Current Topics in Membranes and Transport. Volume 11: Cell Surface Glycoproteins: Structure, Biosynthesis, and Biological Functions (v. 11)

Current Topics in Membranes and Transport Volume 11 Ce llSutfaee Glycoproteins Structure, Biosynthesis, and Biological ...

Author: Felix Bronner | A. Kleinzeller

4 downloads 667 Views 29MB Size Report

This content was uploaded by our users and we assume good faith they have the permission to share this book. If you own the copyright to this book and it is wrongfully on our website, we offer a simple DMCA procedure to remove your content from our site. Start by pressing the button below!
Report copyright / DMCA form

DOWNLOAD PDF

Current Topics in Membranes and Transport Volume 11 Ce llSutfaee Glycoproteins Structure, Biosynthesis, and Biological Functions

Advisory Board

I . S . Edelman Alvin Essig Franklin M . Harold James D. Jamieson Anthony Martonosi Shmuel Raxin Martin Rodbell Aser Rothstein Stanley G . Schultz Contributom

Richard W. Compans Lloyd A . Culp Gordon G . Forstner R. L. Juliano Maurice C . Kemp Michelle Letarte Mario Moscarello Kenneth D. Noonan John R. Riordan Aser Rothstein Harry Schachter Jennifer Sturgess Michael j . A. Tanner

Current Topics in Membranes and Transport Edited by Felix Bronner

Amort Kleinzeller

Department of Oral Biology University of Connecticut Health Center Farmington, Connecticut

Department of Physiology University of Pennsylvania School of Medicine Philadelphia, Pennsyloania

VOLUME 11 Cell Surface Giycqproteinx Structure, Biosynthesis, and Biological Functions Guest Editors R. 1. Juliano

h e r Rothrtein

Research Institute The Hospital for Sick Children Toronto, Ontario, Canada

Research Institute The Hospital for Sick Children Toronto, Ontario, Canada

1978

Academic Prom

New Yo&

San Fmncisco

London

A Subsidiary of Harcourt Brace jovanovich, Publishers

COPYRIGHT @ 1978,BY ACADEMIC PRESS,INC. ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATTON MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.

ACADEMIC PRESS, INC. 111 Fifth Avenue, New

York, New York 10003

United Kingdom Edition published by ACADEMIC PRESS, INC. (LONDON) LTD. 24/28 Oval Road, London N W l 7DX

LIBRARY OF CONGRESS CATALOG CARD NUMBER:70- 11709 1 ISBN 0-1 2-15331 1-5 PHINTED IN THE UNITED STATES OF AMERICA

79808182

9 8 7 6 5 4 3 2

List of Contributors, ix Preface, xi Contents of Previous Volumes, xiii The Cell Membrane-A

Short Historical Perspective

ASER ROTHSTEIN Text, 1 References, 12

The Structure and Biosynthesis of Membrane Glycoproteins JENNIFER STURGESS, MARIO MOSCARELLO, AND HARRY SCHACHTER I. 11. 111. IV. V. VI.

Carbohydrate Asymmetry Across the Membrane Bilayer, 16 Glycoprotein Structure, 19 Model Membrane Systems, 34 The Glycosylation Reaction, 37 Subcellular Sites of Glycosylation, 61 Membrane Biogenesis, 67 References, 85

Techniques for the Analysis of Membrane Glycoproteins

H. L. JULIANO I. 11. 111. IV. V.

Introduction, 107 Identification of Cell Surface Glycoproteins, 108 Fractionation of Membrane Glycoproteins, 117 Chemical Analysis of Membrane Glycoproteins, 129 Genetic Analysis of Membrane Glycoproteins, 133 References. 134

Glycoprotein Membrane Enzymes JOHN R. RIORDAN AND GORDON G. FORSTNER I. Introduction, 146 11. Specific Enzymes, 147 111. Membrane Association, 188 IV. Structure, 1% V

vi

CONTENTS

V. Functional Interrelationships, 201 VI. Biosynthetic and Developmental Aspects, 206 VII. Are All Ectoenzymes Glycoproteins?, 210 References, 210

Membrane Glycoproteins of Enveloped Viruses RICHARD W.COMPANS AND MAURICE C. KEMP I. 11. 111. IV. V.

Membranes of Lipid-Containing Viruses, 233 Components of Viral Membranes, 236 Arrangement of Viral Envelope Components, 240 Structure and Function of Viral Glycoproteins, 242 Assembly of Viral Membranes, 260 References, 268

Erythrocyte Glycoproteins MICHAEL J. A. TANNER I. 11. 111. IV. V. VI.

Introduction, 279 The Origin and Turnover of the Erythrocyte, 280 The Glycoproteins of the Erythrocyte Membrane, 281 Organization of the Glycoproteins in the Erythrocyte Membrane, 285 Structure of the Glycoproteins, 288 Functions of Glycoproteins, 304 References, 316

Biochemical Determinants of Cell Adhesion LLOYD A. CULP I. 11. 111. IV.

Introduction, 327 Cell Substrate Adhesion, 328 Cell-Cell Adhesion, 356 Conclusion, 381 References, 383

Proteolytic Modification of Cell Surface Macromolecules: Mode of Action in Stimulating Cell Growth KENNETH D. NOONAN I. 11. 111. IV. V. VI. VII.

Introduction, 398 The Erythrocyte Membrane, 399 Stimulation of Cell Division in a Resting Lymphocyte Population, 404 Protease Induction of Cell Division in Fibroblasts, 412 Effects of Proteases on Fibroblast Surface Structure, 421 Protease-Induced Transmembrane Events, 432 Limited Autolysis as a Mechanism for Inducing Cell Division, 437

CONTENTS

VIII. Role Media Components Play in Protease-Stimulated Cell Division, 444 IX. Summary, 450 References, 452 Note Added in Proof, 461 Glycoprotein Antigens of Murine lymphocytes

MICHELLE LETARTE I. Introduction, 464 11. Methods of Analysis of Cell Surface Antigens, 467 111. Isolation and Characterization of H-2 Antigens, 477

IV. V. VI. VII. VIII.

Isolation and Characterization of IA Antigens, 487 Isolation and Characterization of Thy-1 Antigen, 493 Preliminary Characterization of Tla Antigens, 500 Preliminary Characterization of Ly-2,3 Antigens, 502 Conclusion, 503 References, 505

Subject Index, 513

vii

This Page Intentionally Left Blank

Numbers in parentheses indicate the pages on which the authors’ contributions begin. Richard W. Companr, Department of Microbiology, University of Alabama at Bir-

mingham, The Medical Center, Birmingham, Alabama 35294 (233) Lloyd A. Culp, Department of Microbiology, School of Medicine, Case Western Reserve

University, Cleveland, Ohio 44106 (327) Gordon 0. Forrtner, The Research Institute, The Hospital for Sick Children, and Depart-

ment of Physiology, University ofToronto, Toronto M5G 1x8, Ontario, Canada (145) R. 1. Juliano, The Research Institute, The Hospital for Sick Children, Toronto M5G 1x8,

Ontario, Canada (107) Maurice C. Kemp, Department of Microbiology, University of Alabama at Birmingham,

The Medical Center, Birmingham, Alabama 35294 (233) Michelle Letarta, Department of Biological Research, The Ontario Cancer Institute,

Toronto, Ontario, Canada (463) Department of Biochemistry, The Research Institute, The Hospital for Sick Children, Toronto M5G 1x8, Ontario, Canada (15)

Mario Morcarello,

of Biochemistry and Molecular Biology, JHM Health Center, University of Florida, Gainesville, Florida 32610 (397)

Kenneth D. Noonan, Department

John R. Riordan, The Research Institute, The Hospital for Sick Children, and Depart-

ment of Clinical Biochemistry, University of Toronto, Toronto M5G 1x8, Ontario, Canada (1) Aser Rothrtein, The Research Institute,The Hospital for Sick Children, TorontoM5G 1x8,

Ontario, Canada (1) Harry Schaehter, Departments of Biochemistry and Pathology, The Research Institute,

The Hospital for Sick Children, Toronto M5G 1x8, Ontario, Canada (15) Jennifer Sturgerr, Department of Pathology, The Research Institute, The Hospital for

Sick Children, Toronto M5G 1x8, Ontario, Canada (15) Michael J. A. Tanner, Department of Biochemistry, University of Bristol, Bristol BS8

lTD, United Kingdom (279)

ix


Due to the rapid pace at which our field is expanding and its increasing reach, the Editors, the Advisory Board, and the Publishers have decided to include in the continuing broad coverage of our series topic-centered volumes which will explore in depth emerging research areas of particular significance. Cell surface glycoproteins are such a topic. We were fortunate that our colleagues, R. L. Juliano and A. Rothstein, were willing to serve as Guest Editors to bring together authors whose work has dealt with various aspects of this topical subject and to structure these contributions into a special volume of Current Topics in Membranes and Trunsport. The introductory chapter presents an historical review and a general discussion of the cell membrane, while the other chapters deal with structure, biosynthesis, and function of the glycoproteins. For our part we are particularly pleased that the first of our special, topic-centered issues deals with a membrane component that appears to function in cellular recognition and assembly. This volume may therefore contribute to the molecular understanding of cellular differentiation, one of biology’s major puzzles. We hope our readers will share our excitement at this eleventh volume of this serial publication.

