Table Of ContentEdited by
M. Alfert, Berkeley, Calif. K. R. Po ...., Boulder, Colo.
W. Beennam, Tiibingen P. Sitte, Freiburg i. Br.
.L. Goldstein, Lexington, Ky.
Advi50ryBoard
J. Brachet, Bruxelles N. Kamiya, Okazaki
D. Branton, Cambridge, Mass. G. Rudkin, Philadelphia, Po.
H. G. Callan, St. Andrews G. F. Springer, Evanston, III.
E. C. Cocking, Nottingham L Stockinger, Wien
W. W. Franke, Heidelberg B. F. Trump, Baltimore, Md.
Volume 10
Springer-VerI.,
WiM •• fork
mUlti_
Cltemistry, Metabolism
and 'unction
Edited by I. Scltauer
Springer-Verlag
Wien .err 'o,1c
Prof. Dr. ROLAND SCHAUER
Biochemisches Institut, Christian-Albrechts-Universitat, Kiel
Federal Republic of Germany
This work is subject to copyright.
All rights are reserved. whether the whole or part of the material is concerned,
specifically those of translation, reprinting, fe-use of illustrations, broadcasting,
reproduction by photocopying machine or similar means, and storage in data banks.
1982 by Springer-VerIagJWien
Softcover reprint of the hardcover 1st edition 1982
With 66 Figures
Library of Congress Cataloging in Publication Data. Main entry under title: Sialic acids-chemistry, metabolism and function. (~ell
biology monographs; v. 10.) Includes bibliographical references and index.!. Sialic acids. I. Schauer, R. (Roland), 1936-. II. Series.
[DNLM: 1. Sialic acids. WI CEI28H v. IO/QU 84 S562.] QP801.S47S55. 1982. 574.19'2482. 82-19326
ISSN 0172-4665
ISBN-13 :978-3-7091-8682-4 e-ISBN-13:978-3-7091-8680-0
DOI: 10.1007/978-3-7091-8680-0
To Elfriede
Preface
Rapid progress in the field of sialic acids has made it desirable to collect the new
data about these unique sugars and to continue the series of books on this topic. In
1960, A. GOTTSCHALK wrote "The Chemistry and Biology of Sialic Acids and
Related Substances" (Cambridge University Press) and in 1976, A. ROSENBERG
and C.-L. SCHENGRUND published "Biological Roles of Sialic Acids" (Plenum
Press). In this book emphasis is given to various modern methods used in the
isolation and analysis of sialic acids. New approaches to the synthesis of free and
bound sialic acids are described and the vast field of occurrence and metabolism of
these substances is reviewed. Sialidoses are dealt with in one of the chapters,
because sialidases have been recognized as factors of pathophysiological im
portance. As knowledge is increasing about the involvement of sialic acids in many
aspects of cell biology, another chapter is devoted to these phenomena. With this
book I intend to demonstrate modern trends in sialic acid chemistry and
biochemistry, and I hope that it will be of practical use and find its place in
laboratories rather than in libraries.
This publication offers an opportunity to thank all colleagues in many
countries, including my coworkers at the universities of Bochum and Kiel, for
their cooperation, stimulating discussions and, very important, useful criticism.
The continuous cooperation with J. F. G. VLIEGENTHART and his coworkers,
Utrecht, has been rewarding in many respects.
I am sorry that my thanks to the authors of this book cannot be extended to
C. F. A. CULLING, who died in July 1982.
The help of WIGBERT BERG, GABRIELE GUTSCHKER-GDANIEC, ERNST MOLLER,
ULRICH N OHLE, GERD REUTER, CORNELIA SCHRODER, and ASHOK K. SHUKLA, who
assisted in proof-reading of the manuscripts and in the preparation of the subject
index, is very much appreciated.
