Glycolipids

GLYCOLIPIDS

New Comprehensive Biochemistry

Volume 10

General Editors

A. NEUBERGER London

L.L.M. van DEENEN Utrecht

ELSEVIER AMSTERDAM. NEW YORK . OXFORD

G1ycolipids

Editor

H. WIEGANDT Marburg/ Lahn

1985

ELSEVIER AMSTERDAM. NEW YORK.OXFORD

0

1985 Elsevier Science Publishers B.V. (Biomedical Division)

All rights reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise without the prior written permission of the publisher, Elsevier Science Publishers B.V. (Biomedical Division), P.O. Box 1527, lo00 BM Amsterdam, The Netherlands. Special regulations for readers in the USA: This publication has been registered with the Copyright Clearance Center Inc. (CCC), Salem, Massachusetts. Information can be obtained from the CCC about conditions under which the photocopying of parts of this publication may be made in the U.S.A. All other copyright questions, including photocopying outside of the U.S.A., should be referred to the publisher. ISBN for the series: 0-444-80303-3 ISBN for the volume: 0-444-80595-8

Published by: Elsevier Science Publishers B.V. (Biomedical Division) P.O. Box 211 lo00 AE Amsterdam The Netherlands

Sole distributors for the USA and Canada: Elsevier Science Publishing Company, Inc. 52 Vanderbilt Avenue New York, NY 10017 U.S.A.

Library of Congress Cataloging in Publication Data Main entry under title: GIycolipids. (New comprehensive biochemistry; v. 10) Bibliography: p. Includes index. 1. Glycolipids. 1. Wiegandt, H. 11. Series. [DNLM: 1. Glycolipids. W1 NE372F v. 10/ QU 85 G56861 QD415.N48 vol. 10 574.19’2 s [574.19’247] 84-21266 [QP752.G56] ISBN 0-444-80595-8 Printed in The Netherlands

Preface

Fundamental to all living cells is the utilization of membranes, that basically comprize of lipid as the main barrier to an aqueous environment. Therefore, a multiplicity of regulated modulations of the interphase between lipid and water is necessary to enable the membrane to perform its specialized functions; amongst many others these include provisions for cell communications and membrane rearrangements. The glycolipids, as judged by the ubiquity of their occurrence in all cells, and their special physicochemical properties as well as their strategic positioning (frequently at the outer cell surface membranes), appear to be molecules particularly well suited to serve as links at the lipid-water membrane interphase. Indeed, glycolipids are enabled to mediate between the hydrophilic and the lipophilic environments because of their unique constitution, the molecular combination of a hydrophdic carbohydrate and a lipophilic aliphatic hydrocarbon chain residue. Positioned in the lipid bilayer, the glycolipids can, with their lipid ‘tails’, dramatically influence the properties of biological membranes, as exemplified in the haloand thermophilic organisms. In addition, many glycolipids carry very complex carbohydrates that may enable highly specialized interactions towards the aqueous environment. The obvious multitude of modulatory requirements at the membrane interphase is possibly reflected by the diversity and variability of the structural constitutions of the glycolipids. Still, most glycolipids can be classified into three major groups, and are distinguished by the molecular entity to which the carbohydrate moiety is directly linked. These groups are: the sphingo-lipids, including their sialic acid-containing components, the gangliosides; and furthermore, the glycero- and isoprenol-glycolpids. Whereas the functional significance of the isoprenol-glycolipids may reside in their ability to mediate the transport of carbohydrates through lipid membranes as part of the biosynthesis of glycoproteins, the sphmgo- and glyceroglycolipids appear to serve more directly as fundamental membrane constituents.

VI

Glycolipids have received special attention in several areas of medical interest. Besides their participation in the immunological expression of cells (they may be involved in storage diseases), they have been implicated to play an important role in the regulation of the social behavior of cells, including cancer, and some of them have been even found to be therapeutically useful agents in the treatment of neurological disorders, such as peripheral nerve injury and other peripheral as well as central neuropathies. The advances in biochemical methodology in recent years has also considerably increased knowledge of the glycolipids; in fact, to such an extent that it is becoming difficult to find an easy access to all available information. With the present volume, we have attempted to describe, the main groups, and to collate the present knowledge of the glycolipids. This has been done, however, not only with the intention of reviewing the more recent advances, but also to allow for the interested nonspecialist reader to become introduced to the respective fields. In addition, in accordance with the title of the New Comprehensive Biochemistry series, some attempt was made to make the present volume as comprehensive as seemed reasonable, in order to be useful as a reference source of the most relevant hitherto published data on glycolipids.

H. Wiegandt Department of Biochemistry School of Medicine Philipps University Marburg an der Luhn F.R. G.

Contents Preface by H . Wiegandt . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

V

Chapter 1. Glycosphingolipids, by A . Makita and N . Taniguchi . . . . . . . . . . . . .

I

........................................

..........................

1 2 4

3.4. High performance liquid chromatography . . . . . . . . . . . . . . . ............................ 3.5 Determination of GSL constituents

6

1. Introduction

.....

3.2. Fractionation.

3.7.

............................

3.5.2. Sphingoid bases

.....................

6

Mass spectrometry of whole GSLs .

.....................

9

3.11. Radiolabelling of GSLs ........................ pports and macromolecules . . . . 3.12. Covalent attachment of 3.13. Immunological procedu ................ 4. The lipophilic moiety of GSLs ........................................ 4.1. Long-chain bases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

........................ 5.1. Gala series . . . . . . . . . . . . . . . . . . . . . . . . . .

15 15 16

...............

16

.................

19

5.2.1. Glucosylceramide . . . . . . . . . . . . . . . . . 5.2.2. Glucocerebroside-ester . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.3. Lactosylceramide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. Globo and isoglobo series . . . . . . . . . . . ........................... 5.3.1. Globotriaosylceramide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

20 20

VlII 5.3.2. Globoisotriaosylceramide. . . . . 5.3.3. Isoglobotetraosylceramide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

5.4.

Ganglio series . . . . 5.4.1. Gangliotriaosy

5.5.1. 5.5.2. 5.5.3. 5.5.4. 5.5.5. 5.5.6.

...............................

Lactotriaosylceramide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Neolactotetraosylceramide. . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lactotetraosylceramide . . . .. ... 1V3-a-Galactosyl-neolactotet ........................... IV3-~-Galactosyl-neolactotetraosylceramide . .. . ...... . . . .. . . . . . . . . . . . . IV4-a-Galactosyl-neolactotetraosylceramide. . ... . .

...............................

5.5.9. Neolactodecaglycosylceramide

........ .........

5.6.2. Lactosylceramide sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.6.3. Sulfated tri- and tetraglycosylceramides . . . 5.7.

Fucolipids . . . . . . . . . . . . . . . . .

........ ...........

5.9.1. Phosphorus-freeglycosphingolipids

...................................... 6.3. 6.4. 6.5.

........................ ...........

Biosynthesis of glucosylceramide . . . . . Biosynthesis of di- and trihexosylceramides . . . . . . . . . . a and 8-N-Acetylgalactosaminyltransferasesinvolved in

...........

....................................

substance . . . . . . . . . . . . . .

6.9.

Galactosylceramide sulfotransferase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1 .lCeramidase .

... . , .. ..... . ... . . . .. .

7.1.7. a-Fucosidase

................... ................................

.

....................................

7.2.2. Activator protein for the hydrolysis of 8-glucosides . .

. . . . .... . . . . .

21 21 21 22 22 23 23 23 24 24 24 25 25 25 26 26 26 26 26 27 27 27 28 36 38 41 42 43 43 44 45 46 46 47 48 48 50 50 51 51 51 52 53 53 54 54 55 55 56 56

IX 7.2.3. Activator protein for the hydrolysis of ganglioside II'NeuAc-Gg,Cer ....................... 7.2.4. Activator for arylsulfatase A 7.2.5. Transfer proteins ........................................ 7.3. Metabolic disorders of glycosphingol ................. ........................... 8. Glycosphingolipids in immunology . . . . . 8.1. Human blood group systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ................ 8.1.1. ABO system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.1.2. Lewis system .......................................... 8.1.3. lisystem . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . ................................ 8.1.4. P system . . . . . . . 8.2. Heterophile antigen lycosphingolipids . . . . . 8.3. Stage-specific embryonic antigens . . ............................ .... , and effects on the antigens of lectins and 8.4. GSL antigen marke ...................... differentiation inducers . . . . . . . , . . . . . . . . . . . . . . 8.5. Antigenicity of simple glycosphingolipids and possible olvement of neutral and acidic ... GSLs in autoimmunization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9. Glycosphingolipid changes in transformation and malignancy . . . . . . . . . . . . . . . . . . . . . . . . 9.1. Glycosphingolipid pattern and metabolism in transformed cells and their possible relationship to cell behaviors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2. Glycosphingolipid changes in tumor tissues and GSLs as possible tumor markers . . . . . . ................................ References . . . . . . . . . . . . . . , . .