FELIXBRONNER

ARNOST KLEINZELLER

xi


Contents of Previous Volumes Volume 1 Some Considerations about the Structure of Cellular Membranes MAYNARD M. DEWEYAND LLOYDBARR The Transport of Sugars across Isolated Bacterial Membranes H. R. KABACK Galactoside Permease of Escherichia coli ADAM KEPES Sulfhydryl Groups in Membrane Structure and Function ASER ROTHSTEIN Molecular Architecture of the Mitochondrion DAVID H. MACLENNAN Author Index-Subject Index

Volume 2 The Molecular Basis of Simple Diffusion within Biological Membranes W. R. LIEB AND W. D. STEIN The Transport of Water in Erythrocytes ROBERT E. FORSTER Ion-Translocation in Energy-Conserving Membrane Systems B. CHANCE AND M. MONTAL Structure and Biosynthesis of the Membrane Adenosine Triphosphatase of Mitochondria

ALEXANDER TZAGOLOFF Mitochondria1 Compartments: A Comparison of Two Models HENRYTEDESCHI Author Index-Subject Index

Volume 3 The Na+, K+-ATPase Membrane Transport System: Importance in Cellular Function ARNOLD SCHWARTZ, GEORGEE. LINDENMAYER, AND JULIUS C. ALLEN Biochemical and Clinical Aspects of Sarcoplasmic Reticulum Function ANTHONY MARTONOSI The Role of Periaxonal and Perineuronal Spaces in Modifying Ionic Flow across Neural Membranes W. J. ADELMAN,JR. AND Y. PALTI Properties of the Isolated Nerve Endings GEORGINARODdGUEZ DE LORES ARNAIZ AND

EDUARDODE ROBERTIS Transport and Discharge of Exportable Proteins in Pancreatic Exocrine Cells: In Vitro Studies J. D. JAMIESON

xiii

xiv

The Movement of Water across Vasopressin-Sensitive Epithelia RICHARD M. HAYS Active Transport of Potassium and Other Alkali Metals by the Isolated Midgut of the Silkworm WILLIAM R. HARVEYAND KARL ZERAHN Author lndex-Subject lndex

Volume 4 The Genetic Control of Membrane Transport CAROLYNW. SLAYMAN Enzymic Hydrolysis of Various Components in Biomembranes and Related Systems MAHENDRA KUMAR JAIN Regulation of Sugar Transport in Eukaryotic Cells HOWARDE. MORGANAND CAROLF. WHITFIELD Secretory Events in Gastric Mucosa RICHARD P. DURBIN Author Index-Subject lndex

CONTENTS

OF PREVIOUS VOLUMES

A Macromolecular Approach to Nerve Excitation ICHIJI TASAKIAND EMILIO CARBONE Subject Index

Volume 6 Role of Cholesterol in Biomembranes and Related Systems MAHENDRAKUMAR JAIN Ionic Activities in Cells A. A. LEV AND W. McD. ARMSTRONG Active Calcium Transport and Caz+Activated ATPase in Human Red Cells H. J. SCHATZMANN The Effect of Insulin on Glucose Transport in Muscle Cells TORBENCLAUSEN Recognition Sites for Material Transport and Information Transfer HALVORN. CHRISTENSEN Subject lndex

Volume 7 Volume 5 Cation Transport in Bacteria: K+, Na+, and H+ FRANKLIN M. HAROLDAND KARLHEINZ ALTENWRF Pro and Contra Carrier Proteins; Sugar Transport via the Periplasmic GalactoseBinding Protein WINFRIEDBoos Coupling and Energy Transfer in Active Amino Acid Transport ERICH HEINZ The Means of Distinguishing between Hydrogen Secretion and Bicarbonate Reabsorption: Theory and Applications to the Reptilian Bladder and Mammalian Kidney WILLIAM A. BRODSKYAND THEODOREP. SCHILB Sodium and Chloride Transport across Isolated Rabbit Ileum STANLEYG. SCHULTZ AND PETER F. CURRAN

Ion Transport in Plant Cells E. A. C. MACROBBIE H+ Ion Transport and Energy Transduction in Chloroplasts RICHARD A. DILLEYAND ROBERTT. GIAQUINTA The Present State of the Carrier Hypothesis PAUL G. LEFEVRE Ion Transport and Short-circuit Technique WARRENS. REHM Subject lndex

Volume 8 Chemical and Physical Properties of Myelin Proteins M. A. MOSCARELLO The Distinction between Sequential and Simultaneous Models for Sodium and Potassium Transport AND R. P. GARAY P. J. GARRAHAN

xv

CONTENTS OF PREVIOUS VOLUMES

Soluble and Membrane ATPases of Mitochondria, Chloroplasts, and Bacteria: Molecular Stnicture, Enzymatic Properties, and Functions RIVKAPANETAND D. RAO SANADI Competition, Saturation, and Inhibition-Ionic Interactions Shown by Membrane Ionic Currents in Nerve, Muscle, and Bilayer Systems ROBERTJ. FRENCH AND WILLIAMJ. ADELMAN,JR. Properties of the Glucose Transport System in the Renal Brush Border Membrane R. KINNE Subject Index

Volume 9 The State of Water and Alkali Cations within the Intracellular Fluids: The Contribution of NMR Spectroscopy MORDECHAISHPORER AND MORTIMER M. CIVAN Electrostatic Potentials at MembraneSolution Interfaces STUARTMCLAUGHLIN A Thermodynamic Treatment of Active Sodium Transport S. ROY CAPLANAND ALVIN ESSIG Anaerobic Electron Transfer and Active Transport in Bacteria WIL N. KONINGS AND JOHANNESBOONSTRA Protein Kinases and Membrane

Phosphorylation M. MARLENEHOSEYAND MARIANO TAO Mechanism and Physiological Significance of Calcium Transport across Mammalian Mitochondria1 Membranes LEENA MELA Thyroidal Regulation of Active Sodium Transport F. ISMAIL-BEIGI Subject Index

Volume 10 Mechanochemical Properties of Membranes E. A. EVANSAND R. M. HOCHMUTH Receptor-Mediated Protein Transport into Cells. Entry Mechanisms for Toxins, Hormones, Antibodies, Viruses, Lysosoma1 Hydrolases, Asialoglycoproteins, and Carrier Proteins DAVIDM. NEVILLE,JR. AND TA-MINCHANG The Regulation of Intracellular Calcium ERNESTOCARAFOLI AND MARTIN CROMPTON Calcium Transport and the Properties of a Calcium-Sensitive Potassium Channel in Red Cell Membranes VIRGIL10 L. LEW AND HUGOG. FERREIRA Proton-Dependent Solute Transport in Microorganisms A. A. EDDY Subject Index


Current Topics in Membranes and Transport volume 11

Cell Surface Glycoproteins: Structure, Biosynthesis, and Biological Functions


CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME

The Cell Membrane-A

11

Short Historical Perspective

ASER ROTHSTEIN Research Institute The Hospital for Sick Children Toronto, Cunudu

This volume reviews several selected topics concerning membrane glycoproteins. Although it is a relatively large volume, it does not offer a complete and comprehensive coverage of existing knowledge on this subject. The incomplete coverage reflects the intense and diverse interests in glycoproteins of large segments of the biological research community. Although soluble glycoproteins have been studied for many years, interest in membrane-bound glycoproteins is of relatively recent origin. For example, in a general review on glycoproteins published in 1970 (Spiro, 1970), only 2 pages out of 39, and 15 out of 274 references, were devoted to membrane glycoproteins. Today, however, membrane glycoproteins (and membrane proteins in general) are a central theme of membrane research. A major shift in interest has occurred that can be illustrated by the following observations. In 1968, when the membrane literature was reviewed for the previous year in theAnnua1 Reuiew of Physiology (Rothstein, 1968), about 400 references were used, but an estimated total of about 600 membrane papers had been published. A breakdown of the topics covered (Table I) indicated that the large majority of papers were concerned with membrane transport activities (65%). Proteins and glycoproteins, per se, were not a recognizable category of membrane study, with the exception of papers concerned with membrane ATPases. The membrane, at that time, was largely the domain of the physiologist, the biophysicist, and the electron microscopist. Table I indicates that by 1976, however, biochemically oriented studies constituted the major thrust of membrane research (42% of the total), with a large proportion of such studies devoted to glycoproteins or to membrane receptors known or implied to be glycoproteins. Such studies now constitute a large interdisciplinary area of research, encompassing major compo1 Copyright 0 1978 by Academic Press. Inc. All rights of reproduction in any form reserved. ISBN 0-12-153311-5

