Cell Surface Glycolipids 9780841205567, 9780841206953, 0-8412-0556-6

Table of contents :
Title Page......Page 1
Copyright......Page 2
ACS Symposium Series......Page 3
FOREWORD......Page 4
PdftkEmptyString......Page 0
PREFACE......Page 5
Preparation and analysis of benzoylated cerebrosides......Page 6
Analysis of ceramides......Page 7
Quantitative analysis of neutral glycosphingolipids......Page 8
Preparative HPLC of per-0-benzoylated glycosphingolipids.......Page 13
Literature Cited......Page 16
2 High Performance Liquid Chromatography of Membrane Glycolipids Assessment of Cerebrosides on the Surface of Myelin......Page 19
Procedures......Page 20
Results......Page 25
Discussion......Page 32
Literature Cited......Page 36
3 Analysis of Glycosphingolipids by Field Desorption Mass Spectrometry......Page 38
Materials and Methods......Page 42
Results and Discussion......Page 43
Conclusions......Page 55
Literature Cited......Page 56
Methods......Page 58
Results and Discussion......Page 59
References......Page 67
5 Glycophosphoceramides from Plants......Page 68
PSL-I: The Major Glycophosphoceramide from Tobacco Leaves......Page 71
Methylation Analyses......Page 75
Acknowledgements......Page 79
Literature Cited......Page 80
6 Glycolipids of Rat Small Intestine with Special Reference to Epithelial Cells in Relation to Differentiation......Page 82
Non-Epithelial Tissue......Page 83
Epithelial Cells......Page 87
Differences between the two compartments and between strains......Page 95
Epithelial Cells of Different Location and Maturity......Page 98
Discussion......Page 101
References......Page 106
7 Galactoglycerolipids of Mammalian Testis, Spermatozoa, and Nervous Tissue......Page 108
Nomenclature, Classification and Tissue Distribution......Page 109
Chemical Characterization of Galactosylalkylacylglycerols......Page 110
Biosynthesis of Testicular and Other Galactoglycerolipids......Page 114
Catabolism of SGG......Page 116
Appearance of Sulfatides During Testicular Development......Page 117
Attempted Labelling of Galactoglycerolipids Using Galactose Oxidase......Page 119
Sulfogalactolipids of the Testis of Various Species......Page 120
Galactoglycerolipids of the Nervous System......Page 122
Conclusion......Page 124
Literature Cited......Page 125
Materials and Methods......Page 129
Results......Page 130
Discussion......Page 135
Literature Cited......Page 136
9 Glycosphingolipids of Skeletal Muscle......Page 137
DEAE-Sephadex Column Chromatography......Page 138
Enzymatic Hydrolysis Employing Glycosidases......Page 139
Results......Page 140
Discussion......Page 145
Acknowledgment......Page 148
Literature Cited......Page 149
10 Glycosphingolipids and Glyceroglucolipids of Glandular Epithelial Tissue......Page 151
The Glycosphingolipids of Gastric Mucosa and Salivary Glands......Page 152
Glycolipids of Mucous Secretion......Page 168
The Nature of ABH Blood Group Antigens in Mucous Secretion......Page 172
Literature Cited......Page 175
11 Fucolipids and Gangliosides of Human Colonic Cell Lines......Page 179
Materials and Methods......Page 180
Results......Page 181
Discussion......Page 184
Summary......Page 186
Literature Cited......Page 187
12 Biosynthesis of Blood-Group Related Glycosphingolipids in T- and B-Lymphomas and Neuroblastoma Cells......Page 189
Materials and Methods......Page 190
Results and Discussion......Page 199
Acknowledgments......Page 210
Literature Cited......Page 211
Materials and Methods......Page 215
Thin Layer Chromatography.......Page 216
Results......Page 217
Discussion......Page 222
Literature Cited......Page 223
Effects of Butyrate on Cell Morphology......Page 224
Induction of GM3 Biosynthesis by Butyrate......Page 225
Induction of Choleragen Receptors by Butyrate......Page 229
Evidence that GM1 is the Choleragen Receptor......Page 231
Effects of Cycloheximide......Page 234
LITERATURE CITED......Page 238
Introduction......Page 241
Methods......Page 246
Results and Discussion......Page 248
Conclusions......Page 259
Literature Cited......Page 260
Plasma Glycosphingolipids and Lipoproteins......Page 264
Glycosphingolipids and Lipoproteins......Page 266
Metabolism of Glycosphingolipids in Cultured Human Fibroblasts......Page 268
Glycosphingolipid and Lipoprotein Metabolism in Cultured Human Fibroblasts......Page 270
Biochemical Characterization of Fibroblasts......Page 272
Incubation of Co-cultured Cells with [3H] Galactose......Page 274
Measurement of Egress of Glycosphingolipids into the Culture Medium......Page 275
Glycosphingolipid Composition of Normal and Familial Hypercholesterolemic Cells......Page 276
Incorporation of Radioactive Leucine, Thymidine and 2-Deoxyglucose in Normal and Familial Hypercholesterolemic Fibroblasts......Page 278
Metabolic Labeling Pattern of Glycosphingolipids in Familial Hypercholesterolemic Heterozygous Fibroblasts and Co-cultured Normal and Familial Hypercholesterolemic Homozygous Fibroblasts......Page 281
Effects of Incubation of Fibroblasts with Lipoprotein Deficient Medium on the Incorporation of Radioactivity Derived from [3H] Galactose into Cellular Glycosphingolipids and Cell Culture Medium......Page 285
Discussion......Page 288
Literature Cited......Page 298
Footnotes......Page 301
17 Biochemical, Morphological, and Regulatory Aspects of Myelination in Cultures of Dissociated Brain Cells from Embryonic Mice......Page 302
Materials and Methods......Page 303
Results......Page 304
Discussion......Page 316
Literature Cited......Page 317
Chemical and physico-chemical properties of gangliosides: a molecular introduction to ganglioside behavior in cell plasma membranes.......Page 319
Ganglioside interactions......Page 322
Gangliosides, sialidase and sialyltransferase in the membranes surrounding nerve endings ( synaptosomal membranes )......Page 324
Ganglioside contribution to the supramolecular organization of synaptosoaml membranes......Page 329
An experimental model for the study of ganglioside behavior in synaptosomal membranes......Page 332
Literature cited......Page 339
19 Specificity and Membrane Properties of Young Rat Brain Sialyltransferases......Page 342
Results and Discussion......Page 343
Acknowledgement......Page 353
Literature Cited......Page 354
20 Modulation of Ganglioside Synthesis by Enkephalins, Opiates, and Prostaglandins Role of Cyclic AMP in Glycosylation......Page 356
Methods......Page 357
Results and Discussion......Page 358
Conclusions......Page 363
Literature Cited......Page 368
21 Gangliosides as Receptors for Cholera Toxin, Tetanus Toxin, and Sendai Virus......Page 370
Ligand methods......Page 371
Interaction between cholera toxin and ganglioside GMI......Page 372
Sendai virus......Page 380
Literature Cited......Page 384
Materials and Methods......Page 388
Effects of Glycolipids on Antiviral Activity of Fibroblast Interferon.......Page 390
Effects of Saccharides on Antiviral Activity of Fibroblast Interferon.......Page 392
Effect of Gangliosides on Antigrowth Activity of Fibroblast Interferon.......Page 394
Effects of Glycolipids on T-cell interferon (19).......Page 396
Effect of T-cell Interferon on Growth of L-1210S and L-1210R Cells (19).......Page 398
Discussion......Page 400
Literature Cited:......Page 401
Antibodies As A Bridge Between Structure And Function......Page 403
Antibodies To Ganglioside Interfere With CNS Functions Selectively......Page 405
Mechanisms By Which Antibodies May Perturb CNS Functions......Page 406
Antibodies To GM1 Ganglioside Inhibit Dendritic Development......Page 408
Conclusions......Page 410
Literature Cited......Page 411
Effects of Exogenously Added Gangliosides on In Vitro Assays of Cellular Immunity......Page 414
Effects of Specific Gangliosides on Lymphoblastic Transformation.......Page 416
Discussion......Page 425
ACKNOWLEDGEMENTS......Page 426
LITERATURE CITED......Page 427
Materials And Methods......Page 429
Results And Discussion......Page 430
Literature Cited......Page 436
26 The Immunology and Immunochemistry of Thy-1 Active Glycolipids......Page 438
Materials and Methods......Page 439
Results......Page 441
Discussion......Page 450
Literature Cited......Page 451
Methods......Page 453
Results......Page 454
Discussion......Page 462
LITERATURE CITED......Page 464
A......Page 466
B......Page 467
C......Page 469
E......Page 473
F......Page 474
G......Page 476
H......Page 482
I......Page 483
L......Page 484
M......Page 485
O......Page 487
P......Page 488
R......Page 489
S......Page 490
T......Page 492
W......Page 494

Citation preview

Cell Surface Glycolipids Charles C. Sweeley,

EDITOR

Michigan State University

Based on a symposium sponsored by the Division of Carbohydrate Chemistry at the 178th Meeting of the American Chemical Society, Washington, D.C. September 11-14, 1979.

ACS

SYMPOSIUM AMERICAN

SERIES

CHEMICAL

W A S H I N G T O N , D.

C.

SOCIETY 1980

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

128

Library of CongressCIPData Cell surface glycolipids. (ACS symposium series; 12 Bibliography: p. Includes indexes. 1. Glycolipids—Congresses. 2. Membranes (Biology) —Congresses. I. Sweeley, Charles Crawford, 1930. II. American Chemical Society. Division of Carbohydrate Chemistry. III. Series: American Chemical Society. ACS symposium series; 128. [DNLM: 1. Cell membrane—Congresses. 2. Glycolipids—Congresses. QU85 C393 1979] QP572.G56C44 574.87'5 80-15283 ISBN 0-8412-0556-6 ACSMC8 128 1-504 1980

Copyright © 1980 American Chemical Society All Rights Reserved. The appearance of the code at the bottom of thefirstpage of each article in this volume indicates the copyright owner's consent that reprographic copies of the article may be made for personal or internal use or for the personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per copy fee through the Copyright Clearance Center, Inc. for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to copying or transmission by any means—graphic or electronic—for any other purpose, such as for general distribution, for advertising or promotional purposes, for creating new collective works, for resale, or for information storage and retrieval systems. The citation of trade names and/or names of manufacturers in this publication is not to be construed as an endorsement or as approval by ACS of the commercial products or services referenced herein; nor should the mere reference herein to any drawing, specification, chemical process, or other data be regarded as a license or as a conveyance of any right or permission, to the holder, reader, or any other person or corporation, to manufacture, reproduce, use, or sell any patented invention or copyrighted work that may in any way be related thereto. PRINTED IN THE UNITEDSTATESOFAMERICA

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

ACS

S y m p o s i u m Series M . Joa

Advisory Board David L. Allara

W . Jeffrey Howe

Kenneth B. Bischoff

James D . Idol, Jr.

Donald G . Crosby

James P. Lodge

Donald D . Dollberg

Leon Petrakis

Robert E. Feeney

F. Sherwood Rowland

Jack Halpern

Alan C. Sartorelli

Brian M . Harney

Raymond B. Seymour

Robert A . Hofstader

Gunter Zweig

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

FOREWORD The ACS S Y M P O S I U M SERIES founded i 1 9 7 4 t provid a medium for publishin format of the Series parallels that of the continuing A D V A N C E S I N C H E M I S T R Y SERIES except that in order to save time the papers are not typeset but are reproduced as they are submitted by the authors in camera-ready form. Papers are reviewed under the supervision of the Editors with the assistance of the Series Advisory Board and are selected to maintain the integrity of the symposia; however, verbatim reproductions of previously published papers are not accepted. Both reviews and reports of research are acceptable since symposia may embrace both types of presentation.

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

PREFACE

T

his collection of papers is a record of the proceedings of a symposium on the chemistry, metabolism, and biological functions of glycolipids. It is also a review of some topics presented at an earlier meeting on the same subject, held in Honolulu, Hawaii in October, 1977 under the auspices of the Japan-United States Science Exchange Program. Both meetings were convened with a hope that aspects of glycolipid biochemistry at the cell these meetings have provide insigh inspiratio to understand the role of these interesting substances in nature. The reader will note substantial progress and new information about the chemistry and metabolism of the glycolipids, with especially comprehensive material on their separation and characterization. The antigenic behavior and the cell surface receptor role of glycolipids are discussed in some detail, providing a sound basis for future investigations of cell surface specificity to particular molecules and supramolecular systems. I am indebted to the foreign speakers; Lars Svennerholm, KarlAnders Karlsson, Guido Tettamanti, Robert Murray, and Yoshitaka Nagai generously consented to participate in this symposium with little financial support from the organizer or the society. Their contributions were an especially important part of the symposium. I am grateful as well to the American participants, who attended the meeting largely with their own funds and made the meeting a success. Although Professor Egge of the University of Bonn could not attend the meeting, he kindly provided an important chapter on high resolution proton N M R of glycosphingolipids, for which I am grateful. Finally, I wish to thank Ms. Dorothy Byrne, Ms. Paula Allen, and Ms. Susan Leavitt for their dedicated assistance in the organization of the meeting, correspondence with speakers, and preparation of the final manuscripts for this book.

East Lansing, Michigan December, 1979

CHARLES

S. SWEELEY

vii In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

1 Preparative and Analytical High Performance Liquid Chromatography of Glycolipids R. H. MC CLUER Eunice Kennedy Shriver Center, Waltham, MA 02154 M. D. ULLMAN Center for Disease Control, Atlanta, GA 30333 High performance liqui use of reusable columns use of pumps for uniform solvent flow operated at high pressures if necessary and automatic on-line sample detection. The availability of a large variety of microparticulate column packing materials, efficient column packing techniques, high pressure-low volume pumping equipment and various types of highly sensitive detectors have led to the development of sensitive, rapid and quantitative methods, analogous to that available for volatile materials by gas-chromatography, for the isolation and analysis of a large variety of relatively high molecular weight substances of biological interest (1). We have attempted to utilize these tools of modern liquid chromatography to develop rapid and highly sensitive methods for the analysis of glycolipids. Our early experience with HPLC techniques indicated that the analysis of glycolipids becomes interestingly sensitive and practical if derivatives are prepared that allow the use of ultraviolet detectors and that exhibit good chromatographic properties. We have primarily studied the preparation of the benzoyl derivatives of glycolipids for the development of analytical and preparative HPLC methods. The following is a concise review of studies with neutral glycosphingolipids with emphasis on recent work in which we have utilized p-dimethylaminopyridine (DMAP) as a catalyst to effect benzoylation with benzoic anhydride. Preparation and analysis of benzoylated cerebrosides We initially demonstrated that brain cerebrosides, galactosylceramides containing hydroxy fatty acids (HFA) and nonhydroxy fatty acids (NFA), could be completely derivatized by reaction with 10% benzoyl chloride at 60OC for 1 hour (2). After removal of excess reagents by partition between hexane and alkaline aqueous methanol, the perbenzoyl derivatives were seen to separate into two completely resolved components (HFA and NFA 0-8412-0556-6/ 80/ 47-128-001 $5.00/ 0 © 1980 American Chemical Society In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

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C E L L SURFACE GLYCOLIPIDS

c e r e b r o s i d e s ) by adsorption chromatography on a p e l l i c u l a r s i l i c a column support ( Z i p a x , E . I . DuPont) w i t h methanol i n pentane as the e l u t i n g s o l v e n t and 280 nm d e t e c t i o n . Hexane-ethyl acetate was subsequently shown to be a s u p e r i o r e l u t i n g solvent because regeneration of the column adsorbant a c t i v i t y was more r e p r o ducible (3). Attempts to recover the parent cerebrosides by treatment of the benzoyl d e r i v a t i v e s w i t h m i l d a l k a l i was successful w i t h the HFA-cerebrosides, but the NFA-cerebroside d e r i v a t i v e gave r i s e to benzoyl psychosine as well as the parent NFA-cerebroside. This was demonstrated to r e s u l t from the d i a c y l amine s t r u c t u r e of the perbenzoyl NFA-cerebroside. NMR s t u d i e s of the two cerebroside d e r i v a t i v e s i n d i c a t e d the presence of s i x benzoyl groups i n each case and the presence of an amide proton i n the H F A - d e r i v a t i v e which was absent i n the NFA-cerebroside d e r i v a t i v e . As reported by Inch and F l e t c h e r (4) f o r the diacylamine d e r i v a t i v e s of amino s u g a r s , the N-acyl groups are randomly removed durin other s p h i n g o l i p i d s whic a c e t y l amino sugars cannot be recovered i n high y i e l d s because a l k a l i n e h y d r o l y s i s of the perbenzoyl d e r i v a t i v e r e s u l t s i n the formation of N-benzoyl compounds as well as the parent N-acyl s p h i n g o l i p i d . Benzoylation of cerebrosides w i t h 10% benzoic anhydride i n p y r i d i n e was shown to lead only to the formation of 0 - a c y l d e r i v a t i v e s and the parent g l y c o l i p i d s could be recovered a f t e r a l k a l i n e methanolysis; however, t h i s r e a c t i o n was s l u g g i s h and r e q u i r e d treatment a t 110 C f o r 18 hours f o r completion. S u l f a t i d e s were shown to be completely converted to benzoylated cerebrosides during t h i s anhydride r e a c t i o n . We chose the benzoyl c h l o r i d e r e a c t i o n f o r a n a l y t i c a l purposes because r e a c t i o n times were shorter and s u l f a t i d e s do not d e s u l f a t e under c o n d i t i o n s r e q u i r e d f o r cerebroside d e r i v a t i z a t i o n . Because s p h i n g o l i p i d s which c o n t a i n only hydroxy f a t t y a c i d s as N-acyl s u b s t i t u e n t s form the same d e r i v a t i v e with e i t h e r the benzoyl c h l o r i d e or the anhydride r e a c t i o n , they can be e a s i l y d i s t i n g u i s h e d from nonhydroxy f a t t y a c i d c o n t a i n i n g s p h i n g o l i p i d s which form d i f f e r e n t d e r i v a t i v e s , d i s t i n q u i s h a b l e by HPLC, when benzoylated with the c h l o r i d e as compared to the anhydride r e a c t i o n . A n a l y s i s of

ceramides

A q u a n t i t a t i v e HPLC method f o r the a n a l y s i s of s p h i n g o l i p i d s as t h e i r perbenzoyl d e r i v a t i v e s was f i r s t developed f o r ceramides (5). Ceramides can be conveniently d e r i v a t i z e d w i t h benzoic anhydride i n p y r i d i n e (3 hrs at 110°C) and the products formed have been u t i l i z e d f o r the q u a n t i t a t i v e a n a l y s i s of NFA and HFA ceramides i n normal and F a r b e r ' s disease t i s s u e . Iwamori and Moser a l s o u t i l i z e d t h i s procedure f o r the a n a l y s i s of ceramides i n F a r b e r ' s disease u r i n e ( 6 ) . More r e c e n t l y Iwamori and Moser (7) e s t a b l i s h e d that the ceramide d e r i v a t i v e s formed by r e a c t i o n with benzoyl c h l o r i d e or benzoic anhydride are analogous to those formed with c e r e b r o s i d e s . They a l s o c h a r a c t e r i z e d the behavior

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

1.

MCCLUER AND ULLMAN

Liquid

Chromatography

3

of ceramides t h a t contain phytosphingosine and described the use of estrone as an i n t e r n a l s t a n d a r d . These published ceramide methods u t i l i z e d 280 nm d e t e c t i o n and the s e n s i t i v i t y of the procedures could e a s i l y be increased to the pmole l e v e l by d e t e c t i o n a t 230 nm i f a v a r i a b l e wave length d e t e c t o r i s u t i l i z e d . A l s o , the speed of d e r i v a t i z a t i o n could undoubtedly be g r e a t l y increased by the use of the DMAP as a c a t a l y s t as described below f o r neutral g l y c o s p h i n g o l i p i d s . Samuelsson (8) u t i l i z e d elegent gas chromatograph-mass spectrometric (GC-MS) methods f o r the a n a l y s i s of ceramide molecular s p e c i e s , but HPLC methods o f f e r the advantages of n o n - d e s t r u c t i v e measurement so that components can e a s i l y be c o l l e c t e d f o r determination of r a d i o a c t i v i t y or f o r f u r t h e r a n a l y s i s . Q u a n t i t a t i v e a n a l y s i s of n e u t r a l g l y c o s p h i n g o l i p i d s An HPLC method f o r designed f o r the a n a l y s i s of human plasma g l y c o l i p i d s ( 3 ) , which c o n s i s t p r i m a r i l y of glucosylceramide, l a c t o s y l c e r a m i d e , g l o b o t r i a o s y l c e r a m i d e and globotetraosylceramide ( g l o b o s i d e ) . Conditions f o r the simultaneous d e r i v a t i z a t i o n of t h i s group of compounds, which provided maximal y i e l d s f o r g l o b o s i d e , were s e l e c t e d to be 37 C f o r 16 hours i n 10% benzoyl c h l o r i d e i n p y r i d i n e , s l i g h t l y d i f f e r e n t from those p r e v i o u s l y u t i l i z e d f o r cerebrosides. S a t i s f a c t o r y chromatographic c o n d i t i o n s , which provided base l i n e r e s o l u t i o n of these four d e r i v a t i v e s i n a minimum of t i m e , were found w i t h the Zipax column and a gradient of e t h y l acetate i n hexane and 280 nm d e t e c t i o n . With t h i s chromatographic system the standard g l y c o l i p i d d e r i v a t i v e s could be separated and q u a n t i t a t e d i n l e s s than 20 min and column a c t i v i t y could be r e p r o d u c i b l y regenerated i n e i g h t min.. Less than 20 nmole of each g l y c o l i p i d could be e a s i l y q u a n t i t a t e d w i t h t h i s procedure. The u t i l i t y of ethyl acetate i n t h i s chromatographic system was e x c e l l e n t but prevented the use o f d e t e c t i o n below 260 nm. Because the max of the benzoyl d e r i v a t i v e s i s a t 230nm we sought chromatographic solvents which could be u t i l i z e d a t t h i s wavel e n g t h , s t i l l provide adequate chromatographic r e s o l u t i o n and a l l o w r a p i d l y r e - e q u i l i b r a t i o n of column adsorbant a c t i v i t y a f t e r gradient e l u t i o n . A dioxane-hexane s o l v e n t system proved adequate except r e s i d u a l l i g h t absorption due to the dioxane produced an undesirable r i s i n g base l i n e during the g r a d i e n t . The r i s i n g base l i n e was e l i m i n a t e d by d i r e c t i n g the s o l v e n t flow through a precolumn p r e - i n j e c t o r high pressure reference c e l l . This path generates a h o r i z o n t a l b a s e l i n e with a negative and p o s i t i v e d e f l e c t i o n a t the beginning and end of the g r a d i e n t r e s p e c t i v e l y . With t h i s system r e l i a b l e q u a n t i t a t i o n of l e s s than 50 pmoles of each of the four major plasma g l y c o s p h i n g o l i p i d s can be obtained (9). For the a n a l y s i s of plasma g l y c o l i p i d s i t i s necessary to f i r s t i s o l a t e a g l y c o l i p i d f r a c t i o n by s o l v e n t e x t r a c t i o n , b

x

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

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chromatography on small U n i s i l columns and treatment w i t h m i l d a l k a l i as o r i g i n a l l y described by Vance and Sweeley (10). C o n s i s t e n t r e c o v e r i e s of the g l y c o l i p i d s i s dependent upon maintaining a f i x e d r a t i o between sample s i z e and the q u a n t i t y of U n i s i l employed. Accuracy o f the method i s improved by the u t i l i z a t i o n of an i n t e r n a l standard such as N-acetylpsychosine which i s added to the plasma samples p r i o r to the i n i t i a l l i p i d e x t r a c t i o n . One ml plasma samples are now r o u t i n e l y used f o r g l y c o l i p i d a n a l y s i s although s e n s i t i v i t y of the HPLC procedure t h e o r e t i c a l l y should a l l o w a n a l y s i s o f l e s s than 0.1 m l . However, the i s o l a t i o n o f such small q u a n t i t i e s of g l y c o l i p i d p r i o r to d e r i v a t i z a t i o n present d i f f i c u l t recovery problems. The high s e n s i t i v i t y of the d e t e c t i o n system employed f o r the a n a l y s i s of pmole q u a n t i t i e s a l s o r e q u i r e s precautions so t h a t UV absorbing contaminates are not introduced during processing of the samples. A l l glassware should be scrupulously c l e a n and HPLC grade s o l v e n t s should be used f o r a l l step This HPLC procedure has a l s o been u t i l i z e d f o r the a n a l y s i s of neutral g l y c o s p h i n g o l i p i d s from a v a r i e t y o f sources. Human e r y t h r o c y t e s , p e r i p h e r a l leukocytes and l i v e r have been s a t i s f a c t o r i l y a n a l y z e d , but i t should be recognized t h a t each d i f f e r e n t t i s s u e source may r e q u i r e d i f f e r e n t e x t r a c t i o n c o n d i t i o n s and modified s o l v e n t g r a d i e n t e l u t i o n i n order to o b t a i n maximal r e c o v e r i e s and optimal chromatographic r e s o l u t i o n of the t i s s u e c h a r a c t e r i s t i c g l y c o s p h i n g o l i p i d s . F l e t c h e r , Bremer and Schwarting (11) have optimized the procedure f o r the a n a l y s i s of e r y t h r o c y t e g l y c o l i p i d s and demonstrated t h a t e r y t h r o c y t e s from blood group P-| i n d i v i d u a l s c o n t a i n more g l o b o t r i a o s y l c e r a m i d e and l e s s l a c t o s y l c e r a m i d e than e r y t h r o c y t e s from blood group ?2 i n d i v i d u a l s . The dramatic sex d i f f e r e n c e i n mouse kidney g l y c o l i p i d s and the occurrence of l a r g e amounts of g l y c o l i p i d s i n male mouse u r i n e was r e a d i l y demonstrated by these HPLC methods. The l i g h t ear ( l e / l e ) mouse pigmentation mutant was shown to have storage of g l y c o l i p i d s i n t h e i r kidneys which i s apparently due to an abnormality i n the s e c r e t i o n of m u l t i l a m e l l a r lysosomal bodies t h a t c o n t a i n l a r g e amounts of g l y c o s p h i n g o l i p i d s (12) . Thus, the a n a l y t i c a l HPLC method f o r g l y c o l i p i d s i s proving useful f o r a v a r i e t y o f s t u d i e s r e l a t e d to g l y c o s p h i n g o l i p i d f u n c t i o n and metabolism. Other useful a n a l y t i c a l HPLC procedures f o r the a n a l y s i s of d e r i v a t i z e d g l y c o l i p i d s have been developed. Nanaka and Kishimoto (13) have devised an HPLC procedure which allows the t i s s u e l e v e l s of NFA c e r e b r o s i d e , HFA c e r e b r o s i d e , NFA s u l f a t i d e , HFA s u l f a t i d e , and monogalactosyl d i g l y c e r i d e to be determined s i m u l t a n e o u s l y . This procedure i n v o l v e s benzoyl a t i o n of t o t a l l i p i d e x t r a c t s , d e s u l f a t i o n w i t h m i l d a c i d and subsequent chromatography w i t h the r a d i e n t of isopropanol i n hexane. S u s u k i , Honda and Yamakawa 14) prepared a c e t y l a t e d g l y c o l i p i d which were subsequently reacted w i t h p - n i t r o b e n z o y l c h l o r i d e to form the O - a c e t y l - N - p nitrobenzoyl d e r i v a t i v e s which have good chromatographic

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

1.

MCCLUER AND ULLMAN

Liquid

Chromatography

5

p r o p e r t i e s and can be detected w i t h high s e n s i t i v i t y w i t h a s i n g l e wavelength detector a t 254 nm. While o f f e r i n g these advantages, t h i s procedure cannot be u t i l i z e d w i t h g l y c o l i p i d s t h a t c o n t a i n only a-hydroxy f a t t y a c i d s and no amino sugar. A l l of the benzoylated or O - a c e t y l - N - p - n i t r o b e n z o y l d e r i v a t i v e s can be u s e f u l l y separated i n t o molecular species by reverse phase chromatography ( 1 1 , 1 2 ) . We have r e c e n t l y shown t h a t the use o f the c a t a l y s t N - d i methylaminopyridine (DMAP) w i t h benzoic anhydride g r e a t l y a c c e l e r a t e s the d e r i v a t i z a t i o n w i t h t h i s reagent (13). Reaction w i t h DMAP and the anhydride avoids amide a c y l a t i o n , forms s i n g l e products w i t h s a t i s f a c t o r y chromatographic p r o p e r t i e s and parent g l y c o s p h i n g o l i p i d s can be regenerated by m i l d a l k a l i n e h y d r o l y s i s . For a n a l y t i c a l purposes, t h i s r e a c t i o n has been u t i l i z e d f o r the a n a l y s i s of plasma n e u t r a l g l y c o s p h i n g o l i p i d s . The g l y c o l i p i d s were reacted w i t h 20% benzoic a c i d a n h y d r i d e , 5% DMAP i n p y r i d i n e at 37°C f o r four hours each gave s i n g l e r e a c t i o n products w i t h maximum y i e l d s w i t h r e a c t i o n times between 2 and 6 hours. Excess reagents were removed from the products by p a r t i t i o n between hexane and aqueous a l k a l i n e methanol as described p r e v i o u s l y f o r the benzoyl c h l o r i d e products. The products were than analyzed w i t h the Zipax column and dioxane g r a d i e n t a l s o as p r e v i o u s l y described ( 3 ) . The chromatographic a n a l y s i s o f the per-O-benzoylated glycosphingol i p i d standards and plasma g l y c o s p h i n g o l i p i d s are shown i n F i g . 1 along w i t h the e l u t i o n p a t t e r n of plasma g l y c o l i p i d d e r i v a t i v e s obtained by r e a c t i o n w i t h benzoyl c h l o r i d e . The d e r i v a t i v e s obtained by r e a c t i o n with benzoic anhydride have longer r e t e n t i o n times when compared to the benzoyl c h l o r i d e products. We have p r e v i o u s l y shown t h a t galactosylceramide which contains a-hydroxy f a t t y a c i d s i s not N-benzoylated w i t h benzoyl c h l o r i d e and r e a c t i o n w i t h benzoic anhydride or benzoyl c h l o r i d e r e s u l t s i n an i d e n t i c a l product. S i m i l a r r e s u l t s have been obtained w i t h anhydride i n the presence of DMAP as i l l u s t r a t e d i n F i g . 2. The behavior of peak b" which we have shown to be derived from a-hydroxy f a t t y a c i d c o n t a i n i n g g l u c o s y l and galactosylceramides i s i l l u s t r a t i v e . The UV response from each of the standard GSLs benzoylated by the anhydride and by the benzoyl c h l o r i d e method were compared. The r e l a t i v e responses ( c h l o r i d e / a n h y d r i d e ) f o r the mono, d i , t r i and t e t r a - h e x o s y l ceramide were found to be 1.18, 1.15, 0.94 and 1.03 r e s p e c t i v e l y . These values were not s i g n i f i c a n t l y d i f f e r e n t from c a l c u l a t e d r a t i o n s 1.20, 1.12, 1.09, and 1.15, based on the assumption t h a t the anhydride method avoids amide b e n z o y l a t i o n . The y i e l d s of the per-O-benzoylated products were s i m i l a r to those obtained f o r the products o f the benzoyl c h l o r i d e method reported p r e v i o u s l y . The parent GSLs can be regenerated from t h e i r p e r - O benzoylated products by treatment w i t h m i l d a l k a l i . Globoside was benzoylated by both methods, the products subjected to HPLC, and the peaks c o l l e c t e d and t r e a t e d w i t h 0.5N methanolic sodium 11

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

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0

2

4

6

8

10

12

14

16

TIME (min) Figure 1.

HPLC of benzoylated standard and plasma glycosphingolipids

The derivatized glycosphingolipids were injected onto a Zipex column (2.1 mm X 50 cm) and eluted with a 13-min linear gradient of 2.5-25% dioxane in hexane with detection at 230 nm. A. Standard glycosphingolipids (GSL) per-O-benzoylated with benzoic anhydride and 4-dimethylaminopyridine (DMAP). B. Plasma GSL per-O-benzoylated with benzoic anhydride and DMAP. C. Plasma GSL perbenzoylated with benzoyl chloride. Glycosphingolipid peaks are identified as: (1) glycosylceramide, (2) lactosylceramide, (3) galactosyl-lactosylceramide, (4) N-acetylgalactosaminylgalactosyllactosylceramide. Peak A is unidentified, and peak B is hydroxy fatty acid containing galactosylceramide.

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980. Preparative isolation of liver glycolipids

A glycosphingolipid fraction (15 mg) was benzoylated with DMAP and benzoic anhydrydride, and the derivatives were chromatographed on a LiChrosorb SI 100 column with an ethyl acetate in hexane gradient as described in the text. Detection was at 280 nm. Components eluting at 7, 9, and 49 min are unidentified.

Figure 2.

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hydroxide f o r 1 hour a t 37°C. A f t e r s o l v e n t p a r t i t i o n , i n C/M/H 0 ( 8 / 3 / 3 ) the lower phase l i p i d products were examined by TLC and v i s u a l i z e d under UV l i g h t and w i t h o r c i n o l and s u l f u r i c a c i d spray reagents. Only a s i n g l e product, with no UV absorption was obtained from the anhydride benzoylated g l o b o s i d e . Methanolysis and GLC a n a l y s i s of f a t t y a c i d s from t h i s product revealed t h a t t h e i r composition was unchanged compared to t h a t of the parent globoside. The r a t i o of 24:1 to 24:0 f a t t y a c i d s was 0.51 i n the o r i g i n a l sample and 0.50 i n the debenzoylated sample. The use o f benzoic anhydride w i t h DMAP as a c a t a l y s t provides a convenient means f o r the p r e p a r a t i o n o f the p e r - O benzoylated d e r i v a t i v e s of GSLs. These d e r i v a t i v e s can subsequently be u t i l i z e d f o r a n a l y t i c a l and p r e p a r a t i v e HPLC because parent GSLs can be c o n v e n i e n t l y recovered i n high y i e l d s by m i l d a l k a l i n e h y d r o l y s i s . 2

P r e p a r a t i v e HPLC of per-O-benzoylate We d e s c r i b e here a procedure f o r the convenient d e r i v a t i z a t i o n and p r e p a r a t i v e i s o l a t i o n o f g l y c o l i p i d s by HPLC w i t h UV d e t e c t i o n a t 280 nm. Previous t h i n - l a y e r chromatography (TLC), l i q u i d chromatography (LC) and HPLC procedures have been encumbered by the l a c k of a convenient n o n - d e s t r u c t i v e method of d e t e c t i o n f o r the components o f i n t e r e s t . F u r t h e r , TLC i s o l a t i o n s are hampered by the small load c a p a c i t i e s of each p l a t e which r e q u i r e s the s t r e a k i n g , scraping and e l u t i o n of compounds from m u l t i p l e p l a t e s and by the a m b i g u i t i e s introduced by l i g h t l y spraying of each p l a t e w i t h a n o n - d e s t r u c t i v e spray such as methanol-water 1:1 ( v / v ) or p r i m u l i n e (15). T h i c k - l a y e r TLC, which allows l a r g e l o a d s , f r e q u e n t l y y i e l d s poor r e s o l u t i o n because the streaked sample tends to " f l a r e " as i t penetrates the separation bed, thus causing s i g n i f i c a n t overlap of bands d u r i n g m i g r a t i o n . Larger q u a n t i t i e s of g l y c o l i p i d s have been separated by L C , w i t h v a r y i n g degrees o f success, on such column packing m a t e r i a l s as alumina ( 1 6 , 1 7 ) , A n a s i l S ( 1 8 , 1 9 , 2 0 ) , F l o r i s i l ( 2 1 , 2 2 , 2 3 , 2 4 , 2 5 ) , Iatrobeads (26), s i l i c i c a c i d (27,28,29) S i l i c a gel G ( 3 0 ) , and U n i s i l ( 3 1 , 3 2 ) . A l l of these LC procedures are hampered by the absence of an adequate d e t e c t i o n system. Although the l a c k of o n - l i n e d e t e c t i o n has impeded the adaptation of the LC procedures to HPLC, p r e p a r a t i v e HPLC of g l y c o l i p i d s has been performed on s i l i c a SI 60 w i t h post-column, o f f - l i n e TLC d e t e c t i o n (30) and w i t h a moving w i r e detector ( 3 1 ) . The procedure described below f o r p e r - 0 - b e n z o y l a t i o n of g l y c o l i p i d s w i t h benzoic anhydride i n p y r i d i n e and DMAP as c a t a l y s t avoids N-benzoylation problem and provides a convenient method f o r the d e t e c t i o n and p r e p a r a t i v e i s o l a t i o n of g l y c o l i p i d s . The a p p l i c a t i o n of t h i s procedure f o r the i s o l a t i o n of 15 mg of g l y c o l i p i d s i n a s i n g l e HPLC run i s d e s c r i b e d . a . I s o l a t i o n of crude l i v e r n e u t r a l g l y c o l i p i d s . Total l i p i d s were i s o l a t e d from human l i v e r by the method of Folch e t a l . (33).

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

1.

MCCLUER AND ULLMAN

Liquid

Chromatography

9

Crude n e u t r a l g l y c o l i p i d s were i s o l a t e d from the l i p i d e x t r a c t e s s e n t i a l l y by the procedure of Vance and Sweeley (10). The l i p i d s were d i s s o l v e d i n chloroform and placed on a s i l i c i c a c i d column (25 gm packing to 1 gm l i p i d e x t r a c t ) the n e u t r a l l i p i d s and f a t t y a c i d s were e l u t e d w i t h chloroform (20 ml/gm p a c k i n g ) . Crude n e u t r a l g l y c o l i p i d s were then e l u t e d from the column w i t h acetone-methanol (9:1 v / v ) (40 ml/gm p a c k i n g ) . The acetonemethanol e l u a t e was c o l l e c t e d i n 200 ml f r a c t i o n s , so t h a t monoand d i - h e x o s y l ceramides were i n greater c o n c e n t r a t i o n i n the e a r l i e r f r a c t i o n s r e l a t i v e to the t r i - and t e t r a - h e x o s y l ceramides. The acetone-methanol from each of the d e s i r e d f r a c t i o n s was evaporated to dryness and exposed to m i l d a l k a l i n e m e t h a n o l y s i s . The content of n e u t r a l g l y c o l i p i d s i n the f r a c t i o n s of i n t e r e s t was determined by the q u a n t i t a t i v e HPLC method o f Ullman and McCluer ( 3 ) . b. P e r - Q - b e n z o y l a t i o n c o n d i t i o n under a stream of n i t r o g e n and which contained approximately 15 mg of n e u t r a l g l y c o l i p i d s from l i v e r , were t r a n s f e r r e d i n t o a 20 mm x 150 mm screw capped c u l t u r e tube and d e s s i c a t e d over P2O5 f o r a t l e a s t three hours. A 1.5 ml p o r t i o n of f r e s h l y prepared 20% benzoic anhydride i n p y r i d i n e (w/v) was added to the c u l t u r e tube followed by a 1.5 ml p o r t i o n of l $ DMAP i n p y r i d i n e (v/v).The tube was b r i e f l y flushed w i t h n i t r o g e n , capped t i g h t l y , and incubated at 37°C f o r two hours. The tube was then placed i n a water bath maintained a t room temperature and the p y r i d i n e was removed w i t h a stream of n i t r o g e n . Three ml of hexane was added to the r e s i d u e and the suspension was washed four times with 1.8 ml of a l k a l i n e methanol. The a l k a l i n e methanol was prepared by the a d d i t i o n of 1.2 gm Na2C03 to 300 ml of methanol-water 80:20 ( v / v ) ( a l l of the Na2C03 d i d not d i s s o l v e ) . Each time the lower phase was withdrawn and d i s c a r d e d . F i n a l l y , the hexane l a y e r was washed once w i t h 1.8 ml of methanol-water 80:20 and a f t e r removal of the lower phase the hexane was evaporated w i t h a stream of n i t r o g e n at room temperature. The sample was then d i s s o l v e d i n 5 ml of methanol and placed onto a reverse phase r a p i d sample p r e p a r a t i o n column (Sep PakTM,Waters A s s o c . ) t h a t had been preconditioned w i t h 30 ml of methanol. An a d d i t i o n a l wash of 5 ml of methanol was added to the column and the per-O-benzoylated g l y c o l i p i d s were e l u t e d w i t h 10 ml of methanol-acetone 9:1 i n a 20 mm x 150 mm screw cap c u l t u r e tube. This f r a c t i o n was d r i e d at room temperature w i t h a stream of n i t r o g e n and r e d i s s o l v e d i n 4% e t h y l acetate i n hexane ( v / v ) f o r i n j e c t i o n . c. HPLC. Per-O-benzoylated n e u t r a l g l y c o l i p i d s were a p p l i e d to a 4.6 mm x 25 cm LiChrosorb SI 100, 10y p a r t i c l e w i t h a loop i n j e c t o r and 4% e t h y l acetate i n hexane pumped a t 0.5 m l / m i n . The d e r i v a t i v e s were then e l u t e d i s o m a t i c a l l y w i t h 18% e t h y l acetate i n hexane f o r 30 minutes and then w i t h a l i n e a r g r a d i e n t of 18 to 45% e t h y l acetate i n hexane over t h i r t y minutes. The

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

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flow r a t e was 3 ml/min and UV d e t e c t i o n was performed a t 280 nm. Each peak was i s o l a t e d and p a r t i a l l y c h a r a c t e r i z e d f o r recovery and p u r i t y by a n a l y t i c a l HPLC. A f t e r the i s o l a t i o n was complete the i n i t i a l s o l v e n t and adsorbant c o n d i t i o n s were regenerated w i t h a three minute reverse g r a d i e n t . d. R e s u l t s . E x c e l l e n t r e s o l u t i o n of the major g l y c o l i p i d peaks ( F i g . 2) was o b t a i n e d . Because of the mono-, d i - , t r i - , and t e t r a - h e x o s y l ceramides d i d not maintain a constant r a t i o i n each f r a c t i o n during the i s o l a t i o n of the crude g l y c o l i p i d f r a c t i o n from U n i s i l , the chromatogram i n Figure 2 i s not r e p r e s e n t a t i v e of the d i s t r i b u t i o n of l i v e r g l y c o l i p i d s . E t h y l a c e t a t e was s e l e c t e d as a s o l v e n t because of the high l i p i d s o l u b i l i t y of the d e r i v i t i z e d g l y c o l i p i d s because i t i s e a s i l y removed a f t e r c o l l e c t i o n of the d e s i r e d f r a c t i o n s , and because column adsorbant a c t i v i t y i s r a p d i l y reestablished a f t e r gradient e l u t i o n . Detection at 280nm avoide l a r g e q u a n t i t i e s of g l y c o l i p i d s were a p p l i e d to the column. Larger columns and greater amounts o f g l y c o l i p i d s could have been used by s e t t i n g the d e t e c t o r to a s l i g h t l y higher wavelength thus decreasing the s e n s i t i v i t y . Although c a p a c i t y o f the column was not determined i n these experiments, there was no change i n r e t e n t i o n time or peak shape when 5, 10, or 15 mg of g l y c o l i p i d s were chromatographed. Each sample was c o l l e c t e d so t h a t approximately 5% of the l e a d i n g and t a i l i n g edges of the peaks were omitted from the c o l l e c t i o n . A f t e r a sample was c o l l e c t e d , the i s o l a t e d f r a c t i o n was d r i e d under n i t r o g e n , d i s s o l v e d i n a known volume of hexane and an a l i q u o t t e s t e d f o r p u r i t y by a n a l y t i c a l HPLC. The sample was then d r i e d and the residue was subjected to m i l d a l k a l i n e h y d r o l y s i s (to recover natural g l y c o l i p i d ) , perbenzoylated i n 10% benzoyl c h l o r i d e i n p y r i d i n e , r e d i s s o l v e d i n hexane and again analyzed by q u a n t i t a t i v e HPLC. The two methods f o r e v a l u a t i o n of f r a c t i o n p u r i t y were i n good agreement. U s u a l l y , i s o l a t e d sample peaks were greater than 98% free of g l y c o l i p i d contaminates and i t was not uncommon to i s o l a t e peaks t h a t contained no other glycolipids. I f necessary, f u r t h e r p u r i f i c a t i o n could be obtained by rerunning each of the i s o l a t e d samples i n an i s o c r a t i c s o l v e n t system with a s o l v e n t composition near the e l u t i n g composition of the g r a d i e n t . Each f r a c t i o n was shown to be free of non-UV absorbing i m p u r i t i e s by m i g r a t i o n of the i s o l a t e d natural g l y c o l i p i d s o n s i l i c a Gel G TLC p l a t e s i n chloroform-methanol water (65:25:4) w i t h d e t e c t i o n by c h a r r i n g w i t h 55% H2SO4 i n water (w/w) To determine the recovery of the neutral g l y c o l i p i d s a crude g l y c o l i p i d was per-O-benzoylated and 0.1% of the sample was i n j e c t e d onto the a n a l y t i c a l chromatograph to determine the amount of each component. The remainder was i n j e c t e d onto the p r e p a r a t i v e column and the t o t a l e f f l u e n t , e x c l u d i n g the s o l v e n t f r o n t was c o l l e c t e d , d r i e d under n i t r o g e n , d i s s o l v e d i n i n i t i a l s o l v e n t and

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

1. MCCLUER AND ULLMAN

Liquid Chromatography

11

0.1% reinjected onto the analytical column. Two different experiments averaged 84, 84.5, 86, and 89% recovery for mono-, di-, t r i - , and tetra- hexosyl ceramides, respectively. After completion of the preparative run, the column was regenerated and reused several times with little loss of efficiency. The use of the rapid sample preparation column in the isolation of the per-O-benzoylated derivatives was necessitated by the large quantity of dark brown impurities in the original lipid preparation, which also eluted from the Unisil column with the crude glycolipid fraction. The use of a Sep-Pak column greatly reduced this discoloration. The isocratic portion of the column elution was utilized to obtain better resolution of the other, early-eluting peaks which were assumed to be monohexosyl-containing sphingolipids. The structures of these components are understudy and presumably are similar to the "a" and "b" components see with th plasm glycolipid i Fig 1 Conclusions Derivatives of glycosphingolipids which have large extinction coefficients can be prepared and separated according to their carbohydrate content by adsorbtion chromatography. This use of modern HPLC equipment allows quantitation of less than 50 pmole quantities of these compounds so that small amounts of body fluids tissue or tissue culture cells can be readily analyzed for the major nuetral glycosphingolipid components. Such components can be further separated into molecular species by reverse phase chromatography. The use of DMAP as a catalyst for derivatization with benzoic acid anhydride allows the convenient preparation of per-0-benzoyl derivatives. Parent glycosphingolipids can be regenerated from these derivatives by treatment with mild alkali. Thus, modern liquid chromatographic techniques with on-line detection can be utilized for the isolation of the neutral glycosphingolipids.

m

Literature Cited 1. Snyder, L.R., and Kirkland, J . J . , "Introduction to Modern Liquid Chromatography"; Wiley-Interscience: New York, N.Y., 1974. 2. McCluer, R.H.; Evans, J.E. Preparation and analysis of benzoylated cerebrosides. J. Lipid Res., 1973, 14, 611. 3. Ullman, M.D., McCluer, R.H. Quantitative analysis of plasma neutral glycosphingolipids by high performance liquid chromatography of their perbenzoyl derivatives. J. Lipid Res., 1977, 18, 371-377. 4. Inch, T.D.; Fletcher, H.C. N-acyl derivatives of 2-acylamino2-deoxy-D-glucopyranose. J. Org. Chem., 1966, 31, 1815. 5. Sugita, M.; Iwamori, M; Evans, J.; McCluer, R.H.; Moser, H.W.; Dulaney, J.T. High-performance liquid chromatography of ceramides: application to analysis in human tissues and demonstration of ceramide excess in Farber's disease. J. Lipid Res., 1974, 15, 223.

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

12 6.

7.

8. 9. 10. 11. 12. 13.

14.

15. 16. 17. 18. 19. 20.

21.

CELL SURFACE GLYCOLIPIDS

Iwamori, M.G.; Moser, H.W. Above normal urinary excretion of urinary ceramides in Farber's disease, and characterization of their components by high performance liquid chromatography. Clin. Chem., 1975, 21, 725. Iwamori, M.; Costello, C.; and Moser, H.W. Analysis and quantitation of free ceramide containing nonhydroxy and 2-hydroxy fatty acids, and phytosphingosine by high-performance liquid chromatography. J. Lipid Res., 1979, 20, 86. Samuelsson, K. Identification and quantitative determination of ceramides in human plasma. Scand. J. Clin. Lab. Invest., 1971, 27, 371. Ullman, M.D.; McCluer, R.H. Quantitative microanalysis of perbenzoylated glycosphingolipids by HPLC with detection at 230 nm. J. Lipid Res., 1978, 19, 910. Vance, D.E.; Sweely, C.C. Quantitative determination of the neutral glycosyl ceramide 1967, 8, 621-630. Fletcher, K.S.; Bremer, E.G.; Schwarting, G.A. P blood group regulation of glycosphingolipid levels in human erythrocytes. J. Biol. Chem., in press. McCluer, R.H.; Gross, S.K.; Sapirstein, V.S.; Meisler, M.H. Testosterone effects of kidney and urinary glycolipids in the light eared mouse mutant. FASEB Proc., 1979, 38, 405. Nanaka, G.; Kishimoto, Y. Simultaneous determination of picomole levels of gluco- and galactocerebroside, monogalactosyl diglyceride and sulfatides by high performance liquid chromatography. Biochim. Biophys. Acta, 1979, 572, 423. Suzuki, A.; Handa, S,; Yamakawa, T. Separation of molecular species of higher glycolipids by high performance liquid chromatography of their O-acetyl-N-p-nitrobenzoyl derivatives. J. Biochem., 1977, 82, 1185. Skipski, V.P. Thin-layer chromatography of neutral glycosphingolipids. Methods in Enzymology, 1975, 35, 396-425. Svennerholm, E.; Svennerholm, L. Isolation of blood serum glycolipids. Acta Chem. Scand., 1962, 16(5), 1282-1284. Gray, G.M. A comparison of the glycolipids found in different strains of Ascites tumour cells in mice. Nature, 1965, 207(4996), 505-507. Siddiqui, B.; McCluer, R.H. Lipid components of sialosylgalactosylceramide of human brain. J. Lipid Res., 1968, 9(3), 366-370. Puro, K. Isolation of bovine kidney gangliosides. Acta Chem. Scan., 1970, 24(1), 13-22. Siddiqui, B.; Hakomori, S. A ceramide tetrasaccharide of human erythrocyte membrane reacting of the anti-type. IV. pneumococcal polysaccharide antiserum. Biochim. Biophys. Acta., 1973, 330, 147-155. Yamakawa, I.; Irie, R.; Iwanaga, M. The chemistry of post-hemolytic residue of stroma of erythrocytes. IV. silicic acid chromatography of mammalian stroma glycolipids. J. Biochem. 1960, 48, 490-507.

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

1.

22. 23.

24.

25. 26.

MCCLUER AND ULLMAN

Liquid Chromatography

Radin, N.S. Florisil chromatography. Methods Enzymol., 1969, 14, 268-272. Rouser, G.; Bauman, A.J.; Kritchevsky, G.; Heller, D.; O'Brien, J. Quantitative chromatographic fractionation of complex lipid mixtures: brain lipids. J. Amer. Oil. Chem. Soc., 1961, 38, 544-555. Rouser, G.; Kritchevsky, G.; Heller, D.; Lieber, E. Lipid composition of beef brain, beef liver, and the sea anemone; two approaches to quantitative fractionation of complex lipid mixtures. J. Amer. Oil. Chem. Soc., 1963, 40, 425-554. Saito, T.; Hakomori, S. Quantitative isolation of total glycosphingolipids from animal cells. J. Lipid Res.,1971, 12(2) 257-259. Ando, S.; Isobe, M.; Nagai, M. High performance preparative column chromatograph Iatrobeads . Biochim Yamakawa, T.; Yokoyama, S.; Kiso, N. Structure of main globoside of human erythrocytes. J. Biochem., 1962, 52, 228-231. Svennerholm, E.; Svennerholm, L. The separation of neutral blood-serum glycolipids by thin-layer chromatography. Biochim. Biophys. Acta., 1963, 70, 432-441. Martensson, E. Neutral glycolipids of human kidney. Isolation, identification, and fatty acid composition. Biochim. Biophys. Acta, 1966, 116, 296-308. Viswanathan, C.V.; Hayashi, A. Ascending dry-column chromatography as an aid in the preparative isolation of glycolipids. J. Chromat., 1976, 123, 243-246. Ullman, M.D.; McCluer, R.H. Isolation and quantitative analysis of neutral glycosylceramides by high performance liquid chromatography (HPLC). FASEB Proceedings, 1976. Tjaden, U.R.; Krol, J.H.; Van Hoeven, R.P.; Oomer-Meulemans, E.P.M.; Emmelot, P. High pressure liquid chromatography of glycosphingolipids (with special reference to gangliosides) J. Chromat.,1977, 136, 233-243. Folch, J . ; Lees, M.; Sloane-Stanley, G.H. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem.,1957, 266, 497-509. (R)

27. 28. 29. 30. 31. 32.

33.

13

RECEIVED

December 10, 1979.

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

2 High Performance Liquid Chromatography of Membrane Glycolipids Assessment of Cerebrosides on the Surface of Myelin SHOJI YAHARA and YASUO KISHIMOTO The John F. Kennedy Institute and Department of Neurology, Johns Hopkins University School of Medicine, Baltimore,MD20205 JOSEPH PODUSLO Neuroimmunology Branch, National Institutes of Health, Bethesda, MD 20205 1

Carbohydrates occu in the form of glycoprotein the outer surface of the cell or membrane may have important biological functions as adhesion sites in terms of cell recognition, as receptors for hormones, toxins, or viruses, or as specific immunological determinants or antibody receptors. The presence of galactose or galactosamine as a terminal carbohydrate in these membrane surface glycolipids or glycoproteins has been determined by a procedure which utilizes the reaction with galactose oxidase (1). The enzyme converts the terminal primary alcohol group of these carbohydrates to an aldehyde. This aldehyde group can then be reduced by NaBH to the original alcoholic group in glycoproteins or glycolipids. By this series of reactions, part of the membrane galactolipids or galactoproteins are labeled with tritium (Chart 1). Since galactose oxidase is not permeable to membrane, the identification of H-labeled galactolipids or galactoproteins in extracts from cells or membranes has been considered acceptable evidence for locating these compounds on the surface of cells or membranes. This procedure has been useful for identifying a variety of carbohydrate-bearing macromolecules (in particular glycoproteins) on the surface of cell membranes (2, 3, 4). There are, however, two major disadvantages to studying glycolipids in this manner. First, many lipids other than galactolipids are also labeled by this procedure. The exact nature of the labeling has not been elucidated, but, at least some double bonds are reduced with tritium and some ester linkage is cleaved yielding radioactive saturated lipid and radioactive alkyl alcohols, respectively. The exchange of hydrogen with tritium may also be occurring. Pretreatment of cells or membranes with nonradioactive NaBH prior to galactose oxidase treatment helps to circumvent this problem to some extent but cannot make this procedure free from this complication. High levels of such non-specific reduction were observed Current address: Department of Neurology, Mayo Clinic, Mayo Foundation, Rochester, Minnesota 55901 3

4

3

4

1

0-8412-0556-6/80/ 47-128-015S5.00/ 0 © 1980 American Chemical Society In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

C E L L SURFACE GLYCOLIPIDS

16

when the i n t a c t myelin sheath p r e p a r a t i o n was t r e a t e d w i t h N a B ^ i n the absence of galactose oxidase (5j). A major r a d i o a c t i v e peak was observed near cerebroside a f t e r s e p a r a t i o n of the lower phase l i p i d s by TLC. Further e v a l u a t i o n of t h i s m a t e r i a l by hyd r o l y s i s showed no r a d i o a c t i v i t y i n g a l a c t o s e , f a t t y a c i d s or s p h i n g o s i n e . In a d d i t i o n , by v a r y i n g the s o l v e n t system, t h i s r a d i o a c t i v e peak could be separated from the c e r e b r o s i d e s . Cons e q u e n t l y , such n o n - s p e c i f i c r e d u c t i o n can e a s i l y r e s u l t i n e r r o neous i n t e r p r e t a t i o n of surface membrane c o n s t i t u e n t s . A second disadvantage of t h i s procedure i s t h a t i t does not have q u a n t i t a t i v e c a p a b i l i t i e s f o r determining surface g l y c o lipids. I t merely demonstrates whether a p o r t i o n o f a given g l y c o l i p i d i s on the surface but not the r a t i o o f surface l i p i d to i n a c c e s s i b l e l i p i d . C u s t o m a r i l y , a l a r g e amount of r a d i o a c t i v i t y i s used f o r such l a b e l i n g but o n l y a very small f r a c t i o n of i t i s incorporated i n t o the l i p i d . T h i s was p a r t i c u l a r l y the case f o r the nonhydroxycerebrosid beled g a l a c t o s e observe l i p i d was o n l y a minor percentage o f the t o t a l l a b e l (5). Interp r e t a t i o n of such low l e v e l s of r a d i o a c t i v i t y may be u n r e l i a b l e f o r a s s e s s i n g surface membrane g l y c o l i p i d s , s i n c e i t may, i n f a c t , represent damage to the membrane b i l a y e r or s p l i t t i n g of the l a m e l l a r . We have r e c e n t l y developed a s e n s i t i v e and s p e c i f i c method f o r the q u a n t i t a t i v e and q u a l i t a t i v e determination of c e r e b r o s i d e s and s u l f a t i d e s using high performance l i q u i d chromatography (16, ]_). In order to q u a n t i t a t e cerebrosides on a s u r f a c e , we developed an a d d i t i o n a l new method t h a t s e p a r a t e l y compares the amount of surface cerebrosides w i t h the remaining cerebrosides by using high performance l i q u i d chromatography. T h i s method a l s o uses galactose o x i d a s e , but i n s t e a d o f reducing the aldehyde formed by N a B r U , the aldehyde i s converted to 2 , 4 - d i n i t r o p h e n y l hydrazone followed by p e r b e n z o y l a t i o n . The product produces separate peaks from t h a t of perbenzoylated c e r e b r o s i d e . Thus, the r a t i o of o x i d i z e d and unoxidized cerebrosides can be d i r e c t l y compared by high performance l i q u i d chromatography. In t h i s manuscript, we w i l l f i r s t d e s c r i b e the newly d e v e l oped high performance l i q u i d chromatography of c e r e b r o s i d e , s u l f a t i d e , and o t h e r minor g a l a c t o l i p i d s . T h i s method a l l o w s complete a n a l y s i s o f a very small amount of these g l y c o l i p i d s i n c e l l or membrane p r e p a r a t i o n s . T h i s w i l l be followed by a des c r i p t i o n of our new method of determining surface g a l a c t o l i p i d s and i t s a p p l i c a t i o n to myelin c e r e b r o s i d e s . 3

3

Procedures M a t e r i a l s . Galactose oxidase was Biochemicals ( F r e e h o l d , N J ) . 1 was (Arlington Heights, I L ) . A l l solvents products of B u r d i c k - J a c k s o n (Muskegon, 125

purchased from Worthington obtained from Amersham ( g l a s s - d i s t i l l e d ) were the M I ) . P y r i d i n e was stored

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

2.

YAHARA ET A L .

Chromatography

of Membrane

Glycolipids

17

over KOH p e l l e t s and used without f u r t h e r p u r i f i c a t i o n . Benzoyl c h l o r i d e was obtained from A l d r i c h Chemicals (Milwaukee, WI) and 70% HClOu (double d i s t i l l e d from Vycor) from 6. F r e d e r i c k Smith Chemical (Columbus, OH). T r y p s i n was obtained from Worthington, w h i l e the t r y p s i n i n h i b i t o r (turkey egg w h i t e ) , phospholipase C ( C I . W e l c h i i type I ) , c a t a l a s e and phosphatidyl c h o l i n e (egg yolk) were a l l obtained from Sigma Chemical Co. ( S t . L o u i s , MO). T h i n l a y e r chromatographic p l a t e s precoated w i t h 0.25 mm t h i c k S i l i c a Gel GF were purchased from Anal tech (Newark, DE). M y e l i n was prepared e i t h e r from b r a i n s o f young Sprague-Dawley r a t s (Charles R i v e r CD, Charles R i v e r B r e e d i n g , Wilmington, MA) or from s p i n a l cords of a d u l t Osborn-Mendel r a t s ( V e t e r i n a r y Resources Branch at the National I n s t i t u t e s of Health) according to Norton and Poduslo ( 8 ) . Standard 6-dehydrocerebrosides were prepared by o x i d i z i n g with g a l a c t o s e oxidase according to Radin (9). Instrumentation. Th 740 s o l v e n t d e l i v e r y system programmer, Model 714 pressure monitor and a Model 755 sample i n j e c t o r ( a l l from S p e c t r a - P h y s i c s , Santa C l a r a , CA). The column used was 25 cm x 3 mm i . d . s t a i n l e s s s t e e l tube packed w i t h either Spherisorb s i l i c a 5 y or Spherisorb ODS 5 y . Detection was made with a Schoeffel Instrument Corporation (Westwood, NJ) Model SF 770 spectromonitor. Peak areas were measured by the cut and weight method. R a d i o a c t i v i t y was measured by d i r e c t measurement i n a S e a r l e Model 1185 Automatic Gamma System. Determination o f c e r e b r o s i d e s , s u l f a t i d e s and o t h e r g a l a c t o 1 i p i d s i n myelin by HPLC. T o t a l l i p i d s were e x t r a c t e d w i t h chloroform/methanol ( 2 / 1 ) , washed according to Folch et al_. (10), and then subjected to b e n z o y l a t i o n - d e s u l f a t i o n as described p r e viously (6). The t o t a l l i p i d s were heated with 20 y l benzoyl c h l o r i d e and 100 y l p y r i d i n e and d e s u l f a t e d w i t h 0.2 M HCIO^ i n a c e t o n i t r i l e (prepared by mixing 0.17 ml 70% HCIO^ and 10 ml a c e t o n i t r i l e ) . With t h i s procedure, cerebrosides were converted to perbenzoyl d e r i v a t i v e s w h i l e s u l f a t i d e s were converted to p a r t i a l l y benzoylated cerebroside i n which the hydroxyl group a t g a l a c t o s e - 3 i s f r e e (Chart 2 ) . A p o r t i o n of the r e a c t i o n mixture d i s s o l v e d i n a known volume o f hexane was i n j e c t e d i n t o the HPLC equipped with Spherisorb s i l i c a 5 y column. The column was e l u t e d w i t h hexane/isopropanol ( 9 9 . 5 / 0 . 5 , v / v ) i s o c r a t i c a l l y f o r the f i r s t 3 min followed by g r a d i e n t e l u t i o n from 0.5 to 10% i s o p r o panol i n hexane i n 20 m i n . The flow r a t e was maintained a t 1.2 ml/min throughout. Peaks o f g l u c o c e r e b r o s i d e , nonhydroxycerebros i d e , hydroxycerebroside, monogalactosyl d i g l y c e r i d e , nonhydroxys u l f a t i d e , and h y d r o x y s u l f a t i d e were separated from each other under these c o n d i t i o n s , and c o n c e n t r a t i o n s o f these l i p i d s were determined from the peak s i z e . Peaks f o r minor nonpolar g a l a c t o 1 i p i d s , namely cerebroside e s t e r s and 1 - 0 - a l k y l isomers o f monogalactosyl d i g l y c e r i d e s overlap w i t h one of the above peaks.

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

18

C E L L SURFACE GLYCOLIPIDS

Products of Benzoylation-Desulfation CH2OBZ

H

Bz

0-CH2-C-N-CO-CH2-R' iBz

H

CH2OBZ H

C

H H

n

R

,

B

BzO-?^ H

z

CH2OBZ

NS

HS

H

R

Bz

BzQ/~C\p-CH2-C -N-CO-CH2 BzO-C^NR )Bz H CH2OBZ H H OBz BZO^°NX)-CH2-C-N-CO-CH-R'

OBz

BzO-C^

^

R

H

CH2OBZ

GC

OBz

Bz0y-0xJ)-CH2-C -N-C0-CH-R'

H

?z

^-CH -C-N-C0-CH2 2

B z O > ^ BzO-CV N ^ R OBz H CH2OBZ H GD Bz(^g^-CH2-C-0-C0-R' OBz

CH2-O-CO-R'

Bz= Benzoyl.

R

=

C 1 3 H 2 7

Rralkyl-

Chart 2

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

2.

YAHARA ET A L .

Chromatography

of Membrane

Glycolipids

19

E l u t i n g the column i s o c r a t i c a l l y w i t h hexane/tetrahydrofuran ( 9 0 . 2 5 / 9 . 7 5 , v / v ) provides s e p a r a t i o n of the above overlapped peaks (11). Determination o f homolog compositions of c e r e b r o s i d e s , s u l f a t i d e s , monogalactosyl d i g l y c e r i d e , and i t s 1 - 0 - a l k y l ether isomer by reverse phase HPLC. The above b e n z o y l a t i o n - d e s u l f a t i o n product i s placed on a TLC p l a t e coated with S i l i c a Gel GF. At l e a s t 1 nmol (approximately 1 yg) i s r e q u i r e d to o b t a i n s a t i s f a c t o r y r e s u l t s f o r each i n d i v i d u a l l i p i d . The p l a t e i s developed w i t h hexane/isopropanol ( 9 8 / 2 , v / v ) once or twice depending on the r e l a t i v e a c t i v i t y of the p l a t e . A f t e r the f i r s t development, the p l a t e was examined under UV l i g h t . I f each component i s s u f f i c i e n t l y separated, as shown i n F i g . 1, a second run i s not necessary. The spots were marked under the UV l i g h t a l l o w i n g 1/2 height of the spot on the top and bottom of each spot (or band) so t h a t any p a r t i c u l a r powder from the spot was scraped and mixed w i t h 0.5 ml of 95% e t h a n o l . The mixture was sonicated i n a sonic cleaner bath f o r 2 m i n . 1.5 ml Of ether was added to the mixture and then shaken v i g o r o u s l y f o r 30 min w i t h a W-8 Twist A c t i o n Shaker (New Brunswick Instrument, New Brunswick, N J ) . The mixture i s then c e n t r i f u g e d , and the supernatant i s evaporated to dryness. The residue i s d i s s o l v e d i n a known volume of cyclohexane, and a p o r t i o n i s i n j e c t e d to HPLC equipped w i t h Spherisorb ODS 5 y column. Although spots of perbenzoylated nonhydroxycerebroside, monogalactosyl d i g l y c e r i d e , and hydroxycerebroside, and p e r benzoyl ated-desul f a t e d nonhydroxy- and h y d r o x y s u l f a t i d e are w e l l separated from each o t h e r , the spot o f benzoylated d e r i v a t i v e of 1 - 0 - a l k y l etherisomer o f monogalactosyl d i g l y c e r i d e overlaps w i t h t h a t of benzoylated nonhydroxycerebroside. The amount of the 1 - a l k y l , 2 - a c y l , 3 - m o n o g a l a c t o s y l g l y c e r o l i s normally so small t h a t i t w i l l not i n t e r f e r e s i g n i f i c a n t l y with the a n a l y s i s of nonhydroxycerebroside. However, i f the a n a l y s i s of t h i s minor g l y c o l i p i d i s d e s i r e d , the m a t e r i a l e l u t e d from the band o f benzoylated nonhydroxycerebroside can be rechromatographed on another TLC system, such as the use of hexane/tetrahydrofuran on S i l i c a Gel GF p l a t e . These two benzoylated l i p i d s separate w e l l from each other under t h i s c o n d i t i o n . I f the examination of homolog composition of monogalactosyl d i g l y c e r i d e and i t s 1 - 0 - a l k y l ether isomers i s d e s i r e d , a l a r g e r amount of b r a i n sample i s r e q u i r e d , s i n c e t h e i r c o n c e n t r a t i o n i s much s m a l l e r than cerebrosides and s u l f a t i d e s . HPLC of g a l a c t o l i p i d s from a membrane t r e a t e d w i t h galactose o x i d a s e . The membrane t r e a t e d w i t h galactose oxidase i s extracted with chloroform/methanol as described above. The e x t r a c t cont a i n i n g up to 1 mg of t o t a l l i p i d s i s shaken w i t h a s o l u t i o n of 2 mg of d i n i t r o p h e n y l h y d r a z i n e HC1 i n 100 y l p y r i d i n e f o r 2 h at room temperature. The s o l v e n t i s evaporated to dryness under a

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

20

C E L L SURFACE GLYCOLIPIDS

n i t r o g e n f l o w , and the r e s i d u e i s f u r t h e r d r i e d i n an evacuated desiccator f o r 1 h r . To the d r i e d r e s i d u e , 30 y l of benzoyl c h l o r i d e and 150 y l of dry p y r i d i n e i s added, and the mixture i s heated at 60° f o r 1 h . The r e a c t i o n mixture i s evaporated to d r y ness under a n i t r o g e n f l o w , and the r e s i d u e i s f u r t h e r d r i e d i n an evacuated desiccator f o r 30 min. The residue i s d i s s o l v e d i n 2 ml hexane. The hexane s o l u t i o n i s washed once w i t h 1 ml o f 3% aqueous sodium carbonate followed twice by a c e t o n i t r i l e / w a t e r ( 4 / 1 , v / v ) and then evaporated to dryness. The residue i s d i s s o l v e d i n a known volume o f hexane and i n j e c t e d i n t o the HPLC system equipped with Spherisorb S i l i c a 5 y column. The column was f i r s t e l u t e d i s o c r a t i c a l l y f o r the f i r s t 5 min w i t h hexane/isopropanol ( 9 9 . 5 / 0 . 5 , v / v ) and then by i n c r e a s i n g l i n e a r l y the p r o p o r t i o n of isopropanol i n the next 20 min reaching the f i n a l c o n c e n t r a t i o n of hexane/isopropanol ( 9 6 / 4 , v / v ) . The flow r a t e was maintained a t 1.2 ml/min throughout. Two peaks due to perbenzoylate of o x i d a t i o n products from nonhydroxy- and hydroxycerebrosides appear a f t e r the peak of benzoylated hydroxycerebrosides under these c o n d i t i o n s . Treatment w i t h galactose o x i d a s e . O x i d a t i o n of m y e l i n with galactose oxidase was performed as described p r e v i o u s l y f o r s i m i l a r o x i d a t i o n of r a t s p i n a l cord preparations ( 4 ) . T y p i c a l l y , myelin c o n t a i n i n g 0.2-1.1 mg p r o t e i n i s incubated w i t h 100-500 u n i t s of galactose oxidase i n 1-3 ml of phosphate buffer (10-100 mM, pH 7 . 2 - 7 . 4 ) w i t h o r without c a t a l a s e . A f t e r the i n c u b a t i o n at room temperature to 30°C f o r the d u r a t i o n of 30 min to o v e r n i g h t , myelin i s recovered by c e n t r i f u g a t i o n , washed, and l y o p h i l i z e d . Total l i p i d s were e x t r a c t e d from the d r i e d r e s i d u e and the o x i d i z e d cerebroside as well as unaltered cerebrosides were analyzed as described above. A l t e r n a t i v e l y , the i n c u b a t i o n was stopped by the a d d i t i o n of 5 volumes of chloroform/methanol ( 2 / 1 , v / v ) and mixed. The lower l a y e r a f t e r c e n t r i f u g a t i o n of the mixture i s washed and then evaporated to d r y n e s s , and the t o t a l l i p i d s obt a i n e d were analyzed as described above. R a d i o i o d i n a t i o n of galactose o x i d a s e . The chloramine T procedure (12) was used f o r the r a d i o i o d i n a t i o n of galactose o x i d a s e . The enzyme, s o l u b i l i z e d i n 0.01 M sodium phosphate b u f f e r , pH 7 . 4 , was l a b e l e d using 1 mCi Na I (13-17 m C i / n g l ) , 0.42 mM chloramine T (Eastman), and 1.14 mM sodium m e t a b i s u l f i t e ( B a k e r ) . Unreacted i o d i d e was separated from the i o d i n a t i o n enzyme by d i a l y s i s , and the enzyme was d i l u t e d i n 0.1 M sodium phosphate b u f f e r , pH 7 . 4 , c o n t a i n i n g 0.001 M c u p r i c s u l f a t e . 1 2 5

P r e p a r a t i o n of liposomes. The method f o r the liposome prepa r a t i o n was a m o d i f i c a t i o n of C o s t a n t i n o - C e c c a r i n i , et a l ^ . , (13). A mixture o f 0.3 mg nonhydroxycerebroside, 0.2 mg of hydroxyc e r e b r o s i d e , and 5 mg o f egg y o l k l e c i t h i n was swollen i n 1 ml of

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

2.

