody inhibited the IL-10 expression. Consequently, SIGNR1 is essential for LLC-induced polarization to M2 macrophage sub-phenotype. The polarized M2 phenotype macrophages in a tumor-associated microenvironment induce the LLC migrative potentials, although the migration is prevented by SIGNR1-specific antibody. In addition, LLC-activated macrophages inhibited the activated T cell growth and IFN-γ-mediated Th1 response, while SIGNR1 inhibition rescued Th1 cell functions. Thus, murine SIGNR1 expressed in LLC-educated macrophages induces macrophage phenotype change to M2 polarization and helps lung cancer evasion.

4.5.6

SAMP and N-Glycan-Dependent Modulation of Inhibitory T Cell Receptors to Suppress T Cell Functions

SA recognition of hosts commences with the immune response during sepsis. SA-bearing SAMPs suppress T cell-driven cytotoxic activity in cancer cells. SA-bearing SAMPs activate potential of immune scape and growth of tumor cells. SAMPs elicit Siglec-engaged immune evasion of cancers from T cells [132]. The cancer immunotherapy has recently been shifted into the immune regulation strategy using immune checkpoint inhibitors. The inhibitory immune checkpoints are CTLA4- and PD-1-specific antibodies with clinical benefits. Therapeutic efficiency of these immune checkpoint inhibitors is dependent on their target immune receptors. This circumstance restricts the tumor therapeutic outcomes obtainable for only certain cancer patients, and unfortunately this indicates unexpected secondary resistances. This indicates new targeting pathways for tumor-specific T cell suppression. For compatibility to meet the desired success, T cell-specific new receptors are candidates such as the CD33-related Siglecs as PRRs of immune cells, because the sialoglycans act as SAMPs to suppress autoimmune responses. Siglecs are expressed in both normal T cells and tumor-infiltrating T cells. For example, Siglec-9expressing T cells coexpress PD-1. If the sialoglycan-SAMP/Siglec pathway is targeted, anticancer immunity is increased. Siglec-9 is a dominant inhibitory Siglec in cancer patients, and Siglec-9 ligands have also high expression in tumor environment. In human patients of cancer, the Siglec-9 expressed in T cells reduces cancer patient survival rate. Thus, the sialoglycan-SAMP/Siglec pathway is a target for T cell activation in tumor immunotherapy. Siglec-9 is an inhibitory CD33rSiglec. Interaction between SA-based SAMPs on the tumor cells and Siglec-9 suppresses NK cell-driven killing of cancer cells and myeloid cell-mediated cancer progression. Sig9 + CD8+ T cells express inhibitory receptors including general immune

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checkpoint proteins. When stimulated by co-stimulatory receptor antibodies, Sig9 + CD8+ TILs are highly activated compared with Sig9-TILS. Functional PD-1-hiCD8+ TILs and Sig9 + CD8+ TIL cells are tumor-specific CD8+ TILs. In the role of SA-SAMP/Siglec-9 in tumor environment, SA-SAMP/Siglec-9 interaction decreases T cell activation in tumor microenvironments, contributing to tumor immune evasion. Thus, targeting Sia-SAMP/Siglec-9 can be a way of cancer immunotherapy. SA-SAMP/Siglec-9 interaction has been also demonstrated in tumor-bearing mice models. Siglec-E, murine counterpart of Siglec-9, increased in tumor-infiltrating T cells, and murine SigE+CD8+ TILs is similar to Sig9 + CD8+ TILs of cancer patients. Tumor growth rate is faster in HS9 CD4-Cre mice with hSig-9 in CD4+ and CD8+ T cells, indicating inhibitory Siglecs in T cells as tumor evasion pathway. In another mice model with Siglec-E16, T cells are more activated and tumor growth was suppressed. Thus, Siglec-E on T cells potentiates immune evasion through inhibition of T cells. In lung carcinoma, Siglec-9 induces lowered survival rate, and polymorphism of Siglec-9 gene is associated with lung cancer development. SA-SAMP/Siglec-9 binding is indeed an immune checkpoint inhibitor in T cell activation, which is a target of cancer immunotherapy. The co-inhibitory receptors act in a N-glycan-dependent manner. For example, T cell inhibitory receptor of CTLA-4 consists of two N-glycan sites, and the N-glycan confers its T cell surface retention and controls CD80-CD28 binding on APCs [133]. The CTLA-4 N-glycans and PD-1 N-glycans regulate the inhibitory functions. Other N-glycan-dependent co-inhibitory receptors include T cell immunoreceptor with Ig and ITIM domains (TIGIT), mucin-domain containing molecule-3 (Tim-3) and lymphocyte activation gene 3 (Lag-3) [134]. Thus, N-glycans activate T cell functions through TCR-coreceptor signaling. PD-1 is also a T cell inhibitory receptor for immune inhibition towards the “T cell exhaustion” [135]. PD-1 and Tim-3 expression is increased by the core fucosylation of FUT8 [136]. The PD-1 core fucosylation inhibition induces an anticancer immune response through activation of T cells, indicating a new antitumor immunity, because the PD-1 glycosylation is implicated in T cell immunosuppression. The PD ligand-1 (PD-L1) known as a specific ligand of PD-1 stabilizes its cells. The binding of non-glycosylated form of PD-L1 to glycogen synthase kinase 3β (GSK3β) responsible for glycogenesis induces the PD-L1 degradation [137]. In triple-negative breast cancer MDA cell line, the β1,3-GlcNAc-transferase (B3GNT3), a poly-LacNAc chain-synthetic enzyme, is essential for the PD-1 binding to PD-L1 [138]. The glycosylated PD-L1 form-specific antibody blocks the PD-1 and PD-L1 binding, resulting in its digestion and internalization and consequently inducing tumor regressive activity against triple-negative breast cancer. Thus, normal Tregs show the different N-glycosylation patterns, compared to CD4+ T cells. The branched complex N-glycan level is correlated with the protein production associated with Treg suppressive roles. The candidate proteins are PD-1, PD-L1, and CTLA-4 [139]. CTLA-4 consists of multiple sites of N-/O-glycosylation, and these sites control its T cell surface-retained time for T cell function [133]. The TCR is activated by β1,6-GlcNAc branches on CTLA-4 N-glycans, because the β1,6GlcNAc-mediated N-glycosylation increases T cell-surfaced CTLA-4 retention,

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consequently suppressing T cell-driven immune tolerance [140]. Thus, the Thr17Ala polymorphism observed in CTLA-4 of human decreases the N-glycosylation sites, limiting T cell-surfaced CTLA-4 retention [141]. Exogenously supplemented vitamin D and GlcNAc residues enhance branched N-glycan synthesis, upregulating the surfaced CTLA-4 retention time and the immunosuppression.

4.5.7

Galectins in Suppression of T Cell Functions

In suppression of T cell function, galectins are also another crucial checkpoint in T cell activity. Galectin-1, galectin-3, and galectin-9 are associated with T cell immune suppression. Galectin-1 is produced by CD4 + CD25+ T cells and tolerogenic DCs [142, 143], inducing T cell apoptosis upon interaction with N-/O-glycans linked to CD45/CD43 SAMP and N-glycan-dependent modulation of inhibitory T cell receptors to suppress T cell functions and CD7 or upon FAS-mediated death of resting T cells [111]. The Th-1/Th-17-activated cells are highly susceptible to apoptotic cell death when treated with galectin-1 if the activated cells synthesize the galectin-1interacting carbohydrates, whereas Th-2 cells express SAα2,6-glycans to protect from the cell death [117]. Certain tumors can produce galectin-1 to induce immunosuppression via a bias towards TH2 cytokines as well as tolerogenic activation by DCs, which express IL-27 cytokine and type 1 Treg cells, which express IL-10 cytokine [144]. Galectin-3 role is still ambiguous in T cell functions. Cytoplasmic galectin-3 protects the cells from apoptosis through antiapoptotic pathway with increased Bcl-2 expression [145]. However, extracellular galectin-3 rather causes the activated T cell death by galectin-3 binding to T cell glycosyl receptors. The dual ambiguous pathway is distinct from galectin-1 [54]. Moreover, galectin-3 can recognize CTLA-4 N-glycans to prolong the CTLA-4 inhibitory signals [54] and also to the CD8+ CTLs Lag-3 to suppress the CD9+ T cell functions [146]. Finally, galectin-9/Tim-3 interaction diminishes glycan-dependently the activities of CD8+ CTLs, Th-1 cells, and Th-17 cells [147, 148]. However, galectin-9 which binds to other receptors may regulate proinflammatory cytokine expression [149]. Galectin-1 is known as an immune-modulator in EAE. Galectin-1 (Lgals1
/
) KO mice induces TH1 and TH17 responses, causing EAE [117]. Galectin-1 also controls the CD8+ CTL activity. Galectin-1 binding to Fas ligand keeps its retention at the CTL cell surface, and the retention hampers the cytotoxic capacity of the T cells [150]. Thus, GBPs and glycoprotein binding are important to motivate a T cell response.

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4.5.8

135

Glycans Regulate T Cell-Mediated Immune Suppression and Tolerance in Tumor Progression

The glycosylation is crucial for T cell development and functions, as glycans develop auto-immunities and cancer survival. Consequently, the bindings suppress the host immune responses. Glycans regulate T cell-mediated immune suppression and tolerance in tumor progression. If abnormally glycosylated pattern of cancer cells are targeted, introduction of antitumor immune response will be a future promising strategy. Tumor cells express aberrant glycan structures in sialylation and branch formation [151]. The aberrant glycosylation affects tumorigenesis and progression in tumor cell dissociation, angiogenesis, adhesion, growth, invasion, and metastasis; hence they escape from tumor immunoediting or immune surveillance [152]. GBPs expressed in immune cells bind to altered carbohydrate structures on cell surfaces of tumor to introduce the glycan information to immune cells towards immune stimulation or immune inhibition. Tumor cells abnormally express Tn and Lewis antigens carrying sialylglycans, and these Tn and Lewis sialylglycans are bound to macrophage and immature DCs DC-SIGN. The binding of MGL to terminal GalNAc residue appeared on CD45 glycoprotein downregulates TCR signaling, consequently decreasing T cell growth and increasing T cell apoptosis [108]. The Lewis Fuc residues induce TH2, follicular, and Treg responses [153]. DCs recognize DC-SIGN-binding Leb/x formulated liposomes and internalize and consequently activate CD4+ Th and CD8+ CTL [154]. Macrophage galactose binding lectin (MGL) recognizes Tn antigen and GalNAc residue with TLR-2 binding; ultimately IL-10 and TNF-α are secreted [155]. Moreover, tumorinfiltrating macrophages (TIL) express IL-10. If the TIL are blocked, CD8+ CTL effectively responds. During chronic infection, IL-10 elevates the Mgat5 gene expression to yield N-glycan branches on CD8+ CTLs. The N-glycan branches of CD8+ CTLs suppress T cells, and hence the infected virus or pathogens can acquire their prolonged infection status [156]. Mgat5-branched N-glycans synthesized by IL-10 suppress CD8+ CTLs in tumor. Fuc residues in Lex and Ley antigens are present on tumor surface proteins like carcinoembryonic antigen (CEA) [157] and induce the IL-10 and IL-27 production, known as anti-inflammatory cytokines, in APCs. CLRs such as MGL and DC-SIGN alert glycosylation changes in some CEA, and MUC1, tumor-associated antigens, occur during onco-transformation or tumorigenesis. The alterations of glycosylation are seen in the Lewis blood group antigenic epitopes including LeX and LeY linked with poor cancer prognosis. Sialylglycans also suppress immune responses via DCs-expressing Siglecs due to their inhibitory domains. For example, DCs SiglecE binding to sialylantigens increases in antigen-specific Treg response and decreases in antigen-specific Teff cell responses. Teff suppresses tumor proliferation and growth [158, 159]. The sialylated tumor mucin antigens of Sialyl-Tn (STn) and Sialyl-T (sT) are such examples. For example, MUC1 leads to tumor immune tolerance. The Siglec-9 binding to MUC1-ST on tumor-infiltrating macrophages initiates MEK-ERK signaling towards immune inhibition [160]. Furthermore, Siglec

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binding to Mucin sTn enables DC maturation and DC-induced FOXP3+ Treg cell activation. In addition, Siglec-Mucin sTn binding decreases the INFγ production of T cells [161, 162]. In fact, CD8 + TIL express Siglec-9 in NSCLC patients and protect the NSCLC from immune surveillance, consequently reducing survival rate. Consequently, Siglec-9 polymorphisms alert the danger signal of lung and colon cancers. Siglec-9+ CD8+ TILs also express Lag3/TIM-3/PD-1/CTLA-4 as inhibitory receptors. Defected synthesis of sialyl glycans in tumor cells reduces tumor growth through the infiltration levels of CD4+ T cells and CD8+ CTLs [163]. Hence, from the innate immune cells, several lectins of CTLs, galectins, and Siglecs interact with each relevant molecule as GBPs in immune responses [164, 165].

4.6

Abnormal N-Glycosylation in Autoimmunity

Abnormal N-glycosylation-associated human autoimmunity is also seen in MS patients. PBMC from MS patients exhibits the decreased Golgi-resident β1,6GlcNAc-transferase (termed core 2 GlcNAc-T) activity, in contrast to normal individuals [166]. In addition, MGAT5 polymorphisms are also associated with level of MS progression [167] with MGAT1, IL2R, and IL7R SNPs [168]. In the IBD progression, T cells in lamina propria region prepared from patients of ulcerative colitis (UC) lack β1,6-GlcNAc-T-catalyzed formation of branches in N-glycan structures because of MGATt gene deficiency [169]. When the branch levels in N-glycosylation of intestinal TCR of T cells are profiled using colon tissue biopsies obtained from human patients of UC, UC patients have a dysregulated N-glycosylation in TCR on lamina propria T lymphocytes. Severe UC patients carry defected N-glycan branches in T cells due to a reduced MGAT5 transcription. N-glycan branch-deficient TCR is a new UC pathogenic factor, which can be used as a potential biomarker for therapeutics. If the UC patients lack the branched N-glycans, standard therapeutic trials will be failed [170]. When T cells resident in intestinal region isolated from human patients of UC and mice of colitis are supplementarily enforced with GlcNAc, β1,6-branched N-glycan structures linked to cell-surfaced proteins on T cells are increased, and consequently, TCR signaling and TNF-α and IFN-γ synthesis, which are proinflammatory cytokines, are suppressed. IBD and MS can be clinically modulated by N-glycans due to T cell immune response [51, 122], towards the development of clinical agents [119, 122]. N-glycosylation analysis can primarily be performed in autoimmune diseases, as muscle-associated muscular dystrophy [171] and congenital disorders of glycosylation are known [172]. The relationship between muscle cell surface glycosylation alteration and inflammation is related to the interaction between muscle glycocalyx and the extracellular molecules in autoimmune diseases including idiopathic inflammatory myopathies (IIM) [173]. Glycans function as determinants in auto-responses by regulating T cells because abnormal glycoantigens unleash an autoimmune response.

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In mouse MRL-lpr model, the mouse phenotypes are accepted as a wellestablished mice SLE. The MRL-lpr mouse accumulates agalactosyl bi-antennary glycans and high-Man and pauci-Man contents in the kidney [174]. Moreover, in Mgat1f/fSyn1 mice, which are characteristic of the Mgat1 gene deletion at the Synapsin I-positive cells, and Synapsin I is abundantly expressed in neuronal area of the brain, and neuronal functions are defected with caspase-3-mediated neuronal apoptosis [175]. The apoptotic events activate immune responses, as frequently observed in autoimmune responses. In rare autoimmune diseases, abnormal N-glycosylation induces the diseases. For example, IIM belong to rare types of autoimmune diseases [176]. Glycoproteins of muscle cell surface display a muscle homeostasis and function. The GNE enzyme as a specific ManNAc kinase is essential in the NeuAc biosynthesis, and the GNE gene disruption yields a broken synthesis of NeuAc and blocking of sialylation of glycoproteins. In this case, supplementation with ManNAc as sialic acid precursor prevents ITM-like hereditary inclusion body myositis (hIBM) [177]. Thus, N-glycosylation is perspectively important in formation of autoantigens, since autoantigens are N-glycosylated with multiple N-glycosylation sites.

4.7

Glycan Regulation of NK Cell Receptors

NK cells are the first defense line in tumor immunosurveillance. Changes in the glycosylation pattern on surfaces of malignant tumor cells influence tumor immune responses through direct interaction with each receptor, glycan-binding protein, and lectin expressed on the immunomodulatory cells. The NK cell activation signals are delivered from their surface molecules such as adhesion molecules known for LFA-1. The NK cell co-stimulatory family receptors include NKG2D, DNAM-1, and SLAM as well as activating receptors bearing the ITAMs, TCR-ζ, DAP12, and FcεRI-γ [177, 178]. The NK cell activating receptors also include NKp30/NKp46. Innate immune NK cells exhibit directly cytotoxic cell killing against MHC-negative cancer cells, stressed cells, and virus-infected cells [179]. Inhibitory receptors of NK cells include NKG2A (CD94) and KIRs as well as Tim-3, etc., providing tolerance of immune checkpoint via the MHC-I recognition in the normal cells. There is imbalanced expression in the activating and inhibitory NK cell receptors, providing the NK cell dysfunction. Among human NK cell subsets, NK cell subset like CD56dim NK cells of CD56dimCD16 + KIR+ occupies 90% more spleen, and peripheral NK cells contain granzymes and perforin. CD56dimCD16 + KIR+ cells are the main cytotoxic cells [180], while CD56bright NK cells of CD56brightCD16dim/–KIR– are the main NK cells in the tonsils and lymphatic nodes [181]. NK cell is activated by KIRs including 2B4, KIR2DS, KIR2DL4, KIR3DS, NKG2D, NCRs, and NKp80 in humans. The predominant ITAM-bearing activating receptors include CD94/NKG2C, FcγRIIIa/CD16, KIR receptor subfamily (KIR2DS and KIR3DS), and NCRs in human NK cells.

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NCRs on NK Cells

NCRs include three distinct types of NCR-1 (NKp46 or CD335), NCR-2 (NKp44 or CD336), and NCR-3 (NKp30 or CD337). The three NCRs are produced by the ncr-1 gene, ncr-2 gene, and ncr-3 gene, respectively. Each NCR selectively performs target cell lysis through perforin, granzymes, and IFN-γ [178]. The NCRs have been isolated by Alessandro and Lorenzo Moretta during the 1990s [182–184] as type I transmembrane glycoproteins. They are characterized to be activating receptors and termed NKp30, NKp44, and NKp46 depending on each molecular weight. The NCRs bind to carbohydrate ligands, which are the targets for recognition of NK cells. The NKp44 and NKp30 genes (ncr2 and ncr3) are located on human chromosomal 6-MHC-III locus, and the NKp46 gene (ncr1) is loaded on the chromosome 19-leukocyte regulatory complex in humans [182, 185]. Interestingly, the ncr1 gene product, NKp46, is also expressed in mice and rats [186, 187]. NKp46 is a marker of all NK cells of human and mouse and present in certain ILC and T cell subpopulations [103, 185]. Of interests, NKp46 is absent in human and mouse CD1d-restricting invariant NKT cells. NKp46 recognizes and kills various tumor target cells. In mice, NKp46 develops type 1 diabetes [185]. For tumor survival through immune suppression, tumor microenvironments display the decreased NKp46 expression on NK cells by a tryptophan metabolite, 1-kynurenine, synthesized by IDO enzyme, which is the indoleamine 2,3-dioxygenase [188]. NKp30 is also present on most human NK cells, as NKp46 does. NKp30 mediates the NK cell and DC crosstalk through stimulating the immature DCs to mature DCs with cytotoxic activity. NKp30 and NKp46 expression is induced by IL-2, IFN-α, and prolactin. But cortisol and methylprednisolone suppress the expression of NKp30/NKp46 receptors [189]. The NKp30/NKp46 expression is also suppressed “memory-like” or “adaptive” NK cells, as shown in cytomegalovirus-infected patients [190]. TGF-β suppresses the NKp30 expression in NK cells [191]. NKp44 is quite different from other NCRs in humans because it is present constitutively on only CD56 bright NK cells. NKp44 is distinctly present in IL-2-induced NK cells in higher primates [184]. Cytokines IL-15/IL-1β/IL-2 enhance the NKp44 synthesis. IL-3 increases the NKp44 expression in pDCs [192]. PGE2 and prednisolone suppress IL-2-mediated NKp44 expression on NK cells [193]. NCRs bear Ig-like domains as the Ig superfamily. NCRs lacks ITAM and transduce signals through adaptor proteins having ITAMs [194]. NKp30 and NKp46 receptors are present in the activated and rested types of NK cells [183]. Three NCRs have different structures, but functions are only similar together, where NCR extracellular domains contain one Ig-like region for the NKp30 and NKp44 receptors and also two Ig-like regions for NKp46, which bind to ligands [185]. NKp30 and NKp44 are homodimerized with NKp30 to generate an I-type Ig-like complex. Two NKp44 V-type Ig-like domains are dimerized with unique disulfide bridging [195]. The NKp46 shows two C-2-type Ig-like domains like the Ig-like domains of KIRs [196]. All the NCD transmembrane domains carry a basic

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Lys residue for NKp44 or Arg residue for NKp30 and NKp46. They bind to Asp residues in DAP12 transmembrane region for NKp44 or TCR-ζ transmembrane region for NKp30 and FcεRI-γ transmembrane region for NKp46. NKp30 and NKp46 expressions are decreased in memory-like or adapted NK cells due to the lacked expression of FcεRI-γ [190]. Thus, adaptors are crucial for receptor transport to the NK cell surface. Thereafter, the transmembrane adaptors also activate the NCR signaling because the adaptor’s cytoplasmic ITAM is phosphorylated and recruits and activates the downstream Syk and ZAP-70 protein tyrosine kinases [185]. NKp44 expression requires three transmembrane charged residues recognition with DAP12. NKp44 extracellular domain contains a V-type Ig-like domain for ligand binding [197, 198], a cytoplasmic ITIM, and a Lys residue-bearing transmembrane domain, where Lys links to a dimer of the ITAM-bearing adaptor DAP12 [199]. Therefore, NKp44 has a dual function of either inhibitory or activating signaling in a ligand dependency. NKp44 induces cytokine release and cytotoxic activity in human NK cells. NKp44 also bind to self-ligands of PCNA and MLL5 alternatively spliced form [200]. Variously spliced variant forms such as ncr2 and ncr3 which are generated for NKp44 and NKp30 have been found, providing NKp44- and NKp30-medited inhibitory signaling.

4.7.2

NCR Ligands

NCR extracellular domains bind to carbohydrates, surfaced proteins, and cytoplasmic proteins that are exposed on infection or transformation (Table 4.2). In viral ligands, influenza viral hemagglutinin (HA) on the infected cells surface binds to branched SAα-2,3-linked and SAα-2,6-SA-likned O-glycans attached to NKp46 protein. Then, human NK cells kill the influenza-infected target cells through a NKp46 signaling [202]. Influenza virus interaction with NK cells or HA interaction with NK cells reduces NCR cytotoxic activity through TCR-ζ expression inhibition [203]. DCs infected by influenza virus are activated to induce IFN-γ release in the NKp46-dependent and HA-dependent manner in NK cells. In fact, NKp46-KO mice easily die upon influenza virus infection [204]. NKp46D2 and NKp44 recognize HA-neuraminidase (NA) protein of the Sendai virus, Paramyxoviridae. NKp44 binds to viral hemagglutinin of the influenza virus, Orthomyxoviridae [205], and the envelope glycoprotein (E-protein) of Flaviviridae [206–208]. Influenza and Sendai virus HAs specifically bind to NKp44 proteins but not NKp30 species. NKp44+ NK cells can also destroy the influenza- and Sendai virus-infected cells [209]. NKp46D2 and NKp44 α2,6-sialylglycans bind to the ligands on tumor cells [210] and envelope-coated membrane glycoproteins decorated on dengue and West Nile viruses [208]. NKp44 binds to charged and IdoA2SGlcNS6S heparin carbohydrates [197]. Similarly, NKp46 and NKp44 bind to avian Newcastle disease virus HA and kill paramyxovirus-infected target cells [211]. HA species isolated from the vaccinia virus of humans, orthopoxviruses, and ectromelia virus of mouse bind to both

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Table 4.2 Lignads and roles of natural cytotoxicity receptors (NCRs) Ligands Hemagglutinin (HA) Main tegument protein pp65 DBL-1α domain HS BAT3/BAG6 B7-H6 HA HA Domain III of WNV envelope protein NKp44L PCNA Cytoskeleton type III vimentin HA HA HA DBL-1α domain HS GAGs HS/HSPGs

Location Human vaccinia virus Human CMV P. falciparum Membrane Membrane Tumor cell surface Influenza and Sendai viruses Avian Newcastle virus West Nile/dengue virus

NCRs NKp30 NKp30 NKp30 NKp30 NKp30 NKp44 NKp44

Tumor membrane Plasma membrane Infected cell surface Influenza virus Avian Newcastle disease Human vaccinia virus

Surface

Ligand role Inhibiting Inhibiting Activating Activating Activating Activating

References [35] [37] [46] [201] [42] [43] [31]

NKp44 NKp44

Activating Activating

[34] [26]

NKp44 NKp44 NKp46 NKp46 NKp46

Activating Inhibiting Activating Activating Activating

[38] [41] [40] [25] [34]

NKp46 NKp46 NKp46 All NCRs

Activating Activating Activating Cis ligand

[35] [46] [201] [70]

NKp46 and NKp30. NKp30 protein expressed in the NK cells less kill vaccinia virus-infected cells, which are present at the late stage of life cycle, compared to normal cells, and due to the presence of viral HA in the target cells [212]. The vaccinia virus-infected cell HA binds to NKp30 to suppress NK cell’s activating function or to stimulate NK cell inhibitory response, whereas NKp46 binding to vaccinia viral HA on host cells kills the host cells. NKp44 recognizes the West Nile and dengue viral envelope glycoproteins [213]. NKp44 protein directly recognizes WNV envelope protein domain III but not viral HA due to NKp44 sialylation. Cells infected with West Nile virus easily recognize the soluble NKp44 protein and consequently activate NK cell degranulation to release cytokine IFN-γ. Dengue viral non-structural protein expressed in the host cells prevents NK cell cytotoxic action due to their MHC-I expression [214]. NKp30 directly binds to pp65 of human cytomegalovirus (HCMV) [215], and consequently, the HCMV-infected host cells are resistant against NK cell cytotoxicity. However, when the pp65-negative HCMV infects the cells or when anti-NKp30 MAbs are treated, the targeted host cells are readily killed. Soluble pp65 treatment with NK cells releases the TCR-ζ protein from the associated NKp30. pp65 disrupts activation signaling through NKp30 in HCMV infection.

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Intracellular protein ligands are moved to cell surfaces in order to function as surfaced ligands of NCRs on cells. NKp44L is a ligand for NKp44. GP41 envelop protein of CD4+ T cells, which are infected with HIV-1, induces expression of NKp44L [216]. The NKp44L is initially generated as a spliced variant protein of mixed-lineage leukemia 5 (MLL5) [200]. The MLL5 protein with a full-length form is located as a nuclear protein in the nucleus, but the spliced NKp44L variant form is located in the tumor cellular PM and transformed cell PMs but not in normal tissues. NKp46 mediates the NK cell-driven cytolysis of Mycobacterium tuberculosisinfected monocytes due to binding to vimentin expressed in infected monocytes [217], where vimentin protein belongs to a cytoskeleton type III intermediate filament. NK cells readily kill vimentin-expressing target cells transfected. NKp44 binds to PCNA of target cells when PCNA transports to the cellular PM region and inhibits cytotoxic cell death and IFN-γ production of NK cells [218]. NKp44 binds to PCNA of target cells when PCNA transports to the PM of target cells and inhibits cytotoxicity and NK cells’ IFN-γ production. The PCNA-NKp44-induced cytotoxicity inhibition is involved in the cytoplasmic ITIM-like sequence of NKp44. The PCNA-NKp44-induced cytotoxicity inhibition is involved in the cytoplasmic ITIMlike sequence of NKp44. PCNA-NKp44 binding induces a conformation shift to transduce an inhibitory signal in ILCs and pDCs [192]. PCNA-NKp44 binding induces a conformation shift to transduce an inhibitory signal in ILCs and pDCs. NKp30 binds to the Bcl2-associated anthanogene 6 (BAG6) and HLA-B-associated transcript 3 (BAT3) and induces NK cell cytotoxicity [219]. BAG6 and BAT3 proteins are mainly present in the nuclear region but transported to the PM of heatshocked cells or secreted into exosomes, which was formed by stressed and tumor cells. BAG6/BAT3-producing exosomal vesicles induce cytokine secretion by NK cells during their NKp30 recognitions. DCs BAT3/BAG6 activate NK cells to interact with DCs. NKp30 binds to a cell surface B7 family, B7-H6 [220]. Interaction between NKp30 and B7-H6 promotes NK cells’ cytotoxic activity and cytokine expression. Although normal cells do not express B7-H6, human tumor cells produce it or upon TLR ligand induction of monocytes and neutrophils or inflammatory cytokines [221]. Certain tumor cells prevent NKp30 interaction through shedding B7-H6 with the ADAM-10 and ADAM-17 metalloproteinases [222] as well as by reduced NKp30 production by peritoneal NK cells, because of chronic recognition of ligand in breast cancers [223].

4.7.3

Interaction of NCRs Ligands with Pathogens

In pathogens, NCRs bind to bacterial and parasite pathogens. NKp30 and NKp46 directly recognize the Duffy binding-like (DBL)-1α region of erythrocytic cellular membrane protein-1 produced in Plasmodium for malaria-infected erythrocyte lysis [224]. NKp44 directly binds to Mycobacterium bovis BCG [225], and BCG increases NKp44 production in CD56 bright NK cells [226]. Nocardia farcinica

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and Pseudomonas aeruginosa bind to NKp44. NKp44 stimulation activates NK cellmediated autoimmunity in chronic cartilage inflammation.

4.7.4

Interaction of NCRs Ligands with Self-Ligands

Recombinant NKp46 and NKp44 extracellular domains (rNKp46, rNKp44) strongly recognize and bind to K562 cells, where the Fuc-transferase (FUT)-3 gene synthesizes the sLeX epitope of NeuAcα2,3Galβ1,4(Fucα1,3)GlcNAc-R sugar structure [227]. The rNKp46 and rNKp44 bind to sLeX-positive transferrin secreted by human hepatoma cells such as HepG2 cells, but not NKp30 (rNKp30) [228, 229]. In a recent paper [230], Globo-A also binds to rNKp44 and induces NKp44-mediated NK cell cytotoxicity. Globo-A binds to rNKp44. rNKp44, but not rNKp30 and rNKp46, recognizes Globo-A although the binding affinity is weak. Globo-A with the carbohydrate structure of GalNAcα1,3(Fucα1,2) Galβ1,3GalNAcβ1,3Galα1,4Galβ1,4Galβ1-Cer sugar structure is generated by the specific enzyme of 3-α-GalNAc-transferase, which transfers GalNAc residue to Globo-H structure of Fucα1,2Galβ1,3GalNAcβ1,3Galα1,4Galβ1,4Galβ1-Cer. Globo-A species is found in the kidney [231] and erythrocytes [231, 232]. The Globo-A sugar structure resembles with the blood A antigenic determinant of ABH blood group, which has the GalNAcα1,3(Fucα1,2)Galβ1- structure. NKp44 does not bind to Globo-H but the terminal part of NKp44 binds to Globo-A. The A antigen of the ABH blood group is present in many human tissues and associated with invasive progression of cancer cells. The NKp44 protein synthesis in NK cells is also associated with the autoimmune reactions. For example, the SCLC and breast cancers [233] exhibit the disappeared antigen of the blood group A, and this phenotype accelerates cancer invasiveness and poor prognosis. Therefore, NKp44 to blood group A antigen interaction would be a key mechanism of tumor progression. In addition, NKp44 and other NCRs directly bind to HS or Hp on HSPGs. HSPG present in NK cells binds to NKp44 present on NK cells itself in a cis type [234, 235]. However, binding of NK cell NKp44 to target cells HSPG is a trans type. The cis interaction NKp44 and the HSPG syndecan of NK cells affects distribution and function of NKp44 and the NKp44 on the membrane [236]. As NKp44, NKp46 and NKp30 are bindable to HS, all the NCRs preferably bind sulfated HS [177, 197, 237–239]. HS on the mammalian cells is predominantly supplied from the syndecan HSPG. KIR2DL4, NKG2D [240–242], and all three NCRs [178, 243] also recognize HSPGs. NCRs recognize distinct HS structures [197]. Exogenous HS interferes with endogenous binding NCR and HS present in the NK cell surface. NKp44-HS interactions regulate the NKp44 function of NK cells. Syndecan in B lymphocytes also result in an “activated”-like phenotype of BCR stimulation. The membrane proximal domain 3 of CD19 binds to HS. CD19 forms BCR synapse via cytoskeleton organization in B cells [244]. HS and heparin

4.7 Glycan Regulation of NK Cell Receptors

143

mimics can suppress progression and metastasis of tumors via NK cell antitumor responses. NK cells also self-modulate its receptor function through the cis “masking.” For example, a “-cis” interaction of NK cell receptor with its ligand can be seen in the Ly49 receptor binding to MHC-I ligand in the NK cells of mice. The Ly49 protein is normally “masked” by the MHC-I of NK cells in a cis-type recognition [245], suppressing the inhibitory potential. The –cis binding of Siglec 7 known as CD328 and α2,8-disialyl ligand on NK cell surfaces is another type of cis interaction [235, 245, 246]. Among GAGs, HS species can bind to KIR2DL4 protein, another NK cell receptor. The binding regulates receptor signaling [247]. NKp44 binds to the HSPGs known as syndecan-4 of the NK cells in a cis type on the NK cells and modulates the receptor distribution and function. HSPG cis recognition regulates KIR2DL4 and NCR functions via masking target cell trans-recognition of HS species or other ligands present in cells, alerting the NCR trafficking to cytosolic degradation site and recycling derived from internalization [248]. Thus, cis-NCR and HSPG recognition influences the cell function. BAT3 is the HLA-B-associated transcript 3 (BAT3), and BAG6 is the Bcl2associated anthanogene 6 (BAG6). They are transported from nuclear to membrane or exosomes. DBL-1α domain is an erythrocyte membrane protein-1 of P. falciparum. The alternatively spliced variant form, NKp44L, is the variant of the mixed-lineage leukemia-5 protein present in the nuclear region. Proliferating cell nuclear antigen (PCNA) is also the nuclear protein and transported to membrane. Vimentin is the intracellular protein of cytoskeleton type III protein.

4.7.5

NK Cells MHC-I-Independent Inhibitory Receptors Siglec-7 and Siglec-9

Siglec-7, which is the p75/AIRM1 protein, and Siglec-9 belong to MHC-I-independent inhibitory receptors expressed in NK cells of human. In NK cells, Siglec-9 predominantly presents in CD56dim NK cells [249], and this selectivity indicates the matured type of NK cells with chemotactic potential. In tumor patients, the level of Siglec-9-expressing NK cell subsets is reduced [250]. Siglec-9 is an emerging immune regulator. NK cell functions are strictly regulated by the surfaced receptors. In virus-infected patients, Siglec-9-expressing NK cells are stimulated with activation receptors including NKG2D, NKp30, and NKp46, but with inhibition receptor of NKG2A. Such receptors resemble known inhibitory receptors such as PD-1 [251]. Siglec-7 or Sigelc-9 ligand expression on malignant tumor cells also strongly and directly activates primary NK cell activity in humans. Siglec-7 and Siglec-9 bind to certain gangliosides such as GD2, GD3, and GT1b produced by human tissues and neuroectodermal origined tumor cells, because these cells and tissues contain Siglec-7- and Siglec-9-specific ligands [252, 253]. Using recombinant Siglec-IgG1 Fc fusion, Siglec-7 and Siglec-9 were demonstrated to bind to melanoma cells or

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lesions as well as AML of CLL leukemia of patients in humans [254]. The Siglec-7Fc protein strongly binds to the GD3 synthase-transfected P815 cells and enhanced NK cell cytotoxicity but not inhibit due to Siglec-7-independent efficacy [246]. Hence, diverse ligands for Siglec-7 and Siglec-9 function in distinct tumor forms for protection from NK cell cytotoxicity. Interaction between Siglec-7/Siglec-9 with their ligands is applicable for the therapeutic, diagnostic, and prognostic biomarkers of malignant cancers in humans. Human tumor-expressed Siglec-7/Siglec-9 ligands are recognized by NK cells. Cytotoxic NK cell-produced Siglec-9 is regarded as a pan-NK cell biomarker [255, 256], while Siglec-9 is specifically present in the type of CD56dim NK cell subset [257], and peripheral Siglec-9-positive NK cell subpopulation level is decreased in cancer patients. The CD56dimSiglec-9+ NK cells exhibit the receptor expression of inhibitory KIRs and ILT2 and consequently decreasing target cell killing capacity. The chemotactic activity of NK cells, which express the CD56dimSiglec-9, tropic to IL-8, is stronger than that of CD56dimSiglec-9-negative type of NK cells. The expression level of chemokine receptors such as CXCR1 known as IL-8 receptor or CX3CR1 is much high in CD56dimSiglec-9+ NK cell subset. Siglec agonists/antagonists are beneficial for targeting or cell-based therapies [258, 259]. Therefore, the Siglec-7/Siglec-9-producing NK cells or their ligands would be a potential strategy for NK cell-derived therapy to antitumor immunity [260]. For example, Siglec-9 expressed on the tumor-associated macrophages binds to sialyl O-glycan-bearing MUC1 (MUC1-ST) on tumor cells to acquire tumorassociated microenvironment in invaded tissue [160]. Stem cell transplants, which are allogeneic KIR-mismatched, in human leukemic diseases can be beneficial if Siglec-7 and Siglec-9 expression is suppressed in donor NK cells [261]. Siglec-7/ Siglec-9 agonists as NK cell inhibitory receptors contribute to improved graft survival in solid organ transplantation [262]. Siglec-2, Siglec-3, and Siglec-8 are targeted with autoimmune and allergic diseases as well as non-Hodgkin lymphoma, hair cell type leukemia, and AML [258, 259]. Siglec roles in NK cell subsets are not well understood, because Siglec-7 and Siglec-9 ligands are present even in cancer cells or healthy cells [263, 264]. Siglec-9 binding in neutrophils makes its quiescence in the bloodstream [265], blocking its recruitment and oxidative burst as well as cell death in inflammatory milieu or cancer [266]. Siglec-9 is a biomarker for the lowered CD56dimSiglec-9-positive NK cells in tumor. Siglec-9 is specifically present in CD56dim NK cells. Thus, ligand analysis specific for Siglec-7/Siglec-9 in immune cells like CD56dimSiglec-9+ NK cells can define distinct functionalities. For example, the ganglioside DSGb5 expressed on renal carcinoma cells is a Siglec-7 ligand [264]. GD3 or DSGb5 expression does not influence on NK cell function, while neuraminidase pretreatment of NK cells inhibits NK cell cytotoxic activity due to unmasking Siglec-7. The secreted or membrane glycosylated tumor antigens including CA125, CA199, CEA, and MUC1 are recognized as tumor markers. MUC16 is a specific ligand for Siglec-9 [257]. MUC16 on human epithelial ovarian cancer cells or shed MUC16 are observed in serum or peritoneal fluid as the CA125 cancer marker. MHC-I-independent Siglec-7/Siglec-9 leads to NK cell inhibition in aberrant sialoglycan ligand-

4.8 Carbohydrate Recognition of Target Antigens by DCs During Infection and. . .

145

bearing tumor cells. Siglec-7/Siglec-9 expression in human NK cells [246] resembles the typical inhibitory receptors like KIRs, CD94/NKG2A, and Ig-like transcripts (ILTs), because they have one or more ITIMs. NK cell receptors recognize the MHC-I, indicating the “missing-self” theory to support the rejection concept of the infected/stressed and tumor cells, deficient for MHC-I, and discrimination between normal self and abnormal cells. Siglecs as ITIM-bearing and MHC-independent inhibitory receptors negatively transduce downstream signalings to NK cells, regardless of the condition that the cells lost the MHC class I molecule, which is a missing-self status. Hence, virus-infected and tumor cells require Siglecs, and therefore they evade the immune surveillances of hosts as a strategy of immune escape. For example, hepatitis B viral patients exhibit the reduced Siglec-9 presence in NK cells. Thus, Siglec-9 blocking activates NK cell function [267]. Inhibitory receptors in innate NK cells and adaptive CD8+ CTL immunity strengthens anticancer activity. For example, blocking KIRs, CTLA-4, or PD-1 controls inhibitory or activating signal transduction in innate and adaptive immune cells to regress cancer cells [260, 268, 269]. In addition, for telomeric length, CD56dim NK cells undergo cell divisions with 5–10 times more than that of CD56bright NK cells, causing a shortened telomeric length with the 50–100 bp lengths per each cell division [270]. But telomeric length difference in the two cell types of CD56dimSiglec-9-positive and CD56dimSiglec-9-negative NK cell types was not detected, suggesting a growth-independent Siglec-9 expression [254].

4.8

Carbohydrate Recognition of Target Antigens by DCs During Infection and Inflammation

Inflammation is a process of self-defense from invasive agents or allergens. Therefore, this protective and alert reaction is involved by invasion of foreign agents or pathogenic infection such as virus, viroids, prions, bacteria, yeasts, fungi, helminths, and larger organisms like macroparasites or protists into tissues or organs of host organisms. During the infection of foreign agents, hosts fight against those infectious agents for defense using innate or acquired immune responses of hosts. Considering the side of infectious agents, the host cells or organisms have to react in some ways like altering the inflamed sites or positive defense. One of such ways during pathogenic infectious events is the systemic induction of changes in host glycosylation level, especially in its cell surfaces. The resulting changes in glycosylation status in cells, tissues, organs, and organ systems of host organisms are directly linked to changes in functions and structures of lipids, proteins, and membranes. It means that host cells are ready to allow distinct functions against attacks. Especially, inflammation events accompany the altered glycosylation status of lipids and proteins and are responsible for cellular functions, giving capabilities to adapt of host immune system in mammals. In pathological description, the status is termed “infectious diseases” or “immune dysfunction

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diseases” when the host immune systems are not normally functioning. In the case of targeting host immune system, pathogenic agents unable the host immune system. With regard to the glycosylation-based pathology, the Glyco-Evasion hypothesis suggests that invasive and pathogenic agents regulate host glycosylation events to promote infection by making host immune system malfunctioning. The GlycoEvasion hypothesis has been suggested by Kreisman and Cobb (2012) [271], indicating that invasive and pathogenic agents regulate host glycosylation events to accelerate infection through host immune retardation [21]. Thus, pathogens have been evolving to modulate host immune responses by controlling its glycosylation, indicating that pathogens regulate the host immune response via glycosylation [272]. Surfaces of mammalian cells are covered by glycocalyx including glycolipids, glycoproteins, glycophospholipids (GSLs), GAGs, and proteoglycans. Glycocalyx is synthesized and matured in the ER and Golgi apparatus. Some of them are transported to the cell PMs. As glycocalyx is biologically important for development, growth, communication, and recognition of cells, this glycome is recognized by surrounded or neighbored cells to interact and communicate for the multicellular societies. This process confers the dynamic system of construction in tissues, organs, organ systems, and individuals. If the state is in non-normal or pathological dangerous outcome, the process that DCs migrate to peripheral tissues requires the molecular recognition of the counterpart target cells which is operated in vascular fluids and lymphatic nodes.

4.8.1

Lewis Ligand Recognition by DCs

In the initial studies on sialic acids, sialyl ligands have been demonstrated to modulate the leukocyte homing or trafficking. During the 1980s, factor H was the sole intrinsic SA-binding protein. SA residue in the SAα2,3Galβ1,3/4(Fucα1,3/4) GlcNAcβ1-R (SLeX/A) is used as intrinsic selectin ligands [273]. The sLeX/A motifs are cooperated with the more negatively charged sulfates attached to the Gal residue or GlcNAc residue for L-selectin ligands (Fig. 4.2a, b). Such sulfation is also found on adjacent tyrosine residues for P-selectin ligands, allowing solely recognition sites on mucin-type O-glycoproteins [274]. The N-terminal sulfoglycopeptide motif on PSGL-1 is a key P-selectin ligand [275]. Although Sias are negative charge carriers and typical ligands for selectins, the esterified sulfate at the C3 of galactose is also used as selectin ligands [276]. Likely, although the α2,3sialyltransferases and α1,3/4 fucosyltransferases synthesize the selectin ligands, GlcNAc sulfotransferases and tyrosine sulfotransferases are also alternates [277]. Certain 6-O-sulfated GAGs including HSe are also alternate selectin ligands [278]. Functionally, Sias are a negative charged pattern served in innate immunity. SAs are the SAMPs [279]. The different amounts of sialic acid on erythrocytes of different mice strains may reflect the control extents of the alternative complement pathway activation [280]. Ficolin is a soluble lectin of the lectin pathway to activate complement system

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(A) Sialyl LeX

Fuc-α1 p 3 NeuAc-α-(2→3)Gal-β-(1→4)-GlcNAc-β-(1→3)Gal-β-1-R

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in circulation and recognizes sialic acids on the pathogenic bacterial surfaces with sialylated glycans [281]. For molecular recognition of the endothelial barriers, specifically expressed “selectin ligands” in DCs bind to its receptors, E-selectins/P-selectins, present in vascular endothelia with attachment to the endothelial lining [282]. Selectins also function in the DC trafficking to peripheral tissues. Immatured DCs, not matured DCs, recognize the E-selectins/P-selectins for the migration into inflammatory skin or tissues. Lymphocytes are also migrated into the peripheral node HEV endothelium. The representative ligands of E-selectins/P-selectins are the glycan determinants of SLeX or SLeA. SLeX or SLeA is peripherally present as sugar oligomers attached on O-glycans, complex N-glycans, or glycolipids synthesized by several glycosyltransferases such as GalT, FucT, GalNAcT, and Sia-T (Fig. 4.3). Sialyl Lewisa (type I) and sialyl Lewisx (type II) determinants are structurally similar in their linkages (Fig. 4.4). Some difucosyl Lex are also known, and this ligand is

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4 Glycans in Glycoimmunology

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: GlcNAc

: Fucose

: NeuAc

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Fig. 4.4 Structures and synthetic enzymes of sialyl Lewisa (type I) and sialyl Lewisx (type II) determinants. (a) Schematic structure. (b) Synthetic enzymes

synthesized by α(1,3)Fuc-Ts and α(2,3)-Sialyl-T from Lex and Galβ1,4GlcNAcβ1,3Galβ1,4GlcNAcβ1-R-, NeuNAcα2,3, Galβ1,4GlcNAcβ1,3Galβ1,4GlcNAcβ1-R- and Galβ1,4GlcNAcβ1,3(Fuc-α1,3)Galβ1,4GlcNAcβ1-R-, or Galβ1,4GlcNAcβ1,3Galβ1,4GlcNAcβ1(Fuc-α1,3)-Rprecursor structures. The respective β1-3 and β1-4 galactosyltransferases determine the sialyl Lewisa (type I) and sialyl Lewisx (type II) determinants, respectively (Fig. 4.5a, b). Terminal type II disaccharides are further modified by several enzymes including CHST2, CHST4, and CHST6; GlcNAc6ST3; FUT3–FUT7, FUT9–FUT11, and FUT1–FUT2; ST3Gal III, ST3Gal IV, and ST3Gal VI; Gal3ST2, Gal3ST3, and Gal3ST4; and ST6Gal I and ST6Gal II (Fig. 4.5c). Sialyl-difucosyl LeX is synthesized from the VIM-2 glycan structure (NeuNAcα2,3Galβ1,4GlcNAcβ1,3Galβ1,4GlcNAcβ1(Fuc-α1,3)-R) or sialyl-LeX structure (NeuNAcα2,3Galβ1,4GlcNAcβ1,3(Fuc-α1,3)Galβ1,4GlcNAcβ1-R) from the precursor glycan structure of NeuNAcα2,3Galβ1,4GlcNAcβ1,3Galβ1,4GlcNAcβ1-R. Sialyl-Lea structure (NeuNAcα2,3Galβ1,3GlcNAc(Fuc-α1,3)-R) synthesized by α(1,3/1,4)Fuc-Ts from NeuNAcα2,3Galβ1,3GlcNAc-R as the precursor glycan, where Galβ1,3GlcNAc-R is converted by α(2,3)Sialyl-T. Lea is directly synthesized by α(1,3/1,4)Fuc-Ts from the precursor glycan Galβ1,3GlcNAc-R (Fig. 4.6a, b, c).

4.8 Carbohydrate Recognition of Target Antigens by DCs During Infection and. . .

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4.8.2

VIM Ceramide Dodecasaccharide

VIM-3 epitope has a structure of ceramide dodecasaccharide 4c and is known as CD65. Historically, the VIM-2 or CD65 epitope has been known from the fucosylation mutants. There are several mammalian α(1,3)fucosyltransferases (α1,3Fuc-Ts) [283]. In order to study the regulation of α1,3FucT expression, the CHO mutants of α1,3FucT-VI-expressing LEC11, LEC11A, and LEC11B were established. The two genes of Fut6A and Fut6B genes are known [284]. The genetic mutation of the two genes produces SLeX with the structure of SAα2,3Galβ1,4 (Fucα-1,3)GlcNAc-, VIM-2 (CD65s) of the SAα2,3Galβ1,4GlcNAcβ1,3,Galβ1,4 (Fucα1,3)GlcNAc-R structure, and LeX with the Galβ1,4-(Fucα1,3)GlcNAc-structures linked to glycoproteins. Other mutants of LEC12 and LEC29 can not be recognized by anti-SLeX antibody, because they do not fucosylate α2,3sialylated LacNAc [285], due to non-expression of the Fut6A, Fut6B, or Fut7 genes. The LEC12 and LEC29 exhibit different patterns of fucosylation. While LEC12 cells produce the LeX and VIM-2 antigens, LEC29 cells are negative for the VIM-2 synthesis [285]. α1,3FucT-IX expression produces the VIM-2 antigen as oncofetal epitope on the cell surfaces. The Fut9 gene in LEC29 mutant cells results in VIM-2 expression, as enhanced by transfection with a Fut9 cDNA. Thus, VIM-2 expression is dependent on the α1,3FucT-IX. Using the substrate of nLc6 glycolipid, Galβ1,4GlcNAcβ-1,3Galβ1,4GlcNAcβ1,3Galβ(1,4)Glcβ1,1-ceramide, the α1,3FucT-IX enzyme in LEC12 cells catalyzes preferentially the fucosylation on the terminal GlcNAc [286]. Human α1,3FucT-IX prefers LacNAc as substrate, compared to di-LacNAc, and also prefers the terminally located GlcNAc residue not the internal location or proximal location of GlcNAc residue of di-LacNAc [287]. Among the tri-LacNAc [288] or tetra-LacNAc [287], human α1,3FucT-IX

150

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α(1,3)Fuc-Ts

Sialyl-LeX α(1,3)Fuc-Ts

Fuc-α1 p 3 NeuNAcα2→3Galβ1→4GlcNAcβ1→3Galβ1→4GlcNAcβ1→R

NeuNAcα2→3Galβ1→4GlcNAcβ1→3Galβ1→4GlcNAcβ1→R

C)

Sialyl-Lea

Lea α(2,3)Sialyl-T Fuc-α1 Fuc-α1 p p 4 4 NeuNAcα2→3Galβ1→3GlcNAc→R Galβ1→3GlcNAcβ1→R

α(1,3/1,4)Fuc-Ts

α(1,3/1,4)Fuc-Ts α(2,3)Sialyl-T NeuNAcα2→3Galβ1→3GlcNAc→R

Galβ1→3GlcNAc→R

Fig. 4.6 Synthesis of difucosyl LeA/X (a), sialyl-difucosyl LeA/X (b), and sialyl-LeA/X (c) as selectin ligands during the inflammatory response towards endothelium interaction

4.8 Carbohydrate Recognition of Target Antigens by DCs During Infection and. . .

151

also prefers the terminal GlcNAc residue. In contrast, for the substrate of α(2,3) sialylated polylactosamines, human α1,3FucT-IX prefers fucosylation of internal GlcNAc residues, which is far from the SA [287]. Thus, VIM-2 is the glycolipid class bearing internally fucosylated sLex isomer. The VIM-2 antigenic epitope contains an SAα-2,3- moieties with two LacN units, where the α-1,3-fucosylated moiety is lined to the GalNAc residue of the internal LacN units. VIM-2 on glycolipids is a binding determinant for E-selectin, and, therefore, VIM-2 carbohydrates potentiate E-selectin binding to the Fut-9-expressing cells [289]. The VIM-2 has the carbohydrate structure of Galβ1,4GlcNAcβ1,3Galβ1,4 (Fucα1,3)GlcNAcβ1,3Gal β1,4GlcNAc β1,3Gal β1,4GlcNAc β1,3Gal β1,4Glcβ1Cer. The VIM-2 epitope (CD65) also includes the carbohydrate structure of NeuAcα2-3Galβ1-4GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAc. This VIM-2 structure is frequently sulfated, and the VIM-2 epitope (CD65) is a putative ligand, but a minor ligand of E-selectin (CD62E). CD65 is the solely independent risk factor of AML for leukemic extravascular dissemination. The Mab VIM-2 recognizes the human myelomonocytic lineage of blood cells [290]. VIM-2 Mab binds to the LacNAc unit in β1,3-conjugated repeats [291]. CD65 carries single Fuc residue. CD65s is α2,3-sialylated ceramide dodecasaccharide 4c (VIM-2) as the VIM2specific antigenic epitope from CML cells both with sialylation and fucosylation [292, 293]. VIM-2 also binds mucins of gastrointestinal tumor such as adenocarcinoma [292]. Galectins can bind to the tandem-repeat sugars in CD65. In addition, a sialyl CD65s is a form of the 2,3-sialylated ceramide dodecasaccharide 4c or 2,3-sialyl VIM-2, having a structure of α(1,3)-fucosylated sialyllactosamines on the Neu5Ac α2,3Galβ1,4GlcNAcβ1,3Galβ1,4-(Fuc α1,3)-GlcNAc-β 1,3Galβ1,4GlcNAcβ1,3Galβ1,4GlcNAc β1,3Gal β1,4Glc β1-Cer. The Fucα1,3linked to GlcNAc is specific for the penultimate LacNAc residue of a terminal polylactosaminyl glycan of NeuAc(SA)α2,3Galβ1,4GlcNAc-β1,3Gal-β1,4 (Fucα1,3)GlcNAc-R. Because AML is a blast cell type resided of the bone marrow and peripheral blood. Both acute lymphoblastic leukemia (ALL) and AML are extravascularly infiltrated. Although extravascular infiltration is predominant in ALL, the extravascular infiltrative disease in AML is rare. As peripheral blood leukocytes traverse the vascular endothelium, AML blast cells also undergo the property in order to escape from the vasculature [294]. Interaction between leukocytes and vascular endothelium is regulated by adhesion molecules. In the adhesive extravascular infiltration by AML, a major E-selectin ligand CD15s is involved. Secondly, a minor E-selectin ligand CD65 is also involved in leukemic infiltration. In AMLs extravascular leukemic cell infiltration utilizes the CD65. When foreign agents or pathogens are invaded, systematic glycomics are changed because of the molecular adaptation of the cells or organisms as well as changes in glycan-transferring, modifying, and hydrolyzing enzymes. This process allows foreign agents or pathogenic invasion to alarm the host organism to inflammation through changes in carbohydrate expression and immunity. During pathogenic infection and inflammatory reaction, the glycans or carbohydrate structures and compositions in lipids and proteins of cells are changed. The recently made word named “glycomics or lipidomics” covers the systemic glycan structures linked to the

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glycoproteins/glycolipids/glycosaminoglycans of cells and tissues or organs. For example, pathogenic infection and consequent inflammation result in altered glycosylation of hosts. This process in modulation of host glycosylation leads to evolution and adaptation to trigger the host defense. Therefore, the evolutionary adaptation of the protein, lipids, and function of the host is carried out by altered glycosylation. As such phenotypes, the cells inform the invaded status to the bodies via immune cells in immune system, as a mode of inflammation or alarms or alerts. Many glycosyltransferase expressions are regulated during the monocytic differentiation into macrophages or DCs [295, 296]. The glycosyltransferases synthesize cell surface glycoproteins and glycolipids to regulate various cellular responses including host defense, tissue homeostasis, immunity, inflammatory responses, or cancer behaviors [297, 298]. Thus, the carbohydrates responsible for cellular regulations such as endocytosis, intracellular trafficking of proteins, folding, cell interaction, adhesion, communication, signaling, apoptosis, and proliferation are recently subjected to elucidate the functions and structures [80, 299]. Especially, the structural basis due to their diverse complexity of glycans is the next step of functional analysis to understand cell-cell recognition and interaction [300]. Moreover, those carbohydrates, glycans, function as receptors for specific ligands such as lower organisms of viruses, bacteria, and helminths during infection and toxins of GM1-specific cholera toxin [301]. Glycans are directly associated with host immune system even in innate immunity and adaptive immunity. In addition, the glycans are co-evolved to antigen presentation of MHC, priming of T cells, leukocyte homing to inflammation sites, and lectin receptor function. Changes in surface gangliosides of cells are also known with regard to function of immune cells.

