Sialic Acids and Sialoglycoconjugates in the Biology of Life, Health and Disease [1 ed.] 0128161264, 9780128161265

Table of contents :
Cover
SIALIC ACIDS AND
SIALOGLYCOCONJUGATES
IN THE BIOLOGY OF LIFE,
HEALTH AND DISEASE
Copyright
Preface
Acknowledgments
Abbreviations
1
Sialic acid and biology of life: An introduction
Introduction
Occurrence and function
Functions of sialic acid
Structure diversity of sialic acids
O -acetylated sialic acid
Enzymes in O -acetylated sialic acid metabolism
Functions of O- acetylated sialic acids
N -acetylation of sialic acids
Other modifications of sialic acids
Sialic acid and the living world
Sialic acid and bacteria
Bacterial sialylation and host immune system
Archaea
Virus
Fungi
Plants
Invertebrates
Protozoa
Cnidarians
Helminths
Annedida
Arthropoda
Mollusca
Echinoderms
Vertebrates
Sialic acid-binding protein
Siglecs
Sialic acid-binding lectins
Selectins
Sialylation and disease
Tumor-associated carbohydrate antigens
Sialic acids and therapeutics: Where we stand
Therapeutic antibodies
Carbohydrate-based vaccines
Discussions
References
Further reading
2
Sialoglycans and genetically engineered plants
Introduction
N -glycosylation in plants
Sialylation and recombinant proteins produced in plants
Fc glycoengineering in plants
Applications
Mucin type O -glycans and plant expression
Introducing helminth glycosylation into plants
Detection
Discussion
References
Further reading
3
Sialic acid, sialoglycans, sialylation: A study in insects
Introduction
Insect physiology and development
Insects as vectors of human and animal diseases
N -Glycosylation: A comparison between mammals and insects
Sialic acids and insects
Sialylation in Drosophila
Sialylation and Spodoptera frugiperda (Sf9)
Sialylation and Ae. aegypti
Sialylation and silkworms
Ticks
Function of sialylation in insects
Genetic engineering approaches and sialylation
Discussion
References
Further reading
4
Sialoglycoconjugates and their role in physiology
Introduction
Nutrition
Reproduction
Ion channels
Nervous system
Stem cells
Cardiac function and disorders
Obesity
Complement
Dendritic cells
Others
Discussion
References
5
Pathogens, infectious disease biology and sialic acid
Introduction
Bacteria
Parasitic protozoa
Virus
Siglec sialic acid and infection biology
Prions
Discussion
References
Further reading
6
Sialic acids in autoimmune disorders
Introduction
Autoimmunity and tolerance
T cells
B cells
Sialic acid
Autoimmunity, tolerance, and sialic acid
Infectious pathogens, sialic acids, and autoimmune diseases
Immunoglobulins, sialic acids, and autoimmune diseases
Complements
Siglecs, HMGB-1, CD24, and autoimmunity
Neu5Gc and chronic inflammation
Selectins, sialic acids, and autoimmunity
Gangliosides and autoimmune diseases
Sialyltransferases and autoimmunity
Sialic acid, Glycobiotechnology, and application in autoimmune disorder therapy
Sialic acids and the therapeutic use of intravenous immunoglobulins
Discussions
References
Further reading
7
Lysosomal storage disease: Disorders related to glycans and sialic acid
Introduction
Lysosomes
Lysosomal storage diseases (LSD)
LSDs and defective glycan degradation
Defective glycoprotein degradation
Human α -mannosidosis
Human β -mannosidosis
Fucosidosis
α -N-Acetylgalactosaminidase deficiency: Schindler disease
Aspartylglucosaminuria
Defective glycolipid degradation
Fabry’s disease
Gaucher disease
Tay-Sachs disease
Defective synthesis and metabolism of gangliosides
Niemann-Pick C Disease
Krabbe disease
Multiple sulfatase deficiency (MSD)
Defective degradation of glucosaminoglycan (GAG)
MPS type IV disease or Morquio syndrome
The Sanfilippo syndrome, or MPS III
MPS I
MPS II or Hunter syndrome
MPS VI
MPS VII
MPS IX
Sialic acid-related disease
Salla disease
Free sialic acid storage disorders (FSASD)
Infantile sialic acid storage disorder (ISSD)
Sialidosis
Glycogen degradation defect diseases
Pompe’s disease
Other Glycogen storage disorders
Other LSDs
Cobalamin F-type disease
Cobalamin F-type disease
Danon’s disease (DD)
Treatment of LSDs
Discussions
References
8
Sialic acids and sialoglycoconjugates in cancer
Introduction
Sialic acid and serum sialylation as biomarkers in cancer
Gangliosides
Structure and synthesis
Gangliosides in tumors
Gangliosides and biotherapies
Sialic acid-Siglec axis and cancer
Sialyl Tn in cancer
Sialylransferase and cancer
Sialidase as cancer targets
Selectins
Discussion
References
Further reading
9
Sialic acids and sialoglycans in endocrinal disorders
Introduction
Sialylations and endocrines
Sialylations in endocrinal disorders
Ovary
Pancreas
Diabetes
Thyroid
Adrenal cancer
Pituitary cancer
Discussions
References
10
Sialic acid and xenotransplantation
Introduction
Xenotransplantation
Major problem associated with xenotransplantation and ways to circumvent them
Physiology
Rejection
Gal epitope
Complement
Thrombin
Glycosyltransferase transgenes
Acute humoral xenograft rejection
Cellular rejection
Infection
Sialic acid and xenotransplantation
Role of Neu5Gc
Role of α -gal antigen
Sialoadhesin
The Hanganutziu-Deicher (H-D) antigen
Xenograft acceptance and strategies based on sialylation
Animal xenotransplantation models and sialic acid
Strategies for knockouts
Sialylation related knockouts and health of donor animals
Discussion
References
Further reading
11
Nanotechnology and sialic acid biology
Introduction
Nanotechnology
Glyconanotechnology
Sialic acid and nanotechnology
Glycans and nanotechnology
Role of nanotechnology in detection and quantitation of sialic acid
Detection of gangliosides
Sialic acid in therapy
Nanotechnology bioimaging application, detection of cellular sialic acid expression and targeting
Discussion
References
Glossary
Index
A
B
C
D
E
F
G
H
I
K
L
M
N
O
P
Q
R
S
T
U
V
W
X
Z
Back Cover

Citation preview

SIALIC ACIDS AND SIALOGLYCOCONJU­GATES IN THE BIOLOGY OF LIFE, HEALTH AND DISEASE

SIALIC ACIDS AND SIALOGLYCOCONJU­GATES IN THE BIOLOGY OF LIFE, HEALTH AND DISEASE

SHYAMASREE GHOSH, PhD

Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, United Kingdom 525 B Street, Suite 1650, San Diego, CA 92101, United States 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom © 2020 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library ISBN 978-0-12-816126-5 For information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Andre G. Wolff Acquisition Editor: Linda Versteeg-Buschman Editorial Project Manager: Sandra Harron Production Project Manager: Debasish Ghosh Designer: Miles Hitchen Typeset by SPi Global, India

Preface In the words of Albert Einstein “Imagination is more important than knowledge. Knowledge is limited, imagination encircles the world.” Although the history of the first discovery of sialic acids is not very well documented, literature records its existence over millions of years ago but their documentation and discovery were revealed only about half a century ago. Their discovery can be traced from two sources namely the glycolipids from a cerebroside fraction and glycoproteins. The earliest history of sialic acid research has been recorded back to 1955 in the works of Gunnar Blix who was working in Sweden, Zilliken working in the United States in 1958, and Gottschalk working in Germany in 1960. In 1927 Landsteiner and Levene in the United States and Walz in 1927 in Germany independently observed sugar-like components in animal lipid preparations. Ernst Klenk in 1935, in Cologne, observed brain glycolipid and termed it ganglioside. He also discovered and coined the term neuraminic acid. Blix in 1957 discovered sialic acid. Klenk and Faillard in 1954 found crystallizable sialic acids from bovine submaxillary mucin and bovine colostrum and from mucolipids or gangliosides (Blix and Odin, 1955; Rosenberg and Chargaff, 1956). With the history of the discovery of sialic acid, the journey of sialobiology began and now the reader would be completely amazed to learn the importance of this molecule in health and disease. The molecule is known to play a major role in cell signaling and cell-cell communication in the normal healthy individuals while aberrations in its synthesis and degradation may lead to diseases. Sialic acid and its conjugates also of immense importance as biomarkers in a number of diseases and as therapeutic targets in several diseases.They are known to play important roles in the biology of transplantation. Recently research is focused in the domain of their application in effective targeting of diseases. This book is focused on the different roles played by sialic acid in health, physiology, and disease biology. Over the last two decades considerable advances in the field of molecular biology approaches and their application in plant biology have enabled the engineering of complex protein sialylation in plant systems. Advances in glycan engineering have enabled the development of ZMapp, a mixture of three anti-Ebola monoclonal antibodies in Nicotiana benthamiana line called ΔXF. ix

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Preface

We do hope the readers enjoy reading the book, and gets an idea about the fascinating and immense potential of this molecule and its conjugates, and find new avenues for further research in this exciting filed of biology and tries to answer questions, hitherto unanswered in the field of sialobiology. Shyamasree Ghosh, PhD

Acknowledgments In the words of Nobel Laureate Prof.Venkataraman Ramakrishnan “Science is not just modern gadgets. Science is any evidence-based knowledge. There is plenty of old world knowledge that stands true even today.” The human mind since time immemorial has been observing natural phenomena and trying to decipher the unknown and treading and discovering the untrodden land. The scientific, intellectual, ethical, philosophical, and political concepts of the West perhaps lay its origin in the thoughts and observation of the ancient Greeks and their desire for knowledge.The book Greek Thought: A Guide to Classical Knowledge highlights the works of scholars on science and philosophy of Ancient Greek. Understanding molecules and their functions in health and disease is a major goal of research across the world. With ongoing research across the globe, sialic acids are now considered molecules important in cell-cell communication and cell signaling pathways with vital role in health and disease. More extensive research is unfolding the importance of this molecule. The scientific efforts of scientists and technologist in the field of sialobiology in deciphering the role of sialic acid in health, physiology, and disease are acknowledged. My interest in the field of sialic acid is drawn from my guide Dr. Chitra Mandal, Ex-Director Indian Institute of Chemical Biology (IICB) under CSIR, India and Dr. Chabbinath Mandal while working with ­9-O-acetyl sialic acids and their overexpression in leukemic blasts of childhood acute lymphoblastic leukemia (ALL) patients, and my guide is sincerely acknowledged. Prof. Ronald Schauer, Biochemisches Institut, Christian-AlbrechtsUniversität, Kiel, Germany, Prof. Reinhard Vlasak, Department of Molecular Biology, University of Salzburg, Billrothstrasse, Salzburg, Austria, Prof. Reinhard Schwartz-Albiez, Department of Translational Immunology, German Cancer Research Center, Heidelberg, Germany with whom I have coauthored papers working in the domain of sialic acids and learnt about sialic acid, are sincerely acknowledged. Director, National Institute of Science Education and Research (NISER), Bhubaneswar, India, Prof. Dhrubajyoti Chattopadhyay,VC Amity University, Kolkata, India and Prof. Ashoke Ranjan Thakur, Ex-VC,WBUT, India, are acknowledged. xi

xii

Acknowledgments

Protein Data bank (pdb, https://www.rcsb.org/) comprising Protein Data Bank archive-information about three-dimensional (3D) shapes of proteins, nucleic acids, and complex assemblies from protein synthesis to health and disease is acknowledged. The entire publication team of Elsevier and immense help from senior acquisition editor, Mrs. Linda Versteeg, Mrs. Swapna Praveen, and editorial project manager Mrs. Sandra Harron are deeply and sincerely acknowledged. It is with their help that the dream came into a reality. Last but not the least my parents and my son are sincerely and deeply acknowledged, who always encouraged in my studies. Shyamasree Ghosh, PhD

Abbreviations Ae. aegypti 4-MPBA-AuNPs 9-O-AcSA 9-O-AcSGPs A. mellifera A. thaliana Ab ABC ACD AD ADCC AECs AF Ag AGL AGN AGP AHXR ALDOA ALG ALL alpha 2,6-ST ALS AML AMR anti-OAcSA AP APBA APBA-QDs APC APIs

Aedes aegypti 4-mercaptophenylboronic acid functionalized AuNps 9-O-acetyl sialic acid 9-O-acetylated sialoglycoproteins Apis mellifera Arabidopsis thaliana antibody accelerated blood clearance Angiokeratoma Corporis Diffusum Alzheimer’s disease Antibody-dependent cell-mediated cytotoxicity Aortic endothelial cells Amniotic fluid Antigen Glycogen debranching enzyme Acute glomerulonephritis α-1-acid glycoprotein Acute humoral xenograft rejection Aldolase A Asparagine-linked N-glycosylation enzymes Acute lymphoblastic leukemia Alpha 2,6 sialyltransferase Anti-lymphocyte serum Acute myeloid leukemia Antibody mediating rejection Anti-O-acetylated sialic acid alternative pathway 4-aminobenzeneboronic acid 3-aminophenylboronic acid functionalized QDs Antigen presenting cells Adult pig islets xiii

xiv

Abbreviations

apo-CIII APP ASD ASGR-1 Asialo-rhuEPO AuNPs B. dorsalis B. mori/Bm BBB BChE BCR BD BDNF BFP BSA BTP-Neu5Ac BY2 C C Domain C. nenuphar C. pulicaria C. thermocellum C6S Ca2+ CAD CD CD CDC CDG Cdot Cer CGD CHO CHS CL CMAH CMIs CMP

apolipoprotein C-III Acute phase proteins Autism spectrum disorder Asialoglycoprotein receptor 1 Asialoerythropoietin Gold nanoparticles Bactrocera dorsalis Bombyx mori Blood-brain barrier Human butyrylcholinesterase B-cell receptor Bipolar disorder Brain-derived neurotrophic factor Body fat percentage Bovine serum albumin Benzothiazolylphenol-based sialic acid Bright yellow 2 Complement Constant domain Conotrachelus nenuphar Chaetocnema pulicaria Clostridium thermocellum Chondroitin-6-sulfate Calcium ions Coronary artery diseases Cluster of differentiation Circular dichroism Complement-dependent cytotoxicity Congenital disorders of glycosylation Carbon dot Ceramide Chronic granulomatous disease Chinese hamster ovary cells Chediak-Higashi syndrome Corpus luteum Cytidine monophospho-N-acetyl Neuraminic acid hydroxylase Cell-mediated immune responses Cytidine monophosphate



CMP-NeuAc CMV CNS CNTs COD COOH-QDs CPS CRAC CRISPR-CAS9

CSAH CSAS CST CT CTLs CTP Ctx CVD D. plexippus DAMPs DC DENV DIG DKO Dm DMB DmSAS DP DTMM DTT DXM DXT/FT

E. cucumeris

Abbreviations

xv

Cytidine 5′-monophospho-Nacetylneuraminic acid Cauliflower mosaic virus Central nervous system Carbon nanotubes Calcium oxalate dihydrate Carboxyl groups modified CdSe/ZnS Quantum dots Sialylated capsular polysaccharide Calcium release activated channel Clustered regularly interspaced short palindromic repeats)-associated protein-9 nuclease CMP-sialic acid hydroxylase CMP-sialic acid synthetase CMP-sialic acid transporter Computed tomography Cytotoxic T cells Cytidine 5′-triphosphate Cholera toxin Cardiovascular disease Danaus plexippus Damage-associated molecular patterns Dendritic cells Dengue virus Differences in glycosylation Double knockout Drosophila melanogaster 1,2-diamino-4,5-methylenedioxybenzene Drosophila sialic acid synthase Double-positive cells 4-(4,6-dimethoxy-1,3,5-triazin2-yl)-4-methylmorpholinium chloride Dithiothreitol Dexamethasone Nicotiana benthamiana glycosylation mutants lacking plant-specific core b1,2xylose and a1,3-fucose residues Epitrix cucumeris

xvi

Abbreviations

E. coli ECM EDC EE EGF EGF EGFR ENO3 EOC EPO EPO-Fc EPOR ER ER ESC ESI-QTOF MS EV-D68 EVs F. nucleatum FACS FADD FasL Fc FcγR FD FGE FGFR FH fHbp FITC FSASD FSNPs FT Fuc G. melonella GAA GAGs

Escherichia coli Extracellular matrix 3-dimethylaminopropyl)-carbodiimide early endosomes Epidermal growth factor Epidermal growth factor Epidermal growth factor receptor Enolase 3 Epithelial ovarian cancer Erythropoietin Recombinant human erythropoietin fused to an IgG heavy chain fragment EPO receptor Endoplasmic reticulum Estrogen receptor Embryonic stem cells Electrospray ionization quadrupole timeof-flight mass spectrometry Enterovirus D68 Extracellular vesicles Fusobacterium nucleatum Fluorescence-activated cell sorter Fas-associated protein with death domain Fas ligand Heavy chain fragment Fc-gamma receptor Fabry disease Formylglycine-generating enzyme Fibroblast Growth Factor receptor Complement factor H Factor H-binding protein (fHbp) Fluorescein isothiocyanate Free sialic acid storage disorders Fluorescent silica nanoparticles Flow through Fucose Galleria mellonella Acid alpha-glucosidase Glycosaminoglycans



Gal GalNAc GalNAc-T GalNAcT2 GalNAc-transferase GALNS GalNT GALT GalT-KO GBM GBS GBS GBS GCE GD GD GD3 GD3S GDM GlcCer GlcNAc GlcNAc Glu-C GM3 GMCSF GNA GNE GO GPI GPI GPNE gRNA Grz GS2

Abbreviations

xvii

Galactose N-acetylgalactosamine N-acetylgalactosaminyltransferase GalNAc-transferase 2 UDP-GalNAc:polypeptide NAcetylgalactosaminyltransferase N-acetylgalactosamine-6-sulfate sulfatase GalNAc-transferases α-1,3-galactosyltransferase Knockout for the enzyme α1,3-galatosyltransferase Group B meningococcus Guillain-Barré syndrome Group B streptococci Guillain-Barré syndrome Glassy carbon electrode Gaucher disease Gangliosides Disialoganglioside 3 GD3 synthase Gestational diabetes mellitus Glucosylceramide N-acetyl-d-glucosamine N-acetylglucosamine Endoglucosaminidase C Monosialoganglioside 3 Granulocyte-macrophage colony-­ stimulating factor Galanthus nivalis Glucosamine (UDP-N-acetyl)-2epimerase/N-acetylmannosamine kinase Graphite oxide Glycosylphosphatidylinositol Glycophosphatidylinositol N-acetylglucosamine-6-phosphate 2′-epimerase guide RNA Granzyme Griffonia simplicifolia II agglutinin

xviii

Abbreviations

GSLs GT-1 GYG1 H H. ducreyi H. influenza H. pylori H. volcanii H. liguirrens H1N1 H3N2 HA HA HA HAR HBCD HBE hCSAT HD hEPCR hEPO HF HGF HGS HHA hHO-1 HR HI HIV HLA HN HPIV3 HPLC HPSA HR-NBs

Glycosphingolipids Alpha-1,3-galactosyltransferase-1 Glycogenin-1 Heavy chains Haemophilus ducreyi Haemophilus influenza Helicobacter pylori Haloferax volcanii Hemipyrellia ligurriens Influenza A virus subtype Human influenza virus X31 Hemagglutinin Hyaluronic acid Hemagglutinin Hyperacute rejection Highly-branched α-glucuronic acidlinked cyclic dextrins Human bronchial epithelial human CMP-Sia transporter Hanganutziu-Deicher human endothelial cell protein C receptor human erythropoietin Hydrops fetalis Hepatocyte growth factor High-grade serous Hippeastrum Hybrid (Amaryllis) human heme oxygenase-1 Hinge region Hashimoto’s thyroiditis Human immunodeficiency virus Human leukocyte antigen Hemagglutinin neuraminidase glycoprotein Human parainfluenza virus type 3 High-performance liquid chromatography Hydrophobically modified polysialic acid High-risk neuroblastoma



HS HSC hTFPI HufH HUVEC Hyp IAV ICAM-1 ICP-AES ICP-MS IDDM IEMs IF IFNγ Ig IgAN IGFBP IIMs IL-2 ILT ILT3 isotretinoin ISSD ITAM ITIM IVIG K(+) KD kdn KIR KLK6 KO

Abbreviations

Hunter syndrome Hematopoietic stem cells human tissue factor pathway inhibitor Human factor H Human umbilical vein endothelial cells Hydroxyproline Influenza virus Intercellular adhesion molecule-1 Inductively coupled plasma-atomic emission spectroscopy Inductively coupled plasma mass spectrometry Insulin dependent diabetes mellitus Inborn errors of metabolism Intercellular fluid Interferon gamma Immunoglobulin IgA nephropathy Insulin-like growth factor-binding protein Idiopathic inflammatory myopathies Interleukin-2 Immunoglobulin-like transcript Immunoglobulin-like transcript 3 13-cis-retinoic acid Infantile sialic acid storage disorder Immunoreceptor tyrosine-based activation motif Immune receptor tyrosine-based inhibition motif Intravenous injection potassium Krabbe disease Deaminoneuraminic acid Killer cell immunoglobulin-like receptor Kallikrein 6 Knockout

xix

xx

Abbreviations

KS L. amazonensis L. braziliensis L. major L. mexicana L. tropica L. donovani LacDiNAc LacNAc LAMP2 LC-ESI-MS LCMS LDHA LDL LE ILR LLO LOS LPS LPS LSA LSD M. catarrhalis mAb Mac-1 MAG Man Man3GlcNAc2 ManNAc ManNAc MCP-1 MERS-CoV MHC MIC MIPs MLS MMP9

Keratan sulfate Leishmania amazonensis Leishmania braziliensis Leishmania major Leishmania mexicana Leishmania tropica Leishmania donovani N,N′-di-N-acetyllactosediamine N-acetyllactosamine Lysosome-associated membrane protein 2 LC-electro-spray ionization-MS Liquid chromatography-mass spectrometry Muscle lactate dehydrogenase Low-density lipoprotein Lupus erythematosus Immunoglobulin-like receptor Lipid linked oligosaccharide Lipooligosaccharide Lipopolysaccharide Lipopolysaccharide Lipid associated sialic acid Lysosomal storage disorders Moraxella catarrhalis Monoclonal antibody Macrophage antigen complex-1 Myelin-associated glycoprotein Mannose Tri-mannosyl core N-acetyl-d-mannosamine N-acetylmannosamine Monocyte chemoattractant protein-1 Middle east respiratory syndrome coronavirus Major Histocompatibility complex MHC class I chain-related protein Molecularly imprinted polymers Maroteaux-Lamy syndrome Matrix metallopeptidase 9



MNPs MPBA MPS MRI MRnS MS MS MSD MSNs MTX MUC MuV MWCNTs N. gonorrhoeae N. meningitides N. tabacum N. benthamiana Na(+) NANA nano-LC/MS NANP NANS NBD NCAM NCs NDV NE NETs Neu5Ac Neu5Gc NeuAc NGal-Ab NHP NHS NIHF NIReg NK NMDARs

Abbreviations

Magnetic nanoparticles Mercaptophenyl boronic acid Mucopolysaccharidosis Magnetic resonance imaging Magnetic relaxation nanosensors Mass spectrometry Multiple sclerosis Multiple sulfatase deficiency Mesoporous silica nanoparticles Methotrexate Mucin Mumps virus multiwalled CNTs Neisseria gonorrhoeae Neisseria meningitides Nicotiana tabacum Nicotiana benthamiana Sodium N-acetylneuraminic acid nano-liquid chromatography/mass spectrometry Sialic acid 9-phosphate phosphatase Sialic acid 9-phosphate synthase Nitrobenzoxadiazole neural cell adhesion molecule Nanocapsules Newcastle Disease Virus Neuroectodermal Neutrophils extracellular traps N-acetylneuraminic acid N-glycolylneuraminic acid N-acetyl-d-neuraminic acid Non-Gal antibodies Nonhuman primates N-hydroxysuccinimide Nonimmune hydrops fetalis Neuroimmune regulatory proteins Natural killer N-methyl-D-aspartate-receptors

xxi

xxii

Abbreviations

NO NOS NPC NPC NPPCCs NPs NPSNPs NQAD NSC NSCLC NTHi NulOs O2- OAcGD2 ORF OS OST P. aeruginosa P. haemolytica P. spumarius P4H PA PAMPs PARP PBA PBL PBMC PCA pCECs PCR PD PD PDAC PDB PDGFR pECs pECs PEG PEI

Nitric oxide Nitric oxide synthase Niemann-pick type C Niemann-pick disease-type C Neonatal porcine islet-like cell clusters Nanoparticles Nanoporous silica nanoparticles Nano Quantity Analyte Detector Neural stem cell Non-small-cell lung cancer Nontypeable Haemophilus influenzae Nonulosonic acids Superoxide anion O-acetyl-GD2 Open reading frame Oxidative stress Oligosaccharyltransferase Pseudomonas aeruginosa Pasteurella haemolytica Philaenus spumarius Prolyl-4-hydroxylases Polysialic acid Pathogen-associated molecular patterns Poly (ADP-ribose) polymerase Phenylboronic acid Peripheral blood lymphocytes Peripheral blood mononuclear cells Principal component analysis Pig corneal endothelial cells Polymerase chain reaction Parkinson’s Disease Pompe disease Pancreatic ductal adenocarcinoma Protein database Platelet-derived growth factor receptor Porcine endothelial cells Pig epithelium cells Polyethylene glycol Polyethylenimine



pERVs PGAM2 PHKA PKFM PLCγ PLGA PLS-DA PM pni PNS PrPSc PRRs PSA-NCAM PTM PYGL PYGM pyr QA QDs QDs-PBA RA RBC RES rhEPO RMSF RNAi ROS RS RSA S. frugiperda S. littoralis S. multistriatus SABL SABP SAGNP SAM SAMPs SAMs

Abbreviations

Porcine endogenous retroviruses Muscle phosphoglycerate mutase Phosphorylase kinase Muscle phosphofructokinase Phospholipase Cγ Poly(D,L-lactide-co-glycolide Partial least-squares discriminant analysis Plasma membrane Perineural invasion Peripheral nervous system Pathogenic prion protein Pathogen recognition receptors PSA-neural cell adhesion molecule posttranslational modification Liver glycogen phosphorylase Muscle glycogen phosphorylase Pyruvate Quercetin 7-O-sialic acid Quantum dots QDs modified with PBA Rheumatoid arthritis Red blood cells Reticuloendothelial system recombinant human erythropoietin Rocky Mountain spotted fever RNA interference Reactive oxygen species Raman spectroscopy Rhizoctonia solani agglutinin Spodoptera frugiperda Spodoptera littoralis Scolytus multistriatus Sialic acid-binding lectin Sialic acid-binding glycoprotein Sialoglyco-NP Self-assembled monolayer Self-associated molecular patterns Sialic acid mimetics

xxiii

xxiv

Abbreviations

SAP SAR SAS SASD SASD SBHA SBL SCID SCLC SCNT SD Se SEAP SECs SEM SERS SGAG SHMT1 SIAE SiaT Siglec siRNA SLA class I SLAM SLE SLe(x) Sn SNA SNA-I SNP SP SPIN SPIO NPs SS SSA

SLAM-associated protein Sialic acid-binding receptor Sialic acid synthase Sialic acid storage disorder Sialic acid storage diseases Sialic acid-binding hemagglutinin Sialic acid-binding lectin Severe combined immunodeficiency Small cell lung cancer Somatic cell nuclear transfer Salla disease Selenium Secreted alkaline phosphatise Swine endothelial cells Scanning electron microscopy Surface-enhanced Raman scattering Sulfated glycosaminoglycan Serine hydroxymethyltransferase 1 Sialic acid acetyl esterase Sodium sialic acid symporter Sialic acid-binding immunoglobulin-like lectin Small interfering RNA Swine leukocyte antigen class I Signaling lymphocyte activating molecule Sialyl Lewis Sialyl-Lewis(x) Sialoadhesin Sambucus nigra agglutinin Sambucus nigra lectin Single nucleotide polymorphisms Single positive Subambient pressure ionization with nanoelectrospray Super paramagnetic iron oxide nanoparticles Sanfilippo syndrome Sambucus sieboldiana agglutinin



SSI ST St6gal1 STLS sTn SWCNTs SZ T. cruzi T eff TAA TALENs TBA TCRs Tc-TS TEM TFs TI-2 TLC TLRs TMC TME TMV TNF TNF-α TRA TRAIL Tregs TRIF TSA TSD TSP TYMV UDP-GlcNAc2-epimerase ULBP UPEC

Abbreviations

xxv

Sonic spray ionization Sialyltransferases β-galactoside α2,6-sialyltransferase-1 Siglec-engaging tolerance inducing antigenic liposomes sialyl-Tn or Sialyl-Thomsen-nouveau Single-walled CNTs Schizophrenia Trypanosoma cruzi T effector cells Tumor-associated antigens Transcription activator-like effector nucleases Thiobarbituric acid T-cell receptors T. cruzi trans-sialidase Transendothelial migration Tissue factor T-cell-independent type-2 Thin-layer chromatography Toll-like recptors Trimethyl chitosan Tumor microenvironment Tobacco mosaic virus Tumor necrosis factor Tumor necrosis factor-α Tumor-rejection antigens TNF-related apoptosis-inducing ligand T regulatory cells TIR-domain-containing adapter-­ inducing interferon-β Total sialic acid Tay-Sachs disease Total soluble protein Turnip yellow mosaic virus Uridine diphosphate-Nacetylglucosamine-2-epimerase UL-binding protein Uropathogenic Escherichia coli

xxvi

Abbreviations

UPLC-MS/MS

UPLC-Q-TOF-MS

US FDA UV V. cholera V domain VATs VCAM-1 VcN VEGF VEGFR VL VLA VSV WGA WHO wt WT1 XenoAbs Xyl ZFNs ZP αGal β4GalNAcTA βGlcNAc ΔXTFT

ultrahigh-performance liquid chromatography-hybrid mass spectrometry ultrahigh-performance liquid chromatography/time-of-flight mass spectrometry United States, Food and Drug Administration Ultraviolet Vibrio cholerae Variable domain Visceral adipose tissues Vascular cell adhesion molecule-1 Vibrio cholerae neuraminidase Vascular endothelial growth factor Vascular endothelial growth factor receptor Visceral leishmaniasis Very late antigen Vesicular stomatitis virus Wheat germ agglutinin World Health Organisation Wild type Wilms tumor 1 Xenoreactive antibodies Xylose Zinc-finger nucleases Zona pellucid alpha Gal xenoantigen β1,4-N-acetylgalactosaminyltransferases A N-acetyl glucosamine β1-4 N-acetyl glucosamine N. benthamiana glycosylation mutant deficient in β1,2-xylosyltransferase and core α1,3-fucosyltransferase

CHAPTER 1

Sialic acid and biology of life: An introduction 1 Introduction Biomolecules including monosaccharides of carbohydrates, amino acids of proteins, fatty acid of lipids, and nucleic acids including DNA and RNA play a significant role in the growth, development, and proper function of the body. Although proteins, nucleic acids, lipids, and small molecules form the major constituents of human cell, the last decade has evidenced considerable progress in the study of glycans on human cells and their role in cell-cell interaction, signaling, host-pathogen interaction, and carbohydrates contributing to important biological functions in cells. Attached to lipids and proteins, carbohydrates comprise glycoproteins and glycolipids, respectively, and play diverse myriads of roles in development, signaling, host-parasite interaction, and immune system in different organisms [1–5]. The study of sialylation in bacteria is a relatively new domain which is of immense importance in understanding and targeting host-parasite interaction in infectious diseases. Plants and insects are being exploited for the production of human recombinant glycoconjugated proteins of therapeutic importance and therefore construction of recombinant organisms capable of synthesis of glycosylated proteins as therapeutic agents in humans holds importance. Altered glycosylation and sialylation have been associated with several diseases in humans including cancer and finds importance in disease targeting. Although with the advent of new technology, it has become possible to study the complex glycans and decipher their biological role in health and disease in greater details [1–6], a lot remains unknown. Sialic acids or N-acetylneuraminic acids (Neu5Ac) are a diverse group of 9‑carbon carboxylated monosaccharides synthesized in animals, present at the outermost end of N-linked and O-linked carbohydrate chains and in lipid-associated glycoconjugates (Fig. 1, 1–6) and lack in plants [6]. Some bacterial species can de novo synthesize sialic acids while some can acquire sialic acid from host and therefore finds relevance from the point of view Sialic Acids and Sialoglycoconjugates in the Biology of life, Health and Disease https://doi.org/10.1016/B978-0-12-816126-5.00001-9

© 2020 Elsevier Inc. All rights reserved.

1

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Sialic acids and sialoglycoconjugates in the biology of life, health and disease

7,8-di-O-acetyl 7,9-di-O-acetyl 8,9-di-O-acetyl 7,8,9-tri-O-acetyl

8-O-sulfate

7-O-acetyl 8-O-acetyl 9-O-acetyl CH3

8-O-methyl

O HO

CH3

S

C

1-tauryl

O O

NH

CH2

CH2

SO3H

9-O-lactyl CH3

C

C

OH O

R8 O R9

9-O-phosphate

OH 6

O

8

9

R5

HN

OH HO

R7 O

P

2

7

5

O

HO

5-hydroxyl

C O

N-acetyl R

HN

1 3

4

R4

O CH3

COOH

O

CH3 HOH2C

C O

C O

4-O-acetyl

N-glycolyl

7-amino, 9-deoxy

Fig. 1  The family of naturally occurring sialic acid. (Image adapted with permission from Schauer R., Srinivasan G.V., Wipfler D., Kniep B., Schwartz-Albiez R. O-acetylated sialic acids and their role in immune defense. In: Wu A. (editor) The molecular immunology of complex carbohydrates-3. Advances in experimental medicine and biology, vol 705. 2011, Springer, Boston, MA.)

of host-parasite interactions revealing evolutionary relationship [7, 8]. Sialic acid-like saccharides termed as legionaminic acid have been reported to occur in Archaea [9]. The negative charge and hydrophilic properties of sialic acid enable its role in different normal and pathological processes, acting as binding sites for various pathogens and toxins wherein pathogen-binding protein recognizes sialic acids present in specific linkages which are thought to have evolved in vertebrates through evolution. Molecular mimicry exhibited by pathogens, by decorating with sialic acids, has been known to enable them to evade the host immune system [1–6, 10–12]. Sialic acid content of the human brain is the highest among other organisms, and may be associated with evolutionary advancement of an organism [1–6, 13–18]. In Neu5Gc or N-glycolylneuraminic acid, the terminal sialic acid residue is linked by the hydroxy group of the glycolic acid unit, synthesized from Neu5Ac catalyzed by CMP-N-acetylneuraminic acid (CMP-Neu5Ac) hydroxylase (CMAH) in animals including lower animals [19–22]. Nue5Gc is absent in humans as they lack CMAH gene. Oxygen



Sialobiology and life

3

and reduced pyridine nucleotide play vital roles in enzyme activity together with an effective cofactor NADH and the substrate CMPNeu5Ac, and are activated by cytochrome b5. Neu5Gc is expressed in extraneural tissues but very low, absent, or reveal suppressed expression in the vertebrate brain [21].

2  Occurrence and function Glycoconjugates are constituents of outer surface of animal cells, and their carbohydrate structures change dramatically during development. They are expressed characteristically in different stages of differentiation and are recognized by specific antibodies. In the mature organism, the expression of distinct carbohydrates is eventually restricted to specific cell types. Aberrant expression of cell surface carbohydrates in human is very often associated with malignant transformation. Sialic acids are found in glycoconjugates of some bacteria, virus, protozoa, and fungi and in animals of the deuterostome lineage [3] constituting glycoproteins and glycolipid-like gangliosides and glycosaminoglycan in mammals and lower vertebrates [3, 5, 23], acting as ligands or receptors for cell-cell communication or host-parasite interaction (1–6, Fig. 1–3). Sialic acid has been reported to occur in Drosophila melanogaster (D. melanogaster) and other insects [24, 25]. N-acetylneuraminic acid is reported to occur in cicada of Philaenus spumarius (P. spumarius) [26]. Sialic acid has been reported to occur in mollusca Arion lusitanicus (A. lusitanicus) and Arion rufus (A. rufus) [27]. Sialoglycoconjugates deuterostome lineage of the echinoderms including starfish and sea urchin is an indicative of an origin of >500 million years ago compared to higher mammals [28]. Sialic acid content in insects and molluscan gastropods is low [28–30] and lack in plants [29]. In mammalian cells, the most common sialic acids are Neu5Ac and Neu5Gc but Neu5Gc is completely absent in normal human cells [30]. Gangliosides are sialylated glycolipids that act as receptors for pathogenic bacterial infection on the gut epithelial cells [3]. Acetylated and sulfated sialic acids act as receptors for viral infections while methylated sialic acids are not found to act as receptors.

2.1  Functions of sialic acid Sialic acids affect the structure and function of glycoconjugates and act as ligands for lectins, antibodies, and enzymes. They mediate cell-cell recognition, communication, aggregation, development, carbohydrate-­protein interactions, controlling the lifetimes of glycoconjugates in organisms,

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Sialic acids and sialoglycoconjugates in the biology of life, health and disease

UDP-GlcNAc

Frc-6-P

Glc

ManNAc UDP-GlcNAc 2-epimerase ADP /ManNAc kinase

ATP

ManNAc-6-P Sia-9-phosphate synthase

PEP

Pi

Sia-9-P ( Sia 9-phosphate phosphatase

CMP-Sia synthetase

-9-P)

Pi

Sia ( PPi

)

CTP

Nucleus

CMP-Sia (CMP-

)

CMP

Golgi lumen

CMP-Sia transporter CMP

CMPSialyltransferase

CMP

Sialoglycoproteins Sialolycolipids

Fig.  2  Synthesis of sialoglycoconjugate. Sialic acid (Sia, purple diamond) is synthesized from Glc through UDP-GlcNAc as a key intermediate for sialic acid metabolism. UDP-GlcNAc is changed to ManNAc 6-phosphate (ManNAc-6-P) by UDP-GlcNAc 2-­epimerase/ManNAc kinase. Sia-9-phosphate synthetase then condenses ManNAc-6-P with phosphoenolpyruvate (PEP) to give Sia-9-P. After dephosphorylation by Sia-9phosphate phosphatase, Sia is activated to CMP-Sia by CMP-Sia synthetase (CMAS) in the nucleus. CMP-Sia is transported into the Golgi apparatus and many sialyltransferases (STs), such as ST3, ST6, and ST8, transfer Sia residues onto the glycoproteins and glycolipids forming sialoglycoproteins and sialoglycolipids (gangliosides). (Adapted with permission from Sato C, Hane M, Kitajima K. Relationship between ST8SIA2, polysialic acid and its binding molecules, and psychiatric disorders. A. Biosynthetic pathways of sialoglycoconjugates, Biochim Biophys Acta 2016,1860:1739-1752.)

­ ediating bacterial and viral infections, tumor growth and metastasis, with m role in immunology, microbiome, cell signaling, reproduction, and biology of nervous system (Fig. 3). They play a vital role in RBC stabilization and in preventing blood component aggregation by their negative charge and hydrophilicity. Sialic acids are known to affect the stability and function of hormones and enzymes. They play a significant role in reproduction, development, and sialylation of follicle-stimulating hormone (FSH) and human chorionic gonadotropin (hCG), and contribute to their stability and function [1, 33–35].



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Fig. 3  Biological and pathological roles of sialic acids. The negative charge and hydrophilicity enable sialic acids to confer neural plasticity, glomerular filtration, or blood cell charge repulsion. They act as binding sites for pathogens and toxins, wherein a ­pathogen-binding protein or extrinsic receptor recognizes sialic acid forms in specific linkages to a defined underlying sugar chain. They also act as ligands for intrinsic receptors such as Siglecs and factor H. Sialic acids enable ‘molecular mimicry,’ by which microbial pathogens incorporate host sialic acids, thereby escaping the host immune responses. Abbreviations: L1CAM, L1 cell adhesion molecule; PILR, paired ­immunoglobulin-like receptor. (Adapted with permission from Ajit Varki Sialic acids in human health and disease Trends Mol Med. 2008; 14(8): 351–360.)

They function as ligands for glycan-binding proteins, including animal lectins like selectins and siglecs, viral lectins like hemagglutinins (HAs), and bacterial lectins like adhesins and toxins. Sialosides interaction with lectins regulates immune response, immune cell functions, and cell growth and survival. Human influenza A viral HA preferentially binds to α2,6-linked sialic acid and avian viral HA preferentially binds to α2,3-linked sialic acid on cell surface glycoproteins thus enabling viral attachment and entry. Thus sialic acid-binding specificity of HA determines viral tropism and host specificity [34]. Bacterial lectins, from Helicobacter pylori (H. pylori) and Mycobacterium tuberculosis (M. tuberculosis) adhesins or Cholera and Tetanus toxins, can interact with host cells by their sialic acid-containing ligands during infection [35]. Sialic acids are known to play a significant role in the development of the central and peripheral nervous system by controlling neuronal cells

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Sialic acids and sialoglycoconjugates in the biology of life, health and disease

function [23, 36]. Sialylated glycoconjugates like Lewis antigens interact with selectins, affect cell adhesion, lymphocyte homing, leukocyte migration to inflamed sites, observed in inflammation, angiogenesis, metastasis, thrombosis, and cancer [37–39]. Host cell’s sialic acids are used by microbial pathogens Trypanosoma cruzi (T. cruzi) to mask their antigenic sites and to prevent recognition and elimination by host immune cells [40, 41].

3  Structure diversity of sialic acids Modifications of sialic acids include diverse forms differing in position 5 of an amino group of neuraminic acid derivatives or an hydroxyl group of 3-deoxy-D-glycero-D-galactononulosonic acid (Kdn), different acylations of the NH2 at position 5 (glycolyl, acetyl), and various substituent of the different hydroxyl groups including phosphate, sulfate, methyl, acetyl, etc. [3]. In the still growing family of sialic acids, >50 different derivatives [3, 5, 42] (Table 1) have been reported of which the two most commonly expressed members of sialic acid family are Neu5Ac and Neu5Gc followed by KDN (2-keto-3-deoxy-nononic acid) and Neu (neuraminic acid) [43]. Modifications to core structures of sialic acid by O-acetylation, ­O-methylation, or introduction of O-lactyl groups, sulfate, or phosphate esters at positions 4, 7, 8, and/or 9, generated by enzymes, have been reported. O-acetyltransferases catalyze the synthesis of O-acetylated sialic acid derivatives. Sialic acids can be O-acetylated at positions C-4, C-7, C-8, and C-9 of the hydroxyl groups (Table 1). The monosaccharide and its linkage to other sugars in three main configurations such as α-2,3, α-2,6, and α-2,8 contribute to the diversity. Although mono-O-acetylated forms are predominant, combinations of acetyl groups at two or more positions generate oligo-O-acetylated derivatives. O-acetylation at positions C-7, C-8, and C-9 forming N-acetyl-7, 8, and 9-O-acetyl sialic acid (O-AcSA) are most common. 9-O-acetyl sialic acid (9-OAcSA) and 4-O-acetylated sialic acid (4 O-AcSA) function as ligands for the agglutinin of influenza virus C [78] and murine hepatitis S virus [81], respectively.

3.1  O-acetylated sialic acid Eukaryotic sialic acid anabolism is a complex enzymatic process in the cytosol leading to the activation of free Neu5Ac by CTP in the nucleus. The resulting CMP-Neu5Ac and CMP-Neu5Gc (synthesized by a c­ytosolic CMAH) serve as donor substrates in various acceptor-substrate-­specific ST reactions in the Golgi. Finally, sialate-O-acetylation occurs at the ­α-­glycosidically linked sialic acid (Fig. 4) [82].



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Table 1  Naturally occurring sialic acid family members [5, 44–80, 83–88] Name

Abbreviation

5-N-Acetyl-4,9-di-O-acccyl-neuraminic acid Neu4,5,9Ac3 1,7-lactone 1,7lactone 1-Tauryl 5-N-acetyl-neuraminic amide Neu5Ac1Tau 5-N-Glycolyl-neuraminic acid Neu5Gc 4-O-Acetyl-5-N-glycolyl-neuraminic acid Neu4Ac5Gc 7-O-Acetyl-5-N-glycolyl-neuraminic acid Neu7Ac5Gc 8-O-Acetyl-5-N-glycolyl-neuraminic acid Neu8Ac5Gc 9-O-Acetyl-5-N-glycolyl-neuraminic acid Neu9Ac5Gc 4,7-Di-O-acetyl-5-N-glycolyl-neuraminic acid Neu4,7Ac25Gc 4,9-Di-O-acetyl-5-N-glycolyl-neuraminic acid Neu4,9Ac25Gc 7,9-Di-O-acetyl-5-N-glycolyl-neuraminic acidk Neu7,9Ac25Gc 8,9-Di-O-acetyl-5-N-glycolyl-neuraminic acid Neu8,9Ac25Gc 4,7,9-Tri-O-acetyl-5-N-glycolyl-neuraminic acid Neu4,7,9Ac35Gc 7,8,9-Tri-O-acetyl-5-N- glycolyl-neuraminic acid Neu7,8,9Ac35Gc 4,7,8,9-Tetra-O-acetyl-5-N-glycolyl-neuraminic acid Neu4,7,8,9Ac45Gc 5-N-Glycolyl-9-O-lactyl-neuraminic acid Neu5Gc9Lt 4-O-Acetyl-5-N-glycolyl-9-O-lactyl-neuraminic acid Neu4Ac5Gc9Lt 7-O-Acetyl-5-N-glycolyl-9-O-lactyl-neuraminic acid Neu7Ac5Gc9Lt 8-O-Acetyl-5-N-glycolyl-9-O-lactyl-neuraminic acid Neu8Ac5Gc9Lt 4,7-Di-O-acetyl-5-N-glycobyl-9-O-lacytl-neuraminic Neu4,7Ac25Gc9Lt acid 7,8-Di-O-acetyl-5-N-glycolyl-9-O-lactyl-neuraminic Neu7,8Ac25Gc9Lt acid 5-N-Glycolyl-8-O-methyl-neuraminic acid1 Neu5Gc8Me 4-O-Acetyl-5-N-glycolyl-8-O-methyl-neuraminic acid Neu4Ac5Gc8Me 7-O-Acetyl-5-N- glycolyl-8-O-methyl-neuraminic Neu7Ac5Gc8Me acid 9-O-Acetyl-5-N-glycolyl-8-O-methyl-neuraminic acid Neu9Ac5Gc8Me 4,7-Di-O-aceryl-5-N-glycolyl-8-O-methylNeu4,7Ac25Gc8Me neuraminic acid 7,9-Di-O-acetyl-5-N-glycolyl-8-O-methyl-neuraminic Neu7,9Ac25Gc8Me acid 5-N-Glycolyl-9-O-methyl-neuraminic acid Neu5Gc9Me 5-N-Glycolyl-8-O-sulfo-neuraminic acid Neu5Gc8S 5-N-Glycolyl-9-O-sulfo-neuraminic acid Neu5Gc9S 5-N-(O-Acetyl)glycolyl-neuraminic acid Neu5GcAc 5-N-(O-Methyl)glycolyl-neuraminic acid Neu5GcMe 2-Deoxy-2,3-didehydro-5-N-glycolyl-neuraminic acidg Neu2en5Gc 8-O-Acetyl-5-N-glycolyl-9-O-lactyl-neuraminic acid Neu8Ac5Gc9Lt 4,7-Di-O-acetyl-5-N-glycolyl-9-O-lactyl- neuraminic Neu4,7Ac25Gc9Lt acid 7,8-Di-O-acetyl-5-N-glycolyl-9-O-lactyl-neuraminic Neu7,8Ac25Gc9Lt acid Continued

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Sialic acids and sialoglycoconjugates in the biology of life, health and disease

Table 1  Naturally occurring sialic acid family members [5, 46–82, 85–90]—cont’d Name

Abbreviation 1

5-N-Glycolyl-8-O-methyl-neuraminic acid 4-O-Acetyl-5-N-glycolyl-8-O-methyl-neuraminic acid 7-O-Acetyl-5-N-glycolyl-8-O-methyl-neuraminic acid 9-O-Acetyl-5-N-glycolyl-8-O-methyl-neuraminic acid 4,7-Di-O-acetyl-5-N-glycolyl-8-O-methyl-neuraminic acid 7,9-Di-O-acetyl-5-N-glycolyl-8-O-methyl neuraminic acid 5-N-Glycolyl-9-O-methyl- neuraminic acid 5-N-Glycolyl-8-O-sulfo-neuraminic acid 5-N-Glycolyl-9-O-sulfo-neuraminic acid 5-N-(O-Acetyl)glycolyl-neuraminic acid 5-N-(O-Methyl)gIycolyl-neurammic acid 2-Deoxy-2,3-didehydro-5-N-glycolyl-neuraminic acidg 9-O-Acetyl-2-deoxy-2,3-didehydro-5-N-glycolylneuraminic acidg 2-Deoxy-2,3-didehydro-5-N-glycolyl-9-O-lactylneuraminic acidg 2-Deoxy-2,3-didehydro-5-N-glycolyl-8-O-methyl neuraminic acidg 2,7-Anhydro-5-N-glycolyl-neuraminic acidg 2,7-Anhydro-5-N-glycolyl-8-O-methyl-neuraminic acidg 4,8-Anhydro-5-N-glycolyl-neuraminic acidj 5-N-Glycolyl-neuraminic acid 1,7-lactone 9-O-Acetyl-5-N-glycolyl-neuraminic acid 1,7-lactone 7-Acetamido-9-O-acetyl-7-deoxy-5-N-glycolylneuraminic acid 7-Acetamido-8,9-di-O-acetyl-7-deoxy-5-N-glycolylneuraminic acidm 2-Keto-3-deoxy-nononic acid 5-O-Acetyl-2-keto-3-deoxy-nononic acid 7-O-Acetyl-2-keto-3-deoxy-nonomic acid 8-O-Acetyl-2-keto-3-deoxy-nononic acid 9-O-Acetyl-2-keto-3-deoxy-nononic acid 4,5-Di-O-acetyl-2-keto-3-deoxy-nononic acid 4,7-Di-O-acetyl-2-keto-3-deoxy-nononic acid 5,9-Di-O-acetyl-2-keto-3-deoxy-nononic acid 7,9-Di-O-acetyl-2-keto-3-deoxy-nononic acid 8,9-Di-O-acetyl-2-keto-3-deoxy-nononic acid 2-Keto-3-deoxy-5-O-methyl-nononic acid 2-Keto-3-deoxcy-9-O-phospho-nononic acidg,n

Neu5Gc8Me Neu4Ac5Gc8Me Neu7Ac5Gc8Me Neu9Ac5Gc8Me Neu 4,7Ac25Gc8Me Neu7,9Ac25Gc8Me Neu5Gc9Me Neu5Gc8S Neu5Gc9S Neu5GcAc Neu5GcMe Neu2en5Gc Neu2en9Ac5Gc Neu2en5Gc9Lt Neu2en5Gc8Me Neu2,7an5Gc Neu2,7an5Gc8Me Neu4,8an5Gc Neu5Gc1,7lactone Neu9Ac5Gc1,7lactone Neu9Ac5Gc7NAc Neu8,9Ac25Gc7NAc Kdn Kdn5Ac Kdn7Ac Kdn8Ac Kdn9Ac Kdn4,5Ac2 Kdn4,7Ac2 Kdn5,9Ac2 Kdn7,9Ac2 Kdn8,9Ac2 Kdn5Me Kdn9P

Adapted with permission from Schauer R, Kamerling JP. Exploration of sialic acid world Chapter 1, Advances in carbohydrate chemistry and biochemistry, vol. 75, 2018, Elsevier.



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Fig.  4  (A) Naturally occurring O-acetylated sialic acids. Sialic acid O-acetylation can take place at the positions C-4, C-7, C-8, and C-9. They can be mono-O-acetylated, oligo-O-acetylated, combined acetylation at C-7 and C-9 leading to di-O-acetylated Neu5,7,9Ac3 form. (B) Model of side-chain O-acetylation of sialic acids. Sialic acids are transported in their CMP-activated form into the Golgi serving as substrates for different sialyltransferase (ST). Acetyl-CoA (AcCoA) enters the Golgi by an AcCoA-transporter and provide it for the O-acetyltransferase (OAT), which transfers the acetyl moiety to the glycosidically bound sialic acid probably at position C-7. After a migration to position C-9, which could be enzymatically catalyzed, an additional O-acetylation reaction can take place at position C-7. (C) Disease-specific glycotope Neu5,9Ac2-GPs. expressed on PBMCALL [PBMC of childhood acute lymphoblastic leukemia (ALL)]. Superimposed three-dimensional (3D) structures of all the sialic acid derivatives which were used as inhibitors of the binding of Neu5,9Ac2-GPsALL to Achatinin-H. (Source: (B) Reproduced from Schauer R, Schmid H, Pommerencke J, Iwersen M, Kohla G. Metabolism and role of O-acetylated sialic acids. Adv Exp Med Biol 2001;491:325-42 with permission. (C) Adapted with permission from Ghosh S, Bandyopadhyay S, Mukherjee K, Mallick A, Pal S, Mandal C, Bhattacharya DK, O-acetylation of sialic acids is required for the survival of lymphoblasts in childhood acute lymphoblastic leukemia (ALL). Glycoconj J 2007, 24:17-24.)

3.1.1  Enzymes in O-acetylated sialic acid metabolism The metabolism of O-AcSA can be divided into two parts. (i) It is initiated by the formation of acetyl esters at different hydroxyl groups of sialic acids by the transfer of activated acetyl groups from acetyl coenzyme A (AcCoA) by O-acetyltransferases (Fig. 4).

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Sialic acids and sialoglycoconjugates in the biology of life, health and disease

Differing in their r­egioselectivity, two groups of sialate-O acetyltransferases exist: (a) acetyl-CoA:sialate 7 [9]-O-acetyltransferase responsible for the frequently found O-acetylation side chain and (b) acetyl-CoA:sialate 4-O-acetyltransferase which leads to the pyranose ring-O-acetylation at C4. (ii) Several enzymes degrade free or glycosidically bound O-AcSA acids such as esterases for Neu5,9Ac2 and Neu4,5Ac2, respectively, which are found in influenza C virus, lysosomes of mammalian cells, mouse hepatitis virus, and in horse liver. 3.1.2  Functions of O-acetylated sialic acids In the past three decades, O-acetylated sialic acids (O-AcSA) have been shown as important cell membrane components that play fundamental roles in the development, immune regulation, cancer processes, and many other biological and pathophysiological events [17, 82, 84]. The biological effect depends on the position relative to the 9‑carbon scaffold in Neu5Ac or Neu5Gc. The O-acetyl group being a more hydrophobic moiety when introduced into the sialic acid molecule the parameters like size, net charge, hydrogen bonding and conformation of glycoconjugate alters and the terminal location enables their involvement in different functions such as cell-cell adhesion, signaling, differentiation and metastasis [85–88]. The extended nature of oligosaccharide chains, and possibly their negative charges, plays an important role in cell-cell and cell-matrix interactions [86]. O ­ -acetylated sialic acids reported to occur in bacteria and parasites are known to act as receptor determinants for some viruses, well-known cancer markers in childhood acute lymphoblastic leukemia (ALL), and also regulate ganglioside-mediated apoptosis. Sialic acid-specific O-acetyltransferases and O ­ -acetylesterases regulate their synthesis [89]. Modifications of sialic acids exhibit tissue-specific and developmentally regulated expression. O-AcSA occurrence is predominant in growing and developing tissues of neuroectodermal origin and in B and T lymphocytes [90], 9-O-Ac-GD3 distribution on the normal tissues is largely restricted to brain [91] while 9-O-acetylsialylated GT3 ganglioside [92] expression is developmentally regulated in rat embryonic cerebral cortex. Sialic acids acetylation is selectively developmentally regulated in the central and peripheral nervous system, the retina, and the medulla of kidney [93] in rat tissue. O-acetylated GD3 on human lymphocytes [94] is reported as a marker for human CD8+ T helper cells [95]. Sialyl-Le x and Sialyl-Le a ligands for selectins are known to play a major role in the early steps of leukocyte r­olling on



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endothelial cells [96]. 9-O-acetylations on immune cells have been reported to regulate CD22 β adhesion negatively through masking of the carbohydrate epitope. 9-O-acteyl sialoglycoconjugates (9-O-Ac-SGs) on erythrocytes in visceral leishmaniasis (VL) serve as diagnostic and prognostic markers [97, 98] and are known to activate the alternate complement pathway enhancing hemolysis accounting for anemia [99]. Sialic acids on Leishmania donovani (L. donovani) amastigotes and two 9-O-AcSGs with Mw 158,000 and 150,000 have been identified as components of amastigote cell surface [99–101]. O-acetyl GD3 is not expressed on human normal tissues [102]. 9-O-Ac-GD3 is considered as an oncofetal marker in animal and human tumors like neuronal tumors, melanoma, basalioma or breast cancer (BC), and in psoriatic lesions [88]. 9-O-acetylated sialic acid and glycoconjugates have been reported as diagnostic and prognostic markers in childhood ALL [83, 103–107]. 9-Oacetylations have been reported to be upregulated in basal cell carcinoma tissues than in the surrounding skin [108]. O-acetylation of disialoganglioside GD3 by human melanoma cells has been reported to be a unique antigenic determinant [109]. BC cells have recently been reported to express b-series gangliosides GD3 and GD2, and O-acetylated GD2 (O-AcGD2) in which 9-O-acetyl-N-acetylneuraminic acid (Neu5,9Ac2) is predominant O-acetylated sialic acid species of GD2 [110]. Two disease-specific 9-O-Ac-SGs, of molecular weight 90 and 120 kDa, have been reported on PBMC of patients from childhood acute lymphoblastic leukemia (PBMCALL) [103, 111] with diagnostic and prognostic importance [103, 111–114] and biological function [83, 103–107], which have been demonstrated by Achatinin-H, a lectin isolated from Achatina fulica (A. fulica) snail, with 9-O-AcSAα2–6GalNAc2 as its lectinogenic epitope. 9-O-AcSA-specific IgM and IgG antibodies have been reported in ALL patients and finds importance in the detection and monitoring of patients [115, 116].

3.2  N-acetylation of sialic acids The N-acetyl group of Neu5Ac at fifth position is known to originate from AcCoA [117–120] (Fig. 5) during conversion of GIcNH2-6-P to GlcNAc6-P, the precursor of UDP-GlcNAc. This then converts to ManNAc by epomerization reaction, and finally to CMP-Neu5Ac [117–119, 121]. Neu5Ac is transferred to macromolecules from the nucleotide sugar, which can later be released into the lysosomes, and exported into the cytosol [122, 123]. The N′-acetyl group can be converted to N′-glycolyl group by a specific hydroxylase [124].

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Sialic acids and sialoglycoconjugates in the biology of life, health and disease

Endocytic Pathways Pre-lysosomal compartment

Lysosome Neu5Ac

Neu5Ac

2

Neu5Gc

Neu5Ac

ManNGc

CMP-Neu5Gc

Neu5Gc

Cytosol

: Glycoconjugate 2: Neuraminidase

?

Plasma membrane secretory pathways

Neu5Gc

Neu5Ac

Neu5Ac

Neu5Gc

2

GlcNAc ManNAc

2?

Neu5Gc

1 CMP-Neu5Ac

Neu Neu5Gc

GOLGI

?3

? Neu5Ac

1: Hydroxylase 3: de-N-acetylase?

ER

Fig. 5  Steps in N-acetylation of sialic acid. (Adapted from Varki A. Diversity in the sialic acids. Glycobiology, 1992, 2:25–40 with permission.)

3.3  Other modifications of sialic acids Unsaturated sialic acids occur in nature or biological fluids generated by enzymes or chemical processes such as 2,7-anhydro sialic acids released by sialidases [125–129], 2,3-didehydro 2,6-anhydro from mild CMP-sialic acids [130], and 4,8-anhydro compounds from release or deacetylation of 4–0-acetylated compounds [131, 132]. The phosphate group of Neu5Ac9P emerges from ManNAc-6-P. (i) Deaminated neuraminic acid (KDN) although reported to occur in vertebrates and bacteria, is predominantly expressed in lower vertebrates. KDN is linked to most glycan structures in place of Neu5Ac and is found to occur as glycoconjugates, including glycolipids, glycoproteins, and capsular polysaccharides. They exhibit linage types such as α2,3, α2,4, α2,6, and α2,8 bearing similarity to Neu5Ac. KDN de novo biosynthesis involves mannose as a precursor, activated to CMPKDN and transferred to acceptor sugar residues. Predominant KDN expression has been reported to occur in fetal human red blood cells and ovarian tumor tissues as compared to adult RBC and normal individuals. KDNase, which cleaves KDN linkages, is reported to occur in bacteria [133]. KDN could arise from sequential deacetylation and deamination of Neu5Ac. C8 acidic sugar 3-deoxy-d-manno-2-octulosonic acid (Kdo) is reported to occur in Gram-negative bacteria lipopolysaccharide (LPS) and plant cell pectic rhamnogalacturonan II. In the light of recent discoveries ­although hitherto unknown, sialic acid expression in algae Kdo has been



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reported. de novo biosynthesis of the deaminated sialic acid, 3-­deoxyd-glycero-d-galacto-2-nonulosonic acid (Kdn), has been reported to occur in Prymnesium parvum (P. parvum) with probable indications of role in host pathogen [134]. ( ii) Other types of substitutions of the hydroxyl groups arise from use of the appropriate donors such as S-adenosylmethionine for methylated sialic acids and 3′-phosphoadenosine 5′-phosphosulphate for sulfated molecules. However, not many studies have been done on other modifications like O-lactyl groups. Sialic acid family has now been designated as subclass of the superfamily of naturally occurring non-2-ulosonic acids (NulOs), the parent molecule of the family being neuraminic acid (Neu), which is not found in free form in nature due to its immediate cyclization to form an internal Schiff base, and is a nine‑carbon-containing monosaccharide, comprising 2-keto-carboxylic acid, a deoxysugar, and an aminosugar. Other members of the NulO superfamily include 5,7-diamino3,5,7,9-­tetradeoxynon-2-ulosonic acids, with the mother molecules pseudaminic acid (Pse), legionaminic acid (Leg), 4-epi-legionaminic acid (4eLeg), 8-epilegionaminic acid (8eLeg), acinetaminic acid (Aci), and 8-epi-acinetaminic acid (8eAci), found in bacterial polysaccharides and glycoproteins [5] (Fig. 6). UDP-α-D-GlcNAc

UDP-α-D-GlcNAc

H2O

H2O

UDP-GlcNAc 2-epimerase/ ManNAc kinase

UDP

UDP-GlcNAc 2-epimerase

D-ManNAc

UDP-GlcNAc 2-epimerase/ ManNAc kinase

ATP ADP

PEP Pi

Neu5Ac9P Neu5Ac 9-phosphate phosphatase

(A)

UDP

D-ManNAc

Neu5Ac synthase

GDP-α-D-6dGlcNAc4NAc

D-Man

PEP

ATP Mannokinase

Pi

D-ManNAc6P

Neu5Ac 9-phosphate synthase

GDP-α-D-GlcNAc

D-Man6P

CTP PPi

Kdn 9-phosphate synthase

CMP-β-Neu5Ac

H2O Pi

(B)

D-6dManNAc4NAc

PEP

Leg5,7Ac2 synthase

Pi

(C)

PEP Pi

Leg5,7Ac2

Kdn9P Kdn 9-phosphate phosphatase

H2O GDP

ADP

Neu5Ac CMP-Neu5Ac synthase

GDP-6dGlcNAc4NAc hydrolase/2-epimerase

H2O

CMP-Leg5,7Ac2 synthase

Pi

CTP PPi

(D)

Fig. 6  Metabolism of non-ulosonic acids: (A) Neu5Ac in vertebrates, (B) Neu5Ac in bacteria, (C) KDn in vertebrates, and (D) Leg 57Ac2 in bacteria. (Adapted with permission from Schauer R and Kamerling JP Exploration of sialic acid world Chapter 1, Advances in carbohydrate chemistry and biochemistry, vol. 75, 2018, Elsevier.)

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Sialic acids and sialoglycoconjugates in the biology of life, health and disease

4  Sialic acid and the living world 4.1  Sialic acid and bacteria Glycoconjugates occur on both prokaryotic and eukaryotic cell surfaces with important biological functions like cell-cell and small molecule-cell recognition and communication. Sialic acids are predominantly found on eukaryotic cell surfaces. Pathogens (Table  2) reveal property of acquiring host sialic acid on cell surfaces, thereby mimicking the host and escaping the host immune responses. Neu5Ac and its structural variants such as substitutions at carbon 5, or covalent modifications of the hydroxyl groups in sugars find importance in this context. Bacteria can either express sialic acid by de novo biosynthesis or acquire sialic acid from their environment and transport in the cell surface using transporters, thereby leading to the formation of sialic acid-acquired surfaces that can affect the host-parasite interaction. Bacteria can process them by common pathways for sialic acid metabolism and use sialic acid in different roles such colonization owing to its disease causing property. Table 2  Pathogens expressing sialic acids on their surfaces Pathogen

Major disease

Sialic acid synthesized by pathogen

Neisseria meningitidis B Escherichia coli K1 Group B Streptococcus Campylobacter jejuni

Meningitis Neonatal meningitis Neonate and infant infections Enteritis, Guillian-Barré syndrome

Host sialic acid taken up by pathogen

Hemophilus influenza Hemophilus ducreyi

Respirator infections Chancroid

Host sialic acid transferred by trans-sialidase

Trypanosoma cruzi Corynebacterium diphtheria

Chagas disease Diphtheria

Host CMP-sialic acid used by sialyltransferase

Neisseria gonorrhoea Neisseria meningitides group A

Gonorrhoea Meningitis

Source of sialic acid un known

Sporotrichium schenkii Aspergillus fumigates

Skin infection Opportunistic infections

Adapted with permission from Ajit Varki Sialic acids in human health and disease Trends Mol Med. 2008; 14(8): 351–360.



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De novo synthesis of sialic acid is reported in Escherichia coli (E. coli) K1, Neisseria meningitidis (N. meningitidis), and Campylobacter jejuni (C. jejuni) [8] wherein UDP-GlcNAc acts as sialic acid biosynthesis precursor and finds importance in cell wall biosynthesis, and NeuC and NeuB proteins enable Neu5Ac by ManNAc conversion (Figs. 7 and 8). Some bacterial pathogens acquiring sialic acid from host secrete sialidase that releases sialic acid from host [135]; but Haemophilus influenzae (H. influenzae), lacking sialidase genes [136], acquires free host sialic acid by other sialidase-expressing bacteria in the host [137], or by the action of host sialidases [138, 139], activated during disease/inflammation. Bacteria use specific transporters to take in this free sialic acid including NanT sialic acid transporter from E. coli K-12 [8] that transports Neu5Ac uptake [8]. H. influenza and Pasteurella multocida (P. multocida) use high affinity tripartite ATP-independent periplasmic (TRAP) transporter, SiaPQM [140–142] and extracytoplasmic solute receptor (ESR) protein for transport; Haemophilus ducreyi (H. ducreyi, SatABCD), causing chancroid, uses a high affinity ABC transporter [143] for the transport of sialic acid. Neisseria gonorrhoeae (N. gonorrhoeae) uptakes activated form of sialic acid CMP-Neu5Ac by secreting the enzymes that sialylates its LPS contributing to virulence factor. Neu5Ac-inducible porin NanC (YjhA) from E. coli K-12 has recently been studied for playing a role in growth on Neu5Ac even though lacks both OmpC and OmpF porins [144]. H. influenza and E. coli can also utilize the transported sialic acid as a carbon and nitrogen source [8, 156], the Nacetylneuraminate aldolase NanA cleaves Neu5Ac to ManNAc and pyruvate (Fig. 7). ManNAc is later converted to fructose 6-phosphate and ammonia by NanK, NanE, NagB, and NagA proteins for further metabolism [8]. After either de novo synthesis or acquiring from host, sialic acid is converted to activated form CMP-Neu5Ac by CMP-sialic acid synthetases, while linkage-specific sialyltransferases enable their addition to appropriate acceptors. N. gonorrhoeae uses outer membrane-associated sialyltransferase to scavenge CMP-Neu5Ac directly from host [145]. In E. coli, NeuA activates Neu5Ac before incorporation into the K1 and K92 capsules while the N. meningitidis ortholog enables both capsule and LPS synthesis. In E. coli, NeuS acts as polysialyltransferase adding Neu5Ac to oligosialic acid receptors to form the polysialic acid (PSA) polysaccharide capsule, exported through the Kps system [8]. After synthesis sialic acid in the PSA capsule of both N. meningitidis and E. coli can be modified by O-acetylation [146–148] like O-acetyltransferases NeuO and NeuD in

Sialic acids and sialoglycoconjugates in the biology of life, health and disease

Fig. 7  See figure legend on opposite page.

16



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Fig.  7, Cont’d Overview of the major pathways for sialic acid utilization in bacterial pathogens. Sialic acid/Neu5Ac. Black arrow: The site of interaction of factor H (fH) on the gonococcal cell surface. Monomeric O-acetylated Neu5Ac produced by E. coli NeuD is believed to enter the normal PSA biosynthetic pathway via NeuA/NeuS [164]; light-green discontinuous arrow: LPS is exposed on the outer membrane. Asterisk: O-acetylation of the disialylated LPS of C. jejuni; catalyzed by the product of the gene orf11, or sialic acid O-acetyltransferase (SOAT) [157]. IM, inner membrane; OM, outer membrane; Neu5Ac, N-acetylneuraminic or sialic acid; ManNAc, N-acetylmannosamine; GlcNAc, N-acetylglucosamine; GlcN, glucosamine; Fru, fructose; PSA, polysialic acid; PEP, phosphoenolpyruvate; Lst, Neisseria LPS sialyltransferase; NanC, E. coli Neu5Acspecific porin; Kps, E. coli PSA capsule export system; SatABCD, H. ducreyi Neu5Ac ABC (ATP-binding cassette) transporter; SiaPQM, H. influenzae/P. multocida Neu5Ac TRAP (tripartite ATP-independent periplasmic) transporter; NanT, E. coli Neu5Ac MFS (major facilitator superfamily) transporter; SiaB and NeuA, respectively H. influenzaem and E. coli CMP-Neu5Ac synthetases; Lic3A, Lic3B: H. influenza sialyltransferases; SOAT, C. jejuni Neu5Ac O-acetyltransferase; NeuC, E. coli UDP-GlcNAc 2-epimerase; NeuB, E. coli Neu5Ac synthase; NeuS, E. coli polysialyltransferase; NeuO, E. coli PSA O-acetyltransferase; NeuD, E. coli Neu5Ac O-acetyltransferase; NanA, Neu5Ac aldolase; NanK, ManNAc kinase; NanE, ManNAc-6P epimerase; NagB, GlcNAc-6P deacetylase; and NagA, GlcN-6P deaminase (the catabolic pathway is present in several bacteria: 8). (Image Reproduced with permission from Severi E et  al. Sialic acid utilization by bacterial pathogens. Microbiology. 2007;153(Pt. 9):2817-22.)

E. coli can modify PSA and monomeric Neu5Ac, respectively, the latter can be deacetylated by NeuA acting as a bifunctional enzyme [148–150]. Streptococcus agalactiae (S agalactiae) is the only Gram-positive b­ acteria causing serious infections in newborns, and can produce sialic acid-­containing capsule by using sialyltransferase (CpsK) adding terminal α-2,3-linked Neu5Ac to galactose within the capsule’s oligosaccharide repeat [151]. Neu5Ac can be modified by O-acetylation [152]. Sialylation of the LPS is mediated by linkage-specific sialyltransferases. Both N. meningitidis and N. gonorrhoeae sialylate their LPS by α-2,3 sialyltransferase L. LPS sialylation is reported to occur in Pasteurellaceae, including H. ducreyi, H. influenzae, Haemophilus somnus (H. somnus), and P. multocida [153]. Lic3A, sialyltransferase adding α-2,3-Neu5Ac [154], is associated with bacterial survival [155]. Lic3B can add mono- or disialic acid to the LPS acceptor [153]. C. jejuni possesses mono- or bifunctional LPS sialyltransferases transferring either α-2,3-Neu5Ac or disialic acid [156]. Terminal sialic acid residue in the disialylated LPS can also be modified by an O-acetyltransferase (Figs. 7) [157] as identified in C. jejuni.

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Sialic acids and sialoglycoconjugates in the biology of life, health and disease

Fig. 8  Example of N-glycans from invertebrates including parasitic or free-living organisms, hosts, or vectors for parasites. A non-exhaustive selection of core and antennal epitopes is shown in the inset: core difucosylation, core ‘GalFuc,’ Lewis X (LeX), fucosylated and non-fucosylated LacdiNAc (LDN), and blood group A. (Image adapted with permission from Paschinger K, Wilson IBH. Comparisons of N-glycans across invertebrate phyla. Parasitology 2019, 3:1-10.)

Sialylated LPS and PSA capsules confer protection to the bacteria to escape host immune responses by ‘molecular mimicry’ [158] as was observed in studies on neisseria and hemophilus bacteria. Neisseria spp., H. influenza and C. jejuni, reveal reversible on to off switching leading to variable expression and also O-acetylation of the PSA capsule of E. coli K1 [148, 159]. 4.1.1  Bacterial sialylation and host immune system The PSA capsule of N. meningitides serogroup B and E. coli K1 is poorly immunogenic and PSA reveal structural similarities to mammalian neuronal cell adhesion molecule, NCAM [160]. The sialylated capsule of S. agalactiae inhibits phagocytosis, impairs C3 deposition on the cell surface, and prevents complement alternative pathway [161] activation.



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LPS sialylation inhibits the complement alternative pathway in both N. gonorrhoeae and non-typable H. influenza (NTHi) [162, 163]. Gonococcal sialylated LPS increases the binding of bacteria to factor H (fH), a complement alternative pathway inhibitor [163], thus conferring protection from C3 attack [163]. In NTHi, LPS sialylation inhibits deposition of C3 without fH binding [162].

4.2 Archaea N-glycosylation is a posttranslational modification that occurs in all three domains. In Archaea, N-linked glycans reveal diversity which is not observed in either Eukarya or Bacteria with the lack of expression of nonulosonic acids (NulOs), sialic acids, pseudaminic acids, and legionaminic acids, in contrast to Eukarya and Bacteria. In haloarchaea Halorubrum sp. PV6 includes an N ­ -formylated legionaminic acid and a biosynthetic pathway [9].

4.3 Virus Viral sialic acid-recognizing lectins or HAs can agglutinate RBC. Viruses use sialic acids linked to glycoproteins and gangliosides to attach to host cells, followed by their entry, for example, corona virus, DNA tumor viruses, hepatitis virus, influenza viruses (A, B, and C), mouse polyoma virus, mumps, Newcastle disease virus (NDV), norovirus, parainfluenza viruses, rotavirus, and Sendai virus. HAs from influenza A, C, NDV, and polyoma viruses have been crystallized. Sialic acid-recognizing lectins from adenoviruses and picornaviruses have not been identified. Some of these viruses carry neuraminidase or sialyl-O-acetyl-esterase that destroys the receptor, promotes virus release from infected cells, and removes sialic acid on host cell affecting cell surface binding of the virus. Influenza A virus enters the host by using host surface sialic acids. Influenza C virus HAesterase specific for 9-O-acetylated sialic acids can break down 9-O-acetyl ester. HA-esterase from mouse hepatitis virus is specific to sialic acids substituted by O-acetyl group at the C-4 position (Neu4,5Ac2). HA-neuraminidase of NDV84 and parainfluenza viruses perform vital functions in infection biology [6, 165].

4.4 Fungi Sialic acids have been reported to occur in some pathogenic fungal cells such as Candida Cryptococcus neoformans (C. neoformans), Aspergillus fumigatus (A. fumigatus), and Sporothrix schenckii (S. schenckii). Neu5Ac, Neu5Gc, and Neu5,9Ac2 were reported to have been expressed. It is hypothesized that probably fungi may have unique ways to synthesize sialic acid however they may acquire it from the environment [166–169].

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Sialic acids and sialoglycoconjugates in the biology of life, health and disease

A. fumigatus pathogenic variety reveals greater sialic acid density as compared to that of non-pathogenic Aspergillus species [170]. Scedosporium apiospermum (S. apiospermum), Scedosporium aurantiacum (S. aurantiacum), Scedosporium minutisporum (S. minutisporum), and Lomentospora prolificans (L. prolificans) reveal lack of sialic acid [171, 172]. A. fumigatus has been known to express a sialidase termed as KDNase that prefers sialic acid substrate, 2-keto-3-deoxy-D-glycero-D-galacto-­ nononic acid (KDNase) and plays an important role in maintaining cell wall integrity and virulence [173]. Core 1 O-linked glycan-specific lectin, Hericium erinaceus lecin (HeL), has been isolated from the fruiting body of the mushroom Hericium erinaceus (H. erinaceus), which acts as a natural source for a sialic acid-binding lectin (SABL) [174]. A lectin has been reported to occur in Australian indigenous mushroom Psathyrella asperospora (P. asperospora) termed as PAL with cyotoxic properties on human colon cancer HT29 and monkey kidneyVERO revealing binding preference toward N-acetylglucosamine (GlcNAc) and sialic acid (Neu5Ac) [175]. Most Fusarium lectins exhibit binding affinity to d-ribose, l-fucose, d-glucose, l-arabinose, d-mannitol, d-galactosamine hydrochloride, d-galacturonic acid, N-acetyl-d-galactosamine, N-acetylneuraminic acid, 2-deoxy-d-ribose, fetuin, asialofetuin, and bovine submaxillary mucin (BSM) [176].

4.5 Plants Plants lack sialic acid but studies on recombinant plant glycoproteins are being conducted. Neu5Ac however has been reported to occur in buckwheat using mass spectrometry. R-keto acids in plants include 3-deoxyD-­arabinoheptulosonic acid 7-phosphate (DAHP) and Kdo in cell wall polysaccharides. Arabidopsis thaliana (A. thaliana) geneome revealed lack of genes for biosynthesis, activation, or transfer of sialic acid [177–179].

4.6 Invertebrates N-glycosylations have been reported to occur in invertebrates (Fig. 8). Sialic acid is synthesized and expressed by different invertebrates. We discuss in brief the expression of sialic acid in invertebrate animals and its functions. 4.6.1 Protozoa Parasitic protozoa are known to reveal sialoglycoconjugates which play an important role in their biological function (Table 3) and T. cruzi (Fig. 9), the causative agent of life-threatening Chagas disease in South America, is



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Table 3  Biological role of sialoglycoconjgates in parasitic protozoa [99–101, 186–210] Parasite

Sialoglycoconjugates

Biological relevance

Trypanosoma cruzi

Acquisition of siallct acids by mucins

Trypanosoma bruœl

Acquisition of sialic acids by procyclic repetitive proteins (PARPs). Appearance of sialic acid during encystation and gangliosides in trophozoites. Absence of Neu5Ac, instead sialic acidbinding protein EBA175 is present. Sialic acid-specific lectin identified in parasite culture supernatant Uptake of fetuin from the culture medium. Polyanionic adsorption

(a)  Invasive determinant (b) Modulation of host immune response (c) Protection against cytolytic agents. (a)  Invasion of host cells (b) Survival within insect vector.

Entamoeba histolytica

Plasmodium falciparum

Trichomonas vaginalis and Trichomonas foetus Toxoplasma gondii Leishmania donovani

(a) Decreases parasite adherence to target cens thereby reducing its cytolytic activity. Ligands for EBA-175 are essential for erythrocyte invasion. Enhances parasite adhesion to mucosal surfaces Not known (a) Determinant of virulence (b) Complement mediated cell lysis

Adapted from Chava AK, Bandyopadhyay S, Chatterjee M, Mandal C. Sialoglycans in protozoal diseases: their detection, modes of acquisition and emerging biological roles. Glycoconj J 2004a. 20:199–206 with permission.

known to express sialic acids transferred from the host glycoconjugates to the terminal β-galactopyranosyl residues of mucin-like molecules on parasite surface by using the enzyme trans-sialidase [180]. These sialic acids might play a role in conferring protection from the recognition and response by the host immune system. Sialic acid such as Neu5Ac, Neu5Gc, Neu5,7Ac2, and Neu5,9Ac2 have been reported to occur in Dictyostelium discoideum, a trypanosome Crithidia fasciculata (C. fasciculata), piroplasmid Theileria sergenti (T. sergenti), and amoebae Entamoeba invadens (E. invadens) and Entameoba histolytica (E. histolytica) [6, 181–185].

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Sialic acids and sialoglycoconjugates in the biology of life, health and disease

(A)

(B) Host sialoglycoconjugate [α2-3]

Cell surface membrane Culture medium (FCS)

Trans-sialidase

Polyanionic adsorption

Adsorbed sialoglycans (α2-3 & α2-6 )

Parasite sialoglycan (α2-3)

(C) Glycoconjugate ST

Golgi Nucleus

Sialoglycoconjugate

CMP-Neu5Ac Neu5Ac ( )

Cytosol

ManNAc Glc

UDP-GlcNAc

- β - Gal - Sialic acid/Neu5Ac

– Sialoglycoconjugate

ST - Sialyltransferase

Fig. 9  Representative profile of sialic acid acquisition by protozoa: (A) with the help of trans-sialidase in T. cruzi and Trypanosoma brucei (T. brucei), (B) polyanionic adsorption of serum sialoglycans by L. donovani promastigotes, and (C) de novo synthesis of sialic acid in E. histolytica. (Adapted from Chava AK, Bandyopadhyay S, Chatterjee M, Mandal C. Sialoglycans in protozoal diseases: their detection, modes of acquisition and emerging biological roles. Glycoconj J 2004;20:199–206.)