YAHARA ET A L .

Chromatography

of Membrane

Glycolipids

21

a s o l u t i o n c o n t a i n i n g 130 mM KC1 and 10 mM T r i s - H C l pH 7.4 f o r 30 min. The tube was f l a s h e d w i t h n i t r o g e n and sonicated f o r 30 min. The sonicated mixture was c e n t r i f u g e d at 50,000 x£ f o r 15 min to remove s o l i d c e r e b r o s i d e s . The liposome s o l u t i o n obtained cont a i n e d 165 yg of nonhydroxycerebroside and 110 yg o f hydroxycerebroside i n 1 ml when measured by high performance l i q u i d chromatography. Results Myelin g a l a c t o l i p i d a n a l y s i s by HPLC. F i g . 2 and 3 show HPLC chromatograms obtained from myelin l i p i d s on S i l i c a column and reverse phase column, r e s p e c t i v e l y . Reverse phase HPLC of monog a l a c t o s y l d i g l y c e r i d e and i t s 1 - 0 - a l k y l ether homolog was not examined but t y p i c a l chromatograms of these l i p i d s obtained from c a l f b r a i n stem were presented p r e v i o u s l y (1J_). Myelin was obt a i n e d from 25 d a y - o l d r a O x i d a t i o n o f myelin surface cerebrosides by g a l a c t o s e oxidase. F i g . 4 shows s i l i c a HPLC of a mixture c o n t a i n i n g benzoylated-nonhydroxy and hydroxycerebroside and benzoylated d e r i v a t i v e s of 2 , 4 dinitrophenylhydrazone of o x i d a t i o n products from nonhydroxy- and hydroxycerebroside. Standard curves o f two 6 - d e h y d r o - d e r i v a t i v e s were shown i n F i g . 5. These standard curves demonstrate t h a t the response of the benzoylated dinitrophenylhydrazones are l i n e a r between 0.025 nmol and 0.6 nmol. Since cerebrosides c o n t a i n i n g 5 nmol can be determined without t a i l i n g to these peaks, t h i s method should a l l o w the determination of as l i t t l e as 0.5% of the o x i d a t i o n product. The f a c t t h a t each curve i n t e r s e c t s 0 p o i n t i n both the a b s c i s s a and o r d i n a t e i n d i c a t e s t h a t even s m a l l e r amounts of these compounds can be detected by t h i s technique. We obtained unexpected f i n d i n g s using t h i s method to study m y e l i n : the o x i d a t i o n by galactose oxidase of myelin-bound cerebrosides could not be d e t e c t e d . The o x i d a t i o n d i d not occur e i t h e r w i t h the i n t a c t s p i n a l cord p r e p a r a t i o n , w i t h i s o l a t e d m y e l i n , or even w i t h l y o p h i l i z e d m y e l i n . In one experiment, l y o p h i l i z e d myelin c o n t a i n i n g 5 mg each of dry weight was i n c u bated w i t h 100, 200, and 500 u n i t s of g a l a c t o s e oxidase f o r 60 min a t room temperature, and no cerebroside o x i d a t i o n o c c u r r e d . To examine whether the enzyme i s a c t i v e under the same c o n d i t i o n s , we coated 0.1 mg each o f nonhydroxy- and hydroxycerebrosides on 10 mg C e l i t e ( A n a l y t i c a l grade) and incubated i t w i t h 100 u n i t s of g a l a c t o s e oxidase f o r 60 min at room temperature. The r e s u l t i n d i c a t e d t h a t 5.6 nmol and 3.5 nmol each o f nonhydroxy and hydroxycerebrosides (approximately 4.6 and 3.0% each were o x i d i z e d . O x i d a t i o n of the same cerebrosides by the same galactose oxidase i n a t e t r a h y d r o f u r a n / w a t e r mixture as described by Radin {9) r e s u l t e d i n n e a r l y complete o x i d a t i o n . To f u r t h e r examine the i n a b i l i t y of galactose oxidase i n o x i d i z i n g myelin-bound c e r e b r o s i d e s , one mg each of l y o p h i l i z e d

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

CELL SURFACE GLYCOLIPIDS

22

Figure 1. TLC of myelin lipids after treatment with perbenzoylation-desulfation. Line S, standard; line M, myelin lipids. Spots 1 through zoylated-desulfated derivatives of: (1) glucocerebroside, (2) nonhydroxycerebroside, (3) mono galactosyl diglyceride, (4) hydroxycerebroside, (5) nonhydroxysulfatide, and (6) hydroxysulfatide, respectively. See text for details of TLC conditions.

Figure 2. Silica HPLC of myelin lipids. NC, nonhydroxycerebroside; HC, hydroxycerebroside; NS, nonhydroxy sulfatide; HS, hydroxy sulfatide; and GD, monogalactosyl diglyceride. See text for details of TLC conditions.

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

2.

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Chromatography

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c

Figure 3. Reverse-phase HPLC of (A) perbenzoylated nonhydroxycerebroside, (B) hydroxycerebroside, (C) perbenzoylated-desulfated nonhydroxy sulfatide, and (D) hydroxy sulfatide. Each homolog peak was identified by fatty acid symbols, carbon numbers followed by number of double bonds.

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

23

C E L L SURFACE GLYCOLIPIDS

0 Figure 4.

10

5

15

min

Silica HPLC of perbenzoylated derivative of dinitrophenylhydrazone 6-dehydrocerebrosides.

of

50 fig of nonhydroxycerebroside and 30 fig hydroxycerebroside is mixed with equal amounts of 6-dehydroderivatives of hydroxy- and nonhydroxycerebroside. These mixtures were subjected to dinitrophenylhydrazone-benzoylation as described above, and 1/20 of each reaction mixture was injected into silica-HPLC. NC, nonhydroxycerebroside; HC, hydroxycerebroside; NA, 6-dehydrononhydroxycerebroside; HA, 6-hydrohydroxycerebroside.

t

1

1

1

r

n mol Figure 5. Standard curve of perbenzoylated derivative of dinitrophenylhydrazone of 6-dehydrocerebrosides as analyzed by silica HPLC. Open and closed circles: derivatives from nonhydroxycerebroside and hydroxycerebroside, respectively.

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

2.

YAHARA ET A L .

Chromatography

of Membrane

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25

myelin was wetted w i t h 0.25 ml o f benzene o r benzene c o n t a i n i n g 167 nmols and 397 nmols each o f nonhydroxy and hydroxycerebrosides, r e s p e c t i v e l y . These were again l y o p h i l i z e d . Each d r i e d r e s i d u e was incubated with 150 u n i t s of g a l a c t o s e oxidase i n room temperature o v e r n i g h t . The r e s u l t s i n d i c a t e d t h a t the r e a c t i o n p r o d uct from the l y o p h i l i z e d myelin which was r e l y o p h i l i z e d w i t h benzene alone showed no d e t e c t a b l e o x i d a t i o n . On the other hand, the product of the same myelin p r e p a r a t i o n but "coated" with cerebrosides showed 12.5 nmol and 8.8 nmol o f nonhydroxy- and hydroxycerebroside o x i d i z e d by the enzyme r e a c t i o n , as shown i n F i g . 6 and F i g . 7. One p o s s i b l e e x p l a n a t i o n f o r the l a c k of o x i d a t i o n by g a l a c tose oxidase was thought to be s t e r i c hindrance. To i n v e s t i g a t e t h i s p o s s i b i l i t y , l y o p h i l i z e d m y e l i n c o n t a i n i n g 5.45 mg p r o t e i n i n 5 ml 0.1 M phosphate b u f f e r , pH 7 . 4 , was mixed with 0.5 ml of the same b u f f e r s o l u t i o n c o n t a i n i n g 1400 u n i t s of t r y p s i n and incubated f o r 1 h a t 37°C the same b u f f e r was adde 30 min at the same temperature. Galactose oxidase (942 u n i t s ) i n 1 ml of the same b u f f e r was next added to the above m i x t u r e , and the i n c u b a t i o n continued f o r 1 h at room temperature. This experiment, however, gave no evidence t h a t o x i d a t i o n by galactose o x i dase o c c u r r e d . In another experiment, 3.3 mg of l y o p h i l i z e d mye l i n was incubated w i t h 3.3 mg o f phospholipase C (6 units/mg) i n 10 ml of b u f f e r c o n t a i n i n g 10 mM T r i s - H C l pH 7 . 4 , 1 mM C a C l a t 37°C f o r 2 h (T4). The r e a c t i o n mixture was c e n t r i f u g e d at 44,000 x£ f o r 1 h and the p e l l e t s obtained were rehomogenized i n 1 ml o f 10 mM pH 7.2 phosphate b u f f e r . The homogenate was then incubated w i t h 200 u n i t s o f g a l a c t o s e oxidase at room temperature o v e r n i g h t . The i n c u b a t i o n product d i d not show any d e t e c t a b l e oxidation. The i n a b i l i t y of galactose oxidase to o x i d i z e myelin-bound cerebrosides may a l s o be due to the a b s o r p t i o n of the enzyme by m y e l i n . We examined t h i s p o s s i b i l i t y by l a b e l i n g g a l a c t o s e o x i dase w i t h I . In one experiment, f r e s h l y prepared myelin cont a i n i n g 2.09 mg p r o t e i n was incubated w i t h 3.77 u n i t s of Il a b e l e d g a l a c t o s e oxidase c o n t a i n i n g 226,500 cpm at room temperat u r e f o r v a r i o u s periods o f t i m e , and the mixture was c e n t r i f u g e d a t 16,000 rpm. The r a d i o a c t i v i t y i n the supernatant was counted by a y - c o u n t e r . In another experiment, the same amount o f myelin was incubated under i d e n t i c a l c o n d i t i o n s except t h a t 192.2 u n i t s of g a l a c t o s e oxidase c o n t a i n i n g the same amount o f r a d i o a c t i v i t y was used. The r e s u l t s of these experiments, shown i n Table 1, demonstrate t h a t the galactose oxidase indeed was bound to myelin. The binding appears to be s a t u r a t e d w i t h i n 5 min i n c u b a t i o n . With 3.77 u n i t s of g a l a c t o s e oxidase used, the average o f 1.67 u n i t s (44.3% of added enzyme) was bound to myelin c o n t a i n i n g 2.09 mg p r o t e i n . On the other hand, when 192.2 u n i t s of the enzyme was i n c u b a t e d , an average of 13.5% which i s 25.9 u n i t s , was bound to the same amount of m y e l i n . 2

1 2 5

1

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

2

5

CELL SURFACE GLYCOLIPIDS

26

Figure 6.

Silica HPLC of product from myelin, which was treated with benzene alone and lyophilized. See caption to Figure 4 for peak identification.

Figure 7. Silica HPLC of product from galactose oxidase-treated myelin which were "coated" by cerebrosides. See caption to Figure 4 for peak identification.

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

YAHARA ET A L .

Chromatography

of Membrane

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). About 20% of the t o t a l l i p i d c o n s i s t s of cerebroside and s u l f a t i d e . Because o f the l i p o p h i l i c nature of the ceramide moiety and the h y d r o p h i l i c nature of g a l a c t o s e , i t has been p o s t u l a t e d t h a t the galactose moiety of myelin c e r e b r o s i d e s i s f a c i n g the surface w h i l e the ceramide moiety i s buried w i t h i n the b i l a y e r . Even c o n s i d e r i n g the m u l t i l a m e l l a r s t r u c t u r e o f m y e l i n , a t l e a s t several percent of the cerebrosides should be present on the myelin s u r f a c e . The method described i n t h i s manuscript should a l l o w l i t t l e as 0.5% o f the t o t a l c e r e b r o s i d e s . Unexpectedly, we found t h a t myelin cerebrosides are not o x i d i z a b l e by g a l a c t o s e o x i d a s e , a t l e a s t not i n a d e t e c t a b l e degree. We have attempted to modify the myelin s t r u c t u r e so t h a t g a l a c t o s e oxidase would have a c c e s s i b i l i t y to the c e r e b r o s i d e s . These manipulations included l y o p h i l i z a t i o n , s o n i c a t i o n , hypotonic treatment, t r y p s i n d i g e s t i o n , and phospholipase C d i g e s t i o n . D i s r u p t i o n o f the myelin s t r u c t u r e u s i n g these treatments has been reported (17J. In f a c t , the e f f e c t of phospholipase C was obvious from the examination of l i p i d s by t h i n - l a y e r chromatography; n e a r l y a l l phosphatidyl c h o l i n e and ethanolamine were degraded. This i n a b i l i t y of g a l a c t o s e oxidase to o x i d i z e cerebrosides i s a d i r e c t c o n t r a d i c t i o n to a recent r e p o r t by L i n i n g t o n and Rumsby (18J. In t h e i r s t u d y , cerebrosides which were not o x i d i z e d by g a l a c t o s e oxidase were compared w i t h c h o l e s t e r o l by GLC. The cerebroside determination was made by measuring g a l a c t o s e a f t e r the m e t h a n o l y s i s ; o x i d i z e d cerebroside y i e l d e d 6-dehydrogalactose which was found unstable under methanolysis c o n d i t i o n s . By measuring the l o s s of g a l a c t o s e r e l a t i v e to the c h o l e s t e r o l c o n t e n t , they found t h a t approximately 36-50% of the cerebrosides i n myelin were attacked by g a l a c t o s e o x i d a s e . The reason f o r t h i s discrepancy between our present study and the f i n d i n g of L i n i n g t o n and Rumsby i s not c l e a r . Our enzyme was very a c t i v e . I t o x i d i z e d n e a r l y a l l cerebrosides when reacted i n a t e t r a h y d r o f u r a n / b u f f e r system. When cerebrosides were coated on c e l i t e or m y e l i n , galactose oxidase a t t a c k e d them. Two p o s s i b l e causes f o r the i n a b i l i t y of t h i s enzyme to o x i d i z e c e r e b r o s i d e i n i s o l a t e d myelin were c o n s i d e r e d . The f i r s t cause may be due to the absorption of galactose oxidase by myelin by e i t h e r i o n i c or hydrophobic i n t e r a c t i o n s . I f a p o r t i o n o f galactose oxidase i s hydrophobic, i t i s p o s s i b l e t h a t the enzyme can be incorporated i n t o the l i p i d m a t r i x . A c c o r d i n g l y , we l a b e l e d g a l a c t o s e oxidase with I and found that such a b s o r p t i o n was i n s i g n i f i c a n t 1 2 5

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

2.

Chromatography

YAHARA ET A L .

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Glycolipids

31

compared to the t o t a l amount of enzyme present during the i n c u b a tion. The second cause may be due to s t e r i c hinderance of neighboring components w i t h i n the myelin sheath. However, we found t h a t d i g e s t i o n of t r y p s i n o r phospholipase C cannot a l l e v i a t e the problem. In a d d i t i o n , hypotonic treatment of myelin which a f f e c t s the i n t e g r i t y of the b i l a y e r and a l s o causes the s p l i t t i n g of the l a m e l l a e a t the e x t e r n a l a p p o s i t i o n (19, 2 0 ) , d i d not r e s u l t i n the o x i d a t i o n of the c e r e b r o s i d e s . Even cerebrosides i n liposomes made from pure phosphatidyl c h o l i n e could not be o x i d i z e d . I n c i d e n t a l l y , t h i s f i n d i n g a l s o c o n t r a d i c t s L i n i n g t o n and Rumsby who reported s i g n i f i c a n t o x i d a t i o n of cerebrosides i n liposomes made from myelin l i p i d s . At t h i s t i m e , our o n l y a l t e r n a t i v e e x p l a n a t i o n to our f i n d ings i s t h a t galactose oxidase may not be able to o x i d i z e the "bound" form of cerebrosides p o s s i b l y because of s i z e r e s t r i c t i o n s at the a c t i v e s i t e o f th i n membranes, lyposomes r e c e n t l y determined the p r o p o r t i o n of 1actosylceramide and g l o b o s i d e l o c a t e d i n e r y t h r o c y t e surface membranes (T. Matsubura and S. Hakomori, personal communication). In t h i s s t u d y , they t r e a t e d e r y t h r o c y t e s w i t h galactose o x i d a s e , reduced i t by NaBDi* t r e a t ment, and measured the r a t i o of the deuterated l i p i d a g a i n s t undeuterated l i p i d w i t h mass spectrometry. They found somewhat more o x i d a t i o n ; 2-3% o f 1actosyl ceramide, and approximately 10% of globoside were l a b e l e d w i t h deuterium. T h e r e f o r e , i t i s l i k e l y t h a t the longer the saccharide chain to which galactose or galactosamine i s a t t a c h e d , the higher the r a t e of o x i d a t i o n t h a t can be achieved by galactose o x i d a s e . Although, as described above, L i n i n g t o n and Rumsby reported up to 50% of the o x i d a t i o n of myelin cerebrosides by g a l a c t o s e o x i d a s e , they a l s o reported very l i t t l e l a b e l i n g of myelin cerebrosides by the galactose oxidase — NaBH>4 method. They o x i d i z e d myelin (75 mg of dry w e i g h t ) , which presumably contained approximately 10 mg of cerebrosides i n 50 mg of t o t a l l i p i d s , w i t h 900 u n i t s of galactose oxidase and reduced i t w i t h 5 mCi of NaB Hi+. A f t e r 5 hrs of i n c u b a t i o n , they obtained 2,261,450 dpm (approximately 1 y C i ) of H i n c e r e b r o s i d e s . Although the s p e c i f i c a c t i v i t y of N a B ^ used was not g i v e n , the cerebrosides l a b e l e d could be about 0 . 1 - 0 . 2 y g , assuming the s p e c i f i c a c t i v i t y was i n the range o f 5-15 Ci/mmol as reported by Poduslo et a l _ . , (4j and a l s o assuming t h a t t h i s r a d i o a c t i v i t y represents s p e c i f i c l a b e l ing of the g a l a c t o s e moiety. T h i s amount of c e r e b r o s i d e , t h e r e f o r e , represents o n l y 0.001-0.002% o f the t o t a l c e r e b r o s i d e . A number of s i m i l a r s t u d i e s on c e l l surface g a l a c t o l i p i d s have been based on t h i s g a l a c t o s e oxidase-NaB Hn r e d u c t i o n procedure. However, i t i s now apparent t h a t o n l y a very small p o r t i o n , l e s s than 0.5% i f any, of the cerebrosides i n membranes are o x i d i z a b l e by galactose o x i d a s e . T h e r e f o r e , cerebrosides and p o s s i b l y o t h e r g a l a c t o l i p i d s p r e v i o u s l y i d e n t i f i e d by the surface l a b e l i n g 3

3

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CELL SURFACE GLYCOLIPIDS

technique apparently represent only a small portion of the total surface galactolipids, and the results of such studies should be interpreted with caution. Abstract An HPLC method is described which determines the quantity and elucidates the homolog composition of cerebrosides and sulfatides in small tissue samples. Total lipids from the tissue were subjected to benzoylation-desulfation, and the product was analyzed quantitatively by silica HPLC. Another aliquot of the product was further fractionated by TLC. Spots of benzoylated cerebrosides and desulfated sulfatides were analyzed by reverse phase HPLC for the homolog compositions of these sphingolipids. Less than 1 mg of fresh brain or nerve tissue is sufficient for complete analysis. A new procedure has been developed which assesses the topographical distribution of cerebroside method involves the treatmen galactose oxidase followed by extraction of the total lipids with chloroform-methanol. The lipids were then reacted with 2,4dinitrophenylhydrazine HCl in pyridine, and the reaction products were benzoylated and analyzed by silica HPLC. The cerebrosides which are oxidized by the enzyme resulted in perbenzoylated derivatives of 6-dehydrocerebrosides which yielded separate peaks behind the unoxidized perbenzoylated cerebrosides. Consequently this procedure would distinguish surface membrane cerebrosides from the unreactive "inaccessible" cerebrosides. This technique was applied to myelin from the central nervous system, and unexpectedly, myelin cerebrosides were found unoxidizable by galactose oxidase. Modifications of myelin, such as lyophilization, hypotonic treatment, trypsin digestion, and phospholipase C digestion, were not effective in exposing myelin-bound cerebrosides. Moreover, we also found that cerebrosides bound to brain microsomes, cytosol, or even in liposomes with lecithin were not oxidized by the enzyme. On the other hand, cerebrosides coated on Celite or myelin were oxidized by the enzyme. These results suggest that cerebrosides bound in a bilayer structure may not be available for oxidation by galactose oxidase. Acknowledgement This study was supported in part by Research Grants NS-13559, NS-13569 and HD-10891 from the National Institutes of Health, United States Public Health Service. Literature Cited 1.

Steck, T.L., "Membrane Research"; Academic Press, New York, 1972; pp. 71-93.

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

2. YAHARA ET AL. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

Chromatography of Membrane Glycolipids 33

Steck, T.L. and Dawson, G. J. Biol. Chem. 1974, 249, 21352142. Gahmberg, C.G. and Hakomori, S. J. Biol. Chem. 1973, 248, 4311-4317. Poduslo, J.F.; Quarles, R.H. and Brady, R.O. J. Biol. Chem. 1976, 251, 153-158. Poduslo, J.F. Adv. Exp. Med. Biol. 1978, 100, 189-205. Nonaka, G. and Kishimoto, Y. Biochim. Biophys. Acta 1979, 572, 423-431. Yahara, S.; Moser, H.W.; Kolodny, E.H. and Kishimoto, Y. J. Neurochem. in press. Norton, W.T. and Poduslo, S.E. J. Neurochem. 1973, 21, 749757. Radin, N.S. Methods Enzymol. 1972, 28, 300-304. Folch, J.; Lees, M. and Sloane-Stanley, G.H. J. Biol. Chem. 1957, 226, 497-509 Yahara, S. and Kishimoto Greenwood, F.C.; Hunter, , 1963, 89, 114-123. Cestelli, A.; White, F.V. and Costantino-Ceccarini, E. Biochim. Biophys. Acta 1979, 572, 283-292. Feinstein, M.B. and Felsenfeld, H. Biochemistry 1975, 14, 3041-3048. Kawamura, N.; Yahara, S.; Kishimoto, Y. and Toutelotte, W.W. manuscript in preparation. Norton, W.T. and Poduslo, S.E. J. Neurochem. 1973, 21, 759773. Smith, M.E. and Benjamins, J.A. "Myelin"; Plenum Press, New York, 1977, pp. 447-488. Linington, C. and Rumsby, M.G. Adv. Exp. Med. Biol. 1978, 100, 263-273. Finean, J.B. and Bunge, R.E. J. Mol. Biol. 1963, 1, 672-682. McIntosh, T.J. and Robertson, J.D. J. Mol. Biol. 1976, 100, 213-217.

RECEIVED

December 10, 1979.

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

3 Analysis of Glycosphingolipids by Field Desorption Mass Spectrometry 2

C. E. COSTELLO, B. W. WILSON, and K. BIEMANN Massachusetts Institute of Technology, Cambridge, MA V. N. REINHOLD Harvard Medical School, Boston, MA 02115 Conventional electro spectrometry requires tha this is clearly a limiting factor in the analysis of many bio­ chemically significant compounds. A newer ionization technique, field desorption (FD) (1,2) removes this requirement and makes it possible to obtain mass spectrometric information on thermally unstable or non-volatile organic compounds such as glycoconjugates and salts. This development is particularly significant for those concerned with the analysis of glycolipids and we have therefore explored the suitability of field desorption mass spectrometry (FDMS) for this class of compounds. We have evaluated experimen­ tal procedures in order to enhance the efficiency of the ioniza­ tion process and to maximize the information content of spectra obtained by this technique. In FDMS, the desorption surface is a 10 μwire covered by a dense growth of microneedles (Figure 1) produced by slowly heating the wire in a high electric field and an atmosphere of benzoni­ trile (3). The microneedles possess much three-dimensional detail and terminate in many fine tips (Figures 2,3). The material to be analyzed is applied either by dipping the emitter into a solu­ tion of the sample or by transferring a few microliters of the solution directly to the wire by means of a syringe (4). The sample-laden emitter (Figure 4) is placed directly in the ion source (Figure 5) and an electric field of about 10 kV is applied. Under these conditions field strengths approaching 10 to 10 V/cm are generated at the microneedle tip and the sample then undergoes ionization and desorption. Heating of the sample may be required. Most significant, this ionization process intro­ duces very little excess energy into the desorbed molecules and the spectra therefore frequently consist of molecular ions showing little or no fragmentation. Other ionization-desorption processes may be observed which correspond to the addition of H , Na or K or similar cationic attachment. It is thus possible to obtain Current address: Battelle Pacific Northwest Laboratories, Richland, Washington. 7

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0-8412-0556-6/80/ 47-128-03555.00/ 0 © 1980 American Chemical Society In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

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CELL SURFACE GLYCOLIPIDS

Figure 1. Electron micrograph of field desorption emitter prepared by activation of 10-fx tungsten wire in a benzonitrile atmosphere. Distance between posts is 5 mm.

Figure 2.

Electron micrograph of activated emitter showing dendrite growth along 10-p. wire

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

COSTELLO ET A L .

Figure 3.

Field Desorption

Mass

Spectrometry

Electron micrograph of region at dendrite tips on activated emitter

Figure 4. Electron micrograph of activated emitter to which sample has been applied by dipping emitter into a solution and allowing the solvent to air dry.

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

C E L L SURFACE GLYCOLIPIDS

38

Figure 5.

Field desorption ion source showing the position of the emitter during the analysis. A: Push rod.

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

3.

COSTELLO ET A L .

Field Desorption

Mass

Spectrometry

39

molecular weight information about n o n v o l a t i l e or f r a g i l e molecules even without d e r i v a t i z a t i o n . By c a r e f u l c o n t r o l of e x p e r i mental c o n d i t i o n s , i t i s a l s o p o s s i b l e to b r i n g about some ( p o s s i b l y thermal) fragmentation and thereby o b t a i n a d d i t i o n a l s t r u c t u ral information. There are a number of obvious advantages to i o n i z a t i o n by f i e l d desorption. F i r s t , d e r i v a t i z a t i o n i s not r e q u i r e d (although as we s h a l l demonstrate below, i t i s sometimes advantageous). This avoids two c o m p l i c a t i o n s : p o s s i b l e l a c k of sample s t a b i l i t y during chemical manipulations and the increase i n mass as a r e s u l t of d e r i v a t i z a t i o n , which leads to molecular weights t h a t often approach or even exceed the mass range of the instrument. Second, the g r e a t e r abundance of parent ions r e l a t i v e to fragment ions makes p o s s i b l e a s e m i - q u a n t i t a t i v e assessment of molecular d i s t r i bution i n complex m i x t u r e s . Phospholipids have been s t u d i e d by t h i s method (5_,6_,7J and the f i e l d desorption mass spectrum of a g l y c o s p h i n g o l i p i d , galactoceramide f e a s i b i l i t y of d i r e c t a n a l y s i s of p o l a r samples without chemical d e r i v a t i z a t i o n and the presence of abundant high mass ions i n the spectra make f i e l d desorption an a t t r a c t i v e approach f o r mass analysis. We r e p o r t here a study to assess the usefulness of FDMS i n the a n a l y s i s of s p h i n g o l i p i d s and g l y c o s p h i n g o l i p i d s , part of a c o l l a b o r a t i v e e f f o r t w i t h the goal of developing a b e t t e r understanding of the abnormal metabolism of these compounds i n mammal i a n t i s s u e s and t h e i r i m p l i c a t i o n i n storage d i s e a s e s . Since benzoylation has been shown to be useful i n the p u r i f i c a t i o n of s p h i n g o l i p i d s by high pressure l i q u i d chromatography (HPLC) ( 8 ) , i t a l s o was of i n t e r e s t to i n v e s t i g a t e the c h a r a c t e r i s t i c s of these d e r i v a t i v e s i n FDMS. The f i e l d desorption mass spectra of carbohydrates, s p h i n g o l i p i d s and g l y c o s p h i n g o l i p i d s of i n c r e a s i n g l y complex s t r u c t u r e s have been obtained at d i f f e r e n t e m i t t e r currents. In a d d i t i o n , permethyl, p e r a c e t y l , p e r t r i f l u o r o a c e t y l and h e p t a f l u o r o b u t y r y l d e r i v a t i v e s have been prepared and the r e s u l t s compared to those obtained using the u n d e r i v a t i z e d compounds. M a t e r i a l s and Methods Sphingomyelin, bovine c e r e b r o s i d e s , psychosine and sphingos i n e were obtained from Supelco, Inc. Dihydrolactocerebroside and dihydroglucocerebroside were obtained from M i l e s Yeda L t d . Psychosine and a new analog thereof were e x t r a c t e d from human b r a i n t i s s u e and separated by HPLC as t h e i r biphenyl carbonyl derivatives. F i e l d desorption mass spectra were obtained on a Varian MAT 731 instrument (Florham P a r k , NJ) f i t t e d w i t h the combined E I / F I / F D ion source. E m i t t e r s were prepared i n the Varian apparatus according to Schulten and Beckey (_3), or were p r e t r e a t e d before a c t i v a t i o n by soaking i n a s a t u r a t e d s a l t s o l u t i o n (9).

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

40

C E L L SURFACE GLYCOLIPIDS

Samples were d i s s o l v e d i n a s u i t a b l e s o l v e n t (CHC13 or 2:1 CHC13/CH30H) (1-10 u g / u l ) and loaded by d i p p i n g the FD e m i t t e r or by adding 1-3 y l of the s o l u t i o n to the e m i t t e r with a micro s y r i n g e . In both cases the s o l v e n t was removed by a i r d r y i n g . The instrument was operated under the f o l l o w i n g c o n d i t i o n s : A c c e l e r a t i n g v o l t a g e , 8 or 6 KV, counter e l e c t r o d e v o l t a g e , 3-6 KV, ion source temperature, 100°C, e m i t t e r c u r r e n t increased manually to a l l o w the r e c o r d i n g of spectra at several temperat u r e s . Low r e s o l u t i o n spectra were recorded by e l e c t r i c a l scanning at a r e s o l u t i o n M/aM 1000 to 2500 depending on the molecular weight of the compound; assignments of mass were made using the instrument mass marker which had been c a l i b r a t e d a g a i n s t PFK. High r e s o l u t i o n spectra were recorded on I0N0MET photoplates at M/aM 5000. Some accurate mass measurements were obtained by peak matching at M/aM 8000. Results and D i s c u s s i o n F i e l d desorption spectra of the s p h i n g o l i p i d s and g l y c o s p h i n g o l i p i d s i n v e s t i g a t e d featured intense protonated molecular ions at moderate e m i t t e r c u r r e n t s (19 to 22 ma). At the best anode temperature (BAT), the molecular ion c l u s t e r s c o n s t i t u t e d the base peak i n many of the s p e c t r a . The assignment of the (M + H ) s t r u c t u r e to the (M + 1 ) ion species was confirmed by accurate mass measurement of the sphingenine ion at m/e 300 (measured 300.2859; c a l c u l a t e d 300.2902) (Figure 6 ) . Attachment of a p o s i t i v e l y charged metal ion ( u s u a l l y Na or K) to a n e u t r a l molecule forming a p o s i t i v e l y charged complex ( c a t i o n i z a t i o n ) was observed f o r several of the compounds and f o r some, the c a t i o n i z e d species i n s t e a d of the MH c o n s t i t u t e d the base peak of the spectrum. This complex may a r i s e because s a l t s are e x t r a c t e d with the sample d u r i n g i s o l a t i o n , or may be due to a s s o c i a t i o n of the sample w i t h c a t i o n s present on the e m i t t e r surface when s a l t - s a t u r a t e d e m i t t e r s are used ' ( 9 ) . For example, Figure 7 shows t h a t spectra of the biphenyl carbonyl d e r i v a t i v e s of psychosine and a new analogue thereof are dominated by the cationized species. These m a t e r i a l s had been p u r i f i e d by HPLC p r i o r to a n a l y s i s by mass spectrometry. The M H , (M + N a ) and (M + K ) ions i n the f i e l d desorption spectrum made i t apparent t h a t these compounds d i f f e r e d i n the degree of u n s a t u r a t i o n , thereby answering the question of s t r u c t u r a l m o d i f i c a t i o n i n the new compound. C a t i o n i z a t i o n o b v i o u s l y does not prevent successful determination of molecular weights and i t has even been suggested (10) to generate i t d e l i b e r a t e l y to r e s o l v e a m b i g u i t i e s . C a t i o n i z a t i o n was not observed f o r compounds whose exchangeable hydrogens had been replaced by d e r i v a t i z a t i o n . The s i m p l i c i t y of FD spectra obtained at low e m i t t e r c u r r e n t s made p o s s i b l e the a n a l y s i s of complex mixtures of g l y c o l i p i d s to o b t a i n i n f o r m a t i o n about molecular weight d i s t r i b u t i o n s . Figure 8 shows the f i e l d d e s o r p t i v e mass spectrum obtained f o r a mixture +

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In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

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COSTELLO ET A L .

CH (CH ) 3

2 (2

Field Desorption

150

171/

Spectrometry

41

MH*

239

50

Mass

250

4 Figure 6. Field desorption mass spectrum of sphingenine, recorded at 20 ma

Figure 7. Field desorption mass spectra recorded at 22-23 ma for a biphenyl carbonyl derivative of psychosine and a biphenyl carbonyl derivative of a new compound isolated from human brain tissue. Structure indicated for the unknown was assigned on the basis of this spectrum and chemical evidence relating the unknown to psychosine. Both samples were purified by HPLC prior to FDMS.

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

CELL SURFACE GLYCOLIPIDS

Figure 8. Field desorption mass spectrum obtained at 22 ma for a mixture of cerebrosides from bovine brain. Assignments of MH* are discussed in the text and summarized in Table 1.

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

3.

Field Desorption

COSTELLO ET A L .

Mass

Spectrometry

43

of bovine c e r e b r o s i d e s . Assignment of the s t r u c t u r e s to the FD peaks was made under the assumption t h a t the sample c o n s i s t e d of a mixture of cerebrosides v a r y i n g i n the nature of t h e i r s i d e chains and t h a t each cerebroside gave r i s e to a protonated molec u l a r i o n . (For the most abundant compound, C-jgOH, an ion at m/e_ 785 {MH + K } was a l s o observed. Analogous ions are probably a l s o present at the same r e l a t i v e i n t e n s i t i e s f o r a l l other compounds i n the m i x t u r e , but the lower abundance of the components makes them l e s s o b v i o u s . ) In Table I , i n f o r m a t i o n r e g a r d ing a c y l groups obtained from t h i s s i n g l e spectrum i s compared to t h a t obtained by gas chromatographic a n a l y s i s of e s t e r s of the f a t t y a c i d s obtained by m e t h a n o l y s i s . As can be seen from the T a b l e , the r e s u l t s of the two methods are q u i t e c o n s i s t e n t . Several compounds not found by GC were detected at low l e v e l s by FDMS. (On the b a s i s of low r e s o l u t i o n data a l o n e , i t i s not p o s s i b l e to d i s t i n g u i s h between C and C - i : 0 s i d e c h a i n s . ) F i e l d desorption a n a l y s i s t h e r e f o r e o f f e r s an o p p o r t u n i t y to survey b i o l o g i c a l e x t r a c t compounds without n e c e s s i t a t i n g e x t e n s i v e chemical workup. At higher e m i t t e r c u r r e n t s , fragment ions became more s i g n i f i c a n t i n the s p e c t r a . A survey of compound types y i e l d e d the spectra shown i n Figures 9 and 10A+B, obtained f o r psychosine, N-stearoyl dihydroglucocerebroside and N - s t e a r o y l d i h y d r o l a c t o cerebroside, respectively. The c h a r a c t e r i s t i c fragments, which have been observed i n t h i s f i e l d desorption study i n a d d i t i o n to the molecular i o n s , are summarized i n Scheme A . The r e s u l t s of Cleavages A , B , C and F are fragments r e l a t e d to the a l i p h a t i c m o i e t i e s of the s p h i n g o l i p i d s , w h i l e D and E are c h a r a c t e r i s t i c of the p o l a r headgroup. The non-binding o r b i t a l s of the heteroatoms i n these molecules provide s i t e s f a v o r a b l e f o r e l e c t r o n removal, which leads to i o n i z a t i o n and subsequent fragmentation i n order to s t a b i l i z e the p o s i t i v e charge. The s t r a t e g i c l o c a t i o n s of several heteroatoms i n s p h i n g o l i p i d s t r u c t u r e s (the b i o s y n t h e t i c consequences of conjugation) introduce p o i n t s of bond l a b i l i t y . The r e s u l t i n g fragments are important f o r s t r u c t u r e determination because they d e l i n e a t e the b u i l d i n g blocks of the molecule. Cleavage A w i t h charge r e t e n t i o n on the oxygen-containing p o r t i o n provides a fragment i o n which makes i t p o s s i b l e to d i s t i n g u i s h between sphingenine and sphinganine d e r i v a t i v e s by the presence of an ion at m/e 239 or 241, r e s p e c t i v e l y . For some compounds, a complementary ion may be observed f o r charge r e t e n t i o n on the n i t r o g e n - c o n t a i n i n g p a r t . Cleavage B or C w i t h charge r e t e n t i o n by the amide p o r t i o n of the molecule y i e l d s important i n f o r m a t i o n about the length of the acyl chain attached to the amino group by i t s degree of u n s a t u r a t i o n or h y d r o x y l a t i o a Cleavage D seems confined to the g l y c o s p h i n g o l i p i d s . The mass d i f f e r e n c e between t h i s ion and the molecular ion i s of value i n determining the s i z e of the carbohydrate p o r t i o n of the molecule. In the spectra of the d i s a c c h a r i d e s , Cleavage E leads to one of +

n

n

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

C E L L SURFACE

TABLE 1. M/E MH

SIDE CHAIN

+

716

758

786

SIDE CHAIN

3%

1%

C :0

19%

25%

4)GlcUA(al+6)^ 2

No f u r t h e r work was reported on c h a r a c t e r i z a t i o n of the more complex members i n t h i s s e r i e s o f p h y t o g l y c o l i p i d s from p l a n t s . Wagner, et_ a K (6) reported to have i s o l a t e d from peanuts a p h y t o g l y c o l i p i d - l i k e m a t e r i a l f o r which a t e n t a t i v e s t r u c t u r e was proposed as f o l l o w s :

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

5.

LAINE ET A L .

Glycophosphoceramides

from

67

Plants

Cer-phosphate-Inos(?-4)G1cUA(al->3)GlcNH (l->?)(Gal, 2

A r a , Man).

C a r t e r and Koob (70 i s o l a t e d a p h y t o g l y c o l i p i d f r a c t i o n from bean leaves (Phaseolus v u l g a r i s ) . They e x t r a c t e d these g l y c o phosphoceramides by r e f l u x i n g i n hot 70% ethanol (0.1 N i n HC1) f o r 20 min. This a c i d i c e x t r a c t i o n procedure may have caused p a r t i a l breakdown o f these complex compounds. Wagner, i t ?lTnos f o r the PSL-I c a r b o x y l reduced t r i s a c c h a r i d e . Periodate o x i d a t i o n experiments to d e t e r mine the l i n k a g e between g l u c u r o n i c a c i d and m y o i n o s i t o l were c a r r i e d out on the i n t a c t PSL-I (12). The phospho-alcohol product from m y o i n o s i t o l was separated from other products by anion exchange chromatography and the f i n a l d e r i v a t i v e examined by chemical i o n i z a t i o n mode o f gas chromatography/mass spectrometry was shown to be e r y t h r i t o l , i n d i c a t i n g t h a t the g l u c u r o n i c a c i d was attached to the C-2 p o s i t i o n o f the m y o i n o s i t o l r i n g (Figures 2 , 3 a , 3 b ) . T h i s completed the c h a r a c t e r i z a t i o n o f PSL-I as

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

Glycophosphoceramides

LAINE ET A L .

RGC 100

from

Plants

>250 X 6 (M^-lnot

2,3,6-Q-M»-Glc

J s b '

O H i s b 2 0 b 2 5 6 ^ SPECTRUM

" 3 0 b 3 E * i e b i s k "

NUMBER

PSL-I Carboxyl-Reduced Trisaccharide: 61cNAcp(M)01cp(M)Inoi

Figure 1. Methylation linkage analysis of PSL-I by GC/MS: total ion chromatogram of partially methylated alditol and myoinositol acetates (PMAA) from PSLrl carboxyl-reduced trisaccharide by gas chromatography/mass spectrometry in electron-impact mode. Peaks identified: penta-O-methyl-mono-O-acetylmyoinositol derived from mono-linked myoinositol, 2,3,6-tri-O-methyl-l,4,5-tri-O-acetylglucitol derived from a 4-linked glucose, and 3,4,6-tri-0-methyl-l,5,di-0-acetyl-2-acetamido-2-N-methylglucitol derived from a terminal N-acetylglucosamine. The PMAA sample was chromatographed on a 1.5 m X 2 mm ID column packed with 3% OV-210 in a Finnigan automated GC/MS model 3300/6110. Temperature program: 150° to 215°C at 6°C/min.

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

CELL SURFACE GLYCOLIPIDS

PERIODATE OXIDATION Possible s u b s t i t u t i o n s on myoinositol

-P-ceramide

Alcohol product from myoinositol

threitol

Biochemistry Figure 2. Possible substitutions on myoinositol by glucuronic acid. Shown are the bonds susceptible to periodate oxidation (wavy lines) and the predicted corresponding final myoinositol-derived alcohol products after periodate oxidation, followed by NaBD, reduction, hydrolysis, anion exchange chromatography and dephosphorylation (12). f

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

5.

LAINE ET A L .

Glycophosphoceramides

from

Plants

71

TOtT

H H-C-OAC H-C-OAC Cl(CH ) MH-HOAC H-C-OAC * •231 H-C-OAC H M>290 AUTHENTIC ERYTHRITOL PERACETYLATED 4

D H-C-OAC CI (CH ) H-C-OAC H-C-OAC H-C-OAC • D M-292 DIDEUTERATED ALCOHOL PRODUCT PERACETYLATED 4

100

M/E 231

M/E 233

4-HOAC =233

15i

251 M/E

Figure 3. Chemical ionization (methane) GC/MS of the acetylated final product derived from periodate oxidation of the myoinositol ring in PSL-I. (a): Total ion chromatogram of co-injected mixture of the unknown dideuterated alcohol product and the authentic erythritol. (b): Chemical ionization spectrum of peak indicated by an arrow in (a). Inset diagrams depict the fragmentation.

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

72

C E L L SURFACE GLYCOLIPIDS

GlcNAcp(al->4)G1cUAp(a1->2)Inos-l-0-phosphoceramide

(Figure 4 ) ( 1 2 ) .

Major O l i g o s a c c h a r i d e s Prepared from the Carboxyl-Reduced Conc e n t r a t e o f Glycophosphoceramides o f Tobacco Leaves For the remaining components i n the c o n c e n t r a t e , Hsieh (22) prepared a mixture o f o l i g o s a c c h a r i d e s from the carboxyl-reduced (23) glycophosphoceramide concentrate. A l a r g e number of c h r o matographic c o n d i t i o n s were examined f o r optimal f r a c t i o n a t i o n . A s e r i e s of c l o s e l y r e l a t e d o l i g o s a c c h a r i d e s w i t h i n c r e a s i n g complexity and i n decreasing abundance were observed on r e v e r s e phase high pressure l i q u i d chromatography as the p e r a c e t y l a t e d d e r i v a t i v e s [procedure adapted from those o f Wells and L e s t e r (24) ] . Combinations o f both reverse-phase and normal-phase columns were used under v a r i o u s sol vent c o n d i t i o n s to achieve i s o l a t i o n o f the major o l i g o s a c c h a r i d e s . M e t h y l a t i o n Analyses M e t h y l a t i o n l i n k a g e a n a l y s i s of the p a r t i a l l y methylated a l d i t o l acetates gave the f o l l o w i n g d e r i v a t i v e s : Major t r i s a c c h a r i d e : 3 , 4 , 6 - t r i - 0 - m e t h y l - 2 - d e o x y - 2 - m e t h y l a m i nogluci t o l 2,3,6-tri-0-methylgluci tol 1,3,4,5,6-penta-0-methylinositol Major t e t r a s a c c h a r i d e :

(Figure

5)

2,3,4,6-tetra-0-methylgalacti tol 3,6-di-0-methyl-2-deoxy-2-methylami nogluci t o l 2,3,6-tri-0-methylgluci tol 1 , 3 , 4 , 5 , 6 - p e n t a - 0 - m e t h y l i nosi t o l Minor t e t r a s a c c h a r i d e : 2,3,4,6-tetra-0-methylmannitol 3 , 4 , 6 - t r i - 0 - m e t h y l - 2 - d e o x y - 2 - m e t h y l a m i nogluci t o l 2,3,6-tri-0-methylgluci tol tetra-O-methylinositol

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

LAINE ET A L .

Glycophosphoceramides

from

CH

3

(CH ) 2

HC II

Plants

8

CHo AND I CH 2

0

'3 HCOH I HCOH ' I I , Q

0~Na

+

Biochemistry Figure 4.

Proposed structure of PSL-I: GlcNAcp(al-*4)GlcUAp(al-*2 sitol-!-O-phosphocer'amide (12)

myoino-

RGC 100 2,3,6-O-Mo-Glc 2,3,46-O-Me-Gal

3,6-O-Me-GlcNAc

(Me) - Inos 5

soiob'

1 5 0 2 0 0 2 5 0 ' 3 0 0 3 5 o "

i00450 '

SPECTRUM NUMBER Major Tetrasaccharide: Galp(l-4)GlcNAcp(l-»4)G1cp(1+2)Ir»os

Figure 5.

Methylation linkage analysis of the major tetrasaccharide from tobacco glycophosphoceramide concentrate

Total ion chromatogram: penta-O-methyl-mono-O-acetylmyoinositol derived from monolinked myoinositol, 2,3,4,6-tetra-O-methyl-l ,5-di-O-acetylgalactitol derived from a terminal galactose, 2,3,6-tri-O-methyl-l ,4,5-tri-O-acetylglucitol derived from a 4-linked glucitol, and 3,6-di-0-methyl-l,4,5-tri-0-acetyl-2-acetamido-2-N-methylglucitol from a 4linked N-acetylglucosamine.

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

CELL SURFACE GLYCOLIPIDS

Major coaponent

«alpO-*L Minor coaponent:

^6alpn^)61ctWcp(1->4)61cpn->2)Inos Araf(l-3r

Figure 6.

RGC 100

Preliminary methylation linkage analysis of the major pentasaccharide from tobacco glycophosphoceramide concentrate

(Mt^-lnos

2,3,4-O-Nto-Gal

2,3,5-O-Mc-Aro

3,6-O-M*- GlcNAc

Major coeponent: Araf(1-»6)8a1e(1-»4)81dlAcp(1-»4)61cp(1-2)liio» Araf(1*3)

Figure 7.

Preliminary methylation linkage analysis of the minor pentasaccharide from tobacco glycophosphoceramide concentrate

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

LAINE ET A L .

Glycophosphoceramides

from

Plants

1 Tobacco leaves I Solvent extraction Crude concentrate of glycophosphoceramides PSL-IA, PSL-IB, PSL-IC PSL-IIA, PSL-IIB, PSL-IIC -PSL-I:

GlcNAc-GlcUA-Inos-l-O-P-Ce

PSL-II: GlcNH -GlcUA-Inos-1-O-P-Ce 2

M>PSL-I:

GlcNAcp(ol->4)GlcUAp(aU2)Inos-l-0-P-Cer

,

i

|COOH-reduced oligosaccharide mixture] **** Major trisaccharide: Major tetrasaccharide: Minor tetrasaccharide:

GlcNAcp(al->4)Glcp(oU2)In6s Galp(Bl-*4)GlcNAcp( l-^)Glcp(aU2)Inos GlcNAcp(al-»4)Glcp(aU?)[ManpGiU?)]Inos

Major pentasaccharide:

Galp(l-^6)Galp(W)GlcNAcp(H4)Glcp(H2)Inos

Minor pentasaccharide:

Araf(l+6)Galp(l-*4)GlcNAcp(l-*4)Glcp(M)Ins

a

*

Kaul and Lester (1975)

**

Kaul and Lester (1978)

***

Hsleh, et al_. (1978)

****

Hsleh, et al_. (1979)

Figure 8.

Summary of structural characterization of glycophosphoceramides fi tobacco leaves

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

76

C E L L SURFACE

GLYCOLIPIDS

Sequence A n a l y s i s The carbohydrate sequence o f the major t e t r a s a c c h a r i d e was determined by examining the n i t r o u s a c i d deamination products (25) as permethylated d i s a c c h a r i d e s by chemical i o n i z a t i o n mode o f gas chromatography/mass spectrometry. The products were i d e n t i f i e d as hexosyl-2,5-anhydromannitol and h e x o s y l m y o i n o s i t o l , i n d i c a t i n g t h a t the major t e t r a s a c c h a r i d e had the sequence Galp(l-»4)GlcNAcp(l-»4)Glcp(l+2)Inos ( F i g u r e 5 ) . Anomeric C o n f i g u r a t i o n A d d i t i o n a l i n f o r m a t i o n on the composition and anomeric conf i g u r a t i o n s were obtained by gas chromatography o f a l d i t o l acet a t e s prepared from the o l i g o s a c c h a r i d e s w i t h and without CrO3 o x i d a t i o n . In the major t r i s a c c h a r i d e , and i n the minor t e t r a s a c c h a r i d e , 80-100 i n d i c a t i n g a l l a c o n f i g u r a t i o n o f the anomeric bonds. In the major t e t r a s a c c h a r i d e , however, the y i e l d f o r galactose was 29% s u r v i v a l , w h i l e the o t h e r sugars showed 80-100% s u r v i v a l . This data suggested the f o l l o w i n g s t r u c t u r e s : Major t r i s a c c h a r i d e : GlcNacp(al->4)Glcp(al->2)Inos Major t e t r a s a c c h a r i d e : Galp(sl-*4)GlcNAcp(al-*4)Gl p(al-*2)Inos C

Minor t e t r a s a c c h a r i d e GlcNAcp(al->4)Glcp(aU?)[Man(al->?)]Inos Thus, the major t r i - a n d t e t r a s a c c h a r i d e were completely c h a r a c t e r i z e d ( F i g u r e 8) (22). The l i n k a g e s i t e s on the myoinos i t o l o f the minor t e t r a s a c c h a r i d e remain undetermined due to the i n s u f f i c i e n t amount of sample a v a i l a b l e . Higher oligomers are being f r a c t i o n a t e d . P r e l i m i n a r y data i n d i c a t e t h a t a major pentasaccharide has the f o l l o w i n g s t r u c t u r e Galp(l->6)Galp(l->4) GlcNAcp(l-*4)Glcp(l+2)Inos and a minor pentasaccharide Araf(l->6) Galp(l->4)GlcNAcp(1^4)Glcp(l->2)Inos (Figures 6, 7 ) . A summary o f the r e s u l t s i s shown i n Figure 8. Acknowledgements This i n v e s t i g a t i o n was supported i n part by Research Grant PCM7609314 from the National Science Foundation , P r o j e c t KTRB053 from the Tobacco and Health Research I n s t i t u t e , U n i v e r s i t y o f Kentucky, and Grant IR0IGM23902 from the National I n s t i t u t e s of Health.

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

5. LAINE ET AL.

Glycophosphoceramides from Plants

11

Abstract Chemical structures of certain glycophosphoceramides from tobacco leaves were studied. The structures which have been characterized to date are as follows: (1) (2)

major glycophosphoceramides PSL-I: GlcNAcp(α1->4)GlcUAp(α1->2)Inos-1-O-P-Cer the oligosaccharides isolated from the glycophosphoceramide concentrate after carboxyl-reduction: (a) major trisaccharide: GlcNAcp(α1->4)Glcp(α1->2)Inos (b) major tetrasaccharide: Galp(β1->4)GlcNAcp(α 1->4)Glcp(α 1->2)Inos (c) minor tetrasaccharide GlcNAcp(α1->4)Glcp( 1->?)[Manp( 1->?)]Ino (d) major pentasaccharide: Galp(1->6)Galp(1->4)GlcNAcp(1->4)Glcp(1->2)Inos (e) minor pentasaccharide: Araf(1->6)Galp(1->4)GlcNAcp(1->4)Glcp(1->2)Inos

Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

Carter, H.E., Celmer, W.D., Galanos, D.S., Gigg, R.H., Lands, W.E.M., Law, J.H., Mueller, K.L., Nakayama, T., Tomizawa, H.H., and Weber, E. J. Am. Oil. Chem. Soc., 1958, 35, 335. Lester, R.L., Smith, S.W., Wells, G.B., Rees, D.C., and Angus, W.W. J . Biol. Chem., 1974, 249, 3388. Kaul, K., and Lester, R.L. Plant Physiol., 1975, 55, 120. Carter, H.E., Brooks, S., Gigg, R.H., Strobach, D.R., and Suami, T. J . Biol. Chem., 1964, 239, 743. Carter, H.E., Strobach, D.R., and Hawthorne, J.N. Biochemistry, 1969, 8, 383. Wagner, H., Zofcsik, W., and Heng, I. Z. Naturforsch, 1969, 24, 922. Carter, H.E., and Koob, J.L. J. Lipid Res., 1969, 10, 363. Wagner, H., Pohl, P., and Munzing, A. Z. Naturforsch, 1969, 24, 360. Carter, H.E., and Kisic, A. J. Lipid Res., 1969, 10, 356. Kaul, K., and Lester, R.L. Biochemistry, 1978, 17, 3569. Taylor, R.L., Shively, J.E., Conrad, H.E., and Cifonelli, J.A. Biochemistry, 1973, 12, 3633. Hsieh, T.C.-Y., Kaul, K., Laine, R.A., and Lester, R.L. Biochemistry, 1978, 17, 3575. Björndal, H., Lindberg, B., and Svensson, S. Carbohyd. Res., 5, 433.

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

78

14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.

CELL SURFACE GLYCOLIPIDS

Björndal, H., Lindberg, B., Pilotti, A., and Svensson, S. Carbohydrate Res., 1970, 15, 339. Hakomori, S. J. Biochem. (Tokyo), 1964, 55, 205. Stellner, K., Saito, H., and Hakomori, S. Arch. Biochem. Biophys., 1973, 155, 464. Hancock, R.A., Marshall, K., and Weigel, H. Carbohyd. Res., 1976, 49, 351. Laine, R.A., Hodges, L.C., and Cary, A.M. J. Supramol. Struct., 1977, 5, Suppl. 1, 31. Hoffman, J., Lindberg, B., and Svensson, S. Acta Chem. Scand., 1972, 26, 661. Laine, R.A., and Renkonen, O. J. Lipid Res., 1975, 16, 102. Albersheim, P., Nevins, D.J., English, P.D., and Karr, A. Carbohyd. Res., 1967, 5, 340. Hsieh, T.C.-Y., Ph.D. dissertation: "Chemical Characterization of Glycophosphosphingolipid fro Tobacco" Universit of Kentucky: Lexington Taylor, R.L., Shively, J . E . , and Conrad, H.E. Methods in Carbohyd. Chem., 1976, 7, 149. Wells, G.B., and Lester, R.L. Anal. Biochem., 1979, 97, (in press). Bayard, B., and Roux, D. FEBS Lett., 1975, 55, 206.

RECEIVED

December 10, 1979.

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

6 Glycolipids of Rat Small Intestine with Special Reference to Epithelial Cells in Relation to Differentiation M. E. BREIMER, G. C. HANSSON, K.-A. KARLSSON, and H. LEFFLER Department of Medical Biochemistry, University of Göteborg, Göteborg, Sweden Saccharides may b the variation in type an heterocyclic carbohydrate monomer may vary in ring size, the glycosidic bond may have both different positions and configurations, and there is often branching of the saccharide chains. A great variability may also mean a rich biochemical language (provided there is specificity of expression) and this is one of the reasons why cell surface carbohydrates are being considered in biological recognition (1, 2). The membrane-bound carbohydrates exist as glycoproteins and glycolipids. Although the functional importance of these substances is far from proven they appear to be essential parts in phenomena such as cellular adhesion, control of differentiation and cell growth, and the binding by cells of enzymes, hormones and toxins. One system that we consider of great interest for the study of cell surface glycolipids is the small intestine. Firstly, the epithelial cells lining the intestine exist in a great number on the enlarged surface area and each cell has in itself a large cell surface involved in transport processes and recognition phenomena. Secondly, these cells, arranged as a single columnar layer on the basement membrane, are rapidly renewed (1-3 days) and undergo a successive maturation on their way from the crypt depth to the villus tip (3). Thirdly, these cells are possible to prepare by a gentle washing technique (4), the oldest, less strongly adhered cells (villus tip) being obtained in the first, and the youngest, cells (crypt) obtained in the final fractions. Lastly, the concentration of complex glycolipids is high in relation to protein (see 5), which may be explained by a large amount of surface membrane in relation to other membranes. Our study was divided into two different parts and applied on two separate strains of rat, which were shown to differ in blood groups. In the first stage, following improvement and adaptation of methods, glycolipids were prepared and characterized from pooled whole small intestine of the black and white strain. In the second stage, the knowledge of the general glycolipid 0-8412-0556-6/80/ 47-128-079S6.50/ 0 © 1980 American Chemical Society In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

80

C E L L SURFACE GLYCOLIPIDS

composition allowed a c h a r a c t e r i z a t i o n on a smaller s c a l e of e p i t h e l i a l c e l l s and n o n - e p i t h e l i a l r e s i d u e , and a comparison of the two s t r a i n s . The d i f f e r e n t components of t i s s u e are v i s u a l i z e d i n F i g . 1. The experience obtained has now been used f o r a s i m i l a r i n v e s t i g a t i o n on human m a t e r i a l ( i n p r e p a r a t i o n ) . Methods The animals used were from inbred s t r a i n s of white, and black and white r a t . The p r e p a r a t i o n of e p i t h e l i a l c e l l s i n separate stages of d i f f e r e n t i a t i o n was modified from the technique of Weiser (4). The completeness of removal of e p i t h e l i a l c e l l s from n o n - e p i t h e l i a l residue was checked by conventional microscopy. The p r e p a r a t i o n of t o t a l g l y c o s p h i n g o l i p i d s f r e e of contaminants has been improved to an important extent but i s based on conventional steps suc a l k a l i n e degradation, d i a l y s i s on DEAE-cellulose and s i l i c i c a c i d . T h i n - l a y e r chromatography was done on HPTLC p l a t e s with s i l i c a g e l 60 (Merck). Conditions f o r mass spectrometry (6,7) and NMR spectroscopy (8, 9_ 10) have been described. Gas chromatography a f t e r degradation of n a t i v e or permethylated g l y c o l i p i d s was done according to standardized t e c h niques (11) except that the a n a l y s i s was performed on c a p i l l a r y columns. 9

Non-Epithelial Tissue The non-acid p a t t e r n of the r e s i d u e a f t e r exhaustive washing and removal of e p i t h e l i a l c e l l s from small i n t e s t i n e i s shown i n F i g . 2, f o r the b l a c k and white ( B ) and white (W ) s t r a i n , r e s p e c t i v e l y . The two samples look i d e n t i c a l with a major compoment corresponding to four sugars. Most of the g l y c o l i p i d s have been i s o l a t e d and c h a r a c t e r i z e d . To present an overview the t o t a l g l y c o l i p i d s of white r a t were subjected to a novel a p p l i c a t i o n of mass spectrometry (7) a f t e r permethylation and r e d u c t i o n with L i A l H ^ . F i g s . 3 and 4 show some of the r e s u l t s . The mixture of g l y c o l i p i d d e r i v a t i v e s i s introduced i n t o the i o n source and s u c c e s s i v e l y heated (5°C/min) as shown on the s c a l e below the curves. Scans (each scan producing a mass spectrum such as that of F i g . 7) were taken each 38 sec, and the change i n r e l a t i v e i n t e n s i t y of s e l e c t e d ions f o r separate g l y c o l i p i d s was r e produced as curves along the temperature and scan s c a l e s . In t h i s case the ions s e l e c t e d contained the complete saccharide and the f a t t y a c i d as shown i n the e x p l a i n i n g formulas ( u s u a l l y r e l a t i v e l y abundant i o n s , which i s demonstrated f o r the A a c t i v e g l y c o l i p i d s i n F i g s . 7 and 8). Curves corresponding to s p e c i f i c ions f o r nine separate g l y c o l i p i d s are reproduced. Two s e r i e s of compounds are shown, one without ( F i g . 3) and the other with hexosamine ( F i g . 4 ) . The curve i n F i g . 3 f o r m/e 516 (monohexosylg

s

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

6.

BREIMER ET A L .

Epithelial

Cells

Figure 1. Acid and non-acid glycosphingolipids were prepared and characterized from different compartments of rat small intestine: non-epithelial residue, total epithelial cells, and epithelial cells of different maturity (crypt, intermediate, and villus fractions).

Figure 2.

Thin-layer chromatogram of non-acid glycolipids of small intestine of black and white (B) and white (W)rat

The following samples were applied: 40 fig of total glycolipids (t); glycolipids corresponding to 4 mg protein of non-epithelial residue (s); glycolipids corresponding to 2 mg protein for epithelial cells of villus (v), intermediate (i), and crypt (c) fractions. Figures in the margins indicate number of sugars. Anisaldehyde was used for the detection, and the solvent was chloroform-methanol-water 60:35:8 (by volume).

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

81

82

C E L L SURFACE

-CH-CH-CH-C

-CH-^H-CH-C.-H,,

Hex-

Hex—o—Hex-

1 3

-Hex-o-Hex-

Hex-o-Hex-

-Hex-

GLYCOLIPIDS

-Hex-o—CH CH2

-Hex-o—Hex—o—CH CH2

-Hex-o-cH C H -

-CH-CH-CH-C

1 3

H-

7

-CH-CH=CH-C

1 3

H_

7

1536 (CH ) 2

Hex-

-Hex-o-Hex-o—Hex—o—Hex-

-Hex-o—CH.CH-

1 4

Figure 3. Selected ion monitoring from mass spectrometry of a permethylatedreduced mixture of non-acid glycolipids from non-epithelial residue of the white rat The curves reproduced correspond to relative abundance of saccharide plus fatty acid ions (see formulas) of glycolipids lacking hexosamine as a function of evaporation temperature. A total of 200 fig was evaporated by a temperature rise of 5°C/min, and spectra were recorded each 38 sec. The electron energy was 34 eV, acceleration voltage 4 kV, trap current 500 fiA, and ion source temperature 280°C.

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

BREIMER ET A L .

Epithelial

Cells

HexN-o—Hex—o—Hex—o—Hex-

-Hex—o—Hex—o—Hex—o—CH C H -

Hex N - o - H e x -

(CH ) 2

HexN-o-Hex-

-Hex—o—Hex—o—Hex-

1 4

-Hex—o—CH_CH-

-1359(x3)5 150°

Figure 4.

' ' I " 21 200°

37 250°

Selected ion monitoring of saccharide plus fatty acid ions of hexosaminecontaining glycolipids from the same experiment as for Figure 3

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

84

C E L L SURFACE GLYCOLIPIDS

ceramide) appears at lower temperature while those f o r higher members ( F i g s . 3 and 4) come up l a t e r , i n some cases i n d i c a t i n g a complete s e p a r a t i o n of g l y c o l i p i d s p e c i e s . The r e l a t i v e i n t e n s i t i e s of the separate bands on the chromatogram ( F i g . 2) are not d i r e c t l y comparable with the i o n curves as the r e l a t i v e abundance of ions decreases r a p i d l y with i o n mass. The space a v a i l a b l e does not allow a more d e t a i l e d presenta t i o n . Mass spectra and s e l e c t e d i o n monitoring of the permethylated (non-reduced) mixture supplement the information with sequence data (6, 7) that allow the formulas w r i t t e n i n F i g s . 3 and 4. The nature of the o l i g o m e r i c hexosylceramides was f u r t h e r s u b s t a n t i a t e d by NMR spectroscopy and degradation of some pure or p a r t i a l l y p u r i f i e d f r a c t i o n s . The hexosamine-lacking compounds were separated from those c o n t a i n i n g hexosamine by use of a c e t y l ated d e r i v a t i v e s and s i l i c i c a c i d column chromatography. F i g . 5 shows NMR s p e c t r a of d e r i v a t i z e and a mixture ( f r a c t i o and f u c o s y l t e t r a h e x o s y l c e r a m i d e . e two ^"resonances and the ex resonance at about 5.0 ppm ( f r a c t i o n A) are comparable with those of trihexosylceramide of human e r y t h r o c y t e membrane (8). Theref o r e , the second a-resonance at about 5.1 ppm (the sharp s i g n a l c l o s e to that i s due to ethanol) may o r i g i n a t e i n a terminal Gal (the r a t i o of G a l r G l c as shown by degradation was 3:1). One Gal i s bond 1->3 (5.1 ppm) and the other 1->4 (5.0 ppm). The spectrum of the mixture (B) shows the same s i g n a l s but the second Gala has now about doubled i n i n t e n s i t y compared with the f i r s t Gala, suggesting that the major pentahexosylceramide i s f o r m a l l y der i v e d from the tetrahexosylceramide by a d d i t i o n of another Gala1-K3. Therefore, the o l i g o m e r i c hexosylceramides (we have det e c t e d by mass spectrometry and t h i n - l a y e r chromatography up to eight hexoses) may be formed by a s e q u e n t i a l a d d i t i o n of Gala1->3 to g l o b o t r i a o s y l c e r a m i d e ( F i g . 6). The minor f u c o l i p i d i s probabl y a l s o derived from the tetrahexosylceramide, i n t h i s case the fucose having caused an u p f i e l d l o c a t i o n of the two Gala resonances ( i n d i c a t e d by d o t s ) . The t e t r a g l y c o s y l c e r a m i d e with terminal hexosamine ( F i g . 4) was shown to c o n s i s t of about one t h i r d of c y t o l i p i n K and two t h i r d s of c y t o l i p i n R ( g l o b o t e t r a o s y l - and i s o g l o b o t e t r a o s y l ceramide, r e s p e c t i v e l y , see F i g . 6). The higher members detected i n t h i s s e r i e s ( F i g . 4) are probably formed by an e l o n g a t i o n of g l o b o t r i a o s y l c e r a m i d e as f o r the f i r s t s e r i e s and a termination by GalNAcg1-*3 ( F i g . 6). Of p a r t i c u l a r i n t e r e s t was the i d e n t i f i c a t i o n of a blood group B a c t i v e hexaglycosylceramide based on galactosamine ( F i g . 6). The g l y c o l i p i d s detected i n n o n - e p i t h e l i a l t i s s u e are summarized i n F i g . 6. The g a n g l i o s i d e composition w i l l be commented on below. Epithelial

Cells

The g l y c o l i p i d p a t t e r n of e p i t h e l i a l c e l l s i s d i s t i n c t l y

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

6.

BREIMER ET A L .

Epithelial

Cells

85

5.0

4,0 6 (ppm)

•

5^0

4.0 5(ppm)

Figure 5. NMR spectra of two permethylated-reduced glycolipid samples (A and B) lacking hexosamine and isolated from whole intestine of black and white rat; 2 mg in 0.5 mL chloroform and 2300 pulses at 40°C (sample A), and 1 mg in 0.5 mL chloroform and 5300 pulses at 40°C (sample B).

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

86

CELL SURFACE GLYCOLIPIDS

Figure 6.

Thin-layer pattern with deduced chemical formulas of non-acid glycolipids of white rat non-epithelial residue (cf. Figure 2).

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

6.

BREIMER ET A L .