4.9

Glycan-Specific Trafficking Receptors in DC Maturation

DCs with the antigen induce only a primary immune response in resting naïve T lymphocytes, priming a naive helper T cells. To B cells, DCs keep the B cell function in control. Thus, antigen-specific DCs function as a tolerance initiator, and DCs are regarded as adaptive immune response player and memory. During activation stage of DCs in such condition of pathogenic infection, (i) DCs uptake antigens and activate signaling pathway to induce DC maturation and receptor-mediated endocytosis that maintains self-tolerance, micropinocytosis, and phagocytosis, and (ii) DCs proceed to maturation step where immune stimuli induce changes in the phenotypes and functions. In maturation stage, the levels of antigen uptake and lysosomal acidification are decreased, while levels of co-stimulatory receptors of MHC-II/ CD86/CD80 and DCs-specific inflammatory cytokine production are increased. For the final stage of migration, cDCs or inflammatory DCs with antigen are migrated to T cell area. Chemokine-mediated cells are recruited to the lymphoid target site with increased adhesion to the endothelium by adhesion molecules

4.9 Glycan-Specific Trafficking Receptors in DC Maturation

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(integrin, selectin, and its ligands). When foreign agents or pathogens invade the host organisms, immune cell trafficking to inflamed sites is a fundamental action for detection and search of inflammatory site. To do this in inflammatory site, attractive trafficking molecules are synthesized from the immune surveillance cells and host target cells. By a basic mechanism of immune cells binding with trafficking molecules expressed on the host cells of tissues and immune cells can enter inflammatory site. To interact with target tissues for entering and escaping tissues, interaction of molecule to molecule is the most fundamental recognition. Then, the question is raised. What is the recognition molecule? The answer is that they are carbohydrate molecules expressed in DCs and host cells together. First, ligand-specific selectin of DCs is recognizing the glycosylation patterns. Before rolling or homing, DC cells have resting integrin structures. In the first process of tethering and rolling of DCs, non-specific glycoproteins or glycolipids and PSGL-1, on basement membrane of DC cell surfaces are attached with selectin’s ligand of sLex or sLeA. They are interacted with sLe sugar’s counterparts of E-/P-selectins present in the surfaces of endothelial cells. Also, L-selectin, 47 integrin, and 41 integrin expressed on DCs surfaces interact with PNAd, MAdCAM, and VCAM-1 expressed on endothelial cell surfaces. In the second stage of activation of DCs, cells have active integrins and chemokine signals, and these trigger the cell responses. More specifically, the G protein-coupled receptor (GPCR) present in DCs or leukocytic cells is activated for downstream signaling pathway by several low molecules including chemoattractants, chemokines, complement, PAF, LTB4, formyl peptides, and other minor molecules secreted to plasma fluids from inflammatory and injured sites. Next, leukocyte arrest to the inflammatory sites is operated by help of activated GPCR signaling. For the arrest, several known molecules were known for common trafficking molecules. They are LFA-1, Mac-1, activated 47 integrin, and activated 41 integrin expressed on DC cell surfaces, and they are interacted with ICAM-2/ICAM-1/MAdCAM, and VCAM-1 present on the surfaces of endothelial cells [302] (Fig. 4.7). When DCs are differentiated into mature forms by antigens, PSGL-1’s sLex expression is decreased for the easy DC migration to lymphatic nodes, processing, and antigen presentation. This is the reason why DCs decrease sLex expression in PSGL-1. For final stage, the arrested cells undergo polarization, diapedesis, and junctional rearrangement from the endothelial cells of endothelium. The cells entered into the tissue sites which induce proteolytic degradation of basement membrane by matrix metalloproteinases (MMP) and then progressed to interstitial migration after interaction with cytokinestimulated parenchymal cells. Finally, the DCs make a clean through clearance of the damaged or inflamed tissues, and the DC cells are migrated to draining lymph node and lymph vessel. In mature DCs, CAMs of CD44 variants [303] and MMP-2 and MMP-9 in the extracellular area are expressed [304]. If the O-glycosylated patterns of CD44 and MMP-9 are changed, their functions will also be altered.

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4 Glycans in Glycoimmunology SLeX

SLeA

SiaD2,3GalE1,4GlcNAcE1,3Gal1-R

SiaD2 ,3GalE1,3GlcNAcE1,3Gal1-R

&%

FucD1,3

FucD1,4

Tethering

PSGL-1

E-selectin

Rolling

L-selectin

Activation

Integrin Integrin DE DE

Arrest

GPCR LFA-1

Mac-1

Activated Activated Integrin Integrin DE DE

%JGOQCVVTCEVCPVU

%JGOQMKPG %QORNGOGPV 2#( .6$ HQTO[NRGRVKFG

Sialyl LeX,A

Glycoprotein / glycolipid

P-selectin

PNAd

Hepara sulfate proteoglycan

MAdCAM VCAM-1

ICAM-2

.WOGP

ICAM-1 MAdCAM

VCAM-1

Extravasation

Endothelium

Fig. 4.7 Common trafficking molecules during DC recruitment during general inflammation and migration step

4.10

Glycan Ligands in Trafficking of DC Migration

The trafficking of mature DCs to the drained lymphatic nodes is essential for the primary immune cell functions. Considering that phagocytosis-based antigen presentation is one of the actions of DCs, DCs activate adaptive immunity of T lymphocytes. The so-called antigen-presenting immune cells (APCs) indicate the macropinocytosis by receptor-mediated endocytosis or receptor-independent endocytosis. The presentation of foreign antigens activates T cell stimulators. In order to play that DCs function as an innate immunity actor and make a link of innate immunity to adaptive immune response, DCs have to recognize trafficking molecules expressed at the cells injured site and enter to the site and consequently uptake antigens (Fig. 4.7) [305]. Then, finally DCs home to adjacent lymph node to translate their antigen information to adaptive immune response and present antigens to T cell and B cell. This stage is called “cell migration step.”

4.10.1 sLex-PSGL-1 Glycans in DC Trafficking The most important trafficking molecules are glycans or carbohydrates called sLex attached mainly on O-glycan, and these capture immune cells to move to inflammatory site. In a minor case, N-glycan is also attached with sLex ligand. The receptor P-selectin binds to sLex as peripheral carbohydrate in order to capture immune cells migrated to the inflammatory site. P-selectin strongly binds to PSGL-1 expressed on

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Glycan Ligands in Trafficking of DC Migration

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Fig. 4.8 Biosynthesis of sialyl Lewis antigens from Tn antigen originated from Thr/Ser residues on polypeptides by sLex-synthesizing glycosyltransferases such as C2GnT1 and C2GnT2. In tumor cells, from core 1 direction, tumor-specific sialyl 6 T, sialyl 3 T, and disialyl T antigens are produced. The name of the carbohydrate structure is indicated. An extended O-glycan contains core 2, which carries variable lengths of stem region caused by LacNAc motif repetition (n  0) and SLeX tetrasaccharide motif. Ser/Thr, serine or threonine

immune cells. During differentiation and maturation of DCs, sLex expression pattern in PSGL-1 decides the cell migration levels. Immune trafficking aptitude of DCs is thus explained by sLex pattern and sLex-synthesizing glycosyltransferase expression. Relationship between triple factors of glycosyltransferase expression pattern, sLex expression pattern, and PSGL-1-P-selectin signal pathway is a parameter of “DC maturation.” DCs are APCs, translating innate information to adaptive immune response, surveilling microenvironment, carrying foreign invasive antigens to adjacent lymph node, and presenting to T cells. For example, sLex expression level is decreased during differentiation and maturation of DCs (Fig. 4.7). This is regulated by sLex-synthesizing glycosyltransferases such as C2GnT1 (1,6GlcNAc-T or core 2 synthase and 1,3GlcNAc-T (C2GnT2) (Fig. 4.8). The gene expression of the two enzyme genes is increased during differentiation, but decreased in maturation stage, while ST3Gal-1 expression as a key factor is increased during differentiation and maturation of DC [306]. For molecular structure of carbohydrate ligands, DCs express sLex on PSGL-1 for the above missions. The PSGL-1 carries sLex as a key ligand molecule.

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4.10.2 Ganglioside Recognition by DC Receptors in Trafficking In the cell membrane, for example, in innate immunity, GSL glycans play pivotal roles to induce differentiation responses of primary defensing cells. Membraneassociated GSLs confer structural integrity to the plasma membrane, GSLs agglomerate into cholesterol dense microdomains, or lipid rafts participate in affordable recognition, adhesion, and signal transduction of cells. Signaling via lipid raft microdomain-associated GSLs is an important process especially in myeloid lineage cells for innate immune responses as well as lymphocytes and osteoclasts [307]. For example, GM3 has well been known to regulate lipid raft- and microdomainassociated cell signaling and cell adhesion in human lymphocytes [308, 309]. Increased synthesis of GM3 is also a defining characteristic that marks the differentiation and maturation of myeloid lineage precursors into monocytes and macrophages, a process that can be promoted by addition of GM3 [310]. In addition, the membrane GSLs play crucial roles in invasion and infection of extracellular infectious agents such as virus. For example, enveloped virus enters into host cells through host cell attachment and fusion into membrane. Virus adsorption occurs at the recognition of specific receptor molecules of viral attachment molecules. Gangliosides are also functional components of the plasma membrane, especially GM3- and 3SL-containing gangliosides. Sialic acid residues in gangliosides expressed in the viral coats also function as capture ligands of pattern recognition receptors of DCs [311]. Because virus captures the cell-surfaced sialyl residues on gangliosides, the gangliosides GM3, GM1, GM2, and GD1 are expressed on viral membrane coats or envelops, as well as known in several viruses including HIV, SFV, VSV, and MuLV [312, 313]. Gangliosides of the gangliotetraose series bearing the sialic acid in α2-3 linkage of GD1a, GT1b, and GQ1b, and neolacto-series gangliosides are also known to the receptors for Sendai virus [311]. Human parainfluenza viruses 1 and 3 recognize Nacetyllactosaminoglycan branches with Neu5Acα2-3Gal. Sialylylated glycan residues of gangliosides have been reported to function as cell adhesion molecules as receptors due to their hydrophilic properties. Considering that sialic acid residues on gangliosides function as host cell receptors for pathogenic bacterial toxins such as cholera toxin [550] and several viruses [314–316], the reverse biology is the case of ligand function. Accordingly, the sialic acid derivatives or analogs can be designed to inhibit the sialidase and/or receptor-sialic acid binding activity which are future antiviral strategies. Integrin-mediated binding of DCs with target cells or antigens is influenced by ganglioside. In the membrane, gangliosides localize with proteins for specific amino acid sequences. For example, GD3 is clustered with β1 integrin and affects properties controlled by integrin-mediated signaling. Chemokine receptor type 9 (CCR9)positive immune cells are enriched in the small intestine, and integrin α4β7-positive cells are enriched in the small intestine and colon. Gangliosides regulate immune cell signaling, as gangliosides are organized into microdomain (lipid rafts) and serve as

4.10

Glycan Ligands in Trafficking of DC Migration

Resting T-cells D 2,3

Disialyl T (NeuAcD2,3GalE1,3 [NeuAcD2,6]GalNAcD-Thr/Ser)

E1,3

Activated T-cells

D 2,6

E1,4

T antigen E 1,3

GalE1,3GalNAcD-Thr/Ser

Core 2 E 1,3

E1,6

GlcNAc E1,6 Thr/Ser GalE1,3GalNAcD-Thr/Ser

E1,6 GlcNAc-T (Core2-synthase)

GalE1,3GalNAcD-Thr/Ser Thr/Ser E 1,3Gal-T

Tn antigen

E1,6

D 2,3NeuAc-T

E1,4Gal-T

Thr/Ser

D2,6NeuAc-T (ST6GalNAc-I, II)

GalE1,3GalNAcE -Thr/Ser

GalE1,4GlcNAc E1,6

Thr/Ser

D 2,6 D

NeuAcD2,3GalE1,4GlcNAc E1,6

E1,6

Thr/Ser

E1,3 E 1,3

E1,4

D2,3 E1,3

D

Sialyl 6T

157

GalNAcD D-Thr/Ser Thr/Ser

Sialic acid (SA) Galactose (Gal) N-Acetylglucosamine (GlcNAc) N-Acetylgalactosamine (GalNAc) Fucose (Fuc)

DGalNAc-T

Thr/Ser

Fig. 4.9 Different glycan structures of O-glycan in resting T cells or activated T cells. Sialylation direction of T cells determines regulation of the relevant T cell responses where they become resting T cells or activated T cells

signaling molecules and receptor trafficking. For example, in T cells, glycoforms of SGL-1 in resting cells and activated cells are quite different in their O-glycans (Fig. 4.9). T lymphocyte activation pathway involves the sialylation of integrin proteins and requires interaction between membrane gangliosides and sialylated O-glycans (Fig. 4.9) [317, 318]. Sialylation direction of T cells determines regulation of the relevant T cell responses where they become activated or resting T cells. Core 2 glycan synthetic direction of Tn antigens on O-glycosylated proteins is operated in the activated T cells. The O-glycosylation enzymes are 1,6 GlcNAc-T (Core2 synthase), 1,4Gal-T, and 2,3NeuAc-T to synthesize the Gal1,3(GlcNAc1,6) GalNAc-Thr/Ser, Gal1,3(Gal1,4GlcNAc1,6)GalNAc-Thr/Ser, and Gal1,3 (NeuAc2,3Gal1,4GlcNAc 1,6)GalNAc-Thr/Ser (Fig. 4.9). Disruption of lipid rafts displaces cellular signaling molecules and alters immunoreceptor signal transduction. Sphingolipid depletion inhibits GPI-anchored protein trafficking in microdomains. For example, inflamed intestinal mucosa has decreased ganglioside content. Enrichment of intestinal mucosa with ganglioside causes a reduction in cholesterol content. Cholesterol depletion disrupts membrane microdomain structure and inhibits generation of proinflammatory mediators. Ganglioside inhibits signals caused by proinflammatory stimuli TNF-α and IL-1β in rats. Ganglioside protects the gut by attenuating proinflammatory signals.

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4 Glycans in Glycoimmunology

Chemokine Receptors in DC Trafficking

4.11.1 Chemokine Chemokine molecules are small ranged, between 8 and 10 kDa, chemoattractant cytokines and render the chemoattraction of migratory cells [319]. They have a similar tertiary structure. Chemokine superfamily includes chemotactic proteins as modulators of leukocyte trafficking [320]. Chemokine signaling occurs through chemokine receptors for immune reactions. Currently, 50 chemokines and 20 chemokine receptors are discovered from humans. The chemokines and chemokine receptors are associated within cellular responses such as homeostasis and inflammation [321]. Discovery of chemokines has been ascended to the initial GAG-binding protein of platelet factor 4 (PF-4, currently termed CXCL4) which is known for more than 40 years. The CXCL4 neutralizes Hp in coagulation [322] by heparin affinity interaction [323]. When IFN-γ-dependent cytokine known as IP-10/ CXCL10 has been discovered in 1985 [324]. The CXCL4, CXCL10, and plateletderived protein β-thromboglobulin/CXCL7 commonly contain four Cys residues in their sequences [325]. Since IL-8/CXCL8 has been defined as a neutrophil chemoattractant, it has been classified as chemokine that denotes chemoattractant cytokines [326]. Using the signature cysteine sequence, 50 more chemokines were isolated as the largest cytokine sub-class. Chemokines consist of conserved Cys residues with classification into four subfamilies of chemokines C, C-C, C-X-C, and C-X3-C, depending on the pattern of Cys residues present in the ligands [644]. Chemokines C-X-C type (termed α-chemokines) bear two Cys residues, which are distinctly specific with a single variable amino acid residue and action on neutrophils, B and NK cells, and T cells. Among them, C-C chemokines known as β-chemokines contain two adjacent Cys residues and associate with monocytes, macrophages, T cell, NK cells, basophils, and eosinophils in the inflammatory sites. The C chemokines known as γ-chemokines which are further classified to lymphotactin α (XCL1) and lymphotactin β (XCL2) have one Cys residue and act on T cells only. The last, chemokine C-X3-C known as δ-chemokine in human CX3CL1 or fractalkine, has N-terminal Cys residues which are distinctive of three variable amino acid residues. CX3CL1 is found as a soluble protein and a membranebound protein attached to a mucin domain. Interestingly, all the chemokine receptors are the types of GPCR having a seven-transmembrane domain. The non-GPCR forms are called the “decoy” receptors or atypical chemokine receptors, and these are found in scavenger receptors [327]. In communication between chemokines and their receptors, chemokine receptors bind to multiple Zchemokines, even in dimerization with other receptor species [328]. In fact, the chemokines of CCR5 and CXCR4 form their heterodimers with selectins, CD4, and integrins [329]. The chemokines and receptor expressions are spatially and temporally controlled in immune cells.

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Chemokine Receptors in DC Trafficking

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4.11.2 Chemokine Receptor Chemokine receptor nomenclature has been made. For example, CXCL8 was previously IL-8 and now is a CXC ligand, while CCL2 was MCP-1 and now called a CC chemokine. Currently, 23 human chemokine receptors are known [330]. Among them, 18 receptors are the GPCR family, whereas 5 receptors are “atypical receptors” such as ACKR1-4 and CCRL2. The atypical receptors exhibit chemokine scavenging and transport functions [331, 332]. PTMs including Tyr sulfation, alternative splicing, proteolysis, ligand modification, and ligand-receptor dimerization alters receptor ligand recognition [330]. The flexible N-terminal region is central to receptor activation in altered leukocyte activity. Different ligands can activate distinct signaling pathways following binding to the same receptor. In fact, both CCL19 and CCL21 activate chemotaxis of CCR7-expressing cells, while CCL19 induces receptor downregulation [324, 325].

4.11.3 Chemokine-GAG Interaction as a Type of Protein-Glycan Interactions Proteins relatively large in their sizes are frequently membrane anchored and are glycosylated. In case of glycans, glycans are extremely heterogeneous and often hard to characterize using the isolated glycans. Proteoglycans consist of one or more GAG chains and core protein parts. GAGs carry various disaccharide units. GAGs are also subclassified to several subgroups, depending on their composition of disaccharide units. The subclasss includes chondroitin, heparin, heparan sulfate (HS), dermatan sulfate (DS), hyaluronan, and keratan sulfate (KS). Protein-glycan interactions (PGIs) are being understood in cellular function from molecular analysis. Interaction between proteins and glycans is widespread in cellular environments, as glycan-protein bindings are being analyzed at a molecular level and challenged. The challenging interests are in the field of chemokine-GAG interaction as a type of protein-glycan interactions in order to understand GAG functions in transduction of signals through proteins that are large, membrane anchored, and often glycosylated. Because glycans are heterogeneous and difficult to isolate and characterize, new technologies including nuclear magnetic resonance (NMR) and mass spectrophotometry (MS) provide detailed structure information on moderately sized systems as well as qualitative information with the precise structural basis. NMR specifically contributes to qualitative receptor ligand bindings. Using MS, qualitative information can be precisely obtained from less material but with few size limitations. Chemokine-GAG interaction as well as N-glycosylated Ig-receptor interaction has been analyzed using the analytic technology [333]. GAG binding of chemokines is involved in leukocyte extravasation because GAG chains are involved in leukocyte transmigration. GAGs mediate cell recruitment where tissue-produced chemokines bind to cell surface GAGs. Circulating leukocytes first recognize selectins and are

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rolling circle; second, they adhere to integrin ligands on the leukocytes and transmigrate into the tissue. Third, GAG and chemokine receptor recognitions promote cell migration [334–337]. Chemokine receptor recognition as well as chemokineGAG recognition on the migrating cells determine the leukocyte type during inflammatory response. The most abundant endothelial cell GAG is HS, occupying up to 90% of total endothelial GAGs [337]. Various proteins including cytokines, adhesion molecules, proteases, and growth factors bind to HS. Endothelial cell-associated CXCL8 binds to GAG through its C-terminal GAG-binding domain for neutrophil migration. For example, chemokines CCL5, CXCL8, and CXCL12γ bind to HS through GAG-binding regions. HS-chemokine recognition has a merit to protect from proteolysis [338]. Chemokine oligomerization increases chemokine activity [339]. For example, the extracellular HSPGs such as perlecan, agrin, and type XVIII collagen bind and sequester chemokines to allow leukocyte migration, contributing to leukocyte diapedesis [340]. Hence HS-chemokine interaction is a key step of leukocyte extravasation because neutrophil migration is depended on the CXCL8-HS binding [341]. Chemokines require immobilization, upon oligomerization and GAG interactions, on cell surface GAGs to transmigrate from leukocyte circulation. GAGs are on cell surfaces or shed as soluble ectodomains. The chain lengths of GAGs range between 1 and 25,000 disaccharide units with different sulfation patterns. The most abundant form are syndecans having a TM domain on the surfaces of cells. However, the glypicans are anchored to the GPI anchors, and the other three (agrin, collagen XVIII, and perlican) are not embedded to the cell membrane but instead associated [342]. GAG-bearing six disaccharide units have been estimated to have at least 12 billion more different disaccharide sequences. The GAG sequence diversity is 100 times more than that calculated from a hexapeptide and 2,000,000 times more than that calculate from DNA [343]. Therefore, chemokine-GAG complexes even in a single chemokine are heterogeneous with large scale for their structure diversity, even for a single chemokine [319]. Additionally, only hyaluronan does not exist in C. elegans among these GAGs. Chondroitin proteoglycans regulate cell division of C. elegans. HS PG is present on the cell surfaces and ECM of cell membranes and regulates signaling pathways such as Hedgehog, TGF-βl, FGF, and Wnt pathways involved in development. Heparan sulfate chains also regulate self-renewal, ES, and pluripotency of ESCs of mice.

4.11.4 Molecular Motifs in Chemokine for GAG Recognition The GAG-binding motif present in chemokines is the C-terminal XBBXBX in CC chemokines [344] or C-terminal XBBBXXBX, where B indicates basic amino acids, while X indicates any non-basic amino acid [345]. For several chemokines, GAG-binding motifs indicate the “BBXB” motif, in which B also indicates a basic amino acid residue and X indicates any amino acid residue. For an instance, CCL5

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(RANTES) interaction with CS hexamer provides BBXB motif recognition with a 10 μM of KD. CCL5 has the 40s-loop BBXB cluster, 44RKNR47, as the crucial GAG binding epitope [346]. Tetramer model can extend to the higher oligomers than the tetramer. This information can rationalize interactions between GAG and receptor proteins [347, 348]. Interaction between chemokine CCL5 and GAGs potentiates adhesion on GAGs expressed cell surfaces for the initiation of the transmigration. Oligomerization and GAG recognition are essential for transmigration of cells. CCL5 dimerization has been experimentally evidenced [349]. However, E66S mutation of CCL5 restricts oligomerization for such CCL5 dimer. In the CCL2, the binding motif is rather the non-BBXB sequence with amino acid residues of R18, K19, R24, and K49 [346]. These residues are important for receptor binding and bind to sulfate groups attached to the GAG and Tyr sulfation-modified receptors [350]. The GAG-binding motifs are different from the receptor binding domain. Although positively charged chemokines favor negatively charged GAGs, non-specific electrostatic forces are also involved. For example, acidic chemokines of CCL3 and CCL4 also bind GAGs due to hydrogen bond and Van der Waals forces [695]. The GAG binding chemokines are crucial for chemokine activity [344, 351–353]. For example, CXCL8 truncated in its C-terminal HS-binding region cannot bind to Hp and activate leukocyte and receptor binding [354] as well as transcytosis across endothelial cells and luminal surface presentation to blood leukocytes, resulting in blocked leukocyte transmigration [355]. The HS binding domains are well established [345]. CCL5 is chemotactic for monocytes and CXCL8 for neutrophils, and CXCL12γ has a HS-binding C-terminus and stimulates migration of lymphocytes. Although CCL4/MIP-1β and CCL3/MIP-1α contain the acidic amino acids on proteins, most chemokines contain basic amino acids on proteins. This is the reason why these exhibit the highest affinity for HS and Hp among other GAGs, which have low sulfation levels, as in CS or DS [356]. Chemokines bind to receptors as monomers and also oligomers. Many chemokines oligomerize [351, 357]. CC type of chemokine CCL2/MCP-1 holds the canonical tertiary structure [358] with “CC dimers.” Other CXC chemokine forms and CXCL8 associate with a “CXC dimer” from the identical monomeric forms [350]. CXC chemokine dimer helps to select its appropriate CXC receptor and CC chemokine dimer for CC receptors. Disulfidebonded CXC chemokines are also dimerized. Chemokine monomers can be acted as receptor agonists. When dimeric form of CC chemokine and CXC dimers cannot recognize receptors, chemokine oligomers are important for GAG recognition to many chemokines. GAGs stabilize dimers and chemokine oligomers [359– 361]. Chemokine-GAG binding and chemokine oligomerization enforce each other to concentrate chemokines near inflammatory sites [362]. Each chemokine displays monomer state (CCL7/MCP-3), tetramer state (CXCL4), and polymer state (CCL5) [363, 364]. Although CCL7/MCP-3 does not take its oligomeric forms, the CCL7/MCP-3 binds GAGs due to its dense GAG binding epitopes, compared to oligomerizing chemokine CCL2. Chemokine oligomer formation and GAG recognition are crucial for migration capacity of cells [365–367].

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Each chemokine exhibits affinity for each GAG. GAG affinities of CXCL4, CXCL11, CCL5, and CCL21 are high, whereas the GAG affinities of CCL2 and CXCL8 are intermediate. GAG affinities of acidic CCL3 and CCL4 chemokines are weak [367]. The CXCL12 isoform, CXCL12α, forms dimers and polymers [368], while another CXCL12 alternatively spliced variant, CXCL12γ, does not oligomerize, but CXCL12γ has 30 amino acid C-terminal extension with BBXB motifs with high affinity for GAGs [369]. Seemingly, the CCR7 receptor ligand, CCL21, contains a basic 40-amino acid extension in C-terminus for GAG immobilization and DC recruitment [370]. Thus, alternative GAG interaction controls chemokine- and GAG-dependent migrative behavior [369]. GAG sulfation is crucial, as Hp 2-O-desulfation loses chemokine-binding affinity. Although HS prefers CCL2 dimers, Hp-bound CCL2 tetramers are rather stabilized [339, 363]. Apart from homo-oligomers, chemokines heterodimerize [371–373] for GAG receptor activation.

4.11.5 C-C Type Chemokine Receptor 4 (CCR4) and Specific Ligand 17 (CCL17) and Specific Ligand 22 (CCL22) CCL17 and CCL22 are specific ligands for C-C type chemokine receptor 4 (CCR4). CCR4 was found from basophiles of human [374]. The murine orthologue is predominantly present in the thymus, lymph node T cells, and peripheral T cells [375]. CCR4 species is mainly present in T cell subpopulations of the activated types Th-2/Treg/T cells. Th-1, Th-2, and polarized Th cells exhibit different chemokine receptors with migration activities. Th1 cells produce CXCR3/CCR5, but Th2 cells produce CCR4/CCR8 [376, 377]. Human and mouse Th17 cells express CCR4 [378]. Memory Th17 cells of human coexpress CCR4 and CCR6. CCR4 expression is restricted to DCs, platelets, NK cells, monocytes, and macrophages [374, 379– 381]. CCR4 is involved in various diseases. CCR4 expression is also seen in T cell, which are skin-homed, and in Th-2 phenotype for skin-involved allergic immune responses [382]. The Ccr4 gene-deficient KO mice do not exhibit its phenotype changes in a Th-2-dependent inflammation [380]. In addition, Th2 cells express CCR4 in allergic disease, and CCR4+ T cells produce Th2 cytokines in asthmatic patients. CCR4 is involved in activation of innate immune response and Th2-involved immune responses [383, 384]. CCR4 is also involved in sepsis, as Ccr4 KO mice survive even in LPS-activated endotoxic condition by decreasing the expression of proinflammatory cytokines and function of macrophages [381]. For CCL17, in 1996, a C-C chemokine gene has been reported as activationregulated chemokine (TARC) and thymus-type chemokine. It has been renamed C-C chemokine ligand 17 (CCL17). The CCL17 gene is expressed constitutively in the thymus and activated PBMCs. CCL17 and its receptor CCR4 are expressed on DCs and macrophages. CCL17 is expressed in murine bone marrow-derived DCs [385]. DCs express CCL17 in homeostasis and inflammation [386]. Among classical

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Chemokine Receptors in DC Trafficking

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or conventional cDCs and pDCs, only cDC types express MHC-II antigens, which are engaged in phagocytosis and antigen presentation. cDCs are further divided, depending on antigen expression of its surface marker CD11b. The CD11bexpressing cDCs stimulate CD4+ T cells. Also, CD8α-expressing cDCs act as cross-presenting cells [387]. In other sides, DCs can also be subdivided into DC-1 and DC-2 subsets, depending on their induction abilities of Th-1 and Th-2 cell differentiation, respectively. CCL17 is expressed predominantly by a CD11bpositive cDC subset in lymphatic organs, not by the spleen. TLR also induces the expression of CCL17 gene in cDC subset, which express CD11b antigen, in lymphatic nodes, not in the spleen. α-GalCer-activated NK T cells in mice stimulate CD8α + DCs and produce CCL17 in the cells. CCL17 is involved in various diseases. CCL17 induces immune reactions of contact hypersensitivity, allograft rejection event, IBD, atopic dermatitis, and atherosclerosis [386, 388]. The reduced atherosclerotic level in Ccl17-deficient KO mice is modulated by Treg cells. The CCL17 expressed in DCs reduces the Treg cell subset level. CCL17 reduces the cell numbers of Treg cells; however it activates the IL-12/IL-23 cytokine releases in DCs. The second ligand specific for CCR4 is the C-C chemokine ligand, CCL22. The CCL22 is also a macrophage-derived chemokine and shares 37% homology with CCL17. Interestingly, the genes encoding CCL17 and CCL22 are proximally present to the close CX3CL1 gene location on chromosome 16q13 of human [385]. The two CCL17 and CCL22 are present in the myeloid cells and thymus. M2 type macrophages involved in Th-2 responses express CCL22 [389]. Monocyte-derived DCs also express CCL22 and thus DCs bind to activated T cells. Chemokinedependent T cell interaction with DCs indicates T cell priming [390]. LPS and IL-1β, TNF-1, and CD40 ligand induce CCL22 expression in DCs. DCs which are activated contain CCL22 and N-terminally truncated CCL22. CCL22 is indeed a chemoattractant for antigen-presented T cells or NK cells, DCs, and monocytes [391]. CCL22 expression is stimulated by the IL-4/IL-13, which are the Th2 cytokines, in myeloid cells and suppressed by the IFN-γ known as a Th-1 cytokine and responding to polarized Th-2. In contrast, IL-4/IL-13 inhibit IFN-γ- and TNF-α-induced CCL22 expression in keratinocytic cells. This indicates a cell type expression of CCL22 gene [392]. CCL22 is also involved in various diseases. CCL22 is involved in allergy and autoimmunity to tumor. In the lung, alveolar macrophage and DC depletions save severe inflammatory mouse caused by IL-13 action, through inflammation protection by reduction in the CCL17 and CCL22 production [393]. CCL22 immune modulation in autoimmunity is based on regulation of the Treg cells. CCL22 expression of pancreatic β cells in IDDM diabetes eliminates Treg cells’ autoimmune attack [394]. CCL22-mediated control of Treg cells is observed in tumor cells in humans, because cancer cells and tumor-associated microenvironmental macrophages express the CCL22 and effectively recruit the Treg cells, Therefore, CCL22 inhibits tumor-mediated T cell immunity. CCL22 indicates an immune escape response of tumor [395]. Expression patterns of chemokine receptors and chemokines also depend on the cell status of maturation of each cell. Conventional cDCs are continuously supplied

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by bone marrow-derived DC precursors (BMDCs) called pre-DCs. The pre-DCs traffic through the BM-blood vessel-blood-peripheral tissue transmission. The born pre-DCs are heterogeneous in subset population, and the currently known pre-DCs are cDC subset-committed progenitors called pre-cDC1 and pre-cDC2. Pre-DC subsets are continuously trafficking into their peripheral locations to afford the host immune responses against immune SAMPs or PAMPs. However, the relation between pre-cDC1 and pre-cDC2 in trafficking, homeostasis, and fighting against PAMPs is not explained. A recent report discriminated the relation between the two subsets. The pre-cDC1 but not pre-cDC2 expresses the Th1-associated chemokine receptor CXCR3. Moreover, the CXCR3 played a cell-specific role in the trafficking of pre-cDC1 to melanoma. Thus it is considered that each pre-cDC1 trafficking differs from others such as pre-cDC2, indicating the different DC lineage determinants [396]. For the case, CCR7 together with MHC as a chemotactic receptor is activated and induced. These two coreceptors allow the DCs to move from the antigen uptake region to the blood stream-based spleen or the lymph node. Such acted CCR7 and MHC stimulate the cytokine production. Chemokines and their receptors, for example, CCL21 and CCR7 [397], have active roles in the migration behavior [398]. PSGL-1 is also a functional secondary receptor responsible for CCL21 of T cells of mice, homing to lymph node [399]. Interestingly, O-glycosylation levels of PSGL-1 modulated the level of the T cell homing event. Intestinal DCs include two subset populations based on the presence of CD103 and C-X3-C motif-bearing chemokine receptor 1 (CX3CR1) proteins [400]. CD103+CX3CR1
cells are a major cell type of migratory intestinal DCs and activate regulatory T cells. In contrast, CD103
CX3CR1+ DCs are the luminal antigen-interacting resident cells, initiating local immune responses. Similar mechanism is also observed in the expression of CCR7 (C-C chemokine receptor type-7) and MMP-9 responsible for the DC migration. In case of PGE2 (prostaglandin E), PGE2 is known to stimulate DC migration by induction of the expression of the above genes. Glycosyltransferase inhibitor, BGN-treated DC, has been known to decrease the DC migration effect in chemotaxis.

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Glycan Structure-Recognizing Selectins in DC-Endothelium Interaction During Infection and Inflammation

Circulating DCs are continuously interacted along with the endothelium of vascular vessel. This is to avoid any damages from the hemodynamic shear forces to have anti-shear power resistance by binding between vascular walls and binding molecules. The binding and interacting molecules are called selectin. The selectin is defined as single-chain transmembrane glycoprotein, and three different selectins such as L-, P-, and E-selectins are known. Selectins interact with ligands. Selectins belong to C-type lectin type, which is a family of CAMs and recognizes and binds to

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specific glycan determinants in a Ca2+-dependent fashion. For selectin binding, specific carbohydrate ligand is needed [401]. Selectins are present in platelets, endothelium, or leukocytes, and they are E-selectin, P-selectin, and L-selectin. They recognize the SA- and Fuc-containing tetrasaccharide, where SLeX is the main form. Ligands of selectins are present mainly in immune and endothelial cells, which are predominantly expressed in the condition of inflammatory responses. sLex is expressed in neutrophils, lymphocytes, and DCs as functional selectin ligand. Using anti-sLex antibody, sLex was known to mediate the DC-selectin binding. The selectin ligand SLeX is present in glycolipids or glycoproteins. The protein-linked SLeX, PSGL-1, is known in moDCs. During chemokinedriven migration of monocytes and DC progenitors, SLeX species-coated PSGL-1 proteins are the binding ligand to P-selectin species [402].

4.12.1 3 Species of Selectins: E-, L-, and P-selectins The storage pore of P-selectin molecules is the endothelial Weibel-Palade body and moves to the surfaces during proinflammatory responses [403]. Cell-cell interactions mediated by molecular recognition of P-selectin with PSGL-1 are well known. Cellcell interactions driven by P-selectin-PSGL-1 binding mediate leukocyte rolling process on inflamed endothelial cells or adherent, activated platelets. Neutrophils, or PMNs, or other leukocytes can also move through rolling by P-selectin recognition. PSGL-1-P-selectin interaction also mediates adhesion of platelets to myeloid leukocytes to form mixed cell aggregate, and this is a responsible mechanism of vascular rupture and atherosclerosis. In addition, the adhesion of platelets to leukocytes mediated from the PSGL-1 and P-selectin interaction generates aggregation in vascular endothelial cells, and this is a reason why tumor cells adhere to platelets and endothelial cells. E-selectin is newly synthesized at every need without storage pools, but L-selectin is newly generated in stimulation-dependent leukocytes only. Selectin structures are similar in their protein architecture with the three distinct domains structures of complement-binding protein like domain, EGF domain, and lectin domains. They are transmembrane-anchored through the plasma membranes, and the domain structures are topologically shredded in the extracellular regions (Fig. 4.10). In cancer cells, SLeX or SLeA is a carbohydrate ligand, which are interacted with E-selectin present in blood vessel endothelial cells. Cancer cell SLeX or SLeA epitope stimulates the cancer cell attachment to endothelial cells. The adhesion is enhanced upon TNF-α-stimulation to endothelial cells. SLeX or SLeA present in cancer tissues helps hematogenous metastasis with cancer prognosis (Figs. 4.11 and 4.12) [402]. Both selectins of E-/P-selectins are mainly present in the endothelial cell surfaces upon inflammatory activation, and they bind to SLeX of the carbohydrate structure of NeuAcα2,3Galβ1,4(Fucα1,3)GlcNAc-R and SLeA structure of NeuAcα2,3Galβ1,3 (Fucα1,4)GlcNAc-R epitopes as well as the sulfated derivatives. The original roles

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Complement-binding protein like domain

w“ˆš”ˆG”Œ”‰™ˆ•Œ

EGF domain

j ›–š–“Š

lŸ›™ˆŠŒ““œ“ˆ™G

jG

Lectin domain

uG

sTzŒ“ŒŠ›•G

lTzŒ“ŒŠ›•G

wTzŒ“ŒŠ›•G

Fig. 4.10 Structure of selectins. Selectins’ structures are similar in their protein architectures with the three distinct domain structures of complement-binding protein like domain, EGF domain, and lectin domains

Normal cells

Cancer cells

#FJGUKQP

TNF-D D (-)

Cultured cancer cells

TNF-D (+)

Cultured endothelial cell monolayer with TNF-D

Fig. 4.11 Tumor cell adhesion to endothelial cells stimulated with TNF-α

of the epitopes include leukocytic homing, where E-/P-selectins recognize cancer cells harboring SLeX or SLeA antigens. The recognition initiates attachment to the endothelial cells and transmigration to the invasive sites of target tissue.

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Infiltration

Intravasation

Detachment

Circulating

Adhesion

Metastasis

Cancer Cells Sialyl LeX Sialyl Lea

Endothelium

E-Selectin

Fig. 4.12 Schematic process of the complex and multistep process of hematogenous metastasis of cancer

Hematogenous metastasis of cancer is carried out by a multistep process. Cancer cells acquire the cell motility and invade blood vessels [404]. Through these steps, the cancer cells migrate across the stroma and blood vessels. SLeX- or SLeAexpressing cancer cells in the invasive sites invade a blood vessel and circulate to organs. SLeX or SLeA selectin recognition is crucial for the construction of metastatic foci at each organ. SLeA or SLeA expressed on cancer cells adheres to selectins present on endothelial cells and gradually provides strong attachment and transmigration. Thus, blood-circulating cancer cells with SLeX or SLeA enhance the metastatic potential. SLeX or SLeA expressed at the invasive sites is a leading factor for postoperative cancer recurrence of metastatic cancer cells. Hematogenous metastasis events of cancer cells are mediated through binding of SLeX- or SLeA-positive tumor cells to E-selectin expressed on the endothelial surfaces. Blocking biosynthesis of SLeX or SLeA as E- or P-selectin ligand prevents the hematogenous metastatic potential of cancer cells. SLeX or SLeA antigen is biosynthesized on glycolipids or glycoproteins by various glycosyltransferases. The essential reactions are completed by distinct α1,3 or α1,4-fucosyltransferases and α2,3-STs. Currently, in humans, six fucosyltransferases of FucT-1 to FucT-7 and FucT-9 enzymes [405] and six α2,3-STs of ST3Gal-I to ST3Gal-VI [406] catalyze the formation of sialyl Lewis antigens using type I epitope of the Galβ1,3GlcNAcβ1-R or the Galβ1,4GlcNAcβ1-R type II substrates in the Golgi complex compartments. The α1,2Fuc-transferase (FUT1) (E.C. 2.4.1.69) disturbs terminal α2,3-sialylation and consequently prevents the SLeX or SLeA synthesis [407]. Thus, FUT1 intercepts the type II precursor substrates before the type II precursors are trafficking to the α2,3-sialyltransferase-resident compartment.

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4.12.2 Representative Selectin Ligand PSGL-1 and Role of PSGL-1 O-Glycan SLex antigen is a tetrasaccharide sugar with SA, Gal, GlcNAc, and Fuc residues as specific carbohydrate structures and determinants. Tetrasaccharide sLex is known to bind three P-, E-, and L-selectins (Fig. 4.13). For selectin binding activity, addition of the sLex tetrasaccharide to O-linked glycan is needed. SLex attached to O-glycans present in the cell surfaces importantly function in cell-to-cell binding events, as SLex is present in monocytes and immatured DCs but not in matured DCs. P-selectin and PSGL-1 primarily mediate the rolling phase of the adhesion cascade. In cancer patients, selectin ligands in cells are circulated in the plasma of blood. Many selectin ligands exist as O-glycans of mucin-type sialylated and thus classified as sialomucin glycoproteins, because they contain sialic acid in PSGL-1, CD34, and GlyCAM-1 carrying sLex. In protein level of PSGL-1 and P-selectin, the two amino-terminal domains are also interacted during homing and rolling. PSGL-1 in cell membranes has a disulfide-bridged dimerization form (s-s bond) in confirmation, and it contains

A)

SLeX

B)

N-linked

O-linked

ST3Gal-4 E1,4GalT-1 D1,3FucT-4, FucT-7

ST3Gal-1 Core 1 E1,3GalT

Core 1

E3GlcNAcT n E1,4GalT-1

n

n

Core 2 GlcNAcT (E1,6) D GalNAcT

Core 2 Thr/Ser

Thr/Ser

Thr/Ser

Core 2

Core 4

Asn SO4

6-SO4-SLeX GlcNAc-6-Sulfo-T-1/2

TNF-D PGE2

ST3Gal-1 Core 2 GlcNAcT

SLeX

Sialic acid (SA) SiaD2,3GalE1,4GlcNAcE1,3Gal1-R Galactose (Gal) N-Acetylglucosamine (GlcNAc) FucD1,3 N-Acetylgalactosamine (GalNAc) Fucose (Fuc) SLeA SiaD D2 ,3GalE1,3GlcNAc E1,3Gal1-R FucD1,4

INF-J

Core 2 GlcNAcT

Fig. 4.13 The biosynthesis of carbohydrate selectin ligands, sialyl Lewis ligands, as forms of N-glycan and O-glycan in DCs. (a) Simplified biosynthetic pathway of SLeX-decorated core 1/core 2 O-glycosylations. Simple pathway of SLeX-bearing O-type glycans. Modulation of DC’s glycosyltransferases by maturation stimuli. PGE2, Prostaglandin E2. (b) Sialyl Lewis ligands as forms of N-glycan and O-glycan. The name of the carbohydrate structure is indicated. An extended O-glycan contains core 2 O-glycans. Ser/Thr, serine or threonine

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the sLex component in broad ranges of carbohydrate length and residues. In monocytes and immature DC, only PSGL-1 is the glycoprotein carrying sLex. sLex as glycan acts in extravasation through selectin recognition which is expressed on PSGL-1 in monocytes and immature DC. DCs express sLex glycan ligands present in PSGL-1, and these ligands recognize P-selectin receptor for their functional migration to lines of endothelial cells on vascular endothelium. When DCs are maturated by specific antigens and inflammatory cytokines, the level of PSGL-1’s sLex expression is decreased. By decreasing sLex expression in PSGL-1, DCs can migrate to the lymph node for processing antigen presentation. For P-selectin interaction with PSGL-1 ligand, O-glycan attached to PSGL-1 displays importantly in the P-selectin adhesion. Thus, if maturation of DCs is over, sLex expression is lost, even though these cells retained expression of PSGL-1. This suggests specific affinity of P-selectin counter-receptor present in myeloid lineage cells. To express function as APCs, DCs and their precursors such as progenitor DCs require translocation and transfer from circulating blood to the damaged peripheral tissues. Further, when foreign antigens induce DC activation, they have to migrate using O-glycans produced in the inflamed tissue sites to the drained lymphatic nodes. In a similar mode, the O-glycans are also crucially associated with T cell trafficking. Therefore, it is certain that sLex expression on PSGL-1 O-glycan is modulated during each step of differentiation and maturation of DCs. PSGL-1 O-glycan is an enforcing factor during the white blood cell recruitment to inflamed sites raised by infection or inflammatory agents.

4.12.3 Glycosyltransferases for Biosynthesis of PSGL-1 O-Glycan DC maturation is involved in the changed production levels of GTs required in O-glycosylation process. Biosynthesis of selectin ligands is well explained in DCs. The mechanistic perspectives of how they are modulated during diverse environmental situation are still under investigation through many glycoimmunists [402]. Biosynthesis of sLex-decorated O-glycans has been studied and synthesis of mo-DC’s glycosyltransferases is modulated during maturation stimuli. As the most significantly changeable glycosylation, sialylation event in DCs is increased in DCs, and this event consequently results in the increased sialylation contents because it is an important issue in the maturation process [408]. Sialyltransferases and sialidases are differentially expressed during differentiation and maturation of DCs. For example, ST3Gal-1, ST6Gal-1, Neu1, and Neu3 activities are increased during DCs differentiation. ST6Gal-1 mediates the transfer of SA residue by an α-2,6-SA linked to a terminal Gal residue of type 2 (Galα1,4GlcNAc) disaccharide. ST6Gal-1 enzyme is mainly present in the Golgi apparatus, extracellular region, and cell membrane [409]. After maturation, DCs show the increased α2,3-sialyl glycans and decreased α2,6-sialyl glycans. Especially, the ST3Gal-4 and ST3Gal-6 activities

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are required for the adhesion-involved sLex antigens, and these glycans allow the DC cell rolling and homing. The expressed sialic acids function as sialic acidrecognizing DC receptors or the effector cells such as T cells. C2GnT1 mRNA downregulation and enzymatic activity is correlated with ST3Gal I and ST6GalNAc II mRNA upregulation. This change in glycosylation leads to lack in the core 2 structures and sLex structures, eventually default in the reduced P-selectin ligand [410]. GT expression related to O-glycan synthesis is increased in differentiation and maturation of DCs. The O-linked glycans must undergo posttranslational modifications in order to function (a counter-receptor for P-selectin) in enzymatic reaction of α1,3-Fuc-transferase and α2,3-ST. For fucosylation of SLex antigen, α ! 1,3fucosyltransferases of FUT4 and FUT7 are known as key enzymes in leukocytes. Interestingly, the glycosyltransferase expression is mediated by PGE2, which is important for DC migration of human. O-glycosylation profile of human mDCs and the O-glycan pattern present in mature cells are similar to the naive T cellexpressing O-glycan types. Thus, O-glycans are suggested to exhibit a common role in the different events of DC migration and T cell homing. PSGL-1 carries both two types of Asn N-glycan and Thr/Ser O-glycan of glycoproteins. Each glycan has the sLe carbohydrate ligand to bind with selectins. In addition, some of carbohydrate selectin ligands is sulfonylated (such as 6-SO4-sLex) [403] (Fig. 4.13). Specific tyrosine residues in PSGL-1 protein are sulfated, and the SO3-Tyr can be strongly bound with the endothelial or platelet P-selectin when the sLe ligands are located on the surrounded region. Thus, the interactions between tyrosine sulfate residue, sialic acid, and fucose residues in O-glycan lead to strong binding capacity. The interactions with tyrosine sulfate residues of PSGL-1 and the sialic acid and fucose residues of the core 2 O-glycan are basically suggested. From sialidase or neuraminidase treatments, the PSGL-1 activity is regulated [411]. PSGL-1 treated with sialidase showed the abolished binding capacity to P-selectin, while PSGL-1 treated with peptide N-glycosidase F had no effect on recognition to P-selectin. PSGL-1 treated with the O-sialoglycoprotease, which degrades sialylated mucins, blocks interactions with P-selectin. For example, HL60 cells treated with benzyl-alpha-GalNAc, which inhibits extension of O-glycans, reduce binding of cells to P-selectin. PSGL-1 treated with endo-ß-galactosidase, which degrades type 2 polylactosamine repeats [-3Galß1 ! 4GlcNAcß1-]n, reduces binding to P-selectin. Thus, selectin ligands are actively interacted with its receptors for the immune cells to potentiate trafficking and migration in inflammation; they directly control the inflammation, leukocyte signaling, rolling, adhesion, extravasation, and inflammation-promoting factors. They are key molecules of the cell communication in forms of glycolipids and glycoproteins. Pathogens or proinflammatory cytokines equally lead to DCs maturation and their migration to lymphoid tissue by adhesion to endothelium and chemotaxis. This event is dependent on selectin recognition to sialofucosylated glycans. SAs also are involved in the firm arrest events which are mediated by chemokine receptors. SAα2,8-polysialylation formed by ST8Sia-4 of neuropilin-2 contributes to chemokine-mediated migration potentials for lymphatic nodes.

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ST3Gal-4 is not related for chemokine-mediated DC homing events. ST6Gal-1 gene-deficient KO mice exhibit impaired DC migration to the drained lymphatic nodes. Moreover, ST6Gal-1-deficient mice (together with α-2,6-cell surface sialic acid-deficient lymphocytes) exhibited reduced B cell proliferation and decreased antibody genesis during presentation of T cell-dependent and T cell-independent antigens [412]. These results indicate that low or no B lymphocyte ST6Gal-1 synthesis, together with low or no α-2,6- cell surface sialic acid production, is associated with a reduced immunoinflammatory effect. A) B).

4.12.4 Designation of Carbohydrate Glycomimetic Drugs and Natural Inhibitors of Selectins Novel carbohydrate drugs are designed and synthesized to inhibit selectin ligand binding to generate glycomimetic drugs. All cells are coated with carbohydrates. Carbohydrates contain much structural information used in various kinds of molecular recognition events. Carbohydrate structures are not directly determined by genomics. The possible number of branched and linear isomers of a hexasaccharide is more than 1.05 x 1012. Over 1 x1012 different structures are theoretically possible from a hexasaccharide (Table 4.3). Considering the possible number of isomers of a hexapeptide, only 46,656 different structures are possible. This dense structural information is used for molecular recognition [413]. This circumstance causes the isomer barrier, and the isomer barrier is the reason why single method is not possibly utilized to apply for determination of whole oligosaccharide structures in low quantity under 100 nmol amounts, which are obtained from 100 pmol amounts by single tools such as a Edman peptide method or Sanger DNA dideoxy-sequencing approach. Difficulty in oligosaccharide synthetic approach is therefore equally found by the extremely multiple number. Therefore, a new class of synthetic carbohydrate drugs are based on mainly the rational design through the structural conformation of functional carbohydrates. Complex carbohydrates contain much structural information in a small space. Recently, glycobiology and the functional carbohydrate analysis invite the possible development of novel therapeutics against human diseases to generate new pharmaceutical industry. The rational cases of carbohydrate synthesis with pharmacological activities are described. P-selectin functions as a Table 4.3 Possible number of branched and linear isomers of oligosaccharides

Saccharide name Monosaccharide Disaccharide Trisaccharide Tetrasaccharide Pentasaccharide Hexasaccharide

Hexose number 1 2 3 4 5 6

Isomer number 2 256 38,016 7,602,176 2,633,600,000 1,053,045,031,000

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E, P-selectin ligand

A)

B) P-selectin ligand

sLeA (CA19-9) sLeX (CD15s)

P-selectin is stored in αgranule

Glycomimetics P-selectin

leukocyte E,P-selectin

platelet

platelet

Cancer cell P-selectin

Endothelial cell

Endothelial cell

Activation

activation

unactivation

P-selectin store granules, Weibel-Palade bodies

Fig. 4.14 (a) Selectins E and P recognize the sLeX/A. Ligands for carbohydrate-binding proteins during leukocyte homing to activated endothelial cells. (b) Selectin E and P ligands. sLeX/A present in cancer cells recognizes the two selectins E and P present in endothelial cells to facilitate metastatic invasiveness

CAM present in the activated endothelial cell surfaces and activated platelets. In unactivated endothelial cells, P-selectin is granulated for cellular storage, termed Weibel-Palade bodies, while in the unactivated platelets, the α-granules contains the P-selectin for storage. Platelets bind to tumor cells via P-selectin, which increase the potential for the tumor cells to reach a distant site. It is important to ensure the effective arrest in capillaries and to facilitate the extravasation of the tumor cells. Therefore, P-selectin inhibitors are candidates. For example, selectins have been considered to be therapeutic targets for inflammatory diseases and cancers (Fig. 4.14). PSGL-1 belongs to a native ligand for selectins and contains both sulfated Tyr residues and SLex for pan selectin binding activity. New generation inhibitors block both the carbohydrate- and sulfate-binding domains on P-selectin. E- and P-selectins bind to sulfated Tyr residues and SLeX present in the native ligand PSGL-1 protein. PSGL-1 known as a typical mucin-type O-glycan expressed in the myeloid cell surfaces is the counter-receptor for binding of P-selectin. The PSGL-1 interaction with the P-selectin needs Ca2+ ions. Sulfated tyrosine residue and the SLeX epitopes present on the O-glycans attached to PSGL-1 are necessary. Both Pand L-selectins require proper sLex glycosylation and Tyr sulfation of PSGL-1 for strong affinity binding. The sulfotyrosine-carrying PSGL-1 O-glycans with SLex constitutes the P-selectin binding site [414]. Therefore, sulfated moiety-based molecular designation can be used for inhibitory candidates of P-selectin. During the sLeX modification with E-selectin-selective recognition, bioactive conformation of parent glycomimetic 9669a has been evolved. Consequently, GMI-1014 series show the higher stacking and is followed by 69669a during preorganization of the bioactive conformation with selectin inhibitory activity [415]. The carbon 2-position modification of Gal residue enhances binding affinity for E-selectin [416].

4.12.5 Glycomimetic Drug Candidates The developed GMI-1070 is a pan selectin antagonist. GMI-1070 has been synthesized through convergent steps and large scale-up chemistry development

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[417]. GMI-1070 is a rationally designed agonist as a small compound, which is a pan selectin inhibitor. Among them, the GMI-1070 antagonist molecule, which was rationally and empirically designed, binds to all three selectins of E, P, and L types. Scalable chemical synthesis enables to synthesize currently in kilogram scale. Several carbohydrate companies are involved in large-scale process of production, and they include Carbogen AC (Switzerland), Cambridge Major Laboratories (USA), SynphaBase AG (Switzerland), Glycosyn (New Zealand), and Carbosynth (UK). For the GMI-1070 phase I clinical trial of healthy volunteers, pharmacokinetics of a single dose of GMI-1070 have been obtained in healthy volunteers. In addition, phase 1 healthy volunteers showed unremarkable safety profile for both PH1 studies. This indicates clean safety profile in phase 1 clinical trials. Summary of the GMI-1070-101 in a single dose described that there is no serious adverse events (SAEs). All adverse events (AEs) were mild or moderate within grades 1 and 2. In the multiple doses of GMI-1070-102, there was also no SAEs, and all AEs were mild or moderate within grades 1–2, as equally described between drug group and placebo controls. Testing effects of GM1-1070 have been demonstrated on vaso-occlusive crisis of sickle cell patients in clinical trials. In phases I and II, sickle cell patients improved the vaso-occlusive crisis (VOC) with no serious adverse events (SAES) and serum half-life of 7 to 8 hrs in humans. Changes in relevant biomarkers (PMA) have been certificated as GMI-1070 hits target in patients. This indicates no major metabolic breakdown with 90% more excretion to extrabody. Sickle cell VOC in animal models is specifically featured with aberrant cell adhesion. This indicates that selectins are associated with the genesis of the vasoocclusions for the VOC expression. Homing, rolling, trafficking, and cell adhesion of leukocytes are basic behaviors to vascular endothelial cells, which are activated by several inflammatory agents or mediators. In addition, other cellular adhesion molecules such as integrins are also conformationally changed to forms of high affinity binding capacities and eventually bind to other blood cells such as RBC and platelets to enhance the formation of occlusions [418]. Sickle cell disease (SCD) known since 1910 is a feature of peculiar appearance of the RBC. The disease originates from an abnormal hemoglobin. Sickle RBC stimulates endothelial cells directly by adhesion. The activated endothelial cells abnormally recruit rolling and adherent leukocytes with chemokines, selectins, and immunoglobulin families, contributing to vascular occlusions. In sickle model mice, vaso-occlusion occurs and is prevented by blocking leukocyte adhesion. Because the molecular discovery of sickle cell disease is crucial in modern biological science using the modern tools of molecular and cellular biological technology, this type of the carbohydrate-based advances will innovate the field. GMI-1070 extends survival in sickle cell mice with VOC. Treatment model for testing the effects of GMI-1070 on VOC is determined by IVM. Age- and gender-matched SCD mice identical and genetic cohorts are created by transplanting bone marrow cells from Berkeley SCD mice containing human sickle hemoglobin into irradiated C57BL/6 male mice. In the VOC mice induced by administration of TNF-a, once VOC is established, GMI-1070 is administered. The VOC mice exhibited relevant improvements with the increased survival, improved blood flow, reduced leukocyte-endothelial interactions, and reduced

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leukocyte-RBC interactions. Effects of GMI-1070 in sickle cell mice in VOC have been confirmed even by intravital microscopy [419]. GMI-1070 has been evidenced in pre-clinical models. In the GMI-1070-administered pre-clinical animal model, thrombus formation has been inhibited. Other positive effects of GMI-1070 include the increased survival in multiple myeloma and the inhibited relapse in multiple sclerosis (EAE). In the treatment of VOC in sickle cell mice, the GMI-1070 treatment exhibited several improvements including the inhibition of infarct size in myocardial infarct model, inhibition of delayed-type hypersensitivity reaction, inhibition of epileptogenesis induced by pilocarpine, protection of mice treated with chemotherapy, and cell cycle inhibition of hematopoietic stem cells. Currently, the phase II trials for clinic of the sickle cell patients in crisis are being tried in 20 hospital centers in the USA and Canada. In the industrial level, potential glycomimetic drugs are validated from the pharmaceutical industry. For example, Pfizer signs $340 million licensing pact with GlycoMimetics. GlycoMimetics initiated a global licensing agreement with Pfizer for GMI-1070, which is mimetic drug for an experimental treatment for vaso-occlusive crisis of erythrocytic sickle disease of human patients. However, the contract deal seems to call for GlycoMimetics to complete the phase II trial study for the mimetic drug. If the issue is solved, Pfizer may further develop and commercialize it. GlycoMimetics has been known to calculate their royalty income worth as much as $340 million [420].