2

ASER ROTHSTEIN

TABLE I ANALYSISOF NUMBERS AND CONTENT OF PAPERS ON MEMBRANES PUBLISHED I N 1968 AND 1976 1968"

1976b

Topic

Number

Percent

Number

Percent

Structure Fractionation Models (real and theoretical) Physical properties Transport Membrane biosynthesis Proteins and glycoproteins Enzymes Receptors (hormones, antibodies, and carriers) Lipids and glycolipids Miscellaneous Total

11 10 36

3 2 9

336 120 324 131 996 144 300 516c 420

9 3 9 3 27 4 8 14 12

156 156 35w

4 4

-

-

265 6

65 1 11 9

47 36

411d

From Table 1 in Rothstein (1971);papers cited in Rothstein (1968). Averages from three randomly selected issues of Biological Membrane Abstracts, Information Retrieval Ltd., London. About one-half of the papers on enzymes involve ATPase, and one-quarter CAMP. Only two-thirds of the papers collected were cited, thus the total is about 600.

nents of biochemistry, immunology, endocrinology, embryology, cell biology, and physiology. The change in emphasis in membrane research is reflected not only in the shift toward a biochemical-cell biological approach, but also in a massive increase in the amount of research effort. It is estimated that, from 1948 to 1968, the number of publications in the membrane field increased at a uniform rate of about 8% per year based on the cumulative number of references cited in the biennial reviews in the Annual Review of Physiology. (During that period, the reviews tended to more or less cover the field of membranes in general.) In view of the fact that this was a period of rapid expansion of biological science, growth of the membrane field was, in all probability, not quite keeping pace with that of other biological fields. Between 1968 and 1976 a major discontinuity in the trend had occurred; the annual rate of production of papers jumped from about 600 to about 3600. Much of the increase took place in the 5-year period between 1968

CELL MEMBRANE-SHORT

HISTORICAL PERSPECTIVE

3

and 1973, because by 1973 the annual rate was already over 2500 (based on the number of abstracts appearing annually in Biological Membrane Abstracts, Information Retrieval Ltd., London). In the field of transport alone, Andreoli and Schaffer (1976) estimated that 3000 papers appeared between 1973 and 1976. It is an astounding fact that, in 1977 alone, almost as many papers on membranes were published as in the 20-year period 1948-1968. The increase in productivity is also reflected in a variety of ways: more journals publishing papers; new journals largely devoted to original papers on membranes or to biomembrane reviews; numerous books on membranes; numerous membrane symposia, workshops, and training courses; and so on. The growth reflects not only an expanded interest in the more classic kinds of membrane studies (transport and membrane structure), but the attraction into the field of investigators from other disciplines (biochemistry, immunology, endocrinology, genetics, and cell biology). The membrane field has become attractive to many new investigators willing to invest their efforts, to journals willing to devote their pages, and to funding agencies willing to donate their money. As an introduction to this volume, we try to analyze the reasons for the rapid growth of interest in membranes and also for the shift in emphasis toward studies relating to proteins and glycoproteins. Because current trends have their origins in the past, we try to place the analysis in historical perspective by briefly outlining past achievements, with appropriate attention paid to both conceptual and technological development. The membrane field, like many fields of biology, is often technology-limited, so that perhaps as many major advances have resulted from technical as from conceptual breakthroughs. The membrane field is relatively old as far as experimental biology is concerned, dating back to the middle of the nineteenth century. Simple observations with the microscope on the extent of shrinking (plasmolysis) of plant cells in osmotic gradients led to the realization that the cells obeyed the laws of osmosis. It was therefore concluded that the cells must be covered by an invisible, semipermeable membrane that allowed water, but not solutes, to pass during the period of observation. Thus the biophysical approach was born. Later in the century, it became clear that osmotically shrunken cells in some solutes slowly recovered their normal volume, and that normal cells swelled (and hemolysis occurred in the case of erythrocytes), leading to the conclusion that some solutes could also penetrate the membrane. By the end of the nineteenth century, Overton and others had systematically determined the rates of penetration of a variety of nonelec-

4

ASER ROTHSTEIN

trolytes and reported a strong corrleation between the measured permeabilities of solutes and their lipid solubilities (partition coefficients). He concluded that the hypothetical, invisible cell membrane must be largely lipid. He also observed that solutes of small molecular size penetrated faster than would be predicted from their lipid solubilities. It was therefore proposed that the lipid membrane might have aqueous membrane “patches” through which small nonelectrolytes could pass. This concept of a mosaic membrane was the progenitor of the modern lipid mosaic membrane theory, formalized by Singer and Nicolson (1972), according to which a continuous lipid matrix is penetrated by transmembrane proteins that may provide permeation pathways for hydrophilic solutes. In quantitative terms, the penetration of nonelectrolytes and the consequent swelling or shrinking of cells were expressed in terms of derivations of the laws of diffusion, with the membrane considered a resistance to diffusion defined in terms of a permeability constant. The concept of permeability in these rigorous terms was refined and developed during the period 1900-1940. A finding that was to lead to the next major advance in membranology was made in the 1920s. Gorter and Grendel(l925) reported that the red blood cell contained just sufficient lipid to form a continuous bilayer over its surface. This observation formed the starting point for the general concept of a continuous lipid bilayer membrane as proposed by Danielli (1943).This model elegantly explained a large variety of permeation data on the basis of lipid partition coefficients and on the basis of the energy barriers to solute flow that existed at the water-lipid interface and within the membrane interior (viscosity). At this time, the membrane was a hypothetical lipid structure invented to explain permeability and osmotic behavior of cells, largely in relation to nonphysiological nonelectrolytes. No functional role was assigned to proteins, although they were presumed to be associated in some way with the surfaces (particularly the inner surface) of the bilayer. It was recognized that physiological substrates such as sugars could be taken up and used by cells or absorbed in the intestine and kidney, but membrane activities relating to metabolites were not extensively explored because meaningful permeability coefficients could not be assigned. By the operational criteria used, the cells were considered relatively impermeable to electrolytes. The next 20 years (1940-1960) represented a revolutionary period in membrane work. A major stimulus was a technical breakthrough, the production of radioactive isotopes and of counting devices which allowed the study of movements of ions, particularly Na+ and K+,

CELL MEMBRANE-SHORT


5

Ca’+, and anions, and of substrates such as sugars and amino acids. The entire emphasis shifted from nonfunctional nonelectrolytes to the functionally important ions and metabolites. It became clear that cells could maintain nonequilibrium distributions of cations despite the fact that they were permeable to these ions. Most of the clarification of ion transport mechanisms came from a rigorous analysis, in thermodynamic terms, of ion flows and distributions across membranes, with appropriate consideration given to the role of the membrane potential as well as to the concentration gradients as driving forces. It was noted that, in addition to the expected fluxes of ions down their electrochemical gradients, fluxes could also take place in the uphill direction against electrochemical gradients. The energy to drive such movements was obviously derived from cell metabolism. The process became known as active transport. An important technical breakthrough was the short-circuit technique developed by Ussing (1954) for use with frog skin (and later with other tissues). With this technique, all external driving forces (electrical and chemical) for ion flow across the membrane could be eliminated, so that only active transport generated by metabolic events within the skin resulted in net movements. During the same period, the thermodynamic approach was used to develop the foundations of modern electrophysiology. In nerve (and muscle), it became clear that membrane potentials and electrical conduction were determined largely by cation gradients (Na+ and K+) maintained by active transport systems, and by cation permeabilities. Technical advances, such as the development of microelectrodes, and the voZtage clamp procedure (similar in concept to the short-circuit technique) were used to describe in detail the cation permeability changes occurring during conduction (Hodgkin, 1958). While flows and forces were being defined in thermodynamic terms, the mechanisms of permeation and transport were being determined by the application of enzyme kinetics. As in the case of enzyme activity, many transport activities reached limiting (maximal) rates as solute concentrations were increased. Furthermore, pairs of chemically related substrates appeared to compete for transport. It was therefore proposed that transport must involve a reversible interaction of the transported molecule with a hypothetical membrane site called a carrier. The substrate-carrier complex was assumed to move across the membrane and was therefore considered a mobile carrier (Wilbrandt and Rosenberg, 1961). On the structural side, another major technical breakthrough was the development of the electron microscope. With its use, an abstract