Epithelial

Cells

87

d i f f e r e n t from that of n o n — e p i t h e l i a l t i s s u e ( F i g , 2), Bands corresponding to one and three sugars are dominating. In a d d i t i o n , there are a number of compounds that have been prepared from pooled whole i n t e s t i n e s of the black and white s t r a i n . Two s e r i e s of f u c o l i p i d s were i d e n t i f i e d , one with blood group H and one with blood group A determinants. Mass spectra of permethylatedreduced d e r i v a t i v e s of two of the A - g l y c o l i p i d s are shown i n F i g s . 7 and 8, r e s p e c t i v e l y . In a d d i t i o n , a 6-sugar A a c t i v e compound was c h a r a c t e r i z e d , thus completing a s e r i e s with 4, 6 and 12 sugars. Concerning the 12 sugar compound the mass spectra of the permethylated d e r i v a t i v e (not shown) and of the permethylatedreduced d e r i v a t i v e (Fig.8) are remarkable i n that they together a f f o r d a c o n c l u s i o n on the type, number and sequence of sugars i n c l u d i n g branching of the chain, i n a d d i t i o n to ceramide s t r u c t u r e (to be p u b l i s h e d ) i n the i n t e r v a l m/e 2835-297 hexoses, f i v e hexosamines, two fucoses and a v a r y i n g f a t t y a c i d , mainly from 16:0 (m/e 2835) to 24:0 (m/e 2947) nonhydroxy f a t t y a c i d , but a l s o 24:0 hydroxy a c i d (m/e 2977). In the spectrum of the non-reduced d e r i v a t i v e (not shown) m/e 396 showed that the dominating base i s phytosphingosine. According to the r e l a t i v e i n t e n s i t y of the s e r i e s of peaks at m/e 2835-2977 the major molecular species contained phytosphingosine and 20:0 nonhydroxy f a t t y a c i d . Evidence f o r the sequence and branching point was obtained by the absence or presence of several i o n s . Some primary and secondary ( l o s s of methanol, mass 32) ions with a successive increase i n the number of sugars from the non-reducing end are shown up to nine sugars (m/e 1915). The absence of sequence ions between m/e 871 and 1915 speaks against a l i n e a r sequence between these two fragmentation p o i n t s . (There were analogous ions obtained from the non-reduced d e r i v a t i v e ) . The absence of ions f o r smaller saccharides with two fucoses ( i n spectra of both d e r i v a t i v e s ) i s evidence f o r fucose l o c a t i o n i n separate c h a i n s . F i n a l l y , there i s a number of rearrangement ions c o n t a i n i n g the f a t t y a c i d and an i n c r e a s i n g part of the saccharide from the ceramide end (some of them i n d i c a t e d below the formula). These ions have taken up one or two hydrogens depending on the l o c a t i o n of the branch (see peaks at m/e 614, 818, 1049, 1239, 1470, 1848, 2093, 2324). Therefore, the evidence obtained from the two d e r i v a t i v e s i s c o n c l u s i v e concerning the sequence of the 12-sugar g l y c o l i p i d . This substance represents the l a r g e s t biomolecule s t r u c t u r a l l y determined by mass spectrometry thus f a r . Compared with the B - a c t i v e g l y c o l i p i d found i n n o n - e p i t h e l i a l t i s s u e , the f u c o l i p i d s i n e p i t h e l i a l c e l l s were based on glucosamine i n s t e a d of galactosamine (see F i g . 9). The H a c t i v e f u c o l i p i d s of b l a c k and white r a t had three and f i v e sugars, r e s p e c t i v e l y . The g l y c o l i p i d s found i n e p i t h e l i a l c e l l s of the two s t r a i n s are summarized i n F i g . 10.

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

60

40

t

d

80

100

300

246

400

296 409- 1 502

613^ 1 614 /

500

I , i 1.1,., 600

501*1

700

800

828 \

844

0—Hexose•

1000

T

991 992 980 924

900

900< 901

I

NMe

1036

1

0

I

1 I

I

0

V

V

1200

1081

1209

1300

1267

1400

n-1

1409

1500

AIaL

n-1

1521

Figure 7. Mass spectrum of permethylated-reduced derivative (60 fig) of a blood-group A active tetraglycosylceramide. Electron energy was 44 eV, acceleration voltage 4 kV, trap current 100 fiA, ion source temperature 290°C, and probe temperature 215°C.

200

187

Hexos ami ne-

246

1600

oo

m O r *< o o r

n

>

c

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00

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

1700

1900

2000

2100

M2300 i*.

2891-624*1 2268

2200

\

2400

V

(2947-885-885)

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900

2700

2800

2900

|

2891

1300

3000

2947

1200

1500

1600

2947-855-624^2 M 7 0

.H„

Journal of the American Chemical Society

2835

lMl,ii.,i,4. 1000 1 100

2600

m/e

4lll

1400

Me Me

I I I ICH — C, _

976-58 1237*2 918 1032-58 048* 1 1 049 1 239 \ 974 855 \ | 992*1 1181*2 1915-624* 1 993 1 183 ,871 * 292 \

2500

JJi

2947-624* 1 2324

700

640

81 7* 818 761*1

1237

I

CH,

2947 j

Figure 8. Mass spectrum of permethylated-reduced derivative (90 fxg) of a blood-group A active dodecaglycos^lceramide. Electron energy was 29 eV, acceleration voltage 2.9 kV, trap current 500 fiA, and evaporation temperature 305°C. In-beam technique was used (23).

1800

1915

2947-855* 1 2093

2891-855*1 2037 ( /

600

\

\

IliniiLl

500

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i

o!

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Hexose-}-0 — Hexosami

llllUuililltii 11 200 300 400

2947-855-246*2 1848

100

J

101 X0.6

Hexosamine-

M - 3232

1700

90

CELL SURFACE GLYCOLIPIDS

A

(min)

Figure 9. Open tubular gas chromatogram of partially methylated alditol acetates obtained from blood-group A active tetraglycosylceramide (A) and hexaglycosylceramide (B), respectively. Stationary phase was OV-1, and carrier gas was N . Column temperature was kept at 175°C for 14 min, then raised l°C/min. The designation above the peaks indicate actual binding positions. 2

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

Figure 10.

Thin-layer pattern with deduced chemical formulas of non-acid glycolipids of epihtelial cells of the two rat strains (cf. Figure 2)

> r

92

CELL SURFACE GLYCOLIPIDS

D i f f e r e n c e s between the two compartments and between s t r a i n s As shown, the g l y c o l i p i d patterns of e p i t h e l i a l c e l l s and n o n - e p i t h e l i a l residue are d i s t i n c t l y d i f f e r e n t . G l y c o l i p i d s with one, two, three and four hexoses e x i s t i n both compartments. Concerning the two globosides these are present only i n the none p i t h e l i a l f r a c t i o n , which i s demonstrated both by chromatography and by the absence of s p e c i f i c ions at mass spectrometry and s e l e c t e d i o n monitoring of e p i t h e l i a l g l y c o l i p i d s . The g l y c o l i p i d s with f i v e to e i g h t hexoses are a l s o present only i n n o n - e p i t h e l i a l t i s s u e , as are the g l y c o l i p i d s with one hexosamine and a v a r y i n g number of hexoses. F u c o l i p i d s are present i n both compartments. However, the blood group B a c t i v e compound of n o n - e p i t h e l i a l c e l l s (absent i n e p i t h e l i a l c e l l s ) i s based on GalNAc while the H and A a c t i v e substances s p e c i f i c f o e p i t h e l i a l c e l l hav GlcNA i t h e i core saccharide. In f a c t epithelial glycolipids fucolipi tetrahexosyl ceramide (as i n d i c a t e d i n f r a c t i o n B of F i g . 5) was obtained from pooled t i s s u e . This g l y c o l i p i d has been shown to be l o c a t e d i n the e p i t h e l i a l c e l l s . In both compartments there are minor slow-moving substances on t h i n - l a y e r chromatography. For example, when p u r i f y i n g and e n r i c h i n g the 12-sugar A a c t i v e g l y c o l i p i d from black and white r a t there appeared more p o l a r m a t e r i a l i n very low amounts, probably being g l y c o l i p i d s having more than 12 sugars. The d i f f e r e n c e between the two s t r a i n s of r a t , the black and white and the white s t r a i n , seems r a t h e r c l e a r . The n o n - e p i t h e l i a l t i s s u e i s i d e n t i c a l f o r the two, i n c l u d i n g the blood group B a c t i v e substance. The d i f f e r e n c e i s found i n the e p i t h e l i a l c e l l s and only concerning f u c o l i p i d s . This i s i l l u s t r a t e d i n F i g . 11 by s e l e c t e d i o n curves a f t e r mass spectrometry and summarized i n F i g . 10. There i s a q u a l i t a t i v e d i f f e r e n c e i n the blood group A type g l y c o l i p i d s with 4, 6 and 12 sugars, these being absent i n e p i t h e l i a l c e l l s of the white r a t . In F i g . 11 there are curves f o r the 4- and 6-sugar compounds (m/e 1125 and 1560, r e s p e c t i v e l y ) i n the black and white but not i n the white r a t . However, the 3and 5-sugar H-type g l y c o l i p i d s (m/e 894 and 1329) e x i s t i n both samples. The 10-sugar H-type g l y c o l i p i d , present i n the white r a t does not show up i n the black and white r a t , probably due to a complete GalNAcd g l y c o s y l a t i o n of the 10-sugar but not of the 3and 5-sugar g l y c o l i p i d s . For some reason the H-type 3-sugar g l y c o l i p i d i s r e l a t i v e l y more abundant i n black and white than i n white r a t ( F i g s . 2, 10 and 11). These r e s u l t s obtained by chemical means were confirmed by immunology, which showed the black and white r a t g l y c o l i p i d s to be blood group A a c t i v e , while those of the white r a t were non-active (Table I ) .

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

y

Figure 11. Selected ion monitoring from mass spectrometry of glycolipids of epithelial cells of the two rat strains. A total of 200 fig each of the permethylated-reduced mixture was evaporated by a temperature rise of 5°C/min, and mass spectra were recorded each 38 sec. Electron energy was 34 eV acceleration voltage 4 kV, trap current 500 jiA, and ion source temperature 290°C.

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

:c

Antisera d i l u t i o n

Antisera d i l u t i o n

1:0

1:1

ND means: not determined

"

"

Non-acid g l y c o l i p i d s (mg)

Blood group B a c t i v i t y

Blood group H a c t i v i t y

Blood group A a c t i v i t y

8.2

2+

1.14

Epithelial Cells

10.9

+

ND

1.19

Non-Epithelial Residue

White Rat (8 r a t s )

The Blood Group A c t i v i t i e s Concern G l y c o l i p i d F r a c t i o n s .

14.6

8.2

+

ND

-

4+ ND

1.26

Non-Epithelial Residue

1.32

Epithelial Cells

Black and White Rat (7 r a t s )

Some C h a r a c t e r i s t i c s of D i f f e r e n t Compartments of Rat Small I n t e s t i n e .

T o t a l p r o t e i n (g)

Table I.

6.

BREIMER ET A L .

Epithelial

95

Cells

Gangliosides The s i t u a t i o n f o r g a n g l i o s i d e s i s a l s o complex, with a number of separate s p e c i e s . However, two of these are q u i t e dominating, and are hematoside with N-acetyl and N - g l y c o l o y l s u b s t i t u t i o n , r e s p e c t i v e l y . F i g . 12 shows that the N - a c e t y l type e x i s t s i n none p i t h e l i a l while the N - g l y c o l o y l type i s mostly present i n e p i t h e l ial cells. E p i t h e l i a l C e l l s of D i f f e r e n t L o c a t i o n and M a t u r i t y E p i t h e l i a l c e l l s of small i n t e s t i n e were prepared i n a f r a c t i o n a l way (4), the o l d e r , l e s s adherent v i l l u s t i p c e l l s being washed out by EDTA-containing phosphate b u f f e r f i r s t , while mitot i c crypt c e l l s appeared i n the f i n a l f r a c t i o n s . The enzyme c h a r a c t e r i s t i c s of th followed conventional c r i t e r i l e s s d i f f e r e n t i a t e d (crypt) c e l l s (3, h). The thymidine kinase a c t i v i t y decreased from crypt to v i l l u s while the a c t i v i t y of a l k a l i n e phosphatase increased ( F i g . 13). The c e l l s obtained were pooled i n three f r a c t i o n s , a v i l l u s ( v ) , an intermediate ( i ) , and a crypt (c) f r a c t i o n . The patterns of g l y c o l i p i d s of these are shown i n F i g . 2 (non-acid) and F i g . 12 ( a c i d ) . The only s i g n i f i c a n t d i f f e r e n c e s between the three l o c a l s concern the three major g l y c o l i p i d s and are a succ e s s i v e i n c r e a s e o f monoglycosylceramide ( F i g . 2) and hematoside ( F i g . 12) from crypt to v i l l u s , but a successive decrease of t r i hexosylceramide ( F i g . 2). These f a c t s have been n o t i c e d before (12, 13). Other d i f f e r e n c e s e x i s t but we have not y e t r e s o l v e d and q u a n t i t a t e d a l l minor bands to allow comments on t h i s . There i s a l s o a change i n r e l a t i v e i n t e n s i t y o f the two bands of each of mono- and trihexosylceramide ( F i g . 2 ) . The slower-moving band i s i n c r e a s i n g towards the v i l l u s . Analogous changes are a l s o apparent f o r minor g l y c o l i p i d s . The reason f o r the two bands i s a heterogeneity i n the ceramide p o r t i o n , mainly concerning 2-hydr o x y l a t i o n of the f a t t y a c i d . As the base i s almost e x c l u s i v e l y phytosphingosine a change i n the mass s p e c t r a l fragments f o r ceramide i n d i c a t e d by the formula of F i g . 14 should r e f l e c t the f a t t y a c i d change. Monitoring of these ions through the temperature i n t e r v a l shown should give the composition of a l l g l y c o l i p i d s present. However, as mono- and trihexosylceramides dominate the two major peaks i n d i c a t e d a t about 190°C and 225°C mainly r e f l e c t these two g l y c o l i p i d s , r e s p e c t i v e l y . One should a l s o bear i n mind that the r e l a t i v e p r o p o r t i o n of these two substances changes between the two f r a c t i o n s (see F i g . 2, f r a c t i o n s B and B ) . With t h i s knowledge one may i n t e r p r e t e from the curves of F i g . 14 a r e l a t i v e lengthening o f the f a t t y a c i d and an increased hydr o x y l a t i o n from crypt to v i l l u s . The r e l a t i v e i n c r e a s e i n c h a i n length i s shown by m/e 722 (24:0 hydroxy) compared with m/e 666 (20:0 hydroxy) and m/e 610 (16:0 hydroxy) i n the two f r a c t i o n s , y

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

c

C E L L SURFACE

Figure 12.

GLYCOLIPIDS

Thin-layer chromatogram of gangliosides of small intestine of black and white (B) and white (W) rat

The fractions and amounts were analogous to those of Figure 2, except for the total fractions (t), where 20 fig glycolipid were used. Bands for N-acetyl (a) and 'N-glycoloyl (b) type of hematoside are indicated. Resorcinol was used for the detection, and the solvent was methyl acetate-2-propanol-CaCl (8 mg/mL)-NH (5M) 45:35:15:10 (by volume). 2

3

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

1PD WPHVdS

S

IV 13 H3WI3HH

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

98

CELL SURFACE GLYCOLIPIDS

but a l s o by m/e 636 (20:0) which i s q u i t e dominating i n the t r i hexosylceramide peak of the crypt f r a c t i o n (225°C) while m/e 692 (24:0) i s the most abundant ion of the v i l l u s f r a c t i o n . The change i n h y d r o x y l a t i o n i s not c l e a r from the curves of Fig.14 without an i n t e g r a t i o n . However, from e a r l i e r experience of the behaviour of molecular species of g l y c o l i p i d s on t h i n - l a y e r chromatography (14) and knowledge of major f a t t y acids present ( F i g . 14) one may conclude that the two bands ( F i g . 2) are mainl y composed of 20, 22, 23 and 24 carbon nonhydroxy acids (upper band) and 20, 22, 23 and 24 carbon hydroxy f a t t y acids (lower band). The change i n f a t t y a c i d composition may be shown f o r separ a t e major or minor g l y c o l i p i d s i n the mixture by s e l e c t i n g fragments s p e c i f i c f o r the species i n question, namely saccharide plus f a t t y a c i d ions which are r e l a t i v e l y abundant (see spectra of F i g s . 7 and 8). One exampl ceramide i n F i g . 15. Th g l y c o l i p i d s ( F i g . 14). , analogou 4-sugar A-type g l y c o l i p i d (compare F i g . 7) d i d not demonstrate that c l e a r d i f f e r e n c e i n chain length between v i l l u s and crypt cells. Discussion Small i n t e s t i n e i s r e l a t i v e l y r i c h i n g l y c o s p h i n g o l i p i d s (Table I ) . Compared to myelin, a m e t a b o l i c a l l y s t a b l e p o l y membrane s t r u c t u r e (15), a l s o with a high content of g l y c o l i p i d (one sugar), small i n t e s t i n e has a p a t t e r n o f t e n dominated by complex f u c o l i p i d s (5, 16). Of p a r t i c u l a r i n t e r e s t i s the f i n d i n g i n t h i s and recent works (5, 16) of the l o c a l i z a t i o n of these more complex substances to e p i t h e l i a l c e l l s which are s t r u c t u r a l l y complex and asymmetrical. These c e l l s are involved i n important transport and r e c o g n i t i o n processes and have a r a p i d turnover ( 3 ) . This s i t u a t i o n has provided us with an i n t e r e s t i n g object f o r the study of s t r u c t u r e - f u n c t i o n r e l a t i o n s h i p s of g l y c o s p h i n g o l i p i d s . Although there i s strong evidence f o r one p a r t i c u l a r g a n g l i o s i d e being the s p e c i f i c receptor f o r c h o l e r a t o x i n (1), there i s at present no good idea about a p h y s i o l o g i c a l f u n c t i o n of a g l y c o l i p i d . A p o s s i b l e exception i s s u l f a t i d e , the only substance with a rather consequent s t o i c h i o m e t r i c r e l a t i o n to a surface membrane f u n c t i o n , i n t h i s case Na and K transport (17, 18). Although the postulated r o l e ( s e l e c t i o n of K ions, 17) i s due to the s u l f a t e group, the sugar part c a r r y i n g t h i s group may be s p e c i f i c a l l y r e quired c l o s e to the membrane matrix. +

+

+

In our i n i t i a l studies reported here of g l y c o l i p i d s of r a t small i n t e s t i n e , p r e p a r a t i v e and s t r u c t u r a l methods were adapted to c h a r a c t e r i z e e p i t h e l i a l and n o n - e p i t h e l i a l t i s s u e and e p i t h e l i a l c e l l s of d i f f e r e n t l o c a t i o n and l e v e l of d i f f e r e n t i a t i o n . The two compartments were d i s t i n c t l y d i f f e r e n t with core saccharides with GalNAc being r e s t r i c t e d to n o n - e p i t h e l i a l c e l l s while those

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

Villus

ia

0i

-CH-CH— C H

CryPt

Non-hydroxy f a t t y acids

Hydroxy f a t t y acids 580

610 636

666

20:0

Figure 14. Selected ion monitoring from mass spectrometry of villus and crypt epithelial glycolipids of the black and white rat. Relative abundance of ceramide ions was reproduced. A total of 100 fig each of the permethylated mixture was evaporated by a temperature rise of 5°C/min, and spectra were recorded each 38 sec. Electron energy was 49 eV, acceleration voltage 4 kV, trap current 500 fiA, and ion source temperature 290°C.

2

Carbohydrate chain-j-CH CH

16:0

692

722

24:0

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

2

1

I' 21 200°

1 1

''I'' 37 250°

o

o

scan

5 150°

— 1158

— 1128

Crypt

Hydroxy f a t t y acids 1158

1128

• • i• • 21 200°

Non-hydroxy f a t t y acids

16:0

37 250°

1214

1184

20:0

Figure 15. Monitoring from mass spectrometry of saccharide plus fatty acid ions of tetrahexosylceramide of villus and crypt epithelial cells of black and white rat. Data'were retrieved from 200 /xg of the permethylated-reduced mixtures of total glycolipids, were evaporated at 5°C/min. Spectra recorded each 38 sec. Electron energy was 34 eV, acceleration voltage 4 kV, trap current 500 yiA, and ion source temperature 280°C.

5 150°

•'l' '

— 1158

Villus

Hex—o—Hex—o—Hex-o—Hex—o—CH.CH-

NMe

CH

1270

1240

24:0

6.

BREIMER ET A L .

Epithelial

Cells

101

with GlcNAc were confined to e p i t h e l i a l c e l l s . An unusual blood group B a c t i v e hexaglycosylceramide based on GalNAc and r e s t r i c t ed to n o n - e p i t h e l i a l c e l l s may be i d e n t i c a l with a g l y c o l i p i d detected i n r a t macrophages and granuloma (19). A l l other fucol i p i d s were found i n e p i t h e l i a l c e l l s and based on GlcNAc or l a c k ing hexosamine. Two s e r i e s of f u c o l i p i d s were found i n the black and white s t r a i n , one H a c t i v e with 3, 5 and 10 sugars, and one A a c t i v e with 4, 6 and 12 sugars. The f u c o l i p i d s with 3 and 4 sugars are novel species and based simply on l a c t o s y l c e r a m i d e , demonstrating that the simple d e r i v a t i v e s of reducing l a c t o s e found i n milk (20) have counterparts i n membrane g l y c o l i p i d s . In l a r g e i n t e s t i n e of r a t we have detected d i f u c o s y l substances which are absent from small i n t e s t i n e (unpublished). Further work may show i f these a l s o are analogous to the simple l a c t o s e saccha r i d e s i n milk (20). I t w i l l be i n t e r e s t i n lipids i n non-epithelia a d d i t i o n of Gala, have s p e c i f i c immunological p r o p e r t i e s or can bind c e r t a i n l e c t i n s . Apparently, the two s t r a i n s of r a t both have these g l y c o l i p i d s but d i f f e r i n e p i t h e l i a l c e l l s being blood group A p o s i t i v e or negative. The d i f f e r e n c e between the two s t r a i n s may be explained by the absence of an a-N-acetylgalactosaminyltransferase i n the white s t r a i n . Of i n t e r e s t i s the lack of A a c t i v i t y i n red c e l l s and red c e l l g l y c o l i p i d s of the black and white r a t , which i s s t r o n g l y A p o s i t i v e i n i n t e s t i n a l g l y c o l i p i d s . Both s t r a i n s had, however, B a c t i v i t y both i n t h e i r i n t a c t red c e l l s and i n red c e l l g l y c o l i p i d s . Whether t h i s B a c t i v i t y i s based on the same g l y c o l i p i d as found i n n o n - e p i t h e l i a l t i s s u e remains to be shown. We have p r e l i m i n a r y evidence that t h i s g l y c o l i p i d i s a major component of the complex g l y c o l i p i d s of r a t l i v e r . According to Table I, the e p i t h e l i a l c e l l s of the black and white s t r a i n were r i c h e r i n g l y c o l i p i d s , and according to F i g s . 10 and 11 the same s t r a i n contained more f u c o l i p i d expressed as the Htype 3-sugar g l y c o l i p i d . In view of current d i s c u s s i o n s on a p o s s i b l e r o l e of c e l l surface saccharides i n c o n t r o l of growth and d i f f e r e n t i a t i o n (1, 2), the changes found i n e p i t h e l i a l c e l l s undergoing a successive maturation from crypt to v i l l u s t i p are, as a f i r s t impression, s u r p r i s i n g l y small. An increase i n monoglycosylceramide and hematoside and a decrease i n trihexosylceramide, the three major g l y c o l i p i d components, was found. A l s o , the ceramide of these g l y c o l i p i d s undergoes a successive change from crypt to v i l l u s with a chain lengthening and a 2-hydroxylation of the f a t t y a c i d . Concerning the more complex f u c o l i p i d s , these are present already i n the crypt c e l l s (see F i g . 2 f o r 10- and 12-sugar compounds) i n d i c a t i n g "a need" f o r these surface saccharides already i n crypt c e l l s . An extension of the saccharide chains p a r a l l e l to the process of maturation (1, 2) was t h e r e f o r e not found. One should, however, bear i n mind the extreme complexity of the e p i t h e l i a l c e l l being h i g h l y asymmetric with a surface

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C E L L SURFACE GLYCOLIPIDS

membrane (where g l y c o l i p i d s are supposed to be located) d i v i d e d mainly i n t o a brush border, f a c i n g the i n t e s t i n a l lumen, and a b a s o l a t e r a l membrane, being i n contact with other e p i t h e l i a l c e l l s and the b a s a l membrane. So f a r we have only studied whole c e l l s and not yet r e s o l v e d minor components f o r a p r e c i s e q u a n t i t a t i o n . A s u b c e l l u l a r f r a c t i o n a t i o n i n t o separate type of surface membrane and g l y c o l i p i d a n a l y s i s may r e v e a l i n t e r e s t i n g both q u a l i t a t i v e and q u a n t i t a t i v e d i f f e r e n c e s . In f a c t , Lewis and coworkers (21) have shown that preparations of brush border and b a s o l a t e r a l membranes of guinea-pig small i n t e s t i n e had d i f f e r e n t g l y c o l i p i d p a t t e r n s . The g l y c e r o l i p i d s of the two regions were f a i r l y s i m i l a r but t r i - and t e t r a g l y c o s y l c e r a m i d e s were more concentrated i n the b a s o l a t e r a l membranes, whereas mono- and diglycosylceramides and s u l f a t i d e were enriched i n the brush border membranes. For human (16) and dog small i n t e s t i n e 05, 22) i t has been shown that globoside an located i n n o n - e p i t h e l i a e p i t h e l i a l c e l l s . This i s s i m i l a r to the f i n d i n g s of t h i s paper. A l s o , g l y c o l i p i d s of e p i t h e l i a l c e l l s (5, 16^, 22) had a more hydroxylated ceramide (phytosphingosine and 2-hydroxy f a t t y acid) than n o n - e p i t h e l i a l c e l l s (sphingosine and nonhydroxy f a t t y a c i d ) . An analogous s i t u a t i o n was found f o r r a t small i n t e s t i n e , a l though the d i f f e r e n c e s were not that c l e a r c u t , as nonhydroxy a c i d s were a l s o present i n e p i t h e l i a l c e l l s and phytosphingosine was a l s o present to some extent i n n o n - e p i t h e l i a l c e l l s . The extent of 2-hydroxylation increased from crypt to v i l l u s t i p ( F i g s . 2 and 14). The meaning of these d i f f e r e n c e s i n ceramide h y d r o x y l a t i o n (from one to three hydroxy groups) i s not known. A model has, however, been proposed, with a system of l a t e r a l l y o r i e n t e d hydrogen bonds along the membrane at t h i s l e v e l of ceramide i n the membrane matrix (17). The e p i t h e l i a l c e l l s of i n t e s t i n e , e s p e c i a l l y those of the v i l l u s , are exposed to an i n t e s t i n a l content of h i g h l y v a r y i n g composition (both h y d r o p h i l i c and hydrophobic) and may need a more t i g h t and s t a b l e surface membrane produced by an increased h y d r o x y l a t i o n of ceramide. As already mentioned the e p i t h e l i a l c e l l s of small i n t e s t i n e are involved i n a number of enlarged transport processes and a l s o i n b i o l o g i c a l r e c o g n i t i o n . S u r p r i s i n g l y , the a c i d g l y c o l i p i d f r a c t i o n of r a t small i n t e s t i n e lacked the animal s u l f a t i d e (ceramide g a l a c t o s e - 3 - s u l f a t e ) , which i s a major component of human i n t e s t i n e (16) and a l s o of small i n t e s t i n e of s e v e r a l animals (cat, guinea-pig, hen and r a b b i t , unpublished). As f o r the r a t , t h i s l i p i d was absent i n small i n t e s t i n e of mouse and cod f i s h (unpublished). The lack of s u l f a t i d e i s unexpected i n view of the postulated r o l e of t h i s l i p i d as a K receptor i n Na and K transport (17, 18) and the dominance of N a t r a n s p o r t i n small i n t e s t i n e as a primary d r i v e f o r the transport of a number of other molecules. However, i n these cases the molecule may be r e placed by the g l y c e r o l - b a s e d s u l f a t i d e , which i s removed i n the +

+

+

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standard procedure of mild alkaline degradation. The recognition processes of interest in relation to cell surface saccharides and intestinal epithelial cells are of at least two kinds. One is the exposure of primarily the brush border membrane for a number of foreign molecules and microorganisms (or products of these) in the intestinal contents. A role for carbohydrate in the binding of bacteria in the mechanism of infection in epithelia has been postulated (1). The second kind of recognition is the association of autologous cells with each other, which should take place in the alteral membranes, and the attachment of the cells to the basal membrane during movement from crypt to villus tip. As a first step in the study of small intestine the present work has defined to some extent the difference concerning cell surface glycolipids between epithelial and non-epithelial cells and between whole epithelial cells of different maturity to investigate the compositio membranes. Also, the large intestine of the same strains of rat, with a somewhat separate profiel of functions, may profile supplementary information. Acknowledgement The work was supported by a grant from the Swedish Medical Research Council (No. 3967). References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Hughes, C.L.; Sharon, N. Trends Biochem. Sci. 1978, 3, N 275. Marchesi, V.T.; Ginsburg, V.; Robbins, P.W.; Fox, C.F.; Eds. "Cell Surface Carbohydrates and Biological Recognition"; Alan R. Liss, Inc.; New York, 1978. Lipkin, M. Physiol. Rev. 1973, 53, 891. Weiser, M.M. J.Biol. Chem. 1973, 248, 2536. McKibbin, J.M. J. Lipid Res. 1978, 19, 131. Karlsson, K.-A. InWitting,L.A., Ed. "Glycolipid Methodology"; American Oil Chemists' Society: Champaign, Illinois, 1976; p. 97. Breimer, M.E.; Hansson, G.C.; Karlsson, K.-A.; Leffler, H.: Pimlott, W.; Samuelsson, B.E. Biomed. Mass Spectrom. 1979, 6, 231. Falk, K.-E.; Karlsson, K.-A.; Samuelsson, B.E. Arch. Biochem. Biophys. 1979, 192, 164. Falk, K.-E.; Karlsson, K.-A.; Samuelsson, B.E. Arch. Biochem. Biophys. 1979, 192, 177. Falk, K.-E.; Karlsson, K.-A.; Samuelsson, B.E. Arch. Biochem. Biophys. 1979, 192, 191.

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11.

Laine, R.A.; Stellner, K.; Hakomori, S.-i. In Korn, E.D., Ed. "Methods in Membrane Biology"; Plenum Press: New York, 1974; Vol. 2, p. 205. Bouhours, J.-F.; Glickman, R.M. Biochim. Biophys. Acta 1976, 441, 123. Glickman, R.M.; Bouhours, J.-F. Biochim. Biophys. Acta 1976, 424, 17. Karlsson, K.-A.; Samuelsson, B.E.; Steen, G.O. Biochim. Biophys. Acta 1973, 306, 317. Morgan, I.G.; Gombos, G.; Tettamanti, G. In Horowitz, M.I.; Pigman, W.; Eds. "The Glycoconjugates"; Academic Press: New York, 1977, Vol. I, p. 351. Falk, K.-E.; Karlsson, K.-A.; Leffler, H.; Samuelsson, B.E. FEBS Lett. 1979, 101, 273. Karlsson, K.-A. In Abrahamsson, S.; Pascher, I.; Eds. "Structure of Biologica 1977, p. 245. Hansson, G.C.; Heilbronn, E.; Karlsson, K.-A.; Samuelsson, B.E. J. Lipid Res. 1979, 20, 509. Hanada, E.; Handa, S.; Konno, K.; Yamakawa, T. J. Biochem. 1978, 83, 85. Kobata, A. In Horowitz, M.I.; Pigman, W.; Eds. "The Glycoconjugates"; Academic Press: New York, 1977, Vol. I, p. 423. Michell, R.H.; Coleman, R.; Lewis, B.A. Biochem. Soc. Trans. 1976, 4, 1017. Smith, E.L.; McKibbin, J.M.; Karlsson, K.-A.; Pascher, I.; Samuelsson, B.E. Biochim. Biophys. Acta 1975, 388, 171. Dell, A.; Williams, D.H.; Morris, H.R.; Smith, G.A.; Feeney, J.; Roberts, G.C.K. J. Am. Chem. Soc. 1975, 97, 2497.

12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.

RECEIVED

December 10, 1979.

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

7 Galactoglycerolipids of Mammalian Testis, Spermatozoa, and Nervous Tissue ROBERT K. MURRAY, RAJAGOPOLIAN NARASIMHAN, MARK LEVINE, and LES PINTERIC Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada M5S 1A8 MARGARET SHIRLEY, CLIFFORD LINGWOOD, and HARRY SCHACTER Department of Biochemistry, Hospital for Sick Children, Research Institute, Toronto, Ontario, Canada M5G 1X8 The two major classe cells are glycosphingolipid with certain members of the latter class that this article is concerned. Glycoglycerolipids are well established constituents of plant and bacterial cells (2,3,4). Galactosyl- and digalactosyldiacylglycerols are the major glycoglycerolipids found in plant cells, although trigalactosyldiacylglycerol, 6-O-acylgalactosyldiacylglycerol and sulfoquinovosyldiacylglycerol have also been described (4). In bacteria, mono- and di- glycosyldiacylglycerols occur most frequently, with the latter generally being the major species. Glucose, galactose and mannose are the usual sugars present in these compounds. Certain of these lipids also contain uronic acids. Halobacterium cutirubrum contains a glycolipid with galactose-sulfate, mannose and glucose linked to a phytanyl diether glyceride (5). Acyl substitutions on the sugar residues of diglycosylglycerolipids have also been described, as have phosphoglycoglycerolipids (4). The presence of glycoglycerolipids in mammalian tissues, specifically nervous tissue, has been known since 1963 (6). Most of the mammalian glycoglycerolipids have been found to contain galactose as their sole sugar; however, the presence in gastric juice and saliva of a novel series of glucoglycerolipids has been described recently (7,8,9). Of the galactoglycerolipids, galactosyl- and digalactosyl- diacylglycerols have received especial attention. An analog of galactosyldiacylglycerol, galactosylalkylacylglycerol, was also found in brain (10) shortly after the initial report of the presence of the diacyl compound in that organ (6). Interest in mammalian galactoglycerolipids accelerated when i t was discovered that the sulfated derivative of the lipid described by Norton and Brotz (10) was the major glycolipid of rat (11) and boar (12) testis. This sulfated galactolipid was partially characterized in a number of studies (e.g. 13 and 14) and has subsequently been fully characterized as l-O-alkyl-2-0acyl-3-0-$-D-(3'-sulfo)-galactopyranosyl-sn-glycerol (15). A variety of topics emerging from the study of this particular glycolipid have been reviewed previously (16). The present article will 0-8412-0556-6/ 80/ 47-128-10555.25/ 0 © 1980 American Chemical Society In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

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concentrate p r i m a r i l y on features concerning t h i s and s e v e r a l c l o s e l y r e l a t e d g a l a c t o g l y c e r o l i p i d s that have a r i s e n s i n c e the above mentioned review was w r i t t e n i n mid 1975. Many aspects of the biochemistry of the v a r i o u s s u l f a t e - c o n t a i n i n g g l y c o l i p i d s found i n mammalian t i s s u e s have r e c e n t l y been reviewed by Sweeley and S i d d i q u i (1), Dulaney and Moser (17) and Farooqui (18). Nomenclature, C l a s s i f i c a t i o n and

Tissue D i s t r i b u t i o n ,

Both l - 0 - a l k y l - 2 - 0 - a c y l - 3 - 0 - g - D - ( 3 - s u l f o ) - g a l a c t o p y r a n o s y l s n - g l y c e r o l and i t s non-sulfated species are major g l y c o l i p i d s of the t e s t i s and spermatozoa of a number of higher animals, i n c l u d ing humans (16). Despite the previous usage of names such as s e m i n o l i p i d (12), s u l f o g l y c e r o g a l a c t o l i p i d (19) and s u l f o g a l a c t o g l y c e r o l i p i d (20) to describe the s u l f a t e d species, i t now appears that s u l f o g a l a c t o s y l a l k y l a c y l g l y c e r o l i s the most chemically informative t r i v i a l nam This a r i s e s from the f a c certainly l-0-acyl-2-0-acyl-3-0-3-D-(3 -sulfo)-galactopyranosyls n - g l y c e r o l , has been i s o l a t e d from b r a i n (21,22). The term s u l f o g a l a c t o g l y c e r o l i p i d would not d i s t i n g u i s h between these two compounds, p a r t i c u l a r l y when r e f e r r i n g to an organ such as r a t brain, i n which they c o - e x i s t (22,23). Hence, i t i s more p r e c i s e to r e f e r to the ether-containing l i p i d as s u l f o g a l a c t o s y l a l k y l a c y l g l y c e r o l (SGG) and to the d i a c y l - c o n t a i n i n g l i p i d as s u l f o g a l a c t o s y l d i a c y l g l y c e r o l (22). The non-sulfated species of these two l i p i d s w i l l be r e f e r r e d to as g a l a c t o s y l a l k y l a c y l g l y c e r o l (GG) and galactosyldiacylglycerol respectively. f

A c l a s s i f i c a t i o n of mammalian g a l a c t o g l y c e r o l i p i d s below. Table I. C l a s s i f i c a t i o n of Mammalian D i a c y l Sub-Class (A) G a l a c t o s y l d i a c y l g l y c e r o l (B) S u l f o g a l a c t o s y l d i a c y l g l y c e r o l (C) D i g a l a c t o s y l d i a c y l g l y c e r o l

i s given

Galactoglycerolipids A l k y l a c y l Sub-Class

(D)

Galactosylalkylacylg l y c e r o l (GG) (E) S u l f o g a l a c t o s y l a l k y l a c y l glycerol (SGG) (F) D i g a l a c t o s y l a l k y l a c y l glycerol

Several features of t h i s c l a s s i f i c a t i o n merit comment. Six l i p i d s have been included i n the Table, but the i d e n t i f i c a t i o n of two of them [(C) and (F)] i s not f i r m l y e s t a b l i s h e d . L i p i d (C) was t e n t a t i v e l y i d e n t i f i e d i n human b r a i n (24); e x t r a c t s of r a t b r a i n appear to be able to c a t a l y s e i t s formation when incubated under appropriate c o n d i t i o n s (25) ( t h i s i s discussed i n more det a i l subsequently). L i p i d (F) was detected i n human t e s t i s and sperm (26), and e x h i b i t e d chromatographic and other p r o p e r t i e s corresponding to what would be expected from a d i g a l a c t o s y l -

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containing a l k y l a c y l g l y c e r o l . The systematic names f o r l i p i d s (A) and (C) are l-0-acyl-2-0-acyl-3-0-$-D-galactopyranosyl-sn-glycero l and l-0-acyl-2-0-acy1-3-0-[a-D-galactopyranosyl(l-*?)B-D-galact o p y r a n o s y l ] - s n - g l y c e r o l ; these l i p i d s are s t i l l widely r e f e r r e d to as monogalactosyl and d i g a l a c t o s y l d i g l y c e r i d e r e s p e c t i v e l y . Systematic names f o r l i p i d s (B), (D) and (E) were i n d i c a t e d above. I t i s premature to assign a systematic name to l i p i d ( F ) ; i t w i l l be of i n t e r e s t to determine whether the anomeric natures of the two g a l a c t o s y l residues are s i m i l a r to those i n l i p i d (C). With regard t o t h e i r d i s t r i b u t i o n i n mammalian t i s s u e s , compounds (A), (B) and (C) have been detected only i n nervous t i s s u e , compounds (D) and (E) i n both nervous t i s s u e and t e s t i s and spermatozoa, and compound (F) only i n human t e s t i s and spermatozoa. However, p r e l i m i n a r y evidence has been obtained (M. Levine and R.K. Murray, unpublished observations), suggesting that small amounts of compounds (A) and (B) may be present i n dog t e s t i s along with l a r g e r amount E x t r a c t i o n of

Galactoglycerolipids

We have found the method of Suzuki (27) to be s a t i s f a c t o r y f o r e x t r a c t i n g these l i p i d s from t e s t i s , sperm and b r a i n . A moderate l o s s of s u l f a t e - c o n t a i n i n g g a l a c t o g l y c e r o l i p i d s i n t o the upper phase of t h e F o l c h e x t r a c t employed i n t h i s method occurs. Using the method of column chromatography on s i l i c i c a c i d d e v e l oped by Vance and Sweeley (28), the g a l a c t o g l y c e r o l i p i d s shown i n Table I are a l l e l u t e d by acetone subsequent to i n i t i a l e l u t i o n of the column by chloroform. A f t e r evaporation of the acetone, i n d i v i d u a l g l y c o l i p i d s can be p u r i f i e d by p r e p a r a t i v e t h i n l a y e r chromatography. I f the g l y c o l i p i d composition of the t i s s u e under study i s complex ( c f . human t e s t i s (26)), f r a c t i o n a t i o n of these l i p i d s by chromatography using DEAE-cellulose (29) i s u s e f u l . Chemical C h a r a c t e r i z a t i o n

of G a l a c t o s y l a l k y l a c y l g l y c e r o l s

Table I I l i s t s the main procedures that have been used to q u a n t i t a t e the amounts of these l i p i d s present i n t e s t i s , sperm and b r a i n and to determine t h e i r chemical s t r u c t u r e s . Reference to some of the techniques a p p l i e d to the c h a r a c t e r i z a t i o n of s u l f o g a l a c t o s y l d i a c y l g l y c e r o l are a l s o included. One technique that we have found u s e f u l i n p e r m i t t i n g an i n i t i a l d i s t i n c t i o n between g l y c o s p h i n g o l i p i d s , g a l a c t o s y l a l k y l a c y l g l y c e r o l s and g a l a c t o s y l d i a c y l g l y c e r o l s i s the use of b r i e f h y d r o l y s i s i n m i l d a l k a l i ( c f . 20). T h i s can be a p p l i e d to e i t h e r the t o t a l g l y c o l i p i d e x t r a c t or to p u r i f i e d g l y c o l i p i d s . T y p i c a l r e s u l t s of t h i s procedure using a member of each of the above three c l a s s e s of g l y c o l i p i d s are shown i n F i g u r e 1. I t should be apparent that the use of t h i s treatment to remove a l k a l i - l a b i l e contaminating l i p i d s (e.g. phospholipids) from a g l y c o l i p i d ext r a c t i s unwise, u n t i l a f t e r a p r e l i m i n a r y a n a l y s i s has been

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Table I I . Procedures Used to Quantitate and C h a r a c t e r i z e Galactoglycerolipids Procedure

General Analyses: Determination of sugar, g l y c e r y l ethers and f a t t y a c i d s by GLC S i m i l a r analyses by GLC-MS Elemental a n a l y s i s Q u a n t i t a t i o n by HPLC Analyses of the S u l f a t e Moiety: Detection of [35s] s u l f a t e Benzidine method Sodium r h o d i z i n a t e metho Estimation of l i p i d - b o u n d sulfate IR spectroscopy Removal of s u l f a t e by hydrol y s i s i n mild acid Removal of s u l f a t e by s o l v o l y s i s i n dioxane Removal of s u l f a t e by a r y l sulfatase A Elution i n salts fraction during DEAE-cellulose chromatography Permethylation * Determination of attachment to galactose by r e s i s t a n c e to treatment with p e r i o d a t e Analyses of the Galactose Moieties: Determination of anomeric l i n k age by IR and NMR Determination of anomeric l i n k age by use o f (S-galactosidase E s t i m a t i o n of amount u s i n g galactose dehydrogenase Estimation of amount u s i n g fluorimetry Analyses of G l y c e r y l E t h e r s : Determination of isomers by TLC Stereochemical a n a l y s i s by optical rotatory dispersion

Compound Studied

Reference

Rat t e s t i s SGG Boar t e s t i s SGG

(11) (12)

Human t e s t i s SGG Rat b r a i n SGG Boar t e s t i s SGG Rat t e s t i s SGG

(15) (23) (12) (30)

Rat t e s t i s SGG

(11)

Rat t e s t i s SGG

(11)

Boar t e s t i s SGG Rat t e s t i s SGG

(12) (11)

Rat t e s t i s SGG

(19)

Rat t e s t i s SGG

(31)

Rat b r a i n SGG

(20)

Boar t e s t i s SGG Rat b r a i n s u l f o galactosyldiacylglycerol

(12) (21)

Boar t e s t i s SGG

(12)

Rat b r a i n SGG

(23)

Guinea p i g t e s t i s SGG

(13)

Human t e s t i s SGG

(15)

Rat t e s t i s SGG

(14)

Human t e s t i s SGG

(15)

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

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Galactoglycero lip ids

Table I I (continued) Procedure

Compound Studied

S u s c e p t i b i l i t y to d e - a c y l a t i o n by m i l d a l k a l i Other A n a l y s i s : I s o l a t i o n of g a l a c t o s y l glycerol

Guinea p i g t e s t i s SGG Rat b r a i n SGG

Use of Spray Reagents to Exclude Other C o n s t i t u e n t s : Ninhydrin (free amino group) Benzidine (sphingosine) 2,4-dinitrophenylhydrazine (plasmalogenic linkage) B i a l ' s o r c i n o l reagent ( s i a l i c acid) A c i d molybdate (phosphate)

Reference

(13) (20)

galactosyldiacyl glycerol

Boar t e s t i s SGG Rat t e s t i s SGG Rat t e s t i s SGG

(12) (11) (11)

Boar t e s t i s SGG

(12)

Rat t e s t i s SGG

(11)

*This procedure n a t u r a l l y a l s o y i e l d s information on the nature of the galactose m o i e t i e s . The methods r e f e r r e d to i n t h i s Table have been used to q u a n t i tate and c h a r a c t e r i z e the t e s t i c u l a r SGG and GG species and a l s o the corresponding l i p i d s and s u l f o g a l a c t o s y l d i a c y l g l y c e r o l from b r a i n . References to s t u d i e s performed p r i o r to 1972 that c h a r a c t e r i z e d the g a l a c t o g l y c e r o l i p i d s of nervous t i s s u e have not been i n c l u d e d . Abbreviations: GLC, g a s - l i q u i d chromatography; MS, mass spectrometry; HPLC, high performance l i q u i d chromatography; IR, i n f r a - r e d ; DEAE, d i e t h y l a m i n o e t h y l ; NMR, nuclear magnetic resonance; TLC, t h i n l a y e r chromatography.

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

C E L L SURFACE

GLYCOLIPIDS

FR.

OR.

1

2 3 4

5

6

Figure 1. Schematic of the effects of brief treatment with mild alkali on the thinlayer chromatographic migrations of three types of glycolipids. 1,2: control and alkali-treated neutral glycosphingolipid; 3, 4: control and alkali-treated galactosylalky lacy Iglycerol; 5, 6: control and alkali-treated galactosyldiacylglycerol. OR: origin; FR: solvent front. The neutral glycosphingolipid is represented as a characteristic double band. We have not observed galactoglycerolipids to migrate as double bands on thin layer chromatography. Glucosyl- and lactosyl-ceramides exhibit the behavior (i.e., lack of effect of mild alkali on their migrations) of the compound shown in channels 1 and 2. SGG and GG behave in the same way as the compound represented in channels 3 and 4; the slower migrating product in channel 4 in the case of these two compounds would correspond to lyso-SGG and lyso-GG, respectively. Both galactosyl- and digalactosyldiacylgycerols show the behavior of the compound in channels 5 and 6; the product migrating at the origin in channel 6 in the case of these two compounds would correspond to galactosyIglycerol and digalactosylglycerol, respectively. Cerebroside esters are one type of glycosphingolipid whose migration would be affected by the above treatment. Conversely, if galactosyIdialkyglycerols exist in mammalian ceils, their chromatographic migrations would not be affected by treatment with mild alkali.

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performed on c o n t r o l and a l k a l i - t r e a t e d samples to determine i f the chromatographic migrations of any of the g l y c o l i p i d s present are a f f e c t e d by i t . The i n i t i a l c h a r a c t e r i z a t i o n s t u d i e s of the SGG derived from r a t (11) and boar (12) t e s t i s revealed the presence of approximately s t o i c h i o m e t r i c amounts of s u l f a t e , galactose, f a t t y a c i d and g l y c e r y l ether. Using NMR spectroscopy, the study of Ishizuka et a l . (12) a l s o suggested the 3 nature of the g a l a c t o s i d i c l i n k age to g l y c e r o l and the p o s i t i o n of the a c y l chain on carbon 2 of g l y c e r o l . In a d d i t i o n , analyses of the products of permethylation i n d i c a t e d that the s u l f a t e was attached to the 3 p o s i t i o n of gal a c t o s e . Measurement of the o p t i c a l r o t a t o r y d i s p e r s i o n of the g l y c e r y l ether moiety (15) e s t a b l i s h e d the d e f i n i t i v e s t r u c t u r e of SGG. Perhaps the most remarkable f e a t u r e of the SGG derived from t e s t i s i s i t s extremely r e s t r i c t e d a l k y l and a c y l composition In the case of the SGG o and human (15,26) t e s t i s t i o n i s comprised of saturated 16 carbon moieties [ g l y c e r y l - 1 hexadecyl ether (chimyl a l c o h o l ) and hexadecanoic a c i d ( p a l m i t i c acid) r e s p e c t i v e l y ] . The SGG present i n r a t b r a i n appears to have a l e s s r e s t r i c t e d a l k y l and a c y l composition (20). 1

Biosynthesis

of T e s t i c u l a r and

Other G a l a c t o g l y c e r o l i p i d s

35 A number of s t u d i e s (11,13,14,32) have shown that [ S] s u l f a t e i s incorporated i n v i v o i n t o t e s t i c u l a r SGG. With regard to the mechanism i n v o l v e d , both Knapp et_ a l . (19) and Handa et a l . (32) have demonstrated formation of t h i s l i p i d i n v i t r o from GG by t r a n s f e r of s u l f a t e from 3 -phosphoadenosine-5'-phosphosulfate (PAPS), i n analogy with the pathway of b i o s y n t h e s i s of s u l f o g a l a c tosylceramide from galactosylceramide (reviewed i n 17). Other g l y c o l i p i d s with a terminal 3 - g a l a c t o s y l residue ( g a l a c t o s y l - and l a c t o s y l - ceramides and g a l a c t o s y l d i a c y l g l y c e r o l ) were found to be s u l f a t e d by the enzyme preparations employed, whereas compounds with a terminal ot-galactosyl residue ( g a l a c t o s y l g a l a c t o s y l g l u c o sylceramide and d i g a l a c t o s y l d i a c y l g l y c e r o l ) were not. Both of these s t u d i e s suggested that p r i m a r i l y one s u l f o t r a n s f e r a s e was involved i n the s u l f a t i o n of the v a r i o u s g l y c o l i p i d substrates; however, t h i s i s s u e i s not s e t t l e d c o n c l u s i v e l y . The sulf©transferase a c t i v i t y i n r a t t e s t i s (19) was markedly enriched i n a G o l g i apparatus f r a c t i o n of that organ, confirming the i n v o l v e ment of that o r g a n e l l e i n both s u l f a t i o n processes (33) and i n the b i o s y n t h e s i s of g l y c o l i p i d s (34,35). W e l l before the above s t u d i e s were performed, the biosynthes i s of g a l a c t o s y l d i a c y l g l y c e r o l i n r a t b r a i n had been examined by Wenger et_ a l . (36). These workers found a 3 - g a l a c t o s y l t r a n s f e r a s e a c t i v i t y capable of c a t a l y s i n g the f o l l o w i n g r e a c t i o n : f

1 , 2 - d i a c y l g l y c e r o l + UDP-gal •> G a l a c t o s y l d i a c y l g l y c e r o l +

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

UDP

112

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Wenger £t a l . (25) l a t e r described the presence i n r a t b r a i n of an a - g a l a c t o s y l t r a n s f e r a s e a c t i v i t y , that used the product of the above r e a c t i o n as substrate and UDP-gal as donor to c a t a l y s e the formation of a second l i p i d , t e n t a t i v e l y assigned the s t r u c t u r e of d i g a l a c t o s y l d i a c y l g l y c e r o l (see e a r l i e r d i s c u s s i o n ) . Subsequently, Flynn et^ a l . (21) demonstrated the presence i n r a t b r a i n of a s u l f o t r a n s f e r a s e a c t i v i t y capable of s u l f a t i n g g a l a c t o s y l d i a c y l g l y c e r o l . The p r o p e r t i e s of t h i s enzyme a c t i v i t y were d e s c r i b ed i n more d e t a i l by Subba Rao et a l . (37). I n t e r e s t i n g l y , s i g n i f i c a n t d i f f e r e n c e s were observed between the formation of s u l f o g a l a c t o s y l d i a c y l g l y c e r o l and s u l f o g a l a c t o s y l c e r a m i d e , when c a t a l ysed by the enzyme preparation used. The data d i d not n e c e s s a r i l y lead to the conclusion that two sulf©transferases were present, but they d i d i n d i c a t e how c e r t a i n f a c t o r s (e.g. ATP and M g conc e n t r a t i o n s ) could c o n t r o l the r e l a t i v e amounts of these two l i p i d s that were synthesized In analogy with th syldiacylglycerol i n brain a b i l i t y of r a t t e s t i c u l a r e x t r a c t s to c a t a l y s e the f o l l o w i n g r e action: 2 +

1 , 2 - a l k y l a c y l g l y c e r o l + UDP-gal •> GG +

UDP

So f a r , although a v a r i e t y of c o n d i t i o n s of incubation have been i n v e s t i g a t e d , convincing evidence f o r the occurrence of t h i s r e a c t i o n i n r a t t e s t i s has not been obtained. The s i g n i f i c a n c e of a negative f i n d i n g of t h i s nature i s l i m i t e d , as i t may only r e f l e c t a f a i l u r e to s e l e c t appropriate c o n d i t i o n s . A l t e r n a t i v e l y , the p u t a t i v e g a l a c t o s y l t r a n s f e r a s e may be extremely l a b i l e or present i n very low a c t i v i t y . However, i t i s a l s o p o s s i b l e that another pathway, using a d i f f e r e n t acceptor molecule and/or a d i f f e r e n t g a l a c t o s y l donor, may be i n v o l v e d . The u t i l i z a t i o n of galactose f o r the i n v i v o b i o s y n t h e s i s of GG and SGG by r a t t e s t i s has a l s o been examined (38,39). [ C ] galactose was i n j e c t e d i n t o the t e s t e s of adult r a t s and the speci f i c a c t i v i t i e s of the g a l a c t o s y l moieties of these two l i p i d s determined at v a r i o u s time i n t e r v a l s . L a b e l l e d galactose appeared i n GG by 10 minutes, the peak s p e c i f i c a c t i v i t y o c c u r r i n g by 2 h a f t e r i n j e c t i o n , and d e c l i n i n g t h e r e a f t e r r e l a t i v e l y r a p i d l y . In c o n t r a s t , the appearance of r a d i o a c t i v e galactose i n the SGG was much slower (detectable by 1 h ) , i t s peak s p e c i f i c a c t i v i t y occurr i n g by 72 h a f t e r i n j e c t i o n . Moreover, the s p e c i f i c a c t i v i t y of the SGG subsequently d e c l i n e d very slowly over the f o l l o w i n g 2 weeks. These r e s u l t s are c o n s i s t e n t with the hypothesis that GG i s the precursor of SGG i n v i v o ; however, they do not prove t h i s , nor do they i n d i c a t e from which precursor GG i t s e l f i s s y n t h e s i z e d Thus, although i t appears reasonable to assume that s u l f a t i o n i s the f i n a l step i n the b i o s y n t h e s i s of t e s t i c u l a r SGG, l i t t l e i s known of the e a r l i e r steps. The pathway of b i o s y n t h e s i s of the g l y c e r y l ether backbone of 1 4

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the t e s t i c u l a r SGG a l s o remains unexplored; i n view of the h i g h l y r e s t r i c t e d a l k y l and a c y l composition of the l i p i d , i t would be of i n t e r e s t to determine the substrate s p e c i f i c i t i e s of the enzymes involved i n formation and t r a n s f e r of these moieties. Catabolism of

SGG

Yamato et a l . (31) p u r i f i e d a r y l s u l f a t a s e A from boar t e s t i s . The s p e c i f i c a c t i v i t i e s of the enzyme preparation towards three substrates - 4 - n i t r o c a t e c h o l s u l f a t e , SGG and s u l f o g a l a c t o s y l c e r amide - increased at almost the same r a t e through the v a r i o u s p u r i f i c a t i o n steps employed. The optimal pH f o r a c t i o n on both of the g l y c o l i p i d substrates was 4.5. The a c t i v i t y of the enzyme was somewhat greater using the s p h i n g o l i p i d as s u b s t r a t e , as compared with SGG. A v a r i e t y of procedures i n d i c a t e d that the two glycol i p i d s were both substrates f o r the enzyme I t was suggested that SGG may be the p h y s i o l o g i c a testis. Essentially simila a l . (40), who examined the a c t i o n of the same enzyme, but p u r i f i e d from human u r i n e , on r a t t e s t i c u l a r SGG and on s u l f o g a l a c t o s y l c e r amide. Neither SGG nor c l a s s i c a l s u l f a t i d e was a substrate f o r a r y l s u l f a t a s e B. Again, these workers concluded that SGG appears to be a p h y s i o l o g i c a l substrate f o r a r y l s u l f a t a s e A. F l u h a r t y et_ a l . a l s o pointed out that a r y l s u l f a t a s e A has been found i n r a b b i t sperm acrosomes, i n which i t was suggested that i t might be i n volved i n the p e n e t r a t i o n of spermatozoa through the investments of the ovum (41). An i n t e r e s t i n g extension of the above work was performed by Yamaguchi et a l . (42). They compared the a c t i v i t i e s towards n i t r o catechol s u l f a t e , SGG and s u l f o g a l a c t o s y l c e r a m i d e of enzyme ext r a c t s from normal human b r a i n and from two cases of a l a t e i n f a n t i l e form of metachromatic leukodystrophy (MLD). The a c t i v i t i e s towards a l l three substrates were markedly d e f i c i e n t (1-5% of cont r o l a c t i v i t i e s ) i n the e x t r a c t s from the diseased b r a i n s . The authors concluded that the enzyme d e f i c i e n c y i n the type of MLD studied was due to a s i n g l e s u l f a t a s e , c a t a l y s i n g the degradation of a l l three substrates used. I t has so f a r not proven p o s s i b l e to determine whether SGG accumulates i n the t e s t e s of a d u l t s with l a t e developing forms of MLD. Nor has i t been e s t a b l i s h e d whether SGG can accumulate i n human b r a i n i n t h i s c o n d i t i o n ; indeed, two s t u d i e s have f a i l e d to demonstrate i t s presence i n that organ (_20,23). However, i t i s p o s s i b l e that the l i p i d could have a very r e s t r i c t e d l o c a t i o n i n human b r a i n . R e i t e r et_ a l . (43) have shown that a second enzyme can a l s o act to degrade SGG. They found that secondary lysosomes from r a t l i v e r contained not only a r y l s u l f a t a s e A, but a l s o a l i p a s e a c t i v i t y that could act to de-acylate SGG. Under the c o n d i t i o n s used, more product was formed by the a c t i o n of the l i p a s e on SGG than by the a c t i o n of a r y l s u l f a t a s e A. These workers a l s o found that the l a t t e r enzyme could use the lyso-SGG as a substrate. I t would be

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of i n t e r e s t to study the a c t i v i t y of the l i p a s e on GG and a l s o on the d i a c y l - c o n t a i n i n g g a l a c t o g l y c e r o l i p i d s . At the present time, the r e l a t i v e p h y s i o l o g i c a l s i g n i f i c a n c e of the two pathways of degradation of SGG has not been e s t a b l i s h e d . However, the l i p a s e a c t i v i t y has not so f a r been reported to be present i n t e s t i s . Further steps i n the catabolism of SGG i n t e s t i s - e.g. removal of the g a l a c t o s y l residue and degradation of the g l y c e r y l ether moieties - have apparently not yet been examined. I t has been shown that a B-galactosidase (E.C. 3.2.1.23) from Charonia lampas i s capable of removing the g a l a c t o s y l residue from both the GG and g a l a c t o s y l d i a c y l g l y c e r o l species d e r i v e d from r a t b r a i n (23). Appearance of S u l f a t i d e s During T e s t i c u l a r Development One approach towards determining the p a r t i c u l a r c e l l stage at which phenotypic products ( i n the present case c e r t a i n s p e c i f i c g l y c o l i p i d s ) of d i f f e r e n t i a t i o that organ from animal ance of the compound(s) under study with the appearance of a p a r t i c u l a r c e l l type as determined by h i s t o l o g i c examination. The time of appearance i n the t e s t i s of the v a r i o u s c e l l types i n v o l ved i n spermatogenesis has been p a r t i c u l a r l y w e l l e s t a b l i s h e d i n the case of the r a t by Clermont and Perey (44). Using t h i s approach , K o r n b l a t t et_ a l . (14) found that primary spermatocytes appeared to be the e a r l i e s t spermatogenic c e l l s to contain high l e v e l s of the SGG. Examination of the l e v e l s of SGG i n the t e s t e s of immature r a t s , hypophysectomized r a t s and normal and s t e r i l e mice i n d i c a t e d that the m a j o r i t y of the SGG was l o c a t e d i n the germinal (spermatogenic) c e l l s (as opposed to non-germinal c e l l s , such as S e r t o l i and Leydig c e l l s ) of the t e s t i s . Another f i n d i n g that r e i n f o r c e s the probable germ c e l l l o c a t i o n of the SGG was made by Suzuki et^ a l . (30). These workers fed a d u l t r a t s a d i e t d e f i c i e n t i n v i t a m i n A f o r 46 days. This r e s u l t e d i n a d e c l i n e of SGG to 13% of i t s l e v e l i n the t e s t e s of appropriate c o n t r o l animals. T o t a l l i p i d , p h o s p h o l i p i d and DNA (expressed a p p r o p r i a t e l y ) were only s l i g h t l y reduced. H i s t o l o g i c examination showed that the t e s t e s were aspermatogenic. Vitamin A i s o b v i o u s l y necessary f o r the maintenance of germ c e l l maturat i o n ; i t would be of great i n t e r e s t to determine i f i t p l a y s any s p e c i f i c r o l e i n the b i o s y n t h e s i s of SGG. A dramatic i n c r e a s e (approx. 50-fold) of the a c t i v i t y of the s u l f o t r a n s f e r a s e i n v o l v e d i n the b i o s y n t h e s i s of the SGG a l s o occurred when spermatocytes f i r s t began to appear i n r a t t e s t i s (19); the r i s e i n the a c t i v i t y of t h i s enzyme preceded by s e v e r a l days a marked r i s e i n the amount of the SGG. Studies on p r e p u b e r t a l human t e s t i s (which i s temporarily blocked i n spermatogenesis at a stage p r i o r to the appearance of primary spermatocytes) have shown that n e i t h e r SGG nor GG i s present (15,26). S i m i l a r l y , the t e s t i s of the p r e - p u b e r t a l fowl a l s o lacks sulfogalactosylceramide, the s u l f a t i d e found i n mature fowl t e s t i s (26). A l l of these f i n d -

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ings are c o n s i s t e n t with the hypothesis that s u l f a t i d e s are synthesized i n the t e s t i s of a v a r i e t y of species when e a r l y spermatocytes appear i n that organ. L e t t s et^ a l . (45) have attempted to answer the question of which c e l l population i n r a t t e s t i s synthesizes the SGG by using methods that allowed f r a c t i o n a t i o n of d i f f e r e n t t e s t i c u l a r c e l l types. T h e i r r e s u l t s i n d i c a t e d that s u l f a t i o n of SGG occurred a t a c e l l stage p r i o r to the l a t e (pachytene and diplotene) spermatocyte stage. L e t t s et a l . (45) a l s o assayed the amount of r a d i o a c t i v e SGG i n e x t r a c t s of t e s t i s and epididymis at i n c r e a s i n g times a f t e r the i n j e c t i o n of [ S ] s u l f a t e i n t o the t e s t e s of adult r a t s . The epididymis showed no r a d i o a c t i v e SGG f o r 4 weeks f o l l o w i n g i n j e c t i o n , but e x h i b i t e d a dramatic appearance of the [35s]-labelled compound at 5 weeks. From previous studies on the k i n e t i c s of spermatogenes i s i n r a t s , i t was p o s s i b l e f o r these workers to conclude that s u l f a t e i n c o r p o r a t i o n i n t o SGG must occur p r i o r to the spermatid stage. These workers a l s t e s t i s decreased s t e a d i l y injectio Kornblatt (46) has made s i m i l a r observations to the above. With respect to the l a s t p o i n t , she found that the rate of the decrease of [35s]-labelled SGG i n t e s t i s coincided e x a c t l y with the rate of decrease of [3H]thymidine-labelled DNA l e v e l s i n t e s t i s . This i n d i c a t e s that the l o s s of l i p i d was due to c e l l death and that there was minimal turnover of SGG i n s u r v i v i n g c e l l s . To summarize, the r e s u l t s from both of these studies suggest that the SGG i s s u l fated at the e a r l y primary spermatocyte stage. The s u l f o l i p i d then appears to undergo l i t t l e or no turnover i n the germinal c e l l s during spermatogenesis and e v e n t u a l l y appears i n the spermatozoa. This i s an i n t r i g u i n g f i n d i n g which implies that the l i p i d appears i n t e s t i s at a c e l l stage w e l l before the spermatozoon and p e r s i s t s i n a m e t a b o l i c a l l y s t a b l e form throughout a l l the complex c e l l mode l l i n g processes that precede and accompany the appearance of the h i g h l y s p e c i a l i z e d sperm c e l l . I t should be noted, however, that the above studies with [35s] s u l f a t e do not exclude the p o s s i b i l i t y that other moieties of the SGG - e.g. the a c y l group - could exh i b i t turnover. Suzuki et a l . (13) showed that boar spermatozoa possessed l i t t l e or no c a p a c i t y to incorporate [35s] s u l f a t e i n t o SGG. Narasimhan et^ al.(39) have confirmed the very l i m i t e d , i f not negl i g i b l e , capacity of sperm to synthesize SGG by incubating bovine spermatozoa with l a b e l l e d g l y c e r o l and galactose. No r a d i o a c t i v i t y was detected i n the SGG f o l l o w i n g incubation with these compounds. R a d i o a c t i v i t y from these compounds was, however, found to be i n corporated i n t o SGG when they were i n j e c t e d i n t o the t e s t e s of mature r a t s . A l s o relevant to the appearance of SGG during t e s t i c u l a r d i f f e r e n t i a t i o n were the r e s u l t s of a study performed by Ishizuka and Yamakawa (47). These workers analysed the g l y c o l i p i d composition of three human seminoma ( t e s t i c u l a r ) tumors. Unlike the c o n t r o l human t e s t i c u l a r t i s s u e , no SGG or GG was detected i n the tumors. 3 5

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As many malignant tumors resemble f e t a l t i s s u e i n t h e i r biochemic a l composition, t h i s r e s u l t i s c o n s i s t e n t with the observed absence of SGG from immature human t e s t i s (15,26). Another t e n t a t i v e i n t e r p r e t a t i o n of t h i s f i n d i n g i s that seminoma c e l l s d e r i v e from a c e l l stage p r i o r to that of the primary spermatocyte, thus accounting f o r t h e i r i n a b i l i t y to synthesize SGG. Subcellular

L o c a t i o n of SGG

i n T e s t i s and

Spermatozoa

Both L e t t s et a l . (45) and Kornblatt (46), using s u b c e l l u l a r f r a c t i o n a t i o n techniques, have obtained evidence i n d i c a t i n g that at l e a s t some of the SGG i n t e s t i s i s present i n the plasma membrane f r a c t i o n of germinal c e l l s . Further s t u d i e s of t h i s subject are i n progress i n the l a b o r a t o r i e s of these workers. Levine et_ a l . (38) have i s o l a t e d head and t a i l f r a c t i o n s of bovine spermatozoa f o l l o w i n g mild treatment of these c e l l s with pronase; the SGG was found to be d i s t r i b u t e result i s c o n s i s t e n t wit membrane, as t h i s s t r u c t u r e i s continuous around the spermatozoon. I t i s apparent that treatment with a r y l s u l f a t a s e A might y i e l d information on the exposure of the s u l f a t e group of the SGG on the surface of these c e l l s and could a l s o provide a u s e f u l t o o l f o r studying the e f f e c t s on spermatozoal f u n c t i o n of modifying the s t r u c t u r e of the l i p i d . However, p r e l i m i n a r y attempts to use the a r y l s u l f a t a s e A of p i g t e s t i s (31) to d e s u l f a t e the SGG of i n t a c t bovine spermatozoa have not been s u c c e s s f u l (M. Levine and R.K. Murray, unpublished o b s e r v a t i o n s ) , d e s p i t e the f a c t that the enzyme p r e p a r a t i o n was very a c t i v e when i s o l a t e d SGG was used as a s u b s t r a t e . The production of an antiserum to SGG (48,49) may permit the a p p l i c a t i o n of immunocytochemical methods to determine both i t s c e l l u l a r and s u b c e l l u l a r l o c a t i o n s . Attempted L a b e l l i n g of G a l a c t o g l y c e r o l i p i d s Using Galactose Oxidase The s t u d i e s of Gahmberg and Hakomori (50) and Steck and Dawson (51) demonstrated the a b i l i t y of galactose o x i d a s e , i n conj u n c t i o n with NaB3H4,to l a b e l at l e a s t c e r t a i n galactose and Nacetylgalactosamine residues of c e l l surface g l y c o p r o t e i n s and g l y c o l i p i d s . In a n t i c i p a t i o n of employing t h i s method to determine whether the galactose moieties of the SGG and GG of t e s t i c u l a r c e l l s and spermatozoa are exposed on the surface of these c e l l s , Lingwood (52) has used t h i s method to attempt to l a b e l seve r a l p u r i f i e d g a l a c t o g l y c e r o l i p i d s i n v i t r o . Using c o n d i t i o n s that r e s u l t e d i n extensive l a b e l l i n g of galactosylceramide, GG was found to l a b e l to a maximum of 10% of the r a d i o a c t i v i t y i n c o r p o r ated i n t o the s p h i n g o l i p i d . In a d d i t i o n , very low l a b e l l i n g of SGG, g a l a c t o s y l d i a c y l g l y c e r o l and s u l f o g a l a c t o s y l c e r a m i d e was a l so observed, i n comparison with galactosylceramide. The l a b e l l i n g of the l a t t e r compound was not i n h i b i t e d i n the presence of GG, SGG or s u l f o g a l a c t o s y l c e r a m i d e .