Kiel, September 1982 ROLAND SCHAUER
Contents
Abbreviations . . . . XV
List of Contributors. XIX
A. Introduction . . .
(ROLAND SCHAUER and JOHANNES F. G. VUEGENTHART)
B. Occurrence of Sialic Acids. . . . . . . . . . . 5
(ANTHONY P. CORFIELD and ROLAND SCHAUER)
I. Introduction 5
II. Viruses . . . . . . . . . . 9
III. Bacteria. . . . . . . . . . 9
IV. Fungi, Algae, and Plants 10
V. Protozoa ........ . 11
VI. Porifera, Platyhelminthes, Nemertinea, Ctenophora, and Coelenterata. 11
VII. Sipuncula, Annelida, Arthropoda, and Mollusca . .... . 12
VIII. Bryozoa, Brachiopoda, Chaetognatha, and Echinodermata 12
IX. Hemichordata, Urochordata, and Cephalochordata 13
X. Vertebrata . 13
1. Pisces .. 13
2. Amphibia 16
3. Reptilia . 17
4. Aves . .. 17
5. Mammalia 18
a) Introduction 18
b) 0 ligosaccharides . 18
at) Milk and Colostrum. 18
~) Urine ...... . 19
y) Nucleotide Sugars 20
c) Glycoproteins .... . 20
at) Serum ...... . 20
~) Epithelial or Mucus Glycoproteins . 21
y) Membrane Glycoproteins ..... . 23
8) Brain Glycoproteins . . . . . . . . . 24
e) Milk, Colostrum and Urine Glycoproteins and Glycopeptides 24
Q Hormone and Enzyme Glycoproteins 25
1) Lipoproteins . 26
it) Miscellaneous. . 26
d) Gangliosides . . . . 27
XI. The Nature of Sialic Acids 32
1. Introduction. . . . . . . 32
2. Free Sialic Acids . . . . 32
3. Glycosidically Linked Sialic Acids 33
x Contents
a) The Type of Sialic Acid . 33
b) Sialyloligosaccharide Structural Considerations. 33
IX) Terminal Linear Position . . . . . . . . . . . . 33
~) Terminal "Side-Chain Position" . . . . . . . . 34
y) Internal Sialic Acids in Oligosialylglycoconjugates and Sialylpoly-
saccharides . . . . . . . . . . . . . . . . . . 35
a) Internal Sialic Acids in the Echinodermata. 37
XII. Evolution of the Sialic Acids . 37
Bibliography . . . . . . . . . . . . . . . . . . 39
C. Isolation and Purification of Sialic Acids . 51
(ROLAND SCHAUER and ANTHONY P. CORFIELD)
I. Introduction . . . . . . 51
II. Acid Hydrolysis . . . 51
III. Enzymic Hydrolysis . . 53
IV. Lipid Extraction . . . . . . 54
V. Ion-Exchange Chromatography. 54
VI. Cellulose Chromatography 55
VII. Crystallization . 56
VIII. Other Methods. 56
Bibliography . . . . . . . . 56
D. Synthesis of Sialic Acids and Sialic Acid Derivatives . . . . 59
(JOHANNES F. G. VLIEGENTHART and JOHANNIS P. KAMERLING)
I. Introduction . . . . . . . . 59
II. Glycosides of Sialic Acids. . . . . . . . . . . . 59
III. Sialodisaccharides . . . . . . . . . . . . . . . . 64
IV. 2-Deoxy-2,3-dehydro-N-acylneuraminic Acids. 65
V. Methylated Sialic Acids. . . . . . . . . 66
VI. Isotopically Labelled Sialic Acids. . . . 67
VII. Substrates for Sialidase Investigations . 68
VIII. ~- and Cs-Analogues of Sialic Acids . 69
IX. Miscellaneous Sialic Acids and Sialic Acid Derivatives. 70
Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
E. Colorimetry and Thin-Layer Chromatography of Sialic Acids. 77
(ROLAND SCHAUER and ANTHONY P. CORFIELD)
I. Introduction . . . . . . . . . . 77
II. Colorimetry and Fluorimetry . 78
I. Diphenol Reagent Assays 78
a) Orcinol. ....... . 78
b) Resorcinol ...... . 80
IX) Resorcinol/Cu2+ Assay 80
~) Periodate/Resorcinol/Cu2+ Assay. 80
2. Periodic AcidfThiobarbituric Acid Assays . 81
a) The Warren and Aminoff Methods ... 81
b) Interference . . . . . . . . . . . . . . . . 83
3. Periodic Acid/Methyl-3-Benzothiazolinone-2-Hydrazone Method 84
4. Fluorimetric Assays. . 85
5. Enzymic Assays. . . . 86
6. O-Acyl Group Assay . 86
Contents XI
7. Automatic Procedures . . . . . . . . . . 87
8. High Performance Liquid Chromatography 87
9. Other Methods ............... . 88
10. Quantitative Problems in Sialic Acid Release. 88
III. Thin-Layer Chromatography. 88
1. Chromatographic Systems. . . 88
2. Visualization . . . . . . . . . . 91
3. Two-Dimensional Techniques. 91
a) Individual Sialic Acid Determination 91
b) O-Acyl Group Identification ..... 91
c) Sialic Acid Identification in Oligosaccharides 91
4. Paper Chromatography 92
Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
F. Gas-Liquid Chromatography and Mass Spectrometry of Sialic Acids. 95
(JOHANNIS P. KAMERLING and JOHANNES F. G. VLIEGENTHART)
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . 95
II. Gas-Liquid Chromatography. . . . . . . . . . . . . . 96
1. Quantitative Sugar Analysis, Including Sialic Acid 97