6

ASER ROTHSTEIN

membrane conceptualized from permeability and transport data became a real structure that could be seen as a “railroad track” on a photographic print (Robertston, 1959). The spacings of the images obtained with metal stains were consistent with the Danielli (1943) continuous bilayer model. The bilayer structure was also confirmed by x-ray diffraction analysis (Finean, 1961). On the biochemical side, progress was slow. It was recognized that membrane entities (carriers) must be involved in the many kinds of transport and that metabolic reactions must provide the energy for active transport, but there seemed to be no way to identify these hypothetical substances and reactions. The membrane was therefore accepted as a “black box,” albeit a lipid one, across which certain substances could flow in a characteristic manner and which could transform metabolic energy into uphill flows of certain solutes. Although it was generally accepted that the basic structure of the membrane was a lipid bilayer, studies on water permeability suggested that flow through “pores” might be involved (Paganelli and Solomon, 1957) (this was a revival of the mosaic membrane concept and another preview of the modern lipid mosaic model). No one knew as yet what to do about membrane proteins, although it was clearly demonstrated during this period that several enzymes were localized on the membrane outer surface (Rothstein, 1954). In the period 1960-1970, the momentum of membrane research continued to increase along lines established during the previous 20 years, but with intensified interest in membrane proteins, and with the beginning of an interest in membrane glycoproteins. The kinetic analysis of transport resulted in more complex and sophisticated models. Asymmetric behaviors were identified, and the substrate-carrier binding was found to be influenced by interactions at other sites (regulator or modifier sites). Thus the structure and behavior of proposed carriers became more and more complicated. On the thermodynamic side, a major conceptual advance was the application of irreversible thermodynamics, which formalized and quantitated the interactions between the flow of energy, solutes, and solvent and which allowed a more rigorous definition of transport (Kedem and Katchalsky, 1961). It became clear that the membrane could be considered an energy transducer in which the flow of a solute could be coupled to the flow of energy (active transport), to the flow of a solvent (water), or to the flow of another solute. Such coupling could account, for example, for the relationship of Na+ and fluid transport in epithelia, for the dependence of sugar and amino acid transport on Na+, and for the dependence of solute transport on proton gradients.

CELL MEMBRANE-SHORT


7

In addition to the fuller exploitation of thermodynamic and kinetic concepts of transport, this period saw development of the foundations of the molecular and biochemical approach to membrane structure and function. Increasing attention was paid to the biochemistry of membrane lipids and to the behavior of lipid models (monolayers, bilayers, and liposomes). Investigations on membrane proteins were stimulated by several observations:

1. Membrane ATPases were identified as components of the Na+-

K+ transport system (Skou, 1965). 2. Sugar transport systems (permeases) in bacteria were found to be under genetic control, presumably through mutations in membrane protein structures; proteins associated with two forms of sugar transport in bacteria, i.e., M protein (Fox and Kennedy, 1965)and the phosphotransferase system (Roseman, 1972),were directly identified. 3. Many kinds of permeation were found to be perturbed by agents that react with proteins (sulfhydryl reactive agents) (Rothstein, 1970).

4. Hormone-binding sites on the cell surface were identified as proteinaceous (glycoproteins) (Kahn, 1976), and CAMP, part of the hormone response system, was identified (Robison et al., 1970). Much of the work on membrane proteins had become possible because of technological success in preparing, identifying, and purifying plasma membrane fractions. Gross analytical determinations indicated that at least half of the total membrane content was protein, that many of the proteins contained substantial quantities of carbohydrate, and that many metabolic enzymes were associated with the membrane. These findings raised many questions about the architectural arrangements of proteins in the membrane and about their functional roles. It was established that membrane functions included not only the interchange of materials between the cell and its environment, but also the recognition of and response to signals from the environment, such as hormones, antibodies, and other cells. The membrane therefore became the focus of attention not only for physiologists and biophysicists interested in transport function, for anatomists interested in structure, and for biochemists interested in membrane lipids, enzymes, and other proteins, but also for endocrinologists, immunologists, embryologists, and cell biologists. This growing interest was matched at the same time by an unusual number of important technical advances that allowed startling progress to be made in the 1970s. They included: (1) improved procedures for marking, isolating, and purifying membranes; (2) sophisticated use of detergents for “dissolv-

8

ASER ROTHSTEIN

ing” membrane proteins; (3) development of covalent, nonpenetrating probes for determining the “sidedness” of membrane components; (4) development of specific probes to “mark” functional proteins and functional sites; ( 5 )use of proteolytic enzymes as probes of membrane-bound proteins; (6) development of subcellular vesicle systems derived from specific membranes; (7) reconstitution of functional proteins in model membranes; (8)development of cross-linking agents to investigate “near neighbors” among components; (9)sophisticated technologies such .as microcalorimetry, nuclear magnetic resonance, electron spin resonance, infrared spectroscopy, circular dichroism, and fluorescence analysis for evaluation of the physical state of the membrane; (10)acrylamide gel electrophoresis for separating and identifying membrane proteins; (11)gel filtration procedures for fractionating vesicles and macromolecular membrane components; (12) the freeze-fracture technique of electron microscopy; and (13)use of purified lectins to explore the sugar arrays of surface glycoproteins. On the conceptual side, research was also stimulated by formulation of the lipid mosaic model of membrane structure, which described the molecular arrangements of proteins in the membrane with respect to the lipids (Singer and Nicolson, 1972). It was proposed that the lipid bilayer forms a continuous phase that is interrupted or partially interrupted by a class of relatively hydrophobic (intrinsic) proteins inserted into the bilayer and held tenaciously by hydrophobic interactions. Such proteins can be removed from the membrane only by the use of detergents. They can be visualized as “particles” within the bilayer by the use of the freeze-fracture technique of electron microscopy. Other proteins (extrinsic) are associated largely with the inner face of the membrane by ionic or hydrogen bonding. They can be extracted by alterations in ionic strength or pH, by the use of cation chelating agents, or by protein perturbants. One of the key features of the membrane is its asymmetry. The lipid compositiion of the two halves of the bilayer is not the same, so that the preponderance of positively charged head groups faces inward (in red blood cells). The asymmetry with respect to proteins is especially pronounced. All the neutral sugar moieties and all the sialic acid groups of the glycoproteins (and glycolipids) are exposed on the outer face. The polypeptides bearing them are intrinsic proteins embedded in the lipid. In contrast, the extrinsic proteins are largely or entirely located on the inner face of the membrane. At least some of the intrinsic proteins pass all the way through the bilayer (span the membrane), so that they are exposed to both the external and internal environments. Because the internal segments of such intrinsic proteins may

CELL MEMBRANE-SHORT


9

be closely associated with some of the extrinsic proteins at the cytoplasmic face of the membrane, the membrane-spanning proteins may play an essential role in providing connections and communications across the bilayer that are important in transport phenomena and in the responses of cells to external stimuli. Much recent membrane work has involved the elucidation of detailed molecular architecture, the relationships of lipids to membrane proteins, interactions between membrane proteins, the identification and characterization of specific proteins, and the molecular mechanisms underlying functional activities. Some interest has also developed in the ways in which cell membranes are assembled, and how their structure is regulated so that they respond appropriately to external signals, growth, and changes in physiological state. From these manifold but interrelated investigations, patterns are beginning to emerge which we briefly outline, with emphasis on the glycoproteins which may play a key role in some membrane phenomena. Only highlights are considered. Detailed discussions of certain aspects are found in the following chapters. The glycoproteins of the cell membrane are largely intrinsic proteins, anchored by hydrophobic interactions in the lipid bilayer with the carbohydrate groups facing toward the outside. Conversely, many intrinsic plasma membrane proteins are glycosylated. The sugar groups form complex arrays with considerable variation from protein to protein. The polypeptide portions may be anchored in the lipid by a sequence of hydrophobic amino acid residues in a-helical form that may traverse the bilayer, but in some cases several polypeptides may collectively form a complex that traverses the membrane. The important point, from a functional point of view, is that protein continua span the bilayer so that they are in contact with both the external and cytoplasmic environments. They can thus provide a means of communication across the bilayer either for flow of solutes and water, or for signals” in response to external “messengers” such as hormones, antibodies, or other cells. Because under normal in vivo conditions the lipid bilayer is fluid, the intrinsic proteins can be considered to “float” in it. Although the proteins can migrate laterally under certain conditions, they may also be relatively fixed with respect to each other because of their interactions with a matrix of extrinsic proteins on the inner surface (and perhaps with each other). Thus their arrangement is not only relatively stable but can be unique. The particular arrays of glycoproteins on the surface may provide for structural features of the cell, and they may also provide specific loci for interaction with external factors. The surfaces of cells contain several exoenzymes, such as phospha6‘