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The chemical explanation f o r the poor l a b e l l i n g of the g a l a c t o g l y c e r o l i p i d s has not been e l u c i d a t e d . However, i t does not appear to be due to decomposition of the borohydride or to degrada t i o n of these l i p i d s during the l a b e l l i n g procedure. P o s s i b l y , some s u b t l e d i f f e r e n c e i n the p h y s i c a l s t a t e s of the g a l a c t o g l y c e r o l i p i d s as compared with the g a l a c t o s p h i n g o l i p i d s i s i n v o l v e d . I t i s a l s o of i n t e r e s t that s u l f a t i o n of the galactose residue i n h i b i t s the a c t i o n of galactose oxidase. In view of these f i n d i n g s , Lingwood (52) p o i n t s out than an i n a b i l i t y to be l a b e l l e d by the galactose oxidase procedure does not n e c e s s a r i l y mean that a g a l a c t o l i p i d i s absent from the c e l l s u r f a c e . These r e s u l t s suggest that the galactose oxidase technique - at l e a s t as p r e s e n t l y employed - i s u n l i k e l y to be u s e f u l i n determining the p o s s i b l e surface l o c a t i o n of g a l a c t o g l y c e r o l i p i d s i n t e s t i c u l a r c e l l s and spermatozoa. Antiserum

to T e s t i c u l a

The p i o n e e r i n g s t u d i e s of Rapport and h i s colleagues (53) c l e a r l y demonstrated the a n t i g e n i c i t y of v a r i o u s g l y c o l i p i d s . Subsequent work has shown that a n t i s e r a to g l y c o l i p i d s may be used to determine t h e i r c e l l u l a r and s u b c e l l u l a r l o c a t i o n s (54). A n t i bodies to SGG and GG could thus prove u s e f u l i n i n v e s t i g a t i n g the c e l l u l a r and/or s u b c e l l u l a r l o c a t i o n of these l i p i d s i n t e s t e s and spermatozoa. The production of a n t i b o d i e s (complement-fixing) to s u l f o g a l a c t o s y l c e r a m i d e has been reported p r e v i o u s l y (55,56). Lingwood et_ a l . (48,49) have thus attempted to produce a n t i b o d i e s to SGG i n r a b b i t s . The animals were i n j e c t e d by the intravenous route with liposomes c o n t a i n i n g SGG. Antibodies to SGG were detected by a complement f i x a t i o n assay. C o n t r o l sera showed no anti-SGG a c t i v i t y , but d i d show low antibody a c t i v i t y to GG, s u l f o galactosylceramide and galactosylceramide. A l l of the anti-SGG a c t i v i t y was l o c a t e d i n the IgG f r a c t i o n . Anti-SGG was p u r i f i e d by adsorption to and e l u t i o n from c h o l e s t e r o l p a r t i c l e s coated with SGG. The e l u t e d anti-SGG reacted with SGG but not with s u l f o galactosylceramide or galactosylceramide; a low t i t e r towards GG remained. These s t u d i e s demonstrate the f e a s i b i l i t y of preparing a n t i b o d i e s to SGG. I t remains to be seen i f these a n t i b o d i e s w i l l prove u s e f u l f o r immunohistochemical approaches towards determining the l o c a t i o n of SGG i n t e s t i s and spermatozoa. S u l f o g a l a c t o l i p i d s of the T e s t i s of Various

Species

The g l y c o l i p i d s of the t e s t i s of a number of animals have been analysed to determine whether SGG i s a u n i v e r s a l c o n s t i t u e n t of t e s t i c u l a r t i s s u e of a l l chordates. The r e s u l t s of these studi e s are summarized i n Table I I I . At l e a s t four p o i n t s concerning these r e s u l t s merit comment: (1) SGG has been detected i n the t e s t e s of a l l of the l i m i t e d number of mammals so f a r examined

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Table I I I . D i s t r i b u t i o n of S u l f o g a l a c t o l i p i d s i n the T e s t i s (or Sperm) of Various Chordata Animal

Class

Human Rat Mouse Guinea P i g Rabbit Boar Duck Fowl Salmon (milt) Trout Puffer f i s h Skate f i s h Green Monkey

Mammalia

Dog B u l l (sperm) Opossum Turtle B u l l frog

SGG

SGC

SGGC

+ +

(15,26) (11,14)

+ + Aves

+ +

Osteichthyes

+ + +

Chondrichthyes Mammalia

+ + + Reptilia Amphibia

References

+ +

(11) (12) (26) (26) (26) (26) (57) (26) M. Levine (unpublished observations)

+ + +

The presence or absence of each of the three s u l f o g a l a c t o l i p i d s studied i s i n d i c a t e d by + or - r e s p e c t i v e l y . I t i s p o s s i b l e that trace amounts of one or other of the three g l y c o l i p i d s l i s t e d may be present i n c e r t a i n of the t e s t i c u l a r t i s s u e s marked as as i n most cases the estimates were based on v i s u a l examinations of a p p r o p r i a t e l y stained t h i n l a y e r chromatograms. A b b r e v i a t i o n s : SGC, sulfogalactosylceramide; SGGC, s u l f o g a l a c t osylglucosylceramide.

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(2) SGG has not been detected i n the t e s t e s of the l i m i t e d numbers of b i r d s , f i s h , r e p t i l e s and amphibians analysed (3) In the t e s t e s of these l a t t e r c l a s s e s of animals that l a c k SGG, two other s u l f o g a l a c t o l i p i d s were found to be the major g l y c o l i p i d s - i . e . s u l f o g a l a c t o s y l - and s u l f o g a l a c t o s y l g l u c o s y l ceramides (4) The two sphingosine-containing s u l f a t i d e s are a l s o found i n the t e s t e s of c e r t a i n mammals - f o r instance, human t e s t i s cont a i n s both of them, i n a d d i t i o n to SGG. These observations i n d i c a t e that i t should be r e v e a l i n g - i n terms of i n c r e a s i n g understanding of the mechanisms that operate to regulate the s u l f a t i d e p r o f i l e of a t i s s u e - to compare the c a p a c i t i e s of t e s t i c u l a r e x t r a c t s from one or more animals of each of the c l a s s e s l i s t e d i n Table I I I to synthesize the v a r i o u s c o n s t i t u e n t parts of the above three l i p i d s . As p a r t i a l l y d i s c u s sed e a r l i e r , the b i o s y n t h e s i s of these s u l f a t i d e s can be c o n s i dered to occur i n 3 stages i . e . ceramide and p o s s i b l (2) g l y c o s y l a t i o n and (3) s u l f a t i o n . The s p e c i f i c i t y of the s u l f a t i o n r e a c t i o n appears to be r e l a t i v e l y low, as the s u l f o t r a n s ferase involved i n the b i o s y n t h e s i s of SGG w i l l s u l f a t e a number of l i p i d s with a terminal (3-galactosyl residue, i n c l u d i n g GG, g a l a c t o s y l - and g a l a c t o s y l g l u c o s y l - ceramides (19,32). As human t e s t i s contains each of these three l i p i d s (15,26), t h i s can exp l a i n , at l e a s t i n p a r t , why i t e x h i b i t s a l l three s u l f a t i d e s . I t thus seems more l i k e l y that the v a r i e d s u l f a t i d e p r o f i l e s d i s played by the t e s t i s of the animals l i s t e d i n Table I I I w i l l be explained by d i f f e r i n g p o t e n t i a l s , among s p e c i e s , of that organ to synthesize the l i p i d moieties, and by the s p e c i f i c i t i e s f o r both the l i p i d acceptors and the sugar donors of the g l y c o s y l t r a n s f e r a s e s i n v o l v e d i n the second stage of s u l f a t i d e biosynthesis (cf.32). Two other p o i n t s a r i s i n g from t h i s l i n e of work a l s o deserve b r i e f d i s c u s s i o n . F i r s t l y , i t i s r e l e v a n t to mention that s u l f o quinovosyl d i g l y c e r i d e has been reported to be the major g l y c o l i p i d of the spermatozoa of sea urchins (58). I t w i l l thus be of i n t e r e s t to extend s t u d i e s of the comparative biochemistry of t e s t i c u l a r g l y c o l i p i d s to lower c l a s s e s of animals as w e l l as to f u r t h e r members of the c l a s s e s l i s t e d i n Table I I I . Secondly, i t i s apparent that, whatever the p r e c i s e phylogenetic d i s t r i b u t i o n of g l y c o l i p i d s i n t e s t i s may turn out to be, the r e s u l t s to date s t r o n g l y support the hypothesis that s u l f a t i d e s p l a y an important r o l e i n t e s t i c u l a r and/or spermatozoal f u n c t i o n i n chordates. G a l a c t o g l y c e r o l i p i d s of the Nervous System As t h i s i s a r e l a t i v e l y l a r g e subject area, i t w i l l only be touched upon i n s o f a r as i t r e l a t e s to work performed by the authors. G a l a c t o s y l d i a c y l g l y c e r o l was reported to be a c o n s t i t u ent of b r a i n i n 1963 (6); subsequently, the same l i p i d derived

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from bovine s p i n a l cord was thoroughly c h a r a c t e r i z e d (59). Also i n 1963, GG was detected i n r a t b r a i n (10); a l a t e r study confirmed the presence of GG i n the b r a i n s of s e v e r a l other species (60). Both of these g l y c o l i p i d s are a l s o present i n p e r i p h e r a l nerves ( c f . 61). A compound corresponding i n i t s p r o p e r t i e s to d i g a l a c t o s y l d i a c y l g l y c e r o l was a l s o found to be a c o n s t i t u e n t of human b r a i n (24). Pathways f o r the b i o s y n t h e s i s of both g a l a c t o s y l - and d i g a l a c t o s y l - d i a c y l g l y c e r o l s by e x t r a c t s of r a t b r a i n have been r e f e r r e d to e a r l i e r . The presence of GG i n r a t b r a i n suggested to Levine et a l . (20) that SGG might a l s o be l o c a t e d i n that organ. These workers d i d indeed f i n d small amounts of SGG (approx. o n e - f i f t e e n t h the amount of sulfogalactosylceramide) i n adult r a t b r a i n . They a l s o detected small amounts of the same l i p i d i n r a b b i t b r a i n , but not i n a p o r t i o n of the f r o n t a l lobes of human b r a i n . In a d d i t i o n , evidence was obtained i n t h e i r study suggesting that a l e s s e r amount of s u l f o g a l a c t o s y l d i a c y l g l y c e r o l might a l s o be present i l y e s t a b l i s h e d by Flyn who provided unequivocal evidence, i n c l u d i n g the i s o l a t i o n of s u l f o g a l a c t o s y l g l y c e r o l , f o r the presence of that compound i n r a t b r a i n . These workers found l a r g e r amounts of the d i a c y l - than of the a l k y l a c y l - c o n t a i n i n g g a l a c t o g l y c e r o l i p i d ; however, i n cont r a s t to Levine et a l . (20), they used immature (approx. 22 day old) animals. Ishizuka et a l . (23) confirmed that both l i p i d s were present i n r a t b r a i n and they developed appropriate methodology, i n c l u d i n g analyses by g a s - l i q u i d chromatography-mass spectrometry, f o r thoroughly c h a r a c t e r i z i n g them. They a l s o showed that the d i a c y l - c o n t a i n i n g l i p i d was the predominant compound i n the b r a i n s of r a t s of age up to 19 days, but t h e r e a f t e r the a l k y l a c y l type predominated, c o n s i t u t i n g 85% of the sum t o t a l of these two l i p i d s by 68 days of age. SGG was detected i n cod b r a i n , but n e i t h e r s u l f o l i p i d was detected i n normal human b r a i n nor i n the b r a i n of a case with metachromatic leukodystrophy. The s t u d i e s of Levine £t a l . (20) revealed that the turnover of the SGG i n r a t b r a i n was s i m i l a r to that of s u l f o g a l a c t o s y l ceramide. This suggested that the SGG l i k e the c l a s s i c a l s u l f a t i d e (62), might be l o c a t e d predominantly i n myelin. P i e r i n g e r et a l . (22) demonstrated that the d i a c y l form of the s u l f o g a l a c t o g l y c e r o l i p i d s present i n r a t b r a i n had a f a s t e r turnover than that of the a l k y l a c y l form. Because previous s t u d i e s (63,64) had i n d i c a t e d that the g a l a c t o s y l d i a c y l g l y c e r o l of r a t b r a i n was an e x c e l l e n t marker metabolite f o r myelination, these workers a l s o studied the p o s s i b l e a s s o c i a t i o n of the two s u l f o g a l a c t o g l y c e r o l i p i d s ( i . e . the d i a c y l and the a l k y l a c y l species) with myelinat i o n . Support f o r the a s s o c i a t i o n of these two compounds with myelination was found by showing that they were absent from r a t b r a i n before 10 days of age and that they accumulated i n that organ between 10 and 25 days of age (the period of maximum myelinat i o n ) . Further support was derived from the f i n d i n g that the s u l f o t r a n s f e r a s e involved i n the b i o s y n t h e s i s of the d i a c y l - c o n -

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t a i n i n g g a l a c t o l i p i d (and presumably, but not c o n c l u s i v e l y e s t a b l ished, a l s o of the a l k y l a c y l species) increased maximally i n act i v i t y during the same time p e r i o d . In a d d i t i o n , the amounts of the s u l f o g a l a c t o g l y c e r o l i p i d s and the a c t i v i t y of the s u l f o t r a n s ferase were g r e a t l y decreased i n the b r a i n s of non-myelinating jimpy mice. Ishizuka et a l . (23) a l s o found that synthesis of r a t b r a i n SGG was most a c t i v e around 18 days of age. Conclusion It i s evident that knowledge of the g a l a c t o g l y c e r o l i p i d s has grown i n recent years. Instead of being recognized s o l e l y as quant i t a t i v e l y r e l a t i v e l y minor g l y c o l i p i d s of nervous t i s s u e , members of the a l k y l a c y l sub-class are now a l s o seen to c o n s t i t u t e major g l y c o l i p i d s of mammalian t e s t i s and spermatozoa. Nevertheless, t h e i r t i s s u e d i s t r i b u t i o n i s extremely r e s t r i c t e d i n comparison with that of the g l y c o s p h i n g o l i p i d s determine whether a d d i t i o n a i a n c e l l s and a l s o i f other types of g l y c o g l y c e r o l i p i d s e x i s t . In t h i s respect, as mentioned e a r l i e r , the Slomianys (2_ ^ 9) have provided evidence that a novel s e r i e s of g l y c e r y l e t h e r - c o n t a i n i n g g l u c o g l y c e r o l i p i d s may e x i s t i n g a s t r i c j u i c e , s a l i v a and perhaps other s e c r e t i o n s . However, t h e i r r e s u l t s have not as yet r e c e i v e d independent confirmation ( c f . 65,66). With respect to f u n c t i o n , one~wonders i f the common l o c a t i o n of GG and SGG i n the b r a i n and t e s t i s of c e r t a i n species r e f l e c t s some p h y s i o l o g i c a l e n t i t y that both of these organs share - f o r instance, a blood b a r r i e r . However, the apparent absence of SGG from human b r a i n (20,23) does not support t h i s conjecture. Simil a r l y , the sharing of GG and SGG by these two "sequestered" o r gans r a i s e s thoughts as to whether t h i s could be of immunological s i g n i f i c a n c e i n some s i t u a t i o n s . Yet another l i n e of s p e c u l a t i o n i s whether the presence of r e l a t i v e l y l a r g e amounts of g l y c e r y l e t h e r - c o n t a i n i n g g a l a c t o g l y c e r o l i p i d s i n t e s t i s may somehow be r e l a t e d to the f a c t that the t e s t e s of most mammals are confined i n a scrotum maintained at a temperature lower than the r e s t of the body. These surmises r e f l e c t the humbling f a c t that there i s as yet very l i t t l e understanding of the functions of the v a r i o u s non-sulfated and s u l f a t e d g a l a c t o l i p i d s present i n mammalian c e l l s . A ray of hope f o r t h i s area i s provided by the hypothesis of Karlsson and h i s colleagues (67,68) that sulfogalactosylceramide may act as a c o f a c t o r s i t e f o r the a c t i v i t y of Na K ATPase. The p o s s i b i l i t y that SGG could be involved i n such a s i t e i n spermatozoa has been r a i s e d (16,69). 9

+

9

+

The most u s e f u l f u n c t i o n of t h i s review w i l l be i f i t stimul a t e s f u r t h e r research i n t h i s area. For t h i s reason, i t seems appropriate to conclude by posing a number of f a i r l y obvious but nevertheless b a s i c - questions, that w i l l h o p e f u l l y be answered i n future i n v e s t i g a t i o n s . What p h y s i c a l d i f f e r e n c e s e x i s t between a l k y l a c y l and d i a c y l g a l a c t o g l y c e r o l i p i d s , between g a l a c t o -

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glycerolipids and galactosphingolipids and between sulfated and non-sulfated galactolipids (assuming in all cases that the pairs of lipids mentioned differ only with respect to the specified moieties)? Assuming that physical differences do exist, what are their functional implications? What are the details of the pathway of biosynthesis of the testicular galactoglycerolipids and what factors (e.g. genetic, hormonal, enzyme specificity etc.) control the expression of this pathway during testicular differentiation? Can this pathway be interfered with by pharmacological agents (e.g. analogs of glyceryl ethers), and if so, what effects could that have on testicular and possibly nervous system function? What are the precise cellular and/or subcellular locations of the galactoglycerolipids in testicular cells and spermatozoa? Finally, what is the function of the SGG in mature spermatozoa is it involved in ion transport, in motility, in sperm-ovum interactions or is it merely a passenger molecule, having fulfilled its function at some earlie tifully specialized cells Acknowledgements We thank Ms. B. Palmer for her excellent technical assistance in a number of the studies whose results are summarized above. The authors are grateful for support from the Ford Foundation, from N.I.H. (Grant No. RO-1HD07889 from the National Institute of Child Health and Human Development) and from the Medical Research Council of Canada. The patience and care displayed by Ms. Stephanie Amos during the typing of this manuscript is warmly acknowledged. Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9.

Sweeley, C.C.; Siddiqui, B. in "The Glycoconjugates" (Horowitz, M.; Pigman, W., eds.), Academic Press, New York, N.Y., 1976, vol. 1, pp.459-540. Carter, H.E.; Johnson, P.; Weber, E.J. Annu. Rev. Biochem., 1965, 34, 109-142. Kates, M. Advan. Lipid Res., 1970, 8, 225-265. Sastry, P.S. Advan. Lipid Res., 1974, 12, 251-340. Kates, M.; Palameta, B.; Perry, M.P.; Adams, G.A. Biochim. Biophys. Acta, 1967, 137, 213-216. Steim, J.M.; Benson, A.A. Fed. Proc., 1963, 22, 299 (abstr. no. 830). Slomiany, B.L.; Slomiany, A.; Glass, G.B.J. Eur. J. Biochem., 1977, 78, 33-39. Slomiany, B.L.; Slomiany, A.; Glass, G.B.J. Biochemistry, 1977, 16, 3954-3958. Slomiany, B.L.; Slomiany, A. Biochem. Biophys. Res. Commun. 1977, 79, 61-66.

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10. Norton, W.T.; Brotz, M. Biochem. Biophys. Res. Commun., 1963, 12, 198-203. 11. Kornblatt, M.J.; Schachter, H.; Murray, R.K. Biochem. Biophys. Res. Commun., 1972, 48, 1489-1494. 12. Ishizuka, I.; Suzuki, M.; Yamakawa, T. J. Biochem., 1973, 73 , 77-87. 13. Suzuki, A.; Ishizuka, I.; Ueta, N.; Yamakawa, T. Japan. J. Exp. Med., 1973, 43, 435-442. 14. Kornblatt, M.J.; Knapp, A.; Levine, M.; Schachter, H.; Murray, R.K. Canad. J. Biochem., 1974, 52, 689-697. 15. Ueno, K.; Ishizuka, I.; Yamakawa, T. Biochim. Biophys. Acta, 1977, 487, 61-73. 16. Murray, R.K.; Levine, M.; Kornblatt, M.J. in "Glycolipid Methodology" (Witting, L.A., ed.), Amer. Oil. Chem. Soc., Champaign, Ill., 1976, pp.305-327. 17. Dulaney, J.T.; Moser H.W in "The Metabolic Basis of Inherited Disease" (Stanbury D.S., eds.), McGraw-Hill pp.770-809. 18. Farooqui, A.A. Int. J. Biochem., 1978, 9, 709-716. 19. Knapp, A.; Kornblatt, M.J.; Schachter, H.; Murray, R.K. Biochem. Biophys. Res. Commun., 1973, 55, 179-186. 20. Levine, M.; Kornblatt, M.J.; Murray, R.K. Canad. J. Biochem., 1975, 53, 679-689. 21. Flynn, T.J.; Desmukh, D.S.; Subba Rao, G; Pieringer, R.A. Biochem. Biophys. Res. Commun., 1975, 65, 122-128. 22. Pieringer, J . ; Subba Rao, G.; Mandel, P.; Pieringer, R.A. Biochem. J., 1977, 166, 421-428. 23. Ishizuka, I.; Inomata, M.; Ueno, K.; Yamakawa, T. J. Biol. Chem., 1978, 253, 898-907. 24. Rouser, G.; Kritchevsky, G.; Simon, G.; Nelson, G.J. Lipids, 1967, 2, 37-40. 25. Wenger, D.A.; Subba Rao, K.; Pieringer, R.A. J. Biol. Chem. 1970, 245, 2513-2519. 26. Levine, M.; Bain, J.; Narasimhan, R.; Palmer, B.; Yates, A.J.; Murray, R.K. Biochim. Biophys. Acta, 1976, 441, 134-145. 27. Suzuki, K. J. Neurochem., 1965, 12, 629-638. 28. Vance, D.E.; Sweeley, C.C. J. Lipid. Res., 1967, 8, 621-630. 29. Kates, M. in "Laboratory Techniques in Biochemistry and Molecular Biology" (Work, T.S.; Work, E., eds.), 1972, American Elsevier, New York, N.Y., vol.3, part II, pp.269-600. 30. Suzuki, A.; Sato, M.; Handa, S.; Muto, Y.; Yamakawa, T. J. Biochem., 1977, 82, 461-467. 31. Yamato, K.; Handa, S.; Yamakawa, T. J. Biochem., 1974, 75 , 1241-1247. 32. Handa, S.; Yamato, K.; Ishizuka, I.; Suzuki, A.; Yamakawa, T. J. Biochem., 1974, 75, 77-83 . 33. Young, R.W. J. Cell Biol., 1973, 57, 175-189. 34. Fleischer, B.; Zambrano, F. Biochem. Biophys. Res. Commun. 1973, 52, 951-958.

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35. Keenan, T.W.; Morré, D. J.; Basu, S. J. Biol. Chem., 1974, 249, 310-315. 36. Wenger, D.A.; Petitpas, J.W.; Pieringer, R.A. Biochemistry 1968, 7, 3700-3707. 37. Subba Rao, G.; Norcia, L.N.; Pieringer, J.; Pieringer, R.A. Biochem. J., 1977, 166, 429-435. 38. Levine, M.; Narasimhan, R.; Pinteric, L.; Murray, R.K. Proced. XIth Internatl. Congress Biochem., 1979, p. 408 (abstr. no. 05-9-R49). 39. Narasimhan, R.; Levine, M.; Murray, R.K. Proced. Vth Internatl. Sympos. on Glycoconjugates, 1979, G. Thieme-Verlag, Stuttgart, in press. 40. Fluharty, A.L.; Stevens, R.L.; Miller, R.T.; Kihara, H. Biochem. Biophys. Res. Commun., 1974, 61, 348-354. 41. Yang, C.H.; Srivastava, P.N. Proc. Soc. Exp. Biol. Med., 1974, 145, 721-725. 42. Yamaguchi, S.; Aoki 1975, 24, 1087-1089 43. Reiter, S.; Fischer, G.; Jatzkewitz, H. FEBS Lett., 1976, 68, 250-254. 44. Clermont, Y.; Perey, B. Amer. J. Anat., 1957, 100, 241-267. 45. Letts, P.J.; Hunt, R.C.; Shirley, M.A.; Pinteric, L.; Schachter, H. Biochim. Biophys. Acta, 1978, 541, 59-75. 46. Kornblatt, M.J. Canad. J. Biochem., 1979, 57, 255-258. 47. Ishizuka, I.; Yamakawa, T. J. Biochem., 1974, 76, 221-223. 48. Lingwood, C.; Murray, R.K.; Schachter, H. Proced. Soc. for Complex Carbohydrates, 1979, abstr. no. 28. 49. Lingwood, C.A.; Murray, R.K.; Schachter, H. Proced. XIth Internatl. Congress Biochem., 1979, p. 491 (abstr. no. 07-3H99). 50. Gahmberg, C.G.; Hakomori, S. J. Biol. Chem., 1973, 248, 43114317. 51. Steck, T.L.; Dawson, G. J. Biol. Chem., 1974, 249, 2135-2142. 52. Lingwood, C.A. Canad. J. Biochem., in press. 53. Rapport, M.M.; Graf, L. Prog. Allergy, 1969, 13, 273-331. 54. Marcus, D.M. in "Glycolipid Methodology" (Witting, L.A., ed.), 1976, Amer. Oil Chem. Soc., Champaign, Ill., pp. 233-245. 55. Zalc, B.; Jacque, C.; Radin, N.S.; Dupouey, P. Immunochem., 1977, 14, 775-779. 56. Hakomori, S. J. Immunol., 1974, 112, 424-426. 57. Ueno, K.; Ishizuka, I.; Yamakawa, T. J. Biochem., 1975, 77, 1223-1232. 58. Nagai, Y.; Isono, Y. Japan. J. Exp. Med., 1965, 35, 315-318. 59. Steim, J.M. Biochim. Biophys. Acta, 1967, 144, 118-126. 60. Rumsby, M.G.; Rossiter, R.J. J. Neurochem., 1968, 15, 14731476. 61. Singh, H. J. Lipid. Res., 1973, 14, 41-49. 62. Norton, W.T. in "Basic Neurochemistry" (Albers, R.W.; Siegel, G.J.; Katzman, R.; Agranoff, B.W.,eds.), 1972, Little Brown, Boston, Mass., pp. 365-386.

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63. Inoue, T.; Desmukh, D.S.; Pieringer, R.A. J. Biol. Chem., 1971, 246, 5688-5694. 64. Desmukh, D.S.; Inoue, T.; Pieringer, R.A. J. Biol. Chem., 1971, 246, 5695-5699. 65. Narasimhan, R.; Bennick, A.; Murray, R.K. Fed. Proc., 1979, 38, p. 404 (abstr. no. 925). 66. Narasimhan, R.; Bennick, A.; Palmer, B.; Murray, R.K. Proced. Soc. for Complex Carbohydrates, 1979, abstr. no. 35. 67. Karlsson, K.-A.; Samuelsson, B.E.; Steen, G.O. Eur. J. Biochem., 1974, 46, 243-258. 68. Karlsson, K.-A. in "Structure of Biological Membranes" (Abrahamsson, S.; Pascher, I., eds.), 1977, Plenum Press New York, N.Y., pp.245-274. 69. Hansson, C.G.; Karlsson, K.-A.; Samuelsson, B.E. J. Biochem., 1978, 83, 813-819. RECEIVED

December 10, 1979

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

8 Structural Studies of Neutral Glycosphingolipids of Human Neutrophils by Electron Impact/Desorption Mass Spectrometry BRUCE A. MACHER and JOHN C. KLOCK Department of Medicine, University of California, San Francisco, CA 94143 Electron impact mass spectrometr sensitive tool for detaile glycosphingolipids. Several kinds of information can be obained with samples of 10-300 µg, including: the number and sequence of carbohydrate residues, the major fatty acid and long chain base species, the number of branching points, and in some cases the molecular weight and information on the position of glycosidic linkage (1-6). We have utilized a variation of electron impact mass spectrometry in the analysis of neutral glycosphingolipids of human neutrophils, which has been referred to as electron impact/desorption mass spectrometry (for a review, see ref. 7). This technique has allowed us to obtain the same type of structural information as outlined above, but with sample amounts of 1-5 µg. A lower source temperature probably leads to less thermal decomposition of the sample and thus increased sensitivity. We have been able to conclude from the spectra obtained by this method that human neutrophils containat least four neutral glycosphingolipids which have the following partial structures: Hexose-0-Cer, Hexose-0-Hexose-0Cer, Hexosamine-0-Hexose-0-Hexose-0-Cer, Hexose-0Hexosamine-0-Hexose-0-Hexose-0-Cer. The ceramide moiety in these four compounds is characterized as a 4-sphingenine with an N-linked palmitic, lignoceric or nervonic acid. Materials and Methods Isolation of human neutrophils. Leukocytes were obtained from normal donors by leukapheresis with an IBM 2997 Blood Cell Separator (8). Normal mature neutrophils were purified from this mixed leukocyte preparation by dextran sedimentation and Ficoll-Hypaque gradient centrifugation as previously described (9). The Wright-stained smears of the preparation showed that the cells were over 95% neutrophils.

0-8412-0556-6/80/ 47-128-127$5.00/ 0 © 1980 American Chemical Society In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

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Extraction and purification of neutrophil glycosphingolipids. Purified human neutrophils were extracted with 20 volumes of each of the following solvent mixtures: chloroform/methanol 2/1, 1/1, 1/2, (v/v). After evaporation of the organic solvents in vacuo, the residue was dissolved in approximately 5 volumes of chloroform/methanol/water (30/60/8, v/v) and mixed with 0.5 g of DEAE-Sephadex A25 (Pharmacia, Piscataway, N.J.) acetate form (10). The sample was allowed to absorb to the column packing for 20 min and was then applied to a column of the same material. Neutral and acidic lipid fractions were eluted as described by Ando and Y u (11). Neutral glycosphingolipids were further purified on a column of BioSil A , 100-200 mesh (Bio Rad, Richmond, C A ) . The neutral lipid fraction was dissolved in 5-10 ml of chloroform/methanol (1/1, v/v), applied to a column of BioSil A (2x30 cm), and eluted as 100 ml fractions with solvent mixtures of increasing polarit methanol). Final purificatio was by preparative thin-layer chromatography using Silica Gel 60 High Performance Plates (EM Laboratories Inc., Cincinnati, OH) in solvent system A (chloroform/methanol/water, 60/35/8, v/v). Glycosphingolipids were visualized by a brief exposure to iodine, eluted with chloroform/methanol/water (50/50/10, v/v) and rechromatographed in solvent A or B (chloroform/methanol/water, 100/42/6, v/v) to demonstrate homogeneity. Direct probe mass spectrometry. Glycosphingolipids (30-100 jjg) were permethylated as described (12). The samples (less than 5 \i g) were subjected to electron impact/desorption analysis with a Varian M A T C H - 5 D F mass spectrometer under the following conditions: emission current, 300JJ A; electron energy, 70^ eV; acceleration voltage, 3KV; ion source temperature, 160 C; emitter wire current, programed from 0 to 35mA. Results Isolation of the neutra^ glycosphingolipids. In a typical extraction procedure, 10 purified human neutrophils yielded 100-150 mg of total glycosphingolipids. As shown in Table I, glycosphingolipids account for approximately 10% of the total cellular dry weight of the neutrophil. Separation of the total neutrophil lipids by DEAE-sephadex and silicic acid column chromatography yielded 70-100 mg of neutral glycosphingolipids from 1 0 c e l l s . iW

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8.

MACHER A N D K L O C K

Electron

Impact Mass

Spectrometry

129

Figure 1. Thin-layer chromatography of fractions I-IV isolated from human neutrophils. The separation is on a plate of silica gel 60 (HPTLC) in solvent system A. S: erythrocyte glycosphingolipid standards; 1-4: human neutrophil glycosphingolipid fractions.

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CELL SURFACE GLYCOLIPIDS

Figure 2. Mass spectra of the intact permethylated glycosphingolipids of fractions I (a), II (b), III (c), and IV (d). See Materials and Methods for conditions.

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

Electron Impact Mass

MACHER A N D K L O C K

200

Spectrometry

300

350

n-n

c=o I I

Hexost-O-Hexosamine-O-Hexose-O-Hexose-O-C^--CH

40"

660

CH-CHrCH-C^^ 253

20200

• •i,U | kJJ. I •, ,Jj.,.l. I , |N>,., 250

300

80

20

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C E L L SURFACE GLYCOLIPIDS

After repeated preparative thin-layer chromatography, four fractions were obtained as shown in Figure 1. When the thinlayer chromatographic mobilities of fractions I-IV were compared to standard glycosphingolipids isolated from human erythrocytes the following relationships were found: fractions IIV had similar mobilities to G l c C e r , LacCer, GbOse^Cer and GbOse^Cer, respectively.

Table I: Percent distribution of neutrophil lipid components

Fraction

% total cell

Total lipid

30

Phospholipids

14.3

Neutral lipids

5.7

Total glycosphingolipids Neutral glycosphingolipids

10.0 7.2

^average of three determinations

Direct probe analysis. The spectra of the methylated derivatives of fractions I-IV are shown in Figure 2 (a-d), together with abbreviated structural formulas and indications of some fragments (Refs. J.-6 were consulted for comparison). Only fraction I gave ions indicative of the entire molecule at m/z 894 (M-l) and m/z 863 (M-32) for a monoglycosyl-ceramide containing C ^ , Q fatty acid and C j « ^ long chain base. Peaks corresponding to the permetnylated carbohydrate portions of the glycosphingolipid fractions are the following: Fraction I, m/z 187, 219, 292, 278, 530, 640 and 642; Fraction II, m/z 187, 219, 422, 496, and 847; Fraction III, m/z 228, 260, 432, 464, and 636; Fraction IV, m/z 182, 187, 228, 432, 464 and 668. To our knowledge the spectrum presented for the methylated derivative of fraction III is the first to be presented for a naturally occurring compound of this structure.

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8.

Electron

MACHER A N D K L O C K

Impact Mass

Spectrometry

Peaks corresponding to the fatty acid residue and part of the long chain were seen at m/z 294, 322, 404 and 406. Peaks for 4sphingenine appeared at m/z 253, 294, 312 and in some scans m/z 364. The entire ceramide fragment with C ^ . Q fatty acid and 24-0 / ^ ^ ^> respectively. C

f

a

t

t

v

a

c

i

d

w

a

s

s

e

e

n

a

t

m

z

66

Discussion The results presented in this report confirm and extend previous studies on the structure of neutral glycosphingolipids of human neutrophils. On the basis of carbohydrate compositional data and T L C properties structures have been prosposed for neutral glycosphingolipids prepared from whole blood including glucosylceramide (13-14) lactosylceramide (13,14,15); galabiosylceramide (l3T; lactoneotetraosylceramide (T4]f~ and globotetraosylceramide (_13) not been published and therefor are still tentative. The studies discussed above have all dealt with glycosphingolipids isolated from neutrophils that were derived from normal whole blood. Wherrett (16) has presented detailed structural analysis of a tetraglycosylceramide isolated from polymorphonuclear leukocytes obtained from the urine of a patient with a urinary tract infection. This glycosphingolipid was determined to be lactoneotetraosylceramide. The data presented in this report allow the assignment of partial structures for four neutral glycosphingolipids of human neutrophils: Hexose-O-Cer, Hexose-O-Hexose-O-Cer, Hexosamine -O-Hexose-O-Hexose-O-Cer and Hexose-O-Hexosamine-O-Hexose0-Hexose-0-Cer. This information was obtained from samples of less than 5 ug by electron impact/desorption direct probe mass spectrometry. On the basis of complete structural analyles, to be presented elsewhere, we have been able to determine that the four fractions isolated thus far actually contain six different glycosphingolipids with the following structures: GlcBl + lCer GalB1+4GlcB1 +1 Cer G l c N Ac B1-* 3Gal B1 ->4Glc B1 1 Cer Gal B1 ->4GlcN Ac B1+3Gal B1 +4Glc B1 +1 Cer

GalBl + lCer Gal a 1+4GalBl + l C e r

In addition to these structures, human neutrophils also contain eight to twelve gangliosides and a few species of glycosphingolipids with more than four saccharide units. Purification and structural analyses are currently underway in our laboratory.

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CELL SURFACE GLYCOLIPIDS

Acknowledgements This study was supported in part by a grant from the Leukemia Research Foundation and by cancer research funds from the University of California. Mass spectrometric analyses were provided by Mr. Lawrence R. Phillips and Ms. Betty Baltzer of the Michigan State University - NIH Mass Spectrometry Facility, and are sincerely appreciated. Abbreviations Glc, glucose; Gal, galactose; GlcNAc, N-acetylglucosamine; TLC, thin-layer chromatography; GlcCer, glucosylceramide; GalCer, galactosylceramide; LacCer, lactosylceramide; GbOse^Cer, globotriaosylceramide; and GbOse^Cer, globotetraosylceramide. Individual d t hav the D configuration and t Literature Cited 1. 2. 3. 4. 5. 6. 7.

8. 9. 10. 11. 12. 13. 14. 15. 16.

Karlsson,K.-A. Biochemistry, 1974, 13, 3643. Karlsson,K.-A. Prog. Chem. Fats Other Lipids, 1978, 16, 207. Leeden,R.W.; Kundu,S.K.; Price,H.C.; Fong,J.W. Chem. Phys. Lipids. 1974, 13, 429. Egge,H. Chem. Phys. Lipids, 1978, 21, 349. Karlsson,K.-A. Biomed. Mass Spectrom., 1974, 1, 49. Hanfland,P.; Egge,H. Chem. Phys. Lipids, 1976, 16, 201. Sweeley,C.C.; Soltman,B.; Holland,J.F. in "High Performance Mass Spectrometry: Chemical Applications. Gross,M.L.Ed. American Chemical Society: New York,1978; p.209. Hester,J.P.; Kellogg,R.M.; Mulzet,A.P.; Kruger,V.R.; McCredie,K.B.; Freireich,E.J. Blood, 1979, 54, 254. Klock,J.C.; Bainton,D.F. Blood, 1976, 48, 149. Ledeen,R.W.; Yu,R.K.; Eng,L.F. J. Neurochem. 1973, 121, 829. Ando,S.; Yu,R.K. J. Biol. Chem. 1977, 252, 6247. Hakomori,S.-I. J. Biochem. 1964, 55, 205. Hildebrand,J.; Stryckmans,P.; Stoffyn,P. J. Lipids Res., 1971, 12, 361. Narasimham,P.; Murry,R.K. Biochem. J. 1979, 326, 63. Miras,C.J.; Mantzos,J.D.; Levis,G.M. Biochem. J. 1966, 98, 782. Wherrett,J.R. Biochem. Biophys.Acta, 1973, 326, 63.

RECEIVED

December 10, 1979.

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

9 Glycosphingolipids of Skeletal Muscle JAW-LONG CHIEN and EDWARD L. HOGAN Department of Neurology, Medical University of South Carolina, Charleston, SC 29403

It is currently hel in cell surface membrane events as receptor interactions (1,2), permeability change (3), cellular adhesion (4) and cellular recognition (5). The likelihood of their localization in sarcolemma and possible role in myogenesis including cell fusion or in conduction of the action potential prompted us to begin their study with isolation and characterization of the gangliosides and neutral glycosphingolipids in chicken and human skeletal muscle. Although muscle comprises approximately 40% of the body weight, there have been only a few studies of glycosphingolipids of muscle. Puro and coworkers (6) studied the qualitative and quantitative patterns of gangliosides in several extraneural tissues including skeletal and cardiac muscles of rat, rabbit and pig, but did not purify the individual gangliosides. Lassaga et al. (7) isolated four gangliosides from the hind leg and back muscle of the rabbit. One had the molar composition of hematoside (GM3) but the structures of the others - two disialo- and one trisialoganglioside - were not fully clarified. Svennerholm et al. (8) did a more complete study of human skeletal muscle. They isolated four major gangliosides and determined their composition by gas chromatography to be consistent with GM3, GM2, GDla and a sialosyltetraglycosylceramide. Recently, Levis and coworkers (9) examined the glycosphingolipids in human heart and found that human cardiac muscle contains the same gangliosides as those of human skeletal muscle. However, the distribution of gangliosides was quite different. In heart, GM3 (23%), GD3 (22%) andGM1(16%) are the major ganlgiosides while in skeletal muscle GM3 (67%) predominates. Neutral glycosphingolipids have also been studied in human skeletal (8) and cardiac (9) muscle. In skeletal muscle, lactosylceramide is the predominant glycolipid (38.4%) followed by globotriaosylceramide (26.3%) and globoside (12.4%); while in heart, globoside predominates (43.0%) followed by globotriaosylceramide (32.0%). 0-8412-0556-6/ 80/47-128-13555.00/ 0 © 1980 American Chemical Society In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

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The r e s u l t s presented here show that the g l y c o s p h i n g o l i p i d s of chicken p e c t o r a l muscle d i f f e r from those o f human s k e l e t a l and c a r d i a c muscles. In a d d i t i o n , we are p r e s e n t i n g the s t r u c tures o f two glucosamine-containing g a n g l i o s i d e s which were c h a r a c t e r i z e d by enzymatic h y d r o l y s i s and methylation s t u d i e s . Materials P e c t o r a l muscle from a d u l t Leghorn chickens was obtained from a l o c a l supermarket (the main source) o r d i s s e c t e d immedia t e l y a f t e r s a c r i f i c e . Human s k e l e t a l muscle was obtained four hours post mortem from the p e c t o r a l and i l i o p s o s a s muscles of a 70-year-old b l a c k male who d i e d o f a gunshot wound to the head. Precoated s i l i c a g e l p l a t e s ( s i l i c a g e l 60) were purchased from S c i e n t i f i c Products. B i o - S i l A (200 - 400 Mesh) was obtained from Bio-Rad L a b o r a t o r i e s Fatty a c i d methyl e s t e r s sphingosine and dihydrosphingosine 10% DEGS-PS, 3% SP-234 N-acetyl and N - g l y c o l y l neuraminic a c i d , DEAE-Sephadex A50 and neuraminidase type IX were obtained from Sigma Company. Ganglios i d e standards from human b r a i n and n e u t r a l g l y c o s p h i n g o l i p i d standards from bovine erythrocytes were prepared i n t h i s l a b o r a tory. 3-galactosidase was i s o l a t e d from papaya and $-hexosaminidase was prepared from j a c k bean meal (10). a-N-acetylgalactosaminidase was a generous g i f t of Dr. Y.-T. L i of Tulane U n i v e r s i t y . E x t r a c t i o n of g l y c o s p h i n g o l i p i d s The muscles were f r e e d by gross d i s s e c t i o n of extraneous t i s s u e which was mainly f a t and p e r i p h e r a l nerves, and then stored at -40°C. For an experiment, approximately 1 kg t i s s u e was macerated by a meat g r i n d e r and homogenized i n ten volumes of tetrahydrofuran:0.01 M KC1 (4:1, v / v ) , s t i r r e d f o r 3 hours, and f i l t e r e d through a Buchner f u n n e l . The e x t r a c t i o n was repeated twice and the f i l t r a t e s then combined and concentrated i n a r o t a r y evaporator. One l i t e r of chloroform-methanol (2:1, v/v) was added to the l i p i d e x t r a c t which has the appearance and consistency of syrup. Gangliosides were p a r t i t i o n e d i n t o the upper l a y e r by the a d d i t i o n o f 200 ml of water (11) and the lower layer extracted two a d d i t i o n a l times with t h e o r e t i c a l upper phase c o n t a i n i n g 0.027% KC1. The combined upper l a y e r s were then concentrated and d i a l y z e d e x h a u s t i v e l y a t 4°C with f i v e changes o f d i s t i l l e d water. DEAE-Sephadex Column Chromatography DEAE-Sephadex A-50 (Cl-form) was converted to the acetate form by the f o l l o w i n g procedure: The g e l was washed f i r s t with f i v e volumes of 0.1 N NaOH, and a f t e r r i n s i n g with d i s t i l l e d

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

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water, i t was converted i n t o the acetate form by washing with IN a c e t i c a c i d . The sedimented g e l was then r i n s e d repeatedly with water u n t i l n e u t r a l , washed with methanol and packed i n t o a column (1.8 x 21 cm). The l i p i d e x t r a c t obtained f o l l o w i n g d i a l y s i s was d i s s o l v e d i n C:M (2:8, v/v) and a p p l i e d to t h i s column which had been p r e v i o u s l y e q u i l i b r a t e d i n the same s o l v e n t . The n e u t r a l l i p i d s were eluted by the C:M (2:8, v/v) and gangliosides then e l u t e d by methanol containing sodium acetate i n the f o l l o w i n g concentrations: 0.01 M ( f r a c t i o n I ) , 0.02 M ( f r a c t i o n I I ) , and 0.2 M ( f r a c t i o n I I I ) . The f r a c t i o n s e l u t e d were concentrated and s a l t removed by d i a l y s i s . B i o - S i l A Column Chromatography of Ganglioside

Fractions

B i o - S i l A was a c t i v a t e d a t 110°C overnight, suspended i n chloroform and packed i n t o a column (1.5 x 45 cm) Fraction I (eluted from DEAE-Sephade solved i n C:M (2:1, v/v applie Ganglioside were e l u t e d with a C:M:H20 solvent system of i n c r e a s i n g p o l a r i t y . We have been using the f o l l o w i n g mixtures: Solvent I - C:M:H Q (130:70:12, v / v ) , 0.4 C:M:H 0 (120:70:14, v / v ) , 0.5 l i t e r . 2

l i t e r and solvent 2 -

2

F r a c t i o n s o f 6 ml volume were c o l l e c t e d and 50 u l a l i q u o t s used to i d e n t i f y the gangliosides by TLC. Four gangliosides have been p u r i f i e d from f r a c t i o n I of chicken s k e l e t a l muscle d i r e c t l y from the column. S i l i c a - g e l G Column Chromatography of Neutral

Glycosphingolipids

The n e u t r a l l i p i d f r a c t i o n from the DEAE-Sephadex A-50 column was combined with the lower phase obtained a f t e r Folch p a r t i t i o n of the t o t a l l i p i d e x t r a c t and the combined l i p i d s d r i e d . To the same f l a s k , 100 ml of 0.6 M NaOH i n methanol was added. The mixture was incubated at 37°C f o r 5 hours. Five v o l umes of acetone were then added and stored overnight a t 4°C. The p r e c i p i t a t e was c o l l e c t e d by c e n t r i f u g a t i o n a t 4°C and d i s s o l v e d i n C:M (4:1, v / v ) . A f t e r a p p l i c a t i o n to the column (2.0 x 25 cm), the column was washed with chloroform. Neutral g l y c o l i p i d s were then e l u t e d with tetrahydrofuran: H2O (10:1). F r a c t i o n s c o n t a i n ing n e u t r a l g l y c o s p h i n g o l i p i d s were pooled and t h e i r g l y c o l i p i d content examined by t h i n - l a y e r chromatography. Enzymatic Hydrolysis Employing

Glycosidases

The sequence and anomeric c o n f i g u r a t i o n of the o l i g o s a c c h a r i d e chain was determined by step-wise h y d r o l y s i s with s p e c i f i c glycosidases. The conditions of incubation f o r h y d r o l y s i s are the same as those p r e v i o u s l y described (12). For the hydroly-

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

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s i s o f s i a l i c a c i d from g a n g l i o s i d e s , 30 yg of the g a n g l i o s i d e was d i s s o l v e d i n 150 y l of 0.05 M sodium acetate b u f f e r at pH 5.0, and incubated overnight a t 37°C with 4 m i n i u n i t s of neuraminidase from C l o s t r i d i u m p e r f r i n g e n s . For h y d r o l y s i s of a s i a l o gangliosides and n e u t r a l g l y c o s p h i n g o l i p i d s , 30 yg of g l y c o l i p i d was incubated overnight with 0.3 - 1.0 u n i t of $-galactosidase or 3-hexosaminidase at 37°C. A f t e r the r e a c t i o n was complete, the product was p a r t i t i o n e d t o the lower l a y e r by the a d d i t i o n of 5 v o l . of C:M(2:1, v / v ) , and the upper phase washed twice with t h e o r e t i c a l lower phase. The combined lower l a y e r s were then resolved by TLC. Permethylation methods The g l y c o s y l linkages were determined using methylation technique. In b r i e f , the p u r i f i e d g l y c o l i p i d s were exposed to d i m e t h y l s u l f i n y l i o n an The methylated d e r i v a t i v 24 cm) which had been packed and e l u t e d with acetone (14). The combined methylated g l y c o l i p i d s were hydrolyzed with 0.6N H2SO4 i n 80% aqueous a c e t i c a c i d a t 80°C f o r 18 hours, reduced and a c e t y l a t e d according to Bjorndal e t a l . (15). P a r t i a l l y methylated g a l a c t i t o l and g l u c i t o l acetates were separated i s o t h e r m a l l y at 180°C u s i n g a column packed with 3% OV-275 Supelcoport (100-120 Mesh). Amino sugar d e r i v a t i v e s are separated by a 3% 0V-17 Supelcoport (100-120 Mesh) column over a range of 180° - 200°C with a temperature increment r a t e of 2°/min. (16, 17, 18). Other Methods F a t t y a c i d methyl e s t e r s were extracted from the methanolysate with hexane and analyzed a t 190°C by GC using a 10% DEGS column. Sphingosine bases were determined a f t e r h y d r o l y s i s (19) as trime t h y l s i l y l d e r i v a t i v e s by GC u s i n g a 3% SE-30 column (20). S i a l i c a c i d was determined by the r e s o r c i n o l method (21) as modified by M i e t t i n e n and Takki-Luukainen (22). Species of s i a l i c a c i d (NANA, NGNA, etc.) were analyzed by TLC (23) and gas chromatography (24). Sugar composition and hexosamines were determined as a l d i t o l acetates using GC (15). Results Comparison of g l y c o s p h i n g o l i p i d s from human and chicken s k e l e t a l muscle. The e l u t i o n of g a n g l i o s i d e s from DEAE-Sephadex A 50 column with these 0.01, 0.02 and 0.2 M sodium acetate conc e n t r a t i o n s separated the g a n g l i o s i d e s i n t o mono-, d i - and p o l y sialo- fractions. The g a n g l i o s i d e s of human muscle are shown i n F i g . 1A. The monosialogangliosides GM3, GM2 and GM1 were e l u t e d with 0.01 M sodium acetate i n methanol (lane 2), GD3 and GDla with the 0.02 M s o l v e n t (lane 4) and others with 0.2 M acetate

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

9.

CHIEN AND

Figure 1A.

HOGAN

Skeletal

Muscle

139

Thin-layer chromatogram of human and chicken muscle ganglioside fractions

Lane 1, standard gangliosides from human brain. Lanes 2 and 3, gangliosides eluted by 0.01M, lanes 4 and 5 by 0.02M, and lanes 6 and 7 by 0.2M sodium acetate in methanol. Lanes 2, 4, and 6 from human muscle; lanes 3, 5, and 7 from chicken muscle. Solvent system: C:M:0.25% CaCl (60:40:9) 2

Figure IB.

Thin-layer chromatogram of

human and chicken neutral glycolipids Lane 1 contains (from the top) standard lactosylceramide, globotriaosylceramide, neolactotetraosylceramide, and lactopentaosylceramide prepared from bovine erythrocytes. Lane 2 contains the neutral glycosphingolipids of human skeletal muscle, and lane 3 contains those of the chicken. Solvent system: C:M:H 0 (60:40:9, v/v). 2

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

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(lane 6). When the upper phase g a n g l i o s i d e f r a c t i o n from chicken p e c t o r a l muscle was resolved i n a s i m i l a r way, four bands were e l u t e d with the 0.01 M acetate i n methanol. The major one had a m o b i l i t y corresponding to GM3. The others had Rf values c l o s e to those of GM1, GD3 and GDla r e s p e c t i v e l y ( F i g . 1A, lane 3 ) . However, a l l contained one s i a l i c a c i d per mole. The d i s i a l o g a n g l i o s i d e f r a c t i o n o f chicken muscle was a l s o considerably d i f f e r e n t from that o f human. Both contained GD3, but the other major d i s i a l o g a n g l i o s i d e s i n chicken migrated between GDla and GDlb; and GDlb and GT1 r e s p e c t i v e l y . Human muscle a l s o contained appreciable t r i s i a l o g a n g l i o s i d e of which very l i t t l e was found i n the chicken. The n e u t r a l g l y c o s p h i n g o l i p i d s o f human and chicken s k e l e t a l muscle were a l s o remarkably d i f f e r e n t ( F i g . IB). Human muscle contained l a c t o s y l c e r a m i d e as the major g l y c o l i p i d followed by globotriaosylceramide and globoside (17) while i n chicken muscle the major n e u t r a l g l y c o s p h i n g o l i p i nLcOse^Cer and IV^Gal-nLcOse^er of galactose and N-acetylgalactosamine and one mole of glucose, and was converted to globoside by the a-N-acetylgalactosaminidase from limpet (25). Thus, i t appears t o be a Forssman-active g l y c o l i p i d . G a s - l i q u i d chromatographic a n a l y s i s of the other n e u t r a l g l y c o l i p i d s was c o n s i s t e n t with the molar composition of galactosylceramide (20%), lactosylceramide (12%), g l u c o s y l c e r a mide (9%), globoside (8%) and globotriaosylceramide (3%). B i o - S i l A column chromatography and g l y c o s y l composition of the g a n g l i o s i d e s o f chicken muscle. The e l u t i o n p a t t e r n of monosialogangliosides from a B i o - S i l A column i s shown i n F i g . 2. Under these c o n d i t i o n s (see t e x t ) , the four g a n g l i o s i d e s separated w e l l . The f r a c t i o n s c o n t a i n i n g the same g a n g l i o s i d e were pooled and the p u r i t y confirmed by repeat TLC. When developed with C:M:0.25% C a C l (60:40:9, v / v ) , the four monosialoganglios i d e s comigrated with GM3, GM1, GD3 and GDla g a n g l i o s i d e s t a n dards i s o l a t e d from human b r a i n ( F i g . 3A). But i n another and a l k a l i n e solvent C:M:0.25N NaOH (60:40:9, v / v ) , three o r a l l except g a n g l i o s i d e I (lane 2) were obviously d i f f e r e n t i n m o b i l i ty ( F i g . 3B). G a n g l i o s i d e I I (lane 3) moved w e l l ahead of b r a i n GM1, g a n g l i o s i d e I I I (lane 4) was behind GD3, and the Rf of g a n g l i o s i d e IV (lane 6) was s l i g h t l y l e s s than that of the GDla. These d i f f e r e n c e s i n Rf must d e r i v e from the d i f f e r e n c e s i n sugar composition i n comparison to the standards from b r a i n (Table I ) . The composition of g a n g l i o s i d e I was the same as b r a i n GM3. Ganglioside I I d i f f e r e d from GM1 i n c o n t a i n i n g N-acetylglucosamine r a t h e r than N-acetylgalactosamine. Ganglioside I I I was a n o v e l s i a l o g l y c o l i p i d with a molar composition of s i a l i c a c i d : N-acetylgalactosamine: galactose: glucose: sphingosine of 1:1:3:1:1. Ganglioside IV had the same sugar composition as that of s i a l o s y l h e x a g l y c o s y l c e r a m i d e from human spleen (26) and bovine e r y t h r o c y t e s (12) with a molar r a t i o of s i a l i c a c i d : N-acetylglucosamine: g a l a c t o s e : glucose: sphingosine of 1:2:3:1:1. 2

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Figure 2. Elution pattern from Bio-Sil A column of monosialogangliosides prepared from chicken pectoral muscle. Column was eluted with C:M:H 0 (130:70:12, v/v) and changed to C:M:H 0 (120:70:14, v/v) at the arrow. 2

2

Figure 3.

Thin layer chromatograms of monosialogangliosides purified from chicken muscle

Lanes 1 and 5, human brain ganglioside standards; lanes 2, 3, 4, and 6 fractions from a Bio-Sil A colume (Figure 2). Solvent systems: (A) CHCl :MeOH:0.25 CaCl (60:49:9, v/v); (B) CHCl :MeOH:0.25M NHfiH (60:40:9, v/v). 3

2

3

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

Gal 1.07 2.16 2.80 2.96

1 1 1 1

I

II

III

IV

GANGLIOSIDE

GLc

1.83

1.18

GlcNAc

0.94

GalNAc

1.04

1.1

1.08

1.09

NANA

SUGAR COMPOSITION OF CHICKEN MUSCLE MONOSIALOGANGLIOSIDES

Table I

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Muscle

143

Characterization of saccharide u n i t . G a n g l i o s i d e I was h y d r o l y z e d by n e u r a m i n i d a s e t o a n e u t r a l g l y c o l i p i d w h i c h was f u r t h e r c l e a v e d by ( 3 - g a l a c t o s i d a s e t o become g l u c o s y l c e r a m i d e . The s e q u e n c e o f t h e two g l u c o s a m i n e - c o n t a i n i n g g a n g l i o s i d e s ( I I and I V ) a r e shown i n F i g . 4 a n d 5 r e s p e c t i v e l y . G a n g l i o s i d e I I was h y d r o l y z e d b y n e u r a m i n i d a s e w i t h no d e t e r g e n t t o a compound with R corresponding to that of Neolactotetraosylceramide which was s u b s e q u e n t l y c o n v e r t e d t o l a c t r i a o s y l - , l a c t o s y l - , a n d g l u cosylceramide by the consecutive a c t i o n s of 3-galactosidase, ^ - h e x o s a m i n i d a s e and 3 - g a l a c t o s i d a s e ( F i g . 4 ) . G a n g l i o s i d e I V was h y d r o l y z e d t o become a n e u t r a l h e x a g l y c o s y l c e r a m i d e when i n c u b a t ed w i t h n e u r a m i n i d a s e f r o m C I . p e r f r i n g e n s w i t h o u t d e t e r g e n t . The a s i a l o g l y c o l i p i d was i n t u r n c l e a v e d b y a l t e r n a t e t r e a t m e n t w i t h 3-galactosidase and 3-hexosaminidase to y i e l d g l u c o s y l c e r a mide ( F i g . 5 ) . G a s - l i q u i d chromatographic a n a l y s i s of the p a r t i a l l y methylated h e x i t o l acetate d e r i v a t i v e s produced 2 , 4 , 6 tri-O-methyl-galactito glucitol-l, 4,5-triacetate methyl-acetamidoglucitol-l,5-diacetate. There was no 2 , 3 , 4 , 6 tetra-O-methyl g a l a c t i c o l - 1 , 5-diacetate produced i n d i c a t i n g t h a t t h e s i a l i c a c i d i s a t t a c h e d a t the t e r m i n a l n o n r e d u c i n g end of t h e s a c c h a r i d e u n i t . f

F a t t y acids and sphingosines. The l i p i d c o m p o s i t i o n of t h e f o u r m o n o s i a l o g a n g l i o s i d e s i s shown i n T a b l e I I . The major f a t t y a c i d s a r e p a l m i t i c , s t e a r i c and o l e i c a c i d s . The l o n g c h a i n base i s composed m a i n l y o f C - 1 8 s p h i n g o s i n e w i t h l e s s t h a n 20% o f d i h y d r o s p h i n g o s i n e . Discussion The s t r u c t u r e s o f t h e g l y c o s p h i n g o l i p i d s o f s k e l e t a l m u s c l e h a v e b e e n s t u d i e d i n human (8) a n d r a b b i t s k e l e t a l m u s c l e (7) and i n human c a r d i a c m u s c l e ( 9 ) . Q u a l i t a t i v e l y , human s k e l e t a l and c a r d i a c m u s c l e c o n t a i n t h e same n e u t r a l g l y c o s p h i n g o l i p i d s and g a n g l i o s i d e s though t h e g a n g l i o s i d e s o f r a b b i t s k e l e t a l muscle are q u i t e d i f f e r e n t . R a b b i t d o e s n o t c o n t a i n GM2 o r t h e g l u c o s a m i n e - c o n t a i n i n g g a n g l i o s i d e r e p o r t e d i n human s k e l e t a l muscle. We h a v e u s e d a D E A E - S e p h a d e x c o l u m n t o s e p a r a t e g a n g l i o s i d e s i n t o three groups, the mono-, d i - and p o l y s i a l o g a n g l i o s i d e s and t h i s e n a b l e d a more d e t a i l e d c o m p a r i s o n o f g a n g l i o s i d e s . Both h u m a n a n d c h i c k e n s k e l e t a l m u s c l e c o n t a i n GM3 a s t h e m a j o r g a n g lioside. The o t h e r m o n o s i a l o g a n g l i o s i d e s o f human m u s c l e a r e GM2 a n d GM1 b u t t h e s e t w o g a n g l i o s i d e s w e r e n o t d e t e c t e d i n c h i c k e n . I n s t e a d , g a n g l i o s i d e s c o n t a i n i n g glucosamine and a s i a losylpentaglycosylceramide constitute the other monosialoglycol i p i d s . C h i c k e n m u s c l e a l s o d i f f e r s f r o m human i n c o n t a i n i n g a n o v e l d i s i a l o g a n g l i o s i d e m i g r a t i n g between GDla and GDlb i n a l k a l i n e c o n d i t i o n s ( F i g . 3 B ) . The major n e u t r a l g l y c o s p h i n g o l i p i d of c h i c k e n muscle i s a Forssman-hapten p e n t a g l y c o s y l c e r a m i d e

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

CELL SURFACE

Figure 4.

GLYCOLIPIDS

Enzymatic hydrolysis of ganglioside II (lane 3 in Figure 3)

Lane 1, standard brain gangliosides. Lane 7, standard neutral glycolipids from bovine erythrocytes. Lane 2, ganglioside II. Lane 3, 2+ neuraminidase. Lane 4, 5+ f3-galactosidase. Lane 5, 4+ ^-hexosaminidase. Lane 6, 5+ j3-galactosidase.

Figure 5.

Enzymatic hydrolysis of ganglioside IV (lane 6 in Figure 3)

Lanes 1 and 9, ganglioside and neutral glycosphingolipid standards as in Figure 4. Lane 2, ganglioside IV. Lane 3, 2+ neuraminidase. Lane 4, 3+ j3-galactosidase. Lane 5, 4+ ^-hexosaminidase. Lane 6, 5+ fi-galactosidase. Lane 7, 6-f ft-hexosaminidase. Lane 8, 7+ /3-galactosidase.

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

Skeletal

CHIEN A N D HOGAN

Muscle

Table I I FATTY ACIDS AND LONG CHAIN BASES OF CHICKEN MUSCLE MONOSIALOGANGLIOSIDES

Gangliosides Fatty Acids

I

II

III

IV

C 16:0 C 16:1

1.8

2.2

3.0

2.5

C 18:0

33.5

24.7

39.2

33.4

C 18:1

21.5

38.5

31.8

24.2

C 18:2

5.2

2.1

1.2

3.7

C 20:0

2.5

1.2

2.2

3.0

C 20:1

1.5

1.8

0.5

1.4

C 21:0

1.0

—

1.0

3.5

C 22:0

2.4

1.0

—

1.1

C 22:1

5.2

2.0

1.7

2.1

C 24:1

2.2

8.4

4.0

8.5

d 18:0

18.0

16.3

14.7

10.7

d 18:1

82.0

83.7

85.3

89.3

Sphingosines

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

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146

GLYCOLIPIDS

i d e n t i c a l to that p r e v i o u s l y described i n horse kidney and spleen (27), sheep e r y t h r o c y t e s , (28), and canine kidney and i n t e s t i n e (18). In c o n t r a s t , lactosylceramide predominates i n human muscle and c o n s t i t u t e s 38% of the t o t a l n e u t r a l g l y c o l i p i d s . Such s t r i k i n g d i f f e r e n c e s i n these compounds - both n e u t r a l and a c i d i c - suggests that species s p e c i f i c i t y p r e v a i l s over organ or t i s s u e c o n t r a i n t s upon the p a t t e r n or r a t i o of these l i p i d s . The use o f ion-exchange chromatography enabled the subsequent greater r e s o l u t i o n of i n d i v i d u a l gangliosides by s i l i c i c a c i d chromatography with B i o - S i l A and c h a r a c t e r i z a t i o n of the major monosialogangliosides i n chicken p e c t o r a l muscle by g l y c o s y l composition, enzymatic sequencing and methylation a n a l y s i s . The s t r u c t u r e s i d e n t i f i e d included GM3 (ganglioside I , NeuAca2-K3Gal$l->-4Glc-»Cer), s i a l o s y l - l a c t o - N - n e o t e t r a o s y l c e r a m i d e or I V aNeuAc-nLc0se4Cer (ganglioside I I , NeuAca2-*3Gal$l-*4GlcNAc 31->3Gal31->4Glc->Cer), s i a l o s y l lacto-N-neohexaglycosylceramide or Vl3 aNeuAc-nLc0se6Ce 4GlcNAc31->3Gal31->4GlcNAc31^3Gal31->4Glc->Cer) sylpentaglycosylceramide (ganglioside I I I ) . The sequence of t h i s ganglioside has been determined by step-wise h y d r o l y s i s using s p e c i f i c glycosidases to be NeuAca-»Gal3- GalNAc3-*Gala->Gal 3Glc->Cer (V NeuAc, IV Gal-Gg0se4Cer) and i s to our knowledge the f i r s t g l y c o l i p i d o f the globo-series c o n t a i n i n g s i a l i c a c i d . There are evidences f o r i m p l i c a t i n g glycoconjugates on the muscle c e l l surface i n myogenesis (29, 30). Whatley e t a l , (29) for example found a t h r e e - f o l d increase i n GDla concentration j u s t p r i o r to f u s i o n i n a r a t myoblast c e l l l i n e while GM3, GM2 and GM1 d i d not change. McEvoy and E l l i s (30) found an increased b i o s y n t h e s i s o f s e v e r a l n e u t r a l g l y c o s p h i n g o l i p i d s and g a n g l i o s i d e s j u s t p r i o r to f u s i o n i n primary c u l t u r e s of chick embryo myoblasts. A r o l e f o r g l y c o l i p i d s i n such i n t e r c e l l u l a r regul a t i o n i s a l s o c o n s i s t e n t with the reported promotion of c e l l adhesion i n Hela c e l l s i n the presence of g l y c o l i p i d s p a r t i c u l a r l y those with tetraose chain length and a terminal galactose (5). I t would seem important to consider the s t r u c t u r e s of chicken muscle g l y c o s p h i n g o l i p i d s i n r e l a t i o n to the study of myogenesis i n view of the wide usage o f embryonic chick muscle as a model system. 3

>

Acknowledgment This work was supported i n part by the Muscular Dystrophy A s s o c i a t i o n . We thank Mrs. Eve Thrasher f o r s e c r e t a r i a l support and typing t h i s manuscript.

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

9. CHIEN AND HOGAN

Skeletal Muscle

147

Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.

Mullin, B. R., Fishman, P. H., Lee, G., Aloj, S. M., Ledley, F. D., Winand, R. J., Kohn, L. D. and Brady, R. O. Proc. Natl. Acad. Sci. U.S.A., 1976, 73, 842-846. Lee, G., Aloj, S. M., Brady, R. O. and Kohn, L. D. Biochem. Biophys. Res. Comm., 1976, 73, 370-377. Glick, J. L. and Githens, S. Nature, 1965, 208, 88. Roseman, S. Chem. Phys. Lipids, 1970, 5, 270-297. Huang, R. T. C. Nature, 1978, 276, 624-626. Puro, K., Maury, P. and Huttunen, J. K. Biochim. Biophys. Acta, 1969, 187, 230-235. Lassaga, F. E., Lassaga, A. G. and Caputto, R. J. Lipid Research, 1972, 13, 810-815. Svennerholm, L . , Bruce, A., Mansson, J.-E., Rynmark, B.-M. and Vanier, M.-T Biochim Biophys Acta 1972 280 626-636. Levis, G. M., Karli, Moulopoulos, Lipids, 1979, 14, 9-14. L i , S.-C. and L i , Y.-T. J. Biol. Chem., 1970, 245, 51535160. Folch, J., Lee, M. and Sloane Stanley, G. H. J. Biol. Chem., 1957, 226, 497-509. Chien, J.-L., L i , S.-C., Laine, R. A. and Li, Y.-T. J. Biol. Chem., 1978, 253, 4031-4035. Hakomori, S. J. Biochem. (Tokyo), 1964, 55, 205-208. Yang, H. and Hakomori, S. J. Biol. Chem., 1971, 246, 1192-1200. Bjorndal, H., Lindberg, B. and Svensson, S. Acta Chem. Scand., 1967, 21, 1802-1804. Stellner, K., Saito, H. and Hakomori, S. Arch. Biochem. Biophys., 1973, 155, 464-472. Stoffel, W. and Hanfland, P. Hoppe-Seyler's Z. Physiol. Chem., 1973, 354, 21-31. Sung, S.-S., Esselman, W. J. and Sweeley, C. C. J. Biol. Chem., 1973, 248, 6528-6533. Gaver, R. C. and Sweeley, C. C. J. Am. Oil. Chem. Soc., 1965, 42, 294-298. Carter, H. E. and Gaver, R. C. J. Lipid Res., 1967, 8, 391-395. Svennerholm, L. Biochem. Biophys. Acta, 1957, 24, 604611. Miettinen, T. and Takki-Luukainen, I. T. Acta Chem. Scand., 1954, 13, 856-859. Granzer, E. Z. Physiol. Chem., 1962, 328, 277-279. Yu, R. K. and Ledeen, R. W. J. Lipid Res., 1970, 11, 506-515. Uda, Y., L i , S.-C. and L i , Y.-T. J. Biol. Chem., 1977, 252, 5194-5200. Wiegandt, H. Eur. J. Biochem., 1974, 45, 367-369. American Chemical Society Library 1155 16th St. N. W. Washington, D. C. 20036 In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

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Siddiqui, B. and Hakomori, S. J. Biol. Chem., 1971, 246, 5766-5769. Fraser, B. A. and Mallette, M. F. Immunochemistry, 1974, 11, 581-585. Whatley, R., Ng, K. D., Rogers, J., McMurray, W. C. and Sanwal, B. D. Biochem. Biophys. Res. Comm., 1976, 70, 180-185. McEvoy, F. A. and Ellis, D. E. Biochem. Soc. Trans., 1977, 5, 1719-1721.

RECEIVED

December 10, 1979.

