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
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ISBN for the series: 0-444-80303-3
ISBN for the volume: 0-444-80595-8

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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