4.12.6 GAG-Glycomimetic Drugs On the other hand, the natural P-selectin inhibitor, heparin as GAG, has been known to inhibit P-selectin. Due to high anticoagulant activity, it can potentially cause hemorrhage. With regard to natural inhibitors, marine invertebrates are rich sources of heparin-like molecules and sulfated polysaccharides, but less is known about the anti P-selectin activity of sulfated fucans and sulfated galactans from sea urchins (Fig. 4.15). Sea urchin sulfated polysaccharides such as sulfated fucan and sulfated galactan function as a P-selectin inhibitor. Sulfated polysaccharides inhibit binding of tumor cells to P-selectin and consequently prevent interaction of tumor cells with platelets in vivo and in vivo through P-selectin-dependent manner in inflammatory cell recruitment. The sulfated polysaccharides inhibit leukocyte recruitment in P-selectin knockout mice. Fucan from S. franciscanus does not present antimetastatic activity. The very similar sulfated polysaccharides isolated from urchins are composed of 2-O-sulfated monosaccharide units. They also have different anticoagulant effects. To prevent selectin-mediated metastasis, mouse models of P-selectin-dependent tumor progression and inflammation have been used [421].

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A)

Strongylocentrotus franciscanus Fucan 1→ 3 linked 80kDa

Strongylocentrotus droebachiensis Fucan 1 → 4 linked 100kDa

Echinometra lucunter Galactan 1 → 3 linked 80kDa

B)

Hematogeneous metastasis

Sulfated polysaccharides Hematogeneous metastasis Fig. 4.15 Sulfated fucan and sulfated galactan from sea urchin

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Chapter 5

Pathogen-Host Infection Via Glycan Recognition and Interaction

5.1

Lectin Recognition of Glycans on Cell Surface and Soluble Glycans

The immune system selects the targets through immune recognition. Lectins in innate immune cells recognize oligosaccharides of cell surface and soluble glycans that encode complex information. Lectin repertoires are extremely diverse in their nonself and self-recognitions. For example, how is diversity in “nonself” and “self”recognition achieved? Binding to targets is primary in innate immunity through the innate immune cell populations of myeloid cells, NK cells, and innate lymphoid cells. In certain circumstances, nonimmune cells and ancient type of humoral complements can also bind to targets. How extensive are the lectin repertoires in a certain species? Have they evolved through the functional and structural diversities? Lectins recognize cell-surfaced and soluble glycans. Representatively, CTLs like MBL in innate immunity directly recognize microbes with opsonic effect and activation of complement pathways. Oligosaccharide structures of cell surface and soluble glycans encode complex information. Like the stepwise recognition, cellular information is being decoded by carbohydrate-binding molecules, and these carbohydrates regulate the interaction between cells and cells or interaction between cells and ECM and, eventually, cellular functions. The recognition occurs in early development as a type of “self-recognition” and innate immunity as a type of “nonself and self-recognition” (Table 5.1). The most specific aspects of the lectins are in their diversity expressed as lectin repertoires in self and nonself recognition, although it is not fully understood yet how is diversity in “self”- and “nonself” recognition achieved? Although innate immunity eliminates most pathogens, certain pathogens are not eliminated because the pathogens produced virulence factors. Innate immune responses are not specific originally but adapted. How extensive or broad are the lectin repertoires in a given species? And have they evolved during early evolutionary phase in their functional diversity? Innate immune receptors include a variety of lectin families as forms of soluble lectins and membrane lectin © The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2022 C.-H. Kim, Glycobiology of Innate Immunology, https://doi.org/10.1007/978-981-16-9081-5_5

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Table 5.1 Differentiation of innate and adaptive immune responses Innate immune response Fast (minutes to hours) Diverse receptors “Hard-wired” in the germline Locally distributed Macrophage, DCs, PMN, and NK cells Non-clonal cell specificity Non-immunologic memory

Adaptive immune response Slow (4–7 days or more latently) Distinct receptors of TCR and Ig Genetic recombination widely to count-react Distinct tissue distribution B and T cells Clonal cell specificity Immunologic memory

receptors. Innate immune system expresses distinct receptors known as PRRs, which directly recognize PAMPs. The direct recognizers are, for example, CD14, DEC205, and collectins. Complement receptors and Toll receptors bind to PAMP recognition products. Because of the nonspecificity of innate immune responses, alternative complementation cooperates with the PRRs. The PRR receptors recognize specific pathogenic components such as glycans because glycans face the outmost world with diversity in the glycan structures and patterns as the nonself and selfrecognition basis. Cell-surfaced receptors as the first defense line alarm the pathogenic presence. Soluble lectins include ficolins, lung surfactant, pentraxins, Man-binding lectins (MBL), etc. [1]. Currently, three known C-reactive protein (CRP), serum-amyloid P protein (SAP), and long pentraxin 3 (PTX3) are the group of pentraxins. Lectin membrane receptors or membrane-associated lectins include mannose receptor (ML), DC-SIGN, Dectin, NK cell receptors, Scavenger receptors, Complement receptors, and TLRs. Membrane-associated lectins consist of transmembrane domain, complement-binding domain, EGF-like domain, and carbohydrate recognition domain (CRD), whereas soluble or humoral lectins are comprised of Cys-rich domain, collagen-like domain, and CRD. During the microbial infection, host innate or acquired immune defense involves the systemic changes in host glycosylation in cell surfaces. In sides of pathogens, they evolve to modulate host immune defenses by modulating its glycosylation. The “self”- and “nonself” glycans are distinguished by lectins. SAs on cell surfaces in prokaryotes are the targets for attack, but they are eukaryotes are SAMPs. Many receptors are found to be pathogen-recognizing receptors in innate immune cells, including TLR, C-type lectins, ML, and Siglecs. Most TLRs are localized to the cell surfaces as immune receptors, and certain types of TLRs are also intracellularly located as cytosolic compartments like the endosome. TLRs recognize PAMPs and DAMPs to afford immune responses in innate immunity. TLRs are interacted with PAMPs or DAMPs, allowing affordable recruiting of Toll/IL-1R (TIR) domain-carrying adaptor proteins. The well-defined TIR-adaptor protein is MyD88, which mediates diverse signal transduction pathways in order to protect them from the microbial infection. However, if TLRs are not sufficiently regulated or negatively regulated in the condition of excessive immune responses, abnormal immune responses are displayed. The resulting diseases include autoimmunity and inflammation.

5.1 Lectin Recognition of Glycans on Cell Surface and Soluble Glycans Fig. 5.1 Lectin classification

201

R-Type Lectin I-Type Lectin

L-Type Lectin

Lectin P-Type Lectin

Galectin C-Type Lectin

Table 5.2 The animal lectin families and lectin domains with known three-dimensional structure Family Galectins Pentraxins CTL I-type or Siglecs P-type lectin Calnexin ERGIC TNF HGF (NK1) Ym1 Cys-MR/FGF2 Tachylectin 2

Carbohydrate specificity Strict Gal/Lac Often noncarbohydrate Diverse Sialic acid Man-6-P Glc Man Chitobiose Heparin/heparin sulfate Heparin/heparin sulfate Sulfated glycans GlcNAc/GalNAc

Fold type β-Sandwich, S-motif β-Sandwich, multi-domain CTL C-motif Ig, Ig-like domain M6P β-sandwich, P-motif β-Sandwich Legume lectin β-sandwich TNF-β-sandwich Basic amino acids Chitinase, basic amino acids β-Trefoil β-Propeller

Ca2+ No Yes Yes No Dependently

No No

The “self”- and “nonself” glycans are recognized and distinguished by glycanbinding proteins, named lectins. This indicates that lectins are variable in their structural and functional aspects (Fig. 5.1). Lectin is a glycan-recognizing protein that can recognize various glycoconjugates on cell surfaces and extracellular matrices, ranging from the mediation of cell adhesion and promotion of cell-cell interaction to the pathogenic recognition (Table 5.2). Therefore, it is ruled out that glycans are information-carrying and third life chains, where its counterreceptors are lectins that are tools to recognize them as their colleagues to interact. Lectins characterize the cellular glycophenotype, phenotype-associated features, transforming faces, and disease-associated alterations. Therefore, it is simply described that lectins are saccharide-binding proteins. Based on structure of the CRD, animal lectins are divided into C-type, galectins, P-type, I-type, and others including heparin-binding proteins, pentraxin, and rhamnose-binding lectins. In addition, rhamnose-binding lectins found in fish eggs have been regarded as one of the lectin families. Most known animal lectins are currently one of the hot subjects of studies on immune responses. Most known animal lectins function in the tissues or blood plasma, but skin mucus lectins function externally to combat with the dermatic invasives. Similar mode to MBP, skin mucus lectin may act together with other humoral factors that exert innate immunity as immunoglobulin (Ig) and complement. The biological functions of skin mucus lectin have not yet well elucidated. However, the

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5 Pathogen-Host Infection Via Glycan Recognition and Interaction

Table 5.3 Intracellular and extracellular lectins in mammalian lectins Intracellular lectins L-type lectins P-type lectins Calnexin

Extracellular lectins CTLs (selectin, DC-sign, ASGPR, Dectin, MBP, etc.) I-type lectins (Siglecs, Ig superfamily) R-type lectins Galectins

Table 5.4 Mammalian lectins as native carbohydrate-binding proteins. Currently, 100 more mammalian lectins or carbohydrate-binding proteins are found Lectin groups C-type Galectins P-type (Mannose-6-phosphate receptor) I-type Pentraxins Heparin-binding type

Ca2+ Yes No

Specificity Variable β-galactosides

Sequence specific and features C-type specific motif S-type specific motif

Variable

Mannose-6-P

P-type specific motif

No Most No

Variable PC/galactosides Heparin/heparanSO42–

Ig-like domains Multimeric recognition motif Basic amino acid sequence motifs

information of animal lectins is quite well studied to date. For example, some animal lectins potentiate the classical complement pathway by help of MBP in innate immunity. These lectins play crucial roles in host defense together with humoral defense factors such as Igs, complement components, CRP, lysozyme, and hemolysin [2]. To date, 100 more mammalian carbohydrate-binding proteins as lectins are reported as forms of intracellular and extracellular lectins (Table 5.3). Currently, 100 more mammalian lectins or carbohydrate-binding proteins are found. Mammalian lectins as native carbohydrate-binding proteins have specific structural features and motifs to binding their carbohydrate ligands (Table 5.4). Vertebrate SA-specific lectins including humans are summarized (Table 5.5). Among them, C-type lectins are most diverse in their functions as mosaic molecules. In innate immunity, C-type lectins directly recognize of microbes, opsonic effect, and activation of complement pathways. For example, MBL is a microbial surface carbohydrate-recognizing C-type lectin. From the diversity of lectin repertoires, diversity in recognition of lectins is determined by tandemly arrayed CRDs with different carbohydrate specificity, multiple isoforms which are formed by conversion, homologous recombination, alternative splicing, and other mechanisms. The lectins bear their distinct binding properties and oligomeric quaternary structures with variable binding avidity and CRDs from different lectin families and effector domains. Thus, lectins have been diverse via coevolution in order to mediate different biological roles, immune response in inflammation and autoimmunity, opsonization of microbial pathogens, fertilization, and cell adhesion.

5.1 Lectin Recognition of Glycans on Cell Surface and Soluble Glycans

203

Table 5.5 Vertebrate SA-specific lectins. Siglec genes are from Homo sapiens. OBBP obesitybinding protein, AIRM adhesion inhibitory receptor molecule, MAG myelin-associated glycoprotein.a CD33rSiglecs are distinctly located compared to the Siglec gene cluster Lectin (synonyms) Selectins E-selectin (CD62E;ELAM1) P-selectin (CD62P; GMP-140; PADGEM) L-selectin (CD62L; Mel 14 antigen) Siglecs Siglec-1 (sialoadhesin/Sn; CD169) Siglec-2 (CD22) Siglec-3 (CD33) Siglec-4 (MAG) Siglec-5 (OBBP2)

SA specificity

Expression cells

sLex, sLea

Activated endothelial cells

sLex, SLea

Activated endothelial cells, platelets

60 -sulfo sLex

Leucocytes

Neu5Acα2,3Gal > Neu5Acα2,6Gal > Neu5Acα2,8

Macrophages

Siaα2,6Gal

B cells

Siaα2,6Gal > Siaα2,3Gal

Myeloid progenitor cells, monocytes, macrophages Oligodendrocyte, Schwann cells Monocytes, neutrophils, B cells, macrophages Trophoblasts, B cells Monocytes, NK cells Eosinophils, basophils, mast cells

Neu5Acα2,3Gal Siaα2,6Gal, Siaα2,3Gal > Neu5Acα2,8

Siglec-6 (OBBP1) Siglec-7 (AIRM-1) Siglec8 (mouse SiglecF) Siglec-9 (mouse Siglec-E)

Siaα2,6GalNAc (sialylTn)

Siglec-10 (mouse Siglec-G)

Siaα2,3Gal, Siaα2,6Gal

Siglec-11a Siglec-12 Siglec-14 Siglec-15 Siglec-16

Neu5Acα2,8 >> Siaα2,6Gal > Siaα2,3Gal Siaα2,3Gal > Siaα2,6Gal

Siaα2,3Gal, Siaα2,6Gal

Neu5Acα2,8Neu5Ac – Siaα2,6Gal, Siα2,6Gal Siα2,6Gal Siα2,8Gal

Monocytes, neutrophils, NK cells, B cells Monocytes, NK cells, eosinophils, B cells Macrophages Macrophages Unknown Macrophages, DCs Macrophages, DCs (continued)

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5 Pathogen-Host Infection Via Glycan Recognition and Interaction

Table 5.5 (continued) Lectin (synonyms) Others Complement factor H Interleukin-1α Interleukin-1β Interleukin-2 Interleukin-4 Interleukin-7 L1

SA-binding proteins Laminin Sarcolectin Calcyclin Calreticulin cSBL

5.2

SA specificity

Expression cells

Sia

Blood

Biantennary, Neu5Acα2,3Gal1,4GlcNac Neu5Acα2,3Gal1-Cer (GM4) GD1b Neu5Ac1,7lactone Siaα2,6GalNAc (sialylTn) Neu5Acα2,3

Sia

Blood Blood Blood Blood Blood Neurons, CD4+ T cells, monocytes, B cells Rat sperm

Siaα2,3Gal1,4GlcNAc Neu5Ac, Neu5Gc Neu5Gc Neu5Gc, Neu5Ac Sia

Extracellular matrix Placenta Bovine heart Ovine placenta Frog egg

Innate Immune-Specific and Host Defensing Lectins of Fungal, Protozoa, Invertebrate, and Lower Vertebrates

Functions of lower fish lectins do exist in the skin mucus of certain lower vertebrates such as fish including the windowpane flounder Lophopsetta maculata, the Arabian Gulf catfish Arius thalassinus, the conger eel Conger myriaster, the dragonet Repomucenus richardsonii, the loach Misgurnus anguillicaudatus, the kingklip Genypterus capensis, and the pufferfish Fugu rubripes. Certain lower vertebrates such as the African clawed frog (Xenopus laevis) produce galectins in the skin mucus. Agglutinin in the moray eel (Lycodontis nudivomer) and the Arabian Gulf catfish (A. thalassinus) and lectin in the dragonet (R. richardsonii) in the skin mucus belonged to Galectins with Galactose specificity [1]. In the Galectin-type lectins of eel skin mucus, Conger eel congerins I and II in the skin mucus are galactoside- and lactose-specific lectins. The lactose (Lac)recognizing lectins are known for AJL-1/AJL-2, which are isolated from the mucosal skin tissues of the Japanese eel of A. japonica, are structurally studied as a Galectin family [2]. They are expressed in the skin only, showing selective resistance to infectious diseases with agglutinating activity against Gram-positive bacterium, Streptococcus difficile [3, 4]. Therefore, the lectins are classified to a defensive factor. Congerins belong to the Galectin family, but no homology with eel galectin AJL-2. For functional aspect, kin mucus galectin, congerin, agglutinates a marine

5.2 Innate Immune-Specific and Host Defensing Lectins of Fungal, Protozoa,. . .

205

Table 5.6 Protozoa SA-specific lectins Species Trypanosomatidae Trichomonadidae

Plasmodiidae

Babesiidae

Trypanosoma cruzi Tritrichomonas mobilensis T. foetus Plasmodium falciparum

P. knowlesi Babesia divergens B. bovis and B. equi

Lectin Inactive TS (Tyr342His) TML TFL

SA-binding specificity CD43 (leukosialin on CD4+ T cells), (Neu5Acα2,3 > Neu5Acα2,6 > sLex) Neu5Acα2,6 > Neu5Acα2,3 > Neu5Ac Neu5Ac > Neu5Gc > Neu5Acα2,3/6

EBA-175 EBA-140, BAEBL, PfEBP2 RfRh1, NBP1 β protein

Neu5Acα2,3Gal (glycophorin A) > Neu5Acα2,6Gal Sia (glycophorin C), Sia (glycophorin B), Sia (receptor E) Sia (receptor Y) Sia (rhesus erythrocytes) Sia (glycophorin A and B) Neu5Acα2,3/6 and Neu5Acα2,3, respectively

pathogenic bacterium Vibrio anguillarum; however, it does not inhibit the growth of V. anguillarum. AJL-2 also agglutinates and suppresses cell growth of E. coli K12. Thus, skin mucus lectin participates in first line of host defense to inhibit the bacterial growth in the mucus. Naturally occurring Galectins with different substrate specificities are considered to effectively respond to a wide range of pathogens. Tandem-repeated Galectin type was purified from the Oncorhynchus mykiss species known as rainbow trout, and the coding gene was isolated from head kidney-derived cDNA library. The tandem-repeated type Galectin exhibits homologies of 40–55% with other known Galectin-9 of mammal sources. Its homologies have weak 19–25% ranges in the N-terminal region or 15–20% in the C-terminal region with galectins isolated from conger and electric eels. The Galectin production is increased when LPS was treated, and the expression is associated with the innate immune response in eel [3]. Several types of SA-specific lectins produced by protozoa such as Trypanosoma cruzi, Tritrichomonas mobilensis, Plasmodium falciparum, and Babesia divergens and fungi including mushroom and pathogenic fungal species are described (Tables 5.6 and 5.7). In addition, SA-specific lectins of invertebrate SA-specific lectins including arthropoda, mollusca, echnodermata, and urochordata are described (Table 5.8).

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5 Pathogen-Host Infection Via Glycan Recognition and Interaction

Table 5.7 Fungal SA-specific lectins. a Also binds GlcNAc. b Phytopathogenic fungus Species Mushroom

Pathogenic fungi

5.3 5.3.1

SA-binding specificity Neu5Gc > Neu5Ac and Neu5Acα2,6Galβ1,4Glc/GlcNAc, respectively Neu5Acα2,3Galβ1,4GlcNAca and Neu5Acα2,3Galβ1,4Glc, respectively

Penicillium marneffei

Lectin HEL abd PSA PVL and ACG –

Aspergillus fumigatus

HA-A

Histoplasma capsulatum Macrophomina phaseolinab

– MPL

Neu5ACα2,6GalNAc/laminin, fibronectin, fibrinogen, collagen Neu5Ac/laminin Neu5Acα2,3Galβ1,4GlcNAc

Hericium erinaceum and Polyporus squamosus Psathyrella vetutina and Agrocybe cylindracea

Neu5Ac/laminin and fibronectin

How Do Hosts Interact with Pathogens? Lectin-Carbohydrate Interaction

Pathogenic microbes in intestines interact with intestinal environment. The pathogenic surface glycoconjugates recognize their interacting molecules of host cells. The pathogenic glycans have hugely diverse structures in a species-dependent manner and act as host cell-binding ligands, giving their pathogenic species specificities. In the host cell infection, tight barrier of host cells protects against microbial pathogens, but bacterial infection occurs through two major routes of trans-cellular type and para-cellular type (Fig. 5.2). Cell-surfaced SAs in eukaryotes and prokaryotes are the targets for attack and SAMPs. SAs and their receptor patterns have been evolving rapidly to adapt and survive in biological environments. Mammalian host species have also evolved selfsialic acids as “self-patterns.” There are several surfaced sugars including Gal, Man, Fuc, and GAGs, which function in immune responses. However, SAs are positively adopted for natural selective roles in immunity. Pathogens initially bind to host cells for the infection. Their binding target molecules are complex carbohydrates or glycans surfaced on the outmost coat cells even in single cells or multiple cells in organisms. Binding machinery molecules are lectins that recognize and interact with specific binding target molecules or glycan patterns on host cell surfaces, mainly on plasma membranes [5]. The lectins and lectin-recognizing glycan or carbohydrate structures represent “receptors” and “ligands.” In eukaryotic self, ligand glycans are frequently disaccharides on glycoconjugates including glycoproteins, glycolipids, or glycosaminoglycans on the host cell membrane that are biosynthesized via ER-Golgi apparatus by glycosyltransferases and glycosidases [6, 7]. The receptors have long been recognized as lectins. Therefore, host-pathogen interactions are the most complex events during the host tissue and pathogen interaction and infection, because cells

Urochordata

Echinodermata

Mollusca

Species Arthropoda

Echinoidea

Gastropoda

Bivalvia

Crustacea

Chelicerata

Halocynthia pyriformis

Limulus polyphemus Carcinoscorpius rotundicauda Heterometrus granulomanus Paratelphusa jacquemontii Cancer antennarius Scylla serrata and Liocarcinus depurator Homarus americanus Litopenaeus setiferus Tracheata Allomyrina dichotoma Modiolus modiolus and Crassostrea gigas Anadara granosa Cepaea hortensis and Achatina fulica Pila globose and Limax flavus Hemicentrotus pulcherrimus Strongylocentrotus purpuratus

Lectin Limulin Carcinoscorpin Scorpin HA-A HA-A HA-A Lobster agglutinin I LsL Allo A-II HA-A HA-A AFL and agglutinin I Achatinin H and PAL LFA 350-kDa sperm-binding protein 350-kDa sperm-binding protein

Table 5.8 Invertebrate SA-specific lectins. There is no available structural information on the lectins below

Neu5Ac, Neu5Gc

SA-binding specificity Neu5Ac, Neu5Gc Neu5Gc, Neu5Acα2,6GalNAc-ol Neu5Ac, Neu5Gc O-AC-Neu5Ac Neu5,9Ac2, Neu4,5Ac2 Neu5Gc and O-Ac-Neu5Ac, respectively Neu5Ac Neu5Ac, O-Ac-Neu5Ac Neu5Acα2,6Gal1,4GlcNAc Neu5Ac Neu5Gc Neu5,9Ac2 Neu5Gc and Neu5Ac > Neu5Gc, respectively Neu5AcGlcCer,(Neu5Ac)2GlcCer Neu5AcGlcCer,(Neu5Ac)2GlcCer

5.3 How Do Hosts Interact with Pathogens? 207

208

5 Pathogen-Host Infection Via Glycan Recognition and Interaction

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are coated with carbohydrates and express carbohydrate-binding proteins (CBPs) or glycan-binding proteins (GBPs). The immune responses are mediated by many GBPs or lectins. Representative lectins known are the galectins, CTLs and Siglecs. The lectin families are directly involved in host innate immunities and adaptive immune responses. In innate immune response, APCs can discriminate self, nonself, stressed self, and altered-self through lectin-glycan interactions. For example, the well-studied lectins as GBPs or PRPs include Siglec-1, Dectin-1, DC-SIGN, Galectin-9, and Galectin-3. Among them, Galectin-3 and Galectin-9 are watersoluble forms as PPRs. The GBPs act as PRRs recognize exposed glycans of nonself on infectious pathogens such as bacteria, yeasts, viruses, and parasites. CLRs such as DC-SIGN particularly exhibit glycan specificity. Lewis glycans and mannosylated glycans bind to multiple pathogens. The pathogens are diverse from viruses like HIV, bacteria like H. pylori and M. tuberculosis, and helminths like Schistosoma mansoni to yeasts like C. albicans. Lectins receive and clear pathogens because virus and bacterial lectins bind host surfaced carbohydrates. To protect hosts, host cells express soluble glycoproteins to block the pathogen lectin to avoid the host cell interaction. Host lectins also remove or disturb pathogen-host binding event. However, due to the structural similarity, the host lectin-pathogen glycan binding potentiates rather invasive endocytosis of the pathogenic agents to receive the host cells. In fact, most surfaced immune-related receptors are glycoproteins such as PRRs. PRRs include chemokine receptors, cytokine receptors, NOD-like receptors, TLRs, MHC-I/MHC-II proteins, co-receptors of T cells/B cells, TCR, and BCR. For example, blocking of T-cell N-glycan synthesis increases TCR signaling with T-cell activation [8]. Silencing of MHC-II N-glycans prevents the presentation of bacterial polysaccharide antigens to T cells [9], whereas experimentally silencing in glycosylation site of TLRs dysregulates the signaling [10, 11].

5.3 How Do Hosts Interact with Pathogens?

209

Mammalian lectins are subclassified into two major types of (i) intracellular lectins, which are resident in cytosolic endosomes and include the M-type lectins (CRT calreticulin and CNX calnexin) in the ER-Golgi glycosylation organelle, and (ii) membrane-type lectins, which are membrane anchors and include the C-type lectins and Siglecs. M-type lectins play crucial roles in eukaryotic glycoprotein and glycolipid secretions by step by step and protein maturation quality control. In contrast, Siglecs and C-type lectins function in elimination of foreign invaders and pathogenic agents as well as innate and acquired immune response to self-host defenses [12].

5.3.2

Bacterial Glycoconjugates Interact with Host Lectins

Various genome data have been obtained from different research consortia including the Korea Microbial Genome Project or EU-MetaHit and the Human Microbiome Project. Bacterial pili and adhesive molecules also interact with the environment in the gut [13, 14]. Glycoconjugates involve in bacteria-host interactions. Glycoconjugates consist of lipopolysaccharides (LPS), capsular polysaccharides (CPSs), exopolysaccharides (EPS), lipooligosaccharides, lipoglycans, glycoproteins, peptidoglycans, and teichoic acids with diversity [15] (Fig. 5.3). The capsule CPS and EPS are biosynthesized by the regulation of capsule synthesis (rcs) system (Fig. 5.4). RcsF is essential for bacterial biofilm formation and pathogenicity. RcsF is a lipoprotein regulator of the rcs system. RcsF modulates the RcsC-D-A/B signaling cascade as the complex pathways. RcsF lipoprotein anchored to the outer membrane activates the rcs phosphorelay for synthesis of EPS and CPS, cell motility, antibiotic resistance, and virulence [16]. The core of the rcs system is the RcsB and the sensor kinase RcsC. RcsB is a DNA-binding protein and is activated via an N-terminal phosphoreceiver domain. The histidine kinase RcsC is complexed with RcsD. RcsC and RcsD are structurally similar with a sensor domain, a transmembrane-spanning motif, a phosphorylation domain of C-terminal region, and a histidine kinase domain. RcsC phosphoreceiver domain holds the phosphoryl group and transmits to the RcsB domain in N-terminal region via the RcsD domain in C-terminal region. Activated RcsB binds to the RcsB box [17]. rcs-dependent promoters synthesize EPS through the RcsAB box motif bound by a RcsB and RcsA complex [18]. These glycoconjugates are so-called surface-glycan barcodes or face signature, giving unique specificities to the host. Thus, the host innate immune cells interact with bacterial glycans through lectins such as DC-SIGN (Fig. 5.5), “sensing” the presence of bacteria.

5.3.2.1

Glycoproteins

Bacterial glycoproteins are also a recently recognized group of glycoconjugates. They are glycosylated virulence factors of bacterial pathogens. Bacterial GTs attach

210

5 Pathogen-Host Infection Via Glycan Recognition and Interaction < Microbial glycoconjugates> Lipid

Bacterial O-Glycosylation Ser

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Ser

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e.g. DC-SIGN

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Adhesin/Lectin

: Glc

: Mono sugar

: Amino acid

Glycoprotein

Fig. 5.3 Bacterial glycoconjugates of flagella, glycoproteins, pili, CPS, EPS, and LPS. Modified from Ref. [13] Tytgat HLP, de Vos WM. 2016. Trends Microbiol. 24(11), 853–861 Fig. 5.4 Regulation of the Rcs system for biosynthesis of capsular polysaccharides (CPSs) or exopolysaccharide (EPS)

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5.3 How Do Hosts Interact with Pathogens?

211

Fig. 5.5 A schematic structure of DC-SIGN Carbohydrate recognition domain (CRD)

Neck domain

Transmembrane domain Tyrosine based motif Triacidic cluster Di-leucine-based motif

glycosylation are known in Burkholderia cenocepacia, Campylobacter jejuni, Helicobacter pylori, Escherichia coli, Neisseria meningitidis, Francisella tularensis, Haemophilus influenzae, Pseudomonas aeruginosa, and Porphyromonas gingivalis [20–28]. Their representative roles in protein glycosylation include bacterial virulence, protein stability, and immune regulation from host immunity or protein substrates. In addition, bacterial protein glycosylation provides ligand functions to interact with the gut host. In the aspect of acquisition of glycans, some intestinal commensal bacteria including Bacteroidetes and Firmicutes [29] acquire and incorporate host-produced glycans into their bacterial surface glycoproteins. The most unique sugar moiety is incorporated into the bacterial surfaces L-fucose residue [30]. Some lactobacilli’s glycoprotein, SlpA of Lactobacillus acidophilus NCFM, specifically recognize DC-SIGN in order to downregulate DCs and T-cell functions [31]. Serine-rich repeat proteins (SRRPs) known as adhesin molecules are also glycoproteins, which are frequently found in streptococci strains such as Streptococcus parasanguinis, S. gordonii, S. pneumoniae, and S. agalactiae as well as staphylococci strains such as Staphylococcus aureus. Such adhesins are O-GlcNAcylated to attach GlcNAc, by GtfA and GtfB enzymes [32]. After O-GlcNAcylation, GtfC and Gtf3 enzymes further attach Glc residues. The glycosylated proteins are delivered by the SecA2-SecY2 system upon reaction by GalT1 enzyme [33]. This type of bacterial O-glycosylation in adhesins highly resembled to the eukaryotes (Fig. 5.3). Similarly, bacterial N-glycosylation is anticipated. As L-fucose residues are abundant in the gut and mediate host-microbe symbiosis, some commensal bacteria release fucose residues from the mucus.

212

5.3.2.2

5 Pathogen-Host Infection Via Glycan Recognition and Interaction

Bacterial Capsule of Capsular Polysaccharide (CPS)

Some bacterial surfaces are coated with their bacterial capsules, which are the structural architectures decorated on the cell surfaces of bacteria and fungi, for their survivals. They prevent the microbes from the immune recognition surveillance of the host and allow the microbes to invade the host. Most of the bacteria have capsule polysaccharides. But the only exception is Bacillus anthracis which have a PGA capsule. Capsules are mainly polysaccharides with huge diversity. Streptococcus pyogenes are unique due to their one-capsule structure. In addition, within the same microbial species, capsule structures are different. The diversity of capsules is based on the different immune mechanisms. The capsule-biosynthetic genes are classed into common gene family and the CPS type-specific gene family. The common gene family includes the genes for capsule transportation to the cellular membrane. The CPS type-specific gene family includes the genes necessary for each type of capsule synthesis. Even in the same species of bacteria, different genes generate their capsules. In addition, different bacteria bear same or similar synthesizing genes of capsules. The capsule-synthetic pathway can be different among microorganisms. The common pathway of capsule synthesis is in that all capsules are generated through the membrane-associated acceptors. Three synthetic pathways such as WZY-, ABC transporter-, and synthetic enzyme-dependent pathways are involved in the genesis of capsules. Such generated bacterial capsules act as a virulence factor and an immune escape factor for bacteria. In human application, the capsules are used as a vaccine antigen. Negatively charged glycoconjugates of capsular and slime polysaccharides are distinct in microbiological world. Bacterial CPSs are produced from both types of Gram-negative and Gram-positive bacteria. Bacterial CPS species are structurally heterogeneous. However, some bacterial CPSs produced by Gram-positive strains of Streptococcus and Staphylococcus families as well as Gram-negative bacteria of Klebsiella, Neisseria, and Haemophilus families are rather homogeneous in their structures with acidic polysaccharides [34]. Gram-positive bacterial CPSs are structurally similar and immunogenic in hosts, providing the concept of CPS-based vaccination [35]. Bacterial capsules are strictly attached, not released, as the outmost glycoconjugates, and they are also produced by fungal cells. Other slime polysaccharides are detached or released from the cell surfaces. Capsule polysaccharides (CPSs) include the mucus polysaccharide such as colanic acid or M antigen and some pathogenic alginic acids [34, 36]. In 1928, Griffith in his “Griffith’s Experiment” [37] reported avirulent “rough” (unencapsulated) and virulent “smooth” (encapsulated) strains of S. pneumoniae. The encapsulated bacterial colonies are called “smooth” type, while the unencapsulated colonies are called “rough” type [37, 38]. Later, Avery et al. identified the capsules as the “genetic marker” to confirm the genetic element [39]. CPS capsules found in S. pneumoniae stimulate host immune responses against the pathogens, conferring the concept of CPS vaccination to encapsulated bacteria such Group B Streptococcus (GBS) [34]. Several prosthetic

5.3 How Do Hosts Interact with Pathogens?

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Fig. 5.6 Three types of CPS synthesis pathway. (1) WZY-, (2) ABC transporter-, and (3) glycosyltransferase-dependent pathways are known. (1) In the Wzy pathway, the WbaP enzyme starts the synthesis of sugar-Und-P attached to the lipid carrier Und-P. CPS-repeated units are produced. The Wzx flippase and Wzy enzyme further elongate the periplasmic repeat units. The matured CPSs are penetrated via the periplasm by Wzc and Wza and localized at the bacterial surface by the Wzi protein [40]. (2) E. coli group 2 CPS KfiABCD and KpsCSMTED proteins synthesize a translocation complex in the ABC transporter pathway. (3) In the glycosyltransferase pathway, a series of KfiA, KfiB, KfiC, and KfiD enzymes are involved. Then, CPSs are delivered by the KpsD/KpsE transporter [42, 47]

groups including O-formyl, O-acetyl, or pyruvate ketosidic groups are substituted with the bacterial CPSs. In fact, some bacterial CPSs are frequently substituted with O-acetyl ester linkage, O-formyl ester linkage, or pyruvate ketoside linkage [40]. The reducing sugars present in CPSs are also diesterized with phosphatidic acids. For example, the CPSs produced by E. coli group 2, H. influenzae type b strain, and N. meningitidis group B species produce the diesterized CPSs, having the phosphatidic acid [34]. The CPSs produced by E. coli K5 species are linked with phosphatidyl-Kdo structure [41]. The CPSs produced by E. coli K30 strains, E. coli K40 species, and Klebsiella family are LPS lipid A-bound CPS known as KLPS forms [42]. S. agalactiae serotype III CPS contains polySialic acids with NeuNAc and GlcNAc residues [43]. CPSs are larger than the LPS O antigen polysaccharides in E. coli [26] with up to 100 kDa per chain. For the CPS synthesis, three pathways of WZY, ABC transporter, and glycosyltransferase pathway are known. First, the Wzy pathway for CPS biosynthesis is also known to block polymerization in the K. aerogenes DD45 strain as the first description [44]. The CPS-produced K. aerogenes DD45 strain contains repeated and multiple sugar units. The CPS synthesis resembled with other LPS and peptidoglycans [35] (Fig. 5.6). For the initiation step of the CPS synthesis in the K. aerogenes DD45 strain, Gal-l-P reside is transferred to the C55-lipid undecaprenyl phosphate by the WbaP protein [44]. Sugar length elongating enzyme, Wzy, sequentially assembles the CPS backbone through linking each repeat in the

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5 Pathogen-Host Infection Via Glycan Recognition and Interaction

bacterial periplasmic space [45]. Polysaccharide-specific transport protein translocates the polysaccharides to the next space [46]. In Gram-negative bacteria, secondary transportation proteins of Wzc and Wza transport the produced CPSs to the periplasmic space and outer membranes [47]. Wzc is a membrane periplasm accessory (MPA) protein present in cytoplasmic region, while Wza is an outer membraneassociated accessory protein (OMA). Wzi is an initial tethering point protein [48]. For example, S. pneumoniae CPSs are polymerized by the Wzy pathway, where each specific glycosyltransferase is involved in the CPS synthesis and the synthesized CPSs are tethered to the bacterial cell wall with covalent bonds [49]. Secondly, most Gram-negative bacterial CPSs are synthesized by the ABC transporter pathway [48]. In the E. coli K5 CPS as a representative case, the K5 CPS polysaccharide is specifically synthesized and polymerized by four different proteins including KfiA, KfiB, KfiC, and KfiD [50]. The KfiA protein and KfiC protein are GlcNAc-transferase and GlcA-transferase, respectively [51, 52]. KfiD protein has an UDP-glucose dehydrogenase enzyme activity and forms the UDP-GlcA substrate from the substrate UDP-glucose [53]. KfiB protein formulates the KfiA/KfiB/KfiC complex form [51]. The final product, CPS-lipid cap, is delivered to the cytoplasm membrane by action of the ABC transporter with the KpsT and KpsM [54, 55]. In E. coli K1 strain, the KpsT protein recognizes the poly-SA structure, called Kl-CPS [56]. The periplasmic CPSs are delivered to the outer membrane and then to the bacterial surface by the continued transporters of KpsE and KpsD associated on membranes [57]. Lastly, a specific pathway, named synthase-dependent synthesis, is known in some Gram-positive bacterial S. pyogenes and S. pneumoniae types which add repeatedly saccharide units to the reducing end [58]. For example, S. pyogenes HasA has two enzyme activities of a GlcA-transferase enzyme and GlcNAc-transferase. Similarly, HasB is the UDP-Glc dehydrogenase enzyme [59]. CPS cap structures are formed via a diester linkage to the reducing sugars with phosphatidic acid or lipids. The E. coli K5 phosphatidylKdo is the example of CPS capping [41]. In the CPSs produced by Klebsiella strains, E. coli K30 strain and E. coli K40 strain, lipid A core structure, is attached at their reducing end of CPS [42]. In the Gram-positive bacteria, which lack for membranes, CPSs are linked to the peptidoglycan or the membrane molecules. CPS produced in the serotype III strain of S. agalactiae, the peptidoglycan, or GlcNAc is attached to the CPS [60].

5.3.2.3

Function of Bacterial CPS

For the function of CPCs, they are virulence factors for bacterial encapsulated pathogens. CPSs also facilitate bacterial adherence and activate dissemination. Also, CPSs as shields protect from host recognition, complement-mediated opsonization, and phagocytosis by innate immune cells, allowing evasion during infections [61]. CPSs can induce IgM-type antibody through the T-cell-independent immune response. The immunogenicity of CPSs depends on the chain length. Certain SA-containing CPSs produced by E. coli K, E. coli K92, N. meningitidis

5.3 How Do Hosts Interact with Pathogens?

215

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group B, N. meningitidis group C, and S. agalactiae species disrupt the complement B as well as complement C3b binding. Therefore, complement cascade pathway of host defense system is avoided [62]. Some CPS structures are similar to host carbohydrates, giving a molecular mimicry, providing an evasion of such pathogenic bacteria from the host immunity. In addition, CPSs produced by E. coli K1 and N. meningitidis group B strains are similar to the terminal poly-SA structures of the neuronal cell adhesion molecule (NCAM) of the human brain [63]. The CPS of E. coli K5 strain contains the N-acetylheparosan sugar structure [34]. The S. pyogenes CPS contains hyaluronic acid found in human tissues [39]. The S. agalactiae type III CPS SAs affect mice B-cell repertoires [64]. The CPS produced by S. agalactiae type V strain lacks SA species and can induces IgM-toIgG switch turnover to allow immunoprotection in Rhesus macaques [65]. CPS is a bacterial decoy for antimicrobial peptides produced by hosts [66]. Anionic bacterial CPSs purified from the encapsulated strain K. pneumoniae increase the minimum inhibitory concentration of polymyxin B. Anionic CPSs also block the bactericidal activity of antimicrobial peptides by binding them in the naturally released CPSs, which are produced by the pathogenic bacteria such as K. pneumoniae, S. pneumoniae, and P. aeruginosa. To prevent host recognition, P. aeruginosa adsorbs SAs released from bacterial cultures to utilize the SAs to block complement deposition in cell surfaces. Unfortunately, the molecular mechanism(s) underlying the P. aeruginosa strains survived in the host-defense system is not known. CPSs can also bind to polymyxin B (Fig. 5.7). K. pneumoniae CPS can also impede the antimicrobial peptide, β-defensin (BD) expression in airway epithelial cells. The CPSs released bind to antimicrobial

216

5 Pathogen-Host Infection Via Glycan Recognition and Interaction

Klebsiella pneumoniae

Lipopolysaccharide (LPS; O antigen)

CPS Pathogenicity factor

- Fucose - O-acetylation

Lipid chain

Periplasmic space GlcNAc Other monosaccharides

WcaI (kp3706) fucosyl transferase WcaG (kp3709) GDP-fucose synthesis Atf (kp3712) fucose acetylation

Kdo

Outer Membrane Polysaccharide surface layer

ugd gnd wbaP wcaI, wcaH wcaG gmd

atf Wzy (magA) ptf wzc wzb wza

β-Defensins : antimicrobial activity hBD1 : constitutively expression hBD2,3 : expression by cytokine, pathogens

CPS

iE-DAP

,κBα

Capsular polysaccharides (CPS; K antigen) Glc GlcA Fuc

CYLD

NF-κB signaling pathway hBD3

MAPK signaling pathway

hBD2

CPS anchorage protein

MyD88

AP : antimicrobial peptide

hBD2

NOD1 p38

X

JNK

hBD2, hBD3

P44/42

hBD3

Blocking bactericidal activity MKP-1

Klebsiella pneumoniae outer membrane

TLR : Toll-like receptor NOD1 : Nucleotide binding and oligomerization domain containing protein 1 NF-κB : Nuclear factor kappa-light-chain-enhancer of activated B cells MAPK : Mitogen-activated protein kinase iE-DAP : D-glutamyl-meso-diaminopimelic acid

Fig. 5.8 K. pneumoniae CPS facilitates pathogen survival in the hostile environment. Modified from [67] Moranta D et al. 2010. Infect Immun.78(3), 1135–46

peptides to block the bactericidal activity (Fig. 5.8). The question of how CPSs protect bacteria for survival in the hosts has been answered by the recent documentation [67]. CPS inhibits activation of the signaling pathway involved in β-defensin expression. The outer membrane CPS of K. pneumoniae is a toxic factor and reduces phagocytosis of DCs or macrophages. BD is an antimicrobial peptide produced in airway epithelial cells. Among human BDs (hBDs) of hBD1, hBD2, and hBD3, hBD1 is constitutively produced by the respiratory tract lined with epithelial cells, whereas the hBD2 and hBD3 expressions are induced by cytokines or pathogens. K. pneumoniae causes pneumonia, and CPS in the outer membrane is a toxic factor and inhibits phagocytosis of macrophages. The CPS binds to antibacterial peptide AP to block the bactericidal action. Therefore, CPS protects bacteria through suppression of the hBD expression in the lungs. The CPS mutant exhibits the low survival rate and is vulnerable to β-defensin. CPS exerts a resistant activity against hBD3. β-Defensin expression is increased only in CPS-lacking mutants as CPS inhibits activation of the β-defensin signaling. Two signalings are known to increase the expression of β-defensin. The first is the NF-κB signaling pathway to induce β-defensin expression, cell survival, and immune responses to infection. The second signaling pathway is activated by NF-κB as the MAPK signaling pathway to increase the hBD2 and hBD3 levels. NF-κB signaling pathway which controls BD expression is inhibited. Apart from the NF-κB signaling, the second MAPK signaling pathway including P38, JNK, and p44/p42 was also inhibited, as the two signaling pathways are commonly activated by TLR molecules. Activation of the two signaling pathways is controlled by TLR. TLR activates through MyD88. MyD88-TLR contributes to hBD2 activation. MyD88 expression is not associated

5.3 How Do Hosts Interact with Pathogens?

217

with the expression of hBD3. Similarly, NOD1, which activates MAPK/NF-κB axis signaling, involves in BD3 expression in humans. CPS is a bacterial decoy for antimicrobial peptides. Antimicrobial peptides (APs) are cationic, and its action is initiated through interactions with the anionic bacterial surface. Humans live with various bacterial populations associated with mucosal surface, termed the microbiota. Microbial glycoconjugates interact with host cells and are species-specific as ligands for host cells. Pathogenic microbes have evolved to evade host immune surveillance and innate immune response and phagocytosis clearance of hosts. Mostly, host and microbial cell surface are coated with carbohydrates, resulting in microbial pattern recognition and governing normal immune cell activities. A major strategy regarding pathogen is to define sugars which mimics or interferes with host’s immune functions. Endogenous sialoglycans represent the “SAMPs” which prevent myeloid immune cell activation and maturation, including leukocytes. Some pathogens can mimic sialyl glycans of hosts, and the SA-containing glycans make pathogenic bacteria to engage inhibitory Siglecs of hosts, attenuate immune clearance, and dampen leukocyte activation. Engagement of inhibitory Siglecs and SA mimicry have been coevolved with changes in Siglecbinding specificity or host sialic acid repertoire. Carbohydrate can have a major role in immune response rather than protein which is known for a major component of immune response. If SAs were incorporated into cell surface CPS, the events help them evade the host immune responses. As a shield, bacterial pathogens including E. coli K1 strain, H. influenzae strain, and S. agalactiae strain have evolved to bypass host immunity. As bacterial virulence factor, K. pneumoniae CPSs are the infection determinants. The high molecular weight polysaccharide CPSs contribute to the muco-phenotype. CPS helps the bacteria evade phagocytosis and protects from bacterial clearance. Sialic acid of CPS in K. pneumoniae strains is antiphagocytic, as Siglec-9 functions as a receptor for the MHC-I expressed in neutrophils, which bind to SAs and transduce the downstream signaling to dampen and suppress inflammation [68]. Sialylated CPSs also provide a virulence factor. Sialic acids in CPSs promote the factor H (FH)-binding activity of the alternative complements, restricting the recognition of C3b and FB (Fig. 5.9) [69]. If carrier proteins are conjugated to CPSs, the conjugated CPSs activate the T-cell-dependent immune response to produce IgG class. CPSs are currently used for vaccination of several pathogenic bacteria including H. influenzae, N. meningitidis, S. typhi, and S. pneumoniae [70]. In addition, the CPS conjugation is also applied for some encapsulated Klebsiella and Pseudomonas [34]. Nonetheless, the CPS vaccination is not successful in younger children under age 2 [71]. CPS-protein conjugates thus improve the CPS immunogenicity in younger children [72]. So-called CPS conjugate vaccines are the current licensing replacements of encapsulated pathogenic bacteria including S. pneumoniae, H. influenzae, and N. meningitidis. The vaccination of Hib vaccine is a representative for the H. influenzae type b [73].

218

5 Pathogen-Host Infection Via Glycan Recognition and Interaction SAα2-3 Galβ1-4GlcNAcβ1 Without sialic acid, Factor B combines with C3b to make a convertase which induces a cleavage of C3 = onset of opsonization and phagocytosis

Kiebsiella Penum oniae (KP-M1) High Level of Sialic Acid in CPS

Factor H binding to C3b protects the bacteria from opsonization and phagocy tosis

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Fig. 5.9 Sialylated CPS of K. pneumoniae is involved in bacterial resistance to host neutrophil phagocytosis. Modified from [68] Lee CH et al. 2014. Virulence 5(6), 673–9

5.4

Pathogen-Producing Lectins as Receptors to Bind to the Host Carbohydrates

Many pathogens use host glycans as targets for adhesion. Therefore, the blockers of carbohydrate-binding adhesins are fighters of infections. Thus, rapid glycan microarray approach can assess the bacterial adhesion. Pathogens are recognized by recognition with GBPs including C-type lectins. Recent progress in glycan arrays has accelerated the rapid deciphering of lectin-binding carbohydrate and glycans as ligand specificity. The essential role of lectins is believed to decipher the glycocode by specific recognition of carbohydrates. The human GIT covered by mucosal enteroepithelial cells provides a mucosal defense barrier decorated by the glycocalyx. Glycan-enriched glycocalyx is bound to plasma membranes of cells or excreted into the extracellular milieu. Interaction between lectins and carbohydrate ligands contributes to various biological responses and pathologic immune responses. Conversely, many pathogenic viruses, bacteria, fungi, and parasites utilize host glycans as targets for adhesion or for secreted toxins. Proteincarbohydrate recognition covers many biological events and diseases. Glycan-binding proteins from pathogens are well known as bacterial lectins, adhesins, or toxins. These molecules are produced during coevolution. They specifically recognize oligosaccharides expressed on host cell surfaces [74, 75]. Molecular ligands for glycan-binding proteins can be used as therapeutic application [76]. A special feature of protein-carbohydrate interactions is the wide prevalence of multivalency [77]. Bridging lectin-recognizing sites by multivalent saccharides can increase binding or inhibitory levels [78]. Bacteria and bacterial toxin’s SA-specific

5.4 Pathogen-Producing Lectins as Receptors to Bind to the Host Carbohydrates

219

Table 5.9 Bacteria and bacterial toxin’s SA-specific lectins. HA-A hemagglutinin activity observed Species Gram-negative Escherichia coli Helicobacter pylori Haemophilus influenzae Pasteurella haemolytica Bordetella bronchiseptica and B. avium Neisseria meningitidis and N. subflava Pseudomonas aeruginosa Moraxella catarrhalis Gram-positive

Mycoplasma Bacterial toxins

Streptococcus gordonii Streptococcus mutans S. mitis and S. suis Mycoplasma gallisepticum Vibrio cholerae Vibrio mimicus Clostridium botulinum Clostridium tetani C. perfringens and E. coli Bordetella pertussis

Lectin

SA-binding specificity

SfaI, SfaII, SFaS K99 fimbriae SabA or HP-NAP HiFA

Neu5Gcα2,3Gal; Neu5Acα2,8Neu5Ac Neu5Gcα2,3Galβ1,4Glc Siaα2,3 or Neu5Acα2,3Galβ1,4GlcNAcβ1,3Galβ1,4 GM3,GM1,GM2, GDIa, GD2, GD1b

Adhesin

Neu5Ac

SBHA and HA-A

Neu5Ac and GD1a (or GT1b), respectively.

OpcA (Opa; NHBA) and Sia-1 –

Neu5Ac and Neu5Acα2,3Galβ1,4Glc, respectively.

Fimbrial protein GspB and Hsa

GM2

PAc SABP

Sialyl-Lex or Siaα2,6

Siaα2,3  Siaα2,6 and Neu5Acα2,3Gal, respectively. Siaα2,6

HA-A

Neu5Acα2,3Galβ1,3GalNac and Neu5Acα2,3Galβ1,4G1cNAcβ1-3Gal, respectively Neu5Acα2,3Galβ1,4GlcNAcb1,3

Cholerae toxin

GM1

Haemolysin Neurotoxin A-F Tetanus toxin

GD1a, GT1b 1b series gangliosides

Delta toxin and heat-labile enterotoxin Pertussis toxin

GM2 and GM1, respectively.

GT1b, GQ1b

GD1a; Neu5Acα2,6Galβ1,4GlcNAc

lectins are described (Table 5.9). Gram-negative bacterial strains including E. coli, H. pylori, H. influenzae, Pasteurella haemolytica, Bordetella bronchiseptica, B. avium, N. meningitidis, N. subflava, P. aeruginosa, and Moraxella catarrhalis

220

5 Pathogen-Host Infection Via Glycan Recognition and Interaction

produce their specific lectins. Gram-positive bacteria include S. gordonii, S. mutans, S. mitis, and S. suis. Mycoplasma gallisepticum also bear its specific lectin-like protein. Bacterial toxins produced by Vibrio cholerae, V. mimicus, C. botulinum, C. tetani, C. perfringens, E. coli, and Bordetella pertussis have also lectin activities. Glycomic analysis studies using glycan microarrays have resolved the glycobiological aspects in lectin-binding sites. Multivalent carbohydrate-lectin interactions between host and pathogen establish infections. Therefore, pathogen adhesion-preventive competitive agents can be potential antimicrobial drugs. Such non-covalent carbohydrate-protein binding is very weak with disassociating constants ranging from 103 to 104 [79]. However, nature compensates this restricted limitation through the multivalent glycan interaction in ligand-receptor recognition. The representative is lectin with “glycan multivalent” effect in enhanced binding capacity. The multivalent mimics of natural glycans are inhibitors of carbohydrateprotein interaction as therapeutic drugs in usages of antimicrobial, antiinflammatory, and antitumor potential [80]. Such developed multivalent scaffolds with carbohydrate epitopes are glycodendrimers, glycopeptides, glycopolymers, glycodynamers, fullerenes, calixarenes, etc. [79]. The known human ABH blood group glycoconjugates are also used as ligands of bacterial lectins. ABH blood group antigens are fucosylated glycans in endothelial and erythrocytic cells. In addition, Lewis antigenic epitopes are such categories as tissue histo-blood types. The ABO and Lewis type sugars were correlated in human population-dependent susceptibility to certain pathogenic bacterial diseases in humans [81]. For example, the blood O-phenotype individuals are susceptible to cholera toxins [82] and Norwalk viral gastroenteritis [83].

5.4.1

Uropathogenic E. coli (UPEC), Enterohemorrhagic E. coli (EHEC), and Enterotoxigenic E. coli (ETEC)

E. coli strains are classified, in general, to facultative anaerobic bacteria, which are originally discovered and isolated from the gastrointestinal tract (GIT) of humans. From the virulence consideration, E. coli strains are further subclassified to the two distinct parameters of pathogenic or virulent E. coli group and nonpathogenic or avirulent E. coli group. Pathogenic virulent E. coli group is zoonotic for wide infectious diseases including diarrhea, sepsis, and meningitis. Currently, the pathogenic E. coli family is further subclassified into five subfamilies of enterotoxigenic E. coli (ETEC), enterohemorrhagic E. coli (EHEC), enteroaggregative E. coli (EAggEC), enteroinvasive E. coli (EIEC), and enteropathogenic E. coli (EPEC) [84]. From the five groups, the EHEC group is a major causing group for epidemically and sporadic E. coli infections.