4.6.2 Cnidarians Mechanoreceptors in sea-anemone tentacles are activated on binding to acetylated sugars and proline from prey [211] while tentacles of certain sea anemones bind N-acetylneuraminic acid (NANA) predisposing



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c­ ontact-sensitive mechanoreceptors (CSMs) [212–215] to trigger discharge upon physical contact of prey and cyclic AMP (cAMP) has been reported to be involved in NANA-sensitized nematocyst discharge [212, 216]. NnL lectin from a jellyfish Nemopilema nomurai (N. nomurai) revealed hemagglutinating activity that was inhibited by N-acetyl-D-galactosamine NANA and Neu5Gc [217]. 4.6.3 Helminths Lymphatic filariasis reveals altered IgG glycosylation, and while decreased galactosylation bears relation with inflammation, increased sialylation is associated with anti-inflammatory responses [218]. Mucins of Haemonchus contortus (H. contortus) or Teladorsagia circumcincta (T. circumcincta) parasites in sheep revealed fucose, glucosamine, galactose, and galactosamine and minute amounts of sialic acids [219]. Schistosoma bovis (S. bovis) a parasite of wild and domestic ruminants lack sialic acid expression but expressed complex-type N-glycans and immunogenic GalNAcβ1–4GlcNAc (LDN) terminate antennae on excretory-secretory (ES) glycoproteins [220]. Dogs infected with adult tapeworms of Echinococcus granulosus (E. granulosus) release fecal antigens (coproantigens) constituting α-D-mannose and/or α-d-glucose, β-­ galactose and N-acetyl-β-glucosamine, N-acetyl-β-glucosamine, and sialic acid residues [221] and antigenic properties occur in cyst-derived glycilipids [222]. Sialic acids were identified in the acidic fraction of glycolipids of E. granulosus metacestode tissue [221]. The altered glycosylation of intestinal mucins of mouse infected with Nippostrongylus brasiliensis (N. brasiliensis) revealed a transferase adding a terminal GalNAc to sialic acid-containing epitope in rat [223]. The sialylation of mucins during a 13-day infectious cycle in Sprague-Dawley rats infected with N. brasiliensis parasite revealed a relative decrease in Neu5Gc compared with Neu5Ac by decreased expression of a CMAH hydroxylase [224]. The removal of adult worms of parasite N. brasiliensis from the small intestinal goblet cell mucins of mice is hypothesized to be possibly associated with terminal GalNAc and sialic acid residues of the small intestinal goblet cell mucins prior to infection [225]. Recently Schistosoma mansoni (S. mansoni), causing human schistosomiasis, has been reported to be rich in fucose, containing terminal beta-­GalNAc residues but lack sialic acid [226, 227]. The expressed fucosylated glycans containing Lewis x (Le(x)) antigen common to human leukocytes and other tissues produce autoantibodies, thereby probably playing a role in affecting lymphocyte functions. Triantennary- and biantennary-like ­complex-type asparagine-linked glycoproteins with the expression of ­mannose, fucose,

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Sialic acids and sialoglycoconjugates in the biology of life, health and disease

N-acetylglucosamine, and N-acetylgalactosamine in S. mansoni have been associated with a role in the host immune response to infection [228]. A cysticercus membrane glycoprotein antigen has been identified with hexoses and sialic acids [229]. 4.6.4 Annedida Very few studies have reported the observation of sialic acid in earthworm. Glycolipid fraction of earthworm Lumbricus terrestris (L. terrestris) has been reported to include cerebrosides and sulfatides containing glucose and galactose, and gangliosides containing glucosamine and sialic acid [230]. 4.6.5 Arthropoda Sialylation pathway in Drosophila reveals similarities in the initial steps to the mammalian sialylation pathways indicating a probable common evolutionary origin [231, 232]. N-glycan processing in insects reveal similari9*ty at early steps with differences in subsequent steps to mammalian N-glycan synthesis with the insect cell lines not processed to terminally sialylated complex-type structures but modified to paucimannosidic or oligomannose structures thus differing from the mammalian cells due to the lack of enzymes including glycosyltransferases involved in generating complex-type structures and appropriate sugar nucleotides [233]. The baculovirus-insect cell expression system has been used to produce recombinant therapeutic glycoproteins [234]. Neu5Ac has been reported to occur in larvae of the cicada Philaenus spumarius (P. spumarius) [26]. 4.6.6 Mollusca Sialic acid-binding lectins (SABLs) SgSABL-1 and SgSABL-2 of Solen grandis (S. grandis) have been revealed to have functions like pattern-recognition receptor (PRRs) and hypothesized to be involved in the innate immune response of S. grandis [235, 236]. Siglec gene or CgSiglec-1 has been characterized from the Pacific oyster, Crassostrea gigas (C. gigas) [237]. Gastropod Haliotis tuberculata (H. tuberculata) foot epithelium reveal N-glycoproteins rich in fucose and mannose while secretory cells reveal expressions of acidic sulfated glycoconjugates such as glycosaminoglycans and mucins, enriched with galactose, N-acetylgalactosamine, and Nacetylglucosamine but foot epithelium lack sialic acid [238]. Lectins were known to participate in immune recognition and host defense and Chsalectin, a novel sialic acid-binding lectin, was reported to occur in Crassostrea hongkongensis (C. hongkongensis) with a role in immune ­recognition and



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host defense against bacterial infection caused by C. hongkongensis [239]. SABL from Manila clam Venerupis philippinarum, VpSABL [240], has been reported. A novel sialic acid-specific lectin (MCsialec) was detected from Manila clam hemocytes infected with Perkinsus olseni that plays a vital role during pathogenic infection [241]. Scalarin from the eggs of Pomacea canaliculata (P. canaliculata, Lamarck, 1822) and Pomacea scalaris (P. scalaris, d’Orbigny, 1835) revealed expression of terminal sialic acid residues possibly resistant to neuraminidase and O-linked residues derived from the T and Tn antigens [242]. N-linked oligosaccharides are expressed in the nacreous layer of Japanese pearl oyster Pinctada fucata (P. fucata) [243]. A novel α/β-galactoside α-2,3-­sialyltransferase was expressed by luminous marine bacterium, Photobacterium phosphoreum JT-ISH-467, isolated from the Japanese common squid (Todarodes pacificus, T. pacificus) [244]. Garden snail Cepaea hortensis (C. hortensis) [245, 246] has been reported to express sialic acid-specific lectin. C. hortensis agglutinin-I (CHAI) lectin binds to O-linked sialic acids [247]. Newly hatched Hawaiian squid Euprymna scolopes (E. scolopes) rapidly become colonized by the bioluminescent marine bacterium Vibrio fischeri which exhibited the unusual ability to migrate to nucleosides, nucleotides, and sialic acid, a component of squid mucus [248]. Neu5Ac and Neu5Gc have been reported to occur in slug Arion lusitanicus (Gastropoda) revealing specificity toward MAA [249]. Eye lenses of common squid (T. pacificus) and Pacific octopus (Octopus vulgaris, O vulgaris) has been known to express gangliosides including gangliotetraose species and c-series gangliosides [250]. Acidic lipids from T. pacificus and O. vulgaris revealed expression of Neu5Ac. Lipid-bound sialic acid in cerebral ganglia were significantly lower as compared to the expression in hepatopancreatic tissues indicative of ganglioside expression in protostomia [167]. Achatinin, a 9-O-acetyl sialic acid (9-O-AcSA) binding lectin, has been isolated from A. fulica snails with sugar specificity toward 9-O-AcSAα2→6GalNAc [251]. Sialic acids have been reported to occur in two marine bivalves, the Pacific oyster C. gigas and the horse mussel Modiolus modiolus (M. modiolus) [252]. A heterogeneous SABL with affinity toward bacterial LPS was expressed in hemolymph of M. modiolus [253]. The M. modiolus SABL is reported to agglutinate erythrocytes and bacterial LPS and react with sialoconjugates [254]. A cDNA library of Limax flavus (L. flavus) was constructed and screened for sialic acid-specific lectin [255]. A total of 16 lectins have been reported to be expressed in the digestive gland of the bivalve mollusc Mytilus galloprovincialis (M. galloprovincialis) [256]. Salmonella djakarta (S. djakarta) and Salmonella isaszeg (S. isaszeg) LPS were investigated

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Sialic acids and sialoglycoconjugates in the biology of life, health and disease

for neuraminic acid expression [257]. Neu5Gc-specific lectin (AFL) has been isolated from the foot muscles of the marine clam Anadara granosa (A. granosa) [258]. A Neu5Gc-specific lectin (PAL) has been isolated from apple snail, Pila globosa (P. globosa) [259]. A sialic acid lectin is expressed in slug Limax [260]. Sialic acid-containing substrates as intracellular calcium receptors have been reported to be involved in transmitter release [261]. 4.6.7 Echinoderms In echinoderms including starfish and sea urchin, expression of Neu5Gc is predominant [50, 59, 60, 64]. Di-sialoglycoconjugates have additional Neu5Gc, O-methyl-Neu5Gc, N-acetyl-O-methylneuraminic acid, and ­N-glycoloyl-O-methyl neuraminic acid [262, 263]. Starfish Asterias rubens (A. rubens) reveals expression of 8-O-methyl-5-Neu5Gc (Neu5Gc8Me) [264] with Neu5Ac, Neu5Gc, and their O-acetylated derivatives, while starfish additionally possess 8-O-methylated sialic acids [264, 265]. Neu5Gc is formed by CMP-Neu5Ac hydroxylase found in gonads of starfish A. rubens revealing similarities to the mammals. However, the echinoderm hydroxylase reveals differences from the mammalian counterpart in membrane association and a requirement for high ionic strength for optimal activity [266, 267] which has been cloned [266]. 4.6.8 Vertebrates Intestinal glycoconjugates of the blunthead pufferfish Sphoeroides pachygaster (S. pachygaster) and gray triggerfish Balistes capriscus (B. capriscus) reveal expression of GalNAc and GlcNAc residuals with GalNAc residuals in S. pachygaster subterminal to sialic acid [268]. Zebra fish Danio rerio (D. rerio) has been studied extensively for glycosylation [269–271] and development of vertebrates and reported the expression of protein and lipid-associated alpha2–8-linked oligosialic acid motifs in the early development [272]. CMP-Sia synthetase (CMAS) has been reported to occur in D. rerio (dreCmas) [273]. PSA action has been reported during axon growth and pathfinding in the developing zebra fish CNS [274]. Sialic acid acetylesterase (SIAE) removes acetyl moieties from the carbon 9 and 4 hydroxyl groups of sialic acid and adult fish reveal expression of siae mRNA in heart, eye, muscle, liver, brain, kidney, and ovary revealing their role in immune system function and the development of central nervous system [270]. The epidermis of sea lamprey Petromyzon marinus (P. marinus) reveals expression of glycoconjugates including sulfated glycosaminoglycans



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­(N-acetylglucosamine and N-acetylgalactosamine) and N-glycoproteins rich in mannose in the mucous.The skin cells, a unique cell of lampreys, reveal expression of l-fucose and sialic acid residues that is lost with metamorphosis [275]. Sialidase removes sialic acids from glycoconjugates and medaka sialidase Neu1 has been reported to exhibit desialylation of α2–3 sialic acid linkage [276]. Gangliosides has been reported to occur in fish containing only ­N-acetylneuraminic acid/sialic acid, while beef, chicken, and pork contained GD1a/b species that incorporated both Neu5Ac and Neu5Gc and hydroxylated fatty acids [277]. Mucin O-glycosylation of five freshwater acclimated Atlantic salmon have been reported to contain sialylated intestinal mucins, Neu5Ac and sialylated core 5 was the most dominant structure with a probable role in host-pathogen interactions [278]. Fish skin mucus reveals NANA/sialic acid, glucose, N-acetylglucosamine, N-acetylgalactosamine, galactose, and fucose residues [278].Trisialyllactosylceramide, GT3, containing an O-acetylated sialic acid has been reported to occur in cod fish brain [279]. In fish neu4 sialidase, neu4 gene has been reported and was cloned from medaka brain mRNA with a probable role in embryonic development [280]. Polysialic acid (PSA) linked to neural cell adhesion molecule NCAM1 forms PSA-NCAM1 by polysialyltransferases STX with functions during the development of vertebrate nervous systems including axon extension and fasciculation. NCAM1 and NCAM2 have been reported in tetrapods and fishes [281]. PSA-NCAM plays a vital role in neuronal differentiation, maintenance, plasticity, and regeneration. In zebra fish homologues of STX (St8sia2) and PST (St8sia4) have been studied and found that PSA-NCAM regulates motility for cerebellar neuronal progenitors [282] and NANS-mediated synthesis of sialic acid was essential for the development of brain and skeleton [283]. Sialyltransferase gene found in the tunicate Ciona intestinalis (C. intestinalis) reveals a possible ortholog of the common ancestor of galactose α2,3-sialyltransferases and ST3Gal II gene from the bony fish Takifugu rubripes (T. rubripes) [284]. Polysialoglycoprotein (PSGP) in salmonid fish egg is a unique glycoprotein bearing α2,8-linked PSA on its O-linked glycans and two α2,8-polysialyltransferases (α2,8-polySTs), PST (ST8Sia IV) and STX (ST8Sia II), have been reported for PSA biosynthesis on N-glycans of glycoproteins in mammal [285]. Unambiguous orthologs of mammalian siglec-4, exclusively expressed in the nervous system, has been identified from fugu and zebra fish. As in mammals, fish siglec-4 is expressed by nervous tissue. Fish Siglec-4 recombinant protein, fish siglec-4 has been reported to bind to sialic acids with a

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Sialic acids and sialoglycoconjugates in the biology of life, health and disease

specificity similar to the mammalian orthologs indicating that siglec occurs in the nervous system of all vertebrates [286]. Infectious salmon anemia virus (ISAV) causes infections in farmed Atlantic salmon. Purified ISAV hydrolyzed free 5-N-acetyl-4-O-acetyl neuraminic acid and the e­ nzymatic activity of the HA-esterase of ISAV revealed similarities to sialate-4-O-­ esterases of murine coronaviruses and related group 2 coronaviruses [287]. PSA-NCAM facilitates axon growth. In lizard, retinal ganglion cell axons have been reported to be transiently PSA-NCAM positive, whereas in goldfish retinal ganglion cell (RGC) axons are PSA-NCAM negative. PSANCAM is negative both in normal animals and throughout regeneration with the exception of a PSA-NCAM-positive fascicle arising from newly generated RGCs [288]. Epidermal, branchial, and digestive mucous cells, and the gastric glands of larvae/postlarvae of three fish species (two teleostean and a chondrostean) revealed negative Con A lectin staining, but oesophageal mucous cell of sturgeon revealed expression of mannose -Man- and/or glucose -Glc-, L-fucose -Fuc-, N-acetyl-D-galactosamine -GalNAc-, N-acetyl-Dglucosamine, -GlcNAc-, and/or sialic acid-NANA-residues [289]. Fish type III antifreeze protein is homologous to the C-terminal region of mammalian sialic acid synthase [290]. In all 18 gangliosides were isolated from dogfish Squalus acanthias (S. acanthias) brain, including GM2, GQ1c, GP1c, and GD2 [291]. CRPs in Labeo rohita (L. rohita) reveal microheterogeneity [292]. Cichlid fish and rat brains also contained GM1b-, GT1b-, and GQ1c-synthase and sialyltransferase activities [293]. α2→8-Linked PSA chains terminate O-linked oligosaccharide chains on Salmonidae fish egg polysialoglycoproteins (PSGPs) the expression of which are developmentally regulated [294]. Gangliosides expression is reported to occur in electric organ of Torpedo marmorata: synaptosomes, presynaptic membranes, postsynaptic membranes, and synaptic vesicle membranes [295]. A 9-O-actetylated GT2 has been reported to occur in cod fish brain [296]. A trisialyllactosylceramide GT3 was found in cod fish brain with a chemical structure of II3(9-O-Ac-NeuAc2–8NeuAc2–8NeuAc2–3)Lac Cer [297]. Trout liver has been reported to express sialate cytidylyltransferase activity. The sialic acid fraction of trout liver after hydrolysis is composed of N-acetylneuraminic acid, N-acetyl-9-O-acetylneuraminic acid, and ­N-acetyl-9-O-lactoylneuraminic acid [298]. The luminal surface of the saccular macula in the rainbow trout revealed expression of a glycoconjugate constituting glucose, galactose,



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fucose, mannose, N-acetylglucosamine, N-acetylneuraminic acid, and ­N-acetylgalactosamine [299]. Carbohydrate-rich sialoglycopolyprotein was isolated from the fertilized eggs of the Medaka fish species, Oryzias melastigma (O. melastigma), as a member of the L-hyosophorin family [300]. The mucin-like keratan sulfate glycopolymer has been reported to occur in ampullae of lorenzini [301]. Amphibians have been studied for molecules with anticancer properties. Onconase, amphinase, cSBL from Rana catesbeiana (R. catesbeiana) eggs, and jSBL from Rana japonica (R. japonica) eggs, which belong to the RNase A family, were purified from the oocyte cells and eggs of three amphibians, and they were found to induce cytotoxicity by degrading cellular RNA [302]. Xenopus embryos revealed expression of polysialo-­ ganglioside including asialo-GMI as the core structure of the ganglioside XI and palmitic and oleic acid as the fatty acids of the ceramide moiety [303]. N-glycan structures have been identified from snake venoms [304]. Four different sialidase forms are known in vertebrates: the lysosomal NEU1, the cytosolic NEU2 and the membrane-associated NEU3 and NEU4. Sialidase orthologs from 21 different organisms have been identified and shown distribution in the evolutionary tree including Metazoa relative, marine choanoflagellate Monosiga brevicollis (M. brevicollis), early Deuterostomia, precursor of Chordata, and Vertebrata (teleost fishes, amphibians, reptiles, avians and early and recent mammals [305]. Mucins in the alimentary tract of the grass snake, Natrix natrix (N. natrix), has revealed expression of mannosylated sialosulfomucins and fundic mucosa of stomach reveal expression of sialomucins with terminal sialic acid linked to galactose, with neck cells expressing sialomucins with mannose, N-acetylglucosamine, galactose, N-acetylgalactosamine, and fucose-α- (1,2)-linked residues [306]. A serine protease with thrombin-like activity (TLBan) expressed by Bothrops andianus (B. andianus) commonly called Andean Lancehead has been reported to contain both N-linked carbohydrates and sialic acid [307].Asian pit viper venom reveals expression of partially O-acetylated NeuAcα2–8NeuAcα2– 3Galβ1-4GlcNAcβ1-terminal epitope [308]. Platypus revealed expression of Neu5Ac. Among nine reptiles and one turtle, Neu5Gc was expressed in the egg and an adult basilisk, belonging to the lizard family. BLAST analysis of platypus, chicken, and zebra finch genomes did not reveal similarity to CMAH structure. Monotremes including Platypus and Sauropsids including birds and reptiles lacked Neu5Gc synthesis machinery. Neu5Gc found in eggs may probably have been acquired from diet or by an a­lternative pathway [309]. Carbohydrate components of the ductus ­ epididymis

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Sialic acids and sialoglycoconjugates in the biology of life, health and disease

­epithelium of a lizard revealed location differences [310]. DM43, an opossum serum protein inhibitor of snake venom metalloproteinases, revealed constitution of N-acetylglucosamine, mannose, galactose, and sialic acid forming four biantennary N-linked chains [311]. The expression of PNAbinding glycoproteins has been reported to occur in lizard lymphocytes [312]. A heavily glycosylated protein fraction was isolated from cobra venom containing both O– and N-linked oligosaccharides; 1 N-linked chain for every 8–10 O-linked oligosaccharides and the O-linked chains revealed expression of fucose, galactose, and N-acetylglucosamine more than N-acetylgalactosamine; with minute level of sialic acid lacking sulfate esters [313]. N-CAM has been shown to undergo decrease in sialic acid content during embryonic to adult conversion with an increase in binding efficacy thus regulating morphogenesis [314]. Sialic acids are found to be constituents of milk oligosaccharides, components of glycoproteins in blood, sera, or plasma of mammals. Human sera reveal expression of Neu5Ac and Neu5Ac9Lt (Lt = lactoyl) and minute quantities of O-acetylated sialic acid derivatives, Neu5,9Ac2 [89]. Sialic acid derivatives in serum glycoproteins reveal differences in expression in mammalian species and level and position of O-acetylation and Neu5Gc expression [2]. Sialic acid and its acetylated derivatives have been reported to occur in epithelial and mucous glycoproteins revealing the presence of Neu5Ac, Neu5,9Ac2, and Neu5,7,9Ac3 [2]. Gangliosides have also been reported. Pathogens are known to bind to sialic acid on human cell surfaces (Table 4).

Table 4  Pathogens that bind to sialic acids on human cell surfaces Pathogen

Binding protein

Known target sialylated sequence

Human Influenza A Avian Influenza A Human Influenza C

Siaα2–6Gal(NAc) Siaα2–3Galβ19-O-Ac-Siaα2-

Vibrio cholera

Hemagglutinin Hemagglutinin Hemagglutininesterase Toxin

Plasmodium falciparum

EBA-175

Clostridium botulinum Helicobacter pylori

Toxin SabA

Galβ1–3GalNAcβ1,4(Siaα2–3) Lac-Cer Siaα2–3Galβ1–3(Siaα2–6) GalNAc-OPolysialogangliosides Siaα2–3Gal on gangliosides

Table adapted with permission from Ajit Varki Sialic acids in human health and disease Trends Mol Med. 2008; 14(8): 351–360.



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Sialic acid-binding protein

Sialic acid components of oligosaccharide side chains in glycoconjugates occur in most higher animals and a few microorganisms act as ligands in glycobiological interactions on binding to a specific sialic acid-binding protein acting as receptors [315]. Siglecs

Siglecs are sialic acid-binding immunoglobulin (Ig)-type lectins which are the members of the immunoglobulin superfamily that act as transmembrane cell surface immune regulatory receptors predominantly found on hematopoietic cells containing an N terminal V-set Ig-like domain with sialic acid-binding sites that recognizes different sialylated glycoconjugates, leading to the activation or inhibition of the immune response. Siglecs include (a) CD33-related Siglecs and (b) Siglec-1 (Sialoadhesin), Siglec-2 (CD22), Siglec-4 (myelin-associated glycoprotein, MAG), and Siglec-15. Phylogenetic studies in higher vertebrates including fishes, amphibians, birds, reptiles and mammals have revealed that Siglecs are conserved in evolution [316]. A loss of Siglec genes in rodents have been reported. The cytoplasmic domain of most Siglecs contain immune receptor ­tyrosine-based inhibitory motifs (ITIMs) that recruit tyrosine phosphatases SHP-1 and SHP-2 and function as inhibiting receptors,inhibiting signal transduction. Siglec-14, Siglec-15, and Siglec-16 associate with t­yrosine-based activation motif (ITAM) adaptor DAP12 and act as activating receptors by recruiting SYK kinase. Siglecs are known to play a vital role in immune regulation in host-pathogen interaction in infectious diseases, inflammation, neurodegeneration, autoimmune diseases, and cancer [317]. The sialic acid-Siglec axis has been recently reported to be exploited by tumors and pathogens for the induction of immune tolerance [318]. Sialic acid-binding lectins

SABLs are lectins that specifically recognize sialic acid residues [319]. They have been reported to occur in plants and animal sources with diverse specificity and are being exploited for analytical properties (Table 5). Selectins

Selectins are a diverse group of calcium-dependent, type I transmembrane molecules that bind to sialylated, fucosylated carbohydrates, function in vascular adhesion, and play a significant role in inflammation, immunity, hemostasis, and cancer progression. Selectin ligand reveal overexpression of

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Sialic acids and sialoglycoconjugates in the biology of life, health and disease

Table 5  Vertebrate sialic acid-specific lectins [18, 320–348] Names (synonyms)

Expression (source)a

Binding specificity

E-selectin (ELAM-1, CD62E) L-selectin (MEL 14 antigen, CD62L) P-sclectin (GMP 140, PADGEM. CD62P)

Activated endothelium

sialyl Lex, sialyl Lea

6′-sulfo sialyl Lex, heparan sulfate Activated endothelium, sialyl Lex, sialyl Lea, heparan platelet sulfate (HS)

Leucocyte

Siglecs

Siglec-1 (sialoadhesin)

Macrophage

Siglec-2 (CD22) Siglec-3 (CD33) Siglec-4 (MAG)

B-cell Myeloid precursor, Mobc Glial cells

Siglec-5

Mo, Gr

Siglec-6 (OB-BP1) Siglec-7 (AIRM-1)

Placenta, B-cell NK cells, Mo, Gr

Siglec-8 Siglec-9 Siglec-10 Siglec-11

Eosinophil, basophil Mo, Gr B-cell Fibroblasts

Neu5Acα2– 3Gal > Neu5Acα2 – 6Cal Siaα2–6Gal Siaα2–6Gal ≥ 2 Siaα2–3Gal Neu5Acα2–3Gal on complex gangliosides Siaα2–6Gal ≈ Sioα2–3Gal, Neu5Acα2–8 Siaα2–6Gal NAc Siaα2–6Gal ≈ Siaα2–3Cal (3-Ig isoform), Siaα2– 6Gal (2-Ig isoform) Siaα2–3Gal ≥ Siaα2–6Gal Siaα2–6Gal ≈ Siaα2–3Gal Siaα2–6Gal ≈ Siaα2–3Gal Neu5Acα2–8Neu5Ac

Others

Complement factor H

Blood

CD83 LI Interleukin-1α

Dendritic cell Mouse neuron Blood

Interleukin-1β

Blood

lnterleukin-2 Interleukin-4 Interleukin-7 Laminin

Blood Blood Blood Extracellular matrix

Sialic acid-binding protein Sarcolectin

Endometrium

Sia; C7–C9 side chain is a part of epitope Sia Neu5Acα2–3 (on CD24) Neu5Acα2–3Galβ1– 4GlcNAc, biantennary Neu5Acα2–3Galβ1-Cer (GM4) GD1b Neu5Ac1,7lactone Neu5Acα2–6GalNAc Neu5Acα2–3Galβ1– 4GlcNAc (α2–3 > α2–6) Neu5Gc > Neu5Ac

Placenta

Neu5Ac, Neu5Gc



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Table 5  Vertebrate sialic acid-specific lectins [18, 323–351]—cont’d Names (synonyms)

Expression (source)a

Binding specificity

Calreticulin

Ovine placenta

Sialic acid-binding protein Calcyclin Sialic acid-binding protein Ganglioside binding protein⁎ Hemagglutinin⁎

Rat uterus

Neu5Gc > Neu5Ac, prefer O-acetyl Sia

Bovine heart Frog egg

Neu5Gc Sia

Rat brain myelin

Gangliosides (CT1b, CQ1b, GD1b) Neu5Ac, Neu5Gc

Rat brain

a

Expression (source) is in human, unless otherwise stated. Mo, monocyte; Gr, granulocyte. c Most of the proteins are cloned and/or purified to homogeneity, except for the entities marked with an asterisk (*). Adapted with permission and updation from Angata T,Varki A. Chemical diversity in the sialic acids and related α-keto acids: an evolutionary perspective. Chem Rev 2002;102:439–469. b

tumor cells leading to increased metastasis, poor prognosis, mediate tumor cell adhesion, extravasation during metastasis, and activate signaling cascade in tumor. Selectins play a vital role in leukocyte homing. L-Selectin binding to ligands on leukocytes activates leukocytes. Selectins enable interactions with platelets and endothelial cells. The interaction between selectin ligand P-selectin glycoprotein ligand 1 PSGL-1 on leukocytes and P-selectin on platelet or E-selectin on endothelial cells triggers intracellular signaling in leukocytes. Selectins and their ligands play an important role in the human implantation. L-selectin on interaction with its ligands plays a critical role in the adhesion of the blastocyst to the endometrium at the maternal-fetal interface. P-selectin and E-selectin play a vital role in human implantation [349]. PSGL-1 is now known as a major participant in inflammation, thrombus, along with cancer [350]. Due to their role in cancer immune system modulation they are being studied as possible targets for controlling tumor immunity [351]. 4.6.9  Sialylation and disease Although sialic acids are found to occupy the terminal position of glycans of all cell types, in disease states, like cancer or immunological disorders, the sialylation profile of cells in affected tissues manifest altered downregulation or overexpression—or neoexpression of certain glycan structures [352].

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Sialic acids and sialoglycoconjugates in the biology of life, health and disease

Aberrant sialylation of oligosaccharide branches of N-glycans (β1,6-­ GlcNAc branching) or affected terminal glycan sequences, like premature sialylation of truncated saccharide units, are reported to occur due to ­affected or altered enzyme expression [352]. In rheumatoid arthritis, an incomplete IgG glycosylation with galactose and sialic acid is observed to lead to immune disorder [353]. But upregulated expression of sialyltransferases like ST3Gal-I is observed in human BC [354] and upregulated ST6Gal-I is reported in human BC and colon cancer. Ganglioside overexpression in cancer has been reported such as CNS-specific GD3s from tumor tissues [355]. The overexpression of 9-O-acetylated sialic acid in BC [356], childhood ALL [83, 103–107] are reported. Altered cell sialylation in cancer affects tumor cell interactions with other cells affecting cell adhesion, migration, and metastasis [357–359] Sialylation affects natural killer (NK) cell cytotoxicity [360]. Tumor cell hypersialylation has been observed to enable tumor cell to evade recognition by NK cells, thus escaping the immune responses. Hypersialylation of tumor cells enables it to escape the immune surveillance [358]. Sialic acid ligand-protein interaction is being exploited for design and development of therapeutics and anticancer therapies containing sialoside that targets siglecs [361]. Nanoparticle formulations are being tested to deliver potential therapeutic agents to the target cell [317]. 4.6.10  Tumor-associated carbohydrate antigens Upregulated sialyltransferases and fucosyltransferases lead to the overexpression of tumor-associated carbohydrate antigens (TACA), being mucin related to Thomsen nouvelle (Tn) antigen (Tn), the sialyl-Thomsen nouvelle (sTn) antigen, Thomsen-Friedenreich antigen (TF-Ag), the blood group-related Thomsen-Lewis antigens LewisY, Sialyl LewisX and Sialyl LewisA, and LewisX (or specific embryonic antigen-1, SSEA-1), the glycosphingolipids Globo H, stage-specific embryonic antigen-3 (SSEA-3), sialic acid-containing glycosphingolipids, the gangliosides GD2, GD3, GM2, fucosyl GM1, and Neu5GcGM3, and PSA [362] that have been implicated in tumor, metastasis, and poor prognosis. Some TACAs are also expressed in fetal tissue, and termed as oncofetal antigens [362]. TF-Ags increased the expression in pancarcinoma and carcinomas of the breast, colon, bladder, prostate, liver, and stomach as compared to normal cells and its role in metastasis renders it as an important tumor target [362].



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The overexpression of LewisY in ovarian, breast, prostate, colon, and epithelial cell lung cancers than in the normal cells makes them potential targets. The overexpression of SLeX in breast, ovarian, melanoma, colon, liver, lung, and prostate cancers and SLeA in breast, colon, and pancreas cancers, and in melanomas due to the upregulated expression of ST3Gal-III and FucT-III enzymes, synthesizing sLea and ST3Gal-IV, ST3Gal-VI, and FucTVII, catalyzing the synthesis of sLex, or due to deficiency in the enzymes responsible for sulfation, and their overexpression acting as E-selectin ligands contribute to metastasis and poor prognosis makes them potential targets. The treatment of these antigens with antibodies inhibited metastasis in pancreatic tumor mouse models. PSA is overexpressed in small cell lung cancer (SCLC), rhabdomyosarcoma, Wilms tumor, and neuroblastoma and PSA-NCAM is involved in increased tumor growth and metastasis with decreased patient survival. Endoneuraminidase N, cleaving PSA could stop cell growth in rhabdomyosarcoma and neuroblastoma cells [362]. Downregulated sialyltransferases and fucosyltransferases lead to reduced cell surface sialylation and reduced TACA expression, decreasing adhesion and migration potency and metastatic activity [363]. sTn antigen generated by sialylation of Tn antigen [364] by ST6GalNAc I occurs rarely in healthy tissues but has been observed in epithelial cancer cells and breast tumors [365]. 4.6.11  Sialic acids and therapeutics: Where we stand Strategies like enzyme inhibitors for disease-associated carbohydrates are being studied, designed, and developed. Inhibitors for the c­ arbohydrate-binding receptors are also being conceived as a strategy [366]. X-ray crystallographic structures of mammalian sialyltransferases (STs) complexed with their ligands are enabling us to better understand the substrate specificities and inhibitor design [367]. Inhibition of STs, glycosyltransferases, is a strategy to reduce cell surface sialylation particularly in cancer [368]. Lack of ST3Gal-IV and ST3Gal-VI reduces sialic acid containing ligands for selectin interactions, thus affecting leukocyte homing and leukocyte recruitment at inflammation sites [369]. Streptococcus pneumonia leads to septicemia, meningitis, and community-­ acquired pneumonia in human. Although prophylactic vaccines are the strategies available for disease targeting, Streptococcus pneumonia (S. pneumonia) sialidases, including NanA and NanB, implicated in the pathogenesis of S. pneumonia and design of novel NanB-selective inhibitors are being tested for drug targets [370].

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Sialic acids and sialoglycoconjugates in the biology of life, health and disease

Binding with selectin antagonists, like bimosiamose (TBC-1269) and GSC-150, sialoside-selectin interactions can be blocked; selectin antagonists also find application in the treatment of inflammatory disorders, autoimmune diseases and metastatic cancers [371]. 11a sLex-derived inhibitor of E-selectin, CGP69669A, with affinity higher than natural ligand sLex has been designed [372]. Modified sLex-containing glycopeptide analogues for E-selectin inhibition has been designed [373]. P-selectin antagonist, GMI1070, is in phase II clinical trials for the treatment of sickle cell crisis, associated with sickle cell disease (SCD) [374]. Bimosiamose (TBC-1269), a P-selectin antagonist, has been reported to have successfully completed phase II clinical trials for psoriasis and chronic obstructive pulmonary disease (COPD) [375]. N-acetylglucosamine 2-epimerase and N-acetylneuraminic acid aldolase allow conversion of simple and sugars into different sialic acid-related compounds from whole cell extracts enabling large scale and economical synthesis of sialic acid and sialyloligosaccharides [376]. Low molecular weight antagonists, targeting sialoside interaction with the lectin MAG, have been designed as a therapeutic strategy to promote axon regeneration after neuronal injury [377]. Zanamivir (Relenza) and oseltamivir (Tamiflu) are designed as sialic acid-based inhibitors of the viral enzyme neuraminidase for targeting influenza infections [378]. But design of carbohydrate-derived drug molecules faces challenges such as pharmacokinetic profiles, poor bioavailability, requirement for active transport through membranes, short plasma half-life, poor metabolic stability, and are rapidly excreted [379, 380]. However, this area of biology is developing with studies or approaches to improve their pharmacokinetic profiles like systematic structural modifications such as the replacement of certain moieties, the introduction of hydrophobic moieties, and/or the incorporation of a prodrug strategy leading to compounds that mimic the biological activity of their carbohydrate precursors called as ‘glycomimetics’ like neuraminidase inhibitors zanamivir and oseltamivir [381, 382]. Drugs targeting glycan-lectin interactions need to be designed by synthetic introduction of moieties that increase the affinity. Inhibition of enzymes in the synthesis of tumor-associated glycans is another strategy [383]. Liposomal nanoparticle are being exploited in selective delivery of drugs and therapeutics like chemotherapeutic doxorubicin in B cell lymphoma, wherein high-affinity ligand, 9- N-biphenylcarboxyl (BPC)-Neu5Acα2,6-Gal-β1,4-GlcNAc, on the liposomal surface confers the selectivity being recognized by CD22 (Siglec-2), exclusively expressed on B cells and



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trisaccharide derivative BPC-Neu5Ac-α2,6-Gal-β1,4-GlcNAc conjugated to pegylated lipid is used in vesicle formation with encapsulated doxorubicin thereby selectively targeting CD22-expressing cells, including B cell lymphoma [384–386]. Selective ligands targeting the lectins CD22 and CD33 are being designed [387–389]. 4-Cyclohexyl-1,2,3-triazole residues at position 5 of sialic acid in combination with meta-substituted moieties at position 9 have been reported to improve affinity and selectivity profile [390]. Therapeutic antibodies

Therapeutic antibodies find application in leukemia and lymphoma. CD33 in leukemic cells and CD22 in B-cell lymphoma are being exploited for targeting. Anti-siglec-monoclonal antibodies conjugated to toxins or chemotherapeutic agents are being designed like gemtuzumab (Mylotarg), an anti-CD33-antibody in acute myeloid leukemia (AML), approved by the FDA in 2000 but faces challenges of safety issues [390, 391] and epratuzumab, an anti-CD22- antibody, is being designed for targeting non-­ Hodgkin lymphoma (NHL) and the autoimmune disorder systemic lupus erythematosus (SLE) [392]. Carbohydrate-based vaccines

Carbohydrate-based vaccines designed based on altered cell surface glycans in disease as compared to normal cells are the latest strategy for targeting cancer, developed over the last two decades, and are used after conventional therapeutic modes of surgery, radiation, and chemotherapy [390, 392]. TACA are being exploited as vaccine targets for their elimination. However, they suffer challenges of being poorly immunogenic self-­antigens and strategies to improve antigenicity like conjugating to a carrier protein, for example, keyhole limpet hemocyanin (KLH), and attachment sites for carbohydrate antigen conjugation [390, 393].Vaccine constructs need to be designed to activate both cell miated and humoral immune responses against specific carbohydrate epitopes of tumor cells [390, 394, 395]. Theratope, a sTn-KLH conjugate, is in clinical practice since 2002 for metastatic BC and colorectal cancer, however, is still in phase III clinical trials since 2003 [390, 396, 397]. Globo H, cancer vaccine against a carbohydrate epitope is unique to target prostate and ovarian cancer cells, such as TACA, including Globo H, sTn, Tn, TF, and Ley antigens attached to a MUC1 glycopeptide backbone conjugated to KLH [362, 390, 394, 398, 399] (Fig. 10). Mimicking of the

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Fig.  10  Structures of the mucin carbohydrate antigens Tn, Sialyl Tn, and TF, and the Lewis blood group-related antigens. (A) LewisY, Sialyl LewisX and Sialyl LewisA, and LewisX. (B) Structures of the glycosphingolipids, the globo series Globo H and SSEA-3, and the gangliosides GM2, GD2, GD3, and Fucosyl GM1. (C) Structures of the additional sialic acid-containing compounds. NeuGcGM3 and polysialic acid. (Adapted with permission from Heimburg-Molinaro J, Lum M, Vijay G, Jain M, Almogren A, Rittenhouse-Olson K, Cancer vaccines and carbohydrate epitopes. Vaccine 2011;29:8802–26.)

tumor cell’s natural carbohydrate epitope composition is hypothesized for a more effective immune response. Sialic acid containing SSEA3 (Gb5) and SSEA4 detected on BC cells and other types of cancer [390, 400–402] finds application in anticancer vaccine development. Hypersialylation has been observed to increase their



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serum half-lives with increased stability of luteinizing hormone (LH) and erythropoietin (EPO) [390, 403]. Ligand for Siglec-7 is being exploited in anticancer therapy. Efficiency of Gp120 immunogens (MN- and A244-rgp120) used in the RV144 trial conferring protection against HIV were improved by enriching the sialic acid content and lacking N-linked glycosylation sites needed for binding by bN-mAbs [404]. Thus sialic acid is offering advantages in designing therapeutics and in targeting diseases like cancer and other immunological disorders.

5 Discussions Sialic biology, with huge diversity in forms, structures, expression, metabolism, and functions in the diverse living world, is a fascinating science and our knowledge in this domain is growing day by day. While the pathogens primarily use sialic acid to mimic the host and escape immune responses, it has different functions in mediating cell-cell interaction, signaling, immune reactions, development and function in animals, and completely lacks in plants. Glycoengineering is enabling modification of plant glycoslation pathways toward the synthesis of mammalian glycoproteins which we have discussed in subsequent chapters. Insect sialylation revealing differences from the mammalian sialylation pathway has been discussed in the subsequent chapters. In the cancer cells, overexpression of sialylation and enzymes involved is reported and forms an interesting area in cancer targeting. Glycomimetics is another such developing domain in the designing of sialic acid-related therapeutics. Nanobiotechnology and its application in sialic acid biology is an emerging domain and has been discussed in subsequent chapters. At this point although a lot of studies has been done, the scope of applications of sialic acid in therapy is an emerging science which seems to be just the tip of the iceberg for the immensely important potential of sialic acid biology.

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[309] Schauer R, Srinivasan GV, Coddeville B, Zanetta JP, Guérardel Y. Low incidence of N-glycolylneuraminic acid in birds and reptiles and its absence in the platypus. Carbohydr Res 2009;344:1494–500. [310] Desantis S, Labate M, Labate GM, Cirillo F. Evidence of regional differences in the lectin histochemistry along the ductus epididymis of the lizard, Podarcis sicula Raf. Histochem J 2002;34:123–30. [311] Neves-Ferreira  AG, Perales  J, Fox  JW, Shannon  JD, Makino  DL, Garratt  RC, Domont GB. Structural and functional analyses of DM43, a snake venom metalloproteinase inhibitor from Didelphis marsupialis serum. J Biol Chem 2002;277:13129–37. [312] Mansour  MH, Negm  HI, Saad  AH, Zahran  AY, Badir  N. Identification of peanut agglutinin-binding glycoproteins on lizard lymphocytes. Zoolog Sci 1995;12:79–85. [313] Gowda DC. Davidson EA Isolation and characterization of novel mucin-like glycoproteins from cobra venom. J Biol Chem 1994;269:20031–9. [314] Hoffman S, Chuong CM. Edelman GMEvolutionary conservation of key structures and binding functions of neural cell adhesion molecules. Proc Natl Acad Sci U S A 1984;81:6881–5. [315] Zeng FY, Gabius HJ. Sialic acid-binding proteins: characterization, biological function and application. Z Naturforsch C 1992;47:641–53. [316] Bornhöfft  KF, Goldammer  T, Rebl  A, Galuska  SP. Siglecs: A journey through the evolution of sialic acid-binding immunoglobulin-type lectins. Dev Comp Immunol 2018;86:219–31. [317] Macauley MS, Crocker PR, Paulson JC. Siglec-mediated regulation of immune cell function in disease. Nat Rev Immunol 2014;14:653–66. [318] Lübbers  J, Rodríguez  E, van Kooyk  Y. Modulation of immune tolerance via ­siglec-sialic acid interactions. Front Immunol 2018;9:2807. [319] Lehmann F,Tiralongo E,Tiralongo J. Sialic acid-specific lectins: occurrence, specificity and function. Cell Mol Life Sci 2006;63:1331–54. [320] Lasky LA. Selectin-carbohydrate interactions and the initiation of the inflammatory response. Annu Rev Biochem 1995;64:113–39. [321] Varki  A. Selectin ligands: will the real ones please stand up? J Clin Invest 1997;99(2):158–62. [322] Brinkman-van der Linden  ECM, Varki  A. New aspects of siglec binding specificities, including the significance of fucosylation and of the sialyl-Tn epitope. Sialic acid-binding immunoglobulin superfamily lectins. J Biol Chem 2000;275:8625–32. [323] Crocker PR, Mucklow S, Bouckson V, McWilliam A, Willis AC, Gordon S, Milon G, Kelm S, Bradfield P. Sialoadhesin, a macrophage sialic acid binding receptor for haemopoietic cells with 17 immunoglobulin-like domains. EMBO J 1994;13:4490–503. [324] Powell LD, Varki A. The oligosaccharide binding specificities of CD22 beta, a sialic acid-specific lectin of B cells. J Biol Chem 1994;269:10628–36. [325] Powell LD, Jain RK, Matta KL, Sabesan S,Varki A. Binding of human plasma sialoglycoproteins by the B cell-specific lectin CD22. Selective recognition of immunoglobulin M and haptoglobin. J Biol Chem 1995;270:7523–32. [326] Freeman  SD, Kelm  S, Barber  EK, Crocker  PR. Characterization of CD33 as a new member of the sialoadhesin family of cellular interaction molecules. Blood 1995;85:2005–12. [327] Collins BE,Yang LJS, Mukhopadhyay G, Filbin MT, Kiso M, Hasegawa A, Schnaar RL. Sialic acid specificity of myelin-associated glycoprotein binding. J Biol Chem 1997;272:1248–55. [328] Collins BE, Ito H, Sawada N, Ishida H, Kiso M, Schnaar RL. Enhanced binding of the neural siglecs, myelin-associated glycoprotein and Schwann cell myelin protein, to Chol-1 (alpha-series) gangliosides and novel sulfated Chol-1 analogs. J Biol Chem 1999;274:27893–9.

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[390] Bauer J, Osborn HM. Sialic acids in biological and therapeutic processes: opportunities and challenges. Future Med Chem 2015;7:2285–99. [391] http://www.fda.gov/. [392] O'Reilly  MK, Paulson  JC. Siglecs as targets for therapy in immune-cell-mediated disease. Trends Pharmacol Sci 2009;30:240–8. [393] Wu J, Guo Z. Improving the antigenicity of sTn antigen by modification of its sialic acid residue for development of glycoconjugate cancer vaccines. Bioconjug Chem 2006;17:1537–44. [394] Zhu J, Warren JD, Danishefsky SJ. Synthetic carbohydrate-based anticancer vaccines: the memorial sloan-kettering experience. Expert Rev Vaccines 2009;8:1399–413. [395] Astronomo RD, Burton DR. Carbohydrate vaccines: developing sweet solutions to sticky situations? Nat Rev Drug Discov 2010;9:308–24. [396] Zeichner  SB. The failed theratope vaccine: 10  years later. J Am Osteopath Assoc 2012;112:482–3. [397] Adis International Ltd. Cancer vaccine THERATOPE- Biomira. Drugs R&D 2003;4(4):236–40. [398] Buskas  T, Thompson  P, Boons  GJ. Immunotherapy for cancer: synthetic ­carbohydrate-based vaccines. Chem Commun (Camb) 2009;36:5335–49. [399] Zhou Z, Liao G, Mandal SS, Suryawanshi S, Guo Z. A fully synthetic self-adjuvanting globo H-based vaccine elicited strong T cell-mediated antitumor immunity. Chem Sci 2015;6:7112–21. [400] Lou YW, Wang PY, Yeh SC, et al. Stage-specific embryonic antigen-4 as a potential therapeutic target in glioblastoma multiforme and other cancers. Proc Natl Acad Sci U S A 2014;111:2482–7. [401] Danishefsky SJ, Shue Y-K, Chang C-H. Development of globo-H cancer vaccine. Acc Chem Res 2015;48:643–52. [402] Cheung  SK, Chuang  PK, Huang  HW, et  al. Stage-specific embryonic antigen-3 (SSEA-3) and β3GalT5 are cancer specific and significant markers for breast cancer stem cells. Proc Natl Acad Sci U S A 2016;13:960–5. [403] Byrne B, Donohoe GG, O'Kennedy R. Sialic acids: carbohydrate moieties that influence the biological and physical properties of biopharmaceutical proteins and living cells. Drug Discov Today 2007;12:319–26. [404] Doran RC, Tatsuno GP, O'Rourke SM,Yu B, Alexander DL, Mesa KA, Berman PW, Doran RC, Tatsuno GP, O'Rourke SM,Yu B, Alexander DL, Mesa KA, Berman PW. Glycan modifications to the gp120 immunogens used in the RV144 vaccine trial improve binding to broadly neutralizing antibodies. PLoS One 2018;13(4):e0196370.

Further reading [405] Campo S, Andreone L, Ambao V, Urrutia M, Calandra RS, Rulli SB. Hormonal regulation of follicle-stimulating hormone glycosylation in males. Front Endocrinol 2019;10:17. [406] Velásquez JG, Canovas S, Barajas P, Marcos J, Jiménez-Movilla M, Gallego RG, Ballesta J, Avilés M, Coy P. Role of sialic acid in bovine sperm-zona pellucida binding. Mol Reprod Dev 2007;74:617–28. [407] Paschinger K, Wilson IBH. Comparisons of N-glycans across invertebrate phyla. Parasitology 2019;3:1–10. [408] Zhu J, Wan Q, Ragupathi G, George CM, Livingston PO, Danishefsky SJ. Biologics through chemistry: total synthesis of a proposed dual-acting vaccine targeting ovarian cancer orchestration of oligosaccharide and polypeptide domains. J Am Chem Soc 2009;131:4151–8.

CHAPTER 2

Sialoglycans and genetically engineered plants 1 Introduction N-glycosylation proteins enable proper protein folding and provides stability to the protein, efficient protein targeting for activity of lysosomal enzymes, protein-protein or protein-carbohydrate interaction, effector functions and biological activity of proteins, and control of protein half-life (Fig. 1). Plants are being engineered to develop large quantities of biopharmaceutical proteins (Table 1) in a cost-effective manner and several technical, veterinary, and pharmaceutical proteins made in plants have been successfully made available commercially [2]. Since plant N-glycosylation pathway differs at some stages with the human N-glycosylation pathway, it is important to manipulate the plant N-glycosylation pathway to render them more appropriate expression systems for human N-glycosylated proteins of desired properties. Solanaceous species under genus Nicotiana, such as Nicotiana tabacum (tobacco) and Nicotiana benthamiana (N. benthamiana, Fig. 2) have been extensively exploited in molecular farming due to the following advantages: (i) ease of cultivation, (ii) high biomass, (iii) genetic tools for trait manipulation, (iv) application of new plant breeding techniques (CRISPR/Cas9); and (v) nonfood status of the plant minimizing chances of contamination, (vi) possibility of natural insertion in the RNA-dependent RNA polymerase 1 gene [1, 3], which leads to a reduced level of gene silencing [1, 4]. Although the recombinant proteins of human origin are generated in plants with proper folding and it is possible to assemble complex proteins within the plant machinery, conventional expression systems for the production of recombinant biopharmaceutical proteins suffers from the limitation of proper synthesis of glycan structure in glycoconjugated molecules leading to the production of aberrant mixture of glycoforms that bear no resemblance to human glycans or are important from the point of view of therapy. Sialic Acids and Sialoglycoconjugates in the Biology of life, Health and Disease https://doi.org/10.1016/B978-0-12-816126-5.00002-0

© 2020 Elsevier Inc. All rights reserved.

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Common precursor: Man8 Och1

Asn

Asn

MnTs Mnsl

Asn

Asn GnTl

Yeast-type highmannosidic N-glycans

Asn MnsIl Asn

Asn

HEXO 3

GnTIl 6

FT , FT

Asn

Insect-type paucimannosidic N-glycans FT6

Asn GlcNAc

Galactose Sialic acid

Asn

GaIT

Mannose Xylose Fucose

FT3, XT

Asn

Plant-type complex N-glycans

ST Asn

Mammalian-type complex N-glycans Fig.  1  N-glycosylation pathway represented from common precursor in plant human, yeast, and insect cells. The common endoplasmic reticulum (ER)-resident oligosaccharide precursor Man8 acts as initiating point for further modifications in the Golgi apparatus. Och1: a1,6-mannosyltransferase; MnTs: mannosyltransferases; Mns: mannosidase; GnT: N-acetylglucosaminyltransferase; GalT: a1,­4-galactosyltransferase; ST: a2,6-­ sialyltransferase; HEXO: hexosaminidase (N-acetylglucosaminidase); XT: β 1,­2-xylosyltransferase; and FT: core fucosyltransferase. Fucose can be transferred in a1,3-linkage (typical of plant) and a1,6-linkage (typical of mammal). (Reproduced with permission from Loos A, Steinkellner H IgG-Fc glycoengineering in non-mammalian expression hosts, Arch Biochem Biophys 2012;526:167–173.)

Table 1  Plant-made pharmaceuticals and clinical trials Product

Host

Application

Clinical trial

Status

Sponsor

Taliglucerase alfa; Recombinant glucocerebrosidase (prGCD) ZMapp

Carrot cell culture

Gaucher disease

NCT00376168

Phase 3 completed (2012); FDA approved (2012)

Protalix, Karmiel, Israel

Tobacco

Ebola Virus

NCT02363322

Phase 1 and 2 (2015)

PRX-102

Tobacco cell culture Tobacco

Fabry Disease

NCT01769001

Phase 1 and 2 (2014)

National Institute of Allergy and Infectious Diseases (NIAID), Bethesda, MD, USA Protalix, Karmiel, Israel

Malaria

NCT02013687

Phase 1 (2015)

Vaccine Recombinant protective antigen HAI-05

Tobacco

Anthrax

NCT02239172

Phase 1 (2014)

Tobacco

H5N1 Vaccine

NCT01250795

Phase 1 (2011)

Recombinant human intrinsic factor H5-VLP + GLA-AF Vaccine

Vitamin B12 Arabidopsis deficiency thaliana (A. thaliana) Tobacco Influenza A Subtype H5N1 Infection Tobacco HIV

NCT00279552

Phase 2 Completed (2006)

Center for Molecular Biotechnology, Plymouth, MI, USA University in Aarhus, Aarhus, Denmark

NCT01657929

Phase 1 Completed (2014)

Infectious Disease Research Institute, Seattle, WA, USA

NCT01403792

Phase 1 Completed (2011)

University of Surrey, Guildford, UK

VaccinePfs25 VLP

P2G12 Antibody

Center for Molecular Biotechnology, Plymouth, MI, USA Center for Molecular Biotechnology, Plymouth, MI, USA

Reproduced with permission from open access article under the Creative Commons Attribution License (CC BY 4.0):Yao J, Weng Y, Dickey A, Wang KY. Plants as factories for human pharmaceuticals: applications and challenges. Int J Mol Sci 2015;16(12):28549–65.

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Fig. 2  Nicotiana sp. (Reproduced from Wikipedia Source Wikipedia: Joachim Müllerchen— Own work Multi-license with GFDL and Creative Commons CC-BY 2.5 https://commons.wikimedia.org/wiki/File:Tabak_9290019.JPG.)

Thus the synthesis of glycans resembling human glycan structure and its huge complexity remain a major challenge in glycoengineering of plant cells [6]. This is all more challenging due to the complexity and diversity of the glycans in different therapeutic recombinant proteins. Targeted manipulation of the plant N-glycosylation pathway has enabled the production of human-like oligosaccharides and enabled the generation of functional and effective biopharmaceuticals. In the recent years plant revealing a simple N-glycosylation pathway but lacking the Oglycosylation pathway have been reported to be better and potential glycan expression systems over the conventional ones. Plant systems are being used as effective expression systems of complex sialoglycans and N-glycans and different strategies are being used for the expression of complex therapeutic sialylated glycoforms in plant systems.



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2  N-glycosylation in plants Recently different posttranslational modifications (PTMs) have been reported for peptide maturation and activation, including proteolytic processing, tyrosine sulfation, proline hydroxylation, and hydroxyproline glycosylation [7] in plants. While glycan epitopes of human complex Nglycans are often targets of lectins important for cell-cell communication, the role of plant N-glycans finds importance in protein folding, and other biological functions including salt stress responses, cellulose biosynthesis, microtubule association, and biogenesis of several receptor-like kinases [8]. N-glycosylation is a major post-translational modification PTM in eukaryotes and is important in maintaining cell viability, where the attached core N-glycans enables proper protein folding of secreted glycoproteins and membrane proteins in the endoplasmic reticulum (ER). Although the studies of N-glycosylation in human is extensive, the knowledge in plants is restricted due to limited N-linked glycan and mutant phenotypes, limited methods to modify and target N-glycans at specific sites, and limited understanding of protein dynamics within secretory system. Glycoproteins move from the ER to the Golgi apparatus, where the N-glycan moieties undergo further maturation or may exit the ER via an alternative route to vacuoles retaining high-mannose N-glycan structures bypassing the modifications in the Golgi. In eukaryotes, N-glycans processing is initiated in the ER where the precursor Glc3Man9GlcNAc2 (Man9) is converted to Man8GlcNAc2 (Man8) and processing of Man8 in Golgi leads to the formation of complex Nglycans (Fig. 1). N-glycan processing is identical in plants and mammals till the formation of vital intermediate GlcNAc2Man3GlcNAc2 (GnGn). In mammals, GnGn oligosaccharides enables diversification of N-glycosylation but in plants, the GnGn structures are arranged with ­1,2-xylose and core 1,3-fucose residues (GnGnXF3). Although in mammals core fucosylation occurs in 1,6-linkage, the fucose residues in plants (Fig. 3) are in 1,3-linkage. Plant cells extend the GnGnXF3 by attaching 1,3-galatose and 1,4-fucose to form Lewis-a epitopes (Lea). Plants reveal formation of paucimannosidic structures due to the removal of terminal GlcNAc residues from GnGnXF3 by endogenous hexosaminidases similar to insects. As compared to human complex N-glycans, N-glycans of the plant systems lack sialic acid but contain core α1,3-fucose (Fuc) and β1,2-xylose (Xyl) modifications, and may contain terminal Lewis-a epitopes (β1,­3-galactose (Gal) and α1,4-Fuc

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Fig.  3  (A) Schematic overview of complex N-glycan processing in plants. Golgiα-mannosidase I (MNS1/2), N-acetylglucosaminyltransferase I (GnTI), Golgi-αmannosidase II (GMII), N-acetylglucosaminyltransferase II (GnTII), β1,2-xylosyltransferase (XylT), core α1,3-fucosyltransferase (FUT11/12), β1,3-galactosyltransferase (GALT1), and α1,4-fucosyltransferase (FUT13). (B) Representative view of N-glycan processing in mammalian cells. Golgi α-mannosidase I (GMI), core α1,6-fucosyltransferase (FUT8), N-acetylglucosaminyltransferase IV (GnTIV) and V (GnTV), β1,4-galactosyltransferase (B4GalT1), and α2,6-sialyltransferases (ST). (C) Optimized N-glycan engineering approach: generation of xylt, fut11, fut12, and galt1 knockouts results in the formation of GnGn structure which serves as acceptor for GnTIV, GnTV, B4GalT1, and ST resulting in fully processed complex N-glycans. Sialylation in plants requires the co-expression of the Golgi CMP-sialic acid transporter (CST) and proteins for CMP-sialic acid biosynthesis. (Reproduced with permission from open access article under a Creative Commons Licence: Schoberer J Strasser R, Plant glyco-biotechnology.Semin Cell Dev Biol. 2018 80:133-141.)



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linked to terminal N-acetylglucosamine, GlcNac (Fig. 3). Human complex N-glycans are often sialylated containing different epitopes, including Lewis x, N-acetyllactosamine (LacNAc), and N,N′-di-N-acetyllactosediamine (LacDiNAc). Although the plants and mammals reveal differences in the N-glycan structures they share high degree of homology in the secretory pathway.

3  Sialylation and recombinant proteins produced in plants Glycoengineering aims at the production of recombinant glycoproteins with a defined glycosylation profile, in order to study the impact of glycosylation and for the production of therapeutic agents. The plant expression systems are being designed to generate therapeutically important glycoproteins. Plant systems find importance as they are biologically safe, cost effective, and convenient. However, as plant N-glycosylation pathway differs in many aspects as compared to human N-glycosylation, modification of N-glycosylation pathway in plants is needed to avoid immunological challenges and get humanized authentic N-glycosylated molecules. Plants reveal highly conserved secretory pathway with folding, assembly, and posttranslational modifications of proteins similar to the mammals. Animal sialyltransferases (STs) consist of four conserved motifs, namely large (L), small (S), very small (VS), and motif III. Although sialic acid has not been detected in plants, three orthologues containing sequences similar to the ST motifs have been identified in Arabidopsis thaliana L. The At3g48820 gene with gene id 824,043 codes for a Golgi resident protein but lacks the ability to transfer sialic acid to asialofetuin or Galβ1,3GalNAc and Galβ1,4GlcNAc oligosaccharide acceptors [10]. Strategies to produce humanized therapeutic glycoproteins in plants involves (i) retaining of the recombinant glycoproteins in ER, where N-glycans undergo modification, (ii) inhibiting the plant endogenous Golgi glycosyltransferase, and (iii) adding new glycosyltransferase from mammals. Different approaches have been used to modify the N-glycosylation pathway in different plant species, using T-DNA insertion mutants [11], RNA interference (RNAi) [12–14], chemical mutagenesis [15], and targeted nuclease [16–18] approaches. N. benthamiana finds importance in molecular farming as the transient expression of proteins is fast and yields antibodies [19] by different transient expression systems, including the MagnICON system [20], the pEAQ vector [21], and the pTRA vector [22]. Zinc finger nucleases (ZFNs) [23] transcription activator-like effector nucleases

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(TALENs) [24] have enabled easy knockout of multiple genes. In N. benthamiana, the two XylT genes and two of the five FucT genes were knocked out with TALENs to completely eliminate the β-1,2-xylosyltransferase activity and reduce core α-1,3-fucosyltransferase activity by 60%. CRISPR/ Cas9 system has been used to knockout two β-1,2-xylosyltransferase and four α-1,3-fucosyltransferase genes in N. benthamiana [25]. Sia and polysialic acid (polySia) play a vital role in biological functions and therapeutic use. Expression system in plants has been designed with multigene vectors enabling the controlled in  vivo synthesis of sialylated structures in the human sialylation pathway (Fig.  4) that sialylate glycoproteins in α2,6- or α2,3-linkage and transient coexpression of human α2,8-polysialyltransferases lead to the production of active and functional polySia structures [26].

UDP-GlcNAc

GNE

ManNAc

GNE

ManNAc-6-P

NANS Neu5Ac-9-P Neu5Ac

ST +CMP

GT

CMAS

Golgi CMP

CMP-Neu5Ac

CMP-Neu5Ac Nucleus

CST

Fig. 4  Strategy to engineer human sialylation pathway in plants using the endogenously present metabolite UDP-GlcNAc. Enzymes involved are: UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine-kinase (GNE), N-acetylneuraminic acid ­phosphate-synthase (NANS), CMP-sialic acid (Neu5Ac) synthetase (CMAS), CMP-Neu5Ac transporter (CST), β1,4-galactosyltransfease (GT), and α2,6-sialyltransferase (ST). In planta protein sialylation was achieved by the coordinated expression and correct subcellular deposition of genes/proteins for (i) biosynthesis (GNE, NANS), (ii) activation (CMAS), (iii) transport (CST), and (iv) transfer of Neu5Ac to terminal galactose (ST). (Reproduced with permission from open access article under a Creative Commons License: Loos A, Steinkellner H IgG-Fc glycoengineering in non-mammalian expression hosts, Arch Biochem Biophys 2012;526:167–173.)



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4  Fc glycoengineering in plants The conserved secretory pathway between plants and mammals enable the production of IgGs and IgAs efficiently. En block transfer of the Glc3Man9GlcNAc2 precursor onto the growing protein and subsequent trimming in ER and cis/medial-Golgi compartments reveal similarities between mammals and plants up to the synthesis of GnGn structures (Fig. 1). After this, in mammals, GnGn structures undergo intensive elongation/ modification processes unlike in plants, which add xylose in β1,2-position to the innermost mannose residue and fucose in α1,3-position to the innermost GlcNAc residue of the GnGn core oligosaccharide (Fig. 1) which are absent in mammalian cells. Monoclonal antibodies (mAbs) in plants exhibit a N-glycosylation profile with a single dominant oligosaccharide structure, GnGnXF3. The humanization of the plant pathways were thoroughly investigated by Palacpac et al. [27] and Bakker et al. [28]. They overexpressed the human β1,4-glacatosyltransferase (GalT) in tobacco plants to elongate the plant-typical GnGnXF3 by β1,4-galactose leading to the formation of galactosylated structures and drastically reduced the degree of xylosylation and fucosylation. Nut production of mAbs (mAbs) suffered from challenges of formation of unexpected glycoforms and incompletely processed and hybrid structures [29, 30]. Mutants lacking plant-specific β1,2-xylose and core α1,3-fucose achieved by the elimination of endogenous enzymes, β1,­2-xylosyltransferase (XT) and core α1,3-fucosyltransferase (FT3, Fig.  2) by knockdown and knockout approaches for the respective genes, and generated mutant plant lines of A. thaliana, Lemna minor, N. benthamian, moss Physcomitrella patens, DXT/FT plants (N. benthamiana glycosylation mutants lack plant-specific core β1,2-xylose and α1,3-fucose residues) were generated and found importance in the production of different mAbs and therapeutics. A schematic diagram of Fc glycoengineering is represented in Fig. 5. Fc-N-glycosylation profiles of these mAbs achieved by the elimination of β1,2-xylose and core α1,3-fucose leading to the synthesis of human-type structures containing dominant GnGn with no detectable β1,2-xylose or α1,3-fucose residues revealed unaltered antigen binding and c­omplement-dependent cytotoxicity CDC activity and enhanced antibody-­ dependent cell-mediated cytotoxicity ADCC, effector functions of antibody. This also enabled the generation of increased galactosylation, ­ sialylation, branching, bisecting GlcNAc, or fucosylation.

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1

2 ∆XT/FT

FT

3

Asn

Gn

6

TII

I Asn

6

3 Asn

Asn

GaIT

4 Asn ST 5 Asn

Fig. 5  Fc-glycoengineering in plants. Overview of mAb glycoforms generated in glycoengineered N. benthamiana. IgG N-glycans generated (1) in Wild type plants: GnGnXF3 [14]; (2) in DXT/FT3 plants: GnGn [14]; (3) in DXT/FT + FT6: GnGnF6 [31]; (4) in DXTFT + GalT: AA [32, 33], (5) in DXT/FT along with six mammalian genes of the mammalian sialic acid pathway: NaNa [29]; and (6) in Wild type + GnTIII: GnGnXF3bi [33]. FT6: a1,­6fucosyltransferase, GalT: b1,4-galactosyltransferase, ST: a2,6-sialyltransferase, and GnTIII: N-acetylglucosaminyltransferase III (1). (Figure and legend reproduced with permission from open access article under a Creative Commons License: Loos A, Steinkellner H. IgG-fc glycoengineering in non-mammalian expression hosts. Arch Biochem Biophys 2012;526:167–73.)

GalT when ­targeted to a late Golgi compartment significantly improved β1,­4-galactosylation in DXT/FT, transgenic plants. mAbs produced in such glycoengineered plants exhibited a single dominant Fc-N-glycan, digalactosylated AA structures which is predominant in serum IgG and mAbs as against HIV produced in these glycoengineered plants which exhibited improved anti-viral activity. GlcNAc bound in b1,4-position to the innermost mannose residue called bisecting GlcNAc is reported to enhance ADCC activity of mAb CAMPATH-1H, glycoengineered Rituxan and Herceptin with increased bisecting structures due to decreased 1,6-fucosylation caused by the blocking of the fucosyltransferase. Contrasting reports exist that in CHO cells the overexpression of Nacetylglucosaminyltransferase III (GnTIII) done with the hypothesis to increase bisecting GlcNAc, produced typical hybrid structures instead with significantly reduced core-fucose content. In the DXT/FT mutant lacking plant-specific core modifications, less of bisecting glycoforms were synthesized as compared to Wild type plants. Glycomodified DXT/FT plants produced mammalian-type core α1,6-­ fucosylation by overexpressing core α1,6-fucosyltransferase, generating mAbs with and without fucose with identical N-glycosylation. Plant based antibody 2G12 batches exhibited glycosylation profiles containing a predominant N-glycan structure, and GnGnXF3, GnGnF6, GnGn, and digalactosylated AA structures with binding similar to FccRI,



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FccRIIa, and FccRIIb. 2G12 glycoforms lacking core fucose mediated antiviral activity against various lentiviruses including HIV-1. The most complex step of human N-glycosylation is terminal sialylation and difficult to accomplish in plants as they lack the enzyme cascades. But in planta the sialylation of mAbs has recently become possible [12, 34] by the introduction of enzymes of the mammalian pathway into plants, allowing the biosynthesis of sialic acid its activation, its transport into the Golgi, and finally its transfer onto terminal galactose and mAbs coexpressed with engineered human sialylation pathway carried up to 80% sialylated structures [30]. Six mammalian enzymes were overexpressed in plants [34].

5 Applications Advancement has been made in the design and development of plant expression systems for the generation of recombinant N-glycans glycoproteins by glycoengineering. N-glycosylation affects many properties of recombinant glycoproteins produced in planta including efficient plant-made antibodies for passive immunization but with shorter half-life in the blood due to a higher clearance rate [35–38]. The removal of the core fucose residue from mammalian α-1,6-fucose or the plant α-1,3-fucose from the N-glycan of an antibody has been reported to increase the antibody-­dependent cellular cytotoxicity (ADCC) [1, 36, 37] thus proving as an effective biopharmaceutical. The Food and Drug Administration (FDA) has approved first plant-made pharmaceutical protein for human parenteral administration including taliglucerase alfa [38], also named as Elelyso, produced by Protalix Biotherapeutics for the application as a replacement therapy for Gaucher disease, which is advantageous due to the structure of the exposed terminal mannose residues on α-1,3-fucose- and β-1,2-xylose-containing N-glycan structures generated in plant cell vacuoles [1] that are required for the efficient uptake of the enzyme into macrophages. N. benthamiana has been extensively researched for the production of mucin-type O-glycans [39] and N-glycans [40] of recombinant proteins. As plant cells lack β1,4-galactosylated and sialylated glycan, which have important biological functions in animal cells [1, 26], transgenic human β1,4-galactosyltransferase producing tobacco BY2 suspension-cultured cells were developed [1, 27]. Two genes encoding human CMP-Nacetylneuraminic acid synthetase and CMP-sialic acid transporter expressed in tobacco suspension-cultured cell to enable sialic acid biosynthesis in plants can act as bioreactor for mammalian glycoprotein production.

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Human butyrylcholinesterase (BChE) is a tetrameric human serum sialylated protein that finds therapeutic importance as a candidate bioscavenger of organophosphorus nerve agents. N. benthamiana has been engineered for the expression of sialylated protein by transient co-expression of BChE cDNA by vectors [41] leading to the generation of rBChE expressing mono- and di-sialylated N-glycans in the intracellular fluid with similarity to the human protein orthologue. β1,4-N-acetylglucosaminyl-transferase IV overexpression in the recombinant engineered plant enabled the generation of branched N-glycans, with tri-sialylated structures with better and effective novel therapeutic role [42]. Glycoprotein hormone erythropoietin (EPO) finds importance in the maintenance of hematopoiesis and providing tissue protection and recombinant human EPO (rhuEPO) find application in the treatment of anemia. However, rhyEPO at higher doses can cause harmful increase in the RBC masses and reveals limited role in tissue protection. Asialoerythropoietin (asialo-rhuEPO), which is a desialylated form of rhuEPO, has been reported to lack hematopoietic activity, but retain cytoprotective activity. But chemically enzymatic desialylation of rhuEPO suffers from not being cost effective. Although plants are known to synthesize complex N-glycans, they lack enzymes to transfer sialic acid and β1,4-galactose to N-glycan chains, therefore serve as a potential expression for generation of asialoerythropoietin. Asialo-rhuEPO is being designed to be produced in plants by introducing human β1,4-galactosyltransferase as the penultimate β1,4-linked galactose residues regulating its in  vivo biological activity. Co-expression of human β1,4-galactosyltransferase and EPO genes in tobacco plants has been reported to accumulate asialo-rhuEPO confirmed by its specificity to Erythrina cristagalli lectin column, revealing expression of N-glycan structures with terminal β1,4-galactose residues and a functional co-expressed GalT. Asialo-rhuEPO has been reported to interact with the EPO receptor (EPOR) with similar affinity as rhuEPO with desired biological function [43]. N-glycans with terminal Neu5Ac residues are important for the biological activities and half-lives of recombinant therapeutic glycoproteins in humans but the fact that plants express negligible amounts of free or protein-bound Neu5Ac presents a major disadvantage for their application as biopharmaceutical expression system. Thus to synthesize Neu5Ac-containing N-glycans, plants need to synthesize Neu5Ac and its ­nucleotide-activated derivative,cytidine monophospho-N-­acetylneuraminic acid. Transgenic A. thaliana plants expressing three key enzymes of the mammalian Neu5Ac biosynthesis pathway, UDP-N-acetylglucosamine



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­2-epimerase/N-acetylmannosamine kinase, N-acetylneuraminic acid phosphate synthase, and CMP-N-acetylneuraminic acid synthetase, has been designed and developed and their simultaneous expression has led to the generation of significant Neu5Ac amounts in planta, which could be further converted to cytidine monophospho-N-acetylneuraminic acid by the coexpression of CMP-N-acetylneuraminic acid synthetase leading to the production of Neu5Ac-containing glycoproteins in plants [44]. Neu5Ac could be synthesized in the plant cytosol by the expression of microbial Neu5Ac-synthesizing enzymes including Neu5Ac lyase from Escherichia coli and Neu5Ac synthase (neuB2) from Campylobacter jejuni in two model plants including Bright Yellow 2 (BY2) tobacco cells and Medicago sativa [45]. Human CMP-N-acetylneuraminic acid (NeuAc) synthase (HCSS) and α2,6-sialyltransferase (HST) enable sialylation of N-linked glycans in mammalian cells. HCSS synthesizes CMP-NeuAc, which HST uses as a donor substrate to transfer NeuAc to the terminal position of N-linked glycans. HCSS and HST genes could be inserted and expressed by the ­suspension-cultured tobacco BY2 cells to enable sialylation pathway in plants, producing mammalian-type sialoglycoproteins with terminal NeuAc residues in plants [46]. Two engineered constructs containing either the native signal peptide from human lactoferrin or the signal peptide from sweet potato sporamin fused to human lactoferrin has been reported to produce N-terminal sequences of rhLf purified from tobacco identical to Lf from human milk for both constructs [47]. The natural insertion of N. benthamiana into the RNA-dependent RNA polymerase 1 gene [1, 49] enables rapid production of high-value hormones, enzymes, and antibodies, and is successful in the production of ZMapp which is a cocktail of neutralizing mAb c13C6 and two chimeric antibodies c2G4 and c4G7, which were applied during the 2014–15 Ebola outbreak [48], and for the efficient production of vaccines against seasonal flu [49]. The intravenous immunoglobulin therapeutic application of ZMapp involves direct reaction to the virus and bind as lock and key leading to its deactivation and provides simulated immune response against Ebolaviral proteins Ebolavirus. Genes of the Ebola antibodies needed for the drug are inserted into Agrobacterium, then tobacco plants are injected or infused with the engineered viral vector-encoding Ebola antibodies, and plants produce the antibodies which are later isolated to form the drug known as ZMapp (Figs. 6 and 7, Table 2) [48].

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Sialic acids and sialoglycoconjugates in the biology of life, health and disease

Foreign DNA is cut by the same enzyme

Restriction cleavage site

The plasmid is reinserted into bacterium

T-DNA Agrobacterium The plasmid is removed from the bacterium and T-DNA is cut by a The foreign DNA is restriction enzyme inserted into the T-DNA od the plasmid.

Tobacco plant grown in a tightyly-controlled environment

Zmapp Drug is obtained

Further processing is done

The plant cells treat the new Plants are injected or infused with the genes as one of engineered virus their own, and start manufacturing the antibody

Fig. 6  Production of ZMapp through tobacco plant. (Reproduced with permission from Zahara K, Bibi Y, Ajmal M, Sadaf HM, Bibi F, Sardar N, Riaz I, Laraib S. J Coast Life Med 2017;5:206–11.)

Nucleoprotein (N) VIRION

Transcription factor VP30

Glycoprotein (GP)

Polymerase cofactor VP35

Monabs bind to EBV epitope by glycoproteins EBV-Monab activates complement c1q

Polymerase (L)

VP24 Matrix VP40

Ebola virus outline

(A)

FcγRI is activated

Monab Ebola

FcγRI

This leads to complement cascade activation ultimately leading to cell lysis

Ebola virus interaction with monab

This leads to activation of adaptive immune response including CD8+ T cell activation.

(B) Fig. 7  (A) Ebola and (B) Ebola and interaction with ZMapp. ((A) Reproduced with permission Ebola picture Source ViralZone, SIB Swiss Institute of Bioinformatics.)

Host

Lead product

Mapp Biopharmaceutical/ LeafBiol, USA Protalix, Carmiel, Israel

Tobacco leaves

ZMapp for Ebola crisis

Carrot or tobacco cell culture Nicotiana benthamiana leaves Rice seeds

ELELYSO (taliglucerase alfa) Enzyme replacement Vaccine for non-Hodgkin’s Lymphoma

Icon Genetics, München, Germany Ventria Bioscience, Junction City, KS, USA Greenovation Biotech GmbH, Heilbronn, Germany

Moss

Kentucky BioProcessing, Owensboro, KY, USA

Nicotiana benthamiana leaves Algae

PhycoBiologics Inc. Bloomington, IN, USA Medicago, Québec, QC, Canada

Vaccines Growth Factor and enzymes Vaccine for influenza, Pandemic market, Rabies and Rotavirus

Advantage

Website references

MagnICON Transient expression ProCellEx Stable Expression

Speed

[52]

Quality

[53]

MagnICON Transient expression Express Tec Stable Expression

Speed and Personalization

[54]

Scale Cost

[55]

Moss Physcomitrella patens-based Broytechnolgy

Speed Scale and Customized

[56]

Geneware Transient expression

Speed

[57]

Microalgae expression Proficia Transient Expression; Stable Expression

Speed Scale

[58]

Speed

[59]

Continued

77

Nicotiana benthamiana Alfalfa

VEN150 for HIVassociated chronic inflammation Moss-GAA for Pompe Disease, Moss-GBA for Gaucher’s Disease, MossAGAL for Fabry Disease Contract service

Expression technology

Sialoglycans and genetically engineered plants

Company



Table 2  Plant produced human pharmaceuticals and industrial production

78

Advantage

Website references

Speed Quality

[60]

Scale Cost

[61]

Influenza vaccine

LEX system Stable expression Stable Nuclear Expression Transient expression

Speed

[62]

Serum albumin

Stable Expression

Quality Scale

[63]

CaroRx for dental caries; PBI-220 antibody for anthrax; DPP4-Fc for MERS coronavirus infection

Stable Expression

Quality Scale

[64]

Company

Host

Lead product

Synthon, Nijmegen, The Netherlands Fraunhofer IME, Aachen, Germany Fraunhofer CMB/iBio, Newark, DE, USA

Duckweed LeafyBiomass Tobacco leaves

Antibody for nonHodgkin’s Lymphoma HIV Antibody

Nicotiana benthamiana leaves Rice seed Tobacco leaves

Healthgen, Wuhan, Hubei, China PlanetBiotechnology, Hayward, CA, USA

Expression technology

Reproduced with permission from open access article under Creative Commons Attribution License (CC BY 4.0):Yao J,Weng Y, Dickey A,Wang KY. Plants as Factories for Human Pharmaceuticals: Applications and Challenges. Int J Mol Sci. 2015;16(12):28549–65.

Sialic acids and sialoglycoconjugates in the biology of life, health and disease

Table 2  Plant produced human pharmaceuticals and industrial production—cont’d



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6  Mucin type O-glycans and plant expression As plants lack endogenous glycosyltransferase that lead to Ser/Thr glycosylation as in mammals, plants find importance for the engineering of O-glycosylation as there are no endogenous glycosyltransferases that can act upon engineered enzymes for the synthesis of O-glycans.Transient expression of mucin-type OGalNAc and core 1 O-linked glycan structures on recombinant human erythropoietin fused to an IgG heavy chain fragment (EPO-Fc) have been reported to be synthesized in N. benthamiana plants. Sialylated core structure constructs encoding human polypeptide:­ N-­acetylgalactosaminyltransferase, Drosophila melanogaster core 1 β1,3-­galactosyltransferase, human α2,3-sialyltransferase, and Mus musculus α2,6-­sialyltransferase have been reported for their transient co-­ expression in N. benthamiana together with EPO-Fc leading to the synthesis of mono- and disialylated O-linked glycans and biantennary structures with terminal sialic acid residues [65]. Engineering of O-linked glycans is not much developed in plant systems as the O-glycosylation pathways in plants are different from that in human. In mammals O-glycans on secretory proteins are formed by the attachment of N-acetylgalactosamine (GalNAc) to serine or threonine residues (mucin-type O-glycosylation) which are further modified by the addition of different monosaccharides such as galactose, GlcNAc, sialic acid, forming mucin-type core Oglycan structures that is important in different biological processes [66] (Fig. 8). In plants, unlike mammals, proline residues are converted to hydroxyproline (Hyp) by prolyl-4-hydroxylases (P4H) that are linked with arabinose residues. Knockout of P4H genes could eliminate O-glycosylation in P. patens, thereby helping in the modification of recombinantly expressed EPO [1, 6, 67]. Overexpression of human polypeptide GalNAc-transferase 2 (GalNAcT2) in Arabidopsis, tobacco BY2 cells, and N. benthamiana [68–70], initiating OGalNAc formation on different recombinant glycoproteins (including EPO and IgA1 antibodies) [71], has been reported. This GalNAc residue acts as a substrate for subsequent elongation with β1,3-galactose by overexpressing β1,3-­ galactosyltransferase (C1GalT1) and expression of C1GalT1 and genes for the human sialylation pathway enabled the synthesis of sialylated O-glycans [6, 72].