10

ASER ROTHSTEIN

tases, polysaccharide hydrolases, cholinesterase, and aminopeptidase. These enzymes seem to have the simple function of digesting certain external substrates. Although the enzymes may be glycosylated, there is no evidence that the sugar moieties are involved in the recognition and binding of the substrate, or in the enzyme process. Such enzymes are held in the lipid bilayer by a hydrophobic segment of the polypeptide, but the insertion seems to serve only to anchor the enzymes in the membrane, playing no role in the catalytic activity. In the case of sucrase in the brush border of intestinal cells, no direct insertion into the bilayer is evident. Sucrase, however, is tightly bound to another enzyme, isomaltase, which has a hydrophobic anchor. The protruding portion containing the substrate binding site can in some cases be cleaved from the anchoring portion by proteolytic enzymes without loss of activity. The transport of solutes and of water is perhaps the most studied membrane function. It is certainly the one that has occupied our interest for the longest time. Although it is generally accepted that proteins are essential components of most transport or permeation systems (those in which a specific carrier seems to be involved), only a few specific transport proteins have as yet been identified, e.g., the cationtransporting ATPases for Na+, K+, and Ca+, the sugar-transporting system of bacteria, and the anion and sugar transport systems of the red blood cell. Characterization of these few identified systems is far from complete. Nevertheless, a general pattern is emerging which allows interesting speculations to be made. At least some polypeptides of each system (more than one polypeptide chain seems to be involved in the systems so far examined at this level) are highly hydrophobic and are inserted into the bilayer so that some protein segments are exposed to the outside and also to the cytoplasmic environment. Thus they form a protein continuum through the lipid through which transport can occur. The transmembrane portion is probably arranged with hydrophobic groups toward the lipid chains and with hydrophilic groups internalized (forming a largely aqueous channel through the bilayer). The arrangement of such protein channels with respect to the inside and outside of the cell is highly asymmetric. Each system contains a carrier site that specifically binds the transported entity and allows its translocation to occur. That site is therefore accessible from either side of the membrane. Translocation probably does not involve a rotational movement of the whole protein, but a small part of the protein containing the carrier site may be mobile, undergoing a conformational change associated with transport. In active transport systems, the movement is associated with enzymic, reactions. Other sites

CELL MEMBRANE-SHORT


11

that bind cofactors (such as ATP and magnesium), inhibitory substances, or perhaps regulatory substances are asymmetrically arranged, facing either outside or inside. The outward-facing segments of transport proteins may be glycosylated (so they are technically glycoproteins), but no evidence indicates that sugar moieties contribute to the specificity of binding of substrates or to transport characteristics per se. Not much is known about the molecular details of the transfer of macromolecules across the membrane. One mechanism of uptake involves endocytosis. A first step is the specific binding of the macromolecule. Recognition in this case may involve the sugar moieties of the transported molecule and of the membrane. For example, desialated glycoproteins are recognized by glycoproteins on the surfaces of liver and kidney cells as a prelude to their uptake and destruction by the cells. The set of functions in which glycoproteins seem to play an important and specific role is the response of cells to environmental substances. The response involves three types of interactions associated with three different parts of the glycoprotein molecule. The first is the recognition (specific binding) of an extracellular molecule (hormone, antibody, lectin, soluble glycoprotein, or the glycopeptide portion of another cell). The second involves a triggering reaction (or form of communication) across the membrane-spanning segment. The third involves interactions at the inner face of the membrane between the membrane gl ycoproteins and other proteins. This interaction may involve various responses, e.g., release of CAMP, phosphorylation of protein, release of Ca2+,or modulation of the microtubular network. As a result, the cell may respond in a variety of complex ways involving shifts in metabolic paths, changes in permeability, endocytosis, protein synthesis and growth, movement, and so on. No direct role can presently be given for glycoproteins in membrane movement or flow, or in the maintenance of special forms of membrane architecture, although all these processes may be modulated by external factors through the mediation of surface glycoproteins. In summary, the ubiquity of glycoproteins in cell membranes is well established. Knowledge of their chemical structure and of their mode of synthesis is considerable, as evidenced by the information presented in this volume. The real deficit in our information concerns the specific functional roles of the many glycoproteins. In proteins that function as exoenzymes or play a role in transport, the carbohydrate moiety seems to play no role in the binding of substrates. In a

12

ASER ROTHSTEIN

few cases, the carbohydrate containing segments can even be digested away without loss of function. Why, then, are these proteins glycosylated? The only functions in which the carbohydrate moieties of glycoproteins are directly implicated are those related to recognition phenomena. Even here, hard evidence is relatively sparse and, in the case of cell-cell interactions, limited largely to cells of lower organisms. Presumably this deficit of information will be rectified in the future, for much research activity is evident in this area. The period from 1970 until today has moved membrane studies ahead by a quantum jump. In the next period of time, one can foresee that the functional role of many additional membrane proteins will be established and elucidated, allowing an analysis of the mechanisms of the various functions at a molecular level. More membrane proteins will be characterized in detail, and sequence analysis will also be involved. The relationship of membrane proteins to each other (membrane topology) will become a more popular topic as technology allows, because it is becoming increasingly evident that many functions involve systems of proteins arranged in the membrane in orderly arrays. Perhaps the most interesting studies one can anticipate will be concerned with the assembly of membranes and the control of this assembly in response to growth, differentiation, and physiological state. This kind of information is central to the understanding of embryological development, responses of cells to normal and abnormal conditions, and cell pathologies. It is safe to predict that the study of membranes is still a growth area in biological science. REFERENCES

A few literature citations are listed below that refer to some of the older studies. No references are given to support the many inferences drawn from more recent findings. The chapter covers such a wide array of information that even a selective list of references would be far longer than the text. The tentative conclusions expressed must therefore be considered to b e one man’s unverified opinion. Andreoli, T. E., and Schaffer, J. A. (1976).Mass transport across cell membranes: The effects of antidiuretic hormone on water and solute flows. Annu. Rev. Physiol. 38,

451-500. Danielli, J. F. (1943).The theory of penetration of a thin membrane. I n “The Penneability of Natural Membranes” (by H. Davson and J. F. Danielli), pp. 341-352. Cambridge Univ. Press, London. Finean, J. B. (1961).“Chemical Ultrastructure in Living Tissues.” Thomas, Springfield, Illinois. Fox, C . F., and Kennedy, E. P. (1965).Specific labeling and partial purification of the M protein, a component of the P-galactoside transport system of Escheri>hia coli. Proc. Natl. Acad. Sci. U.S.A. 54,891-899. Gorter, E.,and Grendel, F. (1925).Bimolecular layers of lipoids on chromocytes of blood.]. Erp. Med. 41,439-443.

CELL MEMBRANE-SHORT


13

Hodgkin, A. L. (1958). Ionic movements and electrical activity in giant nerve fibers. Proc. R . SOC., Ser. B 148, 1-37. Kahn, C. R. (1976). Membrane receptors for hormones and neurotransmitters. J . Cell Biol. 70,261-286. Kedem, O., and Katchalsky, A. (1961).A physical interpretation of the phenomenological coefficients of membrane permeability. J . Gen. Physiol. 45, 143-179. Paganelli, C. V., and Solomon, A. K. (1957). The rate of exchange of tritiated water across the human red cell membrane.J. Gen. Physiol. 41,259-277. Robertson, J. D. (1959).T h e ultrastructure of cell membranes and their derivatives. Biochem. SOC. Symp. 16,3-43. Robison, G. A., Schmidt, M. J., and Sutherland, E. W. (1970).“Cyclic AMP.” Academic Press, New York. Roseman, S. (1972).Carbohydrate transport in bacterial cells. I n “Metabolic Pathways” (C. Hokin, ed.), 3rd Ed., Vol. 6, pp. 41-89. Academic Press, New York. Rothstein, A. (1954).The enzymology of the cell surface. Protoplamatologia 2, E4. Rothstein, A. (1968).Membrane phenomena. Annu. Reu. Physiol. 30, 15-72. Rothstein, A. (1970).Sulfiydryl groups in membrane structure and function. Curr. Top. Membr. Transp. 1, 135-176. Rothstein, A. (1971). Fashions in Membranology. In “Intestinal Transport of Electrolytes, Amino Acids and Sugars” (W. McD. Armstrong and A. S. Nunn, Jr., eds.), pp. 3-11. Thomas, Springfield, Illinois. Singer, S. J., and Nicolson, G. L. (1972).The fluid mosaic model of the structure of cell membranes. Science 175,720-731. Skou, J. C. (1965). Enzymatic basis for active transport of Na+ and K+ across the cell membrane. Physiol. Reo. 45,596-618. Spiro, R. G. (1970).Glycoproteins. Annu. Reu. Biochem. 39,599-638. Ussing, H. H. (1954).Active transport of inorganic ions. Symp. SOC. E x p . Biol. 8,407-422. Wilbrandt, W., and Rosenberg, T. (1961).The concept of carrier transport and its corollaries in pharmacology. Pharmacol. Rev. 13, 109-183.