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

10 Glycosphingolipids and Glyceroglucolipids of Glandular Epithelial Tissue BRONISLAW L. SLOMIANY and AMALIA SLOMIANY Department of Medicine, New York Medical College, New York, NY 10029

A major problem encountered in the analysis of glycolipids is the assurance that glycolipids are removed from the tissue in high yield. Utilization of the classical methods for the isolation of lipids have introduced some limitations with regard to the extractibility of highly polar glycosphingolipids and hence have led to many false statements and to misconceptions that the glycosphingolipid compositions are well explored. Our systematic investigations into the nature of ABH antigens of gastric mucosa have resulted in the isolation and identification of a number of fucolipids, Forssman glycosphingolipid variants and sulfated glycosphingolipids, which have differences in their internal composition, anomeric configuration, length of oligosaccharide chains and degree of complexity. The successful isolation of these glycolipids was the result of a new methodological approach that considered the effect of carbohydrate moiety on the solubility of glycosphingolipids and their tight association with other membrane components. Our extensive studies on glycosphingolipids of gastric mucosa indicate that in order to obtain complete solubilization of this class of compounds, entirely different methodological approaches must be considered. In spite of the assumption that the mucous glycolipids and glycoproteins are similar to, or possibly derived from those found on cell surfaces, glycosphingolipids have not been found to be constituents of mucus secretions. However, the presence of a new type of glycolipid (glyceroglucolipid) has been demonstrated. This implies that glycosphingolipids are confined to membranous structures of the cell in which they may vary in composition, content and expression, and that this may be essential for certain specialized functions of the cell. A protective role of glyceroglucolipids in the cell may be speculated from their localization in the gastric mucous barrier and their resistance to chemical and biological degradation in the most obnoxious of environments. 0-8412-0556-6/80/47-128-14957.00/ 0 © 1980 American Chemical Society In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

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CELL SURFACE

GLYCOLIPIDS

In t h i s a r t i c l e we review developments i n methodological approaches f o r i s o l a t i o n of g l y c o s p h i n g o l i p i d s i n high y i e l d s ; demonstration of g l y c o s p h i n g o l i p i d complexity as w e l l as species, i n d i v i d u a l and organ s p e c i f i c i t y ; demonstration of d i s t i n c t i v e features o f the e p i t h e l i a l t i s s u e versus i t s s e c r e t i o n ; and desc r i p t i o n o f a new group of g l y c o l i p i d s which are confined to mucous s e c r e t i o n s . The G l y c o s p h i n g o l i p i d s

of G a s t r i c Mucosa and S a l i v a r y Glands

In e a r l y attempts to i s o l a t e blood group ABH antigens the idea of " l i p i d - h a p t e n " has been c r i t i c i z e d since the antigens were not e x t r a c t a b l e by ether o r alcohol-ether mixtures (1_, 2) , but i n stead the ABH a c t i v i t y was found i n more p o l a r solvents (3). The presence o f complex g l y c o s p h i n g o l i p i d s i n animal t i s s u e s and t h e i r e x t r a c t i b i l i t y were not known henc th s o l u b i l i t propertie were s u f f i c i e n t to suppor are g l y c o p r o t e i n s . In y , immunologically l i p i d s were obtained by s o l u b i l i t y d i f f e r e n c e s i n organic solvents and by p r e c i p i t a t i o n as metal conrplexes (£, 5). More r e c e n t l y , the i s o l a t i o n and c h a r a c t e r i z a t i o n of g l y c o s p h i n g o l i p i d s has been g r e a t l y advanced, but the problem with complex components has not been completely solved. In contrast t o the well-known blood group a c t i v e g l y c o p r o t e i n s of g a s t r i c mucosa and g a s t r i c s e c r e t i o n , l i t t l e was known about these g l y c o s p h i n g o l i p i d s except f o r the e a r l y work of Masamune and Siojima (6). E x t r a c t i o n with Chloroform/Methano1. E x t r a c t i o n of hog g a s t r i c mucosa with the conventional mixture o f chloroform/methan o l (2/1, v/v) r e s u l t e d i n the i s o l a t i o n o f s e v e r a l glycosphingol i p i d s . The blood-group a c t i v e g l y c o s p h i n g o l i p i d s p u r i f i e d from t h i s e x t r a c t contained up to seven carbohydrate residues i n the molecule (Table 1, s t r u c t u r e s 1,2,3,4). The heptahexosylceramides 1 and 2 e x h i b i t e d strong blood group A a c t i v i t y and d i f f e r e d from each other i n linkages of the subterminal galactose to N-acetylglucosamine residues (type 1 and type 2 chains) and i n f a t t y a c i d composition (7,8). G l y c o s p h i n g o l i p i d 1 with the type 1 chain had 11.4% hydroxylated acids and 15.7% of C22-C24, whereas glycos p h i n g o l i p i d 2 with type 2 chain had 35.4% hydroxylated f a t t y acids and 44.4% of C 2-Co. f a t t y a c i d s . The d i f f e r e n c e s i n f a t t y acids apparently were s u f f i c i e n t to a f f e c t chromatographic m o b i l i t y of these compounds, and permitted t h e i r i s o l a t i o n as two d i s t i n c t bands. This enabled us to show f o r the f i r s t time the existence o f two types o f chains i n A - a c t i v e g l y c o s p h i n g o l i p i d s , s i n c e only type 2 chains were found i n A and H g l y c o s p h i n g o l i p i d s o f the erythrocytes (9,10). A l s o , the i s o l a t e d g l y c o s p h i n g o l i p i d s d i f f e r e d from those o f human erythrocytes by having an a d d i t i o n a l galactose residue adjacent to the l a c t o s y l p o r t i o n o f the carbohydrate chain. 2

Further studies of blood group a c t i v i t y of various i s o l a t e d g l y c o s p h i n g o l i p i d s l e d to i s o l a t i o n o f components 3 and 4

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

10.

SLOMIANY A N D SLOMIANY

Glandular

Epithelial

Tissue

151

( T a b l e I ) . The h e x a h e x o s y l e e r a m i d e 3 was A - a c t i v e b u t l a c k e d N - a c e t y l g l u c o s a m i n e ; i t s a c t i v i t y i n A - a n t i - A s y s t e m was somewhat d i m i n i s h e d as compared t o t h a t o f t h e h e p t a h e x o s y l c e r a m i d e s 1 a n d 2 (11). T h e a b s e n c e o f N - a c e t y l g l u c o s a m i n e was a l s o d e tected i n tetrahexosylceramide 4, which manifested H a c t i v i t y ( 1 2 ) b u t a g a i n e x h i b i t e d weaker a n t i g e n i c p o t e n c y t h a n t h a t shown f o r H - a c t i v e p e n t a - , o c t a - and decahexosylceramides of the erythrocytes (10).This decrease i n antigenic a c t i v i t y apparently r e s u l t s from t h e p r o x i m i t y o f t h e a n t i g e n i c determinant and hydrophobic p o r t i o n o f t h e s e g l y c o s p h i n g o l i p i d s , o t i t may b e d u e t o t h e a b s e n c e o f N - a c e t y l g l u c o s a m i n e , w h i c h i n some s u b t l e w a y i n f l u e n c e s the a c t i v i t y of the antigenic determinants. The f u c o s e - c o n t a i n i n g g l y c o s p h i n g o l i p i d s , so abundant i n hog g a s t r i c mucosa, were n o t d e t e c t e d i n r a t s u b l i n g u a l and s u b m a x i l l a r y glands (13), although both tissues are f u n c t i o n a l l y s i m i l a r . O n l y t r a c e s o f f u c o s e were found i n crude g l y c o s p h i n g o l i p i d f r a c tions p r i o r to t h i n - l a y e t r a c e s o f f u c o s e were a l s l i n g u a l (14) a n d s u b m a x i l l a r y g l a n d s ( 1 5 ) . The absence o f f u c o s e c o n t a i n i n g g l y c o s p h i n g o l i p i d s supports~The f i n d i n g s o f Kent and S a n d e r s ( 1 6 ) , who h a v e s t u d i e d t h e d i s t r i b u t i o n o f b l o o d g r o u p A antigen throughout the d i g e s t i v e t r a c t o f r a t and found i t s highest content i n large i n t e s t i n e . Their data, together with our r e s u l t s , suggest gradient d i s t r i b u t i o n of fucose-containing glyc o s p h i n g o l i p i d s and glycoproteins throughout the d i g e s t i v e t r a c t o f t h e r a t and p o s s i b l y o f o t h e r mammalian s p e c i e s . The n e u t r a l g l y c o s p h i n g o l i p i d s (Table I I ) contained g l u c o s e , g a l a c t o s e and N a c e t y l g a l a c t o s a m i n e . N - a c e t y l g l u c o s a m i n e was f o u n d o n l y i n v e r y s m a l l amounts i n t h e g a n g l i o s i d e f r a c t i o n s o f t h e g l a n d s . The s u b m a x i l l a r y and e s p e c i a l l y s u b l i n g u a l glands e x h i b i t e d a h i g h cont e n t o f t h e s u l f a t e d g l y c o s p h i n g o l i p i d s . These were composed o f mono- and d i - h e x o s e s u l f a t i d e , w i t h t h e former b e i n g predominant i n b o t h types o f g l a n d s . The h i g h content o f s u l f a t e d g l y c o s p h i n g o l i p i d s i s i n agreement w i t h h i s t o l o g i c a l s t u d i e s o f P r i t c h a r d a n d Rusen (17) and P r i t c h a r d (18) o n t h e d i s t r i b u t i o n o f r a d i o s u l f a t e i n r a t s a l i v a r y g l a n d s . The abundance o f s u l f a t e d g l y c o s p h i n g o l i p i d s i n s a l i v a r y g l a n d s may i n d i c a t e t h a t t h e y p a r t i c i p a t e i n the secretory processes of these glands. Buffered Tetrahydrofuran. In 1973, Tettamanti et a l . (19) d e s c r i b e d a n i m p r o v e d p r o c e d u r e f o r t h e e x t r a c t i o n , s e p a r a t i o n and p u r i f i c a t i o n of b r a i n g a n g l i o s i d e s . In t h i s method, the b r a i n t i s s u e was s u b j e c t e d t o h o m o g e n i z a t i o n a n d e x t r a c t i o n w i t h b u f f e r e d ( p o t a s s i u m phosphate b u f f e r , pH 6.8) t e t r a h y d r o f u r a n . F o l l o w i n g c e n t r i f u g a t i o n , d i e t h y l e t h e r was added a n d t h e m i x t u r e s e p a r a t e d i n t o o r g a n i c and aqueous phase. The g a n g l i o s i d e s , r e c o v e r e d e x c l u s i v e l y i n t h e aqueous p h a s e , were t h e n f r e e d o f r e s i dual phospholipids and other minor contaminants ( i . e . peptides)by c o l u m n c h r o m a t o g r a p h y on s i l i c a g e l . T h i s p r o c e d u r e , as shown b y t h e a u t h o r s , w a s s u p e r i o r t o t h e commonly u s e d chloroform/methanol

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

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GLYCOLIPIDS

Table I . S t r u c t u r e s o f the g l y c o s p h i n g o l i p i d s c h a r a c t e r i z e d from hog and dog g a s t r i c mucosa.

Glycolipid Structure 1. GalNAcal->3GalBl->3GlcNAc61^3Gal61->4Gal61->4Glcei^lCer 2 t laFuc 2.

GalNAcal-K5Gal Bl+4GlcNAcei+3Gal gl-»4Gal gl-*4Glc31->lCer 2 t laFuc

3.

GalNAcal-K3Gal ei+3Gal Sl+4Gal 01+4G l c W C e r 2 laFuc

4. 5.

Fucal+2Galal->3GalBl->4Glcl-*lCer G alNAcal+3G a l g 1+4G1 cNAc £1+3G a l 61+4G 1 eg 1->1 Ce r 2 laFuc

6.

7.

8.

9.

3 laFuc

GalNAcal-K5Gal 31+3/ 4G1 cNAc l+3Gal 1+4G1 c 1+lCer 2 laFuc Gall+4GlcNAcl-*3Gall+4Glcl+lCer 3 t lFuc GalNAcal+3Gall+3Gall->4Glcl+lCer 2 i laFuc GalNAcal+SGalNAcBl+SGalal^Gaiei^Glcl+lCer

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

Glandular

SLOMIANY AND SLOMIANY

10.

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153

G a INAc a l +3G a INAc 0.

\

3 Galal+4GalBl->4Glcl+lCer 4

/ GalBl->3GalNAc31 11.

GalNAcal+3GalNAcei

\ 3 Galal+4GalBl+4Glcl+lCer 4

/ Fucai->2GalBl->3GalNAc3l 12.

GalNAcal+3Gall+4GlcNAc 2 \ + f 3 6 laFuc Gal1+4G1cNAcl+4G1cNAcl+3Gal1+4 6 Glcl+lCer

/ GlcNAcl+4GlcNAcl lGlcNAc 4

13.

Gal 1 4 lGlcNAc

GalNAcal+3Gall+4GlcNAcl 2 + 1 Fuc

V + 3 6 Gall+4GlcNAcl+4GlcNAcl+3Gall+4 6 4 Glcl+lCer / f G a INAca 1+3G a 11+4G1 cNAc 1 1 2 GlcNAc f 4 laFuc t lGlcNAc 14.

GalNAcal+3Galgl+4Galgl 3

t 1 Fuc

Gal31+4Gaiei+4Glc31+lCer

/ GalNAcal+3Gal3l

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154

CELL SURFACE

15.

GLYCOLIPIDS

GalNAcal+3Gal 3l+4Gal 31 2 +

\ 3

laFuc

Gal 31+4Glc31+lCer

GalNAcal+3Gal3r 16.

GalNAcal+3Gal31+4Gal31 2 \ t 3 laFuc Gal31+4Glc31+lCer 6 GlcNAc31+4Gal31

17.

/

GalNAcal+3Gal31+4Gal3 2 \ laFuc

\ 3 Gal31+4Glc31+lCer 6

/ Galgl 18.

Gal31+4GlcNAc3l+4Gal31

*

\

f

3 Gal31+4Glc31+lCer 6

laFuc

GlcNAc31+4Gal3l 19.

/

GalNAcal+3Gal3l+3/4GlcNAc3l \ laF uc

3 G a 131+4G1 cNAc 3 1+3G a 131+4 6 Gal31+4Glc31+lCer

/ Fucal+2Gal3l+3/4GlcNAc31 2 0.

GalNAca1+3G a 131+3/4G1cNAc31

I f

>»

laFuc

3 G a 131+4G1 cNAc3 1+4G1 cNAcfB 1+3 6 Galgl+4GlcBl+lCer

Fucal+2Gal3l+3/4GlcNAc3l^

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SLOMIANY A N D SLOMIANY

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BGal 1

21.

4 3G 1 cNAc 1 GalNAcal+3Gal31+3/4GlcNAc3\ + 2 3 6 t Gal31+4GlcNAc31+3Gal31+4 laFuc 6 Gal31+4Glc31->lCer Fucal+2Gal3l+3/4GlcNAc31

22.

GalNAcal+3Gal31+3/4GlcNAc31 2 \ i 3 laFu Fuca 1+2G a 131+3/ 4G1 cNAc 31 *

6 1 3GlcNAc 4 1 3Gal

Table I I The Composition and Molar Ratios o f Carbohydrates gual and Submaxillary G l y c o s p h i n g o l i p i d s .

Glycosphingolipid

G

l

c

G

a

RSL RSM RSL Glucosylceramide Galactosylceramide Dihexosylceramide Trihexosylceramide Tetrahexosylceramide Pentahexosylceramide Monosialoganglioside Disialoganglioside Monohexose s u l f a t i d e Dihexose s u l f a t i d e

GlcNAc

l

RSM

RSL

o f Rat S u b l i n -

GalNAc RSM

RSL

RSM

1,.0 1,.0 1. 0 0.97 1. 95 1. 92 3. 02 1. 02 0.98 1. 0 1 .0 1,.0 0.96

1 .0 1 .0 1 .0 1 .0 1 .0 1 .0

1,,0 1,.0 1,.0 1,.0 1,.0 1,.0

1.0 0.99 1.91 1.90 2.95 0.97 0.28 0.95 t r . 1.0 0.98

1.01 0.98 0.06 0.31 0.10 t r .

RSL, r a t s u b l i n g u a l : RSM. r a t submaxillary; t r . , t r a c e s (From Ref. 13 )

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

0.94 0.97 0.05 0.09

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GLYCOLIPIDS

/water p a r t i t i o n systems (20,21). A p p l i c a t i o n o f b u f f e r e d tetrahydrofuran e x t r a c t i o n to g a s t r i c mucosa, followed by c a r e f u l examination o f the aqueous phase f o r various g l y c o s p h i n g o l i p i d s , i n d i c a t e d that t h i s phase contained s i a l o g l y c o s p h i n g o l i p i d s and considerable q u a n t i t i e s of n e u t r a l g l y c o s p h i n g o l i p i d s (22). These were separated from the a c i d i c g l y c o s p h i n g o l i p i d s by DEAE-Sephadex column chromatography (23). The n e u t r a l g l y c o s p h i n g o l i p i d f r a c t i o n of hog g a s t r i c mucosa was shown to c o n s i s t mostly o f f u c o l i p i d s C 4,25), whereas that o f dog g a s t r i c mucosa e x h i b i t e d a high content of N-acetylgalactosaminecontaining g l y c o s p h i n g o l i p i d s (26). Of the eight f u c o l i p i d s p u r i f i e d from the n e u t r a l g l y c o l i p i d f r a c t i o n o f the aqueous phase o f b u f f e r e d tetrahydrofuran e x t r a c t of hog g a s t r i c mucosa, four were found to be i d e n t i c a l with those (Table I, compounds 1-4) i s o l a t e d p r e v i o u s l y by the chloroform/methanol e x t r a c t i o n procedure (7^,8^,1^,12) and the e l u c i d a t e d s t r u c t u r e s o f four new compounds (5-8) are l i s t e d i n Tabl ted blood group A - a c t i v i t y the A anti-A, B anti-B o r H anti-H systems. In f u c o l i p i d 6, the subterminal galactose was l i n k e d t o the next sugar i n the chain, N-acetylglucosamine by both 1+3 (40%) and 1+4 (60%) l i n k a g e s . F u c o l i p i d 8 was s t r u c t u r a l l y r e l a t e d to compound 3 and, although i t e x h i b i t e d blood group A - a c t i v i t y , i t s carbohydrate chain was devoid o f N-acetylglucosamine. F u c o l i p i d 7 had a carbohydrate chain i d e n t i c a l i n s t r u c t u r e to that i n the g l y c o l i p i d from normal and human adenocarcinoma t i s s u e (28). Thus, i t became apparent that t h i s g l y c o s p h i n g o l i p i d i s not only present i n human glandular t i s s u e but a l s o i n glandular t i s s u e o f other species and may not n e c e s s a r i l y be a s p e c i f i c antigen of malignant c e l l s . 2

The carbohydrate chain o f f u c o l i p i d 5 contained seven sugar residue and d i f f e r e d from the known A - a c t i v e g l y c o s p h i n g o l i p i d s by the presence o f a second fucose residue, l i n k e d to C-3 o f the i n t e r n a l N-acetylglucosamine. I d e n t i f i c a t i o n o f t h i s glycosphingol i p i d provided the f i r s t evidence f o r the existence o f d i f u c o s y l blood group A - a c t i v e g l y c o s p h i n g o l i p i d s with carbohydrate s t r u c tures i d e n t i c a l to d i f u c o s y l o l i g o s a c c h a r i d e s o f g l y c o p r o t e i n o r i g i n (29), suggesting that the same carbohydrate chains may be l i n k e d to a l i p i d o r p r o t e i n core. A s i m i l a r g l y c o l i p i d was i s o l a t e d l a t e r from dog i n t e s t i n e (30). Examination o f the n e u t r a l g l y c o l i p i d s i n the aqueous f r a c t i o n o f a b u f f e r e d tetrahydrofuran e x t r a c t o f dog g a s t r i c mucosa i n d i c a t e d presence o f g l y c o s p h i n g o l i p i d s containing s i g n i f i c a n t amounts of N-acetylgalactosamine, but only traces o f fucose (26). A s i m i l a r conclusion as to the content o f f u c o l i p i d s i n dog gast r i c mucosa was reached e a r l i e r by McKibbin and L y e r l y (31). Extensive p u r i f i c a t i o n o f the g l y c o s p h i n g o l i p i d s present i n the n e u t r a l f r a c t i o n r e s u l t e d i n the i s o l a t i o n o f three d i s t i n c t g l y c o l i p i d s e x h i b i t i n g Forssman a n t i g e n i c a c t i v i t y (Table I, compounds 9,10,11). T h i n - l a y e r chromatographs o f these g l y c o l i p i d s i s i l l u s t r a t e d i n F i g . 1. Chemical analyses o f the p u r i f i e d

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

SLOMIANY

A N D SLOMIANY

Glandular

Epithelial

Figure 1.

Tissue

157

Thin-layer chromatography of

Table II; (3) glycolipid 10, Table I. Solvent system: chloroform/methanol/water (60/ 35/8, v/v/v). Visualization: orcinol reagent. (26)

Figure 2. Thin-layer chromatogram of purified glycolipid 11 (Table I) and its enzymatic hydrolysis products (1) Native glycolipid; (2) glycolipid obtained by treatment of the native compound with a-fucosidase; (3) glycolipid obtained by the sequential treatment of the native compound with a-fucosidase, /? galactosidase and / ? - N acetylhexosaminidase; (4) glycolipid obtained by treatment of the defucosylated compound (from line 2) with a-N-acetylgalactosaminidase and f3-N-acetylhexosaminidase; (5) glycolipid obtained by treatment of the defucosylated compound (from line 2) with j3-galactosidase, a-N-acetylgalactosaminidase and /3-N-acetylhexosaminidase; (6) glycolipid from line 5 after incubation with a- and /?galactosidase. Standards: (7a) glucosylceramide; (7b) lactosylceramide; (7c) triglycosylceramide; (7d) glycolipid 9, Table 1; (7e) glycolipid 10, Table 1. Solvent system: chloroform/methanol/water (65/35/8, v/v/v). Visualization: orcinol reagent. (33)

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GLYCOLIPIDS

compounds i n d i c a t e d t h a t the carbohydrate moiety of g l y c o l i p i d 9 c o n s i s t e d o f glucose, galactose and N-acetylgalactosamine i n a molar r a t i o o f 1:2:2. The same carbohydrates, but i n a molar r a t i o o f 1:3:3, were present i n g l y c o l i p i d 10, whereas g l y c o l i p i d 11 contained glucose, fucose, galactose and N-acetylgalactosamine i n a molar r a t i o o f 1:1:3:3 (26). Further s t r u c t u r a l s t u d i e s (32,33) revealed that the carbohydrate moiety o f g l y c o l i p i d 9 i s chemicall y i d e n t i c a l with that of Forssman hapten, c h a r a c t e r i z e d prev i o u s l y from kidney and i n t e s t i n e o f dog (34,35,36) and from spleen and kidney of horse (37,38). The r e s u l t s o f chemical and enzymatic analyses of g l y c o l i p i d 10 suggested that t h i s compound contains two terminal sugar r e s i d u e s , galactose and N - a c e t y l galactosamine, and thus has a branched s t r u c t u r e . S u s c e p t i b i l i t y of g l y c o l i p i d 10 to g l y c o s i d a s e degradation i n the sequence: 3-galactosidase, 3-N-acetylhexosaminidase, and the sequence: a-N-acetylgalactosaminidase 3-N-acetylhexosaminidase i n d i c a t e d that the s i d e chains ar GalNAc d i s a c c h a r i d e s . P a r a l l e of such enzymic degradation products e s t a b l i s h e d that the above d i s a c c h a r i d e chains are l i n k e d by 1+4 and 1+3 bonds, r e s p e c t i v e l y , to the galactose residue adjacent to lactosylceramide o f the gly-* c o l i p i d core. The presence o f two s i d e chains, aGalNAc(l+3)gGalNAc and aFuc(l+2)3Gal(l+3)3GalNAc, i n g l y c o l i p i d 11 was c l e a r l y demonstrated with the a i d of g l y c o s y l h y d r o l a s e s ( F i g . 2) and permethylation a n a l y s i s . Furthermore, the n a t i v e g l y c o l i p i d 11 exhib i t e d both Forssman and H a n t i g e n i c a c t i v i t i e s . D e f u c o s y l a t i o n o f t h i s g l y c o l i p i d (0.1 M t r i c h l o r o a c e t i c a c i d , 100°C f o r 2 h) r e s u l t e d i n the l o s s o f i t s H - a c t i v i t y but had no e f f e c t on i t s r e a c t i v i t y with Forssman anti-serum o r on i t s a b i l i t y to i n h i b i t hemagglutination i n the A/anti-A system. The l a t t e r a c t i v i t y , shared by a l l three compounds (9,10,11), i s thought to be due to the presence of a terminal a-N-acetylgalactosamine residue i n the Forssman antigen and blood group A determinant (39). I t has been suggested e a r l i e r (39) that Forssman antigen may not be a s i n g l e compound. This view was supported by Gahmberg and Hakomori (40) who i s o l a t e d two polymorphic v a r i a n t s of Forssman g l y c o l i p i d from hamster f i b r o b l a s t s . Both v a r i a n t s , however, shared the common terminal s t r u c t u r e composed o f three sugar r e sidues, GalNAc(al+3)GalNAc(31+3)Gal. Our data i n d i c a t e that t h i s terminal s t r u c t u r e i s not only common f o r the Forssman g l y c o l i p i d v a r i a n t s c o n t a i n i n g s t r a i g h t carbohydrate chains, but a l s o can be located on the t e r m i n i o f g l y c o l i p i d s with branched s t r u c t u r e s which c a r r y more than one a n t i g e n i c determinant. I s o l a t i o n o f the Forssman hapten v a r i a n t s from the aqueous phase o f b u f f e r e d t e t r a hydrofuran l i p i d e x t r a c t s i n d i c a t e s that g l y c o l i p i d s bearing Forssman antigen may e x h i b i t considerable water s o l u b i l i t y . This behavior may be d i r e c t l y r e l a t e d to the r e l a t i v e l y strong antigen i c p r o p e r t i e s of Forssman hapten under the p h y s i o l o g i c a l condit i o n s . In accord with these r e s u l t s the term "Forssman antigen" should r e f e r to g l y c o s p h i n g o l i p i d s bearing a terminal

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

10.

SLOMIANY AND

SLOMIANY

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Epithelial

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159

GalNAc (al+3) GalNAc s t r u c t u r e and should not be used with r e f e r e n c e to one p a r t i c u l a r chemical e n t i t y , i . e . globopentaglycosylceramide. S u l f a t e d g l y c o s p h i n g o l i p i d s (41,42) from the b u f f e r e d t e t r a hydrofuran l i p i d e x t r a c t were also i n v e s t i g a t e d i n our l a b o r a t o r y . In l i p i d e x t r a c t s o f hog g a s t r i c mucosa these g l y c o l i p i d s were found mainly i n the organic phase. A f t e r r i g o r o u s p u r i f i c a t i o n , three s u l f a t e d g l y c o s p h i n g o l i p i d s were obtained i n a homogeneous form. These were i d e n t i f i e d as galactosylceramide s u l f a t e , l a c t o sylceramide s u l f a t e and t r i g l y c o s y l c e r a m i d e s u l f a t e . The s t r u c tures of these compounds are presented i n Table I I I . The presence of g a l a c t o s y l and lactosylceramide s u l f a t e s i n g a s t r i c mucosa and the small i n t e s t i n e has been reported e a r l i e r (31), whereas the i s o l a t e d t r i g l y c o s y l c e r a m i d e s u l f a t e (compound 3, Table I I I ) , not reported h e r e t o f o r e , provided the f i r s t i n d i c a t i o n that s u l f a t e d carbohydrates a l s o occur i n more complex g l y c o s p h i n g o l i p i d s . Succ e s s f u l i s o l a t i o n o f t h i s s u l f a t e d g l y c o l i p i d represents another example o f the s u p e r i o r i t over the conventional chloroform/methano Butanol E x t r a c t i o n . Development o f a butanol ext r a c t i o n procedure f o r the i s o l a t i o n o f complex g l y c o s p h i n g o l i p i d s from e r y t h r o c y t e membrane (43) prompted us to apply t h i s method, with m o d i f i c a t i o n , to hog g a s t r i c mucosa. The aqueous phase, a f t e r n-butanol e x t r a c t i o n , was subjected to a l k a l i n e treatment to degrade the g l y c o p r o t e i n s s u s c e p t i b l e to the 3 - e l i m i n a t i o n r e a c t i o n . The products of a l k a l i n e degradation were d i a l y z e d and the prot e i n s separated from the g l y c o l i p i d s by chromatography on C e l l e x P column (44). The g l y c o l i p i d f r a c t i o n was then a c e t y l a t e d , chromatographed on a F l o r i s i l column and p u r i f i e d to homogeneity ( i n the a c e t y l a t e d form) by t h i n - l a y e r chromatography. Although s e v e r a l g l y c o l i p i d bands were detected, only two i n d i v i d u a l compounds were s u c c e s s f u l l y p u r i f i e d to homogeneity (Table I, compounds 12,13). Both g l y c o l i p i d s e x h i b i t e d blood group A - a c t i v i t y and t h e i r carbohydrate p o r t i o n s were h i g h l y enriched i n N - a c e t y l glucosamine. Results of chemical analyses (44) i n d i c a t e d that g l y c o l i p i d 12 contained twelve sugar u n i t s , and g l y c o l i p i d 13 contained eighteen sugar u n i t s . In g l y c o l i p i d 12 one residue of f u cose, one residue o f N-acetylgalactosamine and two out o f s i x r e sidues of N-acetylglucosamine were l o c a t e d at non-reducing t e r m i n i . G l y c o l i p i d 13 contained two terminal residues o f fucose, two r e sidues o f N-acetylgalactosamine and two terminal residues o f Nacetylglucosamine. A n a l y s i s o f the g l y c o l i p i d fragments recovered a f t e r three complete steps of Smith degradation o f glycosphingol i p i d s 12 and 13 showed, i n both g l y c o l i p i d s , the presence of g l u cose, galactose and N-acetylglucosamine i n the molar r a t i o s of 1:2:2. P a r t i a l a c i d h y d r o l y s i s o f these fragments r e s u l t e d mainly i n the formation o f Gal+Glc+ceramide, GlcNAc+Gal+Glc+ceramide and GlcNAc+GlcNAc+Gal+Glc+ceramide (44) . T h i s suggested that the seq u e n t i a l arrangement of the sugar u n i t s i n the saccharide chains adjacent to the ceramide core i n both g l y c o l i p i d s was

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Table I I I S u l f a t e d G l y c o s p h i n g o l i p i d s of G a s t r i c Mucosa

Glycolipid 1. 2. 3. 4. 5.

Structure

S03H+3Gal+ceramide S0 H+3Gall+4Glc+ceramide SOH+3G a l 1+4G a 11+4G1 c+cer amide S0 H+6G1 cNAcB 1+3G a l 31+4G1 c+cer amide Gal3l+4GlcNAc(6^S0 H) 3l+3Gal3l+4Glc+ceramide 3

3

3

3

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Gal+GlcNAc+GlcNAc+Gal+Glc+ceramide. In each g l y c o l i p i d , one r e s i due o f galactose present i n the backbone pentasaccharide was i n volved i n branching. Among other f e a t u r e s , noted f o r the f i r s t time i n g l y c o s p h i n g o l i p i d s was the occurrence of d i - ( N - a c e t y l ) c h i t o b i o s e . T h i s sequence, o r i g i n a l l y reported i n the carbohydrate chains o f porcine (45,46) and horse (47) blood group a c t i v e g l y c o p r o t e i n s , was a l s o r e c e n t l y found i n tKe complex g l y c o s p h i n g o l i pids o f e r y t h r o c y t e membrane (48). The presence of g l y c o l i p i d s with carbohydrate s t r u c t u r e s i d e n t i c a l to those found i n o l i g o saccharides of g l y c o p r o t e i n o r i g i n l e n t f u r t h e r support f o r the existence o f a common pathway f o r the b i o s y n t h e s i s o f blood groupa c t i v e g l y c o p r o t e i n s and g l y c o s p h i n g o l i p i d s . Sodium Acetate E x t r a c t i o n . In our f u r t h e r s t u d i e s o f f u c o l i p i d s o f hog g a s t r i c mucosa, we have found that the residue l e f t a f t e r very thorough e x t r a c t i o n o f l i p i d s Cchloroform/methanol, 2/1, twice f o r 24 h at derable q u a n t i t i e s o were e x t r a c t a b l e with a mixture o f methanol/chloroform/water cont a i n i n g sodium a c e t a t e . A c c o r d i n g l y , we have developed a procedure which i n v o l v e s i n i t i a l p r e - e x t r a c t i o n o f mucosa scrapings with chloroform/methanol (2/1, v/v) to remove simple g l y c o l i p i d s , f o l lowed by e x t r a c t i o n o f the r e s i d u e with sodium acetate i n methanol/ chloroform/water (60/30/8, v / v / v ) . The h i g h e s t y i e l d of g l y c o l i p i d s was obtained with 0.4 M sodium acetate i n the above methan o l / chloroform/ water system (49). G l y c o s p h i n g o l i p i d s recovered i n such e x t r a c t s i n c l u d e d n e u t r a l g l y c o l i p i d s c o n t a i n i n g fucose as well as a c i d i c g l y c o l i p i d s c o n t a i n i n g both s i a l i c a c i d and s u l f a t e . Separation of these g l y c o l i p i d s i n t o n e u t r a l and a c i d i c components was accomplished by DEAE-Sephadex column chromatography (23). The n e u t r a l g l y c o l i p i d f r a c t i o n was then p e r a c e t y l a t e d and chromatographed on a F l o r i s i l column. The f u c o l i p i d s were contained mainly i n the 1,2-dichloroethane/acetone (1/1, v/v) e l u a t e . T h i s f r a c t i o n , a f t e r extensive p u r i f i c a t i o n on t h i n - l a y e r p l a t e s , y i e l d e d f i v e i n d i v i d u a l f u c o l i p i d s (49,50), four o f which e x h i b i t e d blood group A - a c t i v i t y (Table I, compounds 14-17) and one (compound 18) i n a c t i v e i n the ABH system. Common f e a t u r e s o f a l l f i v e f u c o l i p i d s were a carbohydrate chain with two branches and h i g h enrichment of g a l a c t o s e . In f u c o l i p i d s 14-17, one of the branches was t e r minated by the blood group A-determinant, while the others terminated e i t h e r with a-N-acetylgalactosamine (compounds 14 and 15), 3-N-acetylglucosamine (compound 16) or 3-galactose (compound 1 7 ) . F u c o l i p i d 18, which lacked ABH blood group determinants, a l s o contained two branches, one terminating with 3-galactose and the other with 3-N-acetylglucosamine. The f a c t that only one type of complex g l y c o s p h i n g o l i p i d (enr i c h e d i n galactose) was obtained may have r e f l e c t e d the procedure of p u r i f i c a t i o n , e s p e c i a l l y the choice o f a F l o r i s i l column and the solvents used f o r e l u t i o n of a c e t y l a t e d compounds. This p o s s i b i l i t y became obvious when the n e u t r a l g l y c o l i p i d f r a c t i o n o f the

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sodium acetate e x t r a c t s of hog g a s t r i c mucosa was subjected ( i n the a c e t y l a t e d form) to chromatography on a s i l i c i c a c i d column (51,52). The 1,2-dichloroethane/acetone (1/1, v/v) eluate from t h i s column contained two a d d i t i o n a l f u c o l i p i d s (each 12 sugar residues long), whereas the acetone f r a c t i o n contained f u c o l i p i d s with 14 sugar u n i t s . The subsequent f r a c t i o n , e l u t e d with acetone/ methanol (1/1, v / v ) , contained f u c o l i p i d s w i t h 18-24 sugar u n i t s ; and the l a s t f r a c t i o n , e l u t e d with methanol/chloroform/water (90/10/2), c o n s i s t e d o f f u c o l i p i d s with 28-36 sugar residues (53). The i s o l a t e d f u c o l i p i d s i n t h e i r n a t i v e form, d i d not migrate on t h i n - l a y e r p l a t e s i n the solvent systems used f o r p u r i f i c a t i o n o f the p r e v i o u s l y described blood group ABH f u c o l i p i d s (22,54). However, i n the a c e t y l a t e d form a l l o f the compounds studied e x h i b i ted good m o b i l i t i e s i n s e v e r a l solvent systems ( F i g . 3 and 4 ) . The proposed s t r u c t u r e s f o r g l y c o l i p i d s p u r i f i e d from 1,2-dichloroethane/acetone (compounds 19,20) and acetone (compounds 21,22) f r a c t i o n s are given i these four f u c o l i p i d s were minants (A and H) on the same g l y c o l i p i d molecule and the s i m i l a r i t y o f the o l i g o s a c c h a r i d e chains to those present i n the blood group (A+H) a c t i v e g l y c o p r o t e i n s (45,46). The carbohydrate and sphingosine composition o f the major f u c o l i p i d s p u r i f i e d from the acetone/methanol and methanol/chloroform/water f r a c t i o n s are given i n Table IV. F u c o l i p i d s 23-25 were present i n the acetone/methanol f r a c t i o n , whereas the methanol/ chloroform/water eluate contained f u c o l i p i d s 26-28 (53). In hemag g l u t i n a t i o n - i n h i b i t i o n assays a l l s i x compounds were potent i n h i b i t o r s o f a g g l u t i n a t i o n o f human group A - c e l l s by anti-A serum (1.5-3.1 yg/0.1 ml) and human 0 - c e l l s by a n t i - H l e c t i n (2.1-4.5 yg/0.1 ml), i n d i c a t i n g that the carbohydrate chain o f each f u c o l i p i d bears two types o f blood group determinant, A and H. Although the s t r u c t u r e s o f these f u c o l i p i d s are not yet e l u c i d a t e d , c e r t a i n features o f the saccharide chains can be suggested on the b a s i s o f carbohydrate a n a l y s i s , immunological assays and the s u s c e p t i b i l i t y o f the n a t i v e and d e f u c o s y l a t e d g l y c o s p h i n g o l i p i d s to the action o f s p e c i f i c exoglycosidases. These data i n d i c a t e that the carbohydrate chain o f f u c o l i p i d 23 contains four branches, two terminated by 3-galactose, one by the blood group A (GalNAcal+3[Fucal->2]Gal-) a n t i g e n i c determinant and one by the blood group H (Fucal->2Gal-) determinant; f u c o l i p i d 24 contains two branches terminated by the blood group A determinant, one by the H and one by 3-galactose; f u c o l i p i d 25 contains two branches terminated by the A determinant, one by H and two by 3-galactose; f u c o l i p i d 26 contains three branches terminated by the A determinant, one by H and two by 3-galactose; f u c o l i p i d 27 contains three branches terminated by the A determinant, one by H and three by 3-galactose; and fucol i p i d 28 contains three branches terminated by the A determinant, two by H, two by 3-galactose and one by 3-N-acetylglucosamine. L i p i d s e x t r a c t e d from hog g a s t r i c mucosa with 0.4 M sodium acetate i n methanol/chloroform/water were a l s o i n v e s t i g a t e d f o r

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Figure 3.

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Thin-layer chromatography of the acetylated blood group (A-\-H) complex fucolipids

(1) Fucolipid 19, Table I; (2) fucolipid 20, Table I; (3), fucolipid 21, Table I; (4) fucolipid 22, Table I. Solvent system: chloroform/acetone/methanol/water (52/40/00/4, by volume), plate A; 1,2-dichloroethane/methanol/ water (80/25/2, v/v/v), plate B; 1,2-dichloroethane/acetone/methanol/water (50/40/10/4, by volume), plate C. Visualization: orcinol reagent. (52)

Figure 4.

Thin-layer chromatography of

the acetylated highly complex fucolipids from hog gastric mucosa Left plate, developed in chloroform/methanol/2M NH,OH (40/15/1.5, v/v/v). (1) Fucolipid 19, Table I; (2) fucolipid 24, Table IV; (3) fucolipid 23, Table IV; (4) fucolipid 26, Table IV; (5) fucolipid 25, Table IV. Right plate, developed in chloroform/methanol/water (60/40/10, v/v/v). (1) Fucolipid 26, Table IV; (2) fucolipid 27, Table IV; (3) fucolipid 28, Table IV. Visualization: orcinol reagent (53)

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Table IV. Molar Ratios o f Sphingosin Complex F u c o l i p i d s from G a s t r i c Mucosa.

Fucolipid

23 24 25 26 27 28

a

Molar r a t i o s a Fuc Gal Glc

GlcNAc

2.01 7.86 2.84 7.50 2.90 9.81 3.81 9.77 3.85 11.78 4.63 12.40

5.92 6.77 7.85 9.68 11.89 13.60

1.0 1.0 1.0 1.0 1.0 1.0

GalNAc Sphingosine No. o f sugar residues 1.0 18 1.05 20-21 0.9 1.78 24 2.02 0.8 3.12 28 0.8 32 0.9 2.79 35-36 3.04 0.7

R e l a t i v e to Glc=l

(From Ref. £ 3 )

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the presence of s u l f a t e d g l y c o s p h i n g o l i p i d s . For t h i s , the a c i d i c g l y c o l i p i d s , e l u t e d from DEAE-Sephadex with sodium acetate i n methanol/chloroform/water, were a c e t y l a t e d and separated on a s i l i c i c a c i d column i n t o s e v e r a l f r a c t i o n s (55,56). The 1,2-dichloroethane and 1,2-dichloroethane/acetone eluates contained mainly s i a l o g l y c o s p h i n g o l i p i d s , together with traces of the d i and trihexose s u l f a t i d e s described p r e v i o u s l y (41). F r a c t i o n s e l u t e d with more p o l a r solvents contained s e v e r a l new s u l f a t e d g l y c o s p h i n g o l i p i d s . Some of these g l y c o l i p i d s contained s u l f a t e and s i a l i c a c i d . Whether these compounds represent homogeneous g l y c o s p h i n g o l i p i d s containing both s i a l i c a c i d and s u l f a t e on the same molecule or are a mixture of s u l f a t e d and s i a l y l a t e d glycos p h i n g o l i p i d s remains to be e s t a b l i s h e d . However, two of the c h a r a c t e r i z e d s u l f a t e d g l y c o s p h i n g o l i p i d s (55,56) were devoid of s i a l i c a c i d and contained glucose, galactose, N-acetylglucosamine and s u l f a t e i n molar r a t i o s of 1:1:1:1 and 1:2:1:1, r e s p e c t i v e l y . The proposed s t r u c t u r e Table I I I (compounds 4 These newly i d e n t i f i e d compounds d i f f e r from p r e v i o u s l y c h a r a c t e r i z e d s u l f a t e d g l y c o s p h i n g o l i p i d s (41,42) with respect to sugar composition, length of the carbohydrate chain and the s i t e of s u l f a t i o n . Results of periodate o x i d a t i o n and permethylation analyses showed that both compounds contain N-acetylglucosamine 6 - s u l f a t e . To our knowledge, s u l f a t e d g l y c o s p h i n g o l i p i d s cont a i n i n g s u l f a t e d N-acetylglucosamine have not been p r e v i o u s l y desc r i b e d i n mammalian g a s t r i c mucosa or other t i s s u e s . However, N-acetylglucosamine 6 - s u l f a t e was found i n blood group (A+H) s u l f a t e d g l y c o p r o t e i n s of hog g a s t r i c mucosa (45,46). This again i n d i c a t e s that i n glandular e p i t h e l i a l t i s s u e the same o l i g o saccharides may be l i n k e d to a l i p i d or p r o t e i n core. New Approach to I s o l a t i o n of G l y c o s p h i n g o l i p i d s . Prog r e s s i v e d i s c o v e r i e s of more complex g l y c o s p h i n g o l i p i d s , r e v e a l i n g l y s i m i l a r i n s t r u c t u r e to g l y c o p r o t e i n s , i n d i c a t e that current techniques f o r the i s o l a t i o n of g l y c o s p h i n g o l i p i d s are inadequate and do not permit complete recovery of a l l c o n s t i t u e n t s by any one procedure. S i z e and complexity of the carbohydrate p o r t i o n governs e x t r a c t i b i l i t y and lends to some of these g l y c o s p h i n g o l i p i d s the p r o p e r t i e s of g l y c o p r o t e i n s . Therefore, they are e i t h e r l e f t behind during the e x t r a c t i o n or are c l a s s i f i e d as g l y c o p r o t e i n s . To overcome the problem of g l y c o p r o t e i n - l i k e p r o p e r t i e s of complex g l y c o s p h i n g o l i p i d s and at the same time to i s o l a t e short-chain g l y c o s p h i n g o l i p i d s which may be i n strong a s s o c i a t i o n with other components of the c e l l membrane, we have r e c e n t l y introduced a new approach f o r the i s o l a t i o n of g l y c o s p h i n g o l i p i d s (unpublished). In t h i s procedure, g a s t r i c mucosa i s homogenized i n s o l u b i l i z i n g buff e r (sodium s u l f i t e ) and t r e a t e d s e q u e n t i a l l y with RNA-ase and DNA-ase to decrease the v i s c o s i t y o f the homogenate, and then subj e c t e d to a l k a l i n e degradation (3-elimination) and pronase digest i o n . The r e s u l t a n t t i s s u e d i g e s t i s extracted with chloroform/

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methanol (2/1, v/v) to remove s h o r t - c h a i n g l y c o s p h i n g o l i p i d s and the aqueous phase i s adjusted to 1% with a z w i t t e r i o n i c detergent. A f t e r c e n t r i f u g a t i o n , the c l e a r supernatant f r a c t i o n i s subjected to g e l f i l t r a t i o n (Bio-Gel P-60) and chromatography on DEAE-Sephadex. Following molecular s i z i n g the Bio-Gel P-4 and/or P-6 columns, the g l y c o l i p i d s are a c e t y l a t e d and p u r i f i e d to i n d i v i d u a l components by chromatography on t h i n - l a y e r p l a t e s o r on Bio-Beads SX-1 columns. Since the e n t i r e process o f i s o l a t i o n i s conducted i n a s o l u t e phase and i n the presence o f a detergent, the a r t i f a c t u a l entrapment o f g l y c o s p h i n g o l i p i d s i s e l i m i n a t e d . Glycolipids

of Mucous S e c r e t i o n

The o r a l , g a s t r o i n t e s t i n a l , b r o n c h i a l , pulmonary and reproductive t r a c t s o f higher animals secrete copious q u a n t i t i e s o f viscous mucus which f u n c t i o n s mainly as a l u b r i c a n t and p r o t e c t i v e agent. The v i s c o u s p r o p e r t i e s u l t o f the presence o mucins (57). These g l y c o p r o t e i n s have been s t u d i e d e x t e n s i v e l y (see f o r review r e f . £7,^58); however, u n t i l r e c e n t l y no informat i o n was a v a i l a b l e on g l y c o l i p i d s of mucous s e c r e t i o n s . Furthermore, the general assumption was that both mucous g l y c o p r o t e i n s and g l y c o l i p i d s are s i m i l a r to, o r p o s s i b l y d e r i v e d from, those found on the c e l l surfaces (59). To provide data on the nature o f g l y c o l i p i d s of mucous s e c r e t i o n s , we have analyzed g l y c o l i p i d cons t i t u e n t s o f the l i p i d extracts derived from g a s t r i c s e c r e t i o n , g a s t r i c mucous b a r r i e r , s a l i v a and a l v e o l a r lavage. Analyses o f the l i p i d extracts from human g a s t r i c s e c r e t i o n revealed that g l y c o l i p i d s c o n s t i t u t e about 30% o f the l i p i d f r a c t i o n (60), whereas i n g a s t r i c s e c r e t i o n s from dog Heidenhain pouch and from l i g a t e d r a t stomach, the g l y c o l i p i d f r a c t i o n comp r i s e s up to 50% o f the t o t a l l i p i d s (61). On t h i n - l a y e r chromatography, the g l y c o l i p i d f r a c t i o n from human s e c r e t i o n could be separated i n t o nine i n d i v i d u a l components, f i v e g l y c o l i p i d components were present i n the g a s t r i c s e c r e t i o n o f dog, and four i n the g a s t r i c s e c r e t i o n o f r a t (61,62). Each o f the p u r i f i e d g l y c o l i p i d s contained f a t t y a c i d s , glucose and g l y c e r y l - monoethers. In a d d i t i o n , two g l y c o l i p i d s from human g a s t r i c s e c r e t i o n were s u l f a t e d . None o f these g l y c o l i p i d s contained sphingosine, phosphorus o r alkenyl ethers (61,63). A l l o f the g l y c o l i p i d s Were susc e p t i b l e to d e a c y l a t i o n under m i l d a l k a l i n e c o n d i t i o n s , indicating the presence o f e s t e r - l i n k e d f a t t y a c i d s , and the s u l f a t e d compounds were a l s o s u s c e p t i b l e to a c i d s o l v o l y s i s ( F i g . 5). Results of s t r u c t u r a l analyses performed on the major g l y c o l i p i d components o f human g a s t r i c s e c r e t i o n i n d i c a t e d that the g l y c o l i p i d s o f g a s t r i c s e c r e t i o n are composed of one or more glucose residues l i n k e d to a monoalkylmonoacylglycerol l i p i d core (64,65). The proposed s t r u c t u r e s f o r g l y c o l i p i d s o f human g a s t r i c s e c r e t i o n are presented i n Table V.

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167

Figure 5. Thin-layer chromatogram of the major sulfated glycolipid from human

(1) Native glycolipid, compound 4, Table V; (2) desulfated glycolipid; (3) desulfated and deacylated glycolipid; (4) digalactosyl diglyceride standard. Solvent system: chloroform/ methanol/water (65/25/4, v/v/v). Visualization: orcinol reagent. (64)

Table V . Glyceroglucolipids

Glycolipid 1. 2. 3. 4. 5.

o f Human G a s t r i c

Secretion.

Structure

Glcal+3-1,(3)-0-alkyl-2-0-acylglycerol Glcal->6Glcal+6Glcal+6Glcal+6Glcal->6Glcal+3-l,

(3)-0-alkyl-2-0acylglycerol Glcal+6Glcal->6Glcal+6Glcal->6Glcal->6Glcal+6Glcal->6Glcal->3-l, (3)-0-alkyl-2-0-acylglycerol SO„H-6G1ca1+6G1ca1+6G1ca1+3-1,(3)-0-alkyl-2-0-acylglycerol S0 H-6Glcal+6Glcal+6Glcal+6Glcal+3-l,(3)-0-alkyl-2-0-acylglycerol 3

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Our s t u d i e s on the o r i g i n o f g l y c e r o g l u c o l i p i d s i n g a s t r i c s e c r e t i o n e s t a b l i s h e d that these compounds are present not only i n the s o l u b l e p o r t i o n o f g a s t r i c s e c r e t i o n ( d i s s o l v e d mucin), but a l s o i n the g a s t r i c mucous b a r r i e r and i n the preformed i n t r a c e l l u l a r mucus contained w i t h i n the s e c r e t o r y granules o f the e p i t h e l i a l c e l l s (66). Furthermore, we have demonstrated that i n s t i l l a t i o n of v a r i o u s noxious agents such as ethanol and hyperosmotic NaCl causes d e p l e t i o n o f g l y c e r o g l u c o l i p i d s from g a s t r i c mucous b a r r i e r (67). S i m i l a r d e p l e t i o n o f g l y c e r o g l u c o l i p i d s was observed i n various g a s t r o i n t e s t i n a l d i s o r d e r s ( g a s t r i t i s , g a s t r i c u l c e r s ) (68). These data c l e a r l y e s t a b l i s h the importance o f glyceroglucol i p i d s as an e s s e n t i a l component o f g a s t r i c s e c r e t i o n and suggest the p o s s i b i l i t y o f t h e i r involvement i n the defense mechanism against the i n j u r y o f the mucosal s u r f a c e s . In f u r t h e r studies on the g l y c o l i p i d s of mucous s e c r e t i o n s , we have d i r e c t e d our a t t e n t i o n to s a l i v a (69,70) Since glycoprot e i n s o f s a l i v a r y and g a s t r i t u r a l and immunologica to determine whether the g l y c o l i p i d s o f s a l i v a resemble those o f g a s t r i c s e c r e t i o n . Accordingly, we have i s o l a t e d a g l y c o l i p i d f r a c t i o n from l i p i d e x t r a c t s of whole human s a l i v a and s t u d i e d the composition and s t r u c t u r e o f seven i n d i v i d u a l g l y c o l i p i d components ( F i g . 6). A l l seven compounds were found t o contain g l u cose, f a t t y acids and glyceryl-monoethers. One o f the g l y c o l i p i d s a l s o contained s u l f a t e (70). Results o f chemical analyses (Table V I ) , i n d i c a t e d that these g l y c o l i p i d s are s t r u c t u r a l l y r e l a t e d to those o f g a s t r i c s e c r e t i o n , i . e . they contain p o l y g l u c o s y l carbohydrate chains l i n k e d to monoalkylmonoacylglycerol. Again, glycos p h i n g o l i p i d s were not detected. Our data are c o n s i s t e n t with the r e s u l t s o f e a r l i e r studies on the b i o s y n t h e s i s o f carbohydratec o n t a i n i n g substances i n the s a l i v a r y glands of mice (73), i n which s t i m u l a t i o n with i s o p r o t e r e n o l increased the synthesis o f g l y c o l i p i d o f g l y c e r o g l y c o l i p i d type. A l s o , P r i t c h a r d ' s studies (74) on s u l f o l i p i d formation i n r a t submandibular glands have demonstrated the presence o f a s u l f o t r a n s f e r a s e c a t a l y z i n g the t r a n s f e r o f l a b e l l e d s u l f a t e from 3 -phosphoadenosine-5 -phosphos u l f a t e to an endogenous l i p i d acceptor. This r a d i o - l a b e l l e d s u l f o l i p i d produced by submandibular gland was shown to be o f the g l y c e r o g l y c o l i p i d type. Our recent s t u d i e s (75,76) on the o r i g i n o f g l y c e r o g l u c o l i p i d s i n the s a l i v a i n d i c a t e that tEese compounds are elaborated by the p a r o t i d and submandibular glands and that t h e i r l e v e l s are elevated i n the s a l i v a r y s e c r e t i o n s derived from i n d i v i d u a l s with a high r a t e o f s a l i v a r y c a l c u l u s formation. Whether there i s a d i r e c t a s s o c i a t i o n between the g l y c e r o g l u c o l i p i d content o f the s a l i v a and the development o f plaque, c a l c u l u s and p e r i o d o n t a l disease remains to be e s t a b l i s h e d . For the a n a l y s i s o f e x t r a c e l l u l a r g l y c o l i p i d s o f r e s p i r a t o r y t r a c t , we have chosen the a c e l l u l a r m a t e r i a l l i n i n g the a l v e o l i of mammalian lungs. This unique l i p i d - p r o t e i n mixture, r e s p o n s i b l e f o r the r e d u c t i o n o f a l v e o l a r surface f o r c e s during r e s p i r a t i o n , f

1

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Figure 6. Thin-layer chromatogram of the glycolipids purified from human saliva (see Table VI for structures) (1) Glycolipid 1; (2) glycolipid 2; (3) glycolipid 3; (4) glycolipid 4; (5) desulfated glycolipid 5; (6) glycolipid 6; glycolipid 6; (7) glycolipid 7. Solvent system: chloroform/methanol/water (65/35/8, v/v/v). Visualization: orcinol reagent. (10)

Table VI. G l y c e r o g l u c o l i p i d s of Human S a l i v a .

Glycolipid

Structure

1. Glcal+3-1, (3) - 0 - a l k y l - 2 - 0 - a c y l g l y c e r o l 2,3.Glcal+6Glcal+3-l,(3)-0-alkyl-2-0-acylglycerol 4. Glcal+6Glcal+6Glcal+3-l, ( 3 ) - 0 - a l k y l - 2 - 0 - a c y l g l y c e r o l 5. S0 H-6Glcal+6Glcal+6Glcal+3-l, ( 3 ) - 0 - a l k y l - 2 - 0 - a c y l g l y c e r o l 6. Glcal->6Glcal->6Glcal+6Glcal->6Glcal+6Glcal->3-l, (3)-0-alkyl-2-0acylglycerol 7. Glco:.l-*6Gl c 1+6G1 ca 1+6G 1 ca 1+6G 1 ca 1+6G1 cal+6G 1 cal+6G 1ca1+3-1, C3)-0-alkyl-2-0-acylglycerol 3

a

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includes the s u r f a c e - a c t i v e phospholipids and other moieties such as n e u t r a l l i p i d s , p r o t e i n s and carbohydrates (77,78,79). I n v e s t i gations on the nature o f the carbohydrate component of pulmonary s u r f a c t a n t i n d i c a t e d that t h i s m a t e r i a l i s not only a s s o c i a t e d with a p r o t e i n but also i s present i n the l i p i d e x t r a c t (80). Analyses o f the l i p i d e x t r a c t s from a l v e o l a r lavage o f r a b b i t , p e r formed i n our l a b o r a t o r y (81,82), showed that the carbohydrate component a s s o c i a t e d with l i p i d s c o n s i s t s e x c l u s i v e l y o f glucose. About 60% o f t h i s carbohydrate was a s s o c i a t e d with n e u t r a l glycol i p i d s and 40% with a c i d i c g l y c o l i p i d s . Extensive p u r i f i c a t i o n o f the g l y c o l i p i d s present i n these f r a c t i o n s r e s u l t e d i n the i s o l a t i o n o f four i n d i v i d u a l components. Three o f these g l y c o l i p i d s contained glucose, f a t t y acids and glycerl-monoethers, whereas the major a c i d i c g l y c o l i p i d , i n a d d i t i o n t o the above components, cont a i n e d s u l f a t e e s t e r (82). The s t r u c t u r e s o f these g l y c o l i p i d s are shown i n Table VII Our data (81,82) o of r a b b i t lungs c l e a r l g a s t r i c s e c r e t i o n and s a l i v a , belong to the g l y c e r o g l u c o l i p i d c l a s s . Thus, i t appears that an a c e l l u l a r g l y c o l i p i d s i n the sec r e t i o n s o f the alimentary t r a c t and i n the a l v e o l a r l i n i n g l a y e r o f mammalian lungs are e n t i r e l y d i f f e r e n t from those found i n c e l l membranes. The p h y s i o l o g i c a l importance o f s e c r e t o r y g l y c o l i p i d s i s s t i l l unknown. G l y c e r o g l u c o l i p i d s present i n mucous s e c r e t i o n s of the alimentary t r a c t are p a r t o f the p r o t e c t i v e l i n i n g o f the surface e p i t h e l i a l c e l l s and i n s a l i v a they may be involved i n the process o f tooth p e l l i c l e formation, whereas i n the a c e l l u l a r m a t e r i a l l i n i n g the surfaces o f a l v e o l i g l y c e r o g l u c o l i p i d s may p a r t i c i p a t e i n spreading o f the s u r f a c t a n t l a y e r w i t h i n the a l veolus. The Nature o f ABH Blood Group Antigens i n Mucous S e c r e t i o n The occurrence and nature o f blood s p e c i f i c antigens i n t i s sue and i n mucous s e c r e t i o n s has been studied by a number o f i n v e s t i g a t o r s (£3,84,85^,86^,87); the e a r l y data suggested that mucous s e c r e t i o n s contain water-soluble antigens whereas red c e l l s and most o f the other t i s s u e s contain only the a l c o h o l - s o l u b l e a n t i gens. In s p i t e o f t h a t , the d i s c o v e r y o f blood group-active glycos p h i n g o l i p i d s and g l y c o p r o t e i n s from the same source (see f o r r e view r e f . 2j2,5£,58,8^,89,9£) l e d to the proposal o f t h e i r coexistence i n the t i s s u e s and to the assumption (59) that secret i o n s represent a l s o a mixture o f blood group-active glycosphingol i p i d s and g l y c o p r o t e i n s . Furthermore, evidence was presented on the g l y c o p r o t e i n nature of ABH antigens o f erythrocytes (91,92, 93), which were known t o contain antigens o f the glycosphingol i p i d character only. Our s t u d i e s on g l y c o l i p i d s o f g a s t r i c s e c r e t i o n (62,63,64,65) and s a l i v a (69,70) showed that these s e c r e t i o n s do not contain g l y c o s p h i n g o l i p i T s ; i n s t e a d g l y c e r o g l u c o l i p i d s were found. To

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Table V I I . G l y c e r o g l u c o l i p i d s o f A l v e o l a r Lavage from Rabbit.

Glycolipid 1. 2. 3. 4.

Structure

Glcal->3-l, ( 3 ) - 0 - a l k y l - 2 - 0 - a c y l g l y c e r o l Glcal+6Glcal+6Glcal+6Glcal+6Glcal+3-l,(3)-0-alkyl-2-0-acylglycerol Glcal->6Glcal+6Glcal->6Glcal->6Glcal->6Glcal+3-l, (3)-0-alkyl-2-0acylglycerol S0 H-6Glcal+6Glcal+6Glcal->6Glcal->3-l, ( 3 ) - 0 - a l k y l - 2 - 0 - a c y l 3

Table V I I I . ABH blood group a c t i v i t y i n human s a l i v a and g a s t r i c s e c r e t i o n . Assay 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

Material

Activity*

Native g a s t r i c s e c r e t i o n Native s a l i v a Delipidated gastric secretion Delipidated saliva Native and d e l i p i d a t e d g a s t r i c s e c r e t i o n t r e a t e d with 0.5 M NaOH, (60 h, room temperature) Native and d e l i p i d a t e d s a l i v a t r e a t e d with 0.5 M NaOH (60 h, room temperature) A l k a l i n e degradation o f g a s t r i c s e c r e t i o n i n the presence o f A - a c t i v e g l y c o s p h i n g o l i p i d A l k a l i n e degradation o f s a l i v a i n the presence of A - a c t i v e g l y c o s p h i n g o l i p i d L i p i d extract of g a s t r i c secretion L i p i d extract of s a l i v a Glycolipid fraction of saliva Glycolipid fraction of gastric secretion Blood group A - a c t i v e g l y c o s p h i n g o l i p i d i n the presence o f l i p i d e x t r a c t from s a l i v a or g a s t r i c secretion

* (+) s i g n i f i e s i n h i b i t i o n o f hemagglutination (-) i n d i c a t e s hemagglutination

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+ + + +

+ +

+

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determine the nature of blood group ABH antigens i n s a l i v a and g a s t r i c s e c r e t i o n , the n a t i v e and d e l i p i d a t e d samples, t o t a l l i p i d s , and p u r i f i e d g l y c o l i p i d s were t e s t e d f o r a n t i g e n i c a c t i v i t y . The lack o f i n h i b i t i o n o f a g g l u t i n a t i o n with t o t a l l i p i d s and with p u r i f i e d g l y c o l i p i d s c l e a r l y i n d i c a t e d that the a n t i g e n i c propert i e s o f s a l i v a and g a s t r i c s e c r e t i o n were not r e l a t e d t o the l i p i d p o r t i o n o f these s e c r e t i o n s (94,95). The n a t i v e a c t i v i t i e s o f sal i v a and g a s t r i c s e c r e t i o n were abolished by treatment with a l k a l i which i s known to destroy blood group-active g l y c o p r o t e i n s , but i s completely i n e f f e c t i v e i n degradation o f g l y c o s p h i n g o l i p i d s . However, n e i t h e r a l k a l i nor the presence o f n a t i v e g l y c o l i p i d s from s a l i v a o r g a s t r i c s e c r e t i o n were capable o f d i m i n i s h i n g the a n t i g e n i c potency o f added blood group A g l y c o s p h i n g o l i p i d s (Table V I I I ) . A l s o , the removal o f l i p i d s p r i o r to the hemagglut i n a t i o n - i n h i b i t i o n assay d i d not decrease the n a t i v e a c t i v i t y o f the samples; t o the contrary a s l i g h t increase i n potency per mg o f residue was noted. These data c l e a r l y glycoprotein (water-solubl antigens) are r e s p o n s i b l e f o r the blood group a c t i v i t y o f the sec r e t i o n s and t h e i r presence i n s e c r e t o r y t i s s u e i s only temporary, whereas g l y c o s p h i n g o l i p i d s thus f a r i s o l a t e d from a number o f t i s sues represent antigens which are an i n t e g r a l p a r t o f the c e l l membranes (94,95). T h i s d i s t i n c t i v e feature o f e p i t h e l i a l - s e c r e t o r y t i s s u e versus i t s s e c r e t i o n does not e x p l a i n the o r i g i n o f blood group antigens o f the e r y t h r o c y t e s . The coexistence o f g l y c o s p h i n g o l i p i d and g l y c o p r o t e i n ABH antigens i s s t i l l disputed. According to Koscielak e t a l . (96) the erythrocyte stroma i s only equipped with antigens o f g l y c o s p h i n g o l i p i d nature. This i s s t r o n g l y opposed by others (91,92,95) who have provided evidence that erythrocyte membrane antigens are o f dual o r i g i n . I t i s poss i b l e that r i g o r o u s s e p a r a t i o n o f blood group-active g l y c o p r o t e i n s and g l y c o s p h i n g o l i p i d s between s e c r e t i o n and s e c r e t o r y t i s s u e i s not a p p l i c a b l e to erythrocytes, which represent an unusual type o f tissue entirely.

Acknowledgement. This study has been supported by Grants AM No. 21684-02 and 25372-01 from N a t i o n a l I n s t i t u t e o f A r t h r i t i s , Metabolism and D i g e s t i v e Diseases, N a t i o n a l I n s t i t u t e s of Health, United States P u b l i c Health S e r v i c e .

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Literature Cited 1. Hallauer, C., Z. Immunitaetsforsch. Exp.Ther., 1934, 83, 114. 2. Kossjakow, P.N. and Tribulew, G.P., Z. Immunitaetsforsch. Exp. Ther., 1940, 98, 261. 3. Stepanov, A.V., Jusin, A., Makajeva, Z. and Kossjakow, P.N., Biokhimiya, 1940, 5, 547. 4. Hamasata, Y. Tohoku J. Exp. Med., 1950, 52, 17. 5. Masamune, H., Maekara, T. and Hakomori, S., Tohoku J. Exp.Med. 1954, 59, 225. 6. Masamune, H. and Siojima, S., Tohoku J. Exp. Med., 1951, 54, 319. 7. Slomiany, A. and Horowitz, M.I., J. Biol. Chem. 1973, 248, 6232. 8. Slomiany, A., Slomiany, B.L. and Horowitz, M.I., J. Biol.Chem. 1974, 249, 1225. 9. Hakomori, S., Stellner K and Watanabe K. Biochem.Biophys Res. Commun., 1972 10. Stellner, K., Watanabe 1973, 12, 656. 11. Slomiany, B.L., Slomiany, A. and Horowitz, M.I., Biochim.Biophys. Acta, 1973, 326, 224. 12. Slomiany, B.L., Slomiany, A. and Horowitz, M.I., Eur. J. Biochem., 1974, 43, 161. 13. Slomiany, A., Annese, C. and Slomiany, B.L.,Biochim.Biophys. Acta, 1976, 441, 316. 14. Moschera, J. and Pigman, W., Carbohydr. Res., 1975, 40, 53. 15. Keryer, G., Herman, G. and Rossignol, B., Biochim. Biophys. Acta, 1973, 306, 446. 16. Kent, S.P. and Sanders, E.M., Proc. Soc. Exp. Biol. Med.,1969, 132, 645. 17. Pritchard, E.T. and Rusen, D.R., Arch. Oral. Biol., 1972, 17, 1619. 18. Pritchard, E.T., Arch. Oral Biol., 1973, 18, 1. 19. Tettamanti, G., Bonali, F., Marchesini, S. and Zambotti, V., Biochim. Biophys. Acta, 1973, 296, 160. 20. Folch-Pi, J., Lees, M. and Sloane-Stanley, G.H., J. Biol. Chem., 1957, 226, 497. 21. Suzuki, K., J. Neurochem., 1965, 12, 629. 22. Slomiany, A., Slomiany, B.L. and Horowitz, M.I., in Glycolipid Methodology (Witting, L.A., ed.) Am. Oil Chem. Soc., Champaign, IL., 1976, pp. 49-74. 23. Yu, R.K. and Ledeen, R.W., J. Lipid Res., 1972, 13, 680. 24. Slomiany, A. and Slomiany, B.L., Biochim. Biophys. Acta., 1975, 388, 135. 25. Slomiany, B.L., Slomiany, A. and Horowitz, M.I., Eur. J. Biochem., 1975, 56, 353. 26. Slomiany, A., Slomiany, B.L. and Annese, C., FEBS Lett.,1977, 81, 157. 27. Slomiany, B.L., Slomiany, A. and Horowitz, M.I., Eur. J. Biochem., 1975, 56, 353.