56 57 57 51 59 60 60 62 63 65 67 68 69 12 73 13 77 82

Chapter 2. G[ycoglycerolipids,by I. Ishizuka and T. Yamakawa . , . . . . . . . . . . 101 1. Introduction . . . . . . . . . . . .

2. Structure . . . . . . . . . . .

................... ...............

. . . . .... ....................... .......................

..................... ....................... 3.1. Plant . . . . . . . . . . . . . . 3.1.1. Tissue distribution 3.1.3. Differentiation . . . . . .

................. ....................

..............

........................ ......................

3.2.5. Growth stage . . . . . . . . . . .

.......... ........................... ......................... ............ 3.3.2. Regional distribution . . . . . . . . . . . . . . . . . . . . . . .

101 101

104 104 105 105 106 106 112 121 122 125 127 129 132 132 132 133 134 135 135 137 138 139 140 141 141 141

X 3.3.3. Developmental variations. . . ...................... .. 3.3.4. Turnover of lipophilic domain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... ..... ... 3.3.5. Location in myelin 3.3.6. Hormonal regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Germcell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.1. Distribution 3.4.2. Location in g ..............................

142 142 143 144 144 144 145 145 146 3.5. Secretion of animal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147 3.6. Molecular evolution of glycoglycerolipids . . . . . . . . . . . 147 3.6.1. The concept of molecular evolution ........... 147 3.6.2. Evolutionary convergence and adap ....................... 148 3.6.3. Phylogenetic divergence of domains in glycoglycerolipids . . . . . . . . . . . . . . . . . . 149 4. Metabolism 149 ................................ 150 4.1.1. Synthesis of lipophlic domain . . ..................... 150 4.1.2. Transfer of reducing carbohydrates . . . . . . . . . . . . . . . . . . . . . . 153 4.1.3. Transfer of sulfate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155 4.1.4. Specificity of transferase to lipophilic domain . . . . . . . . . . 156 4.1.5. Transfer of sn-glycerol-1-phosphate and ribitol e. . . . . . . . . . . . . . . . . 158 4.1.6. Location of enzyme activity . . . . . . . . . . . . . . . . . . . . , , . . . . . . . . . . . . . . . . . 160 162 164 4.2. Biodegradation 166 166 166 . . . . . . . . . 168 5. Biological property . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169 170 5.1.1. Lipophilic domain . . . . . . . . . . . . . . . 170 5.1.2. Micelles . . . . . . . . . . . . . . . . . . . . . 170 5.1.3. Unsaturation of lipophlic domain . . . . . . . . . . . . _ _ _ _ . . . 171 . 5.1.4. Electric charge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172 5.1.5. Seminolipid . . . . . . . . . . .................................. 172 5.1.6. Macroglycolipid . . . . . . . . . . . . . . . . . 172 173 5.2.1. Integration of membrane . . . . . ............................... 173 5.2.2. Ion trap . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175 5.3. Interaction with protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175 176 177 178 179 ....................... 179 180 180 5.5.1. Interaction with cations . . . . . . . . . . . . . . . . . 180 181 182 Acknowledgement . . . . . . . . . . . . . . . . . . 183 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184

XI

Chapter 3. Gangliosides, by H. Wiegandt . . . . . . . . . . . . . . . . . . . . . . . . . . . 199 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Chemistry, physics and methods of preparation and analysis . . . . . . . . . . . . . . . .

2.4.1. Alteration of the ceramide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2.6. Physicochemical characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6.1. General . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6.2. Gangliosides in solution . . ....................

.........................................

es . . . . . . . . . . .

3.2.3. Peripheral nerves . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Cellular localisation . . 4. Metabolism . . . . . ...................... 4.1. Biosynthesis 4.2. Biodegradatio 4.3.1. Developmental changes . . . . . . ... . .. ......... 4.3.2. Changes after nerve stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.3. Temperature-adaptive changes in the brain . . . . . . . . . . 5. Immuno-properties of gangliosides . . . . . ............. 5.1. Gener. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

5.2. Involvement in disease .............................. 5.2.1. Animal models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.2. Anti-ganglioside immune activities in human pathology . . . . . . . . . . . . 6. Ligand-binding properties of gangliosides . . . . . . . . . . 6.1. General . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. Ganglioside complexing with ligand protein 6.3. Interaction with lectins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4. Interaction with toxins, hormones, interferon and cell growth and differentiation factors . 6.5. Interactions with .......................... 7. Concludingremarks . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

199 200 200 202 205 205 205 208 210 210 211 219 219 220 221 224 224 224 226 226 226 221 228 229 229 230 232 232 233 234 234 234 238 238 239 239 239 240 240 240 241 241 244 245 245

Chapter 4. Glycosyl phosphopolyprenols, by F. W. Hemming . . . . . . . , . . . . . . . 261 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Polycis-isoprenoid alcohols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

261 262

XI1

s. nomenclature and methods

.......................... ........

2.1.3. Eukaryotic polycis-prenols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Metabolism of polycis-prenols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.1. Formation and hydrolysis of phosphoryl derivatives . . . . 2.3. Biosynthesis of polycis-prenols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2

2.3.2. Eukaryotic cells

...............................................

3.1. Nomenclature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. General principles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Phosphopolycis-prenols in prokaryotic glycosyl transfer . . . . . . . . . . . . . . . . . . . . . . . . 3.3.1. General principles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.2. Formation of peptidoglycan . . . . . . . . ...... 3.3.3. Formation of 0-antigen determinants and capsular ex 3.3.4. Formation of teichoic acids and related compounds . . . . . . . . . . . . . . . . . . . . . . 3.3.5. The formation of other bacterial polysaccharides . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Phosphopolycis-prenolsin eukaryotic glycosyl transfer . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.1. General . . . . . . . . . . . . . . . . . . . . . . . . . ......... 3.4.2. N-Glycosylation of proteins in animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.3. N-Glycosylation of proteins in plants . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.4. 0-Glycosylation in plants .................................... 3.4.5. Miscellaneous glycosyl phosphodoli 3.5. Phosphoretinol in glycosyl transfer . . . . . . . .......................... 4 . The control of phosphopolyprenol-mediatedglyc 4.1. The significance of controlling the process 4.2. Manipulation by administration of antibioti ................ 4.2.1. Bacitracin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.2. Tunicamycin ....................................... 4.2.3. 2-Deoxyglucose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.4. Other antibiotics . . . . . . . . . . . . . . . . . . . . ........ 4.3, Variation in the concentration of phosphopolypre 4.3.1. General . . . . . . . . . . . . . . . . . . . . . . . ....................... 4.3.2. Control of the biosynthesis of phosphopol 4.3.3. The association of phosphopolyprenols wi 4.3.4. Changes in concentration of phosphodolichol during development . . . . . . . . . . . . 4.4. Changes in phosphopolyprenol-mediatedglycosylation in mutant cell lines . . . . . . . . . . . 4.5. The effect of analogues of natural phosphopolyprenols on glycosylation 5 . Summary . . . . . ............................... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

SubjectIndex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

262 262 263 263 265 265 268 268 268 271 271 271 273 213 273 275 277 278 279 279 280 286 287 288 289 290 290 291 291 291 294 294 295 295 295 296 297 297 298 298 299

307

Wiegundt (ed.) Glvcolrprd~~ I985 Elseoier Science Publisher.\ B. V. (Biomedical Diursron)

3:

CHAPTER 1

Glycosphingolipids * AKIRA MAKITA and NAOYUKI TANIGUCHI Hokkuido University School of Medicine, Sapporo 060, Japan