CURRENT TOPICS IN MEMBRANES AND TRANSPORT. VOLUME

11

The Structure and Biosynthesis of Membrane GIycoproteins JENNZFER STURGESS. MARZO MOSCARELLO. AND HARRY SCHACHTER Departments of Biochemistry and Pathology The Research Institute The Hospital for Sick Children Toronto. Ontario. Canada

I . Carbohydrate Asymmetry across the Membrane Bilayer . . . . . . . I1 . Glycoprotein Structure . . . . . . . . . . . . . . . . A . Glycophorin . . . . . . . . . . . . . . . . . . B Rhodopsin . . . . . . . . . . . . . . . . . . . C . Epiglycanin . . . . . . . . . . . . . . . . . . D . Murine Histocompatibility Antigens . . . . . . . . . . . E Ashwell’s Mammalian Lectin . . . . . . . . . . . . . F. The LETS Glycoprotein . . . . . . . . . . . . . . G . Enveloped Viruses . . . . . . . . . . . . . . . . H . Cytochrome b, and Cytochrome b. Reductase . . . . . . . . I . Glycoproteins of Myelin . . . . . . . . . . . . . . J . Proteolipid Protein Fraction of Myelin . . . . . . . . . . K . General Comments on Membrane Protein Structure . . . . . . I11. Model Membrane Systems . . . . . . . . . . . . . . . IV . T h e Glycosylation Reaction . . . . . . . . . . . . . . . A . Nucleotide Sugar Formation . . . . . . . . . . . . . B . Polyprenol Phosphate Sugar Formation . . . . . . . . . . C . Dolichol Pyrophosphate Oligosaccharides and the Assembly of Asn-GlcNAc Core Oligosaccharide . . . . . . . . . . . D . Elongation of N-Acetyllactosamine-type Oligosaccharides . . . . E . Assembly of Ser(Thr)-GalNAcOligosaccharides . . . . . . . V. Subcellular Sites of Glycosylation . . . . . . . . . . . . . A Autoradiographic Evidence . . . . . . . . . . . . . B. Subcellular Localization of Glycosyltransferases . . . . . . . C . The Role of the Ribosome . . . . . . . . . . . . . . VI . Membrane Biogenesis . . . . . . . . . . . . . . . . A . Biogenesis of Plasma Membranes . . . . . . . . . . . B. Biogenesis of Intracellular Membranes . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . .

.

.

.

16 19 24 25 26 26 27 28 28 29 30 32 33 34 37 37 42 46 53 58 61 61

64 64 67 70 83 85 15

Copyright @ 1WB hy Academic Press. Inc. All rights of reproduction in any form reserved. ISBN 0-12-153311-5

16

J. STURGESS, M. MOSCARELLO, AND H. SCHACHTER

Glycoproteins are a large and heterogeneous group of macromolecules and serve a variety of different functions. They occur as soluble secretory molecules such as plasma glycoproteins (a,-acid glycoprotein, transferrin, haptoglobin, and so on), protein hormones (gonadotrophins, thyroglobulin), enzymes (RNase, DNase, amylase, and so on), immunoglobulins, mucins, blood group glycoproteins, acid mucopolysaccharides (glycosaminoglycans), collagens, and basement membranes. The current interest in membrane structure has drawn the attention of many researchers to the fact that glycoproteins also occur in an insoluble form as components of cell membranes. The fluid mosaic model of membrane structure (Singer and Nicolson, 1972) emphasized the important role of proteins as both integral and peripheral components of membranes; the structures of some of these membrane proteins have recently been studied, and it has been realized that many of them are glycoproteins. Some membrane glycoproteins have been isolated by extraction of the membrane with dissociating reagents (Table I); the presence of other membrane glycoproteins has been detected by more indirect methods, e.g., the use of lectins (see Noonan, this volume) and of other carbohydrate-detecting reagents has established the presence of glycoproteins on the surfaces of most cells. The receptors for several hormones (insulin, luteinizing hormone, chorionic gonadotrophin, ACTH) are probably glycoproteins. This chapter reviews the structure and biosynthesis of these membrane-bound glycoproteins; since more information is available on secretory glycoproteins than on membrane glycoproteins, it will occasionally be necessary to extrapolate from the former to the latter. I. CARBOHYDRATE ASYMMETRY ACROSS THE MEMBRANE BILAYER

One of the most interesting concepts arising out of the study of membranes is that there is asymmetry across the membrane bilayer (Rothman and Lenard, 1977; Bretscher, 1973; Singer, 1974). This asymmetry is absolute for proteins and carbohydrates, at least at the sensitivity level of the techniques available to study this problem; i.e., every polypeptide and every glycoprotein molecule has the same orientation across the lipid bilayer. Phospholipids show only partial asymmetry in that every phospholipid is present on both sides of the bilayer but in a different amount. The carbohydrate of the cell surface membrane is always oriented toward the external environment; this fact and the analogous carbohydrate asymmetry across intracellular

17

STRUCTURE A N D BIOSYNTHESIS OF MEMBRANE GLYCOPROTEINS

TABLE I

MEMBRANEGLYCOPROTEINS

Membrane glycoprotein Intrinsic Erythrocyte membrane glycophorin Erythrocyte membrane coomassie blue band 111 Bovine retina rhodopsin Epigl ycanin Murine histocompatibility antigen (H-2) Human histocompatibility antigen (HL-A) Mammalian liver lectin LETS glycoprotein VSV G protein Cytochrome b5 Cytochrome b, reductase Human myelin lipophilin Mouse liver nucleotide pyrophosphatase Oviduct membrane glycoprotein Infectious mononucleosis heterophile antigen (Paul-Bunnell antigen) Peripheral Sarcoplasmic reticulum calsequestrin I Mitochondria1 Caz+-binding glycoprotein Platelet glycocalicin

Nonpolar residues (%)

35-40

-

MW

31,000 90,000

47

-

35,000 100,000-500,000 43,000-47,000

-

30,000

51 -

-

38

64 40

-

40,000; 48,000 210,000-270,000 67,000 16,700 33,000 25,000-28,000 130,000

References"

1-8 4 9, 10 11 12 13, 14

15 16, 17 18-20 21 22 23,24 25

56

25,000 25,700

26 27

43

46,000

28

25

33,000

29

57

148,000

30

(1)Winder (1972);(2) Marchesi et al. (1972); (3) Segrest et al. (1973); (4) Marchesi et a!. (1976); (5) Tomita and Marchesi (1975);(6) Springer et al. (1966); (7) Cleve et al. (1972); (8) Morawiecki (1964); (9) Plantner and Kean (1976); (10) Heller (1968); (11) Codington et al. (1975a,b); (12) Nathenson and Cullen (1974); (13) Tanigari et al. (1973); (14) Creswell et al. (1973); (15) Kawasaki and Ashwell (1976a,b); (16) Hynes (1976); (17) Hunt and Brown (1975);(18) Morrison and Lodish (1975);(19) Knipe et al. (1977a);(20) Toneguzzo and Ghosh (1975); (21) Ozols (1972); (22) Strittmatter (1971); (23) Moscarelloet al. (1973);(24) Moscarello (1976);(25) Evans et al. (1973);(26) Chen and Lennarz (1976);(27)Merricket al. (1977);(28) MacLennan (1975);(29)Carafoli and Sottocasa (1974); (30) Okumura et al. (1976).

18

J. STURGESS, M. MOSCARELLO, AND

H. SCHACHTER

membranes are essential in understanding membrane glycoprotein biosynthesis. The close association of carbohydrate with the cell membrane has been demonstrated using cytochemical techniques such as the periodicacid-Schiff (PAS)reagent (Leblond, 1950)and shown to be a characteristic feature of all cell surfaces except in the region of junction complexes (Rambourg et al., 1966). The density of PAS-reactive carbohydrates varies; for instance in intestinal epithelial cells the apical surfaces stain heavily and the lateral surfaces stain only moderately. Carbohydrate staining always shows asymmetry in that it is distributed on the external surface but not on the cytoplasmic surface of the plasma membrane (Rambourg and Leblond, 1967). The reactive 1,2glycol groups are presumably due to membrane-associated glycoproteins, since glycolipids are extracted during tissue preparation (Winzler, 1970). Electron-dense markers such as colloidal iron (Gasic and Berwick, 1962; Benedetti and Emmelot, 1967), colloidal thorium (Rambourg and Leblond, 1967), ruthenium red (Luft, 1971), and cationized ferritin (Danon et al., 1972; Hackenbrock and Miller, 1975) have demonstrated the presence of acidic or anionic sites at the cell surface; these sites are usually attributed to the carboxyl groups of protein-bound sialic acid residues. The reactive groups detected by these cytochemical techniques are believed to be on carbohydrate residues attached to the polypeptide portions of integral membrane proteins exposed at the external surface of the cell (Winzler, 1969). Similar cytochemical approaches have been applied to subcellular membrane fractions to study the distribution and intracellular localization of glycoproteins. Among intracellular membranes, staining of carbohydrate residues has implicated the Golgi complex in a central role in glycoprotein biosynthesis. Cytochemical staining of the Golgi complex with periodic acid-silver methanamine and with phosphotungstic acid at low pH has shown that glycoproteins are localized mainly in Golgi membrane saccules, cytoplasmic vesicles, and lysosomes. The reaction product occurs at the inner surface, directed into the intracisternal or intravesicular space (Rambourg et al., 1969). This asymmetry of membranes is the reverse of that observed at the cell surface. Recently, carbohydrate residues have been demonstrated also on the cytoplasmic surface of the membranes; for instance, anionic binding sites have been demonstrated on the outer face of the Golgi complex by the binding of cationized ferritin (Abe et al., 1976). However, the membrane-associated carbohydrates on the cytoplasmic surface are of lower density than those observed on the inner membrane face. In rough endoplasmic reticulum, the binding of conca~