7  Introducing helminth glycosylation into plants Parasitic helminths secrete immunomodulatory with certain N-glycan epitopes including Lewis X and LDN-F glycan motives that find importance in treatment of allergies and autoimmune diseases. Overexpression of glycosyltransferases including FucTs, GalTs, and GalNAcTs in N. benthamiana

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Fig.  8  (A) Schematic representation of plant-type O-glycosylation. Proline residues adjacent to O-glycosylation sites are converted to hydroxyproline (Hyp) by prolyl-4-­ hydroxylases (P4Hs). Hyp residues are further elongated (e.g., by arabinosyltransferases—­ AraTs). (B) Mucin-type O-glycan biosynthesis pathway in mammals. Polypeptide GalNAc-transferases (GalNAc-Ts), β1,3-galactosyltransferases 1 (C1GalT1), Cosmc (chaperone), sialyltransferases (ST6GalNAcIII/IV, ST3GalI). (C) Mucin-type O-glycan-engineering in plants. Strategies involve the knockout of P4Hs to prevent Hyp formation and expression of mammalian GalNAc-Ts, Drosophila melanogasterc1galt1, and STs. (Reproduced with permission from Schoberer J Strasser R, Plant glyco-biotechnology.Semin Cell Dev Biol. 2018 80:133-141.)

enabled the reconstruction of Lewis X and LDN-F motives [73] that find importance in the development of anti-helminthic vaccines.

8 Detection Detection of low-level monosaccharides in the glycoprotein hydrolyzate are accomplished by derivatization prior to high-performance liquid chromatography (HPLC)-fluorescence and liquid chromatography (LC)-sonic spray ionization (SSI)-mass spectrometry (MS) analyses. LC-SSI-MS has been employed to identify the compositional monosaccharides including glucosamine, glucose, mannose, arabinose, xylose, and sialic acid found in the transgenic corn [74].



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9 Discussion In plant biomanufacturing of human proteins of importance, glycosylation, is one of the most addressed PTMs, as it affects protein homogeneity and functionality. Different engineering expression systems have been designed to control glycosylation and generate engineered N- and O-linked glycans with targeted sugar profiles and their various applications in the generation of human therapeutics [1–78]. Despite advances in the study of N-glycosylation pathways in plants, the study is far from complete and not completely known as compared to the human N-glycosylation pathway. The N-glycosylation pathway is not completely known for model plant organism A. thaliana and other different plant species. Although intra-Golgi glycosyltransferases are reported in A. thaliana, their functions remain unknown [79] but is assumed to play a vital role in synthesis of O-glycosylated proteins like arabinogalactan proteins. Studies from A. thaliana and rice have indicated that N-glycans enable growth under stress. However, complete genome sequencing of different plants will enable better understanding of the N-glycan pathway in plants and their efficient modifications and research in this exciting field of biology with human applications in the generation of therapeutics compatible to the human body is increasing across the globe.

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[29] Castilho A, Strasser R, Stadlmann J, Grass J, Jez J, Gattinger P, Kunert R, ­Quendler H, Pabst M, Leonard R, Altmann F, Steinkellner H. In planta protein sialylation through overexpression of the respective mammalian pathway. J Biol Chem 2010;285(21):15923–30. [30] Ko K, Tekoah Y, Rudd PM, Harvey DJ, Dwek RA, Spitsin S, Hanlon CA, et al. Function and glycosylation of plant-derived antiviral monoclonal antibody. Proc Natl Acad Sci 2003;100:8013–8. [31] Forthal  DN, Gach  JS, Landucci  G, Jez  J, Strasser  R, Kunert  R, Steinkellner  H. Fc-­ glycosylation influences Fcγ receptor binding and cell-mediated anti-HIV activity of monoclonal antibody 2G12. J Immunol 2010;185:6876–68824. [32] Strasser  R, Castilho  A, Stadlmann  J, Kunert  R, Quendler  H, Gattinger  P, Jez  J, Rademacher T, Altmann F, Mach L, Steinkellner H. J Biol Chem 2009;284:20479–85. [33] Castilho A, Bohorova N, Grass J, Bohorov O, Zeitlin L,Whaley K, Altmann F, Steinkellner H. Plos One 2011;6. [34] Loos A, Steinkellner H. Plant glyco-biotechnology on the way to synthetic biology. Front Plant Sci 2014;5:523. [35] Castilho A, Strasser R, Stadlmann J, Grass J, Jez J, Gattinger P, Kunert R, Q ­ uendler H, Pabst M, Leonard R, Altmann F, Steinkellner H. In planta protein sialylation through overexpression of the respective mammalian pathway. J Biol Chem 2010;285:15923–30. [36] Shields  RL, Lai  J, Keck  R, O’Connell  LY, Hong  K, Meng  YG, Weikert  SHA, et  al. Lack of fucose on human IgG1 N-linked oligosaccharide improves binding to human FcγRIII and antibody-dependent cellular toxicity. J Biol Chem 2002;277:26733–40. [37] Zeitlin L, Pettitt J, Scully C, Bohorova N, Kim D, Pauly M, Hiatt A, et al. Enhanced potency of a fucose-free monoclonal antibody being developed as an Ebola virus immunoprotectant. Proc Natl Acad Sci 2011;108:20690–4. [38] Tekoah Y, Tzaban S, Kizhner T, Hainrichson M, Gantman A, Golembo M, Aviezer D, et al. Glycosylation and functionality of recombinant β-glucocerebrosidase from various production systems. Biosci Rep 2013;33:e00071. [39] Strasser R. Engineering of human-type O-glycosylation in Nicotiana benthamiana plants. Bioengineered 2013;4:191–6. [40] Castilho A, Pia G, Grass J, Jez J, Pabst M, Altmann F, Gorfer M, Strasser R, Steinkellner  H. N-Glycosylation engineering of plants for the biosynthesis of glycoproteins with bisected and branched complex N-glycans. Glycobiology 2011;21:813–23. [41] Schneider  JD, Castilho  A, Neumann  L, Altmann  F, Loos  A, Kannan  L, Mor  TS, Steinkellner  H. Expression of human butyrylcholinesterase with an engineeredglycosylation profile resembling the plasma-derived orthologue. Plant Cell Rep 2012;31:1233–43. [42] Schneider  JD, Marillonnet  S, Castilho  A, Gruber  C, Werner  S, Mach  L, Klimyuk  V, Mor TS, Steinkellner H. Oligomerization status influences subcellular deposition and glycosylation of recombinant butyrylcholinesterase in Nicotiana benthamiana. Plant Biotechnol J 2014;12:832–9. [43] Kittur FS, Hung CY, Darlington DE, Sane DC, Xie J. N-Glycosylation engineering of tobacco plants to produce asialoerythropoietin. Plant Cell Rep 2012;31:1233–43. [44] Misaki R, Fujiyama K, Seki T. Expression of human CMP-N-acetylneuraminic acid synthetase and CMP-sialic acid transporter in tobacco suspension-cultured cell. Biochem Biophys Res Commun 2006;339:1184–9. [45] Kajiura H, Misaki R, Fujiyama K, Seki T. Stable coexpression of two human sialylation enzymes in plantsuspension-cultured tobacco cells. J Biosci Bioeng 2011;111:471–7. [46] Navarre  C, Smargiasso  N, Duvivier  L, Nader  J, Far  J, De Pauw  E, Boutry  M. N-­ glycosylation of an IgG antibody secreted by Nicotiana tabacum BY-2 cells can be modulatedthrough co-expression of human β-1,4-galactosyltransferase.Transgenic Res 2017;26:375–84.

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[47] Salmon  V, Legrand  D, Slomianny  MC, el Yazidi  I, Spik  G, Gruber  V, Bournat  P, ­Olagnier B, Mison D, Theisen M, Mérot B. Production of human lactoferrin in transgenic tobacco plants. Protein Expr Purif 1998;13:127–35. [48] Qiu X,Wong G, Audet J, Bello A, Fernando L, Alimonti JB, et al. Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp. Nature 2014;514:47–53. [49] D’Aoust MA, Couture MM, Charland N, Trépanier S, Landry N, Ors F, et al. The production of hemagglutinin-based virus-like particles in plants: a rapid, efficient and safe response to pandemic influenza. Plant Biotechnol J 2010;8:607–19. [50] Zahara K, Bibi Y, Ajmal M, Sadaf HM, Bibi F, Sardar N, Riaz I. Laraib S. J Coast Life Med 2017;5:206–11. [51] Ebola picture Source ViralZone, SIB Swiss Institute of Bioinformatics. [52] Mapp Biopharmaceutical, Inc. Home Page. Available online: http://mappbio.com. [53] Protealix Biotherapeutics. Home Page. Available online: https://www.protalix.com. [54] Icon Genetics GmbH. Home Page. Available online: https://www.icongenetics.com. [55] Ventria Biosciences. Home Page. Available online: https://www.ventria.com. [56] Greenovation Biotech GmbH. Home Page. Available online: https://www.greenovation.com. [57] Kentucky BioProcessing Home Page. Available online: https://www.kbpllc.com. [58] PhycoBiologics Inc., Home Page. Available online: https://www.phycotransgenics.com. [59] Medicago. Home Page. Available online: https://www.medicago.com. [60] Synthon. Home Page. Available online: http://www.synthon.com. [61] Fraunhofer IME Home Page. Available online: http://www.ime.fraunhofer.de. [62] Fraunhofer CMB. Home Page. Available online: http://www.fhcmb.org. [63] Healthgen Biotechnology. Home Page. Available online: http://www.oryzogen.com. [64] Planet Biotechnology Inc. Home Page. Available online: http://www.planetbiotechnology.com. [65] Castilho A, Neumann L, Daskalova S, Mason HS, Steinkellner H, Altmann F, Strasser R. Engineering of sialylated mucin-type O-glycosylation in plants. J Biol Chem 2012 Oct 19;287(43):36518–26. [66] Schjoldager KT, Clausen H. Site-specific protein O-glycosylation modulates proprotein processing—deciphering specific functions of the large polypeptide GalNAc-­ transferase gene family. Biochim Biophys Acta 1820;2012:2079–94. [67] Parsons J, Altmann F, Graf M, Stadlmann J, Reski R, Decker EL. A gene responsible for prolyl-hydroxylation of moss-produced recombinant human erythropoietin. Sci Rep 2013;3:3019. [68] Daskalova SM, Radder JE, Cichacz ZA, Olsen SH, Tsaprailis G, Mason H, et al. Engineering of N. benthamiana L. plants for production of N-acetyl-galactosamine-­ glycosylated proteins-towards development of a plant-based platform for production of protein therapeutics with mucin type O-glycosylation. BMC Biotechnol 2010;. 6750-10-62. [69] Castilho A, Windwarder M, Gattinger P, Mach L, Strasser R, Altmann F, et al. Proteolytic and N-glycan processing of human alpha1-antitrypsin expressed in Nicotiana benthamiana. Plant Physiol 2014;166:1839–51. [70] Yang  WH, Aziz  PV, Heithoff  DM, Mahan  MJ, Smith  JW, Marth  JD. An intrinsic mechanism of secreted protein aging and turnover. Proc Natl Acad Sci U S A 2015;112:13657–62. [71] Dicker M, Maresch D, Strasser R. Glyco-engineering for the production of recombinant IgA1 with distinct mucin-type O-glycans in plants. Bioengineered 2016;2016(7):484–9. [72] Castilho  A, Neumann  L, Daskalova  S, Mason  HS, Steinkellner  H, Altmann  F, et  al. Engineering of sialylated mucin-type O-glycosylation in plants. J Biol Chem 2012;287:36518–26.



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[73] Wilbers  RH, Westerhof  LB, van Noort  K, Obieglo  K, Driessen  NN, Everts  B, ­Gringhuis SI, Schramm G, Goverse A, Smant G, Bakker J, Smits HH,Yazdanbakhsh M, Schots A, Hokke CH. Production and glyco-engineering of immunomodulatory helminth glycoproteins in plants. Sci Rep 2017;7:45910. [74] Wang T, Nam PK, Shi H, Ma Y. Compositional monosaccharide analysis of transgenic corn glycoproteins by HPLC with fluorescence detection and LC-MS with sonic spray ionization. J Chromatogr Sci 2007;45:200–6. [75] Castilho  A, Pabst  M, Leonard  R, Veit  C, Altmann  F, Mach  L, Glössl  J, Strasser  R, ­Steinkellner H. Construction of a functional CMP-sialic acid biosynthesis pathway in Arabidopsis. Plant Physiol 2008;147:331–9. [76] Chen M, Liu X, Wang Z, Song J, Qi Q, Wang PG. Modification of plant N-glycans processing: the future of producing therapeutic protein by transgenic plants. Med Res Rev 2005;25:343–60. [77] Paccalet  T, Bardor  M, Rihouey  C, Delmas  F, Chevalier  C, D’Aoust  MA, Faye  L, ­Vézina  L, Gomord  V, Lerouge  P. Engineering of a sialic acid synthesis pathway in transgenic plants by expression of bacterial Neu5Ac-synthesizing enzymes. Plant Biotechnol J 2007;5:16–25. [78] Nikolovski N, Rubtsov D, Segura MP, Miles GP, Stevens TJ, Dunkley TP, Munro S, ­Lilley KS, Dupree P. Putative glycosyltransferases and other plant Golgi apparatus proteins are revealed by LOPIT proteomics. Plant Physiol 2012;2012(160):1037–51. [79] Parsons HT, Christiansen K, Knierim B, Carroll A, Ito J, Batth TS, Smith-Moritz AM, Morrison S, McInerney P, Hadi MZ, et al. Isolation and proteomic characterization of the Arabidopsis Golgi defines functional and novel components involved in plant cell wall biosynthesis. Plant Physiol 2012;2012(159):12–26.

Further reading [80] Strasser R. Plant protein glycosylation. Glycobiology 2016;26:926–39.

CHAPTER 3

Sialic acid, sialoglycans, sialylation: A study in insects 1 Introduction Class Insecta comprises the greatest diversity of animals including more than 750,000 species, with body morphology such as three pairs of legs, two pairs of wings originating from the middle and thoracic regions, head with a pair of antenna, and a pair of compound eyes [1].The great adaptive radiation and occurrence in almost all niches ranging from terrestrial to subtidal sea waters by insects is indicative of the success in evolution. Although the property of flight has been developed in birds, reptiles, and mammals, the power of flight was first developed in insects. The evolution of flight in insects enabling their dispersal, escape from predators, and accessibility to food or favorable environmental conditions offers them advantages as compared to other terrestrial invertebrates. Insects offer a great ecological and economical significance as pollinators such as bees, butterflies, wasps, moths, and flies. Mosquitoes, ticks, lice, fleas, bedbugs, however, cause human misery and insects also act as vectors for diseases such as mosquitoes transmitting malaria, yellow fever, elephantiasis, tsetse fly transmitting sleeping sickness, lice transmitting typhus and relapsing fever, fleas transmitting bubonic plague, housefly transmitting fever and dysentery [1], and ticks transmitting Babesiosis, Ehrlichiosis, Lyme disease, Rocky Mountain spotted fever (RMSF),Tularemia, Crimean-Congo hemorrhagic fever, relapsing fever (borreliosis), Rickettsial diseases (spotted fever and Q fever), Tick-borne encephalitis (Table 1). Insects are also known to transmit diseases in plants such as ash-gray leaf bugs (Piesma sp.) transmit beet leafcurl virus, sugarbeet savoy virus, and beet latent rosette disease. Aphids members of superfamily Aphidoidea transmit beet mosaic, cabbage black ringspot, cauliflower mosaic virus (CMV), cherry ringspot, cucumber mosaic virus, onion yellow dwarf, pea wilt, potato Y, tobacco mosaic virus (TMV), tomato spotted wilt, and turnip yellow mosaic virus (TYMV). Leafhoppers belonging to the family Cicadellidae transmit aster yellows, beet curly top, blueberry stunt, dwarf disease of rice, Sialic Acids and Sialoglycoconjugates in the Biology of life, Health and Disease https://doi.org/10.1016/B978-0-12-816126-5.00003-2

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Table 1  Insect causing diseases in human.

Mosquitoes

Aedes sp.

Anopheles sp. Culex sp. Sandflies Ticks

Triatomine bugs Tsetse flies Fleas Black flies Lice

Chikungunya Dengue fever Lymphatic filariasis Rift Valley fever Yellow fever Zika Malaria Lymphatic filariasis Japanese encephalitis Lymphatic filariasis West Nile fever Leishmaniasis Sandfly fever (phelebotomus fever) Crimean-Congo hemorrhagic fever Lyme disease Relapsing fever (borreliosis) Rickettsial diseases (spotted fever and Q fever) Tick-borne encephalitis Tularaemia Babesiosis Ehrlichiosis Rocky Mountain spotted fever (RMSF) Chagas disease (American trypanosomiasis) Sleeping sickness (African trypanosomiasis) Plague (transmitted by fleas from rats to humans) Rickettsiosis Onchocerciasis (river blindness) Typhus and louse-borne relapsing fever

phony peach, and Pierce disease in grapes. Plant hoppers belonging to superfamily Fulgoroidea transmit cereal tillering disease, maize mosaic, oat sterile dwarf, rice stripe, and sugarcane Fiji disease. Whiteflies member of family Aleyrodidae transmit yellow mosaic diseases in cowpeas, roses, soybeans, and tomatoes, leaf curl virus in cotton, potato, tomato, and tobacco plants. Treehoppers belonging to family Membracidae transmit pseudo curly top disease in eggplants. Psyllids members of family Psyllidae transmit greening disease in citrus. Mealy bugs belonging to Pseudococcidae family



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transmit cocoa swollen shoot virus and cocoa mottle leaf virus. Apple maggots, belonging to family Tephritidae, are known to transmit bacterial rot in apples. Flower thrips, members of family Thripidae, transmit Tomato spotted wilt virus. Cabbage maggots belonging to family Anthomyiidae cause fungal infection. Leafminer flies belonging to family Agromyzidae transmit TMV. Leaf beetles, members of family Chrysomelidae, transmit broad bean mottle, TYMV. Potato flea beetles (Epitrix cucumeris) transmit pathogen of potato scab. Corn flea beetles (Chaetocnema pulicaria) cause bacterial infection in corn. Bark beetles, members of family Scolytidae, cause fungal infection.The elm bark beetle (Scolytus multistriatus) causes Dutch elm disease, chestnut blight. Conotrachelus nenuphar belonging to family Curculionidae cause fungal infection. Honey bees, Apis mellifera, members of family Apidae, cause bacterial infection while ants belonging to family Formicidae and bees spread fungal infection [2]. These infections in crops and plants cause losses to the economy of a country and therefore understanding of the physiology and biochemistry of insects finds so much importance.

1.1  Insect physiology and development Insects are categorized as protostomes, differing developmentally from the deuterostomes. In the protostomes, the mouth on the anterior end develops from the blastopore and anus develops later at the termination of the alimentary canal. Holoblastic determinate, spiral cleavage that determines the fate of blastomeres, with mesodermal tissue formation from single blastomere 4d cell, development of ventral nerve cord, and development of coelom schizocoely from mesodermal cell mass are the hallmark features of protostome development in insects [1]. Insects reveal diverse feeding habits and modified mouth parts [3]. The evolutionary history of the diverse class Insecta are based on the our knowledge from taxonomic palaeobiodiversity. Based on this paleodiversity studies on morphology of mouthpart of Hexapoda revealed diversity and extinctions in the Late Carboniferous, Middle and Late Triassic, CallovianOxfordian, Early Cretaceous, and Albian-Cenomanian but the transitions of Permian-Triassic, Triassic-Jurassic, and Cretaceous-Cenozoic did not impact the diversity and mouthpart strongly. The nector feeding type of mouth part has been documented to be originated during the Cretaceous Terrestrial Revolution. Phytophagy together with gymnosperm diversity has been linked to the diversity of origin of insect mouthparts during the Middle and the Late Triassic [4].

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They reveal differences in ultrastructure of antenna. Scanning electron microscopic (SEM) studies on the antenna of Hemipyrellia ligurriens (H. liguirrens) [5] under family Calliphoridae revealed diversity and ultrastructural variation between the two sexes. Studies on adaptive radiations [6] revealed an evolutionary significance in insect diversity.

1.2  Insects as vectors of human and animal diseases Insects are the vectors of a host of parasites such as virus, protozoans, bacteria, and nematodes causing several infectious diseases [7–10] and are some of the major concerns of public health across the globe. Mosquitoes, sandflies, triatomine bugs, blackflies, ticks, tsetse flies, mites, and lice spread infectious agents and cause human diseases. World Health Organization (WHO) has recorded more than 700,000 deaths from diseases such as malaria, dengue, schistosomiasis, human African trypanosomiasis, leishmaniasis, Chagas disease, yellow fever, Japanese encephalitis, and onchocerciasis, globally [10]. Vector-borne diseases, contribute to about 17% of all infectious diseases with the maximum disease burden reported from tropical and subtropical areas affecting largely the economically poor. Since 2014 onwards, major outbreaks of insect-borne diseases such as dengue, malaria, chikungunya yellow fever, and Zika have created a major havoc to the public health. Vector-borne disease distribution is determined by demographic, environmental, and social factors. Travel and trade, urbanization, climate change, growth of urban slums, lack of adequate sanitation and improper waste management, generation of breeding grounds for insects and vectors are known to affect the population increasing the disease causing pathogens and causing diseases. Vectors transmit infectious diseases between humans or from animals to humans mostly by bloodsucking insects, through blood meal from infected individual. Mosquitoes are the best known disease vector. We discuss in this chapter the (i) N-glycosylation in the insects compared to mammals (ii) sialylation in insects. 1.2.1  N-Glycosylation: A comparison between mammals and insects N-Glycosylation in most eukaryotes (Fig. 1) occurs within the endoplasmic reticulum (ER) that initiates with the generation of a lipid-linked oligosaccharide (LLO) by asparagine-linked N-glycosylation enzymes (ALG). The oligosaccharide is then transferred to the polypeptide by oligosaccharyltransferase (OST) and proteins reveal modification at Asn residues containing the N-X-S/T sequence after which further processing takes place in the Golgi apparatus.



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Common precursor: Man8 Och1

Asn

Asn

MnTs Asn

Mnsl Asn GnTl

Yeast-type highmannosidic N-glycans

Asn Mnsll Asn

Asn

HEXO

GnTll

FT3, FT6

Asn

Insect-type paucimannosidic N-glycans FT6

Asn GlcNAc Mannose Xylose Fucose Galactose Sialic acid

FT3, XT

Asn

GalT Asn

Plant-type complex N-glycans

ST Asn Mammalian-type complex N-glycans

Fig. 1  Insect glycosylation and comparison with yeast, plants, mammalian glycosylation. (Reproduced with permission from Loos A, Steinkellner H. IgG-Fc glycoengineering in non-mammalian expression hosts. Arch Biochem Biophys 2012;526(2):167–73. https:// www.who.int/news-room/fact-sheets/detail/vector-borne-diseases.)

Developmental stage-specific expression and lifelong expression of sialylation enzymes have been reported in different insects. While Drosophila (Fig.  2) sialyltransferase shows tissue- and stage-specific expression [11], silkworm sialyltransferase were lifelong and constitutively expressed in the body [12]. But structure and complexity of sialylated moieties vary in the insect and mammalian system due to differential expression of multiple

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Sialic acids and sialoglycoconjugates in the biology of life, health and disease

Fig. 2  (A) Female Drosophila. (B) Protein database (pdb) image of 5A01, O-GlcNAc transferase from Drosophila melanogaster [16]. (Reproduced from Wikipedia: Acharya S. Own work, CC BY-SA 4.0, file: Drosophila melanogaster Proboscis.Jpg. Wikimedia commons.)

processing enzymes including glycosyltransferases [13,14]. N-Glycans from insect cell lines revealed modified paucimannosidic or oligomannose structures and the sialylation process shows marked difference between the insect and the mammalian system. While the silkworm sialyltransferase shows localization in Golgi apparatus, the mammalian counterparts show nuclear localization [15]. Mammalian processing pathways—In humans and other mammals, glycans containing galactose and sialic acid N-linked glycosylated with branched structures by adding N-acetylglucosamine (GlcNAc) sugars to the outer mannose (Man) residues of the tri-mannosyl core (Man3GlcNAc2) appear. GlcNAc can be added to Man residue by GlcNAc transferase (GNTIII) generating branches which may be further extended by galactose (Gal) residues by β-1,4 linkages. GlcNAc-Gal extensions (LacNAc), a second Gal by α-1,3 linkage, can add to the structural complexity of glycans in humans. The β-1,4-linked-Gal residues may be linked with sialic acid by α-2,3 or α-2,6 linkages. A number of sialic acids linked by α-2,8 linkages can lead to polysialylated moieties as in neural cell adhesion molecule (NCAM) and other proteins. Insect cells—Insect glycans reveal truncated or paucimannosidic glycans (Man1–3GlcNAc2) or oligomannosidic glycans (Man5–9GlcNAc2) due to the effect of mannosidases and N-acetylglucosaminidase that chop the Man



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and GlcNAc residues. Insects also revealed less complexity in glycan structure as compared to human (Fig. 1). Insect N-linked glycosylation revealed up to two core fucose (Fuc) residues linked to innermost GlcNAc in α-1,6and α-1,3-linkages. Although α-1,6 fucosylation is present in mammals, the α-1,3 linked Fuc residue is potentially immunogenic to ­humans [17]. 1.2.2  Sialic acids and insects Sialic acids, negatively charged cyclic monosaccharide neuraminic acid, composed of nine carbon backbone with carboxylic acid group at the C1 position are conjugated to proteins or lipids by different acceptors by different linkages including α-2,3, α-2,6, or α-2,8-linkages, by specific sialyltransferases (ST, 20). Sialylated molecules in eukaryotes play a vital role in development, immunity, cell-cell interaction, signaling pathways, cell death and differentiation, host cell-virus interactions [13,18]. Biological masking conferred by sialic acid can shield recognition sites like penultimate sugar moiety in receptors and antigens, and generate a self-like property to cells on one hand and on the other, play a significant role in recognition by functioning as ligands for hormones, lectins, antibodies, and inorganic cations [19] and participate in cellular adhesion, inflammation, immune response, embryogenesis of the nervous system [20]. Studies on sialylation in insects have been done on silkworm Bombyx mori (Bm), family Bombycidae [15,21], the model organism Drosophila melanogaster [13,18], belonging to family Drosophilidae, mosquito Aedes aegypti (Fig. 3), member of family Culicidae [22], honeycomb moth, Galleria mellonella (G. mellonella) belonging to family Pyralidae [23], and is reported from concrement vacuoles of the Malpighian tubules larvae of the cicada meadow froghopper or meadow spittlebug Philaenus spumarius (P. spumarius) belonging to family Aphrophoridae [24], and oriental fruitfly Bactrocera dorsalis (B. dorsalis), under family Tephritidae [25]. The taxonomic positions are highlighted in Table 2.

Fig. 3  Aedes aegypti. (From Wikipedia: https://en.wikipedia.org/wiki/Aedes_aegypti.)

Table 2  Taxonomic positions of insects studied for sialylation.

Classification

Drosophila melanogaster

Aedes aegypti (Ae. aegypti)

Bombyx mori (Bm)

Bactrocera dorsalis (B. dorsalis)

Kingdom

Philaenus spumarius

Spodoptera frugiperda

Ixodes ricinu (I. ricinu)

Animalia

Phylum

Arthropoda

Class Order Family Genus Species

Galleria mellonella

Insecta Diptera Drosophilidae Drosophila D. melanogaster

Diptera Culicidae Aedes Ae. aegypti

Lepidoptera Bombicidea Bombyx Bombyx mori

Diptera Tephritidae Bactrocera Bactrocera dorsalis

Lepidoptera Pyralidae Galleria Galleria mellonella

Arachnida Hemiptera Aphrophoridae Philaenus Philaenus spumarius

Lepidoptera Noctuidae Spodoptera Spodoptera frugiperda

Ixodida Ixodidae Ixodes I. ricinus



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In insect sialylation revealed common steps with mammalian sialylation which may later add sialyl residues [26]. GalNAcα-O-Ser/Thr followed by Ga1β1,3GalNAcα-O-Ser/Thr core-1 during O-glycosylation of molecules in many lepidopteran insect cell lines are reported leading to the production of glycoproteins that can accept sialic acid residues [26,27]. α2,6-­Sialyltransferase in silkworms has been reported to transfer sialic acid to Galβ1-R [15]. While in D. melanogaster, sialylated macromolecules has been reported to play dominant role in development [13], in Ae. aegypti they have been reported to play an important role in dengue infection and in determining competence of vector flies [22]. Sialylation in D. melanogaster showed considerable differences with de novo sialylation pathway in mammals [13,18]. 1.2.3  Sialylation in Drosophila In Drosophila, sialylated macromolecules contribute less than 0.1% of total N-glycan profile [28]. They are mostly reported from embryos and adult heads [29,30]. Drosophila sialyltransferase showed significant homology with the mammalian sialyltransferase with specificity toward GalNAcbeta1– 4GlcNAc and show preference toward glycoproteins [31] with expression restricted in neurons of central nervous system (CNS) throughout the development [31]. In Drosophila, sialyltransferase bearing homology to mammalian ST6Gal sialyltransferase enables [32] modification of N-glycans with α2,6-linked sialic acid moieties [12,31] leading to sialylation of macromolecules. β1,4-N-acetylgalactosaminyltransferases A (β4GalNAcTA), a glycosyltransferase involved in the biosynthesis of complex and hybrid N-glycans, mutants revealed affected sialylated structures, indicative of the fact that N-glycans and sialylations are involved in the development and regulation of D. melanogaster nervous system [33–35]. Mutations targeted toward the inactivation of sialylation pathway reveal the role of sialylated N-glycans in the regulation of the nervous system during development. Mutations in Drosophila sialyltransferase revealed progeny with reduced life span, ­locomotion difficulties and temperature-sensitive (TS) paralysis, defective neuronal excitability, and inactivation of D. melanogaster voltage-gated Na+ channel [31]. CMP-sialic acid synthetase gene (CSAS) coding for enzyme producing CMP-Sia is a sugar donor for sialyltransferase [36] in Drosophila. CSAS mutants revealed phenotypic similarity with sialyltransferase mutants indicative of the fact that sialylations are involved in controlling voltage-gated channels [37]. Sialyltransferase and β4GalNAcTA interactions indicate that sialic acid molecules have a probable role in masking the interaction of LacNAc terminal

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structures of N-glycans with lectins [35] thus playing a dominant role in regulating the nervous system. In Drosophila sialylated glycans have been reported to be developmentally regulated [13] and mediate neural regulation [28]. During early developmental stages in Drosophila, homopolymers of α2,8-linked Sia or Neu5Ac, polysialic acid moieties are expressed [13]. Mutation studies have revealed that α2,6-sialylated N-glycans modulate neural transmission [28] and constitutive expression of sialic acid synthase (D. melanogaster SAS) controls sialic acid biosynthesis in CNS neurons [37] in Drosophila. Paucimannosidic N-glycan structures in S2 cells were reported. β-1,4-galactosyltransferase (GalT) expression and GlcNAcase suppression on N-glycan patterns indicated that a complete functional human glycoprotein in engineered Drosophila S2 cells by suppressing GlcNAcase and coexpressing additional glycosyltransferases of N-glycosylation pathway [38]. 1.2.4  Sialylation and Spodoptera frugiperda (Sf9) Spodoptera frugiperda (Sf9) has found application in understanding the sialylation process in insects [39–41]. Transgenic Sf9 cells namely Sfβ4GalT/ST6 cells could express mammalian β1,4-GalT and α2,6-sialyltransferase genes constitutively [41,42], and revealed a salvage pathway of sialic acid synthesis in lepidopteran insects synthesizing sialic acid in the absence of the source CMP-sialic acid [43,44] or low sialic acid [41,45]. Although studies in the last decade in insect glycosylation have primarily focused on the role of glycans in insect nervous system development, recent studies by fluorescent conjugated lectins including Galanthus nivalis (GNA) and Hippeastrum Hybrid (Amaryllis) (HHA), specific toward mannose, Rhizoctonia solani agglutinin (RSA) and Sambucus sieboldiana agglutinin (SSA), specific toward GalNAc/Gal, wheat germ agglutinin (WGA) and tobacco (Nicotiana tabacum) agglutinin or Nictaba specific toward GlcNAc and Sambucus nigra lectin (SNA-I) with specificity toward Neu5Ac(α-2,6)Gal/ GalNAc have enabled the visualization, localization, and detection of glycan on cell surface of gut and different zones of the midgut of insect pest cotton leafworm Spodoptera littoralis, larva and primary cell lines by confocal microscopy. Apical differentiated columnar cells with microvillar brush b­ order revealed dominant presence of GalNAc/Gal, terminal GlcNAc moieties are indicative of strong binding with SSA and RSA and WGA respectively, while the undifferentiated midgut stem cells revealed predominant binding of GNA and Nictaba and microvilli revealed strong binding with SSA, HHA, WGA, and SNA-I (Fig. 4) indicative of polarized glycan expression and distribution and may bear relation to midgut function and development [46].

A

Binding ratio stem cell/columnar cell microvilli GNA Nictaba RSA HHA SNA-I WGA SSA 0.1

1

10

Fig.  4  Confocal microscopy studies on lectin binding to cell surface of the midgut stem cells of Spodoptera littoralis. (A) Ratio of fluorescence intensity due to lectin binding indicative of differences in glycan distribution in stem cells and microvilli of columnar cells. (B–H) Representative confocal images of stem cells incubated with FITClabeled lectins: GNA (B), Nictaba (C), RSA (D), HHA (E), SNA-I (F), WGA (G), and SSA (H). (Reproduced with permission from Walski T, De Schutter K, Cappelle K, Van Damme EJM, Smagghe G. Distribution of glycan motifs at the surface of midgut cells in the cotton leafworm (Spodoptera littoralis) demonstrated by lectin binding. Front Physiol 2017;8:1020.)

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1.2.5  Sialylation and Ae. aegypti Ae. aegypti (Fig.  5) tissue showed sialic acid expression and α-2,6-linked sialoglycocongugates on mosquito cell membranes and are reported to contribute to vector competence in transmittance and infection [22] of dengue. Ae. aegypti has been found to express endogenous sialic acid in tissues (Fig. 5) detected from head, salivary glands, and midgut revealed by binding to SNA.Treatment with sialidase prior to treatment with SNA removed the SNA binding, indicative of the specificity of the expression of α-2,6-linked SNA lectin

Merge

Salivary gland

DAPI

SNA lectin

Merge

Midgut

Head

Abdomen

Midgut

Gut

Salivary gland

DAPI

(A)

(B) Midgut 0.5 IU sialidase

Head 0.5 IU sialidase

Aedes aegypti

Aedes aegypti

Drosophila melanogaster

DAPI

SNA lectin

Salivary gland 0.5 IU sialidase

(C) Fig.  5  Lectin histochemistry of Ae. aegypti tissues revealing (A) expression of α-2,6linked sialoglycoconjugates in Ae. aegypti salivary gland, midgut, and head incubated with SNA lectin stained with FITC. (B) D. melanogaster abdomen, gut, and midgut served as positive control (C) SNA staining of mosquito salivary gland and midgut pretreated with 0.5IU sialidase for 30 min before SNA incubation, removed SNA binding indicating specificity of α-2,6-linked sialoglycan expression with D. melanogaster acting as reaction controls. Nucleus revealed blue color, stained with DAPI and SNA-FITC produced green color. (Reproduced as under creative common license source. Cime-Castillo J, Delannoy P, Mendoza-Hernández G, et al. Sialic acid expression in the mosquito Aedes aegypti and its possible role in dengue virus-vector interactions. Biomed Res Int 2015;2015:504187.)



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glycans (Fig. 5). DENV interaction with Ae. aegypti salivary glands (SG) and the role played by sialylated glycans has been shown in Fig. 6. Ae. aegypti SGs were incubated with DENV and stained with anti-DENV E antibody. Preincubation of SGs with ConA, LCH, and SNA lectins followed by incubation of DENV revealed blocking of SG interaction with DENV by sialic acid recognizing lectins. DENV-SG interaction was affected by sialidase pretreatment and blocked by sialic acid competitors, fetuin (1 mM), and free sialic acid (200 nM). SGs pretreatment with trypsin prior to adding DENV LcH

SNA

Lectin

ConA

DAPI

DAPI

DENV

DENV-SG

(A)

(B) Trypsin

(C)

Fetuin

5

Free sialic

15

30

DENV

Sialidase

DAPI

DAPI

DENV

DENV-SG

(D)

Fig.  6  Confocal microscopic images revealing DENV interaction with Ae. aegypti salivary glands. (A) DENV interaction with Ae. aegypti SGs wherein SGs are incubated with DENV and stained with anti-DENV E antibody and rhodamine-coupled anti-IgG antibody producing red color. (B) DENV-SG competence assays using ConA, LCH, and SNA lectins, which were preincubated with salivary glands prior to incubation with DENV thereby blocking interaction with DENV by sialic acid specific lectins. (C) DENV-SG interaction in the absence or presence of sialidase. SGs untreated or pretreated with sialidase before adding DENV revealing specificity of sialic acid in the DENV-SG interaction. Sialic acid competitors, fetuin, and free sialic acid could block DENV-SG interaction. (D) DENV-SG interaction in SGs pretreated with trypsin for 5, 15, or 30 min before adding DENV, decreasing the DENV-SG interaction after 15 min, and completely lost at 30 min. Nuclei is stained blue by DAPI, green: (FITC) SNA lectin interaction, red color due to rhodamine-coupled anti-IgG antibody, recognizing anti-DENV E antibody. (Reproduced with permission as under creative common license source. Cime-Castillo J, Delannoy P, Mendoza-Hernández G, et al. Sialic acid expression in the mosquito Aedes aegypti and its possible role in dengue virus-vector interactions. Biomed Res Int 2015;2015:504187.)

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is reported to decrease or completely prevent DENV-SG interaction indicating the role of sialoglycoproteins in DENV-SG interaction. The sialyltransferase gene sequence bear homology with the ST6Gal, α2,6-sialyltransferase (ST6Gal) family [47] and Drosophila sialyltransferase [32]. The sialyltransferase gene sequence bear homology with the ST6Gal, α2,6-sialyltransferase (ST6Gal) family [47] and Drosophila sialyltransferase [32]. Cytidine-5′-monophosphate CMP-sialic acid synthase (CSAS) plays a significant role in sialylation. Ae. aegypti sialyltransferase and sialylated moieties revealed a probable role in dengue virus (DENV)-vector competence [22] and infection. 1.2.6  Sialylation and silkworms α2,6-Sialyltransferase in Bombyx mori (Fig. 7) is localized in the Golgi apparatus and play a vital role in N-glycan modification [15]. Expressed in Sf9 cells, it is found to transfer sialic acid to the nonreducing terminus of Galβ1-R with high affinity toward GalNAcβ1,4-GlcNAc-R [49]. Gene product of 30Kc19 revealing a sialyltransferase activity in silk worm hemolymph could increase the sialylation of recombinant secreted human placental alkaline phosphatase, promote transfer of sialic acid to the glycoprotein [21], and enable synthesis of bi-, tri-, tetra-, and penta-­ antennary branching in recombinant protein human erythropoietin in the transfected Chinese hamster ovary (CHO) cells [50]. A silkworm-baculovirus system has been engineered to produce recombinant glycoproteins. To circumvent the problems of differences in N-glycan structures of silkworm producing pauci-mannose type N-glycans without sialic acid or galactose residues unlike in mammals, galactosylation and sialylation pathways were engineered in silkworm, coexpressing

Fig. 7  Silkworm [48].



Insect sialobiology

Target protein Viral infection Glycan related enzyme Substrate

101

α2,35wT ® a2,3α2,65wT ® a2,6-

Injection/oral administration

+ b1,4-GaIT

+ SialT

+ GnT2

+CMP-sialic acid or sialic acid & CSS

Fig.  8  N-Glycan sialylation in a silkworm-baculovirus system. (Reproduced with permission from Suganuma M, Nomura T, Higa Y, Kataoka Y, Funaguma S, Okazaki H, Suzuki T, Fujiyama K, Sezutsu H, Tatematsu KI, Tamura T. N-glycan sialylation in a silkworm-­ baculovirus expression system. J Biosci Bioeng 2018;126:9–14.)

GalT and sialyltransferase, thereby producing sialylated N-glycoproteins. Based on the type of sialyltransferase, α2,3/α2,6 sialylation to N-glycans were produced. N-Acetylglucosaminyltransferase II coexpression enabled synthesis and expression of di-sialylated N-glycans (Fig. 8) [51]. Silkworm (Bombyx mori) is being studied extensively for the role played by sialic acid in the development and aging revealing changes based on liquid chromatography-mass spectrometry (LCMS) and lectin immunohistochemistry with Maackia amurensis agglutinin (MAA) and SNA. Sialic acid was observed in all stages of the development, decreasing with development and aging, revealing similarity with that of mammals but with increased expression in pupa [52]. 1.2.7 Ticks Recently sialylated N-glycans has been detected in partially fed Ixodes ricinu (Table 2) using matrix-assisted laser desorption/ionization time-of-flight/ time-of-flight mass spectrometry from salivary glands and gut probably from the host (N5).

1.3  Function of sialylation in insects α2,6-Sialyltransferase in Drosophila has been reported to play a vital role in the synthesis of sialic acids and their transfer to glycoconjugates and sialylated N-glycan structures have been shown to modulate cell excitability, exerting prominent effects on voltage-gated Na+ and K+ channels and in the regulation of the nervous system [28]. Drosophila CSAS provide a sugar donor for the sialyltransferase. CSAS, developmentally regulated, is predominantly

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located in the adult head [53]. Drosophila sialyltransferase regulate neural excitability in the CNS throughout development and control neurological functions of life span, locomotor abnormalities, TS paralysis, excitability of neuromuscular junctions, and function of a voltage-gated sodium channel [28] in adults. Drosophila sialic acid synthase (D. melanogaster SAS) also plays a significant role in neuronal regulation as neurons from SAS mutants revealed higher incorporation of sialic acid at the soma than at the neurite, perinuclear regions, and the plasma membrane affecting the biosynthesis and distribution of sialic acid in Drosophila CNS neurons [37] together with decreased longevity, temperature-induced paralysis, locomotor abnormalities, and defects in neural transmission at neuromuscular junctions [36]. While mammalian CSAS showed nuclear localization in Drosophila CSAS was predominantly localized in the Golgi compartment [36]. In Ae. aegypti sialylated molecules in a salivary gland have been reported during DENV internalization in mammalian cells [22] indicating the role of sialic acid in vector competence.

1.4  Genetic engineering approaches and sialylation Insect cell lines have been engineered to generate sialylated recombinant products and to express mammalian proteins in insect cell lines. Insect cell lines lack the ability to synthesize the sialic acid (Sia) donor molecule CMP-Sia. Engineered Sf9 cells have been reported to synthesize CMP-Sia, by engineering the sialic acid synthesis pathway genes using baculovirus technology. Engineering and coexpression of a sialuria mutant UDP-GlcNAc-2-epimerase/ManNAc kinase (EKR263L), Wild type sialic acid 9-phosphate synthase (SAS), and Wild type CSAS in the presence of GlcNAc enable synthesis of CMP-Sia to enable sialylation of N-glycans on glycoproteins [54] in insect cell lines. CMP-Sia transporter functions were established in Sf9 cells with the engineering of six mammalian genes to generate a new cell line, SfSWT-4, transformed again with human CMP-Sia transporter (hCSAT) gene to produce the SfSWT-6 cell line, and revealed high levels of recombinant glycoprotein sialylation, when cultured in the presence of N-acetylmannosamine at low concentrations indicating that hCSAT expression impacted glycoprotein sialylation [49]. Escherichia coli (E. coli), N-acetylglucosamine-6phosphate 2′-epimerase (GNPE), with functions of sialic acid degradation, when transfected with a set of N-glycoprotein sialylation genes efficiently produced sialic acid, CMP-Sia, and sialylated recombinant N-glycoproteins without N-acetylmannosamine revealing that GNPE expressing insect cells



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could produce sialylated N-glycoproteins without N-acetylmannosamine supplementation [55]. Tn-4h cell line derived from cabbage looper Trichoplusia ni, a moth in family Noctuidae and DpN1 cell lines derived from monarch butterfly Danaus plexippus (D. plexippus) from family Nymphalidae revealed the activity of sialylating secreted alkaline phosphatase (SEAP) when infected with a baculovirus expression vector. SEAP from SfSWT-1 cell line revealed stronger sialylation as compared to SEAP from Tn-4h.The SfSWT-1 cells were able to sialylate the recombinant protein GST-SfManI [56]. Genetic engineering techniques in insects have enabled the expression of glycosyltransferases and enzymes generating CMP-N-acetylneuraminic acid, required for sialylation and inhibition of N-acetylglucosaminidase in the processing and production of N-glycans [44].

2 Discussion Insects play an important role in human lives from pollinators to forensic flies detecting crimes [57–61]. Sialylation in insects is a relatively new field of biology [14,27,41,62,63]. The sialyltransferases and fucosyltransferases have been known to be regulated by enzymes [58] in mammalian system, however, their regulation in insect physiology and development is not yet well understood. Studies have shown that sialic acid salvage pathway in insect cells and other transgenic insect cell lines can sialylate recombinant glycoproteins in the absence of CMP-Sia [41]. Although stage-specific expression of N-glycans has been now reported from D. melanogaster and B. mori, factors regulating their stage-specific expression and developmental regulation are not known in insects. Studies on the huge diversity of insects and their sialylation pattern or enzymes controlling sialylation are not known. Blast search with D. melanogaster ST (NCBI Genbank id: NP_726474.1) followed by the distance tree of results and the neighbor-joining method revealed homology within members of Drosophila genus and with closely related insects and flies such as mosquito, housefly, spiders, and other species such as Eudicots and lizards [14], indicating its evolutionary relationship. Considerable progress has been made in developing insect expression systems expressing mammal-like proteins and further research is going on across the globe in these lines. It remains to be seen whether sialylation associated with developmental regulation could be exploited in insect control thereby controlling the threat they pose to the human, plant, and animal lives.

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[23] Karaçalı S, Kırmızıgül S, Deveci R, Deveci O, Onat T, Gürcü B. Presence of sialic acid in prothoracic glands of Galleria mellonella (Lepidoptera). Tissue Cell 1997;29:315–21. [24] Geib SM, Calla B, Hall B, Hou S, Manoukis NC. Characterizing the developmental transcriptome of the oriental fruit fly, Bactrocera dorsalis (Diptera:Tephritidae) through comparative genomic analysis with Drosophila melanogaster utilizing mod ENCODE datasets. BMC Genomics 2014;15:942. [25] Malykh YN, Krisch B, Gerardy-Schahn R, Lapina EB, Shaw L, Schauer R. The presence of N-acetylneuraminic acid in Malpighian tubules of larvae of the cicada Philaenus spumarius. Glycoconj J 1999;16:731–9. [26] Marz L, Altmann F, Staudacher E, Kubelka V. Protein glycosylation in insects. In: Montreuil  J, Vliegenthart  JFG, Schachter  H, editors. Glycoproteins. vol. 29a. Amsterdam: Elsevier; 1995. p. 543–63. [27] Marchal I, Jarvis DL, Cacan R,Verbert A. Glycoproteins from insect cells: sialylated or not? Biol Chem 2001;382:151–9. [28] Scott H, Panin VM. The role of protein N-glycosylation in neural transmission. Glycobiology 2014;2014:407–17. [29] Aoki K, Perlman M, Lim JM, Cantu R, Wells L, Tiemeyer M. Dynamic developmental elaboration of N-linked glycan complexity in the Drosophila melanogaster embryo. J Biol Chem 2007;282:9127–42. [30] Koles K, Lim JM, Aoki K, Porterfield M, Tiemeyer M, Wells L, Panin V. Identification of N-glycosylated proteins from the central nervous system of Drosophila melanogaster. Glycobiology 2007;17:1388–403. [31] Repnikova E, Koles K, Nakamura M, Pitts J, Li H, et al. Sialyltransferase regulates nervous system function in Drosophila. J Neurosci 2010;30:6466–76. [32] Koles K, Repnikova E, Pavlova G, Korochkin LI, Panin VM. Sialylation in protostomes: a perspective from Drosophila genetics and biochemistry. Glycoconj J 2009;26:313. [33] Haines  N, Irvine  KD. Functional analysis of Drosophila beta1,4-N-­ acetlygalactosaminyltransferases. Glycobiology 2005;15:335–46. [34] Haines N, Stewart BA. Functional roles for beta1,4-N-­acetlygalactosaminyltransferase-A in Drosophila larval neurons and muscles. Genetics 2007;175:671–9. [35] Nakamura  M, Pandey  D, Vladislav  M. Panin 2012 genetic interactions between Drosophila sialyltransferase and β1,4-N-acetylgalactosaminyltransferase-A genes indicate their involvement in the same pathway. G3 (Bethesda) 2012;2:653–6. [36] Islam  R, Nakamura  M, Scott  H, Repnikova  E, Carnahan  M, Pandey  D, Caster  C, Khan  S, Zimmermann  T, Zoran  MJ, Panin  VM. The role of Drosophila cytidine monophosphate-sialic acid synthetase in the nervous system. J Neurosci 2013;33:12306–15. [37] Granell  AE, Palter  KB, Akan  I, Aich  U, Yarema  KJ, Betenbaugh  MJ, Thornhill  WB, Recio-Pinto E. DmSAS is required for sialic acid biosynthesis in cultured Drosophila third instar larvae CNS neurons. ACS Chem Biol 2011;6:1287–95. [38] Kim  YK, Kim  KR, Kang  DG, Jang  SY, Kim  YH, Cha  HJ. Expression of β-1,4-­ galactosyltransferase and suppression of β-N-acetylglucosaminidase to aid synthesis of complex N-glycans in insect Drosophila S2 cells. J Biotechnol 2011;153:145–52. [39] Angata T, Varki A. Cloning, characterization, and phylogenetic analysis of siglec-9, a new member of the CD33-related group of siglecs. Evidence for co-evolution with sialic acid synthesis pathways. J Biol Chem 2000;275:22127–35. [40] Kim K, Lawrence SM, Park J, Pitts L,Vann WF, Betenbaugh MJ, Palter KB. Expression of a functional Drosophila melanogaster N-acetylneuraminic acid (Neu5Ac) phosphate synthase gene: evidence for endogenous sialic acid biosynthetic ability in insects. Glycobiology 2002;12:73–83. [41] Hollister J, Conradt H, Jarvis DL. Evidence for a sialic acid salvaging pathway in lepidopteran insect cells. Glycobiology 2003;13:487–95.

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[42] Hollister  JR, Jarvis  DL. Engineering lepidopteran insect cells for sialoglycoprotein production by genetic transformation with mammalian b1,4-galactosyltransferase and a2,6-sialyltransferase genes. Glycobiology 2001;11:1–9. [43] Hooker AD, Green NH, Baines AJ, Bull AT, Jenkins N, Strange PG, James DC. Constraints on the transport and glycosylation of recombinant IFN-gamma in Chinese hamster ovary and insect cells. Biotech Bioengr 1999;63:559–72. [44] Tomiya  N, Narang  S, Lee  YC, Betenbaugh  MJ. Comparing N-glycan processing in mammalian cell lines to native and engineered lepidopteran insect cell lines. Glycoconj J 2004;21:343–60. [45] Lawrence SM, Huddleston KA, Pitts LR, Nguyen N, Lee YC,Vann WF, Coleman TA, Betenbaugh MJ. Cloning and expression of the human N-acetylneuraminic acid phosphate synthase gene with 2-keto-3-deoxy-D-glycero-D-galactonononic acid biosynthetic ability. J Biol Chem 2000;275:17869–77. [46] Walski T, De Schutter K, Cappelle K, Van Damme EJM, Smagghe G. Distribution of glycan motifs at the surface of midgut cells in the cotton leafworm (Spodoptera littoralis) demonstrated by lectin binding. Front Physiol 2017;8:1020. [47] Petit  D, Mir  AM, Petit  JM, Thisse  C, Delannoy  P, Oriol  R, Thisse  B, Harduin-­ Lepers A. Molecular phylogeny and functional genomics of beta-galactoside alpha2,6-­ sialyltransferases that explain ubiquitous expression of st6gal1 gene in amniotes. J Biol Chem 2010;285:38399–414. [48] https://pixabay.com/en/silkworm-summer-share-in-mulberry-3576503/. [49] Mabashi-Asazuma H, Shi X, Geisler C, Kuo CW, Khoo KH, Jarvis DL. Impact of a human CMP-sialic acid transporter on recombinant glycoprotein sialylation in glycoengineered insect cells. Glycobiology 2013;23:199–210. [50] Park JH, Wang Z, Jeong HJ, Park HH, Kim BG, Tan WS, Choi SS, Park TH. Enhancement of recombinant human EPO production and glycosylation in serum-free suspension culture of CHO cells through expression and supplementation of 30Kc19. Appl Microbiol Biotechnol 2012;96:671–83. [51] Suganuma  M, Nomura  T, Higa  Y, Kataoka  Y, Funaguma  S, Okazaki  H, Suzuki  T, Fujiyama K, Sezutsu H, Tatematsu KI, Tamura T. N-glycan sialylation in a silkworm-­ baculovirus expression system. J Biosci Bioeng 2018;126(1):9–14. [52] Soya S, Şahar U,Yıkılmaz MS, Karaçalı S. Determination of sialic acids in the nervous system of silkworm (Bombyx mori L.): effects of aging and development. Arch Biol Sci 2017;69:369–78. [53] Viswanathan  K, Tomiya  N, Park  J, Singh  S, Lee  YC, Palter  K, Betenbaugh  MJ. Expression of a functional Drosophila melanogaster CMP-sialic acid synthetase. Differential localization of the Drosophila and human enzymes. J Biol Chem 2006;281:15929–40. [54] Viswanathan K, Narang S, Betenbaugh MJ. Engineering sialic acid synthesis ability in insect cells. Methods Mol Biol 2015;1321:171–8. [55] Geisler C, Jarvis DL. Innovative use of a bacterial enzyme involved in sialic acid degradation to initiate sialic acid biosynthesis in glycoengineered insect cells. Metab Eng 2012;14:642–52. [56] Hillar  A, Jarvis  DL. Re-visiting the endogenous capacity for recombinant glycoprotein sialylation by baculovirus-infected Tn-4h and DpN1 cells. Glycobiology 2010;20:1323–30. [57] Medvedova L, Knopp J, Farkas R. Steroid regulation of terminal protein glycosyltransferase genes: molecular and functional homologies within sialyltransferase and fucosyltransferase families. Endocr Regul 2003;37:203–10. [58] Aslan CE, Liang CT, Galindo B, Hill K,Topete W.The role of honey bees as pollinators in natural areas. Nat Areas J 2016;36:478–88. [59] Hung KJ, Kingston JM, Albrecht M, Holway DA, Kohn JR.The worldwide importance of honey bees as pollinators in natural habitats. Proc Biol Sci 2018;285(1870).



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[60] Chakraborty A, Banerjee D, Ghosh S, Ansar W. Thermophilic pupal endoparasitoids: Brachymeria minuta (Hymenoptera: Chalicididae) on forensic indicator Sarcophaga (Parasarcophaga) albiceps. Prommalia 2015;3. [61] Joseph I, Mathew DG, Sathyan P,Vargheese G. The use of insects in forensic investigations: an overview on the scope of forensic entomology. J Forensic Dent Sci 2011;3: 89–91. [62] Warren  L. The distribution of sialic acids in nature. Comp Biochem Physiol 1963;10:153–71. [63] Tomiya N, Ailor E, Lawrence SM, Betenbaugh MJ, Lee YC. Determination of nucleotides and sugar nucleotides involved in protein glycosylation by high-performance anion-exchange chromatography: sugar nucleotide contents in cultured insect cells and mammalian cells. Anal Biochem 2001;293:129–37.

Further reading [64] https://www.who.int/news-room/fact-sheets/detail/vector-borne-diseases. [65] Acharya S. Own work, CC BY-SA 4.0, file: Drosophila melanogaster Proboscis.Jpg.Wikimedia commons. [66] https://en.wikipedia.org/wiki/Aedes_aegypti. [67] Vancova M, Sterba J, Dupejova J, Simonova Z, Nebesarova J, Novotny MV, Grubhoffer L. Uptake and incorporation of sialic acid by the tick Ixodes ricinus. J Insect Physiol 2012;58:1277–87. [68] Ghosh S, Waliza A, Banerjee D. Diagnosis of crime reporter flies in forensic entology. Indian J Entomol 2018;80:158–76. [69] Schauer R. Sialic acids as link to Japanese scientists. Proc Jpn Acad Ser B Phys Biol Sci 2016;92:109–20.

CHAPTER 4

Sialoglycoconjugates and their role in physiology 1 Introduction Sialic acid has been known to regulate the development, physiology, immunology, and other vital functions of the human body. Sialic acids in the brain in the form of gangliosides and polysialic acid (PSA) has been known to play a role in the development of the nervous system, stability, regeneration, and has been associated with disease due to their aberrant expression [1]. Sialic acids have been reported to affect the functions of the dendritic cells (DCs) and its vital functions of antigen uptake, processing, and presentation [2]. Sialic acids have been reported to be a vital nutrient playing a role in brain development and cognition [3]. Altered sialylation has been associated with affected functions of the ion channel [4]. In this chapter, we discuss the different roles of sialic acid in the different physiological process and developmental processes in the body highlighting its role in nutrition, development, nervous system, ion channels, DCs, reproductive cells, and immune responses.

1.1 Nutrition Human milk with oligosaccharides [5] has been reported to constitute of sialic acid, mostly comprising of sialyllactose and sialic acid conjugated to glycoproteins and glycolipids including monosialoganglioside 3 (GM3) and disialoganglioside 3 (GD3). Human milk contains sialic acid content more than in bovine milk and serves as an important component of nutrition [6,7], with probable roles in the development of the brain and nervous system and cognition. It is known to compete with pathogenic bacteria and inhibits adhesion of bacterial toxins, promotes the growth of bifidobacteria and lactobacilli, in infant intestinal flora and confers protection of infants from infection. Free sialic acid in breast milk has been reported of nutritional value [7]. GM3 in human milk has been reported to increase, while GD3 concentration has been reported to decrease during lactation [6]. The increased expression of sialyltransferase ST6Gal I in mouse and rats m ­ ammary gland Sialic Acids and Sialoglycoconjugates in the Biology of life, Health and Disease https://doi.org/10.1016/B978-0-12-816126-5.00004-4

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is reported to be mediated by 5′UTR exon (L) during lactation, but not detected in the lactating bovine or human mammary gland [7]. Sialic acid found in breast milk forms components of brain gangliosides and PSA on neural cell adhesion molecule (NCAM) which play important role in CNS functions of cell-cell communication, outgrowth of neurons, controlling synaptic connectivity, and formation of memory and sialic acid in milks and plays a role in the development of brain of the infants [8–10]. Ganglioside supplementation in mother’s diet can provide nutrients influencing fetal and infant brain development and function and play a role in the injury of neurons and disease biology [11].

1.2 Reproduction Sialic acid-binding glycoprotein (SABP) can bind to 25 kDa protein on the plasma membrane of human noncapacitated sperm head leading to increase in superoxide anion (O2 − ) production [12] and intracellular Ca2+ level with a probable role in  vitro sperm capacitation and acrosome reaction [13]. Sialoglycoconjugates with varied distribution have been reported from human sperms during capacitation, in postacrosomal region of the sperm head to the tail middle piece, in incapacitated sperms and postacrosomal regions in capacitated sperm indicating their role in capacitation [14]. Sialic acid forms a major constituent of sperm glycocalyx and female reproductive mucosal surface and plays an important role in regulating sperm migration, uterotubal reservoir formation, and oocyte binding and Siglecs including 1, 2, 5, 6, 10, and 14 with roles in binding of sialic acid and cellcell interactions and signaling has been localized in sperms in human and bulls [15]. Urinary tract infections caused by uropathogenic Escherichia coli (UPEC) in mice has been reported to reveal loss of Neu5Ac in epididymal spermatozoa and epithelial cells and reduced sialic acid bound to the surface of spermatozoa in men indicative of glycocalyx remodeling by sialidases during this infection [16]. Galectin-1 binding is reported to be inhibited by α2,6-sialylation in the human corpus luteum (CL) during luteolysis, with increased expression of ST6GAL1 and galectin-3 during luteolysis and loss of progesterone synthesis. Luteotrophic hormones have been reported to differentially regulate galectin-1 and galectin-3/α2,6-sialylation in granulosa lutein cells [17]. Ultrasensitive analysis of the human zona pellucida (ZP) has shown N-glycans with terminal sialyl-Lewis(x) [sLe(x)] sequence, selectin ligand, and they are known to play a role in protein-protein interactions in



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­human-gamete interactions. sLe(x) a ligand for siglec-9, may transmit inhibitory signals and may play a role in human ZP in immune recognition of the egg [18,19]. Alterations in the N-glycan branching and expression of sialic acid on amniotic fluid α-1-acid glycoprotein (AGP) with increased tri- and tetra-antennary sialylated and N-glycan branching and expression of α2,3 linked sialic acid in the third trimester of pregnancy has been reported to contribute to natural innate defense to fetus and mother [18].

1.3  Ion channels Sialylation on voltage-gated potassium or K+ and sodium or Na+ channels and can cause channel deactivation, gating mechanism, conformational changes due to negative charges of sialic acid [4] and can affect electrical signaling in the brain and heart leading to disorder including arrhythmias, epilepsy, and paralysis [20]. Klotho the antiaging protein expressed in secreted and membrane-bound form reveals predominance in brain choroid plexus, kidney, and parathyroid glands in which secreted Klotho reveals sialidase activity and can regulate ion channels removing terminal sialic acids from N-glycan chains of TRPV5 Ca2+ channel and the renal K+ channel ROMK and exposing disaccharide galactose-N-acetylglucosamine, a ligand for galectin-1 which on binding leads to accumulation of the channels on the plasma membrane [21]. Sialic acid residues on glycosylated cardiac Na+ and K+ channel has been studied to play an important role in preventing early after-depolarization events in cardiac arrhythmias [22].

1.4  Nervous system Glycocalyx of cells is composed of glycolipids, glycoproteins, and proteoglycans, and are terminated with sialic acids, playing a role in molecular interactions, cell-cell interactions, and gangliosides and PSA in the brain and nerve cells [1]. The regulatory mechanisms involved in the control of CNS innate immunity include neuroimmune regulatory proteins (NIReg) such as CD95L, CD200, CD47, complement regulatory proteins including CD55, CD46, fH, C3a, HMGB1, and sialic acid [23]. Polysialic acid (PSA) consisting of α2,8-linked sialic acid is synthesized by two complementary sialyltransferases, ST8SiaII and ST8SiaIV. PSA plays a role in development and maintenance, promotes cancer metastasis, tissue regeneration, and repair, and is implicated in psychiatric diseases. PSA reveals antiadhesive property impacting cell adhesion and signaling, can bind to neurotrophins, growth factors, and neurotransmitters. It finds importance in the designing of meningitis vaccine [24]. PSA is involved in

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p­ lasticity-related responses in the adult CNS, including changes in circadian and hormonal patterns, adaptations to pain and stress, and aspects of learning and memory, and thought to improve the repair of adult CNS tissue [25].Variation of human ST8SIA2 gene is linked to schizophrenia (SZ) and other neuropsychiatric disorders. SynCAM 1 and neuropilin-2 have been identified as novel acceptors of PSA in NG2 cells of the perinatal brain and in DCs of the immune system [26]. Dysregulated polysialylation can lead to brain disease and neuropsychiatric disorders [27]. The major carrier of PSA is the NCAM that play a role in the development and plasticity of the vertebrate CNS. NCAMs play role in cell-cell interactions, developmental processes involving cell migration, cell survival, axon guidance, and synaptic targeting, both in the young and adult brain, enabling plastic processes and cognitive abilities [25]. Rapid neural growth in the postnatal period reveals axon fasciculation, cell migration, neurite outgrowth, and synaptic plasticity. Sialic acid as PSA form NCAMs and human milk serves as the source of sialic acid. NCAM carrying the linear homopolymer of alpha 2,8-linked sialic acid PSA (PSA-NCAMs) and neural gangliosides both play critical roles in mediating cell-cell interactions important for neuronal outgrowth, synaptic connectivity, and memory formation [28–39]. NCAM is considered a signaling receptor that responds to both homophilic and heterophilic cues, mediates cell-cell adhesion [28–37], and promotes plasticity in the nervous system and interactions mediated by PSA-NCAM activates signaling cascades and regulates cell shape, growth, and migration [37,38], mediating synaptic functions by interaction with proteoglycans glutamate receptors, restraining activity of GluN2B-containing NMDA receptors and facilitating subset of AMPA receptors activity. Altered polysialylation and/ or NCAM expression has been associated with SZ, depression, anxiety, and Alzheimer’s disease (AD) [3]. PSA-NCAM has been associated with poor clinical prognosis and aggressive and invasive disease in many cancers, including lung cancer, neuroblastoma, and gliomas [40]. NCAM and PSA-NCAM are thought to play role in the neuroprotective response in neurodegeneration by reducing of AMPA/NMDA receptors sensitivity to glutamate and facilitating disconnection of cell-cell interactions, protecting from excitotoxic damage and promote dendritic/spine re-growth and thus PSA-NCAM has been implicated in neurodegenerative diseases and has a therapeutic application [41]. PSA, NCAM, PSA-NCAM play an important role in the remodeling of the synapse, with functions of dynamic cell interactions, axonal growth, terminal sprouting, and target innervation [42]



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and is developmentally regulated, playing the role of neural, neuroglial, and structural plasticity of the embryonic and adult nervous system and neurogenesis, axonal growth, fasciculation, and cell migration [43]. The adult hypothalamo-neurohypophysial system (HNS) neurons and glia are known to express PSA-NCAM that negatively regulates adhesion and is more expressed on HNS neurons and astrocytes surface and tenascin-C glycoprotein with adhesive and repulsive properties [35,36]. Neurotrophins including brain-derived neurotrophic factor (BDNF) in the CNS and play a role in signaling and is involved in cell growth, differentiation, survival and normal brain function, memory consolidation, synaptic plasticity, and adult neurogenesis, and glycan-mediated BDNF release has been reported to play a role in psychiatric disorders [44–47]. Synaptic N-methyl-d-aspartate-receptors (NMDARs) is important in the induction of synaptic plasticity, cell survival, but extrasynaptic NMDARs activation has been associated with inhibition of long-term potentiation and triggering of neurodegeneration. Polysialylated neural cell adhesion molecule (PSA-NCAM) has been reported to regulate extrasynaptic signaling [46] impacting on synaptic plasticity and cognitive functions [46]. Gangliosides, sialylated glycosphingolipids (GSLs), including GM1, GD1a, GD1b, and GT1b play a role in the development and function in the CNS. ST3GAL5 and B4GALNT1, gene codes for enzymes required for brain ganglioside biosynthesis, but mutations lead to an early-onset seizure disorder, with motor and cognitive decay and hereditary spastic paraplegia, intellectual deficits respectively. Gangliosides also affect the aggregation of Aβ (Alzheimer’s disease, AD) and α-synuclein (Parkinson’s disease, PD) in neurodegenerative diseases [48]. Single nucleotide polymorphisms (SNPs) of ST8SIA2/STX, polysialyltransferase genes leading to impaired structure and function of PSA has been associated with psychiatric disorders including SZ, bipolar disorder (BD), and autism spectrum disorder (ASD) as PSA is the regulator for BDNF and dopamine, involved in psychiatric disorders [49]. Guillain-Barré syndrome (GBS) is an autoimmune disorder affecting the peripheral nervous system (PNS) revealing autoantibodies against gangliosides [50]. Gangliosides with O-acetyl sialylations have been reported in neuroectodermal tumors [51]. Microglia are the resident immune cells of the CNS and Siglecs on microglia play role in inhibitory Siglec signaling, leading to neuroprotective effects, preventing the oxidative burst [52]. Triggering of immunoreceptor tyrosine-based activation motif (ITAM), lead to microglial activation, migration, and phagocytosis and immunoreceptor ­tyrosine-based inhibition

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motif or ITIM-signaling receptors, like Siglecs that recognize sialic acids of neurons leading to ITIM signaling downregulating microglial immune responses and phagocytosis. Desialylated neurons are phagocytosed by microglial complement receptor 3 or CR3 signaling through ITAM containing adaptor protein [28]. Siglecs on microglia and oligodendrocytes can recognize the neuronal and glial sialic acid through an ITIM thus enabling cell homeostasis [24]. Microglia express recognition receptors including toll-like (TLRs), Fc, complement, and cytokine receptors. Carbohydrate-binding receptors including the sulfated glycosaminoglycan (SGAG)-binding receptors recognize anionic structural motifs within SGAG chains and Siglecs recognize sialic acid cap of the intact glycocalyx, galectins that recognize lactosamine and selectins play a role in microglial repair function, leading to pro-inflammatory cytotoxic or an antiinflammatory ­repair-promoting responses, that can regulate phagocytosis during neurodegenerative or neuroinflammatory processes [52]. Siglec-4/myelin-associated glycoprotein (MAG) is expressed on oligodendrocytes and Schwann cells and Siglec-4 is known to play a role in conferring protection to neurons from acute toxicity by interacting with sialic acid. Siglec-11 expressed by microglia has been known to prevent neurotoxicity by interaction with α2.8-linked sialic acid exposed on the neuronal glycocalyx. Microglial Siglec-E is a mouse CD33-related Siglec member inhibiting microglial phagocytosis and oxidative burst. Human Siglec-3/CD33 polymorphisms are associated with late-onset AD. Siglec-3 on microglia producing inhibitory signaling decreases uptake of amyloid-β aggregates and is known to play a role in health and disease [24]. Neu5Gc is consistently expressed at negligibly nil to absent in the brains of all vertebrates [53]. GSLs are abundant in the brain and glucosylceramide (GlcCer), forms the source of most GSLs, including sialic acid-containing GSLs (Figs. 1 and 2) [39]. Gangliosides composed of sialic acid-containing GSLs and their expression levels and patterns in the brain are known to vary during development. Gangliosides are predominant in the outer surface of plasma membranes and exist in microdomains with sphingomyelin and cholesterol with a role in cell adhesion, cell-cell recognition, and signal transduction [54]. Altered ganglioside expression leads to neural disorders, like seizures and axon degeneration. Brain gangliosides function, by interacting with a ­ganglioside-binding lectin, MAG on the myelin sheath, enhances axon-­ myelin stability and inhibits axon outgrowth after injury [55]. Dietary gangliosides influences neonatal brain development [56] Gangliosides, function



Sialoglycoconjugates and their role in physiology

O

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OH O

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OH

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O 8

COO–

OH O

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P T P S

(C)

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NCAM-180

Fig. 1  Representative schematic representations of PSA, PSA-NCAM, and PSA-synCAM. (A) α2-8linked PSA where R = NHCOCH3, Neu5Ac; NHCOCH2OH, Neu5Gc, OH, KDN. In the brain, polySia is polyNeu5Ac. (B) Molecular modeling of PSA which is linked to Gal at the C3-position, (C) PSA-NCAM-180 and polySia-synCAM-1 with NCAM (left) with five immunoglobulin domains (IgI~IgV) and two fibronectin type-III (FNIII) domains in its extracellular domain and in the IgV domain, two of the three N-glycosylation sites (triangle) are polysialylated while SynCAM-1 Tright has three Ig domains. In the IgI domain, one of the three N-glycosylation sites (triangle) is occupied by PSA, represented by black circles (sialic acid). (Reproduced with permission from open-access article under Creative Commons Attribution License: Sato C, Kitajima K. Impact of structural aberrancy of polysialic acid and its synthetic enzyme ST8SIA2 in schizophrenia. Front Cell Neurosci 2013;7:61.)

as receptors in cell-cell recognition, regulating natural killer cell cytotoxicity mediated by Siglec-7, myelin-axon interactions mediated by Siglec-4, and inflammation mediated by E-selectin, regulating the responsiveness of signaling proteins including insulin, epidermal growth factor (EGF), and vascular endothelial growth factor (VEGF) receptors, in the immune ­system,

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Antiadhesive effect

Regulation of ion transport

polySia-NCAM

AMPA Ca2+ channels

(A)

Control of signals

Retain or reservoir

Growth factors

(B)

Control of ion transport and signal

Neurotrophins Neurotransmitters

NT-Rs FGF-Rs Control of signals

(C)

Control of signals

Fig. 2  Schematic representation of PSA and functions. (A) Antiadhesive effect by PSANCAM and negatively regulate cell-cell interactions. (B) Regulates ion transport. (C) Acts as reservoir of biologically active molecules like neurotrophins, neurotransmitters, and growth factors. PSA-NCAM can regulate signal modes. (Reproduced with permission from open-access article under Creative Commons Attribution Licence: Sato C, Kitajima K. Impact of structural aberrancy of polysialic acid and its synthetic enzyme ST8SIA2 in schizophrenia. Front Cell Neurosci 2013;7:61.)

in the nervous system, in metabolic regulation, and has been associated with Parkinson's disease (PD) and in cancer progression [56,57]. The sialic acid component in brain gangliosides and the PSA that modify NCAM [3,9]. Storage of gangliosides, sialic acid-containing GSLs, mostly found in the central nervous system (CNS), is a hallmark of neuronopathic forms of the disease, that include GM1 and GM2 gangliosidoses, Gaucher type II and III, and Niemann-Pick C [33]. Gangliosides play a role in cell-cell recognition, adhesion, and signal transduction and are integral components of cell surface microdomains or lipid rafts along with proteins, sphingomyelin, and cholesterol, playing a role in signaling events affecting neural development and the pathogenesis of certain diseases [30]. Disruption of ganglioside synthase genes in mice induces developmental defects and neural degeneration and thus targeting ganglioside metabolism forms a novel therapeutic strategy for intervention



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in some diseases [30]. Gangliosides, with O-acetylated sialic acid, 9-OAc-GD3, is known as an oncofetal marker in animal and human tumors including breast cancer, melanoma, neuronal tumors, and psoriatic lesions (Fig. 3) [51]. Sialic acids removal is catalyzed by a sialidase including NEU1, NEU2, NEU3, and NEU4 that play a role in cellular functions and pathologies [58]. Tagging of sialic acids in biological systems with contrast agents for magnetic resonance imaging (MRI), is enabling functional imaging of glycans and study its molecular basis. Biochemical engineering approaches involving sialic acids by using small molecule metabolic substrates holds promise for the manipulation of sialic acids and offers the potential for the development of novel therapies for neurological disorders [38,59].

1.5  Stem cells The mammalian CNS is organized by a variety of cells such as neurons and glial cells, originating from the neural stem cell (NSC) with the capacity for self-renewal and multipotency and express glycoconjugates and c­arbohydrate antigens in NSCs, with an emphasis on stage-­specific

CER

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CER

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GM1

CER

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GD1a

CER

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GD1b

GT1b

Key Glucose

GalNAc

Galactose

Sialic acid

Fig.  3  Schematic representation of series-a and series-b ganglioside synthesis. (Reproduced with permission from open-access article under Creative Commons Licence (CCBY): Forsayeth J, Hadaczek P. Ganglioside metabolism and Parkinson’s disease. Front Neurosci 2018;12:45.)

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e­mbryonic ­ antigen-1, human natural killer antigen-1, PSA-NCAM, ­prominin-1, gp130, chondroitin sulfate proteoglycans, heparan sulfate proteoglycans, cystatin C, galectin-1, glycolipids, and Notch [60,61]. Stem cell glycans have been reported to play a role in maintaining its potency and differentiation into different lineages [62]. Stem cells reveal expression of glycans and their differentiation is modulated by FGF-2, Wnt, and Notch signaling. Glycan epitopes like stage-specific embryonic antigens (Lewis X/ SSEA-1, SSEA3-4) and tumor-rejection antigens (TRA1-60, 1-81) find importance as markers for lineage-specific cell identification, characterization, and isolation [60,63]. Neu5Gc has been observed on the surface of human embryonic stem cells due to incorporation from animal products used for their culture (Fig. 4) [60]. Umbilical cord blood (UCB) contains normal CD34+ hematopoietic stem cells (HSCs), with enhanced cell surface expression of α2,3-linked sialic acid, P- and E-selectins, and intercellular adhesion molecule and reduced expression of L-selectin, reduced O-acetylated sialoglycoproteins, low activity of sialylate-O-acetyltransferase, and high sialidase activity [64].

PSA-NCAM

Lewis X

= Glc

Tra 1-60 (KSPG) 6S

6S 6S

6S

6S

CD34

NG2 and 473HD (CSPG) 6S

6S

6S

4S

SSEA-3

4S

SSEA-4

= Gal = Man = GlcNAc = GalNAc = GlcA = IdoA = Fuc = Xyl = Sia

Fig. 4  Representative profile of schemes of glycans markers in stem cell identification and purifications, including TRA 1–60, NG2, 473HD, SSEA-3 and SSEA-4, Lewis X, PSANCAM, CD34, GalNAc. (Reproduced with permission from Lanctot PM, Gage FH, Varki AP. The glycans of stem cells. Curr Opin Chem Biol 2007;11:373–80.)



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1.6  Cardiac function and disorders Serum sialic acid has been associated with predicting coronary heart disease, stroke mortality, atherosclerotic process, and severity of coronary lesions. However, differences in serum sialic acid content have been reported from different populations globally from atherosclerotic individuals. While most reports have correlated increased serum sialic acids with atherosclerosis, some reports suggest reduced sialic acid content of platelets, erythrocytes, and lipoproteins in atherosclerosis [65]. Increased total serum sialic acid has been reported from hypothyroid patients as an atherosclerotic risk factor [66]. Oxidative stress-induced desialylation occurs in atherogenic modification, and therefore prevention and control of oxidative stress and/or inflammatory reactions may prevent cardiovascular disease (CVD). Quercetin 7-O-sialic acid (QA) with the cardiovascular protective effect of quercetin and antioxidant and antiatherosclerosis effect of sialic acid has been reported to protect human umbilical vein endothelial cells (HUVEC, EA.hy926) against hydrogen peroxide (H2O2) or oxidized low-density lipoprotein (LDL)-induced oxidative damage by reducing the production of reactive oxygen species (ROS), stopped H2O2-mediated desialylation of HUVEC and lipoproteins, decreased lipopolysaccharide (LPS) induced secretion of tumor necrosis factor-α (TNF-α) and monocyte chemoattractant p­ rotein-1 (MCP-1) and reduced expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), TNF-α and MCP-1 and enabled overall antioxidation, antiinflammation, cholesterol efflux promotion and biomolecule protection against desialylation and therefore could control CVD [67]. Decreased expression of ST6GAL1 in vascular endothelium was reported in the atherosclerosis development process. ST6GAL1 has been reported to inhibit monocyte-transendothelial migration and preventing initiation of atherosclerosis and transendothelial migration of monocytes suggesting the role of ST6GAL1 in atherosclerosis prevention and treatment [68]. Recently studies of metabolomics has enabled identification of sialic acid in acute myocardial infarction, and targeting neuraminidase-1 could be a therapeutic strategy for coronary artery diseases (CAD) [69].

1.7 Obesity Adipose finds importance in obesity and diabetes and CVD. Downregulation of β-galactoside α2,6-sialyltransferase-1 (St6gal1), responsible for α2,6linked sialic acid synthesis was reported from visceral adipose tissues (VATs)

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from obese mice [70]. Targeting GM3 biosynthesis has been studied to counteract obesity-related metabolic disorders [71,72]. Sialic acid has been reported to be indicative of cardiovascular mortality in adults and serum sialic acid has been correlated with body fat percentage (BFP) in obese children [72].

1.8 Complement Deficiency of sialylated glycans by genetic ablation of CMP-sialic acid synthase (CMAS) has led to the death of mice embryo around day 9.5 post coitum (E9.5) and therefore sialic acid is reported to be important for early development of the embryo and plays role in fetal-maternal immune homeostasis during pregnancy and confers protection to allograft implant against attack by the maternal innate immune system [73]. Complement factor H (FH), regulates alternative pathway (AP) of complement revealing glycan binding sites mediating recognition of α2–3-linked sialic acid by domain 20 and glycosaminoglycans by domains 6–8 and 19–20. FH binds the complement C3-activation product C3b [74].

1.9  Dendritic cells Although glycans have been reported to play important roles in immunity, the detailed mechanism of glycans regulating overall immune response is not known in details. Modified sialic acid is known to affect physiological function, cellular adhesiveness, and cell trafficking, role through selectins and the siglecs. DCs enable antigen, uptake, processing and its presentation to lymphocytes, triggering the adaptive immune response. Sialic acid-­modified structures are involved in all DC functions, including antigen uptake, DC migration, and priming of T cell responses. Sialic acid content changes may play a role in DC functions by altering antigen endocytosis, pathogen and tumor cell recognition, cell recruitment, and capacity for T cell priming. DC surface sialylation holds, therefore, the promise of improvising the DCbased therapies [2].