2. Hydrolytic Release of Sialic Acids from Sialobiopolymers 99
3. Preparation and Analysis of Trimethylsilylated N,O-Acylneuraminic Acid Methyl
Esters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
4. Preparation and Analysis of Pertrimethylsilylated N,O-Acylneuraminic Acids 99
5. Analysis of Acetyl and/or Glycolyl Residues of Sialic Acid. 100
III. Gas-Liquid Chromatography/Mass Spectrometry. . . . . . . . . . . . . . . . . . 100
1. Mass Spectrometric Identification Procedure. . . . . . . . . . . . . . . . . . . 100
2. Analysis of (O-Acylated) N-Acylneuraminic Acids Isolated from Biological Mate-
rial. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
3. Sialic Acids and Methylation Analysis. . . . . . . . . . 114
4. Miscellaneous Sialic Acids and Sialic Acid Derivatives . 117
5. Quantitative Analysis of Sialic Acids by g.l.c./m.s. . . . 122
6. Sialooligosaccharides, Sialoglycolipids, and Sialoglycopeptides. 123
Bibliography. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
G. NMR Spectroscopy of Sialic Acids. 127
(JOHANNES F. G. VLIEGENTHART, LAMBERTUS DORLAND, HERMAN VAN HALBEEK,
and JOHAN HAVERKAMP)
I. Introduction. . . . . . . . . . . . . . . . . . . . 127
II. IH-NMR Spectroscopy. . . . . . . . . . . . . . 128
1. N-Acetyl-and N-Glycolylneurarninic Acid. . . . . . . . . . 128
2. O-Acetylated Neuraminic Acid Derivatives . . . . . . . . . 132
3. Analysis of Sialic Acid Linkage Types in Glycoconjugates . 133
a) The Anomeric Configuration of Sialic Acid . . . . . . . 134
b) Sialic Acid in Carbohydrate Chajns of the N-Acetyllactosarnine Type 137
c) Sialic Acid in O-Glycosidically Linked Carbohydrate Chains 144
d) CMP-N-Acetylneuraminic Acid and Degradation Products 146
4. Enzymic and Chemical Conversions . 149
5. Complexes of Sialic Acids with Ca2+. . . . . . . . . . . . . . . 154
III. 13C-NMR Spectroscopy. '. .............................. 154
1. N-Acetyl-and Glycolylneuraminic Acid. . 154
2. O-Acetylated Neuraminic Acid Derivatives . . . . . . . . . . . . . . . . . .. 156
XII Contents
3. Polysaccharides. . . . . . . . . 157
4. Esterified Polysaccharides . . . 163
5. CMP-N-Acetylneurarninic Acid and 2-Deoxy-2,3-dehydro-N-acetylneuraminic
Acid .................... '.' . . . . . . . . . . . . . . . . . 166
6. Complexes of Sialic Acids with Ca 2 +. . . . . . . . . . . . . 167
7. BC-Relaxation Studies on Sialic Acids and Sialyllactoses. . . . . . . . . . . . 168
IV. Analytical Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168
I. Instrumentation. 168
2. Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170
H. Histochemistry of Sialic Acids. . . . . . . . . 173
(CHARLES F. A. CULLINGt and PHILIP E. REID)
I. Introduction . . . . . . . . . . . . . . 173
II. Histochemical Identification of Sialic Acids. 174
I. General . . . . . . . . . . . . . . . . . . . . 174
2. Methods Based upon Staining with Cationic Dyes 175
a) Staining at Different pH's. . . . . . . . . . . . . . . 176
b) Removal of Specific Components Contributing to the Staining 176
0<) Use of Enzymatic Procedures. . 176
~) Use of Chemical Procedures. 178
i) Acid Hydrolysis. . . . . . . . 178
ii) Methylation. . . . . . . . . . 179
c) Inhibition of Staining with Alcian Blue in the Presence of Varying Concentra-
tions of Inorganic Salts (C.E.C. Methods). . . . . 179
3. Methods Based upon the Use of Periodate Oxidation. . . . . . . . 180
a) The PAS Reaction. . . . . . . . . . . . . . . . . . . . . . . . . 180
b) The Periodic Acid/phenylhydrazinejSchiff (PAPS) Procedure 180
c) The Use of Very Dilute Periodic Acid . . . . . . . . 182
d) Identification of Side Chain O-Acylated Sialic Acid. 182
0<) PBTjKOHjPAS Technique 183
~) PATjKOH/pAS Procedure . . . . 185
y) 9-0-Acyl Sialic Acids . . . . . . . 185
4. Methods Based upon the Use of Lectins . 186
III. Staining and Control Procedures. 187
l. Fixation and Processing. . 187
2. PAS Technique. . . . . . . 187
3. PBTjKOHjPAS Technique 188
4. PATjKOH/pAS Technique 188
5. High Iron DiaminejAlcian Blue Method. 189
6. Alcian Blue (pH 2.5). . . 190
7. Alcian Blue (pH 1.0). . . 190
8. Neuraminidase Digestion 190
9. Acid Hydrolysis. 191
Bibliography. . . . . . . . . . 191
I. Metabolism of Sialic Acids 195
(ANTHONY P. CORFIELD and ROLAND SCHAUER)
I. Introduction . . . . . . . . . . . . . . 195
II. De novo Biosynthesis of Sialic Acids . 196