I . Introduction Glycosphingolipids (GSLs) are composed of a long-chain base (sphingoid), a fatty acid, and a carbohydrate. The hydrophobic moiety, which is a ceramide, consists of the long-chain base substituted at the amino group by a fatty acid. The carbohydrate moiety is linked at the primary hydroxy group of the sphingoid base, e.g., sphingosine (sphing-4-ene): N - Acyl-sphingosine (ceramide)

Glycosphingolipid

CH,(CH,),,-CH=CH-CH-CH-CH, CH,(CH,),,-CH=CH-CH-CH-CH, I l l I l l OH NH OH

I

R

=

Fatty acid

OH NH 0

I

I

co

co sugar

I R

I R

The lipophilic moiety of GSLs shows microheterogeneity, and the GSLs of particular animal species and organs have characteristic lipid distribution patterns. Although the GSLs of mammalian tissues and cells have been most extensively studied, GSLs are also known to be present in organisms other than vertebrates, such as molluscs, plants and even microorganisms, in which their constituents and structure differ in varying degrees from those of mammals (see Section 5.9). GSLs are both species and tissue specific with regard to their qualitative and quantitative distribution patterns. In cells GSLs exist mainly as components of cellular membranes, especially of cell surface membranes. Their hydrophobic moiety embeds in the lipid bilayer, while the carbohydrate moiety extends to the outside. Essentially all of the GSLs are antigenically active, and one of their biological

* Sialic acid-containing glycosphingolipids (the gangliosides) are discussed in a separate chapter (see Chapter 3).

2

properties is that they act as immunogens (see Section 10). Some of the GSLs play roles as cell receptors for bacterial toxins and possibly also for bacteria and virus (see the chapter on gangliosides). Although all the biological properties of GSLs are intimately associated with their carbohydrate moiety, their lipid moiety, which makes the GSL molecule amphipathic, is essential to strengthen such biological properties as antigenicity [1,2], selective glycosylation [3,4], and possibly the organization and orientation of the carbohydrate chains [ 5 ] .

2. Classification and nomenclature GSLs usually are classified with respect to the chemical structures found in their carbohydrate moiety. This includes the number and species of the constituent monosaccharides, their sequence, positional and anomeric linkages, and other components such as sulfate (“sulfatides”) or sialic acid (“gangliosides”). The latter group will be dealt with in a separate chapter. Although numerous GSLs have been assigned trivial names derived from their history, the nomenclature and abbreviations recommended by the IUPAC-IUB Nomenclature Commission (1977) [6] cover semi-systematically the structures of most GSLs and are used in this chapter as much as possible. The GSL series with two novel core carbohydrate sequences have been demonstrated recently in nonvertebrates (Section 5.9). These GSLs are classified newly into arthro (a name derived from arthropod) series and mollu (a name derived from mollusc) series in this chapter. In this nomenclature system, the principal classifications based on the skeletal structure of the carbohydrate moiety are indicated by prefixes as follows: Prefix

Abbreviation

Structure

globo

Gb

Gal( /?1 + 3)GalNAc( j3l 3)Gal(a1 4)Gal(b l + 4)Glc -+

-+

isoglobo

iGb

GalNAc( bl + 3)Gal(a1 + 3)Gal(/3l

lacto

Lc

(Gal( /3l 3)GlcNAc),,(bl + 3)Gal(/3l+ 4)Glc

neolacto

nLc

(Gal(b1- 4)GlcNAc)”(/3l-+3)Gal( pl-+ 4)Glc

-+

4)Glc

-+

GalNAc( /3l + 4)Gal( pl 3)GalNAc(81 .-, 4)Gal( /31--$4)Glc

ganglio

-+

gala

Ga

Gal( a1

arthro

Ar

GalNAc( a1 + 4)GalNAc( 81 -+ 4)GlcNAc( p l + 3)Man( P l - 4)Glc

mollu

MI

Fuc( a1 + 4)GlcNAc( bl-+ 2)Man(a1 + 3)Man( bl 4)Glc

-+

4)Gal

-+

3 The number of monosaccharide units in an oligosaccharide is indicated by the suffixes “-biaose”, “-triaose”, “-tetraose” etc. [6]. For example, globoside is designated as globotetraosylceramide, and the corresponding GSL with one less monosaccharide is globotriaosylceramide (refer to Table 1.3). Differences in linkage position (e.g., 1 -,4 uersus 1 -, 3) in an otherwise identical sequence are indicated by the prefixes “iso-” or “neo-”, as in isoglobotetraosylceramide (refer to Tables 1.1 and 1.3). The prefixes tabulated above imply the entire structure of the root oligosaccharide (family) of the GSLs, including the order of the sugars and the position and anomeric configuration of the glycosidic linkages. With regard to shorthand notations for GSLs, the symbols Cer for ceramide, Sph for sphingoid base [6], and the recommended symbols for the hexoses (Gal, Glc, etc.) [7] have been adopted. Galactosylceramide therefore is abbreviated GalCer, and lactosylceramide LacCer. For complex GSLs, oligosaccharides are represented by specific symbols in which the number of monosaccharide units (-oses) is indicated by Ose,, preceded by two or three letters of the family name of the oligosaccharide (Gg, nLc, etc.); for example, gangliotriaosylceramide is GgOse,Cer which may also be abbreviated as Gg,Cer. Examples of GSLs representing the structure and abbreviation in this way are shown in Table 1.1. For GSLs with five or more glycose units of either straight or branched sugar chains, the nomenclature and abbreviations [6] recommended for fucolipids and gangliosides are employed in this chapter. The location of a glycose residue is indicated by a Roman numeral (counting from the ceramide end) designating the position at which the residue is attached to parent oligosaccharide, and by an Arabic numeral superscript indicating the position within that parent sugar residue to which the glycose is attached. The anomeric symbol follows the Roman numeral and precedes the (specified) “glycosyl-”. Therefore, 4N-acetylglucosaminylfll -, 3galactosylpl -, galactosylal + 3galactosylfll 4glucosylceramide (refer to Table 1.3) is written as IV3-a-galactosylneolactotetraosylceramide and abbreviated as IV3aGal-nLc,Cer, while galactosylal -, 3galactosyl

-

TABLE 1 . 1 Examples of names and abbreviations of di-, tetra- and pentaglycosylceramides Structure

Name of GSL

Abbreviation

GalPl + 3GalNAcPl+ 3Galal + 4Gal/31+ 4GlcCer G a l N A c P l - + 3Galal- 3GalPI + 4GlcCer GalPl + 3GlcNAcPl+ 3 G a l P l - + 4GlcCer G a l P l + 4GlcNAcPl+ 3Gal/31+ 4GlcCer GalNAcPl + 4GalPl + 3GalNAcPl+ 4GalPl+ 4GlcCer Gala1 + 4GalCer Fucal + 4GlcNAcPl 2Manal + 3ManPl + 4GlcCer GalNAcal + 4GalNAcPl -+ 4GlcNAcPI 3Manpl -,4GlcCer

Globopentaosylceramide lsoglobotetraosylceramide Lactotetraosylceramide Neolactotetraosylceramide Gangliopen taosylceramide Galabiaosylceramide Mollupentaosylceramide

Gb, Cer iGb,Cer Lc,Cer n Lc, Cer Gg,Cer Ga,Cer MI Cer

Arthropentaosylceramide

Ar,Cer

-

-+

A A sole mollupentaosylcerarnide has not been isolated, but the lower and higher homologues are shown in Table 1.5.