1.10 Others Sialoglycoconjugates are known to play a role in aging of red blood cells (RBCs) [75]. Defective sialylation has been associated with impaired podocyte maturation [76]. Renal cells sialoglycoconjugates determine the nucleation of calcium oxalate dihydrate (COD), the most common crystal in human urine leading to kidney stone formation [77].



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2 Discussion We have highlighted the role of sialic acid in the nutrition, physiology, and development of the body [1–81]. While sialic acid has a profound role in neuronal development and generation of cognition skills, proper function of ion channels, proper function of the DCs of the immune system, deficiency or malformation may lead to diseases, thus proving the importance of sialic acid in physiological processes of the body. They also find importance as markers for stem cells and development and cell differentiation into different lineages. However, there are a large number of interesting questions in this field of biology. Although the importance of sialic acid in immune regulation is being reported the downstream pathways of silica regulation of immune pathways are not completely known. The regulation of differentiation of stem cells and role played by sialic acid in signaling such pathways are yet not well understood. It would, therefore, remain important if studies are made in this domain in the years to come.

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CHAPTER 5

Pathogens, infectious disease biology and sialic acid 1 Introduction In bacteria, Neu5Ac and polysialic acids (PSAs) as homopolymers of Neu5Ac find importance as virulence factors [1]. Cytidine 5­′-monophosphate (CMP)-Neu5Ac synthetase (CSS) that synthesizes CMP-Neu5Ac occurs in both eukaryotes and prokaryotes like pathogenic bacterial species such as Neisseria meningitides (N. meningitides), Escherichia coli (E. coli), group B streptococci (GBS), Haemophilus ducreyi (H. ducreyi), and Pasteurella haemolytica (P. haemolytica) and an enzyme from nonpathogenic bacteria, Clostridium thermocellum (C. thermocellum) is reported. The presence of sialic acids predominates on vertebrate cells and occasionally is mimicked by bacterial pathogens using homologous biosynthetic pathways and has been thought to have emerged by convergent evolution or horizontal gene transfer in pathogens [2]. GBS colonizing human respiratory tract has been revealed to contain polysaccharide with terminal sialic acid in an α2,3-linkage in capsules [3]. Gram-positive and Gram-negative bacteria reveal similar enzyme properties of pH, temperature, divalent cation requirement, and catalysis with enzymes revealing acetylhydrolase and sugar nucleotidyltransferase activity [2–4]. Host factors can determine growth and influence factors for mucosal colonization, penetration, interference, and host-derived cytidine 5′-monophospho-N-acetyl neuraminic acid and lactate has been reported to influence gonococci, meningococci, and Haemophilus influenzae [5] pathogenesis. Bacterial pathogen enables them to colonize mucous surfaces, penetrate, grow in the environment of the host, inhibit or avoid host defenses, and damage the host, and are determinants of pathogenicity, bacterial activities [4]. Sialylation of the lipopolysaccharide (LPS) of gonococci in vivo cytidine 5′-mono-phospho-N-acetyl neuraminic acid from host and lactate from host affect pathogenicity [5, 6] of parasite. Meningococci bind to factor H by factor H-binding protein (FHbp) that enables it to evade complement-mediated killing of parasite and finds importance as candidate as meningococcal vaccine [7]. Concerns of usage of PSA, the capsule of Sialic Acids and Sialoglycoconjugates in the Biology of life, Health and Disease https://doi.org/10.1016/B978-0-12-816126-5.00005-6

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Group B meningococcus (GBM) and E. coli K1, as vaccine candidates exists as they are component of mammalian glycopeptides and could induce immunopathology, but is being tested in clinical trials as conjugate vaccines [4–7]. In this chapter, we study the role of sialic acid in different organisms such as bacteria, protozoa, and virus

2 Bacteria Different pathogenic organisms are known to synthesize or acquire sialic acids from the host. A few Gram-negative bacteria like H. influenzae, Pasteurella multocida, and H. ducreyi derive sialic acid from their medium of growth whereas GBS, a Gram-positive bacteria and Gram-negative bacteria such as E. coli K1, N. meningitides, and Campylobacter jejuni (C. jejuni) can synthesize sialic acids [8–21]. Genotyping of C jejuni strain containing sialic acid-positive lipo-oligosaccharide or LOS (class A, B, or C) has been reported to be associated more frequently with patients revealing bloody diarrhea and prolonged disease symptoms [22, 23]. Sialidases produced by some host microbiota can promote growth of Clostridium difficile, Salmonella, and E. coli that lack endogenous production of sialidases [24]. O-acetyl ester modifications of sialic acids protects against sialidase action. Gut bacteria can also produce sialylate-O-acetylesterases to remove them thus playing a major role in infection. [24]. Mimicry of sialic acid enables some pathogenic bacteria to evade host defenses. Fusobacterium nucleatum (F. nucleatum) NeuB putative Neu5Ac synthase can synthesize Neu5Ac from N-acetylmannosamine and phosphoenolpyruvate in vitro and it encodes a functional CMP-sialic acid synthetase [25]. Due to molecular mimicry, C jejuni LOS can induce a cross-reactive antibody response to nerve gangliosides, which leads to Guillian Barre Syndrome (GBS) [26]. Glycan interactions play a major role in infection biology, colonization, and disease progression. Gram-negative N. meningitides, causing meningococcal sepsis and meningitis, and Neisseria gonorrhoeae (N. gonorrhoeae), causing sexually transmitted infection gonorrhea, leading to morbidity and mortality globally. N. meningitidis displays capsule polysaccharide, LOS and O-linked glycoproteins but N. gonorrhoeae lacks a capsule and can scavenge host sialic acids and few N. meningitidis serogroups can synthesize sialic acid [27]. Cell surface expressed sialic acid can confer serum resistance and survival in the host. Neisseria protein adhesins such as Opc and NHBA on binding to host glycans causes adherence and colonization of host cells [27, 28].



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Complement activation is tightly controlled by inhibitors, which are activated by invading pathogens. N gonorrhoeae and N. meningitidis, expressing glycans, including Neu5Ac, mimic host structures to evade host immunity [4]. Neu5Ac inhibits complement, by enhancing binding of the complement inhibitor factor H (FH) through C-terminal ­domains [19, 20] on FH. A point mutation in FH domain 19 is reported to ­prevent hemolysis caused by unmodified FH18–20, but retained binding to gonococci [4]. Both N gonorrhoeae and N. meningitidis bind to FH which enhances their ability to evade complement-dependent killing. Porin, the ligand for h ­ uman FH on gonococci and meningococci uses a lipoprotein, FHbp to bind to FH and enhances its ability to evade ­complement-dependent killing and finds importance as meningococcal vaccine candidate antigen [7]. N. meningitidis LOSs reveals 12 immunotypes, heterogeneity in antigenic structures detected by monoclonal antibody that recognizes lacto-N-neotetraose [LNnT, (Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc)] ­ sequence and lectin, Maackia amurensis leukoagglutinin (MAL), specific for NeuNAcalpha2-3Galbeta1-4GlcNAc. N. meningitidis LOSs mimic glycosphingolipids, such as paragloboside (LNnT-ceramide) and sialyl paragloboside, and some host glycoproteins having LNnT and N-acetyllactosamine sequences with or without α2–3-linked sialic acid that might play a significant role in N. meningitides pathogenesis [29]. The molecular mimicry of host structures by LOS or LPS is reported from N. gonorrhoeae, H. ducreyi, H. influenzae, Moraxella catarrhalis (M. catarrhalis), C jejuni, and Helicobacter pylori (H. pylori) [29]. Polysaccharides and glycolipid membrane expressed by Neisseria downregulate complement activation thereby play a significant role in evading the host immune response [6, 30]. Human factor H (HuFH), a key inhibitor of the alternative pathway of complement, binds to N. gonorrhoeae and contributes to complement evasion [31] of human immune system. Immunochemical studies of the LOS of the N. gonorrhoeae and N. meningitidis have revealed their role in pathogenesis. Sialylation of lactoneoseries LOS on N gonorrhoeae when grown in the presence of cytidine ­5′-monophospho-N-acetyl-neuraminic acid (CMP-NANA) [32] suggested their role in infection. Mammalian cell surfaces reveal sialic acid conjugated glycoconjugates which can be used by the commensal and pathogenic bacteria, but require a functional sialic acid transporter to import the sugar into the cell and uptake

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and utilization of sialic acid. The sodium sialic acid symporter (SiaT) from Staphylococcus aureus (S. aureus SiaT) has been revealed to rescue an endogenous sialic acid transporter lacking E. coli strain in the presence of sialic acids, Neu5Ac, or Neu5Gc in growth media. Strategies to develop new inhibitors targeting these transporters could inhibit bacterial growth [33]. Sugar molecules on both microbial and mammalian cells play a major role in cellular communication, govern microbial virulence, and modulate host immunity and inflammatory responses. N-glycosylation has been reported in all three domains, with the most complex diverse forms from Archaea but no expression of nonulosonic acids (NulOs), nine‑carbon sugars such as sialic acids, pseudaminic acids, and legionaminic acids although they are reported from Eukarya and Bacteria (Fig. 1) [34]. N-linked glycan of the S-layer glycoprotein of the haloarchaea Halorubrum sp. has been reported. PV6 includes an N-formylated legionaminic acid. Halorubrum sp. PV6 genome reveals sequences predicted for legionaminic acid biosynthesis pathway and transcription of pathway genes together with the activities of LegI, catalyzing condensation of 2,4-di-N-acetyl-6-deoxymannose and phosphoenolpyruvate to generate legionaminic acid, and LegF, catalyzing the addition of cytidine monophosphate (CMP) to legionaminic acid, both heterologously expressed in Haloferax volcanii and the genes-encoding enzymes of the legionaminic acid biosynthetic pathway are clustered together with sequences-encoding components of the N-glycosylation pathway [35]. FH and properdin interact with glycosaminoglycans (GAGs) and sialic acid, regulating complement cascade [36]. C3 and C3b, complement components, bind covalently to bacterial surfaces. Sialylated LOS of both N. meningitidis and N. gonorrhoeae show limited C3 deposition [37]. Phylogenomic analyses of microbial biosynthetic pathways for sialic acids reveal higher sugars derived from 5,7-diamino-3,5,7,9-­ tetradeoxynon-2-ulosonic acids and that NulO types were expressed by various organisms and both sialic acid and related pseudaminic acid expression reveal that bacterial pathogens express sialic acid due to adaptation of legionaminic acid biosynthesis [38]. Infection of Pseudomonas aeruginosa (P. aeruginosa) reveals a significant role of sialic acids and its incorporation with decreased complement deposition on the bacteria, contributing to bacterial pathogenicity and hostcell interactions by reduced complement deposition and Siglec-dependent recognition [39]. The sialic acid-Siglec interaction enabled innate immune escape in P. aeruginosa [39, 40] and sialic acids on bacteria and their role in host recognition through human Siglecs have been reported. P. aeruginosa on



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The core nonulsonic acid biosynthetic (NAB) pathway NAB-2 6C hexosamine

UDPGlcNAc

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Fig. 1  Representative picture of synthesis of nonulosonic acids (NulO). (Reproduced with permission from Lewis AL, Desa N, Hansen EE, et al. Innovations in host and microbial sialic acid biosynthesis revealed by phylogenomic prediction of nonulosonic acid structure. Proc Natl Acad Sci U S A. 2009;106 (32):13552–7.)

interaction with human Siglec-9 on neutrophils reduce innate immune responses [39–41]. Neu5Ac, Neu5Gc, and O-acetylated form (Neu5,9Ac2) are reported on P. aeruginosa surface [31, 39, 40]. P. aeruginosa expressed surface α2,6-linked Neu5,9Ac2 was reported through binding studies with a lectin, Achatinin-H having preferential affinity toward Neu5, 9Ac2α2, 6GalNAc

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sialoglycotope [21], and 9-O-acetylated sialic acids were reported from intact bacteria and its membrane fraction [39, 40]. P. aeruginosa, biosynthetic pathway for sialic acid synthesis is absent and is probably acquired from environment [39, 40]. Sialic acids acquisition by pathogens is associated with subverting the host immunity by acting as a molecular mimic [34, 42]. Sialic acids content of type III GBS strains also inhibits C5a production [43]. P.  aeruginosa adsorbed sialic acids from host serum (PA+Sias) and has been reported to lower anti-C3 binding as compared to P. aeruginosa cultured in sialic acids-free medium (PA-Sias) indicating a direct relationship between the sialic acids levels and C3 deposition on P. aeruginosa with a probable role in escaping the host serum [39]. Adsorbed sialic acids in parasites from the host serum are reported to inhibit C3 deposition [39] and associates with immune cells and Siglec-dependent recognition. P. aeruginosa associates with neutrophils by sialic acids-Siglec-9 interaction producing less ROS and elastase by neutrophils and with reduced neutrophils extracellular traps (NETs) formation [39, 40] impairing the neutrophil-mediated killing and reducing the host innate immune response. Neutrophils express more Siglec-9 and Siglec-5 on its surface. PA+Sias revealed more binding with neutrophils and sialidase-treated neutrophil as compared to that of PA-Sias [39, 40]. Blocking Siglec-9 with anti-Siglec-9 antibody on neutrophils can reduce binding of PA+Sias indicating a major role of Siglec-9, while antibody against Siglec-5treated neutrophils revealed no such decrease in binding highlighting the interaction through sialic acids-Siglec-9 in infection [39, 44]. Noninvasive enteric bacterium Vibrio cholerae (V. cholera) causes cholera only in humans with neuraminidase (VcN) and the AB5 cholera toxin (Ctx) as the virulence factors. Release of host intestinal epithelial sialic acids by VcN enables synthesis of GM1 monosialoganglioside to which B subunit of Ctx binds, delivering CtxA subunit into host epithelia, triggering fluid loss by cAMP-mediated activation of anion secretion and inhibition of electroneutral NaCl absorption. Loss of Neu5Gc and excess Neu5Ac adds to pathogenesis [45].

3  Parasitic protozoa Parasitic diseases such as Kala azar [visceral leishmaniasis (VL)], Chagas disease human (American trypanosomiasis), and African sleeping sickness (African trypanosomiasis) affect people worldwide and mostly considered as important neglected diseases with no vaccination, and treatment is based on chemotherapeutic drugs, and recently novel small molecule-based therapeutic approaches are being tested. Drugs currently used for the ­



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treatment of Kala azar include pentavalent antimonials, Amphotericin B, Miltefosine, and Paromomycin. Liposomal formulations such as AmBisome find importance in the treatment of Kala azar. In Chagas disease benznidazole and nifurtimox-based chemotherapeutic approaches are used and proteases, sterols, and sialic acids are potential promising drug targets. Suramin, pentamidine, melarsoprol, and eflornithine are well-established drugs to treat African sleeping sickness. Combination therapy of eflornithine and nifurtimox in Chagas disease is being tested [46]. Leishmania donovani (L. donovani), causes VL containing cell surface glycoconjugates that play an important role in parasite virulence and infectivity. Sialoglycans and their acquisition and biological roles of ­anti-O-acetylated sialic acid (anti-OAcSA) antibodies have been reported to influence in this disease [20]. Enhanced expression of 9-O-acetylated sialoglycoproteins on erythrocytes in VL [47–52] activates an alternative complement pathway-­ mediated hemolysis leading to anemia [47]. Glycosylated bacterial ABCtype phosphate transporter has been isolated from the PBMC of VL patients and 9-O-acetylated sialoglycoproteins (9-O-AcSGPs) of molecular weight 19, 56, and 65 kDa have been associated with the disease [50]. 9-O-AcSA has been reported from a virulent (UR6) and virulent (AG83 and GE1) promastigotes of L donovani with UR6 revealing lower infectivity and phagocytic index than AG83, indicating that 9-O-AcSA on promastigotes of virulent L donovani enables parasite entry in macrophages [50, 53]. Sialic acids are acquired by parasite and act as a virulent factor reducing immune responses thereby enabling an establishment of infection [22, 39, 53]. L. donovani promastigotes and amastigotes contain α2,6- and α2,3-linked sialic acids such as Neu5Ac, Neu5Gc, Neu5,9Ac2 on their cell surface [22, 53]. Leishmania tropica (L. tropica K27), Leishmania major (L. major, JISH118), and Leishmania mexicana (L. mexicana, LV4) causing cutaneous, Leishmania braziliensis (L. braziliensis L280) and Leishmania amazonensis (L. amazonensis, LV81) causing diffuse, and Leishmania infantum (L. infantum, MON29) causing infections reveal differential distribution of sialic acid despite similar pathogenesis. K27, JISH118, L280, and MON29 reveal high sialic acid content with enhanced 9-O-acetyl sialic acid (9-O-AcSA) whereas LV4 and LV81 expressed reduced sialic acid. 9-O-AcSA excess promastigotes revealed significant viability as compared to their de-O-acetylated forms after exposure to NaNO2 suggesting the involvement of 9-O-AcSA in conferring nitric oxide (NO) resistance [22]. Chagas disease or American Trypanosomiasis, a parasitic infection borne by triatomine bugs, affects millions of people in Latin America. Trypanosoma,

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a major genus of kinetoplastida, possessing highly sialylated and surface glycosylphosphatidylinositol (GPI)-anchored dense mucin layer [54]. Trypanosoma cruzi (T. cruzi), causing Chagas disease lacks sialic acids synthesis machinery and trans-sialidase (Tc-TS, Fig. 2) enzyme facilitates the transfer of glycosidically bound sialic acids from serum sialoglycoconjugates by cleaving the sialic acids from the glycoconjugates on the host cell and transfers it to the terminal β-galactopyranosyl residues of mucin-like molecules on parasite cell surface [55, 56] and can sialylate/re-sialylate glycoconjugates expressed on the surface of host cells. Tc-TS lacks any analogue in the vertebrate host. The enzyme also helps in escape of the immune response and enables the establishment of persistent chronic infection [57, 58].

Fig.  2  Representative profile of the TcTS/sialoglycoproteins system on T. cruzi trypomastigotes. (A) Scanning electron micrograph (SEM) of a bloodstream form T. cruzi trypomastigote (www.fiocruz.br/chagas_eng/cgi/cgilua.exe/sys/start.htm?sid=13) (B) GPI-anchored TcTS. Sialic acid present on T. cruzi sialoglycoproteins (C) is transferred from host sialoglycoconjugates to parasites (D) by TcTS activity. (E) TcTS transfers sialic acid residues from Siaα2–3Galβ1-R containing donors and attaches them in α2–3 linkage to terminal β-Galp containing acceptors but without carbohydrate acceptor, Tc-TS irreversibly transfers sialic acid to a water molecule. (Reproduced with permission from Freire-de-Lima L, Oliveira IA, Neves JL, Penha LL, Alisson-Silva F, Dias WB, and Todeschini AR Sialic acid: a sweet swing between mammalian host and Trypanosoma cruzi Front. Immunol., November 29, 2012.)



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Current chemotherapy based on the nitroaromatic compounds, benznidazole and nifurtimox suffers from side effects and low efficacy [59] and new strategies for disease targeting are under research.

4 Virus Human respiratory paramyxoviruses, parainfluenza virus type 3 (HPIV3), causing common respiratory diseases both in infants and a subset of adults, recognize sialic acid-containing receptors on host cells. Human influenza viruses (IAVs) reveals preferences for α2,6-linked sialic acid, whereas HPIV3 reveals preferences for α2,3-linked sialic acids. Other human paramyxoviruses are able to recognize the capsular sialic acid of GBS. GBS attaches to transfected BHK cells expressing the HN protein of mumps virus (MuV) on their surface by sialic acid-dependent way. This emphasizes coinfection synergism by respiratory pathogens, by α2,3-linked sialic acid recognition with increased bacterial adherence to airway cells contributing to disease severity [60]. IAV infections remain an important cause of morbidity and mortality and challenges of recurrence of pandemic influenza remains. Strategies of vaccination with the inactivated, intramuscular influenza vaccine have reduced morbidity and mortality associated with influenza infection. Live, attenuated influenza vaccines administered intranasally have been studied in clinical trials with stronger mucosal and cell-mediated immune responses and effective for inducing protective immunity in children or the elderly are being developed. Two major classes of antivirals include adamantanes and the neuraminidase inhibitors [61]. The putative receptor for the IAV-binding protein, hemagglutinin (HA), contains the sialic acid residues which play a significant role in the infection process [62]. Crystal structures of neutralizing and protective antibodies complexed with H7 HA could recognize overlapping residues surrounding the receptor-binding site of HA [63]. L4A-14 antibody were reported to bind into sialic acid-binding site and made contacts with HA residues that were conserved in the great majority of 2016–17 H7N9 isolates [61]. The HA proteins of influenza A and B (IAV and IBV), or the hemagglutinin-esterase-fusion (HEF) proteins of ICV, are known to bind sialic acid, causing endocytosis [62]. Human IAV reveals preferences to α-2,6-linked sialic acid-containing receptors, and mutations are reported to alter binding preference, virus infectivity, and host tropism. Human influenza A (H3N2) viruses carbohydrate-binding patterns revealed variation over time. Viruses causing major outbreak in 1999 and 2003 revealed binding preferences to both α-2,3 and α-2,6 sialylated

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glycans. Twenty amino acid substitutions reported from antigenic sites reveal changes in glycan-binding patterns and might contribute to H3N2 virus evolution [64]. Newcastle disease virus (NDV), belonging to the Paramyxoviridae family, causing infectious disease in birds. Hemagglutinin neuraminidase glycoprotein (HN) plays a role in infection and its inhibition is a target for the development of new drugs based on sialic acid glycals, with the 2-deoxy-2,3-didehydro-d-N-acetylneuraminic acid (Neu5Ac2en) [63]. Enterovirus D68 (EV-D68) can cause severe respiratory disease and genome-wide haploid screening revealed genes involved in sialic acid biosynthesis, transport, and conjugation associated with infection [65]. Group A rotaviruses causes diarrhea in many mammalian species. A porcine intestinal GM(3) ganglioside receptor has been reported to be essential for sialic acid-dependent rotavirus recognition of host cells [66].

5  Siglec sialic acid and infection biology Host pathogen interacting molecules often include cell membrane glycoproteins or glycolipids. Siglecs on immune cells act as ligands for sialic acids on several pathogenic bacteria. Cis interaction of Siglecs in a cell undergoes changes to trans interaction due to cellular activation or vicinity with the sialylated pathogens. Membrane sialidase activity and change in glycosylation pattern or plasma membrane reorganization can regulate dynamic cis-trans interactions of Siglecs[39]. In resting B cell, inhibitory receptor Siglec 2 (CD22) revealed masked condition while activated condition reveals unmasking [67–71]. Cis interaction of Siglecs is known to modulate cellular functions by inhibiting the nonspecific interaction of Siglecs [72–74]. NK cell inhibitory receptor, Siglec-7 with specificity toward Neu5Acα2,8Neu5Ac reveals negligible cytotoxicity on target cell due to masking effect in cis interaction, sialidase treated NK cells unmasks Siglec-7 from cis interactions thereby upregulating cytotoxic activity [72–74]. Porcine reproductive and respiratory syndrome virus (PRRSV) uses envelope sialic acids for infecting pig alveolar macrophages through Siglec-1 [75]. N. meningitidis, C. jejuni, GBS, and T. cruzi exhibit sialic acid-mediated interactions with Siglec-1 and CD33-related Siglecs on host cell [76–78]. Sialylated capsular polysaccharide (CPS) of various GBS serotypes are known to recognize Siglec-5, -7, -9 on human leukocytes during infections [21, 77]. Sialylated glycan of GBS serotype III and neutrophil Siglec-9 interaction subverts the host innate immune response [76, 77]. Sialylated LOS on C. jejuni and Siglec-1 interaction is important in Guillain-Barre’ syndrome (GBS) [78, 79].



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C. jejuni and Siglec-7 interacts and modulates inflammatory and immune responses of host [78, 79]. Neutrophils undergo “respiratory burst” during infection and ingestion of microbes leading to increased oxygen consumption [80] and reactive oxygen species or ROS is generated in the phagosome of neutrophil combat the invading pathogens. PA+Sias through sialic acids-Siglec-9 neutrophil produces ROS indicating impairment of host’s first line of defense [39, 40]. Reduced or absence of oxidative burst of neutrophil is associated with chronic granulomatous disease (CGD) patients infected with pathogens such as Staphylococcus sp., Salmonella, Aspergillus sp., and Candida species [81–83]. Siglec-9 interacts with sialic acids on PA+Sias leading to reduced release of antimicrobial agent elastase from neutrophil [39, 84]. Elastase can degrade toxins of Shigella, Salmonella, and Yersinia and can kill them in its phagosomes [85]. Sialic acid-siglec interaction can be blocked by either specific antibody or removal of sialic acids leads to release of elastase and enabling its antimicrobial function [39]. Outer membrane protein A of E. coli, Klebsiella pneumoniae, and P. aeruginosa strain (H103) degrades neutrophil elastase during infection. Ecotin, a serine protease inhibitor of E. coli shows a protective activity against neutrophil elastase [86, 87]. Neutrophils play a dominant role in mediating inflammation and bacterial defense by three processes such as phagocytosis, degranulation, and neutrophil extracellular traps or NETs formation [81]. NETs include extracellularly released chromatin material, serine proteases, and cytoplasmic proteins. Serine proteases in neutrophils include elastase, cathepsin G, azurocidin, and proteinase 3 that play an essential role in acute infections and inflammation. Both Gram-negative and Gram-positive bacteria and their virulent proteins are degraded extracellularly by NETs [84, 85]. PA+Sias shows resistance toward NETs-mediated killing by neutrophils. Gram positive S. aureus, Shigella flexneri, Streptococcus pyogenes, Bacillus anthracis, Mycobacterium tuberculosis, Candida albicans, and L. amazonensis undergo lysis by NETs formation [39, 88–90]. Siglec-E, in mouse on dendritic cells, macrophages and neutrophils contribute to defense against T. cruzi [39]. T. cruzi pathogenic strains exhibits enhanced binding with Siglec-E-Fc chimera compared to nonpathogenic strain. T. cruzi expresses a modified structure of sulfated glycan, a ligand for Siglec-E [39, 90–92]. Sialic acid-Siglec-1 interaction is observed in T. cruzi interaction with tissue macrophages [91]. In this infection dendritic cells are known to reduce MHC (major histocompatibility complex) class-I

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antigen presentation in vitro [93] and produce interleukin (IL)-12 [93] and Siglec-1 may play a role in phagocytosis of T. cruzi by macrophages and immunoregulation by ITIM-bearing CD33-related Siglecs [90–93].

6 Prions Neuroinflammation is a hallmark feature associated with pathogenic infection in neurodegenerative diseases such as Alzheimer’s, Parkinson’s, or prion diseases. In prion diseases, pathogenic prion protein or PrPSc can lead to microglial-mediated neuroinflammatory responses and is affected by nature of terminal carbohydrate groups like sialic acids on the surface of PrPSc particles (Fig. 3). Partial cleavage of sialic acid residues by neuraminidase treatment could boost the inflammatory response of microglia to PrPSc [94]. PrPSc with reduced sialylation did not cause prion disease indicative of the fact that sialylation of the prions is associated with prion infection [24] prion replication barrier, affected prion structure, animal-to-human transmission, prion lymphotropism, toxicity, strain interference, function of PrP(C), and affected etiology [95–99]. The transmissible spongiform encephalopathies, is a prion disease, associated with aggregation of disease-related isoforms of the prion protein

Fig. 3  Illustration of sialylation control of PrPSc. (A) The sialylation of PrPC is controlled by STsi. (B) NEUs do not appear to affect the steady-state sialylation level of PrPC. (C) Sialoglycoforms of PrPC are recruited into PrPSc selectively by their sialylation status and is strain specific. (D) PrPSc is a subject of post-conversion sialylation by STs. Sias are represented in red diamonds. (Reproduced with permission from Baskakov IV and Katorcha E Multifaceted Role of Sialylation in Prion Diseases, Front. Neurosci., August 08, 2016.)



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(PrP(Sc)) or the cross-linkage of PrP(C), involving sialic acid-containing GPI anchor clustering leading to altered membrane composition, activation of cPLA(2), and synapse damage [80, 98, 99]. PrP(Sc) a proteinaceous infectious agent includes misfolded and aggregated form of a sialoglycoprotein or PrP(C) with two sialylated N-linked carbohydrates [99]. The terminal sialic acid residues in PrP(Sc) create a negative charge on the prion surface leading to electrostatic repulsion between sialic residues affecting the structure and thereby creating a prion replication barrier controlling replication rate and glycoform ratios [98].

7 Discussion Parasites adopt several methods to escape the host immune response and establish an infection. Sialic acids have been observed to play a dominant role in the infection process and are finding applications as promising candidates to targeting of paratistic diseases. T cruzi uses trans-sialidase (TcTS) to transfer sialic acid from glycoconjugates of host to parasite mucins [100]. Membrane HA of IAV type A is known to recognize sialylglycoconjugate receptors on the host cell surface during infection process, and HA inhibitors are promising candidates for novel antiviral drugs [101]. In C jejuni-induced GBS, molecular mimicry between C. jejuni LOS and host gangliosides leads to the production of cross-reactive antibodies attacking nerves of the host [78]. Sialic acid reveal to be virulence factors in otitis media pathogenesis due to H. influenzae. [102]. Disease specific overexpression of 9-O-acetylated sialoglycoproteins (9-O-AcSGPs) on PBMC of VL patients as compared to healthy individuals has been demonstrated using a lectin, Achatinin-H [49]. Sialic acids have been reported to enhance pneumococcal biofilm formation in vitro, providing insights into treatment strategies [10]. Avian pathogen (M synovia) revealed sialidase activity suggesting an important role in virulence of the organism [103]. Pathogenic avian H5N1 strain and escalating human infections by the virus is globally alarming. A sialidase fusion construct, DAS181, has been able to cleave sialic acid receptors used by both human and avian IAVs [104]. Sialidase activity and serum total sialic acid and haptoglobin has been reported to be associated with complement activation and development of hypocomplementemia in postinfectious acute glomerulonephritis (AGN) patients [105]. The biology of pathogen, infectious disease, and host parasite interaction is a complex process. With the applications of omics-based approaches,

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more and more details of the parasite host biology and infection are being brought to light. The role of sialic acid in pathogen, host, and infection biology is rather a new and developing domain with insights into infection pathophysiology and possible avenues and candidates for disease targeting.

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[18] Pearce  OM, Varki  A. Chemo-enzymatic synthesis of the carbohydrate antigen ­N-glycolylneuraminic acid from glucose. Carbohydr Res 2010;345:1225–9. [19] Chava AK, Chatterjee M, Sharma V, Sundar S, Mandal C.Variable degree of alternative complement pathway-mediated hemolysis in Indian visceral leishmaniasis induced by differential expression of 9-O-acetylated sialoglycans. J Infect Dis 2004;189:1257–64. [20] Mukhopadhyay S, Mandal C. Glycobiology of Leishmania donovani. Indian J Med Res 2006;123:203–20. [21] Chava AK, Bandyopadhyay S, Chatterjee M, Mandal C. Sialoglycans in protozoal diseases: their detection, modes of acquisition and emerging biological roles. Glycoconj J 2004;20:199–206. [22] Ghoshal  A, Gerwig  GJ, Kamerling  JP, Mandal  C. Sialic acids in different Leishmania sp., its correlation with nitric oxide resistance and host responses. Glycobiology 2010;20:553–66. [23] Mortensen  NP, Kuijf  ML, Ang  CW, Schiellerup  P, Krogfelt  KA, Jacobs  BC, van Belkum  A, Endtz  HP, Bergman  MP. Sialylation of campylobacter jejuni lipo-­ oligosaccharides is associated with severe gastro-enteritis and reactive arthritis. Microbes Infect 2009 Oct;11(12):988–94. [24] Robinson LS, Lewis WG, Lewis AL. The sialate O-acetylesterase EstA from gut Bacteroidetes species enables sialidase-mediated cross-species foraging of 9-O-acetylated sialoglycans. J Biol Chem 2017;292:11861–72. [25] Lewis  AL, Robinson  LS, Agarwal  K, Lewis  WG. Discovery and characterization of de novo sialic acid biosynthesis in the phylum fusobacterium. Glycobiology 2016;26:1107–19. [26] Yu RK, Usuki S, Ariga T. Ganglioside molecular mimicry and its pathological roles in Guillain-Barré syndrome and related diseases. Infect Immun 2006;74(12):6517–27. [27] Mubaiwa TD, Semchenko EA, Hartley-Tassell LE, Day CJ, Jennings MP, Seib KL. The sweet side of the pathogenic Neisseria: the role of glycan interactions in colonisation and disease. Pathog Dis 2017; 75(5): ftx063. https://doi.org/10.1093/femspd/ ftx063. [28] De Vries FP, Cole R, Dankert J, Frosch M, Van Putten JP. Neisseria meningitidis producing the Opc adhesin binds epithelial cell proteoglycan receptors. Mol Microbiol 1998;27:1203–12. [29] Tsai CM. Molecular mimicry of host structures by lipooligosaccharides of Neisseria meningitidis: Characterization of sialylated and nonsialylated lacto-N-neotetraose (Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc) structures in lipooligosaccharides using monoclonal antibodies and specific lectins. Adv Exp Med Biol 2001;491:525–42. [30] Jarvis GA. Recognition and control of neisserial infection by antibody and complement. Trends Microbiol 1995;3:198–201. [31] Shaughnessy  J, Ram  S, Bhattacharjee  A, Pedrosa  J, Tran  C, Horvath  G, Monks  B, Visintin A, Jokiranta TS, Rice PA. Molecular characterization of the interaction between sialylated Neisseria gonorrhoeae and factor H. J Biol Chem 2011;286:22235–42. [32] Mandrell  RE, Apicella  MA. Lipo-oligosaccharides (LOS) of mucosal pathogens: molecular mimicry and host-modification of LOS. Immunobiology 1993;187: 382–402. [33] North Rachel A., Wahlgren Weixiao Y., Remus Daniela M., Scalise Mariafrancesca, Kessans Sarah A., Dunevall Elin, Claesson Elin, Soares da Costa Tatiana P., Perugini Matthew A., Ramaswamy S., Allison Jane R., Indiveri Cesare, Friemann Rosmarie, Dobson Renwick C. J.The sodium sialic acid symporter from Staphylococcus aureus has altered substrate specificity, Front Chem 6 2018, 233 [34] Vimr ER, Kalivoda KA, Deszo EL, Steenbergen SM. Diversity of microbial sialic acid metabolism. Microbiol Mol Biol Rev 2004;68:132–53. [35] Zaretsky M, Roine E, Eichler J. Sialic acid-like sugars in archaea: Legionaminic acid biosynthesis in the halophile Halorubrum sp. PV6. Front Microbiol 2018;9:2133.

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Further reading [106] Freire-de-Lima L, Oliveira IA, Neves JL, Penha LL, Alisson-Silva F, Dias WB, Todeschini AR. Sialic acid: a sweet swing between mammalian host and Trypanosoma cruzi. Front Immunol 2012;3:356.

CHAPTER 6

Sialic acids in autoimmune disorders 1 Introduction Autoimmune disease occurs due to the attack of the immune system on the hosts own cells due to the complete breakdown of immunologic tolerance. The origin and cause of autoimmune disorders (AD) have been known to be associated with genetical, infectious agents, and/or environmental predisposing factors, together with other triggering factors such as viral, bacterial, or chemical factors (Fig.  1) leading to triggering of self-reactivity culminating in tissue destruction. It was almost 50 years back that the pioneering studies of Macfarlane Burnett and his hypothesis of the ‘forbidden clone’ that won the Nobel Prize that the importance of autoimmunity, lymphoid cell development, thymic education, apoptosis and deletion of autoreactive cells was recognized globally as factors that control the immune physiology in autoimmunity [1] (Figs. 1 and 2). Both genetic and environmental factors are known to contribute to AD. An altered pathophysiology in AD include both organ-specific disorders in which antibodies and T cells react to self-antigens expressed by the localized tissue, as in thyroid (Hashimoto thyroiditis), stomach (pernicious anemia and autoimmune atrophic gastritis), adrenal glands (Addison disease), and pancreas (type I or IDDM) and systemic disorders characterized by reactivity of immune cells against a specific antigen/antigens throughout the body/systemically including rheumatoid arthritis (RA) affecting the joints, systemic lupus erythematosus (SLE) a chronic inflammatory disease revealing autoantibody response to nuclear and cytoplasmic antigens, scleroderma a chronic disease affecting the connective tissue disease, multiple sclerosis (MS) [2] affecting the central nervous system (CNS) including the brain and the spinal cord and idiopathic inflammatory myopathies (IIM) affecting the muscles [3–6]. Most autoimmune diseases reveal hallmark features of autoantibodies in circulation and immune complex deposition, antibody-mediated Sialic Acids and Sialoglycoconjugates in the Biology of life, Health and Disease https://doi.org/10.1016/B978-0-12-816126-5.00006-8

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Genetic predisposition like HLA, cytokine

Environmental factors like infection, pathogen

Trauma, injury

Autoreactive T cells Defective regulation

Immune system

Activation and proliferation of autoreactive T cell clones

Fig.  1  Genetic susceptibility, environmental triggers, injury, and defective regulation of the immune system and breakdown of tolerance lead to triggers in autoimmunity. Genetic polymorphisms in HLA, cytokines/receptors, involved in central tolerance, environmental factors, like infection and injury and defective regulation of immune system creating a microenvironment with suppressed or nonfunctional T regulatory cells are factors that enable activation and proliferation of autoreactive lymphocytes.

Fig. 2  T-cell anergy, deletion and suppression and autoimmunity.



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­ psonization and cell lysis by complement, altered cytokine production, o neurohormones, and stress contributing to the disease initiation and pathogenesis [7, 8]. While ThI cytokines including IL-2 and IFNγ are known to predominate organ-specific diseases, with cell-mediated immune responses (CMI) like killing by cytotoxic T cells (CTLs) by cytokines or by IgG and IgM antibodies targeting cell-surface antigens and leading to Fc ­receptor-mediated cell lysis, systemic AD reveal elevated levels of Th2 cytokines including IL-4, -5, and -10 [9]. Despite the complex contribution of both genetic and environmental factors in the development of SLE, single-gene defects, in components of the complement cascade, 3′-5′ DNA exonuclease TREX1 and single-­nucleotide polymorphisms (SNPs) in genes encoding integrin αM (ITGAM), IgG Fc receptors, sialic acid O-acetyl esterase (SIAE), the catalytic subunit of protein phosphatase PP2A (PPP2CA) and signaling lymphocytic activation molecule (SLAM) family members [10] have been reported to lead to SLE indicating that sialic acid plays an important role in SLE [10].

2  Autoimmunity and tolerance Studies from experimental immunology has suggested three mechanisms by which self-reactive lymphocytes are prevented from responding to self-molecules (Fig.  2) therefore contributing to tolerance, including (i) clonal deletion, by which autoreactive lymphocyte are removed, (ii) clonal anergy, that eliminates autoreactive lymphocytes by downregulation of responsiveness, and (iii) suppression or inhibition of autoreactive lymphocytes by interaction with CTLs or NK cells by releasing cytokines with negative regulatory effects, neutralizing specific antigens and inhibiting B-cell function through cross-linking of surface Ig or Fc receptors, or by production of antiautoantibodies or antiidiotypes that can recognize the antigen receptor on autoreactive B lymphocytes [9], thereby dampening the immune system and preventing inflammation reactions. Breakdown of tolerance leads to the origin of autoimmunity. From studies in animal models, T lymphocytes are found to be the key cells in the initiation and maintenance of both spontaneous and chemical-induced AD [9, 11–15].

2.1  T cells T cells originate from the hematopoietic stem cells (HSC) in the bone marrow and mature and develop in the thymus. Initially, the progenitor T cells do not express CD4/8, but the TCR gene undergoes

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r­ earrangement and cells proliferating very fast, develop into CD4+CD8+ double-positive (DP) cells. In the thymic cortical region, these DP T cells encounter epithelial cells expressing MHC class I and II molecules and those cells that fail to recognize self-MHC undergo apoptosis but those T cells that can bind to self-MHC molecules undergo positive selection, survive, and differentiate into single-positive (SP) cells. CD4+ cells can bind to MHC Class II and CD8+ cells, can bind to MHC class I molecule. These cells then undergo migration into the thymic medulla wherein the SP cells are exposed to autoantigens in the presence of self-MHC molecules and cells that bind self-antigens with high affinity undergo negative selection and undergo death. Autoreactive T cells that exhibit low affinity to self-antigens not present in the thymus during normal development undergo anergy or are suppressed in the peripheral lymphoid tissues (Fig. 2). The tolerance in B cells could be therefore due to the lack of T cell help for self-reactive B cells due to successful T-cell tolerance [16].

2.2  B cells B-cell tolerance is mediated by receptor editing, clonal deletion, and anergy mediated by signaling downstream of the BCR by self-antigens [17]. A distinct mechanism of B-cell tolerance involves the repression of antigen receptor signaling, by Lyn Src-family kinase, the SHP-1 tyrosine phosphatase, inhibitory members of the Siglec family, and negative regulatory enzyme called sialic acid acetylesterase and its genetic variants that regulates negatively BCR activation [17, 18] (Fig. 3). BCR signaling on B-1 cells is controlled by several inhibitory receptors, including Siglec-G, that inhibits B-cell signaling by binding to BCR by interaction with sialic acid ligands [19]. T-cell-independent type-2 (TI-2) antigens (Ags) give rise to innate immune responses and B-cell-restricted Siglecs, CD22 and Siglec-G, might contribute to B-cell tolerance to self TI-2 Ags [20]. When B-cells encounter self-antigens, in bone marrow, that cross-link surface Ig receptors with high avidity, their surface Ig is downregulated and undergo clonal deletion by apoptosis. However, some self-reactive B cells at the periphery can encounter soluble antigen that is not capable of cross-linking surface Ig receptors, therefore causing clonal anergy by downregulation of surface IgM (2). However, some pathogens reveal molecular



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Ca+ Siglec-2

CRAC

BCR

P Ca+

PLCy2

BLNK/ SLP65

P BTK

ER

(A) Cross-linking occurs between Siglec-2 and BCR

Siglec-2

BCR

Ca+

SHP-1

P Lyn

ER

(B) Fig.  3  Signal transduction on Siglec 2-BCR interaction. BCR signaling is downregulated by cross-linking of Siglec-2. (A) BCR becomes phosphorylated and activated due to PLCy2, BLNK/SLP65, and BTK signaling molecules, leading to efflux of calcium ions (Ca2+) from ER that enters the cell by the calcium release activated channel (CRAC). (B) After BCR and Siglec-2 cross-linking, Src kinase, Lyn phosphorylates ITIMs of Siglec-2 thereby recruiting of SHP-1 tyrosine phosphatize that binds to two phosphorylated ITIMs. Depletion of intracellular Ca2+, reduces BCR signaling. (Adapted with permission from Eakin AJ, Bustard MJ, McGeough CM, Ahmed T, Bjourson AJ, Gibson DS.Siglec-1 and -2 as potential biomarkers in autoimmune disease. Proteomics Clin Appl 2016;10(6):635–44.)

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mimicry expressing molecules resembling self-antigens of the host and Ig genes under somatic mutation during clonal expansion leading to the generation of self-reactive clones leading to initiation of AD.

2.3  Sialic acid While inhibitory signaling is instrumental in conferring tolerance, loss of inhibitory signaling in the immune system can lead to autoimmune disease. Sialic acid-recognizing Ig superfamily lectins or Siglecs expressed in hematopoietic cells, function as inhibitory receptors which on binding to sialic acid containing ligands can recruit SH2-domain-containing tyrosine phosphatases, leading to the generation of inhibitory signals conferring tolerance. The inhibitory functions of CD22/Siglec-2 and Siglec-G also find importance in tolerance and autoimmunity. We discuss in this chapter (i) role of sialic acids, (ii) antibodies, (iii) complements, and (iv) siglecs in autoimmunity in the context of sialic acid (Fig. 4). Siglec-1

V-set lg-like domain

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Fig.  4  Different siglecs. Reproduced from Siglecs: A journey through the evolution of sialic acid-binding immunoglobulin-type lectins. (Reproduced with permission from Bornhöfft KF, Tom Goldammer T, Rebl A, Galuska SP. Dev Comp Immunol 2018;86:219–31.)



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3  Autoimmunity, tolerance, and sialic acid Neu5Ac or commonly referred to as sialic acid in humans contributes as terminal sugars of N-glycans, O-glycans, and gangliosides, and at terminal points of side chains of glycophosphatidylinositol (GPI) anchors. An ­α-glycosidic bond links the sialic acid at C-2 position with the galactose at C-3 or C-6 position or preterminal N-acetylgalactosamine residues at the C-6 position. In positions lying internal in glycans, one sialic acid is linked to another sialic acid at the C-8 position mediated by specific sialyltransferase enzymes using CMP-sialic acids as high-energy donors. Thus diverse sialic acid linkages to the underlying glycan sugars confer further diversity to the sialic acids and lectins reveal preferential binding for a particular sialic acid with a specific linkage.While sialyltransferases add sialic acid to glycans, neuraminidases, or sialidases remove sialic acids from glycans and both the enzymes control the sialic acid biology in health and disease (Fig. 5). Neu5Ac and Neu5Gc differs by a single 5-position oxygen atom incorporated by cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) [21] and forms the commonly occurring forms of sialic acid in animals. While most mammals express Neu5Gc, humans lack Neu5Gc due to exon deletion in the CMAH gene acquired through evolution [22]. Therefore human pathogens and commensals display Neu5Ac on their surfaces, and their receptors reveal preferences for Neu5Ac instead of Neu5Gc. Siglecs reveal the fastest evolving genes in humans revealing several gene CMAH CMP-Neu5Ac

CMP-Neu5Gc CMP-Neu5Ac Synthase Neu 5Ac

O O HO O

OH COOH OH H O O ~ R SIAE N HO

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a2,3-Gal a2,6-GalNAc a2,8-Gal

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Fig. 5  Sialic acid modifying enzymes. Sialic acids are conjugated to cytidine monophosphate (CMP) by CMP-5-N-acetylneuraminic acid synthetase. Sialyltransferases in the mammalian genome enable the transfer of sialic acid, specific for a particular linkage. Sialidases cleave sialic acids from glycans. Sialic acid acetylesterase (SIAE) cleaves 9-Oacetyl group from sialic acid bearing glycans.

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conversions and pseudogenization with altered Siglec expression profiles in human hematopoietic cells, to adapt to their changing ligands [21–25]. Sialic acids are frequently present in the host and pathogen interaction interfaces, comprising the glycocalyx, occur in mucin secreted by epithelial cells and play role in cell–cell interactions and cell signaling, inhibitory self-signal to the immune system and are known as self-associated molecular patterns, or SAMPs [26, 27].While most microbes lack the expression of sialylated glycans on their surfaces, these terminal sugars or SAMPS are expressed in vertebrates confer a tolerogenic signal in mammals [26]. SAMPS like the pathogen-­associated molecular patterns (PAMPs) and damage associated molecular patterns (DAMPs) are recently reported to recognize signatures of interacting molecules and contribute to autoimmunity and innate immunity respectively [28]. Sialic acids as part of glycan motif are specifically recognized by inhibitory Siglecs as receptors, either in “cis” or in “trans” configuration on innate immune cells. Sialic acid is known to maintain inhibitory signaling by preventing inflammation reaction. However, unlike DAMPs or PAMPs that function by binding to pathogen recognition receptors (PRRs) thereby activating innate immune responses and dendritic cell priming to activate T cells. However, some Siglecs can act as activating receptors (Fig. 6B).

Trans Interaction

Cis Interaction

Fig. 6  Cis and Trans interaction. (Reproduced with permission from Lübbers J, Rodríguez E, van Kooyk Y. Modulation of Immune Tolerance via Siglec-Sialic Acid Interactions. Front Immunol. 2018;9:2807.)



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Some microbial pathogens acquire sialic acid from the host or synthesize them de novo and display sialic acids on their outer surfaces, mimicking the hosts, a property attributed to convergent evolution and thus avoid immune detection [29, 30] and responses. These sialic acids on microbial pathogens can lead to the generation of autoantibodies against host sialic acids [31] (Fig. 7). While hemagglutinins of certain viruses like influenza types A and B can recognize terminal sialic acid on host glycans, those of influenza C virus, recognize 9-O-acetyl sialic acid (9-O-Ac-SA) and thus sialic acid structure and its modification can modulate the immune responses [29, 32–34]. 9-OAc-SA through modulation of Siglec function is associated with autoimmunity [32–36]. Siglecs on immune cells, bind sialic acids [32–34, 36] by sites in their Ig domains, and consist of a transmembrane and a cytosolic domain containing signaling motifs comprising of immunoreceptor tyrosine-based inhibitory motifs (ITIMs) or immunoreceptor tyrosine-based activating motifs (ITAMS) on some siglecs, and play role in cell signaling and adhesion. ITIM-containing Siglecs play a role as inhibitory receptors, thereby conferring tolerance and in mouse models and their loss of function has been associated with autoimmunity. Inhibitory Siglecs include CD22 (or Siglec-2) and Siglec-G expressed on B cells while their ligands on T cells are known to play a role in preventing B-cell-mediated autoimmunity. However, the biology of sialic acid is far more complex contributed by different modifications of sialic acid containing ligands thereby altering their binding specificities generating more than one glycoforms acting as ligands for any given Siglec adding to the diversity and complex biology, of modulation of sialic acid-siglec interaction.

Fig. 7  Self-associated molecular patterns (SAMPs) inhibit but DAMPs and PAMPs activate innate immune receptors and signals.

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O-acetyl groups are added to sialic acids by different acetyltransferases including O-acetylation of α2–8-linked sialic acids by sialic ­acid-specific O-acetyltransferase capsule structure 1 domain containing 1 or CASD1 and removed by sialic acid acetylesterases like SIAE (­sialic acid acetyl esterase [37] that removes 9-O-acetylation modifications from sialic acids. 9-O-acetylation of sialic acid prevents Siglec binding. Siglec-1, -2, and -3 can bind to ligands containing 9-O-acetylated sialic acid. 2–6 linked sialic acid-containing glycoproteins can bind to and activate Siglec-2, both in cis- and trans-configurations. Siglec-2 activation leads to inhibition of BCR activation and dampens the immune response. Crosslinking of Siglec-2 leads to phosphorylation and activation of BCR due to PLCy2, BLNK/SLP65, and BTK signaling molecules, leading to increased calcium ions (Ca2+) from the endoplasmic reticulum (ER) influxed into the cell by calcium release activated channel (CRAC). BCR and Siglec-2 cross-linking leads to phosphorylation of ITIMs of Siglec-2 by Src kinase, Lyn, leading to the recruitment of SHP-1 tyrosine phosphatase that binds to phosphorylated ITIMs. With reduced intracellular Ca2+, BCR signaling to be reduced (Fig. 8). Defective SIAE function, lead to increased 9-O-acetylated sialic acid, leading to reduced Siglec-2 suppression of BCR activation, resulting in increased autoreactive BCR signaling and immunogenicity. SIAE, therefore is reported to regulate B-cell development and play role in conferring peripheral tolerance as studied from rare variants of SIAE mutants and patients with lupus and RA. Mutant SIAE and its pathway have been therefore associated with AD.

4  Infectious pathogens, sialic acids, and autoimmune diseases Several pathogens have evolved strategies like mimicking host cell antigen thereby escaping host immune responses. Neisseria meningitides (N. meningitides) and Hemophilus influenzae (H. influenza) strains can express sialylated lipopolysaccharides [LPS [2, 38]] which can influence susceptibility to bactericidal antibody, decrease or prevent phagocytosis, downregulate complement activation, decrease adherence to neutrophils and oxidative burst response [39]. Nontypeable Hemophilus influenzae (NTHi) has been reported to incorporate host sialic acid into its surface conferring



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SIAE

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Removal of 9-O-acetyl group from sialic acid by SIAE cis binding and activation of siglec-2

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BCR activation Defective SIAE function SIAE inability of siglec-2 to bind 9-O-acetylated sialic acid

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Siglec-2

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BCR activation

Fig. 8  The effect of normal and defective SIAE function on Siglec-2 regulation of BCR activation. (A) demonstrates normal sialic acid acetylesterase (SIAE) function. SIAE is responsible for the removal of 9-O-acetyl groups from sialic acid molecules in sialylated glycoconjugates. Glycoproteins containing sialic acid in α2–6 linkage are capable of binding to and activating Siglec-2, both in cis-, as demonstrated, and trans-interactions. The activation of Siglec-2 inhibits BCR activation and subsequent immune response. (B) demonstrates defective SIAE function, resulting in increased 9-O-acetylated sialic acid. Siglec-1, -2, and -3 are incapable of binding ligands containing 9-O-acetylated sialic acid. A defective SIAE system will, therefore, result in reduced Siglec-2 suppression of BCR activation. This can result in increased autoreactive BCR signaling and immunogenicity. (Adapted and modified with permission from Eakin AJ, Bustard MJ, McGeough CM, Ahmed T, Bjourson AJ, Gibson DS.Siglec-1 and -2 as potential biomarkers in autoimmune disease. Proteomics Clin Appl 2016;10(6):635–44.)

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its ­protection against host adaptive and innate immune responses and enables its sustenance in biofilms. This has also led to the evolution of human antibodies to nonhuman sialic acid antigens like sialylated lipooligosaccharide (LOS) mimicking human antigenic sugars of glycosphingolipids, enables their camouflague and escape from host immune response leading to inflammation and autoimmune disease [39, 40]. Some pathogens have evolved mechanisms to evade the immune system by mimicking host cells by incorporating sialic acids onto their cell surface. Non-vertebrate pathogens or commensal bacteria lack sialic acid and have evolved strategies of molecular mimicry to gather sialic acid from the host and/or synthesize sialic acids [29]. Mannose and sialic acid play important roles in host-pathogen interaction in the Gram-negative bacteria Campylobacter jejuni (C. jejuni). N- and O-linked glycoproteins, expressed on capsule polysaccharide and/or LOS contributes to molecular mimicry of human gangliosides and carbohydrates including [Galbeta1-3GalNAcbeta1-4(NeuAcalpha2–3) Galbeta1-] and has been associated with autoimmune neuropathies like Guillain-Barre syndrome (GBS) [41–43]. C. jejuni requires specific genes to enable sialic acid biosynthesis or transfer [41–43] for such mimicry. Cross-reactive antibodies between C. jejuni LPS and GM1 and GD1 gangliosides has also been reported to contribute to GBS pathogenesis [44]. C. jejuni is known to bind to innate immune receptors signaling through MyD88, NLRP3 inflammasome, TIR-domain-containing adapter-inducing interferon-β (TRIF), Siglecs, macrophage galactose-type lectin (MGL), and Immunoglobulin (Ig)-like receptors (TREM2, LMIR5/CD300b) [41] thereby playing role in innate immune activation. Siglec-1 and Siglec-7 are known to interact directly with gangliosides and Siglec-1 activation has been known to enhance phagocytosis and inflammatory responses [45].

5  Immunoglobulins, sialic acids, and autoimmune diseases IgG molecules playing role in establishment and maintenance of immune homeostasis [46] can function as potent pro-inflammatory mediators promoting effector functions toward infected cells, tumor cells, or healthy tissues in AD. Sugar moieties attached to each IgG constant heavy chain regions fragment, including one conserved N-glycosylation site at Asn 297 is revealed to influence the structure and is critical for its immunomodulatory, pro- and anti-inflammatory functions of IgG [46, 47]. The biantennary core glycan structure may comprise of four N-acetyl-glucosamine



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(GlcNAc) and three mannose (Man) residues, including fucose, a bisecting GlcNAc and terminal galactose (Gal) or galactose with sialic acid adding to the complexity in the glycan architecture of the IgG molecule [48, 49]. While non-galactosylated or agalactosylated IgG antibodies have been associated with pro-inflammatory effects in patients with autoimmune RA, sialylated IgGs used in intravenous injection (IVIG) obtained from purified IgG from pooled human plasma, used as an intravenous injection to treat of autoimmune patients reveal anti-inflammatory effects [49–51]. Alteration in serum IgG glycosylation, sialylation, and altered sialic acid residues has been reported to modulate anti-inflammatory activity, affecting the molecular and cellular pathways [52]. Sialylated Fc IgGs has been reported to reduce FcγR binding and activate anti-inflammatory cascade through the lectin receptor SIGN-R1 or DC-SIGN which leads to upregulated surface expression of the inhibitory FcR, FcγRIIb, on inflammatory cells, thereby attenuating autoantibody-­ initiated inflammation [52, 53] and is a possible mechanism of suppression of autoimmune disease by IVIG [52, 53]. Therefore, antigen-specific galactosylated and sialylated IgGs holds promises as a therapeutic tool for reestablishing tolerance against self-antigens in autoimmune disease or allergy [54]. Patients with IgA nephropathy (IgAN) reveal circulating IgA1 antibodies with deficient/reduced galactose (Gal) on O-linked glycan chains in the hinge region (HR) of their heavy (H) chains and increased exposure of N-acetylgalactosamine (GalNAc) and sialic acid of O-linked glycans recognized by naturally occurring GalNAc-specific antibodies. Thus, in IgAN, immune complexes comprise of Gal-deficient IgA1 molecules as an antigen, and GalNAc-specific IgG and/or IgA1 as an antibody [55] are observed. These aberrant IgA1 glycosylated antibodies are known to fix complement and their expression on mesangial cells can regulate integrin expression, enhance nitric oxide synthase (NOS) activity, decrease endothelial growth factor synthesis, reducing proliferation and increasing apoptosis and may modulate clinical expression and progression of IgAN [56]. Autoantibody responses to endometrial and serum antigens like ­alpha2-Heremans Schmidt glycoprotein and carbonic anhydrase are observed in endometriosis [57]. Depleting carbohydrate moieties from these antigens has been reported to cause loss of antibody binding confirming the involvement of this glycotope in the autoantibody responses with the involvement of at least one sialic acid in the autoantibody binding and that the glycotope involved may be a sialylated T antigen [57, 58].

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6 Complements The factor H (FH) protein is a complement regulatory inhibitory protein of the complement alternative pathway with sites for binding complement component C3b, polyanions like heparan sulfate glycosaminoglycans and sialic acids. It accelerates the decay of C3b on host cell surfaces, and defective FH causes chronic inflammation and tissue injury, leading to various disorders of the kidney or the eye [59]. Rare inherited mutations in the C-terminal domains of Factor H involved in sialic acid recognition, or autoantibodies against factor H that interfere with its complement regulatory function, can lead to hemolytic uremic syndrome or age-related macular degeneration [60]. Both monomeric IgG and IgG immune complexes have been reported to diminish Fc receptor and complement-dependent inflammatory processes [61].

7  Siglecs, HMGB-1, CD24, and autoimmunity Siglecs I-type lectins of the immunoglobulin superfamily are expressed in hematopoietic cells, with antibody-like one V (variable) like domain and one or more C2 (constant) like domains that can discriminate between the self and non-self, and regulate the cell functions in innate and adaptive immune, infectious diseases, inflammation, neurodegeneration, autoimmune diseases, and cancer [62, 63]. CD22 or Siglec 2 and Siglec-G are two B-cell-expressed members of the Siglec that inhibit B-cell signaling through BCR that bind to sialic acid-containing ligands and recruit SH2-domain-containing tyrosine phosphatases to their cytoplasmic tails and deliver inhibitory signals that constrain immune cells, conferring protection to the host from autoimmunity and thus CD22/Siglec-2 and Siglec-G contributes to tolerance [26, 62, 63]. (a) In CD 22, the inhibitory signaling is mediated by ITIMs. CD22 is an inhibitory coreceptor of the BCR, first expressed in the cytoplasm of pro-B and pre-B cells, and maximally expressed on mature B cells [64, 65] and again reveals to be downregulated in plasma cells by Blimp-1 [66, 67]. It is able to modulate B-cell signaling dependent on its proximity to the BCR, governed by its binding to α2,6-linked sialic acid ligands as terminal motifs of soluble and cell surface associated N-glycans.With BCR cross-linking, the CD22 ITIM tyrosines undergo phosphorylation by the Src-family tyrosine protein kinase Lyn. Tyrosine phosphatase SHP-1, a negative regulator of B-cell signaling is recruited and thus regulation of BCR signaling through CD22 modulates peripheral B-cell tolerance. Lack of these inhibitory signals could lead to systemic autoimmunity by B cells [68]. Recently a more complex role for CD22 has emerged,



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including a central role in a novel regulatory loop controlling the CD19/ CD21-Src-family protein tyrosine kinase (PTK) amplification pathway that regulates normal B-cell signaling and intensifies Src-family kinase activation after BCR ligation, thus playing an important role in central regulation of peripheral B-cell homeostasis and survival, promoting BCR-induced cell cycle progression, and potentially regulating CD40 signaling. Alterations in these tightly controlled regulatory activities may influence autoimmune disease [69, 70]. Manipulation of CD22 ligand binding has profound effects on B-cell signaling, and this receptor might play role in mediating autoimmune disease therefore indicating the relation of genetic predisposition in modifying CD22 function and predisposition to autoimmunity [70, 71]. CD22 is a target protein different disorder including B-cell leukemias and lymphomas, and B-cell-mediated autoimmune diseases and finds importance in designing therapeutic strategies in AD and cancer [72]. Immunomodulatory activity of epratuzumab, anti CD22 antibody is finding application in the treatment of SLE [73]. Both antibodies and synthetic chemically modified sialic acids are currently tested to target Siglecs on B cells. (b) Siglec-G is another inhibitory Siglec, expressed on B cells; can recognize α2,3- and α2,6-linked sialic acids and its cis-ligands modulate its ability to inhibit BCR signaling. Its human homolog, Siglec-10, reveal a preference for α2,6-linked sialic acids [35, 74, 75]. CD24 functions as a cis ligand of Siglec-G on dendritic cells and CD24/Siglec-G complex can act as a sensor of endogenous DAMPs. Although specific glycoprotein ligands of Siglec-G on B cells are not well defined, deletion of Siglec-G has been reported to lead to autoimmunity [62]. Siglec-G and CD 24 expressed on some dendritic cell where CD24 GPI-linked sialoglycoprotein cells can impact on central tolerance mechanisms. The absence of either Siglec-G or its cis ligand CD24 has been reported to affect the negative regulation of the response to the danger signal [62, 76]. HMGB1 is an alarmin released by necrotic cells, DCs, and macrophages under pro-inflammatory cytokine TNFα [77] and is upregulated in serum in several autoimmune conditions, promoting inflammation. HMGB1 can bind to CD24, RAGE, thrombospondin, and CXCL12, interact indirectly with TLRs [78]. HMGB1 and Siglec-G are known to function as the signal transducer for endogenous danger signals (alarmins), HSP70 and HSP90, in a ­ CD24-dependent manner thereby dampening inflammation [26]. Siglec-G can inhibit DC responses to PAMPs and DAMPs and

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d­ isruption of the Siglec-G/CD24 complex by microbial sialidases, has led to sepsis [76]. Sialidase inhibitors, therefore, that can preserve the Siglec-G/CD24 interaction may have therapeutic potential in sepsis management [79]. (c) While the cis-ligand-binding activity of CD22 leads to homo-oligomer formation Siglec-G is recruited by Sialic acid binding to the BCR and Siglec-G—mIgM interaction leads to an inhibitory function for B-1 cells and thereby control B-cell tolerance [71].Therefore the loss of both CD22 and Siglec-G, its ligands or its inhibitory pathways can increase the susceptibility for autoimmune diseases, causing spontaneous autoimmunity in mice [80] and mutations in enzymes modifying Siglec ligands are associated with several autoimmune diseases in humans [26]. (d) Human Siglecs 15 have been known for their role in immune regulation and different Siglecs are emerging as potential targets in the diseases including inflammatory, autoimmune, allergic, neurodegenerative, and infectious diseases [36]. This finds importance in circumventing conventional modes of treatment for autoimmune diseases by anti-­ inflammatory and immunosuppressive drugs, to reduce symptoms of the disease to improve quality of life for patients suffering side effects [81]. Murine Siglec-F and human Siglec-8 expressed on eosinophils, and therefore targeting Siglec-8 with an antibody can initiate apoptosis of eosinophils and finds application in targeting allergic asthma [82, 83]. (e) Sialoadhesin (Sn, or Siglec-1 and CD169), a member of the Siglec family, conserved across mammals that is expressed on macrophages in lymph node and spleen and has been known to play role in sialylated pathogen uptake, antigen presentation, and lymphocyte proliferation and to influence both immunity and tolerance. Sn-positive macrophages have been reported in different pathological conditions, including AD, inflammatory infiltrates, and tumors [84]. Siglec-1 and Siglec-2 finds importance as biomarkers and as potential therapeutic targets in AD [85] (Fig. 9). (f) Siglec-1 interacts with Tregs. Siglec-1 positive macrophages have been reported to negatively regulate Tregs as Siglec-1 knock out (KO) macrophages have been reported to trigger Treg production as compared to Wild type macrophages. On the contrary, a decreased number of Siglec-1 positive macrophages leads to increased Tregs leading to lowered disease activity in animal models. Siglec-1 and Tregs interaction is thought to be through the sialylated glycoprotein T-cell surface receptor, CD43, a counter receptor for Siglec-1 [87].



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CD4+ Tregs inhibitsCD4+ Teff proliferation and proinflammatory response

(A)

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Adds Sialic acid in α 2-3 linkage

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(B) Fig.  9  The inhibitory role of siglec-1 on CD4+ Treg cells, the subsequent increase in CD4+ Teff cell proliferation, and enhanced inflammatory response. (A) demonstrates the inhibitory role CD4+ Tregs on CD4+ Teff proliferation and subsequent pro-­ inflammatory response. (B) Kidder et  al. demonstrated an increase in β-galactoside α-2,3-­sialyltransferase (ST3Gal) mRNA expression and α2–3 sialylation of CD4+ Treg cells in a murine model of SLE. (C) Kidder et al. also demonstrated siglec-1 positive macrophages induced the death of CD4+ Treg cells, expressing sialic acid in the α2–3 linkage, by an unknown mechanism. The subsequent reduction in Treg numbers resulted in an increase in Teffs and enhanced inflammation [86].

8  Neu5Gc and chronic inflammation Humans lost CMAH in the course of evolution during divergence from the ancestors the great apes [88] and cannot synthesize Neu5Gc but are exposed to this sialic acid, through diet as Neu5Gc, is abundant in red meat, can be incorporated into tissues [89, 90]. Siglecs, being coded by genes in chromosome 19 for humans and 7 for mice are among the most rapidly evolving gene families in humans undergoing evolution in sequence, gene conversions, changes in gene expression and pseudogenization events, [91]. Therefore Neu5Gc also called as xenoantigen represents a foreign antigen that is incorporated into self-structures and anti-Neu5Gc antibodies are referred to as xeno-autoantibodies [92]. Although dietary Neu5Gc is poorly

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immunogenic, their incorporation into the cell walls of commensal bacteria may convert Neu5Gc into a more immunogenic form [26]. Anti-Neu5Gc antibodies have been linked to a variety of specific human diseases that are not seen in the great apes including cardiovascular disease and cancer [93, 94] and induce chronic inflammation [95].

9  Selectins, sialic acids, and autoimmunity Sialic acids form sialyl LewisX—ligand components for selectins, a family of transmembrane cell adhesion molecules [96, 97], expressed on the surface of leukocytes, activated endothelial cells that regulate the leucocyte adhesion and extravasation from circulation to inflammation sites. During inflammation, leukocytes contact endothelial adhesion receptors, thus rolling along the endothelial lining of the blood vessel [98] enabled by selectin-binding selectins to glycans with sialyl LewisX modify and any disruptions in this process may predispose an individual to autoimmunity. L-selectin blocked by antibodies could prevent experimental allergic neuritis, a rat model of GBS [99].Thus selectin inhibitors finds importance in therapeutic targeting in inflammatory disorders [100–102].

10  Gangliosides and autoimmune diseases Gangliosides are sialic acid-containing glycolipids containing a hydrophobic portion, the ceramide, and a hydrophilic part, the oligosaccharide chain. Reported initially from neural tissue, they are ubiquitous molecules expressed in all vertebrate tissues. Autoantibodies to gangliosides, have been reported in several AD including GBS, MS, lupus erythematosus, Hashimoto’s thyroiditis, and IDDM where the target autoantigens, include protein and acidic glycolipids including sulphatides158 and the gangliosides GT3, GD3, and especially GM2–1 wherein GM2–1 has been reported to be potential targets of IgG autoantibodies, is recognized by cytoplasmic ICA [103]. Development of anti-ganglioside antagonists can serve as targeted therapy for the treatment of GBS with maximum efficacy and specificity [104].

11  Sialyltransferases and autoimmunity Sialyltransferases play role in generating sialic acids with α2–3, α2–6, and α2–8 linkage, based on the underlying N-glycan, O-linked mucin, or glycolipid, and are ligands of the Siglecs. β-Galactoside α-2,6-sialyltransferase-I



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(ST6Gal-I) generates α2–6 linked sialic acid acting as ligands for Siglec-2, playing a role in regulation and induction of B-cell tolerance, autoimmunity. Reports are suggestive of ST6Gal-I deficient mice display impaired immune responses. ST3Gal produces α2–3 linked sialic acids. T cells expressing Siglec-1 ligand had increased ST3Gal-III and ST3Gal-VI and α2–3 sialylation finds importance in Siglec-1 binding and modulation of T-cell activation. Reports suggest that ST3Gal-I null mice exhibit a phenotype displaying relatively reduced levels of CD8+ T cells [105].

12  Sialic acid, Glycobiotechnology, and application in autoimmune disorder therapy Sialic acid with its role as vital regulators of the immune system through Siglecs and aberrant Sialic acid-Siglec interactions are associated with diseases including infection, AD, and cancer. Therefore, controlling the Sialic acid-Siglec axis is being recognized as targets to prevent several diseases. Chemical modifications of the natural Sialic acid ligands have led to the generation of sialic acid mimetics (SAMs) with improved binding affinity and selectivity toward Siglecs. Glycobiotechnology is enabling the designing of SAMs on nanoparticles, polymers, and living cells by bioorthogonal synthesis and thereby enabling the study of sialic acid-Siglec axis in biology and therapy [106]. Antibody-mediated therapy, high-affinity ligand-based probes of Siglec receptors find importance in addressing therapeutic opportunities of Sialic acid-mediated Siglec recognition [107]. Intravenous immunoglobulin (IVIG) has been thought to be replacement therapy in immunocompromised patients and finds application as therapy for IgGdependent AD [108, 109].

13  Sialic acids and the therapeutic use of intravenous immunoglobulins IVIG, an immunosuppressant has been used to treat autoimmune recently reveal a conserved complex biantennary N-glycan in the IgG Fc domain [110] which if terminates in an α2,6-linked sialic acid, imparts a regulatory function to IgG, altering IgFc domain conformation, thereby losing its ability to bind canonical (type 1) Fc receptors, and promoting interaction with type 2 Fc receptors such as the DC-SIGN, on regulatory macrophages, dendritic cells, or other innate immune cells triggering anti-inflammatory

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cascade involving the production of cytokine mediators such as IL-33 and IL-4 [26, 111–113], that alter the ratio of inhibitory to activating type 1 Fc receptors on effector macrophages, and also lead to activation of T regulatory cells [111–113]. Thus, IVIg finds application in the treatment of antibody and T-cell-mediated autoimmunity. Siglecs finds application as therapeutic targets due to their cell-­specific expression, endocytic properties, overexpression on certain cancerous cells, and the ability to modulate receptor signaling. Several antibody-based therapies for targeting of siglecs are in clinical trials of lymphoma/leukemia and AD. [114, 115].The anti-CD33 or Siglec-3 antibody gemtuzumab (Mylotarg) finds application in treating acute myeloid leukemia (AML), and antibodies targeting CD22 (Siglec-2) are currently in clinical trials for the treatment of B-cell non-Hodgkins lymphomas (NHL) and AD [115]. Antibody binding of Siglec-8, Siglec-9, and CD22 has been demonstrated to induce apoptosis of eosinophils, neutrophils, and depletion of B cells and find application in cell-targeted therapies [115].

14 Discussions Environmental, epigenetic factors, somatic mutation, and gut microbiome have also been reported to contribute to AD. Abrogation of immunological tolerance, due to genetic alterations affecting inhibitory signaling and rare genetic variants, loss of function mutations have also been reported to cause exposure of autoantigens, enhancing inflammatory responses, and contributing to autoimmunity with loss of immunological tolerance. Genomic changes in sialic acid biology occurring in human ancestors or hominins has been reported to causes enhanced immunoreactivity, and autoimmunity. Hominins are believed to have lost the enzyme synthesizing the common mammalian sialic acid Neu5Gc, leading to an accumulation of the precursor sialic acid Neu5Ac. This led to an enhanced reactivity by some immune cells and the increased ability of macrophages to kill bacteria, at the cost of increased endotoxin sensitivity. While on one hand, human-specific evolutionary changes in inhibitory and activating Siglecs, immune cell receptors that recognize sialic acids as “self-associated molecular patterns” (SAMPs) has been known to modulate immunity, dietary intake of Neu5Gc (derived primarily from red meats) allows metabolic incorporation of this nonhuman molecule into human cells—has led to “xeno-autoimmunity” involving “xeno-autoantigen” interactions with



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­circulating “xeno-­autoantibodies.” All these factors may contribute to autoimmunity. Although extensive research has been carried out globally, still there are no tools to predict autoimmune disease. Although role for environmental factors, stochastic or epigenetic phenomena are known to be contributory factors, identification of cytokines and chemokines, and their cognate receptors is enabling novel therapies only to block pathological inflammatory responses within the target organ particularly in RA. But this domain of biology is still in its infancy requiring better therapeutic agents and potential targets to target the autoimmune diseases.

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Further reading [116] Bornhöfft  KF, Tom Goldammer  T, Rebl  A, Galuska  SP. Dev Comp Immunol 2018;86:219–31. [117] Ang CW, Noordzij PG, de Klerk MA, Endtz HP, van Doorn PA, Laman JD. Ganglioside mimicry of Campylobacter jejuni lipopolysaccharides determines antiganglioside specificity in rabbits. Infect Immun 2002;70(9):5081–5.

CHAPTER 7

Lysosomal storage disease: Disorders related to glycans and sialic acid 1 Introduction Lysosomes are membrane-bound organelle of animal cells and most plant cells that contain hydrolytic enzymes digesting many kinds of biomolecules and help in waste disposal system of the cell by digesting waste from outside the cell taken up by endocytosis and waste components inside the cells digested by autophagy. Lysosomal enzymes are coded by nuclear genes, mutations of which may lead to enzyme deficiency leading to different human genetic disorders, collectively known as lysosomal storage diseases (LSDs), leading to the accumulation of specific substrates, due to the lack of enzymes to catalyze them, thereby preventing breakdown certain lipids or carbohydrates in the body cells. These genetic defects are related to several neurological disorders, cancers, cardiovascular diseases, and aging-related diseases.We discuss in this chapter the different LSDs associated with glycan and sialic acid storage and breakdown.

2 Lysosomes Lysosomes are membrane-bound compartments within cells that contain enzymes that breakdown large molecules such as proteins, carbohydrates, and fats. Lysosomes are known to contain hydrolases that maintain cellular homeostasis by acting on substrates causing their degradation and cellular processing. Lysosomes contain >50 different hydrolytic enzymes that can hydrolyze the macromolecules. Lysosomal enzymes reveal optimal activity at acidic pH around pH 4.6 and thus are acid hydrolases in nature. The high proton pump H+ATPase is present in the organelles membrane boundaries. Lysosomal membranes contain different glycosylated integral proteins whose carbohydrate chains form protective lining shielding membrane attack by enclosed enzymes. A defect in the catabolic activity of one or more Sialic Acids and Sialoglycoconjugates in the Biology of life, Health and Disease https://doi.org/10.1016/B978-0-12-816126-5.00007-X

© 2020 Elsevier Inc. All rights reserved.

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of these hydrolases, therefore, leads to the accumulation of its substrate in the cells leading to loss of cellular homeostasis causing lysosomal storage disorders [1, 2] (Fig. 1 and Table 1).

3  Lysosomal storage diseases (LSD) Lysosomes are cytoplasmic organelles and lysosomal membrane proteins play a dominant role in lysosomal life cycle, regulating the liposomal functions and events encompassing lysosomal lumen acidification, involved in transportation of a variety of metabolites, are involved in molecular motor recruitment and fusion with other organelles thereby maintaining smooth cellular and physiological processes [20]. Defective transport of small molecules or ions across the lysosomal membrane leads to disorders. Lysosomal proteinases maturation initiates after receptor mediated targeting to prelysosomal compartments, and is completed after lysosomal delivery. The impaired late processing events of proteinase maturation are reported to be biomarkers of inherited LSD and are reported from patients suffering from sialic acid storage disease (SASD) and mucolipidosis II (I-cell disease) [21]. >LJƐŽƐŽŵĞ

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(C) Fig.  1  Lysosomes within cells and functions: (A) cell organelles, (B) lysosomes, and (C) functions of lysosomes.



Table 1  Lysosomal enzymes Substrates

Types

Functions

Acid phosphatases

Phosphomonoesters

Phosphatases

Acid phosphodiesterase

Phosphodiesters

Acid ribonuclease

RNA

Acid deoxyribonuclease

DNA

Cathepsin

Proteins

Collagenase

Collagen

Hydrolases functions by splitting water molecules. Acid phosphatases (APs) belong to the hydrolase family that causes hydrolysis of orthophosphate monoesters under acidic conditions [1] Phosphodiesterase family includes autotaxin, phospholipases C and D, sphingomyelin phosphodiesterase, DNases, RNases, and restriction endonucleases (RE) [3] Involved in RNA metabolism including hydrolysis of single-stranded (ss) RNA, double-stranded (ds) RNA, and RNA hybridized with DNA [4] DNAse [5] degrades DNA by hydrolysis of its phosphodiester backbone Cathepsin are proteases that active in a slightly acidic pH of lysosomes and includes serine proteases cathepsins A and G, the aspartic proteases cathepsins D and E, and the lysosomal cysteine cathepsins [6] Degradies native fibrillar collagen that finds importance to initiate collagen turnover in normal connective tissue [7].

Nucleases

Proteases

Continued

Glycan and sialic acid associated LSDs

Enzymes

175

Table 1  Lysosomal enzymes—cont’d

Iduronate sulfatase β-Galactosidase

Dermatan sulfate GAG hydrolyzing Keratin sulfate, ganglioside enzymes GM1, lactosylceramides, lactose, and various glycoprotein Heparin sulfate

Heparin-N-sulfatase

Types

α-N-acetylglucosaminidase

α-Glucosidase

Glycogen

Fucosidase

Fucosylologosaccharide

α-Mannosidase

Mannosyloligosaccharide

Sialidase

Sialylologosaccharide hydrolyze the nonreducing, terminal sialic acid linkage in various natural substrates, including glycoproteins, glycolipids, gangliosides, and polysaccharides

Polysaccharidase and oligosaccharidases

Functions

Degrades heparan sulfate and dermatan sulfate [8] G enzyme that causes hydrolysis of β-galactosides by breaking the glycosidic bond lying above the galactose molecule [9] Heparin sulfate requires stepwise digestion by (i) iduronate sulfatase, (ii) α-L-iduronisade, (iii) heparin N-sulfatase, (iv) acetyl transferase catalyzing transfer of free amino acid, (v) α-D-Nacetylglucosaminidase, (vi) β-glucurodinase, and (vii) N-acetylglucosamine 6-sulfate sulfatase [10] Hydrolysis of terminal nonreducing N-acetyl-D-glucosamine residues in N-acetylalpha-D-glucosaminides, hydrolyses UDPN-acetylglucosamine [11] Acts on α(1→4) bonds breaks down starch and disaccharides to glucose [12] Tissue α-L-fucosidase is coded in humans by the FUCA1 gene. It breaks down fucose [13] Involved in the cleavage of the α form of mannose [13] Sialidases can hydrolyze non reducing terminal α-(2→3)-, α-(2→6)-, α-(2→8)-glycosidic linkages of terminal sialic residues in oligosaccharides, glycoproteins, glycolipids, colominic acid and synthetic substrates [2]

Sialic acids and sialoglycoconjugates in the biology of life, health and disease

Substrates

176

Enzymes



Ceramide

Glucocerebrosidase

Glycosyl ceramide

β-Hexosaminidase

GM2 ganglioside

Arylsulfatase A

Galactosylsulfatide, cerebroside 3-sulfate

Acid lipase

Triglycerols

Phospholipase (PL)

phospholipids

Sphingolipid hydrolyzing enzyme

Lipid hydrolyzing enzymes

Cleaves fatty acids from ceramide, producing sphingosine (SPH) which is phosphorylated by a sphingosine kinase to form sphingosine-1phosphate [14] The glucosylceramidase activity cleaves, by hydrolysis, the β-glucosidic linkage in chemical glucocerebroside, an intermediate product in glycolipid metabolism [15] Involved in the hydrolysis of terminal N-acetylD-hexosamine residues in N-acetyl-β-Dhexosaminides [16] Coded by the ARSA gene in humans. It breaks down sulfatides, like cerebroside 3-sulfate into cerebroside and sulfate [17] Catalyzes the hydrolysis of cholesteryl esters and triglycerides [18] Hydrolyzes phospholipids into fatty acids and other lipophilic substances. Includes four classes, A, B, C, and D, PLA1—cleaves the SN-1 acyl chain, PLA2—cleaves the SN-2 acyl chain, releasing arachidonic acid, PLB—cleaves both SN-1 and SN-2 acyl chains, PLC—cleaves before the phosphate, releasing diacylglycerol (DAG). PLD—cleaves beyond the phosphate, releasing of phosphatidic acid and an alcohol [19]

Glycan and sialic acid associated LSDs

Ceramidase

177

178

Sialic acids and sialoglycoconjugates in the biology of life, health and disease

Lysosome dysfunction due to gene mutations has been associated with neurodegenerative diseases including LSDs, Alzheimer’s disease (AD), Parkinson’s disease (PD), and frontal temporal dementia (FTD) [22]. (i) Diseases related to glycan degradation include galactosialidosis, mucopolysaccharidosis (MPS) types, Niemann-Pick disease type A, Gaucher’s disease, GM2-gangliosidosis, GM1-gangliosidosis, Fabry’s disease, Niemann-Pick type C (NPC) disease, SASDs, and glycogen storage disorders [22], (ii) diseases due to lipid storage include sphingolipidoses, NPC, Wolman disease, and cholesteryl ester storage disease [23], (iii) defective lysosomal transporters including cystinosis, sialic acid storage disorders, and Salla disease [24], (iv) defects in lysososmal trafficking leading to Chediak-Higashi syndrome (CHS) [23], (v) defects in lysosomal acidification including renal tubular acidosis, dominant deafness-onychodystrophy syndrome, ZimmermannLaband syndrome, autosomal recessive cutis laxa type II, autosomal recessive osteopetrosis, neuronal ceroid lipofuscinosis (NCL), infantile neuronal ceroid lipofuscinosis (INCL), and Wolfram syndrome, and (vi) defects in lysosomal ion channels and transporters including, mucolipidosis type IV, PD, osteopetrosis, and NPC [23].