STRUCTURE AND BIOSYNTHESIS OF MEMBRANE GLYCOPROTEINS

19

navalin A (Con A) occurs exclusively on the inner membrane surface, i.e., the surface opposite that occupied by ribosomes (Nicolson and Singer, 1971; Hirano et al., 1972). In the Golgi complex, Con A binds on the inner membrane surface and, to a limited extent, on the cytoplasmic surface of the membranes (Abe et ul., 1977). This indicates that carbohydrate residues are present, and probably assembled, mainly on the inner aspect of the membrane, but that some may be exposed on the cytoplasmic surface. Similarities in cytochemical reactivity between the Golgi complex and the plasma membrane provide evidence for a biogenetic relationship in which the plasma membrane has a complement of Golgi-type membranes that are everted following fusion of membrane vesicles with the cell surface (Fig. 1).

II. GLYCOPROTEIN STRUCTURE

The number of membrane glycoproteins that have been isolated in a form pure enough for structural studies is quite small (Table I). A discussion of membrane glycopratein structure must therefore be limited, and generalizations based on the available data are obviously subject to error. At the present state of knowledge it appears that the oligosaccharide moieties of membrane-bound glycoproteins are structurally similar to the moieties present on secretory glycoproteins. A brief review of glycoprotein structure is therefore presented, although much of this information is based on secretory glycoproteins. It has become customary to discuss glycoprotein structure on the basis of the carbohydrate-amino acid linkage of the oligosaccharide moiety. This approach has been useful because the carbohydrateamino acid linkage dictates the properties of the associated oligosaccharide group and because a single glycoprotein molecule may contain oligosaccharides of more than one linkage type. Animal glycoproteins contain oligosaccharides of four types of carbohydrate-amino acid linkages (Schachter and Roden, 1973): (1) asparagine-N-acetylglucosamine (Asn-GlcNAc), (2) serine(threonine)-N-acetylgalactosamine [Ser(Thr)-GalNAc], (3)hydroxylysine-galactose, and (4)serinexylose. The hydroxylysine-galactose-type linkage occurs only in collagens and basement membranes, while the serine-xylose-type linkage occurs in the chondroitin sulfates and possibly in other acid mucopolysaccharides (Roden, 1970); these types are therefore not discussed further. The Asn-GlcNAc-type linkage is of very wide distribution in animal glycoproteins and is found in many different secretory proteins (plasma glycoproteins, protein hormones, enzymes, immuno-

20

J. STURGESS,

M. MOSCARELLO, AND H . SCHACHTER

hOS RIBOSOMAL SUBUNIT

-mRNA - 605

RIBOSOMAL SUBUNIT

PEPTIOE HYDROPHOBIC SEOUENCE

mRNA

CY TOPLASMIC JFACE

-9 ???? ? ??? - - 111 111A

LIPID BlLAYER OF ROIJGH - ENDOPLASMIC RETICULUM -

9 9 1 P O ? PP- - 11111111- - -

\ INTRAVESICULAR FACE

RIBOSOME BINOING PROTEIN

8 OSYLTRANSFERASE PLASMA MEMBRANE

n

~

EXTRACELLULAR

FIG.1. Schematic illustration of the biosynthesis of membrane glycoproteins and their insertion into the plasma membrane. (1)Synthesis of membrane protein probably begins on free ribosomes. By analogy to the situation for secreted proteins, it is postulated that the N-terminal sequence of newly synthesized peptide carries a hydrophobic signal sequence (Blobel and Dobberstein, 1975a,b; Schechter et al., 1974) which serves to attach the free ribosome to the endoplasmic reticulum membrane. The evidence that all glycoproteins, including membranous glycoproteins, are assembled on membranebound ribosomes is quite convincing (Schachter, 1974a,b; Morrison and Lodish, 1975; Bergeron et al., 1975), but the presence of a signal N-terminal sequence on a nascent membrane glycoprotein has not yet been demonstrated. However, recent work on the biosynthesis of viral membrane glycoproteins (Rothman and Lenard, 1977; Wirth et al., 1977) suggests that data on the question of a signal sequence will soon be forthcoming. (2) The ribosome becomes attached to the endoplasmic reticulum membrane via a ribsome-binding protein which is postulated to assemble into a hydrophilic channel for


21

globulins) as well as in membrane glycoproteins. The Ser(Thr)GalNAc-type linkage is found in mucins and blood group glycoproteins and has also been described in the major red cell membrane glycoprotein, i.e., glycophorin. Asn-GlcNAc oligosaccharides always have a core structure containing only GlcNAc and mannose residues; this core often, but not always, has the branched structure shown in Fig. 2 (Montreuil, 1975). The further elongation of the core structure can take one of two gen-

’

the passage of nascent peptide through the membrane into the intravesicular space. (3)As the ribosome moves along the mRNA, translation occurs and more and more of the nascent peptide enters the intravesicular space. (4) It is postulated that intrinsic membrane proteins have a hydrophobic sequence which anchors them into the membrane (Marchesi et al. 1976; Singer and Nicolson, 1972). Presumably, when translation of this sequence occurs, the hydrophilic channel somehow dissipates and the hydrophobic sequence anchors the peptide in the membrane. (5)The disappearance of the hydrophilic channel probably will cause release of the ribosome from the membrane. Completion of the C-terminal sequence can take place, but without further movement of nascent peptide through the membrane. Also, a t some point in this sequence of events, the N-terminal hydrophobic signal sequence may be cleaved by a peptidase or “signalase” (Blobel and Dobberstein, 1975a) present on the inside of the endoplasmic reticulum. Carbohydrate incorporation occurs primarily after the release of nascent peptide from the polyribosome complex, but there is evidence that some carbohydrate may also become attached to the nascent peptide (Schachter, 1974a,b; Kiely et ul., 1976). (6) Peptide synthesis is complete, and the peptide detaches from the polyribosome complex; the ribosome falls off the mRNA. The peptide is now incorporated into the membrane as indicated. Carbohydrate incorporation occurs, catalyzed by a battery of enzymes called a multiglycosyltransferase system (Schachter and Rod&, 1973; Roseman, 1970); these enzymes are firmly attached to the endomembrane system, and there is evidence that the active sites are directed toward the intravesicular space. Since some membrane glycoproteins (e.g., red cell membrane band 111; see Table I and Tanner, this volume) traverse the membrane more than once, it is clear that the N-terminus must be capable, in some cases, of reentering the bilayer from the intravesicular side. Such twisting of the polypeptide chain within the bilayer should not interfere with the asymmetrical incorporation of carbohydrate from the intravesicular side. (7)The membrane glycoprotein somehow migrates through the endomembrane system from the rough-surfaced endoplasmic reticulum to the Golgi apparatus. One theory (the membrane shuttle hypothesis) suggests that migration occurs by the movement of discrete vesicles from one part of the cell to the other (Meldolesi, 1974a,b; Steiner et ul., 1974; Jamieson and Palade, 1971), followed by selectioe lateral diffusion of proteins after membrane fusion (Bergeron et ul., 1973); another theory (the membrane flow hypothesis) suggests the flow of complete membrane domains along continuous membrane channels (Morre et ul., 1974). Carbohydrate incorporation is completed within the Golgi apparatus (Schachter, 1974a,b). (8)Vesicles migrate from the Golgi apparatus to the plasma membrane where fusion occurs. (9) Lateral migration causes the insertion of membrane glycoprotens into the cell surface; unused components of the vesicle membrane return to the cytoplasm for reutilization. Many aspects of this scheme are hypothetical; evidence in support of the model is presented throughout this chapter.