4 ( 2 + alfucosyl)/31 + 3N -acetylglucosaminyl/31 3galactosyl/3l 4glucosylceramide becomes IV2-a-fucosyl-IV3-~-galactosyllactotetraosylceramide which is abbreviated IV2aFuc-IV3aGal-Lc4Cer. -+

-+

3. Preparation and analysis Although carbohydrates compose nearly half the molecular weight of a trihexosylceramide, and GSLs having three or more glycose units are soluble in water, GSLs are prepared according to the methods used for the isolation of such lipids as phospholipids. The procedure for the preparation of GSLs consists of lipid extraction from the tissue, removal of lipids other than GSLs, particularly phospholipids, and separation of the individual GSLs. Recent methods for the isolation and characterization of of GSLs [8], including other useful procedures [9,10], have been summarized. 3.1. EXTRACTION

Tissues or cells are homogenized directly with 19 volumes of chloroform-methanol (2 : 1, v/v) [ll]. The residue is often extracted further with chloroform-methanol (1 : 1) and (I : 2) to ensure complete extraction, and the extracts are combined. For a large scale preparation of GSLs, the tissue or erythrocyte ghost is homogenized with acetone, and essentially all the simple lipids are removed by filtration. The acetone powder is extracted for 10-30 min [12] with pure or 90% ethanol near its boiling point. Polyglycosylceramides, which contain very long carbohydrate chains, were extracted with phosphate buffer-butanol from the ethanol-extracted erythrocyte ghosts ~31. 3.2. FRACTIONATION

The chloroform-methanol extract is adjusted to a solvent ratio of 2 : 1 by the addition of chloroform, and 0.2 vol. of water are added [ll]. After partition, most gangliosides and neutral GSLs with long carbohydrate chains are recovered in the upper phase. Mono- to pentaglycosylceramides, most of the cerebroside sulfate and lipids other than GSLs, and a portion of the less polar gangliosides are recovered in the lower phase. Folch’s partition [ll] is best suited for GSL fractionation from adult brain tissue which contains little neutral GSL that has two or more glycose units but contains the more complex and freely water-soluble gangliosides. The lipid extract, which contains the bulk of the phospholipids, is subjected to mild alkaline hydrolysis [ 141, or peracetylation [ 151 followed by Florisil (activated magnesium silicate) column chromatography, after which the native GSLs are recovered by deacetylation with sodium methoxide [16]. Most of the phospholipids can also be removed by chromatography on silicic acid from which neutral GSLs, cerebroside sulfate and considerable amounts of the less polar gangliosides are eluted with

5

acetone-methanol (9 : 1, v/v) while phospholipids remain on the column [17]. Separation of neutral GSLs from acidic GSLs is achieved by chromatography on a diethylaminoethyl (DEAE) Sephadex column. Neutral GSLs are eluted with chloroform-methanol-water (30 : 60 : 8 by volume) and acidic GSLs with chloroformmethanol-0.8 M sodium acetate (30 : 60 : 8 by volume) [18]. It is probably not wise to directly apply GSL mixtures containing large amounts of phospholipids to a DEAE-Sephadex column. A DEAE-silica gel column may also be useful [19]. Sulfoglycosphingolipids in the acidic GSL fraction can be fractionated by chromatography on silicic acid. Non-lipid low-molecular weight substances are removed by dialysis against water or by gel filtration on a Sephadex G-25 column using chloroform-methanol-water (60 : 30 : 4.5 by volume) [20] at any step in the preparation. 3.3. ISOLATION OF INDIVIDUAL GLYCOSPHINGOLIPIDS

Isolation of individual GSLs is almost exclusively achieved by chromatography on silicic acid. The chromatography is performed by either stepwise elution with increasing proportions of methanol in a chloroform-methanol solution, or gradient elution with increasing concentrations of methanol and water in a chloroformmethanol-water system [21]. By the use of porous silica gel spheres (Iatrobeads), clear separations of mono- to tetraglycosylceramides were achieved with linear gradient elutions using a chloroform-methanol-water system [21]. To monitor the chromatographic separation of GSLs on a column, the color reaction of hexose and/or thin layer chromatography are used to examine an aliquot of the eluate. A GSL often appears as a double (or multiple) spot on thin layer chromatography; the fast-moving species include predominantly longer-chain fatty acids with 22-26 carbon atoms, and the slow-moving species with 16-18 carbon atoms and, if present, a-hydroxy acids. Separation of a GSL mixture which migrates closely during chromatography, such as isomers with the same number of glycose residues, can be achieved by repeated chromatography, or by chromatography on a column or a preparative thin layer plate of peracetylated GSLs using a less polar solvent mixture. Galactosylceramide and glucosylceramide are separated on a borate-impregnated thin layer plate [22], while there is no way at present to separate galabiosylceramide from lactosylceramide (for their structure see Table 1.3). For thin layer chromatography of a complex mixture of GSLs, two-dimensional chromatography is useful [23]. A GSL preparation obtained by repeated silica gel chromatography is often contaminated with “soluble” silica gel. Non-lipid contaminants can be effectively removed by passing an aqueous solution of the preparation through a hydrophobic column (Sep-Pak C18) [756]. GSL can be obtained in an amorphous, white solid form by dissolving at warm temperature in a minimum volume of methanol followed by precipitation with acetone. 3.4. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

Due to its rapid and fine separation, high performance liquid chromatography has proven to be of considerable use in the isolation and quantification of GSLs. Most

6

of the presently available apparatus is equipped with a UV detector and a refractometer. Benzoylated GSLs [24] can be used for detection of minute amounts of GSLs (on the order of nmol) using a UV monitor. With the use of high performance liquid chromatography, neutral GSLs containing one to four glycose residues were quantitatively separated as their perbenzoylated [25] and 0-acetyl-N-p-nitro-benzoylated [26,27] derivatives. Without derivatization, both five globo series GSLs with monoto pentaglycosyl residues and five blood group H-active GSLs with penta- to tetradecaglycosyl residues were separated by elution with a 2-propanol-hexane-water system in a high performance liquid chromatography apparatus. However, detection was made manually [28]. For widespread application to the separation and quantification of GSLs, improvement of detectors such as the moving wire flame ionization detector [29] or development of new detection devices seems necessary. 3.5. DETERMINATION OF GSL CONSTITUENTS

3.5.I . Fatty acids The purified GSL is methanolyzed and fatty acid methyl esters are extracted with hexane [576]. The methyl esters are analyzed by gas chromatography [576]. a-Hydroxy acid esters, if present in the GSL, can be separated from the straight-chain acid esters using a preparative thin layer plate * developed with hexane-diethyl ether (85 : 15, v/v) followed by extraction with diethyl ether and acetylation or trimethylsialylation to convert the hydroxy acid esters to volatile derivatives [8].

3.5.2. Sphingoid bases After removal of the fatty acid esters by hexane extraction, the methanolysate of GSL is made alkaline, and sphingoid bases are extracted with diethylether. Following periodate oxidation, the sphingoid bases are analyzed in the form of long-chain aldehydes [30] or trimethylsilyl derivatives [31] by gas chromatography. It should be noted that periodate oxidation of a dihydroxy sphingoid base with n carbon atoms yields the same aldehyde as does a trihydroxy base with n + 1 carbon atoms. The concentration of sphingoid bases can be estimated by colorimetry of a complex with methyl orange [32], or more sensitively either by a fluorometric procedure using fluorescamine after differentiation of the hexosamine amide [33,34] or by a radioisotopic method after N-acetylation with I4C-labeled acetic anhydride [86]. 3.5.3. Carbohydrates The composition of hexoses, including fucose and hexosamines. can be estimated by gas chromatography of their trimethylsilyl methyl glycoside derivatives [35]. In this

* The fatty acid methyl ester fraction is often contaminated with phthalic acid esters, such as plasticizer, which may come from the organic solvents used and which interfere heavily with fatty acid analysis by gas chromatography. Introduction of thin layer chromatography before gas chromatography can remove completely the contaminants, which are left at the solvent front.

7

case, the methanolysate of the GSL, after extraction of the fatty acid esters, is neutralized by an anion exchanger [36], and hexosamines (and sialic acids, if necessary) are re-N-acetylated with acetic anhydride and trimethylsilylated. To estimate the content of individual monosaccharides, the methanolysate is supplemented with a known amount of mannitol as an internal standard, and then processed as above. Another valuable technique for carbohydrate determination is gas chromatographic estimation of alditol acetates [37], whch are prepared by a procedure involving acid hydrolysis of the GSLs, removal of fatty acids, reduction of monosaccharides with NaBH,, and peracetylation. 3.6. DETERMINATION OF CARBOHYDRATE STRUCTURE

The structural determination of complex carbohydrates involves three principles; sequence, linkage position and anomeric configuration. 3.6.1. Analysis of sequence and anomeric configuration