4  LSDs and defective glycan degradation LSDs have been reported to be the common cause of neuro-regressive disorders in children [25] revealing clinical symptom of dysmorphic features and cherry red spot. The pathophysiology and enzyme analysis reveals glycolipid storage disorders and MPS with increased plasma chitotriosidase, overexpression of glycosaminoglycans (GAGs), and deficiency of enzymes associated with mucosaccharide disorders including mucolipidosis-II/II [26]. LSDs are hereditary disorders caused by gene mutations in lysosomal enzymes that function by degradation of glycans, glycoconjugates, and other complex biomolecules in the lysosome in normal individuals. The lysosomal catabolism of glycoproteins maintains the cellular homeostasis of glycosylation. Glycoproteins by endocytosis or autophagy enter lysosomes and undergo catabolic effects by proteases, glycosidases, and lysosomal hydrolases. The proteases include endo and exopeptidases that acts to produce amino acids and dipeptides that are transported across the lysosomal membrane into the cytosol by diffusion and carrier-mediated transport. Glycans of all mature glycoproteins in normal individuals are degraded in lysosomes. Metabolic disorders are associated with the non-synthesis or non-function



Glycan and sialic acid associated LSDs

179

of carbohydrate-processing enzymes in this cell compartment [27]. The defects in the glycan degradation leading to LSDs include four types including (i) defects in glycoprotein degradation, (ii) glycolipid degradation, (iii) glucosaminoglycan degradation, and (iv) glycogen degradation. The catabolism of N-linked glycans has been studied extensively mediated by bidirectional sequential removal of monosaccharides from the nonreducing end by exoglycosidases and proteolysis of the carbohydrate-­ polypeptide linkage at the reducing end. Removal of any core and peripheral fucose is a prerequisite for the peptide N-glycanase aspartylglucosaminidase, action hydrolyzing the glycan-peptide bond which initiates the process and this enzyme requires free α-carboxyl and amino groups on the asparagine residue, prior to proteolysis (Fig. 2). The glycosyl transferase pathway synthesizes oligosaccharides linked O-glycosidically to amino acids serine or threonine, while the dolichol, lipid-linked pathway synthesizes oligosaccharides linked N-glycosidically to amino acid asparagines. In the lysosomes, oligosaccharides are degraded by exoglycosidases acting on the nonreducing termini, and by endo-β-N-­ acetylglycosaminidase and aspartylglucosaminidase that acts at the reducing end. Incorrectly folded or glycosylated proteins undergo degradation in the endoplasmic reticulum (ER) and cytoplasm and degraded cytosolic end products of N-glycans is delivered to the lysosomes. The interaction of lysosome and proteasome critically regulates the biosynthesis and distribution of N-linked glycoproteins. Lysosomal proteases released into the cytosol initiate the lysosomal pathway of apoptosis [28]. Deficiencies or defects in these enzymes cause glycoprotein or oligosaccharide storage diseases and LSD that leads to the accumulation of not digested oligosaccharides that are intermediates in the lysosomal catabolic pathways and intermediates in N-linked glycan synthesis pathway (Fig. 2). Many lysosomal glycosidases can act on N- or O-linked glycans, GAGs, or glycolipids and the monosaccharides released from broken down N- and O-linked glycans are transported across the lysosomal membrane into the cytosol by diffusion and carrier-mediated transport (Figs. 3–5 and Tables 2 and 3). LSDs and congenital disorders of glycosylation (CDGs), among the inborn errors of metabolism (IEMs) can be diagnosed in the second half of pregnancy by analysis of mucopolysaccharides, oligosaccharides, sialic acid, lysosphingolipids, and some enzyme activities from the amniotic fluid (AF). 7- and 8-Dehydrocholesterol, desmosterol, and lathosterol could indicate diagnosis of cholesterol synthesis disorders (CSDs) [44].

180

Sialic acids and sialoglycoconjugates in the biology of life, health and disease

Degradation of N-Glycans (enzymes & disorders due to their deficiencies)

Proteases

Peptides

Asn

α-Fucosidase

α-Fucosidosis Fucose Asn

AspartyI-N-acetylglucosaminidase AspartyIglycosaminiuria

Asn

Endo-β-N-Acetylglucosaminidase MPS III, Type-B Sanflippo syndrome

GIcNac Sialidase Sialidosis

Sialic acid β-Galactosidase Gal

Morquio Syndrome B, GM1 gangliosidosis

β-N-Acetylhexosaminidase Tay-Sachs Disease Sandhoff disease

GlcNac α-Mannosidase α-Mannosidosis Man β-Mannosidase Man

β-Mannosidosis

Fig. 2  Degradation of complex N glycans and associated enzymes and their deficiency disorders.



Glycan and sialic acid associated LSDs

Heparan sulfate degradation

181

Disorders

Substrates

Enzymes

CH2OH

OH

O COOH O OH OSO2H

OH

O NHOSO2H

O

OH

O

O NHOSO2H

α-L-iduronidase

O

O

OH

O

(n)

NAC

S

CH2OH O COOH OH OH

CH2

OSO2H

Iduronic acid 2-sulfatase

OH

OSO2H

COOH O OH

O

MPSII Hunter syndrome

OSO2H

COOH O OH

CH2 O

O

OH

O

OSO2H

(n)

NAC

IdOA CH2OH O OH

OH

O NHOSO2H

heparam N-sulfatase

CH2OH OH

OH

O NH2

O

O

OH

OSO2H

COOH O OH

O

O

O

OH

O

Sanflippo Syndrome C, MPS-IIIC

OSO2H CH2

O

O OH

O

galA

α-N-acetylglucosaminidase

OH

(n)

NAC

OSO2H

NAC

Sanflippo Syndrome A, MPS-IIIA

CH2

COOH O OH

O OH

(n)

NAC

galNac

CH2OH

O

OSO2H

OSO2H

acetylCoA -N-acetylltransferase

OH

CH2

S

O

Hurler Syndrome MPS1, Hurler-Scheie Syndrome

OSO2H

COOH O OH

(n)

NAC

Sanflippo Syndrome B, MPS-IIIB

OSO2H

COOH O OH

CH2 O

O

OH

O

OH

(n)

NAC

β-glucuronidase Sly syndrome (MPSVII)

OH

COOH O OH

Sly Syndrome (MPS IV) OSO2H CH2

O

O OH

O

OSO2H

Glucuronate sulfatase

(n)

NAC

S

Sanflippo Syndrome D MPS-IIID

OSO2H

N-acetylglucosamine 6-sulfatase

CH2 HO

O OH

O

(n)

NAC

S

OH CH2 HO

α-N-Acetylglucosaminidase

O OH

O

(n)

NAC

GlcNac

Fig.  3  Degradation of heaparin sulfate and associated enzymes and their deficiency disorders.

182

Sialic acids and sialoglycoconjugates in the biology of life, health and disease

Dermatan sulfate degradation (enzymes & disorders due to their deficiencies)

OSO2H CH OH 2 O

OH

O O COOH OH OSO2H

COOH O

O

S

Hunter syndrome A (MPSII)

O

O

O

O

OH

(n)

NAC

IdoA Hurler, Hurler-Scheie Scheie syndromes (MPSI)

OSO2H CH OH 2 O O

COOH O

HO

CH2OH O O

O

OH

NAC

(n)

NAC

OH S

N-acetylgalactosamine 4-sulfatase

Maroteaux-Lamy syndrome (MPSVI)

CH2OH O O

OH

CH2OH O

OH

a-L-iduronidase

HO

HO

COOH O

NAC

OH

(n)

NAC

OH

OSO2H CH OH 2 O

O COOH OH OH

O

O

OH

NAC

Iduronate sulfatase

OH

O

COOH O

HO

b-hexosaminidases A, B and S COOH O

HO

gal Nac Tay-Sachs disease Sandoff disease CH2OH O O

O

OH

(n)

NAC

OH

HO

O

O

OH

NAC

CH2OH O

(n)

NAC

OH

gal A b-glucuronidase HO

Sly syndrome (MPSVII) CH2OH O O

OH

(n)

NAC

GalNac

Fig. 4  Degradation of dermatan sulfate, enzymes involved, and associated deficiency disorders.



Glycan and sialic acid associated LSDs

183

Keratan sulfate degradation

HO

OSO2H

OSO2H

CH2

CH2

O

O

OSO2H

OSO2H

CH2

CH2

O

HO

(n)

OSO2H

CH2OH

O

CH2

O O

O

NAC

OH

O NAC

Morquio syndrome A (MPSIVA)

OH

O

O

OH

OH

S Galactose 6-sulfatase

OH

CH2

O NAC

OH

HO

OSO2H

CH2OH O

OH

O

OH

HO O

O

OH

O

(n)

NAC

OH

Gal Morquio syndrome B (MPSIVB)

b-galactosidase OSO2H

HO

CH2

CH2

O

OH

HO

OSO2H

CH2OH

O O

O

NAC

N-acetylglucosamine 6-sulfatase

O NAC

OH

S

HO

(n)

Sanfilippo syndrome D (MPSIIID)

OH CH2

O

OH

HO

OH

OSO2H

CH2OH

O

CH2

O O

O

NAC

O NAC

OH

(n)

Tay-Sachs disease Sandhoff disease

b-N-Acetylhexosaminidases A and B HO

O

OH

GalNac OSO2H

CH2OH

CH2

O OH

O OH

O

OH

O NAC

(n)

N-acetyl glucosamine-6-sulfatase

HO

CH2OH O OH OH

Glc Nac

Fig. 5  Keratan sulfate degradation: enzymes involved and deficiency disorders.

Disorder

Mutation

Transmission

α-L-fucosidase

Fucosidosis

Autosomal recessive

GlcNAc-1-phosphotransferase.

Mucolipidosis II (ML II) N-acetyl-α-neuraminidase MPS I

FUCA1 gene located on chromosome 1p36 [29]. GNPTAB located on 16p13.3 [30]

Deficiency in lysosomal enzyme α-L-iduronidase Deficiency in β-D-galactosidase Disrupted β-D-hexosaminidase A and B activity Deficiency of α-D-mannosidase Deficiency of β-D-mannosidase Defect and deficiency of N-(β-Nacetylglucosaminyl)-1-asparaginase or aspartylglucosaminidase Defect and deficiency of α-Nacetylgalactosaminidase (α-Dgalactosidase B) UDP-N-acetylglucosamine-1phosphotransferase leading to multiple enzyme deficiencies Protective protein/cathepsin A

IDUA gene, located on 4p16.3 [31]

GM1-gangliosidosis Sandhoff disease

GLB1 gene, located on 3p22.3 [32] HEXB gene, located in 5q13.3 [33]

α-Mannosidosis β-Mannosidosis Aspartylglucosaminuria

MAN2B1 gene, location 19p13.13 [34] MANBA gene location 4q24 [35] AGA gene gene location, 4q34.3 [36]

Schindler and Kawasaki disease

α-N-acetylgalactosaminidase gene (α -NAGE) is located chromosomal region 22q13.1 → 13.2 [37] GNPTAB gene, chromosomal location 12q23.2 [38]

I-cell disease and pseudoHurler polydystrophy (mucolipidosis II and III) Galactosialidosis

Cathepsin C

Papillon-Lefevre syndrome

Cathepsin K Defective α-galactosidase A

Pycnodysostosis Fabry disease

Deficiency of iduronate-2-sulfatase (I2S)

Hunter syndrome or MPS 11

CTSA gene, located in chromosome 20q13.12 [39] C TSC gene, located in chromosome 11q14.2 [40] CTSK located on chromosome 1q21 [41] GLA gene, located in chromosome Xq22.1 [42] IDS gene located in Xq28 chromosome [43]

X-linked

Sialic acids and sialoglycoconjugates in the biology of life, health and disease

Enzyme

184

Table 2  LSDs with defects in metabolism of glycans



Glycan and sialic acid associated LSDs

185

Table 3  LSD glycosylation related disorder type and effects Disease

Underlying effect

Mucopolysaccharidosis Glycogen storage diseases Oligosaccharidosis Sphingolipidosis and sulfatidosis Mucolipidosis Sialic acid storage disorders

Defective metabolism of glucoaminoglycans Defective Storage of Glycogen Defective catabolism of glycans in glycoprotein Defective catabolism of sphingolipids and components Defective catabolism of acid mucopolysaccharides Defective synthesis and catabolism of sialic acid

Hydrops fetalis (HF) is a common manifestation in LSDs revealing excessive accumulation of abnormal serous fluid and excess body water in the subcutaneous soft tissues and serous cavities of the fetus as subcutaneous edema, together with effusions in serous cavities including pericardial or pleural effusions, and ascites [45]. The different LSDs including ISSD, MPS VII and IVA, type 2 Gaucher disease (GD), sialidosis, GM-1 gangliosidosis, galactosialidosis, NPC, disseminated lipogranulomatosis (Farber disease), and mucolipidosis II (I-cell disease) reveal the common manifestation of HF and nonimmune hydrops fetalis (NIHF), that is, not caused by red cell alloimmunization [e.g., Rh(D), Kell] which occurs without an immune factors [46] finds importance as prenatal diagnostic factor with risk ascertained prior to birth [47].

4.1  Defective glycoprotein degradation 4.1.1 Human α-mannosidosis α-Mannosidosis is a LSD caused by an autosomal recessive genetic mutation in the gene MAN2B1, located on chromosome 19p13.2-q12, leading to the deficiency of α-D-mannosidase. It causes defective glycoprotein catabolism leading to abnormal levels and excretion of small mannose-rich oligosaccharides. The more severe infantile or type I phenotype includes rapidly progressive mental retardation, hepatosplenomegaly, severe defective bone-related dysostosis, and death in around 3 and 12 years of age. The slowly progressive, milder or juvenile-adult or type II phenotype, reveals 10%–15% survival into adulthood (Figs. 6 and 7). 4.1.2 Human β-mannosidosis β-mannosidosis is an inherited disorder of autosomal recessive inheritance of MANBA gene located in human chromosome 4q21–25 oligosaccharide catabolism, due to deficiency of lysosomal enzyme β-mannosidase activity

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Sialic acids and sialoglycoconjugates in the biology of life, health and disease

Fig. 6  Structures of enzymes involved in glycan degradation from Protein Data Bank (PBD) [48–53], (i) 3W82 human alpha-L-iduronidase in complex with iduronic acid, (ii)  5FQL insights into Hunter syndrome from the structure of iduronate-2-sulfatase, (iii)  4XWH crystal structure of the human N-acetyl-alpha-glucosaminidase, (iv) 6DRV beta-­galactosidase, (v) 1FSU crystal structure of 4-sulfatase (human), and (vi) 1BHG ­human beta-glucuronidase at 2.6 A resolution.

associated with increased storage and excretion of Man(β1 → 4)GlnNAc. Pathophysiology includes status epilepticus, severe quadriplegia leading to death by 15 months, the presence of angiokeratomas, mental retardation, respiratory infections, and hearing losses with associated speech impairments. 4.1.3 Fucosidosis Fucosidosis is an autosomal recessive disorder resulting from a deficiency of the lysosomal hydrolase α-fucosidase (FUCA1). The FUCA1 gene codes for multiple forms of FUCA1 mapped to chromosome 1p24 in normal individuals.The more severe symptoms in the first year of life include the onset of psychomotor retardation, coarse facies, growth retardation, dysostosis multiplex, neurologic retardation, and increase in sweat sodium chloride. The milder phenotypes are characterized by angiokeratoma, longer survival, and sweat sodium chloride at normal levels. The enzyme defect leads to accumulation and excretion of glycoproteins, glycolipids, and o ­ ligosaccharides



Glycan and sialic acid associated LSDs

187

Fig. 7  Structures of enzymes involved in glycan degradation from Protein Data Bank (PBD) [54–59], (i) 2PE4 structure of human hyaluronidase 1, (ii) 1FMI crystal structure of human class I alpha1,2-mannosidase, (iii) 1SNT structure of the human cytosolic sialidase Neu2, (iv) 2ZWY alpha-L-fucosidase, (v) 1O7A human beta-hexosaminidase B, and (vi) 2GJX crystallographic structure of human beta-hexosaminidase A.

containing fucoside moieties. Although the disorder is panethnic, they are mostly reported from Italy, southwestern part of the United States with a high frequency of consanguinity in affected families. 4.1.4  α-N-Acetylgalactosaminidase deficiency: Schindler disease Schindler disease is an autosomal recessive disorder due to d­efective α-N-acetylgalactosaminidase gene located in chromosomal region 22q13.1 →  13.2 leading to deficiency of lysosomal hydrolase α-N-­ acetylgalactosaminidase, also called as α-galactosidase B, leading to accumulation of sialylated and asialoglycopeptides, glycosphingolipids, and oligosaccharides with α-N-acetylgalactosaminyl residues. Type I disease an infantile-onset neuroaxonal dystrophy reveals normal development in the first 8–15 months and rapid neurodegeneration leading to severe psychomotor retardation, cortical blindness, myoclonus, and seizures. Type II disease, an adult-­onset disorder is characterized by angiokeratoma corporis diffusum (ACD) and mild intellectual impairment. Type III disease reveals

188

Sialic acids and sialoglycoconjugates in the biology of life, health and disease

i­ntermediate and v­ ariable form with manifestations ranging from seizures and moderate psychomotor retardation in infancy to a milder autistic presentation with speech and language delay, early childhood behavioral difficulties. Pathophysiology reveals abnormal urinary oligosaccharide and glycopeptide profiles and prenatal diagnosis of enzymatic defect in chorionic villi or cultured amniocytes. 4.1.5 Aspartylglucosaminuria Aspartylglucosaminuria is an autosomal recessive hereditary disorders revealing progressive loss in mental functioning, with disease development after early childhood, revealing delayed speech, mental disability which worsens in adolescence. Caused by mutations in the AGA gene which cannot produce as partylglucosaminidase, enzyme that helps in normal individuals to breakdown complexes of sugar molecules attached to glycoproteins. Thus, in the diseased individuals, excess glycoproteins disrupt the normal functions and lead to cell destruction affecting nerve cells in the brain.

4.2  Defective glycolipid degradation The defects in the glycolipid degradation leading to LSDs have been broadly grouped into (i) degradation of GM1 ganglioside, (ii) degradation of sulfatide, and (iii) degradation of globotriaosyleramide. Sphingolipidoses are human metabolic storage disorders caused by abnormal accumulation of harmful quantities of glycosphingolipids and phosphosphingolipids structurally sharing feature of hydrophobic portion called the ceramide. Glycosphingolipids constitute of oligosaccharides linked to ceramide through glycosidic bonds including glucocerebroside, composed of ceramide and glucose. Expressed on cell membrane and cellular physiological processes and their biosynthesis and metabolism are regulated by enzymes. Defective metabolism due to defective enzyme activity leads to their intracellular accumulation causing LSD [60].They differ in clinical manifestation based on the severity and onset of the disease which are dependent on the enzyme deficiency or minimal activity. In Tay-Sachs disease, ganglioside GM2 accumulates. In Niemann-Pick disease types A and B, sphingomyelin being a phosphosphingolipid accumulates in patients [61]. In Krabbe disease or globoid cell leukodystrophy, galactosylceramide, and galactosylsphingosine accumulates [62]. Glycolipids with less than four carbohydrate that are embedded in intralysosomal membranes, requires the presence of specific activator glycoproteins that are not catalytically functional but act as cofactors for



Glycan and sialic acid associated LSDs

189

enzymes, including GM2-activator protein that are specific for gangliosides and sphingolipid activator proteins or saposins A, B, C, and D, which manipulate the membranes that enables reaching of degradative enzymes to glycolipid substrates. The four saposins are derived from a single precursor protein, prosaposin, synthesized in the ER, transported to the Golgi for glycosylation and then to the lysosomes. While saposin C enables degradation of galactosyl- and glucosylceramide, saposin B enables hydrolysis of sulfatide, globotriaosylceramide, and digalactosylceramide and saposin D stimulates degradation of lysosomal ceramide by acid ceramidase. ­β-­Glucosylceramidase and saposin C enable the generation of the structural ceramides from skin glucosylceramides Catabolism of glycosylceramides glucosylceramide

saposinC

→

glu cos ylceramidase

galactosylceramide

ceramide

saposinC

→

β  galactosylceramidase

ceramide

4.2.1  Fabry’s disease Fabry’s disease, also called ACD, is a X-linked disease, caused by mutation in GLA gene in the X chromosome at position Xq22.1, affecting predominantly the males caused due to deficiency of α-galactosidase A. Clinical symptoms are manifested by abnormal deposits of a glycosphingolipid ceramide trihexoside in the blood vessels affecting the heart and kidney resulting in reduced life expectancy. Distinctive clusters of dark red granules in the skin, abdomen, and knees of victims and blood lipid deposits are observed. Treatment provides relief of the intense burning pain typical of the disease, however, kidney failure may lead to death. 4.2.2  Gaucher disease Gaucher Disease (GD) is an inherited metabolic disorder with pathophysiology of mild to severe form and affecting childhood to adulthood. Mutations in the glucocerebrosidase (GBA) gene located in chromosome 1q22 cause GD. The GBA gene in normal individuals codes for β-GBA that breaks down glucocerebroside into glucose and ceramide. Mutations in the GBA gene reduce or inhibit β-GBA leading to the accumulation of toxic glucocerebroside and related oligosaccharides within cells and tissues, causing tissue damage, characteristic features of GD. Oligosaccharide may contain neutral and acidic oligosaccharides including gangliosides with one

190

Sialic acids and sialoglycoconjugates in the biology of life, health and disease

or more Nue5Ac/sialic acid molecules. The disease includes two types, the common type 1 GD, coined as non-neuronopathic GD manifesting symptoms of hepatospenomegaly, anemia, reduced platelet count or thrombocytopenia, affected bones, arthritis, lung diseases but not affecting brain and spinal cord of the central nervous system (CNS). Types 2 and 3 GD reveal neuronopathic forms revealing affected CNS, abnormal eye movements, seizures, and brain damage. While type 2 GD can be life threatening at infancy, type 3 GD affects primarily the nervous system and worsens slowly than type 2.The perinatal lethal form is the most severe manifestation of the disease revealing life-threatening complications occurring before birth or at infancy, with features of HF, dry, scaly skin or ichthyosis, and other skin abnormalities; hepatosplenomegaly; affected facial features with neurological problems, cardiovascular disorders, calcification of valves, eye abnormalities, and bone disease. GD has been reported in 1 in 50,000–100,000 individuals of which type 1 forms the most common form occurring frequently in people of Ashkenazi, Europe with Jewish heritage with frequency around 1 in 500–1000 people. The treatment suffers from the major limitation of suitable therapeutic agent that can cross the blood–brain barrier (BBB). Research on different therapeutic approaches including enzyme and bone marrow transplantation, gene therapy, substrate reduction, and through chaperons are being carried out. Enzyme replacement therapy has revealed effectiveness in therapy of LSDs including imiglucerase, taliglucerase, and velaglucerase for GD, substrate reduction therapy by using eliglustat for GD has recently been approved [63]. 4.2.3  Tay-Sachs disease Tay-Sachs disease is a rare reveals GM2-gangliosidosis and is caused due to deficiency in enzyme β-hexosaminidase A. β-N-acetylglucosaminidases (GlcNAcases) hydrolyze N-acetylglucosamine-containing oligosaccharides and proteins and they have been known to play an important role in the maintenance of the cellular levels of O-linked GlcNAc and catabolism of ganglioside storage in Tay-Sachs disease [64]. Genetical basis of Tay-Sachs disease includes mutations in the HEXA gene and inheritance pattern is autosomal recessive in nature. The HEXA gene in normal individuals codes for β-hexosaminidase A enzyme, that functions to breakdown GM2 ganglioside and a defective enzyme coded by defective or mutated gene leads to GM2 ganglioside accumulation in the nerve cells and neurons of the brain and spinal cord, causing the neurodegenerative disease with progressive damage of the nervous system.



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Based on the onset of age, Tay-Sachs disease have been characterized into three types including infantile, most common severe form, in which the symptoms are manifested within first few months of life. Symptoms include regressive loss of skills learned, seizures, loss of muscle, and mental functions and children do not survive after early childhood with death due to lung inflammation like bronchopneumonia. Juvenile form reveals a range of severity, with symptoms appearing any time between the ages 2 and 5 with symptoms of behavior problems, gradual loss of skills, recurrent respiratory infections, and seizures and life is not beyond teenage. Infections can cause death. Late onset/adult form or chronic form is the least severe disease, with symptoms appearing between late childhood to adulthood of an individual. Symptoms including clumsiness, loss of coordination, muscle weakness, psychiatric disorders like schizophrenia, gradual loss of skills, difficulty in speaking or swallowing, and uncontrollable muscle spasms and movements leading to the need for mobility assistance, impaired intellect, and behavior, with lifespan varying from shortened to unaffected.While some individuals reveal a shortened lifespan due to the disease others do not. Individuals suffering from this disease inherit one mutation from each of their parents.The parents carrying one mutated allele are the carrier for the disease who do not express any symptoms. However, if both parents are carrier, each child has a (i) 1 in 4 chance to have the disease, (ii) 1 in 2 chance to be an unaffected carrier, and (iii) 1 in 4 chance to be unaffected and not a carrier. Although it is reported most common among the people with Ashkenazi Jewish descent, this disease is also reported from people from other ethnic backgrounds. Tay-Sachs disease is diagnosed by blood test that can detect the absence or very low levels of beta-hexosaminidase A enzyme activity. HEXA gene can also be tested for specific mutations to confirm the disease. Tay-Sachs disease suffers from the lack of therapy for either halting or slowing the progression of the disease. Treatment is aimed to relieve symptoms and increase quality of life. Seizures in children are often treated with anti-seizure medicines, life conditions can be improved by adequate nutrition and hydration to make life better Currently, there is no cure for Tay-Sachs disease, and there is no treatment that stops or slows the progression of the disease. Treatment is focused around relieving of symptoms, reducing and controlling infections and associated complications thereby increasing the quality of life. Anticonvulsants are administered to control seizures in children, and antipsychotic medications to keep psychiatric disorders in adults under check. Search for new therapy is an ongoing research topic in Tay-Sachs disease.

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4.2.4  Defective synthesis and metabolism of gangliosides Defective synthesis and metabolism of gangliosides has been associated with different diseases broadly categorized into two different categories of neuronopathic diseases including those related to ganglioside storage. Conditions such as GM1 gangliosidosis that cause molecules to build up inside the lysosomes are called LSD. GM1 gangliosidosis is rare and reported from 1 in 100,000 to 200,000 newborns and type I is the most frequent disorder. Most individuals with type III are of Japanese descent. GM1 gangliosidosis is caused by the mutations in the GLB1 gene coding for β-galactosidase that enable breakdown and recycling of GM1 ganglioside in normal individuals and also plays critical role in the brain and normal function of nerve cells in the brain. Mutations in the GLB1 gene alter or prevent the β-galactosidase activity and prevent GM1 lysis leading to the accumulation of toxic levels of GM1 in many tissues and organs, including the brain leads to destruction of brain nerve cells. The β-galactosidase expression dictates the severity of symptoms with lower the expression more is its severity. The GLB1 gene encodes elastin-binding protein which on interaction with cathepsin A and neuraminidase 1 leads to the formation of elastin receptor complex, forming elastic fibers as connective tissue components. Thus, mutated GLB1 gene affects the connective tissue in diseased individuals. There is no treatment or cure for GM-1 disease but research is being carried out to substitute the mutated/missing enzyme or decrease the waste accumulation or better the quality of life and an integrated approach of all the three methods will slow, halt, and cure the damage caused by these diseases. (a) Type I GM1-gangliosidosis GM1 gangliosidosis, type I affecting the infants is the most severe appearing at the age of 6 months revealing normal appearance but with slow development and weakened muscle movements, cardiomyopathy, progressively regression of development, with exaggerated startling reaction to loud noises, hepatosplenomegaly, abnormalities of skeleton, gums, seizures, profound intellectual disability, corneal clouding, and loss of vision with gradual deterioration of retina. A cherry-red spot, in the eye, is characteristic of this disorder. Limited life span not more than childhood is seen in patients. (b) Type II GM1-gangliosidosis occurs later in life than the infantile forms showing normal early development, but revealing symptoms around 18 months to 5 years. Symptoms include developmental regression but without cherry-red spots, abnormal facial features, or enlarged organs. Type II disorder leads to short life.



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(c) Type III GM1 gangliosidosis occurs in adult and is chronic in expression, with symptoms expressed in their teens, with features of involuntary tensing of various muscles or dystonia, spinal bone-related abnormalities, reduced life span. 4.2.5  Niemann-Pick C Disease NPC is an autosomal recessive disorder with pathophysiology revealing lack of cholesterol and lipids transport inside cells, leading to their abnormal accumulation in different tissues including brain tissue and damaging it. The age of onset and specific symptoms varies within individuals, and can appear within neonatal period with life-threatening complications to a late onset at adulthood. NPC is caused by mutations in the NPC1 (NPC Intracellular Cholesterol Transporter 1 gene, NPC type 1C, located in 18q11.2 or the NPC2 NPC Intracellular Cholesterol Transporter 1 gene, 2, NPC type 2C, located in 14q24.3). NPC includes two other disorders, including Niemann-Pick disease type A and Niemann-Pick disease type B. caused due to defect in types A and B caused by mutations in the SMPD1 gene, located in 11p15.4 and deficiency of the enzyme acid sphingomyelinase, which does not occur in NPC and NPC types A and B includes deficiency of acid sphingomyelinase. NPC affects neurologic, psychiatric, and visceral functions, accumulation of fluid in the fetal abdomen, cholestasis, jaundice, failure to thrive, and growth deficiency, hepatomegaly, splenomegaly, and accumulation of lipid-­containing foam cells in the lungs, liver, and lung disease causing life-threatening complications with symptoms of neurological disorders at a later age. The disease can affect both males and females equally and can affect individuals of any ethnic background and estimated to occur in 1 in 100,000–120,000 live births. 4.2.6  Krabbe disease Krabbe disease or globoid cell leukodystrophy is a severe neurological condition and is included under the group of disorders termed as leukodystrophies, due to demyelination in the nervous system. Pathophysiology of Krabbe disease reveals abnormal cells in the brain called globoid cells, which are large cells with more than one nucleus. The most common form of Krabbe disease is the infantile form, starting at the age of 1 with signs and symptoms of irritability, muscle weakness, feeding difficulties, episodes of fever, stiff posture, delayed mental and physical development, weakened muscles, affecting the infant’s ability to move, chew, swallow, and breathe,

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with loss of vision and seizures. Due to severity of the disorder, individuals with the infantile form of Krabbe disease do not survive after the age of 2, less commonly, late onset Krabbe disease begins in childhood, adolescence, or adulthood, revealing symptoms of vision problems, walking difficulties. The signs vary considerably among affected individuals. In the United States, Krabbe disease affects about 1 in 100,000 individuals. A higher incidence (six cases per 1000 people) rates have been reported from few isolated communities in Israel. 4.2.7  Multiple sulfatase deficiency (MSD) MSD is a LSD that affects the brain, skin, and skeleton, the signs and symptoms include three types neonatal, late-infantile, and juvenile of which the neonatal type is the most severe form, with symptoms developing soon after birth and the late-infantile type is the most common form and with progressive loss of mental abilities and impaired movement and the juvenile type is rare, with a slow regression of psychomotor development in mid to late childhood. MSD is caused by mutations in the SUMF1 gene located in 3p26.1 and reveals an autosomal recessive inheritance pattern. The SUMF1 gene codes for formylglycine-generating enzyme (FGE) found in the ER, involved in protein processing and transport. MSD reveals clinical manifestations similar to metachromatic leukodystrophy. It also plays role in modifying other sulfatases that breaks down sulfates. FGE converts cysteine into C-α-formylglycine. There is no cure for MSD.

4.3  Defective degradation of glucosaminoglycan (GAG) There are seven subtypes of MPS disease and MPS I is the first subtype [the others are MPS II (Hunter syndrome), MPS III (Sanfilippo syndrome), MPS IV, MPS VI, MPS VII, and MPS IX]. 4.3.1  MPS type IV disease or Morquio syndrome MPS type IV (MPS IV), also known as Morquio syndrome, is a rare metabolic condition wherein which the body fails to breakdown GAGs leading to accumulation of toxic levels of these sugars in lysosomes, leading to this disorder revealed by symptoms of abnormalities of the skeleton, eyes, heart, and respiratory system. The most common observed age of occurrence lies between 1 and 3. There are two forms of MPS IV. >10 mutations, most of which are single-nucleotide mutations (snps) in the GLB1 gene have been found to cause MPS IV. It is of two distinct types including MPS type IVA (MPS IVA or Morquio syndrome, type A), caused



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by mutations in the GALNS gene in chromosome 16q24.3 and MPS type IV B caused by mutations in the GLB1 gene leading to deficiency of lysosomal enzymes—N-acetylgalactosamine-6-sulfate sulfatase (GALNS) and β-galactosidase, respectively. GALNS deficiency leads to accumulation of GAGs, keratan sulfate (KS), and chondroitin-6-sulfate (C6S). The mutations disrupt the breakdown of KS by β-galactosidase but do not affect the degradation of GM1 ganglioside. The lack of β-galactosidase activity leads to the accumulation of KS within lysosomes. Due to the predominant occurrence of KS in cartilage and the cornea, the mutations lead to the accumulation of keratin sulfate leading to skeletal abnormalities and cloudy corneas. Overall pathophysiology reveals affected skeletal system with short stature, knock knees, pectus carinatum, and malformed spine, hips and wrists, respiratory disorders, valvular heart disease, hearing impairment, corneal clouding, dental abnormalities, hepatomegaly, and spinal cord compression. MPS IV is estimated to occur in 1 in 200,000–300,000 individuals and males and females are equally affected. In 2014, the FDA has approved the administration of a recombinant human GALNS enzyme replacement therapy including elosulfase alfa, or Vimizim manufactured by BioMarin Pharmaceutical Inc. for MPS IVA treatment. Supportive therapy is also administered. Surgery to decompress and fuse the bones of the upper neck to the base of the skull can prevent destabilization of the cervical vertebrae and potential damage to the spinal cord. Corneal replacement may be performed to treat corneal opacification causing impaired vision. Genetic counseling for affected individuals and their families, recording of family history is recommended. 4.3.2  The Sanfilippo syndrome, or MPS III The Sanfilippo syndrome, or MPS III, is an LSD caused by mutation in the gene encoding N-sulfoglucosamine sulfohydrolase with impaired degradation of heparan sulfate. MPS III includes four types, of deficiency in enzymes including heparan N-sulfatase (type A) with the affected gene SGSH, located in 17q25.3; α-N-acetylglucosaminidase (type B) with the affected gene NAGLU, located in 17q21.2; that catalyzes hydrolysis of the terminal nonreducing N-acetyl-D-glucosamine residues in N-acetylα-D-glucosaminides and hydrolyses UDP-N-acetylglucosamine; acetyl CoA:alpha-glucosaminide acetyltransferase (type C) with the affected gene HGSNAT, located in 8p11.21-p11.1; and N-acetylglucosamine 6-sulfatase (type D), with the affected gene GNS, located in 12q14.3. The Sanfilippo syndrome reveals pathophysiology of severe degeneration of CNS.

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Clinical features occur between 2 and 6 years, with severe neurologic degeneration occurring in most patients between 6 and 10 years of age, and death in the 20s or 30s of life.Type A reveals most severity, earlier onset, and rapid progression of symptoms and shorter survival. 4.3.3  MPS I Mucopolysaccharidoses (MPS) are genetic disorders in which critical body enzymes are either missing or are present in insufficient amounts. Hurler syndrome is an MPS and is a type of LSD caused by the deficiency of the enzyme, α-L-iduronidase (IDUA) caused by gene mutation in the IDUA gene, located on chromosome 4. IDUA breaks down long chains of sugar molecules but lack of enzymes leads to the accumulation of GAGs in the lysosomes that progressively damage parts of the body. The disease may manifest varying from severe progressive condition involving many bodily systems. Bone deformities, heart, respiratory system, and brain are affected. The child shows defective physical and mental development for his age, trouble in crawling and walking, problems in the joints, causing hands unable to straighten out, heart failure, or pneumonia. Frequency is severe MPS I in 1 in every 100,000 births and is divided into three groups according to the type, severity, and symptoms progress. Attenuated MPS I is less common, occurring in  A) variant and a truncating (c.819 + 1G > A) variant, confirming the diagnosis of Salla disease at age 3.5 years [77]. 4.4.2  Free sialic acid storage disorders (FSASD) FSASD arise out of defective free sialic acid transport from the lysosomes, thereby leading to the accumulation of free sialic acid in the lysososmes.This has been reported to be caused by pathogenic variants of mutations in the SLC17A5 gene, in the gene encoding the lysosomal N-acetylneuraminic acid transport membrane protein sialin [70, 78]. In a recent genomic study,

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from Finland, direct sequencing approaches of SLC17A5 revealed the single-nucleotide polymorphism (snp) homozygosity for the 115C → T (R39C) sequence being the root cause of Salla disease [78]. Other snps are reported from 55 genes SLC17A5 gene from two affected individuals and their parents in the disease locus of 14.9 Mb region on chromosome 6 [78]. While the normal physiologic range of total sialic acid (TSA) level in ­serum/plasma lies within the range of 1.58–2.22 mmol L−1, the free sialic acid constituting 0.5–3 μmol L−1, and the lipid associated sialic acid (LSA) of 10–50 μmol L−1, in Salla disease the Sia levels are extensively increased [75]. FSASD are detected by elevated free Sia and/or or Neu5Ac in urine and/ or CSF detected by fluorimetric thiobarbituric acid (TBA) assay, thin-layer chromatography (TLC), or mass spectrometry (MS) analysis. Management is symptomatic and supportive including rehabilitation to optimize mobility and communication; provision of adequate nutrition; standard treatment of seizures; and is in need of regular monitoring [74]. FSASD are autosomal recessive disorder in inheritance and detection of increased risk is possible by prenatal testing by quantitative estimation of free sialic acid either in chorionic villus biopsy specimens at ~10–12 weeks’ gestation or in amniocytes at ~15–18 weeks’ gestation [74]. 4.4.3  Infantile sialic acid storage disorder (ISSD) ISSD has been reported to be a rare autosomal recessive metabolic disorder caused due to defective lysosomal membrane transport, leading to the free Sia accumulation in lysosomes. The morphological features are reported to be characterized by coarse facies fair complexion, hepatosplenomegaly, and severe psychomotor retardation, nephrotic syndromes in infants, fetal/ neonatal ascites or hydrops, cardiomegaly, respiratory infections, and early deaths [79]. ISSD revealed and original French type sialuria are reported to reveal marked differences in the pathophysiology. Phase microscopy and immunochemical studies revealed abnormal storage of Sia within intracellular inclusions and lysosomes in ISSD cells, while the sialuria cells revealed accumulated free cytosolic sialic acid but no storage in any organelles [82]. Cases studied in ISSD patients have revealed manifestation of fetal ascites [80, 81] detected by ultrasonography with hydropic, dysmorphic features, including sparse white hair, coarse facies, hypertelorism, epicanthal folds, anteverted nostrils, visceromegaly, long philtrum, and vacoulated lymphocytes at birth. Bone marrow with large numbers of foam cells with accumulated sialic acid in neurons, endothelial cells, and Kupffer cells and increased



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free sialic acid in urine [80, 81], serum, liver, heart, and brain tissues [80] and AF [81] are observed. Symptoms in patients with ISSD included in utero with nonimmune hydrops, ascites, and anemia requiring intrauterine transfusion and death from respiratory failure after 2 days with brain hemorrhages, severe cardio- and hepatosplenomegaly, enlarged lysosomes in liver, myocardium, and placenta [81]. 4.4.4 Sialidosis Sialidosis or mucolipidosis I is also an autosomal recessive LSD due to deficiency of neuraminidase that normally cleaves terminal α2 → 3 and α2 → 6 sialyl linkages of several oligosaccharides and glycopeptides that accumulates in tissues and fluids of affected patients. Type I sialidosis is the milder form characterized by the development of ocular ­cherry-red spots and generalized myoclonus in the second or third decade of life, with seizures, hyperreflexia, and ataxia. Type II sialidosis reveals early onset of a progressive, rather severe, MPS-like phenotype with visceromegaly, dysostosis multiplex, and mental retardation. The gene coding for this neuraminidase is located in human chromosome 6p21 within the HLA locus. Patient with sialidosis type I, with cherry red macular spots but mild excretion of bound sialic acid and revealed NEU1 gene (c.699C > A, p.S233R in exon 4 and c.803A > G; p.Y268C in Exon 5 in NEU1transcript NM_000434.3) variants causing impaired protein function [83] has been reported (Fig. 8).

4.5  Glycogen degradation defect diseases 4.5.1  Pompe’s disease Glycogen has been reported to accumulate in the body’s cells in certain organs and tissues, especially muscles in Pompe disease that impairs their ability to function normally. This is due to the deficiency in acid α-­glucosidase (GAA). Three types of Pompe disease, differing in severity and the age of appearance include classic infantile-onset, non-classic ­infantile-onset, and late-onset. The classic form of infantile-onset Pompe disease begins within few months of birth with infants expressing muscle weakness or myopathy, poor muscle tone or hypotonia, hepatomegaly and heart defects, failure to gain weight and to thrive and have breathing problems. The non-classic form of infantile-onset Pompe disease usually appears by age 1, characterized by delayed motor skills, progressive muscle weakness, cardiomegaly, serious breathing problems, and most children with non-classic infantile-onset Pompe disease survive only into

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Gal-GalNAc NANA GM-1 Gangliosidosis

Gal-Glc-Cer [GM1] GM-1 b galactosidase, GM2 activator, SAP B Gal

GalNAc NANA

Gal-Glc-Cer (GM2)

GalNAc-Gal-Gal-Glc-Cer [Globoside] Sandhoff disease α-Hexosaminidase A & B

Tay-Sachs disease β-Hexosaminidase A GM2 Activator

GalNAc

GalNAc

Gal-Gal-Glc-Cer

NANA-Gal-Glc-Cer[GM2) Neuraminidase Or, Sialidase SAP-B Sialidosis

Fabry disease α-Galactosidase

NANA Gal

Gal-Glc-Cer Lac Cer β-Galactosidase SAP-B & SAP-C Gal Glc-Cer Gaucher disease β-Glucocerebrosidase SAP-C

SO3H2 SO3H2Gal-Cer Sulfatide

Gal-Cer

Glc Coline-P

Gal

Ceramide

Metachromatic Krabble disease leukodyntrophy β-Galactosidase Arylsulfatase A SAP-A & SAP-C Farber disease SAP-B Acid ceremidase SAP-A & SAP-D

Phosphorylcholine-Cer [Sphingomyelin]

Niemann-Pick disease Sphingomyelinase

Fatty acid

Sphingosine

Fig. 8  Glycans and sialic acid degradation related LSDs. NANA: N-Acetylneuraminic acid or sialic acid; GalCer: galactosylceramide; Cer: ceramide; GalNac: N-Acetylgalactosamine; Gal: Galactose; GM1, GM2: Gangliosides; Glc: glucose; Lac: Lactose; SAP: Saposin.

early  childhood. Affects  about 1 in 40,000 people in the United States and is reported from different ethnic groups. 4.5.2  Other Glycogen storage disorders The other glycogen storage disorders are enlisted in Table 4 revealing most common signs of hepatomegaly, hypoglycemia, hyperlipidema, and symptoms of muscle-associated problems.



Table 4  Different types of glycogen storage diseases (GSD) Enzyme deficiency (Gene)

Hypoglycemia

Hepatomegaly

Hyperlipidemia

Muscle symptoms

GSD 0

Glycogen synthase (GYS2) Glucose-6-phosphatase (G6PC/SLC37A4) Acid alpha-glucosidase (GAA) Glycogen debranching enzyme (AGL)

Yes

No

No

Yes

Yes

Yes

Muscle cramps are occasional None reported

No

Yes

No

Muscle weakness

Yes

Yes

Yes

Myopathy

No No

Myopathy and dilated cardiomyopathy Exercise-induced cramps, Rhabdomyolysis None reported

GSD I/GSD 1 (von Gierke’s disease) GSD II/GSD 2 (Pompe’s disease) GSD III/GSD 3 (Cori’s disease or Forbes’ disease) GSD IV/GSD 4 (Andersen disease)

Glycogen branching enzyme (GBE1)

No

GSD V/GSD 5 (McArdle disease)

Muscle glycogen phosphorylase (PYGM)

No

Yes, also cirrhosis No

GSD VI/GSD 6 (Hers’ disease)

Liver glycogen phosphorylase (PYGL) Muscle phosphoglycerate mutase (PGAM2) Muscle phosphofructokinase (PKFM)

Yes

Yes

Yes

No

No

No

Yes

Yes

Yes

GSD VII/GSD 7 (Tarui’s disease) GSD IX/GSD 9

Continued

203

Phosphorylase kinase (PHKA2/PHKB/PHKG2/ PHKA1)

Exercise-induced muscle cramps and weakness None

Glycan and sialic acid associated LSDs

Type

204

Type

Enzyme deficiency (Gene)

Hypoglycemia

Hepatomegaly

Hyperlipidemia

GSD X/GSD 10

Enolase 3 (ENO3) Muscle lactate dehydrogenase (LDHA) Aldolase A (ALDOA)

Not reported

Not reported

Not reported

Not reported

Not reported

Not reported

Not reported

In some

Not reported

No

No

GSD XI/GSD 11 GSD XII/GSD 12 (Aldolase A deficiency) GSD XIII/GSD 13 GSD XV/GSD 15

β-Enolase (ENO3) Glycogenin-1 (GYG1)

Not reported No

Muscle symptoms

Exercise intolerance, cramps. In some Rhabdomyolysis. Exercise intolerance, cramps Muscle atropy

Adapted with modification from https://en.wikipedia.org/wiki/Glycogen_storage_disease under Wikipedia: Text of Creative Commons Attribution-ShareAlike 3.0 Unported License.

Sialic acids and sialoglycoconjugates in the biology of life, health and disease

Table 4  Different types of glycogen storage diseases (GSD)—cont’d



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4.6  Other LSDs 4.6.1  Cobalamin F-type disease Cobalamin F-type disease is reported to be caused by defective lysosomal putative cobalamin transporter and a defective transporter with transferase activity of acetyl groups is reported to cause MPS type IIIC A, respectively [20]. 4.6.2  Cobalamin F-type disease Mucolipidosis type IV, caused by mutations of lysosomal cation channel protein, TRPML1 has been reported to be associated with storage of lipids causing one of the channelopathies [20]. Mucolipidosis type 4 affects the processing of certain carbohydrates and fats leading to their accumulation in cells. Most people with mucolipidosis type 4 develop severe affected psychomotor delay after the first year of life and worsened visual impairment, limited or absent speech; intellectual disability; hypotonia, controlling hand movements; impaired production of stomach acids, and iron deficiency, eye abnormalities. Mucolipidosis type 4 is caused by mutations in the MCOLN1 gene and is inherited in an autosomal recessive manner. Treatment is based on the expression of signs and symptoms in individuals. 4.6.3  Danon’s disease (DD) Danon’s disease (DD) is an X-linked dominant skeletal and cardiac muscle disorder revealing symptoms of cardiomyopathy, skeletal myopathy, and intellectual disability. Defect in autophagy in DD due to role of lysosome-­ associated membrane protein 2 (LAMP2) in lysosome/autophagosome fusion and dynein-based centripetal motility [20]. Biochemistry of the disease reveals accumulation of glycogen in muscle tissue as in Pompe disease and is a LSD glycogen storage disease type IIb. Genetic defects have been reported in the lack of expression of LAMP2 gene, encoding the LAMP2 protein which disrupts intracytoplasmic trafficking and leads to accumulation of autophagic material and glycogen in skeletal muscle and cardiac muscle cells. The LAMP2 protein is a type 1 membrane protein predominantly located in the lysosome consisting of a large heavily glycosylated luminal domain, a transmembrane region, and a short carboxy-terminal cytoplasmic tail. The short LAMP-2A cytoplasmic tail acts as a receptor for uptake of certain proteins into the lysosome for degradation, in chaperone-mediated autophagy. The LAMP2 protein isoforms include LAMP-2A, LAMP-2B, and LAMP-2C, differ only at the carboxy-terminal lysosomal transmembrane domain and at the short cytosolic tail. Symptoms include skeletal and cardiac myopathy, cardiac conduction abnormalities, mild intellectual

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difficulties, and retinal disease. Males are more severely affected than females. The disease is X-linked dominant in inheritance.

4.7  Treatment of LSDs LSDs are a group of inherited metabolic disorders and research in the last two decades has enabled development of a variety of innovative therapeutic approaches, including hematopoietic stem cell transplantation, enzyme replacement therapy, pharmacological chaperone therapy using sugar mimetics [84], and gene therapy to replace or substitute the missing enzyme [85–87]. Reducing the synthesis of the stored substrate is another way to stop the disease [86]. Strategies to reduce substrates include application of chemical chaperone based on small molecules with the property to bind and stabilize the misfolded enzymes [87].

5 Discussions Although most LSDs have no treatment available, research is going on to look into the possibility of gene therapy. Pharmacological chaperones including reversible inhibitors of these enzymes, acting as templates for the correct folding and transport of the respective protein mutant, thereby leading to its improved concentration and its lysosomal enzymatic activity. No definitive treatment is currently available for these lysosomal enzyme deficiencies. Identification of affected fetuses by biochemical analysis is reliable and has been demonstrated for each of these abnormalities by means of chorionic villus samples or cultured AF cells. Researchers are underway to devise strategies in targeting these genetic disorders.

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[7] Shingleton WD, Hodges DJ, Brick P, Cawston TE. Collagenase: a key enzyme in collagen turnover. Biochem Cell Biol 1996;74(6):759–75. [8] Demydchuk  M, Hill  CH, Zhou  A, Bunkóczi  G, Stein  PE, Marchesan  D, Deane  JE. Read RJ insights into hunter syndrome from the structure of iduronate-2-sulfatase. Nat Commun 2017;8:15786. [9] Garrett R. Biochemistry. Belmont, CA: Cengage Learning; 2013. p. 1001. ISBN 9781133106296. [10] The biochemistry of glycoproteins and proteoglycans edited by William Lennarz. Springer https://books.google.co.in/books?isbn=1468410067. [11] von Figura K. Human alpha-N-acetylglucosaminidase. 1. Purification and properties. Eur J Biochem 1977;80(2):523–33. [12] Larner J, Lardy H, Myrback K. Other glucosidases. In: Boyer PD, editor. The Enzymes. 2nd ed., vol. 4. New York: Academic Press; 1960. p. 369–78. [13] Fukushima  H, de Wet  JR, O’Brien  JS. Molecular cloning of a cDNA for human ­alpha-L-fucosidase. Proc Natl Acad Sci U S A 1985;82(4):1262–5. [14] Mao C, Obeid LM. Ceramidases: regulators of cellular responses mediated by ceramide, sphingosine, and sphingosine-1-phosphate. Biochim Biophys Acta 2008;1781(9): 424–34. [15] Vielhaber  G, Pfeiffer  S, Brade  L, Lindner  B, Goldmann  T, Vollmer  E, Hintze  U, Wittern KP, Wepf R. Localization of ceramide and glucosylceramide in human epidermis by immunogold electron microscopy. J Invest Dermatol 2001;117(5):1126–36. [16] Hou Y, Tse R, Mahuran DJ. Direct determination of the substrate specificity of the alpha-active site in heterodimeric beta-hexosaminidase. Biochemistry 1996;35(13): 3963–9. [17] Fujii T, Kobayashi T, Honke K, et al. Proteolytic processing of human lysosomal arylsulfatase A. Biochim Biophys Acta 1992;1122(1):93–8. [18] Tovoli  F, et  al. A relative deficiency of lysosomal acid Lypase activity characterizes non-alcoholic fatty liver disease. Int J Mol Sci 2017. [19] D. M.Vasudevan & S. Sreekumari, Textbook of biochemistry (5th ed.) [20] Ruivo R, Anne C, Sagné C, Gasnier B. Molecular and cellular basis of lysosomal transmembrane protein dysfunction. Biochim Biophys Acta 2009 Apr;1793(4):636–49. [21] Mach L. Biosynthesis of lysosomal proteinases in health and disease. Biol Chem 2002 May;383(5):751–6. [22] Pearl P.Y. Lie, Ralph A. Nixon, Lysosome trafficking and signaling in health and neurodegenerative diseases, Neurobiol Dis, 2018 [23] Schulze H, Sandhoff K. Lysosomal lipid storage diseases. Cold Spring Harb Perspect Biol 2011;3(6): https://doi.org/10.1101/cshperspect.a004804. pii: a004804. [24] Mancini GM, Havelaar AC,Verheijen FW. Lysosomal transport disorders. J Inherit Metab Dis 2000;23(3):278–92. [25] Sheth J, Mistri M, Bhavsar R, Sheth F, Kamate M, Shah H, Datar C. Lysosomal storage disorders in Indian children with Neuroregression attending a genetic center. Indian Pediatr 2015 Dec;52(12):1029–33. [26] https://www.ncbi.nlm.nih.gov/pubmed/8750610. [27] Bryan Winchester, Advance Access publication on January 12, 2005, Oxford University Press, 2005. 1R REVIEW Lysosomal metabolism of glycoproteins. Glycobiology vol. 15 no. 6 pp. 1R–15R. [28] Guicciardi  ME, Leist  M, Gores  GJ. Lysosomes in cell death. Oncogene 2004;23: 2881–90. [29] https://www.omim.org/entry/230000. [30] Leroy JG, Cathey S, Friez MJ. Mucolipidosis II. 2008 Aug 26 [Updated 2012 May 10]. In: Adam MP, Ardinger HH, Pagon RA, et al., editors. GeneReviews® [internet]. ­Seattle (WA): University of Washington; 1993–2018.

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[51] Cianfrocco MA, Lahiri I, DiMaio F, Leschziner AE. cryoem-cloud-tools: a software platform to deploy and manage cryo-EM jobs in the cloud. J Struct Biol 2018;203:230–5. [52] Bond  CS, Clements  PR, Ashby  SJ, Collyer  CA, Harrop  SJ, Hopwood  JJ, Guss  JM. Structure of a human lysosomal sulfatase. Structure 1997;5:277–89. [53] Jain S, Drendel WB, Chen ZW, Mathews FS, Sly WS, Grubb JH. Structure of human beta-glucuronidase reveals candidate lysosomal targeting and active-site motifs. Nat Struct Mol Biol 1996;3:375–81. [54] Chao KL, Muthukumar L, Herzberg O. Structure of human hyaluronidase-1, a hyaluronan hydrolyzing enzyme involved in tumor growth and angiogenesis. Biochemistry 2007;46:6911–20. [55] Vallee F, Karaveg K, Herscovics A, Moremen KW, Howell PL. Structural basis for catalysis and inhibition of N-glycan processing class I alpha 1,2-mannosidases. J Biol Chem 2000;275:41287–98. [56] Chavas  LMG, Tringali  C, Fusi  P, Venerando  B, Tettamanti  G, Kato  R, Monti  E, Wakatsuki S. Crystal structure of the human cytosolic sialidase Neu2: evidence for the dynamic nature of substrate recognition. J Biol Chem 2005;280:469–75. [57] http://www.rcsb.org/structure/2ZWY. [58] Maier T, Strater N, Schuette C, Klingenstein R, Sandhoff K, Saenger W. The X-ray crystal structure of human beta-hexosaminidase B provides new insights into Sandhoff disease. J Mol Biol 2003;328:6. [59] Lemieux  MJ, Mark  BL, Cherney  MM, Withers  SG, Mahuran  DJ, James  MN. Crystallographic structure of human beta-Hexosaminidase A: interpretation of Tay-Sachs mutations and loss of GM2 ganglioside hydrolysis. J Mol Biol 2006;359(4):913–29. [60] Boomkamp  SD, Butters  TD. Glycosphingolipid disorders of the brain. Subcell Biochem 2008;49:441–67. [61] Brady RO. Therapy for the sphingolipidoses. Arch Neurol 1998 Aug;55(8):1055–6. [62] Freeze HH, Kinoshita T, Schnaar RL. Genetic disorders of glycan degradation, 2017. In:Varki A, Cummings RD, Esko JD, et al., editors. Essentials of glycobiology [internet]. 3rd ed. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 2015–2017; Chapter 44. [63] Ferreira CR. Gahl WALysosomal storage diseases. Transl Sci Rare Dis 2017;2(1–2):1–71. [64] Zhang R,Zhou J,Song Z,Huang Z.Enzymatic properties of β-N-­acetylglucosaminidases. Appl Microbiol Biotechnol 2017 Nov 16. [65] Natowicz  MR, Short  MP, Wang  Y, Dickersin  GR, Gebhardt  MC, Rosenthal  DI, Sims  KB, Rosenberg  AE. Clinical and biochemical manifestations of hyaluronidase deficiency. New Eng J Med 1996;335:1029–33. [66] Ghosh S. Sialylation and sialyltransferase in insects. Glycoconj J 2018;35(5):433–41. [67] Schauer R. Sialic acids as regulators of molecular and cellular interactions. Curr Opin Struct Biol 2009;19:507–14. [68] Cantz M, Ulrich-Bott B. Disorders of glycoprotein degradation. J Inherit Metab Dis 1990;13(4):523–37. [69] Gopaul KP, Crook MA.The inborn errors of sialic acid metabolism and their laboratory investigation. Clin Lab 2006;52(3–4):155–69. [70] Strehle  EM. Sialic acid storage disease and related disorders. Genet Test 2003;7(2): 113–21. Summer. [71] Mancini  GM, Verheijen  FW, Beerens  CE, Renlund  M, Aula  P. Sialic acid storage disorders: observations on clinical and biochemical variation. Dev Neurosci 1991; 13(4–5):327–30. [72] Misasi R, Dionisi S, Farilla L, Carabba B, Lenti L, Di Mario U, Dotta F. Gangliosides and autoimmune diabetes. Diabetes Metab Rev 1997 Sep;13(3):163–79. [73] Sagné  C, Gasnier  B. Molecular physiology and pathophysiology of lysosomal membrane transporters. J Inherit Metab Dis 2008 Apr;31(2):258–66.

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CHAPTER 8

Sialic acids and sialoglycoconjugates in cancer 1 Introduction Sialic acids are 9-carbon monosaccharide and the most common member of the sialic acid family is Neu5Ac, followed by Neu5Gc and different Oacetylated derivatives, comprising of about 80 neuraminic acid derivatives localized on cell membranes with different biological functions [1] of cell fate decision during development, signaling reprogramming, and cancer progression [2]. Cancer is the largest killer disease of the 21st century claiming lives across the globe in both developing and developed countries. The major challenge in the domain of cancer biology research remains early detection and efficient therapy that can increase the chances of survival. Detection of diagnostic and prognostic markers for early detection and monitoring of patients under therapy, respectively, forms the major highlight of cancer biology research. Suitable markers to detect the minimal residual disease (MRD) and detection of clinical relapse is also a major objective of research in caner biology research. Altered glycosylation has been recognized as one of the hallmarks in different cancers with roles in cancer progression, metastasis, and invasion. Cancer-associated glycosylation changes often involve sialic acids which play important roles in cell-cell interaction, recognition, and immunological response. Hypersialylation and their conjugated moieties find importance as diagnostic and prognostic cancer marker. O-Acetylated sialic acids are known to be cancer markers in childhood acute lymphoblastic leukemia (ALL) [3, 4], with increased O-actyl transferase [5]. Hypersialylation of sialic acid-­binding receptors, including siglecs and selectins, plays role in maintaining inflammation in cancer [6]. Differential expression of glycosylation regulating enzymes including glycosyltransferases, glycosidases, monosaccharide transporters, and aberrant expression of sialic acid in the cancer niche together with increased nonhuman sialic acid Neu5Gc, altered sialyltransferase and sialidase, affected Sialic Acids and Sialoglycoconjugates in the Biology of life, Health and Disease https://doi.org/10.1016/B978-0-12-816126-5.00008-1

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sialic acid-binding lectins (SABLs), inflammation are hallmarks in cancer [7]. Siglecs inhibitory receptors like Siglec 9 and 7 on natural killer (NK) cells are known to inhibit antitumor immunity [8]. Selectins have been used as targets in cancer therapy [9]. Targeting sialic acids is a promising tool in immunotherapy. Incorporation of nonnatural sialosides into the cancer sialome finds importance in immunotherapy for targeted tumor-specific delivery of imaging or therapeutic agent [10].

2  Sialic acid and serum sialylation as biomarkers in cancer Sialylation of glycoproteins and glycopilids finds role in embryonic development, neurodevelopment, reprogramming, oncogenesis, and immune responses. It has recently reported of role in cell fate decision during development, reprogramming, and cancer progression. Altered sialylation levels and patterns are associated with cancer progression, and thus highlight their role as cancer biomarkers [2]. Total serum sialylation including total sialic acid, sialic acid per total protein, cancer-associated glycoproteins, serum acute-phase proteins (APP), and immunoglobulins (Ig), sialic acid containing antigens including CA19-9, sialyl Lewis X, and sialyl Tn on serum proteins and enzymes including overexpressed sialidases and sialyltransferases finds importance as diagnostic and prognostic marker, and reporting on cancer advancement and improving prognosis [11]. Sialic acid, aberrant biosynthesis of sialylglycoconjugates has been reported to correlate with clinical prognosis in head and neck squamous cell carcinoma (HNSCC) [12]. Altered sialylations in the endocrines including ovary, pancreas, thyroid, adrenal, and pituitary gland have been reported to find importance as biomarker in endocrinal cancer [13]. Neu5Gc absent in humans due to deletion in the CMP-Nacetylneuraminic acid hydroxylase (CMAH) gene encoding the hydroxylase converting CMP-Neu5Ac to CMP-Neu5Gc, but Neu5Gc can be metabolically incorporated into human body from red meat, and high-level expression is reported levels in some human cancers. Exposure to Neu5Gccontaining foods early in life may be incorporated by commensal bacteria into lipooligosaccharides leading to generation of ­cross-reactive antibodies against Neu5Gc-containing glycans or xeno-autoantigens forming ­anti-Neu5Gc antibodies or xeno-autoantibodies that are reported from all humans. Neu5Gc-deficient CMAH(-)(/)(-) mouse model resembling humans reveal inflammation due to xenosialitis resulting from a­ ntigen-antibody



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interaction that can promote tumor progression and initiate cancer, emphasizing the role of red meat as a risk factor for carcinoma and human anti-Neu5Gc antibodies has been reported of their importance as cancer biomarkers [14]. Cancer-associated glycoconjugates including globo-H, CA125, CA15.3, and CA27.29 are reported to be important biomarkers in cancer and find importance in diagnosis and therapy [15] but their exact structures remain unknown by conventional methods. New methods of immunological mapping of the glycome have been proposed to detect novel markers in cancer [16]. Overexpression of sialoglycoconjugates in the progression, invasion or metastasis, of cancer including colorectal cancer [17] where expression of mucin (MUC) 1 with specific structure of a sialo-oligosaccharide plays role in metastasis and finds importance as prognostic markers in patients with colorectal cancer [18]. Tumor-associated expression of α 2,3- and α 2,6-­sialylated oligosaccharides in colorectal carcinomas was reported [17]. The structural diversity of sialic acid is rapidly expanding, and several analytical methods find application in detection of 9-O-acetylated N-acetylneuraminic acid (Neu5,9Ac), Neu5Gc, deaminoneuraminic acid (Kdn), O-sulfated sialic acids (SiaS), and di-, oligo-, and polysialic acid in glycoproteins and glycolipids, due to their role in immune system regulation, neural development and function, metastasis, tumorigenesis, and aging [19]. Sulfated sialoglycoconjugates like α2→3 sialylated 6-sulfo-Lewis x acted as ligands for selectins on skin-homing helper memory T cells, and α2→6 sialylated 6-sulfo-LacNAc acted a ligand for CD22/siglec-2 on human naïve B cells and monoclonal antibodies (mAbs) have been designed with specificity to sulfated sialoglycoconjugates to isolate these ligands on human leukocytes [20]. Overexpressed sialylation in carcinoma of the papilla of Vater has been detected by histochemical staining with Maackia amurensis (MAA) leukoagglutinin and Sambucus nigra agglutinin (SNA) [21]. Sialic acid was reported from low-density lipoproteins (LDL) in rats with fibrosarcoma [22]. Sialic acids and various sialoglycoconjugates were reported to be associated with cancer phenotype and metastasis in intestine and kidney [23] cancer. Excessive sialylation were detected by SABLs using MAA and SNA in primary colorectal cancer and lymph node is indicative of poor prognosis [24]. Childhood ALL reveals overexpression of 9-O-acetylated sialoglycoconjugates (9-OAcSGs) [25] on lymphoblasts and immature erythrocytes [26, 27] (Figs. 1 and 2) with concomitant anti-9-OAcSGs and

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Fig.  1  Overexpression of 9-OAcSGs on PBMC of childhood ALL patients. (A) Representative profile of their disease specific expression in PBMC of B-ALL patients revealed by staining cells with FITC Achatinin-H and PE conjugated anti-CD19, anti-CD7, anti-CD13, and anti-CD14 mAbs, respectively. Appropriate isotype matched controls were used as controls. (B) Representative profile of whole blood from normal individuals processed similarly served as the normal controls. (Reproduced with permission from Ghosh S, Bandyopadhyay S, Pal S, Das B, Bhattacharya DK, Mandal C Increased interferon gamma production by peripheral blood mononuclear cells in response to stimulation of overexpressed disease-specific 9-O-acetylated sialoglycoconjugates in children suffering from acute lymphoblastic leukaemia. Br J Haematol 2005,128:35–41.)

immune-complexed 9-OAcSGs finds importance as diagnostic and prognostic biomarkers and the immune complex finds importance in monitoring of disease status [28]. Achatinin-H, alectin isolated from hemolymph of Achatina fulica snails, synthesized in amoebocytes is specific toward a 9-Oacetyl sialic acid (9-O-AcSA). Overexpression of 9-OAcSA-specific IgG2 was reported in ALL [29, 30] with no effector function and O-acetyl sialic acid-specific IgM is reported in childhood ALL [31, 32]. Disease-specific overexpression of 9-OAcSGs on peripheral blood mononuclear cells (PBMC) of children with [ALL, PBMC(ALL)] has been demonstrated using a lectin, achatinin-H, with specificity toward 9-O-AcSAα2-6GalNAc. Stimulation of PBMC(ALL) with achatinin-H through 9-O-AcSGs led to a lymphoproliferative response with a significantly increased interferon-gamma (IFN-γ) production inferring the fact that overexpressed 9-OAcSGs regulate signaling for ­proliferation,



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Fig. 2  Enhanced glycosylation, sialylation, and α2,6-linked sialic acids on erythrocytes in childhood ALL patients. (A) Representative profile of enhanced a glycosylation and (B) sialylation on RBC membranes determined by the DIG glycan detection kit. (C) Densitometry scoring of the spot intensities. (D) Fluorimetric quantitation of sialic acids expressed on RBC membrane and expressed in μg/108 cells. (E) Predominance of α2,6 linkage-specific sialic acid on ALL RBC membranes detected by the DIG glycan differentiation kit using SNA (panel 1) and MAA (panel 2). (F) Densitometric scorings of the intensities of spots. (G) Quantitative estimation of (8)9-O-Ac sialic acid A on RBC expressed in μg/108 cells determined fluorimetrically. (Reproduced with permission from Ghosh S, Bandyopadhyay S, Bhattacharya DK, Mandal C. Altered erythrocyte membrane characteristics during anemia in childhood acute lymphoblastic leukemia. Ann Hematol 2005,84:76–84.)

leading to the release of IFN-γ [33]. Excessive serum IFN-γ induced nitric oxide (NO) production in childhood ALL patients may enable the escape of immune surveillance and enabling survival of O-acetylated lymphoblasts in childhood ALL [34]. Erythrocytes from childhood ALL patients reveal altered expression of sialic acid which may play role in anemia in these patients [35]. Sialoglycoconjugates and glycosphingolipids are reported to be overexpressed in YAC-1 lymphoma [36]. Overexpression of sialoglycoconjugates recognized by α2,3-sialic acid-specific MAA has been reported with role in cancer progression in gastric cancer, Ehrlich tumor cells [37, 38]. Sialoglycoconjugates on pancreatic carcinoma were detected by salic acid-­specific lectin, limulin (Limulus polyphemus Lectin [LPA]; from Limulus polyphemus hemolymph) [39]. Ferritin- and rhodamine-­ conjugated

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LPA was used identify sialoglycoconjugates in human ­ endometrial ­adenocarcinoma [40]. Griffonia simplicifolia II agglutinin (GS2) were used to detect sialoglycoconjugate expression in primary benign mixed tumor (BMT) and a case of recurrent mixed tumor (MT) [41]. Deaminated neuraminic acid (KDN) is a biosynthetically independent member of the sialic acid with overexpression in fetal cord blood cells and some malignant tumor cells [42] including human ovarian teratocarcinoma with elevated activities of enzymes and aberrant presence of mannose, in the biosynthesis of KDN [43]. Sialytransferase (ST, Fig. 3), total and bound sialic acid, and sialoglycoconjugates are reported to be overexpressed in cancerous tissues of liver [46]. STs were reported to be overexpressed from cervix squamous cell carcinoma [47]. Uridine diphosphate-N-acetylglucosamine-2-epimerase (UDP-GlcNAc2-epimerase) which is a key enzyme in sialic acid biosynthesis has been reported to be involved in expression of sialylation in human Burkitt’s lymphoma cell line [48]. Deletion of UDP-GlcNAc 2-epimerase is known to be embryonically lethal and downregulation of sialyltransferase ST6GAL1 leads to decreased reprogramming efficiency [2].

3 Gangliosides Although predominant in the nervous system in healthy adults, complex gangliosides (Figs.  4–6) which are glycosphingolipids containing sialic acid including disialoganglioside with three glycosyl groups GD3 and

Fig. 3  (A) 5BO8: structure of human sialyltransferase ST8SiaIII from pdb (structure of human ST8SiaIII sialyltransferase provides insight into cell-surface polysialylation) [44]. (B) 4JS2 crystal structure of human beta-galactoside alpha-2,6-sialyltransferase 1 in complex with CMP (2015) [45].



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(G)

(H)

Fig.  4  Carbohydrate determinants of gangliosides commonly found in the central nervous system and cancer cells. CNS gangliosides: (A) GT1b. (B) GD1a. (C) GD1b. (D) GM1. Cancer-related gangliosides: (E) GD2. (F) GD3. (G) GM3. (H) Neu5Gc-GM3. Each structure is differentiated by the removal of one or more residues from GT1b, the glycosidic linkages specified on GT1b apply to all of the structures, including Neu5Gc-GM3. N-acetylneuraminic acid/Sialic acid—purple diamond; galactose—yellow circle; N-acetylgalactosamine—yellow square; glucose—blue circle; N-glycolylneuraminic acid/Neu5Gc—light blue diamond. (Reproduced with permission from Agostino M, Yuriev E, Ramsland PA. Antibody recognition of cancer-related gangliosides and their mimics investigated using in silico site mapping. PLoS One 2012,7:e35457.)

d­ isialoganglioside with two glycosyl groups GD2 have been reported to play role in aggressiveness, adhesion, angiogenesis, cell proliferation, invasion, migration, and in preventing immunosuppression of neuroectoderm-derived tumors including melanoma and neuroblastoma [85].

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Fig.  5  Schematic drawing of NeuAc GM3, a common ganglioside in vertebrate tissues. Purple diamond—N-acetylneuraminic acid/Sia, yellow circle—D-galactose, blue circle—D-glucose. (Reproduced with permission from open access article under Creative Commons Attribution License [CC BY]: Krengel U, Bousquet PA. Molecular recognition of gangliosides and their potential for cancer immunotherapies. Front Immunol 2014, 5:325.)

3.1  Structure and synthesis Overexpressed GD3 and GD2 highly reported from gliomas enable increased invasion, mobility, cell growth, with increased S-G2/M phase cells [86].

4  Gangliosides in tumors The b-series disialogangliosides GD3 and GD2 are overexpressed in astrocytoma, medulloblastoma, meningioma, and neuroblastoma [87]. b-Series gangliosides GD1b, GT1b, and GQ1b are less expressed in neuroblastoma tumors than in normal brain associated with an aggressive phenotype and a poor prognosis [87]. GD3 is known as the marker for melanoma overexpressed in primary melanoma tumors. In lung tumors, gangliosides including N-glycolyl-GM3, GM2, GM1, fucosyl-GM1, GD3, 9-O-acetyl-GD3, and GD2 are overexpressed (Table 1). The gangliosides GD3, 9-O-acetyl-GD3, and 9-O-acetyl-GT3 are overexpressed in about 50% of invasive ductal breast carcinoma (IDC) [88]. Gangliosides GD2 and GD3 are highly expressed in sarcomas of children, adolescents, and young adults [89]. The N-glycoslylated ganglioside Neup5Gc-GM3 has been detected in Wilms tumor (pediatric solid kidney tumors) [90], human renal carcinoma reveals expression of globosides and gangliosides. These tumor-associated gangliosides have been reported to regulate the signal transduction by growth-factor receptors including EGFR, FGFR, HGF, and PDGFR in the glycosynapse involving interaction with selectins and siglecs that recognize glycans involved in cell adhesion and immune regulation [91, 92] (Table 2).



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Fig. 6  Structure and representative reactions of ganglioside biosynthesis. (A) Chemical structure of gangliosides. (B) Schematic representation of ganglioside biosynthesis pathway. Published from as open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) [84].

220

Ganglioside

Structure

Cancer type

References

NeuAc GM3

αNeu5Ac(2-3)βDGal(1-4)βDGlc(1-1) Cer αNeu5Gc(2-3)βDGal(1-4)βDGlc(1-1) Cer

Melanoma, NSCLC, breast carcinoma, renal carcinoma Colon cancer, retinoblastoma, melanoma, breast carcinoma, neuroectodermal cancer, Wilms tumor Melanoma, neuroblastoma, SCLC, t-ALL, breast carcinoma, renal carcinoma SCLC, renal carcinoma

[49–55]

NeuGc GM3 GM2 GM1 GD3 GD2

βDGalNAc(1-4)[αNeu5Ac(23)]βDGal(1-4)βDGlc(1-1)Cer βDGal(1-3)βDGalNAc[αNeu5Ac(23)]βDGal(1-4)βDGlc(1-1)Cer αNeu5Ac(2-8)αNeu5Ac(2-3)βDGal(14)βDGlc(1-1)Cer βDGalNAc(1-4)[αNeu5Ac(28)αNeu5Ac(2-3)]βDGal(14)βDGlc(1-1)Cer

[53, 54, 56–59] [49, 50, 54, 55, 60–63] [49, 54, 62]

Melanoma, neuroblastoma, glioma, SCLC, [49, 51, 52, 61, 64–70] t-ALL, breast carcinoma Melanoma, neuroblastoma, glioma, SCLC, [49, 51, 52, 61, 62, 65, t-ALL 67, 68]

Reproduced with permission from open access article under Creative Common Attribution License (CC BY): Krengel U, Bousquet PA. Molecular recognition of gangliosides and their potential for cancer immunotherapies. Front Immunol 2014;5:325.

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Table 1  Gangliosides expressed in human cancer cells.



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Table 2  Gangliosides affecting the growth factor receptors EGFR and VEGFR. Ganglioside

Growth factor receptor

References

GM3 GM1 GM2 GM4 GD3 GD1a GT1b GM3 GD1a GD3

EGFR EGFR EGFR EGFR EGFR EGFR EGFR VEGFR VEGFR VEGFR

[71–74] [74, 75] [76, 77] [76] [76, 78] [74, 79] [74] [80, 81] [81, 82] [83]

Reproduced with permission from open access article under Creative Common Attribution License (CC BY): Krengel U, Bousquet PA. Molecular recognition of gangliosides and their potential for cancer immunotherapies. Front Immunol 2014;5:325.

O-acetyl-GD2 (OAcGD2) and 9-O-acetyl-N-acetylneuraminic acid (Neu5,9Ac2) but not OAcGD3 are reported from breast cancer cells, although none are reported from peripheral nerves [85]. GM3(Neu5Gc) as a tumor-specific antigen finds importance in targeting in for cancer immunotherapy [93]. GD3 synthase (GD3S) the regulatory enzyme of GD3 and GD2 synthesis playing role in tumorigenesis and cancer development finds importance as cancer target and potential new drugs for cancer therapy [94]. Ganglioside (GM3) has been reported from kidney tumor [95]. Overexpression of GD2 has been reported from breast cancer patients [96] (Fig. 7). Danoitti et al. [84] have summarized the main strategies in cancer immunotherapies involving gangliosides (Fig. 8) including (i) vaccination approaches with natural gangliosides or anti-idiotype mAbs, (ii) application of humanized anti-ganglioside mAbs, (iii) application of chimeric T-cell receptors (TCRs), (iv) cancer cell glycoengineering and targeted delivery of mAb and targeted cell killing, and (v) targeted delivery of anticancer drugs, small molecules, and cytotoxic agents using specific mAbs to gangliosides with specific cytolytic activity [84]. GD3 synthase gene (ST8Sia I) transfected in a normal melanocyte cell line enabled conversion of GM3 into GD3 and accumulated both GD3 and its acetylated form, 9-O-acetyl-GD3 and rendered the melanocytes more migratory [97]. Mammalian sialidases including Neu1, Neu2, Neu3, and Neu4 can remove α-glycosidically linked sialic acid from glycoproteins and glycolipids and Neu 3 and 4 play roles in ganglioside hydrolysis thereby

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Fig. 7  Pattern of GD2 localization in breast cancer. Cytoplasmatic (A; arrow) and membrane (B; arrow head) positivity for GD2 in breast cancer specimens. Few GD2 positive cells are visible within negative stroma (C). Strong GD2 staining in spindle cells in a metaplastic sarcomatoid breast cancer section (D). (Reproduced with permission from open access article under Creative Common Attribution License [CC BY]: Orsi G, Barbolini M, Ficarra G, Tazzioli G, Manni P, Petrachi T, Mastrolia I, Orvieto E, Spano C, Prapa M, Kaleci S, D’Amico R, Guarneri V, Dieci MV, Cascinu S, Conte P, Piacentini F, Dominici M. GD2 expression in breast cancer. Oncotarget 2017;8:31592–31600.)

influencing biological processes including cell adhesion, differentiation, growth, and motility have been reported of their pathological roles [98]. Overexpressed GD3S involved in biosynthesis of complex gangliosides, in estrogen receptor (ER)-negative breast cancer tumors, and reported to be associated with decreased survival while tumor necrosis factor (TNF) has been reported to differentially regulate ganglioside expression in breast cancer cell lines thus revealing association between inflammation, ganglioside expression, and tumor development [99]. Gangliosides are known to be shed by tumors and while IFN-γ are known to activate antitumor immune responses, reports suggest that in the tumor microenvironment (TME), gangliosides and IFN-γ might act in immunosuppression thereby promoting tumor progression [100].