5.4 Pathogen-Producing Lectins as Receptors to Bind to the Host Carbohydrates

5.4.1.1

221

Uropathogenic E. coli (UPEC)

Bacterial lectins are basically hairlike proteins, more specifically known to pili or fimbriae. They interact with the cell outmost coats. The Gram-negative E. coli species express type 1 pili or fimbriae as host receptor recognition and attachment proteins [85]. Pathogenic E. coli FimH variations influence bacterial adhesion. The Man-specific adhesin of E. coli or V. cholerae toxin binds to ganglioside GM1 as relevant examples. Such E. coli cells migrate to the kidneys and shed the type 1 fimbriae and are shifted to the alternative expression of PapG lectins on pin-like P pili. For example, mannose-specific adhesin FimH is located on fimbriae. Direct glycan array to mannan or related glycans printed onto glycan microarray wells is applicable for searching the adhesions if we know the target glycan. The fimbrial lectin FimH from UPEC and ETEC binds to nanomolar affinity to Manα1,3Man dimannosides at their nonreducing end, but only with micromolar affinities to Manα1,2Man. FimH develops infection by adherent-invasive E. coli, Crohn’s disease (CD), urinary tract infections (UTI), enteritis, diarrhea, sepsis, and meningitis [86]. In uropathogenic E. coli (UPEC), adhesins are FimH displayed at the type 1 fimbriae tip in a way of a single FimH molecule per fimbria. It binds to terminal mannosides of the mannosylated glycoprotein uroplakin on bladder urothelial cells. The Man-dependent hemagglutination by the strains is indeed an indicator of the presence of type 1 fimbrial adhesins [87]. Uropathogenic E. coli expresses the adhesin FimH lectins as two-domain adhesin on the terminal portion of hairlike “type 1” fimbriae. The FimH lectin is connected to a pilin anchored with FimG and FimF. It specifically binds to Man residue of bladder epithelial cell membranes to potentiate the invasion to the human urinary bladder [88–91]. The fimbrial adhesin or lectin FimH of uro- or enteropathogenic E. coli binds to high-mannosylated glycoprotein (MGP) receptor molecules exposed on the epithelial cell surfaces resided in oropharyngeal, urinary, or GITs [92]. FimH consists of (1) two Ig-like domains of the lectin or carbohydrate recognition domain and (2) the pilin domain [93]. The lectin or carbohydrate recognition domain (amino acids 1–157) acts using a short flexible linker made by Thr158, Gly159, and Gly160. The pilin domain (aa. 161–276) links FimH to the other pilins to form the fimbrial rod. FimH adheres to MGP carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6), which is excessively overexpressed by GIT epithelial cells of the CD patients as well as EHEC and ETEC [94]. FimH also adheres to the MGP uroplakin Ia (UPIa) on the urinary tract epithelial umbrella cells. UPIa contain high-mannosyl N-glycans [95]. Human CEACAM6 contains two high-mannosylated N-glycosylation sites [86]. From analysis of the FimH lectin structure obtained from crystals, ligand recognition of the recognition pocket of FimH lectin evolved. It binds to the terminal Manα1,2-, Manα1,3 and Manα1,6-linked N-glycans, to oligomannose glycans of Manα1,3Man di-Mannosides, and slightly to Manα1,2Man dimannosides. The two dimannoses are important for infection by E. coli. Manα1,2Man shielding the

222

5 Pathogen-Host Infection Via Glycan Recognition and Interaction

Manα1,3Man glycan is the best link for bacterial adhesion and invasion. Among the di-Man (Manα1,2Man and Manα1,3Man), FimH lectin prefers to the dimannose Manα1,3Man, not for Manα1,2Man. There are ligand-binding shifts in the association equilibrium between the FimH lectin and the FimH lectin pilin domain. A single N-glycan binds to three FimH lectin molecules, although cell surface N-glycans favor monovalent FimH lectin binding [96]. A single N-glycan binds up to three FimH lectin molecules, so that excess of N-glycans over FimH on the cell surface strongly binds to FimH. Similarly, F9 pili specifically bind to Gal, GalNAc, or Thomsen-Friedenreich (TF) antigen (Galβ1,3GalNAcα)- epitopes on the kidney and inflamed bladder. FmlH binds to TF within naive or infected kidneys and also to Thomsen nouvelle (Tn) antigen (GalNAc) within the inflamed bladder epithelium. Experimental silencing of FmlH in the urosepsis strains blocks the ability. Furthermore, challenging vaccination with the LD of FmlH (FmlHLD) inhibits the urosepsis [97].

5.4.1.2

Enterotoxigenic E. coli (ETEC)

ETEC is a type of E. coli, and the diarrhea-causing bacterium frequently occurs in the developing countries. ETEC pass through the mucus layer of human intestinal epithelial cells. ETEC infection ranges from symptomless to serious cholera-like diseases. The main hallmarks are enterotoxin and fimbriae, which is an attachment pilus with adhesin and used for attachment to host intestinal cells. ETEC pass through the mucus layer of mucin domain in intestinal epithelial cells. ETEC secretes EtpA proteins to intestinal epithelial cells through fimbriae. EtpAs specifically opsonize RBCs by binding to terminal sugar, GalNAc, of blood type A glycans. Blood agglutination and ETEC enterotoxin cause diarrhea. It has been found that ETEC H10407 strains separated from cholera-like diseases caused type A blood group specificity. ETEC strains secrete EtpA-bearing molecules specific for red blood cells. ETEC pass through the mucus layer of human intestinal epithelial cells and secretes EtpAs to intestinal epithelial cells through fimbriae which specifically opsonize RBCs via terminal GalNAc of blood type A glycan [98]. The specific binding of ETEC EtpA with GalNac mediates bacteria to host interaction in the intestine. The main hallmarks include enterotoxins, fimbriae as the attachment pilus, and adhesin used for attachment to host intestinal cells. EtpA is a lectin, carbohydrate-binding protein, which recognizes the host glycans on the cellular and molecular levels. It mediates attachment and binding of bacteria and viruses to their intended targets. EtpA is a representatively secreted adhesin molecule. Enterotoxins include heat-labile (LT)/heat-stable (ST) forms. During enteropathogen’s attachment and colonization to the epithelium in intestines, enteric pathogens like ETEC use a long proteinaceous fiber termed type IVb pilus (T4bP) [99]. ETEC E. coli-blood group A interactions intensify diarrheal severity. ETEC strain H10407 induces in blood group A human severe diarrhea more than other blood groups [98]. ETEC infection displays serious cholera-like diseases. ETEC H10407 strains cause type A blood group-specific diarrhea. ETEC

5.4 Pathogen-Producing Lectins as Receptors to Bind to the Host Carbohydrates

223

H10407 secretes EtpA proteins to intestinal epithelial cells through fimbriae which specifically opsonize condense and RBCs through binding to terminal GalNAc residue of blood type A carbohydrate. This binding consequently causes diarrhea in humans. Specific interaction of ETEC-secreted EtpA protein with GalNac induces severe ETEC-mediated diarrhea in the intestine. ETEC strain H10407 secretes the EtpA adhesin molecule. In glycan arrays, EtpA is an ETEC blood group A-specific lectin or hemagglutinin. EtpA binds to the glycans on intestinal epithelial cells from blood group A individuals for adhesion and delivery of both the ETEC LT and ST toxins. Therefore, ETEC is defined by the plasmid-encoded LT and/or ST enterotoxins [100]. The toxins are easily transported to cognate receptor molecules expressed on the intestinal epithelial cell surfaces and allowing net salt and water efflux into the lumen of the small intestine. This is diarrhea. ETEC is identified person suffering from severe cholera-like diarrhea. In ETEC and intestinal epithelia interaction, EtpA is conserved among ETEC strains. FUT2 α1,2 fucosyltransferase synthesizes ABO blood group antigens on intestinal epithelia [101]. Blood groupdependent microbial-host interactions indicate specificity of gastrointestinal pathogens. In H. pylori, the bacterial BabA adhesin molecule attaches to the gastric mucosal ABO and Leb. Similarly, V. cholerae infections are linked to the O blood group [102]. Both CT and LT toxins of ETEC share with different binding of blood group antigen. They favor blood group O enterocytes [103]. EtpA enhanced virulence in blood group A hosts. The secreted EtpA lectin acts with two additional glycan-binding tip adhesins of ETEC fimbriae. Genomic FimH as a type 1 pili binds to mannosyl proteins on epithelial cells, and the plasmid CfaE adhesin lectin combined with CFA/I pili binds to sialylglycoproteins. They are not bound to blood groups. EtpA is the only blood group A-specific lectin identified in ETEC [100]. EtpA-producing ETEC pass through the mucus layer of mucin domain in intestinal epithelial cells. Then, the ETEC secretes EtpA proteins to intestinal epithelial cells through fimbriae. EtpA specifically opsonizes RBCs by binding to terminal sugar, GalNAc, of blood type A glycans. Eventually, blood agglutination and ETEC enterotoxin cause diarrhea. However, blood groups N and O do not cause agglutination of RBCs. Or α-N-acetylgalactosidase treatment to RBC abolishes the EtpA’s agglutination capacity, as illustrated in Fig. 5.10. The colonization factor antigen/III (CFA/III) is called a T4bP of ETEC. It bears a minor pilin, CofB, containing an H-type lectin domain. CofB is needed for pilus assembly with a trimeric complex. But bacterial attachment mechanism is not defined. For bacterial attachment, T4bP needs a secreted CofJ encoded on the same CFA/III operon. CofJ binds to CofB by N-terminal extension linked into the glycan-binding site of the CofB H-type lectin domain. The CofJ-CFA/III pilus complex is a target against ETEC infection. Bacterial pathogens have evolved surface organelles to synthesize the filamentous protein polymers, simply to say pili or fimbriae. Enterotoxigenic E. coli (ETEC) causes diarrhea by at least 22 types of pilus-related colonization factors (CFs) of ETEC. They are called CF antigens (CFAs) or coli surface antigens (CSs). Among 22 CFs, 17 are complex polymerized via chaperone-usher (CU) pathway of major and minor pilus subunits named pilins. ETEC pili adhesins have two Ig-like domains and N-terminal receptor-binding lectin

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α-N-Acetylgalatosidase

RBC A

Agglutination

Abolished

NO AGGLUTINATION Æ Blood Group B, O

‹ Bool group glycans of RBCs #

Glycan Terminal sugar : GalNAc

$

GlycanTerminal sugar : Gal

O

Glycan Terminal sugar :-

Fig. 5.10 Enterotoxigenic Escherichia coli (ETEC) EtpA proteins recognize intestinal epithelial cells through fimbriae. EtpAs specifically opsonize RBCs by binding to terminal sugar, GalNAc, of blood type A glycans

domain that recognizes glycoconjugates or glycosphingolipids. Therefore, host glycan and ETEC binding is key step for ETEC infection [104, 105]. CofB C-terminal domain adopts an H-type lectin [106]. As H-type lectins typically bind to N-GalNAc molecules, CofB is a lectin targeting the small intestinal mucosal glycome. In CFA/III-mediated attachment, it requires a secreted protein, CofJ, at the GalNAc-binding sites of the CofB trimer. This is important for ETEC infection.

5.4.1.3

Enterohemorrhagic E. coli (EHEC)

EHEC is a major causing agent of gastrointestinal diseases. During enteric bacterial infection, pathogens such as EHEC recognize gut microbiota known to be relatively resistant to bacterial pathogens. After interaction with the host microbiota, the EHEC attach to the gut intestine via the lectin-glycan interaction to the host cells. For the known EHEC, E. coli as a subpopulation group of Shiga toxin (STx)-forming E. coli (STEC) is a causing agent of systemic hemolytic uremic syndrome (HUS), hemorrhagic colitis (HC), and diarrhea in humans. Two main STx species of EHEC are designated STx1 and STx2, as the virulence factors. Low doses of EHEC infection

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cause disease development. There is no biochemical difference known to distinguish general EHEC from nonpathogenic E. coli. Thus, specific detection of EHEC is difficult. The O:H serotype E. coli strains are associated with STEC infection in humans. Among O:H serotypes, HC and HUS diseases are mainly developed by certain serotypes including O26:H11, O103:H2, O145:H28, O157:H7, and O111: H8 strains. Among EHEC, the E. coli O157:H7 strain defines a major foodborne pathogen as a virulent serotype [107]. Due to its pathogenic property, O157:H7 strain of EHEC E. coli is regarded to be a dangerous bioweapon. Pathogenically, the strain is survived and expresses bacterial virulence gene in the human GIT [108]. EHEC strains cover 400 more serotypes. Among them, O157:H7 is a representative [109]. EHEC colonizes intestinal epithelium by microvilli loss and adhesion to the cell surfaces of hosts. Recently, to investigate the information on the lectin-glycan interaction of EHEC, a genome-wide analysis of putative adhesins or lectin-like proteins has been searched using lectin-glycan interaction network. Several lectin candidates were isolated through comparative transcriptome and proteome analysis in order to allow mucin recognition in EHEC [110]. For example, these lectin-glycan interactions are mediated by glycosylated mucins such as MUC2, MUC5 (A/B), and MUC6 in the human GIT. Bacterial pathogens express lectin-like virulent factors, and the virulent proteins are potentially used for vaccination and drug targets. Lectin-like proteins include pathogenic adhesins with tropism for host cell recognition via distinct architectures, including capsules, enzymes, fimbriae, pili, and vesicles. The lectin-like proteins interact with receptor proteins on host cell surfaces to allow cross-membrane invasiveness. Some adhesions activate host immune responses by binding to Man residue. As lectin-like proteins, E. coli surface antigen 20 (CS20); fimbriae (FimH, Yad) and SfaS; N. meningitidis autotransporter adhesin; S. enterica serovar Enteritidis ShdA, MisL, Sad, and BapA; and S. epidermidis polysaccharide intercellular adhesin (PIA) are known [110]. Structural domain and 3D structure can be integrated into multiple omics with transcriptome microarray information, proteome interaction, and genome data. Because membrane proteins recognize lectin-related proteins, homology analysis and B-cell epitope analysis combined with the integrated multiple omics information will predict successful vaccination or medicine development against pathogens such as EHEC to validate the predicted adhesins. Infection of the O157:H7 strain of EHEC E. coli involves in the colonized mucus layer, evasion of the immune defense, growth, and tissue damage of host [111]. Several types of virulence factors such as intimin, Shiga toxin, intimin, and pathogenicity islands (PAIs) induce the EHEC virulence [112–124]. As the major two different lectins or adhesin proteins, (1) intimin and (2) Shiga toxin are known. More totally, Curli fibers, EPEC intimin, EHEC intimin, EPEC intimin, EHEC O157:H7 CsgA, Lpp proteins, and type III secretion system are known to influence EHEC E. coli O157:H7 or EPEC E. coli internalization into host cells [115– 117]. First, the eae gene encodes the intimin protein of MW 94-kDa, and intimin mediates the adherence and invasion of EHEC E. coli O157:H7 and EPEC E. coli strains to hosts [118]. The adhesin intimin is a primary lectin with a minor long polar

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5 Pathogen-Host Infection Via Glycan Recognition and Interaction

fimbria (Lpf) [119]. Intimin lectin is the bacterial outer membrane protein. It initiates infection of EHEC E. coli strain during translocation of the intimin receptor (Tir) and the intimin interaction with the surface proteins of host cells, which is encoded on the bacteria genome. Intimin is an antibacterial drug target. Virulence of EHEC is related to factors encoded to the locus of enterocyte effacement (LEE), which is a specific pathogenic island for the intestinal mucosal adhesion and the attaching and effacing (A/E) lesion formation. The LEE gene locus encodes the eae gene for the outer membrane intimin and adhesin and also a type III secretion system (T3SS). The LEE products lead to interaction between EHEC (or EPEC) and intestinal epithelial cells [120]. Two Tir and intimin genes are located on 43 kb length sequence of the PAI, as the LEE with the A/E lesion formation [113]. The bacterium and host interaction are very interesting [121]. Intimin expressed by EHEC and EPEC mediates bacterial adhesion to the cell surfaces of hosts and A/E lesion generation [122]. Tir enters into the cells through the T3SS and functions as an intimin receptor expressed on the host cells [122, 123]. The intimin binding to Tir accelerates the pathogen adhesion to host cell [124]. At least 18 intimin subtypes [125] are found. As the adhesin protein, intimin is subdivided into five distinct forms of intimin-α, intimin-β, intimin-γ, intimin-δ, and intimin-ε by their C-terminus domains [126, 127]. Among these, the eae-γ1 subtype is common in O157:H7 and O145:H28 strains of EHEC. However, the eae subtypes of β1-, ε-, and θ- types are common in O26:H11, O103:H2, and O111:H8 strains of EHEC, respectively. The γ-type intimin-γ is the EHEC O157:H7 intimin, whereas the intimin-α is an EPEC type. Tir protein produced from EHEC O157:H7 is distinct from other EPEC-produced forms, in their phosphorylation patterns when they infiltrate into host cells [122]. EPEC intimin binding domain has been analyzed [128, 129]. The Tir-recognition domain in the intimin protein (Int188) produced from O157:H7 strain of EHEC E. coli is elucidated for its structural basis [107]. The four major structural variations have been known between intimins produced by EHEC E. coli and EPEC E. coli strains. From their structures, domain I has been known as an Ig-like domain and domain II is a C-type lectin-like domain. Mucin-digesting zinc metalloprotease named StcE helps infiltrative penetration of the adhesion protein and mucus layer [130]. After colonization, STx lectin is the main virulence factors of EHEC, because it causes the hemolytic symptom in kidneys and brain [131]. STx gene is located on the lambdoid bacteriophage genome. STx consists of five B subunits, which interact with globotriaosylceramide-3 (Gb3) as receptor present in the surfaces of endothelial cells. One catalytic subunit A targets eukaryotic ribosomes to inhibit protein biosynthesis [132]. The STx family consists of the two major types of STx1 and STx2 forms. STx2 is only produced during lytic cycle of phage, whereas STx1 type is produced during phage cycle and an iron-regulated promoter [133]. Therefore, the regulator protein of ferric uptake event, named Fur, suppresses the STx1 gene expression when iron levels are excessive. Thus, EHEC virulence is seen by multiple factors, not by single gene or gene product. EHEC STx lectin or intimin lectin binds to mucin sugars of gut mucus layer. The intestinal epithelium is decorated by secreted oligomeric mucins with heavy

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O-glycan proteins [134]. The main monosaccharides on human intestinal mucins are Gal, GalNAc, Fuc, and Neu5Ac [135], while EHEC consumes Fuc, Gal, and GlcNAc for gut colonization [136]. EHEC strain but not commensal E. coli releases mucin sugars by mucin-degrading enzymes such as metalloprotease StcE [137, 138] and esterase. Esterase is expressed by the prophage-carrying nanS genes known as nanS-p, which is located on the E. coli O157:H7 EDL933 strain genome and E. coli O104:H4 LB226692 strain genome. The esterase deacetylates 5N-acetyl-9-O-acetyl SA (Neu5,9Ac2) present in mucus, and thus the released Neu5Ac form is metabolized by the pathogen. EHEC E. coli has two the nanS-p gene and stcE gene what they are co-expressed with the stx2 gene and LEE gene, respectively. For digestion of sulfated intestinal mucins, sulfatase gene co-regulates the LEE genes in EHEC. Thus, EHEC has evolved acquisition to utilize mucin sugars and to colonize the GIT. For adhesion to the intestine epithelial cells, EHEC enters the mucus layer of epithelium. Adhesion level of EHEC to mucin-coated epithelial cells is higher than the mucin-negative cells, as also observed in other intestinal enteropathogenic E. coli (EPEC) and S. enterica. An interesting aspect is that EHEC binding to mucuspositive cells does not require any specific adhesion. However, flagella must be in the absence for this interaction. The reason is because a ΔfliC mutant adheres effectively rather than the wild types [138], and flagellin-synthesizing genes are not expressed during mucus recognition [139]. Metalloprotease StcE reduces mucin levels since a ΔstcE mutant cannot disrupt the epithelial cell mucus layers [139]. A cytotoxin named subtilase of certain non-O157 EHEC E. coli strains depletes mucin or mucus layers [140]. EHEC-altered mucus layer allows easy interaction with EHEC to bind to enterocytes. EHEC also synthesizes virulence factors for bacterial adhesion. NagC protein represses GlcNAc residue and Gal residue catabolism in E. coli. NagC regulates the LEE gene expression by binding to the LEE1 transcription-regulation region. If GlcNAc or Neu5Ac residues are present in the medium, EHEC adhesion to epithelial cells is decreased.

5.4.1.4

EHEC O157:H7 or EPEC Recognition of Core 2 O-Glycans of Mucin Type on Cell Membrane of Host

The relationship between host glycosylation and infection of the O157:H7 strains of EHEC E. coli or EPEC E. coli strains is still unknown. O-glycans are related to attachment and infection of the O157:H7 of EHEC E. coli and EPEC E. coli to host cells [141]. The O157:H7 of EHEC E. coli or EPEC E. coli infection and invasiveness to HT-29 cells are dependent on the host O-glycosylation status. O-glycans of mucin type have eight major groups (cores 1–8) by linkage of carbohydrate residues. Core 2 O-glycans of mucin type are synthesized by a specific GT enzyme of the core 2 β1,6-GlcNAc-transferase 2 (C2GnT2) [142, 143]. O-glycans are associated with the commensal microbial flora in the distal colon. O-glycans of Galβ1,4GlcNAcβ1,6 (Galβ1,3)GalNAc structure (called core 2) are converted to the Galβ1,3GalNAc structure (called core 1) in the MUC1 synthesis [144]. O-glycans help the intracellular delivery of the glycoproteins in intestinal epithelial cells, which are polarized

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[145]. O-glycans are a binding site for many bacteria [146]. HT-29 cells, which are deficient for the core 2 O-glycan structures, showed the reduced level of EHEC O157:H7 or EPEC adherence. In addition, the upregulated MUC3 in HT-29-Gal cells diminishes EHEC E. coli O157:H7 or EPEC E. coli attachment [147, 148]. Core 2 O-glycans of mucin type produced in epithelial HT-29 cells are the sites for the EHEC O157:H7 or EPEC invasion and infection. Mucin-type O-glycan is synthesized by serial enzyme reaction from GalNAc on Ser/Thr residues on proteins. For example, core 1 synthase (C1GnT) transfers the Gal residue to the acceptors and generates the core 1 sugar of the Galβ1,3GalNAcα1Ser/Thr structure. Similarly, core 3 synthase (C3GnT) transfers the GlcNAc residue to its acceptors and forms the core 3 sugar of the GlcNAcβ1,3GalNAcα1-Ser/Thr structure (termed core 3 structure). The core 1 sugar chains are modified to the core 2 sugar chain through multiple enzymatic conversion of GTs including C2GnT-1, C2GnT-2, and C2GnT-3 enzymes. Among them, the core 3 chain can also be converted to the core 4 chain by the specific GT enzyme of C2GnT-2. If Man, Fuc, Glc, or GlcNAc residues are attached to Ser/Thr protein, the core 5 to core 8 chains are yielded.

5.4.2

Lectins and Glycans of Other Pathogenic Bacteria

5.4.2.1

Legionella pneumophila, K. pneumoniae, S. pneumoniae, and B. cepacia Complex

The PapG lectins specifically recognize the “Gal-based disaccharides” on the terminal or distal ends of glycolipid oligosaccharides of renal cells [89, 91, 149, 150]. If lectins are produced from Legionella pneumophila, K. pneumoniae, S. pneumoniae, P. aeruginosa, and B. pertussis, the lectins specifically recognize the “GalNAc (beta 1-4) Gal” on the human respiratory tract [151–154]. Bacterial lectins can recognize fucosyl human histo-blood group carbohydrates. Human fucosyl oligosacchariderecognizing soluble lectins are reported in P. aeruginosa and B. cepacia complex of B. ambifaria strain and B. cenocepacia strain [154–156]. P. aeruginosa LecB, named PA-IIL, is a tetrameric lectin structure with an affinity for L-Fuc and two Ca2+ ions [157]. B. ambifaria BambL lectins consist of a trimeric structure through β-propeller configuration with two Fuc-recognition sites in each monomer [82]. Both lectins also have higher affinity toward the LeA antigenic epitope, and BambL has a strong affinity for the epitope of H-type 2 antigen of AB(O)H blood group of humans. From the above lectin specificity, the host-pathogen lectin-carbohydrate interactions give some insights into the preventive infection if inhibitors are available. For example, an antiadhesive agents or adhesion-inhibitory agents can disturb or block the GalNAc (beta 1,4) Gal interaction, giving potential anti-pathogeninfection drugs.

5.4 Pathogen-Producing Lectins as Receptors to Bind to the Host Carbohydrates

5.4.2.2

229

Non-typeable H. influenzae and Acinetobacter baumannii

Non-typeable H. influenzae (NTHi) is a host-adapted pathogen present in the human respiratory tract. The incidence of invasive NTHi infections is rather increasing due to the vaccination against H. influenzae b type and S. pneumoniae. NTHi vaccine is absent. NTHi expresses surface factors to adhere to host surfaces. For example, major outer membrane proteins (OMPs) P1 bind to carcinoembryonic antigen (CEA)-related CAM-1 (CEACAM-1) [83]. OMP P4 recognizes the ECM components including fibronectin, laminin, and vitronectin [158]. H. influenzae surfaceadhesin protein E is also known to inhibit the binding activity. Acinetobacter baumannii PilA glycoprotein binds to selectins and CEACAMs of surfaces of host cells. Type IV pili bind to intercellular adhesion molecule 1 (ICAM-1) [159]. Fimbriae bind to human mucins [160, 161]. Adhesins of HMW1 and HMW2 also bind to carbohydrates. HMW1 binds to α2–3 sialyl-lactosamine, although the host receptor for HMW2 is not known. However, simply, various host glycans can act as HMW2 receptors. The results obtained from glycan-binding HMW2 microarray and surface plasmon resonance (SPR) techniques indicate that HMW2 recognizes the NeuAcα2,6 linkage, not the non-human SA form, NeuGc [162].

5.4.2.3

H. pylori

In H. pylori infection, H. pylori has been suggested to evolve for the 30,000 years with the humans as host in a way of coevolution. H. pylori has acquired from the learning to adapt its antigenic structures such as human Lewis antigen to evade the immune surveillance of hosts. In the human host, humans may also adapt to the commensal symbiosis with the pathogen for its contribution to provide antibiotic and probiotic-like components which are produced for regulatory control of H. pylori [163]. H. pylori is classified to the human carcinogenic class 1. H. pylori recognizes human blood group antigens (HBGAs) produced in O-glycan structures of mucous surfaces of human epithelial cells, as a pathogen binding and infection site. Human stomach tissues also express blood group antigen-binding adhesin (BabA). Recently, another LabA lectin has been discovered, and the LabA binds to LacdiNAc structure of GalNAcβ1, 4GlcNAc-carbohydrates. This LabA lectin recognizes a glycan sequence, which is different from those of BabA and SabA [164]. LabA adhesins also recognize LeB and LacdiNAc. This specificity leads to H. pylori colocalization in the mucin MUC5AC in gastric epithelial surfaces, but not in MUC6. These adhesins recognize HBGA-related glycans present in gastric mucosal epithelial cells. HBGA-recognizing adhesin known as BabA and the SA-recognizing adhesin known as SabA are also lectins produced by Helicobacter outer membrane protein group attached to host cells. BabA binds to fucosyl-type 1 glycan structure of the human AB(O)H blood group antigens and Lewis antigens expressed on glycolipids, mucins, and glycoproteins expressed in the GITs [165]. BabA recognizes the Fucα1–2 linked type 1 epitope of Gal1-3GlcNAc core in the O blood group,

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which is the same structure as the H1 antigen in LeB antigen. If Fucα1–4 is linked to the GlcNAc residue present in the type I sugar chain, the sugar antigenic type is called the H1 antigen of ABO blood group. BabA binds to A/B type blood group antigens with the terminated sugar of GalNAcα1,3 and Galα1,3 residues present in the H1 epitope, respectively. They are Lewis b antigens [166]. Contrary to BabA, the SabA binds to sialyl-dimeric LeX (sdiLeX) on glycolipids and GSLs as well as to the monomer SLeX and the derivative SLeA forms [167]. Sialylglycans are not enriched in normal and healthy stomach of humans [168, 169], but gastric inflammatory event and malignantly transformed cells develop sialylated Lewis antigens [170–172] and lost neutral blood group ABH antigens. Simultaneously, pathogens adapt SabA adhesin [167]. Helicobacter exploits the receptors such as selectin mimicry through the sialyl-(di)-LeX/A GSL recognition, because inflammatory intestinal and stomach express sialyl Le glycans expressed in the leukocytic cells, which recognize selectins. The other lectins of OipA, HopZ, and AlpA/B are known [163].

5.4.2.4

P. aeruginosa

In P. aeruginosa, the bacteria adhere to epithelial cells through cell-surfaced adhesins such as LecA known as PA-IL and LecB known as PA-IIL as well as type IV pilus known as T4P. They all recognize carbohydrate ligands present in epithelial cell surfaces [173–175]. Among them, LecA and LecB also activate cell functions of host [175, 176]. Soluble homotetrameric fucophilic lectin LecB forms biofilm, bacterium/host, and bacterium/bacterium interaction potentiating its cytotoxicity and inhibition of ciliated removal [177, 178]. Four subunits of 11.7 kDa, which is composed of 114 amino acids, are bridged with 2 calcium atoms resided in the recognition sites. The two Ca2+ atoms chemically bind to the Fuc residue for a monomeric ligand [179]. LecB binds to LewisA (LeA) 1 [180], 3-fucosyllactose [180], SLeA [181], antigen H [182], LNFP-II, LNnFP-V, Lex [183], SLex [181], and mannan [184]. LecB can bind to both monosaccharides of L-Fuc and D-Man. LecB prefers to recognize Fucα1,4->Fucα1,3-> Fucα1,2-carbohydrates. From the Fuc linkages, the nonreducing end-linked Fucα1,2 carbohydrate prevents binding of LecB to Fucα1,3/4. Carbohydrate structure of Galβ1,3(Fucα1,4)GlcNAcβ1,3Gal-R more potently recognizes the LecB, when compared to the carbohydrate structure of the Fucα1,2Galβ1,3(Fucα1,4)GlcNAcβ1,3Gal-R. The LecB preferentially recognizes the LeA antigens and 3-fucosyllactose (3FL) oligosaccharides [181]. The co-crystal structure of complex LecB and LeA with the structure of Galβ1,3 (Fucα1,4)GlcNAc- suggests that component monosugars in Lea bind to the LecB surface. 3FL oligosaccharide differs from Lex antigen due to loss of the N-acetyl modification on the Glc residue. This difference induces an increased affinity for LecB. Lectin LecA is also a virulence factor in the adhesion, biofilm synthesis, and lung injury [185, 186]. Gram-negative pathogenic P. aeruginosa in dermatitis, keratitis, pancreatitis, urinary infections, and respiratory tract infections causes immunocompromised death. P. aeruginosa lectin LecA binds to glycosylated targets on the cell

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surface [187]. Globotriaosylceramide Gb3 globoseries is indeed a major ligand of LecA lectin and frequently found in epithelial cells of murine lungs and human intestines [188, 189]. Gb3 can activate LecA attachment in a similar fashion of cholera toxin subunit B (CTB), as a hetero-multivalent binding is also observed in CTB [190]. Hetero-multivalent binding of LecA to Gb3 induces a LecA signaling to trigger CrkII phosphorylation [191]. LecA lectin has a homotetrameric structure, and each monomer carries each single carbohydrate-recognition site. LecA lectin carries two pairs of the adjacent recognition site [192]. LecA preferentially binds to α-Gal-terminated glycolipids. Typical one is globotriaosylceramide Gb3 globoseries with the structure of Galα1,4Galβ1,4Glc-Cer as the LecA ligand [193–195]. LecA lectin can also bind to other glycolipids of Galβ- and GalNAc-terminated glycolipids, but with lower affinities than Gb3 [193, 196]. Therefore, inhibition of LecA prevents adhesion of the bacteria. This concept [197] was expanded to the therapeutic Gal solution usage against P. aeruginosa pneumonia in murine models and human patients due to inhibition of the LecA binding to its glycosylated targets [198]. Anti-infectious drugs include antiadhesive molecules in P. aeruginosa through multivalent ligands of LecA and LecB. A series of 27 LecA-targeting glycoclusters were synthesized. A low-nanomolar (Kd ¼ 19 nm) ligand with a Tyr-based linker bridge was found in a study of the structure-activity relationship. Molecular binding between the glycoclusters and the lectin tetramer was elucidated [199].

5.4.2.5

N. gonorrhoeae, N. meningitidis, S. aureus, Chlamydia pneumoniae and Vibrio parahaemolyticus

However, tissues frequently secrete glycan fluids to inhibit the lectins of bacterial and viral pathogens and prevent pathogenic adhesion and recognition to cell membrane glycans [200, 201]. Some representative secreted glycan is the adhesive glycoprotein, human kidney uromodulin known as Tamm-Horsfall protein. The uromodulin carries branched oligosaccharide glycans on lectin glycosyl moiety expressed by some pathogens such as N. gonorrhoeae and S. aureus [202, 203]. Immunomodulatory molecules are associated with cardiovascular diseases, including apolipoprotein-E (Apo-E), CRP, and Man-binding lectin (MBL) [204, 205]. MBL as a circulatory and soluble C-type lectin functions in nonselfversus self-recognition. The lectin MBL distinguishes and identifies dead cells, dying cells, or cancer cells to capture and make clearance by phagocytic cells of host [206]. MBL recognizes PAMPs to destroy pathogen [207]. MBL2 expression is dysregulated in abnormal conditions such as coronary heart disease, and abnormal function of MBL2 increases the host susceptibility to pathogenic infection [208, 209]. In cardiovascular inflammatory pathogen Chlamydia pneumoniae (CP), an obligate intracellular bacterium, it invades human endothelial cells [210]. C. pneumoniae with circulating phagocytes induces plaque forming vascular coronary heart disease. The serum MBL2 in human blood functions as a binding receptor for Man and NAcGlc on the C. pneumoniae membrane. Detected MBL

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contributes coronary disease severity in C. pneumoniae patients [211]. Alternative splicing variants or different promoter-transcribed variants in the MBL2 gene are expressed as altered coding missense SNPs, defecting blood MBL level-based diseases [212]. An associate between C. pneumoniae and MBL2 is indicated within the coronary disease cohort. C. pneumoniae-reactive antibody production in humans is controlled by the transcription promoter variant XY as well as the allele number of MBL2-A and MBL2-B. Therefore, human diseases are developed by the interaction between the host immune cells and pathogen C. pneumoniae. The frequency of the coding missense SNP alleles depends on population origin. MBL2 genotypes are associated with coronary artery diseases [213], derived by the relationship between MBL2 genotypes and the immune response to C. pneumoniae [214]. Meningococcal adhesins are known for pili (type IV fimbriae) and opacity protein (Opc). Meningococcal pili as high molecular heteropolymeric glycoproteins protrude the capsule and have a capacity to recognize host endothelial and epithelial cells [215]. This crucial adhesin, pili, binds to the platelet activating factor receptor of host cells via the pilin-linked glycans [216]. Nonencapsulated pili mutant cannot recognize asialo-GM1 and GalNAcβ1–4 Gal. Therefore, meningococcal pili are essential to bind to the host. Asialo-GM1 is abundant in regenerating epithelia and a host receptor for meningococcal adhesion. In fact, certain pathogens present in the respiratory tract bind to the GalNAcβ1–4 Gal epitope in asialo-GM1 or asialo-GM2 [215]. In P. aeruginosa, pili35 binds to the asialo-GM1. However, Vibrio parahaemolyticus mannose-sensitive hemagglutinin (MSHA)-lacking pilus does not bind to asialo-GM1 [217]. Additionally, the surface components of meningococcal bacteria include capsule, LOS, Opc, and pili. Pathogenic meningococcal bacteria bind to the GAGs for adherence and invasion. The nasopharyngeal epithelial cells are enriched with mucus and mucins [214]. The CNS is also enriched with gangliosides and chondroitin sulfate. Neisseria meningitidis strains consist of 13 capsular serogroups. Six (A, B, C, W, X, Y) capsular serogroups cause diseases [218]. N. meningitidis cause bacterial meningitis and sepsis with about 10% mortality. Meningococcal virulence factors such as Opc 13, 16, Opc 13, Opc 16, and NHBA specifically bind to host glycans. The opacity protein, Opc, is integral and outer membrane protein [219]. Opc 16 binds to sialylated monosaccharides. Opc recognizes human ECM GAGs of the heparan sulfate and the fibronectin and vitronectin of endothelial cells [220]. Neisseria heparin binding antigen (NHBA) binds to heparin residues [221]. Meningococcal Opc attaches to the CNS cells during meningitis. The main chondroitin-6-sulfate GAG expressed in the CNS is recognized by Opc. In fact, Opc binds to chondroitin-6-sulfate structures and lacto-N-neotetraose structures. Lacto-N-neotetraose is also known as a component of lacto-neo series GSLs such as paragloboside. Lacto-N-neotetraose structure is used as the precursor of the ABO and P1 blood group antigens. These GSLs and blood group antigens are also expressed in neuronal and erythrocytes, respectively. The lacto-N-neotetraose structure on nasopharyngeal epithelial cells is the binding site for Streptococcus pneumoniae. Free lacto-N-neotetraose inhibits pneumococcal binding to human and animal nasopharyngeal cells. Capsule-based vaccines can be produced by capsular

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serogroups A/C/W/Y. Capsular B serogroup is used for outer membrane proteinbased vaccine [222]. N. meningitidis glycosyltransferase genes of lst, lgtB, lgtA, and lgtE generate LOS species. The capsule and LOS modulate pathogen-host interactions and virulence. Meningococcal LOS consists of two oligosaccharide chains linked to heptose residues [223] with different types of L1–L12 [224]. LOS L3 immunotype binds to Thomsen-Friedenreich (TF) antigen. The N. gonorrhoeae LOS binds to the ASGPR (asialoglycoprotein receptor) on epithelial cells.

5.4.3

Viral Lectins or Host Lectin-Binding Glycans

For viral infection receptors, many viruses produce lectins for their infection. Currently, well-known virus SA-specific lectins are summarized (Table 5.10). In the case of viruses, most pathogenic virus carries hemagglutinin (HA) lectins, and the term of HA agglutinates red blood cells, as mainly studied on human cases. Viruses have lectin genes to express them on their surfaces for cell targeting. Influenza, noroviruses, and rotaviruses are studied for their lectins. Also, in dengue viruses, they use a DC-SIGN, one of C-type lectin family, expressed on DCs for entrance to the cells and replication [225]. In structural aspects, norovirus-encoded lectins bind to the carbohydrate receptors of HBGA. The surfaced capsid proteins of norovirus function as a lectin-like receptor. Norovirus is a non-enveloped ssRNA virus of the Caliciviridae family. Noroviruses transmit person-to-person spread. The norovirus virion is composed of the assembled structure with 180 capsid protein (VP1) copies. The capsid VPI is dimerized to an icosahedron core [226]. The monomeric protein, capsid VP1, consists of two different structures of domains named (i) shell domain (S) and (ii) protruding (P) domain. P domain involves in lectin-like receptor binding with diversity in strains. The norovirus capsid VP1 is a lectin which binds to sialyl glycans, while human norovirus VP1 P domain binds to polymorphic HBGAs [227–231]. Human noroviruses bind to their HBGA partners, giving their infections and spreading. The noroviral interaction with HBGA is evolutionarily linked with genetic traits. Fuc residue in Lewis antigens and histo-group antigens of humans is a key recognition site for the lectin of the VP1 of GII norovirus strains. For the fucosylation reaction of Fuc residues in the Lewis antigens and ABO blood group antigens, the two known fucosyltransferases of FUT-2 and FUT-3 enzymes catalyze the Fuc residue transfer. For therapeutic application, besides vaccine-based combating the virus, a strategy to prevent norovirus infection is to use human milk oligosaccharides (HMOs). HMOs contain competing agents against virus-producing carbohydrate receptors, which mimic the O-glycans of mucin type, which are reactive to blood group antigens of humans. In the human HMOs, several trisaccharides such as 3SL, 6SL, and 20 -fucosyllactose (2FL) are included in the glycolipid forms or free oligosaccharide forms. Among them, 2FL prevents norovirus binding and has gained market approval [163, 232, 233]. Oligofucoses in hepta- to decasaccharides promote competitive effects on norovirus binding. L-fucose

Reoviridae

Coronaviridae

Polyomaviridae

Paramyxoviridae

Species Orthomyxoviridae

Human coronavirus OC43 Porcine haemagglutinating encephalomyelitis virus Porcine transmissible gastroenteritis corona virus Avian infectious bronchitits coronavirus Murine hepatitis virus Reovirus type 1 Porcine rotavirus group A OSU Human rotavirus KUN, MO

Influenza virus B and C Newcastle disease virus Sendai virus Human parainfluenza virus type 1 and type 3 Porcine rubula virus LPM Murine polyoma virus (large and smallplaque) Simian virus 40 Human polyoma virus JC and BK Bovine coronavirus

Influenza virus A

Human Avian and equine Porcine

Neu5,9Ac2α2,6Gal  Neu5,9Ac2α2,3Gal Neu5,9Ac2 Neu5Gcα2,3  Neu5Acα2,3 Neu5Acα2,3 Neu4,5Ac2 Siaα2,3 Neu5Gc-GM3  Neu5Ac-GM3 GM1

S protein HA-A HE σ1 VP4 VP4

Neu5Acα2,3Gal, Neu5Acα2,6Gal Neu5Acα2,6Gal and Neu5,9Ac2, respectively GM3, GM2, GM1, GD1a, GD1b, GT1b, N-glycans NeuAcα2,3Galβ1,3GalNac/4GlcNAc NeuAcα2,3Galβ1,4GlcNAc and NeuAc/Neu5Gcα2,3/ 6Galβ1,4GlcNAc Neu5Acα2,3Gal Neu5Acα2,3Galβ1,3GalNA; Neu5Acα2,3Galβ1,3 [Neu5Acα2,6]GalNAc GM1 Siaα2,6 and Siaα2,3, respectively. Neu5,9Ac2α2,3Gal  Neu5,9Ac2α2,6Gal

SA-binding specificity Neu5Acα2,6Gal Neu5Acα2,3Gal

VP1 S protein, HE S protein HA-A

HN VP1

HA and HE HN HN HN

Lectin HA

Table 5.10 Virus SA-specific lectins. HA-A HA activity observed, HE HA esterase, HN HA neuraminidase

234 5 Pathogen-Host Infection Via Glycan Recognition and Interaction

Herpesviridae Hepdnaviridae

Parvoviridae

Adenoviridae Picornaviridae

Rhesus rotavirus Bovine rotavirus NCDV Bluetongue virus Adenovirus type 37 Theiler’s murine encephalomyelitis virus Human enterovirus type 70 Bovine parvovirus Adeno-associated virus serotype 4 and 5 Murine and human cytomegalovirus Hepatitis B virus Small S protein

HA-A HA-A

VP4 VP4 VP4 Fiber knob VP2

Neu5Ac > Neu5Gc Neu5Gc-GM3 Neu5Ac, Neu5Gc Siaα2,3 Siaα2,3 Siaα2,3 Neu5Acα2,3Gal Neu5Acα2,3Gal and Neu5Acα2,6Gal, respectively Neu5Ac and Neu5Ac > Neu5Gc, respectively Neu5Ac

5.4 Pathogen-Producing Lectins as Receptors to Bind to the Host Carbohydrates 235

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dendrimers of α-L-fucose residues are natural polysaccharides of polyfucoses or fucans. Fucoidans from brown algae exert beneficial effects [234]. In a blood groupindependent manner, anti-norovirus activities of αL-Fuc residue in fucoidan in algae and desulfated form of Fuc residues present recognition of viral capsid proteins with mucins present in human GIT [163]. SA-containing glycans of influenza virus HA. Hemagglutination allows easy detection of influenza virus strains, as they agglutinate erythrocytes of special avian birds and mammals [235]. For example, distinguishment between the H5N1 avian influenza virus and infectious H1N1 swine influenza virus strains is based on the hemagglutinin experiment. H5N1 lectins specifically bind to the α2,3 sialyl-Gal disaccharide on chicken erythrocyte membranes and airway epithelial cells of the lower human respiratory tract. Lectins of the H1N1 swine influenza virus typically bind to the α2,6 sialyl-Gal on pig erythrocyte membranes and airway epithelial cells, which are lined in the upper human respiratory tracts [236].

5.5

Host Lectin Defense Mechanisms in Lectin-Carbohydrate Interactions

Host defenses against pathogen infections depend on a systemic collaboration of innate (nonspecific) and adaptive (specific) components. The organisms are facing potentially life-threatening pathogenic infections by microbial pathogens. The survival game of the host depends on how they well recognize the pathogenic microbial infections and how to lead to defense responses [237]. The immune response of innate immune cells senses foreign invading agents to follow immune responses. Innate defense is the major host defense line to restrict the pathogenic propagation in host tissues prior to the transduction of adaptive immunity. Serum and tissues contain a series of lectins to recognize and bind the pathogens. The host defense response is initiated by innate immune sensors of danger signals, via pattern recognition receptors (PRRs) since proposal in 2002. Microbial sensors including CTL receptor (CTLR), complement system, TLR, the RIG-I-like helicase, and the nucleotide-binding oligomerization domain-like receptor are all defensing receptors. Ligand-activated PRR triggers to intracellular signaling events with the co-stimulatory molecule expression and the innate immune responses, and eventually induces adaptive immunity of host. Proinflammatory or anti-inflammatory reactions are well associated with the innate immune responses. Although four key life molecules are nucleic acids, proteins, carbohydrates, and lipids, the different subunits of sugars, amino acids, nucleotides, and fatty acids are linked to different component, structure, and distribution. Diverse pathogens consist of diverse PAMPs. The major PAMPS of pathogens include cell wall lipoproteins, lipoteichoic acid, LPS, and peptidoglycans. β-Glucan and mannan are the fungal PAMPs. RNA and DNA frequently mimicked by poly I:C or CpG are targeted by the immune system to recognize virus [237].

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Carbohydrates (glycans) are utilized for antigen formation and innate immune recognition [238]. The glycans are either secreted or located on the cell surfaces. Therefore, they are the components of pathogen-host interaction and pathogen infection as well as host innate responses [74]. Because recognition event is the most basic step for the different hosts-pathogen interaction, glycans show diverse nonself recognition molecules [239]. Carbohydrate recognition during host-microbe interaction is much more elucidated by basis of genome information in hosts and glycan diversity in pathogens. Carbohydrate-lectin interaction bonds are very weak, compared to covalent chemical bonds. Thus, it seems that relatively high numbers of lectins and carbohydrates are interacted together to give the desired physiological cellular interactions. Bacterial cell coats thus present their lectins along with the shafts of fimbriae, as surrounded like fur, to give clustered adhesive affinity and force by multiple lectin-carbohydrate interactions on cell surfaces [240, 241]. Such clustered with glycans are well described in the result that the FimH lectinexpressing E. coli cells readily and strongly bind to mannose on glycoprotein filmcoated micelles [242]. Among the carbohydrate-binding molecules, a representative member is a CLR of DC-SIGN present on the DC surfaces. The DC-SIGN binds to various Man- and Fuc-carrying ligands in the envelope of HIV [243]. Other cases include Siglecs, CD22, and BCR complex, which uptakes antigen and activates B cells [244]. Additionally, the galectins associate with host glycans, to be organized into receptor lattices [245, 246]. In microbes, their immunogens are mainly glycans, where bacterial and virus coats are decorated with a sugar coat known as glycocalyx. For example, gp120 protein of HIV coat protein is a representative glycoprotein. Some bacteria are encapsulated with polysaccharides, glycolipids, or endotoxins. Peptidoglycans of Gram-positive bacteria are also the representatively exposed coated molecule. The PAMPs are such endotoxin LPS, bacterial capsules, muramyl dipeptide, and viral coats. Therefore, pathogenic microbes have evolved to produce host-similar glycans on their surfaces of cells to mimic the cell surfaces of hosts. In addition, such glycans evade immune surveillance [247], indicating the carbohydrate immunogen’ roles in the immune system. As described above, host lectins are often used as receptors of pathogenic infection agents. However, reversely, some host lectins are well designed to avoid and prevent pathogenic infections. Surfaces of most microbes express mannose residues, and some mammals often express mannose-binding lectins to perform the innate immunity [248]. Reversely, however, some pathogens utilize such lectins engaged in the innate immunity rather to invade host cells to cause severe fatal infections. Such a well-known case is the glycans on the Ebola virus envelope, and this lectin readily recognizes the CTL-like domain family 4, member g (Clec4g) known as LSECtin present in endothelial cells of human lymph nodes and liver. The glycan-LSECtin binding induces endocytosis-based infection and death of the host cells [249, 250]. Also, a Man-binding lectin (MBL) binds to the Ebola virus envelope glycoprotein glycan to block the interaction between the glycans and other mannose-binding lectins expressed on host cell membrane [251]. Thus, high doses of MBL can protect mice infected with the Ebola virus [252]. Such inhibitory

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agents of host-pathogen lectin-carbohydrate interactions can be served as antiinfectious agents in humans.

5.6

Pathogenic Glycans to Trigger Innate Immune Enhancement

In infection, microbial pathogens of bacteria or fungi cause pathogen-borne diseases by glycan recognition and intracellular signaling events as well as pathogenic surface expression of mammalian-associated glycans to escape from host’s immune system. Host defense system against pathogens can be improved by immuneenhancing polysaccharides through regulating the levels of cytokines, macrophage, and lymphocyte. Macrophages play important roles especially in innate immune system such as microbial infection, producing inflammatory mediators, cytokines, and phagocytic activities. The mediators of NO, ROS, IL-1β, and TNF-α are increased upon stimulation of macrophages with treatment of LPS. Various host defense functions of activated macrophages are mediated by NO that leads to signaling molecule, and inflammatory mediator mainly regulates immune responses. To trigger cellular signaling response, many external stimuli bind to PRRs which recognizes surface of microorganisms and induce secretion of cytokines or TLRs on surface of DCs or macrophage and then trigger various signaling pathways. With T lymphocytes, NK cells in innate immunity are also important for tumor cells and infectious pathogens by binding to MHC molecule not binding to a specific antigen. Cytotoxic NK cells release IFN-γ cytokine and are the major IFN-γ producer in humans. Deficiency in IFN-γ production of NK cells can cause malignancy and infection. IFN-γ exhibits anti-infection activity through tumor surveillance and induces tumor apoptosis. Moreover, NK cell-generated IFN-γ promotes macrophage, DC, and T-cell activation during inflammation and malignant diseases. NK cells exhibit spontaneous cytotoxicity against transformed cells. NK cells are also upregulated by IFN species or upregulated in an indirect manner by IFN-inducing agents of bacterial immunogens, mitogens, and viral components. IFN-γ-induced NK cell activation and resulting killing activity are crucial for cancer immunotherapy of NK cells. IFN-γ expression is regulated by MAPK and NF-κB pathway, inducing phosphorylation of p38, ERK, and c-Jun NH2-teminal kinase (JNK). NF-κB activates IFN-γ transcription. Glycans or polysaccharides as natural macromolecules are glycosidic bonded carbohydrates or covalently bonded glycoproteins or glycolipids existed in microbes, plants, and animals. Polysaccharides can regulate antimicrobial, antioxidant, antiviral, or antitumor activity. Glycans of plants and bacteria have also immune-enhancing activity among diverse immune responses, as activate innate immune cells specifically. The roles of glycans are recently revisited for the functional diversity and specificity of glycan-recognizing components of the innate immune system. In mammals, glycans have diverse functions including apoptotic

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induction for mutant cells, clearance, self/nonself-discrimination, cell-cell communication, and cell signaling. Glycosylation in humans is frequently linked to inflammation, cellular stress, aging, or cancer, as the importance of glycans is in recognition and interaction. The immunological role of glycans observed in various bacteria and fungi has been defined. Considered function of polysaccharides in immune system, they seem mainly to function in activation of innate immune cell and increasing levels of cytokine, but minor effect of humoral immunity. Because innate and humoral immunities are cooperative, the effect of polysaccharide in innate immunity is regarded as the major point. Among many innate immune cells, polysaccharides activate commonly macrophages or DCs. The first defense line of hosts in pathogenic infections and cancer is macrophage or DC, as they release inflammatory mediators and cytokines upon stimulation with LPS and various polysaccharides. They are IL-6, TNF-α, and IL-1α, which directly elevate the tumoricidal activity of B- and T-cell proliferation as well as macrophage growth. Additionally, from the studies on T-cell-independent antigen, many pathogenic polysaccharides regulate TLRs expressed in macrophage or DC surface and recognize pathogen surface patterns, following signaling pathway-activated MAPK and transcription factor NF-κB. For example, the pneumococcal C-polysaccharide and capsular polysaccharides activate the immune cells and cytokine release upon stimulation of whole blood. Streptococcus pneumonia is mainly causative of bacterial pneumonia, meningitis, and sepsis, causing a pneumococcal invasion disease in humans including adults and children. Bacterial Gram-positive S. pneumoniae strain expresses capsular polysaccharides on the bacterial surface with each specific serotype. In all serotypes, the cell surface wall polysaccharides are structurally similar as a common marker for the species. Because of current lack of effective pneumococcal vaccines and antibiotic resistance, treatment of the pneumococcal disease is dependent on the vaccination. The polysaccharides of S. pneumoniae stimulate class switch promoting T cells and non-T cells like NK cells and monocytes. To date, two different vaccines are designed using the polysaccharides. Immunoglobulin class switching from IgM to IgG takes place in response to the unconjugated polysaccharide vaccine due to T-cell-independent (type 2 thymusindependent) antigen. Some immune-related cells such as CD4, CD4+, NK-like T cells, NK-like T cells, and monocytes are activated by CPSs. These cells protect against pneumococcal infection via B-cell function of antibody production and induction of immunoglobulin class switching, which is a phenomenon in the T-cell-independent antigens such as capsular polysaccharides. One vaccine type contains only the polysaccharides, while the other type contains proteins plus polysaccharides. As the polysaccharides are T-cell-independent antigens, the polysaccharides directly stimulate B-cell responses to produce antibodies. However, as proteins are T-cell-dependent antigen, the protein-conjugated capsular polysaccharides stimulate T-cell response via MHC presentation of protein antigen with memory B/T cells and antibodies. Although such vaccination to pneumonia is available, there is still concern that the capsular polysaccharide and other polysaccharides may associate with infection of host cells and recognize distinct

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immune-related cells. The C-polysaccharide and capsular polysaccharide modulate individual immune cell functions and cytokine expression. On the other hand, some polysaccharides give immune-enhancing activity. For example, the polysaccharides from Cyrtomium macrophyllum (family of plant) or from Paecilomyces cicadae (family of Eumycetes) enhance immune responses through TLR4 activation of macrophage. Anoectochilus formosanus (family of plant like orchid) type II AGAF is a potent immunomodulator, having the signaling actions of immunomodulatory activity, upregulating NK cell-mediated cytotoxicity via expression of IFN-γ to stimulate the innate immune system. NK cell-mediated cytotoxic activity is enhanced by type II arabinogalactan glycans produced by A. formosanus. Thus, different glycans exhibit common activation of innate immune cells including macrophage, NK cell, NK-like cell, or CD+4 cells. From the current glycan-immune cell interaction, the basic question is raised: how glycans activate innate immune cells, if they have distinct mechanism including signaling pathways associated with glycan activation?

5.6.1

Example 1: Polysaccharides with Immune Enhancement of Cyrtomium macrophyllum

Cyrtomium macrophyllum is a family of plant, and the rhizomes of Cyrtomium contain pharmacological anti-parasite, antibacterial, antiviral, and anticancer capacities, giving an immune-enhancing activity. The glycans activate TLR4 of innate immune cells such as macrophage.

5.6.2

Example 2: Activation of Macrophage by Polysaccharide from Paecilomyces cicadae

Polysaccharides isolated from plant Paecilomyces cicadae (PCP) have an immunomodulatory property on the macrophage [253]. PCO increases in interferon-γ production by Peyer’s patch cells, phagocytosis by macrophage, spleen cell proliferation, and dendritic cell maturation with immune-stimulatory activity on macrophage and dendritic cell maturation. Another example, Cordyceps cicadae is a parasitic fungus and functions for curing of malaria, enhancing blood aggregation, and antitumor activity. PCP induced macrophage activation through TLR4 pathway. Polysaccharide cannot penetrate cells, due to their large molecular mass, so the first step in the modulation of cellular events is binding to receptors. The role of TLR4 as the PCP receptor was confirmed in macrophages in C3H/HeJ mice and NO production and cytokine gene expression. Because the activity of PCP is similar to LPS, differences between LPS and PCP were also observed. Aside from activation macrophage and TLR4 receptor, signaling pathways including MAPKs and

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241

NF-κB were activated by binding of TLR4 with PCP. Activation of MAPKs is significant in the induction of NO as it controls the NF-κB activation. Here, it has been confirmed that NF-κB and p38 are important for NO production by macrophages. PCP firstly bound to membrane receptors including TLR4, and activated MAPK and NF-κB signaling, finally increased the production of NO and various cytokines IL-6, IL-1β, and TNF-α.

5.6.3

Example 3: NK Cell-Mediated Cytotoxicity Increased by Arabinogalactan from Anoectochilus formosanus

Anoectochilus formosanus is an Orchidaceae plant having anti-asthma, hepatoprotective, antitumor, and immunomodulatory effects [254]. A. formosanus polysaccharide is characterized by a type II arabinogalactan (AGAF) and has a MW of 29 kDa. The AGAF has an immunomodulatory effect against colon cancer in mice. AGAF promotes splenocyte cytotoxicity and elevates the relative cell number of CD4+ T cell and CD8+ CTLs, giving antitumor potentials because NK cells and CTLs are critical in anticancer activity. The type II arabinogalactan AGAF has the increasing activity of NK cell-mediated cytotoxicity. NK cells also activate the innate immune responses, although the cells can directly kill pathogenic invaders and tumor cells. Tumor cells and virus-infected cells are easily controlled by NK cells through the proinflammatory cytokines of IFN-γ and TNF-α and also the released granules with cytotoxic activity. For signaling pathway associated with IFN-γ, the glycans act as TLR ligands of the innate immune cells and NK cells. These molecules activate NF-κB. The NK cell-mediated cytotoxic activity is regulated by cytokines of IFN-γ/TNF-α and perforin. However, only IFN-γ regulates the NK cell cytotoxic activity enhanced by AGAF. Also, the intracellular signaling through MAPK activates phosphorylation of transcription factors during AGAFelicited IFN-γ gene expression. For example, AGAF induces the NK-92MI cells to express the IFN-γ via the NF-κB. AGAF-elicited IFN-γ expression is correlated with the increased cytotoxic activity, which is associated with JAK2/STAT3 signaling of the NK cells. Therefore, for the mechanistic explanation of the AGAF-activated NK cell function, multiple pathway for the IFN-γ expression and autocrine cytotoxic activity is suggested because the cytokine IFN-γ is an acting factor for the NK cell cytotoxic activity. The polysaccharides have also high affinity for TLR2 and TLR4. Polysaccharides activate macrophages via the TLR2 and TLR4 signalings. Silencing of the TLR genes using siRNA knockdown technology demonstrates that both AGAG upregulate the TLR2 and TLR4 signalings to enhance IFN-γ release and NK cell cytotoxic activity. When the PRRs present in the cell surfaces are stimulated, the MAPK subfamilies such as the ERK, p38, and JNK are activated by innate immune cells. AGAF activates the phosphorylation of IKK and Iκα/β with NF-κB translocation into the nucleus. Moreover, IFN-γ triggers AGAF-elicited NK cell cytotoxic activity via the JAK2/STAT3 pathway. In the downstream pathway of

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IFN-γ during the NK cell cytotoxic activity, another pathway of JAK2/STAT3 is closely involved in the signaling cascade.