Specific exoglycohydrolases are employed to determine simultaneously the sequence and the anomeric configurations of the carbohydrate moiety in a GSL [38,39]. Many of the exoglycosidases so far characterized from invertebrate sources are specific for the anomeric configurations in the respective glycosidic linkages, but do not differentiate positional isomers. By sequential treatment of a GSL with the respective exoglycosidases in the presence of a detergent, and identification of the products (mostly the ceramide-linked products are subjected to thin layer chromatography) with or without inactivation of the glycosidase at each hydrolysis step, the sequence and the anomeric configuration of the GSL are determined simultaneously [38,39]. The enzymes, such as a- and P-galactosidases, P-N-acetylhexosaminidase, a-Nacetylgalactosaminidase, P-glucosidase. and a-L-fucosidase, can be prepared from a variety of sources according to published procedures [9] and are commercially available. An endo-P-galactosidase from Escherichia freundii catalyzes the hydrolysis of the inner P-galactosidic linkages of GSLs of the lacto series [40], and this enzymatic hydrolysis was adopted for characterization of GSL sugar chains containing a repeating N-acetyllactosamine (GalP1 + 4GlcNAc) unit [41]. Treatment of acetylated hexopyranosides with CrO, easily oxidizes the P-glycosidic glycose units but only very slowly oxidizes the a-glycosidic ones, providing a valuable procedure for determination of anomeric configurations [42]. Gas chromatographic analysis of the composition of hexoses and hexosamines in the peracetylated GSLs, before and after CrO, oxidation, reveals that a-linked monosaccharides in the GSLs remain almost intact, whereas P-linked ones are considerably reduced [43]. However, when this oxidation method was applied to the GSLs containing 0-methyl monosaccharides which are found in shellfish GSLs, some demethylation occurred (probably enough to give ambiguous results) [343].

8

173

43

e

61 116 158 129

205 7

100

I

f

61

200

116

147 233

43 I

Fig. 1.l. Mass spectra of partially methylated alditol acetates. Blood group A-active hexaglycosylceraisolated from human lung [SO] was permethylated and mide, IV2Fuccr,lV3GalNAccr-nLcOse4Cer. hydrolyzed. The partially methylated monosaccharides were reduced, peracetylated and subjected to a combined gaschromatography-mass spectrometry. a, 2,3,4,-tri-O-methyl-l,5-di-O-acetyl-fucitol (unsubstituted Fuc); b, 3-substituted Gal: c, 4-substituted Glc: d, 2,3-substituted Gal; e, unsubstituted GalNAc; f, esubstituted GlcNAc.

9 3.6.2. Determination of glycoside position Methylation analysis is the most valuable technique for determination of the positions of glycosidic linkages in complex carbohydrates. A particularly useful application of methylation analysis is based on identification by combined gas chromatography and mass spectrometry of the partially methylated alditol acetates derived from the permethylated glycoconjugates (reviewed in Refs. 44 and 45). The GSL in dimethylsulfoxide is permethylated with methyl sulfinyl carbanion and methyl iodide [46,47]. The permethylated derivative of the GSL is then hydrolyzed, and partially methylated monosaccharides are converted to their alditol acetates and identified by gas chromatography or combined gas chromatography and mass spectrometry [48,49]. Relative retention times of partially methylated alditol acetates on a gas chromatogram and characteristic mass fragments are given in Refs. 44,48 and 49. The mass spectra of the partially methylated alditol acetates derived from III*aFuc, II13aGalNAc-neolactotetraosylceramideof blood group A-glycosphingolipid [50] are shown in Fig. 1.1 as examples. Partially methylated hexosaminitol acetates, present at lower than a certain level, usually give a considerably weaker response in gas chromatography than neutral monosaccharides. However, application of a sample containing more than 0.5 nmol of the hexosamine to a column 1.5 m in length or less can largely prevent the preferential loss of hexosamine derivatives [51] during gas chromatography. Periodate oxidation, a classical method for determining glycosidic positions, is still useful, especially in combination with other analytical methods, for the attainment of such information as the glycosidic position, number and species of periodate-sensitive glycoses in GSLs with very complex carbohydrate chains [52]. 3.7. MASS SPECTROMETRY OF WHOLE GSLS

Direct inlet mass spectrometry of GSLs in the form of their volatile derivatives yields considerable information about the number of glycose residues, the approximate sequence of the glycose units (for example, -hexose-hexosamine-hexose-),the nature of the terminal glycose, and the approximate composition of the fatty acid (chain length, hydroxy or nonhydroxy, and double bonds) and sphingoid base. Although the pertrimethylsilylated [53,54] and peracetylated GSLs [55] can be analyzed, the permethylated GSLs which give more stable mass fragments are the particularly useful derivatives [57-591. A direct inlet mass spectrum of permethylated IV 3GalNAca-globotetraosylceramide (Forssman antigen) is shown in Fig. 1.2. In electron impact mass spectrometry, the molecular ion and the fragment ions in the high mass range are scarcely obtained, unless the amide group in the ceramide moiety, and N-acetylhexosamine if present, is reduced with LiAIH, to a substituted amine [60].On the other hand, mass spectrometry of permethylated GSLs [61-631 by chemical ionization, a soft ionization, provides molecular ions which consist of a number of ions due to the heterogeneous composition of fatty acid and sphingoid base, and rarely gives fragments due to ring opening which are often observed in electron impact mass spectrometry.

10

A GSL mixture of permethylated and LiAlH,-reduced derivatives was subjected to temperature programming of the direct inlet probe, which led to successive evaporation of GSL species mainly according to the number of glycoses [64,65]. The mass spectra and the ion curves for selected mass ions of the mixture could, in most cases, be assigned to specific GSLs which were revealed by thm layer chromatography [64,651. Field desorption mass spectrometry [66-681, fast atom bombardment mass spectrometry [69], and secondary (or sputtered) ion mass spectrometry do not require chemical derivatization of the samples. In these techniques, GSLs yield the fragment ions derived from almost successive cleavages of the glycose units at their glycosidic

HexNAc - 0 - H C x N A c

260

i

-

*,

..

’ -0-Hex- O-Hex-O-Hex-, O+Cer

505;

-449

709:

408-

M’1793

913;

1117;

j660

408

%

0

0

200

160

360

400

I%

’ O O P

0

0 1000

1100

1200

1300

Fig. 1.2. Direct inlet mass spectra of permethylated IV3GalNAccr-Gb,Cer(Forssman antigen). (Performed by Dr. S. Gasa at Hokkaido University, School of Medicine.)

11

linkages. Fast atom bombardment mass spectrometry cleaved the amide linkage of the ceramide moiety in the case of lactosylceramide [69]. 3.8. NUCLEAR MAGNETlC RESONANCE (NMR) SPECTROSCOPY

Proton NMR spectroscopy is a valuable means of determining the stereochemical configurations of the anomeric linkages and of H-1 to H-5 of glycopyranoses, the geometrical positions of CH=CH bonds and amides, and other proton signals or GSLs. Anomeric proton signals are clearly separated from other proton signals, and those of the different monosaccharide residues of GSLs were assigned by their chemical shifts using native GSLs [70,71], a triglycosylsphingosine [72] and oligosaccharides [73-751 derived from GSLs, pertrimethylsilylated GSLs [39] and permethylated GSLs [77-791. The use of a high resolution N M R apparatus equipped with a 200-500 MHz resonance frequency magnet facilitated the analysis of protons other than anomeric protons, such as protons H-1 to H-6 in monosaccharide residues of peracetylated [80], permethylated [77-791 and native GSLs [84,85]. By proton N M R spectroscopy of native GSLs in deuterated dimethylsulfoxide solution, amide protons can also be measured, giving structural information about the lipid moiety as well [85,87]. As shown in Fig. 1.3, chemical shifts of anomeric protons of globotetraosylceramide are resonated in a narrow range between 4 and 5 ppm, amide protons between 7 and 7.5 ppm, and methyl protons of an acetamide group at

Gioboslde 1100

9

8

7

6

5

4

I

I

I

3

2

1

1

0

PPM

Fig. 1.3. Proton NMR spectrum of globotetraosylceramide in dimethyl-d, sulfoxide at 110 O C . N-Ac. methyl proton signal of N-acetyl group; GalNAc-NH and Cer-NH. amide proton signals at N-acetylgalactosamine and ceramide. respectively; Olefinic. olefinic proton signals in ceramide moiety. Others are anomeric protons. (Taken by Dr. S. Gasa at Hokkaido University. School of Medicine.)