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28. Yang, H.J. and Hakomori, S.I., J. Biol. Chem., 1971,246, 1192. 29. Lloyd, K.O., Kabat, E.A. and Rosenfield, R.E., Biochemistry, 1966, 5, 1502. 30. Smith, E.L., McKibbin, J.M., Karlsson, K.A., Pascher, I. and Samuelsson, B.E., Biochim. Biophys. Acta, 1975, 398, 84. 31. McKibbin, J.M. and Lyerly, D.F., Ala. J. Med. Sci., 1973, 10, 299. 32. Slomiany, A. and Slomiany, B.L., Eur. J. Biochem., 1977, 76, 491. 33. Slomiany, B.L. and Slomiany, A., Eur. J. Biochem., 1978,83, 105. 34. Esselman, W.J., Ackerman, J.R. and Sweeley, C.C., J. Biol. Chem., 1973, 248, 7310. 35. Sung, S.J., Esselman, W.J. and Sweeley, C.C., J. Biol. Chem., 1973, 248, 6528. 36. Smith, E.L., McKibbin J.M., Karlsson K.A. Pascher I and Samuelsson, B.E., 37. Siddiqui, B. and Hakomori 5766. 38. Karlsson, K.A., Leffler, H. and Samuelsson, B.E., J. Biol. Chem., 1974, 249, 4819. 39. Rapport, M.M. and Graf, L . , Progr.Allergy, 1969, 13, 273. 40. Gahmberg, C.G. and Hakomori, S.I., J. Biol. Chem., 1975, 250, 2438. 41. Slomiany, B.L., Slomiany, A. and Horowitz, M.I., Biochim.Biophys. Acta, 1974, 348, 386. 42. Slomiany, B.L., Slomiany, A. and Badurski, J., Post. Biochem., 1975, 21, 319. 43. Gardas, S. and Koscielak, J., FEBS Lett., 1974, 42, 101. 44. Slomiany, B.L. and Slomiany, A., FEBS Lett., 1977, 73, 175. 45. Slomiany, B.L. and Meyer, K., J. Biol. Chem., 1972, 247, 5062. 46. Slomiany, B.L. and Meyer, K., J. Biol. Che., 1973, 248, 2290. 47. Newman, W. and Kabat, E.A., Arch. Biochem. Biophys., 1976, 172, 535. 48. Zdebska, E. and Koscielak, J., Eur. J. Biochem., 1978, 91,517. 49. Slomiany, B.L. and Slomiany, A., Biochim. Biophys. Acta, 1977, 486, 531. 50. Slomiany, B.L. and Slomiany, A., Chem. Phys. Lipids, 1977,20, 57. 51. Slomiany, A. and Slomiany, B.L., FEBS Lett., 1978, 90, 293. 52. Slomiany, B.L. and Slomiany, A., Eur. J. Biochem., 1978, 90, 39. 53. Slomiany, B.L., Slomiany, A. and Murty, V.L.N., Biochem. Biophys. Res. Commun., 1979, 88, 1092. 54. McKibbin, J.M., J. Lipid Res., 1978, 19, 131. 55. Slomiany, B.L., Slomiany, A. and Annese, C., J. Am. Oil Chem., 1978, 55, 239A. 56. Slomiany, B.L. and Slomiany, A., J. Biol. Chem., 1978, 253, 3517. 57. Herp. A., Wu, A.M. and Moschera, J., Mol. Cel. Biochem., 1979,

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23, 27. 58. Glass, G.B.J. and Slomiany, B.L., in Mucus in Health and Disease, (Elstein, M. and Parke, D.V., eds.), Plenum Publishing Corp., New York, 1977, pp. 311-347. 59. Pigman, W. and Moschera, J., in Biology of the Cervix (Blandau, R.J. and Moghissi, K., eds.), University of Chicago Press, Chicago, 1973, pp. 143-173. 60. Slomiany, B.L., Slomiany, A. and Glass, G.B.J., Fed. Proc., 1977, 36, 978. 61. Slomiany, A. and Slomiany, B.L., J. Am. Oil Chem. Soc., 1978, 55, 239A. 62. Slomiany, A. and Slomiany, B.L., Biochem. Biophys. Res. Commun. 1977, 76, 115. 63. Slomiany, B.L., Slomiany, A.and Glass, G.B.J., FEBS Lett., 1977, 77, 47. 64. Slomiany, B.L., Slomiany A d Glass G.B.J. Eur J. Bio chem., 1977, 78, 33 65. Slomiany, B.L., Slomiany, . d Glass, G.B.J., Biochemistry, 1977, 16, 3954. 66. Slomiany, A., Yano, S., Slomiany, B.L. and Glass, G.B.J., J. Biol. Chem., 1978, 253, 3785. 67. Slomiany, A., Patkowska, M.J., Slomiany, B.L. and Glass,G.B.J. Internatl. J. Biol. Macromol., 1979, in press. 68. Slomiany, B.L. and Slomiany, A., IRCS Med. Sci., 1979, 7,373. 69. Slomiany, B.L. and Slomiany, A., Biochem. Biophys. Res. Commun 1977, 79, 61. 70. Slomiany, B.L., Slomiany, A. and Glass, G.B.J., Eur. J. Biochem., 1978, 84, 53. 71. Kent, S.P. and Sanders, E.M., Proc. Soc.Exp. Biol. Med., 1969, 132, 645. 72. Lambert, R., Andre, C. and Berard, A., Digestion, 1971, 4, 234. 73. Galanti, N. and Baseraga, R., J. Biol. Chem., 1971, 246,6814. 74. Pritchard, E.T., Biochem. J., 1977, 166, 141. 75. Slomiany, A., Slomiany, B.L. and Mandel, I.D., Submitted for publication. 76. Slomiany, B.L., Slomiany, A. and Mandel, I.D., Submitted for publication. 77. Scarpelli, E.M., Clutario, B.C. and Taylor, F.A., J. Appl. Physiol., 1967, 23, 880. 78. Sanderson, R.J., Paul, G.W., Vatter, A.E. and Filley, G.F., Res. Physiol., 1976, 27, 379. 79. Godinez, R.J., Sanders, R.L. and Longmore, W.J., Biochemistry, 1975, 14, 830. 80. Colacicco, G., Buckelew, A.R. and Scarpelli, E.M., J. Appl. Physiol., 1973, 34, 743. 81. Slomiany, B.L., Smith,F.B. and Slomiany, A., Biochim. Biophys. Acta, 1979, in press. 82. Slomiany, A., Smith, F.B. and Slomiany, B.L., Eur. J. Biochem. 1979, 98, 47.

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83. Schiff, F. and Adelsberger, L., Z. Immunitaetsforsch. Exp. Ther., 1924, 40, 335. 84. Eilser, M. and Mortisch, P., Z. Immunitaetsforsch. Exp.Ther., 1928, 57,

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96. Koscielak, J., Miller-Podraza, H., Krauze, R. and Piasek, A., Eur. J. Biochem., 1976, 71, 9. RECEIVED

December 10, 1979.

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

11 Fucolipids and Gangliosides of Human Colonic Cell Lines BADER SIDDIQUI and Y. S. KIM

Veterans Administration Medical Center and Department of Medicine, University of California, San Francisco, CA 94121 Carcinoma of the larg affluent countries. I get colonic cancer each year and half of them die from it. Cancerous growth of tissues appears to be the result of cells not following the normal differentiation pathway towards formation and maintenance of normal functional organs. One approach to treatment of cancer is to redirect the cellular differentiation pathway toward normal growth with chemical agents. Several chemical agents have been used to modify the differentiation process in cultured tumor cell lines. A variety of chemical compounds, including cyclic AMP, sodium butyrate, dimethylformamide, dimethylsulfoxide, 5-bromodeoxyuridine, and tri-fluoro-methyl-2deoxyuridine can affect morphological and biochemical properties of cells. Some reports demonstrate that the tumorigenicity of cancer cells is markedly reduced or completely abolished by these agents. (See review by Prasad and Sinha, 1.) Butyrate treated Hela cells (2) and KB cells showed marked increases in the amounts of G gangliosides and elevated levels of the enzyme, CMP:sialic acid: lactosylceramide sialosyltransferase, required for its synthesis. Human colonic mucosa and colonic tumors are rich in glycolipids including gangliosides and several fucolipids. These lipids are important because they often determine blood group and other surface properties of cells. To understand better the effects of differentiating agents on tumor cells, we have been concentrating our efforts on the effect of agents like sodium butyrate or dimethylsulfoxide on colonic tumor cell lines. Previous studies in our laboratory have dealt with some of the effects of sodium butyrate on two colonic tumor cell lines, SW-480 and SW-620. This study describes the effect of sodium butyrate on glycolipids from four human colonic tumor cell lines, SKCO-1, HT-29, SW-480 and SW-620 and a human fetal intestinal line, FHS. M3

0-8412-0556-6/ 80/ 47-128-177$5.00/ 0 © 1980 American Chemical Society In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

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Materials and Methods C e l l Lines. The human fetal intestinal c e l l line (FHS) was kindly supplied to us by Dr. Walter A. Nelson-Rees at the Naval Bioscience Laboratory, Oakland, California. The human colonic c e l l line, SKCO-1 developed by Drs. G. Trempe and L.F. Olds, was obtained from Dr. Jorgen Fogh, Sloan Kettering Institute, Rye, New York. The HT-29 c e l l line was developed and obtained from Dr. J . Fogh. SW-480 and SW-620 were developed at Scott and White C l i n i c in Temple, Texas and were obtained from Col. A. Liebovitz. A l l of the c e l l lines are routinely maintained as monolayer in Dulbecco's modified Eagle's medium supplemented to 10% with fetal bovine serum, 100 units/ml of p e n i c i l l i n and 100 mg/ml of streptomycin. Labelling of Cells For labelling experiments Dulbecco's modified Eagle's mediu glucose. Cells were seede medium at 37° and allowed to attach for 20-24 hours. The medium was then replaced with fresh medium containing sodium butyrate, 1.0 mM in case of SKC0-1 c e l l s or 2.5 mM for the other c e l l lines. Medium was changed every 3-4 days. After 8 days, medium containing 50uCi of Q H3-galactose (specific activity 9.1 Ci/m mole, (New England Nuclear Corporation, Boston, Massachusetts)) or £3H]fucose (specific activity 13.2 mCi/m mole, (NEN)) was added with or without butyrate. The c e l l s were further incubated for 20-24 hours. The c e l l s were harvested with 10 mM phosphate-buffered, 0.15 M saline, pH 7.4, containing 2 mM EDTA and washed three times with cold phosphate-buffered saline. Cells were collected by centrifugation. 3

Isolation of Labelled Glycolipids. Cells were sonicated in a small volume of saline and the total protein was determined on an aliquot by the method of Lowry et. a l . (_5) . Total l i p i d s were extracted with 20 volumes of chloroform; methanol (2:1) f i l t e r e d , and the residue re-extracted with 10 volumes of chloroform: methanol: water (1:2:0.15). Extracts were combined and concentrated at 40o under vacuum and dialyzed against d i s t i l l e d water for 2 days at 4°. The dialyzate was dried and applied on a 1 x 10 cms DEAE-Sephadex column (6). Labelled neutral glycolipids, along with other l i p i d s , were eluted with 50 ml chloroform: methanol: water (30:60:8) and the ganglioside fraction, also containing sulfoglycolipids, was eluted with chloroform: methanol: 0.8 M sodium acetate (30:60:8). In some experiments, total l i p i d s were separated into upper phase and lower phase. Each phase was applied separately to columns containing DEAE-Sephadex to isolate three classes of glycolipids: neutral glycolipids, sulfoglycolipids and gangliosides {!). Gangliosides and sulfoglycolipid fractions were dialyzed and lyophilized. Glycolipids were resolved by thin layer chromotography.

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Thin Layer Chromotography. Unless otherwise stated, a l l thin layer chromotography was on plates coated with s i l i c a Gel G (E. Merck, Dramstadt). A l l the solvents were mixed on a volume basis. Neutral glycolipid fractions were developed i n chloroform: methanol: water (60:35:6.5). Labelled fucolipid fractions were developed in chloroform: methanol: water (40:40:10). For separation of gangliosides, chloroform: methanol: 2.5N aqueous NH4OH (60:40:9) was used. Fluorography of TLC Plates. TLC plates were developed in the appropriate solvent system and dried at 50° for 10-15 minutes. The plates were impregnated with the s c i n t i l l a t i n g medium by dipping them into 20% 2,5,Diphenyloxazole (PPO) i n toluene, dried and exposed to X-ray film (Kodak X-Qmat R XR ) for several days at -70°. Fluorgraphs wer Results Effect of Sodium Butyrate on Morphology and C e l l Growth. FHS, SKCO-1, HT-29 did not show any significant morphological changes with sodium butyrate. SW-480 and SW-620 c e l l s produce angular c e l l s rich i n cellular membranes. These processes were pronounced with SW-620 c e l l lines. Cells were seeded at 1 to 2 X 10 cells/75cm flask with sodium butyrate concentrations from 0.5 to 5.0 mM and without butyrate in growth medium. After 8 days, the c e l l s were harvested and protein was determined. Figure 1 shows total milligram protein/T-75cm flasks as plotted against sodium butyrate concentrations. The c e l l protein per flask of SKCO-1 decreased sharply with increased concentrations of butyrate when compared with control culture c e l l s . With SW-480 and SW-620 culture c e l l s , protein was decreased against butyrate concentrations, but the decrease was more pronounced with SW-620 c e l l s . C e l l protein of FHS and HT-29 cultures were unaffected (Fig. 1). 6

2

2

TLC of Ganglioside. Figure 2 TLC patterns of gangliosides obtained from fetal c e l l lines and three colonic cancer c e l l lines. The fetal c e l l lines (track 1) contained uncharacterized gangliosides, a through f; SW-480 (track 4) contained uncharacterized gangliosides, a through h; HT-29 gangliosides (track 3) have a simpler pattern; GJ43 i s the major ganglioside i n the SKCO-1 line (track 2). Labelled Fucolipids. Figure 3 shows fluorograms which were obtained from c e l l s labelled with G*lQ-fucose with and without butyrate treatment. Fucolipids were not found i n fetal c e l l s and, therefore, are not shown here. Figure 3A, track 2, shows fucolipid patterns of SW-480 cells without butyrate. Although

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Figure 1. Total milligrams of T-75 cm flasks vs. millimolar tions of sodium butyrate in growth medium. Total cell protein was determined after cells were incubated in growth medium with sodium butyrate for eight daysat37°C. O, FHS; A, SKCO-1; HT-29; • , SW-480; | , SW-620.

Figure 2.

TLC chromatogram of gangliosides in chloroform:methanol:2.5N

NH, OH t

(60:40:9) 1, FHS; 2, SKCO-1; 3, HT-29; 4, SW-480 cell lines; 5, small intestine gangliosides used as standards; 6, 7 contain human brain standard gangliosides. Apparent discrepancy in mobilities among A and B is because they were obtained from different runs and conditions vary slightly. Gangliosides visualized by spraying with resorcinol, followed by heating at 130°C for 15-25 min. G , NeuAca2 -> 3Galfil -> 4GIB1 l'Cer G , NeuAca2 -> 8NeuAca2 -» 3Galf31 -> 4Glc/31 -» VCer G , GalNAcpl -» 4Gal(3 «- 2aNeuAc)(31 -* 4Glcf31 -» VCer G , Gaipi 3GalNAc/31 -» 4Gal(3 4Glc/31 -> VCer G , NeuAca2 -> 3Gal/31 -> 3GalNA pi -» 4Gal(3 4Glc(31 -> VCer G , Gal/31 3GalNAcf31 -> 4Gal(3 8NeuAca2 -» JGa/jSi -» 4GlcNacf31 -> JGa/y37 -> 4Gfcj8J -> i'Cer m

D3

M2

M1

Dla

C

Dih

Lc

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

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Figure 3.

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Thin-layer chromatographic autoradiograms of labeled fucolipids

A: 1, labeled standard glycolipids GL-2a through Le ; 2, labeled fucolipids from SW-480 control cells. B: 3, labeled standard glycolipids GL-lb through Gl-5a. Labeled fucolipids from cells grown in butyrate-free medium are: HT-29, track 4; SW-480, track 6; SW-620, track 8. Fucolipids from cells grown in butyrate are: HT-29, track 5; SW-480, track 7; SW-620, track 9. A was developed in chloroform:methanol:water (60:35:8); B was developed in chloroform.methanohwater (40:40:10). In B equal amounts of fucolipid activity from both control cultures and butyrate-treated cells were applied. A is the result of direct radioautography of glycolipids. TLC plate B was dipped in 20% PPO in toluene and dried prior to exposure to x-ray film for several days at —70°C. Arrows show reactions of faint bands. b

GL-lb, GL-2a, GL-3a, GL-4a, GL-5a,

Gal/31 -> VCer Galfll -» 4Gkpi -» VCer Galal 4Galf31 4Glc/31 -> VCer GalNAc/31 3Galal 4Galf31 -» 4Glc/31 -> VCer GalNAcal 3GalNA pi -> 3Galal -> 4Galpl -> 4Glcf31 -* C

VCer

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the pattern of SKCO-1 c e l l s are now shown here, there was no change i n the fucolipid patterns of these c e l l s with and without butyrate. Figure 3B shows fucolipid patterns of c e l l s with and without butyrate treatment. Tracks 4 and 5 show fucolipid patterns of HT-29 c e l l s with and without butyrate treatment. In the HT-29 c e l l line fucolipid FL-1 i s not present but there i s a decrease in FL-5 when c e l l s are grown i n butyrate. Track 6 and 8 show fucolipid patterns of SW-480 and SW-620 c e l l lines. There i s a difference i n fucolipid patterns between these two c e l l lines although they were both derived from the same patient. On treatment with sodium butyrate, FL-1 i s markedly decreased or disappears i n these two c e l l lines (Fig. 3B, Track 7 and 9) and reappearance of slow-migrating fucolipids (FL-7 through FL-9). Labelled Gangliosides. Figure 4 shows fluorograms of gangliosides labelled with £*H]\-galactose from c e l l s grown with or without butyrate. In th marked difference betwee a slight difference in the intensities between the two spots of Gj43 (Fig. 4A) . GM3 i s a major ganglioside i n SKCO-1 c e l l s and labelling appeared to be unaffected by butyrate treatment (Fig. 4B). In HT-29 c e l l lines, the amount of G appeared to remain the same; however, the distribution of GJ43 components was affected by butyrate. Minor changes in other gangliosides could be seen (Fig. 4C). Although the overall pattern of ganliosides of SW-480 c e l l s with and without butyrate i s similar, there are some changes i n G143, GM2 and G ^ regions which may be due to alterations in the l i p i d moieties(Fig. 4D). Similar results are also observed with SW-620 c e l l s , as shown in Figure 4E. M 3

M

Labelled Neutral Glycolipids. Neutral glycolipids were labelled with -galactose. As" was seen with the ganglioside, the butyrate affected the neutral glycolipid patterns but the most marked alterations appeared to be due to changes in the l i p i d moeities. Discussion In the present study, sodium butyrate had a differentiated effect on c e l l morphology. Sodium butyrate caused the SW-620 lines to become markedly angular with extension of many membraneous processes. These effects were also seen with the SW-480 c e l l lines but were less pronounced. No morphological changes were observed when SKCO-1, HT-29 and FHS c e l l lines were cultured in sodium butyrate. The concentration of sodium butyrate was observed to have a d i f f e r e n t i a l effect on c e l l growth i n colonic c e l l lines. After culturing for 8 days with 5 mM sodium butyrate, the c e l l protein per flask of the SCK0-1 line was decreased to less than 10% of the control cultures. In the SW-620 culture, c e l l protein per

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

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Figure 4.

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183

Thin-layer chromatographic fluorograms of labeled gangliosides

The plates were developed in chloroform:methanol:2.5N NH OH (60:40:9). There are apparent discrepancies in the mobilities among the fluorograms because each plate was obtained from different runs and the conditions varied slightly. Standard [ H]-gangliosides G , G , and G were used in tracks 1, 4, 7, 10, and 12. A: labeled gangliosides obtained from FHS cell lines with (3) and without (2) butyrate treatment. B: gangliosides of SKCO-1 with (6) and without (5) butyrates. C: gangliosides of HT-29 with (9) and without (8) butyrate. D: gangliosides of SW-480 with (12) and without (11) butyrate. E: gangliosides of SW-620 cell lines with (15) and without (14) butyrate. Bands above G in C, D, and E are sulfoglycolipids. L

3

y3

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M3

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was reduced to 20-25% of the control while the related line SW-480 showed a 50% reduction in c e l l protein. Cell protein of FHS and HT-29 cultures was unaffected. When c e l l s were cultured in labelled fucose or galactose i n the presence or absence of butyrate, alterations i n the labelled glycolipids were observed. Treatment of a l l of the c e l l lines with butyrate did not markedly affect the incorporation of L H J galactose in ganglioside per milligram of c e l l protein. In a l l of the lines except SW-480, butyrate caused a decrease in monoglycosylceramide compared to diglycosylceradmie; however, the changes were not as distinct as the changes in gangliosides. When SW-480 and SW-620 c e l l lines were grown i n the presence of butyrate, the fastest migrating fucolipid disappeared concomitant with the appearance of slow-migrating fucolipids. Although there were few qualitative changes i n the gangioside patterns of the SKCO-1 and FHS lines there were marked alterations of ganglioside lines. The major change ing the Gj43 fraction. In HT-29, SW-480, and SW-620, there was a s h i f t i n GM3 to less polar components suggesting that the carbohydrates may be unchanged but the l i p i d moieties are altered. Alternatively, there may an acetylation of a hydroxyl group i n the carbohydrate moiety since i t has been shown that the butyrate increases the amount of acetylated histones i n Friend erythroleukemic c e l l s (9). The butyrate-induced s h i f t to less polar components i s also seen in the G fraction. The s h i f t i n GM2 and G143 components may be important in disturbing c e l l surface properties. SW-480 and SW-620 showed dramatic morphological alterations when cultured i n butyrate, and these c e l l s had obvious shifts to less polar components within the GM2 and GM3 fractions. In the SKCO-1 and FHS lines, these shifts were not observed and thus there were no morphological changes i n these two c e l l lines i n butyrate. However, since HT-29 c e l l s did not change morphology i n butyrate but also demonstrated the polarity shift in GM2 and GM3 components, the correlation between the two may be more complex, such as being dependent upon concentration or distribution of these components on the c e l l surface. We are currently exploring the effects of butyrate on ganglioside components of other c e l l lines to determine i f this glycolipid shift i s related to morphological alterations and to the malignant properties of c e l l s . 3

M 2

Summary In the present study, we examined the pattern of fucolipids and gangliosides i n cultured c e l l lines and alterations produced by a differentiating agent. A human f e t a l intestinal line (FHS), and four human colonic tumor lines (SKCO-1, HT-29, SW-480 and SW-620) were used. Cells were grown with or without sodium butyrate, (1.0 mM i n SKCO-1 and 2.5 mM in a l l other c e l l lines) i n

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

Fucolipids and Gangliosides

11. SIDDIQUI AND KIM

185

growth medium. After 8 days medium containing 50yCi of C3HJgalactose or Q3Hj-f se was added with or without butyrate, followed by incubation for another 20-24 hours. Glycolipids were purified by column chromatography, characterized by thin-layer chromatography and were detected by radioautography or by conventional staining. Each tumor line revealed a distinct pattern of labelled fucolipids consisting of at least 10 components. No labelled fucolipids were detected in the FHS cell lines. The butyrate treated SKCO-1 cells did not show any change in fucolipid patterns. In HT-29 cell lines, there was a decrease of fucolipid FL-5 when the cells were grown in butyrate. There is a difference in fucolipid patterns between SW-480 and SW-620 cell lines. On treatment with sodium butyrate FL-1 (fast moving fucolipid) is markedly decreased or disappears, and there is appearance of slow migrating fucolipids (FL-7 through FL-9). Gangliosides were labelled with galactose In the fetal cell lines (FHS) and SKCOtreated and untreated cells , , lines, the amounts of GM3 appeared to remain the same, but the distribution of GM3 components was affected by butyrate. These changes, might be due to alterations in the lipid moieties or, alternatively, there might be an acetylation of a hydroxyl group in the carbohydrate moiety, since it has been shown that the butyrate increases the amount of acetylated histones. UCO

Acknowledgements This work was supported in part by the United States Public Health Service Grant CA-14905 from the National Cancer Institute through the National Large Bowel Cancer Project, and by the Veterans Administration Medical Research Service. We are indebted to Dr. J . S. Whitehead for his critical review and valuable discussions in the preparation of this manuscript. We also appreciate the technical assistance of Mr. James Bennett. Literature Cited 1. 2. 3. 4. 5.

Prasad, K.N., and Sinha, P.K. (1978) In "Cell Differentiation and Neoplasia" (G.F. Saunders, ed.), pp 111-141. Raven Press, New York. Fishman, P.H., Bradley, R.M., and Henneberry, R.C. (1976) Arch. Biochem. Biophys. 172, 618-626. Macher, B.A., Lockney, M., Moskal, J.R., Fung, Y.K., and Sweeley, C.C. (1978) Exptl. Cell Res. 117, 95-102. Kim, Y.S., Tsao, D., Siddiqui, B., Whitehead, J.S., Arnstein, P., Bennett, J., and Hicks, J . Cancer, In press. Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275.

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

186

CELL SURFACE GLYCOLIPIDS

6. Ledeen, R.W., Yu, R.K., and Eng, L.F. (1973) J . Neurochem. 21, 829-939. 7. Siddiqui, B., Whitehead, J.S., and Kim, Y.S. (1978) J . Biol. Chem. 253, 2168-2175. 8. Mills, A.D., and Laskey, R.A. (1975) Eur. J . Biochem. 56, 335-341. 9. Reeves, R., and Cserjesi, P. (1979) J . Biol. Chem. 254, 4283-4290. RECEIVED December

10, 1979.

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

12 Biosynthesis of Blood-Group Related Glycosphingolipids in T - and B-Lymphomas and Neuroblastoma Cells MANJU BASU and SUBHASH BASU Department of Chemistry, University of Notre Dame, Notre Dame, IN 46556 MICHAEL POTTER National Cancer Institute, Bethesda, MD 20014 Glycosphingolipid uents of a l l eukaryoti ent classes of glycosphingolipids (1) are commonly found in animal c e l l s : a) GSLs containing mono- or disaccharides; b) GSLs containing a core structure, GalNAcβ-Galβl-4Glc-Cer (Gg series); c) a core struc­ ture of Galα-Galβl-4Glc-Cer (Gb series); d) a core structure of GlcNAcβ-Galβl-4Glc-Cer (Lc series). Short-chain GSLs of the f i r s t three families appear to be ubiquitous among eukaryotic c e l l s . How­ ever, long-chain GSLs of the latter two families are important constituents of the plasma membranes of numerous animal cells (2,3). Cell surface GSLs of the globoside family (Gb series) and blood group family (Lc series) have been implicated in the processes of c e l l - c e l l recognition and growth regulation (4,5), receptor function (6,7), malignant transformation (810), and blood group specificity (11-17). In recent years specific blood group-active glycosphingolipids (A, B , H, Le , Le , P , and I) have been identified in human erythrocytes (11-17). The neolactotetraosylcer­ amide, nLcOse Cer (Galβl-4GlcNAcβl-3Galβl-4Glc-Cer) exists as a common structure in these GSLs. The possi­ b i l i t y that nLcOse Cer is a tumor-associated surface antigen in NIL polyoma-transformed tumor cells was suggested by Hakomori and his co-workers (18,19). Irrespective of blood type, nLcOse Cer has also been identified in normal human erythrocytes (20) and in elevated quantities in the erythrocyte stroma of patients with congenital dyserythropoietic anemia type II (21). Are these changes in the content of nLcOse Cer due to blocked synthesis of higher chain length blood group glycosphingolipids (22) or to ele­ vated activity of UDP-Gal :LcOse Cer (β1-4) galactosyl­ transferase (EC 2.4.1.86) (24) ? In search of answers a

b

1

4

4

4

4

3

0-8412-0556-6/80/ 47-128-187$6.50/ 0 © 1980 American Chemical Society In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

188

CELL SURFACE

GLYCOLIPIDS

t o t h i s q u e s t i o n we have s t u d i e d b i o s y n t h e s i s i n v i t r o of nLc0se4Cer and i t s c o n v e r s i o n t o GMlb(GlcNAcT gangl i o s i d e o r b l o o d group H i - and B - a c t i v e GSLs (see F i g . 1) i n d i f f e r e n t tumor c e l l s o f p r i m a t e and non-primate origin. I t i s now w e l l e s t a b l i s h e d t h a t the T-lymphocytes t h a t d e v e l o p i n the thymus and r e l e a s e d i n the c i r c u l a t i o n have d i f f e r e n t p h y s i o l o g i c a l immune r e s p o n s e s from the a n t i g e n - s t i m u l a t e d , immunoglobulin-secreting B-lymphocytes ( F i g . 2) . I n r e c e n t y e a r s tumors o f t h e mouse l y m p h o r e t i c u l a r system have become the b e s t models f o r the study o f myeloma p r o t e i n s (23). I n the p r e s e n t r e p o r t we have compared the b i o s y n t h e t i c r o u t e s o f the b l o o d g r o u p - r e l a t e d g l y c o s p h i n g o l i p i d s mentioned above i n mouse l y m p h o r e t i c u l a r tumors and n e u r o b l a s t o m a s . The b i n d i n g o f l e c t i n s and t o x i n s t o some o f t h e s e tumo o b t a i n i n f o r m a t i o n abou p r e s e n t on the c e l l s u r f a c e s . M a t e r i a l s and Methods Mouse L y m p h o r e t i c u l a r Tumors. S i n c e 1971 N a t i o n a l Cancer I n s t i t u t e has been f r e e z i n g and s t o r i n g t r a n s p l a n t a b l e mouse l y m p h o r e t i c u l a r tumors. More than 1000 d i f f e r e n t tumors have been d e p o s i t e d . The most common tumor t y p e s a v a i l a b l e a r e l y m p h o c y t i c tumors o f bone marrow and thymic o r i g i n , A b e l s o n v i r u s - i n d u c e d lymphosarcomas and plasmacytomas, and c h e m i c a l l y i n d u c e d plasmacytomas. The tumors under i n v e s t i g a t i o n i n our l a b o r a t o r y a r e l i s t e d i n T a b l e I . Some d a t a on the c e l l s u r f a c e markers have been d e s c r i b e d r e c e n t l y by M a t h i e s o n e t a l . (24). The plasmacytomas are a l s o f r e q u e n t l y cKeckecT~for immunog l o b u l i n p r o d u c t i o n and a n t i g e n - b i n d i n g a c t i v i t y . S i n c e a l l o f the tumors a t Cancer I n s t i t u t e a r e e n t e r ed i n a computer bank (as mentioned i n T a b l e I ) , a d e s c r i p t i v e i d e n t i f i c a t i o n system has been adopted (Table I I ) . The computer name o f a tumor c o n t a i n s four pieces o f information: i ) strain of origin; i i ) mode o f i n d u c t i o n ; i i i ) c e l l t y p e ; and i v ) a c c e s s i o n number. A b e l s o n V i r u s - I n d u c e d Lymphocytic Tumors. A b e l s o n v i r u s (A-MuLV o r MuLV-A) i s a type C RNA v i r u s (25) and e x i s t s i n t h e murine leukemia v i r u s complex. This i s a d e f e c t i v e v i r u s and c o n t a i n s the Moloney l e u k e m i a v i r u s h e l p e r component and a r e p l i c a t i o n - d e f e c t i v e A b e l s o n component (23) t h a t t r a n s f o r m s lymphocytes and 3T3 f i b r o b l a s t s . While A b e l s o n v i r u s i s not

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

BASU ET

Lymphomas

AL.

and Neuroblastoma

: GdNAc-Gol-Gk-Ctr! Gol-Glc-Cer GMP-AoNw Gd-Gte-Cer AcNeu SAT-l

Cells

UDP-GlcNAj

GlcNAc-Gal-Glc-Cer |

f

CMP-AcNu

SAT Gal-Gte-Cer (AcNeu ) 2

(6D3)

GDP-Fuc

j~Ga7-~GaTlW^ GgOM^Ctr

FucT-2

Gal-GlcNAc-GoJ-Gte-Cer AcNeu

Gd-GaNAc-Gal-Glc-Cer AcNeu (GMIo)

foWMGIcNAc)]

-GteNAc-Gol-Glc-Cer

CMP-ACNM|

4

1

;Gal-GalNAc-Gal-Glc-Cer ! SAT iAcNeu Gd-GdNAc-Gal-Gte-Ce (GMIb) AcNeu AcNeu (GDla)

Figure 1.

Gol-GlcNAc-GoJ-Glc-Cer^

.

x

(B ) (

Proposed pathways for glycosphingolipid biosynthesis

Stem Cellt

(Bone marrow)

j

Lymphoid Stem Celli

Prothymocytes

Pre-B

I Thymus cortex I medulla 11

Thymocytes U-Clrculallon

C Ly-i.2,3, e [ Ly-I ; 6

+ +

+4+

;

H-2

+

]

; H-2 ]

B-lymphocytes

+

N^Short lived population(tpleen)

Peripheral T-lymphocytes CLy-1,2,3; e i H - 2 +

[Ly-l e ;

+

;

H-2

+++

]

4++

]

' l

.^-Circulation Antigen driven Stag.. « s t 0

Plasma Cell*

Figure 2.

Development oj B- and T-lymphocytes

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

M

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

Lymphocyte

Type

(a)

Unknown

X-5563

2

(Y b)

IgH

BPC-1

2

(y a)

IgG

CBPC-101

TEPC-824

Spontaneous ileocecal

Mineral o i l or alkane

polymer

TEPC-15

RNA)

Plasmacytomas

Abelson virus CC-type

Induction Condition

TE/109 d a y s AB/89 d a y s IgA

e (leukogenic)

Characteristics

System

ABPL-2

ABLS-140

ABLS-1

C o m p u t e r Name

Lymphoreticular

I

Plasmacytic lymphosarcomas

Bone marrow lymphocytic tumors

Tumor

Tumors o f t h e Mouse

Table

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

Tumor

Type

Lymphocytic tumors o f thymic o r i g i n

Thymic lymphoc y t i c neoplasms

I . (contd.)

Lymphocyte

Table

P-1798

BALENTL-5

BALENTL-3

+

0 ,

Estrogen pellet/521 days

Ethylnitrosourea/173 days

+

+

Chemically induced

Spontaneous

Induction Condition

+

Ly-1,2,3

Ly-2,3

6 ,

+

Ly-2,3

+

+

G ,

+

Ly-l (2 )

L-4946

Characteristics

Ly-1

Name

SAKRTLS-13

Computer

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

Pristane =

3

2,6,10,14-tetramethylpentadecane.

lympho-

LS = lymphosarcoma

MS = mastocytomas

a

X = C H

TE = P r i s t a n e

TL, TS = thymic c y t i c neoplasm

(CSTBl/GXBALB/C^

PC = plasma c e l l

AB = A b e l s o n V i r u s

Type

Cell

Induction

AKR

CB =

BAL = BALB/C

Strain

A b b r e v i a t i o n s used t o I d e n t i f y Tumor L i n e s o f Mouse L y m p h o r e t i c u l a r System

Table II

12.

B A S U ET

AL.

Lymphomas

and Neuroblastoma

Cells

193

i n f e c t i o u s i n a mouse c o l o n y , leukemias can be i n d u c e d i n a d u l t BALB/C mice w i t h i n 21 t o 30 days a f t e r i n j e c tion. Lymphosarcomas 5nd P l a s m a c y t i c Lymphosarcomas. These tumors a r i s e i n bone marrow c a v i t i e s o r lymph nodes under the i n f l u e n c e o f A b e l s o n v i r u s . F o r the p r o d u c t i o n o f p l a s m a c y t i c lymphosarcomas ( P L ) , a p r e i n c u b a t i o n p e r i o d o f 2 t o 3 months w i t h p r i s t a n e i s n e c e s s a r y (26). PL c e l l s a r e d i s t i n g u i s h e d from o t h e r plasmacytomas by t h e i r s i z e and lymphoid c h a r a c t e r . Lymphocytic Tumors of Thymic O r i g i n . The appearance o f spontaneous tumors (e.g. SAKRTLS-13) i n AKR and C58 mice (27) i s q u i t e common. Other thymic tumors can a l s o be i n d u c e d c h e m i c a l l y {28) (BALENTL-3 -5 o r P-1798; T a b l virus C29)). In th they a r e c o n f i n e d t o t h e thymus, b u t l a t e r t h e tumor i s m e t a s t a s i z e d t o the s p l e e n , l i v e r , k i d n e y , and lymph nodes. Plasmacytomas. The t r a n s p l a n t a b l e plasmacytomas a r e d e r i v e d from tumors i n d u c e d i n BALB/C m i c e . These tumors were i n d u c e d by i n t r a p e r i t o n e a l (IP) i m p l a n t a t i o n o f p l a s t i c m a t e r i a l s ( L u c i t e d i s c s or M i l l i p o r e d i f f u s i o n chambers) or by t h e IP i n j e c t i o n o f m i n e r a l o i l s ( l i g h t and heavy m e d i c i n a l m i n e r a l o i l s , B a y o l , F, D r a k e o l GVR) and a l k a n e s such as p r i s t a n e . Plasmacytomas a r i s e i n p e r i t o n e a l t i s s u e s and r e q u i r e a m i n e r a l o i l environment d u r i n g t h e i r e a r l y development. C e l l C u l t u r e . Human neuroblastoma IMR-32 ( p a s s aged t h r o u g h nude mice; the c e l l s were donated by Dr. Steven E. Brooks, K i n g s b r o o k J e w i s h M e d i c a l C e n t e r , Brooklyn) and mouse neuroblastoma c l o n e s NIE-115, NS20, and N*-18) (donated by Dr. Shraga Makover, Hoffmann LaRoche, I n c . , N u t l e y , New J e r s e y ) were m a i n t a i n e d i n our l a b o r a t o r y as d e s c r i b e d p r e v i o u s l y (30,31). Conf l u e n t monolayers (6 t o 8 x 1 0 c e l l s per 250-ml F a l c o n p l a s t i c f l a s k ) were h a r v e s t e d f o r enzymatic s t u d i e s w i t h p h o s p h a t e - b u f f e r e d s a l i n e [7.0 mM p o t a s sium phosphate/0.14 M NaCl - b u f f e r , pH 7.2 ( P i / N a C l ) ] c o n t a i n i n g 0.1% EDTA. A c l o n e o f g u i n e a p i g tumor c e l l s , 104C1 (the c e l l s were donated by Dr. C h a r l e s H. Evans, N a t i o n a l Cancer I n s t i t u t e , Bethesda, MD), was m a i n t a i n e d i n our l a b o r a t o r y (7) on RPMI-1640 medium (Gibco) s u p p l e mented w i t h 10% f e t a l b o v i n e serum ( G i b c o ) . C u l t u r e s were grown i n F a l c o n T - f l a s k s (75 cm ) c o n t a i n i n g 15 6

2

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

194

CELL SURFACE

GLYCOLIPIDS

m l o f m e d i u m a n d i n c u b a t e d u n d e r a w a t e r - s a t u r a t e d 95% air/5% CO2 atmosphere a t 3 7 ° C . T h e m e d i u m was c h a n g e d o n c e b e f o r e h a r v e s t i n g , a n d c e l l s w e r e h a r v e s t e d when t h e y r e a c h e d a p o p u l a t i o n d e n s i t y o f 5 t o 8 x 10^ c e l l s per T - f l a s k . Glycosphingolipids. A c c e p t o r GSLs were isolated f r o m v a r i o u s a n i m a l t i s s u e s {32). Lactosylceramide and GM3 g a n g l i o s i d e w e r e i s o l a t e d f r o m b o v i n e spleen ( 3 3 ) , GM1 a n d GM2 g a n g l i o s i d e s f r o m human b r a i n s ( 3 4 ) , and B - a c t i v e n e o l a c t o p e n t a o s y l c e r a m i d e ( n L c O s e s C e r ; Galal-3GalBl-4GlcNAc31-3Gal31-4Glc-Cer)from rabbit erythrocytes ( 3 5 ^ 3 £ ) and bovine e r y t h r o c y t e s {37). Neolactotetraosylceramide (nLcOse Cer;Gal61-4GlcNAc$l3Gal31-4Glc-Cer) and l a c t o t r i a o s y l c e r a m i d e (LcOse3Cer; G l c N A c 3 1 - 3 G a l $ l - 4 G l c - C e r ) were p r e p a r e d from nLcOsesCer by s e q u e n t i a t o s i d a s e (38,39) an G g O s e 4 C e r was p r e p a r e d f r o m b o v i n e b r a i n gangliosides by m i l d a c i d h y d r o l y s i s a c c o r d i n g t o a p r e v i o u s l y p u b l i s h e d method ( 4 1 ) . The p u r i f i e d g l y c o s p h i n g o l i p i d s were a n a l y z e d b e f o r e u s e as s u b s t r a t e s by gas chromatography-mass spectrometry (42). 4

Glycosphingolipid; Glycosyltransferase Assays. A 25-33% ( v o l / v o l ) h o m o g e n a t e o f mouse t u m o r s o r h a r v e s t e d c e l l s i n 0 . 3 2 M s u c r o s e c o n t a i n i n g 0.1% 2 - m e r c a p t o e t h a n o l a n d 0 . 0 0 1 M EDTA (pH 7 . 0 ) was u s e d a s enzyme s o u r c e . Membrane f r a c t i o n s f o r g l y c o l i p i d : g l y c o s y l t r a n s f e r a s e a s s a y s were i s o l a t e d a t t h e j u n c t i o n of 0.32 M a n d 1.2 M on a d i s c o n t i n u o u s s u c r o s e density gradient (32,43). i) G a l a c t o s y l t r a n s f e r a s e Assays. The complete i n c u b a t i o n m i x t u r e c o n t a i n e d t h e f o l l o w i n g components ( i n micromoles) i n f i n a l volumes o f 0.045 m l : g l y c o s p h i n g o l i p i d a c c e p t o r s , 0 . 0 2 5 ; T r i t o n X - 1 0 0 , 100 y g ; c a c o d y l a t e - H C l b u f f e r , pH 7 . 3 , 1 0 ; M n C l , 0 . 1 2 5 ; U D P [ 1 4 c ] G a l , 2 5 , 0 0 0 cpm ( 1 . 3 x 1 0 cpm p e r y m o l e ) a n d h o m o g e n a t e o f t u m o r o r c e l l s , 0 . 3 t o 0 . 5 mg o f p r o t e i n ( e s t i m a t e d b y t h e m e t h o d o f L o w r y e t a l . (44J u s i n g b o v i n e serum a l b u m i n as s t a n d a r d ) . A f t e r 2 hours at 3 7 ° C , t h e r e a c t i o n was s t o p p e d b y a d d i n g 2 . 5 y m o l e s o f EDTA (pH 7 . 0 ) . 2

6

ii) Sialyltransferase Assays. The complete i n c u b a t i o n m i x t u r e c o n t a i n e d t h e f o l l o w i n g components ( i n micromoles) i n f i n a l volumes o f 0.065 m l : g l y c o s p h i n g o l i p i d a c c e p t o r s , 0 . 0 5 ; T r i t o n CF-54 and Tween-80 ( 2 : 1 ) , 200 y g ; c a c o d y l a t e - H C l b u f f e r , pH 6 . 4 , 9 ; M g C l , 0 . 2 5 ; C M P - [ ^ C ] A c N e u , 6 1 , 0 0 0 cpm ( 2 . 6 x 10& cpm p e r 2

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

12.

B A S U ET A L .

Lymphomas

and Neuroblastoma

Cells

195

ymole); and homogenate o f tumor o r c e l l s , 1.0 t o 1.5 mg of p r o t e i n . A f t e r 2 hours a t 37°C, the r e a c t i o n was stopped by adding 10 y l o f chloroform-methanol (2:1). i i i ) F u c o s y l t r a n s f e r a s e Assays. The complete i n c u b a t i o n m i x t u r e c o n t a i n e d the f o l l o w i n g components ( i n micromoles) i n f i n a l volumes of 0.037 ml: g l y c o s p h i n g o l i p i d a c c e p t o r s , 0.025; d e t e r g e n t , G-3634A ( A t l a s Chemi c a l ) , 100 yg; c a c o d y l a t e - H C l b u f f e r , pH 6.4, 10; M g C l , 0.125; EDTA (pH 7.0), 0.5; GDP-[14c]Fuc, 24,000 cpm (222 y C i per ymole and 3.5 x 1 0 cpm per ymole); and homogenate of tumor o r c e l l s , 0.12 t o 0.28 mg o f protein. A f t e r 1 hour a t 37°, the r e a c t i o n was stopped by adding 10 y l o f chloroform-methanol (2:1). The i n c u b a t i o n m i x t u r e s were assayed by the double chromatographic method (33^,4_0) o r by a c o m b i n a t i o n o f h i g h v o l t a g e b o r a t e e l e c t r o p h o r e s i s and r e v e r s e f l o w chromatography (3_2/^0 chloroform-methanol-H2 The a p p r o p r i a t e areas o f each chromatogram were d e t e r mined q u a n t i t a t i v e l y i n a t o l u e n e s c i n t i l l a t i o n system w i t h a Beckman s c i n t i l l a t i o n c o u n t e r (model LS-3133T). 2

6

1 2 5

1 2 5

B i n d i n g of [ I ] L e c t i n and [ I ] T o x i n t o C e l l Surfaces. F a l c o n T - f l a s k s (75 cm2) c o n t a i n i n g conf l u e n t p o p u l a t i o n s of c u l t u r e d c e l l s (IMR-32, NIE-115, NS-20, N-18, o r 104C1) were washed w i t h PBS (2 x 10 ml) at 15-20°C and i n c u b a t e d w i t h [ l 5 i ] l e c t i n o r [ l 5 l ] t o x i n ( s p e c i f i c a c t i v i t i e s are mentioned i n T a b l e s V I I and V I I I ) i n 3 ml of serum f r e e medium ( E a g l e s MEM f o r human neuroblastoma IMR-32 c e l l s ; Dulbecco's MEM f o r mouse neuroblastoma NIE-115, N-18; and NS-20; and RPMI1640 f o r g u i n e a p i g 104C1 c e l l s ) f o r 15 minutes a t 37° C. The medium was removed; the c e l l l a y e r was washed g e n t l y w i t h PBS (2 x 10 ml) a t 15-20°C and then kept i n an i n c u b a t o r f o r 10-15 minutes a t 37°C i n the presence of 5 ml o f 0.1% EDTA i n PBS (pH 7.2). The l o o s e c e l l s were f i n a l l y d i s p e r s e d and t r a n s f e r r e d t o a 15-ml g r a d u a t e d c e n t r i f u g e tube w i t h a d i s p o s a b l e P a s t e u r pipette. An a l i q u o t (0.5 t o 1 ml) was taken and f i l t e r e d through b o r o s i l i c a t e f i b e r d i s c s (Whatman GF/A, p o r o s i t y , 1.0 ym; d i a m e t e r , 2.4 cm) i n a M i l l i p o r e apparatus. The d i s c s were washed w i t h c o l d 5% t r i c h l o r o a c e t i c a c i d (TCA) o r 5% TCA f o l l o w e d by c h l o r o form-methanol (2:1) and d r i e d a t 100°C f o r 15 m i n u t e s . [125j] c o n t e n t was q u a n t i t a t i v e l y determined i n a t o l u e n e s c i n t i l l a t i o n system i n the presence and absence o f PCS (Amersham/Searle) w i t h a Beckman LS3133T c o u n t e r . P u r i f i e d c h o l e r a t o x i n was purchased from Schwarz/Mann and l a b e l e d w i t h N a I i n the p r e s ence of Chloramine-T a c c o r d i n g t o the method o f 2

2

f

1 2 5

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

N e u r o b l a stoma

Human

T-lymphocytic:

B-lymphocytic:

Mouse

5,282

TEPC-15

IMR-3 2

BALENTL-3

3,270

3,465

15,570

1,251

ABLS-140

(al-3)

(31-4)

595

533

886

1,642

115

pmol/mg p r o t e i n / 2 h r

nLcOse^Cer

Incorporated

LcOse^Cer

14 [ C]Galactose

GM2

82

244

2,075

3,132

194

(31-3)

G a l a c t o s y l t r a n s f e r a s e A c t i v i t i e s i n Mouse and Human Tumor C e l l s

L~4946

Tumor Type

Glycolipid:

Table I I I

12.

B A S U ET

AL.

Lymphomas

and Neuroblastoma

Cells

197

Cuatrecasas (45). The D o l i c h o s b i f l o r u s l e c t i n was a g i f t from Dr. M a r i l y n n E t z l e r (46) . U l e x europeus and B a n d e i r e a s i m p l i c i f o l i a l e c t i n s were p u r i f i e d i n our l a b o r a t o r y and l o d m a t e d w i t h Nal25j. ( c a r r i e r - f r e e ) by Sepharose 4B-bound l a c t o p e r o x i d a s e (47)and d i a z o t i z e d i o d o a n i l i n e c o u p l i n g (48) p r o c e d u r e s , r e s p e c t i v e l y . R e s u l t s and

Discussion

A. B l o o d G r o u p - R e l a t e d G l y c o s p h i n g o l i p i d S y n t h e s i s i n Tumor C e l l s . Mouse l y m p h o r e t i c u l a r tumors c o n t a i n at l e a s t three d i f f e r e n t g l y c o l i p i d : g a l a c t o s y l t r a n s f e r a s e a c t i v i t i e s , which can be d i s t i n g u i s h e d by t h e i r a c c e p t o r s p e c i f i c i t i e s ( T a b l e I I I ) . R e c e n t l y we have shown (22) t h a t c u l t u r e d c e l l s (TSD) from the cerebrum of a Tay-Sachs-diseased f e t u s c o n t a i n high a c t i v i t y of UDP-Gal:Lc0se3Cer ( 3 1 - 4 ) g a l a c t o s y l t r a n s f e r a s e ( F i g . 1, GalT-4 o r EC 2.4.1.86 o f UDP-Gal:GM2(31-3 GalT-3 o r EC 2.4.1.62) (£9). The p r e s e n t study i n d i c a t e s t h a t the l e v e l s of t h e s e two g a l a c t o s y l t r a n s f e r a s e a c t i v i t i e s depend on the d i f f e r e n t i a t e d c e l l t y p e s . In plasmacytomas (TEPC-15) the l e v e l s of t h e s e two enzymatic a c t i v i t i e s are h i g h and a r e a l m o s t comp a r a b l e , whereas i n B - l y m p h o c y t i c tumor o b t a i n e d by i n d u c t i o n w i t h A b e l s o n v i r u s (lymphosarcoma, ABLS-140) the l e v e l of GalT-3 was o n l y 15% t h a t of GalT-4 a c t i vity. I t appears t h a t mouse l y m p h o r e t i c u l a r tumors (B- o r T - l y m p h o c y t i c o r i g i n ) i n d u c e d v i r a l l y (ABLS140, T a b l e I) or c h e m i c a l l y (BALENTL-3, T a b l e I) show a lower c o n t e n t o f GalT-3 a c t i v i t y , whereas h i g h e s t a c t i v i t y i s p r e s e n t i n tumors o b t a i n e d by m i n e r a l o i l i n d u c t i o n (TEPC-15). Among a l l the tumors t e s t e d , the a c t i v i t i e s of GalT-4 and GalT-5 ( F i g . 1, UDP-Gal: n L c 0 s e 4 C e r ( a l - 3 ) g a l a c t o s y l t r a n s f e r a s e o r EC 2.4.1.87) (38) are h i g h e s t i n .spontaneous thymic l y m p h o c y t i c neoplasms (L-4946). The a c t i v i t i e s of t h e s e t h r e e g l y c o l i p i d : g a l a c t o s y l t r a n s f e r a s e s were a l s o compared (under p r e s e n t c o n d i t i o n s i n v i t r o ) i n human n e u r o b l a s t o m a IMR-32 c e l l s . S y n t h e s i s o f GM1 g a n g l i o s i d e from GM2 i s u n u s u a l l y low compared w i t h the s y n t h e s i s of n e o l a c t o t e t r a o s y l c e r a m i d e ( n L c O s e ^ e r ) or B - a c t i v e neolactopentaosylceramide (nLc0se5Cer), as i n ABLS-140 and BALENTL-3 mouse l y m p h o r e t i c u l a r tumors. From our p r e v i o u s s t u d i e s i t appears t h a t s y n t h e s i s of n e o l a c t o t e t r a o s y l c e r a m i d e i s u b i q u i t o u s among v a r i o u s normal (4_0,_53) and tumor c e l l s (7^, 22 , 37_, 5£, 55) grown i n culture. I t i s i m p o r t a n t t o f i n d out whether t h e s e n e u t r a l b l o o d group c o r e s t r u c t u r e s are then t r a n s formed t o any t y p e - s p e c i f i c a n t i g e n i c s i a l i c a c i d - o r f u c o s e - c o n t a i n i n g g l y c o s p h i n g o l i p i d on the s u r f a c e o f tumor c e l l s .

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

198

CELL SURFACE

GLYCOLIPIDS

S i a l y l t r a n s f e r a s e a c t i v i t i e s using three s p e c i f i c g l y c o l i p i d s u b s t r a t e s were a l s o t e s t e d i n t h e s e mouse l y m p h o r e t i c u l a r tumors. The a c t i v i t y o f CMP-AcNeu:GM3 ( a 2 - 8 ) s i a l y l t r a n s f e r a s e (50) was h i g h e s t i n e t h y l n i t r o s o u r e a - i n d u c e d thymic l y m p h o c y t i c tumor BALENTL-3 b u t almost n e g l i g i b l e i n B - l y m p h o c y t i c tumors (Table I V ) . The t r a n s f e r o f s i a l i c a c i d t o the t e r m i n a l g a l a c t o s e of g a n g l i o t e t r a o s y l c e r a m i d e i s catalyzed e f f i c i e n t l y by a s i a l y l t r a n s f e r a s e p r e s e n t i n embryonic c h i c k e n (41,51) and r a t (52) b r a i n s . A G o l g i - r i c h membrane p r e p a r a t i o n i s o l a t e d from b o v i n e s p l e e n (32^33^ a l s o c a t a l y z e s the r e a c t i o n e f f i c i e n t l y . R e c e n t l y we have shown t h a t b o t h embryonic c h i c k e n b r a i n (43) and b o v i n e s p l e e n (33,43) a l s o c a t a l y z e the t r a n s f e r o f s i a l i c a c i d (AcNeu) from C M P - [ C ] A c N e u t o n e o l a c t o t e t r a o s y l ceramide t o form AcNeu-nLcOse4Cer o r GMlb(GlcNAc) We have a l s o c h a r a c t e r i z e l i n k a g e p r e s e n t i n th from t h i s enzymatic r e a c t i o n . 2 Studies with glycoprotein substrate s p e c i f i c i t i e s o f p o r c i n e s u b m a x i l l a r y s i a l y l t r a n s f e r a s e suggest (56) t h a t the enzyme c a t a l y z i n g the t r a n s f e r o f s i a l i c acTd t o Gal61-3GalNAc-Ri g l y c o p r o t e i n a c c e p t o r may not c a t a l y z e s i a l i c a c i d t r a n s f e r t o Gal61-3GalNAc-R g l y c o l i p i d a c c e p t o r ( i . e . GM1) o r Galgl-4GlcNAc-R3 acceptor. U s i n g g l y c o s p h i n g o l i p i d s as a c c e p t o r s ( n L c O s e C e r and G g O s e ^ e r ) f o r the s u b s t r a t e c o m p e t i t i o n s t u d i e s w i t h embryonic c h i c k e n b r a i n and b o v i n e s p l e e n G o l g i - r i c h membrane systems (3_3 ,4_3) , ' i t appears t h a t b o t h r e a c t i o n s might be c a t a l y z e d by the same enzyme. I n the p r e s e n t s t u d i e s , e x c e p t i n TEPC15 and IMR-32, the a c t i v i t i e s w i t h b o t h s u b s t r a t e s ( n L c O s e C e r and GgOse4Cer) are almost comparable i n a l l o t h e r tumor c e l l l i n e s . Further k i n e t i c studies of t h e s e two a c t i v i t i e s are under way. The n a t u r a l o c c u r ence o f AcNeu-GgOse4Cer (GMlb) i n r a t a s c i t e s tumor c e l l s has been r e p o r t e d r e c e n t l y (57). Different glycolipid:fucosyltransferase activities have been r e p o r t e d (32,55,58) t o c a t a l y z e the a d d i t i o n o f f u c o s e t o p o s i t i o n C-2 o f the t e r m i n a l D - g a l a c t o s e and p o s i t i o n C-3 o f the i n t e r n a l N - a c e t y l g l u c o s a m i n e o f nLcOse4Cer t o form b l o o d group H o r human tumors p e c i f i c l i p i d (10). Incorporation of fucose i n t o GgOse4Cer was a l s o r e p o r t e d r e c e n t l y by T a k i et. a l . (59) i n r a t a s c i t e s hepatoma c e l l s (AH 7974F). From our p r e s e n t s t u d i e s i t appears t h a t GDP-Fuc:nLcOse4Cer ( a l - 2 ) f u c o s y l t r a n s f e r a s e a c t i v i t y ( F i g . 1, FucT-2 o r EC 2.4.1.89) i s 10 t o 20 times more a c t i v e i n mouse B - l y m p h o c y t i c tumors (ABLS-14 0 and TEPC-15) than i n T - l y m p h o c y t i c tumors (L-4946 and BALENTL-3). From 14

2

4

2

3

4

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

Neuroblastoma

Human

T-lymphocytic:

B-lymphocytic:

Mouse

IMR-32

BALENTL-3

L-4946

92

534

1,081

307

(a2-3)/(a2-6)

(a2-3)/(a2-6)

90

992

213

< 10

1,554

< 10

623

617

< 10

(a2-8)

GM3

401

pmol/mg p r o t e i n / 2 h r

4

GgOse Cer

nLcOse^Cer

TEPC-15

Cells

14 [ C]AcNeu I n c o r p o r a t e d

A c t i v i t i e s i n Mouse and Human Tumor

316

Sialyltransferase

ABLS-140

Tumor Type

Glycolipid;

T a b l e IV

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

Neuroblastoma

Human

T-lymphocytic:

B-lymphocytic:

Mouse

(310)

1

(124)'

5

BALENTL-3

IMR-3 2

9

103

TEPC-15

L-4946

73

31

(al-2)/(al-3)

(al-2)/(al-3)

(363)

1121)' ]

3

40

140

222

29

-2 cpm x 10 /mg p r o t e i n / h r

nLcOse^Cer

nLcOSe Cer 4

Cells

(15)

14 [ C]Fucose Incorporated

F u c o s y l t r a n s f e r a s e A c t i v i t i e s i n Mouse and Human Tumor

ABLS-140

Tumor Type

Glycolipid:

Table V

a

< 0.1

< 0.1

8

< 0.1

(al-2)

GM1

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

(contd.)

' The complete i n c u b a t i o n m i x t u r e s c o n t a i n e d t h e same components as d e s c r i b e d i n t h e t e x t e x c e p t t h a t h i g h e r c o n c e n t r a t i o n (0.01 ymole p e r 50 y l i n c u b a t i o n volume) and lower s p e c i f i c a c t i v i t y o f G D P - [ - ^ c ] f u c o s e (3.6 x 10^ cpm p e r ymole) was used f o r a) IMR-32 homogenate and b) membrane f r a c t i o n i s o l a t e d a t t h e j u n c t i o n o f 0.32 and 1.2 M sucrose g r a d i e n t . The m i x t u r e s were i n c u b a t e d f o r 2 h r a t 37°C. Values not given i n parentheses represent i n c o r p o r a t i o n of [ l ^ c ] f u c o s e i n t o respective substrates using high s p e c i f i c a c t i v i t y GDP-[l^c]fucose (189 mCi/mmole; New E n g l a n d N u c l e a r ) and c e l l homogenates as d e s c r i b e d i n t h e t e x t .

Table V

202

CELL SURFACE

GLYCOLIPIDS

c o m p e t i t i o n s t u d i e s w i t h a b o v i n e G o l g i - r i c h membrane f r a c t i o n {60) and an IMR-32 membrane f r a c t i o n i t appears t h a t t h e t r a n s f e r o f [ C ] f u c o s e t o nLcOse4Cer and nLcOsesCer i s p r o b a b l y c a t a l y z e d by t h e same enzyme. A c t i v i t i e s w i t h t h e s e two s u b s t r a t e s i n mouse l y m p h o r e t i c u l a r tumors and human neuroblastoma c e l l s a r e almost comparable. However, l i t t l e o r no f u c o s e t r a n s f e r t o GM1 g a n g l i o s i d e o r GgOse4Cer (unpublished) has been o b s e r v e d w i t h t h e s e tumor c e l l s under our present assay c o n d i t i o n s . Higher a c t i v i t i e s o f g l y c o p r o t e i n : (a-2) o r (a-3) f u c o s y l t r a n s f e r a s e a c t i v i t i e s i n human c a n c e r t i s s u e s and i n s e r a o f c a n c e r p a t i e n t s have been r e p o r t e d from d i f f e r e n t l a b o r a t o r i e s (61-64) but t h e f u n c t i o n o f t h e s e f u c o s y l a t e d g l y c o c o n j u g a t e s on m a l i g n a n t c e l l s u r f a c e s i s s t i l l unknown. 1 4

4

1 2 5

1 2 5

B. B i n d i n g o f [ I ] L e c t i n s and [ I ] T o x i n t o Neuroblastoma C e l l In o r d e r t o o b t a i n c o c o n j u g a t e s and t h e i r g r o s s t o p o g r a p h i c a l o r i e n t a t i o n on t h e c e l l s u r f a c e s , we measured t h e b i n d i n g o f l 5 i l a b e l e d l e c t i n s and t o x i n t o human neuroblastoma IMR32 and mouse neuroblastoma NIE-115, NS-20, and N-18 c l o n e s (Tables V I I and V I I I ) . The "5% TCA Wash" column (Table VII) r e p r e s e n t s 5 i - i a b e l e d l e c t i n or toxin bound t o b o t h g l y c o p r o t e i n and g l y c o l i p i d . The "5% TCA p l u s c h l o r o f o r m - m e t h a n o l 2:1 Wash" column r e p r e s e n t s t i g h t l y bound l e c t i n o r t o x i n t o t h e c e l l s u r f a c e s . These r e s u l t s s u g g e s t t h a t , i n a d d i t i o n t o GM1 g a n g l i o s i d e , IMR-32 c e l l s may c o n t a i n some g l o b o p r o t e i n and g a n g l i o p r o t e i n (65) w i t h t e r m i n a l N-acetylgalactosamine (Table VII) and ot-fucose r e s i d u e s (Table V I I I ) (65) . A l t h o u g h we found v e r y l i t t l e a c t i v i t y o f F u c T - 2 ~ T F i g . 1) i n mouse neuroblastoma c l o n e s (.55f60_) i t appears t h a t N-18 has t h e h i g h e s t Ulex europeus [ l 5 i ] l e c t i n b i n d i n g a b i l i t y o f a l l c l o n a l l i n e s t e s t e d . The b i o s y n t h e s i s i n v i t r o o f non-fucose B - a c t i v e n e o l a c t o p e n t a o s y l c e r a m i d e (37,53-55) i n c u l t u r e d mouse (37,54), g u i n e a p i g (53) ,and human tumor c e l l s (55,60) ~TTair~been e s t a b l i s h e d i n our l a b o r a t o r y . I t i s important t o see changes i n i t s appearance on t h e tumor c e l l s u r f a c e s d u r i n g c h e m i c a l l y induced d i f f e r e n t i a t i o n . The b i n d i n g s t u d i e s w i t h B. s i m p l i c i f o l i a [ l ^ i ] l e c t i n t o NIE115 c e l l s ( a f t e r c h e m i c a l d i f f e r e n t i a t i o n ) showed no marked d i f f e r e n c e when compared w i t h c o n t r o l c e l l s (Fig. 3 ) . In the presence of c y t o c h a l a s i n - B the c e l l volume i n c r e a s e s , a change t h a t may r e p r e s e n t i n c r e a s e d b i n d i n g s i t e s o r reduced i n t e r n a l i z a t i o n o f t h e [ l 5 l ] l e c t i n due t o t h e a l t e r a t i o n s o f m i c r o t u b u l e s . 2

1 2

2

2

2

C. Role o f G l y c o s p h i n g o l i p i d s as C e l l S u r f a c e Receptors^ Both B- and T-lymphocytes emerge from bone marrow i n t o t h e l y m p h a t i c t i s s u e s and e n t e r t h e b l o o d

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

l^,

toxin

simplicifolia

2

R,

(n.d.)

113,000 (4)

170,000

84,000 (2)

114,000 (4)

M o l e c u l a r Wt, (Subunits)

3

4

or glycoprotein,

GalNAc(al-3)Gal-R

Fuc(al-2)Gal-R

AcNeu

Gal(31-3)GalNAc-Gal-R.

1

Specificity

Gal(al-3)Gal-R

Sugar

B i n d i n g o f L e c t i n s and T o x i n s

R^, o r R^ = o l i g o s a c c h a r i d e s , g l y c o l i p i d ,

biflorus

europeus

Dolichos

Ulex

Cholera

Bandeiraea

Name

Sugar S p e c i f i c

T a b l e VI

T

A

H

GM1

e

B l o o d Group yp

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

1 2 5

toxin

1 2 5

Surfaces

1,372 2,816 8,731

2,175 3,662 11,572

3.0 12.0

1,407

1,387

10.0

1.5

419

436

222

5.0

cells

5% TCA + C h l o r o f o r m methanol (2:1) wash

cpm p e r 10

5% TCA wash

125 [ I ] L e c t i n o r T o x i n Bound

I J T o x i n t o IMR-32 C e l l

248

1 2 5

2.5

yg/ml

Concentration

I ] L e c t i n and [

125 The s p e c i f i c a c t i v i t i e s o f I - l a b e l e d D o l i c h o s b i f l o r u s l e c t i n and c h o l e r a t o x i n were 1.12 x 10^ cpm and 3.1 x 10^ cpm p e r mg o f p r o t e i n , r e s p e c t i v e l y .

Cholera

biflorus

I]Lectin/Toxin

Dolichos

[

Binding of [

Table VII

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

1 2 5

60

80

o f U l e x europeus

33

40

activity

14

IMR-32

6

cells

110

61

42

NS-20

116

44

14

NIE-115

Mouse Neuroblastoma

Surfaces

125 6 [ I ] l e c t i n was 0.5 x 10 cpm p e r mg o f

ng/10

Human Neuroblastoma

125 [ I ] L e c t i n B i n d i n g t o Neuroblastoma C e l l

20

yg/ml

I ] L e c t i n Added

The s p e c i f i c protein.

[

U l e x Europeus

Table VIII

216

46

36

N-18

CELL SURFACE

206

GLYCOLIPIDS

TIME (hre)

Figure 3. Effect of chemical differentiating agents on radioactive Bandeiraea simplicifolia [ I]lectin binding to mouse neuroblastoma (NltL-115) cell surfaces 125

Falcon T-flasks containing 104CI cells (1.5-2.5 X 10 cells per flask) were incubated with nothing (%), 4 fiM BrdUrd (O), 4 /xM 6-mercaptoguanosine(A),or 1 fig Cytochalasin-B (40) per ml of DMEM containing 10% fetal bovine serum (GIBCO) for the indicated periods. Binding of B. simplicifolia [ I]lectin (9.0 X 10 cpm per mg protein) to cell surfaces of each flask was studied according to the method described in text except that 20 fig of lectin/3mL of serum-free DMEM was used and [ I]lectin-bound cells were harvested from each flask in 5.0 mL of PBS. 6

125

6

u5

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

BASU ET AL.

Lymphomas

and Neuroblastoma

Cells

207

Figure 4. Phase contrast micrographs of 104CI guinea pig tumor cells grown in culture (X100) (a): Control cells grown in 250-mL Falcon T-flask as described in text, (b): Cells incubated with GMl ganglioside (10 ng/mL) as described in text, (c): Cells incubated 30 min at 37°C with cholera toxin (5.0 fig/mL) in serum-free RPMI-1640 medium, (d): Cells incubated 30 min at 37° C with cholera toxin after treatment with GMl ganglioside.

Figure 5.

Phase contrast micrographs of 104CI guinea pig tumor cells grown in culture

(a): Control cells grown in Falcon T-flasks (75 cm ) (see text), (b): Cells incubated 25 min at 37°C with Dolichos biflorus [ l]lectin (12 fig/mL) is RPMI-1640. (c): Cells incubated with lectin as in (b), then washed with PBS (2 X WmL). 2

125

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

208

CELL SURFACE

GLYCOLIPIDS

stream as f r e e , nonadhesive c e l l s c a r r y i n g s p e c i f i c a n t i g e n - b i n d i n g r e c e p t o r s on t h e i r s u r f a c e s . The mouse embryonic thymus has 6 - p o s i t i v e lymphocytes by day 12 o f g e s t a t i o n ; by day 15 the embryonic s p l e e n c o n t a i n s b o t h I g - p o s i t i v e and 6 - p o s i t i v e c e l l s . However t h e s e f e t a l s p l e e n c e l l s do not y i e l d antibody-producing c e l l s when grown i n c u l t u r e . I t i s believed that a n t i gen s e l e c t s programmed c e l l s i n the a d u l t a n i m a l , b u t the mechanism o f e x p r e s s i o n o f t h e s e programmed c e l l s i s not known. I t i s a l s o not known whether a s p e c i f i c c l a s s o f glycoconjugate i s expressed during development o f B- o r T-lymphocytes. Our p r e l i m i n a r y i n v e s t i g a t i o n i n t o the b i o s y n t h e s i s o f b l o o d g r o u p - r e l a t e d g l y c o s p h i n g o l i p i d s i n mouse l y m p h o r e t i c u l a r tumors a t d i f f e r e n t s t a g e s ( F i g . 2) may answer some o f t h e s e questions. M a k i t a and h i s co-workers have r e p o r t e d enhanced a c t i v i t i e ferases (involved i i n hamster thymuses b e a r i n g advanced s t a g e s o f lymphoma growth (66). On t h e ~ F a s i s o f [ I ] l e c t i n and [ I ] t o x i n bindi n g t o tumor c e l l s u r f a c e s we proposed (!) the e x i s t ence o f a - G a l N A c - l i n k e d (Forssman l i k e ) and G M l - l i k e r e c e p t o r s ( g l y c o l i p i d o r g l y c o p r o t e i n ) on the s u r f a c e s o f human n e u r o b l a s t o m a IMR-32 and g u i n e a p i g tumor c e l l s (104C1). E x o g e n o u s l y added Forssman g l y c o l i p i d o r GM1 g a n g l i o s i d e i n c r e a s e d b i n d i n g o f D o l i c h o s biflorus [l 5i]lectin and [ 1 I ] c h o l e r a t o x i n ( 7 ) , r e s p e c t i v e l y , i n a d d i t i o n t o the marked m o r p h o l o g i c a l changes o f t h e s e tumor c e l l s ( F i g s . 4 and 5 ) . A t t h i s s t a g e i t i s m e r e l y s p e c u l a t i v e t o propose t h a t a n t i g e n i c g l y c o l i p i d s (Forssman h a p t e n , n L c O s e ^ e r , o r nLcOsesCer) o r a c i d i c g l y c o l i p i d s (GM3, GM1, GMlb, o r GMlb(GlcNAc)) d e t e c t e d on tumor c e l l s u r f a c e s p l a y some r o l e i n c e l l growth and b e h a v i o r . I t i s possible that the s p e c i f i c p r o t e i n s i n v o l v e d i n growth r e g u l a t i o n (e.g;. , c h o l e r a t o x i n - m e d i a t e d a d e n y l a t e c y c l a s e a c t i v a t i o n (61) o r DNA r e p l i c a t i o n (31) ) a r e a t t a c h e d t o tumor c e l l s u r f a c e s t h r o u g h s p e c i f i c t e r m i n a l sugar residues of glycosphingolipids or glycoproteins. 1 2 5

2

1 2 5

2 5

Acknowledgments T h i s work was s u p p o r t e d by U.S. P u b l i c H e a l t h G r a n t s NS-09541 and CA-14764 and a g r a n t - i n - a i d from M i l e s L a b o r a t o r i e s , I n c . , E l k h a r t , I n d i a n a t o S.B. We would l i k e t o thank Dr. P r a b i r B h a t t a c h a r y a , D r . K a t h l e e n A. P r e s p e r , Dr. J o s e p h R. M o s k a l , Mr. A l e x V u c k o v i c , and Mr. W i l l i a m G. Shanabruch f o r h e l p i n the l e c t i n b i n d i n g s t u d i e s and c e l l c u l t u r e work.