EE PM

RE

Golgi complex

Lysosomes

ER

(A) Cell death

(1) Vaccine Antigens: (a) Natural gangliosides

Immune response

(b) Anti-idiotype monoclonal antibody (2) Humanized anti-ganglioside antibodies

(5) Targeted delivery of cytotoxic agents

(4) Cancer cell glycoengineering

(3) Chimeric T-cell receptors Cer Glc-Cer Lac-Cer GM3 GD3 GM2 GM3NPhAc

(B)

Cytotoxic agents (i.e., saporin)

Fig.  8  Simplified scheme of metabolic pathways of plasma membrane (PM)associated gangliosides and molecular targets for immunotherapies in cancer cells. (A) Representative pathway for intracellular trafficking of gangliosides. Black and red arrows represent the exocytic/biosynthetic and endocytic, recycling, and catabolic pathway, respectively. Dotted arrow and green arrow indicates the vesicular or protein-mediated transport of ceramide between ER and the Golgi complex and remodeling of glycosphingolipids by PM-associated glycohydrolases and glycosyltransferases, respectively. The hypothetical neobiosynthesis of GM3 at the Golgi complex and later transport to PM is represented. De novo-synthesized gangliosides or synthesized at the PM can undergo endocytosis through clathrin-independent vesicles, and recycled back to the PM directly by recycling endosomes (REs) or sorted from early endosomes (EEs) to the Golgi complex, where they may then be reglycosylated, or transported to the lysosomes for total or partial degradation. (B) Potential cancer immunotherapies using gangliosides as molecular targets. Schematic representation depicting the main cancer immunotherapies involving gangliosides (1–5). (Published from Daniotti JL, Vilcaes AA, Torres Demichelis V, Ruggiero FM, Rodriguez-Walker M. Glycosylation of glycolipids in cancer: basis for development of novel therapeutic approaches. Front Oncol 2013;3:306 as open-access article distributed under the terms of the Creative Commons Attribution License (CC BY, 96).)

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5  Gangliosides and biotherapies Gangliosides as tumor-associated antigens are overexpressed in cancers and anti-tumor-associated ganglioside mAbs have been developed for d­ iagnosis, monitoring, and treatment of cancer patients (Table  3). Ganglioside GD2, abnormally expressed on neuro-ectoderm-related cancer cells, melanoma, neuroblastoma, small-cell lung cancer, breast cancer, finds importance in mAb therapy. Dinutuximab, a chimeric, human-murine anti-GD2 mAb, is approved by the US FDA and is used in combination with granulocyte-­ macrophage colony-stimulating factor (GM-CSF), interleukin-2 (IL-2), and isotretinoin (13-cis-retinoic acid) in targeting pediatric high-risk neuroblastoma (HR-NB) [145]. Anti-GD2-based targeting by antibodies and vaccines finds importance in treatment of HR-NB, leading to improvised survival in patients in their first remission and finds application in chemorefractory and relapsed neuroblastoma [145]. Chimeric antibody c.8B6 ­recognizing ­O-acetyl-GD2 finds application in treatment of HR-NB. Racotumomab, an anti-idiotypic antibody known commercially as Vaxira, recognizing particularly Neup5Gc-GM3, overexpressed in a variety of solid tumors [146] is being tested for maintenance therapy for advanced nonsmall-cell lung cancer [147]. Neup5Gc-GM3/very small size proteoliposomes (VSSP) vaccine is in phase I/II clinical trial for treatment of patients with advanced metastatic melanoma in subcutaneous [148] and intramuscle applications in breast cancer patients [148]. NeuGcGM3 ganglioside used as cancer vaccine combined with the outer membrane protein complex of (N meningitides) when administered with adjuvant Montanide ISA51 revealed induction of ­anti-NeuGcGM3 antibodies and efficient as cancer vaccine in breast cancer patients [149].

6  Sialic acid-Siglec axis and cancer Aberrant sialic acid-Siglec interactions have been reported from infection, autoimmunity, and cancer and are an emerging target to target disorders [150]. Siglec interaction with sialic acid on a different cell or protein/particulate is termed as trans interaction or with same cell sialic acids is termed as cis interaction (Fig. 9). Aberrant glycosylation of multiple cancers and its influence on cancer progression and metastasis are well known. Increased sialylation, α2,3; α2,6, and α2,8-linked sialic acids, in cell surface and serum act as serving biomarkers for cancer detection, progression, and treatment responses.

mAb anti-GM3NPhAc 2H3 Glycoengineered GM3NPhAc-KLH and ManNPhAc N-glycolyl GM3 ganglioside vaccine Anti-idiotype mAb (racotumomab) Addition of GM3 mAb DMF10.167.4 (in vitro) mAb DMF10.167.4 Hu-mAb BIW-8962 and KM8927

Passive Active

[101] [102, 103]

Active Active Passive – Passive Passive

[104, 105] [106] [107] [108] [109] [110, 111]

GM2-KLH/QS-21 vaccine mAb R24

Active Passive Active Passive Active Passive – – Passive – Active Passive Passive Passive

[112, 114] [115] [116] [117] [118, 119] [120] [121] [122] [123] [124] [125–128] [129] [123], [130–132] [133, 134]

Type of treatment

GM3

Melanoma

GD3

GD2

Bladder cancer Lymphoma Melanoma and SCLC Multiple myeloma and SCLC Melanoma Melanoma

Melanoma

Neuroblastoma

Anti-GD3 chimeric sFv-CD28/T-cell mAb anti-idiotype (BEC2) R24 anti-anti-idiotype mAb mAb R24-saporin (in vitro) Chimeric 14.18 Ab-IL-2 (in vitro) Hu-mAb L72 Immunotoxin 14.G2a mAb-ricin A (in vitro) mAb anti-idiotype (1A7) Immunocytokine chimeric 14.18 mAb-IL-2 M-mAb 3F8/Hu-mAb 3F8 M-mAb 14.G2a/M-mAb 14.G2a + IL-2

Continued

225

References

Type of tumor

Sialibiology in cancer

Type of acquired immunity

Ganglioside

GM2



Table 3  Immunotherapeutic strategies involving tumor associated gangliosides.

226

Ganglioside

Type of tumor

Fucosyl-GM1

SCLC

9-O-Ac-GD2

SCLC, lymphoma, neuroblastoma, ovarian carcinoma

Type of treatment

Immunotoxins 14.G2a mAb-ricin A/ BW704dgA 14.G2a chimeric T-cell receptors Anti-idiotype mAb (ganglidiomab) Immunocytokine chimeric 14.18 mAb-GMCSF (in vitro) Immunocytokine chimeric 14.18 mAb-IL-2 mAb F12 and F15 Fucosyl-GM1-KLH vaccine mAb 8B6

Type of acquired immunity

References

Passive

[135, 136]

Passive Active –

[137, 138] [139] [140]

Passive Passive Active Passive

[141] [142] [143] [144]

Reproduced from Daniotti JL, Vilcaes AA, Torres Demichelis V, Ruggiero FM, Rodriguez-Walker M. Glycosylation of glycolipids in cancer: basis for development of novel therapeutic approaches. Front Oncol 2013;3:306 as open-access article distributed under the terms of the Creative Commons Attribution License (CC BY).

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Table 3  Immunotherapeutic strategies involving tumor associated gangliosides—cont’d



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Fig.  9  Sialic acids, linkages, and interactions. (A) Chemical structure of sialic acids Neu5Ac and Neu5Gc. (B) α2,3; α2,6, and α2,8-linked sialic acids. (C) Trans and (D) Cis interactions of Siglecs with sialic acids. (Reproduced with permission from Lübbers J, Rodríguez E, van Kooyk Y. Modulation of immune tolerance via siglec-sialic acid interactions. Front Immunol 2018,9:2807.)

Siglec receptors have different binding affinities for different linkage and modifications of sialic acids with most Siglecs revealing preference for either α2,3, α2,6, or α2,8-linked sialic acid, or redundant specificity toward more linkages [151, 152] and the Siglec-sialic acid interaction leads to immune modulatory effect and is regulated through downstream signaling pathways. Siglec-5 to Siglec-11, acting as the inhibitory Siglecs, with immunoreceptor tyrosine-based inhibition motif in their cytoplasmic domains, which can be phosphorylated by the Src family, thereby creating a binding site for the tyrosine phosphatases SHP-1 and SHP-2, causing dephosphorylation of downstream targets and leading to ubiquitination, internalization, and phosphorylation of the receptor [153, 154]. Signaling of different Siglecs through the binding of sialic acids or antibody cross-­linking can lead to both an inflammatory or tolerogenic state in phagocytes. Antibodies against Siglec-3 and -7 inhibit the proliferation of myeloid cells [155] while monocytes treated with Siglec-3 antibodies show increased production of the pro-inflammatory cytokines IL-1β, TNF-α, and IL-8 [70]. Siglec-1 is a non-signaling Siglec that internalizes upon ligand binding. Siglecs can also exert their immune modulatory effects by altering Toll-like receptor (TLR) signaling. Siglec CD169 acts as an

228

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adhesion molecule and a facilitate r­ecognition and internalization of sialic acid decorated apoptotic bodies and exosomes derived from tumors, and thereby stop antitumor immunity [8]. Antibodies against Siglecs are being explored for the treatment of different cancer types. In acute lymphoblastic lymphoma (ALL), the FDA approved inotuzumab ozogamicin (Besponsa), a mAb against Siglec-2 coupled to calicheamicin, a toxic agent targets Siglec-2 positive B-lymphoblasts and causes cell death but reveals adverse effects of myeloid suppression [156]. In acute myeloid leukemia (AML), Siglec-3 coupled to calicheamicin [157, 158] are used to target myeloid blasts but also reveals adverse effects. Siglec-3 targeting chimeric antigen receptor T-cells can induce CD8+ T cell degranulation against primary AML and AML cell lines [113, 159]. Strategies involving modifying the phosphorylation status of Siglec-3 and Siglec-9, like dephosphorylation of the receptos, can lead to increased immunity of moDCs, when treated with dasatinib a nonreceptor tyrosine kinase (SRC) inhibitor that dephosphorylates Siglec-3 and Siglec-9 [160]. Strategies involving blocking of Siglecs could remove inhibitory effects on phagocytes leading to their improved maturation, and cause tumor-­ specific T cell responses [161]. Lübbers et al. in 2018 have summarized the different Siglec-based strategies in control of cancer (Fig. 10). (A) Siglec-2 mAbs linked to immunotoxin can induce apoptosis of Siglec-2-expressing ALL cells thus killing the cancer cells [156]. (B) Antitumor immunity through Siglecs has been tested by targeting HER2 with a mAb fused to a sialidase [163] that specifically removes sialic acid ligands bound by Siglec-7 and Siglec-9 thereby leading to NK cell-mediated killing of HER2 positive tumor cells [163]. Sialidase coupled to the HER2 mAb targeting HER2 expressing cancer cells decreases sialic acid expression, reduce T reg induction and induced T cell activation and initiates NK cell killing [163], (C) nanoparticles designed with sialic acid inhibitor P-3Fax-Neu5Ac targeted to tumor cells inhibit sialic acid expression on the tumor cells, thereby decreasing metastasis and inducing tumor cell killing [164, 165], and (D) sialylated antigens target dendritic cells (DC) to remove T regulatory cells or T regs [166]. (E) Antigen-specific B cell apoptosis induction by Siglec-engaging tolerance inducing antigenic liposomes (STALs) targeting Siglec-2 together with an antigen that inhibits B cell receptor (BRC) signaling on B cells [162] thereby leading to apoptosis of B cells (Fig. 10). Modifications of the natural sialic acid ligand have led to generation of sialic acid mimetics (SAMs). They have been reported to express improved binding affinity and selectivity toward Siglecs. Bioorthogonal chemistry approaches are enabling the generation of cells with different sialic acid



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Siglec-2 Immunotoxin

Targeting Siglec-2

Apoptosis of cancer cells

Acute lymphoblastic leukemia (A) HER2

Sialidase immunoconjugate Targeting Her2

(B)

Sialic acid NK mediated killing

Cancer cells Sialic Acid Inhibitor P-3Fax -Neu5Ac

TRP-1

αTRP-1 Antibody

(C)

Metastasis

Tumor targeting nanoparticles

Siglec-E

(D)

Induction of regulatory T cells (Antigen specific)

Murine DCs

Sialylated antigens

Antigen BCR signalling inhibition

Liposome

Sialylated glycans

STALs Siglec-engaging tolerance-inducing antigenic liposomes

(E)

Siglec-2 B cells

BCR

Apoptosis B cells B cell tolerance (Antigen specific)

Fig.  10  Sialic acid-Siglec axis and cancer, allergies, autoimmune diseases and their treatment (A–E) [162]. (Reproduced from Lübbers J, Rodríguez E, van Kooyk Y. Modulation of immune tolerance via siglec-sialic acid interactions. Front Immunol 2018,9:2807.)

modifications with increased binding toward the different Siglec family members. SAMs when presented decorated on nanoparticles, polymers, and living cells by bioorthogonal synthesis have been reported to enable understanding of their role in immune modulation, signaling pathways, and applications in therapy [150].

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7  Sialyl Tn in cancer Aberrant glycosylation in cancer cells is exemplified by the expression or overexpression of the onco-fetal sialyl-Tn (sTn) antigen or SialylThomsen-nouveau antigen (Fig. 11) which is a truncated O-glycan containing a sialic acid α-2,6 linked to GalNAc α-O-Ser/Thr related with poor prognosis in cancer.They are reported from gastric, colon, breast, lung, oesophageal, prostate, endometrial cancer, and invasive bladder cancer [167]. Sialyltransferase ST6GalNAc1, and loss of heterozygosity (LOH) mutations in the COSMC core (1 β3-Gal-T-specific molecular chaperone gene) are involved in the biosynthesis of sialyl Tn antigen, a glycan structure failing any further processing, and blocking the further posterior elongation of the O-glycan chains and are involved in tumor development and could be potential targets in early stage cancer [168]. Truncated O-glycans in cancer may result from altered expression of glycosyltransferase, overexpression of sialyltransferase ST6GalNAc1, hypermethylation or mutations in COSMC [169–171], mis-localization of GalNAc-transferases from the Golgi to the ER [172], and pH levels alterations [173] and are frequently associated with tumor development. C1Ga1T1 enzyme catalyzes the first elongation step of the Tn antigen to produce the core 1 O-glycan structure (T antigen) and the COSMC chaperone protein folds it [170, 174–176], therefore, LOH mutations in C1GALT1 COSMC

GALNT Ser/Thr

Ser/Thr

Tn antigen

Core extension Complex glycans Normal cells

ST6GalNAc1

Sialic acid Ser/Thr

GalNAc

sTn-antigen Cancer cells

Fig. 11  Schematic representation of biosynthesis of the Sialyl-Tn (sTn) antigen in cancer cells. (Reproduced from Munkley J. The role of Sialyl-Tn in cancer. Int J Mol Sci 2016,17:275, by Creative Commons by Attribution (CC-BY) license (http://creativecommons.org/licenses/ by/4.0/).)



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COSMC gene prevent its proper folding and activity. sTn is an onco-fetal antigen expressed by fetal organs and amniotic fluids, and low-level expression in adult normal tissues. Overexpression of sTns has been reported from gastric, pancreatic, colon, melanoma, cervical, ovarian, and breast cancer. MUC1, CD44, integrin β1, and osteopontin proteins have been identified as sTn carrier proteins with roles in cell adhesion, migration, or chemotaxis. sTn in serum as O-glycoprotein shedded antigen or secreted from tumors is a prognostic marker and has been associated with decreased overall survival, aggressiveness, metastasis, reported from tumor cells in metastatic bladder and colorectal cancers [177] and targeting sTn carrier proteins finds importance in cancer therapy in ovarian cancer [178].

8  Sialylransferase and cancer Certain pathogenic bacteria camoflaging with sialic acid can modulate the innate immune response and evade the immune system through myelomonocytic lineage inhibitory receptor Siglec-9 [179]. Metastasizing mammary tumors in rats have revealed elevated levels of serum sialoglycoconjugate and serum ST which may play role in the escape of immune response against metastasizing tumor cells [180]. NEU3, preferentially acts on gangliosides, is involved in cell differentiation, motility, and tumorigenesis and reported of elevated expression in human cancers, including colon, renal, prostate, and ovarian cancers and finds importance in inflammation and tumor development [181]. CMP-NeuAc: Gal β 1,4GlcNAc α 2,6 sialyltransferase (α 2,6-ST) is regulated in the development process, and reveals tissue-specific expression and plays role in progression of tumor development and metastasis and is reported from human brain tumors. α 2,6-ST and α 2,6-linked sialoglycoconjugates were detected of their expression in meningiomas, chordomas, and craniopharyngiomas by anti-rat α2,6-ST antibody and the α 2,6-linked sialic acid-specific lectin, SNA by histochemical studies, and a human α 2,6-ST-specific cDNA probe for Northern analysis [182]. Siglecs expressed by immune cells can reveal functions of inhibitory receptors that regulate inflammation mediated by damage-­ associated and pathogen-associated molecular patterns (DAMPs and PAMPs) that are involved in innate immune responses, adhesion, and phagocytosis. They can associate with the immunoreceptor tyrosine-based activation motif-­containing DAP12 adaptor and inhibit immune cells both by binding to cis ligands expressed in the same cells and pathogen-derived

232

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s­ialoglycoconjugates enabling maintenance of tolerance in B lymphocytes, modulating the activation of both conventional plasmacytoid DC, and regulate T cell activity. Siglecs modulate immune responses, influencing almost every cell in the immune system, and are of relevance both in health and disease [183].

9  Sialidase as cancer targets Sialidase are known to act on sialiglycoconjugates and removes sialic acid that bears an important role in physiological and pathological processes. Cancers have been reported to overexpress sialidase that finds importance in cancer diagnosis. A novel benzothiazolylphenol-based sialic acid derivative (BTP-Neu5Ac) as a fluorescent sialidase substrate. BTP-Neu5Ac enabled visualization of sialidase activities sensitively and selectively in acute rat brain slices and found importance in histochemical imaging of sialidase [184]. Lysosomal sialidase Neu1 expressed in colony forming unit-­erythroid precursors and K562 erythroleukemic cells is indicative of the differences in sialoglycoconjugate and sialidase expression in tumor cells with that of normal cells [185]. SABL, isolated from oocytes of (R catesbeiana), is leczyme and has both lectin and ribonuclease (RNase) activities and reported of its antitumor activity that is specific to tumor cells and finds importance as potential anticancer drug development [186].

10 Selectins Aberrant glycosylation is associated with promoting tumor progression and invasion, increased immune cell evasion, drug evasion, drug resistance, and vascular dissemination, leading to metastases. Hypersialylation of cancer cells by overexpressed STs causing glycosidic linkages (α2-3, α2-6, or α2-8) to the underlying glycan chain and generation of selectin ligands that requires sialyl-Lewis X and its structural isomer sialyl-Lewis A (Fig.  12), expression synthesized by the combined action of α1-3-­fucosyltransferases, α2-3-sialyltransferases, β1-4-galactosyltranferases, and N-acetyl-β-glucosaminyltransferases is known in cancer [9]. The α2-3-sialyltransferases ST3Gal4 and ST3Gal6 find importance in generating functional E- and P-selectin ligands and these STs have been reported to be overexpressed in solid tumors and multiple myeloma. Thus, targeting selectins and their ligands, use of inhibitors like glycomimetic



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Fig.  12  Structure of SLex (A) and its structural isomer SLea (B). (Reproduced with permission from Natoni A, Macauley MS, O’Dwyer ME. Targeting selectins and their ligands in cancer. Front Oncol 2016;6:93 under the terms of the Creative Commons Attribution License (CC BY, 9).)

drugs and antibodies, enzymes involved in their synthesis are strategies to halt cancer progression [9].

11 Discussion Aberrant glycosylation, increased sialylation, as terminal sugars attached to proteins or lipids, altered sialylation enzymes activity, including expression and activity of STs and sialidases, overexpressed selectins, expression of sTns affecting cell signaling and contributes to the aberrant sialome in cancer and forms the hallmark in cancer, increasing invasiveness and metastatic potential in cancer. Sialic acid as shedded antigen in serum observed in malignant tumors may be due to activity of sialidases. Several sialylation modifications have been reported as diagnostic and prognostic markers. ST inhibitors could effectively modulate cell surface sialylation. Cell surface overexpressed sialylation finds importance in targeting of cancer and

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Sialic acids and sialoglycoconjugates in the biology of life, health and disease

targeted delivery of drugs and anticancer agents are being tested to control cancer and its progression. Drugs and molecules are being researched that can inhibit sialylation and thus prevent cancer metastasis and invasion. Selectin antagonists bimosiamose (TBC-1269) and GSC-150 have been applied in the treatment of inflammatory disorders, autoimmune diseases, and metastatic cancers to reduce leukocyte trafficking to inflamed tissues and to prevent metastasis [187, 188].

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Further reading [189] Agostino  M, Yuriev  E, Ramsland  PA. Antibody recognition of cancer-related gangliosides and their mimics investigated using in silico site mapping. PLoS One 2012;7:e35457. [190] Taniguchi  M, Wakabayashi  S. Shared antigenic determinant expressed on various mammalian melanoma cells. Gann 1984;75:418–26. [191] Frost JD, Hank JA, Reaman GH, Frierdich S, Seeger RC, Gan J, et al. A phase I/IB trial of murine monoclonal anti-GD2 antibody 14.G2a plus interleukin-2 in children with refractory neuroblastoma: a report of the Children’s Cancer Group. Cancer 1997;80:317. [192] Lajaunias  F, Dayer  JM, Chizzolini  C. Constitutive repressor activity of CD33 on human monocytes requires sialic acid recognition and phosphoinositide 3-kinase-­ mediated intracellular signaling. Eur J Immunol 2005;35:243–51.

CHAPTER 9

Sialic acids and sialoglycans in endocrinal disorders 1 Introduction The endocrine network comprises of endocrine glands (Fig. 1) and nerves and is located throughout the body. Endocrine glands synthesize and release a plethora of hormones that play an important role in the regulation of physiological processes of the body, including growth and development, metabolism, electrolyte balances, reproduction, and maintaining homeostasis. The hypothalamus producing different hormones act on the pituitary gland, stimulating the release of pituitary hormones. Some of the pituitary hormones act on several other glands in the body, whereas some of them directly act on their target organs. Other endocrine glands include the thyroid gland, producing thyroid hormone, the parathyroid glands producing parathyroid hormone, adrenal glands producing predominantly cortisol, pancreas producing insulin and glucagon, the gonads including ovaries and testes, which produce sex hormones. Hormones play a role in regulating hormonal cascades including hypothalamic hormone, pituitary hormones, and target gland hormones. Endocrine disorders reveal a wide spectrum of diseases including autoimmune disorders including type 1A diabetes, Graves’ disease, Hashimoto thyroiditis, Addison disease causing autoimmune-mediated tissue destruction and tumors. Cancer still remains to be a major killer across the globe. An estimated total number of cases of 18,078,957 of incidence of cancer have been reported worldwide by the World Health Organization (WHO) in 2018 (Fig. 2). The age-standardized incidence and mortality rates worldwide, both sexes and ages as reported by WHO in 2018 is depicted in Fig. 3 indicating the killer disease and its claim on human lives. Endocrine tumors are a rare group of tumors initiating in the endocrinal cells most of which are benign while some are malignant.They are classified according to the particular endocrine glands involved, type of cell from where cancer initiates and part of the body affected. Endocrine tumors lead Sialic Acids and Sialoglycoconjugates in the Biology of life, Health and Disease https://doi.org/10.1016/B978-0-12-816126-5.00009-3

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Fig. 1  Endocrine glands in human.

Fig.  2  Estimated number of new cases in 2018, worldwide, all cancers, both sexes, all ages. (Reproduced with permission from Ferlay J, Ervik M, Lam F, Colombet M, Mery L, Piñeros M, Znaor A, Soerjomataram I, Bray F. Global cancer observatory: cancer today. Lyon: International Agency for Research on Cancer; 2018. Available from: https://gco.iarc.fr/ and from Lloyd RV, Osamura RY, Klöppel G, Rosai J. World Health Organization classification of tumours of endocrine organs. 4th ed. Lyon: International Agency for Research on Cancer; 2016.)

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Fig. 3  The age-standardized incidence and mortality rates worldwide, both sexes and ages in 2018. (Reproduced with permission from Ferlay J, Ervik M, Lam F, Colombet M, Mery L, Piñeros M, Znaor A, Soerjomataram I, Bray F. Global cancer observatory: cancer today. Lyon: International Agency for Research on Cancer; 2018. Available from: https://gco. iarc.fr/ and from Lloyd RV, Osamura RY, Klöppel G, Rosai J. World Health Organization classification of tumours of endocrine organs. 4th ed. Lyon: International Agency for Research on Cancer; 2016.)

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to disbalance and collapse of homeostatic control mechanisms, with affected and increased hormone secretion. Carcinogenesis although have been understood to be resulting from genetic mutations, several cancers reveal alteration in synthesis, secretion, frequency, and types of different hormones including breast cancer associated with estrogen, progesterone, prolactin hormones, estrogen, testosterone in prostate cancer, estrogen in endometrial cancer and affected thyroid stimulating hormone, TSH, ­tri-iodothyronine (T3), and thyroxine (T4) hormones in thyroid cancer. Thyroid cancer reveals to be the most common endocrine malignancy leading to lethal solid cancers. An estimated total number of cases of 18,078,957 of incidence of cancer has been reported worldwide by WHO in 2018 (Fig.  2). The age-standardized incidence and mortality rates worldwide, both sexes and ages as reported by WHO in 2018 are depicted in Fig. 3. Table 1 highlights the classification of different types of endocrinal tumors by WHO.

Table 1  WHO has classified endocrinal tumors.

Pituitary gland

Pituitary adenoma Somatotroph adenoma Lactotroph adenoma Thyrotroph adenoma Corticotroph adenoma Gonadotroph adenoma Null cell adenoma Plurihormonal and double adenomas Pituitary carcinoma Pituitary blastoma Craniopharyngioma Neuronal and paraneuronal tumors Gangliocytoma and mixed gangliocytoma-adenoma Neurocytoma Paraganglioma Neuroblastoma Tumors of the posterior pituitary Mesenchymal and stromal tumors Meningioma Schwannoma Chordoma Hemangiopericytoma/Solitary fibrous tumor Hematolymphoid tumors Germ cell tumors Secondary tumors



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Table 1  WHO has classified endocrinal tumors—cont’d

Thyroid gland

Parathyroid gland Adrenal cortex

Follicular adenoma Hyalinizing trabecular tumor Other encapsulated follicular-patterned thyroid tumors Tumors of uncertain malignant potential Noninvasive follicular thyroid neoplasm with papillary-like nuclear features Papillary thyroid carcinoma Follicular thyroid carcinoma Hürthle (oncocytic) cell tumors Poorly differentiated thyroid carcinoma Anaplastic thyroid carcinoma Squamous cell carcinoma Medullary thyroid carcinoma Mixed medullary and follicular thyroid carcinoma Mucoepidermoid carcinoma Sclerosing mucoepidermoid carcinoma with eosinophilia Mucinous carcinoma Ectopic thymoma Spindle epithelial tumor with thymus-like differentiation Intrathyroid thymic carcinoma Paraganglioma and mesenchymal/stromal tumors Paraganglioma Peripheral nerve sheath tumors Benign vascular tumors Angiosarcoma Smooth muscle tumors Solitary fibrous tumor Hematolymphoid tumors Langerhans cell histiocytosis Rosai-Dorfman disease Follicular dendritic cell sarcoma Primary thyroid lymphoma Germ cell tumors Secondary tumors Parathyroid carcinoma Parathyroid adenoma Secondary, mesenchymal and other tumors Adrenal cortical carcinoma Adrenal cortical adenoma Sex cord-stromal tumors Adenomatoid tumor Mesenchymal and stromal tumors Myelolipoma Schwannoma Hematolymphoid tumors Secondary tumors Continued

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Table 1  WHO has classified endocrinal tumors—cont’d

Adrenal medulla and extraadrenal paraganglia

Neuroendocrine pancreas

Inherited tumor syndromes

Phaeochromocytoma Extra-adrenal paragangliomas Head and neck paragangliomas Sympathetic paraganglioma Neuroblastic tumors of the adrenal gland Composite phaeochromocytoma Composite paraganglioma Nonfunctioning (non-syndromic) neuroendocrine tumors Insulinoma Glucagonoma Somatostatinoma Gastrinoma VIPoma Serotonin-producing tumors with and without carcinoid syndrome ACTH-producing tumor with Cushing syndrome Pancreatic neuroendocrine carcinoma (poorly differentiated neuroendocrine neoplasm) Mixed neuroendocrine-non-neuroendocrine neoplasms Mixed ductal-neuroendocrine carcinomas Mixed acinar-neuroendocrine carcinomas Multiple endocrine neoplasia type 1 Multiple endocrine neoplasia type 2 Multiple endocrine neoplasia type 4 Hyperparathyroidism-jaw tumor syndrome Von Hippel-Lindau syndrome Familial paraganglioma-phaeochromocytoma syndromes caused by SDHB, SDHC, and SDHD mutations Neurofi bromatosis type 1 Carney complex McCune-Albright syndrome Familial non-medullary thyroid cancer Cowden syndrome and PTEN-related lesions Familial adenomatous polyposis and APC-related lesions Non-syndromic familial thyroid cancer Werner syndrome and Carney complex DICER1 syndrome Glucagon cell hyperplasia and neoplasia

Reproduced with permission from WHO: Lloyd RV, Osamura RY, Klöppel G, Rosai J. World Health Organization Classification of Tumors of Endocrine Organs, 4th ed. IARC, Lyon, 2016.



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2  Sialylations and endocrines Sialylations of glycoproteins have been associated with health and disease. Altered sialylations like acetylated sialylated proteins [3–8] and altered sialyltransferases [9, 10] are reported from different disorders and find importance as disease markers and potential therapeutic targets. While in the normal menstrual cycle of healthy women sialylations reveal an “M” shaped expression [11], sialylated apolipoprotein C-III (apo-CIII) is associated as a risk factor in cardiovascular diseases and pro-inflammatory properties in type 2 diabetic individuals [12]. Overexpression of sialylated moieties have been reported in hypo and hyperthyroidism [13]. Sialylated thyrotropin (TSH) hormone reveals enhanced hormone bioactivity [14]. Pathophysiology of cancer including endocrinal cancer reveals altered sialic acid expression playing a dominant role in enhancing tumor growth, metastasis, evading immune surveillance, escaping the apoptotic pathways, leading to cancer cell survival, and resistance to therapy [3–8]. Deletion in the human gene (cmah) coding for the enzyme cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMP-Neu5Ac hydroxylase) prevents them from synthesizing N-glycolylneuraminic acid (Neu5Gc). However, in malignancy, Neu5Gc-sialoconjugates are reported from the gangliosides [15]. Sialylation as disease marker for endocrinal disorders are reported in cancers of (i) ovary, (ii) pancreas, (iii) thyroid, (iv) adrenal gland, (v) pituitary, and (vi) hypothalamus [16].

3  Sialylations in endocrinal disorders 3.1 Ovary Ovarian cancer forms the major cause of death amongst cancers of the reproductive system and ranks fifth cause of mortality in women, being lethal when diagnosed late and there is need of markers for early detection of the disease and detection of inflammation and progression. Increased sialylation [17–31] and synthesis of branched sialylated moieties by overexpressed sialyltransferase (ST) branching enzyme MGAT5 [18], altered DNA methylation [19], overexpressed serum sialoglycoproteins [28], ST6GalI (β-galactosamide α2,6-sialyltranferase I), overall overexpressed sialylations in the sialome [21], altered mRNA expressions of α2,3-­sialyltransferase ST3GalI, ST3GalIII, ST3GalIV, ST3GalVI, and α2,6-­ sialyltransferase ST6GalI together with increased expression of ST3GalI

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leading to increased α2,3-linked sialylation [22] has been reported in ovarian cancer leading to tumor progression, metastasis, and resistance to therapy thereby indicating the role of sialic acid as biomarker for ovarian cancer detection and monitoring [17–25]. Serum component fucosylation, α2–6 sialylation, β1–4 branching, and β1–6 branching have been reported to be markers in cancers including pancreatic, serous ovarian and prostate cancer [32]. Serine hydroxymethyltransferase 1 (SHMT1) has been reported to promote cancer progression in ovarian cancer and cell migration in culture and mice models [33]. SHMT1 promoter with binding sites of transcription factor Wilms tumor 1 (WT1) revealed their role in ovarian cancer cells. Knock out of WT1 lead to reduced SHMT1 expression and metabolomic analysis revealed reduced levels of several metabolites including Neu5Ac, with downregulated IL-6 and 8 indicative of regulation by SHMT1 in high-grade serous (HGS) ovarian cancer accounting for 90% of all mortality from ovarian cancer [33]. Hypersialylation, altered expression of sialyltransferases (STs) and siglecs in malignant sialome are reported to be associated with tumor progression and suppressing antitumor responses in gynecologic cancers, including ovarian cancer. Therefore the balance between sialosides liberated by specific STs and siglecs expressed on leukocytes might regulate antitumor immunity [34]. Sialyltransferase ST3Gal-I catalyzing transfer of sialic acid from cytidine monophosphate-sialic acid to galactose-containing substrates has been revealed to promote cell migration, invasion, and TGF-β1-induced ­epithelial-mesenchymal transition or EMT and confer paclitaxel resistance in ovarian cancer [35]. Sialylation of platinum drug cisplatin receptors by overexpressed ST6Gal-I in ovarian cancer has been implicated to play a role in metastasis, poor prognosis and conferring chemoresistance, and act as a potential therapeutic target for platinum-resistant tumors [36]. Several ST together with ST6Gal-I (β-galactosamide α2,6-­ sialyltransferase-I) overexpressed in ovarian cancers and the sialome of biological fluids from ovarian cancer patients and cell lines reveal as potential biomarker reported by tandem mass spectrometry (MS) analysis [21]. Overexpression of GM3 synthase (α2,3-sialyltransferase, ST3Gal-V) could inhibit anticancer compound Taxol-mediated signaling of caspase-3 activation. This indicated that GM3 synthase overexpression could prevent apoptosis and provide resistance to apoptotic effects of Taxol [37]. The O ­ -linked glycosylated moieties from ascites fluid from patients with ovarian cancer



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revealed sialylated core 1 and 2 structures, sulfated core 2 structures, terminating disialic acid NeuAc-NeuAc on the O-linked core 1 and 2 oligosaccharides, epidermal growth factor (EGF)-associated O-linked fucose structure, further emphasizing the increased sialylated moieties in ovarian cancer [38]. Using silver nanoparticles (NPs) as substrates sialic acid in the saliva has been detected as a marker in ovarian cancer patients as detected by Raman spectroscopy [39]. Modification of kallikrein 6 (KLK6) with α2,6-linked sialic acid has been reported as a potential biomarker in ovarian cancer [23]. Ascites, plasma, and serum from ovarian cancer patients reveal increased sialic acids, disialic acids, gangliosides, altered sialylation of alpha-1 protease inhibitor, increased lipid- associated sialic acid (LSA) and increased sialylation in the cyst and peritoneal fluids [24–31]. Overexpression of fully sialylated C4-binding protein (FS-C4BP) in the serum in patients with epithelial ovarian cancer (EOC) finds importance as a disease marker in ovarian clear cell carcinoma revealed by studies using liquid chromatography-hybrid mass spectrometry (UPLC-MS/ MS, 42). N ­ -acetylneuraminic acid-9-phosphate, 5′-methioadenosine, uric acid-3-­nucleoside, pseudouridine, L-valine, succinic acid, L-proline, and β-­ nicotinamide mononucleotide identified from urine of ovarian cancer patients by ultrahigh-performance liquid chromatography/time-of-flight mass spectrometry (UPLC-Q-TOF-MS) analysis followed by data analysis by principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) revealed their role in disease progression and finds importance as biomarker in clinical staging of patients [40]. Extracellular vesicles (EVs) are heterogeneous messengers supporting cancer growth and spread by enabling communication between cancer cells and the normal cells and the cancer microenvironment and have gained considerable importance in understanding the biology of cancer establishment and growth. EVs from ovarian carcinoma OVMz cells revealed expression of tumor susceptibility genes Tsg101, CD63, CD9, annexin-I, and galectin-3 binding protein (LGALS3BP) enriched with sialylated N-glycans and membrane-expressed markers including calnexin, GRASP65, GS28, LAMP-1, L1CAM indicative of the potential importance of glycosylated moieties of EV as diagnostic biomarker in ovarian cancer [41]. Serum sialic acid and hydroxyproline finds importance as a diagnostic biomarker as compared to human epididymis protein 4 and carbohydrate antigen 125 in ovarian cancer [42]. Serum sialic acids, glycoproteins, protein bound, hexose, hexosamine, fucose, sialic acid, and carbohydrate are reported to be a diagnostic marker in ovarian cancer [43].

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3.2 Pancreas Overexpression of α2,6-sialic acids in pancreatic adenocarcinoma cell lines mediating increased adhesion to the extracellular matrix (ECM) while overexpressed α2,3-sialic acids contributing to increased migration of cells lead to metastatic effects in cancer [44]. Altered glycosylation of human alpha-acid glycoprotein with seven glycan moieties with α2–6 linked sialic acids, including five fucosylated moieties, have been reported in pancreatic ductal adenocarcinoma (PDAC 48). Reduced expression of Neu2 in different PDAC cells, patient tissues, reveals a correlation with disease pathophysiology. Overexpression of Neu2 induced apoptosis in drug-resistant Human pancreatic cell lines MIAPaCa2 and AsPC1 revealed by decreased apoptotic markers Bcl2/Bax ratio, caspase 3, 6, and 8 activation, which are a family of conserved cysteine proteases playing role in apoptosis, PARP or Poly (ADPribose) polymerase involved in cellular processes including DNA repair, genomic stability, and programmed cell death, CDK2/CDK4/CDK6 which are cyclin-dependent kinases, and cyclin-B1/cyclin-E reduction, overexpression of Fas/CD95-death receptor, FasL, FADD, or Fas-associated protein with death domain, and Bid cleavage confirming cell death by extrinsic pathway of apoptosis, exhibiting reduced cell migration, invasion with decreased Vascular endothelial growth factor (VEGF),VEGFR or receptor for VEGF, and MMP9 or Matrix metallopeptidase 9, a type IV collagenase, belonging to the zinc-metalloproteinases family involved in ECM degradation, levels confirming that α2,6-linked sialylation of Fas helps cancer cells to survive, which is a substrate for Neu2 and that Fas/CD95 could be modulated by Neu2, revealing their potential role in pancreatic cancer [45]. Altered glycosylation of serum proteins including N-glycosylation and sialic acid content in glycoproteins including immunoglobulin gamma chains were reported to indicate a predisposition to pancreatic cancer [46]. The inflammatory milieu of glycosylation in PDAC cells, together with overexpressed tumor-associated sialylated antigens sialyl-Lewis(x) or SLe(x), has been reported to contribute to pancreatic tumor malignancy [47]. Altered sialylation of α2β1 integrin and E-cadherin is thought to play a role in regulating pancreatic cancer cell adhesion and invasion [48]. Overexpression of sialylation and sialic acid in pancreatic carcinoma is associated with altered enzymes [49], like overexpression of GD3-synthase causing overexpression of GD3 could induce cell cycle arrest, disrupt anchorage by integrin-β, and inhibit angiogenesis leading to apoptosis in pancreatic cancer cells [49]. Pancreatic cancer and acute pancreatitis have been reported to be associated with altered concentrations of sialylated N-glycoprotein macromolecules from acute-phase proteins, and immunoglobulins [50].



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Metastasis promoting sialofucosylated selectin mucin 16 (MUC16) ligand has been reported to be overexpressed by metastatic pancreatic cancer cells displayed on O- and N-linked glycans that enable binding to E- and L-selectin but not to P selectin [51]. Polysialylated-neural cell adhesion molecule or PSA-NCAM induced by oncogenic K-Ras is reported to enable a reduction in cell-cell adhesion molecule E-cadherin and cellular adhesion mediated by E-cadherin and therefore facilitates dissemination of tumor cells, preventing cell-cell adhesion [52]. Perineural invasion in pancreatic adenocarcinoma, by which cancer cells invade and intimately contact the endoneurium of pancreatic nerves, causing pain and local disease recurrence is mediated by interaction of differentially glycosylated MUC1, mucin with shortened core I O-glycans for monosialyl and disialyl T antigens and myelin-associated glycoprotein (MAG), expressed on oligodendrocytes and Schwann cells, binds myelin to neurons, preferring ligands derivatives of the monosialyl and disialyl T antigen and that this binding has been reported to be ­sialidase-sensitive [53]. The sialylated carbohydrate antigens, sialyl-Lewisx and sialyl-Lewisa, are expressed in pancreatic tumor cells and fucosyltransferases and alpha 2,3-sialyltransferases ST3Gal III and IV are reported from pancreatic adenocarcinoma cell lines playing a role in the formation of the sialyl-Lewis antigens [54]. Elevated levels serum sialic acids [55] overexpressed sialic acid affecting migration and matrix adhesion in pancreatic adenocarcinoma [56] and overexpressed α2,3 sialyltransferase ST3GalIV has been reported to promote migration and metastasis in pancreatic adenocarcinoma [57]. Overexpressed sialic acid conjugated N-glycans [58] and sialylated MUC1 mucin are reported from the benign pancreas and pancreatic duct adenocarcinoma [58] to be hallmarks in this disorder. Alpha2,8-polysialyltransferase enzymes including ST8Sia IV, ST8Sia II, and ST8Sia III (Fig. 4) enabling incorporation of oligosialic and polysialic acid on various sialylated N-acetyllactosaminyl oligosaccharides, including neural cell adhesion molecule (NCAM) [59, 60] and highly polysialylated NCAM or PSA-NCAM have been reported to play a role in perineural invasion (pni) [60, 61]. Downregulated UDP-N-acetylglucosamine 2-epimerase/N-­ acetylmannosamine kinase (GNE) and N-acetylneuraminic acid 9­ -phosphate synthase by tumor suppressor, p16(INK4a) in pancreatic carcinoma revealed the role of sialylation in pancreatic cancer [63]. The increased metabolic influx of sialic acids [64], genetic alteration [65], overexpression of alpha ST3Gal III [66] in pancreatic cancer has been reported to be associated

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Fig. 4  5BO8 Structure of human sialyltransferase ST8SiaIII Structure of human ST8SiaIII sialyltransferase provides insight into cell-surface polysialylation [62]. (Reproduced from protein database (pdb) Volkers G, Worrall LJ, Kwan DH, Yu CC, Baumann L, Lameignere E, Wasney GA, Scott NE, Wakarchuk W, Foster LJ, Withers SG, Strynadka NC. Nat Struct Mol Biol 2015;22:627–635.)

with progression and metastasis thus highlighting the role of sialic acid as diagnostic markers in pancreatic cancer.

3.3 Diabetes Chronic inflammation plays a key role in both type 1 and type 2 diabetes. Siglec-7 expressed on normal β-cells was downregulated in type 1 and type 2 diabetes and in infiltrating activated immune cells. Experimental Siglec-7 overexpression in diabetic islets reduced cytokines, prevented βcell dysfunction and apoptosis and reduced recruiting of migrating monocytes suggesting the role of human Siglec-7 expression as a novel strategy



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to inhibit immune activation and restore β-cell function and survival [67]. Diabetes mellitus patient reveals increased plasma sialic acid content [68] and sialylated apolipoprotein E [69] and sialylated insulin-like growth factor-­binding protein (IGFBP-3), reveal increased affinity of IGFBP-3 [70]. Decreased sialidase activity of peripheral mononuclear leukocytes in diabetic individuals as compared to normal individuals [71] and decreased glycophorin sialylation lead to increased erythrocyte aggregation leading to pathogenesis of vascular disease in diabetes [72] and higher placental sialidase activity is reported from individuals with gestational diabetes mellitus (GDM), leading to reduced sialic acid content of glycodelin-A(GdA) and defective immunomodulatory effects in GDM pregnancies [73].

3.4 Thyroid Overexpression of cell surface α-2,6, α-2,3, sialic acid, and α-1,6 fucose residues on glycan chains in thyroid cancer cell line anaplastic 8305C, follicular FTC-133, and papillary K1 thyroid carcinoma cells highlighted the importance of sialic acid as a diagnostic marker and potential therapeutic targets in thyroid cancer [74]. Overexpressed thyroid sialyltransferase-I are reported in Graves’ disease [75] and α2,3-sialic acids as compared to α2,6-sialic acids are reported from autoimmune thyroiditis [76] Overexpression of sialylated thyroid stimulating hormone or TSH due to overexpressed α-2,6-sialyltransferase in hypothyroid or fetuses have been associated with their impaired activity [77]. Thyroid malignancy reveal contrasting features of sialylation with reports from both [78, 79] increased α2,3-sialic acids [80] sialylated fibronectin [81] sialic acid masking Lewis (a) antigen [80], serum and total sialic acid [82], and decreased sialylation [83] associated with thyroid malignancy.

3.5  Adrenal cancer An overall overexpressed cellular and cytoplasmic sialic acid content [84] plasma bound form [85] is reported from adrenal cancer.

3.6  Pituitary cancer Altered sialylation, polysialylation of NCAMs [86–88] have been reported as prognostic markers in pituitary cancer. Neuroendocrinal cancer reveals overexpression of polySia-NCAM on cancer cell surface including neuroblastoma and polysialyltransferase (polyST) enzymes including ST8Sia-II and ST8Sia-IV find importance as anti-metastatic drug targets [85, 89–92].

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Polysialylated NCAMs have been associated with tumor invasion in pituitary tumors [93]. NCAM-PSA has been associated with poor differentiation and aggressive clinical behavior in neuroendocrinal lung tumors [94].

4 Discussions Disorders of the endocrines, tumors, and metastatic cancers [1–111] are a concern across the globe and markers for early detection and potential therapeutic targets are being researched across the globe. Alteration of expression and modification of structures of sialic acids have been reported to occur in cancer metastasis and progression. Not much is known about the regulation of GNE producing UDP-GlcNAc2 epimerase by CMPNeu5Ac, in the sialic acid biosynthesis pathway (Fig. 5) [16]. Sialylated glycoconjugates owe their origin from sialic acid/Neu5Ac.This initiates with (i) glucose to UDP-N-acetylglucosamine (UDP-GlcNAc) conversion, (ii) UDP-GlcNAc to N-acetylmannosamine (ManNAc) by the

Fig. 5  Sialic acid metabolism and cancer of the endocrine. (Reproduced from Ghosh S. Sialic acids: biomarkers in endocrinal cancers. Glycoconj J 2015;32(3–4):79–85, as authors own work and with permission.)



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enzyme UDP-GlcNAc2 epimerase (GNE), (ii) ManNAc to ManNAc-6phosphate by ManNAc5 kinase, (iii) ManNAc-6-P to NeuAc-9-phosphate by sialic acid 9-phosphate synthase (NANS), and (iv) NeuAc-9-P converts into Neu5Ac by sialic acid 9-phosphate phosphatase (NANP). Cytosolic Neu5Ac moves into the nucleus and is converted into CMP-Neu5Ac by CMP-Neu5Ac synthase (CMAS). UDP-GlcNAc2 epimerase is negatively regulated by feedback inhibition by cytosolic CMP-Neu5Ac in normal individuals. CMP-Neu5Ac enters the cytosol from the nucleus to the Golgi apparatus and catalyzed by sialyltransferases (ST) are added to glycoconjugate moieties forming sialoglycoconjugates. Neuraminidases/Sialidases catalytically cleaves sialic acid from the sialoglycoconjugates. In endocrine cancer, predominant overexpression of sialyltransferases and downregulation of sialidases are reported suggesting the reason for overall increased sialylation. But the status of negative feedback regulation of GNE producing UDP-GlcNAc2 epimerase by CMP-Neu5Ac, in the sialic acid biosynthesis pathway is not yet worked out for endocrine [16]. The major limitation associated with detection of sensitive markers for early detection of the disease lies in the absence of sensitive methods.While alteration of sialic acid like O-acetylation is reported from other cancer, no such report exists from cancers or disorders of the endocrines. However, our current knowledge definitely emphasizes the role of sialic acid as a diagnostic marker in diseases of the endocrines and endocrinal tumors and cancer. More research is needed in this branch of biology to answer many questions hitherto unanswered.

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[39] Martínez-Martínez  MU, Rámirez-de-Ávila  AL, Hernández-Arteaga  AC, García-Valdivieso  MG, Hernández-Cedillo  A, José-Yacamán  M, Navarro-­ Contreras HR. Determination of sialic acid in saliva by means of surface-enhanced Raman spectroscopy as a marker in adnexal mass patients: ovarian cancer vs benign cases. J Ovarian Res 2018;11:61. [40] Jiang T, Lin Y, Yin H, Wang S, Sun Q, Zhang P, Bi W. Correlation analysis of urine metabolites and clinical staging in patients with ovarian cancer. Int J Clin Exp Med 2015;8:18165–71. eCollection 2015. [41] Gomes J, Gomes-Alves P, Carvalho SB, Peixoto C, Alves PM, Altevogt P, Costa J. Extracellular Vesicles from Ovarian Carcinoma Cells Display Specific Glycosignatures. Biomolecules 2015;5:1741–61. [42] Li PL, Zhang X, Li TF,Wang LL, Du LT,Yang YM, Li J,Wang HY, Zhang Y,Wang CX. Combined detection of sialic acid and hydroxyproline in diagnosis of ovarian cancer and its comparison with human epididymis protein 4 and carbohydrate antigen 125. Clin Chim Acta 2015;439:148–53. [43] Thakkar V, Patel P, Prajapati N, Kaur R, Nandave M. Serum levels of glycoproteins are elevated in patients with ovarian cancer. Indian J Clin Biochem 2014;29:345–50. [44] PBassagañas S, Pérez-Garay M, Peracaula R. Cell surface sialic acid modulates extracellular matrix adhesion and migration in pancreatic adenocarcinoma cells. Pancreas 2014;43:109–17. [45] Nath S, Mandal C, Chatterjee U, Mandal C. Association of cytosolic sialidase Neu2 with plasma membrane enhances Fas-mediated apoptosis by impairing PI3K-Akt/ mTOR-mediated pathway in pancreatic cancer cells. Cell Death Dis 2018;9:210. [46] Krishnan  S, Whitwell  HJ, Cuenco  J, Gentry-Maharaj  A, Menon  U, Pereira  SP, Gaspari M, Timms JF. Evidence of altered glycosylation of serum proteins prior to pancreatic cancer diagnosis. Int J Mol Sci 2017;18(12). [47] Bassagañas S, Allende H, Cobler L, Ortiz MR, Llop E, de Bolós C, Peracaula R. Inflammatory cytokines regulate the expression of glycosyltransferases involved in the biosynthesis of tumor-associated sialylated glycans in pancreatic cancer cell lines. Cytokine 2015;75:197–206. [48] Bassagañas S, Carvalho S, Dias AM, et al. Pancreatic cancer cell glycosylation regulates cell adhesion and invasion through the modulation of α2β1 integrin and E-cadherin function. PLoS One 2014;9:e98595. [49] Mandal C, Sarkar S, Chatterjee U, Schwartz-Albiez R, Mandal C. Disialoganglioside GD3-synthase over expression inhibits survival and angiogenesis of pancreatic cancer cells through cell cycle arrest at S-phase and disruption of integrin-β1-mediated anchorage. Int J Biochem Cell Biol 2014;53:162–73. [50] Kontro  H, Joenväärä  S, Haglund  C, Renkonen  R. Comparison of sialylated N-­ glycopeptide levels in serum of pancreatic cancer patients, acute pancreatitis patients, and healthy controls. Proteomics 2014;14:1713–23. [51] Chen SH, Dallas MR, Balzer EM, Konstantopoulos K. Mucin 16 is a functional selectin ligand on pancreatic cancer cells. FASEB J 2012;26:1349–59. [52] Schreiber  SC, Giehl  K, Kastilan  C, Hasel  C, Mühlenhoff  M, Adler  G, Wedlich  D, ­Menke A. Polysialylated NCAM represses E-cadherin-mediated cell-cell adhesion in pancreatic tumor cells. Gastroenterology 2008;134:1555–66. [53] Swanson BJ, McDermott KM, Singh PK, Eggers JP, Crocker PR, Hollingsworth MA. MUC1 is a counter-receptor for myelin-associated glycoprotein (Siglec-4a) and their interaction contributes to adhesion in pancreatic cancer perineural invasion. Cancer Res 2007;67:10222–9. [54] Peracaula R, Tabarés G, López-Ferrer A, Brossmer R, de Bolós C, de Llorens R. Role of sialyltransferases involved in the biosynthesis of Lewis antigens in human pancreatictumour cells. Glycoconj J 2005;22:135–44.



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[69] Kopitzsch S, Winkler L, Oswald B, Schlag B, Dargel R. The sialylation rate of apolipoprotein E in insulin-dependent (IDDM) and non-insulin-dependent (NIDDM) diabetes mellitus. Z Med Lab Diagn 1990;31:47–52. [70] Nedić  O, Lagundžin  D, Masnikosa  R. Posttranslational modifications of the ­insulin-like growth factor-binding protein 3 in patients with type 2 diabetes mellitus assessed by affinity chromatography. J Chromatogr B Analyt Technol Biomed Life Sci 2012;904:93–8. [71] Waters  PJ, Flynn  MD, Pennock  CA, Corrall  RJ, Greenwood  RJ, Eisenthal  R. Decreased sialidase activity in mononuclear leucocytes of type 1 diabetic subjects: relationship to diabetic complications and glycaemic control. Diabet Med 1995;12:670–3. [72] Rogers  ME, Williams  DT, Niththyananthan  R, Rampling  MW, Heslop  KE, ­Johnston DG. Decrease in erythrocyte glycophorin sialic acid content is associated with increased erythrocyte aggregation in human diabetes. Clin Sci (Lond) 1992;82: 309–13. [73] Lee CL, Chiu PC, Pang PC, Chu IK, Lee KF, Koistinen R, Koistinen H, Seppälä M, Morris HR, Tissot B, Panico M, Dell A, Yeung WS. Glycosylation failure extends to glycoproteins in gestational diabetes mellitus: evidence from reduced α2–6sialylation and impaired immunomodulatory activities of pregnancy-related glycodelin-A. Diabetes 2011;60:909–17. [74] Elkashef SM, Allison SJ, Sadiq M, et al. Polysialic acid sustains cancer cell survival and migratory capacity in a hypoxic environment. Sci Rep 2016;6:33026. [75] Kiljański J, Ambroziak M, Pachucki J, Jazdzewski K, Wiechno W, Stachlewska E, Górnicka B, Bogdańska M, Nauman J, Bartoszewicz Z. Thyroid sialyltransferase mRNA level and activity are increased in Graves’ disease. Thyroid 2002;15:645–52. [76] Janega P, Cerná A, Kholová I, Brabencová E, Babál P. Sialic acid expression in autoimmune thyroiditis. Acta Histochem 2002;104:343–7. [77] Persani L, Borgato S, Romoli R, Asteria C, Pizzocaro A, Beck-Peccoz P. Changes in the degree of sialylation of carbohydrate chains modify the biological properties of circulating thyrotropin isoforms in various physiological and pathological states. J Clin Endocrinol Metab 1998;83:2486–92. [78] Babál P, Janega P, Cerná A, Kholová I, Brabencová E. Neoplastic transformation of the thyroid gland is accompanied by changes in cellular sialylation. Acta Histochem 2006;108:133–40. [79] Krzeslak  A, Gaj  Z, Pomorski  L, Lipinska  A. Sialylation of intracellular proteins of thyroid lesions. Oncol Rep 2007;17:1237–42. [80] Takeyama H, Kyoda S, Okamoto T, Manome Y, Watanabe M, Kinoshita S, Uchida K, Sakamoto A, Morikawa T. The expression of sialic fibronectin correlates with lymph node metastasis of thyroid malignant neoplasmas. Anticancer Res 2011;31:1395–8. [81] Vierbuchen  M, Schröder  S, Larena  A, Uhlenbruck  G, Fischer  R. Native and sialic acid masked Lewis(a) antigen reactivity in medullary thyroid carcinoma. Distinct ­tumour-associated and prognostic relevant antigens.Virchows Arch 1994;424:205–11. [82] Kökoğlu E, Uslu E, Uslu I, Hatemi HH. Serum and tissue total sialic acid as a marker for human thyroid cancer. Cancer Lett 1989;46:1–5. [83] Jiang MS, Passaniti A, Penno MB, Hart GW. Adrenal carcinoma tumor progression and penultimate cell surface oligosaccharides. Cancer Res 1992;52:2222–7. [84] Dwivedi C, Dixit M, Hardy RE. Plasma lipid-bound sialic acid alterations in neoplastic diseases. Experientia 1990;46:91–4. [85] Trouillas  J, Daniel  L, Guigard  MP, Tong  S, Gouvernet  J, Jouanneau  E, Jan  M, Perrin G, Fischer G,Tabarin A, Rougon G, Figarella-Branger D. Polysialylated neural cell adhesion molecules expressed in human pituitary tumors and related to extrasellar invasion. J Neurosurg 2003;98:1084–93.



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[86] Daniel  L, Trouillas  J, Renaud  W, Chevallier  P, Gouvernet  J, Rougon  G, Figarella-­ Branger  D. Polysialylated-neural cell adhesion molecule expression in rat pituitary transplantable tumors(spontaneous mammotropic transplantable tumor in Wistar-­ Furth rats) is related to growth rate and malignancy. Cancer Res 2000;60:80–5. [87] Ozyurt E, Sönmez H, Süer S, Kökoğlu E. The prognostic importance of fibronectin and sialic acid levels in human pituitary adenomas. Cancer Lett 1996;100:151–4. [88] Kökoğlu E, Süer S, Ozyurt E, Siyahhan A, Sönmez H. Plasma fibronectin and sialic acid levels in various types of human brain tumors. Cancer Biochem Biophys 1995;15:35–40. [89] Roth J, Zuber C, Wagner P, Blaha I, Bitter-Suermann D, Heitz PU. Presence of the long chain form of polysialic acid of the neural cell adhesion molecule in Wilms’ tumor. Identification of a cell adhesion molecule as an oncodevelopmental antigen and implications for tumor histogenesis. Am J Pathol 1988;133:227–40. [90] Figarella-Branger  D, Durbec  P, Rougon  G. Differential spectrum of expression of neural cell adhesion molecule isoforms and L1 adhesion molecules on human neuroectodermal tumors. Cancer Res 1990;50:6364–70. [91] Lahr G, et al. Neural cell adhesion molecules in rat endocrine tissues and tumor cells: distribution and molecular analysis. Endocrinology 1993;132:1207–17. [92] Komminoth P, et al. Polysialic acid of the neural cell adhesion molecule in the human thyroid: a marker for medullary thyroid carcinoma and primary C-cell hyperplasia: an immunohistochemical study on 79 thyroid lesions. Am J Surg Pathol 1994;18:399–411. [93] Lantuejoul S, Moro D, Michalides RJ, Brambilla C, Brambilla E. Neural cell adhesion molecules (NCAM) and NCAM-PSA expression in neuroendocrine lung tumors. Am J Surg Pathol 1998;22:1267–76. [94] Park  JJ, Lee  M. Increasing the α 2, 6 sialylation of glycoproteins may contribute to metastatic spread and therapeutic resistance in colorectal cancer. Gut Liver 2013;7:629–41. [95] Tanabe K, Matsuo K, Miyazawa M, Hayashi M, Ikeda M, Shida M, Hirasawa T, Sho R, Mikami M. UPLC-MS/MS based diagnostics for epithelial ovarian cancer using fully sialylated C4-binding protein. Biomed Chromatogr 2018;32(5):e4180. [96] Mancera-Arteu  M, Giménez  E, Balmaña  M, Barrabés  S, Albiol-Quer  M, Fort  E, Peracaula  R, Sanz-Nebot  V. Multivariate data analysis for the detection of human ­alpha-acid glycoprotein aberrant glycosylation in pancreatic ductal adenocarcinoma. J Proteomics 2019;195:76–87. [97] Costa-Nogueira  C, Villar-Portela  S, Cuevas  E, Gil-Martín  E, Fernández-Briera  A. Synthesis and expression of CDw75 antigen in human colorectal cancer. BMC Cancer 2009;9:1–10. [98] Picco G, Julien S, Brockhausen I, Beatson R, Antonopoulos A, Haslam S, Mandel U, Dell  A, Pinder  S, Taylor-Papadimitriou  J, Burchell  J. Over-expression of ST3Gal-I promotes mammary tumorigenesis. Glycobiology 2010;20:1241–50. [99] Lee M, Park JJ, Ko YG, Lee YS. Cleavage of ST6Gal I by radiation-induced BACE1 inhibits golgi-anchored ST6Gal I-mediated sialylation of integrin β1 and migration in colon cancer cells. Radiat Oncol 2012;7:1–10. [100] Park JJ,Yi JY, Jin YB, Lee YJ, Lee JS, Lee YS, Ko YG, Lee M. Sialylation of epidermal growth factor receptor regulates receptor activity and chemosensitivity to gefitinib in colon cancer cells. Biochem Pharmacol 2012;83:849–57. [101] Swindall  AF, Bellis  SL. Sialylation of the Fas death receptor by ST6Gal-I provides protection against Fas-mediated apoptosis in colon carcinoma cells. J Biol Chem 2011;286:22982–90. [102] Kim HJ, Kim SC, Ju W, Kim YH,Yin SY, Kim HJ. Aberrant sialylation and fucosylation of intracellular proteins in cervical tissue are critical markers of cervical carcinogenesis. Oncol Rep 2012;31:1417–22.

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[103] Yang  L, Nyalwidhe  JO, Guo  S, Drake  RR, Semmes  OJ. Targeted identification of metastasis-associated cell-surface sialoglycoproteins in prostate cancer. Mol Cell Proteomics 2011;10(6). M110.007294 1-22. [104] Saldova R, Fan Y, Fitzpatrick JM,Watson RW, Rudd PM. Core fucosylation and alpha 2–3 sialylation in serum N-glycome is significantly increased in prostate cancer comparing to benign prostate hyperplasia. Glycobiology 2011;21:195–205. [105] Glavey SV, Manierm S, Natoni A, Sacco A, Moschetta M, Reagan MR, Murillo LS, Sahin I, Wu P, Mishima Y, Zhang Y, Zhang W, Zhang Y, Morgan G, Joshi L, Roccaro AM, Ghobrial IM, O'Dwyer ME. The sialyltransferase ST3GAL6 influences homing and survival in multiple myeloma. Blood 2014;124:1765–76. [106] Ilić V, Milosević-Jovcić N, Petrović S, Marković D, Stefanović G, Ristić T. Glycosylation of IgG B cell receptor (IgG BCR) in multiple myeloma: relationship between sialylation and the signal activity of IgG BCR. Glycoconj J 2008;25:383–92. [107] Fleming SC, Smith S, Knowles D, Skillen A, Self CH. Increased sialylation of oligosaccharides on IgG paraproteins—a potential new tumour marker inmultiple myeloma. J Clin Pathol 1998;51:825–30. [108] Zhao Y, Li Y, Ma H, Dong W, Zhou H, Song X, Zhang J, Jia L. Modification of sialylation mediates the invasive properties and chemosensitivity of human hepatocellular carcinoma. Mol Cell Proteomics 2014;13:520–36. [109] Shen Y, Kohla G, Lrhorfi AL, Sipos B, Kalthoff H, Gerwig GJ, Kamerling JP, Schauer  R, Tiralongo  J. O-acetylation and de-O-acetylation of sialic acids in human colorectal carcinoma. Eur J Biochem 2004;271:281–90. [110] Mann B, Klussmann E,Vandamme-Feldhaus V, Iwersen M, Hanski ML, Riecken EO, Buhr HJ, Schauer R, Kim YS, Hansk C. Low O-acetylation of sialyl-Le(x) contributes to its overexpression in colon carcinoma metastases. Int J Cancer 1997;72:258–64. [111] Baba AI, Câtoi C. Comparative oncology. Bucharest: The Publishing House of the Romanian Academy; 2007. Chapter 16, Endocrine tumors.

CHAPTER 10

Sialic acid and xenotransplantation 1 Introduction Transplantation is the only resort in the case of acute organ failure, burns, and when no other therapy is available. With the increasing need for organs, despite the available live donors and cadaveric donors (Fig.  1), the list is not sufficient to meet the demands of the people in need of organ transplantation. While the demands on vital organ for saving lives is far more than that what is available has led to the development of possible xenotransplantation that involves transplantation, implantation, or infusion into a human recipient (i) live cells, tissues, organs from a nonhuman animal, or (ii) human body fluids, cells, tissues, or organs that have had ex vivo contact with live nonhuman animal cells. This also finds importance in therapy for certain diseases including neurodegenerative disorders and diabetes, with a dearth of available human replacements. Despite promises of xenotransplantation and only resort when life saving organs are a necessity, major public health concerns dwelling around xenotransplantation include (i) infection of recipients with both known and unknown infectious agents and their transmission to the human population, (ii) chance of cross-species infection by retroviruses, being latent initially with symptoms of disease revealed years later after infection, and (iii) difficulty to identify new infectious agents and unknown pathobiology.

2 Xenotransplantation Xenotransplantation includes transplantation of animal live organs, tissues, and cells to a human who is suffering from organ failure and need replacement of organs [1, 2]. Across the globe, the requisite number of human donors is much less as compared to the people waiting in need of the organs. The only chance of survival in patients with end-stage failure including cardiac, respiratory, or hepatic failure is a transplantation. In 1991 there were Sialic Acids and Sialoglycoconjugates in the Biology of life, Health and Disease https://doi.org/10.1016/B978-0-12-816126-5.00010-X

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9000

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Fig.  1  Number of cadaveric donors and transplants in the UK from April 1, 2007 to March 31, 2017 and patients on active transplant list on March 31. Statistics prepared by NHS Blood and Transplant UK Transplant. United Kingdom Preliminary Data. Bristol: UK Transplant, 2002, www.nhsbt.nhs.uk, Reproduced with permission.

reports of 4815 patients in need of organs for transplantation while in 2000 reports suggest as escalated number of patients in need of solid organ transplants of 6823 including 6154 renal transplants, 494 cardiothoracic, and 175 liver transplants while reports suggest a decrease in the organ donors from 934 to 845 [3, 4]. With the increase in diseases like kidney disorders encompassing diabetes mellitus renovascular disease, glomerulonephritis, polycystic kidney disease, pyelonephritis, in addition to cardiac and respiratory disease, it is almost impossible to tackle the need of organs for transplantation and therefore organs from nonhuman sources and animals (Figs.  2–4) are finding importance and thus xenogeneic materials has immense potential as an alternative human transplant. The history of xenotransplantation dates back to the early origin of the Greeks and documented in Greek literature as strange. In the early 17th-century records of blood transfusions from animals to man exists from England and France which were probably the first attempts of xenotransplantation. Calne was the first to classify donor-recipient combinations in either of the two types including discordant revealing rapid and hyperacute rejection (HAR) and concordant revealing rejection pace similar to allotransplantation, which was later attributed to the presence or absence of preformed antibodies in the 20th-century records of solid organ transplantation in concordant transplants. Reports of survival of kidney transplants from chimpanzee for 9 months posttransplantation exists. [4].



Xenotransplantation sialobiology

Survival of a GTKO pig liver in a baboon for 25 days Shah et al.

Pancreatic islets from hCD46 pigs survived 396 days in baboons Dr. D. Coopery Dr. M.Trucco Production of the first pig with suppression of the α-1,3 galactosyltransferase gene Liangxue Laiy Randal Prather, Compania Immerge Biotherapeutics Transplantation of porcine pancreatic islets combined with Sertoli cells in diabetic children Rafael Valdes y David White First transplant of porcine pancreatic islets in a diabetic patient Carl Gustav Groth

First transgenic pig expressing complement decay-accelerating factor David White, Compania Imutran Cardiac xenotransplant from a baboon in an infant Leonard Bailey

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2009

2001

2001

1993

1992

1984 1969

Liver xenotransplant from baboons in humans Thomas Starzl

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Cardiac xenotransplant from a chimpanzee in a human James Hardy

1963–64 1920 1875 1838 1667

Renal xenotransplants from chimpanzees in humans Keith Reemtsma

Testicular transplants from chimpanzees in humans Serge Voronoff

Skin transplants using rabbit cheeks in humans Houzé de I’Aulnoit

First comeal xenotransplant from a pig in a human Richard Sharp Kissam

Xenotransfusions from lambs in humans Jean-Baptiste Denis

Fig. 2  Timeline of xenotransplantation. Reproduced with permission from Aristizabal AM, Caicedo LA, Martínez JM, Moreno M, Echeverr GJ, Clinical xenotransplantation, a closer reality: literature review, Cir Esp 2017;95:59–120.

Reversal of acute cellular rejection by steroids in high doses was reported. Non-primate donors revealed shorter graft survival in hours or minutes. Pig hepatocytes (Fig. 3) transplantation were attempted and non-primate tissue has found application in the treatment of diseases like diabetes and Parkinson’s disease (PD) [5, 6].

Calf

Hamster

Sheep

Pig

Rabbit Human

Baboon

Fig. 3  Animals and xenotransplantation.

Fig. 4  Pig and xenotransplantation.

Blue shark



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However the major concerns of xenotransplantation encompasses the safety, the spread of infection/deadly viruses like endemic retrovirus and other virus like Malaysian Nipah virus from pigs in the recipient and to the public, closeness of the animal to human physiology for graft acceptance, host immune rejection, and ethical concerns [7–10] (Tables 1 and 2).

Table 1  Database on different xenotrasnplantations across the globe.

– Kidney cells | Hamster | Switzerland – adrenals cortical substance | Pig | Ukraine – Adult cells | Sheep | Germany – Choroid plexus | Pig | New Zealand – Chromaffin cells | Calf | Switzerland – Fetal stem cells | Rabbit | Malaysia – Fetal ventral mesencephalic cell | Pig | USA – Spleen | Pig | Russia – stem cells | rabbit, pig, | Nigeria – Testis organ | Pig | Ukraine Heart | Pig | India Kidney | Pig | Sweden Embryonic stem cells – | blue shark | Mexico Embryonic stem cells – | Pig | USA Embryonic stem cells – | Pig | USA Embryonic stem cells – fetal cells, adult cells | rabbit, cattle, sheep | Mexico Embryonic stem cells – fetal cells, adult cells | cattle, sheep, rabbits | Germany Fetal islet like cell clusters – | Pig | Sweden Hepatocytes – | Pig | USA Hepatocytes – | Pig | France Hepatocytes – | Pig | 11 US and 9 European hospitals Hepatocytes – | Pig | Italy Hepatocytes – | Pig | Germany Hepatocytes – | Pig | Italy Islets of Langerhans – | Rabbit | Russia Islets of Langerhans – | Pig | New Zealand Islets of Langerhans – | Pig | China Islets of Langerhans – | Pig | Russia Islets of Langerhans – | Pig | Ukraine Islets of Langerhans – | unknown | Ukraine Islets of Langerhans – | unknown | Ukraine Islets of Langerhans – | Pig | Ukraine Islets of Langerhans – | Pig | Argentina Islets of Langerhans – Sertoli cells | Pig | Mexico Table adapted from World Health Organization (WHO) website 2012, reproduced with permission from Database: http://humanxenotransplant.org/home/index.php/2012-02-07-14-13-27 WHO 2012.

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Table 2  Categories of Potential Pathogens Resulting from Xenotransplantation and availability of validated microbiological assays.

Common Human Pathogens of Allotransplant Recipients (EBV, CMV, herpes simplex virus, varicella zoster virus, Apergillus species, Listeria monocytogenes, mycobacterial species, Pneumocystis jirovecii) Specific microbiological assays are generally available Traditional Zoonoses: well-characterized clinical syndromes of humans (Toxoplasma gondii) Specific microbiological assays are generally available Species-specific agents: organisms generally thought to be incapable of causing infection outside the xenograft (e.g., porcine CMV) Some specific microbiological assays are available; few standardized assays available for use in humans Potential pathogens: Organisms of broad “host range” which may spread beyond the xenograft (adenovirus) Some specific microbiological assays are available for use in humans; may not be standardized for porcine strains Unknown pathogens: Organisms not known to be human pathogens, not known to be present in the source animals, or for which clinical syndromes and microbiologie assays are poorly described or unknown – New pathogenicity within the new host, while not known to be present or pathogenic (e.g., protozoa or retroviruses) – Viral recombinants resulting from intentional genetic modification of donor diseases resulting from multiple simultaneous infections Reproduced with permission from WHO Second WHO Global consultation on regulatory requirements for xenotransplantation clinical trials, October 17–19 2011, WHO, Geneva, Switzerland.

3  Major problem associated with xenotransplantation and ways to circumvent them 3.1 Physiology The size, longevity, aging in xenotransplanted organs, hormones revealing differences from the human counterpart, incompatible proteins among donor animals and human recipients are a major concern. Differences in human 37°C and porcine body temperature being 39°C pose a challenge to transplantation. Porcine rennin is unable to cleave human angiotensin and porcine erythropoietin is unable to stimulate human erythropoiesis, posing issues of incompatibility and therefore functional disturbances posttransplantation. Incompatibility of liver proteins poses challenges to hepatic xenotransplantation. Effect of differences in body temperature could affect the activity of porcine enzymes.



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3.2 Rejection HAR of xenotransplanted grafts is manifested by rapid onset, within minutes of transplantation revealing pathophysiology of edema, hemorrhage, and vascular thrombosis, due to preformed antibodies.

3.3  Gal epitope Human being lack the gal epitope or nonreducing trisaccharide group, galactosyl α-(1,3)-galactosyl β-1,4-N-acetyl glucosaminyl, lacking the enzyme generating it and therefore recognizing the gal epitope as foreign and generate immune responses against gal epitope forming anti-gal antibodies that target the gal epitope [11] leading to activation of complement by natural killer (NK) cells and affecting the endothelium. The gal epitope is discussed in details in subsequent sections. To make a xenograft acceptable, therefore, a strategy would be to remove all xenograft natural antibodies by plasmapheresis, however, they might return within a few days. α-gal toxin [12] to remove the plasma cells producing the antibody is also attempted. Knockout (KO) animals like mouse strain with disrupted α1,3 galactosyl transferase genes has been generated that lack the ability to synthesize α-gal epitopes and produce natural ­anti-gal antibody.

3.4 Complement Xenograft natural antibodies activate complement thereby activating the endothelial cell releasing cytokines and platelet activating factor thereby causing HAR with pathophysiology of thrombosis, hemorrhage, and infarction. Depletion of complement depletion with cobra venom factor has been used as a strategy to prevent rat to primate and pig to primate transplants [13] C1-inhibitor (C1-INH), playing role in the initial steps of complement activation, overexpressed could prevent hyperacute xenograft rejection. Another major problem associated with the incompatibility of porcine complement regulatory proteins, is circumvented by generation of pigs expressing human complement regulators including—CD55 (decay accelerating factor), CD46 (monocyte chemoattractant protein) and CD49 that could confer protection of cells from complement-induced lysis [14] and increase graft survival in pig-to-primate renal and cardiac transplants [15]. Both transgenic organs and immunosuppression could upscale the graft survivability up to 78 days [16].

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3.5 Thrombin Thombin inhibition has enabled prolong graft survival and strategies for the generation of anticoagulant molecules on the endothelial cells are attempted [15].

3.6  Glycosyltransferase transgenes Transgenic pigs and mice with overexpressed human β-D-mannoside β-1,4-N-­acetylglucosaminyltransferase led to reduced gal epitope expression together with reduced complement-mediated and NK cell ­mediated lysis.

3.7  Acute humoral xenograft rejection Acute humoral xenograft rejection (AHXR), causes delayed xenograft rejection, manifested by endothelial swelling or disruption, vascular thrombosis with blood extravasation and interstitial edema arising within 24 h of transplantation and graft destruction, mediated byIgM with an increase in IgG levels. Depletion of anti-gal antibodies is one such strategy.

3.8  Cellular rejection Xenografts interact with both innate and adaptive immune system. Stimulation of cross-reactive antigens present on the flora of the recipient leads to the generation of antibodies. In alloresponses, the recipient immune system recognizes allogeneic MHC molecules directly by engaging T cell receptors (TCRs) with the MHC molecules. The adaptive immune system recognizes the foreign antigen that is processed and presented by MHC molecules on antigen presenting cells (APC) to TCRs on T cells causing cell lysis (Fig. 5). Recipient T cells recognize donor MHC determinants on donor APC by a process called direct presentation or they can recognize donor MHC peptides, released from donor cells and processed and presented by host APC within self-MHC molecules by a process of indirect presentation (Fig. 6). Graft cell expressing MHC class II antigens can activate the indirect pathway. Alloantigens shed from the graft are treated as an exogenous antigen by recipient APC and is presented by MHC class II molecules to activate recipient CD4+T cells. A semi-direct pathway of antigen presentation has been described recently [20–22] wherein the recipient dendritic cells acquire MHC-antigen complexes from donor dendritic and endothelial cells, and present them by direct antigen presentation to alloreactive T cells. They can stimulate CD4+T cells and CD 8+T cells [17–19] initiating the cellular process of rejection. T cell activation leads to acute rejection of the graft.



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Complement Thrombin

IgG IgM C3a C5n

Gal

TF

nonGal Neonatal pig isiet MAC

Infiltration

Clot

Neutrophils Monocytes

Fig. 5  Representative profile of the immediate host response after xenotransplantation from donor animal. Tissue factor (TF) is expressed, antibody binding to Gal and non-Gal antigens in the neonatal porcine islets leads to complement activation, coagulation cascade, blood clot formation, and cell membrane is damaged by membrane attack complex (MAC), and recruitment of macrophages and monocytes through chemoattractant complement components C3a and C5a. Reproduced with permission from Aristizabal AM, Caicedo LA, Martínez JM, Moreno M, Echeverr GJ, Clinical xenotransplantation, a closer reality: literature review, Cir Esp 2017, 95: 59–120.

(A)

(B)

Fig. 6  Processing and Presentation of Xenogeneic antigen: (A) Direct and (B) Indirect presentations.

Adhesion of NK cells leads to damage of the pig epithelium (pECs, Fig. 7) and endothelial cell damage together with morphological changes on pEC monolayers. NK cells of humans can activate pECs in a cell ­contact-dependent manner, inducing release of E-selectin and IL8 by an NF-κB-dependent pathway and NK cell cytokine secretion including IFNγ

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Fig. 7  NK cytotoxicity against 2A2 pig endothelial cells. (A) Monolayers of porcine pEC incubated with IL2-activated purified polyclonal NK cells (B) After 4 h of coculture with IL2-activated NK cells, the pEC monolayer was destroyed. Reproduced with permission from Puga Yung G, Schneider MKJ, Seebach JD. The role of NK cells in pig-to-human xenotransplantation. J Immunol Res 2017;2017:4627384. Under Creative Commons Attribution License.

and TNF and causing non-MHC restricted direct cytotoxicity and ADCC against pECs in vitro causing lysis of porcine chondrocytes, islets, and embryonic brain-derived cells [20–23]. Immunosuppression is a strategy to circumvent this problem.

3.9 Infection Risk of transmission of infectious agents (Tables 2 and 3) across the species is a major concern. Although sanitization measures are used in feed and drinking water to prevent prion infection, risks of infection by retroviruses like porcine endogenous retroviruses (PERVs) remains. Bovine spongiform encephalopathy and HIV infection in humans are thought to have originated in monkeys.

4  Sialic acid and xenotransplantation All living cells contain glycans conjugated to proteins and lipids on the cell surface glycome mediated by enzymes, taking part in a variety of physiological processes, including recognition of self from that of the non-self molecules and therefore generate immune responses against the nonself or foreign molecules. Many of these glycans have penultimate sialic acids, with diverse linkages to glycan residues thus generating a complex and high diversity of molecules and carriers included under the Sialomes. The entire sialome of the human body includes a diverse complex of sialic acids, sialoglycans,

Table 3  Common microorganisms of swine to be considered among potential causes of infection in immunocompromised pigs and/or human xenograft recipient. Bacteria

Viruses

Actinobacillus species (e.g., pleuropneumoniae) Bordetella bronchoseptica Brucella suis

Adenovirus sp.

Campylobacter species (e.g., coli, jejuni) Chlamydia psittaci Clostridium difficile Corynebacterium species (i.e., pyogenes, suis) Hemophilus species (i.e., parasuis, suis) Klebsiella species (e.g., pneumoniae) Legionella pneumophila Leptospira species Listeria monocytogenes Mycobacterium species (i.e., bovis, tuberculosis, non-tuberculous mycobacteria) Mycoplasma hyopneumoniae (lung transplant) Nocardia species Pasteurella species (i.e., hemolytica, multocida, pneumotropica) Pseudomonas species (i.e., aeruginosa, pseudomallei) Salmonella species (i.e., typhi, typhimurium, cholerasuis) Serpulina hyodysenteriae Shigella species Staphylococcus species (i.e., aureus, hyicus) Streptococcus species (e.g., pneumonia, suis) Strongyloides species (e.g., ransomi) Yersinia species (i.e., enterocolitica, pseudotuberculosis) Parasites Ascaris species Cryptosporidium species (i.e., parvum) Echinococcus Isospora species Neospora Strongyloides stercoralis Toxoplasma gondii Trichinella spiralis

Encephalomyocarditis virus Influenza virus (swine, avian, human) Lymphocytic choriomeningitis virus (LCMV) Nipah (Hendra-like) Menangle virus Porcine circovirus Porcine cytomegalovirus (PCMV) Porcine endogenous retrovirus (PERV) Porcine hepatitis E virus Porcine lymphotropic herpesvirus (PLHV) Porcine parvovirus (PPV) Porcine Reproductive and Respiratory Syndrome virus Pseudorabies virus Rabies virus Rotavirus Torque teno virus Fungi Aspergillus species Candida species Cryptococcus species Histoplasma capsulatum Microsporum species Trichophytum species

Reproduced with permission from WHO Second WHO Global consultation on regulatory requirements for xenotransplantation clinical trials, October 17–19 2011, WHO, Geneva, Switzerland.