5.6.4

Example 4: Streptococcus pneumonia Polysaccharides Activate NK Cells, NK-Like T Cells, and Monocytes

Streptococcus pneumonia glycans activate leukocytes, T lymphocytes, and NK cells, as CWPS (cell wall polysaccharide) and three capsular polysaccharides (types 3, 9, 23) [255]. After activation with CWPS treatments, the CD69 values were observed for monocytes, NK cells, CD4- (negative), CD4+, and CD56+ T cells. CWPS is a stronger stimulator than the capsule. Among the capsules, type 23 is the strongest stimulator followed by type 9 and type 3. With immune cell activation, cytokine secretion is also increased by activated innate immune cell. CWPS stimulation was observed for IL-8 production and followed by TNF-α, IFN-γ, and IL-10. CWPS is also known as teichoic acid, structurally similar (especially in S. pneumoniae) to lipoteichoic acid and a well-known ligand for TLR2. Also, TLRs as innate immune components bind to the conserved molecular patterns or PAMPS on various pathogens. So, TLR was speculated as the main component of downstream mechanism. Capsular polysaccharides are T-cell-independent B-cell antigens, and their capacity to induce protective antibody responses is the main reason they are used for vaccination. It is less clear how the capsular polysaccharides may stimulate the immune cells investigated in this study, since they were not ligands for TLRs. It is known that even highly purified pneumococcal capsular polysaccharides contain some amount of CWPS. Thus, it seems that CWPS is an active component to activate innate immune cells. Activation of cells could only be stimulated by direct TLR contact. NK cells, NK-like T cells, and monocytes express TLRs. However, ordinary (CD56-) CD4- (negative) or CD4+ T cells do not produce TLRs. An indirect activation such as cytokines from directly activated cells may explain the stimulation of T cells which is not expressing TLRs. During vaccination or a live S. pneumoniae infection, TLR-expressing immune cells like macrophages and DCs are present in the peripheral tissues but not in peripheral blood, and they are likely to be activated. In many situations, a combined activation with direct and indirect (via cell-cell contact or secreted mediators) mediators of immune cells occurs.

5.6.5

Example 5: C. macrophyllum Polysaccharides (CMP) Enhance Lymphocyte Proliferation and Macrophage Function

In Cyrtomium macrophyllum, T lymphocyte differentiation is stimulated by polysaccharide. In details, the percentages of CD4+ T/CD8+ CTLs and the CD4+/CD8+

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243

CTL ratio were remarkably increased in the groups treated with CMP [256]. With the increasing of phagocytic activity in macrophage, the production of cytokine was also increased. In detail, the production of IL-2 and IL-6 was significantly higher than control group. Similarly, the concentration of IL-2 was also significantly higher than control group. Surprisingly, not only cytokines but also immunoglobulin levels are also increased in CMP-treated groups in experiment. In detail, mice treated with CMP-L, CMP-M, and CMP-H groups were observed with significant enhancements in IgM and IgG levels. Moreover, the NO production in RAW 264.7 macrophages was increased by CMP. Nitrite secretion was significantly increased by CMP treatment. In addition, CMP-treated-RAW 264.7 cells significantly secreted more TNF-α. Thus, CMP enhances the NO production and cytokine secretion as well as the iNOS protein expression from RAW 264.7 cells with NF-κB (in nuclear) protein expression. iNOS and NF-κB proteins produced by RAW 264.7 cell were significantly upregulated by LPS. In other words, CMP and LPS are thought to stimulate production of cytokines and immunoglobulin via NO which is a critical component of signaling pathway. In immune-enhancing activity of CMP in mice as well as RAW264.7 cells, CY can act as a chemotherapeutic drug for cancers, but long-term administration can cause immune suppression. The effects of immune suppression on the development of immune organs such as the spleen and thymus could be resisted by CMP. Another result, recovery of splenocyte-proliferative responses to both T and B lymphocytes was promoted with CMP in mice. Aside from enhancing the proliferation of immune cells, the IL-2 and IL-6 secretion by Th1/Th2 cells was also stimulated by CMP. IL-2 mediates cellular immunity by promoting proliferation and differentiation of T cell. IL-6 mediates humoral immunity by B-cell growth and Ig production. Especially, IgG subtype and IgM subtype are the major Igs, which activates the complement system, antigen opsonization, and toxin neutralization of toxins as major responses in innate immune system. The levels of IgG and IgM in CY-treated mice were increased by CMP. Humoral immunity as well as innate immunity could be enhanced by CMP. Moreover, production of NO which contributes to killing of infected cells, tumor cells, and some pathogens and production of cytokines were induced by activated macrophage.

5.7

TLR4 Receptor-Activating Glycans Activate NO Production in Macrophage

In Paecilomyces cicadae, PCP (Paecilomyces cicadae polysaccharide) can activate macrophage. Upon exposure to PCP, the NO production was increased by macrophages. Gene expression of iNOS was increased by PCP. The gene expression of IL-16, TNF-α, and IL-1β, which are inflammatory cytokines, was also increased by PCP in a similar pattern with iNOS. So how PCP can be recognized by macrophages despite large molecule size? Because PCP cannot penetrate cells due to its large molecular size, PCP-activated macrophage is followed by PCP binding to specific

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membrane receptors. Among the specific membrane receptors, TLR4 activates macrophages. For the TLR4 downstream signaling pathway, TLR4 signaling is associated with the MyD88- and TRIF, but they commonly activate MAPKs and NF-κB signaling. Both MAPKs and NF-κB signaling play major roles in macrophage activation in a TLR4-dependent mechanism. The phosphorylation of ERK, JNK, and p38 which are associated with MAPK signaling was induced by PCP. PCP can help the nuclear translocation of NF-κBp50/p65 by degradation IκB α/β block protein, as it is demonstrated by the increased levels of nuclear p50/p65 protein. PCP binds to TLR4, followed by the activation of p38 and NF-κB.

5.8

CBPs or GBPs in Antigen Recognition

GBPs expressed on the immune cells regulate immune responses of both innate and adaptive immunities. The families of CLRs, galectins, TLRs, and Siglecs are well known for the GBPs. GBPs contain one or more CRD. CLRs as a group of PRRs influence TLR signaling. CLRs are Ca2+ dependent for their carbohydrate bindings, capturing the carbohydrate ligand and a Ca2+ ion. Macrophages, DCs, and other APCs of innate immunity express the CLTs on their surfaces. The CLRs play a role to deliver immune tolerogenic signals upon antigen recognition, but the mechanisms are not largely known yet. As Ca2+-dependent glycan-binding proteins, they contain two distinct motifs for glycan recognition and Ca2+ ion engagement. The human DC-SIGN (CD209) is present in DCs and macrophages [257, 258]. CLRs contain a Glu-Pro-Asn (EPN) sequence as carbohydrate recognition domains (CRDs), for Man- or Fuc-carrying glycans such as Lea,b,x,y carbohydrate antigens [259]. The CLRs also include the DC-SIGN, mannose receptor (MR), and langerin. Among CLRs, there is another type of Gal-specific CLRs, and these include macrophage Gal lectin (MGL). L-SIGN/DCSIGNR is included with terminal Man- and/or Fuc glycan-binding specificity [260]. However, Gal-binding C-type lectins include MGL and DCASGPR [261]. They have the three amino acid sequence of Gln-ProAsp (QPD) present in the CRD region, with terminal Gal residue or GalNAc residuecontaining glycan-binding specificity. Certain CLRs such as Dectin-1 do not require Ca2+ for glycan recognition. Dectin-1 binds to β-glucan sugars present in yeasts. For the type II subfamily of CLRs, 17 human subfamily is present in APCs of macrophages and DCs and also other endothelial or NK cells [262]. Most C-type lectin family contains cytoplasmic endocytosis motifs for internalization in the tail region and uptake glycosylated antigens [263]. CLRs also function in antigen presentation to the MHC-II for CD4+ T-cell activation specific for antigens [264]. Certain DC C-type lectins intracellularly shuttle foreign antigens to the MHC-I receptor to trigger antigen-specific responses of CD8+ T cells. CLRs consist of both inhibition and activation motifs in the cytoplasmic tail [260, 265]. CLRs regulate processing and presentation of antigens for various immune reactions by T cells. Apart from humans, mice also express C-type lectins of DC-SIGN homologues such as mDC-SIGN, SIGNR1, and SIGNR3 [266]. The mouse SIGN-R1 (CD209b)

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is the homologue of human DC-SIGN known as CD209 [267, 268]. Among several different homologues, three mouse type DC-SIGN species of the SIGN-R1 (CD209b), SIGN-R3 (CD209d), and DC-SIGN (CD209a) are present in DCs and macrophages of mouse [269, 270]. However, the three mouse DC-SIGN species lack glycan recognition sites. Mouse SIGN-R1 species resembles DC-SIGN species of humans, and mouse SIGN-R1 is present in marginal macrophages of spleens and macrophages of lymphatic node medulla. Mouse SIGN-R1 species captures various microbial organisms, which have polysaccharides like CPSs or dextran of S. pneumoniae [271, 272]. SIGN-R1 involves in clearance of infected pathogens in host. Defected SIGN-R1 lacks macrophage phagocytosis ability against pathogens such as S. pneumoniae. In addition, the SIGN-R1 directly helps CPS opsonization by C3. SIGN-R1 defection blocks complement-mediated CPS capture on splenic follicles, resulting in prevalent pneumococcal infection to mice [273, 274]. Moreover, mouse SIGN-R1 recognition with CPS activates the classical complement pathway which is Ig-independent for the C3 opsonization on CPS. SIGN-R1 also removes the apoptotic cells by C1q-SIGN-R1 interaction via the C3 opsonization on the apoptotic target cells, resulting in reduction of autoimmunity [275]. SIGN-R1 has a specificity to recognize α2,6 sialyl-antibodies, but not α2,3 sialyl-antibodies. SIGN-R1 binds to terminal α2,6 SAs through the carboxylate moiety. α2,6 SA-bearing IgG is thus recognized by SIGN-R1. Thus, SIGN-R1 is anti-inflammatory against intravenous Ig (IVIG) engagement. SIGN-R1 expressed in macrophages resident in splenic marginal zones binds to the sialyl-Fc. Defected for of SIGN-R1 species loss its anti-inflammatory response of intravenous sialyl-Fc and IVIG [276]. The SIGN-R1 binding to sialylated Fcs elicits the anti-inflammatory response by a Th2 cytokine pathway [277], because the IVIG has anti-inflammatory activity through the IgG Fc-α2,6-SA [278]. The SIGN-R1 CRD binds to dextran sulfate (DexS) and NeuAc [279]. The sialylated Fc-SIGN-R1 binding is based on the recognition of α2,6-SA, eliciting anti-inflammatory response of IVIG. The sialylated C1q and Ig bind to SIGN-R1 in a Ca2+-dependent CRD manner. The SIGN-R1 captures C1r-bound C1q and C1s-bound C1q by a SIGN-R1 CRD and the α2,6 sialyl C1q, while SIGN-R1 recognizes the pathogen patterns through another carbohydrate-binding site, as the SIGN-R1 in a Ca2+-independent way [279]. Mouse SIGN-R3 binds only distinct ligands [280].

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264. Birkholz K, Schwenkert M, Kellner C, et al. Targeting of DEC-205 on human dendritic cells results in efficient MHC class II-restricted antigen presentation. Blood. 2000;116:2277–85. 265. Osorio F, Reis e Sousa C. Myeloid C-type lectin receptors in pathogen recognition and host defense. Immunity. 2011;34:651–64. 266. Park CG, Takahara K, Umemoto E, et al. Five mouse homologues of the human dendritic cell C type lectin. DC-SIGN Int Immunol. 2001;13:1283–90. 267. Park CG, Takahara K, Umemoto E, Yashima Y, Matsubara K, Matsuda Y, Clausen BE, Inaba K, Steinman RM. Five mouse homologues of the human dendritic cell C-type lectin. DC-SIGN Int Immunol. 2001;13:1283–90. 268. Powlesland AS, Ward EM, Sadhu SK, Guo Y, Taylor ME, Drickamer K. Widely divergent biochemical properties of the complete set of mouse DC-SIGN-related proteins. J Biol Chem. 2006;281:20440–9. 269. Cheong C, Matos I, Choi JH, Dandamudi DB, Shrestha E, Longhi MP, Jeffrey KL, Anthony RM, Kluger C, Nchinda G. Microbial stimulation fully differentiates monocytes to DC-SIGN/ CD209(+) dendritic cells for immune T cell areas. Cell. 2010;143:416–29. 270. Nagaoka K, Takahara K, Minamino K, Takeda T, Yoshida Y, Inaba K. Expression of C-type lectin, SIGNR3, on subsets of dendritic cells, macrophages, and monocytes. J Leukoc Biol. 2010;88:913–24. 271. Kang YS, Yamazaki S, Iyoda T, Pack M, Bruening SA, Kim JY, Takahara K, Inaba K, Steinman RM, Park CG. SIGN-R1, a novel C-type lectin expressed by marginal zone macrophages in spleen, mediates uptake of the polysaccharide dextran. Int Immunol. 2003;15:177–86. 272. Kang YS, Kim JY, Bruening SA, Pack M, Charalambous A, Pritsker A, Moran TM, Loeffler JM, Steinman RM, Park CG. The C-type lectin SIGN-R1 mediates uptake of the capsular polysaccharide of Streptococcus pneumoniae in the marginal zone of mouse spleen. Proc Natl Acad Sci U S A. 2004;101:215–20. 273. Kang YS, Do Y, Lee HK, Park SH, Cheong C, Lynch RM, Loeffler JM, Steinman RM, Park CG. A dominant complement fixation pathway for pneumococcal polysaccharides initiated by SIGN-R1 interacting with C1q. Cell. 2006;125:47–58. 274. Lanoue A, Clatworthy MR, Smith P, Green S, Townsend MJ, Jolin HE, Smith KGC, Fallon PG, McKenzie ANJ. SIGN-R1 contributes to protection against lethal pneumococcal infection in mice. J Exp Med. 2004;200:1383–93. 275. Prabagar MG, Do Y, Ryu S, Park JY, Choi HJ, Choi WS, Yun TJ, Moon J, Choi IS, Ko K. SIGN-R1, a C-type lectin, enhances apoptotic cell clearance through the complement deposition pathway by interacting with C1q in the spleen. Cell Death Differ. 2013;20:535–45. 276. Anthony RM, Wermeling F, Karlsson MCI, Ravetch JV. Identification of a receptor required for the anti-inflammatory activity of IVIG. Proc Natl Acad Sci U S A. 2008;105:19571–8. 277. Anthony RM, Kobayashi T, Wermeling F, Ravetch JV. Intravenous gammaglobulin suppresses inflammation through a novel T(H)2 pathway. Nature. 2011;475:110–3. 278. Kaneko Y, Nimmerjahn F, Ravetch JV. Anti-inflammatory activity of immunoglobulin G resulting from fc sialylation. Science. 2006;313:670–3. 279. Silva-Martín N, Bartual SG, Ramírez-Aportela E, Chacón P, Park CG, Hermoso JA. Structural basis for selective recognition of endogenous and microbial polysaccharides by macrophage receptor SIGN-R1. Structure. 2014;22(11):1595–606. 280. Caminschi I, Corbett AJ, Zahra C, Lahoud M, Lucas KM, Sofi M, Vremec D, Gramberg T, Pöhlmann S, Curtis J. Functional comparison of mouse CIRE/mouse DC-SIGN and human DC-SIGN. Int Immunol. 2006;18:741–53.

Chapter 6

Innate Immunity Via Glycan-Binding Lectin Receptors

Recognition of glycans and transfer of information contained in the glycan structures are performed by carbohydrate-recognizing proteins of lectins or GAG-recognizing proteins. Lectins bind to N-glycans, O-glycans, and GSLs, while GAG-binding proteins easily bind sulfated GAGs. Lectin receptors are innate immune receptors and include Siglec, C-type lectin, galectin, DC-SIGN, and TLRs in DCs during pathogenic infection and immune tolerogenic homeostasis. The CTL roles are to directly recognize invaders of microbes and also contribute to opsonic effect via activation of complement pathways. Innate immune cells survey their habitat to recognize pathogens by means of PRRs, where PRRs selectively bind PAMPs. PAMPs are heterogeneous and homogeneous mannose oligomers and polymers, β-glucans, and chitins in the fungi surface as well as carbohydrate moieties including GlcNAc derivatives in bacteria. Innate immunity system represents our first host defense line where the innate pattern recognition receptors/molecules (PRRs/PRMs) encounter, recognize, and bind conserved motifs of microbial invaders or PAMPs. Therefore, the PRRs/PRMs function as the initiator of innate immunity to microbial invaders. Upon interaction with their invading pattern molecules as ligands, PRRs/ PRMs enter into signal transduction pathways and activate diverse downstream kinases and transcription factors to lead to inflammatory response and immune responses, depending on defense circumstances. For example, LPS, CpG DNA, dsRNA, ssRNA, rRNA, or pathogenic surface glycans bind TLR4, TLR9, TLR3, TLR7/TLR8, or TLR13 to elicit expression of cytokines of type I IFN/TNF-α/IL-6, which are inflammatory. Lectin is defined as proteins that recognize and bind glycan carbohydrates, and the lectin-carbohydrate ligand binding potentiates various biological processes in internalization and intercellular signaling. Lectins as carbohydrate-binding proteins are specific for their binding ligands of sugar moieties and thus specifically recognize and bind with cellular and molecular signaling properties. In intracellular roles, they act as ER-Golgi key regulators of protein glycosylation and maturation. Protein glycosylation-based interaction of calreticulin and calectin are ER-specific. Among many lectin families, Siglec, C-type lectin DC-SIGN, galectin, and TLR are © The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2022 C.-H. Kim, Glycobiology of Innate Immunology, https://doi.org/10.1007/978-981-16-9081-5_6

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representatively studied in intercellular signaling of phagocytosis and inflammatory process of innate immune cells of myelocytic cells. To date, lectin receptors expressed on DCs include Siglecs, dectin-1/dectin-2, MICL, Mincle/MCL, mannose-binding protein (MBP), macrophage mannose receptor, and collectin.

6.1 6.1.1

Glycosylation Effect on Autoimmunity and Inflammation Glycosylation in Immunological Recognition and Inflammation

Glycans are involved in multiple biological events and regulate the immune responses. The diversity of glycans and lectins is also regulated by the immune cells, as well described for DC functions. The changes of DCs’ glycan phenotype are observed during differentiation and maturation. The cell surface glycosylation and lectin-glycan interaction regulate the immune tolerance and homeostasis. Lectinglycan interactions also contribute to immune regulation in mammalian system and activate tolerogenic system towards autoimmunity or antitumor immunity depending on the downstream signaling. In the mammal system, immune cells are kept for their authenticities through the whole organism. Hence, self-reactive T cells or autoreactive immune T cells are strictly controlled to be eliminated via apoptotic events in the thymus. In autoimmune diseases, such thymus works are incomplete. To compensate the undesired autoreactiveness, peripheral regulatory system is operative, and the system dampens the undesired and harmful reactions. Globally, such system is suggested to be homeostasis. Overreaction or undesired responses in immunity should be regulated by immunosuppressive cytokines or inhibitory receptors, totally operative in tolerance system. The tolerance system includes T cell anergy, deletion, and Treg cell expansion. These behaviors eventually prevent tissue damages. In cellular level, regulatory system includes trafficking, clustering, and signaling receptor internalization to control the immune cells [1]. Eukaryotic cells, protein receptors, and immune mediators including MHC-I and MHC-II, TCR and BCR, chemokine and cytokine receptors, and antibodies are normally glycosylated by covalent linkages. This is because glycans are involved in major biological events in almost every biological process. They are directly linked with a number of inflammatory conditions [2], autoimmune diseases, and hematological cancers [3, 4], as glycans seem to be associated with almost every human disease [5]. The discrimination of “self” from “non-self” is the essential driving force and the most important aspect to defend the hosts in organisms. From the glycome diversity and the glycan-biosynthetic immunological, glycosylation is directly associated with immune cell networks. Glycan-related genes are phylogenetically conserved to consist of the glycosylation system. However, variations at the intra- and interspecies levels are observed among synthesized glycans from each organism. This

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reflects that the glycans can act as danger-associated molecular patterns (DAMPs) and PAMPs, as a key parameter of non-self- and self-discrimination in immunity. The lectin-glycan recognition events contribute to pathogen-based immune cell trafficking [6]. The lectin recognition of its ligand is indeed a multidirectional action in detection of the structure, number, and glycan density in multivalent carbohydrates expressed in the cell surfaces. Resultant power is expressed as the type of lectin-glycan interactions [7]. The molecular interactions form multi-dimensional adjustments of glycans and lectins, called “lattices.” Lectins recognize glycans with specific affinity in the very low molar levels. In immune system, lectin-interacting surface glycoproteins include the TCR, BCR, and specific cytokine receptors. GBPs or lectins can enhance or disrupt immune tolerance in DCs, T cells, or B cells, because the roles of glycosylation in pathogen recognition and lectin-glycan interactions are reality in regulation of immune tolerance, autoimmunity, and inflammation. In addition, glycosylation regulates inflammation and autoimmunity by immune homeostasis. Cell surface glycans are changed from normal to inflammation and transformation status [8]. In the inflammation status, the environmental inflammatory changes influence migration and trafficking of innate immune cells to disease sites. The well-defined example is selectins as the recognition lectin receptors for the immune cell migration, because they bind to sialyl- and fucosyl-glycans of sLex and sulphated Le antigens on leukocytes and endothelial cells [9]. Thus, to bloc inflammatory response in anti-inflammatory therapy, interaction of selectins and Lewis glycans on leukocytes and endothelia can be inhibited.

6.1.2

Glycosylation Effect on Autoimmunity

On the other hand, in autoimmunity status, T cells express altered surfaced glycans such as terminally GalNAcylated or Galβ1,4GlcNAcylated structures, exhibiting characteristic of desialylated residues that lack terminal sialic acid residues. For example, terminally desialylated glycans are frequently observed in systemic lupus erythematosus (SLE) and RA [10]. Such altered glycosylation seems to influence the T cell immunological action in terms of TCR synapse behavior, and T cells regulate the adaptive immunity and autoimmune responses. The desialylated glycans are easily targeted by several lectins such as MGL and galectin. The MGL and galectins specifically recognize such glycan structures of terminal GalNAc and Galβ1,4GlcNAc structures without sialic acids, and consequently, TCR downstream signaling is suppressed, and CD45 phosphatase activity is changed [11–13]. Specifically, the GnT-5, mannose β1,6GlcNAcTransferase-5 (or Mgat5), yields the β1,6GlcNAc branches structure on glycoproteins such as TCR. GnT-5 (or Mgat5) adds the β1,6-GlcNAc residue to mannose residue of N-glycan core structures in the Golgi apparatus. β1,6-GlcNAcylation in N-glycan by GnT-5 or Mgat5 yields the galectin-binding ligands on surface glycoproteins. Galectins use the N-acetyllactosamine (LacNAc) repeats as ligand substrates. The galectin-glycan lattice alters surface glycan concentration and consequently affects cell proliferation

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and differentiation. For example, GnT-5-KO T cells exhibit the reduced T cell activation level, and GnT-5-KO mice show the increased delayed-type hypersensitivity and susceptible autoimmune responses. Mgat5-deficient experimental model animals such as mice exhibit severely type 4, delayed-type hypersensitivity and autoimmunity such as autoimmune encephalomyelitis (EAE). The mice exhibit EAE, glomerulonephritis, and immune complex deposition [12]. GlcNAc treatment increased in GnT-5-mediated N-glycan β1,6GlcNAc branches and blocked activation of TCR in autoimmune EAE and T2DM models [14]. For more evidenced results, genetically controlled, EAE-susceptible mice exhibit reduced N-glycan branching in T cells when compared with EAE-resistant mice such as BALB/c mouse [15]. Glycosylation also influences both the adaptive immunity and the autoimmune diseases via innate immune to autoimmune and inflammatory development. Silencing ER-resident α-mannosidase-II (αM-II) impairs N-glycan branching and induces an autoimmune disease in αM-II KO mice like human SLE [16], independent of the adaptive immunity, but dependent of innate immune activation. Endogenous lectins such as mannose receptor (MR) may recognize the mannose-deficient N-glycans and induce innate immune responses even in the condition without infection, developing lupus-like autoimmune disease [16]. Aside from N-glycans in the onset of autoimmune response, O-glycan changes also lead to such similar autoimmune responses. For example, IgA nephropathy is well known to related with the exposed GlcNAc residue linked to O-glycans in IgA because IgA complexes are often deposited in the inflamed kidney glomerular nephritis [17]. Human monomeric IgA1 bears two distinct sites of N-glycosylation in the CH2 domain and C-terminal tail piece of the α-heavy chain. IgA1 contains an extended hinge region with the nine different sites of O-glycosylation in the constant domains. IgA1 produced by the healthy individuals has six sites of the nine O-glycan sites which have mono- or di-sialylated core 1 O-glycan structures. Specific types of Gal-deficient O-glycans are also detected in disease status [18]. The galactosedeficient O-glycans are caused by dysfunctional C1GalT1 or its chaperon Cosmc and aberrantly expressed N-acetylgalactosaminide α2,6-sialyltransferase I/II (ST6GalNAc-I/II) enzyme specific for sialyl Tn synthesis [19]. Considering IgA and IgG glycans, the presence of α2,6 sialic acid in the IgG Fc is a key antiinflammatory decision factor in autoimmune diseases [20]. Totally, changes in Nand O-glycan-branched glycans can result in abnormal innate or adaptive immune responses. In mice, loss of T antigen causes thrombocytopenia, and the human Tn syndrome is displayed in the thrombocytopenia and leukocytopenia, caused by the COSMC gene mutation for β1,3GalT (T-synthase) or core 1 β1,3-GalT (C1β3GalT), for O-glycan biosynthesis [21]. The C1β3GalT enzyme is an evolutionarily conserved enzyme that transfers Gal to a mucin-type O-glycan GalNAcα1-O-Ser/Thr glycan (Tn antigen), to form a Galβ1,3GalNAcα1-O-Ser/Thr (T antigen). The functions of T antigen were known because deficiency of C1β3GalT1 displays many defects in developmental diseases. Representatively in mice, lacking T antigen displays thrombocytopenia [22] and vascular vessel dysfunction [23], while in humans, Tn

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syndrome is caused for thrombocytopenia and hemolytic anemia [24], with dysfunctional Cosmc, a chaperone protein for human C1β3GalT. Tn syndrome also exhibits malfunction in the hematopoietic stem cells, although the roles of T antigen are not known for hematopoiesis. However, T antigen has been suggested to be conserved in blood cell lineages among species. The surface glycan repertoires of immune cell types are also related to immune diseases. T cell glycosylation regulates T cell functions like activation and differentiation through cis- or trans-masking or demasking of the ligands for endogenous lectins. For example, α2,6 SA content is increased on the Th-2 cell surface. However the α2,6 SA levels are not increased in Th-1 or Th-17 cells. The difference in the α2,6 SA contents indicates the T cells’ susceptibility to the GBP galectin-1 that endogenously recognizes the altered glycosylation of cell surface glycoproteins and induces cell death of activated lymphocytes [25]. Therefore, galectin-1 silencing KO mice (Lgals1
/
) express autoimmune status of autoimmune EAE, caused by expansion of antigen-specific Th-1/Th-17 cells and DCs immune response [23, 25]. Thus, galectin-1 therapeutic treatment can restore immune tolerance and consequently inhibit chronic inflammation in autoimmune diseases of diabetes, EAE, hepatitis, IBD, RA, and uveitis. Also, prevention of fetal loss and graft vs. host disease (GvHd) can be obtained through Th1 and Th17 suppression by Treg cell expansion and tolerogenic DC supplementation. Similar to autoimmune diseases, in HIV-infected T cells, T cell surface glycosylation is also altered and increases susceptibility to apoptotic death of T cells, which is induced by galectin-1. Hence, galectin-1 influences the pathogenic features of AIDS. O-glycan modifications of peripheral lymphocytes from AIDS patients are such altered glycosylation types. HIV-1-infected T cells and AIDS patients-derived peripheral CD4 T cells and CD8 T cells exhibited exposed lactosamine residues and the lactosamine residues triggered to enhanced susceptibility of the cytotoxic death of T cells by galectin-1 [24]. Altered surface glycosylation level of T cells caused by infection of HIV-1 contributes to the increased T cell death via galectin-1-mediated signaling. In the galectin-1-interacting proteins, galectin-1 has been known to cause the immature thymocyte death and activated peripheral T cell death by directing recognition with CD7, CD45, and CD43 glycans on T cells. The CD7 and CD45 functional roles are not known when galectin-1 promotes apoptotic T cell death. During galectin-1mediated cell death of T cells, galectin-1 interacts with CD43 glycans, and thus CD43 is suggested to be the subject for the galectin-mediated T cell death. Heavily O-glycosylated CD43 functions as the galectin-1 recognition sites of T cells. Core 1 O-glycosylation or core 2 O-glycosylation structures in CD43 glycoprotein regulate galectin-1-mediated T cell susceptibility, indicating that T cell glycans are the galectin-1-binding targets [26]. The tandem-repeat galectin-9 can also confer such suppression of progressed autoimmune responses via galectin-9 binding to the Tim-3 glycoreceptor [27]. Galectin-9 is a soluble lectin and forms lattices between galectin-9 and glycoprotein of the surfaces. Interaction between galectin-9 and its ligands causes death of CD4-positive Th1 cells, but not of CD4-positive Th-2 cells, simply due to different glycan structures [28]. Galectin-9 is specifically expressed in T cells,

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eosinophils, DCs, endothelial cells, and macrophages. It causes the cell deaths of T cells and thymocytes with specificity to CD4-positive Th-1 cells but spares CD4-positive Th-2. The resistance of CD4-positive Th-2 to galectin-9 is based on the α2,6-SA linkages present in the surfaces of Th2 cells. The cell surface α2,6-sialic acids block galectin-9 recognition to glycan ligands crucial for cell death. C-type lectins can also modulate inflammation and autoimmune diseases. Blocking of mannosyl encephalitogenic peptide inhibits the EAE development, because the immature DCs MR loss its Man ligand. Thus, mannose-supplemented administration can induce oral tolerance and generate IL-10-producing Treg cells through the CLR SIGNR1 on DCs [29]. Also, Siglec-2 (CD22) inhibits B cell signals, as confirmed by the results that Siglec-G/CD22-deficient double KO mouse exhibits the B cell-dependent autoimmune disease spontaneously developed. The double KO mouse generates anti-DNA and anti-nuclear autoantibodies [30].

6.2

Glycosylation Effect on Tumor Immunity of Immune Cells

The tumor-associated microenvironment (TAM) is a symphonic orchestrate of cellular networks of stromal cells, tumor cells, and infiltrating immune cells. In the host, the immune system generally modulates development of cancer cells to a certain extent in host system, as settled down for the immunosurveillance theory [30]. Thus, the host immune system detects and eradicates transformed tumors. However, the immunosurveillance in the current tumor biology does not sufficiently eliminate such transformed tumor cells. Then to explain the tumor survivals from the immune system, tumor immunoediting theory has been newly conceptional [31], where tumor cells acquire the evasion potentials from immune responses through lacking immune recognition as well as increasing immune regression and immunosuppressive microenvironment. In tumor patients, tumor-specific immune status is believed to be a parameter for prediction of prognosis and therapeutic monitoring. The theoretical immunoscore is one of such predictable unit that is calculated by the infiltration of immune cells towards immunotherapeutic consideration. For solid tumors, the immunoscore levels of tumor-infiltrating lymphocytes are associated with the tumor survival rate. High proportional rates of memory T cells or antitumor CD8+ CTLs are indicative of the reduced invasion of solid tumor cells [32]. In parallel, the immunoscore of peripheral-blood lymphocytes is also indicative of the reduced tumor developments [33]. Although CTLs/NK cells induce the cytotoxic killing of tumor cells, in the hypoxic tumor microenvironment, NK cell receptors are not expressed, indicating no cytotoxic effect of NK cells [34]. Therefore, DCs in collaboration with naive CD4+ T cells and CD8+ CTLs predominantly contribute to the adaptive immunity. Certain patterns of glycosylation present in the cell surfaces of transformed cells influence host immune modulation and survival potentials of malignant tumors.

6.2 Glycosylation Effect on Tumor Immunity of Immune Cells

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Tumor cells proliferate through the immune evasion, which is accompanied by immune checkpoint inhibitors like PD-1 of hosts. Transformed cells exhibit the surface glycosylation changes in the plasma membrane-associated glycoproteins. Currently, representatively certain tumor-associated antigens (TAAs) such as CEA and MUC1 are mostly well studied for the altered glycosylation to date [35], although they are present in normal colonic mucosa and epithelial cells. However, the precise roles of tumor glycosylation in immune evasion has not well been defined. However, the aspect describing that aberrant tumor glycosylation potentiates the immune escape from immune surveillance under the host immune system provides valuable insights into consideration. It is noted that consequent immunosuppressive events are obtained via carbohydrate-recognizing receptors. Some carbohydrate patterns expressed on tumor cells is also another type of immune checkpoint, because glycosylation of tumor cells is used as T cell targets to tumorspecific recognition. Then, a recent study describes how the tumor-yielded “glycan code” alters the host immune system, and if it goes through, such targeting glycans are the future avenue for the therapeutic clues [36]. For tumor detection in microenvironment, APCs express diverse glycan-binding receptors like CLRs and Siglecs. CLRs can internalize the bound ligands to APCs and associate to process and present the digested antigens to T cells. The CLRs also modify DC functions and macrophage functions. The macrophage Gal-binding lectin known as MGL/CD301 of human CLR binds to terminal GalNAc residues linked to Tn and STn antigens of the aberrantly expressed O-glycans in tumor cells [37]. MGL is specifically present in tolerogenic or immature DCs or macrophages of myeloid innate immune cells. The MGL expressed in human is a valuable biomarker for TAM presence [38]. An immunomodulatory activity of MGL is also clinically dedicated to the high MGL association in cancer patients with a poor tumor-free survival [39]. For example, Lewis blood group of Lex and Ley expressions indicate poor prognosis of the tumor cells. CLRs of DC-SIGN and MGL on DCs recognize the Lewis glycan structures attached to CEA or MUC1, but not recognize normal type of CEA species or MUC1 produced by colon tissues [39]. CLR-glycan binding can trigger antitumor responses and trigger antigen-targeting autoimmune responses. Another CLR, CLEC9A predominantly expressed on human and mice DCs phagocytically digest dead cells for MHC-I-based presentation of the antigen epitopes and activation of CD8+ CTLs. However, currently, the CLEC9A-binding ligands are not clear yet. The CLEC9A upon antibody-antigen conjugate treatment induces antigen endocytosis to activate CD4+ T cell and CD8+ CTL proliferation. Furthermore, CLEC9A in cancer cells raises T cell-driven rejection of tumor cells [40]. For another case, high Man-modified antigen GP100 of melanoma cells stimulates both T cell types of GP100-specific CD4+ T cells and CD8+ CTLs, since high Man-type structures recognize DC-SIGN, potentiating endosomaldependent antigen presentation [41]. Two other Man receptor-specific ligands, such as sulfated LeA or GlcNAc, enhance the MR activity upon sulfate LeA or GlcNAc ligation with protein antigens, increasing antigen presentation to T cells [42]. Tumor cell exploits lectin-carbohydrate recognition to evade the host immune responses [43]. Tumor-produced galectin-1 can inhibit host immune responses, as

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evidenced by many tumor cells including lung carcinoma cell, Hodgkin’s lymphoma cells, melanoma cells, neuroblastoma cells, and pancreatic carcinoma cells. Galectin-1 modulates T cell and DCs [44–46] through the Th2-dominant cytokines and tolerogenic activation by IL-10-expressing type 1 Treg cells and IL-27expressing DCs [47]. Galectin-9 also increases the number of myeloid suppressor cells with CD11b + Ly-6G+ granulocytic phenotype in order to inhibit antitumor activity [48], while the galectin-3 controls the anergic T cells status [49]. Thus, selectively expressed galectin family is responsible for blocking immunosuppression at tumor growth sites through targeting immunoevasion. Glycosylation-dependent tumor immune escape has also been observed in bladder tumor cells. The bladder tumor cells aberrantly express the core 2 β1,6GlcNAc-transferase (GCNT1) which is encoded by C2GnT gene. The GCNT1 catalyzes a GlcNAc branching on GalNAc in core 2 O-glycans. The tumor cells have highly metastatic potential form because they evade the host NK cell immunity. Galectin-3 binding to poly-LacNAc units attached to mucin-type core 2 O-glycosylations of cancer-involved MHC-I-related chain A (MICA) decreases the NK receptor NKG2D-activating MICA affinity and consequently impairs the NK cell function of anti-cancer activity [50]. Moreover, the tumor cell-produced disialyl GD3 also controls NK cell cytotoxic activity through Siglec-7 signaling [51]. Mucins produced from tumor patients modulate the DC immunogenicity via Siglec-9 in human [52]. The lectin repertoires and the cellular glycosylation patterns in the TAM influence immune cell fate and tumor survivals. The importance of carbohydrates opens new vista on the mechanism of how autoimmunity, development, cancer, lymphocyte differentiation, and host-microbe interaction undergo. The glycan-focused immunology, glycoimmunology, will be interested in the field that carbohydrates influence immune responses because carbohydrates are integral to immune pathways.

6.3

Immune Tolerance and Defense Mechanisms of Innate Immune DCs During Infection

DCs bridge the innate immune response to the adaptive immunity, and this indicates its key regulators of the host immune system. In addition, they determine whether direct immunity or tolerance is generated in the body [53], since its discovery in 1868 by Paul Langerhans [54] at Berlin. Historically, in 1973, DCs were found in the spleens of mice [55]. After another 12 years, Langerhans cells (LCs) were defined as one group of the DCs, as in 1985, Gerold Schuler and Ralph Steinman reported the seminar title of “Epidermal Langerhans cells in murine mature into potent immunostimulatory DCs in vitro” [56]. Thus, a series of discoveries has been recognized as a landmark in milstone [57]. In 1985, DCs meets a new era to shape the LC and other Langerin + DC [58], establishing the “LC paradigm” theory [59]. Immune homeostasis is operated and kept by the coordination of innate and adaptive immune cells and epithelial cells. As the major innate immune cell and

6.3 Immune Tolerance and Defense Mechanisms of Innate Immune DCs During Infection 269

APC, DCs uptake, digest, process, and present foreign antigenic molecules to naive T cells to start specific immune responses against pathogens. In the side of virus, however, DCs are also used as target cells for certain viral infections. This indicates that infected virus-derived immune escape behavior hampers the T cell-activating capacity of DCs. Most of DC subsets have tolerogenic roles. Tolerogenicity of DCs is a fundamental phenotype and induces T cell anergy and Treg and T cell deletion [59]. Tolerogenic DCs have been described ex vivo for the first time, from the finding that UV-irradiated Langerhans cells induce T cell anergy [60]. UV-mediated apoptosis indicates DC induction of tolerance. Immature DCs in peripheral tissues function as immune sensors for pathogens. Pathogens, danger or inflammatory signals, induce maturation and activation DCs, and the matured DCs are now ready to migrate into the draining lymph nodes. DC presentation of pathogenprocessed antigens stimulates T cells by means of costimulation and proinflammatory cytokine production [61]. The most important role of tolerogenic DCs is to control dysregulated T cell responses against harmless or self-antigens. The tolerance capacity in the inflammation status will add new prospects for treating patients with autoimmunity and hypersensitivity. Tolerogenic DCs exhibit an antiinflammatory phenotype through lowered synthesis of co-stimulatory proteins and Treg induction. Most infectious microbes are co-evolved to interact with their hosts, inducing a balance between a pathogen-triggered protective response and immune tolerance to prevent microbial elimination. Non-adapted microbial infections cause the host cell death or are eliminated by the host’s immune reaction. If adapted, the microbes can chronically infect without symptomatic infection by means of the host’s immune tolerance during pathogen-host coevolution. In the inflammation status, effector T cells are functionally dampened by sialyl antigen-accounted DCs. DCs are tolerogenic when they are accounted with soluble sialyl antigens. Sialylated antigen-specific immune tolerance is also induced in the inflammatory status. Sialyl antigen-accounted DCs elicit polarization of naive CD4+ T cells for Tregs. Although DC uptake of sia antigens in vitro is associated with surface marker synthesis, referring to “classic” tolerogenic DCs, DCs are functionally tolerogenic when sialyl antigens are endocytosed. The phenotype of moDCs of human is tolerogenic when the cells are incubated with the highly sialyl pathogens. Sialyl glycan-induced DCs elicit Treg functions and block effector T cell population through binding of receptor to ligand, but not by anti-inflammatory cytokines. Interestingly, the same glycan-pulsed DCs do not affect CD4+ T cells. The immune tolerance adaptation is obtained by innate immune DCs and macrophages because they stimulate immune tolerance and sense antigens. The immune surveillance system of the innate immunity involves in suppressive tolerogenic and proinflammatory responses by extracellular mediators to regulate the suppressive and promotive balances of immune signals. Most food-borne antigens are evolutionarily immune tolerant to commensal microbial antigens. Wnt/β-catenin signaling mediates DC morphogenesis and development [62]. The Wnt signaling in DC function regulates the stromal cells and mucosal cells to activate DCs. For immune tolerance mechanism of DCs, the DCs migrate to lymph nodes to induce the generation of Tregs and cytokines. Then, the Treg cells and the cytokines induce

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the immunologic tolerance of self-antigens or commensal symbionts. In contrast, when inflammatory agents or infectious pathogens are encountered, the immunologic defense system is operated, where DCs and macrophages initiate inflammatory responses to facilitate the activation of adaptive immunity [63]. During invasion and infection, pathogens produce several agonists to bind DC receptors such as TLRs. The agonists induce classical DC maturation, and the matured DCs produce effector T cells and potentiate inflammatory immune responses [64]. For example, DCs, intestinal epithelial cells, and lamina propriaresident leukocytes bind to TLRs as pattern recognition receptors, subsequently allowing innate immune responses. Although the DCs migrated to lymph nodes activate regulatory T cells and cytokine production for immune tolerance of selfantigens or commensal symbionts, if this process is failed, unexpected immune disorder, inflammatory bowel disease (IBD) is the case, as seen in the gut intestine system. This is an exceptional case that an organism’s self-immune cells attack the self-antigens in the intestine. Therefore, classical IBD is classified into the chronic inflammatory disorders with organ-replacing properties, and UC and Crohn’s disease (CD) belong to the disease. Actually, the diseases are severely inflammatory without any drug effectiveness for immunosuppressive agents or steroid-based preventive and therapeutic agents [65]. Thus, ulcerative colitis is clinically cared and treated through surgical removal of the bowel disease-developing organ or region. The diseases are characterized with onset of the familial history as a key risk factor for IBD progression.

6.4 6.4.1

How Are Pathogenic Bacteria Recognized by Receptors of DCs of the Host Immune System? DC Lectins for Glycan Recognition of Invasive Agents

Lectins as glycan recognition proteins display many different biological roles including cell-to-cell recognition and binding. Lectins can be further categorized into many different types depending on their various characteristics. Lectin can recognize various glycoconjugates on cell surfaces and extracellular matrices, ranging from the mediation of adhesion and promotion of cellular recognition pathogens. The different families of animal lectins and lectin domains are known with their three-dimensional structures. Lectins are found from mammals and plants and some lower invertebrates such as nematodes. From genome analysis, each organismspecific lectin profile has been made. Lower organisms have abundantly C-type lectins, and plant-specific lectin domains are not found in animals but found in some lower species such as nematodes. Common lectins between plant and animal origins are legume-like, ricin-B, and class V chitinase-like lectins, and they are also observed in nematodes. Plant lectin-specific sequence is not found in animals. However, they are found in some nematode species. The immunoglobulin

6.4 How Are Pathogenic Bacteria Recognized by Receptors of DCs of the Host. . .

271

(Ig) superfamily is named by Alan F. Williams in 1988 for recognition molecules, which he found then from the immune cells. The Ig region is used to bind molecular structures because of their highly well-rounded fold. Lectin as a carbohydratebinding protein mediates protein-carbohydrate interaction including cellular adhesion or recognition events. Lectin is divided into three classes of C-, P-, and I-types. The Ig-type or I-type lectins belong to the Ig superfamily in recognition with distinct structural diversity of glycan binding. One of them, the Siglecs, are the most wellknown I-type lectins. As the first defense line during pathogen invasion, the innate immune cells effectively respond to inflammation and tissue damage. DCs, macrophages, and non-expert cells including fibroblast cells, endothelial cells, and epithelial cells are involved in pathogen recognition [20]. Innate immune DCs have evolved to sense pathogenic agents via PRRs, which interact with PAMPs to transduce microbial pattern signs into immune cells to trigger their downstream activation. Apart from importance of lectin-glycan recognition, secreted glycoproteins such as antibodies in the immune system exert their functional activities through O- or N-glycans attached to the IgA hinge regions or IgG Fc regions with modulating antibody activity [20, 66], where terminal SAs give an inhibitory signal to immune cells, whereas the defected terminal SAs give activating signals. The glycosylated ligands including N- and O-glycan branching, LacNAc levels, and the balance of α2,3-SA and α2,6-SA regulate lectin recognition with their counter-receptors, indicating crucial roles of the specific glycans in lectin-binding partners in patterns of proper orientation and glycan clustering on multiple side chains. When lectins are released from the cells, they are accumulated on the cell surface matrix to increase in local concentration. Thus, lectins build multiple forms and cross-link glycoconjugates on the cell surfaces to modulate downstream intracellular signaling towards a variety of cellular events. Cellular responses including receptor expression, protein synthesis, Golgi-specific enzyme behavior, glycan biosynthesis, and glycan density regulate lectin-glycan interaction. During biological adaptation and evolution, innate immune cells mostly co-evolve to sense pathogenic microbes through host PRRs, which recognize the conserved PAMPs that independently evolved, and consequently in order to transduce pathogen information into host responses. PRRs also recognize endogenous DAMPs such as alarmins produced from invasion-derived inflammatory responses, autoimmune responses, inflammation, or tumor growth. Representatively, TLRs mediate the recognition events in way of lectin-glycan interactions. However, it became apparent that the glycan epitopes expressed on pathogenic agents are also expressed on the host surfaces and involved in cellular functions. Pathogenic agents basically mimic the host cell surfaced glycans to avoid the host immunity. Then a question is raised. Therefore, the molecular mechanisms responsible for pathogenic glycan binding by host lectins are not clearly discriminated, remaining questions to answer. How? PAMPs are molecular signature of pathogens and PRRs recognize the pathogen patterns. In spite of the TLRs in antigen recognition, lectin-glycan bindings promote pathogen sensing and related responses of immunity. The glycans and lectins are acting elements of pathogen binding and starting the immune responses of

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innate immune cells. Pathogens and host cells produce such acting elements. Endogenous carbohydrate-recognizing proteins or lectins decode the information. The number, density, composition, and distribution of carbohydrate antigens of multivalent carbohydrate moieties in glycoproteins, glycolipids, and GAG decide the fate of lectin-binding affinity. Most patterns are composed of glycans during evolutional adaptation. Currently, several PRRs are identified for TLRs, CLRs, RLRs, NLRs, and the AIM2-like receptor (ALR). The major decoding elements include C-type lectins, galectin, and Siglecs. For the general recognition receptors of DCs and their ligands, C-type lectins such as selectins recognize the glycans of bacteria, while galectins recognize the glycans of bacteria. Siglecs also recognize the glycans of bacteria. Pattern recognition of lectins in innate immune responses indicates a glycan recognition property through a density-dependent manner. In innate immune cells, the surfaced Siglecs and C-type lectins specifically bind to glycans of microbes, while galectins preferentially recognize soluble targets. Apart from the soluble target-recognizing lectins, there are also soluble glycanrecognizing PRRs that include ficolins in which three types of ficolin-1, ficolin-2, and ficolin-3 are known and MBL. The lectin pathway associated with the complement system depends on PPRs to help the clearance of microbial invaders. For example, ficolins belong to a PPR family and lectin pathway component. The ficolins are the secreted components of complement in epithelial cells, endothelial cells, and immune cells of human [67]. Ficolins recognize GlcNAc, GalNAc, and acetyl glycans of target cells [68]. In the side of pathogenic bacteria, they evolved to have complement evasion strategies to mimic and recruit complement factors or degrade complement factors [69]. Both Gram-positive and Gram-negative bacteria can evade complementation by recruitment of complement regulators. The representative regulators are the factor H- and C4b-binding protein (C4BP) [70]. The neonatal meningitis-causing E. coli K1 utilizes the Omp known as outer membrane protein A to protect the bacteria itself from the host killing activity through complements. For example, the C4BP binds to bacterial OmpA protein [71]. Immune evasion is also mediated in pathogenic EAEC strain by proteolytic degradation of host complement proteins using a serine protease known as Pic [72]. Pic protease blocks complement activation by enzymatic inactivation of complement components of C2, C3, C3b, and C4 [73]. Also, biofilm production is a way of evading complement of hosts [74]. In ficolin escape, some bacterial strains show the changed surface composition to avoid binding to ficolin-2, eventually escaping the host complement [75]. On the other hand, two types of collectin-10, termed as CL-10 and CL-L1/ collectin-11, termed as CL-11/CL-K1, also exert the similar type of complement activation [76, 77]. They recognize pathogen-associated PRRs on the microbial pathogenic surfaces and stimulate the lectin pathway via lectin pathway-involved serine proteases or MBL/ficolin-associated ser-proteases (MASPs) [78]. They are indeed soluble pattern recognition molecules, which depend on glycan recognition, the target foreign microorganisms or altered host cells induce the complement cascade reaction of host via the alternative lectin pathway [79]. This is a distinct type of innate immune response of mammals. Once soluble forms of PRRs are

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273

bound to a ligand, the downstream cascade is started by the MASPs, which cleave and activate complement factors C2 and C4, contributing to the C3 convertase formation known as C4b2a. The active C3 convertase enzyme degrades C3 form to C3a form, known as anaphylatoxin, and the opsonin factor C3b activates complement factor C3 [80]. Therefore, in the lectin pathway of the MBP and ficolins, complement activation, therefore, differs from the classical C1q activation pathway [81]. The alternative lectin pathway is thus initiated by C3 degradation and the opsonin factor C3b binding. The C3 can be further degraded by factor D, yielding the C3 convertase, known as C3bBb. The alternative lectin pathway includes C3b genesis, and the increased C3b factor consequently forms the C5 convertase enzyme for the C4b2aC3b/C3bBb3b. This process yields the C5b-9 membrane attack complement (MAC) complex, which has the activity of terminal lysis [75]. The complement activation of classical pathway commences with unique binding of antibody to antigen, and this consequently interacts with the PRR C1q and cleaves C2 and C4 by specific C1r/C1s proteases and deposits C3b opsonin factor.

6.4.2

Toll-Like Receptors

TLRs are historically introduced from the long investigation on innate immunity. The most remarkable milestone in innate immunity study indicates the 2011 Nobel laureates of Drs. Bruce A. Beutler, Ralph M. Steinman, and Jules A. Hoffmann in physiology or medicine for their DC findings and discovery with roles in adaptive immunity as well as for their discoveries concerning the innate immunity activation. TLRs have been termed from their similar protein to the Toll gene in Drosophila [82]. The Toll found in Drosophila regulates development of embryo- and fungispecific immune responses [82, 83]. TLRs have Leu-rich repeated ectodomains and Toll-IL-1R (TIR) domain in the intracellular region. The known TLRs are the TLR1 to TLR10 and the TLR11 to TLR13. The TLR1–TLR10 members are isolated from human and the TLR12, TLR1 to TLR9, and TLR11 to TLR13 are isolated from murine [84]. TLRs recognize PAMPs. TLR2/TLR1 and TLR2/TLR6 recognize lipoproteins as well as diacyl lipopeptides and triacyl lipopeptides. TLR2 binds to fungal zymosan, lipoteichoic acid, and peptidoglycans. TLR3 binds to dsRNA species, TLR5 binds to flagellin, and TLR9 binds to unmethylated CpG DNA. TLR8 also recognizes various synthetic molecules like guanosine analogues and imidazoquinolines. The TLR alone or TLR with co-receptors binds to pattern molecules [84]. For the TLRs of DCs and their ligands, the interactions are outlined and summarized below: – TLR6, TLR2, and TLR1 recognize peptidoglycan (Gram + positive bacteria), lipoprotein, diacyl lipopeptides, triacyl lipopeptides, lipoteichoic acid, lipoarabinomannan (mycobacteria), LPS (Leptospira), LPS (porphyromonas), GPI (Trypanosoma cruzi), and fungal zymosan (yeast).

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– TLR4 and CD14 recognize Gram-negative bacterial LPS, Gram-positive bacterial lipoteichoic acid, and RSV F protein. – TLR3 recognizes dsDNA, dsRNA, a viral product. – TLR5 recognizes flagellin. – TLR7 recognizes small synthetic immune modifiers, imiquimod, R-848, loxoribine, and bropirimine. – TLR9 recognizes unmethylated CpG DNA. – TLR8 recognizes imidazoquinolines and guanosine analogues.

6.4.2.1

DC-Receptor-Specific Pathogenic Ligands

GBPs recognize and bind to pathogens as PRRs through the pathogen-exposed glycans. As PRRs, TLR and Nod-like receptors of DCs recognize bacterial patterns. The well-known bacterial patterns are peptidoglycan as TLR2 ligand, LPS as TLR4 ligand, flagellin as TLR5 ligand, unmethylated CpG DNA sequences as TLR9 ligand, and muramyl di- and tripeptides (Nod1 and Nod2) to date [85]. Other glycans produced by pathogenic agents can also be recognized by other kinds of DC receptors. Those DC receptors are C-type lectins, galectins, and Siglecs. These three kinds of receptors are also expressed by intestinal epithelial cells and APCs [86]. For example, it is reported that DC-SIGN directly binds to α-glucan and mannose-containing glycans of mycobacterial cellular capsules, and these glycans induce immune suppression of host immune cells upon encounter with the mycobacteria. The binding proteins such as DC-SIGN bind Lewis and mannosecontaining carbohydrates exposed on pathogens [87–89]. HIV-1, bacteria M. tuberculosis and H. pylori, helminth Schistosoma mansoni, and yeast Candida albicans are such examples (Fig. 6.1). More specifically, DC-SIGN recognizes their high Man, LeX or LeY, or LDNF. Pathogen glycan-bound CLRs are internalization receptors, due to the pathogen uptake by APCs [90]. Because CLRs thus are PRRs and similar to TLRs. The functional difference between CLRs and TLRs is in that TLRs do not internalize antigens to present by MHC-I or MHC-II. The different signaling function is in that CLRs interfere with Fc receptor (FcR) or TLR activity. CLRs transduce signals via the Raf-1 or Syl signaling pathways during binding to glycans [87, 91, 92]. CLRs form normally complex machines with cytoplasmic signaling motifs and adaptor proteins. Exceptionally, the DC-SIGN elicits expression of target genes upon binding to PRRs. For fungal β-glucans, Dectin-1, a specific CLR, transduces downstream signaling via the Syl tyrosine kinase and ERK-JNKNF-κB axis pathway [87, 93]. This signaling is cooperated with the adaptor CARD9, independently to TLR signaling. Again, DC-SIGN also recognizes Mycobacterium tuberculosis strains through the ManLAM carbohydrates produced by Mycobacterium strains and activates IL-6 and IL-12 releases, which are proinflammatory cytokines, through a Raf-1 downstream signaling. However, if DC-SIGN recognizes Fuc residue, Raf-1-independent signaling induces inflammatory IL-10 expression but inhibits IL-6 and IL-12 gene expressions, which are proinflammatory cytokines [92]. Therefore, each carbohydrate signature of pathogenic agents triggers each

6.4 How Are Pathogenic Bacteria Recognized by Receptors of DCs of the Host. . . Fig. 6.1 DC-SIGN signaling in HIV-1 or M. tuberculosis infection

HIV-1

275

M. tuberculosis DC-SIGN

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distinct signaling response, even via the same CLRs. The GBPs also interact with self-glycans with mannose/fucose or GalNAc glycan structures [94]. Tumor antigen MUC-1 binds to MGL and CEA recognizes DC-SIGN [35]. GBPs bind to single carbohydrate structures. In fact, DC-SIGN does not bind to sialylated carbohydrates but binds to sialylated IgGFc, contributing to the anti-inflammatory response of IVIGs and to the inhibition response of FcR FcyRIIB signaling during the IL-33 expression in Th-2 and IL-4 expression in basophils [20]. Myelin oligodendrocyte glycoprotein (MOG) acts as an autoantigenic factor of the neuro-inflammatory MS event. Hman myelin-located MOG is a fucosyl type of N-glycans, which is interacted with the DC-SIGN present in microglia cells and DCs. The binding of MOG to DC-SIGN during TLR4 activation induces secretion of IL-10 and inhibits T cell growth. Inflammatory oligodendrocytes suppress the fucosyltransferase expression. Fucose residue loss on myelin decreases DC-SIGNdriven homeostasis with activation of inflammasome, growth of T cells, and differentiation of Th17. DC-SIGN ligands include the CEA and CEA-CAM1. The ligands activate the DC response to the TLR4 ligand LPS like M. tuberculosis ManLAM to increase the LPS-induced IL-10 production. Thus, tumor and pathogens can escape immune response via the host receptor tolerogenesis (Fig. 6.2). DC-SIGN is a homeostatic receptor by pathogens and tumors via their glycan structure change [96]. In other example of bacterial pathogen, C. jejuni-produced α2,3-SA-containing glycans can be bound by Siglec-7, which is present in DC cell surfaces. Siglec-7 is compatible for its recognition capability with both α2,3- and α2,8-SA-containing glycans. Therefore, the α2,3-SA-bound Siglec-7 expressed in the DC cell surfaces stimulates T helper-2 responses, whereas α2,8-sialic acid glycan-bound Siglec-7 on the DC stimulates T helper-1 responses [97].