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

12.

BASU ET AL.

Lymphomas and Neuroblastoma Cells

209

Footnotes ^IUPAC-IUB Commission on Biochemical Nomenclature (1976) "Enzyme Nomenclature" (and amendments thereto) Biochim. Biophys. Acta/ 429, 1-45. ^Chien, J. L . , Basu, M . , Basu, S. and Stoffyn, P . , manuscript in preparation. 3 Basu, M . , Basu, S. and Stoffyn, P . , manuscript in preparation. 4 Presper, K. A . , Basu, M. and Basu, S., manuscript in preparation. Literature Cited 1. IUPAC-IUB Commissio "The Nomenclature of Lipids", Lipids, 1977, 12, 455-468. 2. Hakomori, S. I. Biochem. Biophys. Acta, 1975, 417. 55-89. 3. Yamakawa, T . ; Nagai, Y. Trend Biochem. S c i . , 1978, 3, 128-131. 4. Gahmberg, C. G . ; Hakomori, S. I. Proc. Nat. Acad. Sci. U.S.A., 1973, 70, 3329-3333. 5. Lingwood, C. A . ; Hakomori, S. I. Exptl. Cell Res., 1977, 108, 385-391. 6. Gahmberg, C. G . ; Kiehn, D.; Hakomori, S. I. Nature, 1974, 248, 413-415. 7. Basu, M.; Basu, S.; Shanabruch, W. G . ; Moskal, J. R.; Evans, C. H. Biochem. Biophys. Res. Commun., 1976, 71, 385-392. 8. Baumann, H . ; Nudelman, E.; Watanabe, K . ; Hakomori, S. Cancer Res., 1979, 39, 2637-2643. 9. Steiner, S.; Melnick, J. L.; K i t , S.; Somers, K. D. Nature, 1974, 248, 682-684. 10. Hakomori, S. I. Prog. Biochem. Pharmacol., 1975, 10, 167-196. 11. Handa, S. Japan J. Exp. Med., 1963, 33, 347-360. 12. Hakomori, S. I . ; Watanabe, K. "Glycolipid Analysis", Witting, L . A., E d . ; American O i l Chemists' Society, Champaign, Ill., 1976, p. 1347. 13. Gardas, A . ; Kosceilak, J. Eur. J. Biochem., 1973, 32, 178-187. 14. Ando, S.; Yamakawa, T. J. Biochem., 1973, 73, 387-396. 15. Hakomori, S. I . ; Strycharz, G. D. Biochemistry, 1968, 7, 1279-1285.

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

210

10,

CELL SURFACE GLYCOLIPIDS

16. Naiki, M.; Fong, J.; Ledeen, R.; Marcus, D. M. Biochemistry, 1975, 14, 4831-4837. 17. Watanabe, K . ; Hakomori, S. I . ; Childs, R. A . ; F e i z i , T. J. B i o l . Chem., 1979, 254, 3221-3228. 18. Gahmberg, C. G . ; Hakomori, S. I. J. B i o l . Chem., 1975, 250, 2438-2446. 19. Sundsmo, J. S.; Hakomori, S. I. Biochem. Biophys. Res. Commun., 1976, 68, 799-806. 20. Ando, S.; Kon, K . ; Isobe, M.; Yamakawa, T. J. Biochem., 1973, 73, 893-895. 21. Joseph, K. C.; Gockerman, J. P. Biochem. Biophys. Res. Commun., 1975, 65, 146-152. 22. Basu, M.; Presper, K. A . ; Basu, S.; Hoffman, L . M . ; Brooks, S. E. Proc. Nat. Acad. S c i . , U.S.A., 1979, 76, 4270-4274. 23. Potter, M. in "Mosbache Rajewsky, K . , Eds. 1976; p. 141-172. 24. Mathieson, B. J.; Campbell, P. S.; Potter, M . ; Asosky, R. J. Exptl. Med., 1978, 147, 1267-1279. 25. Abelson, H. T . ; Rabstein, L. S. Cancer Res., 1970, 30, 2208-2212; i b i d . , 2213-2222. 26. Potter, M.; Sklar, M, D.; Rowe, W. P. Science, 1973, 182, 592-594. 27. Rowe, W. P. Cancer Res., 1973, 33, 3061-3068. 28. Kirschbaum, A . ; Liebelt, A. G. Cancer Res., 1955, 689-692. 29. Moloney, J. R., Federation Proc., 1962, 21, 19-31. 30. Basu, S.; Moskal, J. R.; Gardner, D. A. in "Ganglioside Function: Biochemical and Pharmacological Implications", Porcellati, G . ; Ceccareli, G.; Tettamanti, G . , Eds.; Plenum Press, New York, 1976; V o l . 71, p. 45-63. 31. Bhattacharya, P.; Simet, I . ; Basu, S. Proc. Nat. Acad. S c i . , U.S.A., 1979, 76, 2218-2221. 32. Basu, S.; Basu, M.; Chien, J. L . J. B i o l . Chem., 1975, 250, 2956-2962. 33. Chien, J. L . Ph.D. Thesis, University of Notre Dame, Notre Dame, Indiana, 1975. 34. Svennerholm, L . in "Comprehensive Biochemistry", Florkin, M . ; Stolz, E . H . , Eds.; Elsevier, Amsterdam , 1969; Vol. 18, p. 201-227. 35. Basu, M . ; Basu, S. J. B i o l . Chem., 1972, 247, 1489-1495. 36. Basu, M. D.Sc. Thesis, University of Calcutta, Calcutta, India, 1974. 37. Moskal, J. R. Ph.D. Thesis, University of Notre Dame, Notre Dame, Indiana, 1977. 38. Basu, M . ; Basu, S. J. B i o l . Chem., 1973, 248, 1700-1706.

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

12. BASU ET AL.

Lymphomas and Neuroblastoma Cells211

39. L i , Y. T.; Li, S. C. Methods Enzymol., 1972, 28, 714-720. 40. Basu, S.; Basu, M.; Moskal, J. R.; Chien, J. L.; Gardner, D. A. in "Glycolipid Methodology", Witting, L. A . , Ed.; American Oil Chemists Society Press, Champaign, Ill., 1976; p. 123-139. 41. Basu, S. Ph.D. Thesis, University of Michigan, Ann Arbor, Michigan, 1966. 42. Bjorndal, H.; Hellerqvist, C. G.; Linderberg, B.; Svensson, S. Angew. Chem. Int. Ed. Engl., 1970, 9, 610-619. 43. Basu, S.; Basu, M.; Chien, J. L . ; Presper, K. A. in "Ganglioside Structure and Function", Svennerholm, L . ; Mandel, P., Eds.; Plenum Press, New York and London; 1979 in press 44. Lowry, O. H.; Rosenbrough Randall, R. J. J. , , , 45. Cuatrecasas, P. Biochemistry, 1973, 12, 3567-3577. 46. Etzler, M. E.; Rabat, E. A. Biochemistry, 1970, 9, 869-877. 47. David, G. S.; Reisfeld, R. A. Biochemistry, 1974, 13, 1014-1021. 48. Hays, C. E . ; Goldstein, I. J. Anal. Biochem., 1975, 67, 580-584. 49. Basu, S.; Kaufman, B.; Roseman, S. J. Biol. Chem., 1965, 240, 4115-4117. 50. Kaufman, B.; Basu, S.; Roseman, S. J. Biol. Chem., 1968, 243, 5804-5806. 51. Kaufman, B.; Basu, S.; Roseman, S. Methods Enzymol., 1966, 8, 365-368. 52. Stoffyn, A.; Stoffyn, P.; Yip, M. C. M. Biochim. Biophys. Acta, 1975, 409, 97-103. 53. Basu, M.; Moskal, J. R.; Gardner, D. A.; Basu, S. Biochem. Biophys. Res. Commun., 1975, 66, 13801388. 54. Moskal, J. R.; Gardner, D. A.; Basu, S. Biochem. Biophys. Res. Commun., 1974, 61, 751-758. 55. Presper, K. A.; Basu, M.; Basu, S. Proc. Nat. Acad. Sci., U.S.A., 1978, 75, 289-293. 56. Rearick, J. I.; Saddler, J. E . ; Paulson, J. C.; Hill, R. L. J. Biol. Chem., 1979, 254, 4444-4451. 57. Hirabayashi, Y.; Taki, T.; Matsumoto, M. FEBS Lett., 1979, 100, 253-257. 58. Pacuszka, T.; Kosceilak, J. Eur. J. Biochem., 1976, 64, 499-506. 59. Taki, T.; Hirabayashi, Y.; Matsumoto, M.; Kojima, K. Biochim. Biophys. Acta, 1979, 572, 105-112. 60. Presper, K. A . , Ph.D. Thesis, University of Notre Dame, Notre Dame, Indiana, 1979. 61. Bauer, C. H.; Kottgen, E . ; Reutter, W. Biochem. Biophys. Res. Commun., 1977, 76, 488-494.

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62. Ip, C.; Das, T. Cancer Res., 1978, 38, 723-728. 63. Khilanani, P.; Chou, T. H.; Lomen, P. L . ; Kessel, D. Cancer Res., 1977, 37, 2557-2559. 64. Chatterjee, S. K.; Bhattacharya, M.; Barlow, J. J. Cancer Res., 1979, 39, 1943-1951. 65. Tonegawa, Y.; Hakomori, S. I. Biochem. Biophys. Res. Commun., 1977, 76, 9-17. 66. Ishibashi, T.; Atsuta, T.; Makita, A. J. Nat. Cancer Inst., 1975, 55, 1433-1436. 67. O'Keefe, R.; Cuatrecasas, P. J. Membrane Biol., 1978, 42, 61-79. RECEIVED

December 10, 1979.

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

13 Altered Glycolipids of CHO Cells Resistant to Wheat Germ Agglutinin PAMELA STANLEY Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY 10461

In recent years thi characterized a variet to different plant lectins (1,2). Mutants which fall into three different genetic complementation groups have been selected by virtue of their resistance to the cytotoxicity of wheat germ agglutinin (WGA). One of the mutants which may be selected with WGA falls into complementation group I and, if isolated from a WGA selection, it is termed Wga . Mutants in complementation group I have been shown to lack a specific N-acetylglucosaminyltransferase activity which appears to provide the biochemical basis of lectin resistance in this genotype (see 3). The other previously described Wga mutants (termed Wga and Wga ) fall into complementation groups II and III respectively, and have been shown to possess decreased sialylation of glycoproteins and the ganglioside GM at the cell surface (4,5). However, an enzymic basis for these genotypes has not been uncovered. A fourth type of Wga CHO cell mutant has now been isolated (Stanley, manuscript in preparation). This mutant is more highly resistant to WGA than the previously described mutants and it has been shown to belong to a new complementation group (group VIII). In this paper, the glycolipids of Wga cells are compared with those of parental CHO cells and the other Wga CHO cell mutants. RI

R

IR I

RIII

3

R

RVIII

R

Materials and Methods Alpha medium (containing ribonucleosides and deoxyribonucleosides) and fetal calf serum (FCS) were obtained from Grand Island Biological Co., U.S.A.. Reagent grade chloroform, methanol and hydrochloric acid were obtained from Fisher Scientific Co., U.S.A. and redistilled before use. Pre-coated silica gel 60 plates (0.25mm) were obtained from E.M. Laboratories, Germany. Alphanaphthol and resorcinol were obtained from Fisher Scientific Co., U.S.A. Resorcinol was twice recrystallized before use. Dowex 1 x 4 (100-200 mesh) chloride form was obtained from Bio Rad Laboratories, U.S.A. and converted to the acetate form according to 0-8412-0556-6/80/ 47-128-213$5.00/ 0 © 1980 A m e r i c a n C h e m i c a l S o c i e t y

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

CELL SURFACE

214

GLYCOLIPIDS

the manufacturers i n s t r u c t i o n s . Neuraminidase of V.cholerae was obtained from Calbiochem, U.S.A. N-acetyl (4,5,6,7,8,9-l^C) neuraminic a c i d was obtained from Amersham, U.S.A. A l l other chemicals were reagent grade. Purif i e d g l y c o l i p i d standards and reagents f o r GLC a n a l y s i s were kind g i f t s from Dr. Samar Kundu, A l b e r t E i n s t e i n C o l l e g e . C e l l Culture. C e l l s were c u l t u r e d i n alpha medium 10% FCS a t 37° i n suspens i o n . The experiments reported were performed with the f o l l o w i n g c e l l l i n e s : Gat"2 (a glycine-adenosine-thymidine r e q u i r i n g auxotroph - the parent from which each o f the mutants was d e r i v e d ) ; Gat"2Wga lN; Gat~2Wga 4C; Gat"2Wga 6F; and G a t - 2 W g a l-3. The nomenclature of these l i n e s i s s i m p l i f i e d i n the t e x t and f i g u r e s to P f o r p a r e n t a l (Gat~2) and to Wga Wga Wga and Wga V I I I f Rl

RlI

RlII

R v l I I

Rl

o

r

Rl1

1

1

1

r e S

Extraction of Glycolipids. Exponentially-growing c e l l s (^2-3x10^) were washed twice with 50-100 volumes of phosphate b u f f e r e d s a l i n e (PBS). A f t e r resuspens i o n i n 50ml PBS, a l i q u o t s were taken f o r c e l l counting and f o r p r o t e i n determination. The c e l l s were c e n t r i f u g e d , resuspended i n 40ml lOmM T r i s - H C l pH 7-4 and c e n t r i f u g e d a t 2000 rpm at 4° i n an I n t e r n a t i o n a l PR2 c e n t r i f u g e . The p e l l e t was e x t r a c t e d with 20 volumes r e d i s t i l l e d chloroform-methanol (C:M) 2:1 by mixing 2 min at low speed i n a Waring blender. The mixture was f i l t e r e d through a s i n t e r e d g l a s s f u n n e l and the residue subsequently e x t r a c t e d with 10 volumes C:M (1:2) - based on o r i g i n a l c e l l p e l l e t volume. The combined f i l t r a t e s were rotoevaporated, r e d i s s o l v e d i n C:M (1:1) at 10** c e l l e q u i v a l e n t s per ml and stored a t -20°. C e l l s which were t r e a t e d with neuraminidase p r i o r to l i p i d e x t r a c t i o n were washed twice i n PBS and resuspended a t 10° c e l l s per ml i n PBS c o n t a i n i n g 50 u n i t s neuraminidase per ml o r i n PBS alone ( c o n t r o l ) . The c e l l suspensions were incubated 5 min at 37°, c e n t r i f u g e d a t 1200 rpm f o r 10 min a t 4° i n an I n t e r n a t i o n a l PR2 and washed once with c o l d PBS. The c e l l s were resuspended i n hypotonic T r i s - H C l , c e n t r i f u g e d and e x t r a c t e d with chloroformmethanol i n the manner d e s c r i b e d above. Thin Layer Chromatography. L i p i d e x t r a c t s (0.3ml) were d r i e d under n i t r o g e n , resuspended i n vL5ul C:M (1:1) and spotted on a c t i v a t e d s i l i c a g e l 60 p l a t e s with p u r i f i e d g l y c o l i p i d standards GM3, GM2, l a c t o s y l c e r a m i d e (LC) and glucosylceramide (GC) c o n t a i n i n g VLOyg s i a l i c a c i d each. L i p i d e x t r a c t s t r e a t e d with neuraminidase were d r i e d under N2 and incubated a t 37° with 50 y l (25 u n i t s ) V.cholerae neuraminidase. A f t e r 16 h r , 1 ml C:M (2:1) was added, the samples were incubated

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

STANLEY

13.

Altered

Glycolipids

of CHO

Cells

215

15 min at 23° and c e n t r i f u g e d at 3000 rpm f o r 15 min i n a PR2 I n t e r n a t i o n a l c e n t r i f u g e . The supernatant was d r i e d under N£, r e d i s s o l v e d i n ^15 y l C:M (1:1) and spotted on a c t i v a t e d TLC p l a t e s . P l a t e s were developed by ascending chromatography i n chloroform: methanol:0.02% CaCl2 (60:40:9). Dried p l a t e s were subsequently stained by a-naphthol/sulphuric a c i d to detect carbohydrate or r e s o r c i n o l to detect s i a l i c a c i d - c o n t a i n i n g g l y c o l i p i d s (6,7). Determination

of S i a l i c A c i d i n L i p i d E x t r a c t s .

Free and l i p i d - b o u n d s i a l i c a c i d were determined i n each l i p i d e x t r a c t by the t h i o b a r b i t u r i c a c i d (TBA) method (8) f o l l o w i n g p a r t i a l p u r i f i c a t i o n of f r e e s i a l i c a c i d on Dowex 1x 4 (100-200 mesh) acetate form (9). L i p i d e x t r a c t s c o n t a i n i n g ^20-30yg s i a l i c a c i d were d r i e d under n i t r o g e n and resuspended i n 2ml H2O. About 10,000 cpm l ^ C s i a l i c a c i d was added to each sample The samples were r e d i s s o l v e d i n 1.0ml r e d i s t i l l e d methano termination of t o t a l s i a l i HC1 (by adding 1.0ml 0.1NHC1 to samples i n methanol) f o r 1 hr at 80°C. The pH of the hydrolyzed samples was adjusted to pH^8.0 with NaOH, and they were heated at 56° f o r 5 min to destroy lactones which might have formed during the h y d r o l y s i s . These preparations were f i l t e r e d through ^2cm g l a s s wool and subsequently loaded onto a 3 cm column of Dowex (acetate form). The sample eluate and a 4 ml wash of d i s t i l l e d deionized water (DDW) were c o l l e c t e d t o gether and a 1 ml a l i q u o t taken f o r s c i n t i l l a t i o n counting. The column was then e l u t e d with 7.5ml IN formic a c i d . These eluates were d r i e d on an evapomix (or l y o p h i l i z e d ) , r e c o n s t i t u t e d i n DDW and assayed f o r - ^ C - s i a l i c a c i d and u n l a b e l l e d s i a l i c a c i d by the TBA assay (performed on d u p l i c a t e or t r i p l i c a t e samples). The determination of f r e e s i a l i c a c i d i n the l i p i d e x t r a c t s was made on samples which had been d r i e d and r e c o n s t i t u t e d i n methanol (1.0ml), 0.1NHC1 and O.lNNaOH (1ml each, added together) and adjusted to pH 8.0. These samples were heated at 56° f o r 5 min and then passed over Dowex (acetate) e x a c t l y as described above. Detection of s i a l i c a c i d i n the form of C M P - s i a l i c a c i d was p r e l i m i n a r i l y examined using the assay of Kean and Roseman (10). Aqueous samples (0.2ml) were incubated with 30 y l c o l d sodium borohydride (100 mg/ml) with a g i t a t i o n f o r 15 min before the a d d i t i o n of 30yl acetone. A f t e r a f u r t h e r 15 min at room temperature, the samples were assayed f o r s i a l i c a c i d by the TBA method. Results The g l y c o l i p i d s of p a r e n t a l CHO c e l l s and the four d i f f e r e n t Wga CHO c e l l l i n e s were compared by t h i n - l a y e r chromatography of l i p i d e x t r a c t s . As described p r e v i o u s l y by t h i s l a b o r a t o r y (5) and by others (11,12), the major g l y c o l i p i d i n CHO c e l l s i s the g a n g l i o s i d e GM3 which has the s t r u c t u r e s i a l i c a c i d galactose i - 3 glucose-ceramide. This i s i n d i c a t e d i n F i g s . 1,3 and 4 by the co-migration of the major carbohydrate-containing band of R

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Figure 1.

GLYCOLIPIDS

Glycolipids of parental and Wga CHO cells stained with a-naphthol R

Lipid extracts from ~3 X 10 cells were compared by TLC (see Methods). Areas that stained blue after the a-naphthol/sulfuric acid are bracketed. The individual glycolipids in the mixture of purified glycolipid standards are also identified (Std). Cell extracts are identified as P (parental cell extract) and W , W W and W " for Wga ', Wga » Wga " and Wga . The major glycolipid band from parental cells ran between authentic GM and GM . However, in other experiments this band was shown to co-migrate with GM (see Figures 2-4). Bands that occur near the origin and precede GM have been shown to co-migrate with free sugars (sialic acid and neutral sugars) and do not correspond to known glycolipids. 7

1

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n t

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p a r e n t a l CHO c e l l s with authentic GM3 marker g l y c o l i p i d . Further e v i dence that t h i s band i s GM3 i s provided i n F i g s . 2 and 3. In F i g . 2 i t i s shown that neuraminidase treatment of a p a r e n t a l c e l l l i p i d e x t r a c t converts most of the band co-migrating with authentic GM3 to two bands which co-migrate with authentic lactosylceramide (LC;gal-glucceramide). The enzyme a l s o converts the GM3 standard to LC but does not change the p o s i t i o n of authentic GM2. I n F i g . 3 i t i s shown that the band i n CHO c e l l e x t r a c t s which co-migrates with GM3 a l s o s t a i n s with the r e s o r c i n o l reagent which i s s p e c i f i c f o r s i a l i c a c i d . Taken together, the data i n F i g s . 1-3 show that GM3 i s the major g l y c o l i p i d i n CHO c e l l s and that other g a n g l i o s i d e s , i f present, a r e i n small amounts not detected by these methods. The g l y c o l i p i d p a t t e r n of each Wga CHO mutant i s a l s o given i n F i g s . 1-3. Three of the four mutants e x h i b i t a l t e r e d g l y c o l i p i d s . As described p r e v i o u s l y (5), W g a H and W g a H I c e l l s possess low amounts of GM3 and increased amounts of LC compared with p a r e n t a l CHO c e l l s c o l i p i d pattern i d e n t i c a b i l i t y of GM3 on the surface of Wga l c e l l s to neuraminidase appears to be s i m i l a r to that o f p a r e n t a l CHO c e l l s ( F i g . 4). This i s p a r t i c u l a r l y i n t e r e s t i n g i n view of the f a c t that the m a j o r i t y of the " a c i d i c or "complex" asparagine-linked carbohydrate moieties of CHO membrane g l y c o p r o t e i n s are a l t e r e d i n Wga I c e l l s to a p a r t i a l l y - p r o c e s s e d intermediate of the s t r u c t u r e Manal,6 [Manal,3] - Manal,6 [Manal,3]-Man31,4GlcNAc$l,4 GlcNAc Asn peptide (13; E t c h i s o n and Summers, manuscript i n p r e p a r a t i o n ) . Thus i t might have been expected that s t e r i c p r o t e c t i o n of membrane GM3 molecules would be reduced a t the Wga I c e l l surface compared with p a r e n t a l CHO c e l l s . The data i n F i g s . 1-4 were obtained from mutants s e l e c t e d independently from those described i n our previous experiments (5) and demonstrate that the a l t e r e d g l y c o l i p i d patterns expressed by these mutants are a s t a b l e phenotypic property d i s t i n c t i v e of each genotype. The new Wga mutant (Wga VIII) a l s o e x h i b i t s a unique g l y c o l i p i d p a t t e r n ( F i g s . 1-3). Like W g a cells, Wga cells possess very low amounts of GMQ. However i n c o n t r a s t to W g a H (and Wga *) mutants, Wga * c e l l s e x h i b i t no concomitant i n crease i n the amounts of LC or GC v i s i b l e on the chromatograms. This suggests that these mutants may be making l e s s GM3 due to a defect p r i o r to the a d d i t i o n of the f i r s t glucose moiety to ceramide. Since the major g l y c o l i p i d of CHO c e l l s i s GM3 and s i n c e t h i s g a n g l i o s i d e contains one mole of s i a l i c a c i d per mole, the d i f ferences i n GM3 contents between the Wga CHO mutants may be quantitated by determining the amount of g l y c o s i d i c a l l y - b o u n d s i a l i c a c i d i n chloroformrmethanol c e l l e x t r a c t s . The r e s u l t s of such an a n a l y s i s a r e given i n Table T. P a r e n t a l and Wga CHO c e l l s c o n t a i n about 1.0 ug g l y c o s i d i c a l l y - b o u n d s i a l i c a c i d per mg c e l l p r o t e i n . L i p i d e x t r a c t s of these c e l l l i n e s a l s o c o n t a i n a small amount of f r e e s i a l i c a c i d (^O.lyg per mg c e l l p r o t e i n ) . Each o f t h e remaining Wga mutants e x h i b i t s decreased l e v e l s of R

R

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R

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CELL SURFACE

GLYCOLIPIDS

Figure 2. Glycolipids of parental and Wga CHO cells after neuraminidase treatment R

Extracts from ~3 X 10 cells and glycolipids GM and GM were treated with neuraminidase and analyzed by TLC in parallel with mixed glycolipid standards (see Methods). Plate was stained with a-naphtholl sulfuric acid; areas that turned blue are bracketed. 7

3

Figure 3.

2

Gangliosides of parental and Wga CHO cells R

Extracts from ~3 X 10 cells and a mixture of GM and GM were compared by TLC after staining with resorcinol. To improve sensitivity of technique, the spray was applied heavily, giving rise to some nonspecific staining. Only those bands that reproducibly stained the characteristic blue of gangliosides are bracketed. 7

S

2

Figure 4. Accessibility of GM in parental and Wga cell membranes to action of neuraminidase 3

Rl

Cells were washed, and half were treated with neuraminidase (see Methods). Other half were treated identically in the absence of enzyme. Lipid extracts later made from neuraminidase-treated and control samples in the usual way, and extracts from ~6 X 10 cells were compared by TLC. Plates stained with a-naphtholl sulfuric acid; blue areas are bracketed. (—): Controls; (+): treated with neuraminidase. 7

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219

Table I S i a l i c A c i d Contents of L i p i d E x t r a c t s of P a r e n t a l and Wga CHO C e l l s R

Cell Line

Bound S i a l i c Acid (ng per m protein Expt. 1

Ave % Bound S i a l i c Acid

Expt. 2

Expt. 1

Expt. 2

1030

910

125

90

1290

990

169

75

94

55

439

435

7%

R

420

560

73

80

50%

R

110

310

741

520

20%

Parental Wga

Free S i a l i c Acid

Rl

Wga

R l 1

Wga IH Wga VHI

117%

S i a l i c a c i d was p a r t i a l l y - p u r i f i e d from l i p i d e x t r a c t s a f t e r h y d r o l y s i s i n 0.05NHC1 ( t o t a l s i a l i c acid) or without h y d r o l y s i s ( f r e e s i a l i c acid) as described i n Methods. The amounts of g l y c o s i d i c a l l y - b o u n d s i a l i c a c i d ( t o t a l minus free) and f r e e s i a l i c a c i d were determined by the TBA assay. The hydrolysed samples were a l s o analyzed using gas l i q u i d chromatography by the method of Yu and Ledeen (15) and shown to possess e s s e n t i a l l y i d e n t i c a l amounts of t o t a l s i a l i c a c i d . However, the crude l i p i d e x t r a c t s were too impure t o make GLC the method of choice i n the absence of extens i v e p u r i f i c a t i o n of the g l y c o l i p i d s .

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R l

g l y c o s i d i c a l l y - b o u n d s i a l i c a c i d as would be p r e d i c t e d , Wga H c e l l s possess ^O.Syg bound s i a l i c a c i d per mg c e l l p r o t e i n (a 50% decrease compared with p a r e n t a l c e l l s ) but e x h i b i t s i m i l a r l e v e l s of f r e e s i a l i c a c i d to that found i n p a r e n t a l c e l l e x t r a c t s . W g a H and Wga VIII c e l l s e x h i b i t the most marked decrease i n bound s i a l i c a c i d having only approximately 10% of that found i n p a r e n t a l CHO c e l l s . S u r p r i s i n g l y , however, both these c e l l l i n e s possess high l e v e l s of f r e e s i a l i c a c i d (^4-8-fold the amounts found i n p a r e n t a l CHO c e l l s ) . The observation of high l e v e l s of f r e e s i a l i c a c i d i n l i p i d e x t r a c t s o f Wga and W g a c e l l s prompted us to examine whether i t might be s i a l i c a c i d complexed with the n u c l e o t i d e cyt i d i n e monophosphate ( i . e . CMP-sialic a c i d ) . C M P - s i a l i c a c i d i s detected as f r e e s i a l i c a c i d i n the TBA assay (10). This question was i n v e s t i g a t e d i n p r e l i m i n a r y e x p e r i ments by comparing the s e n s i t i v i t y of the f r e e s i a l i c a c i d i n W g a H and Wga VIH l i p i Roseman (10) have show f r e e s i a l i c a c i d while l e a v i n g C M P - s i a l i c a c i d i n t a c t and theref o r e capable of r e a c t i n g normally with the TBA reagent. I n f a c t , when the aqueous preparations of f r e e s i a l i c a c i d from Wga H and a V I I I c e l l e x t r a c t s were treated with borohydride, a l l r e a c t i v i t y with the TBA reagent was abolished (data not shown). Thus i t would appear that very l i t t l e ( i f any) of the TBA p o s i t i v e m a t e r i a l i n these preparations i s i n the form of C M P - s i a l i c a c i d . R

R

1

R

1

R v i 1 1

R

R

W g

Discussion Wheat germ a g g l u t i n i n (WGA) may be used to s e l e c t a t l e a s t four d i s t i n c t mutations i n CHO c e l l s , three of which e x h i b i t d i f f e r e n t a l t e r a t i o n s i n g l y c o l i p i d metabolism. I n t h i s paper we have shown that independent i s o l a t e s of the p r e v i o u s l y described mutants Wga , Wga II and W g a exhibit identical glycolipid patterns to other members of t h e i r r e s p e c t i v e complementation groups. In a d d i t i o n we have described a new Wga mutant ( W g a ) which e x h i b i t s yet another g l y c o l i p i d p a t t e r n . W g a cells synthesize reduced amounts of GM3 (the major g l y c o l i p i d i n CHO c e l l s ) and do not synthesize increased amounts of precursor molecules such as LC or GC. This mutant a l s o e x h i b i t s marked a l t e r a t i o n s i n r e s i s t a n c e t o a v a r i e t y of l e c t i n s (Stanley, manuscript i n preparation) suggestive o f extensive s t r u c t u r a l a l t e r a t i o n s i n the carbohydrate moieties of surface g l y c o p r o t e i n s (see 14). S t r u c t u r a l s t u d i e s of the g l y c o p r o t e i n s synthesized by W g a c e l l s a r e i n progress. Two Wga mutants s e l e c t e d from CHO c e l l s have been p a r t i a l l y c h a r a c t e r i z e d by B r i l e s et^ a l . (12). One of the Wga c e l l l i n e s described by these authors i s designated clone 1021 and i t exh i b i t s many p r o p e r t i e s s i m i l a r to those of W g a H c e l l s . The other mutant (clone 13) possesses c e r t a i n p r o p e r t i e s s i m i l a r to W RVIII c e l l s ( f o r example, a very high degree of r e s i s t a n c e to Rl

R

R l 1 1

R V I 1 1

R v i 1 1

R v l 1 1

R

R

ga

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WGA) but clone 13 cells appear to be blocked In the synthesis of GM3 at a step beyond the synthesis of GC, in contrast to Wga VIII cells, which do not appear to synthesize increased amounts of GC. Clearly, more biochemical and genetic studies are required to determine whether these Wga cell lines arise from identical or different mutations. Suffice to say at present that the partial characterization of these mutants has revealed the complexity of their respective phenotypes. Since each of the mutants Wga , Wga , Wga Hl and Wga may be isolated in a single step selection (1; Stanley, manuscript in preparation), it is likely that they all arise from single mutational events. Thus these mutants should prove invaluable in defining the biosynthetic links between glycoprotein and glycolipid metabolism in animal cells. Rl

Rl1

R

Rvl11

Acknowledgements The author wishes t Youkeles for excellent technica supporte by National Science Foundation grant No. PCM76-84293. The author is the recipient of an American Cancer Society Junior Faculty Award. Literature Cited 1. 2. 3.

4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

Stanley, P.; Caillibot, V.; Siminovitch, L. Cell, 1975, 6, 121. Stanley, P.; Siminovitch, L. Somatic Cell Genetics, 1977, 3, 391. Stanley, P. "Surface Carbohydrate Alterations of Mutant Mammalian Cells Selected for Resistance to Plant Lectins". Lennarz, W.J. Ed. In "Biochemistry of Proteoglycans and Glycoproteins", Plenum Publishing Co., New York, (in press). Robertson, M.A.; Etchison, J.R.; Robertson, J.S.; Summers, D.F.; Stanley, P. Cell, 1978, 13, 515. Stanley, P.; Sudo, T.; Carver, J.P. J. Cell Biol. (in press). Siakotos, A.N.; Rouser, G. J. Am. Oil Chem. Soc., 1965, 42, 913. Svennerholm, L. Biochim. Biophys. Acta., 1957, 24, 604. Aminoff, D. Biochem., 1961, 81, 384. Yogeeswaran, G.; Stein, B.S.; Sebastian, H. Cancer Research, 1978, 38, 1336. Kean, E.L.; Roseman, S. J. Biol. Chem., 1966, 241, 5643. Yogeeswaran, G.; Murray, R.K.; Wright, J.A. B.B.R.C., 1974, 56, 1010. Briles, E.B.; Li, E.; Kornfeld, S. J. Biol. Chem., 1977, 252, (3), 1107. L i , E.; Kornfeld, S. J. Biol. Chem., 1978, 253, 6426. Stanley, P.; Carver, J.P. Adv. in Exptl. Med. and Biol., 1977, 84, 265. Yu, R.K.; Ledeen, R.W. J. of Lipid Research, 1970, 11, 506.

RECEIVED

January 2, 1980.

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

14 Induction of Ganglioside Biosynthesis in Cultured Cells by Butyric Acid PETER H. FISHMAN and RICHARD C. HENNEBERRY National Institute of Neurological and Communicative Disorders and Stroke, The National Institutes of Health, Bethesda, MD 20205

The pleiotropic biochemica in culture by butyric aci (1). Although the initial reports on this subject appeared only six years ago, the number of published papers in this area has increased rapidly and is now approaching a hundred. The first reported observations of an effect of this fatty acid on cultured cells involved morphological changes. Ginsburg et al (2) noticed striking alterations in the shape of several lines of cultured cells including HeLa after exposure to butyrate. Independently, Wright (3) reported that butyrate caused morphological changes in Chinese hamster ovary (CHO) cells. Contrary to popular assumption, in neither of these studies was butyrate being used as a control for butyrylated derivatives of cyclic nucleotides. In 1974, our laboratories reported that the activity of CMP-sialic acid:lactosylceramide sialyl transferase and amount of its biosynthetic product ganglioside GM3 (N-acetylneuraminylgalactosylglucosylceramide) increased dramatically in butyrate-treated HeLa cells (4). More recently, we have found that the gangliosideGM1(galactosyl-N-acetyl-galactosaminyl-[N-acetylneuraminyl]galactosylglucosylceramide) is also increased in HeLa cells exposed to butyrate (5). GM1 has been implicated as the cell surface receptor for cholera toxin (6,7). In this article, we will review the effects of butyrate on cell morphology and ganglioside synthesis and provide conclusive evidence thatGM1is the cholera toxin receptor. In addition, we will describe some novel effects of cycloheximide on the turnover of membrane gangliosides. Effects of Butyrate on Cell Morphology In HeLa cells, the striking morphological alterations which follow exposure of the cells to butyrate are characterized by the extension of neurite-like processes (Fig. 1). No significant differences in the fine structure of the cell surface was observed by scanning electron micrography (Fig. 1). In addition to butyrate, propionate and pentanoate but not other homologous 0-8412-0556-6/ 80/ 47-128-223S5.00/ 0 © 1980 American Chemical Society In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

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f a t t y acids a l t e r e d HeLa morphology U , 8 ) . C y c l i c AMP o r i t s but y r y l a t e d d e r i v a t i v e s d i d not induce shape changes i n HeLa (2) although they d i d e f f e c t the morphology o f other c e l l s such as CHO ( 9 J . HeLa c e l l s responded to butyrate i n serum-free medium which by i t s e l f had no e f f e c t on c e l l shape ( 8 ) . In c o n t r a s t , neuroblastoma c e l l s extended long processes when deprived o f serum (10) but a l s o developed these n e u r i t e s when exposed to b u t y r a t e (11). The formation o f the n e u r i t e - l i k e processes appears to be dependent on assembly o f microtubules as c o l c h i c i n e and Colcemid, a n t i m i c r o t u b u l e drugs, prevented shape changes i n the presence o f b u t y r a t e (S £ dia. (figs. 1). C e l l s grown f o r c e r t a i n days i n c u l t u r e i n c o r p o r a t e more r a d i o a c t i v i t y into s u l f a t i d e than into cerebroside during the 16 h r e x p o s u r e t o / f % 7 - g a l a c t o s e . Some c a u t i o n m u s t b e e x e r cised i n attempting to correlate the data of Table I with the a c t u a l c o n c e n t r a t i o n of each l i p i d i n t h e c u l t u r e . The data r e f l e c t t h e balance between t h e s y n t h e s i s o f t h e l i p i d from e x o g e n o u s /_ % 7 - g a l a c t o s e a n d t h e c o n v e r s i o n o f t h e s y n t h e s i z e d l i p i d t o some o t h e r m e t a b o l i t e ( s ) o r degradation products(s) d u r i n g a 16 h r . p e r i o d . They do n o t n e c e s s a r i l y i n d i c a t e c o n c e n t r a t i o n e s p e c i a l l y s i n c e t h e amount o f e a c h l i p i d a c c u m u l a t e d f o r a n u m b e r o f d a y s i n c u l t u r e p r i o r t o t h e e x p o s u r e t o l~ 3H7g a l a c t o s e i s unknown. A l s o i t h a s n o t been e s t a b l i s h e d t h a t t h e e x t e r n a l l y d e r i v e d /_' ^ H 7 - g a l a c t o s e w i l l b e m e t a b o l i z e d t h e same as o r e q u i l i b r a t e d w i t h endogenously s y n t h e s i z e d galactose. V e r y l i t t l e o f (Z^W-galactose precursor was converted to a glucocerbroside o r t o a l a c t o s y l c e r a m i d e under these c u l t u r e c o n d i t i o n s ( 2 1 ) . Thea c t i v i t i e s o f t h e s u l f o t r a n s f e r a s e and g a l a c t o s y l t r a n s f e r a s e , enzymes r e s p o n s i b l e f o r t h e s y n t h e s i s of t h e m y e l i n - a s s o c i a t e d s u l f o - and g l y c o l i p i d s , were a l s o measured a t d i f f e r e n t days i n c u l t u r e ( f i g . 4 ) . The a c t i v i t i e s o f t h e s e enzymes i n homogenates o f c e l l s d e r i v e d f r o m t h e 15 d a y embryo were r e l a t i v e l y l o w a t 8 days b u t i n c r e a s e d u n t i l r e a c h i n g 3

n

t

n

e

m

e

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

5

CELL SURFACE

308

Figure 3. Time course of incorporation of SOf into cerebroside sulfate (cerSO0 and sulfogalactosyl glycerol lipid (SGG-lipid) in dissociated brain cells from 15-day mouse embryos grown 19 days in culture

GLYCOLIPIDS

35

TIME (HOURS)

TABLE I I n c o r p o r a t i o n of dissociated brain c e l l culture.

Days i n Culture

Cer

MGD

SG-Ceram

SGG-Lipid

CPM/mg Protein/16 h r s . 5

275

58

222

69

9

373

59

279

87

15

321

73

484

125

20

1441

322

2537

650

25

515

269

3662

339

28

876

346

6214

615

34

2267

824

19067

1732

41

6116

2143

24826

1400

50

201(192)

56

111(74)

45(41) 1667 (1883) 8(11)

749 (852)

9 59

Cer. ( c e r e b r o s i d e ) ; MGD (monogalactosyl d i a c y l g l y c e r o l ) ; SG-Ceram. ( s u l f o g a l a c t o s y l ceramide); and S G G - l i p i d ( s u l f o g a l a c t o s y l d i a c y l and monacylmonoalkylglycerol). Data i n parentheses are d u p l i c a t e v a l u e s . The c e l l s were grown i n the presence o f the [ % ] - g a l a c tose f o r 16 h r s . (21) B r a i n Research, Table I .

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

17.

PiERiNGER E T A L .

Myelination

in Brain

309

Cells

ι ο

DAYS INCULTURE

Brain Research Figure 4. Activities of cerebroside; PAPS sulfotransferase (CST); monogalactosyl diacylglycerol: PAPS sulfotransferase (MST); and hydroxy fatty acyl ceramide: UDP-galactose galactosyltransf erase (C Gal. T) in reconstituted homogenates of dissociated brain cells from 15-day embryonic mice grown for varying days in cul­ ture. Note that the activity of the galactosyltransferase is expressed on a different scale from that of the sulfotransferase (21).

TO­ DAYS IN CULTURE

Brain Research Figure 5.

Concentration of protein per flask of dissociated cells from 15-day embryonic mice at different days in culture (21)

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

310

CELL SURFACE

GLYCOLIPIDS

a peak a t about 43 days i n c u l t u r e ( f i g . 4). The peak a c t i v i t i e s appeal to be the same order of magnitude found i n preparations from f r e s h b r a i n (30, 29, 28, 5). The change i n a c t i v i t y of these enzymes with i n c r e a s i n g days i n c u l t u r e again c l o s e l y p a r a l l e l s the temporal development o f the r e l a t i v e r a t e of i n c o r p o r a t i o n o f ^ S 0 = d % - g a l a c t o s e from the media i n t o s u l f o - and g l y c o l i p i d s ( f i g . 1 and Table I ) . The concentration o f p r o t e i n i n the c u l t u r e s increased and then plateaued between about the 20th and 56th day i n v i t r o ( f i g . 5). There was no l o s s of p r o t e i n (suggesting the c o n t i n u ed v i a b i l i t y of the c e l l s ) from the s u r f a c e c u l t u r e s during the period i n which the myelin-associated biochemical parameters were most a c t i v e . The b r a i n c e l l s from 15 day embryonic mice undergo i n t e r e s t i n g morphological changes during growth i n v i t r o . The succession of changes with i n c r e a s i n g age i n c u l t u r e had been studied by scanning e l e c t r o day i n v i t r o (DIV) the produce extensive membranes and small aggregates ( f i g . 6a). By approximately the 15 DIV the c e l l aggregates have increased i n s i z e and have coalesced to form nests of c e l l s ( f i g . 6b). During t h i s stage the s u r f a c e i s i n i t i a l l y filamentous but by the 29 DIV appears smoother as i f covered with a m a r t r i x ( f i g . 6c). By the 43 DIV the nests of c e l l s and the f i b r o u s character of the s u r face have disappeared, apparently covered by a membrane- l i k e substance, l e a v i n g a r e l a t i v e l y smooth s u r f a c e ( f i g . 6d). Higher m a g n i f i c a t i o n s o f each o f the panels of f i g . 6 are shown i n f i g . 7 which f u r t h e r emphasizes the s t r i k i n g temporal morphol o g i c a l changes observed i n these c u l t u r e s . Figure 8 i l l u s t r a t e s the general features o f the c e l l nests and t h e i r morophological diversity. The key f e a t u r e of a l l nests o f c e l l groups concerns t h e i r a b i l i t y to extend membranes ( f i g 8b, d, e, f ) . However, the c e l l morphology v a r i e s from being round to f l a t t e n e d . Occasi o n a l l y a group o f c e l l s i s seen to produce t h i c k membrane ( f i g . 8c). The sequence of development o f the membranous m a t e r i a l observed i n c u l t u r e c o r r e l a t e s with the p r o g r e s s i o n o f a c t i v i t i e s of the myelin-associated parameters measured above. Support for the production o f a m y e l i n - l i k e membrane i n these surface adhering, primary c u l t u r e s have come from the s t u d i e s of Yavin and Yavin (25). Using transmission e l e c t r o n microscopy they demonstrated that s i m i l a r primary c u l t u r e s produced myelinated axons. The bimolecular myelin membrane o f the c u l t u r e d e r i v e d preparations appeared to be the same as i n v i v o derived preparations . The c u l t u r e system used i n t h i s study has proven s u i t a b l e for studying the r e g u l a t i o n , e s p e c i a l l y by hormones, of myelination i n v i t r o . I n i t i a l s t u d i e s (35-36), however, showed no e f f e c t o f 3, 5, 3 - t r i i o d o t h y r o n i n e (T-) on s u l f o l i p i d s y n t h e s i s by d i s s o c i a t e d b r a i n c e l l s grown on medium c o n t a i n i n g 20% c a l f a

n

T

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

PiERiNGER E T A L .

Myelination

in Brain Cells

311

Brain Research Figure 6. Microphotographs illustrating the morphological changes associated with different stages of the in vitro growth patterns for cell populations isolated from brains of 15-day old mouse embryos (21) (a): After four DIV a combination of membranous structure and cell aggregates form (X140). (b): After 15 DIV super aggregates develop in addition to nests of small cells (X420). (c): After 29 DIV, aggregates have become very large, and their surfaces are covered with matrix and single cells (X210). (d): By 43 DIV the surface of the aggregates is smooth and characterized by the absence of single cells (X210).

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

CELL SURFACE

GLYCOLIPIDS

Figure 7. Microphotographs illustrating the morphological features associated with changes in Figure 6; at higher magnification topographical detail is clearer. (a): Four DIV, note the membrane covering this aggregate (X700). (b): 15 DIV, note the filar nature of the surface (X1400). (c): 29 DIV, surface topography is less filamentous, cells are still readily visible (X700). (d): 43 DIV, surface topography is quite smooth, absence of filament and individual cells are not readily visible (X700).

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

17.

PiERiNGER ET A L .

Myelination in Brain Cells

313

serum. I t should be noted that the endogenous concentratiens of hormones and growth f a c t o r s i n the serum may be s u f f i c i e n t l y high (36) to preclude observing an e f f e c t by exogenous hormones. This c o n d i t i o n could not be d i f f e r e n t i a t e d from that i n which the added hormone i s completely nonassociated with the process being measured. The r o l e of t h y r o i d hormone may be t e s t e d best by using c a l f serum obtained from a thyroidectomized animal. Depressed a c t i v i t i e s observed i n the hypothyroid s t a t e should be r e s t o r e d by exogenous hormones. This experimental design has been used s u c c e s s f u l l y by Samuels, e_t a_l. (37) to study the e f f e c t of t h y r o i d hormone on the metabolism of p i t u i t a r y tumor c e l l line in culture. The hormone concentrations i n the normal and hypothyroid c a l f sera as determined by radioimmunoassay are given i n Table II. Both thyroxine (T^) and Τ 3 values are f a r below normal i n hypothyroid c a l f serum In the experiments d e s c r i b e d the growing b r a i n c e l l s i c a l f serum and hormone s u l f a t i d e s (cerebroside s u l f a t e and monogalactosyl d i a c y l g l y c e r o l ) was studied by using H/_^ "^S/O^ and [~ H7-galactose as the l a b e l ­ ed p r e c u r s o r s . The s y n t h e s i s of these l i p i d s has been used as an index f o r f o l l o w i n g m y e l i n a t i o n (11, 12, 2^ 4). Table I I I gives the r e s u l t s of an experiment i n which the e f f e c t of serum manipulation on s u l f o l i p i d s y n t h e s i s by d i s s o ­ c i a t e d b r a i n c e l l s was examined. The c e l l s i s o l a t e d from the embryonic mouse b r a i n were grown on the medium c o n t a i n i n g c a l f serum (20%) f o r 3 days by which time most of the c e l l s would have attached to the substratum. On the 4th day, the medium was r e p l a c e d by f r e s h medium c o n t a i n i n g c a l f serum, c a l f serum + Τβ(2 χ 10~8M), hypothyroid c a l f serum, or hypothyroid c a l f serum + ^ ( 1 3 ng/ml). C u l t u r e s were grown f o r another week and then l a b e l e d with 400 y C i H 7/ S704, f o r 16 h. The l i p i d s i s o l a t e d from the c u l t u r e s were analyzed by t h i n l a y e r chroma­ tography and the r a d i o a c t i v i t y was determined. As i s c l e a r from the r e s u l t s , when Τ 3 was added to the c u l t u r e s grown on media c o n t a i n i n g normal c a l f serum, h a r d l y any e f f e c t was d i s c e r n i b l e . On the other hand, presence of hypothyroid c a l f serum caused a r e d u c t i o n i n the s y n t h e s i s of s u l f o l i p i d s . T h i s i n h i b i t i o n could be reversed by i n c l u d i n g Τ 3 i n the d e f i c i e n t medium. In Table IV i s described the e f f e c t of hormone manipulations on myelin l i p i d s y n t h e s i s a t a l a t e r stage, namely, 11th day i n culture. The c u l t u r e s were exposed to the e f f e c t o r s f o r 3 days and then the s y n t h e s i s of g l y c o l i p i d s was followed by l a b e l i n g the c e l l s with H /~ 3 %7θ4 and / " ^ / - g a l a c t o s e . The t o t a l l i p i d e x t r a c t was analyzed f o r c e r e b r o s i d e s , monogalactosyl d i a c y l g l y ­ c e r o l , c e r e b r o s i d e s u l f a t e , and monogalactosyl d i a c y l g l y c e r o l sulfate. As expected, there was about a 3- to 4 - f o l d i n c r e a s e i n the r a t e of s y n t h e s i s of s u l f o l i p i d s on the 15th day as com­ pared to 10 days i n c u l t u r e . The s y n t h e s i s of a l l the four l i p i d c l a s s e s studied appears to be a f f e c t e d by t h y r o i d hormone l e v e l 3

2

35

2

2

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

314

Figure 8. Microphotographs illustrating the morphological features of cell groups/ nests representative of activities after nine DIV. Arrows point to the different membranous structures characteristic of cell nests, (a): X700; (b)-(f): χ2100.

Table I I Values of Thyroxine (T4) and T r i i o d o t h y r o n i n e (T3) i n normal and hypothyroid c a l f - s e r a (22)· Hormone Concentration T4, yg/ml

T3, ng/100 ml

C a l f serum

5.8

110

hypothyroid c a l f serum

1.2

3)calNAc(β, 1->4)gal (β, 1->4)glc. The acidic oligosaccharide is β - g l y c o s i d i c a l l y linked to ceramide, formed by a long chain fatty acid (primarily C 18:0) and a long chain, mainly unsaturated, base (C 18 and C 20) linked together by an amide bond.

0-8412-0556-6/80/ 47-128-321 $5.75/ 0 © 1980 American Chemical Society In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

322

CELL SURFACE

The the of

oligosaccharide portion

high

hydrophilicity

of gangliosides,

of the molecule, displays

interactions: hydrogen bonds

These

bonds

chains ules

of adjacent

than

within

important NMR

are more

role

studies

likely

and ion bonds

gangliosides

(or other

the same ganglioside

in any process

performed

binding

glycoconjugates) Thus

they

association.

molec­ play^gn The

et a l . ( 2 ) on ganglioside

— -in

micellar

this

form-

ganglioside

sialic

acid

explain

as oxygen

(with

galactosamine molecule.

lead to visualize rich

its carboxylic

and the terminal

surfaces

binding

involving

but also

galactose

The occurrence of these

G

C

KAtt

M ]

the cation

group),

sites).

the saccharide

molecule.

of ganglioside

by S i l l e r u d

responsible for

a double potentiality

(cation

to occur between

GLYCOLIPIDS

not only the

the N - a c e t y l -

residue

additional

sites of

present

oxygen

in

the

ligands may

why the affinit

that

exhibited by free

The

oxygen

rich

α -

surfaces

described

in ganglioside

G

à

J

are e x -

4

Mi pected ral

to be present

feature The

in all gangliosides

and to constitute

a gene-

of ganglioside chemistry.

apolar chains of the ceramide portion

of gangliosides are

responsible for the hydrophobic properties of gangliosides and for of

their

availability

sine

and the carbonyl

spread mutual with

association,

other

range

In the presence :

18-50

gliosides from

the

were

core

10 A(see

20 A

weight

10 ,

10

M.

-10

properties showed

and leads

in which

with

water.

molecule

groups

about

solutions

The literature

of gangliosides,

that

ganglioside

G

K i M

on the cylinder

30

(10),

that

of the

groups of

large

for the c r i t i c -

are in the range works

of S c h w a r z -

reporting

investigations

recently performed

A ,

head

micelles

values

for the recent

scattering

( hydration

tFje radius of

by the sugar

( 9 ) and of Formisano et a l . light

gives

structures of gan-

structures

at 3 7 ° C ,

In dilute aqueous

except

volume

the apolar chains radiate

the sugar

In these

is,

are formed.

Laser

chain

them to associate in

quantities of water

packed cylinder

( 4 )

M (4,5,6,7,8),

maj^ri et ^

of each

of an hydrophi lie

concentration (cmc) of gangliosides

rr^icellag -

with

l).

tendency for

association

molecules

and the annulus formed

Figure

molecular

of small

of the rods,

all ganglioside

lipid

properties

described

in contact

formation

would tend to

their

of approximately equal

% ) hexagonally

the center

surface

10

in the ganglioside

strong amphiphilic

water.

acid,

reducing

to promote

The

group of the sphingo-

( 3 ).

an hydrophobic portion

them

of the fatty

chains

and thus

molecules

presence

the 3-hydroxyl

oxygen

the two hydrocarbon

The and

al

to hydrophobic interactions.

an hydrogen bond between

a cmc

of

on the micellar

in our laboratory,

and G ^ ^ in the concentration range

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

18.

TETTAMANTI

ET

Nerve-Ending

AL.

Membranes

323

Figure 1. Dimensions of the saccharide and lipid portion of a ganglioside in a cylinder structure (adapted from Curatolo et al (4))

1

GM1

Figure 2. Laser light scattering of ganglioside G

TURBIDITY

•—• A—A ·-· Δ-Δ

40

ο 30

3x10"

2

CONCENTRATION, M

Mt

in aqueous solution

GM1 GDla GQlb GTIb

20 10

20

40

60

Figure 3. Physicochemical features of mixed aggregates of phosphatidylcholine, phosphatidylethanolamine (PE, used as surface marker), and gangliosides (G i, G , G τ ib, G ) at increasing propor­ tions of ganglioside. Highest value of the outer PE/total PE ratio corresponds to liposomes. Lowering of turbidity and concurrent enhancement of ratio indicate presence of micelles. "Break" point is in­ dicated as the "transition ganglioside/ phospholipid molar ratio." M

Dla

ο 20 40 60 GANGLIOSIDE IN THE MIXTURE MOLAR %

Q]b

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

CELL SURFACE

324 10

-10

Figure to our G

M,

2).

which

was

examined,

Thus the

cmc

should be

measurements t^e micelles,

doubles the

that

like

While

micelles,

low

( J_0 ).

are

formed

and

other

chains,

the

This (on

ellipsoids

weak bonds

Ganglioside

The earliest

These

authors

mixtures

ganglioside that

it

is

At

studied the

sediments

of

A

the

ratios

was

recently

technique

unilamellar

face

sided

sulfonic

proper

the

and till

on the

ity

molar

is

of

to

of

be

TNBS)

in the

micelles

saccharide

and L e s t e r

ganglioside-phospho-

value

of

solvent 0,

the

leads

(over

in our

by

4)

as

mixed

mixtures

laboratory.

phases

F o r this

integrity

al.

(used

the

(

12

as

liposomes.

The

shown

turbidity

In fact

remains are

sur-

dissolved with

type pf

the

aggre-

ratio

the

level

and the

unchanged

being formed

and increase

of

ratio

in Figure

the

gangliosides,

micelles

).

a

were mixed

residue

ganglioside/phospholipid

mixed

At

6-trinitrobenzene

employed.As

absence of

the

micelles.

of

ganglioside/phospholipid molar

to

low

phosphatidyl-

gangliosides

and sonicated.

sided phosphatidylethanolamine

of

at

indicating

Barenholtz et

with 2, 4,

Over

value

that

than 0.05)

and monitoring the

removed,

species

in the

decrease

ratio

undertaken

preparing

outer

that

ratios (lower

on ganglioside-phospholipid

and the various

ganglioside

same as

gradual

of

phases or

60%. by

the

investigation on gan-

done by H i l l

supernatant

study

revealed

at pH 7.

aggregation

the

to

very

phosphatidylcholine bilayer.

defined

depends on the

a certain

cess

is

hydrogen

adjacent

and phosphatidylethanolamine

organic

buffer

gate formed

between

behavior

liposomes described

marker

acid,

together,

are

less

more detailed

Phosphatidylcholine

a

into the

and ganglioside

present.

used

once

with the phosphatidylcholine,

incorporated

interactions we

that

upon ultracentrifugation and observed

intermediate

are

associate

micellar structure.

was

ganglioside/phosphatidylcholine

choline

not

better,disk-

monomers

interactions)

systematic

interactions

ganglioside/phophatidylcholine

high

the

are

or,

interaction

Phospholipids.

lipid

established

which

may support

rapidly to

see

and

a value

This

(

According

the

A ,

micelles)

micelles

hydrophobic

M.

micelles

monomers

stability of

glioside-phospholipids ( JJ_ ).

of

are

enhancing the

60

interpreted assuming

basis

as. micelles

10

structures.

(rodlike

ganglioside

can be

is

ganglioside

dissociation of

the

than

c

molarity,

solutions

but prolate

present

hyo^rodynamic radi^s of

-10

in dilute

micelles.

form

10

exhibited by cylinder

idea that

spherical

at

are

lower

GLYCOLIPIDS

as

of

3 proturbid-

% of at about shown

aminogroups

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

18.

available for med

TNBS.

appears

to

be

side/phospholipid ratio",

varies

to

for

0.

10

acid do

G

content

not

larly

of

,

G_ , Tib

This

.

ecular

the

it:

from

to verify

was

(

in artificial

phospholipids, cooperative

at

the

the

glycophorin

on the

with

microbial

nin,

deal

with

exceeded

acid

the

range,

1 to

0. 2

for

supramol-

gangliosides

in phospholipid

of

the

Gangliosides

toxins,

that

general binding

(

of

the

monomeric for

not

All

Figure

yet

glyco-

Probably

these evidences that

proteins.

are

gangliosides

the

magnitude and/ agents.

A l l published

either pure or mixed, wheat

interactions.

germ aggluti-

Considering

studies,

which

the

abundant-

interactions pertain

gangliosides.

gangliosides

Of

cannot

protein-ganglioside been

immobilizaof

leading to a

by crosslinking

described

levels

glycoprotein,

effect.

4)

As

in

and d e c r e a -

addition

process

interferon,

monomeric

model

The

membrane

in these

for-

interactions, by

ganglioside

gangliosides,

used

toward

gangliosides

gangliosides,

bind

the

through the

to

"clusters",

easily

of

physiological

causes

see

hormones,

has

of

%

tendency

saccharide chains.

divalent cations

ganglioside-protein

molarity,

1.5

likely

crosslinking

layers,

interactions of

(

tendency

gangliosides.

lipid

mean

monomeric)

groups

them being enhanced

does

A

the

erythrocyte

of

(at physiological

mobility of

concentrations.

concentrations 10

the

mobility or

hypothesis

rather than to

not

gangliosides

The presence

in the

micellar

proteins.

cations

a micellar organization.

adjacent

increases

rich

on fluid

Proteins.

ly

G ^ sialic

particu-

liposomal

amounts of

themselves,

between

head

assembly

with

ganglioside

sugar

magnitude

stability of

studies

ions

takes part

tend to form, or

a

0.

show a measurable

interaction

ganglioside

more packed consistent

monovalent

from

low concentrations

among

bonds

this

,

a sialic

enhances

;

would give

terms)

ganglioside

lower

phorin,

at

to^iminuish.

further

tion

of

or Mg

crosslinking ses

even

hydrogen

+

,

G

determine

of

for the

.

\3_)

in molar

consequence

Ca

0.25

the physiological

dynamics of

interaction

layerg tends of

"transition

from

while

for-

ganglio-

by decreasing

phosphatidylcholine bilayers

pHs) gangliosides,

mation of

being

the

divalent cations,

for

presence ,

or

of

made by usin

E P R signals

that

ions

Ca

the

rises

ratio",

0. 45

Ca

of

transition,

it

start

value

gangliosides:

within

0. 2 ^ o

in the

absence

micelles

In addition,

"transition

organization

attempt

for

words

gangliosides.

means that

which, in the

layers

ratio

In other

which

325

The c r i t i c a l

different

at concentrations

double

An

-

the

Membranes

at

sharp.

molar

in||uence the

Ca

4

The point

quite

with

^ lib

almost

a

Nerve-Ending

TETTAMANTI ET A L .

worked out.

course

to

this

interact with (micellar and

In a recent

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

our

CELL SURFACE

326

investigation

on the

binding

of

G ^

ganglioside

with

GLYCOLIPIDS

bovine

serum

Mi albumin,

differential

fugation wed

studies,

that

at

UV

absorption,

associated

least

three

G

k

-albumin

J

fluorescence

and u l t r a c e n t r i -

with chromatographic evidences, complexes

are

formed,

sho-

dif-

M1 fering tion. ^M1

markedly

One form m

o

n

o

m

e

r

s

in their

is

molecular

result

The other

#

./albumin

the

ratio of

one

of

the

weight

and molecular

interaction

two complexes

ganglioside

between

are

conforma-

albumin and

characterized by a

micelle

per

albumin polypep-*

Ml tide chain:

one

other

which

from

one,

complex

hydrophobic

bumin been

micelles,

sides

with

monomers ces,

of

high

gangliosides to

the

sugar

the

apolar portion

for

the

surrounding

be

of

nervous

to

only

to

nerve

to c a r r y

(

be

acceptable

likely

endings

large

a 5-fold

The

all

cell

labelled

form,

In all

exposed

on

molecule

to

ganglio-

inspect

at

Ganglioside to

all

surfa-

these processes

surface,

to

be

indicating

responsible

(

( JJ3 ).

sialic

either

acid from

intrinsic

to

the

plasma

was

of

but

in the

to have a

nervous

sialidase

membranes,

side

similar-

to expose (

Y7J*

(neuraminidase)

or

makes

on these membranes

sialylglycoconjugates membrane

tis-

synaptosomal

appear

membrane

it

shown

separation procedure,

1_9,_20 ) ,

outer

ver-

( 25-30 nmoles of

of

gangliosides

synaptosomal

membranes c a r r y

remove

included-

the

neuronal

the portion

( J J > ),

on the yield

all

displayed by

membrane)

than elsewhere

glycolipids (

chains to

all

gangliosides

in a conventional

the

is

The

mg protein)

based

of

membranes of

However

synaptosomal

enrichment of

surface

in the membranes

membranes )

content

the neurons.

much higher

gangliosides

Synaptosomal to

Ul ).

in the plasma

amounts of

An evaluation,

oligosaccharide

able

(

ganglioside

gangliosides

content

V7_) .

.

to the

result

ganglioside-al-

adhere potentially

synaptosomal

present

cells,

membranes obtained

to

of

and sialyltransferase

endings

are

contains

ganglioside

18_)

which

enabled

monomeric

ganglioside

bound N-acetylneuraminic acid /

ly

availability

included

appear

the

The highest

tissue

surrounding

(

mixed

radioactivity

capable

sialidase

nerve

cells.

membrane

(

actually

in the

walls

groups

Gangliosides tebrate

sue

and i r r e v e r s i b l y

complexes,

interaction.

Gangliosides,

not

The

specific

and plastic

head

two

rearranged

gangliosides.

appear

glass

slowly

These

interactions are

a very

behavior

polymerizes

a dimer.

in whic

extensively

Monomeric

the

is

21

their )·

activity,

-gangliosides

added ( _22,

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

23, *2A) ·

18.

In parallel sent in

Nerve-Ending

TETTAMANTi E T A L .

with

along

the

the

gangliosides

all

neuron

synaptosomal

occurrence

of

a

sialidase.

location branes paratus the

In fact

site for

to

be

:

experimental no

to

endings.

previous on

5

the

red

to

the

no

,

lowered

(d)

as

specific

of

of

the

was

than that

the main

Golgi

)

memap-

approached

strategy of

the Golgi to

same enrichment

to

complex

occur

inside

nerve

ending

on removing

when preparing

which came out,

alsewhere

Then,

as

confirmed

by

(

26

of

obtained

(

).

As

)

shown

appeared,

,

the

and the

density

see

biochemical

-Ach-

showed

gradient

; of

(c)

low

membranes of

parallel

to

C

in the

This

Table this

I ): ex-

markedly

this

membrane reductase origin

), ;

gangliosides and

that

of

authentic

synaptosomal activity,

evidence, by other

nervous

),

absolute c o n -

other

and sialidase, This

and corroborated that

(b)

intracellular

sialy transferase

6.

;

NADPH-Cyt.

gangliosides

see

S'-nucleotidase very

same preparation of

in Figure

(

).

membrane markers

concentration of

sialidase

5

and compa-

activity;

endings

plasma

esterase,

activities

Figure

analyses

( LDH )

nerve

reductase,

hypothesis

28

ner-

perfecting

submitting

( 27,

a series

authentic

specific

The

exposed

with the

and efforts

shock

membranes

substantial

properties be

C

activity of

the

course

to

unruptured

specific

the

of

the

a highly homogeneous preparation

low contamination of

contains

consists

( 26

evidences

ending fraction,

plasma

membrane markers. branes

material

nerve

( NADH-Cyt.

enhancement the

described

activity of

tent,

of

that

of

plasma

the

up the

membranes

hypoosmotic

acetylcholine

a

recently

attention

light

submitted

presence

this proving

the

of

possibility for

lactate dehydrogenase

11

them

and

only

homogeneous.

to

membranes

when

qualifying

markers

been

treated

specific

ATP-ase,

We

be

ending preparation we obtained

fairly

starting

the

enhanced

our of

centrifugations,

"trapped

cludes

(

has

nerve

synaptosomal

(a)

Therefore

The preparation procedure

diffrential

of

the

difficulties

known to

In setting

pre-

synaptosomal

fragments

considered

Therefore

ending fraction

preparation

task.

cortex. we

we focused

hypoosmotically and

devoid of

assessment

in the

is

and cytoenzymatic

contamination

morphologically, nerve

The

is

be enriched

contai

methods,

Figure

).

activity

which

to

intracellula

on this

possible

( J22, 23

activity,

appears

apparatus

an easy brain

approach

endings.

membranes of Relying

ve

not

morphological

preparations

all

should be

is

has

nerve

,

much more technical

the Golgi

with calf

our

the

)

glycosyltransferases.

used

which

problem

sialidase ( 25

sialy(transferase

membranes encountered of

this

surface

membranes

327

Membranes

mem-

displaying

and bearing which should

proofs,

tissue

strongly

sialyltrans-

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

CELL SURFACE

GLYCOLIPIDS

Figure 5. Electron microscopic examination of the "nerve ending fraction" (Xl3,000) and of the "synaptosomal membrane fraction" (χ7150)

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

18.

Nerve-Ending

TETTAMANTi ET A L .

Membranes

TABLE

Biochemical the

sides

are

enzyme med C

characteristics

"Synaptosomal

expressed

substrate

tissue,

in

cases

all

as

the

"Nerve

min"

at

are

the

lower

ending fraction"

from

calf

brain.

International

37°C The

-

30°

data

for

1 0

%

of

of the

(

NADH-,

shown,

mean values

than +

Units

6

1 nmole and

referred

acid

;

transfor-

NADPH-Cyt

to

experiments;

mean

and of

Ganglio-

nmoles bound N-acetylneuraminic

in milli

and L D H ) .

fresh

I

f r a c t i o n " , obtained

activities

reductase

of

329

1 g starting

the

S. E .

was

values.

Nerv fraction

fraction

Parameter Activity (or

total

"Occluded" L D H

specific

4.4

ATP-ase

149

Ach-esterase

Activity

concentration)

1.0

0.

226.

(or total

67

0

0.

specific

003

161.

concentration)

0.O16

865.6

3. 83

18.4

3. 41

4.

5.4

3. 4

5 -nucleotidase

101.2

15.3

13. 2

71.

Gangliosides

171.6

26.

16. 83

90.5

0. 43

2.3

Neuraminidase

NADH-Cyt.

3.

C

165.

17

0

0.48

0

26.0

0. 024

0

34.2

f

Enrichment

3. 50

19.

1

65

3. 48

4.

79

0. 73

1

reductase

NADPH-Cyt.

C

15.

18

2.3

0. 316

1.7

0. 74

reductase

S i a l y l transferase

+

+

Sialyltransferase

119.9

activity

19.0

14.

expressed

as

1

76.3

c.p.m.

4.

min

^of

02

incub-

14 ation

using

C-NeuAc-CMP

and lactosylceramide

as

substrates

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

CELL SURFACE GLYCOLIPIDS

330

100

300

500 PROTEIN, pg

700

900

30 60 90 INCUBATION TIME, min

120

Figure 6. Effect of CMP-NeuAc concentration (V/S), of pH (V/pH), of enzymatic protein concentration (V/protein), and of incubation time (V/t) on the activity of synaptosomal membrane-bound sialyltransferase. Calf brain cortex. Acceptor substrates for sialyltransf erase: (jç) lactosylceramide; (f) desialylated fetuin; (φ) endogenous glycoprotein; (±) endogenous glycolipids.

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

ferase

has,

tosomal the of

Nerve-Ending

TETTAMANTI ET A L .

18.

as one of its sites

membranes .

biochemical

gangliosides

cycle

potentiality

is schematically

other

plasma

transmission. ization mes ion

capable

The

fact

transferase

A biochemical

local

fluctuations

ganglioside,

membranes sialidase support.

at

is shown

and

1/8-

very

were

since

reasonably

ratio

ring of

functionali-

in the nerve

ending and

has a consistent

has been of

not yet a s -

sialyltransferase

( 29, 3Q, 31 ) makes

organization of

membranes,

the specific in the brain

low range.

should

molar

the chemical

ratio,

non neural

of general

cells

value

concentration of different

as much

nature

of both

greater,

layer

l/4.

and

locaof the

This

discussed

molar

above.

phospholipids

the presence

This mem-

within a

asymmetrical

i.e.

ratio"

mat-

in p l a s -

gangliosides

varies

in the outer

can be accounted

of gangliosides

observed

of vertebrates.

animals

of the individual

membranes,

protein.

in this

for synaptosomal

Due to the ganglioside

of the "transition

mg of total

per mg

established

) the highest

phospholipid ratio

be twice

in synaptosoaml

quantities

prepared from rat

0.73-0.93

and 0.073-0. 125 mg of gangliosides

hydrophobic proteins,

tive

of c e l l s

shown ( J_6 ) to contain

is in the range

Likely

chains.

and s i a l y l -

of gangliosides

surface

occurrence

of a number

synaptosomal

the ganglioside/

membrane

hence the

probable.

be considered

phospholipids

tion

The location

is by far ( 5O-100 fold

might

( 4. 0 ),

the optimal

of sialyltransferase

ma membranes obtained from

branes,

pHs

sialyltransferase 8.

membrane

ganglioside/phospholipid

figure

correlate

the reported

surface

purified

cortex,

-

ratio;

membranes

phospholipids

erial

special-

of the saccharide

at acidic

from

neuro-

and in the enzy-

ganglioside

contribution to the supramolecular

synaptosoaml

The

acid /

may

plasma

However,

assignement

Highly

in gangliosides

best

in Figure

The sidedness

Ganglioside

tissue for enabling

of pH

sialidase,

the external

brain

works pHs,

7.

henc

in the outer

certained.

this

the sialic

11

This

membranes differentiate

and crosslinking capacity

at neutral

).

correlation to this functional

enrichment

sialidase

the synap-

sialylglycoconjugates

in F i g u r e

the synaptosomal

ty of these enzymes,

of

location,

membranes feature

for a "sialylation-desialylation c y c l e

as of other

membranes of brain

to modify

that

of subcellular

depicted

is the striking

complexing

331

Thus the synaptosomal

( as well

In conclusion, the

Membranes

occur-

of cholesterol and

for enhancing the r e l a -

required for causing

a bilayer-

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

332

CELL SURFACE

Figure 7.