L

h,

TABLE 1.2 Molar composition of glycosphingolipids by measurement of intensities of amide and anomeric protons a GSL

Required Cer

Glc

GlcCer GalCer LacCer Ga,Cer Gb,Cer Gg3Cer

1

1

Lc,Cer Gb,Cer

GalNAc

GlcNAc

1

1

1 1

1

1

1

1 1

1

1

1

Analyzed Gal

1

1 2 2

1

1

1 2

1 1

Cer

BGlc

1.0’ 1.o 1.0 1.o 1.0 1.0

1.1

1.2 1.2

0.8 1.1 1.1 1.1 1.3

1.0

1.1

1.o

1.0

1.2

1.3

B-Gal

1.1

a-Gal

p-GalNAc

a-GalNAc

8-GlcNAc

1.1

0.9 0.7’ (1.3)

0.9 1.o

1.1

(1.0)

’

(WhC Gg,Cer

1

1

2

1

1.0

1.1

2.3

1.1

(0.9)

nLc,Cer IV ’GalNAcaGb,Cer I ’SO,-GalCer

1 1 1

1 1

2 2 1

1

2

1.0 1.0 1.01

1.4 1.2

2.2

.o

1

1.1 0.9

’

0.9 (0.9)

’

(1.3)

0.8 (0.9)

1.1

The peak intensities of each GSL in a dimethyl-d, sulfoxide solution were integrated in the spectra taken at 110 OC (data taken from Ref. 87). The value was from amide proton. The value was from anomeric proton. For abbreviations of GSLs, see Table 1.3.

a

13

1.85 ppm. The molar composition [87] of ceramide and carbohydrates of GSLs which were measured by integrating the intensities of signals of the amide and anomeric protons, respectively, agrees reasonably with their known compositions (Table 1.2). Spin decoupling in NMR analysis [85] facilitates measurement of particular groups such as the acetyl ester group in a GSL molecule [loll. Carbon 13 NMR spectroscopy, though it requires a fairly large amount of sample, also yields some structural information about GSLs. 13C-NMR spectroscopy of glucosylceramide (300 mg in a CDCl,/CD,OD solution) confirmed the existence of a pyranose ring with /%configuration in the glucosyl moiety, and measured the contents of unsaturated hydrocarbon chains and of a-hydroxy acids in the lipid [89]. The presence of the latter was deduced from the spectra of the geometric orientation. 3.9. INFRARED SPECTROSCOPY

Infrared spectra of GSL commonly show a broad absorption maximum near 3500 cm- due to OH group, whch can be employed to confirm permethylation of GSLs, peaks at 2900 and 2850 cm-' (CH, and CH, respectively), and a peak at 1550 cm-' (NHCO) (Fig. 1.4). Sulfoglycosphingolipids show broad absorption maximum near 1240 (SO,) and 810 cm-' (C-0-S) (Fig. 1.4, lower). Monoglycosylceramides that contain an ester absorb at 1740 cm-' due to the aliphatic ester (Fig. 1.4, middle),

'

s w oI 3000 ,

I

I

V I

I

1

I

I

I

6

I

I

,

I

I

I

10

0

I

700 cm-1

1000 900 800

I

I

I

I

4

2

4000

1400 2000 1600 1200

I

I

I 12

I

I

I

14 B

I

I

I

3000 2000 1000 1600 1400 1200 1000 800 600 (cm-1)

Fig. 1.4. Infrared spectra of GSLs. Upper, glucosylceramide; middle, 6-fatty acyl-glucosylceramide; lower, I 'SO,-GalCer. For absorptions characteristic of functional groups in GSLs, see text.

14

and this peak can be a semi-quantitativeindicator as to whether or not a non-esterified GSL preparation is contaminated with some glycerophospholipids. 3.10. PREPARATION OF OLIGOSACCHARIDES AND OF GLYCOSYLSPHINGOSINE

Whole oligosaccharides are released from intact GSLs in reasonably high yields by oxidative ozonolysis of the olefinic double bond of the sphingenine residue [81,90]. The oligosaccharides can also be prepared by an osmium tetroxide-periodate oxidation procedure [83,91]. Partial acid hydrolysis [52] or alkaline hydrolysis [92] of the intact GSLs in an aqueous medium yields oligosaccharide fragments, and provides a useful technique for characterization of GSLs with very complex carbohydrates. In alkaline hydrolysis in a butanol solution, amide bonds of GSLs are cleaved as well, and glycosylsphingosines(lyso GSLs) are obtained in low yield [93]. 3.11. RADIOLABELING OF GSLr

GSLs can be radiolabeled either on the lipid moiety or on some of the carbohydrate chains. By reduction of GSLs in tetrahydrofuran solution with tritiated KBH,, using PdCl, or Pd on BaSO, as catalysts, GSLs which are labeled at their sphingenine residue with tritium are obtained [94]. The procedure for tritium labeling of GSLs which have galactose or N-acetylgalactosamine at their non-reducing termini involves oxidation with galactose oxidase [95] in the presence of horseradish peroxidase [96] followed by reduction with tritiated NaBH,. In our experience, the use of a solution containing more than 50% tetrahydrofuran in the oxidation reaction does not give labeled GSLs of high specific radioactivity, possibly due to some inactivation of galactose oxidase. The oxidation reaction in a phosphate buffer resulted in the high labeling [97]. 35 S-labeled sulfoglycosphingolipidscan be prepared from the brain and kidney of an animal which was administered inorganic [ 35S]sulfate,either intracerebrally [98] or intraperitoneally. 3.12. COVALENT ATTACHMENT OF GSLr TO SOLID SUPPORTS AND MACROMOLECULES

Peracetylated GSL is oxidized with KMnO, in the presence of a crown ether, dicyclohexyl-18-crown-6, yielding a carboxy-bearing product (‘glycolipid acid’) derived by cleavage of the olefinic double bond of the sphingenine residue [99]. ‘Glycolipid acid’ is also prepared by performic acid oxidation of glycolipid aldehyde [83]. The amide conjugate of the glycolipid acid and an amino-bearing solid support is successfully formed in an approximate yield of 50% or more through coupling of the glycolipid acid with alkylamine glass beads [99] or aminopropyl silica gel [19] in the presence of both N-hydroxy succinimide and dicyclohexyl carbodiimide followed by deacetylation with mild alkali. Deacetylated glycolipid acids can be coupled with aminoethyl-Sepharose, polyacrylic hydrazide or protein in the presence of N-hydroxy succinimide and l-ethyl-3(3-dimethylaminopropyl)-carbodiimideto give the corresponding GSL-polymers [991.

15 3.13. IMMUNOLOGICAL PROCEDURES

GSLs possess an antigenic reactivity whch is specific for the respective carbohydrate structure. To detect the antigenic reactivity of GSLs, one or more immunological methods are employed in the presence or absence of auxiliary lipids such as phosphatidylcholine and cholesterol. These include double immunodiffusion in agar, hemagglutination, immune hemolysis, complement fixation, quantitative precipitin reaction, immunoadherence, and an enzyme-linked immunosorbent assay. The procedures are summarized in Ref. 100. The murine hybridoma technique for producing monoclonal antibodies of Koehler and Milstein [82] is useful to obtain highly specific monoclonal antibodies. Multiple monoclonal antibodies directed against a GSL will each recognize a distinct portion of the carbohydrate moiety. The antibodies can be detected by a binding assay using GSL-antibody complex conjugated with I-labeled protein A of Stuphylococcus aweus in wells of a plastic micro plate [lo21 (solid phase radioimmunoassay). Another recent sensitive, simple method of detecting small amounts of GSL antigens is a solid phase immunoautoradiography assay [lo31 which is a modification of a method developed for the detection of gangliosides that bind to cholera toxin [104]; GSLs separated on a thin-layer plate are reacted with mouse antibody specific for a GSL and then with 1251-labeledF(+> G,,,1 = G,,,2a >> Glril [405-4081 with a preference for gangliosides that carry two sialic acid residues at the non-terminal galactose of gangliotetraose. In one assay system, employing Sephadex-adsorbed tetanus toxin, however, all gangliosides tested, including G,J, G,ril, G,,,l, 2b and 3b as well as unrelated synthetic sialoglycolipids, bound the toxin in a 1 : 1 molar ratio with high affinity, and to a comparable extent [403]. It can at present not be excluded that this preference for binding to certain ganglioside structures is influenced by matrix molecules used in the assay systems, e.g., a cerebroside preparation [405,407], ganglioside-containing liposomes [408] or native membranes of brain [409,410]. A prerequisite for binding to tetanus toxin appears to be the presence of a lipophilic moiety in the sialoglycoconjugate. Ganglioside-derived free sialooligosaccharide could not be shown to bind to tetanus toxin (Wiegandt, unpublished observations). Whereas at the molecular level the specificity of binding of tetanus toxin to certain isolated ganglioside species is less pronounced, its association with cells shows a high degree of selectivity. Cells of central neuronal origin, preferentially bind tetanus toxin [604]. T h s may be related to an exposure of the central neuron-typical di- and trisialogangliosides G,,,2b and 3b at the surface of these cells. It is speculated that, after its fixation to ganglioside centers, the tetanus toxin may be translocated and sequestered by other membranal structures that provide for its further intraaxonal transport to presynaptic terminals [634]. There is obvious parallelism for the binding of tetanus toxin and thyrotropin (thyroid-stimulating hormone) to membranes of the thyroid gland [413]. Ganglioside binding to thyrotropin shows an efficiency similar to that observed for tetanus toxin: G,,,2b >> G,,,3b > G,,,1 > G,ril = CI,,,~> G,,,2a [412,416,417].It was therefore postulated that gangliosides are involved as receptors for both the hormone and the toxin [412,414,415]. Speculations that only gangliosides act as cell receptors for tetanus toxin and thyrotropin are contradicted by the observation that neuroblastoma C 1300 cells, pretreated with neuraminidase and fl-galactosidase, still are able to fix tetanus toxin by a mechanism that may be unrelated to ganglioside [411]. Both effectors also specifically bind to a glycoprotein component from thyroid gland [418]. Perhaps both ganglioside and a specifically binding membrane glycoprotein may be involved