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6 Innate Immunity Via Glycan-Binding Lectin Receptors

Fig. 6.2 DC-SIGN action as a homeostatic receptor in T cell responses. MOG, myelin oligodendrocyte glycoprotein; CEA, carcinoembryonic antigen; CEACAM1, CEA-related CAM-1. Adopted from ref. [95] García-Vallejo JJ et al. J Exp Med. 211(7):1465–83

Pathogens express their own specific glycans. Helminth glycans are carbohydrate structures such as GBPs-binding LDNF with the structure of GalNAcβ1,4(Fucα1,3) GlcNAc-R and LDN with the structure of GalNAcβ1,4GlcNAc-R- [89]. HIV-1 expresses high Man structures not pathogen-specific but recognized by certain GBPs of the CLRs like DCIR, DC-SIGN, Langerin, and MR [98–100]. DC-SIGN, MR, or MGL easily bind endogenous glycans, and thus they are adhesion molecules, antigen uptake mediators, or signaling receptors [101]. DC-SIGN recognizes the ICAM-2 and ICAM-3 during cell-cell interaction and DC homing via LeY epitope expressed in vascular endothelial cells. DC-SIGN also recognizes the LeX and LeY glycans expressed in Mac-1 and CEA-CAM-1 receptors expressed in neutrophils and thus positively regulates adhesion of neutrophils and DCs [102]. GalNAc residue linked to CD45 present in the effector memory cells of CD4+ T cells and CD8+ CTLs recognizes the MGL, a CTL dominantly present in tolerogenic APCs. The recognition events induce apoptotic cell death proliferation of these cells, giving a homeostasis-keeping function of CD45 carbohydrates [13]. Furthermore, similar CTLs such as DC-SIGN, MR, and Langerin also recognize tissue antigens including collagen type I, Fc-IgG, plasma hydrolases, and tissue-type plasminogen activator TPA. DC-SIGN, Langerin, and MR can internalize, serving to homeostatic surveillance by APCs of DCs and macrophages [103]. SAs are frequently expressed on infectious pathogens, as known in many different pathogenic bacteria including C. jejuni, H. influenzae, N. gonorrhoeae, N. meningitidis, and Pasteurella multocida [97, 104–106]. In fact, the incorporation of host-derived SAα2,3-linkage into H. influenzae is reported to be a major virulence factor for experimental otitis [104]. Because the sialyl moieties of the pathogenic bacteria are structural mimics of the human sialyl glycans, these are evolutionized to

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use to avoid or escape the host immune system. Although α2,3-sialyllactose or α2,6sialiclactose motif interacts with TLR4 on DCs, the Gram-negative bacterial negative LPS or sialylated bacterial glycans targets the TLR4 of DCs for their protection from the hosts. For example, α2,3-sialylated LPS on C. jejuni binds to the TLR4 of DCs to escape the host defense immunity [107].

6.4.2.2

Signaling upon Receptor-Ligand Interaction

TLR signaling initiated by TLR dimerization induced by the ectodomain consists of two main pathways of the TRIF/MyD88 pathways [108]. From the knowledge on the interaction of PRRs such as TLRs with PAMPs, the adaptor protein MyD88 is involved in the downstream signaling towards NF-κB activation. NF-κB is consequently activated to express the inflammatory genes of chemokines, immunoreceptors, and cytokines [109]. MyD88 is conserved in TLRs, not for TLR3 [110]. TLR3/TLR4 uses the TRIF pathway, initiated by dsRNA and LPS, respectively [111, 112]. Myd88 factor recruits several adaptor proteins such as IL-1R-associated kinase protein (IRAK)-1/IRAK-4/TNFR-associated factor 6 (TRAF6) [113]. IRAK-1 protein ubiquitinates TRAF6 and stimulates TGF-β-activated kinase 1 (TAK1) [114]. TAK1 kinase elicits NF-κB and MAPK axis with IKK kinase complex with subunits of the IKKα/IKKβ catalysis subunits as well as regulatory IKKγ subunit. The IKK enzyme degrades NF-κB inhibitor IκBα or IκB families like p105. Then, canonical NF-κB family is released and translocated. For example, the complex dimer formed between NF-κB1 p50-cREL and NF-κB1 p50-RELA induces expression of proinflammatory gene. TAK1 also activates MAPK family including ERK1/ERK2, JNK, and p38 to elicit the transcription factor AP-1. TAK1/p38/NF-κB axis signaling is required for stimulation of microneme proteins. TLR extracellular ectodomains expose leucine-rich repeats (LRR) binding to other functional amino acids to confer the domain capacity. In addition, heterodimerization and co-receptor recruitment increase the recognition capability of various ligands. TLR2 and TLR4 extracellular ectodomains carry four and nine N-glycans, respectively. N-glycosylated TLRs represent lectin-binding abilities as protein-carbohydrate interactions (PCI) [115]. The TLR heterodimers include TLR4/ TLR6, TLR2/TLR1, and TLR2/TLR6 [116–118]. Homodimeric TLRs are TLR5/ TLR5, TLR4/TLR4, TLR3/TLR3, and TLR2/TLR2 [119–121]. TLR2 complexes incorporate their co-receptors such as CD14 [122] and CD36 [123], forming three distinct heterocomplexes including CD14-TLR2-TLR1 complex for triacyl lipoproteins as ligand and bacterial Salmonella curli fibers [124], TLR1/TLR2/CD14/CD36 complex for mycobacterial lipoarabinomannan and lipomannan as ligands [125], as well as CD14/CD36/TLR6/TLR2 complex for LTA and diacyl lipoprotein ligands [123]. Currently known PAMPs are the DNA unmethylated with CpG motifs, bacterial LPS, and bacterial peptidoglycans. TLRs binding to PAMPs elicit antimicrobial responses via adaptive Th1 immunity. TLR agonists are subject to study

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prophylactic therapy and therapy to prevent pathogenic infection or neoplastic transformation [126]. The extracellular domain of TLR architecture has the Leu-rich repeats (LRR), and LRR can recognize broad ranges of PAMPs. The recognition event deals with the PAMP structure, the TLR type, the heterodimerization, the recruitment of co-receptors, and carbohydrate-binding lectin of parasites. TLR N-glycans are targeted by the lectin CRD. Lectin binding elicits cell signaling for proinflammatory cytokines, and this lectin response resembles with PAMP recognition response. Thus, the lectins are functional TLRs agonists. PAMP ligands of TLR2 and TLR4 elicit dimer formation of the ectodomains, leading to conformational shifts for homodimer formation of the cytoplasmic TIR domains and assembly with MyD88. The TIR domain-containing adapter molecule (TICAM)-1, which is also known as TRIF, recruits its specific TLRs. TLR2, CD282, recognizes foreign substances of LTA. TLR2 recognizes and develops immune responses against Gram-positive bacteria via peptidoglycan, lipoprotein, and LTA, upregulating expression of the chemokines of CCL2 and CXCL8 to recruit immature subset of DCs via upregulated NOD2 expression [127]. TLR2 is involved in the functions of TLR1 and TLR6 to form heterodimers for bacterial lipoproteins and lipopeptide recognition [128]. TLR2 and TLR6 recognize the diacyl lipoprotein forms produced in Mycoplasma fermentans [129]. TLR4 specifically recognizes the Gram-negative bacterial LPS [130]. Upon activation, TLR4 induces the proinflammatory cytokine expression. TLR2 is expressed on macrophages and DCs. The TLR5 ligand is the bacterial flagellum required for bacterial motility [131]. Of interests, several pathogens including Bartonella bacilliformis, H. pylori, and C. jejuni strains express flagellins but not recognized by TLR5 because of their mutations of amino acid sequences [132]. The TLR family of TLR3, TLR7, TLR8, and TLR9 are not expressed on surfaces, but expressed on intracellular, cytoplasmic endosomes. They recognize DNAs and RNAs. For example, TLR3/TLR7/TLR8 recognize the ds-/ssRNA species. TLR9 recognizes unmethylated CpG DNA [133]. The dsRNA is recognized by TLR3 and this is a viral receptor. TLR7/TLR8 are structurally similar, and they recognize the same ligands such as the nucleic acid species of viruses. TLR7 in murine DCs triggers the regular responses of IFN-α to influenza virus, while TLR8 in human recognizes the oligo-NTPs. Mouse TLR9 binds to the ssRNA virus and vesicular stomatitis virus to induce IFN-α secretion, and they distinguish self-RNA species from non-self-RNA species. TLR7 also recognizes synthesized guanine analogues like imiquimod, loxoribine, and resiquimod, which are applicable for antiviral agents.

6.4.3

Innate Immune Receptors in Malaria Infection

Malaria is caused by Plasmodium parasites that are transmitted to people through the female Anopheles mosquitoes. The five parasite species that generate human malaria include Plasmodium falciparum, P. knowlesi, P. malariae, P. vivax, and P. ovale.

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Plasmodium parasites bind to RBC surface receptors sialylated. Plasmodium knowlesi is a zoonotic parasite that is transmitted from long-tailed and pig-tailed macaques causing malaria in humans in Southeast Asia. There seems to be a congenital resistance in malaria infection, indicating that the malaria infection requires its infection receptors to enter the red blood cells (RBC). Erythrocyte surface receptor, for example, Duffy negative genotypes are resistant to malaria infection. The genotype of Duffy (Fy
Fy
) population frequently found in Africa is resistant to the infection. In contrast, the genotypes of FyaFy
, Fy
Fyb, or FyaFyb are susceptible for infection. In the strains, P. vivax mainly infects reticulocytes, P. malariae prefers to infect the mature RBCs, and P. falciparum infects all RBCs in individuals. Intraerythrocytic factors which are resistant to malaria infection are known. For example, individuals carrying abnormal Hb types including HbS, HbC, HbE, thalassemia, sickler, glucose-6-phosphate dehydrogenase (G-6-PD) deficiency, PABA deficiency, ATP deficiency, etc. are rather resistant to the infection. Therefore, individuals having genetic disorders in RBC function exhibit the resistance against malaria infection, and normal individuals are infected to die, terming “adverse selection.” In addition, immature RBCs are preferentially targeted by malaria infection. The immature RBCs are not easy to be cultured, and the reticulocytes are easy host for the infection once isolated. In addition, because the malaria infection is not successful to infection in animals, the malaria research is not easy compared to other infectious diseases. On the other hand, acquired immunity in malaria is not recognized, because the individuals who were infected once before can be also reinfected by malaria, although antibody responses and cellular responses occur. However, the “premunition” is acceptable for malaria infection, as premunition indicates that the current malaria patients can defense reinfection of the same strain to a less extent. This indicates the species-specific or strain-specific concomitant immunity. In contrast, mixed infection is also observed. Currently, there is no anti-malaria vaccination, but only antigenic proteins and vaccine candidates are known for circumsporozoite surface protein (CSP) that is a sporozoite surface protein antigen, Duffy binding protein (DBP), merozoite surface proteins (MSP-1, MSP-3α, MSP-3β, and MSP-3γ), and apical membrane antigen-1 (AMA-1). One question is raised: why is it difficult to design such vaccination? The answers are reasonably based on the immune evading mechanisms of malaria during infection: (1) intracellular location (within RBC), (2) antigenic variation (from sporozoites to merozoites and gametocytes), (3) immunosuppression (splenomegaly and dysfunction), and (4) interference with immune effecter mechanisms, as the number of macrophages are decreased and antigen presentation level is also decreased, called the “smoke screen theory.” If merozoites are exited from RBCs, the merozoites have high motility and at fast enter to the other RBCs.

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6.4.3.1

6 Innate Immunity Via Glycan-Binding Lectin Receptors

Pathogenic Process in Malaria Plasmodium falciparum Infection

For pathogenesis and pathology, the primary events are involved in destruction of RBC to cause severe anemia and blockage of capillary due to knobbing of infected RBCs which tend to attach to the capillary wall (e.g., brain capillary in P. falciparum). Knobbing events are involved in the severe headache, and the infected RBCs are knobbing to block the blood circulation through adhesion to the capillary blood vessels. The strain of P. falciparum is the most causative agent of such disease phenotypes, but not P. vivax. Secondary events include anoxemic impairment, leading to tissue dysfunction, cellular reactions, and immunopathology (e.g., brain cell damage by TNF-α). During cellular reaction, many chemical compounds such as TNF-α are released, and they damage the neuronal cells for immunopathology. Therefore, knobbing causes brain damage and immunopathology of cerebral malaria. Receptors in innate immunity mediate systemic inflammation. Like most infectious diseases, the pathology of malaria is driven by cytokines. Cytokine production results in the symptoms of malaria that include fever, chills, rigors, headaches, myalgias, lethargy, and more. Several strains of Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, and Plasmodium ovale cause the malaria symptoms. To prevent and treat malaria, the first drug of choice is chloroquine, but the recent chloroquine resistance issue is raised. The second drug is Fansidar which is pyrimethamine or sulfadoxine. The mitochondria-acting agents are Halfan or halofantrine as a 9-phenanthrene with an expensive price. The first discovered drug to prevent and treat malaria was quinine, as the action mechanisms are absolutely the same as that of chloroquine. Thereafter, artemisinin (a sesquiterpene lactone peroxide), artemether, and artesunate were isolated from the Artemisia plants. They are commonly linked with endoperoxides, and this endoperoxide is essential for the anti-malaria efficacy, because malaria protozoan heme molecule interacts with artemisinin, artemether, or aresunate to yield the endoperoxide species, and endoperoxide alkylates the malaria proteins and peroxidates the malaria lipids. At the present time, anti-malaria drugs are representatively prescribed for atovaquone, chloroquine, doxycycline, mefloquine, primaquine, proguanil, and hydroxychloroquine. In vertebrates, malaria are reproduced by a schizogony type, while in intermediate hosts, they reproduce by a gametogony. With regard to the innate immune receptors for malaria parasites, several important aspects should be considered: (1) Define parasite targets for innate immune receptors, (2) identify relevant innate immune receptors, (3) define their role on host/parasite interaction and disease outcome, and (4) elaborate prophylactic/therapeutic interventions employing TLR agonists or antagonists. The malaria pathology has been associated with the three major steps of an excessive production of inflammatory cytokines and septic shock-like syndrome, progress in severe anemia, and adhesive accumulation of the infected RBCs to capillary vessel walls, contributing to the RBC destroy and damaged erythropoiesis. Malaria toxin hypothesis has been suggested in 1889 by Golgi. Fever-causing factor

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Protein

Gal

Gal

Man

Gal

Man

Gal

P

EtN

• PLC: Phosphadyl lipase C • EtN: Ethanolamine

Man

GPI-anchored protein (glycophosphadylinositol)

GlcNAc

Ino

P

Fig. 6.3 The Plasmodium parasite of malaria protozoa is coated with a GPI anchor

has been mentioned to be pyrogenic toxin by a mechanism(s) that Plasmodium parasite constituents activate innate immune receptors to generate the inflammatory mediators. Why do humans get fever during malaria? The search for the “malaria toxin” has been done. What innate immune receptors are activated during disease and what microbial products (“malarial toxins”) activate these receptors? Is the source of inflammation a molecule of the parasite membrane? Do bear malaria like Gram-negative bacterial LPS? Is the source of inflammation a membrane molecule of the merozoite? In other words, is the malaria toxin just like LPS? Plasmodium strains are decorated with specific GPI-Aps, as known for variable surface glycoproteins of other protozoan strains in order to escape from host immunity (Fig. 6.3). GPI-APs activate TLR2 signaling in hosts. For example, the GPI-APs of T. cruzi activate mainly TLR2 signaling and partly TLR4 signaling. P. falciparum GPI-APs also exhibit the TLR stimulating activity in the human myeloid cells.

6.4.3.2

P. falciparum GPI Anchor Glycosylation

General functions of GPI anchors are depicted in the following four main subjects of (1) stable association of proteins with the plasma membrane, but with measurable “off-rates” from the membrane and the potential to be shed by phospholipases, (2) the potential for very high lateral mobility on the plane of the lipid bilayer, (3) the potential to insulate the protein domain from the cell interior (may be important in protozoa), and (4) the potential to participate in signaling through association with other membrane-spanning components in lipid rafts.

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GPI-anchored glycoproteins are mainly located as GPI membrane anchors. In other words, GPI stands for a phosphatidylinositol (PI) phospholipid linked via a glycosyl, or sugar chain, component to the C-terminal protein region. GPI-anchored proteins are processed by GPI transamidase. Proteins are bound to membranes, and many GPI-anchored proteins are functional on the plasma membranes [134]. The overall GPI-anchored protein biosynthetic pathway has been reported with the structural remodeling and transport [135]. GPI is biosynthesized using free PI in the ER region via 10 more steps. The en bloc transferring of proteins requires multiples genes of 20 more genes, which are termed to PIG genes. In the cytosolic region of ER, the two steps are involved in initiation of protein transferring reaction. Thereafter, the next steps take place in the luminal side of ER. Alkylated lipid species initially generated in the peroxisomal organelle are supplied to the ER lumen. The alkylated lipids are then modified to the 1-alkyl-2-acyl group linked to GPIs. Genetic disorders such as PNH- and GPI-deficient diseases are attributed to defects of the PIG-A gene and PIG-M or PIG-V gene, respectively. For linkage of the GPIs and proteins, naïve protein forms are then subjected to modification by GPI through their specific sites for GPI linking. The GPI linking sites consist of a ω site, a space with hydrophilic region, and a hydrophobic region. The native protein forms are biosynthesized independently from the GPI synthesis. The transamidation reaction is catalyzed by the complex enzyme of GPI transamidase. The native protein modification with GPI species is one of the post-translational modifications (PTMs). The GPI remodeling in their structures occurs in the combination of the GPI lipid portions with glycans, by means of catalytic activity of the post-GPI attachment to proteins (PGAPs). A fatty acyl group, which is a palmitic acid in most GPI-Aps of eukaryotes, is cleaved from the acylated inositol by a deacylase termed PGAP-1 enzyme. Thereafter, another PGAP-5 enzyme cleaves to an EtN-P from Man-2 structure. The Man-2 binds to p24 family proteins and efficiently sorts the p24 family proteins to the ER region. During gradual budding out in the ER region, the produced GPI-APs are incorporated to small delivery vesicle of COPII by means of specific transportation proteins such as Sec24C and Sec24D delivery proteins. Sequentially, the GPI-Aps formed are then delivered to the surfaces or membranes via the Golgi complex. In the Golgi complex, the GPI-APs are further modified for their specific structures, mainly in their remodelings of fatty acids [135]. The remodeling of the GPI-APs includes changes in the composition of the GPI lipid parts, to fit lipid raft-competent GPI-Aps of membranes. The free PI is used as a PI source, as the PI consists of diacyl fatty chain and unsaturated lipids at the sn-2 position. The representative diacyl unsaturated lipid is 1-stearoyl, 2-arachidonoyl PI. They are translocated to the ER luminal side by flippase activity, GlcN-PI species are remodeled by fatty acyl groups such as palmitic acid to the inositol, the lipid part is exchanged, and peroxisome-borne alkyl-acyl donors are used. PGAP1 enzyme eliminates a fatty acyl group, which is linked to the inositol of GPI-APs, and the GPI-APs are sorted to the ERES. GPI-APs are delivered to the Golgi apparatus to receive further structural remodeling, termed “remodeling of fatty acids,” for formation of GPI-AP-lipid raft complex [136]. However, the unsaturated lipid group attached to the position of sn-2 is cleaved off by PGAP3, and the saturated lipid

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chain like stearoyl chain is added by PGAP2, although the enzyme activities of PGAP2 and PGAP3 are not yet defined. Transglycosylation reaction results in that a GPI cell wall protein becomes cross-linked to cell wall β1,6-glucan via its GPI glycan [137]. Defect of GPI biosynthesis in tissues abolishes GPI-APs surfaced expression and causes lethality in the early embryonic development. The partially displaying patients have been reported to be caused by a point mutation within the Sp1 transcriptional factor binding site in the PIG-M gene promoter for the mannosyltransferase for Man-1. This point mutation severely reduces the expression levels of the PIG-M protein, displaying the onset of venous thrombosis and seizures. Sodium butyrate, a histone deacetylase inhibitor, improved the PIG-M expression and the surface expression of GPI-APs. Another PIG-V gene encodes the mannosyltransferase for Man-2, and the gene is often mutated in hyperphosphatasia mental retardation (HPMR) syndrome (or Mabry syndrome), an autosomal recessive form of mental retardation with elevated serum alkaline phosphatase. The reduced expression of PIG-V and surface GPI-Aps are associated with the onset of the disease. Human African sleeping sickness is caused by Trypanosoma brucei. Trypanosoma forms in blood smear from patient with African trypanosomiasis by many protein variants in common GPI anchors, where the surface coat is made of a dense monolayer of variant surface glycoprotein (VSG) [138]. In 2004, R. Gazzinelli and his colleagues began purifying P. falciparum parasites to study [139]. Unfortunately, purified P. falciparum, the parasites without the RBC membranes or other debris, failed to induce cytokines from PBMC or mouse macrophages. He found that the malarial parasite is coated with a GPI-Aps.

6.4.3.3

GPI Anchor on the Merozoite Surface in Inflammatory and TLR2, TLR4, and TLR9

The malarial parasites are coated with a variety of GPI anchors. Therefore, the question of “Is the inflammation source, malaria toxin, a component of the outer membrane just like LPS?” was raised for a while? If GPI-APs of P. falciparum bear TLR activation activity, it is now assumed that the GPI anchors present on Plasmodium’ cellular membrane function as LPS that is a “malaria toxin” [140]. GPI anchor on the merozoite surface induces inflammatory response and activates TLR2, but weakly activates TLR4, as GPI anchors from T. cruzi are also TLR2 ligands. Malaria PAMP is a malaria pigment, hemozoin, that is a hemin’s crystalline polymer as a degraded hemoglobin. The produced level of hemozoin indicates the severity of the malaria disease. Then, there was basic question on “What are the innate immune receptors in innate immune recognition system involved on hemozoin recognition?” The question has been raised because the innate immune responses are well conserved even to human through Drosophila. The next question was “How does malaria detoxify protoporphyrin IX (hemin)?” The inflammatory component of hemozoin has been known to be DNA. For the hypothesis, hemozoin is internalized into the phagolysosome, and TLR9 can be recruited to the phagosome thus causing

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for the expression of proinflammatory cytokine of TNF-α (which causes fever). Later, its DNA is liberated from the crystal surface by the proteases and other enzymes. What is hemozoin? Hemozoin is the crystalline breakdown product of hemoglobin. The levels of hemozoin in circulating phagocytes correlates with disease severity. It is how malaria detoxifies protoporphyrin IX, also known as hemin. Hemozoin chemistry made clear in solution that hemin chloride can form β-hematin dimer by bonding between carboxylic groups and heme. However, synthetic hemozoin, if manufactured from highly purified hemin, lacked cytokineinducing activity. There was a big hypothesis as Christiane Nüsslein-Volhard, Nobel laureate (Medicine, 1995), suggested that hemozoin is a potential candidate for a proinflammatory substance made by malaria. Toll receptors are likely to crucially act in the pathogenesis of malaria through the recognition of hypothesis of hemozoin. Her question was “What is a Toll receptor?” Nüsslein-Volhard is associated with the discovery of Toll, which led to the identification of TLRs. In the dictionary, “Toll” means amazing, bodacious, cool, corky, crazy, frantic, furious, great, like blazes, mad, madcap, screaming, super, wild, or wow. Later, Toll was cloned by Schneider and Anderson and recognized to be a type I transmembrane (TM) receptor with a high homology to the cytosolic IL-1R (TIR domain) region [141]. The observation that the Toll had a domain in common with the IL-1R led to the hypothesis that IL-1 is associated with innate immunity. The innate immune responses are actually well conserved between Drosophila and humans. For example, the purple sea urchin has 222 TLRs [142]. Another answer has been quoted from the results that phagocytized hemozoin colocalizes with TLR9 in the lysosomes, where CpG and AT-rich DNA activate different innate immune pathways. TLR9 activates host innate immune response by binding to hemozoin of the malaria pigments [143]. Plasmodium parasites in the red blood cells degrade hemoglobins to a polymeric form known as hemozoin. The hemozoins are exposed in the plasma blood and the reticuloendothelium captures the hemozoins. Hemozoin is immunologically active and modulates the innate immune system. The purified hemozoin is a TLR9 ligand and activates innate immune responses. The hemozoin responses are blocked in TLR9-deficient null mice and MyD88-deficient null mice. However, the hemozoin-induced responses are not found in the several null mice. For example, the Toll-IL-1R domain-containing adaptor-inducing IFN-β, TLR2, TLR4, or TLR7-deficient null mice are not responding to the hemozoin. Chemically pure hemozoin synthesized also activates innate immune responses in vivo by a TLR9. Bacterial DNA is non-methylated, rich in CpG, and immunologically active, but CpG-rich DNA is recognized by TLR9. Natural hemozoin activates cytokine production via TLR9/MyD88. But the stimulatory activity of hemozoin was destroyed by DNase. Thus, the cytokine-inducing component of hemozoin has been suggested to be DNA. Is the DNA on hemozoin originated from human or malarial? PCR analysis of hemozoin showed that most of its DNA is malarial-based. Does malaria DNA activate innate immune responses? Can malaria DNA actually stimulate cells, presumably via TLR9? The cell biology of TLR9 is complex. TLR9 resides in the ER organelle. TLR9 translocates to the endosomal compartment to bind its ligand (CpG-rich DNA). Malaria DNA is

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285

stimulatory for DCs only when introduced into cells directly with the transfection reagent (endosomal compartment). The malaria genome contains 269 CpG repeats. Hemozoin functions to delivery DNAs to a TLR9-recruited vesicles of cytosolic compartments. However, most malaria DNA is AT-rich. The malaria genome contains the motif ATTTTTAC over 6000 times, and AT-rich DNA mimics malarial DNA. Transfection of AT-rich DNA drives a variety of promoter constructs like native DNA and activates IL-6/IL-1β/IFN-β/TNF-α cytokines. Six AT-rich motifs have been studied (AT1-AT6), where AT-2 sequence is GCACACATTTTTACTAAAAC. Microarray analysis of 14 patients with febrile P. falciparum showed an “IFN signature.” Hemozoin and DNA complex rapidly activates IFN-β production from PBMC. From the discovery of Dr. Akira Shizuo, it has been clear that hemozoin constitutes the first non-nucleotide ligand for TLR9. Therefore, the malaria infection is involved in step 1; internalization of hemozoin by phagocytes leads to activation of MyD88 via TLR9, and this event results in IFN-γ production, inflammasome formation, and caspase-1 activation. Therefore, many researchers have assumed that can we interfere with pathogenesis of malaria by blocking MyD88 activation [144]. Step 2: IFN-γ priming of phagocytes induces the enhanced TLR expression, TNF-α production, and maximal production of pro-IL1β. When caspase-1 is cleaved and matured, the inflammatory reaction is progressed.

6.4.3.4

Inflammasome and Sialic Acid Tropism in Malaria Recognition

The next question was that “Is the inflammasome involved in malaria recognition? What is a inflammasome?” And the answer was that the inflammasome is a complex form of multiproteins, which is associated with IL-1β production. Therefore, in activation of IL-1β via caspase-1, malarial AT-rich DNA appears to induce the inflammasome. For the role of fever-causing IL-1β, AT-rich motifs have been suggested to be a major way in which malaria causes fever. Then, the generation of type I interferons has been assumed to involve the inflammasome, because activation of type I interferons requires the inflammasome component Nalp3, but not ASC. Continuously, a basic question is raised. How do the hemozoin-surfaced DNAs migrate from the phagosomes to the cytosolic area? Different to IFN-β, IL-1β activation requires caspase-1. Phagocytosis of inert particles causes leakage of phagolysosomes, releasing several molecules such as asbestos, SAs, uric acid, or amyloid-β peptides (Aβ) known as a plaque rupture factor of Alzheimer’s disease (AD). The Neu5Gc is a factor for P. knowlesi invasion of human RBCs. While macaques synthesize the Neu5Gc, humans are mutated in the CMAH enzyme to synthesize Neu5Gc. P. knowlesi is restricted in its invasion of human RBCs due to the absence of Neu5Gc. Plasmodium infection pathway is involved in attachment, reorientation, and invasion. When the chimpanzee CMAH gene (PtCMAH) has been transfected in human CD34+ hematopoietic stem cells, the transfected human RBCs express different SA variants. P. knowlesi invades Neu5Gc-positive rhesus macaque RBCs but not Neu5Ac-producing human RBCs. P. knowlesi invades Neu5Gc-

286 Fig. 6.4 Human-adapted P. knowlesi invasion is Neu5Gc-independent by the mechanism that has adapted to use NeuAc-binding ligands and SA-independent DBP gene duplication

6 Innate Immunity Via Glycan-Binding Lectin Receptors

Plasmodium Knowlesi, reichenowi (Laverania)

invasion

2. Evolution Plasmodium falciparum

Chimpanzees (Neu5Gc+)

1. CMAH Loss

invasion

Human (Neu5Gc-)

producing PtCMAH cRBCs, but little Neu5Ac-positive cRBCs. Duffy antigen/ chemokine receptor (DARC) is the human receptor for P. knowlesi invasion. Duffy binding proteins (DBP) act as ligands. Glycophorin A (GPA) and GPC are used as glycosylated receptors. For the determinants of SA-dependent invasion in P. knowlesi, the binding domains of the P. knowlesi DBP ligands, PkDBPa, PkDBPb, and PkDBPg, were analyzed. PkDBPb and PkDBPg did not bind to neuraminidase-treated macaque RBC ghosts. PkDBPb and PkDBPg bind to PtCMAH-transfected cells. PkDBPb and PkDBPg are Neu5Gc-binding ligands for invasion into Neu5Gc-expressing RBCs. Thus, P. knowlesi invades human RBCs via surface Neu5Gc, when P. knowlesi strain H(Pk H) adaptation to human RBCs is generated. In the invasion of P. knowlesi strain Pk H into PtCMAH cRBCs, how does Neu5Gc engage in the human-adapted P. knowlesi strain? P. knowlesi strain Pk H invades human and macaque RBCs equally. The question is “How does P. knowlesi evolve to infect human RBCs?” Duplication of the SA-independent PkDBPa gene as well as a complete deletion of the SA-dependent PkDBPg gene was detected in P. knowlesi Pk YH1. However, it evolves to invade human RBCs in a SA-independent manner. Neu5Gc is a key host determinant of the tropism by P. knowlesi. Human-adapted P. knowlesi invasion is Neu5Gc-independent. This is the mechanism that P. knowlesi Pk YH1 has adapted to use NeuAc-binding ligands for human infection, preventing the NeuGc dependence for invasion (Fig. 6.4) [145]. Evolution seems to be progressed to the direction of infected mosquito ! monkey ! human ! human/monkey. In summary, for the malaria infection, (1) infection with Plasmodium leads to a proinflammatory priming and hyper-responsiveness of TLRs; (2) in mice, TLR9 has an important role in promoting this proinflammatory priming, which is mediated by DC-released IL-12 and NK cell/T cell-produced IFN-γ; and (3) treatment with an antagonist of nucleic acid sensing TLRs prevents cytokinemia and lethality in a rodent model of cerebral malaria.

6.4 How Are Pathogenic Bacteria Recognized by Receptors of DCs of the Host. . .

6.4.4

287

Innate Immunity Receptors in Protozoan Parasite Toxoplasma gondii

Toxoplasma gondii, a protozoa parasite, is a group of the phylum Apicomplexa as a coccidian parasite and causes toxoplasmosis. The protozoan parasite hosts include vertebrates such as humans with one third of the worldwide population infection. During T. gondii life cycle, parasites change in cellular stages with characteristic phenotypes including morphology and physiology. The cycled stages are the tachyzoites, merozoites, tissue-type bradyzoites, and sporozoites. Tachyzoite stage is specific in phenotypes such as motile and multiple population in the host. Tachyzoites spread through the whole body and convert to stage of tissue-locating bradyzoites. Merozoites fast multiplies the parasite population within the cat intestine. Bradyzoites convert into merozoites within intestinal epithelial cells. Bradyzoites are the slowly dividing stage of parasites to form tissue cysts. Sporozoite stage indicates the stage of the parasite present in oocysts. Host innate immune cells produce IL-12 upon infection of T. gondii, and the produced IL-12 elicits activation of NK cells. Tryptophan is a key amino acid for T. gondii. IFN-γ elicits the indole-amine-2,3-dioxygenase (IDO) and Trp-2,3-DO (TDO). Both enzymes of IDO and TDO degrade Tryptophan. The strains exhibit their specific endemicity and transmissibility in epidemiological aspects [146], occupying about one third carriers of the T. gondii-infected human population [147]. The major symptoms include immune compromise [148], after the protozoan invasion to host cells through the tachyzoite’s motility via two apically located organelle of rhoptries and micronemes [149].

6.4.4.1

Host Carbohydrate-Binding Domain of Toxoplasma gondii-Secreted Proteins

For the host infection, first, membrane proteins are involved. T. gondii infection of host cells occurs by secreted microneme proteins (MICs) as well as ROP and RON rhoptry proteins. Lac-recognition fraction (Lac+) of T. gondii extracts contains MIC1 and MIC4, and they activate splenic immune cells to express IL-12. Immunization of mice with Lac+, MIC1, or MIC4 protects the hosts from T. gondii infection. The well-known PRRs of TLRs transduce their signals through MyD88 adaptor protein and downstream signaling for NF-κB activation. Thus, the MyD88–67 KO mice are highly susceptible to infection of T. gondii because TLRs act as receptor for the parasitic glycans or antigens, functioning as a modulator of the innate immunity. T. gondii adheres and invades host cell through carbohydrate recognition by MIC as CRD. MICs are expressed as parasite membrane complexes from distinct organelles with MIC1, MIC4, and MIC6. MIC1, MIC4, and MIC6 are preset as complexes with other T. gondii proteins for the host adhesion and invasion as well as the virulence factors. The three MIC molecules of MIC1, MIC4, and MIC6 are

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assembled with adhesin complex resided on cell surfaces. The MIC6 is a parasite surface-embedded form as transmembrane complex. MIC1, MIC4, and MIC6 of T. gondii tachyzoites are trafficking to membrane-containing microneme organelles once complexed in ER. MIC1 and MIC4 are extracellularly exposed and directly bind to surface receptors of host cells. MIC1 and MIC4 play an integral role for the first step of host cell-tachyzoite attachment and adhesion. MIC1 and MIC4 bind to terminal SA residues and Gal residues, respectively, due to their lectin domains. MIC1 [150–152] and MIC4 [150, 153] are soluble adhesins and membranespanning soluble adhesin proteins. The MIC1–MIC4 and MIC6 complex adheres to host cells. The MIC proteins from oligomers with different molecules increase the parasite repertoire to invade host cells. Interaction between MIC1 and MIC4 with the host cells triggers to respond immune cells. MIC1 and MIC4 were later evidenced as TLR2-binding ligand and TLR4-binding ligand. The two MIC molecules of MIC1 and MIC4 elicit IL-12 gene expression in myeloid immune cells. T. gondii MIC1 and MIC4 proteins enhance the expression of IL-12 from adherent cells of splenocytes. Substantially, immunization with the lactose-binding recombinant MIC1 or recombinant MIC4 prevents T. gondii infection in mice. MIC1 and MIC4 activate DCs and macrophages through TLR2 and TLR4 in a way of sugar recognition. They contain CRDs. The MIC1 and MIC4 recognize the N-glycans linked to TLR2 and TLR4. MIC1 is versatile in changing oh host cells for T. gondii infection competency. MIC1 and MIC4 immunomodulate the hosts for Th1 protective behavior. The Th1 cells express IL-12, and the produced IL-12 activates NK/T cells and consequently expresses IFN-γ in Th1 cells and contributes to host protection against intracellularly infected pathogens. This MIC1 and MIC4 activate TLR axis signaling pathway to elicit proinflammatory cytokine release in macrophages and DCs. MIC1 and MIC4 immunization protects against T. gondii infection, due to effects of the microneme MIC proteins such as MIC3, MIC6, MIC8, MIC11, and MIC13 [154]. In molecular level, MIC1 and MIC4 proteins have distinct carbohydrate recognition domains (CRD) [150, 155, 156]. For the IL-12 expression in response to several infections, the innate immune receptors of TLR2 and TLR4 are selected. Especially, TLR2 Leu-rich repeat domain in the extracellular region consists of four N-glycans, and TLR4 contains nine N-glycans [157]. MIC1 and MIC4 can recognize the TLR2 and TLR4 N-glycans on innate immune cells such as APCs and APCs which produce IL-12. Thus, the first step of the parasite infection is that MIC1 and MIC4 bind to TLR2/TLR4. MIC1/MIC4 target TLR2/TLR4 through the lectincarbohydrate interactions or PCI. Among them, the MIC1 lectin is more competent for T. gondii infection and virulence. MIC1 binds to terminal α2,3-SA residue attached to β-Gal [141, 158], while MIC4 binds to terminal β1,4- or β1,3-Gal [150, 151, 159]. Binding of MIC1 or MIC4 activates immune cells. T. gondii molecules activate TLR signaling [154, 160]. In binding specificity of MICs to TLR2, MIC1 binds to the second, third, and fourth numbers of its TLR2 N-glycans, whereas MIC4 specifically recognizes the third number of TLR2 N-glycans. During TLR2 and lipopeptide agonist interaction, heterodimers between TLR2 and TLR1 or TLR6 are formed. Consequent heterodimerization complex increases in the number of accessible TLR2 agonists and potentiates the multiple association with

6.4 How Are Pathogenic Bacteria Recognized by Receptors of DCs of the Host. . .

289

co-receptor CD14, GPI-anchored protein, and B scavenger receptor CD36. CD14 is the known molecule in LPS binding of TLR4 ligand. The CD14 as a co-receptor associates with TLR4 and consequently complexes with LPS, and CD14 recognizes Mycobacterium tuberculosis lipoproteins by TLR2. This event enhances the TLR2mediated signaling. The MIC1 and MIC4 lectin domains bind to α2,3-sialyl-LacNac and β1,3- or β1,4-GalNAc of N-glycans in host surface molecules [150, 151], respectively. MIC1 and MIC4 lectin domains bind to N-glycans linked to the ectodomain parts of TLR2 and TLR4 in host macrophages and DC for IL-12 release. Pathogen lectin binding to TLR2 N-glycans elicits signaling which is well reported in the typical lipopeptide agonists. TLR bindings to PAMPs elicit receptor dimerization and consequently associate with TIR-1 domains towards NF-κB-involved synthesis of chemokines, proinflammatory cytokines, and type I IFNs. RAW264.7 macrophages treated with MIC1 or MIC4 exhibit NF-κB, and the response resembles with the PAMP action. Binding of MIC1 or MIC4 to homodimeric or heterodimeric TLR2 elicits Myd-associated endosome complex. TAK1 activates the canonical MAPK and NF-κB signalings for the IL-12 expression by MIC1 and MIC4. BMDMs incubated with the TAK1 inhibitor and induced by microneme MIC proteins block the IL-12 expression; thus, canonical NF-κB activation, but not non-canonical, is relevant for the MIC1- or MIC4-activated response in cells. For the mechanism to address the TLR role in T. gondii infection, MIC1 or MIC4 elicits formation of TLR2/TLR1 as a heterodimer and heterodimer TLR2/TLR6. TLR2involved heterodimerization stimulates MIC response in TLR2-expressing HEK cells. This is different from the results obtained from Mycobacterium leprae binding to TLR2 homodimer or TLR2/TLR1 heterodimer. Ligand binding to TLR ectodomain induces receptor dimerization.

6.4.4.2

T. gondii GPI-Anchored Protein Recognition of TLR2, TLR4, and Galectins of Host APCs

GPI-anchored proteins are mainly present on the T. gondii tachyzoite surface. Second, GPI-anchored proteins bind to TLR2 and TLR4 as well as galectins on the host APCs [159]. GPI-anchored proteins are mainly present on the T. gondii tachyzoite surface. T. gondii GPI glycans and lipids elicit the TNF-α releases in macrophages through TLR2 and TLR4 in the NF-κB signaling when exposed to T. gondii GPIs. Although CD14 is also involved in TLR2-mediated macrophage behavior against Trypanosoma cruzi GPIs [158], T. gondii GPIs do not affect the CD14 responses during the TNF-α expression in macrophages [161]. Diacylglycerols derived from the T. gondii GPIs cleavage can activate macrophages to produce TNF-α through TLR2 and TLR4. But only the TLR2 is activated by GPIa or the whole GPIs. In contrast, CD14 is not involved in TNF-α expression during T. gondii GPI treatment [161]. T. gondii GPIs act as ligands of galectin-3, where galectin-3 has long glycan-binding sites and binds to a broad range of carbohydrate structures. T. gondii parasite expresses six GPIs on surface. GPIs III and VI contain a GalNAcβ1,4 linked to Man residue of the core glycan. GPI-I,

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GPI-II, GPI-IV, and GPI-V contain an additional Glcα1,4 linked to the GalNAc residue. Binding of GalNAcβ1,4 carbohydrates is unusual to galectins, as galectin-3 only binds to GalNAcβ1–4GlcNAc carbohydrates produced by S. mansoni strain [162]. Galectin-3 associates with its binding to the N-acetyl group in GalNAc O-2 position, but other galectins do not. Lactose prevents galectin-3 recognition with GalNAcβ1,4GlcNAc structure. Galectin-3 binds to the polygalactose (Galβ1,3)n structure seen on Leishmania major lipophosphoglycans, but galectin-1 does not recognize the L. major. Thus, polygalactose structure recognizes the galectin-3 binding site. The polygalactose species are not present in Leishmania donovani strains. Therefore, galectin-3 differentially recognizes the two parasites. However, GPI species present on virulent T. gondii and nonvirulent T. gondii strains are different in their structures, but galectin-3 binds to all the GPIs. The diacylglycerol species linked to the T. gondii GPIs can recognize galectin-1. Galectin-1 recognizes Davanat, having β1,4-D-Man residues and D-Gal residues α1,6 linkage [163]. The β-Gal-binding domain still holds the galectin-1-Davanat complex. Galectin-1 is the tissue plasminogen activator (TPA) receptor. Binding of TPA to galectin-1 is stronger than to galectin-3 [164]. This indicates that galectins are not always binding to β-Gal. GPI-AP species produced by T. gondii strains act as ligands for galectin-3 and induce TNF-α expression in macrophages by galectin-3. Galectin-3 is a TLR-related molecule responding to the T. gondii GPIs. T. gondii GPI-elicited TNF-α expression is mediated by TLR2 and TLR4 in macrophages [161]. S. mansoni- or T. gondiiinfected mice, which are galectin-3 deficient, show high Th-1 cell-mediated immune responses and reduce inflammatory response [165]. The galectin-3 deficiency produced high levels of Th1 cytokines, because galectin-3 controls Th1 cytokine production. Galectin-3-deficient macrophages express lowly TNF-α upon Candida albicans treatment [166]. Galectin-3-deficient macrophages can not produce TNF-α during treatment with T. gondii GPI species. Most immune cells, except for macrophages, highly produce inflammatory Th-1 cell cytokines in galectin-3-negative KO animals. The galectin-3 binds to N-glycans in TLRs [167]. The surfaced expression of TLR2 is increased in the galectin-3 null macrophages, which are negative for the galectin-3 expression [168]. Galectin-1 recognizes protozoan parasites. Expression of the endogenous galectin-1 is enhanced by T. cruzi and also in cardiologic organ shown by chronic Chagas disease [169]. The T. cruzi adhesion to smooth muscle cells or muscle cells of the heart is regulated by GPI, and heart galectin-1 enables parasite invasion [170]. In the human cervical cells, galectin-1 expressed on the epithelial cells acts also as the protozoa Trichomonas vaginalis receptor through binding to parasite lipophosphoglycans. Thus, host galectin-1 attaches to T. gondii. During Candida albicans infection, galectin-3, which binds to β-Gal residues, is associated with TLR2 on PMA-induced human THP-1 macrophage differentiation, and galectin-3 is co-expressed with TLR4 on macrophages derived from BM [171]. The galectin-3-recruited effector cells protect against parasite infection. Indeed, galectin-3 is expressed on neighbored egg cells or worms in S. mansoni infection [162]. Galectin-3 is specifically co-localized with carbohydrate structure of GalNAcβ1,4GlcNAc seen on the egg surfaces to bind to GalNAcβ1,4GlcNAc and

6.4 How Are Pathogenic Bacteria Recognized by Receptors of DCs of the Host. . .

291

subsequent macrophagic phagocytosis. Thus, GalNAcβ1,4GlcNAc is a molecular pattern of parasites in immune recognition by galectin-3. For the galectin-3-deficient mouse infected with S. mansoni strain, their egg sizes are reduced [172]. Galectin-3 deficiency also influences the cell numbers of the spleen-resident B cells and T cells in S. mansoni-infected mouse but not influence the DC cell number. In mice tissues, T. gondii infection elicits galectin-3 expression. T. gondii-infected mouse with galectin-3 deficiency exhibits diminished inflammation responses in tissues. Galectin-3-deficient mice showed high mortality with deficient macrophages and neutrophils in the peritoneal region, while galectin-3-expressing mice, each T. gondii-infected, showed high survival. Galectin-3 modulates through interference with the prolonged life span and neutrophil activations in the T. gondii infections [173]. TLR followed MyD88-deficient mice are easily infected with T. gondii [154] with eliciting innate immune response. From T. gondii, several TLRs agonist candidates are exemplified to date. Profilin has been suggested to be the mouse TLR11-specific ligand and TLR12-specific ligand [174] and also as human TLR5specific ligand [175]. GPI-anchored proteins have also been regarded as TLR2- and TLR4-specific ligands [161]. In addition, certain parasite nucleic acids are known as TLR7 and TLR9 ligands [174]. Until now, the known agonists recognized by T. gondii TLRs include murine TLR11 and TLR12 ligands as well as human TLR-5 ligand and profilin. In addition, human GPI-APs are the ligands for TLR2 and TLR4 activation, and DNAs are for TLR7/TLR9 recognition. MIC1 and MIC4 are ligands of TLR2 and TLR4. In MIC-TLR2 binding, MIC1 binds to the first, second, and fourth N-glycans linked to the receptor, while MIC4 recognizes the third N-glycans linked to TLR2. TLR2 binds to lipopeptide. TLR1–TLR2 or TLT1–TLR6 heterodimerization is formed. TLR co-receptors such as GPI-AP CD14 and B scavenger receptor CD36 also enhance TLR2 signaling. Pathogen and plant lectin binding to glycans elicit their signalings. Like non-canonical ligands including cationic lipids and nickel ions, lectins help the homodimerization to the receptor ectodomains [176]. The TLR immune cells response has been shown in the ArtinM, a plant lectin [177], which is a Man recognition lectin. The lectin binds to the TLR2 N-glycans and its CD14 co-receptor [178]. The first N-glycan-site is found on the LRR solenoid. The second and third N-glycan sites are located on the LRR concave surface. The fourth is located on the LRR16 of the inner surface [157]. TLR2 heterodimerizes with TLR1 or TLR6. ArtinM does not prevent the heterodimerization responses [178]. ArtinM lectin specific for the tri-Man core in N-glycan-induced macrophages expresses IL-12 through ArtinM interaction with N-glycans on TLR2 and CD14. The ArtinM-TLR2 N-glycan or ArtinM-CD14 N-glycan membrane complex undergo cell signaling [179]. CD14 and CD36 are associated with MIC1- or MIC4-involved TLR2/TLR1 or TLR2/TLR6 signaling. Without CD14 or CD36 co-receptors, the TLR2/TLR1- or TLR2/TLR6-expressing HEK293T cells are activated by the MIC protein, through IL-8 and NF-κB signaling [180]. Apart from the above parasite molecules to interact with host cells, some T. gondii proteins TLR-dependently stimulate innate immune cells, but without sugar interaction. For example, profilin (TgPRF) is essentially involved in the

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6 Innate Immunity Via Glycan-Binding Lectin Receptors

T. gondii parasite’s gliding motility via actin polymerization, and T. gondii profilin binds to TLR11 [181] and TLR12 [182, 183]. Also, T. gondii parasite RNA binds to TLR7 of the host cells, and DNA binds to TLR9 as ligands [183]. T. gondii-derived GPIs activate TLR2 and TLR4 [184]. All of the TLRs induced by the T. gondii ligands eventually culminate in MyD88 pathway stimulation towards IL-12 release. Some other T. gondii proteins elicit the proinflammatory IL-12 release, without cooperation of TLRs. In fact, GRA-7 molecule, known as the dense granule protein-7, elicits the MyD88-NF-kB axis signaling towards IL-6/IL-12/TNF-α release [185]. MIC3 elicits TNF-α release and M1 macrophage polarization [186], but T. gondii type II strain GRA15 elicits NF-kB towards IL-12 release. The T. gondii GRA24 elicits the p38 MAP kinase autophosphorylation and downstream signaling towards proinflammatory cytokine and chemokine release. In contrary, T. gondii TgIST prevents IFN-γ release through inhibition of STAT1-involved proinflammatory gene expression. Thus, T. gondii has both induction and suppression activities against the host immunity to control T. gondii pathogenesis in hosts [187]. TLR-exogenous ligand recognition opens new ways for the therapeutic drug design. Lectins are used as TLR agonists to treat infections or tumors in immunocompromised patients. Lectins are also used as adjuvants to boost Th1 and Th17 responses. Paracoccin (PCN) lectin of Paracoccidioides brasiliensis also elicits immune responses through TLR4 and TLR2 N-glycan recognition. The TLR2 N-glycans elicit the PCN response [188]. PCN targets the fourth N-glycan on TLR2. The TLR2 N-glycans in numbers 1 to 4 are attached to Asn116, Asn199, Asn416, and Asn442.

6.5

Pathogen Recognition and Adaptive Immune Responses in Acquired Immunity

Although vertebrates such as mammals are often exposed to infectious pathogens, relevant resistant mechanism(s) are normally explained through the innate immunity and continued adaptive immunity. The two innate and acquired immunities differ functionally in their actions. The innate immune-specific responses are classified to an indeed inborn immunity or the first defense line immunity, which is also regarded as a dispensable defense line. From the recent studies, it has also been argued to have the adaptive immunity-like activity, as it is also distinguishable self-antigens from non-self-antigens as well as activates adaptive immunity. The immune system recognizes conserved molecular pattern structures on the pathogen surfaces such PAMPs, where PAMPS include extracellular components of microbes such as cell wall molecules and DNAs. Molecular recognition is mediated by transmembrane or soluble receptors, in which germline subsets encode the PRR proteins. The bestdefined receptors are the TLRs and the CLRs. Upon engagement of PRRs with PAMPs, immune cells produce humoral mediators of cytokines, chemokines, or

6.5 Pathogen Recognition and Adaptive Immune Responses in Acquired Immunity

293

complement elements to eliminate the pathogen and to initiate the adaptive responses. PRRs recognize microbial elements as well as structure-mimic shapes of damaged cells of hosts. Endogenous ligands for PRR recognition include DAMPs, which are served for the contextual cell death and damage [189]. DAMPS and PAMPs induce proinflammatory and anti-pathogenic signaling for innate myeloid cells, consequently inducing the adaptive immune response [190]. The innate myeloid cells detect microbial products and respond to adaptive CD8 CTLs. The CD8 CTLs strictly control pathogen infections like virus. Naive CD8 CTLs initially respond to molecules alarmed by danger signals such as molecular patterns of dsRNA or LPS. CD8+ T cells contact infected host cells for pathogen peptides presented by MHC-I and target cell proteins, and memory T cells remain when infection is cleared. Maturation of DCs triggers to initiate adaptive response mediated by T cell immunity [191]. PRRs mediate cytokine induction and co-stimulatory molecule expression, as well known for TLRs [189]. The fundamental role of DCs is to load microbial peptides presented to MHC-I or MHC-II receptors, as a type of T cell antigenic presentation [192, 193]. For another role of DCs, they additionally express surfaced co-stimulatory receptors of CD80, which is a B7–1. In addition, another CD86 known as a B7–2, which activate T cell function, is expressed [194]. The third role of DCs is to express cytokines like IL-12, which upregulates function of adaptive immune cells [195]. Collaboratively, the multiple roles of DCs enable naive T cells to differentiate to the effector cell or memory cell types of T cells [191]. For example, upon microbial pattern interaction, TLRs form the myddosome known as cytoplasm supramolecular organizing center (SMOC), and the SMOC activates proinflammatory NF-κB/AP-1 factors [196]. The NF-κB/AP-1 activates adaptive immunity such as T cell activation. Therefore, the innate immunity stimulates the adaptive immune response basically by action of the APCs. Professional DCs as an APC process and present immune-stimulatory peptides on MHC-I molecule or MHC-II molecule. By MHC contexts, regular T cells and Tregs propagate or regulate immune responses. Proteasome can process antigens and present the fragments on MHC-I for CD8 CTLs [197]. T cells are present in a naive functional cell type state prior to antigen interaction [198]. Antigen-exposed CD8 CTLs directly recognize and kill transformed cells or infected cells via MHC-I action [199]. CD8 T cells also express cytokine IFN-γ. Another subpopulation, named NK T cells, utilizes αβTCR chains to target cells and produce cytokine [200]. NK T cell forms contain semi-invariant TCRs to recognize lipid-related antigens rather than protein antigens. MHC-2loaded peptides are presented to CD4 T cells [197, 198], where naive CD4+ T cells are differentiated to effector cell types such as Th-1/Th-2 and Th-17 cells. Two IL-12/IFN-γ cytokines stimulate naive CD4 T cells to initiate the differentiation to Th-1 cells via costimulation with TCR [198, 201], indicating the essential roles of IL-12 and IFN-γ for genesis of the effector Th-1 cells. IL-4 through costimulation

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with TCR induces differentiation of the naive CD4+ T cells to Th-2 cells as the effector cell types. The differentiated Th-1 and Th-2 cells are important for the parasites including helminths and allergy. Then, Th-2 cells express its type 2-related responses through release of the type 2 IL-4/IL-5/IL-13 cytokines [200]. On the other hand, cytokines IL-6/TGF-β with TCR costimulation activate the naive T cells to differentiate into Th-17 cells [202]. The Th-17 type differentiation from the naïve T cells requires IL-1 when fungal-infected conditions are associated with autoimmune responses. Eventually, the Th-17 cells express IL-17 [203]. After differentiation, various effector T cells are phenotypically shifted to the memory T cell types that are ready to reactivate in an antigen-dependent manner upon pathogenic reinfection [204]. The γδ-T cells use the TCR γ and δ chains, which are opposite to the TCR αβ chains constituted for all regular T cells. Such T cells express IL-17 and IFN-γ [205]. Apart from T cell subpopulations, B cell subpopulations can occupy the acquired immunity, recognizing three-dimensional and conformational epitopes of antigens [206]. Specific subpopulations of B cells are generated through hypermutation of the somatic chromosome with class switch recombination adjusted for antigen-B cell receptor interaction towards antibody effector function. Such adapted B cell receptors are secreted as antibodies upon differentiation to plasma cells. The antibodies indicate humoral immunity [207]. Adaptive immune responses protect against many pathogens, but not all. CD+ T cells lead to the adaptive responses to both intracellular and extracellular pathogens because the naive CD4+ T cells are differentiated into the effector-type T cells such as Th1/Th2. The CD4+ T cells provide vast help to other lymphocytes by cytokines and co-stimulatory molecules and generate antibody responses to pathogens with memory B and plasma cells with long life span. The CD4+ T cells do not induce the CTLs against intracellular pathogenic viruses. However, the CD4+ T cells induce to differentiate the memory CD8 T cells towards expansion during secondary exposure to the pathogen. CD4+ T cells are needed to eliminate chronic viral infections. Using secreted cytokines, effector CD4+ T cells can invite monocytes and peripheral neutrophils to the infection sites for inflamed reaction [208]. CD4+ T cells regulate dampening immune responses through the thymus-derived Tregs or antiinflammatory IL-10 cytokine [209]. 1. CD8 T cells---Cytotoxic CD8 CTLs---–Kills virus infected cells or viruses. 2. CD4 T cells are classified into three subsets: ---T helper 1 cell (Th1)----IFN-r—Intracellular pathogens/mycobacteria ---T helper 2 cell (Th2)----IL-4—Parasites ---T helper 17 cell (Th17)---IL-17—Extracellular bacteria/fungi NK T cells---Lipid antigens.