I

t

I

t

Sialylation-desialylation cycle of gangliosides

C2> sialyltransferase

Figure 8.

GLYCOLIPIDS

ο

Location of gangliosides, sialidase, and sialyltransferase at the nerveending membrane

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

18.

micellar

transition.

Gangliosides membrane is

mainly

presence with (

tend

),

ve.

the

ganglioside

crosslinking agents,

stay

Over

the

to

in equilibrium diffusing, critical

greater

Now,

as

being

(

recorded

respect

exceeded

linking

agents.

any

,

signals

we

for

or

the

point

essential of

from

stable

remind that

to

to

( by

be

may easily

the

of

clustering

appropriate

viceversa.

stress

In other

displays

sible

phase transitions. (

Several

the

are

size;

of

mob-

dissolions

or

interactions,

the

of

is

of

Ca

ions

as

point

clusters

view,

is

In this

interact

high

either

or

ganglioside

a defined

points

sites.

ganglioside

with

binding

location on the

which direct

In conclusion

clustering. is

that

non-covalent

and reform

See

as

clusters.

carbohydrate-carbohydrate

focal

this

are

cross-

of

membrane. can

%

would occur

( hydrophobically ),

in addition, are

1.5

abundantly

ganglioside

the

started

membranes appear

stable

sites

quoted

layer

serve

a physiological

the

proteins

9

involved

is

easily

removed,

supramolecular

great flexibility

would

signi-

important

the stress

organiz-

and of

rever-

).

which can

carbohydrate

An

the forces

and could break

when

kind of of

Figure

consequences of

in the

Moreover

ganglioside

characteristics

organization

of

( carrying

as

already

and giving a possible functional

words

ation

cluster

ganglio-

immobilization

gangliosides

in given

in mind,

in their

which could

which have

serve

governing

process

kept

presence

concentration

mutual

Thus proteins

gangliosides

ganglioside

under or

of

of

a certain

and should

mutual

synaptosomal

in preferential

molecules

ficance

in

the

and glycoproteins,

membrane, packing

).

in the

membranes.

the formation

membrane glycoproteins

be

This

surface,

Therefore

should

interactions

or

concentration

membrane embedded proteins

affinity)

process than

molecules,

ganglioside

a ganglioside

the formation

sites,

receptors

gangliosides,

surrounding solution of

and Grant, of

synaptosomal

problem,

respect

In analogy

woul

Sharom

membrane

candidates

One

with

at

in the

the less

which facilitate

to phospholipids.

on the

whether

by

1 _ 3 _ )

present

in

ganglioside

,

is

and on the

membrane of

on the

clusters

than th

reported

investigation

which

with the

such

cations.

concentration

concentration,

dynamically favoured,

ideal

a patch

crosslinking agents

clusters

with

"critical"

of

concentration

capping of

made.

distribution

The formation

crosslinking agents

leads

should be

their

like divalent

suggested for

absence of

laterally

other

even.

thus

dependent on

below a certain

cannot

:

be

been

333

a consideration

clusters

not

of

in the

Membranes

may

side clustering

ile,

However

to form

surface

what has

32

or

it

Nerve-Ending

TETTAMANTI ET A L .

be

carrying

expected from

molecules

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

the

in the

CELL SURFACE

334

membrane

surface.

have

indicated

been

First, as

carbohydrate portions ificity. not

The

of

mutual

perative

would cause easier pid

composition

the

rearrange tion,

in

this

bilayers

toxin,

(

33

of

exposed

matrix

The

the

provided the

the

from

ly

sides.

similar

remainder cal

This

some

for

As

to

the

the l i -

ganglio-

reaching,

This

by

of

or

to

organiza-

Tosteson

study

of

and

channels when

ganglioside

the

were

above view

agent for

,

cholerae

leads

rearrangement of

to the of

the

channels.

ganglioside

ganglioside

the

behavior in

of

of

great

ganglioside

model,

as

of

about

60%

of

gangliosides much more

on this direction,

by

and homogeneous vesicles,

a study The

the

In fact

et

al.

(

gangliosides

inner

side.

Of

the

monolamellar

34j

mimic-

which

lipid

authors

layers ( J^) ,

liposomes gangliosides

are

on

on a substantialouter

course

sided,

the

an asymmetri-

layer

would

above purposes. phospholipid

prepared according

containing

on

sur-

exper-

gangliosides

liposome

the

model

model

quoted

carrying

on the outer

size,

of

help.

on the

precise

phospholipids and

suitable for

using

course,

immobil ization

used

limitations.

dispersions

located on the

distribution

of

availability

would be

determined by Cestaro

being

model

of

multilamellar vesicles,

system,

These

the

important

ked

small

containing

According

F o r this

evidence

location of

V2 ).

report

development

membranes needs,

this

(

till

enabling

at

would be forced

aggregation.

recent

the

a crosslinking

der

of

increase lipids

look

Here

coo-

glycocalyx

areas,

us

a cluster.

The

in the

toxin.

membrane

yielded as

both

seen

as

were prepared from and

value.

hypothesis

liposome.

suffers

to

let

the

proteins

model

supports. cell

Finally

would greatly

describing

synaptosomal

imental

modulating the

oligosaccharide-free ligands.

spec-

might

membranes

cluster

of

by

surface

binding kinetics a

G. clusters. The following MI would result in the formation

experimental

face

is

,

functioning of

also,

the

their

determining

patch organization of

glycerolmonooleate,

synaptosomal

ing

)

surfaces,

polar channel

to cholerae

formation

An

of

and glycoproteins

cell

but,

to

a micellar kind of

sense can be

Tosteson

lipid

ratio

involving

the formation

of

apolar

transition

toward

likely

give

in correspondence

side/phospholipid

at

instruments for

binding

the

the formation with

sites

carbohydrate chains on

receptors

Second,

collision

exceeding,

of

interactions,

nature.

gangliosides

being the

aggregation

only facilitate

extent

both

receptor

GLYCOLIPIDS

We

renwor-

vesicles,

to Barenholtz

phosphatidylcholine (

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

carrying

TETTAMANTI ETA L .

Nerve-Ending

Membranes

335

GANGLIOSIDE GLYCOPROTEIN CLUSTER

GANGLIOSIDE CLUSTER

OUTER MEMBRANE LAYER

GLYCOPROTEIN

Figure 9.

GANGLIOSIDE

Formation of stable ganglioside clusters: role of proteins (hatched irregular circles) and of glycoproteins as focal points of clustering

GANGLIOSIDE CLUSTER

CHANNEL

Figure 10. Formation of a polar channel in correspondence of a ganglioside cluster. Note the presence of proteins and glycoproteins (hatched irregular circles).

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

CELL SURFACE

336

C

choline)

incubation

and phosphatidylethanolamine

in the

presence

tritium ι labelled The process cles

is

ionic

ganglioside

ganglioside

rate

is

as

G

with G

( .

see

equal

(

would be

0.6-0.7

cess of

μιηοίβε )

dependent

the

as

it

saccharide

molar

transition

ratio

glioside

molecules

to vesicles

of

maintain at

the

by

ganglioside

The

to

since

of

phospholipid vesicles

at

37°C

sialidase

lerae

a

D1

the

m

c

e

micellar activity.

which

is

,

e

»

s

By

was

a max­ reached

saturation

then

a

pro­

found

(

34^)

values

this

inserted

substrate for

which

As

incorporated

isolated

and

into

micelles with

are

acid

the

are

lipid

on G

the

incubated

cholerae )

Vibrio

hfgher the

after

submitted

is

than on

present

the

therefore

action

of

in

neuraminidase

the

ganglio­

vesicle

rate of

when phospholipid

incubation a certain

to

Cho­

expression

yielding a mixed

proof,

is

-phospholipid

record of

enzyme;

matrix

mixtures

( NeuAc

Since

likely

this

loss

observed.

when

Vibrio

ganglioside

a further

in­ and

ganglio­

with phospholipid vesicles

into vesicles,

increases.

hours,

was

In fact,

than 50-fold

lowest

interaction

gan­

the

with phospholipid v e s i ­

kinetics.

all

lower

of

significant

vesicles

display

Initially

no

molecules

kinetics

yielding the

allowing

the

treated

more

much

alter

stable for

vesicles,

process.

to

is

association

phosphatidylethanolamine

micelles

sigmoidal

which

The

N-acetylneuraminic

phenomenon.

a better

.were

incorpora­

cases,

incubation with cold

and ganglioside

a

form,

the

a fusion

the™ gmoidal

release

vesicles,

of

amount of

Vibrio

Cholerae

a

sialidase G

,

become

NeuAc

G^^ JD1

of

liposomes V '

in all

a

0.5-

with time

The

significantly

sided

ganglioside

times,

release

following

sides

by

which follows

sialidase

mixed G

likely

different

,

).

0.07,

remain

upon

ganglioside

the

recorded,

outer

of

of

not

radioactivity from

of

for

and from

incorporation

incorporation was

above.

these

separation

insertion of

vesicle,

ratio

does

Moreover,

interaction of

lead

11

expected for

discussed

proportion of

value.

followed

the

cles

vesicles,

%

a constant

sides, of

the

of

tem­ 0.9

chain

the

tegrity

)

the

proportionately

Figure

level

).

upon th

ganglioside/phospholipid than

),

Interestingly,

imum and approximately

( 35

starting from

vesicles

G^.

proceed

) upon

concentration,

instance,

,

mol

(containing

gangliosides

ganglioside

For

by

into phospholipid v e s i ­

monolamellar

concentration

highest

incorporate

pH,

(

into vesicles

ganglioside

tion

strength,

phospholipid (

μηιοίβε of

and

do

( 95/5,

micelles

incorporation

dependent phenomenon.

μη-ιοίβε of

of

ganglioside

gangliosides)

ganglioside

a time,

perature

2

of

of

GLYCOLIPIDS

the

kinetics

are

hyperbolic,

-phospholipid vesicles

prepared

the by

same exhibited

sonication.

This

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

by

mixed

means

TETTAMANTI ET A L .

Nerve-Ending

Membranes

337

Figure 11. Effect of incubation time (at 37°C) and of ganglioside concentration on the incorporation of gangliosides (G , G , G ib) into phosphatidylcholine monolamellar vesicles. Phosphatidylcholine (as vesicles): 9 μmol. Ganglioside: from 0.5 to 2 μ/nol. After incubation the mixtures were passed through a 1 X 20 cm Sepharose 4B column to separate vesicles from ganglioside micelles. M1

Dla

T

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

CELL SURFACE

338

Figure 12.

GLYCOLIPIDS

Time course of NeuAc release from liposome-associated ganglioside G by the action of Vibrio cholerae sialidase Dla

Incubations done at 37°C in 0.05M Tris-HCl buffer, pH 6.8, with 1 IU of enzyme (Behringwerke). Released NeuAc determined by method of Warren (37); available amino groups (carried by phosphatidylethanolamine) by TNBS method (12). Arrow indicates addition of detergent (Triton X-100, 0.5%). Ganglioside pattern during enzyme hydrolysis was monitored by TLC (silica gel plates; solvent: chloroform/methanol/ 0.3% aqueous CaCl , 60/35/8, by vol, 2-hr run; spots detected by spraying with Ehrlich's reagent and heating at 110°C for 10 min). 2

(A) :

liposomes containing phosphatidylcholine, phosphatidylethanolamine, (90/3/7, by mol) and prepared by the sonication method (12)

and G

D l a

(B) : liposomes containing phosphatidylcholine and phosphatidlyethanolamine (90/5, by mol), prepared by sonication, were incubated in 0.05M Tris-HCl buffer (pH 6.8) with G micelles for 1 hr, then separated by Sepharose 4B column chromatography. These liposomes contained about 5% (by mol) of incorporated ganglioside. D l a

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

TETTAMANTI ET A L .

Figure 13.

Nerve-Ending

Membranes

339

Time course of oxidation of the terminal galactose residue of liposomeassociated ganglioside G by the action of galactose oxidase M1

Incubations done in 0.05M Tris-HCl buffer (pH 6.8) at 37°C, with 1 IU of enzyme (Kabi). Oxidation was followed by the coupled o-anisidine peroxidase procedure. Formation of oxidized G was also monitored by thin-layer chromatography, under the conditions described in Figure 12. Note that oxidized G could be reduced to the starting G by NaBH treatment. All other conditions as described in Figure 12. Mt

M1

h

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

Mi

CELL SURFACE

340

that

in both vesicle

tion )

the

insertion

bohydrate chains As

shown

troduced tion

of

vior

about ter

60

%

sided

the

by

which

aminogroups lipids,

the

integrity

in the

G

à

J

course,

becomes

(

available

second

results,

by

terminal

13

).

All

introduced

The

species

this

the

of

mined

by

insertion

as

outer

layer

the of

carried

the

in which

to

of

( the o u -

treatment

TNES

of

0.5

by

measure-

%

inner

is

100

split

which

Triton

G

sided off.

gan-

Con-

%

of

releasable

addition

of

Triton

bevavior,

liposomes

phospholipid

à

was

i

X-100.

were obtained

monitored

as

the

oxidation (

in which

ganglioside

An

initial

ganglioside

see

gangliosides units only in

latter

layer,

the

a certain

the vesicle

structure.

prevent

by

entry the

micelles,

this

leads

of

of

other

mimicking

(

suggested

followed lipid

units

deter-

by

been

resulting hand the

the

stable

matrix of

diffusion

into the

lipid

on

inside

requirements for

across

the

layer.

incorporated into

are

being formed

with-

in a stabilization of acquired

and incorporation

a saturation

is

mainly

ganglioside

weak bonds

this

adhesion

has

is

into the

energetic groups

ganglioside

On the

to

carrying

side,

adhesion,

ganglioside

high

and other

further

or

moieties

polar head

amount

hydrogen

lipid

process

the

large

contact,

vesicles,

outer

carbohydrate chains,

ganglioside

This

monolayer

located on the

the oligosaccharide chains,

side

by

galactose-oxidase

absorption c a r r y

being prevented

charge

that only

aminogroups,

and

liposomes

differential

means that

asymmetrically

lipid

the vesicle, in

by

the

movement of After

galactose

follows.

the v e s i c l e . the

sialidase

enzyme

before

liposomes,

this

formation

occur

releasable

layer.

gangliosides to

of

ac-

beha-

JVl 1

available

are

%

enzyme

on addition

the one to

sialidase

showing

containing

the outer

differential 60

in-

the

a

the

indicated

IV1 1 Figure

^ was

submitted to

expected,

to

during

as

car-

TNB

released

4

Of

remainder NeuAc

versely,

Identical

as

available

Noteworthy,

their

about

meaning,

is

absorpthe

destroys

with

)

is

only

by

leads

in which G are

the following

case

sialidase,

layer

surface.

sided phosphatidylethanolamine

gliosides

NeuAc

layer

liposomes

sialidase

).

sonication or lipid

absorption,

ganglioside

maintain

outer

by

In the first

split

of

when

or

unchanged r e c o r d s .

X-100,

with

12

Cholerae

ganglioside

liposomes ments of give

in Figure

is

in the

to protrude on the

observed.

acid

( prepared by

gangliosides

sonication,

Vibrio

is

sialic

by

species of

GLYCOLIPIDS

)

surface of

process.

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

ganglio-

18.

Nerve-Ending

TETTAMANTi E T A L .

Membranes

341

Conclusion

The of

studies on the

gangliosides

these on

the

recent

years,

involvement

portant

to

expect

in

different

ligands

fields

-

gangliosides

near

out

the

by

developed.

future,

interactions played

by

-

of

at

will

of

biologically and

it

is

reasonable

progress

membrane

provide enough

gangliosides

im-

adequate

gangliosides

the

in

increasing evidences

Thus

integrated

and behavior

more frequent

sophisticated

physico-chemistry

role

the

in a number

more

enzyme events occurring

-glycocalyx

to figure

stimulated

Moreover

in the

properties

membranes became

models have been

that,

membranes;

surely

of

phenomena.

experimental

physico-chemical

in artificial

of in

research artificial

surface; information

in synaptosomal

mem-

branes.

Symbols

The was

used

ganglioside

nomenclature

proposed

by

Svennerholm

(

36

)

fol lowed.

Acknowledgements

The

experimental

supported

by

(

),

C.N.R.

grants

data

from

reported

in this

paper

the Consiglio Nazionale

pertain

to

work

del le Ricerche

Italy.

Literature cited 1. Wiegandt, H. Advances Lip.Res., 1971, 9, 249. 2. Sillerud, L.O.;Prestegard,J.H.;Yu,R. K.;Schafer, D.E.; and Konigsberg, W.H. Biochemistry, 1978,17,2619. 3. Howard, R.E.;and BurtonR.M.Biochim.biophys.Acta, 1964, 84, 435. 4. Curatolo W.;Small D.W.;Shipley G.G. Biochim.Biophys.Acta 1977, 468, 11. 5.Gammack,D.B. Biochem. J., 1963, 88, 373. 6. Rauvala, H. FEBS Lett.., 1976, 65, 229. 7. Yohe, H. C.; and Rosenberg, A. Chem. Phys. Lipids, 1972, 9, 279. 8. Yohe H. C.; Roark, D.E.; and Rosenberg, A. J. Biol. Chem. 1976, 251, 7083. 9. Schwarzmann, G.; Mraz, W.; Sattler, J.; Schindler, R.; and Wiegandt, H. Hoppe-Seyler's Z.Physiol. Chem.1978, 359, 1277

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10. F o r m i s a n o , S . ; Johnson, M. L.;Lee, G.;Aloj, S. M.;and Edel­ hoch, H . Biochemistry, 1979, 18, 1119. 11. Η i l l , Μ. W.;and Lester, R. Biochim. Biophys. Acta, 1972, 282, 18. 1 2 . Barenholtz, Y . ; G i b b s , D.;Sitman, B . J.;Goll, J.;Thompson, T . E.; and C a r l s o n , F. D . Biochemistry, 1977, 16, 2806. 1 3 . Sharom F. J.; and Grant, C. W . M . Biochim.Biophys.Acta, 1978, 507, 280. 1 4 . Holmgren, J. Proceedings C N R S International Symposium ''Structure and function of gangliosides", L e Bischenberg, F r a n c e , A p r i l 1979. 15. Ledeen, R. W. J. Supram. S t r u c t . , 1978, 8, 1. 1 6 . Breckenridge, W. C.;Gombos, G.;and Morgan, I . G . Biochim. Biophys. Acta, 1972 1 7 . Hansson, H . A.;Holmgren A c a d . S c . U S A . , 1977, 9, 3782. 1 8 . Tettamanti , G . ; P r e t i , A . ; C e s t a r o , B.;Venerando, B.;Lombardo, A . ; G h i d o n i , R.;, and Sonnino, S. Proceedings C N R S Interna­ tional Symposium "Structure and function of gangliosides", Le Bischenberg, F r a n c e , A p r i l 1979. 1 9 . Bretscher, M. S. Science, 1973, 181, 622. 2 0 . Yamakawa, T . ; a n d Nagai Y . T I B S , 1978, 3, 128. 2 1 . Rosenberg, A . A d v . E x p t l . M e d . B i o l . 1978, 101, 439. 2 2 . Schengrund C . L . ; a n d Rosenberg, A . J. B i o l . Chem. 1970, 254, 6196. 23. Tettamanti , G.;Morgan, I. G.;Gombos, G.;Vincendon, G . ; a n d Mandel, P . Brain Res. 1972, 47, 515. 2 4 . Tettamanti, G . ; P r e t i , A . ; L o m b a r d o , A.;Suman, T . ; a n d ZambotV. J . Neurochem., 1975, 25, 451. 2 5 . Tettamanti , G.;, P r e t i , A., Lombardo, A.; Bonali, F . ; a n d Zam­ botti,V. B i o c h i m . B i o p h y s . A c t a . , 1973, 306, 466. 2 6 . P r e t i , A.;Fiorilli, A.;Lombardo, A . ; C a i m i , L . ; a n d Tettamanti G . submitted for publication. 2 7 . Ledeen, R. W.;Scrivanek, L. J.;Tirri, R. K . ; M a r g o l i s , R. K.; and Margolis, R. U. A d v . E x p t l . M e d . Biol., 1976, 71, 83. 2 8 . Venerando, B . ; P r e t i , A . ; L o m b a r d o , A . ; C e s t a r o , B.;and T e t t a ­ manti, G . B i o c h i m . B i o p h y s . A c t a , 1978, 527, 17. 2 9 . Shur, B . D.;and Roth, S. Biochim.Biophys.Acta, 1975, 415, 473. 3 0 . P o r t e r , C. W.;and Bernacki, R. J. Nature, 1975, 256, 648. 3 1 . Colombino, L. F.;Bosmann, H. B.;and Mc Lean, R. J. Exptl. Cell R e s . , 1978, 112, 25. 3 2 . Gershon, N . D . P r o c . Natl. A c a d . S c . U S A , 1978, 75, 1357.

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33. Tosteson, M. Y.; and Tosteson, D. C. Nature, 1978, 275, 142. 34. Cestaro, B.; Barenholtz, Y.; and Gatt, S., submitted for publication. 35. Cestaro, B.;Ippolito, G.;Ghidoni, R . ; O r l a n d o , P . ; a n d Tettamanti, G . B u l l . M o l . Biol.Med, 1979, in p r e s s . 36. Svennerholm, L. J. Lipid R e s . , 1964, 5, 145. 37. Warren, L. J. B i o l . C h e m . , 1959, 234, 1971. Received December 10, 1979.

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

19 Specificity and Membrane Properties of Young Rat Brain Sialyltransferases 1

SAI-SUN NG and JOEL A. DAIN Department of Biochemistry and Biophysics, University of Rhode Island, Kingston, RI 02881

Sialyltransferase enzymes that transfer s i a l i tor molecules. The acceptor molecules may be low molecular weight oligosaccharides or higher molecular weight glycolipids and glycoproteins. Twelve a c t i v i t i e s typical of sialyltransferases have been described and these a c t i v i t i e s probably represent eight separate and distinct enzymes (1). Our interest in rat brain s i a l y l transferases stemmed from our work on the ganglioside biosynthetic pathways. These studies by us (2,3) and others (4,5) suggested the existence of more than one pathway for the synthesis of polysialogangliosides depending on when a sialyltransferase is brought into action after other sugar residues have been added to a precursor glycolipid. These early studies had also documented that the rat brain sialyltransferases are mainly membrane-bound. This is of interest because the neuraminidases in brain tissues are also membrane bound. The neuraminidases together with gangliosides have been l o calized in the nerve ending structures (6,7). Theoretically the sialylation and desialylation cycle may mediate a cyclic reaction at a very important locale in a nerve synaptic structure. This hypothetical involvement of s i a l i c acid metabolism in synaptic transmission has gained support from several studies which have suggested a synaptic localization of the glycosyltransferases (8, 9,10,11) and from proposed theoretical models in which the sialoglycolipids are considered an important constituent in the functional units of neuronal membranes (12,13,14). It was apparent, however, that the rat brain sialyltransferases have not been sufficiently characterized for the postulation of a biological role for the sialylation-desialylation cycle. Consequently, we concentrated our efforts to characterizing the general behaviors of the sialyltransferases in their membrane environments in the rat brain. What follows is a summary of our 1

Department of Biochemistry, McGill University, Montreal, Quebec H3C 3Gl, Canada. 0-8412-0556-6/80/47-128-345$5.00/ 0 © 1980 American Chemical Society

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

346

CELL SURFACE GLYCOLIPIDS

results on the properties of rat brain membrane-bound sialyltrans­ ferases, their sub-cellular localization and our initial attempts to solubilize and purify these enzymes. Part of the results have been published previously (15,16). Methods In all experiments to be described, brains of 11-15 day old albino rats (Sprague-Dawley) were used. Young rats of this age were chosen because rapid accumulation of gangliosides and sialoproteins have been reported to occur around this period (17). For most studies, total brain homogenates were centrifuged at 105,000 g for 60 min and the pellet used as the enzyme source. Details of the conditions for the enzymic assays have been re­ ported (15,16). The four reactions below were investigated. The abbreviations for gangliosides are those proposed by Svennerholm (18) . (A)

Endogenous glycolipids Cer-Glc-Gal

^ Cer-Glc-Gal (GM) NeuNAc Cer-Glc-Gal-GalNAc-Gal (GM-ι ) Cer-Glc-Gal-GalNAc-Gal (GD ) I > I I NeuNAc NeuNAc NeuNAc (B) Exogenous glycolipid Cer-Glc-Gal-GalNAc-Gal (GM-. ) Cer-Glc-Gal-GalNAc-Gal (GD-, _ ) I ——> ι I NeuNAc NeuNAc NeuNAc (C) Endogenous glycoproteins Glycoproteins ^ Glycoproteins-NeuNAc (D) Exogenous glycoprotein Desialated (DS) fetuin ^ DS-fetuin-NeuNAc Results and Discussion Kinetic Properties of Sialyltransferases. The sialyltransferase activities with the endogenous glycoprotein and glycolipid acceptors in the standard assays (15) were linear with time for at least 60 min, while those with the exogenously added GMi and DS-fetuin were linear with time only for about 30 min (Figure 1). Activities were directly proportional to the amount of enzyme added up to 0.75 mg protein/assay (Figure 2). The enzyme activities, expressed as nmol of NeuNAc incorpor­ ated per 0.5 mg protein per 30 min at 37C and pH 6.3, were 0.095, 0.039, 0.17 and 0.64 with the endogenous glycolipids, the endo­ genous glycoproteins, the exogenous GM^ and exogenous DS-fetuin, respectively. Incorporation into the endogenous glycolipids was always higher than the incorporation into the endogenous glyco3

la

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

19.

N G A N D DAIN

Rat Brain Sialyltransferase s

347

1.4 Ο

0

10 20 30 40 50

60

TIME (minutes) Journal of Neurochemistry

of NeuNAc into the following substrates was determined: X, endogenous glyco­ proteins; Δ , endogenous glycoproteins plus exogenous DS-fetuin; Ο , endogenous glycolipids; O, endogenous glycolipids plus exogenous GM\. (15).

0.8 Q UJ

S

0.6 h

or ο

CL G p i > G^i > M3 > g l o b o s i d e , r e q u i r i n g i n d i v i d u a l c o n c e n t r a t i o n s o f ]k 30, hS 100 and 1000 yM f o r c o m p l e t e r e v e r s a l o f t h e a n t i v i r a l e f f e c t . 2

a

G

9

$

Effects feron.

of

S a c c h a r i d e s on A n t i v i r a l A c t i v i t y o f

Fibroblast

Inter-

S i n c e t h e c e r a m i d e p o r t i o n s o f more and l e s s I n h i b i t o r y g l y c o l i p i d s are very s i m i l a r , d i f f e r e n t i a l i n h i b i t i o n o f a n t i v i r a l a c t i v i t y o f mouse f i b r o b l a s t i n t e r f e r o n must be r e l a t e d t o t h e i r carbohydrate side chains. We t h e r e f o r e a s s a y e d a n t i v i r a l a c t i v i t y in t h e p r e s e n c e o f v a r i o u s s a c c h a r i d e s c o n t a i n e d i n g a n g l i o s i d e s . As seen i n F i g u r e 3, b o t h N - a c e t y 1 n e u r a m i n y l l a c t o s e and N - a c e t y l neuramlnic a c i d i n h i b i t e d a n t i v i r a l a c t i v i t y , r e q u i r i n g a p p r o x i m a t e l y e q u a l c o n c e n t r a t i o n s t o o b t a i n c o m p l e t e I n h i b i t i o n (60 mM). H o w e v e r , In c o m p a r i s o n t o G M , 6 0 0 - f o l d h i g h e r c o n c e n t r a t i o n s o f t h e s e s u g a r s had t o be e m p l o y e d t o y i e l d c o m p l e t e i n h i b i t i o n o f antiviral activity. N - g l y c o l y l n e u r a m l n l c a c i d and t h e $ - m e t h y 1 3

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

396

C E L L

T-//-1

0

5

1

1

1

r

10

20

40

80

S U R F A C E

G L Y C O L I P I D S

SACCHARIDE CONCENTRATION [mM]

Figure 3.

Effects of mono- and oligosaccharides on antiviral activity of mouse fibroblast interferon

Experimental conditions as in Figure 1. M, Lactose; Δ , N-glycolylneuraminic acid; A. neuraminic acid β-methyl glycoside; O , N-acetylneuraminic acid; · , N-acetylneuraminyl lactose. T: EMC titer in the absence of interferon. This titer was unchanged in the presence of each saccharide up to a concentration of 100 mM.

Figure 4. Binding of mouse fibroblast interferon to Sepharose-ganglioside colums and elution with N-acetylneuraminyl lactose One mL interferon solution (2 Χ 10 IU) in MEM plus 50 y,glmL bovine serum albumin was loaded onto a small column containing 0.2 mL of the Sepharose-ganglioside adduct as described in Materials and Methods. The column was first eluted with MEM-albumin alone. At arrow, elution was continued with a solution of 0.07M N-acetylneuraminyl lac­ tose in MEM-albumin at pH 2. Antiviral activity in each fraction was determined as described in Materials and Methods. A small amount of the antiviral activity (7% ) passed the column unretarded; the remaining por­ tion (89% of that applied) was eluted with N-acetylneuraminyl lactose. 3

4

6

ELUTION VOLUME [ml]

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

8

22.

A N K E L

E T

A L .

Interferon-

Carbohydrate

Interaction

397

g l y c o s i d e o f n e u r a m i n i c a c i d a l s o were i n h i b i t o r y , y e t c o n c e n t r a t i o n s a p p r o x i m a t e l y t h r e e times h i g h e r than those o f the above s a c c h a r i d e s r e s u l t e d in comparable i n h i b i t i o n . In v i e w o f t h e fact that a l l gangliosides contain substituted l a c t o s y l residues i t i s i n t e r e s t i n g t h a t l a c t o s e had no e f f e c t on a n t i v i r a l a c t i v i t y up t o c o n c e n t r a t i o n s o f 100 mM. O t h e r s u g a r s t h a t had l i t t l e o r no e f f e c t a t c o m p a r a b l e c o n c e n t r a t i o n s w e r e : N - a c e t y l g l u c o s a m î n e , N-acetylgalactosamine, mannose, g a l a c t o s e and L - f u c o s e . It i s s u r p r i s i n g t h a t t h e t r i s a c c h a r i d e N - a c e t y l n e u r a m i n y l l a c t o s e was much l e s s p o t e n t i n i n h i b i t i n g a n t i v i r a l a c t i v i t y than the c o r r e s p o n d i n g g l y c o l i p i d G M . T h i s might i n d i c a t e t h a t e i t h e r t h e b i n d i n g s i t e o f mouse f i b r o b l a s t i n t e r f e r o n on G M i n c l u d e s p a r t o f t h e l i p i d p o r t i o n as w e l l , o r t h a t a r r a n g e m e n t o f c a r b o h y d r a t e c h a i n s in g a n g l i o s i d e m i c e l l e s f a v o r s a conformation w h i c h a l l o w s f o r a much t i g h t e r f i t o f i n t e r f e r o n . The f r e e t r i s a c c h a r i d e i n s o l u t i o n , on t h e o t h e r h a n d , m i g h t assume any number o f c o n f o r m a t i o n s , o to i n t e r f e r o n b i n d i n g . 3

3

T h a t i n h i b i t i o n o f a n t i v i r a l a c t i o n i s due t o b i n d i n g , and t h a t t h i s i n v o l v e s t h e c a r b o h y d r a t e s i d e c h a i n s on t h e g a n g l i o s i d e m o l e c u l e , was c l e a r l y i n d i c a t e d by t h e b e h a v i o r o f fibroblast i n t e r f e r o n on a f f i n i t y columns c o n t a i n i n g c o v a l e n t l y bound g a n g l i osides. As seen i n F i g u r e k when mouse f i b r o b l a s t i n t e r f e r o n was p l a c e d on s u c h a c o l u m n , l e s s t h a n 10% o f t h e a n t i v i r a l a c t i v i t y passed through u n r e t a r d e d . The r e m a i n i n g a n t i v i r a l a c t i v i t y was q u a n t i t a t i v e l y e l u t e d w i t h 70 mM s o l u t i o n s o f N - a c e t y l n e u r a m i n y l l a c t o s e a t pH 2. It s h o u l d be n o t e d t h a t t h i s c o n c e n t r a t i o n o f the t r i s a c c h a r i d e a l s o c o m p l e t e l y r e v e r s e d the a n t i v i r a l effect, as i n d i c a t e d i n F i g u r e 3. 9

Effect of Gangliosides Interferon.

on A n t i g r o w t h A c t i v i t y o f

Fibroblast

S i n c e i t has been e s t a b l i s h e d t h a t a n t i v i r a l and a n t i g r o w t h a c t i v i t i e s o f mouse f i b r o b l a s t i n t e r f e r o n r e s i d e i n t h e same m o l e cules (18), one w o u l d e x p e c t t h a t g a n g l i o s i d e s w o u l d I n h i b i t b o t h a c t i v i t i e s In a s i m i l a r f a s h i o n . To I n v e s t i g a t e t h e e f f e c t o f g a n g l i o s i d e s on g r o w t h i n h i b i t i o n , we used mouse l e u k e m i a L - 1 2 1 0 c e l l s , w h i c h grow more r a p i d l y t h a n mouse L - c e l l s . This cell line i s o f a d d i t i o n a l i n t e r e s t s i n c e G r e s s e r , Bandu and B r o u t y - B o y e have I s o l a t e d a s u b l i n e ( L - l 2 1 OR) by c o n t i n u o u s g r o w t h In t h e p r e s e n c e o f mouse f i b r o b l a s t I n t e r f e r o n , w h i c h i s r e s i s t a n t t o i t s a n t i v i r a l and a n t i g r o w t h a c t i v i t i e s ( 6 ) . When g r o w t h o f i n t e r f e r o n - s e n s î t î v e L - 1 2 1 0 c e l l s ( L - l 2 1 O S T was f o l l o w e d f o r k d a y s , t h e number o f c e l l s In c o n t r o l c u l t u r e s was t h r e e t i m e s h i g h e r t h a n t h a t In c u l t u r e s w h i c h c o n t a i n e d mouse f i b r o b l a s t Interferon (Figure 5). Although a d d i t i o n of gangliosides alone Inhibited g r o w t h t o some e x t e n t , t h e e f f e c t o f f i b r o b l a s t i n t e r f e r o n In t h e p r e s e n c e o f g a n g l i o s i d e s was l a r g e l y r e v e r s e d and t h e c e l l number In t h e s e c u l t u r e s a p p r o a c h e d t h a t o f c u l t u r e s grown In t h e

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

398

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Figure 5. Effect of bovine brain ganglio­ sides on growth inhibition of L-l 21 OS cells by mouse fibroblast interferon Cells were grown in RPMI medium plus 5% fetal bovine serum in 1 mL total volume as described in Materials and Methods. At 24hr intervals the cells were counted in a Coul­ ter counter. Control cells; · , cells from cul­ tures containing 1000 lU/mL mouse blast interferon; •, cells from containing bovine brain gangliosides concentration corresponding to 35 μΜ sialic acid; W, cells from cultures containing both interferon (1000 IU/mL) and gangliosides (35 μΜ sialic acid).

1 2

3

4

TIME OF CULTURE [DAYS]

Figure 6. Effects of mousefibroblastin­ terferon L-1210S and L-1210R cells in the presence of increasing ganglioside concentrations Cells were seeded at an original cell density of 8 X 10'> L-1210S cells/mL and 7 X 70* L-l21 OR cells/mL in 0.4 mL total volume. Cells were cultured as in Figure 5 and counted after three days of growth. O, Cells grown in the presence of 1000 IU/mL mouse fibroblast interferon; · , those grown in its absence under identical conditions. Top, L-1210R cells; bottom, L-1210S cells.

0

20

40

60 80

GANGLIOSIDE CONCENTRATION [ μ Μ SIALIC ACID]

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

22.

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presence o f g a n g l i o s i d e s alone. S i n c e t h e g r o w t h - î n h i b î t o r y e f f e c t o f g a n g l i o s i d e s was much l e s s p r o n o u n c e d a f t e r 3 days o f c u l t u r e , we i n v e s t i g a t e d t h e e f fect o fdifferent ganglioside c o n c e n t r a t i o n s on a n t i g r o w t h a c t i v i t y by c o u n t i n g t h e c e l l s a f t e r 3 d a y s . A comparison o f t h e e f f e c t s o f g a n g l i o s i d e s on a n t i g r o w t h a c t i v i t y o f i n t e r f e r o n i n b o t h L-1210R and L - l 2 1 OS c e l l s i s shown i n F i g u r e 6. As e x p e c t e d f r o m t h e o r i g i n a l o b s e r v a t i o n s by G r e s s e r est aj_. (6) t h e L-121 OR cells were n o t i n h i b i t e d by f i b r o b l a s t i n t e r f e r o n and a d d i t i o n o f g a n g l i o s i d e s had l i t t l e e f f e c t , b o t h i n t h e a b s e n c e and i n t h e presence o f i n t e r f e r o n . On t h e o t h e r h a n d , i n t h e L-121 OS c u l t u r e s i n t e r f e r o n p r o d u c e d a p p r o x i m a t e l y 50% r e d u c t i o n i n c e l l number. The c e l l n u m b e r , h o w e v e r , p r o g r e s s i v e l y i n c r e a s e d w i t h increasing concentrations o fgangliosides. Complete r e v e r s a l o f t h e i n t e r f e r o n e f f e c t was o b s e r v e d a t a g a n g l i o s i d e concentration c o r r e s p o n d i n g t o 70 μΜ s i a l i c a c i d . T h i s compares t o a c o n c e n t r a ­ t i o n o f 20 μΜ f o r c o m p l e t mouse L - c e l l s y s t e m ( F i g u r Effects

o f G l y c o l i p i d s on T - c e l l

interferon

(19).

I t h a s been o b s e r v e d i n s e v e r a l l a b o r a t o r i e s t h a t i n t e r f e r o n p r o d u c e d i n m i t o g e n - s t i m u l a t e d spleen c e l l s ( T - c e l l interferon) d i f f e r s from f i b r o b l a s t i n t e r f e r o n i n s e v e r a l o f i t s p h y s i c o chemical p r o p e r t i e s , although t h e b i o l o g i c a l e f f e c t s are q u i t e s i m i l a r t o those o f the f i b r o b l a s t v a r i e t y ( 2 0 ) . P r e l i m i n a r y s t u d i e s u s i n g c r u d e i n t e r f e r o n preparations Tappr. 1 0 lU/mg) o b ­ t a i n e d from c u l t u r e d mouse spleen c e l l s o f BCG s e n s i t i z e d a n i m a l s a f t e r s t i m u l a t i o n w i t h o l d t u b e r c u l i n (21) i n d i c a t e d t h a t g a n g l i o ­ s i d e s were much l e s s i n h i b i t o r y t o t h i s i n t e r f e r o n t h a n t o mouse f i b r o b l a s t i n t e r f e r o n ( 2 2 ) . In a n a t t e m p t t o f u r t h e r e l u c i d a t e whether a f f i n i t y f o r g a n g l i o s i d e s i s indeed a p r o p e r t y n o t shared by T - c e l l i n t e r f e r o n , we have c o l l a b o r a t e d w i t h t h e l a b o r a t o r y o f D r . E r n e s t o F a l c o f f and s y s t e m a t i c a l l y compared t h e e f f e c t s o f g l y c o l i p i d s o n a n t i v i r a l and a n t i g r o w t h a c t i v i t i e s o f mouse T - c e l l and f i b r o b l a s t i n t e r f e r o n s u n d e r i d e n t i c a l e x p e r i m e n t a l conditions u s i n g more h i g h l y p u r i f i e d p r e p a r a t i o n s o f t h e f o r m e r (1.6 χ 1 0 lU/mg; r e f . A s seen i n F i g u r e 7, a t g a n g l i o s i d e c o n c e n t r a ­ 3

5

t i o n s where a n t i v i r a l a c t i v i t y o f f i b r o b l a s t i n t e r f e r o n was com­ p l e t e l y i n h i b i t e d , that o f T - c e l l i n t e r f e r o n remained unchanged. Individual g l y c o l i p i d s that i n h i b i t e d f i b r o b l a s t interferon ( F i g u r e 2) had no e f f e c t when t e s t e d w i t h T - c e l l i n t e r f e r o n u n d e r i d e n t i c a l c o n d i t i o n s a t c o n c e n t r a t i o n s up t o 100 μΜ 0 9 ) . These included G , G , G , G i , G j i b and G . M 3

M 2

M 1

D

a

u

T h a t T - c e l l i n t e r f e r o n does n o t b i n d t o g a n g l i o s i d e s i s d e m o n s t r a t e d by i t s b e h a v i o r o n g a n g l i o s i d e a f f i n i t y c o l u m n s : Under c o n d i t i o n s where o v e r 90% o f mouse f i b r o b l a s t interferon was r e t a i n e d (as shown i n F i g u r e k) T - c e l l i n t e r f e r o n q u a n t i t a ­ t i v e l y e l u t e d i n t h e b r e a k t h r o u g h o f t h e column ( F i g u r e 8). T c e l l i n t e r f e r o n , a f t e r p a s s a g e t h r o u g h t h e a f f i n i t y c o l u m n , was still insensitive to ganglioside i n h i b i t i o n , excluding the p o s s i -

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

400

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Figure 7. Effects of bovine brain gangliosides on antiviral activities of mouse fibroblast and T-cell interferons Experiment was carried out as Figure 1, using 20 IU/mL of both interferons. · , Mouse fibroblast interferon; •, T-cell interferon; M, T-cell interferon after passage through a Sepharose-ganglioside column (see Figure 8). Y : EMC titer in the absence of interferon (19).

0 " 2.5 5 10 20 GANGLIOSIDE CONCENTRATION [AM SIALIC ACID]

Figure 8. Lack of binding of mouse T-cell interferon to Sepharose-ganglioside columns Experiment was carried out at the same time and under the same conditions as described in Figure 4, applying 1 mL of a T-cell interferon solution containing 10 1U. At least 90% of the applied antiviral activity passed the column unretarded (19). 3

2 4 6 ELUTION VOLUME [mL]

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b î l i t y that a non-1nterferon contaminant with high a f f i n i t y f o r g a n g l i o s i d e s had been removed by t h i s p r o c e d u r e (See F i g u r e 7). To e x c l u d e e n z y m a t i c d e s t r u c t i o n o f g a n g l i o s i d e s we i n c u b a t e d a s o l u t i o n o f g a n g l i o s i d e s w i t h T - c e l l i n t e r f e r o n a t an i n t e r f e r o n c o n c e n t r a t i o n 10 t i m e s h i g h e r t h a n t h a t n o r m a l l y used i n t h e a n t i v i r a l a s s a y , f o r 2k h r s a t 3 7 ° , t h e n h e a t - i n a c t i v a t e d t h e T - c e l l i n t e r f e r o n and a s s a y e d t h e t r e a t e d g a n g l i o s i d e s f o r t h e i r i n h i b i t o r y a c t i o n on f i b r o b l a s t i n t e r f e r o n . As c o n t r o l we used a g a n g l i o s i d e s o l u t i o n t r e a t e d i d e n t i c a l l y , but in the absence o f T-cell interferon. A l t h o u g h t h e r e was a s m a l l d e c r e a s e i n i n h i b i t o r y potency o f the g a n g l i o s i d e s o l u t i o n s a f t e r t h i s treatment ( a p p r o x i m a t e l y 30%), t h e r e was no s i g n i f i c a n t d i f f e r e n c e between t h e s o l u t i o n s p r e i n c u b a t e d w i t h T - c e l l i n t e r f e r o n as compared t o t h o s e t h a t were p r e i n c u b a t e d i n MEM a l o n e . Thus i t a p p e a r s t h a t l a c k o f b i n d i n g t o and i n h i b i t i o n by g a n g l i o s i d e s i s due t o t h e T - c e l l i n t e r f e r o n m o l e c u l e i t s e l f and n o t t o c o n t a m i n a t i n g f a c t o r s t h a t e i t h e r compet them t o n o n - i n h i b i t o r y Effect

of T-cell

Interferon

on Growth o f L - l 2 1 OS and L - l 2 1 OR C e l l s

uw. I f t h e a f f i n i t y o f mouse f i b r o b l a s t i n t e r f e r o n f o r g a n g l i o s i d e s r e l a t e s t o i t s f u n c t i o n a l i n t e r a c t i o n w i t h mouse c e l l s , t h e n c l e a r l y T - c e l l i n t e r f e r o n must i n t e r a c t w i t h d i f f e r e n t comp o n e n t s o f t h e s e c e l l s , a s i t does n o t b i n d t o g a n g l i o s i d e s . T h e r e f o r e , i f t h e L - 1 2 1 0 R c e l l s t h a t were s e l e c t e d f o r t h e i r r e s i s t a n c e t o f i b r o b l a s t i n t e r f e r o n (6), had a l t e r e d o r i n a c c e s s i b l e s i t e s on t h e membrane t h a t no l o n g e r a l l o w e d p r o d u c t i v e i n t e r a c t i o n w i t h f i b r o b l a s t i n t e r f e r o n , then T - c e l l i n t e r f e r o n m i g h t s t i l l be a c t i v e w i t h t h e s e c e l l s . That t h i s i s indeed the c a s e i s shown i n F i g u r e 9. L-121 OS c e l l s were f o u n d t o be e q u a l l y s e n s i t i v e to the a n t i g r o w t h a c t i v i t i e s o f both i n t e r f e r o n s . Howe v e r , L-1210R c e l l s , a l t h o u g h i n s e n s i t i v e to f i b r o b l a s t i n t e r f e r o n , were as s e n s i t i v e t o T - c e l l i n t e r f e r o n as L-121 OS c e l l s . In a d d i t i o n and as e x p e c t e d , a n t i g r o w t h a c t i v i t y o f T - c e l l i n t e r f e r o n , l i k e a n t i v i r a l a c t i v i t y , was found t o be r e s i s t a n t t o i n h i b i t i o n by g a n g l i o s i d e s a t c o n c e n t r a t i o n s t h a t c o m p l e t e l y reversed t h e a n t i g r o w t h e f f e c t o f f i b r o b l a s t i n t e r f e r o n ( F i g u r e 8). That r e s i s t a n c e o f L-121 OR c e l l s t o f i b r o b l a s t i n t e r f e r o n was n o t due to g a n g l i o s i d e s (or o t h e r i n h i b i t o r s s p e c i f i c f o r the f i b r o b l a s t v a r i e t y ) shed f r o m t h e s e c e l l s i n t o t h e medium, was c o n f i r m e d by a s s a y i n g t h e a n t i g r o w t h a c t i v i t y o f f i b r o b l a s t i n t e r f e r o n on L - l 2 1 OS c e l l s s u s p e n d e d i n 4-day o l d c u l t u r e medium o f L - 1 2 1 0 R cells. In c o m p a r i s o n t o c o n t r o l L-121 OS c e l l s s u s p e n d e d i n 4-day o l d c u l t u r e medium from L - l 2 1 OS c e l l s , t h e a n t i g r o w t h e f f e c t o f f i b r o b l a s t i n t e r f e r o n was u n c h a n g e d , i n d i c a t i n g t h a t d i f f e r e n t r e s p o n s e s t o b o t h i n t e r f e r o n s by L-121 OR c e l l s i s due t o t h e c e l l s t h e m s e l v e s and n o t t o f i b r o b l a s t i n t e r f e r o n - s p e c î f i c i n h i b i t o r s shed i n t o t h e medium by L - 1 2 1 0 R c e l l s , i n a c c o r d a n c e w i t h r e s u l t s

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

C E L L

402

S U R F A C E

G L Y C O L I P I D S

Figure 9. Antigrowth activities of mouse fibroblast and T-cell interferons on L-1210S (left) and L1210R cells (right) and the effects of gangliosides on antigrowth activity Cells were seeded at an original density of 8 X 10'' L-l 21 OS and L-l 21 OR cells I mL in 0.3 mL total volume. Cells were cultured as in Figure 5 and counted after three days of growth. The cell number in control cultures (100%) was 3.1 X 10 L-1210S cells/mL and 2.6 X 10 L-1210R cells/mL. O , Mouse fibroblast interferon; · , mouse fibroblast interferon plus gangliosides (52 μΜ sialic acid); •, T-cell interferon; T-cell inter­ feron plus gangliosides (52 μΜ sialic acid) (19). s

5

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22.

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previously

A L .

Interferon-

reported

Carbohydrate

403

Interaction

(6).

Discussion Data p r e s e n t e d In t h i s c o m m u n i c a t i o n p r o v i d e t h e f o l l o w i n g evi dence: a) b o t h a n t i v i r a l and a n t i g r o w t h a c t i v i t i e s o f mouse f i b r o b l a s t i n t e r f e r o n a r e i n h i b i t e d by g a n g l i o s i d e s ; b) i n h i b i t i o n i s due t o b i n d i n g o f i n t e r f e r o n t o g a n g l i o s i des ; c) b i n d i n g i n v o l v e s t h e c a r b o h y d r a t e s i d e c h a i n s on t h e g a n g l i o s i d e m o l e c u l e s , and i s a t l e a s t i n p a r t d i r e c t e d t o w a r d s s i a l i c a c i d r e s Îdues ; d) n e i t h e r a n t i v i r a l n o r a n t i g r o w t h a c t i v i t i e s o f T - c e l l i n t e r f e r o n a r e i n h i b i t e d by g a n g l i o s i d e s , and T - c e l l i n t e r f e r o n does n o t b i n d t o g a n g l i o s i d e s ; e) mouse l e u k e m i a fibroblast interferon retai interferon. It i s n o t known w h e t h e r f i b r o b l a s t o r T - c e l l i n t e r f e r o n s o r p a r t s o f them have t o e n t e r t a r g e t c e l l s i n o r d e r t o r e s u l t i n a n t i v i r a l or antigrowth responses. The f a c t t h a t mouse f i b r o blast interferon interacts with carbohydrate constituents of g a n g l i o s i d e m o l e c u l e s and t h a t some t r a n s f o r m e d mouse c e l l s g a i n i n c r e a s e d s e n s i t i v i t y to i t s a n t i v i r a l e f f e c t a f t e r uptake o f exogenous g a n g l i o s i d e s i n t o t h e c e l l membrane (5) tempts us t o speculate that i n t e r a c t i o n o f t h i s type of i n t e r f e r o n with c e l l membrane g a n g l i o s i d e s i s o f f u n c t i o n a l s i g n i f i c a n c e . Clearly, if t h i s were t h e c a s e , t h e n T - c e l l i n t e r f e r o n , a l t h o u g h p r o d u c i n g t h e same b i o l o g i c a l e f f e c t s as f i b r o b l a s t i n t e r f e r o n , must have a d i f f e r e n t mechanism by w h i c h i t i n t e r a c t s w i t h i t s t a r g e t c e l l s , s i n c e i t does n o t b i n d t o g a n g l i o s i d e s and i s a c t i v e w i t h c e l l s s e l e c t e d f o r r e s i s t a n c e to f i b r o b l a s t i n t e r f e r o n . It i s p o s s i b l e t h a t t h e r e a r e two c l a s s e s o f i n t e r f e r o n b i n d i n g s i t e s on t h e c e l l membrane, e a c h s p e c i f i c f o r p r o d u c t i v e i n t e r a c t i o n w i t h o n l y one t y p e o f i n t e r f e r o n . Thus p r o l o n g e d growth o f L-1210 c e l l s in the p r e s e n c e o f f i b r o b l a s t i n t e r f e r o n c o u l d s e l e c t f o r t h o s e c e l l s t h a t have no o r n o n - f u n c t i o n a l b i n d ing s i t e s f o r f i b r o b l a s t i n t e r f e r o n , but s t i l l c a r r y u n a l t e r e d sites for binding of T - c e l l interferon. A l t e r n a t i v e l y , uptake mechanisms f o r b o t h i n t e r f e r o n s o r t h e i r a c t i v e f r a g m e n t s m i g h t be d i f f e r e n t , one i n v o l v i n g g a n g l i o s i d e s , t h e o t h e r one a d i f f e r e n t t y p e o f g l y c o l i p i d o r none a t a l l . T h i r d l y , a l t h o u g h the b i o l o g i c a l r e s p o n s e s t o b o t h t y p e s o f i n t e r f e r o n a p p e a r t o be i d e n t i c a l , t h e r e m i g h t be two ( o r more) d i f f e r e n t mechanisms by w h i c h t h e s e m i g h t be t r i g g e r e d , i n v o l v i n g a c t i v a t i o n o f d i f f e r e n t enzymes o r e n z y m a t i c s t e p s , e a c h s p e c i f i c f o r one t y p e o f i n t e r feron. A t t h e p r e s e n t t i m e t h e r e i s no d i r e c t e v i d e n c e t o d e c i d e which o f these p o s s i b i l i t i e s is the c o r r e c t o n e . There are

two a s p e c t s

of

medical

significance

related

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observations: Firstly it is known that cancer patients ofte have elevated levels of circulating gangliosides which might reflect increased concentrations of these glycolipids in the tumor-surrounding tissue (23,24). Therefore, treatment of such patients with human fibroblast or leucocyte interferon might not be very effective, as these two also bind to gangliosides (5,25). Thus using human T-cell interferon as an alternative treatment in cases where fibroblast or leukocyte interferons fail to show the desired effects could have obvious advantages, provided that indeed the former is comparable to mouse T-cell interferon in its resistance to inhibition by gangliosides. Secondly, the observations of Gresser et_ ah (6) concerning the selection of fibroblast interferon-resistant leukemia cells might be of relevance in interferon therapy of leukemic patients, which likewise might select for resistant cells that would escape from the desired growth inhibition. Our data suggest that alternation between fibroblast and T-cell interferon prevent such selection Acknowledgements This work was supported by grants from the National Science Foundation (PCM 76-84125) and from the National Institutes of Health (AI-15007).

Literature Cited: 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Besançon, F . , and H. Ankel, Nature (London) (1974) 250, 784-786. Besançon, F., and H. Ankel, unpublished observation. Besançon, F., and H. Ankel, Nature (London) (1974) 252, 478-480. Besançon, F . , H. Ankel, and S. Basu, Nature (London) (1976) 259, 576-578. Vengris, V . E . , F.H. Reynolds, J r . , M.D. Hollenberg, and P.M. Pitha, Virology (1976) 72, 486-493. Gresser, I., M.T. Bandu, and D. Brouty-Boye, J. Natl. Cancer Inst. (1974) 52, 553-559. Wietzerbin, J., S. Stefanos, M. Lucero, E. Falcoff, D.C. Thang, and M.N. Thang, Biochem. Biophys. Res. Commun. (1978) 85, 480. Wietzerbin, J., S. Stefanos, M. Lucero, Ε. Falcoff, J. O'Malley, and E. Sulkowski, Gen. Virol., in press. Svennerholm, L.J., Neurochem. (1963) 10, 613-623. Folch, J., M.B. Lees, and G.H. Sloane Stanley, J. Biol. Chem. (1957) 226, 497.

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22.

11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.

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Interferon- Carbohydrate Interaction

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Brunngraber, E.G., G. Tettamanti, and B. Berra, In "Glycolipid Methodology" (L.A. Witting, ed.) American Oil Chemists' Society, pp. 159-186 (1976). Sica, V., I. Parikh, E. Nola, G.A. Puca, and P. Cuatrecasas, J. Biol. Chem. (1973) 248, 6543-6558. Cuatrecasas, P., Biochemistry (1973) 12, 4253-4264. Craighead, J . E . and A. Shelokov, Proc. Soc. Exp. Biol. Med. (1961) 108, 823-826. Svennerholm, L . , Biochim. Biophys. Acta (1957) 24, 604-611. Warren, L . , J. Biol. Chem. (1959) 234, 1971-1975. Ledeen, R.W., and R.K. Yu, in "Glycolipid Methodology" (L.A. Witting, ed.) American Oil Chemists' Society, pp. 187-214 (1976). De Maeyer-Guignard, J., M.G. Tovey, I. Gresser, and E. De Maeyer, Nature (London) (1978) 271, 622-625. Besançon, F . , H. Manuscript in Preparatio Epstein, L.P. in "Interferons and their Actions", W.E. Stewart II, ed., CRC Press, Inc. pp. 91-132 (1977). Sonnenfeld, G., A.D. Mandel, and T.C. Merrigan, Cellular Immunology (1977) 34, 193-206. Besançon, F . , H. Ankel, G. Sonnenfeld, and C.T. Merrigan, unpublished observation. Kloppel, T.M., T.W. Keenan, M.J. Freeman, and D.J. Morré, Proc. Natl. Acad. Sci. USA, (1977) 74, 3011-3013. Portoukalian, J., G. Zwingelstein, N. Abdul-Malak, and J.F. Doré, Biochem. Biophys. Res. Comm. (1978) 85, 916-920. Besancon, F., and H. Ankel, Texas Rep. on Biol. and Med. (1977) 35, 282-292.

RECEIVED December 10, 1979.

In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

23 Perturbation of Behavior and Other CNS Functions by Antibodies to Ganglioside MAURICE M. RAPPORT, STEPHEN E. KARPIAK, and SAHEBARAO P. MAHADIK Departments of Psychiatry and Biochemistry, Columbia University, College of Physicians and Surgeons, New York, NY 10032

From the viewpoin most limitless frontier

,

tion control, has been shown by anatomists and physiologists to be composed of a network of neurons that make contact with one another mostly by release of chemicals at synaptic junctions (neurotransmission).

There are astronomical numbers of these

synaptic junctions, and there is also a complex array of chemical transmitters and chemical modulators involved in neurotransmission. Many of these transmitters and modulators have not yet been identified.

The physiological actions of these substances are diverse

(they both excite and depress activity) so we must also postulate that many different molecular structures are involved in receptor functions even for the very same transmitter or modulator. In this extensive array of synaptic connections lie the mechanisms for plastic adaptations of the brain to the external environment -- modifications that subserve the processes of sensory reception, memory and learning, emotional responses, and abstract thought.

A major task of neurochemists is to sort out molecules

participating in the myriad synaptic connections and to identify them. Antibodies As A Bridge Between Structure And Function Methods of separating and characterizing molecules have developed rapidly in the past twenty years and new ones are appear-

0-8412-0556-6/ 80/ 47-128-40755.00/ 0 © 1980 American Chemical Society In Cell Surface Glycolipids; Sweeley, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

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ing with regularity. Among these, Immunological methods are unique in representing relatively mature methods that s t i l l retain a large measure of unexploited potential. The specificity of antibodies for both large and small chemical structures has been well established during the last 30 to 40 years, and has proven of Inestimable value, especially in the field of endocrinology. However, the application of immunological techniques to study synaptic differences is s t i l l in Its infancy. Our laboratory has been addressing Itself for more than a decade to developing an immunological bridge that will make a connection between specific chemical structures in the synapse and various CNS functions earlier demonstrations that antibodies or antisera can serve as Interventive agents that will perturb CNS functions — for example by inducing alterations in the EE6 or inhibiting performance on various tasks (]_,£,3). We have studied the interventive action of antibodies against an array of different antigens using EEG as well as a number of behavioral paradigms (4,5,6) and have demonstrated to our own satisfaction that an encouraging degree of specificity is associated with the actions of these different types of antibodies. For example, we found that functional alterations were Induced with antisera to gangliosides, to S-100 protein (a brain specific protein found mainly in glial c e l l s ) , and to synaptic membranes. No such effects were seen with antisera to galactocerebroside, to 14-3-2 protein (a neuron-specif1c protein), or to erythrocyte membranes. Furthermore, the antibodies to G ganglioside following intracortical Injection induced EEG spiking (7) and inhibited learning (8,9) whereas antiserum to S-100 protein inhibited learning but did not alter the EEG (10). If we accept this evidence that passive administration of antibodies is capable of at least some degree of discrimination among different CNS functions, the specificity of antibodies for molecular structure provides a bridge between structure and function. M1

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G M I Ganglioside As A Synaptic Target For Antibodies We will make one further assumption in order to reduce the area of investigation to reasonable proportions. We will assume that synaptic contacts are the site of action of these passively transferred antibodies that are able to disrupt CNS functions. Therefore molecules that are directly involved in such contacts and are accessible to the extracellular space (synaptic cleft) become priority targets of our efforts. G^ ganglioside f i t s the category well. It Is a small stable molecule whose chemistry is well-established. It can be prepared in workable quantities by reproducible procedures, and criteria of purity are available and readily met. Ganglioside synaptic membranes,and the ganglioside molecule is accessible to the synaptic cleft as we have shown by labeling intact synaptosomes using enzymic oxidation (galactose oxidase) followed by reduction with tritium-labelled borohydride (]i). However, available knowledge suggests that gangliosides are present in a l l synaptic connections, and i f the hypothesis is correct that disturbances of CNS functions by antibodies result from perturbation of synaptic contacts, we might then expect that all CNS functions would be susceptible to disturbance by these antiganglioside antibodies. If this were true, i t would limit the usefulness of these antibodies as an interventive agent. Ant i ganglioside serum would s t i l l offer two major advantages stemming from 1) the relative ease and reproducibility of the methods for preparing i t and characterizing its antibody content (titer) and specificity and 2) its provision of a rigorous control reagent 1n the form of the antiserum from which antibodies to G M I are absorbed (removed) with pure Gm ganglioside. Antibodies To Ganglioside Interfere With CNS Functions Selectively We have now subjected rats to a number of behavioral tests in which we could show inhibition of learning by small quantities of antiganglioside serum and no inhibition by the absorbed serum.

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Among these may be listed inhibition of passive avoidance learning (9), inhibition of morphine analgesia (12) and blockade of sedation induced by reserpine (unpublished data). Schupf and Williams (13) have added blockade of cholinergic stimulation of drinking. However, and quite unexpectedly, we found the antiganglioslde reagent was not effective in a number of other tests, such as pattern discrimination, fixed-ratio conditioning, selfstimulation, pain threshold and activity levels (all unpublished results), and in the experiments of Schupf and Williams, eating and drinking (personal communication). These results, summarized in Table I, Indicate that despite the widespread distribution of M1 ganglioside in the particular behavior will or will not be affected by administration of antibodies to ganglioside. One infers from these results that G M I ganglioside receptors may provide a chemical basis for discriminating among different behaviors. If this proves to be true for G^n ganglioside, i t may also be true for other ganglioside species as well as for other molecules in synaptic membranes that can serve as "receptors" for antibody ligands. G

Mechanisms By Which Antibodies May Perturb CNS Functions How might one account for this discriminatory capability? One explanation might be found in the differences in topography of "antigenic receptors" in different synaptic contacts, differences both in the number of receptors and in their distribution. Another explanation might be found in differences among synaptic contacts with respect to the type of membrane process that is a l tered by the binding of antibody molecules. The number of such processes is substantial and continues to grow. If we consider that the binding of an antibody ligand to an antigenic site in the membrane may alter membrane conformation and/or membrane fluidity, then as a consequence of such alterations a number of properties of the membrane may change including its permeability to ions, its enzyme activities, and the distribution

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TABLE I BIOLOGICAL EFFECTIVENESS OF ANTIGANGLIOSIDE SERUM EFFECTIVE

INEFFECTIVE

Injection Site Test Procedure Test Procedure Injection Site cortex: sensorihypothalamus l.EEG seizures l.EEG seizures motor, visual; hippocampus; amygdala visual cortex 2.Inhibition of 2.Pattern i.vc. lateral learning discrimigeniculate (passive nation avoidance) 3.Inhibition 3.Activity periacqueductal i .vc. of morphine grey levels analgesia 4. Blockade of 4. Fixed^ratio i.vc. i.vc. reserpine conditioning sedation 5. Developmental lateral 5. Selfi.cist. hypothalamus interference stimulation (DRL behavior; a) PAG dendrogenesis 6. Pain threshold of pyramidal b) i.vc. cells in cortex) lateral 7. Eating and hypothalamus drinking 6. Blockade of lateral cholinergic hypothalamus stimulation of drinking

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of membrane components, Including receptor sites. These in turn may affect release and uptake of neurotransmitters, cause i n creased metabolism of receptor sites or trigger endocytosis. For most of these mechanisms examples are available from studies of various types of cells. In addition the antibody binding can activate the complement system leading to membranolysis. The l i s t of possible mechanisms, indicated in Figure 1, is by no means complete. It does, however, suggest a number of experiments that should be helpful in elucidating the basis for discriminatory capability. Antibodies To GMI Ganglioside Inhibit Dendritic Development The effect of antiganglioside serum on development, indicated in Table I, provides some suggestion that gangliosides may be i n volved in the signaling mechanisms that regulate the sequential developmental processes of dendrogenesis and myelinogenesis in the CNS. It was recently observed (14,15) that intracisternal injection of antiganglioside antibodies into 5 day-old rats caused chemical, morphological, and behavioral changes in the adult animals. Chemical studies of somatosensory cortex revealed decreases of about 30% (p 60 40 Η 20 h 0 60 40 20

U M

0^5

J IHÏÏS

J

J

16^20 2^30 31-40 41-50 >50

LACTOSYL CERAMIDE

La

. . . . . . . . Figure 1. Natural antibodies in normal human and rabbit sera against liposomes containing no glycolipid, galactosylceramide, or lactosylceramide. Glucose release % OF TRAPPED measured from liposomes containing DMPC, GLUCOSE CHOL, RELEASED DCP, and, where indicated, galactosylceramide (150 pg/pjnol PC) or lactosylceramide (150 μg/μmol PC). Closed bars, humans; open bars, rabbits.

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TABLE I . HUMAN AND RABBIT NATURAL ANTIBODIES AGAINST DIGALACTOSYL DIGLYCERIDE AND CERAMIDE TRIHEXOSIDE HUMAN % OF TRAPPED GLUCOSE % RELEASED REACTING

GLYCOLIPID

Digalactosyl diglyceride Ceramide trihexoside

3

6.1±1.7 (17)

76

2.3±3.6 (17)

12

RABBIT % OF TRAPPED GLUCOSE % RELEASED REACTING 3

9.8±

8.1(42)

69

0

46.7110.8(12) 100

0

a

E x p r e s s e d a s : mean±standar ^Present i n the liposomes a t 150 yg per ymole of p h o s p h o l i p i d . P r e s e n t i n the liposomes a t 150 nmoles per ymole of phospholipid. c

Natural a n t i b o d i e s were p u r i f i e d from r a b b i t serum by a f f i n ­ i t y b i n d i n g to liposomes (6,12). B r i e f l y , t h i s i n v o l v e d adsorb­ ing the a n t i b o d i e s from the serum onto liposomes containing the a p p r o p r i a t e g l y c o l i p i d , washing the liposome-antibody complexes f r e e of unreacted serum, then e l u t i n g the a n t i b o d i e s from the liposomes i n 1M Nal. Both anti-CDH and anti-CTH were i s o l a t e d from the same batch of normal r a b b i t serum and were compared for s p e c i f i c i t y . As shown i n Figure 2, the anti-CDH d i d not react with CTH-containing liposomes. In c o n t r a s t , the anti-CTH d i d r e a c t with CDH liposomes (Figure 3 ) , though t o a l e s s e r extent than d i d the anti-CDH. Since no anti-CMH a c t i v i t y was observed i n the whole serum, the p u r i f i e d a n t i b o d i e s were not tested a gainst this antigen. A somewhat d i f f e r e n t pattern o f r e a c t i v i t y was observed with p u r i f i e d a n t i b o d i e s obtained from r a b b i t s immunized with CDH or CTH. As Figure 4 shows, immune anti-CDH a n t i b o d i e s d i d c r o s s - r e ­ a c t with CMH. This observation i s i n contrast to the l a c k of c r o s s - r e a c t i v i t y observed with the n a t u r a l antibody (see above), but i s i n agreement with the r e s u l t s of Arnon et^ a l . (13) ob­ tained with r a b b i t s that were immunized with l a c t o s y l s p h i n g o s i n e conjugated to a polypeptide. The immune anti-CTH studied here showed l i t t l e or no r e a c t i v i t y with CMH. The normal human sera shown i n Figure 1 a l s o were tested a g a i n s t four g l y c o l i p i d s (globoside, Forssman, a s i a l o - G ^ * °M2) having terminal N-acetylgalactosamine residues (Table I I ) . The f i n d i n g that a l l the i n d i v i d u a l human sera had a c t i v i t y against globoside (Table II) was s u r p r i s i n g , since globoside i s the major g l y c o l i p i d of human e r y t h r o c y t e s , and only i n d i v i d u a l s of the r a r e ρ and P^ blood types l a c k globoside (14). Several, but not a l l , of the i n d i v i d u a l s tested a l s o had a c t i v i t y a g a i n s t Forssman g l y c o l i p i d , as reported p r e v i o u s l y (1,15). This l a c k of c o r r e l a -

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against

Glycolipids

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Figure 2. Reactivities of purified natural anti-CDH and anti-CTH antibodies against CTH-containing liposomes. Glucose release measured from liposomes con­ taining DMPC, CHOL, DCP, and CTH (50 nmol/'μίηοΐ PC) (6).

ELUTED ANTIBODIES (μ\) Immunochemistry Figure 3. Reactivities of purified natural anti-CDH and anti-CTH antibodies against CDH-containing liposomes. Glucose release measured from liposomes con­ taining DMPC, CHOL, DCP, and CDH (154 nmol/^mol PC) (6).

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Lu CO