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and sialoglycoconjugates and are known to play a major role in generating human immune recognition and responses. However, pathogens and cancer cells have evolved strategies to bypass the identification by sialic acid and cause havoc disturbances to the host. Sialic acids play a dominant role in inflammation by receptors families including asialoglycoprotein receptor 1 (ASGR-1), Siglec-1 (Sialoadhesin), selectin, integrin, and galectin. Desialylation or alterations in sialyltransferases has been reported to play a role in modulation of cell–cell adhesion, cell activation pathways affecting homeostasis and inflammation associated with xenografts [24]. During xenotransplantation of vascularized tissues, the differences in human and pig membrane glycocalyx sugars and receptors lead to dysregulated endothelial cell activation, disseminated coagulation causing affected vessel wall integrity and generation of innate and adaptive immune responses. α-Gal or non-Gal antigens in pig corneal endothelial cells and keratocytes were detected by immunohistology and mass spectrometry of N-glycans from common adult pig corneal endothelial cells and in  vitro cultured keratocytes.Totally, 34 of the sialylated N-glycans from pig corneal endothelial cells and 27 from pig keratocytes were reported to contain nonhuman sialic acid, NeuGc and NeuAc and 25 of α-galactosylated N-glycan structures from the pig corneal endothelial cells and 18 of that from the pig keratocytes were also reported which may bear direct consequences on xenotransplantation. [25]. Host preformed or natural antibodies responsible for xenograft rejection include human anti-pig antibodies of IgG, IgM, and IgA with specificity toward oligosaccharides with an α Gal terminal residue, including α Gal, α Gall-3 β Gal-, αGall-3 β Gall-4 β GlcNAc, and α Gall-3 β Gall-4 beta Glc. Pig vascular endothelium revealed α Gall-3 β Gall-4 βGlcNAc, α NeuAc2–3 βGall-4 β GlcNAc, and βGall-4 beta GlcNAc expression. Human vascular endothelial cells reveal expression of same lactosamine-ended precursor and sialylated chains as pigs, but instead of terminal α-Gal they express the fucosylated polymorphic ABH blood group epitopes. Galα-1,3-Gal (αGal) and Neu5Gc are two known xenoantigens [26] and humans are known to contain natural anti-αGal, Neu5Gc antibodies, and other antibodies that can bind to the porcine Galactose-α-1,3galactose (αGal), Neu5Gc on pigs and pig cell surface carbohydrate antigens respectively that cause complement activation, leading to HAR of porcine xenografts [27]. Both natural and elicited human antibodies against porcine, glycocalyx non-Gal sugars and non-sugar molecules also play a role in causing delayed vascular rejection of xenograft tissue with endothelial cell



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activation, clotting, and immune responses. Both anti-Gal and non-Gal antibodies have been known to cause detrimental consequences to nonvascular xenografts of pancreatic islets or cornea [24, 25]. Some natural anti-Gal IgEs have been reported to trigger anaphylactic reactions [28]. The roles played by the different sialic acid containing moieties in xenotransplantation are discussed below.

4.1  Role of Neu5Gc Humans, as compared to the other mammals, have lost the ability to synthesize the Neu5Gc, due to the loss of function of the enzymes coded by cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) gene which has been dated back during evolution estimated around 3.5 million years ago [29–31]. CMP-Neu5Ac is converted to CMP-Neu5Gc in the cytoplasm by active CMP-Neu5Ac hydroxylase, encoded by the CMAH gene (Fig. 8). Neu5Gc expression has been reported from echinoderms to mammals with the exception of birds, reptiles, and monotremes including Platypus and Echidna and ferrets, new world monkeys and man (Fig.  9) and the CMAH gene has been recorded from 332 deuterostome genomes [34]. CMAH homologs have been reported from two green algae and a few prokaryotes.Within deuterostomes, putatively functional CMAH homologs were reported from 184 genomes studied, with a total loss of 31 independent gene pseudogenization events [40]. Neu5Gc-containing xenografts when exposed to these human anti-Neu5Gc antibodies lead to the generation of the immune response [41]. Although humans cannot synthesize Neu5Gc, they can acquire Neu5Gc from food like red meat or milk and express on the normal human cell surface, predominant on epithelial and endothelial cells [42, 43] and reported of expression from kidney, liver, skin, and heart. Humans can produce high diversity of anti-Neu5Gc IgG and IgM antibodies that varies with the individual adding to the complexity of types of sialome antibodies against Neu5Gc present as natural antibodies in the serum [44]. Anti-Neu5Gc antibodies have been shown to contribute substantially around 85% against other anti-non-Neu5Gc determinants in human serum and were detected by hemagglutination assays and show almost equal expression of anti-Gal antibody. Anti-Neu5Gc antibodies expressed on some human tissues can cause chronic inflammation termed as xenosialitis and may lead to cancer and atherosclerosis and also play a role in determining the fate of kidney graft survival [44, 45].

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HO HO CH3

(A)

C

HO

COOH

OH O

HN

HO

OH

CMAH gene

OH

O

HOH2 C

C O

Neu 5Ac

COOH

OH O

HN

OH

OH

Neu 5Gc

Parallel losses of Neu5Gc Mustelids and Pinnipeds ~40 Mya

New World Monkeys ~30 Mya

Origin >100 Mya Neu5Gc(+) Neu5Gc(–)

Homo ~3 Mya

(B) Innate self signal receptors CD33 Related siglecs Inhibitory

Conserved

Activating

A

V-set C2-set ITIM ITIM-like

(C)

3

5

6

7

8

9 10 11 XII

14 16

1 2 4

15

Fig. 8  (A) Sialic acids found at the terminal ends of N- and O-glycans, and as polysialic acid of which Neu5Ac and Neu5Gc are two most common form. Neu5Gc is synthesized from Neu5Ac by the CMAH protein. (B) Humans cannot synthesize Neu5Gc, as human CMAH was inactivated over two million years ago [32]. Independent losses of CMAH have recently been found in New World Primates [33] and in Mustelids [34]. (C) Sialic acids are bound by Siglec receptor proteins expressed on most immune cells [35] which inhibits inflammation. Many pathogens bind Siglecs to exploit these immunosuppressive effects, and in response, several immune-activating Siglecs have evolved. Other pathogens use Neu5Gc itself as a receptor on host cells. Several human pathogens have modified their receptor affinity to recognize Neu5Ac following the loss of Neu5Gc in humans. Reproduced with permission from Springer SA, Gagneux P, Glycomics: revealing the dynamic ecology and evolution of sugar molecules, J Proteomics 2016;135:90–100.

Different tissues of pig show differences in the content of Neu5Gc expression based on differential tissue-specific expression of CMAH, therefore, leading to the variation of the Neu5Gc/Neu5Ac ratio [26]. Porcine tissues including the vascular endothelium-cell surface of kidney, liver, heart tissues including valves, cornea, neonatal pancreatic islet-like cell clusters [46–49] and in adult pancreatic islets reveal differences in Neu5Gc expression as compared to Gal while neurons are known to lack Neu5Gc [50] and accordingly responses post xenotransplantation may vary. Devitalized and decellularized animal tissue or organs to reduce their immunogenicity like porcine or bovine biological prosthetic heart valves



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Fig. 9  α Gal antigen, highly reactive, foreign antigen. (A) ∝ gal, on animal glycans, reveals a terminal galactose alpha 1,3 linked with an underlying galactose. (B) ∝1,3galactosyltransferase (∝1,3GT), the enzyme that synthesizes ∝ gal, was inactivated at the common ancestor of Old World Primates. Apes, humans, and Old World monkeys cannot synthesize the terminal ∝ gal linkage. Most mammals, including the New World primates, do synthesize the ∝ gal epitope. (C) Individuals can be immunized against foreign glycans by a pathogen or by diet. The loss of ∝ gal may contribute to Old World primates resistance to infection because anti ∝ gal antibodies target this epitope on enveloped viruses from other mammalian species [36]. Anti ∝ gal antibodies can cause severe allergic reactions in humans that eat red meat [37]. ∝ gal antibodies prevent organ transplantation from other mammal species like ∝ gal-rich pig organs [38]. Anti ∝ gal antibodies can be targeted against pathogens or cancerous cells [39]. Humans predominantly express Neu5Ac in contrast to other mammals predominantly expressing Neu5Gc [27]. Reproduced with permission from Springer SA, Gagneux P, Glycomics: revealing the dynamic ecology and evolution of sugar molecules, J Proteomics, 2016,135:90–100.

(BPHVs), used for human heart valves replacement, and tendons [51, 52]. Pigs KO for the CMAH enzyme and taking Neu5Gc-rich diet has been reported to show a certain level of Neu5Gc in their serum and other tissue [53]. Human antigens injected into animals derive polyclonal antibodies from serum but animal polyclonal IgG molecules expressing the Neu5Gc sialic acid linked to Fc and variable Fab asparagine Asn297 [54] are reported to

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be immunogenic. Humans exposed to rabbit or equine IgGs receiving the anti-lymphocyte serum (ALS), lead to Ig response, leading to serum sickness [55] predominant due to the expression of anti-Neu5Gc antibodies [56, 57]. N- or O– glycosylation sites on the variable domains of some Igs contribute to more immunogenic [58].

4.2  Role of α-gal antigen Humans have lost function of the enzyme alpha-1,3-galactosyltransferase-1 (GT1), due to a loss of function mutation in the GGTA1 gene during evolution and cannot synthesize galactosyl-alpha(1,3)-galactose disaccharide (α-Gal), and therefore α-Gal is not expressed in any human tissue. Since gut microbiome bacteria are known to express Neu5Gc, Gal α1–3Gal, which are not by self, humans recognizes it as a foreign antigen and generate anti-carbohydrate antibodies including anti-Neu5Gc, anti-Gal IgM, and IgA and IgG antibodies in serum [26, 59]. Complement-binding anti-Gal antibodies to the Gal antigen of donor pig organ glycocalyx triggers an immune response and hyperacute xenograft rejection [26, 60].While αGal is represented by a single carbohydrate xenogeneic antigen with structure Galα1–3-Galβ1–4-GlcNAc-R, multiple Neu5Gc glycans are expressed on glycoproteins and glycolipids [24, 27, 61]. Although anti-Gal antibodies in human serum are predominantly of IgM isotype, the anti-Neu5Gc antibodies are predominantly of anti-IgG isotype [62].

4.3 Sialoadhesin Patients with fulminant hepatic failure reveal high mortality rates and use of extracorporeal porcine liver perfusion for liver dialysis is an alternative therapeutic strategy limited by the fact that within 72 h of extracorporeal perfusion with human blood, sialoadhesin of porcine Kupffer cells has been reported to bind to Neu5Ac expressed on human erythrocytes causing and cell lysis [63]. Sialoadhesin, a xenogeneic receptor can mediate fast binding of porcine macrophages to human erythrocyte Neu5Ac as compared to other nonhuman primate erythrocytes due to reduced expression of Neu5Ac indicative of the fact that physiology of extracorporeal porcine liver perfusion is different in human and nonhuman primates and that Porcine-nonhuman primate (NHP) xenotransplantation model is different from Porcine-human model [63–65].

4.4  The Hanganutziu-Deicher (H-D) antigen The HD with terminal NeuGc is absent in birds and human but is distributed in monkeys and apes. HD antigens, widely expressed on endothelial



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cells of all mammals except humans, are found to be potential targets for natural and elicited anti-non-Galα1–3 GaL (Gal) antibodies in humans [66] and the antigenicity is to both Galα1–3Galβ1–4GlcNAc-R (α-Gal) and H-D antigens, involves activation of complement. Molecular cloning of pig CMAH was done to produce H-D KO pigs wherein a pig endothelial cell (PEC) line, MYP30, was used [67]. Reduced expression and antigenicity of H-D antigens were achieved by silencing of pCMAH gene by targeting small interfering RNA (siRNA) for pCMAH gene on a GalT-KO from PEC line, and PEC and fibroblasts. Xenoantigenicity for the human serum of the GalT-KO cells was then confirmed [68]. The α1,3 galactosyltransferase (α1,3GT) activity of islets was determined by high-performance liquid chromatography (HPLC) [48] and in α1,3galactosyltransfease KO the H-D antigen is reported to be the major non-Gal antigen [69].

5  Xenograft acceptance and strategies based on sialylation To make the porcine xenograft acceptable, to the human recipient several approaches have been used including modification of the porcine genome. The different molecular biology approaches for generating pig organ acceptable for xenotransplantation by the human host are included in the foregoing paras. Studies have shown that such modifications either singly or in combinations have been able to improve graft acceptance in the host and prevent long-term immunological injury [70]. However, each strategy is not without drawbacks which are also discussed. (i) The most successful strategy to circumvent the problems posed by sialic acid for xenograft acceptance is to generate α-1,3-­galactosyltransferase (GalT) enzyme KO animals including Neu5Gc KO pigs that may limit xenogeneic injury. (ii) Removal of the αGal epitope from the pig cell surface, by the generation of GT1 knockouts (GT1KO) [71] and glycan analysis of these KO tissues detected no new neo-antigens but a high level of Neu5Gc expression on glycoproteins and glycolipids. But GalT-KO pigs, however, may not completely inhibit antibody mediating rejection (AMR) due to diverse antibody responses to non-Gal pig antigens and predominant l IgG and IgM non-Gal antibodies (NGal-Ab) in nonhuman primates and humans identified by immunoabsorbing serum using Gal-coated Sepharose beads [72]. However, immune suppression may significantly reduce immune responses [72].

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(iii) Generation of transgenic pigs expressing the following might enable better acceptance of graft including: (a) human complement regulatory proteins like CD46 (hCD46) and CD55 (hCD55), to inhibit complement activity, (b) human endothelial cell protein C receptor (hEPCR), (c) human tissue factor pathway inhibitor (hTFPI), and human thrombomodulin (hTBM), to inhibit clotting mechanisms triggered by pig endothelium exposed to human blood are being tested. (iv) Control on processes involved in overall sialylation of cells so as to regulate inflammatory processes, cell adhesion, trafficking during xenotransplantation.

6  Animal xenotransplantation models and sialic acid Pig liver xenotransplantation in human and baboon transplant models suffers from the limitation of thromocytopenia by liver sinusoidal cells that express asialoglycoprotein receptor 1 (ASGR1) a component of the Ashwell receptor expressed on (LSEC) with carbohydrate-binding sites recognizing exposed galactose β1–4 N-acetyl glucosamine (Galβ) and macrophage antigen complex-1 (Mac-1) on Kupffer cells (KC) that recognizes N-acetyl glucosamine β1–4 N-acetyl glucosamine (βGlcNAc) and mediate platelet phagocytosis. Knockdown of these proteins by siRNA approaches can reduce human platelet phagocytosis. Studies have revealed differences in structure between human and porcine ASGR1 lectin-­binding domains with four times more exposed Galβ and βGlcNAc in human platelets as compared to fresh porcine platelets. Porcine platelet Galβ and βGlcNAc moieties not masked by sialic acid and that removal of sialic acid from human platelets increased binding and phagocytosis by LSEC and KC have been reported to contribute to platelet loss in porcine liver xenotransplantation [73]. While mice express both NeuAc and NeuGc gangliosides, severe combined immunodeficiency (SCID) mice express only NeuGc-containing gangliosides, and human cells express only NeuAc-containing gangliosides. The neutral glycosphingolipids of the U87-MG cells comprised of glucosylceramide, galactosylceramide, lactosylceramide, and asialo-GM1 (Gg4Cer) expressed in the xenograft, suggested macrophage infiltration from the SCID host playing role in immunogenic properties of xenografts [74].



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7  Strategies for knockouts Homozygous disruption of the GGTA1 and the CMAH genes in ­liver-derived female pig cells using zinc-finger nucleases (ZFNs) followed by somatic cell nuclear transfer (SCNT) has been reported to enable easy production of healthy cloned piglets lacking both CMAH and GGTA1 gene activities from the genetically modified liver cells [53] with reduced antibody- mediated destruction of xenotransplantation, thus being effective in generating grafts for transplantation [53]. Pig CMAH gene isolated and characterized from pig small intestine revealed an ORF (open reading frame) of 1734 bp, encoding 577 amino acids with 14 exons. The pig CMAH mRNA has been reported to be expressed in pig rectum, tongue, spleen and colon tissues, and predominant in small intestine capable to synthesize the xenoantigen Neu5Gc in humans thus playing a major role in the pig-human rejection of transplant [75]. CMAH KO pigs were generated by ZFNs [69] in combination with donor DNA containing a total homology length of 1600 bp and targeted disruption of GGTA1 in the CMAH KO genetic background. NeuGc epitopes are likely targets of anti-non-Gal Abs. GalT and CMAH double knockout (DKO) mice have revealed that expression of Gal and NeuGc could be completely inhibited in hematopoietic cells, heart, lungs, liver, kidney, and pancreatic tissues detected by flow cytometric and immunohistochemical studies. Thymocytes from Wild type (wt), Single KOs (SKO) including GalT(−/−), CMAH(−/), and DKO mice when used to capture xenoreactive antibodies in human sera from healthy volunteers of different blood group, followed by detection of complement dependent cytotoxicity (CDC), revealed most CDC of human sera in thymocytes of wt, followed by CMAH(−/−) mice, whereas thymocytes of GalT(−/−) were more resistant to lysis and CDC was not detectable from DKO mouse thymocytes [76]. Earlier studies had revealed that human erythrocytes or red blood cells (RBC) are identified by pig Kupffer cells by a carbohydrate-dependent mechanism. Using the 51 chromium assay and inhibition of erythrocyte rosette formation it was reported that pig Kupffer-cell can recognize terminal sialic acid moiety on human RBC irrespective of blood groups indicative of the fact that a sialic-acid receptor plays a role in innate cellular recognition of xenogeneic epitopes [77] and initiating immune responses in graft rejection. Macrophages have been reported to play a major role in the xenogenic rejection. CD33-related Siglecs have been known to contain immunoreceptor

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tyrosine-based inhibitory motif (ITIM) at the cytoplasmic end that can inhibit cytokine production. Human macrophages have been known to express various CD33-related Siglecs. Overexpression of α-2,6-sialyltransferase (2,6-ST) in swine endothelial cells (SECs) could prevent the cytotoxicity by macrophages and therefore inhibit macrophage-mediated xenograft rejection [78]. The new technology of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-associated protein-9 nuclease (Cas9) from (S pyogenes) enables removing, adding or altering sections of the DNA sequence (Fig. 10) thereby editing the genome. The CRISPR-Cas9 system includes two molecules that can introduce a mutation in the DNA, including Cas9 enzyme that cuts the two strands of DNA at a specific location and a RNA or guide RNA (gRNA) of about 20 bases long located within a longer RNA scaffold that binds to DNA and the designed sequence guides the entry of the Cas9 to the exact position in the genome enabling the Cas9 enzyme to cut at precise position in the genome. Although it was first demonstrated in 2012, it has found applications in targeting human, bacteria, C. elegans, Drosophila, mice, monkeys, plants, pigs, rabbits, rats, mice, (X tropicalis), yeast, and zebrafish [79].

Guide RNA Spacer

Target sequence

Scaffold Guide RNA binds to target sequence

Genomic DNA

Cas 9 Endonuclease Mutation

Cas9 enzyme binds to guide RNA

The cut is repaired introducing mutation

Cas9 enzyme cuts both strands of DNA

Fig.  10  Crisper-Cas9 Technology of gene editing. Adapted from Source https://www. yourgenome.org/facts/what-is-crispr-cas9 under Creative Commons Attribution 4.0 CC-BY License.



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A modified porcine glycan model designed through CRISPR/Cas9mediated silencing of the GGTA1 and CMAH genes did not enhance the human-anti-pig cellular response and proved to be an effective model for pig-human xenotransplantation [80]. Pig pancreatic islets are being subjected to Phase I clinical trials on burn patients without any PERV transmission or generation of anti-PERV antibody response in the recipient but has been reported to increase anti αGal and anti-non-αGal IgG or ­anti-Nue5Gc a­ ntibodies from glycan array studies contributing to chronic inflammation [41]. Adult pig islets (APIs) and neonatal porcine islet-like cell clusters (NPCCs) reveal differences in glycosylation and antigenicity from lectin microarray studies on API, NPCCs, and GalT-KO NPCCs. While NPCCs revealed strong expression of ligands for lectins Sambucus nigra (SNA), (S sieboldiana), and Trichosanthes japonica and binding of α2,6 sialic acid, APIs revealed stronger ligand expression for lectins (L tetragonolobus), Aleuria aurantia, (N pseudonarcissus), and (G nivalis) lectins, suggesting that APIs express high levels of high-mannose forms. The NPCCs from a GalT-KO pig indicated downregulation of α-Gal and upregulation of α-GalNAc and α2–6 sialic acid expression and thus having a role in the design of immune-modified pigs with less antigenicity [49]. No reports exist on human or baboon plasma containing antibodies directed against sialic acid or lactosamine, but as human tissues contain both these carbohydrates, these epitopes may not play any role in the vascular rejection of porcine tissue in other primates [81]. TALENS or transcription activator-like effector nucleases a newly developed technology for genome editing includes a nonspecific DNAcleaving nuclease fused to a DNA-binding domain in order to target a desired sequence with tremendous application in basic research and generate potential therapeutic strategies for genetic disorders. Using TALENs, bi-allelically modified GGTA1 cells were generated [82]. TALEN was successfully used to target the CMAH gene coupled with SCNT to make embryos of pigs compatible with xenotransplantation. Gal-deleted cells expressing human tumor necrosis factor receptor I IgG1-Fc (shTNFRI-Fc) and human hemagglutinin-tagged-human heme oxygenase-1 (hHO-1) transfected with a TALEN target for CMAH and cells lacking CMAH were negatively selected using Neu5Gc tagged magnetic beads. Cloned embryos from genetically modified cell clones transferred into three recipients produced three cloned pigs revealing disrupted CMAH gene and lack of its expression, decreased expression of Neu5Gc in piglets produced by SCNT.

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This ­technology was successful in generation of pigs with quadruple modified genes CMAHKO/GTKO/shTNFRI-Fc/hHO-1 with suitability for xenotransplantation by overcoming immune rejection [83]. Neu5Gc is a potential xenogeneic carbohydrate antigen in bioprosthetic heart valves (BHV) that worsens with time. Porcine/bovine pericardium revealed greater expression of Siaα2–3 as compared to Siaα2–6 while the reverse was reported in porcine aortic/pulmonary valve cusps. Quantitative analysis of sialic acid by HPLC revealed increased Neu5Gc expression in porcine/bovine pericardium as compared to that of ­porcine aortic/pulmonary valves, with Neu5Ac at sixfold over Neu5Gc. Affinitypurified human anti-Neu5Gc IgG showing high specificity ­ toward Neu5Gc-glycans on a glycan microarray, strongly bound to all tested commercial BHV, demonstrating Neu5Gc immune recognition in cardiac xenografts [84]. Apart from the Neu5Gc coded by the CMAH gene, Sd (a) antigen coded by the β1,4 N-acetylgalactosaminyltransferase gene in pigs leads to an immune reaction in human. Deletion of both in pigs finds importance in primate complement and coagulation activation leading to increased life of the graft in nonhuman primate recipients’ minutes. [85]. In different study on NeuGc expression in GTKO/hCD46 and GTKO/ hCD46/NeuGcKO pig aortas and corneas, and pRBCs, pPBMCs, aortic endothelial cells (pAECs), corneal endothelial cells (pCECs), and isolated pancreatic islets have been revealed while the absence of NeuGc expression on pig aortas, corneas, and RBCs, peripheral blood mononuclear cells (PBMCs), aortic endothelial cells (AECs) significantly reduces the human antibody binding, but the absence of NeuGc expression on pig CECs, isolated islet cells does not reduce human antibody-binding. NeuGc absent expression on GTKO/hCD46 pAECs does not reduce human platelet aggregation [86]. The pig pancreas as a source of islets for xenotransplantation in patients with type I diabetes.

8  Sialylation related knockouts and health of donor animals KO animals by removing the genes involved in or affecting sialylation may affect the health of the animals. Mice lacking exon 6 of CMAH gene to produce Neu5Gc KO mice have been reported to produce abnormal pancreatic islet with altered metabolic response to a high-fat diet together with impaired skin wound healing [87, 88] Pig KO lacking Gal and Neu5Gc antigens have been produced, by knocking out both GT1 and CMAH genes [69].



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9 Discussion Despite advances in the growth and application of technology and genome editing tools and genetic engineering approaches and generation of KO in solving challenges associated with xenotransplantation, it remains still a major challenge in the field of biological searches. Especially this area remains largely to be worked out the area of the brain and neurodegenerative disorders including Huntington’s disease (HD) and Parkinson’s disease (PD). The role of sialic acid is tremendous in regulating graft rejection and acceptance and therefore, strategies to generate models with better life survival in human recipient needs to be searched for circumventing the challenges posed by sialic acids in xenotransplantation. Protocols to control the spread of unknown infections in the humans from animal donors need to be designed prior to their application in humans and remains a major challenge in this domain.

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[55] Pirofsky B, Ramirez-Mateos JC, August A. “Foreign serum” heterophile antibodies in patients receiving antithymocyte antisera. Blood 1973;42:385–93. [56] Higashi H, Naiki M, Matuo S, Okouchi K. Antigen of “serum sickness” type of heterophile antibodies in human sera: Identification as gangliosides with N-­glycolylneuraminic acid. Biochem Biophys Res Commun 1977;79:388–95. [57] Merrick  JM, Zadarlik  K, Milgrom  F. Characterization of the Hanganutziu-Deicher (­serum-sickness) antigen as gangliosides containing N-glycolylneuraminic acid. Int Arch Allergy Appl Immunol 1978;57:477–80. [58] Zauner G, Selman MH, Bondt A, et al. Glycoproteomic analysis of antibodies. Mol Cell Proteomics 2013;12:856–65. [59] Galili U. Anti-Gal: an abundant human natural antibody of multiple pathogeneses and clinical benefits. Immunology 2013 Sep;140(1):1–11. [60] Petersen B, Carnwath JW, Niemann H. The perspectives for porcine-to-human xenografts. Comp Immunol Microbiol Infect Dis 2009;32:91–105. [61] Varki A. Evolutionary forces shaping the Golgi glycosylation machinery: Why cell surface glycans are universal to living cells. Cold Spring Harb Perspect Biol 2011;3:a005462. [62] Gao B, Long C, Lee W, Zhang Z, Gao X, Landsittel D, Ezzelarab M, Ayares D, Huang Y, Cooper  DKC, Wang  Y, Hara  H. Anti-Neu5Gc and anti-non-Neu5Gc antibodies in healthy humans. PLoS One 2017 Jul 17;12(7):e0180768. [63] Waldman JP, Brock LG, Rees MA. A human-specific mutation limits nonhuman primate efficacy in preclinical xenotransplantation studies.Transplantation 2014;97:385–90. [64] Brock LG, Delputte PL, Waldman JP, Nauwynck HJ, Rees MA. Porcine sialoadhesin: a newly identified xenogeneic innate immune receptor. Am J Transplant 2012;12:3272–82. [65] Waldman JP,Vogel T, Burlak C, Coussios C, Dominguez J, Friend P, Rees MA. Blocking porcine sialoadhesin improves extracorporeal porcine liver xenoperfusion with human blood. Xenotransplantation 2013;20:239–51. [66] Miwa  Y, Kobayashi  T, Nagasaka  T, Liu  D, Yu  M, Yokoyama  I, Suzuki  A, Nakao  A. Are N-glycolylneuraminic acid (Hanganutziu-Deicher) antigens important in pig-to-­ human xenotransplantation? Xenotransplantation 2004;11:247–53. [67] Ikeda  K, Yamamoto  A, Nanjo  A, Inuinaka  C, Takama  Y, Ueno  T, Fukuzawa  M, Nakano  K, Matsunari  H, Nagashima  H, Miyagawa  S. A cloning of cytidine monophospho-N-­acetylneuraminic acidhydroxylase from porcine endothelial cells. Transplant Proc 2012;44:1136–8. [68] Yamamoto A, Ikeda K,Wang D, Nakatsu S,Takama Y, Ueno T, Nagashima H, Kondo A, Fukuzawa M, Miyagawa S. Trial using pig cells with the H-D antigen knocked down. Surg Today 2013;43:782–6. [69] Kwon  DN, Lee  K, Kang  MJ, Choi  YJ, Park  C, Whyte  JJ, Brown  AN, Kim  JH, Samuel M, Mao J, Park KW, Murphy CN, Prather RS, Kim JH. Production of biallelic CMP-Neu5Ac hydroxylase knock-out pigs. Sci Rep 2013;3:1981. [70] Squinto  SP. Genetically modified animal organs for human transplantation. World J Surg 1997;21:939–42. [71]. Salama A, Conchon S, Perota A, Martinet B, Judor J-P, Evanno G, Le-Bas S, Le L, Hervouet J, Minault D, Concordet J-P, Dugast E, Vanhove B, Abadie J, Gaide N, Lagutina I, Duchi R, Lazzari G, Sachs D, Gauthier O, Brouard S, Cozzi E, Blancho G, Perreault H, Bach J-M, Galli C, 3, J-P 11. Characteristics of IgGs Produced in Neu5Gc and α1–3 Gal Double Knock-Out Pigs [abstract]. Am J Transplant. 2015; 15 (Suppl. 3). https://atcmeetingabstracts.com/abstract/characteristics-of-iggs-producedin-neu5gc-and-1-3-gal-double-knock-out-pigs/. Accessed February 21, 2019. [72] Byrne GW, McGregor CGA, Breimer ME. Recent investigations into pig antigen and anti-pig antibody expression. Int J Surg 2015;23(Pt B):223–8. [73] Paris  LL, Chihara  RK, Sidner  RA, Tector  AJ, Burlak  C. Differences in human and porcine platelet oligosaccharides may influence phagocytosis by liver sinusoidal cells in vitro. Xenotransplantation 2012;19:31–9.



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[74] Ecsedy JA, Holthaus KA, Yohe HC, Seyfried TN. Expression of mouse sialic acid on gangliosides of a human glioma grown as a xenograft in SCID mice. J Neurochem 1999;73:254–9. [75] Song KH, Kang YJ, Jin UH, Park YI, Kim SM, Seong HH, Hwang S,Yang BS, Im GS, Min KS, Kim JH, Chang YC, Kim NH, Lee YC, Kim CH. Cloning and functional characterization of pig CMP-N-acetylneuraminic acid hydroxylase for the synthesis of N-glycolylneuraminic acid as the xenoantigenic determinant in pig-human xenotransplantation. Biochem J 2010;427:179–88. [76] Basnet NB, Ide K, Tahara H, Tanaka Y, Ohdan H. Deficiency of N-glycolylneuraminic acid and Galα1-3Galβ1-4GlcNAc epitopes in xenogeneic cells attenuates cytotoxicity of human natural antibodies. Xenotransplantation 2010;17:440–8. [77] Burlak C, Twining LM, Rees MA. Terminal sialic acid residues on human glycophorin A are recognized by porcine kupffer cells. Transplantation 2005;80:344–52. [78] Maeda A, Kawamura T, Nakahata K, Ueno T, Usui N, Eguchi H, Miyagawa S. Regulation of macrophage-mediated xenocytotoxicity by overexpression of alpha-2,6-­ sialyltransferase in swine endothelial cells. Transplant Proc 2014;46:1256–8. [79] Wu X, Kriz AJ, Sharp PA. Target specificity of the CRISPR-Cas9 system. Quant Biol 2014;2:59–70. [80] Butler JR, Wang ZY, Martens GR, Ladowski JM, Li P, Tector M, Tector AJ. Modified glycan models of pig-to-human xenotransplantation do not enhance the human-­antipig T cell response. Transpl Immunol 2016 Mar;35:47–51. [81] Cooper DK, Koren E, Oriol R. Oligosaccharides and discordant xenotransplantation. Immunol Rev 1994;141:31–58. [82] Beaton  BP, Kwon  DN, Choi  YJ, Kim  JH, Samuel  MS, Benne  JA, Wells  KD, Lee  K, Kim JH, Prather RS. Inclusion of homologous DNA in nuclease-mediated gene targeting facilitates a higher incidence of bi-allelically modified cells. Xenotransplantation 2015;22:379–90. [83] Kim GA, Lee EM, Jin JX, Lee S, Taweechaipaisankul A, Hwang JI, Alam Z, Ahn C, Lee  BC. Generation of CMAHKO/GTKO/shTNFRI-fc/HO-1 quadruple gene modified pigs. Transgenic Res 2017;26:435–45. [84] Reuven EM, Leviatan Ben-Arye S, Marshanski T, Breimer ME,Yu H, Fellah-Hebia I, Roussel JC, Costa C, Galiñanes M, Mañez R, Le Tourneau T, Soulillou JP, Cozzi E, Chen  X, Padler-Karavani  V. Characterization of immunogenic Neu5Gc in bioprosthetic heart valves. Xenotransplantation 2016;23:381–92. [85] Cooper  DK. Modifying the sugar icing on the transplantation cake. Glycobiology 2016;26:571–81. [86] Lee W, Hara H, Ezzelarab MB, Iwase H, Bottino R, Long C, Ramsoondar J, Ayares D, Cooper DK. Initial in vitro studies on tissues and cells from GTKO/CD46/NeuGcKO pigs. Xenotransplantation 2016;23:137–50. [87] Kavaler  S, Morinaga  H, Jih  A, et  al. Pancreatic b-cell failure in obese mice with ­human-like CMP-Neu5Ac hydroxylase deficiency. FASEB J 2011;25:1887–93. [88] Hedlund M,Tangroranuntakul P,Takematsu H, et al. N-glycolylneuraminic acid deficiency in mice: implications for human biology and evolution. Mol Cell Biol 2007;27:4340–6.

Further reading [89] UK Transplant. United Kingdom Preliminary Data. Bristol: UK Transplant; 2002, www.nhsbt.nhs.uk. [90] Aristizabal AM, Caicedo LA, Martínez JM, Moreno M, Echeverr GJ. Clinical xenotransplantation, a closer reality: literature review. Cir Esp 2017;95:59–120. [91] Database: WHO, http://humanxenotransplant.org/home/index.php/2012-02-07-1413-27; 2012.

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[92] Second WHO Global consultation on regulatory requirements for xenotransplantation clinical trials, October 17–19 2011, WHO, Geneva, Switzerland. [93] Springer SA, Gagneux P. Glycomics: revealing the dynamic ecology and evolution of sugar molecules. J Proteomics 2016;135:90–100. [94] https://www.yourgenome.org/facts/what-is-crispr-cas9.

CHAPTER 11

Nanotechnology and sialic acid biology 1 Introduction Nanotechnology, the science of matter with dimensions ranging from 1 to 100 nm, with unique size (Fig. 1), physical and chemical properties including compatibility, catalytic, photonic, electronic, or magnetic properties has found myriads of diverse applications as biosensors by detecting minute quantities of matter like microorganisms, detection of pathogens, allergens, unwanted matters in food, detection, imaging and targeting of diseases, as vaccine adjuvants, as biomedicine, in postimplantation monitoring, in nanoarrays to detect toxic molecules and infectious agents, in monitoring of signaling pathways of stem cells, in environmental remediation, in personal care products like products related to improvement of the brightness of teeth, development of optical tags, and nanoplex biotags [1–7].

2 Nanotechnology Nanotechnology encompasses the science of matter at dimensions and tolerances at nanoscales of less than 100 nm, with manipulation of individual atoms and molecules. Their unique size-dependent properties enable their superior applications to human use. Bionanotechnology is the science encompassing the application of biotechnology and nanotechnology and has applications in products used in our everyday lives including personal care products to drugs and medicine. Nanoparticles (NPs) are now gaining much importance due to their application in biology and medicine. The major biological applications include as fluorescent biological labels, detection of pathogens and proteins, probing of the DNA, engineering applications in tissues, targeting tumor and targeted delivery of drugs, genes, and small molecules, identification, estimation separation, purification, and characterization of biological molecules and cells, as magnetic resonance imaging (MRI) contrast enhancement agents and in phagokinetic studies [8]. Sialic Acids and Sialoglycoconjugates in the Biology of life, Health and Disease https://doi.org/10.1016/B978-0-12-816126-5.00011-1

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Fig.  1  Similar dimensions of nanoparticles and biological entities are represented. (Reproduced with permission from M. Marradi, M. Martin-Lomas and S. Penade’s, Adv. Carbohydr. Chem. Biochem., 2010, 64, 211–290.)

NPs with similar size as proteins enable applications of NPs in bio-­ tagging or labeling. To interact with biological target, a biological molecule antibodies, including biopolymers like collagen, or monolayers of small molecules acting as bioinorganic interface rendering the property of biocompatibility. To enable optical detection, NPs with fluorescent properties of that alter their optical properties are applied [8]. Some of the modifications of NPs for biomedical applications are enlisted in Fig. 2.

3 Glyconanotechnology Nanotechnology has been applied to glycobiology forming the new science of glyconanotechnology and is a synergy between nanotechnology and glycans playing role in biological and medical applications [1]. More recently, they have been applied in the sialic acid biology. Glycans occur as surface lining macromolecules and form the first line of contact for any other cell or pathogen and protein-carbohydrate interactions and are known to play role in cell signaling, molecular recognition, immunity, and inflammation. Carbohydrate comprising of wood, insect shells, or cartilage, with mechanical properties serve as biomaterials of importance. Cellulose nanocrystals find importance in processes for degradation of biomass to biofuels and chemicals.



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Antigen detection

Linkers

Functional layers

Protective layer

Fluorescent signalling Biocompatibility

Shape recognition

Fig.  2  Configurations utilized in nano-biomaterials applied to medical or biological problems. (Reproduced with permission from Salata O. Applications of nanoparticles in biology and medicine. J Nanobiotechnol 2004;2:3. Published online April 30, 2004. doi: https:// doi.org/10.1186/1477-3155-2-3.)

Glyconanomaterials (Fig. 3) with properties of nanomaterials of better solubility, biocompatibility, lower cytotoxicity with the uniqueness of their size, chemical properties, surface engineering, surface charge and electronic, photonic, and magnetic like physical properties and properties of glycans of water solubility, biocompatibility, structural diversity, and specific targets [7] have major applications in biology encompassing the domains of (i) as sensitive biological probes in cells and tissues enabling building of different scaffolds, (ii) as imaging agents, (iii) as spectroscopic tools for their detection, (iv) monitoring of cellular systems, and (v) application in vaccination and drug delivery.

4  Sialic acid and nanotechnology Sialic acid has been associated with the disease pathology in several diseases including autoimmune disorders, infection, and cancer. Recently, the sialic acid-Siglec axis discussed in the earlier chapters is revealing to be an emerging target to prevent or affect several diseases. However, the study of deeper role of sialic acid-Siglec axis in immune modulation and therapy suffers from the limitation of suitable sensitive methods.

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Fig. 3  Representative picture of glyconanomaterials made by coupling glycans to the surface of diverse nanomaterials. (Reproduced with permission from Penadés S, Davis BG, Seeberger PH. Glycans in Nanotechnology. 2017. In: Varki A, Cummings RD, Esko JD et al., editors. Essentials of Glycobiology [Internet]. 3rd edition. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 2015–2017. Chapter 58.)

Natural sialic acid ligands can be modified by chemical methods leading to the development of sialic acid mimetics (SAMs) that reveal improved and selective binding affinity toward Siglecs. Glycobiotechnology involving bioorthogonal synthesis is enabling the presentation of SAMs on NPs, polymers, and living cells which finds application in the study of the sialic ­acid-Siglec axis and its role in immune modulation and therapeutic potential [9]. Although sialic acid and its derivatives reveal promises as stealth carriers with properties including targeting ability, cancer inhibition, viral and inflammation recognition, brain targeting and effective targets in several ­disorders



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by in vivo and in vitro studies, design of nanocarrier in drug delivery and targeting, based on sialic acid remains as a major challenge and suffers from the limitations of multi-target side effects and calls in for more research [10]. We discuss in this chapter (i) the glycans and nanotechnology, (ii) role of nanotechnology in detection and quantitation of sialic acid, and (iii) nanotechnology and their applications in sialic acid biology and associated biology of disease, the application, and challenges.

5  Glycans and nanotechnology Glycoproteins or glycolipids that take part in cellular communication, inflammation, and immune responses using carbohydrate-protein or carbohydrate-­carbohydrate and are known for their multivalent interactions [11] with larger variation in affinity/avidity in normal individuals and reported to be disease markers in different diseases as cancer, asthma, and diabetes. Nanotechnology enabling estimation, creating, manipulation of matter at nanoscales, is finding application in study and manipulation of glycans. Various scaffolds such as glycodendrimers or glycopolymers with high surface/volume ratios, allowing better surface contact with improved multivalency effects have been constructed from diverse nanomaterials including semiconductor, carbon-based nanomaterials and have been studied for carbohydrate-protein interactions and find applications in drug delivery, imaging, diagnostics, or sensitive quantitation tools. Polysaccharides nanomaterials including chitosan, dextran, hyaluronic acid, and heparin have been designed drug delivery devices [1–13] and in pharmaceutical application with the advantages of biocompatibility, with reduced toxicity/nontoxicity, prolonged persistence, and drug release. Hybrid substructures are also constructed with metal-cored NPs coated with polysaccharides. Inorganic nanostructures of iron oxide, noble metal, and semiconductors enable formation of synthetic scaffolds to multimerize glycans and enhance the affinity for receptors. Magnetism and fluorescence of hybrid materials finds applications in sensing, delivery, or imaging. Gold nanoparticles (AuNPs) [13, 14] in conjugation with glycans [13–18] enable them with high aqueous solubility/dispersibility, biocompatibility and their high surface area/volume ratio of AuNPs allows enhanced sensitivity. Carbohydrate-lectin analyses have been enabled by the application of AuNPs. Mannosylated AuNPs find application in detection of complement activation and opsonization processes in macrophage-­ mediated endocytosis and to target Escherichia coli–containing type 1 pili mannose-specific receptors.

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Magnetic nanoparticles (MNPs), including iron oxide and manganese oxide NPs, find application as contrast agents for MRI [19, 20]. GlycoMNPs with high surface/volume ratio have enabled detection of early stage disease by mimicking leukocyte recruitment during inflammation [7, 21]. Tetrasaccharide sialyl-Lewis x (sLex)-functionalized MNPs have found application in targeting E-/P-selectins and find application in detection of inflammation [7, 21]. Quantum dots (QDs) including binary cadmium or zinc selenides or sulfides are luminescent semiconducting nanomaterials and can emit light with broader excitation spectrum and sharper emission bands [22, 23]. Glyco-QDs functionalized with carboxymethyldextran and polylysine have found application in study of carbohydrate-protein interactions. QDs can be stabilized with glycodendrimers [7, 21]. Buckminsterfullerene C60, and carbon nanotubes (CNTs) [24–27] glycosylated as in α-D-mannosyl fullerenes and fullerenols have been known to inhibit erythrocyte aggregation [28, 29]. Single-walled CNTs (SWCNTs) and multiwalled CNTs (MWCNTs) linked to C18-lipid tail with α-­GalNAc residues have been applied as probes or radiotracers in developing sensitive in vivo imaging or radiation delivery systems with high radioisotope loading [21, 30]. Glycan-linked graphene has been reported to enable agglutination and inhibition of bacterial motility. Chitosan-based NPs [31] are reported to deliver proteins, oligonucleotides, and plasmid DNA. Multifunctional glycol-chitosan NPs with a near-infrared (NIR) fluorophore for fluorescence imaging [32] have found application in encapsulating anticancer drugs or complex small interfering (siRNA) as drug delivery device. Chitosan-polyethylene glycol (PEG)-coated iron oxide NPs have been reported to make better intracellular delivery of a DNA repair inhibitor (O6-benzylguanine) to glioblastoma multiform cells and enable treatment monitoring by MRI. Dextran enables improved water solubility and stability of iron oxide MNPs while sulfated dextran can electrostatically interact with positively charged polycations. Functionalization of dextran-coated iron oxide NPs with sLex tetrasaccharide has enabled recording of inflammation in mouse brain. Hyaluronic acid and heparin-based NPs offer promises in cancer therapy by targeted, magnetic, photodynamic, and gene therapy [33]. Glycodendrimers enabled formation of three-dimensional (3D) supramolecular sugar scaffolds of sugars with a Ru (bpy)3 core [34] that could bind to E. coli expressing mannose receptor of bacterial pili, displayed on virus-like particles [35] enabled picomolar inhibition of adhesion of Ebola



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virus. Nanoengineered glycan sensors probes of AuNPs and CNTs may help with glycoprotein profiling. Glyconanomaterials [36–38] of gold and silver find application in cancer detection by quantifying cell-surface mannose glycans. Mannan-coated AuNP incubated with a human gastric cell line in the presence of the mannose-binding lectin ConA enabled detection of aberrant glycosylation in cancer [39]. ConA-functionalized CNTs finds application in surface glycan detection [40].

6  Role of nanotechnology in detection and quantitation of sialic acid Sialic acid is known to be present as components of mucin component, glycoproteins, and other microbial polymers in nature food, further emphasizing the need of sensitive tools to detect them even in traces.The glycome of cells and glycoproteins and their detection and estimation finds importance in understanding glycan functions, development of diagnostics tests, and monitoring of glycoprotein pharmaceuticals. Sialic acid-containing carbohydrates, collectively grouped as sialosides, are known to play major roles in the physiology of health and disease-like infections by virus and bacteria, tumor cell metastasis, but limitation of suitable methods to study sialosides forms the major challenge in the study of their structure and function. Appropriate quantitation of sialic acid finds importance in health and disease to understand the levels correlating with the homeostasis and pathophysiology of the body in infection and disease. Although several biochemical tests find importance in detection and quantitative estimation of sialic acid in the body, the detection of minute quantities of sialic acid and the perturbation in disease states is far from complete. Nanotechnology and its diverse application and application in sensitive methods finds importance in the quantitative detection of N-glycans and sialic acid in the body even in very small amounts. Synthetic sialoside chemistry, by chemoenzymatic or stereochemical approach, have produced homogeneous size- and structure-defined sialosides with array application, to mimicking cell-surface display and aids in understanding sialoside-mediated interactions. Application of nanotechnology in sialoside arrays [41] is suggested to lead to promising results in study of sialic acid biology. N-glycans are isolated and characterized by conventional methods of enzymatic treatment, followed by their release and derivatization with a fluorochrome and separation by normal-phase high-performance liquid chromatography (HPLC). Nano Quantity Analyte Detector (NQAD) has been

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designed to quantitate the nonderivatized sialic acid in glycoproteins, separated by hydrophilic interaction chromatography, detected by measuring size differences in dry aerosol and by converting the particle count rate into chromatographic output signal.This sialic acid quantitative sensitive method lacking requirement of active chromophore or fluorophore finds importance over conventional methods and HPLC/NQAD method offers advantage in reproductive results over the conventional HPLC/DMB method. While HPLC/DMB method involves derivatization of glycoproteins using 1, 2-diamino-4, 5-methylenedioxybenzene (DMB) and Dionex-based high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), the HPLC/NQAD method is designed with elimination of the derivatization step and efficient PolyGLYCOPLEX amide column adding to the sensitivity to the detection method [42]. Simultaneous quantification and characterization of the N-glycans including both neutral and sialylated glycans suffers from lack of appropriate methods of identifying them. This is circumvented by applying a weak ­anion-exchange HPLC separation step to fractionate glycans by their sialic acid content followed by a mild acid desialylation step and then resolved by nano-LC-coupled electrospray ionization (ESI)-mass spectrometry with an intercalated nanofluorescence detector by which neutral glycans can be separated and characterized [43]. ESI-MS method finds applications in detection and characterization of the heavily polysialylated N-glycans in human serum with improved sensitivity [44]. Isomeric glycan profiling using nanoLC-MS with porous graphitized carbon (PGC) as the stationary phase has found importance in detection of sialylated serum proteins by high detection sensitivity and chromatographic resolution [45]. Subambient pressure ionization with nanoelectrospray (SPIN) using advanced data processing tools has increased the efficiency and sensitivity and has enabled high-resolution MS in detecting sialic acid polymer chains over conventional ESI-MS [46]. A quick, sensitive fluorimetric detection method by using a sensitive ­lectin-CdTe QDs nanoprobe made by conjugating Sambucus nigra bark lectin (SNA) as probe for sialic acid forming SNA-CdTe QDs has been designed to detect sialic acid in egg products. Sialic acid and SNA-CdTe QDs, interaction lead to generation of fluorescent signal and is able to detect as sialic acid as low as 0.67 ng/mL [47]. N-Glycolylneuraminic acid (NeuGc), is produced in animals, including cattle and mice, but not in human and is considered to be immunogenic in humans. Therefore, NeuGc contamination in human embryonic stem cells



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cultured xenogeneic serum due to accumulation indicated its harmfulness and raised concerns over its safety of cell therapy products. To detect femto level the presence of Neu5GC, a nano-flow liquid chromatography/Fourier transformation ion cyclotron resonance mass spectrometry (nanoLC/FTMS) and nanoLC/MS/MS has been designed with promising results [48].

6.1  Detection of gangliosides Gangliosides (GGs) are involved in many brain functions at the cell and molecular level and their study detection and characterization suffers limitation of suitable sensitive methods for detection and analysis. Sialic acid-coated NPs are finding applications in targeting in cancer [49]. Nanotechnologybased detection of glycans and sialic acid conjugates is finding application in detection of GG composition. In human hemangioma, GG composition and structure has been detected by highly sensitive methods of mass spectroscopy (MS) methods based on fully automated chip-nanoelectrospray (nanoESI) high-capacity ion trap (HCT) and collision-induced dissociation (CID) all integrated in the chip-nanoESI approach revealed detection of the presence of one modified O-Ac-GD2 and O-Ac-GM4 GGs and the presence of GT1a and GT1b isomers and unusual GT1c and GT1d glycoforms in brain hemangioma tumor [50]. The nanotechnology-based method offers advantage over conventional methods in its sensitivity and detection of unusual forms of GGs hitherto undetected by conventional methods in this disorder. In all, 81 GG components were detected in human caudate nucleus (CN) by chip-nanoelectrospray MS performed on a NanoMate robot coupled to a HCT instrument in only 1.5 min revealing structures of mono-, di-, and trisialylated GGs and finds importance in detection of GG in CNrelated neurodegenerative disorders [51]. Sensitive detection of Neu5Gc-containing GGs from Neu5Accontaining analogs was separated from the lymphoma cell line derived from mouse (YAC-1) lymphoma cell monosialoganglioside fraction by nano-high-performance liquid chromatography (nanoHPLC) in online conjunction with ESI quadrupole time-of-flight (ESI-QTOF) mass spectrometry and served as a promising glycolipidomic tool [52].

7  Sialic acid in therapy Evading the reticuloendothelial system (RES) is a major obstacle in drug delivery and targeting in cancer. Sialic acid is known for its reduced interaction with the innate immune system by Siglec, thus regulating p­ hagocytic

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Fig.  4  AuNP distribution and tumor targeting: While (A) unmodified AuNPs undergo phagocytosis by the reticuloendothelial system (RES), (B) modified AuNPs like sialic acidmPEG-AuNPs escape RES phagocytosis and target tumor cells. (Reproduced with permission from Kim YH, Min KH, Wang Z et al. Development of Sialic Acid-coated Nanoparticles for Targeting Cancer and Efficient Evasion of the Immune System. Theranostics. 2017;7: 962–973.)

e­ vasion. Surface engineered AuNPs conjugated to sialic acid have revealed that sialic acid-mPEG-AuNPs can escape uptake by RES and therefore, efficiently target tumor cells and by targeted delivery can enhance accumulation in tumor [49] (Fig. 4). Influenza A infection is initiated by binding of viral envelope hemagglutinin (HA) glycoproteins to cell membrane sialic acid. Free toxic sialic acid monomers cannot block HA adhesion in  vivo. Polyvalent, generation 4 (G4) sialic acid-conjugated polyamidoamine (PAMAM) dendrimer (G4-sialic acid) has been found to inhibit three influenza A subtypes (H1N1, H3N2) indicative of the fact that polysialic acid (PSA) inhibitors have potential in antiviral therapeutics [53]. Amongst dendritic polymeric inhibitors, including spheroidal, linear, linear-dendron copolymers, combbranched, and dendrigraft polymers are known for the ability to inhibit virus hemagglutination (HA) and to block infection of mammalian cells in vitro. Comb-branched and dendrigraft inhibitors revealed to be the most effective inhibitor, with up to 50,000-fold more antiviral activity [54]. Targeted delivery of sialic acid inhibitors like sialic acid-blocking glycomimetic has been reported to block cancer metastasis [7, 21, 55]. Sialic ­acid-blocking glycomimetic [P-3F (ax)-Neu5Ac] coated antibodies a­ llowed targeted delivery into melanoma cells successfully preventing cancer metastasis in murine lung cancer model [55] (Fig. 5).



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Fig. 5  Targeted delivery of a sialic acid-blocking glycomimetic to cancer cells inhibits metastasis. (Reproduced with permission from Christian Büll, Thomas Jan Boltje, Eric A. W. van Dinther, Timo Peters, Annemarie M. A. de Graaf, Jeanette H. W. Leusen, Martin Kreutz, Carl G. Figdor, Martijn H. den Brok, and Gosse J. Adema ACS Nano, 2015, 9(1), pp. 733–745.)

Linkage-specific sialylated glycans could be characterized from reaction with condensation reagent 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-­ 4methylmorpholinium chloride (DMT-MM) in methanol with nanoscale liquid chromatographic separation prior to accurate mass Orbitrap MS analysis and improve separation and enrichment of trisialylated N-glycan fraction from haptoglobin and human plasma, as trisialylated fraction has been linked with cancer-associated changes in the serum N-glycome [56]. The identification of sialylated Thomsen-Friedenreich antigens in proteins finds importance in cancer research. Sialylated antigens in minute quantities (0.1 μg) and plasminogen (1.0 μg) could be detected from gels by reductive β-elimination, permethylated and analyzed by ­nano-LC-matrix

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assisted laser desorption/ionization (MALDI)-TOF-MS and using a computational algorithm to filter spectral noise and enhance/isolate the signals of interest [57]. Integrating a fast preparation protocol of mucins with high-­throughput nanoLC/MS have enabled the study the O-glycosylation of the colon MUC2 mucin from biopsy of sigmoid colon during routine colonoscopy of 25 normal control patients [58]. Negative ion nano-liquid chromatography/mass spectrometry (nano-LC/MS) and tandem mass spectrometry (nano-LC/MS [2], using graphitized carbon as separating medium, could analyze neutral and acidic O- and N-linked oligosaccharide alditols. Automated glycofragment mass fingerprinting using the GlycosidIQ software confirmed the oligosaccharide sequence for both neutral desialylated as well as sialylated structures in membrane proteins from ovarian tissue [59]. Attachment of α-N-acetylneuraminic acid (Neu5Acα) to the terminal glycine residues tetraantennary peptides [glycine (n)-NHCH [2]] [4] C is reported to give rise to water-soluble assembled glycopeptides that can to bind influenza virus multivalently and inhibit adhesion of the virus to cells more effectively is a promising antiviral strategy in the design of multivalent antivirals [60].

8  Nanotechnology bioimaging application, detection of cellular sialic acid expression and targeting Polyacrylamide hydrogel-based lectin microarray with 27 lectins on colorectal cancer (CRC) cell lines SW480, SW620, and HCT116 revealed high glycan expression of d-galactose, d-glucose, and/or sialic acid residues with Uelx Europaeus Agglutinin-I (UEAI) showing specificity to SW480 cells. UEA-I conjugated with silica-coated NaGdF4: Yb3+, Er3+@ NaGdF4 has been reported to be effective designs to target tumor molecule in SW480 tumor detected by upconversion luminescence imaging, T1-weighted MRI, and X-ray computed tomography (CT) imaging [61]. CD22 finds importance as an important drug target in autoimmune diseases and B cell-derived malignancies. Nanoprobe of sialic acid/N-­ acetylneuraminic (NANA) acid conjugated to carboxyl groups modified CdSe/ZnS quantum dots (COOH-QDs) by the NHS/EDC esterification chemistry led to the formation of functionalized QD nanoconjugate which has been applied to target CD22 and the targeting could be detected by fluorescence imaging [62]. An immobilized mercaptophenyl boronic acid



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(MPBA) nanochip with nanocone-array substrate on Au and Ag NPs for dynamic electro-optical by the metal-S bond could detect selective sialic acid as low as 17 μM [63]. Nano-TiO2 has been proved to have cytotoxic and phototoxic effects on different crystalline phases for human skin keratinocytes (HaCaT cells) under ultraviolet (UV) irradiation revealing increased α2,6-sialylated glycans. Although mixture of crystalline P25 revealed highest cytotoxicity and phototoxicity, followed by pure anatase A25, and pure rutile R25 but A25 and R25 did not affect sialic acid expression on HaCaT cells [64]. Nanomaterials find application in tumor targeting and find application in cancer therapy. PEGylated, borate-coordination-polymer-coated polydopamine NP (PDA@CP-PEG) with DOX (Doxorubicin) reveal synergetic targeting of sialic acid-overexpressed tumor cells. Photothermal effect of the polydopamine core and the DOX-loading capacity of the polymer layer enable their potential for chemo-photothermal combination therapy with less toxicity, efficient tumor targeting ability, and chemo-photothermal activity for tumor inhibition with promising potential clinical applications [65]. 4-MPBA, a surface-enhanced Raman scattering (SERS) nanoprobe (glucose-MPBA-AgNPs) prepared with 10 times stronger SERS enhancement ability as compared to MPBA-AgNPs could detect sialic acid ­expression by amplifying their expression on cancer cells, by the differential accumulation of glucose-MPBA-AgNPs on cancer vs normal cells due to the differences of sialic acid expression on cancer vs normal cells enabling detection of cancer cell with diagnostic and prognostic potential [65, 66]. Carbon dot (Cdot) NPs offer promising potential for drug delivery and bioimaging applications. J774.1 macrophages have been shown to take up phenylboronic acid (PBA)-modified NPs as PB binds to sialic acid residues overexpressed on diseased cell surfaces and finds application in drug targeting to macrophages associated with tumors [67]. Sialic acid as a ligand by dexamethasone (DM)-loaded solid lipid NPs has been used for targeting renal ischemia–reperfusion injury (IRI)-induced acute kidney injury (AKI). DM-loaded sialic acid-conjugated PEGylated NPs (sialic acid-NPs) could reduce apoptotic human umbilical vein endothelial cells (HUVECs) via downregulating oxidative stress-induced Bax, upregulating Bcl-xL, and inhibiting caspase-3 and caspase-9 activation being internalized by inflamed vein endothelial cells (VEC) mediated by specific binding between sialic acid and E-selectin receptor expressed on the inflamed VEC and could effectively ameliorate renal functions in AKI mice, causing improved blood biochemical indexes, histopathological changes, oxidative stress

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levels, and pro-inflammatory cytokines proving to be efficient and targeted delivery of DM for ischemia–reperfusion-induced injury-induced AKI, with improved therapeutic outcomes and reduced side effects [68]. β-amyloid (Aβ) plaques in the brain are pathological features of Alzheimer’s disease (AD). NP contrast agents capable of binding with Aβ highly selectively enable early detection of AD. But the major obstacle is provided by the blood brain barrier (BBB) that preclude the entrance of NPs into the brain for Aβ binding. Bovine serum albumin (BSA)-coated NPs are designed with sialic acid (NP-BSAx-Sia) has been reported to overcome the challenges in Aβ imaging in vivo due to biocompatible and high magnetic relaxivity, indicating their suitability as contrast agents for MRI [69]. ConA-conjugated DOX-loaded mesoporous silica nanoparticles (MSNs) find applications as delivery devices in bone cancer treatment as ConA and can recognize and bind sialic acid overexpressed in human ­osteosarcoma (HOS) cell line [70]. Neutrophils by forming neutrophil extracellular traps (NETs) with DNA fibers and histones can combat pathogens and antimicrobial components to kill pathogens, but NETs could lead to pathological conditions like sepsis or acute lung failure due to histone-mediated toxicity. Poly sialic acid NPs with property as an antagonist of the cytotoxic properties of extracellular histones neutralize histone-mediated cytotoxicity and initiate binding of these polysialylated particles to NET filaments [71]. Middle East respiratory syndrome coronavirus (MERS-CoV) targets the epithelial cells of the respiratory tract in human and camel host, binding to the cell-surface receptor dipeptidyl peptidase 4 (DPP4) by S1B and sialic acid by S1A domain. Binding is hampered by modification of sialic acid including 5-N-glycolylation and (7)9-O-acetylation or depletion of cell surface sialic acid by neuraminidase treatment indicating that virus-sialic acid interactions are vital to viral entry and infection [72]. Inhibition of influenza A virus infection by multivalent sialic acid inhibitors is a promising strategy [73]. A red blood cell (RBC) cytosensor has been designed employing sialic acid on a quartz crystal microbalance (QCM) by immobilizing RBCs on a ConA-modified gold chip employing recognition between ConA and mannose. 4-Aminobenzeneboronic acid (APBA)-functionalized gold nanoparticles (AuNPs/APBA) were used to label sialic acid and acted as a signal amplification nanoprobe and find importance in detection of sialic acid in diabetic individuals as compared to the normal individuals [74]. MNPs find importance in molecular targeting therapy in cancer but have limitations in targeting. Sialic acid-binding lectins, wheat germ lectin (WGA) conjugate,



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or ­nanomagnetolectin, can target sialic acids overexpressed in prostate cancer and promoted apoptosis under magnetic field (magnetofection) [75]. Early diagnosis of metastatic cancers can prevent mortality in cancer. As aberrant overexpression of sialic acid has been reported in tumors correlating with progressive metastasis, PBA-installed PEGylated AuNPs coupled with Toluidine blue O (T/BA-GNPs) as SERS probes has been reported to target surface overexpressed sialic acid revealing strong SERS signals from metastatic cancer cell lines (breast cancer; MDA-MB231 and colon cancer; Colon-26 cell lines) [76]. CD22 is a member of the Siglec family and CD22-ligand-targeted NPs with therapeutic functions have proved successful in preclinical settings for blood cancers, autoimmune diseases, and tolerance induction [77]. Biosensors for detection of virus were developed by utilizing plasmonic peak shift phenomenon of the AuNP and viral infection mechanism of HA on virus and sialic acid on animal cells [78]. Molecularly imprinted polymers (MIPs) as artificial receptors; can be designed to bind targets like hyaluronan and sialylatic acid and their conjugates and find application in labeling and imaging of cellular targets. Fluorescent-labeled MIP NPs with glucuronic acid (MIPGlcA) and sialic acid could target extracellular hyaluronan and MIP-coated InP/ZnS QDs could target hyaluronan and sialylation sites in both their intra and extracellular expression. Green and red-emitting QDs functionalized with MIPGlcA and MIPsialic acid, respectively, have been reported to enable multiplexed cell imaging [79]. (3-Aminomethylphenyl)boronic acid (AMPB)-installed hyaluronic acid (HA)-ceramide (HACE)-based NPs, including manassantin B (MB), forming HACE-AMPB/MB NPs (239 nm), when targeted on cancer cells revealed increased cellular accumulation and efficient antitumor activity and were hypothesized to react with sialic acid overexpressed in CD44 receptor-­positive human adenocarcinoma cells [80]. Hydrophobically modified polysialic acid (HPSA) NPs,prepared by 1-ethyl-­ 3-(3-dimethylaminopropyl)-carbodiimide (EDC)/N-hydroxysuccinimide (NHS) coupling between N-deacetylated PSA and 5β-cholanic acid loaded with DOX forming (DOX-HPSA) are reported for its anticancer drug nanocarrier activity, therapeutic efficacy, and specific targeting of cancer cells in A549 cells [81]. Fluorescent-conjugated polymer NPs with their optical properties and low cytotoxicity find applications in imaging and the fluorescent intensity was reported to further improve when these polymers were modified with a

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PBA group, covalently linked with sialic acid, forming a sialic acid-imprinted NPs (30 nm) size with selective staining for DU 145 cancer cells [82] (Fig. 6). Protamine nanocapsules (NCs) linked with PSA acted as drug delivery devices and revealed properties of enhanced stability and facilitated transport of macromolecules across the intestinal epithelial cells in cell line including Caco-2 [83]. Magnetic relaxation nanosensors (MRnS) made by conjugating entry blocker peptides to iron oxide NPs through targeted binding with HA. 2,6and 2,3-sialic acid ligands on cell surface could detect HA variants (H1 and H5) in fats and detect the different influenza subtypes [84].

Fig. 6  (A) Selective binding of sialic acid-imprinted fluorescent conjugated polymeric nanoparticles to cancer cells. (B) Confocal laser scanning microscopic images of DU145 (left) and HeLa (right) cells incubated with sialic acid-imprinted fluorescent conjugated polymeric nanoparticles for 24 h at 37°C. (Reproduced with permission from Liu RH, Cui QL, Wang C, Wang XY, Yang Y, Li LD. Preparation of sialic acid-imprinted fluorescent conjugated nanoparticles and their application for targeted cancer cell imaging. ACS Appl Mater Interfaces 2017;9:3006–15.)



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Sialic acid coatings on polymeric micelle consisting of poly(sarcosine)-­ block-poly(l-lactic acid) (lactosome) targeting the immunosuppressive receptors of Siglec-G and CD22 could prevent accelerated blood clearance (ABC) ­phenomenon due to the reduction of the anti-poly(sarcosine) IgM ­production [85]. Sialoglyco-conjugated NPs synthesized from highly branched α-­ glucuronic acid-linked cyclic dextrins (GlcA-HBCD) forming sialoglyco-NP (Neu5Acα2,6LacNAc-GlcA-HBCDs, sialoglycoNP [SAGNP]) could recognize and interact with human influenza virus strain A/Beijing/262/95 (H1N1) detected by HA inhibition assay and SAG-NP with sialic acid substitution of 30, have been reported to inhibit virus-binding activity [86]. Au-NPs functionalized with sialic acid diluted with a PEG forming the sialic acid functionalized gold nanoparticles could detect soluble form of murine Siglec-E (mSiglec-E-Fc fusion protein) on Chinese hamster ovary cells (CHO cells) and find application in detection of Siglec on mammalian cells [87]. A benzoic group functionalized gold nanoflower was designed as nanoprobes for recognition of target sialic acid and assembly of poly sialic acid by sensitive SERS signal [88]. Fluorescent biocompatible polymeric NPs designed with a hydrophobic monomeric core, fluorescent monomer, and a protein-binding monomer that conjugates lectin to target sialic acid is reported to detect and monitor progression of influenza viral infection by detecting the sialic acid expression level changes in human lung epithelial cells [89]. Fluorescent dye rhodamine and two InP/ZnS QDs emitting in the red and green-MIP particles with D-glucuronic acid (GlcA), a substructure of hyaluronan, and sialic acid capable to localize hyaluronan and sialic acid has been designed for bioimaging of human keratinocytes extracellularly viewed by epifluorescence and confocal microscopy and proves to be a promising tool toward monitoring of disease progression [90]. SNA forms strong bonds with AuNPs as compared to Saraca indica (saracin II), in the ground state as detected by UV–vis absorption, steady state, time-­ resolved fluorescence coupled with circular dichroism (CD) spectral studies, finding application in drug delivery systems [91]. AuNPs with sialic acid-­terminated complex bi-antennary N-glycans, synthesized with glycans isolated from egg yolk, found application as sensor in detection of both recombinant HA and whole influenza A virus particles of the H1N1 subtype [92].Aggregation of 4-mercaptophenylboronic acid functionalized AuNPs (4-MPBA-AuNPs) could bind to sialic acid and detected by colorimetric assays and finds importance in detection of sialic acid in blood serum samples [93].

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PBA conjugated with polyethylenimine (PEI1.8 k) to generate amphiphilic PBA-grafted PEI1.8 k (PEI-PBA) nanovector, encapsulated siRNA to form PEI-PBA/siRNA nanocomplexes with properties of biocompatibility, serum stability, and RNase resistance enabled specific delivery to sialic acid overexpressed target cancer cells and significantly decreased polo-like kinase-1 (PLK)-1 expression in tumors, leading to apoptosis and cell cycle arrest [94]. Monosaccharide-imprinted fluorescent NPs comprising of doped silica NPs with a shell imprinted with sialic acid, fucose, or mannose as the template with probe fluorescein isothiocyanate (FITC) enabled imaging of human hepatoma carcinoma cells (HepG-2) and human primary tumor cell line michigan cancer foundation (MCF-7) derived from mammary gland [95]. Sialic acid incorporation into the GG molecule could increase fourfold anticancer compound paclitaxel loading capacity forming self-­ assembled nanostructures of di- and tri-sialogangliosides [96]. An inductively coupled plasma mass spectrometry (ICP-MS) is used to detect sialic acid on the cancer cell surface, recognized by biotinylated phenylboronic acid (biotin-APBA) AuNPs in HepG2 and MCF-7 cells [97]. Molecularly imprinted NPs were prepared as SERS for imaging cancer cells based on targeting of sialic acid overexpressed on cancer cells [98]. Sialic acid coreshell NPs with nitrobenzoxadiazole (NBD) fluorescent groups allowing environmentally sensitive fluorescence finds application as a biosensor [99]. Sepsis is known to lead to acute respiratory distress syndrome (ARDS). Murine Siglec-E and its human orthologs Siglec-7 and Siglec-9 play role in negatively regulating acute inflammatory responses and may act as targets in sepsis and ARDS treatment. Thus, poly(lactic-co-glycolic acid) NPs linked with Siglec ligand, di(α2 → 8) N-acetylneuraminic acid (α2,8 NANA-NP), induced enhanced oligomerization of the murine Siglec-E receptor on macrophages [100]. Reduced graphene oxide-tetraethylene pentamine-1-butyl-­ 3methylimidazolium hexafluorophosphate (BMIMPF6) hybrids with bimetallic gold platinum alloy nanoparticles (AuPtNPs) with SNA could detect α2,6-sialylated glycans in serum [101]. Sialic acid-modified selenium (Se) NPs conjugated with an alternative peptide-B6 peptide forming B6-sialic acid-SeNPs, can cross BBB and enter into cerebral endothelial cells and can act as nanomedicine in AD detected by laser-scanning confocal microscopy, flow cytometry analysis, and ICP-atomic emission spectroscopy (ICP-AES) [102]. 3-Aminophenylboronic acid functionalized CdSeTe@ZnS-SiO2 QDs (APBA-QDs) probes could detect sialic acid on K562 cells [103].



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PSA can be immobilized on nanoporous silica materials silica nanoparticles (NPSNPs) of MCM-41 type [104] with different applications. Raman spectroscopy (SERS)-based sensing platform was developed for detecting sialic acid on single cell surface by 4-(dihydroxyborophenyl) acetylene (DBA)-linked AuNPs HeLa cell [105]. Super-paramagnetic iron oxide nanoparticles (SPIO NPs), αCD22 Abs and MXD3 siRNA molecules entered leukemia cells and knocked down MXD3, leading to apoptosis in Reh cell line and in primary preB ALL samples with synergistic effects by anticancer agents vincristine or DOX [106]. Avian influenza viruses preferentially bind to sialic acid α-2,3-galactose receptors on epithelial cells and magnetic NPs coated with chitosan and functionalized with Maackia amurensis (MAA) lectin (NP-lectin) could isolate sialic acid α-2,3-galactose receptors from porcine trachea [107]. AuNPs immobilized with graphite oxide (GO), Prussian blue (PB), and PTC-NH2 (an ammonolysis product of 3,4,9,10-perylenetetracarboxylic dianhydride) nanocomposite GO-PB-PTC-NH2 modified glassy carbon electrode (GCE) linked to SNAs could detect α2,6-sialylated glycans in serum [108]. Lectin-tagged fluorescent polymeric NPs (35 nm) could detect cellular sialic acid expression [109]. QDs labeled avian influenza H9N2 virus could enable study of establishment of infection in human bronchial epithelial (HBE) cells using a 3D SPT technique [110]. DM and methotrexate (MTX) entrapped within PSA-trimethyl chitosan (TMC) NPs enabled site-specific targeting in rheumatoid arthritis [111]. QDs modified with PBA (QDs-PBA) could target sialic acid expressed on vesicular stomatitis virus (VSV) enabling the virus labeling [112]. Covalent immobilization of SNA on a mixed self-assembled monolayer (SAM) on planar gold surfaces forming a two-dimensional (2D) sensor and immobilized SNA on mixed SAM layer on AuNPs forming 3D sensor could detect sialic acid [113]. AuNPs functionalized with a thiolated trivalent α2,6-thio-linked sialic acid and a thiolated PEG has been designed to detect the human influenza virus X31 (H3N2) as the trivalent α2,6-thio-linked sialic acid bind to virus hemaglutinin [114]. Multifunctional fluorescent silica nanoparticles (FSNPs) with PBA were designed to label sialic acid on cancer cell surface with high selectivity and sensitivity [115]. Sialic acid conjugated to poly(ethylene oxide)-polycaprolactone polymersomes could interact with influenza viruses by inhibiting viral HA binding to host cell sialic acids, thus preventing viral entry. Targeting by design

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of neuraminidase inhibitor zanamivir into the polymersome core, inhibited viral replication [116]. AuNPs attached to polycrystalline gold modified by an aminoalkanethiol linker layer with covalently immobilized SNA on a mixed SAM formed on AuNPs could detect sialic acid and finds application in arthritis or cancer [117]. Siglec-7 ligand, displayed on liposomal NPs, allowed targeting of Siglec-7 positive cells in peripheral human blood [118]. Signals between QDs and AuNPs-sialic acid-binding proteins (SBPs) and sialic acid moieties, respectively, enable biosensing based on the nanometal surface energy transfer (NSET) and could enable detection of glycosylation linkages (α2–6 vs α2–3), and 9-O-acetyl and N-glycolyl group modifications [119]. Gold nanocluster probe was developed to detect cell surface sialic acid [120]. Sialic acid reduced and stabilized AuNPs synthesized by a simple onepot, green method for colorimetric detection of influenza virus by HAsialic acid binding [121]. The liposomes targeting sialioadhesion or Sn or CD 169 could selectively bind to Sn-expressing cells and macrophages accumulating intracellularly overtime enabling antigen delivery to macrophages for their presentation to T cells [122]. A novel electrochemical strategy for in situ detection of cell surface sialic acids by chemoselective labeling technique and a dual-functionalized nanohorn probe [123] was developed. 3-Aminophenylboronic acid functionalized QDs (APBA-QDs) synthesized by covalently binding APBA to mercaptopropionic acid-capped CdS QDs, and polysialic acid stabilized gold nanoparticles (PSA-AuNPs), were prepared by a one-pot procedure.The APBA-QDs recognized the sialic acid on BGC-823 human gastric carcinoma (BGC) cells and then the PSA on AuNPs, therefore, amplifying signal [124] enabling detection of sialic acid. Semiconductor QDs with small molecular PBA tags enabled labeling of sialic acid and imaging of cells [125]. Sialic acid surface-decorated selenium nanoparticles (sialic acid-Se-NPs) have been reported to penetrate cervical carcinoma cells and induce apoptosis by proapoptotic enzymes caspase-3 and poly(ADP-ribose) polymerase (PARP) cleavage in cancer cells [126]. Sialic acid-terminated glycerol dendron functionalized AuNPs have been reported to inhibit influenza virus infection [127]. Lectin-Au-thionine bioconjugates linked to AuNPs revealed mannose expression and expression of biomarker sialic acid in cancer detection, diagnosis, and treatment [128]. PLGA NP modified with BBB penetrating peptide (similopioid peptide) and a sialic acid residue could cross the BBB



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and ­interact with brain receptors. [129]. Polymeric (poly(D,l-lactide-co-­ glycolide), PLGA) NPs surface modified with sialic could be devised [130].

9 Discussion The application of nanotechnology in the study and biomedical applications of glycosylated molecules and sialic acids and their conjugates on the cells and serum, in human health and disease is a recent development and considerable work has been progressed in the last decade. Different designs of nanoparticles have enabled (i) sensitive detection of sialic acid in free forms and in conjugated form inferring on their structures and discovery of novel molecules, hitherto unknown in health and disease due to their sensitive and improved specialized nature of detection systems and (ii) targeting of sialic acids as drug delivery targets with tremendous application in targeting of infectious, pathogenic diseases and cancer. The ongoing research is their application in biomedicine, imaging, and sensor applications is thought to have a major impact on the human lives.With the advancement of the applications of nanotechnology in sialic acid biology, a new term perhaps needs to be coined now as Sialonanotechnology encompassing the applications of nanotechnology is study of sialic acid biology in health and disease.