6.6 Galactose-Specific C-Type Lectin: Two Major ASGPR and Macrophage Galactose. . .

6.6

295

Galactose-Specific C-Type Lectin: Two Major ASGPR and Macrophage Galactose Lectin (MGL) in the Human

Carbohydrate recognition involves cellular homeostasis in the body by carbohydrate-lectin interaction. SAs act as biological masks. SA is a survival indicator of circulating blood cells and glycoconjugates. STs catalyze SA transfer using the donor substrate of CMP-SA to acceptor substrate in order to terminate the stable negative charged sialic acids and consequently protect them from the apoptosis and clearance. Human C-type lectins are representatively mediating the mission, following cellular clearance events such as endocytosis [210]. This leads to cell-cell interactions because SAs are served as specific lectin ligands of selectins and Siglecs. Circulating blood senescent cells are specifically eliminated by the scavenging machinery of the host cells, which make clear and induce apoptosis [211]. To perform the roles in the cell level, they are expressed on the DCs and macrophage surfaces. Disappearance of surfaced sialic acids accelerates the cell clearance, as in elimination of erythrocytes and plasma glycoproteins by β-Gal recognition. It has been suggested the SAs have dual roles as biological masks and binding sites [212]. For example, the asialo-glycoprotein receptor (ASGPR) known as “Ashwell receptor” is present in hepatocytes [213]. The exposed β-Gal residue which SAs are removed is the ASGPR target. The ASGPR is different from the Gal-binding receptors such as soluble galectins that bind to Gal residues seen on surfaces of cells [214, 215]. Galectins prevent endocytotic internalization of the surfaced proteins of cells in the immune system [216]. Most galectins prefer LacNAc disaccharide ligands as the terminal β-Gal residues without SA residues. However, galectin8 and galectin-9 prefer to bind SAα2,3-LacNAcs [217]. Two major Gal-recognizing CTLs are lectin ASGPR and MGL known in the human [218]. The recognition allows the phagocytosis by the lectin ASGPR [219]. ASGPR-1 is generally expressed by liver Kupffer macrophage cells, liver hepatocytes, and liver sinusoidal endothelial cells and recognizes galactose on human platelets when Neu5Ac species are cleaved off by human sialidases (NEU1–4). SA species removal from the cells leads to exposure of subjected glycans that are then recognized, bound, and interacted with galactose-binding lectin receptors of macrophages such as ASGPR-1. Such status induces phagocytosis of the used human platelets as a life span. C-type lectins of DC-SIGN and Man receptor (MR or CD206) consist of the specific “Glu-Pro-Asn” motif (EPN motif) triamino acids in the CRD region. They preferentially recognize Man residue, L-Fuc residue, and GlcNAc residue [220]. MGL present on the macrophage and DC surfaces stimulate T cell signaling. The mechanistic signaling is further described in the other section [221]. Masking of sugar chains by SA species blocks interaction with the ASGPR, which is an abundant lectin receptor present on hepatocytes and macrophages. ASGPR as a liver-specific C-type lectin is also present in sinusoidal surfaces of hepatocytic cells. The major role of the ASGPR is clearance of apoptotic cells and

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6 Innate Immunity Via Glycan-Binding Lectin Receptors

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Fig. 6.5 Recognition mechanism of macrophage against apoptotic cells in blood and hepatic regions

vascular homeostasis [222], through binding to terminal sugars of Gal/GalNAc residues, which are predominantly present in senescent cells and desialylated carbohydrates of blood glycoproteins and platelets in circulatory track [223]. Hepatic macrophages or hepatocytes recognize apoptotic cells in blood and hepatic regions using poly-N-acetyllactosamine-type glycan receptor or ASGPR, as illustrated in Fig. 6.5. The examples are elimination of erythrocytes and platelets [224, 225], as nonsialylated glycan chains are involved in clearance of senescent blood cells [226, 227]. As β-Gal residue serves as the internal ASGPR ligands, the appearance of the Gal residues upon bacterial infection results in septic condition [228]. The ASGPR recognizes and removes the exposed carbohydrates, which have the β-Gal and GalNAc residues. In addition, they also do not recognize the SAα2,6GalNAc and SAα2,6Gal [229, 230]. Large carbohydrate ligands are endocytosed through the ASGPR present on macrophages [231]. As the reverse demonstration of the role of ASGPR in mice, the ASGPR-depleted macrophages in experimental mice exhibit the level of prolonged circulation of ST3Gal-IV-deficient platelets. Hepatocytes mediate the platelet clearance, which are deficient in sialylation of platelets. Hepatic ASGPRs are directly involved in clearance of cells and desialylated glycoproteins in sera. In addition, hepatic macrophages also endocytose senescent cells for clearance from plasma and desialylated platelets by trans-sialidases or sialidases of bacterial pathogens [232]. The St3gal4 ST gene-deficient mice (ST3Gal-IV
/
mouse)

References

297

expose the terminal Gal residues on cells, resulting in cell clearance by ASGP receptor of macrophages, as the ST3Gal-IV-deficient platelets are eliminated by hepatic macrophages [211]. Depletion of macrophages rather increase the survival rate of the ST3Gal-IV
/
platelets in wild-type mice [228]. ASGPRs expressed in both hepatocytes and macrophages bind to desialylated platelets and clear the defected platelets in sialylation. Compared to another soluble lectin of MGL, the ASGPR has been isolated much earlier than the MGL [233]. Normal ASGPR protein is a heterogenous oligomer, which contains two distinct H1 and H2 subunits [234]. ASGPR ligands are known to be the Gal residue linked to the tri-antennary and tetra-antennary structures of N-glycans [235]. B cells produce a BCR (sIgM) that binds to glycans that enter the tolerization status and are normally eliminated before leaving from the bone marrow. Host SAs can be regarded as “immunosuppressive” for the hosts.

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220. Lee RT, Hsu TL, Huang SK, Hsieh SL, Wong CH, Lee YC. Survey of immune-related, mannose/fucose-binding C-type lectin receptors reveals widely divergent sugar-binding specificities. Glycobiology. 2011;21:512–20. 221. van Kooyk Y, Ilarregui JM, van Vliet S. Novel insights into the immunomodulatory role of the dendritic cell and macrophage-expressed C-type lectin MGL. J Immunobiol. 2015;220:185– 92. 222. Grozovsk R, Begonja AJ, Liu K, Visner G, Hartwig JH, Falet H, Hoffmeister KM. The Ashwell-Morell receptor regulates hepatic thrombopoietin production via JAK2-STAT3 signaling. Nat Med. 2014;21:47–54. 223. Hoffmeister KM. The role of lectins and glycans in platelet clearance. J Thromb Haemostasis. 2011;9:35–43. 224. Kotze HF, van Wyk V, Badenhorst PN, Heyns AD, Roodt JP, Lotter MG. Influence of platelet membrane sialic acid and platelet-associated IgG on ageing and sequestration of blood platelets in baboons. Thromb Haemost. 1993;70:676–80. 225. Greenberg J, Packham MA, Cazenave JP, Reimers HJ, Mustard JF. Effects on platelet function of removal of platelet sialic acid by neuraminidase. Lab Investig. 1975;32:476–84. 226. Bratosin D, Mazurier J, Tissier JP. Cellular and molecular mechanisms of senescent erythrocyte phagocytosis by macrophages: a review. Biochimie. 1998;80:173–95. 227. Sorensen AL, Hoffmeister KM, Wandall HH. Glycans and glycosylation of platelets: current concepts and implications for transfusion. Curr Opin Hematol. 2008;15:606–11. 228. Grewal PK, Uchiyama S, Ditto D. The Ashwell receptor mitigates the lethal coagulopathy of sepsis. Nat Med. 2008;14:648–55. 229. Park EI, Baenziger JU. Closely related mammals have distinct asialoglycoprotein receptor carbohydrate specificities. J Biol Chem. 2004;279:40954–9. 230. Steirer LM, Park EI, Townsend RR, Baenziger JU. The asialoglycoprotein receptor regulates levels of plasma glycoproteins terminating with sialic acid alpha 2,6 galactose. J Biol Chem. 2008;284:3777–83. 231. Rensen PC, Sliedregt LA, Ferns M, et al. Determination of the upper size limit for uptake and processing of ligands by the asialoglycoprotein receptor on hepatocytes in vitro and in vivo. J Biol Chem. 2001;276:37577–84. 232. Tribulatti MV, Mucci J, Van Rooijen N, Leguizamon MS, Campetella O. The trans-sialidase from Trypanosoma cruzi induces thrombocytopenia during acute Chagas’ disease by reducing the platelet sialic acid contents. Infect Immun. 2005;73:201–7. 233. Weigel PH, Yik JH. N-Glycans as endocytosis signals: the cases of the asialoglycoprotein and hyaluronan/chondroitin sulfate receptors. Biochim Biophys Acta. 2002;1572:341–63. 234. Tolchinsky S, Yuk MH, Ayalon M, Lodish HF, Lederkremer GZ. Membrane-bound versus secreted forms of human asialoglycoprotein receptor subunits. Role of a juxtamembrane pentapeptide. J Biol Chem. 1996;271(24):14496–503. 235. Stokmaier D, Khorev O, Cutting B, Born R, Ricklin D, Ernst TO, Böni F, Schwingruber K, Gentner M, Wittwer M, Spreafico M, Vedani A, Rabbani S, Schwardt O, Ernst B. Design, synthesis and evaluation of monovalent ligands for the asialoglycoprotein receptor (ASGP-R). Bioorg Med Chem. 2009;17(20):7254–64. 236. Fu H, Gerhardt JM, McDaniel B, Xia X, Liu L, Ivanciu A, Ny K, Hermans R, Silasi-Mansat S, McGee E, Nye T, Ju MI, Ramirez P, Carmeliet RD, Cummings F, Lupu LX. Endothelial cell O-glycan deficiency causes blood/lymphatic misconnections and consequent fatty liver disease in mice. J Clin Investig. 2008;118:3725–37. 237. Berger EG. Tn-syndrome. Biochim Biophys Acta. 1999;1455:255–68. 238. Solinas G, Schiarea S, Liguori M, Fabbri M, Pesce S, Zammataro L, Pasqualini F, Nebuloni M, Chiabrando C, Mantovani A, Allavena P. Tumor-conditioned macrophages secrete migrationstimulating factor: a new marker for M2-polarization, influencing tumor cell motility. J Immunol. 2010;185:642–52.

Chapter 7

Sialic Acid-Binding Ig-Like Lectins (Siglecs)

During the mid-1980s, in vitro rosette formation between macrophages and sheep erythrocytes was reported; however, sialidase treatment on erythrocytes abolished the rosettes [1]. Rosette formation is based on sialic acid-binding receptors expressed by macrophages. Later, these receptors were identified to be sialoadhesin that binds to sialic acids as ligands [2]. The SA-recognizing property of the innate immune system of vertebrates led to the discovery of SA-bearing glycans as the ligands for lectins. The binding of SA ligands to Siglecs with immune inhibitory properties leads to suppressed immune functions. The SA-to-Siglec recognition implicates immune activation to limit self-recognition and to destroy the defense mechanism in hosts. Siglecs attenuate ‘self’-inflammatory triggers called DAMPs. During the early 1990s, the first sialyl carbohydrate-binding receptor protein called Siglecs was discovered, for example, Siglec-2 (or CD22) present on B cells. Siglec-1, known as sialoadhesin present on macrophage surfaces, was found. The Ig-like domains of such lectins are different from those of other known C-type lectins or Ca2+-dependent lectins. Among them, I-type lectins are named I-type because they belong to the varied immunoglobulin superfamily of proteins. As I-type lectins share similar characteristics with the immunoglobulin superfamily of proteins, they contain Ig folds, which consist of antiparallel β-sheets. I-type lectins can be broken down into many different regions: V-set, C1 and C2 sets, and ITIM and ITIM-like domains. The V-set domain is the primary site of ligand binding and recognition. The C1 and C2 sets act as spacers and are believed to control the entire length of lectins. ITIM and ITIM-like domains (in some cases, ITAM domain) are essentially tyrosine-based signaling motifs that inhibit (or, in the case of the ITAM domain, activate) downstream signaling and thereby modulate cell activities. More than 16 I-type lectins, such as Siglecs, recognize diverse sialoglycans by immune cells. Even among I-type lectins, there are many different subtypes and the best characterized subtype is called Siglecs. Siglecs are SA-binding lectins and they are the most well-studied I-type lectins. Like any other lectins, Siglecs are composed of many different domains, namely, the V-set, C1 and C2 sets, and mostly the ITIM domains.

© The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2022 C.-H. Kim, Glycobiology of Innate Immunology, https://doi.org/10.1007/978-981-16-9081-5_7

311

312

7 Sialic Acid-Binding Ig-Like Lectins (Siglecs)

In B cells, a sialyl ligand-binding receptor such as CD22 was found as a SA-binding lectin. The CD22 ligand binds α2,6-SAs and not α2,3-SAs [3]. SA is widely distributed in animal tissues. α2,3/2,6 sialic acid linkages are general bonds in connection with Gal or GalNAc. α2,3-SA-linked glycans are recognized by Siglec-4, α2,6-linked sialosides by Siglec-2, and α2,8-sialosides by Siglec-7 [4]. In evolution, Siglecs are divided into two subgroups, namely, Sn/CD22/MAG and CD33rSiglecs. The Sn/CD22/MAG group has a single ortholog in many species, whereas the CD33rSiglecs group is not clear in differentiating between primates and rodents in their clear-cut orthologs. The specificity of ligand binding of the Sn/CD22/MAG group is highly conserved with conserved domain structures and minor variations, whereas that in the CD33rSiglecs group is poorly conserved toward rapid evolution. Interestingly, the Sn/CD22/MAG group exhibits a cell type-specific pattern of expression. In fact, CD22 expression is observed in B cells, MAG expression in glial cells, and Sn expression in macrophages. Siglecs are further subdivided into two different subgroups based on the genome similarity of each Siglec. The first of such subgroups is composed of Siglec-1 (termed “Sn”), Siglec-2 (termed “CD22”), Siglec-4 (termed “MAG”), and Siglec-15. Another subgroup is the CD33-related Siglec group (CD33rSiglecs) and is composed of CD33, Siglec-5, -6, -7, -8, -9, -10, -11, -14, and -16. Siglec-1 is associated with the phagocytic clearance of SA-covered pathogens. The CD33related Siglec group suppresses inflammatory reaction through recognition of ligands present on the cell surfaces of the same cells, which is termed “cis-interactions,” or on cell surfaces of other cells, which is termed “trans-interactions.” Most of them consist of ITIM-bearing Siglec-3 and (CD33)-related Siglec groups. Many of such Siglecs are coupled so that one Siglec has inhibitory effects, whereas the other has activating effects on the same cell. These coupled Siglecs work in tandem to modulate downstream signaling. Most Siglecs comprise one or multiple numbers of ITIMs in the cytosolic region. ITIMs often mediate to counteract activation signals that are triggered by immunoreceptor tyrosine-based activation motif (ITAM)-carrying receptors. In the aspect of signal transducing motifs, the Sn/CD22/MAG group is highly specific for the ITIM expression. hCD33rSiglecs are engaged in innate cells via interaction with sialylated antigens to downregulate the roles of proinflammatory APCs. The role of hCD33rSiglecs in T-cell responses is not yet clearly understood. For example, only CD22 contains ITIMs. However, Sn and MAG both do not contain ITIMs. Only MAG contains a Tyr-based amino-acid sequence, which is quite similar to the known CD33rSiglec sequence. In contrast, the typical CD33rSiglecs consist of the conserved common ITIM sequences and ITIM-like sequences. With regard to the structural aspect, the Siglec groups belong to the type I TM proteins, which contain an extracellular region. The extracellular region comprises an N-terminal SA-recognizing V-set Ig-like domain and multiple 1–16 C2-set Ig-like domains. The cytosolic tail regions of all Siglec forms, but not that of Siglec-1 (Sn), have one or more Tyr residues as signaling motifs. Arginine (Arg97) forms a salt bridge complex with the COOH group of SAs. The Siglec V-set region and the adjacent C2-set region both have cysteines to form disulfide bonds in the V-set region and an

7.1 PolySia and Host Sialic Acids Modulate Host Immune Responses as Pathogenic. . .

313

Table 7.1 Mammalian orthologs of Siglecs in baboons, chimpanzees, humans, mice, and rats Human Sn/Siglec-1 CD22/Siglec-2 MAG/Siglec-4 CD33/Siglec-3 Siglec-5 (OBBP-2) Siglec-6 (OBBP-1) Siglec-7 (AIRM-1) 5iglec-8 Siglec-9 Siglec-10 Siglec-llb Siglec-XII (Siglec-L1, SV2)a NF NF

Chimpanzee Sn/Siglec-1 CD22/Siglec-2 MAG/Siglec-4 CD33/Siglec-3 Siglec-Va Siglec-6 Siglec-7 Siglec-8 Siglec-9 Siglec-10 Siglec-11b Siglec-12 Siglec-13 ?

Baboon ? ? ? CD33/Siglec-3 Siglec-5 Siglec-VIa NF Siglec-8 Siglec-9 Siglec-10 ? NF Siglec-13 ?

Mouse and rat Sn/Siglec-1 CD22/Siglec-2 MAG/Siglec-4 CD33/Siglec-3 Siglec-F NF NF Siglec-E Siglec-G NF NF NF Siglec-H (rata)b

AIRM adhesion inhibitory receptor molecule, OBBP, obesity-binding protein, NF V-set domains, not found a Siglec-like proteins lacking the “Arg residue” for binding b CD33rSiglecs located outside the Siglec gene cluster (modified from Glycobiology (2006) 16, 1R-27R) [5]

S–S bond in each domain. The structural characteristics of SAs affect Siglec recognition. Many orthologs of Siglecs have been found in mammalian species such as baboons, chimpanzees, rats, mice, and humans. The structural orthologs corresponding to their mammalian Siglecs such as baboons, chimpanzees, humans, rats, and mice are summarized (Table 7.1) [5].

7.1

PolySia and Host Sialic Acids Modulate Host Immune Responses as Pathogenic Decoys

SAs are terminally present as monosaccharides found in glycans. Aside from this, polysialic acid (PolySia, PSA) is a linear homopolymer of α2,8-linked sialic acid residues that are repeatedly linked on the cell surfaces of proteins. Polysialic acid consisting of α2,8-Neu5Ac units is an essential nutrient in brain development, morphogenesis, and neural systems [6, 7]. Some immune cells also produce polysialic acids in a subpopulation of DCs [8, 9] and T cells [10, 11], where the polysialic acid is attached to neuropilins. The polySia expression on a NCAM has long been studied in the initial stages of neural progenitor cells, and successfully matured neuronal cells express excess amounts of α2,8-linked poly SAs on the NCAM protein (Fig. 7.1). The role of PSA-NCAM in neuronal genesis has been attributed to its antiadhesion property (Fig. 7.2). In neuronal progression, polySia functions as a contact-dependent differentiation inducer. Therefore, a malformed

314

7 Sialic Acid-Binding Ig-Like Lectins (Siglecs)

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7.1 PolySia and Host Sialic Acids Modulate Host Immune Responses as Pathogenic. . .

315

default expression of polySia indicates the prematuration and dysmaturation of neuronal cells simply due to the absence of polySia. PolySia also increases cell proliferation. In the immune system, it increases the migration level of hematopoietic progenitor cells from the bone marrow to the lymphatic node and the thymus. ST8Sia-IV synthesizes N-linked polySia. PolySia also stimulates DC migration to the lymphoid organ. PolySia is relatively large in size with repeated negative charges due to carboxylic acid, and it acts as a cell–cell interaction regulator and inhibitor of adhesion molecule interaction. For a specific condition, such as growth factor, polySia interaction induces signal transduction. For example, polySia stimulates CCL21-dependent migration of DCs to the secondary lymphoid organ. As NK cells express NCAM (CD56), the cells can migrate to the lymphoid organ. In cancer cells and embryonic development, polySia potentiates the metastatic progression of cancer cells including lung carcinomas, myelomas, neuroblastomas, gliomas, and Wilms’ tumor, as well as trophoblastic movement of fused blastocysts to placental locations. In blastosis, polySia is expressed in the early implantation stage. Ig-like lectins (Siglecs) are also abundantly expressed in plants, fungi, and bacteria. Siglecs also trans-recognize SA ligands, on adjacent, neighboring, and encountering cells, or pathogen-expressing ligands. This event leads to Siglec signaling and endocytosis. A series of SA-dependent adhesion receptors has recognized these SAs present on glycoproteins and glycolipids. Siglecs contain variable C2-set domains in all V-set Ig-like domains of the N-terminal region and subsequent extracellular regions where 17 human Siglecs have been confirmed. The moiety that recognizes sialic acid is present in the V-set Ig-like domain, which has two aromatic residues and one arginine motif conserved in all signals. Sialic acid is abundantly present in various cell membranes and can affect the functional properties of cells (cis and trans) by binding to ligands. Host SAs are usually the binding targets or receptors for viruses, toxins, and some bacteria, indicating PolySia as a decoy. Thus, viruses or sialic acid-binding parasites approach the host mucosal surface to encounter mucin-type glycoproteins. Mucins are highly sialylated glycoproteins. As decoys, mucins protect the organism from pathogens [12]. Thus, loss of O-linked mucins increases colitis diseases [13]. SA-binding pathogens encounter the heavily sialylated glycoproteins before reaching their target. Erythrocytes can be used as another type of decoy for the host due to their non-nuclear cells. Erythrocytes occupy about 50% of the total blood volume. A SA-binding virus, influenza virus, enters into the bloodstream and encounters this extensive cell surface. Hemagglutination defines the binding specificity of viruses. Malarial parasites use SAs for invading erythrocytes [14].

316

7.2

7 Sialic Acid-Binding Ig-Like Lectins (Siglecs)

Sialic Acid Recognition by Siglecs for Self- or Nonself-Antigens

Cell surface sialylation and sialoglycan-recognizing receptors function in immune responses of DCs. Sialoglycosylation is thus considered to be important in DC function and in immunological defense of mammals. Mammalian SAs are substituted at the position of R. Like CLRs, Siglecs such as cell surface GBPs have both positive and negative immune responses. Siglecs are members of an immunomodulatory and transmembrane lectin family expressed prominently on leukocytes. SAs are made up of 9-C saccharides that exist on the cell surfaces of glycolipids or glycopeptides. SA induces a regulatory mechanism with antigens through linkages to SAα2,3, SAα2,6, or SAα2,8 in mammalian cells. Desialylation activates signaling pathways related to self-eating, and this activation enhances the ability of autophagy to lessen apoptotic cells [15]. Therefore, sialic acid is an important marker of the self-immune response system. In the recognition of pathogens by receptors, many distinct molecular events mediate adhesion, migration, interaction, costimulation, and inhibition of cell signaling. Siglecs are cell surface proteins that bind SAs and are mostly found on immune cells. Each Siglec has a unique specificity for sialylated ligands, as each protein mediates a distinct, partially overlapping function. Siglecs recognize glycoproteins and glycolipids from pathogens as cell surface molecules. They were initially studied from a macrophage lectin-like adhesion protein, sialoadhesin, later termed “Sn” [1], and CD22, which is B cell-specific [16]. Later, two other members of the Ig superfamily, CD33 and MAG proteins, were discovered [17, 18]. Currently, the above four Siglecs represent sialic acid-recognizing lectins; however, another type, CD83, is found to be expressed on mature DCs, and CD83 is a specific Siglec type with the capacity of sialidase-sensitive interaction [19]. Immune receptors generally exhibit high maturational structural similarity, having the properties of antagonistic signaling known in coupled receptors. Immune receptors are considered to balance immune homeostasis in tolerance and inflammation. Siglecs, a group of the immunoglobulin superfamily, can bind several types of sialylated glycans. The most wellevolved immune receptors of human Siglecs are reported to have 14 types of Siglecs. Mammalian immunity has evolved to distinguish the “self” from “nonself,” and this contributes to protection from pathogenic and foreign infectious agents. When the transformed tumors are accounted for, the immune system eliminates them because they are recognized as “foreign agents” during immunosurveillance [20]. Until now, various receptors for eliminating these foreign agents have been identified. Foreign agents are treated as nonself-molecules or self-mimicking molecules [21, 22]. Thus, most immune-related cells synthesize a series of Ig family receptors, which inhibit the functions of immune cells in the host. Among molecules that regulate the level of inflammation, the Siglec families are mainly present on the surfaces of leukocytes that bind to sialylated glycans and translate glycan binding into changes in immune cell function. Structurally, the representative inhibitory receptors are the SA-recognizing Ig-like lectin (Siglecs) group (Fig. 7.3). Siglecs, as the

7.2 Sialic Acid Recognition by Siglecs for Self- or Nonself-Antigens

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SA-recognizing lectin family, have been recently defined and predominantly found on immune-related cell surfaces and mediate downstream immunomodulatory signaling through ligand bindings. The C2-type Ig domains of the known Siglecs are apically extended from the surfaces of immune cells because they do not have the glycan-recognition capacity. Different numbers of C2-type domains are found in each Siglec. Siglecs as type 1 membrane proteins carry the N-terminal V-set Ig domain and this domain is the SA recognition site. The N-terminal V-set Ig domain consists of multiple C2-set Ig domains. CD33-related Siglecs have diverse binding properties to their ligands, depending on the host species with sequence similarities in the ExT region and a conserved Tyr-based signaling motif in the cytoplasmic region. Different mammalian CD33-related Siglec species include CD22, MAG, sialoadhesin (Sn), and Siglec-15. They differ in sequence homologies from those of CD33rSiglecs. CD33rSiglec receptors recruit Tyr phosphatases by inhibiting the activation signals, which are mediated by ITAM signaling. Siglecs have α-helix structures. They are type 1 membrane polypeptides with glycoprotein and glycolipid sialic acid-binding capacities. Siglecs contain multiple extracellular Ig-like domains that mediate glycan binding and have short cytoplasmic tails. They are characteristically composed of a V-set Ig-like domain in the N-terminal region, which binds SA residues that are abundantly present in mammals but are relatively low in prokaryotes, and a C2-set Ig-like domain [23]. The first V-set Ig-like domain binds to carbohydrate ligands, and the second Ig-like domain may also contribute to the binding. The N-terminal region is linked to multiple Ig C2-set domains, a TM domain, and a tail in the cytoplasmic region. The number of

318

7 Sialic Acid-Binding Ig-Like Lectins (Siglecs)

C2-set immunoglobulin domains is diverse depending on each Siglec. Siglecs are extended by C2-type Ig domains, which have no SA-recognition capacity. Siglecs have different numbers of C2-type domains. Cell surface Siglecs that are normally masked by the self-SA species via cis-recognition regulate the function of cisligands. Thus, endogenous cis-ligand identification is important for Siglecs to elucidate the immune function.

7.3

Classification of Siglecs

The Siglecs are a family of SA-binding Ig-like lectins that recognizes SA-terminated glycans of glycoproteins or glycolipids. Siglec families have been well established in both humans and mice. Siglecs belong to the type 1 membrane proteins. As expected, they bear a V-set immunoglobulin domain in the N-terminal region, and this domain recognizes SA residues on the sialoconjugates. Siglecs are conventionally grouped into two main groups, depending on sequence and evolution conservation [23]. For example, Siglec-1, -2, -4, and -15 have homologues across species and thus belong to the same group. Siglecs are further classified into two groups: (1) inhibitory Siglecs (Siglec-5, -7, -9, and -10), with the transducing capacity of inhibitory signals in cellular functions by intracellular ITIM domains, and (2) activating Siglecs (Siglec-14), with the activating capacity of cells in Siglec-dependent responses by intracellular adaptor proteins such as DAP12 recruitment. To date, 14 mammalian Siglecs have been reported. Most of them are present in specific cell types on immune-related cells. Immune cells express them on their surfaces. Depending on their finding history, name of Siglecs has been made. Although most Siglecs are negative signaling regulators due to ITIMs, others activate the immune cell function. Structurally, Siglecs belong to the Ig superfamily and are classified into two distinct groups, in which the first is the low-homologous group with 25–35% homology. They are Siglec-1 (Sn), -2 (CD22), and -4 (MAG). The common Siglec forms in mammals are Siglec-1, -2, -4, -15, and the Siglec-3 (CD33)-related family. Among them, CD33 (also named Siglec-3) and its related Siglecs, i.e., Siglec-5, -6, -7, -8, -9, -10, -11, -14, -15, and -16, are involved in the innate immune system [24] (Fig. 7.4). CD22 is a B cell-specific glycoprotein and is a membrane-bound glycan-recognition protein like the BCR complex. Thus, CD22 is a SA-recognizing lectin. In B cells, if α2,6-SAs are ablated, the basic immune functions of B cells are effected. For example, BCR signaling suppression, IgM reduction, and antigen responses in T cell-dependent and T cell-independent mechanisms are completely effected [25]. The second distinct group is the fast-evolving high-homologous group with 50–90% homology and is known as CD33-related Siglecs (CD33rSiglecs). There are mammalian orthologs of Siglec-1, CD22, Siglec4, and Siglec-15 with low homology and similarity. Interestingly, the Siglec family is well conserved between the two mammalian Siglecs, Siglec-1 and -2, and also between the vertebrate Siglecs, Siglec-4 and -15. However, a family of a large cluster, which is located on human chromosome 19q, varies in each different species

7.3 Classification of Siglecs

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Fig. 7.4 Siglec family proteins and expression in humans

[26]. This concept led to a new terminology, “CD33-related Siglecs” (or CD33rSiglecs). CD33rSiglecs include human Siglec-3, -5, -7, -8, -9, -10, and -11, and mouse Siglec-14 and -E, -F, -G, and -H. The second group of Siglecs is highly specific for SA-containing glycans toward α2,3-SA, α2,6-SA, and α2,8-SA linkages. They branch from two subsets. The first one is based on their sequence similarities and the second one is based on their evolutionary conservation. (1) The first group includes Siglec-1, -2, -4, and -15. They are highly homologous across species and are conserved from humans to vertebrates as all mammalian species orthologs. They are related in only 25–30% sequence identities of Siglecs and have definite mammalian orthologs. Siglec-1 is also known as sialoadhesin (or CD169) and is a macrophage-specific adhesion lectin. Siglec-2, also called CD22, is expressed on mature B cell surfaces with sialic acid-binding capacity. (2) The second group includes human CD33 (Siglec-3) and CD33rSiglecs as well as Siglec-5, -6, and -7 (another name: adhesion inhibitory receptor molecule 1, AIRM1), Siglec-8, -9, and -10 (in mouse, Siglec-G), -11, -14, -E, -F, Siglec-H, Siglec-16, and Siglec-17. This group is characterized by rapid evolutionary stages and high species variations [23, 24]. Different from Siglec-1, -2, -4, and -15, CD33rSiglecs have 50–99% similarity and are under rapid evolution through gene conversion, duplication, exon acquisition, and exon loss or exon shuffling. CD33Siglecs have evolved from SIGLEC genes. Many mammals express CD33-related Siglecs than mice and rats, showing a loss of Siglec genes in rodents. Moreover, SD33-related Siglecs are difficult-to-classify orthologs. For example, several mouse CD33-related Siglecs have been discovered. As Siglecs exhibit high structural homology, they are recognized as the “Siglec family.” Siglec-7/-9 and -E show a high sequence similarity with mice Siglec-E, where Siglec-7 binds to α2,8 sialic acid and Siglec-9 recognizes α2,3-linked sialic acid, whereas Siglec-E recognizes both α2,3-SA and α2,8-SA. Siglec-8 and Siglec-F

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Fig. 7.5 Human and rodent Siglecs. Conserved Siglecs are found in mammals. CD33-related Siglecs exhibit evolutionary phenotypes. Eosi, eosinophil; Macro, macrophage; Mono, monocyte; Neur, neutrophil; Oligo, oligodendrocyte; Osteo, osteoclast; DCs, dendritic cells; Placen, placental syncytiotrophoblast; Schw, Schwann cell. Siglec-7/-9 and -E show a high sequence similarity with mice Siglec-E, where Siglec-7 recognizes α2,8-SA and Siglec-9 recognizes α2,3-linked SA, whereas Siglec-E binds to both α2,3-SA and α2,8-SA. Siglec-8 and -F share a similar degree of sequences, where Siglec-8 and -F recognize 60 -sulfo-sLeX during eosinophil apoptosis, inhibiting mast-cell-mediator release in eosinophilic asthma. Similarly, Siglec-10 and -G recognize Neu5Gc in α2,3 and α2,6 linkages (modified from Ref. [27] Brown GD, Crocker PR. 2016. Microbiol Spectr. 4(5)

share a similar degree of sequences, whereas Siglec-8 and -F recognize 60 -sulfo-sLeX during eosinophil apoptosis, inhibiting mast-cell-mediator release in eosinophilic asthma. Similarly, Siglec-10 and -G recognize Neu5Gc in α2,3 and α2,6 linkages, as shown in Fig. 7.5 [27]. Among Siglecs, mouse-specific mSiglec-E was isolated by a yeast two-hybrid search utilizing the SHP-1 gene for the fifth mouse Siglec. CD33rSiglecs contain one or more cytoplasmic ITIMs, allowing them to act as inhibitory receptors to suppress receptor ITAM activation signals through the recruitment of Tyr and inositol phosphatase. CD33rSiglecs are divided into two groups: (i) ITIM-carrying cytosolic domains with a tyrosine phosphorylation site, and as a result recruiting tyrosine phosphatases like SHP-1 and SHP-2 [28–31]. This dephosphorylates phosphor-Tyr on receptor kinases. Thus, this type is called “inhibitory CD33rSiglecs” and negatively regulates hyperreactive innate immune responses on SAMPs, thereby inhibiting unwanted responses such as inflammation in self-tissue damage [31]. Some CD33rSiglecs also have other cytosolic ITIM-like domains. In addition, some inhibitory Siglecs function independently of the tyrosine-based motifs [32, 33]. (ii) Contrary to the inhibitory Siglecs, the other type of CD33rSiglecs is conceptional, i.e., the so-called “activating CD33rSiglecs,” which contain amino acids with positive charges in the transmembrane domain. The

7.4 Evolution of Siglecs, Sialic Acids, and Sialic Acid O-Acetylation as Host. . .

321

activating CD33rSiglecs recruit ITAM-containing adapters such as DAP12. DAP12 recruits the Tyr kinase, Syk, to phosphorylate Tyr residues [34, 35]. Therefore, the N-terminal domains serve to regulate the inhibition or activation properties of CD33rSiglecs. The activating Siglecs seem to progressively evolve over bacteria with inhibitory Siglecs [34, 36].

7.4

Evolution of Siglecs, Sialic Acids, and Sialic Acid O-Acetylation as Host Ligands (Receptors) for Microbes and Innate Immunity

Innate immune responses reflect the resistance against pathogenic infections by sensing PAMPs via signaling for proinflammatory cytokine secretion to provide the first line of defense against foreign agents. Siglecs primarily bind “cis-ligands” sialylated on cell surfaces [37]. If sialyl ligands are surrounded on the surfaces of other cells or other soluble molecules, they can competitively bind to the cis-ligands [38, 39]. The V-set Ig-like domain in the N-terminal region consists of a SA binding site with an Arg residue conserved as the binding site to carboxylate the sialylated ligands [40, 41]. Interestingly, the Arg residue is easily subjected to mutation, possibly creating a new type of Siglecs specific to certain species [34, 41] in a random manner because the Arg residue has been suggested to be mutable more than the other codons. For example, the Arg residue once mutated mutates once again for Siglec-5 and -14 in humans and great apes, thus inching toward an evolutionary recognition between sialylated microbes and Siglecs [34]. Viruses and bacteria as well as bacterial toxins recognize sialylated targets for recognition [42]. A Sia-binding virus uses hemagglutinin and sialidase (neuraminidase) to cleave the same receptor [43]. Viruses are basically defined by the hemagglutinin (H), neuraminidase (N), antigenicity, serology, or RNA genotypes. Some viruses bind to O-acetyl-SAs and cleave off the O-acetyl group [44–46]. Some protozoa also employ SA binding (the malarial merozoite) [47–49]. A bacterial SubAb toxin specifically binds Neu5Gc SAs [50]. In fact, H. influenzae utilizes SAs as a C-sourced energy in the breakdown of Neu5Gc to form pyruvic acid and ManNAc residues. ManNAc is further converted to GlcNAc [51]. Free SA can be used as a signaling factor in some infectious pathogens. Pneumococcus [52] forms biofilms to colonize using SAs. For SAs as a Siglec-binding SAMP [22], bacterial desialylation perturbs self-recognition events and, consequently, increases inflammatory responses by desialyl DAMPS [53, 54]. SAs on certain bacterial polysaccharides are O-acetylated, thus dually affecting the host–pathogen interaction. The O-acetyl Sias reduce recognition by CD33rSiglecs and also enhance immunogenicity [55]. Therefore, O-acetylation helps the bacteria survive under certain conditions, protecting them from microbial neuraminidases or bacteriophage-recognizing proteins. One exceptional case is sialoadhesin where O-acetylation blocks sialoadhesin interaction [56]. This helps the bacterium to avoid phagocytosis [55, 57]. Unlike

322

7 Sialic Acid-Binding Ig-Like Lectins (Siglecs)

selectins, Siglecs recognize Sia C7–C9. O-acetylation of Sia C7–C9 blocks Siglec recognition [56, 58]. Thus, sialic acid O-acetylation modulates Siglec function. O-acetylation in the ganglioside GD3 is known for a melanoma-specific antigen [59]. Although T cells also express O-acetyl GD3 and GD3 has apoptosis-inducing potential, O-acetyl GD3 has antiapoptotic effects [60].

7.5

Microbial Sialic Acid-like Molecules Synthesis and Recognition of Microbial Sialic Acids by DCs and Bacteriophages

Some bacterial pathogens express surface Sias [61]. Bacterial sialic acid biosynthesis has adopted a bidirectional parallel evolution of recruitment and modification of bacterial nonulosonic acid synthesis [62–64] to acquire Sias. Evolved sialyltransferases were independently combined to recreate sialylated glycans. Based on the roles of Sias and SAMPs, microbes synthesize host-like sialyl glycans. Organisms synthesize the ancient family of 9-C saccharides, namely, nonulosonic acids (NulO), including legionaminic acid and pseudaminic acid [64]. Bacteria persisted in ancient nonulosonic acid biosynthesis for the production of vertebratelike Sias [62]. Thus, bacterial Sias include nonulosonic acid (NulO). If bacteria express vertebrate cell Sias, then one must question how the immune system distinguishes sialylated pathogens (or nonulosonic acids), even after maintaining tolerance to self-sialyl ligands. CD33rSiglecs easily bind sialylated pathogens and potentiate endocytosis to capture and internalize the cells [65–68]. However, Siglec1 known as sialoadhesin has no signaling capacity, but due to its size, length, and structure, it phagocytoses them. Siglec-1 has a well-conserved and common specificity for Neu5Ac-α2,3 or Neu5Ac-α2,8, but not for the Neu5Gc species, binding; this property explains this concept. Bacteria escape from bacteriophages by binding to bacterial glycans. In addition, bacteriophages evolve much faster than prokaryotes such as bacteria, as certain bacteriophages evolve to bind to SA-containing bacterial capsules [69, 70]. Gram-negative bacterial membranes are characteristic of O- and K-antigens linked by lipids or free surface polysaccharides. These K- and O-antigens consist of a thick layer to cover the cell surface and are called a capsule. In meningitis, pneumonia, and septicemia-causing bacteria, the K- and O-antigen complex capsules determine the pathogenicity [71]. CPSs shield bacteria from the host immune system. Surface CPSs protect bacteria from bacteriophage infection. On the other hand, CPSs increase the pathogenic capacity by suppressing the function of the host innate immune system. Certain pathogenic CPSs are identical to glycans produced by mammalian host cells, resulting in protection by molecular mimicry in the neuroinvasive Neisseria meningitidis serogroup B and E. coli K1. The capsules of both organisms contain α2,8-poly Sia that is abundant on human cell surfaces [72]. PolySia is an α2,8-NeuAc chain polymer that is present on the cell surfaces of eukaryotes and also bacteria. CPSs also serve as receptors for specialized

7.5 Microbial Sialic Acid-like Molecules Synthesis and Recognition of. . .

323

bacteriophages [73]. Bacterial CPSs often interact with bacteriophages. Capsule phages contain spikes and function as an adsorption receptor and as a capsuledegrading enzyme [74]. The degrading enzymes also bind to the capsular polysaccharide. The CPS of enteropathogenic E. coli K92 is composed of poly SAs with SAα2,8 and SAα2,9 linkages. For the E. coli Kl polysialic acid capsule, certain Kl phages recognize the capsular polysialic acid as a cell surface receptor by their capsule-degrading endosialidase. For example, group B Streptococcus (GBS) has evolved to form distinct polysaccharide-producing types, terminating with the sialylated trisaccharide [75]. The GM1-binding cholera toxin B is encoded by nonlysogenic phage in V. cholerae [76]. As one of the viridans streptococci species, the specific strain S. mitis causes infective endocarditis. The bacteria bind to human platelets through the bacterial proteins Pbl-A and Pbl-B, which cause pathogenic disorders [77]. The bacteria recognize the sialyl-oligosaccharides present in host cells and colonize the oral cavity by bacterial adherence to salivary glycoproteins [78]. The SA species linked to the host glycolipids and glycoproteins are used as bacteria-binding receptors for bacterial adhesion. The bacteria attach the SAs of the GP1b glycoprotein present in platelets [79]. As gangliosides serve as receptors for many bacteria or toxins including the cholera toxins, H. influenzae, H. pylori, and N. meningitidis [78], two gangliosides (GM3 and GD3) are predominantly expressed in the membranes of human platelets [80, 81]. The GM3 ganglioside is enzymatically converted to the disialyl form of GD3 in the early step of activation and differentiation of platelets before calcium homeostasis and granule release. The GD3 ganglioside directly participates in FcλRIIa receptor signaling in human platelets [82]. The interaction of platelets with the SF100 protein is predominantly induced by the bacterial proteins Pbl-A and Pbl-B, which target the SAα2,8-linked membrane GD3 present in platelets [40]. The basic innate immunity protects against pathogenic microorganisms such as S. pneumoniae [82] via the classical complement pathway as an activation mechanism. As the key pathway, the C3 convertase forms C3 fragments that bind to microbes for leukocytes such as DCs and macrophages. Therefore, the deposited C3 convertase on the bacterial surface triggers the elimination of microbes. However, if microbes are encapsulated, as frequently found in S. pneumoniae, the complement pathway is restricted by a reduction of C3 deposition [83]. Hosts have, therefore, evolved to acquire an alternative strategy to detect pathogens by PRRs, which distinguish the PAMPs expressed on pathogens and, most alternatively, allow differential binding of pathogens and the released products in the immune response [84]. The obligate human pathogens of N. gonorrhoeae and N. meningitidis have evolved to evade cytotoxicity by the hosts. Therapeutic antibiotic-resistant N. gonorrhoeae strains are increasingly problematic and are classified as a “lifethreatening bacterium” by the Centers for Disease Control (CDC) and CDC Prevention (CDCP). An innovative therapeutic strategy against the strains is necessary for combating antibiotic-resistant N. gonorrhoeae strains. As a complement is the line of defense against N. meningitidis and N. gonorrhoeae, such classical and alternative pathways amplify complement component 3 (C3) deposition. Inhibition of such

324

7 Sialic Acid-Binding Ig-Like Lectins (Siglecs)

pathways increases serum resistance of the strains. The strains have evolved to acquire resistance to evade the complement in an alternative pathway of the host. LOS sialylation of gonococci strains induces serum-sensitive types to acquire serumresistant types, reduces the capacity of target-recognizing antibodies, and evades DCs, neutrophils, and antimicrobial peptides. Sialylation of LOS is pivotal to N. gonorrhoeae survival from the host immune responses. N. gonorrhoeae sialyl LOS protects host complement-mediated cytotoxicity through recognition enhancement of the complement inhibitor, which is known as factor H (FH). Sialylation of LOS contributes to acquisition of N. meningitidis resistance. Diminished LOS sialylation reduces FH binding [85]. N. meningitidis and N. gonorrhoeae LOSs are frequently terminal-modified in lacto-N-neotetraose (LNT) residues with sialic acid. LOS α-2,3 sialyltransferase (Lst) adds sialic acids. Lst enzymes scavenge the host sialic acids to incorporate into their LOS. Gonococci scavenge CMP-NeuAc (SA) from the human host and sialylate the terminal Gal residue of the LOS LNT. The Sia-bound LOSs decrease antibody recognition [86] and increase the host alternative pathway inhibitor, factor H (FH) [87]. N. meningitidis inhibits the alternative pathway using CPSs [88], FH-recognizing proteins, including Neisseria surface protein A (NspA)/PorB2/FH-binding protein (FHbp) [87–91], and Neisseria sialyl LOSs [92]. The sialylation of LOS components protects the N. gonorrhoeae strains from complement-involved cytotoxicity, and thus resistant N. gonorrhoeae strains have evolved to acquire higher Lst activity to combat host immune protection than sensitive strains (Fig. 7.6). Complement pathway Neu5Ac

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7.6 Hematopoietic System in Siglecs

7.6

325

Hematopoietic System in Siglecs

In mammals, surface glycans contain SA residues present in the terminal nonreducing end of glycans, which are located in the distal region. Sialylated glycans on cell surfaces have modulated cellular events for more than 500 million years since the appearance of the first vertebrates in the Paleozoic Era, which is estimated to be approximately 500 million years ago [93, 94]. In early mammals, CD33-related Siglecs likely manifested for more 200 million years [95]. The phylogenetic pedigree of mammalian Siglecs indicates the coevolutionary relationship between Siglec-1, -2, -4, and -15. However, CD33rSiglecs are independently adapted to evolve [96]. The high sequence similarity between CD33rSiglecs is likely caused by pathogenic attraction to SA mimic selection during invasion and infection (called the “Red Queen effect”), as argued by Varki’s group [96, 97]. SA glycoconjugates belong to SAMPs, which are recognized by inhibitory receptors to dampen immune responses via the interaction of Siglecs with distinct sialylated glycans. Eukaryotes and certain prokaryotes can decorate them with distinct sialylglycans, and immune-involved cells recognize sialylglycans as endogenous ligands for self- or nonself-pathogens. Siglecs as immune-regulatory receptors are mainly present on the hematopoietic cells of vertebrates. Detailed prospects and outlines have been made by Varki’s group on specific sialylated glycans expressed on self-endogenous cells and nonself-exogenous cells such as pathogens. The sialylated glycan-recognizing Siglec families have coevolved toward both inhibitory and activating roles, as the Siglec expression is largely elucidated in mice and humans. In addition, Siglec expression is still under study in many vertebrates including amphibians, birds, fishes, and reptiles with a coevolutionary progress [96]. This is why certain Siglec groups, i.e., Siglec-1, -2, -4, and -15, are structurally conserved in the above fish. Bornhöfft et al. (2018) analyzed BLAST data of human and mice sequences and found the presence of Siglecs both in vertebrates and in invertebrates [96], where they found the two Siglec-2 and -15 types from vertebrates as proinflammatory and anti-inflammatory receptors, respectively. They are all found in the five vertebrate species of amphibians, birds, fishes, mammals, and reptiles. Siglec-1 and Siglec-4 have been suggested to be ancient receptors, with the Siglec-2 and -15 forms having the same vertebrate ancestors. Siglec-2 specifically exhibits an autoimmunity-prevention activity [98], whereas Siglec-4 that is expressed on the inner side of the myelin wrap helps in myelin-axon recognition [99]. Siglec-15 regulates bone resorption [100]. Thus, in the vertebrate evolution, Siglec-2, -4, and -15 are suggested to be crosslinked to BCR differentiation, myelination, and bone formation, respectively [96]. The evolution of Siglecs is also related to lactation, which induces the mammalian CD33-related Siglec appearance. All human Siglecs are found on hematopoietic cells, except for MAG (Siglec-2). Siglec-1 promotes intercellular interaction through binding to α2,3-linked SA and is expressed on CD169+ macrophages. Siglec-5 and -14 are myeloid progenitor markers. Siglec-5 is inhibitory, whereas Siglec-14 is an activating receptor. They

326

7 Sialic Acid-Binding Ig-Like Lectins (Siglecs)

bind to the sialyl-Tn structure. In Siglec-7/-9 and -E, Siglec-7 binds to α2,8-linked SA, whereas Siglec-9 binds to α2,3-linked SA. Siglec-E binds to both of them and suppresses proinflammatory cytokines. Siglec-8 and -F bind to 60 -sulfo-sLeX and induce eosinophil apoptosis but inhibit mast-cell-mediator release. Eosinophilic asthma is thus regulated by them. Siglec-10 and -G bind to Neu5Gc in α2,3 and α2,6 linkages. Siglec-10 induces NK cells, whereas Siglec-G induces B cells for antibody production. In Siglec-11 and -16, Siglec-11 inhibits the microglia via α2,8linked SA but Siglec-16 activates it. Siglec-15 binds the sialyl-Tn structure on tumor cells and macrophages and suppresses osteoclast differentiation. Some Siglecs are widely expressed on hematopoietic cells. The V-set Ig-like domains of Siglecs recognize different sialylated glycans and activate or inhibit the immune responses. Siglecs are mainly expressed on hematopoietic cell surfaces, but the extracellular region of the immune cells also produces Siglecs. In fact, Siglec-4, known as MAG, is mainly present on Schwann cells and oligodendrocytes [101]. Sn is preferentially produced in macrophages, whereas CD22 is produced in B cells. However, Siglec-8 is expressed on eosinophils [23]. The two homologues of Siglec9 and -E are differentially present on myeloid-derived DCs (mDCs) of human and mouse mDCs, respectively. However, Siglec-5 is present on human pDCs and Siglec-H is detected on mouse DCs [23]. Siglec coreceptors are FccRIIB1, CD22, paired Ig-like receptor B, and killer cell inhibitory receptor. Except for MAG, all Siglecs are found in the hematopoietic cell lineage. Some Siglec expressions are cell type-dependent. In fact, sialoadhesin is specifically expressed on macrophages and is a type of adhesion molecule. CD22 is an inhibitory receptor of B cells with cellular activation and survival signals. However, CD33rSiglecs are present as complex forms on innate immune cells. CD33-related Siglec–ligand interaction leads to phosphorylation of the Tyr residue of ITIMs, which is catalyzed by Tyr kinases such as Src, and recruits tyrosine phosphatases, namely, SHP-1 and SHP-2. Therefore, Siglec receptor function is modulated by inhibition of Tyr phosphorylation [102]. Human Siglec-10 and mouse homologue of Siglec-G bind to CD24 and distinguish the PAMPs from the DAMPS [31]. Similar to other CLRs, Siglec-H functions as an endocytosis receptor of pDCs, when the cells capture pathogenic viruses and bacteria for internalization to intracellular TLRs and antiviral immune responses [103]. Siglec-H is deficient in Tyr-involved sequences and associates with the adaptor DAP12 [103]. Thus, each specific Siglec differentially functions in immune cell signaling. From the results obtained on Siglecs of various species from evolutionary distinct clades, it has been determined that the conserved forms of Siglec-1, -2, -4, and -15 exist in the same ancestral vertebrates before the division of ancestors into tetrapod species-like mammals such as humans and teleost fishes such as salmon about 400 million years ago [104–106]. This indicates the existence of evolutionary pressure on the four receptors; however, the CD33rSiglec genes are expanded with the coevolution of mammals [107]. On the other hand, the absence of the activating Siglec-14 in some human individuals provokes the group B Streptococcus suppression of neutrophil function [108]. Loss or lack of Siglec-14 increases the risk of prematurity, since the placental amniotic epithelium produces Siglec-14 as the target for the invasive group B Streptococcus [109].

7.7 Structure of Siglecs

7.7

327

Structure of Siglecs

As cell surface receptors, Siglecs are Ig-like lectins of the Ig superfamily of vertebrates that bind sialylated glycans and are involved in many physiological events including glycoprotein turnover, intracellular trafficking, and pathogen interaction. Their extracellular regions comprise multiple numbers of ‘C2-set’ Ig-like domains, which exhibit a conserved IgFc-like sequence, an Ig-like domain in the N-terminal region, and a V-set domain, which is highly similar to the IgV region [110]. This domain has the SA-binding domain [111]. The multiple numbers of C2-set Ig-like domains imply the SA-recognizing tendency of Siglecs expressed on the surfaces of the same cells in a cis-type interaction or on adjacent neighboring cells in a transtype interaction. The known Siglec-1 form consists of 15 Ig domains that bind carbohydrates in a trans-type. The Siglec-3, -8, and -15 forms, having Ig domains, recognize sialyl glycans in a cis-interaction manner [112]. As all the Siglecs carry an Ig-like domain in the N-terminal region with nine β-strands, they are similar to the IgV-set domain. The Arg residue in this IgV-set domain is essential because it forms a salt bridge with the COOH group that is linked to SAs. One salt bridge interacts with SAs. In human Siglec-1 (hSiglec-1), the SA-recognizing domain is thus present in the N-terminal region. The R116 guanidine group mediates a salt-based binding to the NeuAc COOH group. The acetyl group attached to Neu5Ac binds by van der Waals forces to the W21-attached indole ring. In addition, the SA C9-attached glycerol group binds to the W125 aromatic group [96]. The recognition of hSiglec-5 and SAs is mediated by a salt bridge formed with the Arg (R124)– COOH group of Neu5Ac [113], similar to Siglec-1. K132 and S134 also recognize the NeuAc C8 secondary amine and the hydroxyl groups, respectively, by hydrogen bonds. Van der Waals actions occur between the Y133 aromatic group and C9 of NeuAc [113].

7.7.1

Cytoplasmic ITIM and ITAM Domains of Siglecs

In view of the protein structures, Siglecs have generally cytoplasmic ITIMs and they are well conserved in the Siglec forms expressed on the hematopoietic cell system (Fig. 7.7). ITIMs are specialized domains for inhibitory signaling on stimulation with self-antigens, allowing immune tolerance. As representative cases, CD33rSiglecs and CD22 consist of one or multiple cytoplasmic ITIMs to effectively perform the inhibition of tolerance. Generally, ITIMs function as suppressive mediators of activation signals that initiate from immunoreceptor tyrosine-based activation motifs (ITAMs), which recruit tyrosine. Most Siglecs consist of an intracellular domain ITIM and ITIM-like domains with the (I/V/L/S)-X-Y-X-X-(L/V) sequence to counteract immune activation via ITAM-involved signaling inhibition. Among the Siglec forms, Siglec-14/-15/-16 specifically recognize the DNAX activation protein (DAP)10/12 to induce immune responses, where DAP10/12 consists of

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ITAM domains [35, 114, 115], with the amino-acid sequence Y-X-X-L/I-X6-8Y-XX-X-L/I (where X indicates any undefined amino acid) [116]. DAP10/12 recognition is mediated by the transmembrane region’s amino acids with positive charges in Siglecs [115, 117]. Exceptionally, CD33 and Siglec-H of mouse and human Siglec14 and Siglec-15 are deficient in ITIMs for unknown reasons but seem to occur throughout the evolutionary stage. As Siglecs have ITIMs, they can send negative signals through the recruitment of the negatively working region, the SH2 domain, which contains both SHP-1 and SHP-2. The activated ITIM inhibits receptor signaling operated within the ITAM. Therefore, the receptors are called inhibitory receptors due to suppression of activation signals caused by ITAM-carrying receptors through Tyr phosphatase recruitment. Siglec-14, -15, and -16 are closely related to the ITAM activity and the DAP12 adaptor protein by a transmembrane region composed of positive amino acids in Siglecs. The activating receptor signals recruit some spleen tyrosine kinase (SYK). In humans, two different pairs of Siglecs are expressed. Siglecs transmit intracellular signals upon interaction with multivalent “trans-“ligands derived from nonself or self.

