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The Biochemistry of Glycoproteins and Proteoglycans The Biochemistry of Glycoproteins and Proteoglycans Edited by William J. Lennarz The Johns Hopkins University School of Medicine Baltimore, Maryland PLENUM PRESS • NEW YORK AND LONDON Library of Congress Cataloging in Publication Data Main entry under title: The Biochemistry of glycoprotelns and proteoglycans. Includes index. 1. Glycoproteins. 2. Proteoglycans. 3. Biological chemistry. I. Lennarz, William J. QP552.G59B56 574.1'924 79-9176 ISBN 978-1-4684-1008-2 ISBN-13: 978-1-4684-1008-2 e-ISBN-13: 978-1-4684-1006-8 001: 10.1007/978-1-4684-1006-8 © 1980 Plenum Press, New York Softcover reprint of the hardcover 1 st edition 1980 A Division of Plenum Publishing Corporation 227 West 17th Street, New Yark, N. Y. 10011 All rights reserved No part of this book may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise, without written permission from the publisher Contributors Gilbert Ashwell • National Institute of Arthritis, Metabolism and Digestive Diseases, National Institutes of Health, Bethesda, Mary land Paul H. Atkinson • Departments of Pathology and Developmental Bi ology and Canl:er, Albert Einstein College of Medicine, Ikonx, New York John Hakimi • Departments of Pathology and Developmental Biology and Cancer, Albert Einstein College of Medicine, Bronx, New York Rosalind Kornfeld. Department of Medicine, Washington University School of Medicine, St. Louis, Missouri Stuart Kornfeld. Department of Medicine, Washington University School of Medicine, St. Louis, Missouri William J. Lennarz • Department of Physiological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland Elizabeth F. Neufeld • National Institute of Arthritis, Metabolism and Digestive Diseases, National Institutes of Health, Bethesda, Mary land Lennart Roden • University of Alabama in Birmingham, Birmingham, Alabama Saul Roseman • Department of Biology and the McCollum Pratt In stitute, The Johns Hopkins University, Baltimore, Maryland Harry Schachter • Department of Biochemistry, Hospital for Sick Chil dren, Toronto, Canada v vi CONTRIBUTORS Pamela Stanley • Department of Cell Biology. Albert Einstein College of Medicine. Bronx. New York Douglas K. Struck • Department of Physiological Chemistry. The Johns Hopkins University School of Medicine, Baltimore, Maryland Preface Although glycoproteins and proteoglycans have been a subject of re search for many years, it is only during the last five or so years that they have aroused the interest of a very broad cross section of investigators in the biological sciences. The reason for this expanded interest in these molecules is simple: not only are glycoproteins and proteoglycans ubiq uitous, but many are molecules with well-defined and important biological functions. The list of molecules that fall into this category grows daily; interferon, immunoglobulins, certain hormones, many cell surface recep tors, and viral coat proteins are but a few examples. Thus, investigators with interests as diverse as viral replication. cell-cell interactions. poly isoprenoid synthesis, secretory processes, hormone responses, embryonic development, and immunology have become concerned with glycopro teins and proteoglycans. The objective of this book is to summarize the current state of knowledge on the biochemistry of these molecules. Coverage is by no means encyclopedic; rather the thrust is to emphasize the recent ad vances. The first chapter deals primarily with structural work on the oligosaccharide chains of glycoproteins, but it will be apparent in it and in the succeeding two chapters on biosynthesis that not only do structural studies aid biosynthetic investigations, but that studies on biosynthesis often playa major role in elucidation of structure. The fourth chapter emphasizes the utility of the genetic approach in elucidation of the struc ture and function of cell surface glycoproteins, while the fifth deals with the important and complex question of the possible role of these mole cules in the regulation of growth of normal and transformed cells. In the sixth chapter another potential functional role of cell surface molecules, namely, in the binding and uptake of glycosylated ligands, is discussed. vii viii PREFACE Finally, in the last chapter, a comprehensive description of the current state of knowledge of the biosynthesis and the catabolism of the proteo glycans is presented. William J. Lennarz Baltimore Contents Chapter 1 Structure of Glycoproteins and Their Oligosaccharide Units Rosalind Kornfeld and Stuart Kornfeld I. Introduction............................................ 1 2. Isolation and Structural Analysis of Glycopeptides ......... 3 2.1. Methods for Isolation of Glycopeptides ............... 3 2.2. Methods Used in Determination of Oligosaccharide Structure .......................................... 4 3. Glycopeptides Containing Oligosaccharides Linked O-Glycosidically to the Peptide. . . . . . . . . . . . . . . . . . . . . . . . . . . 6 3.1. Oligosaccharides Linked through N-Acetylgalactosamine to Serine and Threonine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 3.2. Oligo saccharides Linked to Serine and Threonine through Sugars Other Than N-Acetylgalactosamine ............ IO 3.3. Oligosaccharides Linked to the Hydroxyl Group of Hydroxylysine and Hydroxyproline .................. II 4. Glycopeptides Containing the N-Acetylglucosaminyl- Asparagine Linkage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 4.1. Structure of the Core Region ........................ 13 4.2. Structure of "Simple"-Type Oligosaccharides ......... 13 4.3. Structure of "Complex"-Type Oligosaccharides ....... 16 4.4. Structure of the Oligosaccharide-Lipid Intermediate ... 22 5. Role of Oligosaccharides in Glycoprotein Synthesis. . . . . . . . . 25 6. References............................................. 27 ix x CONTENTS Chapter 2 The Function of Saccharide-Lipids in Synthesis of Glycoproteins Douglas K. Struck and William 1. Lennarz 1. Introduction............................................ 35 2. Dolichol ............................................... 37 3. Monoglycosyl Derivatives of Dolichol Phosphate . . . . . . . . . . . 38 3.1. Mannosylphosphoryldolichol and N -Acetylglucosaminyl- pyrophosphoryldolichol ............................. 38 3.2. Other Glycosyl Polyprenol Derivatives. . . . . . . . . . . . . . . . 40 4. Assembly of Oligosaccharide-Lipids . . . . . . . . . . . . . . . . . . . . . . 41 5. Transfer of Oligosaccharide Chains to Endogenous Membrane Proteins ............................................... 44 6. Transfer of the Oligosaccharide Chains from Oligosacchar- ide-Lipids to Exogenous, Soluble Proteins ................ 47 6.1. Existence of a Tripeptide Acceptor Sequence. . . . . . . . . . 47 6.2. In Vitro Glycosylation of Denatured Soluble Proteins. . . 50 7. A Model for Glycosylation of Membrane and Secretory Glycoproteins .......................................... 53 8. Regulation of Glycoprotein Synthesis ..................... 58 9. Inhibitors of Glycosylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 9.1. In Vivo Effects of Deoxy Sugars on Protein Glycosylation 61 9.2. Effect of Deoxy Sugars on the Secretion of Glycoproteins 62 9.3. Effect of Deoxy Sugars on Virus Replication .......... 63 9.4. In Vitro Effect of Deoxy Sugars on Protein Glycosylation 63 9.5. Fluorodeoxy Sugars ................................ 64 9.6. Amino Sugars. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 9.7. Bacitracin ......................................... 65 9.8. Tunicamycin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66 10. References............................................. 73 Chapter 3 Mammalian Glycosyltransferases: Their Role in the Synthesis and Function of Complex Carbohydrates and Glycolipids Harry Schachter and Saul Roseman Part A I. Introduction............................................ 85 1.1. Sugar NucIeotides and Transglycosylation . . . . . . . . . . . . . 85 1.2. Glycosyltransferases: General Comments ............. 88 2. Glycosyltransferases Involved in Elongation of N-Glycosidi cally Linked Oligosaccharides of the N-Acetyllactosamine Type.................................................. 92 CONTENTS xi 2.1. Processing of Protein-Bound Oligosaccharide Prior to Elongation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93 2.2. N-AcetylglucosaminyItransferases and Control of Elongation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95 2.3. FucosyItransferases................................. 99 2.4. Galactosyltransferases .............................. 100 2.5. Sialyltransferases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103 2.6. The Golgi Apparatus as the Major Subcellular Site of Elongation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108 3. GlycosyItransferases Involved in Synthesis of O-Glycosidi- cally Linked OIigosaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111 3.1. Synthesis of Serine(Threonine )-N -Acetyl-D-Galactosa- mine Linkage ...................................... 113 3.2. Synthesis of Submaxillary Gland Mucins . . . . . . . . . . . . . . 115 3.3. Synthesis of Human Blood Group Oligo saccharides .... 121 Part B 1. Introduction............................................ 127 2. New Gangliosides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128 3. Occurrence and Subcellular Distribution. . . . . . . . . . . . . . . . . . . 132 4. Gangliosides as Membrane Components. . . . . . . . . . . . . . . . . . . 133 5. Biosynthesis and Degradation. . . . . . . . . . . . . . . . . . . . . . . . . . . . 135 6. Effect of Cell Transformation on Ganglioside Synthesis ..... 139 7. Gangliosides in Development and Differentiation. . . . . . . . . . . 142 8. Gangliosides as Membrane Receptors for Toxins and Hormones ............................................. 143 9. References............................................. 147 Chapter 4 Surface Carbohydrate Alterations of Mutant Mammalian Cells Selected for Resistance to Plant Lectins Pamela Stanley 1. Selection of Cells with Altered Surface Carbohydrate. . . . . . . 161 2. Biochemical Basis of a Surface Carbohydrate Alteration .... 162 3. Genetic and Biochemical Properties of GIt CHO Cells .... 166 1- 3.1. Localization of the Mutated Gene Product ............ 166 3.2. Altered Membrane Properties. . . . . . . . . . . . . . . . . . . . . . . . 167 4. Surface Carbohydrate Alterations of Other Lectin-Resistant (LecR) Cell Lines ....................................... 168 4.1. Ricin-Resistant (RicR) Mouse L Cells ................. 168 4.2. Wheat Germ Agglutinin-Resistant (WgaR) CHO Cells. . . 170