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Glossary 4-Mercaptophenylboronic acid functionalized AuNps used in the selective sensor for colorimetric detection of sialic acid in which the solution color changes from wine red to blue after binding with sialic acid. They find application in detection of sialic acid in blood serum samples 9-O-acetyl sialic acid a 9-O-acetylation modification of human Sia 5-N-acetylneuraminic acid (Neu5Ac) that affects the binding of several Sialic acid-specific lectins and affects the immune system. CASD1 is known to be essential for sialic acid 9-O-acetylation Accelerated blood clearance is a phenomenon where the blood clearance rate of the carrier system is substantially raised Acid alpha-glucosidase catalyzes the breakdown to glycogen in lysosome Acute glomerulonephritis disorder of the kidney revealing inflammation and damage of the glomeruli leading to hematuria, proteinuria, and azotemia Acute humoral xenograft rejection involves acute vascular rejection, of organ transplants, and forms a major challenge in organ xenotransplantation Acute lymphoblastic leukemia (ALL) is a cancer that starts from lymphocytes Acute myeloid leukemia a type of cancer in which the bone marrow makes abnormal myeloblasts, red blood cells, or platelets Acute phase proteins or APPs are proteins whose plasma concentrations increase called as positive APPs or decrease called as negative APPs in response to inflammation leading to the acute phase response Adult pig islets are pancreatic islets from pig pancreas Aedes aegypti belongs to the family Culicidae, commonly called as the yellow fever mosquito that can spread disease pathogens including dengue fever, chikungunya, Zika fever, Mayaro, and yellow fever viruses Aldolase A commonly called as fructose-bisphosphate aldolase is coded by the ALDOA gene on chromosome 16 Alpha 2,3 sialyltransferase catalyzes the transfer of CMP-N-acetylneuraminate (CMPsialic acid) to the b-D-galactosyl-1,4-N-acetyl-D-glucosaminyl termini on glycoproteins Alpha Gal xenoantigen is a xenoantigen in pigs recognized by human natural antibodies Alpha-1,3-galactosyltransferase-1 can catalyze the transfer of galactose with an α-1,3 linkage, on terminal lactosaminide Alternative pathway is one of the three complement pathways that opsonize and kill pathogens. It is triggered by C3b protein directly binding to a microbe Alzheimer's disease is a chronic neurodegenerative disease that worsens over time leading to dementia Amniotic fluid is a clear, yellow fluid found within the first 12  days after conceiving within the amniotic sac Angiokeratoma Corporis Diffusum also called Fabry disease which is an X-linked inherited disorder caused due to deficiency of the lysosomal enzyme alpha-galactosidase Antibody-mediated rejection involves allograft rejection by antibodies against ­donor-specific HLA molecules, blood group antigen (ABO) or endothelial cell antigens

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Antibody also called as immunoglobulin (Ig), is produced mainly by plasma cells that function in the immune system comprising the humoral branch of immunity that neutralizes pathogens like bacteria and viruses Antibody-dependent cell-mediated cytotoxicity cell-mediated effector response of the immune system whereby an effector cell actively lyses a target cell, where surface antigens are bound by specific antibodies Antigen is a foreign agent that can elicit/induce an immune response in the body Antigen presenting cells can process a protein antigen and present them on cell surface class I/Class II MHC molecules to interact with appropriate T cell receptors Anti-lymphocyte serum is a serum containing antibodies against lymphocytes Anti-O-acetylated sialic acid antibodies against O-acetylated sialic acids Aortic endothelial cells are isolated from the human ascending (thoracic) and descending (abdominal) aorta. They find application in cardiovascular function and disease, vascular diseases like thrombosis, atherosclerosis, hypertension, stent-graft compatibility testing Apis mellifera commonly called as honey bees, belonging to family Apidae Apolipoprotein C-III coded by the APOC3 gene that regulates triglyceride metabolism Arabidopsis thaliana belongs to mustard (Brassicaceae) family Asialoerythropoietin is a nonerythropoietic cytokine. It has neuroprotective activity Asialoglycoprotein receptor 1 is encoded by ASGR1 gene that plays a role in serum glycoprotein homeostasis. It mediates endocytosis and lysosomal degradation of terminal galactose or N-acetylgalactosamine residues containing glycoproteins Autism spectrum disorder developmental disorder affecting communication and behavior B-cell receptors are antigen receptors on cells structurally related to secreted antibodies with structural difference in the C-terminal region of the heavy chains containing a short hydrophobic portion in the lipid bilayer of the membrane. They control B-cell activation Bactrocera dorsalis also called as oriental fruit fly causes damage to fruits Benzothiazolylphenol-based sialic acid can act as a fluorescent sialidase substrate Bipolar disorder also referred to as manic-depressive illness, is a brain disorder with unusual shifts in mood, energy, and activity levels Blood-brain barrier is a selective semipermeable membrane separating blood from the brain in the central nervous system (CNS) Body fat percentage is the percentage of body weight constituted by fat Bombyx mori commonly called as a silkworm belongs to order Lepidoptera Bovine serum albumin is serum albumin protein of bovine origin Brain-derived neurotrophic factor is expressed in CNS, gut and other tissues and plays a role in neuronal survival and growth, acting a neurotransmitter modulator, neuronal plasticity, controls learning, and memory responses Bright Yellow 2 or Tobacco BY-2 cells is a cell line of plant cells, generated from a callus induced on a seedling of Nicotiana tabacum Calcium ions control physiology and biochemistry of organisms and the cell with roles in signal transduction pathways, in neurotransmitter release, in muscle contraction, and fertilization Calcium release activated channels are integral membrane proteins critical in cell signaling by generating the calcium influx Carbon dot are small carbon nanoparticles of less than 10 nm



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Carbon nanotubes or CNTs are cylindrical large molecules in hexagonal arrangement of hybridized carbon atoms, existing either in a single sheet of graphene forming ­single-walled CNT (SWCNTs) or in multiple sheets of graphene forming multiwalled CNT (MWCNTs) Carboxyl groups modified CdSe/ZnS quantum dots CdSe/ZnS core-shell quantum dots (QDots) gains water-soluble property when surface is functionalized with carboxylate groups Cardiovascular disease disorders of the heart or blood vessels including coronary artery diseases (CAD), angina and myocardial infarction, stroke, peripheral artery disease, and atherosclerosis Cauliflower mosaic virus belongs to the genus Caulimovirus, family Caulimoviridae are pararetroviruses infecting plants Cell adhesion molecules are cell adhesion proteins involved in binding with other cells or extracellular matrix (ECM) Cell-mediated immune responses involve immune response mediated by immune cells like phagocytes, cytotoxic T-lymphocytes, involving cytokines against pathogen/antigen Central nervous system is the part of the nervous system containing brain and spinal cord Ceramide are waxy lipid molecules, composed of sphingosine and fatty acid, within the cell membrane of eukaryotic cells Chaetocnema pulicaria also called as cornflea beetle Chediak-Higashi syndrome is a rare autosomal recessive disorder due to mutated lysosomal trafficking regulator protein, causing a reduction in phagocytosis, thereby increasing recurrent pyogenic infections, albinism, and peripheral neuropathy like disorders Chinese hamster ovary cells are an epithelial cell line generated from the ovary of the Chinese hamster. Finds application in biological research Cholera toxin is a heat-labile enterotoxin family which is a multimeric protein secreted by Vibrio cholera, causing watery diarrhea and cholera Chondroitin-6-sulfate is sulfated glycosaminoglycans with alternating Nacetylgalactosamine and glucuronic acid and reveals structural similarity with hyaluronic acid Chronic granulomatous disease is a disorder of the immune system Circular dichroism is an absorption spectroscopy based on the differential absorption of circularly polarized light by optically active chiral molecules Clostridium thermocellum anaerobic, thermophilic bacterium Cluster of differentiation is a surface marker enabling identification of a particular lineage of cells Clustered Regularly Interspaced Short Palindromic Repeats-associated protein-9 nuclease or CRISPR/Cas9 enables genome editing in all species, consisting of a nuclease (Cas9) and two short single-strand RNAs (crRNA and tracrRNA) which can be fused for genome editing as single-guide RNA (sgRNA). Cas9-gRNA form a ribonucleoprotein complex binding to genomic DNA.The Cas9–gRNA complex scans the genome to identify a protospacer adjacent motif (PAM) and its adjacent genomic DNA sequence adjacent matching the gRNA sequence to cleave it CMP-sialic acid hydroxylase same as cytidine monophospho-N-acetyl neuraminic acid hydroxylase CMP-sialic acid synthetase is an important enzyme of the sialylation pathway, deficiency in enzyme does not express sialylated molecules

330

Glossary

CMP-sialic acid transporter localized in the medial-trans Golgi that plays a role in the transportation of cytosolic CMP-sialic acid into Golgi where glycosyltransferases function Complement are proteins of the immune system enabling antibodies and phagocytic cells to act and lyse microbes and remove damaged cells, promotes inflammation Complement factor H is a regulator of complement activation pathway Computed tomography is an imaging procedure using special x-ray equipment Congenital disorders of glycosylation involve rare genetic, metabolic disorders due to defects in the process of glycosylation Conotrachelus nenuphar true weevil that can destroy fruits Constant domain part of the Ig molecule that determines the mechanism used to destroy the antigen Coronary artery diseases develop due to damage of major blood vessels supplying the heart with blood, oxygen, and nutrients Corpus luteum forms the last active stage of an ovarian follicle's lifecycle, performs endocrine functions in ovary producing progesterone during early pregnancy. Essential for the maintenance of early pregnancy Cytidine 5′-monophospho-N-acetylneuraminic acid is a substrate for sialyltransferases and plays a role in sialylation of glycans Cytidine 5′-triphosphate consists of a ribose sugar, and three phosphate groups acts as a substrate for RNA synthesis, acts as a coenzyme in the synthesis of glycerophospholipids and protein glycosylation Cytidine monophosphate also known as 5′-cytidylic acid, is a nucleotide used as a monomer in RNA Cytidine monophospho-N-acetyl neuraminic acid hydroxylase is an enzyme that catalyzes the conversion of CMP-N-acetylneuraminic acid or CMP-Neu5Ac into hydroxylated derivative CMP-N-glycolylneuraminic acid of CMP-Neu5Gc Cytotoxic T cells are generated from cytotoxic T cells (Tc cells), are CD8+, and MHC class I restricted and play a role in T cell responses Damage-associated molecular patterns or alarmins are molecules released by stressed cells undergoing necrosis. They are endogenous danger signals that accelerate the inflammatory response. Examples include high mobility group box-1 (HMGB1), S100A8 and S100A9 (MRP8, calgranulin A and MRP14, calgranulin B), and Serum amyloid A (SAA). Increased serum levels of these DAMPs have been reported in arthritis, atherosclerosis, lupus, cancer, Crohn’s disease, and sepsis Danaus plexippus is also called as the monarch butterfly belonging to family Nymphalidae Deaminoneuraminic acid is a member of sialic acids family in which acylamino group at the C-5 position of N-acylneuraminic acid (Neu5Acyl) is replaced by a hydroxyl group Dendritic cells are antigen-presenting cells of the immune system that process and present antigenic peptides to the T cells Dengue virus is, a mosquito-borne flavivirus with a single-stranded RNA positive-strand virus belonging to Flaviviridae family. DENV causes Dengue Fever (DF) and lifethreatening Dengue Hemorrhagic Fever (DHF) or Dengue Shock Syndrome (DSS). Four antigenically different viral serotypes include BENV-1, DENV-2, DENV-3, and DENV-4 DIG Glycan detection kits It enables analysis of carbohydrate structures by the specific binding of selected lectins. Both glycoproteins bound to nitrocellulose membranes and carbohydrate structures on tissue sections can be identified by lectins including



Glossary

331

GNA, SNA, MAA, DSA, and PNA. Detection of digoxigenin-labeled lectins bound to specific carbohydrate moieties on blots is performed using anti-digoxigenin alkaline-­ phosphatase conjugates. The digoxigenin-labeled lectins enable differentiation and detection of complex and high-mannose chains, α(2,3) and α(2,6) linkage of terminal sialic acids, and core disaccharide Galß(1,3)GalNAc of O-glycans Disialoganglioside 3 is predominantly expressed at neuronal development with limited expression in adult tissues Double knockout Knocking out two genes simultaneously in an organism. Finds application in research Double-positive T cells are cells in the development of T cells expressing both CD4 and CD8 Drosophila melanogaster is the common fruit fly and model organism used in biological studies Drosophila sialic acid synthase produces Neu5Ac and 2-keto-3-deoxy-D-glycero-­Dgalacto-nononic acid (KDN). Using N-acetylmannosamine 6-phosphate and mannose 6-phosphate as substrates it can generate phosphorylated forms of Neu5Ac and KDN. Finds importance in sialic acid biosynthesis in neurons Early endosomes endocytic vesicles rapidly targeted to the endocytic organelle Electrospray ionization quadrupole time-of-flight mass spectrometry a mass spectrometry method in which sample is ionized by forcing a solution of the sample through a small heated capillary into an electric field generating a very fine mist of charged droplets. In the first stage, the ions are held in a stable orbit by a quadrupole and in the second stage the kinetic energy and the time taken for each ion to reach a detector at a known distance is measured and time is dependent on the mass-to-charge ratio of the ion Embryonic stem cells are pluripotent stem cells derived from the inner cell mass of a blastocyst of an embryo Endoglucosaminidases catalyzes the formation of glycosidic bond between an N-acetylβ-D-glucosamine and the adjacent monosaccharide Endoplasmic reticulum membrane-bound cell organelle within the cytoplasm of eukaryotic cells. Attached to ribosomes, it is involved in protein and lipid synthesis Enolase 3 is an enzyme coded by the ENO3 gene. There are three enolase isoenzymes reported in mammals. One in adult skeletal muscle cells plays a role in muscle development and regeneration. Genetic mutations have been associated with glycogen storage disease Enterovirus D68 is a member of the Picornaviridae family and is a non-poliovirus, nonenveloped virus with, positive-sense single-stranded RNA and can be transmitted by respiratory and gastrointestinal secretions Epidermal growth factor and Epidermal Growth factor receptor: EGF binds to EGFR and plays a role in cell proliferation, differentiation, wound healing, and survival Epithelial ovarian cancer develops from the cells in the outer surface of the ovary, most being benign. Cancerous epithelial tumors are carcinomas initiating in the tissues lining the ovaries Epitrix cucumeris commonly called as potato flea beetle belong to the family Chrysomelidae Erythropoietin and EPO receptor EPO functions on binding to its high-affinity receptor EPOR. EPO is a hormone produced by the kidneys, playing a role in RBC production Escherichia coli is a Gram-negative, rod-shaped, coliform bacterium

332

Glossary

Estrogen Receptor is activated by estrogen which is a nuclear hormone receptor. Finds application in targeting breast cancer, osteoporosis, and other female endocrine disorders Extracellular matrix includes a three-dimensional network of extracellular macromolecules, including collagen, enzymes, and glycoproteins, providing structural and biochemical support of surrounding cells Extracellular vesicles Heterogeneous group of membranous structures comprising exosomes and microvesicles, released from the cell, originating from the endosomal system, enabling cells to exchange proteins, lipids, and genetic material. They facilitate intercellular communication in immune responses and coagulation Fabry disease a type of lysosomal storage disease (LSD) caused due to genetic mutation leading to aberrant processing of sphingolipids, leading to their accumulation in walls of blood vessels and other organs Factor H binding protein (fHbp) is a lipoprotein of Neisseria meningitidis essential for pathogenesis and allows bacterial survival and growth in human blood by binding the human complement factor H. Finds application in designing of vaccines Fas ligand or FasL or CD95L is a type-II transmembrane protein, member of TNF family that binds to Fas R inducing apoptosis. FasL-FasR interactions play a role in the regulation of the immune system and the progression of cancer Fas-associated protein with death domain is an adapter protein, playing a role in apoptosis and is encoded by the FADD gene on the 11q13.3 of human chromosome 11. It bridges Fas-receptor, to procaspases 8 and 10 forming death-inducing signaling complex (DISC) during apoptosis. Plays a role in the cell cycle, development, and proliferation Fc-gamma receptor or FcγR belong to the immunoglobulin superfamily inducing phagocytosis of opsonized microbes. Includes, FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA, and FcγRIIIB Fibroblast Growth Factor receptor included as a subfamily of receptor tyrosine kinases (RTKs), Members include FGFR1, FGFR2, FGFR3, and FGFR4 and plays a role in regulating cell proliferation and differentiation in development and tissue repair Fluorescein isothiocyanate is a fluorescent molecule finding application in staining antigens/proteins when tagged with antibodies. Finds applications in immunohistochemistry and flow cytometric studies Fluorescence-activated cell sorter is a specialized type of flow cytometry, enabling sorting of cells based on the specific light scattering and fluorescent nature of each cell Fluorescent silica nanoparticles are prepared by techniques by which fluorophores used to modify silica NPs Formylglycine-generating enzyme catalyzes the conversion of cysteine to formylglycine (fGly) Free sialic acid storage disorders are disorders of free sialic acid metabolism. Includes neurodegenerative disorders and increased lysosomal storage of free sialic acid like Salla disease, intermediate severe Salla disease, and infantile free sialic acid storage disease (ISSD) Fucose is a monosaccharide hexose deoxy sugar with the chemical formula C6H12O5 forming a component of mammalian N- and O-linked glycans and glycolipids Fusobacterium nucleatum is a bacterium found in the dental plaque and can cause gum disease Galactose is a monosaccharide with chemical formula C6H12O6, similar to glucose, differing in the position of one hydroxyl group that gives different chemical and biochemical properties from glucose Galanthus nivalis commonly called as snowdrop plants



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Galleria mellonella is commonly called as the honeycomb moth, member of family Pyralidae GalNAc-transferases can catalyze mucin-type O-glycosylation reaction Gangliosides are glycosphingolipids containing sialic acid, with a predominant expression on neuronal cells, and other cells Gaucher disease is a rare genetic disorder, due to the deficiency in glucocerebrosidase enzymes, and leads to deposition of glucocerebroside in macrophage and monocyte cells GD3 synthase or ST8SIA1 regulates the synthesis of GD3 and GD2 Gestational diabetes mellitus is increased glucose levels during pregnancy Glucosamine (UDP-N-acetyl)-2-epimerase/N-acetylmannosamine kinase or Glucosylceramide is a glycosphingolipid and is plasma membrane component that modulates signal transduction Glycan are assembly of sugars, either free or attached to another molecule Glycan-binding proteins proteins that identify and bind to specific glycans and mediate their biological function Glycobiology involves the study of structure, chemistry, biosynthesis, and functions of glycans and their derivatives and their biological role Glycocalyx the cell coat of glycans and glycoconjugates surrounding animal cells Glycoconjugate one or more glycan units are covalently linked to noncarbohydrate molecules like protein/lipid in the same molecule Glycoforms more than one molecular forms of a glycoprotein, resulting from variable glycan structure and/or glycan attachment site occupancy Glycogen debranching enzyme facilitates glycogen breakdown leading to glucose storage in the body Glycogen a polysaccharide with glucose residues in α1–4- and α1–6-linkages Glycogenin-1 catalyzes glycogen biosynthesis Glycolipid a molecule containing a glycan linked to a lipid Glycome the entire glycan profile of an organism Glycomics study and systematic analysis of the glycome Glycomimetics noncarbohydrate molecules that mimic glycan properties Glycopeptide a peptide with one or more covalently attached glycans Glycoprotein a protein containing one or more bound glycans covalently Glycoproteomics analysis of glycoproteins, at systems biology level studying their protein associations, glycosylation sites, and glycan structures Glycosaminoglycans also called as mucopolysaccharides. They are long unbranched polysaccharides with repeating disaccharide unit, consisting of N-acetylglucosamine or N-acetylgalactosamine and a uronic sugar including glucuronic acid or iduronic acid or galactose Glycosaminoglycans polysaccharide side chains of proteoglycans Glycosidase enzyme causing hydrolysis of glycosidic bonds in a glycan Glycoside a glycan with one glycosidic linkage to another glycan or an aglycone Glycosidic linkage bond that links two monosaccharides Glycosphingolipids are components of cell membrane glycolipids/sphingolipids containing the amino alcohol sphingosine Glycosylation the enzymatic attachment of a carbohydrate to a polypeptide, lipid, polynucleotide, carbohydrate, or other organic compounds Glycosylphosphatidylinositol is a glycolipid linked to protein C-terminus during posttranslational modification. They play important roles in different biological processes

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Glossary

Glycosyltransferase enzyme catalyzing transfer of a sugar from a sugar nucleotide donor to a substrate GNE regulates NeuAc, or sialic acid precursor biosynthesis forms a rate-limiting enzyme in the sialic acid biosynthetic pathway Gold nanoparticles gold particles with 1 to 100 nm diameter Granulocyte-macrophage colony-stimulating factor is a cytokine secreted by endothelial cells, fibroblasts, macrophages, mast cells, NK cells, and T cells Granzyme are serine proteases released by cytotoxic T cells and NK cells, inducing apoptosis of the infected/cancerous cell Graphite oxide is a single monomolecular layer of graphite with various oxygen-­ containing groups like epoxide, carbonyl, carboxyl, and hydroxyl Griffonia simplicifolia II agglutinin is isolated from African legume Griffonia simplicifolia seeds that can bind to terminal non-reducing a- or ß-linked N-acetyl-D-glucosamine Group B meningococcus causes infections like meningitis Group B streptococci includes Gram-positive cocci, Streptococcus agalactiae causing illness and death Guide RNA or gRNA finds application in prokaryotic DNA editing by CRISPR/Cas9 system Guillain-Barré syndrome is an autoimmune disease affecting the peripheral nervous system leading to weakness and or paralysis Hemagglutinin neuraminidase glycoprotein exhibits three distinct functions including a receptor-binding or hemagglutinin activity, a receptor-destroying or neuraminidase activity, and a membrane fusion activity that enables the fusion of viral envelope to host cell membrane for infection. It is reported from paramyxoviruses (negative-stranded RNA viruses), including Mumps virus, Human parainfluenza virus 3, and the avian pathogen Newcastle disease virus Haemophilus influenza is the causative agent of influenza, Gram-negative bacteria of Pasteurellaceae family Haloferax volcanii belongs to genus Haloferax of domain Archaea Hanganutziu-Deicher antigen or H-D is a heterophile antigen and is a glycoconjugate containing N-glycolylneuraminic acid Hashimoto's thyroiditis is an autoimmune disease degrading the thyroid gland Heavy chain fragment component of the antibody molecule. Heavy chain exists in the variable and constant region of the Ig molecule Helicobacter pylori is a bacterium that infects the digestive tract and may lead to gastric cancer Hemagglutinin are glycoproteins causing agglutination of RBCs Hematopoietic stem cells are the stem cells which give rise to different types of blood cells Hemipyrellia ligurriens belongs to Diptera under Calliphoridae and is a forensic fly Heparan sulfate a glycosaminoglycan with disaccharide unit (GlcNAcα1–4GlcAβ1– 4/IdoAα1–4), containing N- and O-sulfate esters covalently linked to a proteoglycan core protein Heparin a heparan sulfate-type synthesized by mast cells with the highest amount of iduronic acid and N- and O-sulfate residues Hepatocyte growth factor is a growth factor secreted by mesenchymal cells acting on epithelial, endothelial, hematopoietic progenitor cells and T cells and plays a role in organ development, myogenesis, regeneration, and in wound healing



Glossary

335

High-grade serous carcinoma originates from serous epithelial layer in the ovary. HGSCs reveal the highest mortality rates High-performance liquid chromatography is a form of column chromatography that pumps an analyte mixture or analyte in a solvent/mobile phase at high pressure through a column with chromatographic packing material called as the stationary phase High-risk neuroblastoma forms the most common extracranial solid tumor in children Hinge region a portion of the immunoglobulin molecule with flexible amino acid stretch that links these two chains by disulfide bonds Hippeastrum Hybrid (Amaryllis) includes perennial bulbous plants Human bronchial epithelial cells isolated from the surface epithelium of human bronchi and play a role in lung lubrication, humidity maintenance, and the cleaning of the respiratory tract Human butyrylcholinesterase is a nonspecific cholinesterase enzyme hydrolysing different choline-based esters Human CMP-Sia transporter is coded by the SLC35A1 gene, catalyzes transport of CMP-sialic acid from the cytosol into Golgi where glycosyltransferases function Human endothelial cell protein C receptor is an N-glycosylated type I membrane protein that plays a role in the activation of protein C, belongs to the MHC class I/CD1 and is encoded by the PROCR gene Human heme oxygenase-1 catalyzes the cleavage of the heme ring at the α-methene bridge to form biliverdin which is subsequently converted to bilirubin Human immunodeficiency virus is retrovirus that causes HIV infection Human influenza virus X31 also called H3N2 influenza strain Human leukocyte antigen code for the major histocompatibility complex (MHC) proteins in humans Human tissue factor pathway inhibitor tissue factor (TF) pathway inhibitor (TFPI) inhibits blood coagulation Human umbilical vein are endothelial cells originated from endothelium of veins of umbilical cord Hunter syndrome or mucopolysaccharidosis type II (MPS II), is aLSD caused by a deficiency of the lysosomal enzyme iduronate-2-sulfatase (I2S) leading to accumulation of heparan sulfate and dermatan sulfate in all body tissues Hyaluronic acid is an anionic, nonsulfated glycosaminoglycan predominantly found in connective, epithelial, and neural tissues Hydrops fetalis is a fetal condition in fetus due to abnormal accumulation of fluid in ascites, pleural effusion, pericardial effusion, and skin edema Hydroxyproline constitute collagen and together with proline plays important roles in conferring collagen stability Hyperacute rejection initiated within a few minutes of a transplant due to tissue incompatibility Idiopathic inflammatory myopathies are a group of disorders of the muscle caused by muscle inflammation and difficulty in movement IgA nephropathy also Berger's disease is a kidney disease due to anti IgA antibodies that deposit in kidneys leading to local and affected kidney function Immune receptor tyrosine-based inhibition motif is the conserved sequence of signature amino acids (S/I/V/LxYxxI/V/L) found in the cytoplasmic tails of many inhibitory receptors of the immune system

336

Glossary

Immunoglobulin acts as antibodies, synthesized by B cells, give rise to the humoral branch of the immune system Immunoglobulin-like transcript represent novel Ig superfamily receptors, expressed in dendritic cells, lymphoid and myeloid cells Immunoreceptor tyrosine-based activation motif is a conserved sequence of four amino acids motif of a tyrosine separated from a leucine or isoleucine by any two other amino acids, like YxxL/I. This signature is repeated twice in the cytoplasmic tails of certain cell surface proteins of the immune system and play a role in signal transduction Inborn errors of metabolism is a type of genetic disease involving metabolic congenital disorders Inductively coupled plasma mass spectrometry: ICP–MS is an instrumental analytical technique used as a high-temperature ionization source (ICP) coupled to a mass spectrometer Inductively coupled plasma-atomic emission spectroscopy or ICP-OES, is an analytical emission spectrophotometric technique used to detect chemicals by excited electrons and emitting energy at a given wavelength as they return to ground state characteristic of the atom. As the emitted energy intensity at the particular wavelength is proportional to the concentration of that element, the emitted wavelengths are emitted and their intensities enable qualitative and quantitative estimation of the elements in the sample as compared to the reference materials Infantile sialic acid storage disorder is a Sialic acid storage disease and autosomal recessive neurodegenerative disorder Influenza A virus it is a type of influenza virus, comprising hemagglutinin (H) and the neuraminidase (N) which may be of 18 different H subtypes and 11 N subtypes and different strains of influenza A virus include influenza A (H1N1) and influenza A (H3N2) viruses Influenza virus belongs to Orthomyxoviridae family, of enveloped viruses with segmented negative-sense single-strand RNA segments, including four genera A, B, C, and Thogotovirus, of which, A and B cause influenza in humans Insulin-dependent diabetes mellitus where individuals produce very little or no insulin Insulin-like growth factor-binding protein bind insulin-like growth factors-I and -II (IGF-I and IGF-II) with high–affinity-promoting cell differentiation, growth, proliferation, and survival Intercellular adhesion molecule belong to the immunoglobulin superfamily, with five members, including ICAM-1 to ICAM-5, playing a vital role in inflammation, immune responses, and in intracellular signaling Interferon-gamma (IFN-γ) is a cytokine that plays a role in both innate and adaptive immunity, activates macrophages, induces MHC Class II molecule, and acts against viral, some bacterial, and protozoal infections Interleukin-2 is a cytokine that plays a role in signaling in the immune system and regulates leukocytes, lymphocytes activity, and immunity Intravenous injection is injected directly into the veins Kallikrein 6 coded by the KLK6 gene are serine proteases having diverse physiological functions and has been implicated in cancers Keratan sulfates are sulfated glycosaminoglycans predominant in expression in bones, cartilage, and cornea Killer cell immunoglobulin-like receptor is a type I transmembrane glycoproteins expressed on NK cells and T cells that regulate the killing function of these cells by interacting with MHC class I molecules



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337

Knockout organisms have an existing gene disrupted by replacing it with an artificial DNA Krabbe disease or leukodystrophy or galactosylceramide lipidosis is a rare autosomal recessive disorder and often fatal LSD leading to the damaged nervous system due to aberrant metabolism of sphingolipids Lectin a protein that recognizes and binds to glycans Leishmania donovani intracellular parasites of genus Leishmania, causing leishmaniasis Ligand It is recognized by a specific receptor Lipid-associated sialic acid finds application as a serum marker in cancer Lipid-linked oligosaccharides are the substrates of oligosaccharyltransferase (OST) that transfers oligosaccharide onto the acceptor asparagine during N-glycosylation Lipooligosaccharide are glycolipids comprised of core oligosaccharide (OS) and lipid A components Lipopolysaccharide or lipoglycans and endotoxins consist of a lipid and a polysaccharide composed of O-antigen, outer core, and inner core linked by a covalent bond located in Gram-negative bacteria outer membrane Liquid chromatography-mass spectrometry is an analytical technique that enables physical separation by HPLC and analysis by mass spectrometry (MS) Low-density lipoproteins are lipoproteins that carry cholesterol to and from cells Lupus erythematosus is an autoimmune disease in which the body's immune system attacks healthy tissue Lysosomal Storage disorders about 50 rare inherited metabolic disorders due to defective lysosomal function Lysosome-associated membrane protein 2 coded by LMP2 gene is a lysosome-­ associated membrane glycoprotein Magnetic nanoparticles are nanoparticles made up of substances with magnetic properties like iron, nickel, and cobalt and they can be manipulated in magnetic fields Magnetic resonance imaging noninvasive imaging technology of radiology producing three-dimensional anatomical images of the anatomy and physiological processes of the body Mannose is a C-2 epimer of glucose and finds importance in glycosylation of certain proteins Maroteaux-Lamy syndrome or MPS VI is a rare genetic disorder due to lack of enzyme activity arylsulfatase B Mass spectrometry is an analytical method in which chemicals to be identified are ionized and sorted into a spectrum based on their mass-to-charge ratio Matrix metallopeptidase 9 are zinc-metalloproteinases that are involved in the degradation of the extracellular matrix, coded by MMP9 gene in humans Mesoporous silica nanoparticles are the latest developments of nanoparticles that find application in drug delivery Methotrexate is a drug used in the treatment of arthritis and cancer MHC class I chain-related protein or MICA and MICB are polymorphic proteins induced by stress, damage or transformation of cells that triggers cell killing through NK cells Microheterogeneity Structural variations in a glycan Middle East respiratory syndrome coronavirus or MERS is a viral respiratory disease caused by Middle East respiratory syndrome coronavirus or MERS-CoV Molecularly imprinted polymers are designed recognition materials that mimic biological receptors

338

Glossary

Monoclonal antibodies are antibodies that originate from unique parent cell that they bind to the same epitope Monocyte chemoattractant protein-1 is one of the chemokines regulating migration and infiltration of monocytes and macrophages Monosialoganglioside 3 constitutes more than 70% of total human milk gangliosides Moraxella catarrhalis is a Gram-negative, diplococcus bacteria causing respiratory infections and infectious middle ear, eye, central nervous system, and joints of humans Mucins are heavily glycosylated proteins produced by epithelial tissues in most animals Mucopolysaccharidosis inherited disorders in which body fails to breakdown mucopolysaccharides Multiple sclerosis is an autoimmune disorder affecting the CNS, brain, and spinal cord Multiple sulfatase deficiency is an autosomal recessive disorder, leading to LSD in deficiency of sulfatases causing accumulation of sulfatides, sulfated glycosaminoglycans in the body Multiwalled CNTs hollow, cylindrical allotropes of carbon with a high aspect ratio (length to diameter ratio) and may be of 30 nm diameter Mumps virus affects salivary glands and can lead to fever, swollen jaws Muscle lactate dehydrogenases are cytoplasmic, and isoenzymes of muscle Muscle phosphofructokinase is an enzyme that regulates glycolysis and deficiency is associated with a rare muscular metabolic disorder, autosomal recessive in inheritance Muscle phosphoglycerate mutase is a homotetramer enzyme and is a disorder that affects skeletal muscle in movement Myelin-associated glycoprotein is a type 1 transmembrane protein glycoprotein, expressed in periaxonal Schwann cell and oligodendrocyte membranes, with a role in glial-axonal interactions N-acetyl-d-glucosamine/N-acetylglucosamine is a glucose derivative and is an amide between glucosamine and acetic acid N-acetylgalactosamine also abbreviated as GalNAc is an amino sugar derivative of galactose, forms terminal carbohydrate in antigen of blood group A, connects serine or threonine during protein O-glycosylation. Plays a role in intercellular communication and formation of sensory nerve structures in humans and animals N-acetylgalactosamine-6-sulfate sulfatase acts as hydrolase cleaving the 6-sulfate groups of the N-acetyl-D-galactosamine, chondroitin sulfate N-acetylgalactosaminyltransferase uses two substrates UDP-N-acetyl-D-galactosamine and polypeptide, producing UDP and N-acetyl-D-galactosaminyl-polypeptide. (UDP-Nacetyl-D-galactosamine+polypeptide⇌UDP+N-acetyl-D-galactosaminyl-polypeptide) N-acetylglucosamine-6-phosphate 2′-epimerase catalyzes reaction UDP-N-acetylD-glucosamine⇌UDP-N-acetyl-D-mannosamine N-acetyllactosamine is structurally nitrogen-containing disaccharide, intermediate in Keratan sulfate and N-Glycan biosynthesis N-acetylneuraminic acid forms the predominant sialic acid in human and many mammalian cells Nanocapsules nanodimensional shells, ranging from 10 to 1000 nm in diameter with nontoxic polymeric material with an inner liquid/solid core. With the property of stability, they find application in drug delivery Nanoparticles are particles of nanometre scale of below 100 nm, possessing physical properties of uniformity, conductance, strength, biocompatibility or special optical properties that find application in materials science and biology



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Natural killer cells are innate immune cells with functions like cytotoxic lymphocytes Neisseria meningitidis Gram-negative bacteria causing meningitis, meningococcal disease, meningococcemia, and sepsis Neisseria gonorrhoeae bacteria causing gonorrhoea Neural cell adhesion molecule is a type of glycoprotein expressed on neurons, glia and skeletal muscle Neural stem cell or NSC are multipotent cells originating in the CNS that differentiate into neurons and glial cells Neuroectodermal tumors are malignant tumors of neuroectodermal origin Neuroimmune regulatory proteins or NIRegs including CD95L,TNF, CD200, CD47, sialic acids, CD55, CD46, fH, C3a, and HMGB1 can suppress innate immunity and inflammation. NIRegs may play in controlling lymphocyte, microglia hyperinflammatory responses, neurogenesis, and brain tissue remodeling Neutrophils extracellular traps are extracellular structures composed of chromatin, cellular proteins, granules that bind, trap, and kill pathogens Newcastle disease virus causes bird disease in domestic and wild species N-Glycan a glycan that is covalently linked to an asparagine (Asn) residue of a polypeptide in the consensus sequence of -Asn-X-Ser/Thr N-glycolylneuraminic acid or Neu5Gc is a sialic acid molecule in most nonhuman mammals and absent in humans as they lack CMAH gene although reported in apes N-hydroxysuccinimide or NHS can convert carboxyl groups to amine-reactive NHS esters for bioconjugation, cross-linking of molecules, etc Nicotiana benthamiana closely related to tobacco plant indigenous to Australia Nicotiana tabacum is the scientific name for the tobacco plant Niemann-Pick type C or NPC is a rare progressive genetic disorder caused due to aberrant cholesterol and other fatty acid transport Nitric oxide or NO is cell signaling molecule, playing a role in physiology Nitric oxide synthase catalyzes nitric oxide (NO) production from L-arginine N-methyl-D-aspartate-receptors are glutamate receptor and ion channel protein of nerve cells Non-small cell lung cancer is a type of epithelial lung cancer Non-Gal antibodies react with neuraminic acid terminal N-Acetyl and N-Glycoloyl forms and are present in humans and or nonhuman primates (NHP) and do not cause hyperacute xenorejection Nonhuman primates are primates other than humans including macaques, rhesus monkey, African green monkey, baboons, etc Nonimmune hydrops fetalis is a type of HF, is a severe fetal condition due to excessive fluid accumulation in extravascular compartments and body cavities of fetus Nontypeable Haemophilus influenzae is a Gram-negative bacteria causing mucosal infections, otitis media, sinusitis, conjunctivitis, etc Nonulosonic acids or NulOs are negatively charged nine-carbon α-keto sugars including sialic, legionaminic, and pseudaminic acids O-acetyl-GD2 is the O-acetyl derivative of GD2 ganglioside. Here the outer sialic acid is modified by an O-acetyl ester O-Glycan a glycan glycosidically linked to the hydroxyl group of the amino acids serine, threonine, tyrosine, or hydroxylysine Oligosaccharyltransferase functions like a glycosyltransferase and catalyzes transfer of 14-sugar oligosaccharide from dolichol to protein during glycosylation reaction of proteins

340

Glossary

Open reading frame or ORF is stretch of codons that can be translated. Initiation codon is usually AUG while stop codon is usually UAA, UAG, or UGA Oxidative stress is caused due to an imbalance of free radicals and antioxidants leading to damage of the body Pancreatic ductal adenocarcinoma involves exocrine pancreatic malignancy Parainfluenza virus causes upper and lower respiratory illnesses Parkinson's disease is a disorder of the nervous system that affects movement Pasteurella haemolytica causes respiratory diseases in cow, sheep, and goat Pathogen-associated molecular patterns are signatures expressed by pathogens that are recognized by innate immune receptors PRRs including TLR, NLR, etc. that play a role in the activation of innate immune responses and removal of the pathogen Pathogen recognition receptors can detect signatures of PAMPs of the pathogen. They are components of the innate immune system Pathogenic prion protein is the pathogenic form of prion protein Prp leading to diseases like Creutzfeldt-Jakob disease (vCJD) Perineural invasion invasion of cancerous growth along nerves, and is common in head and neck cancer, prostate cancer, and colorectal cancer Peripheral blood lymphocytes comprise T cells, NK cells, and B cells and they are mature lymphocytes that circulate in the blood Peripheral blood mononuclear cells comprise of lymphocytes (T cells, B cells, NK cells) and monocytes in peripheral blood Peripheral nervous system includes nerves and ganglia outside the CNS, brain, and spinal cord Philaenus spumarius is a vector for plant pest commonly called as meadow froghopper Phosphorylase kinase is a serine/threonine protein kinase that activates glycogen phosphorylase to release glucose-1-phosphate from glycogen Pig corneal endothelial cells are endothelial cells from pig cornea Platelet-derived Growth factor receptor plays a role in cell proliferation, differentiation, growth, development in health and disease like cancer Poly (ADP-ribose) polymerase can catalyze the transfer of ADP-ribose to proteins and is known to play a role in DNA repair, genomic stability, and apoptosis Polymerase chain reaction is a technique that enables making multiple copies of a specific desired DNA Polysialic acid is α2,8-linked sialic acid homopolymer, expressed on embryonic and adult brain neural precursors Pompe disease is an autosomal recessive metabolic disease damaging muscle and nerve cells due to defective glycogen catabolism, due to lacking lysosomal acid alpha-­glucosidase enzyme leading to accumulation of glycogen in the lysosome Porcine endogenous retroviruses is a retrovirus infection of which is a major threat while transplantation of pig tissue to humans Posttranslational modification or PTM is a covalent enzymatic modification of proteins after protein biosynthesis Principal component analysis is a technique used to emphasize variation in dataset and analyzes the patterns Prolyl-4-hydroxylases catalyzes the formation of (2S,4R)-4-hydroxyproline (Hyp) that enables stabilization of collagen triple helix, finds importance in collagen biosynthesis Protein Databank is a resource archiving information about the 3D shapes of proteins, nucleic acids, and complex assemblies for the study of molecular, structural, and computational biology of molecules



Glossary

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PSA-neural cell adhesion molecule or PSA-NCAM is a marker of developing and migrating neurons and of synaptogenesis Pseudomonas aeruginosa is a capsulated, Gram-negative, rod-shaped bacteria causing disease in plants, animals, and humans and especially hospitalized patients Pyruvate the conjugate base, CH3COCOO−, is intermediate metabolic pathways throughout the cell Quantum dots are nanocrystals of a semiconducting material with diameters of 2-10 nanometers Raman spectroscopy is a spectroscopic method based on inelastic scattering of monochromatic light from a laser source Reactive oxygen species are generated during microbial killing by immune cells. The species includes hydrogen peroxide (H2O2), superoxide anion (O2−), hydroxyl radical (•OH), and singlet oxygen (1O2) Red blood cells or erythrocyte, are the cellular component of blood that carry oxygen Reticuloendothelial system is a part of the immune system comprising phagocytic cells located in reticular connective tissue Rheumatoid arthritis is an autoimmune disease, causing inflammation, swelling, and pain affecting the hands and feet Rhizoctonia solani agglutinin is found in mycelium and sclerotia of the pathogenic fungus R. solani with selectivity toward terminal nonreducing N-acetylgalactosamine residue RNA interference or RNAi inhibit gene expression or translation of targeted mRNA molecules Rocky Mountain spotted fever is a bacterial disease spread by an infected tick Salla disease is an autosomal recessive LSD revealing physical impairment and disabled intellect Sambucus nigra agglutinin reveal preferential affinity toward sialic acid attached to terminal galactose in α-2,6 linkage Sambucus sieboldiana agglutinin is obtained from the barks of plants of genus Sambucus with binding preferences for sialylated glycoconjugates containing NeuAc(α2-6)Gal/ GalNAc sequence Sanfilippo syndrome is a rare autosomal recessive LSD with a deficiency in enzymes to break down glycosaminoglycan (GAG) heparan sulfate Scanning electron microscopy is a type of electron microscopy where images of a sample are produced by scanning the surface with a focused electron beam Schizophrenia is a mental disorder appearing in late adolescence or early adulthood, with symptoms of delusions, hallucinations, and cognitive disorders Self-assembled monolayer molecular assemblies of organic molecules formed spontaneously on surfaces by adsorption Self-associated molecular patterns are hypothesized to be inhibitory receptors that dampen autoimmune responses Serine hydroxymethyl transferase plays an important role in cellular one-carbon pathways catalyzing the reversible, conversions of L-serine to glycine and tetrahydrofolate (THF) to 5,10-methylenetetrahydrofolate Severe combined immunodeficiency is a primary immune deficiency, with defective T & B lymphocyte functions Sialic acid mimetics They are chemically modified natural sialic acid ligands with improved binding affinity and selectivity Sialic acid 9-phosphate phosphatase or N-acylneuraminate-9-phosphatase catalyzes N-acylneuraminate 9-phosphate+H2O⇌N-acylneuraminate+phosphate

342

Glossary

Sialic acid 9-phosphate synthase or N-acylneuraminate-9-phosphate synthase catalyzes phosphoenolpyruvate+N-acyl-D-mannosamine 6-phosphate+H2O⇌N-­ acylneuraminate 9-phosphate+phosphate Sialic acid acetyl esterase catalyzes the synthesis of 9-O acetylated sialic acid Sialic acid synthase plays a role in biosynthetic pathways of sialic acids. N-acetylmannosamine 6-phosphate and mannose 6-phosphate are used to generate phosphorylated forms of Neu5Ac and KDN Sialic acids acidic sugars with a nine-carbon backbone, of which N-acetylneuraminic acid is most common in vertebrates Sialoadhesin is a cell adhesion molecule expressed on the macrophage surface Sialylated capsular polysaccharide finds importance in virulence of group B streptococci Sialyl-Lewis(x) is a tetrasaccharide carbohydrate, attached to O-glycans. It plays a role in cell-cell recognition and as a ligand for the selectin on endothelial cells mediate extravasation of neutrophils into sites of injury or infection Sialyl-Tn or Sialyl-Thomsen-nouveau antigen is formed by GalNAc linked to serine or threonine by a glycosidic bond Sialyltransferases are enzymes that transfer sialic acid to oligosaccharide chain and terminal ends of gangliosides or to the N- or O-linked glycoproteins revealing specificity toward particular sugar substrate Siglec-engaging tolerance inducing antigenic liposomes Siglecs/Sialic acid-binding immunoglobulin-like lectin: Sialic acid-binding proteins, members of the I-type lectin family Signaling lymphocyte activating molecule expressed by B and T cells is a CD2-related surface receptor, acts as a self-ligand enhancing T-cell proliferation and IFN-γ production. Defective SLAM-associated protein (SAP) leads to X-linked lymphoproliferative syndrome (XLP) Single-nucleotide polymorphisms include a substitution of a single nucleotide at a specific position in the genome, and each variation exists in a population. Sometimes they can give rise to defective proteins Single-positive at the final stage of thymocyte maturation T cells are single-positive either CD4 or CD8 Single-walled CNTs are hollow, long cylinders of one atomic sheet of carbon atoms in a honeycomb lattice with extraordinary electrical, thermal, and mechanical properties and find application in basic research SLAM-associated protein is an adaptor molecule with a Src homology 2 (SH2) domain and is expressed in T cells and NK cells and binds to SLAM family receptors thereafter recruiting Fyn and leading to downstream signaling pathways Small-cell lung cancer comprises about 10% of all lung cancers Small interfering RNA is a double-stranded RNA, about 20–25 bp interfering with the gene expression of specific genes with complementary nucleotide sequences and by degrading mRNA after transcription Sodium sialic acid symporter is a secondary active transporter, belonging to the family of sodium solute symporter (SSS), that use Na+ gradients for extracellular substrates uptake Somatic cell nuclear transfer is a biotechnology technique by which the nucleus of a somatic cell is transferred to an enucleated egg cytoplasm to create hybrids of the desired quality Sonic spray ionization is a technique to ionize compounds and prepare them for mass spectrometric analysis



Glossary

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Spodoptera frugiperda insect pest belonging to order Lepidoptera Sulfated glycosaminoglycan Sulfation is catalyzed by sulfotransferases (STs). Sulfated glycosaminoglycans enable cell-cell and cell-ECM interactions. Defective sulfation of GAGs can lead to diseases Superparamagnetic iron oxide nanoparticles or SPIONs comprise cores made of iron oxides that can be targeted to the required area through external magnets. With properties of superparamagnetism, high field irreversibility, high saturation field, they find applications in drug delivery into specific target sites Superoxide anion is a reactive oxygen species, is a free radical and paramagnetic properties, product of the one-electron reduction of dioxygen O2. Superoxide dismutase (SOD) protects the cell from its deleterious effects Surface-enhanced Raman scattering is a technique enhancing Raman scattering by molecules adsorbed on rough metal surfaces Swine endothelial cells or pig endothelial cells interact with human body after organ xenotransplantation. Finds application in cases of end-stage organ failure in humans T-cell receptors are antigen receptors on the T cells that play a role in cell-mediated immunity (CMI) in adaptive immune responses T-cell-independent type-2 is a T-independent antigen with highly repetitive structure and can simultaneously cross-link BCR and can produce antibodies by B cells without T-cell involvement T. cruzi trans-sialidase is a virulence factor from Trypanosoma cruzi, playing a role in protozoan biology T effector cells are T-cells that interact with a target cell displaying specific antigen T regulatory cells are T cells that regulate or suppress immune responses Tay-Sachs disease is a genetic disorder leading to nerve cell damage in the CNS Thin-layer chromatography is an analytical chromatographic technique used to separate the mixture using a thin stationary phase supported by an inert backing. Each separated component is calculated for its retention factor (Rf) detected by distance migrated over the total distance covered by the solvent and is calculated by Rf=distance traveled by sample/distance traveled by the solvent TIR-domain-containing adapter-inducing interferon-β is an adapter protein that plays a role in TLR activation Tissue factor is also called platelet tissue factor and is coded by the F3 gene that plays a role in clotting TNF-related apoptosis-inducing ligand is a ligand playing a role in apoptosis Tobacco mosaic virus belongs to genus Tobamovirus and is a positive-sense singlestranded RNA virus infecting plants, like tobacco and other plants making a mosaic-like appearance on the leaves Toll-like receptors or TLRs are type I transmembrane pattern recognition receptors (PRRs) that are innate immune receptors that can sense pathogens molecular signatures (PAMPS) or damage signals (DAMPS) thereby initiating an immune response Transcription activator-like effector nucleases are artificial restriction enzymes (RE) engineered to cut sequences of DNA of interest for experimental purposes Transendothelial migration or TEM or diapedesis involves leukocyte extravasation in which leukocytes squeeze across the endothelial lining of blood vessels to inflammation sites Trimethyl chitosan is a quaternized hydrophilic derivative of chitosan Trypanosoma cruzi is the causative agent of Chagas disease, transmitted to humans by triatomine bugs

344

Glossary

Tumor microenvironment is the environment encompassing a growing tumor including blood vessels, extracellular matrix, etc Tumor necrosis factor is a cytokine playing a role in systemic inflammation and in acute phase reaction Tumor-associated antigens found only on tumor cells, not found in normal healthy cells Turnip yellow mosaic virus belongs to family Tymoviridae, infects cabbages, cauliflower, broccoli, etc UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase they catalyze ­mucin-type linkages (GalNAcalpha1-O-Ser/Thr), transferring GalNAc from the sugar donor UDP-GalNAc to serine and threonine residues UL-binding protein includes a novel family of MHC class I-related molecules (MICs) that can bind to the human cytomegalovirus (HCMV) glycoprotein UL16 Ultraviolet rays-rays from the sun, causing skin burn and can cause skin cancer United States, Food and Drug Administration is a federal agency of the United States Department of Health and Human Services, protecting and promoting public health through the control and supervision of food safety, drugs, and pharmaceutical safety and use, blood transfusions, animal feed, etc Uridine diphosphate-N-acetylglucosamine-2-epimerase catalyzes UDP-N-acetylD-glucosamine⇌UDP-N-acetyl-D-mannosamine Uropathogenic Escherichia coli is the infection by E. coli in urinary tract causing urinary tract infection (UTI) in neonates, preschool girls, sexually active women, and elderly women and men Variable domain It is the region of the Fab or fragment, antigen-binding region of an immunoglobulin molecule. Fab comprises one V domain from each heavy and light chain of the Ig molecule Vascular cell adhesion molecule-1 functions in cell adhesion Vascular endothelial growth factor and Vascular endothelial growth factor receptor play a role in major physiological processes and angiogenesis Very late antigen form members of molecules that play a role in adhesion and embryogenesis vesicular stomatitis virus is a member of family Rhabdovirideae and is negative-sense RNA virus, infecting mammalian and insect cells Vibrio cholerae is a Gram-negative bacterium, the causative agent for cholera Vibrio cholerae neuraminidase plays a role in cholera pathogenesis by removing sialic acid and unmasking gangliosides like GM1, the receptor for cholera toxin (CT) and then CT increases the severity of the infection Visceral adipose tissues fat tissue located deep in the abdomen and around internal organs Visceral leishmaniasis also called as kala-azar caused by protozoan parasites of the genus Leishmania Wheat germ agglutinin is a lectin with binding specificity toward N-acetyl-Dglucosamine and Sialic acid Wild type is that which occurs normally in nature, in contrast to mutant form World Health Organisation is an Organisation which directs international health within the United Nations' system and global health responses Xenoreactive antibodies initiate hyperacute rejection of transplanted organs from pigs to primates Xylose is a monosaccharide of aldopentose type with five carbon atoms and an aldehyde group



Glossary

345

Zinc-finger nucleases are engineered DNA-binding proteins facilitating targeted genome editing by producing double-strand DNA breaks at desired locations. It is a powerful genome editing tool. The two domains include (i) A DNA-binding domain forming a zinc-finger protein and (ii) A DNA-cleaving domain of the nuclease domain of Fok I. The fused domains act as molecular scissors Zona pellucid is the transparent membrane covering the mammalian ovum prior to implantation α-1,3-Galactosyltransferase catalyzes the transfer of galactose α-1-Acid glycoprotein also called as orosomucoid, is an acute phase protein (APP) in blood β1,4-N-acetylgalactosaminyltransferases A or GalNAcT that catalyzes the synthesis of the glycosphingolipids GM2, GD2, and GA2 β-Galactoside α2,6-sialyltransferase-1 catalyzes transfer of sialic acid from CMP-sialic acid to galactose-containing acceptor substrates

Index Note: Page numbers followed by f indicate figures and t indicate tables.

A Aberrant sialylation of oligosaccharide, 34 ACD. See Angiokeratoma corporis diffusum (ACD) Acetylation, sialic acids, 10 Achatinin, 25–26 Acute humoral xenograft rejection (AHXR), 276 Acute lymphoblastic leukemia (ALL), 211 Acute lymphoblastic lymphoma (ALL), 228 Acute myeloid leukemia (AML), 228 Acute respiratory distress syndrome (ARDS), 314 AD. See Autoimmune disorder (AD) ADCC. See Antibody-dependent cellular cytotoxicity (ADCC) Adenocarcinoma, pancreatic, 257 Adrenal cancer, 259 Adult pig islets (APIs), 289 Aedes aegypti, 93f sialylation and, 98–100, 98f African sleeping sickness (African trypanosomiasis), 132–133 AHXR. See Acute humoral xenograft rejection (AHXR) ALL. See Acute lymphoblastic leukemia (ALL) Allotransplantation, 270–271 α-gal antigen, 284 α-mannosidosis, 185 Alpha2,8-polysialyltransferase enzymes, 257 American trypanosomiasis, 132–134 3-Aminophenylboronic acid functionalized QDs (APBA-QDs), 316 Anabolism, eukaryotic sialic acid, 6 α-N-acetylgalactosaminidase deficiency, 187–188 Angiokeratoma corporis diffusum (ACD), 187–189 Animal diseases, 90–101 Animal xenotransplantation models, 286 Annedida, 24 Antibodies, therapeutic, 37

Antibody-dependent cellular cytotoxicity (ADCC), 73 Anti-carbohydrate antibodies, 284 Anti-GD2-based targeting, 224 Antigens α-gal, 284 Hanganutziu-Deicher (H-D), 284–285 mucin carbohydrate, 38f sialylated carbohydrate, 257 sialylated Thomsen-Friedenreich, 307–308 tumor-associated carbohydrate, 34–35 Anti-Neu5Gc antibodies, 281 Archaea, 19 Arthropoda, 24 Asialoerythropoietin (asialo-rhuEPO), 74 Aspartylglucosaminuria, 188 Aspergillus fumigatus, 20 Autoimmune diseases, 147–149, 156–159 gangliosides and, 164 Autoimmune disorder (AD), 147 therapy for, 165 Autoimmunity, 160–162, 164 sialyltransferases and, 164–165 and tolerance, 149–152 Autoreactive T cells, 149–150 Avian influenza viruses, 315

B Bacteria, 128–132 pathogens, 127–128 sialic acid and, 14–19 Bacterial sialylation, 1 and host immune system, 18–19 B cell, 150–152 B cell receptor (BRC), 228 BCR signaling, 150, 151f β-mannosidosis, 185–186 Bimosiamose, 36 Biomolecules, 1 Biosensors, 311 Biotherapies, gangliosides and, 224 Blood brain barrier (BBB), 310

347

348

Index

β1,4-N-acetylgalactosaminyltransferases A (β4GalNAcTA), 95–96 Bombyx mori, sialylation and, 100–101, 100f Bovine serum albumin (BSA)-coated nanoparticles, 310 Brain-derived neurotrophic factor (BDNF), 113 Buckminsterfullerene, 302 Butyrylcholinesterase (BChE), 74

C Calcium oxalate dihydrate (COD), 120 Calcium release activated channel (CRAC), 156 Campylobacter jejuni, 128 Cancer, 211 adrenal, 259 endocrine, 260f neuroendocrinal, 259–260 ovarian, 253–255 pituitary, 259–260 serum sialylation as biomarkers in, 212–216 sialic acid as biomarkers in, 212–216 sialic acid-Siglec axis and, 224–229, 227f, 229f sialidase as cancer targets, 232 sialylransferase and, 231–232 sialylTn in, 230–231, 230f truncated O-glycans in, 230 Cancer-associated glycoconjugates, 213 Cancer-associated glycosylation, 211 Carbohydrate antigens sialylated, 257 tumor-associated, 34–35 Carbohydrate-based vaccines, 37–39 Carbohydrate-rich sialoglycopolyprotein, 29 Carbon dot (Cdot) nanoparticles, 309 Carbon nanotubes (CNTs), 302 Carcinogenesis, 247–250 Cardiac function and disorders, 119 Catabolism of N-linked glycans, 179 CD22, 160–161, 311 CD24, 160–162 CDGs. See Congenital disorders of glycosylation (CDGs) Cell leukodystrophy, globoid, 193–194 Cell-mediated immune responses (CMI), 147–149

Cellular rejection, 276–278 C1Ga1T1 enzyme, 230–231 Chagas disease, 132–134 Chinese hamster ovary (CHO) cells, 100 Chronic inflammation, 163–164, 258–259 Cis interaction of Siglecs, 136–137 CMAH. See Cytidine monophosphate-Nacetylneuraminic acid hydroxylase (CMAH) CMAS. See CMP-sialic acid synthase (CMAS) CMP-N-acetylneuraminic acid (CMP-Neu5Ac), 2–3 CMP-sialic acid synthase (CMAS), 120 CMP-sialic acid synthetase gene (CSAS), 95 CMP-Sia transporter functions, 102–103 Cnidarians, 22–23 Cobalamin F-type disease, 205 Complements, 160 Congenital disorders of glycosylation (CDGs), 179 Contact-sensitive mechanoreceptors (CSMs), 22–23 Crisper-Cas9 Technology of gene editing, 288f CRISPR/Cas9, 63, 69–70 CSMs. See Contact-sensitive mechanoreceptors (CSMs) Cytidine monophosphate-Nacetylneuraminic acid hydroxylase (CMAH), 281 Cytidine 5′-monophosphate (CMP)Neu5Ac synthetase, 127–128 Cytosensor, red blood cell, 310–311 Cytotoxic T cells (CTLs), 147–149

D Damage-associated molecular patterns (DAMPs), 154, 231–232 Danio rerio, 26 Danon’s disease (DD), 205–206 Deaminated neuraminic acid (KDN), 12–13, 215–216 Defective glycan degradation, 178–206 Defective glycolipid degradation, 188–194 Defective glycoprotein degradation, 185–188

Degradation dermatan sulfate, 181f glycan, 178–206 glycolipid, 188–194 glycoprotein, 185–188, 186–187f heaparin sulfate, 181f keratan sulfate, 183f Dendritic cells (DCs), 109, 120 De novo synthesis of sialic acid, 15 Dephosphorylation, 4f Dermatan sulfate, degradation of, 181f Desialylated neurons, 113–114 Desialylation, oxidative stress-induced, 119 Diabetes, 258–259 Disialoganglioside 3 (GD3), 109–110 O-acetylation of, 11 Drosophila melanogaster, 95 sialylation in, 95–96 Drosophila sialic acid synthase (Dm SAS), 101–102 DXT/FT mutant, 72 plants, 71–72, 72f

E Ebola virus, 75, 76f Echinoderms, 26 E. coli, 15–17 EGFR, 221t Endocrine disorders, 247 sialylations in, 253–260 Endocrine glands, 247, 248f sialylations and, 253 Endocrine tumors, 247–250, 250–252t Endoplasmic reticulum (ER), 67 Enterovirus D68 (EVD68), 135–136 Enzymes alpha2,8-polysialyltransferase, 257 lysosomal, 173, 175–177t in O-acetylated sialic acid metabolism, 9–10 Erythropoietin (EPO), 74 Erythropoietin receptor (EPOR), 74 E-selectin, 33, 36 Eukaryotes sialic acid anabolism, 6 sialylated molecules in, 93 Extracellular matrix (ECM), 256 Extracellular vesicles (EVs), 255

Index

349

F Fabry’s disease, 189 Factor H (FH) protein, 160 factor H-binding protein (fHbp), 127–128 Fc-glycoengineering in plants, 71–73, 72f Fc-N-glycosylation, 71–72 Filariasis, lymphatic, 23 Fluorescent biocompatible polymeric nanoparticles, 313 Fluorescent-conjugated polymer nanoparticles, 311–312 Fluorescent silica nanoparticles (FSNPs), 315 Food and Drug Administration (FDA), 73 Free sialic acid storage disorders (FSASD), 199–200 FSNPs. See Fluorescent silica nanoparticles (FSNPs) F-type disease, cobalamin, 205 FUCA1 gene, 186–187 Fucosidosis, 186–187 Fungi, 19–20 Fusobacterium nucleatum, 128

G Galectin-1 binding, 110 Gal epitope, 275 Gangliosides (GGs), 3, 27, 109–110, 113–117, 199, 216–218 and autoimmune diseases, 164 biosynthesis, 219f and biotherapies, 224 carbohydrate determinants of, 217f defective synthesis and metabolism of, 192–193 detection of, 305 expression, 24–25, 28–29, 34 in tumors, 218–222, 220–221t, 222–223f, 225–226t Gangliosidosis, GM1, 192 Gaucher Disease (GD), 73, 189–190 GBA gene, 189–190 GBS. See Guillian-Barre Syndrome (GBS) GD. See Gaucher Disease (GD) GD3 synthase gene, 221–222 Genetic engineering, approaches and sialylation, 102–103

350

Index

GLB1 gene, 192 Globo H, 34, 38f cancer vaccine, 37–38 Globoid cell leukodystrophy, 193–194 Glucosaminoglycan (GAG), defective degradation of, 194–198 Glycan, 120, 202f, 278–280, 298 defects in metabolism of, 184t degradation, 178–206 interactions, 128 markers in stem cell, 118f and nanotechnology, 301–303 synthesis of, 66 Glycan-lectin interactions, 36 Glycan-linked graphene, 302–303 Glycobiotechnology, 165, 300–301 Glycocalyx of cells, 111 Glycoconjugates, 3, 14, 26, 63 cancer-associated, 213 sialylated, 5–6, 260–261 Glycodendrimers, 302–303 Glycoengineering, 66, 69, 73 Fc glycoengineering in plants, 71–73 Glycogen, 201–202 degradation defect disease, 201–202 storage disorders, 202, 203–204t Glycolipids, 188–189, 301 degradation, defective, 188–194 membrane, 129 sialylation of, 212 Glycomimetics, 36, 39 Glyconanomaterials, 299, 300f, 302–303 Glyconanotechnology, 298–299 Glycoproteins, 67, 69, 178–179, 198, 301 degradation, 185–188, 186–187f sialylation of, 212, 253 Glycosidases, lysosomal, 179 Glycosphingolipids (GSLs), 114, 188, 199 Glycosylated bacterial ABC-type phosphate transporter, 133 Glycosylation, 1, 178–179, 211 expression of, 211–212 into plants, 79–80 of serum proteins, 256 Glycosyltransferase, 35 pathway, 179 transgenes, 276 GM1 gangliosidosis, 192

GnGn core oligosaccharide, 71 GnGn oligosaccharides, 67–69 Gold nanoparticles (AuNPs), 301, 313, 315 Golgi apparatus, 67 Gram-negative bacteria, 127–128 Gram-positive bacteria, 127–128 Graphene, glycan-linked, 302–303 Group B meningococcus (GBM), 127–128 Guillain-Barre Syndrome (GBS), 113, 128, 136–137, 158

H Haliotis tuberculata, 24–25 Hanganutziu-Deicher (H-D) antigen, 284–285 Heaparin sulfate, degradation of, 181f HEF. See Hemagglutinin-esterase-fusion (HEF) Helminth, 23–24 glycosylation into plants, 79–80 parasites control, 79–80 Hemagglutinin (HA), 19 Hemagglutinin-esterase-fusion (HEF), 135–136 Hemagglutinin neuraminidase glycoprotein (HN), 135–136 Hematopoietic stem cells (HSC), 118, 149–150 Hereditary disorders, 178–179 HEXA gene, 190 HF. See Hydrops fetalis (HF) HMGB1, 160–162 Hominins, 166–167 Host factors, 127–128 Host immune system, bacterial sialylation and, 18–19 HSC. See Hematopoietic stem cells (HSC) Human butyrylcholinesterase (BChE), 74 Human CMP-N-acetylneuraminic acid (NeuAc) synthase (HCSS), 75 Human CMP-Sia transporter (hCSAT) gene, 102–103 Human diseases, 90–101 Human factor H (HufH), 129 Human influenza A (H3N2) viruses, 135–136 Human milk, with oligosaccharides, 109–110

Human, recombinant proteins of, 63 Human respiratory paramyxoviruses, 135–136 Human Siglecs 15, 162 Hunter syndrome, 196–197 Hurler syndrome, 196 Hyaluronic acid, 302–303 Hydrophilic interaction chromatography, 303–304 Hydrophobically modified polysialic acid (HPSA) nanoparticles, 311 Hydrops fetalis (HF), 185 Hydroxyproline, 80f, 255 Hypersialylation, 211, 254 Hypothalamo-neurohypophysial system (HNS), 112–113 Hypothalamus, 247

I IAVs. See Influenza viruses (IAVs) IDC. See Invasive ductal breast carcinoma (IDC) IEMs. See Inborn errors of metabolism (IEMs) Immunoglobulins, 158–159 therapeutic use of intravenous, 165–166 Immunological tolerance, abrogation of, 166 Immunoreceptor tyrosine-based activating motifs (ITAMS), 155 Immunoreceptor tyrosine-based inhibitory motif (ITIM), 155, 287–288 Inborn errors of metabolism (IEMs), 179 Infantile sialic acid storage disorder (ISSD), 200–201 Infectious pathogens, 156–158 Infectious salmon anemia virus (ISAV), 27–28 Inflammation, chronic, 163–164, 258–259 Influenza vaccines, 135–136 Influenza viruses (IAVs), 135–136 Insects, 87–89 causing diseases in human, 88t physiology and development, 89–90 sialic acids and, 93–95 sialylation function in, 101–102 as vectors of human and animal diseases, 90–101

Index

351

Intravenous immunoglobulins (IVIG), therapeutic use of, 165–166 Invasive ductal breast carcinoma (IDC), 218 Invertebrates, 20–39 Ion channels, 111 Islets, pancreatic, 280–282, 289–290 ISSD. See Infantile sialic acid storage disorder (ISSD) ITIM. See Immunoreceptor tyrosine-based inhibitory motif (ITIM) IVIG. See Intravenous immunoglobulins (IVIG)

K Kala azar, 132–133 KDN. See Deaminated neuraminic acid (KDN) Keratan sulfate degradation, 183f Knockout (KO) mice, 287, 290 Knockout (KO) pigs, 287 Krabbe disease, 193–194

L Lectins, 20, 24–25. See also Sialic acid-binding lectins (SABLs) NnL, 22–23 viral sialic acid-recognizing, 19 Legionaminic acid, 1–2 Leishmania donovani, 133 Leukodystrophy, globoid cell, 193–194 Ligand-protein interaction, sialic acid, 34 Ligands, 37, 300–301 Linkage-specific sialylated glycans, 307 Lipopolysaccharide (LPS), sialylation of, 17, 19, 127–128 Liposomal nanoparticle, 36–37 LSD. See Lysosomal storage disorders (LSD) LSDs. See Lysosomal storage diseases (LSDs) L-selectin, 33, 164 Lymphatic filariasis, 23 Lysosomal enzymes, 173, 175–177t Lysosomal glycosidases, 179 Lysosomal proteinases maturation, 174 Lysosomal storage diseases (LSDs), 173–178, 205–206 Lysosomal storage disorders (LSD), 173–174, 178–206

352

Index

Lysosomes, 173–174 within cells and functions, 174f dysfunction, 178

M Maackia amurensis (MAA) lectin, 315 Macrophages, 287–288 MAG. See Myelin-associated glycoprotein (MAG) Magnetic nanoparticles (MNPs), 302 Magnetic relaxation nanosensors (MRnS), 312 Mammalian cell surfaces, 129–130 Mannan-coated AuNP, 302–303 Mannosylated AuNPs, 301 Maroteaux-Lamy syndrome, 197 Mechanoreceptors, in sea-anemone tentacles, 22–23 Membrane sialidase activity, 136–137 4-Mercaptophenyl boronic acid (MPBA), 309 Mesoporous silica nanoparticles (MSNs), 310 Metabolism gangliosides, 192–193 glycans, 184t non-ulosonic acids, 13f O-acetylated sialic acid, 9–10 sialic acid, 260f Metastasis, 257 Microbial pathogens, 155 Microglia, 113–114 Siglec-E, 114 Siglec-3 on, 114 Middle East respiratory syndrome coronavirus (MERS-CoV), 310 Minimal residual disease (MRD), 211 MIPs. See Molecularly imprinted polymers (MIPs) MNPs. See Magnetic nanoparticles (MNPs) Molecularly imprinted polymers (MIPs), 311 Molecular mimicry, 2, 5f, 18 Mollusca, 24–26 Monoclonal antibodies (mAbs), 71 Monosaccharide, 6 detection of low-level, 80

imprinted fluorescent nanoparticles, 314 Monosialoganglioside 3 (GM3), 109–110 Morquio syndrome, 194–195 Mosquito, 87, 88t, 90, 98–100, 98f MPS. See Mucopolysaccharidoses (MPS) MRD. See Minimal residual disease (MRD) MRnS. See Magnetic relaxation nanosensors (MRnS) MSD. See Multiple sulfatase deficiency (MSD) MSNs. See Mesoporous silica nanoparticles (MSNs) Mucin 16 (MUC16), 257 Mucin carbohydrate antigens, 38f Mucin-like keratan sulfate glycopolymer, 29 Mucin type O-glycans and plant expression, 79 Mucolipidosis, 201, 205 Mucopolysaccharidoses (MPS), 196 MPS I, 196 MPS II, 196–197 MPS III, 195–196 MPS IV, 194–195, 197 MPS VII, 197–198 MPS IX, 198 Multiple sulfatase deficiency (MSD), 194 Multiwalled CNTs (MWCNTs), 302 Mumps virus (MuV), 135–136 Mutants, 71 Myelin-associated glycoprotein (MAG), 114

N N-acetylation of sialic acids, 11, 12f N-acetylglucosamine 2-epimerase, 36 N-acetylglucosaminyltransferase III (GnTIII), 72 N-acetylneuraminic acids (Neu5Ac), 1–3, 10, 14, 95–96, 127–129, 153–154 aldolase, 36 N-acetyl group of, 11 Nanobiomaterials, 299f Nanocapsules (NCs), protamine, 312 Nanoparticles (NPs), 297–298 bovine serum albumin (BSA)-coated, 310 carbon dot, 309 chitosan-based, 302–303 dimensions of, 298f

fluorescent biocompatible polymeric, 313 fluorescent-conjugated polymer, 311–312 gold, 301 hydrophobically modified polysialic acid, 311 liposomal, 36–37 monosaccharide-imprinted fluorescent, 314 sialoglyco-conjugated, 313 Nano Quantity Analyte Detector (NQAD), 303–304 Nanotechnology, 297–298 bioimaging application, 308–317 glycans and, 301–303 sialic acid and, 299–301, 303–305 NCAM. See Neural cell adhesion molecule (NCAM) Neisseria gonorrhoeae, 15, 129 Neisseria meningitidis, 129 Neonatal porcine islet-like cell clusters (NPCCs), 289 Nervous system, 111–117 NET. See Neutrophil extracellular traps (NETs) Neu5Ac. See N-acetylneuraminic acids (Neu5Ac) NeuGc. See N-glycolylneuraminic acid (NeuGc) Neu5Gc. See N-glycolylneuraminic acid (Neu5Gc) Neural cell adhesion molecule (NCAM), 110, 112–113 Neural stem cell (NSC), 117–118 Neuraminic acid, deaminated, 12–13 Neuraminidase, 19 Neuroendocrinal cancer, 259–260 Neuroinflammation, 138 Neurons, desialylated, 113–114 Neuropilin-2, 111–112 Neurotrophins, 113 Neutrophil extracellular traps (NETs), 130–132, 310 Neutrophils, 137–138, 310 Newcastle disease virus (NDV), 135–136 N-glycans, 18f, 74–75, 91–92, 303–304 complex, 67–69, 68f degradation of complex, 180f

Index

353

mono- and di-sialylated, 74 in plants, 67–69 sialylation in silkworm-baculovirus system, 100–101, 101f synthesis, 24 N-glycolylneuraminic acid (Neu5Gc), 2–3, 10, 26, 117–118, 153–154, 163–164, 212–213, 280–282, 282f, 284, 290 epitopes, 287 role, 281–284 N-glycolylneuraminic acid (NeuGc), 304–305 N-glycosylation, 19, 67, 69–71, 73, 130 c-N-glycosylation, 71–72 diversification of, 67–69 mammals and insects, 90–93, 91f pathway, 63, 64f, 66, 69–70, 81 in plants, 67–69 proteins, 63 Nicotiana sp., 63, 66f N. benthamiana, 63, 69–70, 72f, 73–75, 77–78t, 79 N. tabacum, 63 Niemann-Pick disease, 188 Niemann-Pick C Disease (NPC), 193 Nippostrongylus brasiliensis, 23 N-linked glycans, catabolism of, 179 N-linked glycoproteins, 179 N-linked oligosaccharides, 24–25 N-methyl-D-aspartate-receptors (NMDARs), synaptic, 113 NnL lectin, 22–23 Nontypeable Hemophilus influenzae (NTHi), 156–158 Nonulosonic acids (NulO) metabolism, 13f synthesis, 131f NQAD. See Nano Quantity Analyte Detector (NQAD) NSC. See Neural stem cell (NSC) NulO. See Nonulosonic acids (NulO) Nutrition, 109–110

O O-acetylated sialic acid (O-AcSA), 6–11, 9f, 211 functions, 10–11 metabolism, 9–10

354

Index

9-O-acetylated sialoglycoconjugate (9-O-AcSGs) overexpression, 214–215, 214f O-acetylation of disialoganglioside GD3, 11 O-acetyl ester, 128 9-O-acetyl sialic acid (9-O-AcSA), 25–26 9-O-acteyl sialoglycoconjugates (9-O-AcSGs), 10–11 Obesity, 119–120 O-glycans, mucin type, 79 O-glycosylation, 79, 80f pathway, 66 Oligosaccharide, 179 aberrant sialylation of, 34 GnGn, 67–69 GnGn core, 71 human milk with, 109–110 O-linked glycans, 79 Ovarian cancer, 253–255 Oxidative stress-induced desialylation, 119

P PAMPs. See Pathogen-associated molecular patterns (PAMPs) Pancreas, 256–258 Pancreatic adenocarcinoma, 257 Pancreatic islets, 280–282, 289–290 Parainfluenza virus type 3 (HPIV3), 135–136 Parasites, 133, 139 Parasitic diseases, 132–133 Parasitic protozoa, 20–21, 21t, 132–135 Pathogen-associated molecular patterns (PAMPs), 154, 231–232 Pathogen-binding protein, 5f Pathogen recognition receptors (PRRs), 154 Pathogens, 2, 30t expressing sialic acids, 14t infectious, 156–158 Paucimannosidic N-glycan, 96 Peripheral nervous system (PNS), 113 Petromyzon marinus, 26–27 Pharmaceuticals and industrial production, 77–78t Pituitary cancer, 259–260 Plant-made pharmaceuticals and clinical trials, 65t Plants, 20 Fc-glycoengineering in, 71–73, 72f

glycosylation into, 79–80 N-glycans processing in, 67–69 N-glycosylation in, 67–69 recombinant proteins in, 69–70 transmit diseases in, 87–89 Polysaccharides, 129 Polysialic acids (PSAs), 27, 35, 70, 109, 111–112, 115–116f, 127–128, 315 Polysialoglycoproteins (PSGPs), 28 Polysialylated neural cell adhesion molecule (PSA-NCAM), 27–28, 112–113, 115–116f Pompe’s disease, 201–202 Porcine rennin, 274 Porcine reproductive and respiratory syndrome virus (PRRSV), 136–137 Posttranslational modifications (PTMs), 67 Prions, 138–139 Prolyl-4-hydroxylases (P4H), 79 Protamine nanocapsules (NCs), 312 Protein Data Bank (PBD), 186–187f Proteins N-glycosylation, 63 sialic acid-binding, 31 Protein tyrosine kinase (PTK) amplification pathway, 160–161 Protozoa, 20–21, 21t parasitic, 132–135 sialic acid acquisition by, 22f PRRs. See Pathogen recognition receptors (PRRs) PRRSV. See Porcine reproductive and respiratory syndrome virus (PRRSV) PSA-NCAM. See Polysialylated neural cell adhesion molecule (PSA-NCAM) PSAs. See Polysialic acids (PSAs) P-selectin, 33, 36 Pseudomonas aeruginosa, 130–132 PTMs. See Posttranslational modifications (PTMs)

Q Quantum dots (QDs), 302 semiconductor, 316

R Raman spectroscopy, 315 Recombinant human EPO (rhuEPO), 74

Recombinant proteins of human, 63 in plants, 69–70 Red blood cell (RBC) cytosensor, 310–311 Renal cells sialoglycoconjugates, 120 Reproduction, 110–111 Reticuloendothelial system (RES), 305–306

S SABLs. See Sialic acid-binding lectins (SABLs) SABP. See Sialic acid-binding glycoprotein (SABP) Salla disease, 199 Sambucus nigra bark lectin (SNA), 304, 313–315 covalent immobilization of, 315–316 SAMPs. See Self-associated molecular patterns (SAMPs) SAMs. See Sialic acid mimetics (SAMs) Sanfilippo syndrome, 195–196 Sialic acid-related disease, 198–201 SASD. See Sialic acid storage disease (SASD) Schindler disease, 187–188 Schistosoma bovis, 23 Schistosoma mansoni, 23–24 Schistosomiasis, 23–24 SCNT. See Somatic cell nuclear transfer (SCNT) Sea-anemone tentacles, mechanoreceptors in, 22–23 Selectins, 31–33, 164, 232–233, 233f Self-associated molecular patterns (SAMPs), 154, 155f, 166–167 Self-reactive lymphocytes, 149 Semiconductor quantum dots, 316 Sepsis, 314 Serine hydroxymethyltransferase 1 (SHMT1), 254 Serine proteases, 137–138 Serum proteins, glycosylation of, 256 Serum sialic acid, 119, 255 Serum sialylation, as biomarkers in cancer, 212–216 SHMT1. See Serine hydroxymethyltransferase 1 (SHMT1)

Index

355

Sialic acid, 152. See also specific types of sialic acids acetylation, 10 acquisition by protozoa, 22f and bacteria, 14–19 biological and pathological roles, 5f as biomarkers in cancer, 212–216 De novo synthesis of, 15 detection of cellular, 308–317 functions of, 3–6 and insects, 93–95 ligand-protein interaction, 34 metabolism, 260f modifications of, 6, 12–13 N-acetylation of, 11, 12f and nanotechnology, 299–301 nanotechnology in detection and quantitation of, 303–305 negative charge and hydrophilic properties of, 2 occurrence and function, 3–6, 7–8t Siglec, 136–138 structural diversity of, 6–13, 213 and therapeutics, 35–39 in therapy, 305–308, 306–307f utilization in bacterial pathogens, 17f Sialic acid acetylesterase (SIAE), 26, 156, 157f Sialic acid-binding glycoprotein (SABP), 110 Sialic acid-binding lectins (SABLs), 24, 31, 32–33t, 232 Sialic acid-binding protein, 31 Sialic acid mimetics (SAMs), 228–229 Sialic acid-Siglec axis and cancer, 224–229, 227f, 229f Sialic acid-Siglec interaction, 130–132 Sialic acid storage disease (SASD), 174 Sialidase, 27, 128 as cancer targets, 232 Sialidosis, 201 Sialoadhesin, 162, 284 Sialoglycans, 133 Sialoglyco-conjugated nanoparticles, 313 Sialoglycoconjugates, 110, 120, 215–216 deuterostome, 3 overexpression of, 213 renal cells, 120 synthesis, 4f

356

Index

Sialoglycopolyprotein, carbohydrate-rich, 29 Sialosides, 5 Sialylated carbohydrate antigens, 257 Sialylated glycans deficiency of, 120 linkage-specific, 307 Sialylated glycoconjugates, 5–6, 260–261 Sialylated molecules, in eukaryotes, 93 Sialylated Thomsen-Friedenreich antigens, 307–308 Sialylation, 1, 69–70, 111, 285–286, 290 and Aedes aegypti, 98–100, 98f in bacteria, 1 control of PrPSc particles, 138, 138f and disease, 33–34 in Drosophila, 95–96 in endocrinal disorders, 253–260 and endocrines, 253 function in insects, 101–102 genetic engineering approaches and, 102–103 of glycopilids, 212 of glycoproteins, 212, 253 of lipopolysaccharide, 17, 19, 127–128 of oligosaccharide, 34 and silkworms, 100–101, 100f and Spodoptera frugiperda (Sf9), 96, 97f taxonomic positions of insects for, 94t Sialyl-Lewisa, 257 Sialyl-Lewisx, 257 Sialyl-O-acetylesterase, 19 SialylTn in cancer, 230–231, 230f Sialyltransferase (ST), 4f, 17, 35, 69, 95–96, 198, 216, 216f, 254 and autoimmunity, 164–165 and cancer, 231–232 Siglec, 31, 160, 163–164 Cis interaction of, 136–137 inhibitory receptors, 211–212 receptors, 227–228 sialic acid and infection biology, 136–138 Siglec-3, 114 Siglec-4, 114 Siglec-11, 114 Siglec-E, 114 Siglec-G, 161–162 signaling, 113–114

Siglec-engaging tolerance inducing antigenic liposomes (STALs), 228 Signal transduction, on Siglec 2-BCR interaction, 151f Silkworm-baculovirus system, N-glycan sialylation in, 100–101, 101f Silkworms (Bombyx mori), sialylation and, 100–101, 100f Single nucleotide polymorphisms (SNPs), 113 Single-walled CNTs (SWCNTs), 302 Sly syndrome, 197–198 Sodium sialic acid symporter (SiaT), 129–130 Somatic cell nuclear transfer (SCNT), 287, 289–290 Sphingolipidoses, 188 Spodoptera frugiperda (Sf9), sialylation and, 96, 97f Spodoptera littoralis, 97f Spongiform encephalopathies, transmissible, 138–139 ST. See Sialyltransferase (ST) STALs. See Siglec-engaging tolerance inducing antigenic liposomes (STALs) Stem cells, 117–118 glycans markers in, 118f ST6GAL1, 119 sTn antigen, 35 Streptococcus agalactiae, 17 Stress-induced desialylation, 119 Subambient pressure ionization with nanoelectrospray (SPIN), 304 Sulfated sialic acids, 3 Super-paramagnetic iron oxide nanoparticles (SPIO NPs), 314 Surface-enhanced Raman scattering (SERS), 314 nanoprobe, 309, 311 signal, 311, 313 Swine endothelial cells (SECs), 287–288 Synaptic N-methyl-D-aspartate-receptors (NMDARs), 113 SynCAM 1, 111–112

T TACA. See Tumor-associated carbohydrate antigens (TACA)

TALENs. See Transcription activator-like effector nucleases (TALENs) Tay-Sachs disease, 190–191 T cell, 149–150 T cell receptors (TCRs), 276 T cruzi uses transsialidase (TcTS), 139 TcTS/sialoglycoproteins system, 134f Therapeutics antibodies, 37 sialic acids and, 35–39 Theratope, 37 Thrombin, 276 Thyroid, 259 cancer, 247–250 Ticks, 101 Tissue factor (TF), 277f Toll-like receptor (TLR) signaling, 227–228 Transcription activator-like effector nucleases (TALENs), 69–70, 289–290 Transgenes, glycosyltransferase, 276 Transgenic A. thaliana plants, 74–75 Transmissible spongiform encephalopathies, 138–139 Transmit disease, in plants, 87–89 Transplantation, 269 Truncated O-glycans, in cancer, 230 Trypanosoma cruzi, 133–134, 134f Tumor-associated carbohydrate antigens (TACA), 34–35, 37 Tumors, gangliosides in, 218–222, 220–221t, 222–223f

U UDP-GlcNAc, 4f UDP-GlcNAc2 epimerase, 260–261 Umbilical cord blood (UCB), 118 Unsaturated sialic acids, 12 Urinary tract infections, 110 Uropathogenic Escherichia coli (UPEC), 110

Index

357

V Vaccines carbohydrate-based, 37–39 influenza, 135–136 Vector-borne diseases, 90 Vectors of human and animal diseases, 90–101 VEGFR, 221t Vertebrates, 26–33 Viral sialic acid-recognizing lectins, 19 Virus, 19, 135–136 Visceral leishmaniasis (VL), 132–133

W Wilms tumor 1 (WT1), 254

X Xenoantigen, 163–164 Xenoautoantibodies, 163–164 Xenograft acceptance, 285–286 natural antibodies, 275 Xenotransplantation, 269–273, 271–272f, 273–274t complement, 275 Gal epitope, 275 infection, 278, 279t models, 286 physiology, 274 rejection, 275 sialic acid and, 278–285 of vascularized tissues, 280 X-linked disease, 189

Z Zebra fish, 26 Zinc-finger nucleases (ZFNs), 69–70, 287 ZMapp, 75, 76f Zona pellucida (ZP), 110–111