7.7 Structure of Siglecs

7.7.2

329

Adaptor Proteins Associated with Siglecs

Among the three Siglecs, Siglec-15 is well characterized as DAP12- and DAP10associated Siglecs. The Asp residue in the DAP12 transmembrane domain is known to interact with the Lys residue in the Siglec-15 transmembrane domain [35]. Upon Siglec-15 interaction with sialylated glycans, the Src kinases phosphorylate the Tyr residues found in the ITAM, which then serves as a docking site for the ZAP70 SH2 domains, and, as a result, Syks exert immune activation [35, 116]. With regard to the functional and immunological roles of DAP-associated Siglecs, for example, the Siglec-15 form regulates the fate of osteoclastic differentiation [118] in bone resorption [119]. Siglec-15 binding to DAP12 after SA recognition induces differentiation of osteoclasts into multinucleated cell types that are functional for bone resorption [118]. Anti-Siglec-15 antibodies block the differentiation of osteoclasts through dimerization, internalization, and degradation of Siglec-15 dimers [120]. On the other hand, apart from the DAP-involved Siglecs, ITIM-having Siglecs or ITIMcontaining Siglecs can silence ITAM-triggered immune reactions. The interaction of sialyl glycans phosphorylates the intracellular Tyr residues of Siglecs by Src kinases. Phospho-Siglecs can recruit SHP-1 and SHP-2, capable of receptor interaction, and can inhibit kinase-dependent pathways [23]. For example, antibody production is inhibited in a Siglec-mediated manner, when the BCR binds to a counterpart antigen. B cells differentiate into Ab-expressing plasma cells to produce antibodies. However, if the target antigen is copresented with sialyl glycans on endogenous cells, the B-cell Siglec-2 binds to sialyl glycans [121, 122]. Then, the BCR and Siglec-2 clusters recruit SHP-1 and SHP-2 and, consequently, the kinase-dependent signaling pathway is inhibited, thus resulting in blocked antibody production against the autoantigen. On the other hand, Siglec-2 can also recruit several activator proteins such as GFR-bound protein 2 (GRB2), PLC-γ2, PI3K, and the SH2-domaincontaining transforming protein C (SHC), indicating the dual capacity of Siglec-2 to act on the respective B cells [122]. For the ITIM-having Siglecs, Siglec-9-suppressed immunity was reported by Varki’s group [123]. Siglec-9 suppresses neutrophil function upon recognition of sialyl glycoproteins on erythrocytes, indicating that sialyl glycans function as a SAMP on erythrocytes. This can be applied to tumor cells for Siglec-involved roles. AML cells and CLL cells largely express sialylated glycans as Siglec-7/-9 ligands, residing on the surfaces of NK cells. Therefore, Siglec-7/-9-specific SAs ligands present on tumor cells suppress the activation of NK cells, allowing cancer growth [100, 124, 125]. In addition, it is important to keep a balance in the Siglecrelated action to maintain a constant healthy status, as any imbalance may provoke any disease status like neurodegenerative diseases [126–129]. Representatively, Siglec-3 is a possible risk factor for Alzheimer’s because microglial cells known as tissue macrophages of the neuronal system increasingly express Siglec-3, resulting in an insufficient capture of amyloid-β plaques [130, 131]. Moreover, the polymorphic Siglec-8 gene pattern is linked to the onset of allergic asthma [132], as Siglec-8 is known to be mainly expressed on human eosinophils. Siglec-8–ligand

330

7 Sialic Acid-Binding Ig-Like Lectins (Siglecs)

interaction on eosinophils in tissues promotes eosinophil apoptotic cell death. In an airway inflammation animal model induced by ovalbumin, mucins and epithelial cells contain an excess level of Siglec-F ligands, where Siglec-F is a mice homologue of human Siglec-8, allowing a reduced level of the invading eosinophils [100, 133]. ITIM-bearing Siglecs control the immune response and may have therapeutic potential. However, pathogens can also utilize Siglecs. As mentioned earlier, pathogenic microorganisms including N. meningitides, H. influenza, C. jejuni, P. aeruginosa, and group B Streptococcus express sialylglycans to interact with Siglecs [134]. More specifically, group B Streptococcus produces a capsular SAα2,3-Galβ1,4-GlcNAc ligand for Siglec-9. The binding of Sia α2,3-Galβ1,4GlcNAc to Siglec-9 dramatically reduces the function of the neutrophil–myeloid– oxidative axis to increase the survival rate of pathogens [68]. Mutation results from CD33-related Siglecs show that the ITIM is predominantly dominant over the other ITIM-like domains and that it recruits the adaptor phosphatases SHP-1 and SHP-2. The recognition capacity of SHP-1 and SHP-2 needs the phosphor-Tyr residue to be attached to the ITIM and ITIM-like domains. However, Siglec-5 weakly recognizes SHP-1, but it is not phosphorylated due to Ala replacement from Tyr [135].

7.7.3

SA-Recognition Tropism of Siglecs

SAs on immune cells can be defined as biological masking agents and cell patternrecognizing agents. SA-containing carbohydrates present on DCs are recognized by Siglec receptor-holding effector cells. Although there are many SA species in vertebrates, and they have good interactions with Siglecs [136] (Table 7.2), two common SA glycans recognized as ligands by Siglecs are shown (Fig. 7.8a, b). For example, high α2,6-SA contents of tolerogenic DCs and immature DCs are recognized by inhibiting Siglecs produced by effector T cells and this is indeed a host tolerance-induced mechanism. The enhanced binding capacities of Siglec-1, Siglec2, and Siglec-7 are correlated with the high SA levels of mature DCs. DCs also express themselves as Siglecs on their surfaces. For biological masking functions, SAs shield the host cells from pathogenic binding and thus consequently prevent autoimmune responses. Therefore, the concentration of SAs expressed on human cell surfaces is relatively increased. High SA contents are caused as a result of acutephase responses. SAs also upregulate immune cell activities and discriminate the “self-” antigens from the “nonself” ones. The majority of DC Siglecs bind in the cistype. However, sialidases can inhibit Siglec–SA binding through the extrinsic or intrinsic action of sialidases. More specifically, sialidases can improve phagocytosis operated by human monocyte-derived DCs (mo-DCs) and mature mo-DCs. Sialidase treatment increases the immune response of MDDCs [137]. E. coli phagocytosis is also improved by SA moieties. Sialidase treatment improves the capacity of mo-DCs to phagocytose pathogenic E. coli isolates, as removal of SAs from the surfaces of mo-DCs highly sialylates induced mo-DC maturation and decreased

FYNkinase site ITIMlike

ITIM ITIM ITIM

Sia α2,3-Gal

Sia α2,6-Gal

Sia α2,8Sia Sia α2,6-GalNac Sia α2,6-GalNac Sia α2,8Sia2,3Gal

Oligodendrocytes Schwann cells

Myeloid progenitors Macrophages Monocytes (microglia, Granulocytes) Neutrophils, monocytes (B cells)

Trophoblasts (B cells) NK cells (monocytes, mast cells)

Siglec-4 (MAG), 4 Ig D

Siglec-3 (CD33), 1 Ig D

Siglec-6 (CD327), 2 Ig D Siglec-7 (CD328), 2 Ig D

Siglec-5 (CD170), 3 Ig D

ITIM

Sia α2,6-Gal

B cells

Siglec-2 (CD22), 6 Ig D

Tyrosine motifs –

Sialyl linkage Sia α2,3Gal>α2,6

Cognate glycan structure

Distributed cells Macrophages (activated monocytes)

Siglec (CD number), No. of Ig domains Siglec-1, sialoadhesin (CD169), 16 Ig D

Plasmodium falciparum merozoite EBA-175

Human influenza A

Pathogen binding Avian influenza A

Maackia amurensis hemagglutinin (MAH)

Sambucus nigra agglutinin

Lectin Maackia amurensis leukoagglutinin (MAL)

, Sialic acid (SA);

(continued)

Cancer C. jejuni infection

Preeclampsia

Group B Streptococcus infection

Acute myeloid leukemia (AML), Alzheimer’s disease

Related disease Autoimmunity HIV-1, PRRSV infection C. jejuni, group B streptococcus infection Lymphoma, leukemia, SLE, rheumatoid arthritis Neurodegeneration

Table 7.2 Human Siglec family and binding of sialic acids to SA-recognition molecules. Words in red indicate CD33-related Siglecs. , galactose (Gal); , N-acetylglucosamine (GlcNAc); , N-acetylgalactosamine (GalNAc); , fucose (Fuc)

7.7 Structure of Siglecs 331

E-selectin

Sia α2,3Gal1,4GlcNAc (Fucα1,3)β1,3Gal1-R

Sia α2,8Sia2,3Gal

–

Viral infection Neuroinflammation

Osteopetrosis

–

Osteoclasts Macrophages, dendritic cells Macrophages Microglia

Siglec-15

Siglec-16

Group B Streptococcus infection COPD

–

Sia α2,8Sia α2,3Gal Sia α2,6-Gal Sia α2,6-Gal

Neutrophils Monocytes

Siglec-14, 2 Ig D

Lymphoma, leukemia, eosinophilia, allergy Neuroinflammation

Related disease Eosinophilia, asthma

ITIM

–

Lectin

Chronic lung inflammation

Pseudomonas aeruginosa mucoid strain 8830 Anaplasma phagocytophilum

Pathogen binding

ITIM

Tyrosine motifs ITIM

ITIM

Sia α2,3GalGalNac Sia α2,6 Sia α2,6-Gal

Sialyl linkage Sia α2,3-Gal

Sia α2,8Sia α2,3Gal

Distributed cells Eosinophils, (mast cells, basophils) Neutrophils, monocytes, dendritic cells (NK cells) B cells (monocytes, Eosinophils)

Macrophages (microglia)

Cognate glycan structure

Siglec-11, 4 Ig D

Siglec-10, 4 Ig D

Siglec-9 (CD329) 2 Ig D

Siglec (CD number), No. of Ig domains Siglec-8, 2 Ig D

Table 7.2 (continued)

332 7 Sialic Acid-Binding Ig-Like Lectins (Siglecs)

7.7 Structure of Siglecs

R’

A)

333 CO2H

OH

HN

R

HO

CO2H

OH

OH HN

O

O

Neu5Acα α 2,6Gal-glycans

HO

HO

HO

CO2H

OH

HO

O

OH HO HN

O01IN[ECPU

O

C O

HO

R

R’

CH3 OH CH2OH OH CH3 OAc CH2OH OAc

C O

HO

R

Neu5Ac Neu5Gc Neu5,9Ac Neu5Gc9Ac

O

HO

B)

Sialic acid R

OH

O

Neu5Acα 2,3Gal-glycans O

O01IN[ECPU

HO

HO

C O

Fig. 7.8 Sialic acid recognition of Siglecs. (a) SA is substituted in mammals at the positions of R and R0 . (b) SA–Gal linkages: Recognition of two SA ligands by many Siglecs. The sialic acid species are diverse in vertebrates. The sialic acid structure is substituted at the carbon positions of R and R0 by amino group halogenation and acylation. Various sialic acids are found in vertebrates (adopted from Crocker PR et al. 2007. Nat Rev Immunol. 7(4), 255–66 [23] with a slight modification)

micropinocytosis [138]. ST6Gal-1-null BMDCs have a higher phagocytic activity compared to that of normal cell types [23]. Desialylated MDDCs increase the phagocytic capacity of E. coli. Treatment of MDDCs with sialidase increases the level of NF-κB activation and cytokine gene expression, consequently increasing the phagocytic capacity of the cells, indicating that desialylated mo-DCs enhance immunological functions including cytokine gene expression and NF-κB activation, as well as IFN-γ gene production by T cells. E. coli phagocytic capacity is influenced by the SA species. BMDCs isolated from ST6Gal-1 KO mice increase the phagocytic activity against E. coli. Neuraminidase influences the reorganization of the cytoskeleton and activates Rho-GTPases. Phagocytosis remodels the actin cytoskeleton and activates Rho small-type GTPases [137].

334

7.8

7 Sialic Acid-Binding Ig-Like Lectins (Siglecs)

Inhibitory Signaling of DCs

SA-containing glycans cover the DC surface to be implicated in DC biology. Sialoglycans regulate DC function. Siglecs are variable in their ligand-binding specificity for SA ligands. Their immune cell expression is of relevance both in health and in disease. In the historical aspect, the Siglec-type CD22 was first reported for the CD22-encoding cDNA clone, which is primarily expressed on B cells, indicating that it bears multiple Ig-like domains [16] and that CD22 binds to two sialoglycoproteins, as reported in 1991 [139]. In the same year, the sheep erythrocyte receptor on macrophages was reported to be renamed sialoadhesin due to its sialoglycoconjugate-binding capacity [2]. Sialoadhesin and MAG were also reported as SA-binding lectins and a type I membrane protein integrated into the PM. They have Ig-like extracellular domains [140, 141]. The lectin CD22 belongs to the sialoadhesin lectin family [141], Ig-like lectin family of the I-type lectin family [142], which is different from C-type lectins or Ca2+-depending lectins. On the basis of the work by Crocker and Varki in 1998, the family of SA-binding Ig domaincarrying lectins is termed “Siglecs” [143]. Certain Siglecs, however, do not bind to sialyl ligands. Detailed information on the specificity of Siglecs is currently in the editing stage in a step-by-step process due to their rapid evolution [104]. Some Siglecs are classified as inhibitory receptors of innate immune cells due to their regulation property of inflammation caused by DAMPs and PAMPs. They are involved in adhesion and phagocytosis through association with the ITAM-carrying DAP12 adaptor. As activating receptors, Siglecs inhibit immune cells by recognizing cis-ligands, which are expressed in identical or endogenous cells, as well as by interacting with pathogenic sialoglycans. They also maintain tolerance in B cells, regulate conventional and plasmacytoid DCs, and regulate T-cell functions [117]. Desialylation with neuraminidases induces the response of DCs to endotoxin LPSs and T-cell activation [144–148]. Thus, SAs limit DC response to TLR-mediated signaling. For example, a synthetic and fluorine derivative SA mimetic, Ac53FaxNeu5Ac, is reported to block SA synthesis in human mo-DCs [149]. Blocking the SA expression increases the response of mo-DCs to TLR signaling and T-cell activation. Mo-DCs express sialic acids and also the immune inhibitory receptors of Siglecs-3, -5, -7, -9, and -10 that bind to sialic acids. Some Siglecs have inhibitory signaling sequences as amino-acid motifs in their cytosolic region. Such Siglecs are crucial molecules in inhibitory signaling, which suppress immune responses and dampen inflammatory responses in specialized conditions like lymphocyte exhaustion or tissue cell apoptosis. Inhibitory signaling motif-lacking Siglecs are involved in other roles in sialic acid recognition. This discriminates how Siglecs control immune responses. For example, human cells mostly express two pairs of Siglecs. CD33rSiglecs and CD22 consist of one or multiple cytoplasmic immune receptors known as ITIMs. In mice, Siglec-E is an inhibitory CD33rSiglec receptor. The presence of Siglec-E is mainly observed in macrophage and neutrophil subsets. Generally, ITIMs act as inhibitors of activation signaling induced by immunoreceptor tyrosine-based activation motifs (ITAMs).

7.8 Inhibitory Signaling of DCs

335

ITAMs recruit Tyr-based molecules. The inhibitory receptors of Siglecs regulate TLR signaling, as, recently, CD33rSiglecs have been considered to have evolved and are mainly present on clusters of innate immune cells. They consist of an ITIM in the cytoplasmic region and an ITIM-like domain. SA recognition by inhibitory Siglecs is mediated in a cis-interaction manner with SAs in leukocytes. Siglecs can also recognize SA ligands in a trans-manner on adjacent cells or ligand-carrying pathogens to elicit Siglec-dependent endocytotic signaling [149]. Exceptionally, murine Siglec-H and CD33 as well as human Siglec-14 and -15 are deficient in ITIMs. Due to ITIMs, Siglecs can send a negative signal through recruitment of the SHP-1- and SHP-2-containing SH2 domain. Activator receptors are based on spleen tyrosine kinase (SYK) recruitment. A model mouse Siglec-E is predominantly present on tissue macrophages, neutrophils, and splenic DCs [150, 151], as inhibitory CD33-related Siglec, Siglec-E, and its human homologue Siglec-9 regulate TLR-4-involved cytokine production in DCs and macrophages. In murine BMDMs, binding of Siglec-E to antibodies decreases the production of IL-6, TNF-α, and RANTES upon LPS stimulation [152]. An enhanced expression of Siglec-9 of human origin in RAW264 and THP-1 macrophages decreases proinflammatory cytokine production upon LPS treatment [153]. Siglec-E also suppresses proinflammatory cytokine expression in macrophages during treatment of sialyl capsules of group B Streptococcus [154]. TLRs can directly interact with Siglec-E [155]. The cis-type Siglec-E binding to TLR-4 is a prerequisite for TLR-4-mediated endocytotic internalization during E. coli capture and downregulates TLR-4-mediated inflammatory responses [155, 156]. Siglec-E induces the differentiation of macrophages upon LPS treatment, thus weakly modulating TLR-4-involved signaling in macrophages or DCs [157]. Siglecs expressed on DC cell surfaces present ITIMs in their cytosolic portion. Since DCs modulate both B and T cells, Siglecs regulate host tolerances (Fig. 7.7). Thus, such inhibitory activated signals can induce immunomodulatory roles in the cells. For example, ST6Gal-I is one such representative. Some pathogens mimic masking to evade the immune system. In case of recognizable cell patterns of Trypanosoma spp., they are recognized by several cell surface receptors like CLRs and Siglecs. Two big Siglecs are members of the CD22 family and the CD33-related family. These recognize and bind ligands in a trans- and cis-manner. In addition, Siglecs present ITIMs or ITAMs, regulating and discriminating between SAMPs and PAMPs. In a knockout mice study, ST6Gal-1 KO mice showed decreased humoral immune responses. As CD22 recognizes ST6Gal-1-mediated glycans, sialic acid 2,6 is known to regulate several B-cell functions. In addition, the soluble form of ST6Gal-1 regulates myelopoiesis upon acute inflammation. In the case of ST3Gal1 KO mice, α2,3-sialyl-O-glycans maintain CD8+ T-cell homeostasis. Human and mouse Siglecs in immune cells have been well studied (Table 7.3). Siglec families have been well established with SA-binding specificities, C2 Ig domain numbers, and ITIM structures (Table 7.4). Human Siglecs and their mouse orthologs are known. Sequences of Siglec-1, -2, -4, and -15 exhibit conserved common homologies in both mice and humans. Other Siglecs such as members of the CD33 (Siglec-

Mouse

Human

B cells Siglec-2 (CD22) Siglec-5 (CD170) Siglec-6 (CD327) Siglec-9 (CD329) Siglec-10 CD22/Siglec-2 Siglec-G

T cells Siglec-7 Siglec-9

Siglec-E

NK cells Siglec-7 Siglec-9 Siglec-10

Monocytes CD33 Siglec-5 Siglec-7 Siglec-9 Siglec-10, Siglec-14 Siglec-E

Macrophages Sialoadhesion CD33 Siglec-5 Siglec-11 Siglec-15, Siglec-16 Sialoadhesion Siglec-F

Table 7.3 Patterns of Siglec expressions on different immune cells in humans and mice DCs CD33 Siglec-7 Siglec-9 Siglec-10 Siglec-15 Siglec-E Siglec-H

CD33 Siglec-E

Neutrophiles Siglec-5 Siglec-9 Siglec-14

Siglec-F

Eosinophiles Siglec-8 Siglec-10

Basophiles Siglec-5 Siglec-8

336 7 Sialic Acid-Binding Ig-Like Lectins (Siglecs)

7.8 Inhibitory Signaling of DCs

337

Table 7.4 SA-binding specificities, C2 Ig domain numbers, and ITIMs or SA-binding residues of different Siglecs

Name Sn/Siglec-1 (CD169) CD22/Siglec-2 MAG/Siglec-4 Siglec-15 CD33/Siglec-3 Siglec-5 Siglec-6 Siglec-7 Siglec-8 (mouse Siglec F) Siglec-9 (mouse Siglec E) Siglec-10 (mouse Siglec G) Siglec-11 Siglec-12 Siglec-14 Siglec-16

Distributed cells Macrophage

SA specificity α2,3 > α2,6

C2-Ig domain number 16

B cells Myelin Macrophage, DCs Myeloid progenitors, monocytes Neutrophiles, monocytes Trophoblast NK cells Eosinophils

α2,6 6S α2,3 > α2,6 α2,6 α2,6 > α2,3

6 4 1 1

ITIM None Lys ITIM

α2,3

3

ITIM

α2,6 α2,8 > α2,6 > α2,3 α2,3 > α2,6

2 2 2

ITIM ITIM ITIM

2

ITIM

B cells

α2,6 ¼ α2,3 (prefers sulfated residues) α2,6 ¼ α2,3

4

ITIM

B cells Macrophages Unknown Macrophage, DCs

α2,8 No recognition α2,6, α2,8 α2,8

4 2 2

ITIM ITIM Arg

Monocytes, neutrophils, DCs

ITIM or SA-binding residue None

3) family and their mice orthologs are named as Siglec-E. CD33 and its related Siglecs are broadly expressed in the innate immune system (Fig. 7.8). Sialoglycan structures are present in epithelial cells, most immune cells, and tumor cells. Some microbial agents take up SAs from the host cells to escape from the Siglec-based immune recognition of hosts. The well-defined examples include GBS, C. jejuni, and N. meningitides. They recognize CD33rSiglecs [66]. Siglecs are involved in cis- and trans-binding to sialylated glycans. The cis-bindings of Siglecs are often masked by sialyl ligands with low affinities toward the adjacent receptors and they cannot block trans-binding to other cells [23].

338

7.9 7.9.1

7 Sialic Acid-Binding Ig-Like Lectins (Siglecs)

Siglec-1 (CD169, Sialoadhesin/Sn) General SAbinding Specificity of Siglec-1

Siglec-1/sialoadhesin (Sn/CD169) belongs to a cell surface CAM expressed on macrophages among immune cells. In the beginning of the 1980s, historically, Kraal and Janse discovered Siglec-1/CD169+ macrophages using mAb MOMA-1 against mouse lymph node cells [158]. At that time, Siglec-1/CD169, known as Sn, was initially recognized as an unopsonized sheep erythrocyte-binding receptor present in stromal macrophages in tissues [1, 159]. The Siglec-1 (CD169) gene was isolated as the sole Siglec form activated during LPS-treated DC maturation. Currently, Siglec-1 is renamed as a specific receptor for some exosomes [160, 161], certain retroviruses [162–164], and sialylated bacterial pathogens [165, 166]. Like other Siglecs, Sn, Siglec-1/CD169 binds to SAs and prefers SA conjugates such as SAα2,3-Gal-linked forms. CD169/Siglec-1 binds to α2,3-Neu5Ac-sialylated molecules found on pathogens and the host’s glycosphingolipid GM3 [112]. Siglec-1 is an I-type lectin because it has 17 Ig domains, i.e., 1 variable domain and 16 constant domains, as the Ig superfamily. For binding of Siglec-1/CD169 to sialyl ligands, proteins form a salt bridge between a conserved Arg residue from the V-set domain to the α2,3-sialyl linkage and the carboxylate group in SAs. Since Siglec-1/CD169 binds to SAs with its N-terminal IgV domain, it is named the Siglec family. Siglec-1/ CD169 predominantly recognizes neutrophils and also recognizes monocytes, B cells, NKs, and CTL subpopulations through interaction between SA ligands on cell surfaces. Siglec-1/CD169+ macrophages trans-signal to undergo cell-to-cell communication and to maintain hematopoietic stem cell retention at the niche. Siglec-1-positive macrophages are located on the interspaced area between circulating fluids and the local tissue, allowing easy interaction with target molecules in the tissue and consequent transduction of information to the adjacent immune cells [167]. At the interspace of the circulating fluids and tissue, Siglec-1/CD169+ macrophages capture and internalize blood- and lymphoid-produced molecules in the lymphatic organs. Antigen information transduced by Siglec-1/CD169+ macrophages to the adjacent immune cells elevates antimicrobial immune responses upon recognition of the invasive pathogen. In tumor antigen recognition, Siglec-1/ SC169+ macrophages transduce information to immune cells to exert both antitumor immunity and tolerance. Macrophages are indeed distinguished from conventional macrophage subsets to ontogeny-based macrophage subpopulations. Therefore, a precise classification of the macrophages is performed using certain parameters of functional analysis including function, localization, and surface phenotype [168]. From the characteristic properties of Siglec-1/CD169+ macrophages differentiated during antigen stimulation, Siglec-1/CD169+ cells utilize distinct endogenous transcription factors and exogenous local signals generated from the tissues for their existence. Therefore, the specific expression of the Siglec-1/CD169+ cell phenotype is dependent on local tissues. Then, a question arises: how are tissuespecific phenotypes displayed or developed? To answer this question, specific

7.9 Siglec-1 (CD169, Sialoadhesin/Sn)

339

transcription factors develop Siglec-1/CD169+ macrophages, as reported from the fact that liver X-receptor α-/β-lacking mouse do not develop Siglec-1/CD169+ marginal zone macrophages [169]. Lymphotoxin (LT) α1β2, a B cell-derived cytokine, functions during the phenotype change in lymphatic node-resident macrophages but not in medullar sinus-resident macrophages and Siglec-1/CD169positive gut macrophages [170]. In inflammatory responses, Siglec-1/CD169+ macrophages modulate immune responses and maintain tolerance in the spleen. Siglec-1/CD169+ macrophages capture apoptotic cells to induce cellular antigen-specific tolerance. They also exhibit the proinflammatory phenotype, although proinflammatory cytokines IL-6/ IL-23/IL-1β are expressed in low levels in Siglec-1/CD169+ macrophages than in Siglec-1/CD169 cells. Inflammatory cells are rarely infiltrated in the absence of Siglec-1/CD169+ macrophages. CCL2 is the monocyte chemoattractant and is not expressed differently in both Siglec-1/CD169-positive and Siglec-1/CD169-negative macrophages. In the colon tissue, CCL8 expression, which is a monocyte chemoattractant protein 2, is enhanced in Siglec-1/CD169+ macrophages and for CCL8 mRNA expression. In the intestinal tract, Siglec-1/CD169+ macrophages exacerbate inflammation by CCL8 production to recruit monocytes. Therefore, Siglec-1/CD169+ macrophages can be used in tissue-targeting therapies for treating both immune and nonimmune diseases [167]. Siglec-1/CD169 mediates innate-like lymphocytic adhesion in the sinus lymphatic nodes and blocks the loss of cellular adhesion capacity during flow through the lymphoid line [171]. However, Siglec-1/ CD169-deficient mice show a normal phenotype and are fertile [172]. Siglec-1/ CD169+ macrophages express the chemokine CCL8 as a chemoattractant to recruit inflammatory monocytes. For example, CCL8 expression in Siglec-1/CD169+ macrophages, but not CCL2, recruits monocytes during inflammation. CCL8 regulates Langerhans cell, small intestinal macrophage, or Th2 cell trafficking [173, 174], regardless of Siglec-1/CD169 and Siglec-1/CD169+ populations. Therefore, CCL8 is regarded as a therapeutic target candidate for inflammatory disorders such as colitis. Siglec-1/CD169+ macrophage-produced CCL8 aggravates colitis, although pathogenic bacteria and endogenous adjuvants lead to CCL8 expression. Bone marrow cells cultured with BMDMs produce Siglec-1/CD169. LPSs and poly (I:C) also induce CCL8 expression in BMDMs. A DAMP, namely, HMGB1 of necrotic cells, also elicits CCL8 expression in BMDMs. Siglec-1/CD169+ macrophages can express CCL8 upon treatments with both PAMPs and DAMPs. To block the CCL8 role in vivo, CCL8-specific antibodies block the transmigration of monocytic cells toward CCL8. An intraperitoneal CCL8-specific antibody injection suppresses colitis development with decreased tissue injury in mice. Therefore, it is concluded that the Siglec-1/CD169-positive macrophage–CCL8 axis is a future candidate for injured inflammatory responses in the mucosal tissues. In cancer immune responses, CD169+ macrophages potentiate antitumor immunity. Siglec-1/CD169+ macrophages also induce antigen-targeting immune responses at the cellular level [175]. Therefore, CD169+ macrophages are prognostically important if they reside in the tumor-draining lymphatic nodes. CD169 expression level is a prognostic factor in tumor-specific survival. CD169+

340

7 Sialic Acid-Binding Ig-Like Lectins (Siglecs)

macrophages in the lymphatic nodes enhance anticancer immunity by expanding CD8+ T cells [176]. Subcutaneous injections of tumor cells are subjected to capture by sinus-lining Siglec-1/CD169+ macrophages. They internalize dead tumor cell antigens to present their information to the adjacent CD8+ CTLs to suppress tumor growth. Siglec-1/CD169+ macrophages recruit monocytes by CCL8 expression; therefore, the depleted Siglec-1/CD169+ macrophages decrease in monocyte level. Siglec-1/CD169-positve macrophages that are present in the tumor-draining lymphatic nodes decrease the number of cancer survivals [177–181]. Siglec-1/CD169 expression does not indicate prognosis in human breast cancer patients. However, the expression indicates the level of tumor-infiltrating CD8 T cells [182]. It is known that CD169-expressing lymph node sinus macrophages present in the regional lymphatic nodes elicit an antitumor immune response in the host. The lymph node macrophagic CD169 expression is linked to longer cancer cell survival. Thus, Siglec-1/CD169+ macrophages indeed indicate the level of antitumor immunity [183], as described by a result that a subcutaneous injection of cancer antigen leads to internalization by medullary sinus macrophages and inhibition of tumor growth [184, 185]. The level of tumor-infiltrating lymphocytes in a cancerous tissue and the macrophagic Siglec-1/CD169 expression are linked in cancer patients. The lymph node macrophagic expression of Siglec-1/CD169 and indolamine 2,3-dioxygenase is increased by IFNs [185]. Therefore, Siglec-1/CD169 expression in macrophages contributes to immune responses against human tumors. The Siglec1/CD169 expression level can be evaluated to check anticancer immunity. Siglec-1/ CD169+ macrophages in the lymphatic node sinus activate anticancer immunity. The structural basis of Siglec-1/CD169 is that the cytoplasmic tail is not conserved with other known Siglecs and lacks tyrosine-based signaling motifs such as ITIMs. Sn has its own specific structure bearing 17 distinct Ig-like domains in its extracellular region. The extracellular region has been suggested to extend its length of approximately 40 nm from the cell surface areas. This basis suggests that Siglec-1/ CD169 is involved more specifically in ligand recognition and cell–cell communication than in intracellular signaling to elicit immune responses. As Siglec-1 (CD169) is indeed a family of macrophage-restricted Siglecs, inflammatory and autoimmune diseases are regulated by Siglec-1/CD169. The CD169/Siglec-1 expression level is also increased upon type I IFN treatment on innate immune cells of macrophages and DCs [186]. For example, Siglec-1/CD169 levels are frequently increased in macrophages activated in the inflamed local organs and tissues of known inflammatory diseases [187]. The inflammatory examples are RA, experimental autoimmune encephalomyelitis (EAE), systemic lupus erythematosus (SLE), Sjögren’s syndrome, and experimental autoimmune uveoretinitis (EAU) [112, 188]. Rheumatoid arthritis (RA) patients highly express Siglec-1/CD169 on macrophages of the local tissues. Siglec-1/CD169-deficient mice exhibit reduced disease levels of inflammatory diseases [188]. These syndromes develop autoantibodies by IFN signaling and type I IFN-affected Siglec-1/CD169 gene expression. How is Siglec-1/CD169 expressed by IFNs and how do the phenotype changes of macrophages occur to the express the Siglec-1/CD169 gene? TLR ligation of macrophages with certain antigens induces an IFN-affected gene

7.9 Siglec-1 (CD169, Sialoadhesin/Sn)

341

expression such as Siglec-1/CD169. CD45 + CD11c + and CD45 + CD11c- human leukocytes obtained from patients with autoimmune congenital heart blocks express high levels of Siglec-1/CD169 by type I IFN stimulation [189]. In certain pathogenic bacteria, sialic acids are decorated on their surfaces. Such sialic acids on pathogens aid in pathogenic virulence through complement inactivation or Siglec engagement on host leukocytes. Siglec-1/CD169 is involved in the engagement of Siglecs on macrophages with pathogenic sialyl ligands to provoke inflammation. Sia α(2,3)Gal glycans are frequently found on pathogenic bacterial surfaces. Siglec-1/CD169+ macrophages internalize sialylated N. meningitidis via a Siglec-1/CD169–sialic acid interaction. In GBS-causing C. jejuni strains, Sia α(2,3) Gal glycans attached on the LOSs are associated with the pathogenicity of C. jejuni, causing autoimmune GBS and Miller–Fisher syndrome (MFS) [65]. Siglec-1/ CD169 captures the killed sialylated C. jejuni, thus activating proinflammatory cytokine production and responses to type I IFNs [190]. However, Siglec-1 and invasive sialylated pathogen binding is not demonstrated during infection. In sialylated GBS pathogens, Siglec-1/CD169 exerts bactericidal activity via Siglec1/CD169–sialyl ligand recognition. Sialoadhesin on the spleen marginal zone metallophillic macrophages captures circulating GBS and prevents the GBS from manifesting to the adjacent tissues or organs. This effect prevents GBS mortality from tissue to tissue [191]. In the marginal sinuses, marginal zone macrophages highly express Siglec-1/CD169 to recognize sialylated GBS. Currently, Siglec-1/ CD169 recognizes sialylated ligands expressed on several sialylated pathogens, including protozoa [192], bacteria [65, 187], and enveloped viruses [193–195]. In a virus, HIV-1 recognizes Siglec-1/CD169 via gp120-attached sialic acids. This interaction stimulates infection and transinfections in IFN-α-induced CD14+ monocytes. The gene expression of Siglec-1/CD169 is increased in CD14+ monocytes obtained from HIV-1-positive patients [193]. When mo-DCs are treated with LPSs, the inflammatory IL-1β/TNF-α cytokines, TGF-β1 and Siglec-1, are produced with an increased capturing ability of HIV particles. CD169 is the major receptor for HIV-1 infection and is produced by DC-associated mucosal environments. DCs capture the HIV virus and transmit it to the host cells. TGF-β1 induces the expression of CD169, but not DC-SIGN or other Siglecs on DCs, in the HIV-1 virion capture by the APCs [196].

7.9.2

Siglec-1 Is a Pathogen-Binding Receptor

Siglec-1 is expressed on the myeloid lineage in humans, mice, rats, and pigs. Its presence is restricted to macrophage subpopulations, and, thus, sialoadhesin is a macrophage-specific receptor that binds to sialyl glycan ligands on the host cells and pathogens. However, the functional roles of pathogen engagement by Siglec-1 are unclear. Siglec-1 is largely detected on marginal zone macrophage subpopulations in the spleen tissue and also on subcapsular sinus macrophages of the lymphatic nodes in mice [197]. Siglec-1 is physiologically involved in many types of signaling, but its

342

7 Sialic Acid-Binding Ig-Like Lectins (Siglecs)

role is still under study. It was regarded as a hematopoiesis regulator; however, KO mice results showed only changes in T- and B-cell populations [172]. Siglec-1 may modulate inflammatory and autoimmune responses. It is also involved in different processes such as viral infection, viral transinfection, or viral clearance with sialylated pathogenic HIV [198], as Siglec-1 is a porcine viral infection receptor in alveolar macrophages. However, a question arose in the study of Siglec-1 KO pigs [199]. Both hSiglec-1 and mSiglec-1 capture the murine leukemia virus (MLV) in a ganglioside-requiring manner [200]. Siglec-1 as a receptor also recognizes and takes up bacteria such as N. meningitides, C. jejuni, and T. cruzi, as well as, protozoa, which are sialylated on their cell surfaces [201]. For defining the roles of Sn in the interaction and capture of bacterial C. jejuni, Siglec-1-expressing mice are used [201]. The Gram-negative spiral bacterium, C. jejuni, shares interesting glycosylation properties with CPSs, lipooligosaccharides (LOSs), and sialylated glycoconjugates, which are capable of modulation in host immune responses. The bacterium has been specially outlined in serious neurological complications, namely, the Miller–Fisher and Guillain–Barré syndromes. Two protein glycosylation events are known in the pathogenic bacteria, C. jejuni human strains. First, an O-glycosylation event is found in Ser/Thr residues attached to flagellin proteins. Second, a N-glycosylation event is observed in Asn residues linked to various C. jejuni proteins. A lack of N-glycosylation activates inflammatory responses in hosts. Among them, the CPS species belong to the serotype-determining polysaccharides. In fact, C. jejuni strains deficient for CPS biosynthesis are negative for serotype determination, which is detectable by the Penner Scheme [202], and inhibit inflammation reaction, as a CPS-deficient C. jejuni strain produces cytokines [203]. Surface glycolipid LOSs consist of a lipid A attached to oligosaccharides. C. jejuni strains produce LPS-like high-molecularweight components, where lipid A is an inflammatory endotoxin. Synthesis of sialylated LOSs in bacteria enhances bacterial capture and synthesis of inflammatory cytokines in the host immune cells [204]. Interestingly, the sialyloligosaccharides produced by C. jejuni are similar in structural basis to GM1 gangliosides produced in humans. The sialosaccharides cause the axonal Guillain–Barré syndrome (GBS) due to ganglioside-specific antibodies. GBS-causing C. jejuni strains interact with sialoadhesin (Siglec-1) [205]. In addition, disialylated LOS-expressing C. jejuni strains can recognize Siglec-7 [66]. Siglec-7 is also reported to specifically recognize C. jejuni associated with GBS or MFS oculomotor weakness [206]. Sn rapidly takes up the C. jejuni GB11 strain, which synthesizes GD1a- and GM1-like carbohydrates. Sialylated bacteria rapidly migrate to the spleen and liver tissues and produce high cytokine levels of IFN-β/TNF-α. Sn recognizes pathogens as a pathogen-recognition molecule for sialylated bacteria with type I IFN responses that are crucial in host defense. C. jejuni sialylation is essential for macrophage capture and Sn-dependent expression of cytokines. The immune responses were well explained in C. jejuni sialylation in a previous mouse model [201]. Sn-dependent cytokine production can also be observed in other bacterial sialylations including GBS and N. meningitidis, as the Sn receptor cooperates with TLRs. Sn-dependent cytokine responses may develop postinfectious GBS.

7.9 Siglec-1 (CD169, Sialoadhesin/Sn)

343

In the V-set, Siglec-1, Arg97, and Trp-2 or Trp-106 amino-acid residues interact with SAs. Siglec-1 binds Neu5Ac in an α2,3 linkage to the D-Gal residue but not Neu5Gc-Gal or Neu5Ac9Ac-Gal linkages [56]. The ganglioside sialyl glycan head groups contain the HIV-1 binding sites as confirmed by human Siglec-1-arranged liposomes [207]. Two different gangliosides, GM3 and GM2, which contain sialyllactose (SL) consisting of a SA-Gal-Glc link to ceramides, are recognized by HIV/Siglec-1 [208, 209]. Viral membranes carry sialylated gangliosides synthesized from the host ER–Golgi system. There are many examples known in retroviruses such as HIV-1, murine leukemic virus (MLV), Semliki forest virus, and stomatitis virus. For example, during MLV infection, MLV is captured by Siglec-1 in mDCs. In the capture process of viral infection, Siglec-1 functions as a receptor for molecular binding of several enveloped viruses via mDC viral captures. Moreover, Siglec-1 is involved in the antivirus responses of immune cells to antigen-captured immune cells. Moreover, Siglec-1-positive myeloid cells efficiently take up the vesicular stomatitis virus with antivirus immune responses of B cells and prevention of virus neuroinvasion through type I IFNs. Elimination of sialyllactose-bearing GM3 or larger gangliosides, rather than that of monosialosyl motives, from viral budding coats or sialidase treatment with desialylating activity of viral membrane gangliosides, inhibits capacities of the mDC capture and immune recognition. In another case of sialic acids, certain influenza viruses are often resistant to ganglioside GM3 due to the viral neuraminidase enzyme activity in HA types. Therefore, it is interesting to see whether the SA-defective events are related to escape from immune recognition via Siglecs, such as Siglec-1, expressed on innate immune cells of the host. This is explained by the fact that sialylglycans are ligands for Siglecs produced on mDC surfaces, allowing pathogen capture and clearance. mDC capacity to capture sialic acid-containing gangliosides in viral coat membranes via Siglec-1 is believed to be an evolved result of sialylated pathogens.

7.9.3

Siglec-1 Recognizes HIV and Is a Transinfection Receptor Expressed on mDCs

Siglec-1/CD169 is also an HIV-1-capturing receptor expressed on mDCs [210]. Siglecs interact with gp120 on host cells and on monocyte-derived macrophages (mo-Ms). Siglec–gp120 binding to HIV-1 leads to host infections [195]. Macrophage cell surfaces produce many CTLs like DEC-205 or MR, which capture HIV-1 through the oligoman glycan link to the envelope protein, leading to virus endocytosis or infection. Siglecs bind to surface mucins, which heavily glycosylate, and mucin-like sequences. For glycans in infection, the gp120 mutation conferring the defected N-glycan sites on HIV results in noninfectious viruses due to higher sensitivity to neutralization. Thus, viral glycans are crucial for shielding against host immune detection. In addition, multiple glycan mutations increase lower infection activity or syncytia-generating capacity rather than the WT virus types, implying

344

7 Sialic Acid-Binding Ig-Like Lectins (Siglecs)

glycans’ role in viral entry. Siglecs bind gp120 of HIV-1. The surface envelop protein gp120 on the CCR5- (R4) and CXCR4-tropic (X-4) strains of the HIV-1 virus consists of more than 20 N-glycosylation sites with terminal SAs. SAs linked to gp120 glycoproteins potentiate virus adhesion, attachment, and virus entry via interaction with Siglecs. Results obtained from a direct interaction between the gp120 protein and monocytic Siglec-1, -3, -5, -7, and -9 of humans show that Siglec-1- and Siglec-9-Fc-fused receptors are reported to bind to the gp120 envelope protein, with affinities between 0.01 and 1 μM [211]. HIV-1 uses its SA-containing glycans linked to the envelope protein for virus interaction with Siglec-1 on macrophages and for improving the recognition level with CD4 for constant entry. In addition, HIV-1 glycans shield HIV from host immune surveillance and also serve as an infection tool for host entry. gp120 glycans are the sites for complex, high mannose, and hybrid glycan types. Glycans linked to envelope glycoproteins of gp41 and gp120 trimers of HIV-1 are distributed in CD4 complex forms. The four domains in the CD4 protein are located on the complex associated with the two domains in the N-terminal region. The number of blood CD169-positive cells is increased in HIV-infected patients. HIV-1-infected humans exhibit CD169 expression on monocytes in peripheral blood. In addition, the CD169 level is increased in the early infectious stage [212]. Furthermore, a recent report [195, 210] has updated a key function of CD169-positive cells in retroviral infection spread [212]. Therefore, CD169 can be a marker of the infectious pathogen lenti virus, leading to HIV-1 pathogenic progression. Thus, CD169 is a receptor of lentiviral infections and accelerates HIV-1 pathogenesis in vivo. Unmasking Siglecs on the surfaces of cells increases their recognition with virus SAs. Between Siglec-1 and Siglec-9, Siglec-1 exists as an unmasked type because of its relatively large (17) Ig domains. Moreover, Siglec-9 expresses better adhesiveness only after neuraminidase digestion despite its affinity toward SAs, indicating that surface receptors expressed on cells are masked by SAs. In addition, type I IFN-induced CD169 diminishes the antivirus capacity of type I IFNs via HIV infection enhancement in myeloid cells [213]. When monocytic THP-1, as a model cell, is incubated with IFN-α and the induced expression level of CD169 is examined, then normal-type HIV-1 amplification is increased even in the condition of IFN-α treatment. CD169 increases viral fusion and entry to host cells. The IFN-α-activated antivirus condition helps to evade mo-Ms and CD169 involved in HIV-1 fusion in MDMs. Moreover, CD169 overexpression on inflammatory DCs increases at the time of virus entry by a DC-involved mechanism, such as a transinfection event. In addition, the CD169 expression potentiates viral amplification in the host CD4+ T cells, regardless of the presence of type I IFNs. This indicates the important roles of type I IFN-induced CD169 and the suppressive roles of antivirus type I IFN in myeloid cells, which inhibit the viral amplification of HIV-1. CD169 takes up CD169-bound HIV-1 and HIV-1 associated with membranes of THP-1 cells and DCs as intermediate hosts. HIV-1 and CD169 clustering events increase in complex formation between viruses and host cells to enhance the efficiency of HIV-1 viral endocytosis to hosts. Apart from cis-type infection,

7.9 Siglec-1 (CD169, Sialoadhesin/Sn)

345

HIV-1 can enter the host CD4+ T cells through DC-involved transinfection events. HIV-1 transinfection or transmission between T cells and DCs takes place at the tightly joined “cell-to-cell junction,” also known as the viral synapse, which concentrates viral particles of HIV-1 to enhance the fusion between HIV-1 and the host T cells [214]. CD169 macrophages are constitutively present in lymphatic node tissues, including the subcapsular sinus, and perifollicular and medullary macrophages. CD169+ cells like tissue-resident macrophages are involved in viral dissemination even in the condition of IFN-α. Lymphatic node-resident macrophages take up most pathogenic agents, and CD169 is important for the GM3-dependent uptake of HIV-1 [186, 209, 215, 216]. The SL portion of viral GSLs is bound during mDC HIV-1 uptake. The adhesion and uptake receptor is a SA-binding molecule. Siglecs bind to sialyl ligands, driving cell recognition and immune responses. mDCs, not iDCs, in lymphatic tissues, efficiently transmit the HIV-1 virus to the host T cells. The virus–host interaction is the key transmission factor in the highly dense lymphatic tissues. The recognition of mDCs with CD4 + T cells creates an infectious synapse environment. Although the DC-SIGN level is decreased during DC maturation, the HIV-1 capture-based infection is increased. Mannan or DC-SIGN-specific antibodies such as DC-SIGN blockers exhibit only low activities on HIV capture-based infection by mDCs. However, DC-SIGN-transfected mDCs show completely blocked HIV infection. Moreover, although DC-SIGN is not present in myeloid DCs of blood and Langerhans cells, these cells take up and transinfect HIV. Thus, the DC-SIGN receptor is not required for HIV virus uptake in mDCs, indicating that the HIV capture by mDCs is performed by other molecules because viral envelope glycoproteins are not important for mDC HIV-1 capture and DC-SIGN recognizes only the HIV-1 gp120 glycoprotein [217]. Viral envelope proteins are not linked for mDC uptake. Instead, other components on viral surfaces will be more important for mDC capture. When HIV-1positive or exosome-positive cells are treated with GSL-synthesizing enzyme inhibitors, the mDC capture capacity is decreased. Thus, sialic acid-containing glycosphingolipids are important for mDC capture and infection of HIV-1, indicating the capture capacity of ganglioside-dependent mDCs and sialylated carbohydrate as the recognition region [207]. Sialic acids on cellular membranes are used as a binding and adhesion receptor by several pathogenic agents and toxins. Removing sialic acid from viruses by sialidase treatment or asialo-ganglioside-reconstituted exosomes abolishes mDC uptake because SA is important for recognition of mDCs. mDC capture of a virus is observed when the PMs contain GM1, GM2, or GM3 gangliosides. Interestingly, GM4, a SA-bound Gal moiety, has no capacity to capture a virus by mDCs. The fact that GM3, GM1, and GM2, SA-bound to the lactose head group, have mDC-recognition capacity indicates that the sialyllactose head group is responsible for mDC recognition. In fact, sialyllactose prevents HIV-1 uptake by mDCs, although an efficient capture needs membrane gangliosides, sialyllactose-bound ceramides. Between GM1 and GM3, GM3 is strongly recognized by mDCs. When HIV-1 is exposed to human genital mucosal epithelial cells, the cells mediate DC maturation by thymic stromal lymphopoietin (TSLP). Then,

346

7 Sialic Acid-Binding Ig-Like Lectins (Siglecs)

DCd-involved HIV-1 infection is accelerated in the host cells, CD4+ T cells, where Siglec-1 drives the infection of HIV-1 to vaginal Langerhans cells or DCs. In contrast, intact viruses are endocytosed in a CLR-independent manner. When mucosal inflammation is raised by other bacteria, fungi, or viruses, maturation of the resident DCs or newly migrated DCs can be stimulated by binding to the pathogens or by chemokines and inflammatory cytokines [218]. These events accelerate the transinfection events of HIV-1. Chronic inflammation or systemic inflammation is also the key inducer of increased infection of HIV-1, and various proinflammatory agents activate Siglec-1 production, thus increasing HIV-1 transinfection. LPSs induce HIV-1 infection, stimulating the systemic maturation of DCs and enhancing viral spread. IFN-α is an antivirus cytokine generated by pDCs upon HIV-1 infection due to IFN-α-stimulated Siglec-1 generation in mDCs or monocytes. Therefore, although IFN-α is an antiviral agent, it induces HIV-1 transinfection even in antiviral conditions. A higher HIV-1 viral titer is linked to increased Siglec-1 expression in monocytes, as enhanced by plasmacytoid DCs, which express IFN-α upon HIV-1 infection and elicit DC maturation [186].

7.10

CD22/Siglec-2

7.10.1 General and Structural Aspects of CD22/Siglec-2 Among immune cells, only B cells predominantly express Siglec-2/CD22, and Siglec-2 regulates the septic balance of immune responses by B-cell modulation. It also functions for adjusting chemokine production. B cells undergo humoral immune responses through BCR signaling. Therefore, Siglec-2/CD22 is a specific modulator of B cells among Siglecs. Functionally, Siglec-2/CD22 belongs to a B-cell inhibitory receptor, which modulates various types of B-cell behaviors in both survival and activation thresholds. Positive activating BCR signaling frequently participates in lectin–glycan interactions, which are managed by sialoglycans and sialic acid-binding lectins, namely, Siglecs. In contrast, negative inhibitory BCR signaling is also found. For example, two different Siglec forms such as human Siglec-2 (CD22) and human Siglec-10 (mouse Siglec-G) are BCR inhibitory coreceptors because they control B-cell activation and peripheral tolerance. Therefore, such BCR inhibitory receptor-negative individuals are susceptible to autoimmunity. CD22 is indeed a BCR-signaling suppressor in conventional B cells, B2 cells. For example, mice lacking CD22 or Siglec-G or those lacking both develop symptoms of autoimmune diseases [219]. Genetically deficient mice with congenital mutations in the biosynthesis of CD22 sialic acid ligands are well known for conferring an autoimmune status on humans [220]. CD22 is a membrane glycoprotein receptor of the immunoglobulin (Ig) superfamily (IgSF) and is predominantly expressed on mature B lymphocytes and not on premature cells. CD22 is a glycoprotein of a 140 kDa and has a single-spanning region that spans the plasma membrane. From the elucidated human CD22 crystal structure, it is known that the

7.10

CD22/Siglec-2

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binding specificity for α2,6-SA ligands originates from a β-hairpin structure for recognition [221]. The D1 domain has been shown to adopt a V-type fold as well as C1 and C2 strands to yield a β-hairpin structure. The extracellular domain of CD22 has hairpin-like Ig regions and 12 N-glycosylation sites. The outermost domains of CD22 are named D1 and D2. Both D1 and D2 that are the most N-terminally located Ig domain regions are binding sites for α2,6-linked SAs [104, 222]. Structurally, the SA-binding domain has nine β-strands and Ig V-set domains. The V-set domain specifically recognizes the α2,6-sialyl linkages on glycan structures. The β-hairpin structure is involved in the binding of sialic acids (Fig. 7.9) [223]. The surfaceexposed arginine residues in the glycan-binding receptor family are key factors for binding NH2 to form a salt bridge with the COOH group linked to SAs. Thus, the Arg residue in the receptor and the sialic acid residue in the ligand are counterparts. CD22 is distinct in terms of its structure and is not homologous to other known Siglecs, although many Siglecs in all mammals have been reported to date. In fact, the four Siglecs, Sd, CD22, MAG, and Siglec-15, which are expressed in all mammals, are not homologous. Regarding the aspect of sequence homology, Siglecs are classified into two major groups and are subject to investigation to determine their evolutionary adaptation [224]. For example, CD33-related Siglecs in many organisms are diverse in their structural homologies due to constant evolution, keeping the conserved region in tyrosine-based signaling motifs at the extracellular domain regions. CD22 has long been regarded as an adhesion molecule of SAs [225], with a specificity to various α2,6-linked SAs as ligands [226]. The preferred ligand for human CD22 is the N-acetylneuraminic acid (Neu5Ac) form of SAs on N-/O-glycans and glycolipids. Specifically, in humans and mice, 9-O-acetylated sialic acid is known as the common form of sialic acids and is linked to autoimmune response and substitution [227]. The specific binding of CD22 to 9-O-acetylated SAs is a possible explanation for why SA acetylation on self-antigens can prevent

348

7 Sialic Acid-Binding Ig-Like Lectins (Siglecs)

CD22 recognition and consequently increase the level of B cell-mediated autoimmune responses. Human CD22 is known to preferentially bind to Neu5Ac, whereas murine CD22 specifically binds to N-glycolyl neuraminic acid (Neu5Gc), also known as the nonhuman type of sialic acid. This indicates that recognition of CD22 SA ligands has evolved in each species in a CD22-dependent evolutionary manner [228]. CD22 functions depend on its ligand-binding ability. CD22 (Siglec-2) regulates B cell-dependent immune responses including prevention of autoimmunity. In B-cell circulation, CD22 strictly controls the B-cell homing event to the bone marrow in recirculating B-cell subsets, as the CD22 (Siglec-2) expression is observed only in B cells. In addition to its ligand-binding affinity to sialic acids, CD22 plays many roles in B cells. For example, CD22 modulates BCR signal transduction as the most important function in B cells [225]. BCR signaling is initiated by interactions with specific antigens. When antigens bind to BCRs, BCRs initiate the signaling pathway to activate related transcription factors including c-Myc and ATF-2 to upregulate gene expressions toward activation of B cells [225]. CD22, upon binding to the soluble sialic acid ligand, activates various signaling protein molecules to transduce inhibitory signals downstream of the BCR signaling pathway. The known recruiting adaptor molecules such as SHIP and SHP-17 have been well studied in inhibitory BCR signaling. The final inhibition of BCR signaling is directed by the so-called ITIMs. ITIMs are suppressed by a Src-family kinase, Lyn, upon CD22 activation of BCRs, consequently activating and recruiting SHP-1 [228]. CD22 is known to have at least two cytoplasmic, three inhibitory domains, ITIMs. The cytoplasmic tail of ITIMs inhibits BCR signaling. Using ITIMs, CD22 can activate and differentiate B cells into any specific cell type [229]. In B cells, SHP-1 initiates and maintains the BCR-induced Ca2+-dependent pathway [229]. CD22 interaction with glycoprotein ligands expressed in T cells also downregulates downstream signaling of T cells. Using ITIMs, CD22 recruits various dephosphorylases such as phosphatases that remove phosphorus from signaling protein molecules, which are subjected to kinase action during the BCR signal transduction of B cells. Thus, BCR signaling is deactivated and terminated [230], indicating that CD22 is an apical regulator of homeostasis in adaptive immunity. It is summarized that CD22 is a keeper of B-cell suppression and a holder of adaptive immunity. Lectin CD22 is regulated by the membrane-bound glycan ligands on the same cells, and these types of ligands are called cis-ligands. Biologically, it is an inhibitory coreceptor of BCRs because it downregulates diverse B-cell functions. As a B lymphocyte-restricted membrane receptor, it recognizes α2,6-linked SAs as endogenous self-ligands. CD22 has been a model receptor of cis-ligands, as an inhibitory coreceptor of a BCR specifically recognizes α2,6 sialic acids with a lectin domain capable of binding α2,6 sialic acids in the extracellular region. The cytoplasmic region contains ITIMs to negatively regulate BCR signaling [231]. The functional CD22 domain seems likely to be occupied by cis-ligands. If the sialyltransferase ST6Gal-I gene that is responsible for α2,6 sialylation is deficient in B cells or if B cells are deficient in CD22 lectin activity that is responsible for sialic acid

7.10

CD22/Siglec-2

349

recognition due to genetic mutation of the CD22 gene, then BCR signaling will be terminated [232]. Such an assumption can be made by the fact that cis-ligands regulate CD22 functions. As described above, the extracellular Ig domain-1 of CD22 binds to Sia α2,6Galβ1,4 residues. CD22 is also self-sialylated and can form cis-homo-oligomers on the surfaces of B cells. CD22 functions are operated through the intracellular recruitment of phosphatases for stimulatory coreceptor dephosphorylation. Apart from the self-sialylation on CD22, CD45 is a known CD22 ligand in the cis-form [121]. At a cellular level, the affinity for Sia α2,6-Galβ1,4 is known to be low [233]. Furthermore, because of the high contents of SAs on B- cell surfaces, the Sia α2,6-Galβ1,4 binding sites of CD22 are masked in a cis-manner by SAα2,6-Galβ1,4 residues expressed on B-cell surfaces [234].

7.10.2 CD22 I Associated with Development of Autoimmune Diseases CD22 is indeed a surface glycoprotein, which is restrictively expressed on B cells, and is a component of the BCR complex. CD22 has now been recognized as a SA-binding lectin. CD22 is present on B cells, and, when subjected to certain ligands such as sialic acids, it can inhibit the B-cell receptor signaling pathway by activating certain phosphatases. This mechanism is shown to be crucial in many defense mechanisms of our bodies. First and foremost, CD22 plays a crucial role in preventing autoimmunity by stopping B cells from producing antibodies against one’s own body. B cells are important for protecting our bodies from harmful pathogens by producing highly specific antibodies against various pathogens (Fig. 7.10). However, B cells sometimes recognize “self” as a harmful agent and thus produce autoantibodies, which cause autoimmune diseases [235]. CD22 that plays an inhibitory role in B cells also affects autoimmunity. The loss of CD22 in a certain breed of mice can increase susceptibility to autoimmune diseases [236]. For example, an autoimmune SLE is categorized as a member of the autoimmune disease family, and antinuclear antibodies produced by B cells in patients with SLEs are the hallmarks of the disease [237]. As self-recognizing antibodies cause damage to patients with SLE, many researchers are focusing on manipulating B cells to cure SLE. Epratuzumab is a CD22 IgG antibody that binds to CD22 and enables the B-cell receptor to be internalized, thus resulting in the reduction of B-cell activities [238]. Since epratuzumab reduces the activities of B cells by modulating CD22, it can be used to treat many different autoimmune diseases such as SLE but not restricted to only SLE. Although the efficacy and safety of epratuzumab are still questionable, many clinical trials are being undertaken to examine the efficacy of CD22 antibodies against various autoimmune diseases including bowel diseases and autoimmune arthritis [239–241]. As a result, CD22 is a specialized receptor for regulating B-cell functions and immune responses mediated by B cells. In highly

350

7 Sialic Acid-Binding Ig-Like Lectins (Siglecs)

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