243 in the mechanism of reception, and the mediation of effector information [412]. Further doubt was cast on the assumption that gangliosides might serve as thyroid-stimulating hormone receptors by the finding that neurarninidase treatment of thyroid cells converting more complex gangliosides to ganglioside G,,,1 did not change binding of the hormone. Furthermore, down regulation of its receptors by thyrotropin has no effect on the distribution of gangliosides [549]. Cholera toxin binds specifically and multivalently to ganglioside G,J. The ganglioside G,,,2b binds some ten-times less strongly to the toxin as compared to G,,,1 [626] (for reviews, see Refs. 425, 385, 426, 486, 550, 616). Of the two cholera toxin promoter subunits, the A-protein, carrying ADP-ribosyltransferase activity, and the pentamer B-protein, only the latter binds to ganglioside [427]. In the binding, only part of the monosialogangliotetraose, the carbohydrate moiety of ganglioside G,,,l, is specifically involved [428-430,6281. Alteration of the 113-monosialogangliotetraose by substitution of the terminal galactose by fucose in 2-position, or reduction of the sialic acid-carboxyl group, as well as removal of galactose or sialic acid lead to a loss of binding capacity for cholera toxin. Integrity of the glucose residues appears not to be necessary for the toxin binding. Detailed studies showed that with cholera toxin only the sugar moiety of ganglioside G,,,1 is involved, whereas the ceramide provides an anchorage for the toxin at the cell membrane [429,431]. At present the mechanism of the ganglioside-dependent transmembrane events induced by the cholera toxin are not yet known in detail. After attachment to a cell-surface membrane the disulfide bond between the two subunits ( M , , 24 000 and 5400) of the A-protein is broken, and the larger peptide, that is hydrophobic, penetrates deeply into the lipid bilayer [551,623,624]. Specific disulfide bond reduction therefore appears necessary for the choleragenic action of the toxin. Since cholera toxin can induce redistribution of membrane constituents that are believed to be connected with the cytoskeletal system, it is also speculated that ganglioside membrane protein interactions could be involved [432-434,552,5531. A possible role of membrane ganglioside as receptor for interferon has also been suggested [435,437,554,555].Similar to tetanus toxin or thyrotropin, the specificity of binding to interferon is not unequivocal and restricted to one ganglioside, but decreases in effectiveness in the following order; G,,1 >> G,,,3b > G,,,l >> Gte12a> G,aJ *. Interferon-ganglioside interaction, however, appears not to be a general property of all types of interferon. Of the two interferon species detected in mouse fibroblasts, only one (type I) binds to ganglioside [438,555,572]. There is some indication that gangliosides may possibly function as receptors for certain cell growth and differentiation factors. One example for this is an L-cellderived factor that can stimulate the clonal growth of granulocyte macrophage ~~

* See, however, Ref. 555: mouse interferon type 1 is neutralized in the following order: G,,,3b. G1,,2b >> Glr,l: no binding by G,J or neutral glycosphingolipid. Also Ref. 554: Glr,l, G , J and Lac-Cer, all neutralize the antiviral action of interferon.

244 progenitor cells. Tlus factor is fixed by ganglioside in the following order of efficiency: G,,,1 > G,,1 > Gtet2a> G,,,3b [439]. Other findings are also in support of a possible receptor function of gangliosides for growth factors. Culturing cells in media that have been passed over a gangliotetraosylceramide or a ganglioside GJ-affinity column do not support the growth of 3T3-mouse fibroblast cells [440]. In addition, pretreatment of 3T3-cells with monovalent antibodies to gangliotetraosylceramide or to ganglioside G,,,l inhibits growth stimulation by serum. This might be interpreted as a masking of serum growth factor reception sites [440]. Another interesting example of the possible role of gangliosides as receptors for factors that influence cell behavior may concern lymphokine action on macrophages [556]. The macrophage migration inhibition factor (MIF) and macrophage activation factor (MAF) activities of culture supernatants of concanavalin A-stimulated lymphocytes can be abolished with a total brain ganglioside fraction [529]. Further indication of the involvement of ganglioside is the report that macrophages show an enhanced responsiveness to MIF after incubation with ganglioside-containing liposomes [529]. Even though the putative ganglioside receptor has not yet been characterized, it is believed to carry a terminal a-fucose residue, since this sugar is inhibitory for the MIF [557,558]. Sialooligosaccharide structures at the cell surface present receptors for viruses, such as paramyxovirus, influenza virus, encephalomyocarditis virus and Sendai virus [597,598]. A specific function of ganglioside in the cell reception for a virus was described in the case of Sendai virus [522]. Whereas sialidase treatment of cells makes them resistant to infection, incubation with ganglioside carrying a NeuAc-Gal-GalNActerminus restores susceptibility for the virus [522]. Due to their highly amphiphilic nature, gangliosides can act as rather sticky molecules. It is for this property that “receptor” functions may be observed for gangliosides that indeed are of no true biological significance. An example for this perhaps is reflected in the ability of gangliosides to inhibit a fibronectin-mediated cell attachment to collagen- or fibronectin-coated substrates in a nonspecific manner [5 88- 5901. 6.5. INTERACTION WITH NEUROTROPIC AGENTS

Wolley and Gommi [441,442] originally observed that the serotonin sensitivity of a neuraminidase-treated fundus preparation could be restored by adding ganglioside, in particular G,,,2. The involvement of sialic acid conjugates in the serotonin transport system could also be demonstrated in rat brain synaptosomes [445]. The question, however, whether or not gangliosides constitute serotonin tissue receptors has not yet been answered unequivocally (for review, see Ref. 383). Serotonin not only binds to gangliosides but also to other sialoglycoconjugates, e.g., fetuin [443]. Whereas the ion permeability of ganglioside-containing liposomes was not changed [443], release of glucose could be effected with serotonin and other biogenic amines

245

[a]. Tamir et al. [559]could see no binding of gangliosides to serotonin at relevant concentrations. These authors, however, made another interesting observation that may shed more light on the heretofore equivocal subject. In the presence of other lipids, e.g., lecithin and Fe2+, ganglioside, especially G ,ac2, strongly enhances the fixation of serotonin by the serotonin-binding protein [559]. It was speculated that possibly the interaction of ganglioside with serotonin-binding protein may regulate the concentration of the biogenic amine in the synapse. Other drugs that bind to ganglioside are d-tubocurarine [446], chlorpromazine [447] and colchiceine [448]. 7. Concluding remarks It is obvious that gangliosides appear to be involved in an embarrassing multitude of biological phenomena. Still, it is not yet possible to name clearly one universal role played by gangliosides in the life of cells, singly or in a tissue. The same holds true for the neutral members of the glycosphingolipid family, for which also no unified explanation of their biological significance can be offered at present. Even though t h s review attempts to keep the gangliosides “ under surveillance”, it may perhaps not be justified to consider the possible physiological function of the sialic acid-containing species only. However, considering the ubiquity of distribution of these plasma membrane constituents, future research of gangliosides is encouraged by the intriguing possibilities inherent in the complexity of these molecules as an expression of cell differentiation properties.

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Wiegandt (ed.) Gi)coliptds I985 Elsevier Science Publishers B. V. (Biomedical Division)

261

Q

CHAPTER 4

Glycosyl phosphopolyprenols FRANK W. HEMMING Department of Biochemistry, Queen’s Medical Centre, The University of Nottingham, Nottingham NG7 2 UH,England

I . Introduction Glycosyl phosphopolyprenols (see Section 3.1 for nomenclature of phosphorylated derivatives) differ from other glycolipids in a number of ways that are significant biochemically. The polyisoprenoid moiety has distinctive properties important in the interactions of these compounds with membranes and with specific glycosyl transferases. The lipid is also clearly derived by a very different biosynthetic route sharing parts of the pathway leading to sterols and polyisoprenoidquinones and hence is sensitive to some of the factors that control steroidogenesis. The linkage between sugar and lipid by a phosphate bridge is unique in glycolipids. Since t h s involves a sugar-l-phosphate bond, the transfer potential is comparable with that of the nucleotide diphosphate sugars. This leads to a difference in function: whereas the glycolipids are seen primarily as end products of metabolism, or as intermediates solely as acceptors of further sugar residues, the glycosyl phosphopolyprenols readily donate their glycosyl residues to some appropriate acceptor and have earned the term lipid-linked intermediates. This coenzymic role of phosphopolyprenols to some enzyme systems concerned with the transfer of sugars from nucleoside diphosphate sugars to form some glycosylated polymers is found in all living systems investigated so far. The type of polymer formed via lipid-linked intermediates differs in different kinds of organisms. In bacteria phosphopolyprenols are involved in the formation of the complex polysaccharides of the cell wall. In all eukaryotic cells the process of protein N-glycosylation is mediated by this type of coenzyme. In yeasts and fungi the coenzyme also functions in protein 0-mannosylation while in green plants their role also encompasses the formation of glucans. As yet a function in animal cells other than that in protein N-glycosylation has not been observed. Before discussing the details of the coenzymic role of phosphopolyprenols it is necessary to consider their chemical nature, for it will be seen that subtle differences of structure of these compounds, dependent upon their biological source, may be significant to their function. Acquaintance with phosphorylated derivatives of polyprenols will lead naturally into an account of their formation and metabolism;

aspects that will be particularly relevant to an appreciation of the discussion of the control of protein N-glycosylation towards the end of the chapter.

2. Polycis-isoprenoid alcohols 2.1. CHEMISTRY

2.1.1. General structures, nomenclature and methods

The chemistry of polyisoprenoid alcohols has been reviewed in detail by the author previously [l].The main features will be discussed here supplemented with more recent information. Polycis-isoprenoid alcohols possess the general structure 1 in which n varies from 5 to 24 depending upon the natural source. From any one source the range in the numerical value of n is relatively small giving rise to a family of isoprenologues spanning a difference in size of four or five residues. Most of the isoprene residues have cis-substituted double bands, and only two or three at the wend of the chain are in the trans configuration giving rise to the term di trans- (or tri trans-)polycis-isoprenoid alcohol. In this respect they differ from those polyprenols, such as solanenol, which are precursors of the side chains of plastoquinones, ubiquinones and menaquinones and are all-trans. Some polycis-isoprenoid alcohols contain one or a small number of saturated isoprene residues. Dolichols have a saturated a-residue (i.e., are 2,3-dihydro-polycis isoprenoid alcohols). They are also usually very hydrophobic molecules in which n is towards the upper end of the range (Greek SoXiKoa (dolikos): long). The structure of individual polyprenols has been established by using a combination of data from mass and infra red spectrometry, proton magnetic resonance, thin layer chromatography, hydrogenation and ozonolysis [ 11. Shibaev [2] has reported that ''C nuclear magnetic resonance allows assignment of the stereochemistry of each individual isoprenoid residue in the chain and this has since been taken further [2al. The term polycis isoprenoid alcohol is often abbreviated to polycis prenol or polyprenol depending on the context. The most sensitive and straightforward assay for unesterified polyprenols is by high performance liquid chromatography using a UV detector set at 210 nm [3]. This wavelength is a compromise between the peaks of absorption of the isolated double bonds and the absorption characteristics of appropriate solvents. Accurate assay of less than 100 pmol of mammalian dolichols is possible. In principle the method is also directly applicable to determination of glycosylated and phosphorylated derivatives. The concentration of polycis-prenols in tissues does not necessarily reflect the concentration of the functional form, the monophosphate. It may be more a measure of the phosphatase activity or the rate of metabolism of the liberated polyprenols. Nevertheless, the Occurrence of polycis-prenols can generally be taken either as positive evidence of the presence of the monophosphate and of phosphatase activity, or of a dietary source. It also indicates a potential to form the phosphate derivative using a polyprenol kinase, a phenomenon that will be discussed later.

263 2.1.2. Prokaryotic polycis-prenols All bacteria appear to contain ditrans, polycis-prenols containing between ten and 12 isoprene residues. The main component, undecaprenol, is sometimes called bactoprenol. The presence of phosphoundecaprenol and its glycosylated derivatives in baceria has been described on many occasions. The accumulation of unesterified bactoprenol to appreciable quantities has been described in only a few bacteria, notably Staphylococcus aureus [4] and various Lactobacilli [5]. In S. aureus over 90% of the undecaprenol (0.03 rnol g weight of cells) is unesterified at stationary phase. 2.1.3. Eukaryotic polycis-prenols 2.1.3.1. Ficaprenols and betulaprenols. Green leaves of higher plants contain tritrans-polycis-prenolsin which n (Fig. 4.1) usually varies from ten to 13 with the major components of the mixture containing eleven or twelve isoprene residues. Some of the earliest samples were obtained from Ficus elastica, giving rise to the trivial names ficaprenol-11 and ficaprenol-12 [6]. Small quantities of leaf polycis-prenols containing fewer than three trans residues have been observed to accompany the tri trans-polycis prenols. The concentration of these alcohols usually rises as the leaf ages, most of the increase occurring in osmiophilic globules of the chloroplast [7] with some also in the cell wall [ 81. Pine needles of several conifers accumulate tritrans, polycis-prenols ranging in size from 13 to 21 isoprene residues [9-111, and mainly as acyl esters. Non-photosynthetic tissue of hlgher plants sometimes yields slightly different polycis-prenols. These are ditrans, polycis-prenols comprising six to nine isoprene residues and are found mainly as fatty acyl esters. For example, the wood of silver birch, Betula oerrucosa, contains betulaprenols-6 to -9 [9]. 2.1.3.2. Dolichols. Most eucaryotic cells contain 2,3-dihydro, polycis-prenols which have been called dolichols. Most of these carry two trans residues at the wend of the chain. The distribution of dolichols among vertebrate animals is summarised in Table 4.1. Only human and rat tissues have been observed in detail. It can be seen that concentrations of dolichol vary markedly from one tissue to another. Human tissues appear to be a much richer source of dolichols than are rat tissues. Also, whereas the best human sources are testis, pituitary and adrenal, the richest rat tissues are spleen, pancreas and liver. The composition of the family of dolichols varies slightly from one species to another but in the rat the variation from one tissue to another is relatively small. Most of the unesterified dolichol in pig liver cells was found, in early work, to be associated with a subcellular fraction rich in mitochondria, whereas the major part of that esterified to fatty acids was located in a crude nuclear fraction [19]. More CH3

I

CH2-C=CH-CH2-

1 '

CH3

-

-CH2An 2

w - residue

Fig. 4.1. Polyisoprenoid alcohol - general structure

C=CH-CH2-OH

u-residue

264 TABLE 4.1 Distribution of dolichols in some vertebrate tissues Source

Concentration

Main components

Esterified % of total

Refs.

130- 170 200

20,19,18 ' ND

25 5

10,11 12

> 0.5 182 164- 316

ND ND ND

ND ND ND

13 13 15

ND ND ND 20,21, 19 ND ND ND ND ND ND ND

20 ND