22


Gal

I Sialic Acid

OLIMMANNOSIDE TYPE

N-ACETYLLACTOWMNE TYPE

FIG. 2. Schematic representation of a typical Asn-GlcNAc-type oligosaccharide (Montreuil, 1975).The core structure can be elongated to become either an N-acetyllactosamine- or an oligomannoside-type oligosaccharide.

era1 directions. If GlcNAc residues are incorporated into the core, the structure becomes the N-acetyllactosamine type; sialic acid residues are often attached to the galactose residues in this structure (Fig. 2). If, however, further mannose residues are incorporated into the core, the structure becomes the oligomannoside type (Fig. 2). When AsnGlcNAc oligosaccharides contain fucose residues, these are usually found attached to the GlcNAc residue nearest the asparagine residue. In general, the oligomannoside structure does not contain galactose or sialic acid residues, and neither type of Asn-GlcNAc oligosaccharide contains GalNAc residues. The N-acetyllactosamine-type oligosaccharide may contain only two arms, as shown in Fig. 2, or further branching may occur by the attachment of two GlcNAc residues to a single mannose residue of the core; thus a single Asn-GlcNAc oligosaccharide may contain zero, one, two, three, or possibly four residues of sialic acid. A single glycoprotein molecule (e.g., thyroglobulin) may contain both N-acetyllactosamine and oligomannoside oligosaccharides (Arima and Spiro, 1972; Arima et al., 1972). Ser(Thr)-GalNAc oligosaccharides vary greatly in length and complexity. Ovine submaxillary mucin contains mainly Ser(Thr)-GalNAc and Ser(Thr)-GalNAc-sialic acid groupings, whereas the blood group glycoproteins isolated from the fluids of ovarian cysts carry much more complex branched oligosaccharide structures containing GalNAc, GlcNAc, galactose, and fucose residues (Lloyd et al., 1968; Rovis et al., 1973). Ser(Thr)-GalNAc oligosaccharides have never been reported to contain mannose residues but may carry sulfate ester groups.


23

Neither Asn-GlcNAc nor Ser(Thr)-GalNAc oligosaccharides have ever been reported to contain glycuronic acids, and the finding of these sugar acids usually indicates the presence of acid mucopolysaccharides. The occurrence of glucose is a more complex problem. The only well-characterized glycoproteins proved to contain glucose are collagens and basement membranes. However, recent studies on the biosynthesis of Asn-GlcNAc oligosaccharides indicate that lipid pyrophosphate oligosaccharides believed to be intermediates in the biosynthetic process contain not only GlcNAc and mannose residues but also glucose residues (Behrens et al., 1973; R. G. Spiro et al., 1976; M. J. Spiro et al., 1976a,b; Herscovics et al., 1977a,b). The role of lipid intermediates in glycoprotein synthesis is discussed in Section IV,C; the important point to be noted at this time is that M. J. Spiro et al. (1976a,b) have reported the presence of glucose residues in lipid pyrophosphate oligosaccharides isolated from calf thyroid, kidney, and thymus, and from hen oviduct, have shown the transfer in thyroid slices of glucose-containing oligosaccharides from lipid intermediates to endogenous protein acceptors, and have found glucose to be a constituent of the glycoprotein fraction of various thyroid membrane preparations. The glucose residues appear to be part of an oligomannoside Asn-GlcNAc oligosaccharide and are present in internal positions between an outer oligomannoside sequence and an internal core structure (R. G. Spiro et al., 1976). It is possible that this glucose-containing structure will prove to be a constituent of the membrane glycoproteins of many tissues, but further structural work is required to establish this hypothesis. The presence of Ser(Thr)-GalNAc oligosaccharides on a glycoprotein can readily be demonstrated by using mild alkali hydrolysis in the presence of borohydride (Carlson, 1968) to effect p-elimination of the oligosaccharide. Asn-GlcNAc oligosaccharides are resistant to the reaction and there is in fact no method available for cleaving the AsnGlcNAc linkage of a glycoprotein without damaging both the oligosaccharide and the polypeptide backbone. Recently, endo-P-N-acetylglucosaminidases have been reported (Koide and Muramatsu, 1974; Tarentino and Maley, 1974; Ito et al., 1975). These can cleave the asparagine-linked GlcNAc-GlcNAc sequence (Fig. 2) but most appear to act efficiently only on glycopeptides and not on the intact glycoprotein. Table I lists the major membrane glycoproteins that have been isolated to date. The integral membrane glycoprotein G present in the membrane of vesicular stomatitis virus (VSV) is included as an example of an enveloped virus glycoprotein; these interesting viruses are

24


discussed further by Compans in this volume. The plasma membrane enzyme nucleotide pyrophosphatase is included as an example of a membrane glycoprotein enzyme; further examples are discussed by Riordan and Forstner in this volume. Murine histocompatibility antigens are glycoproteins, as are other antigens on the lymphocyte surface; this topic is discussed by Letarte in this volume. The human red cell membrane contains two major glycoproteins, glycophorin and coomassie blue-staining band 111; these proteins are discussed by Tanner in this volume. The following discussion deals with structural aspects of interest to the problem of biosynthesis. A. Glycophorin

Glycophorin is the major sialic acid-containing glycoprotein of the human red cell membrane. This protein does not stain with coomassie blue after electrophoresis in sodium dodecyl sulfate (SDS) polyacrylamide gels but can be detected with the PAS stain. Both glycophorin and coomassie blue band I11 are glycoproteins which span the lipid bilayer. Glycophorin is an amphipathic polypeptide chain comprised of three sections: (1)a hydrophilic N-terminal sequence which carries all the oligosaccharide groups and which is exposed at the outer side of the plasma membrane, (2) a middle hydrophobic section which interacts with the lipid bilayer, and (3) a hydrophilic C-terminal section which is exposed at the inner surface of the plasma membrane. Glycophorin contains both Ser(Thr)-GalNAc and Asn-GlcNAc oligosaccharides clustered together at the N-terminal end of the molecule (Tomita and Marchesi, 1975). Thomas and Winzler (1969a,b) studied the structures of oligosaccharides released from glycophorin by mild alkaline hydrolysis in the presence of borohydride; the major products were the tetrasaccharide shown in Fig. 3 and smaller chains lacking one or both sialic acid residues or the galactose residue. Ser(Thr)GalNAc oligosaccharides larger than the tetrasaccharide have been isolated from glycophorin and are believed to be responsible for the MN blood group antigenic activities of red cells (Springer and Yang, 1977). The Asn-GlcNAc oligosaccharide present on glycophorin has a typical N-acetyllactosamine structure (Fig. 2; Thomas and Winzler, 1971; Kornfeld and Kornfeld, 1969; Kornfeld and Kornfeld, 1970). Sialic AcidBGalNAc-OH IPl.3

Sialic AcidSGal FIG.3. Structure of a tetrasaccharide isolated from glycophorin by the mild alkaliborohydride elimination reaction (Thomas and Winzler, 1969a,b).


25

8. Rhodopsin

Rhodopsin is a glycoprotein found in the outer segment cells of mammalian retinal rods. It is involved in the molecular events of vision, and therefore information on its structure is important. It is an intrinsic membrane protein and is solubilized only by detergents (Osborne et al., 1974). Considerable variation has been found in the reported MWs which vary between 27,000 and 40,000 (Lewis et al., 1974). However, most values are about 30,000, based on amino acid analyses, gel electrophoresis, agarose column chromatography, and analytical ultracentrifugation. The carbohydrate moiety has been studied in various laboratories. Heller and Lawrence (1970) found that bovine rhodopsin contained a covalently linked carbohydrate unit consisting of three GlcNAc and three mannose residues; this was based on the isolation of a single glycopeptide of nine amino acids from a peptic digest. All the carbohydrate was linked through an Asn-GlcNAc linkage. The surface location ofthe oligosaccharide chain was determined b y Con A and wheat germ agglutinin binding (Renthal et hl., 1973). The role of the carbohydrate groups in the chromophoric properties of the protein was studied by periodate oxidation which would be expected to cleave between C-3 and C 4 of GlcNAc and between C-2 and C-3, and C-3 and C-4, of the mannose residues. The 500-nm absorption band characteristic of rhodopsin was not affected by periodate oxidation and was regenerable after bleaching. It was concluded (Renthal et al., 1973) that the intact carbohydrate moiety was not essential for the chromophoric properties of rhodopsin and that the role of the carbohydrate might be to orient rhodopsin in the disk membrane, such that the oligosaccharide protrudes into the hydrophilic environment. Plantner and Kean (1976) reinvestigated the carbohydrate composition of bovine rhodopsin and found 8-9 moles of mannose and 5 moles of GlcNAc per mole of visual pigment. The MW of the rhodopsin apoglycoprotein was calculated to be 38,000 on the basis of amino acid analysis. Bovine rhodopsin carries two oligosaccharide groups per mole at asparagine residues 2 and 15 from the N-terminal end (Hargrave, 1977). Further, like glycophorin, it appears to have a hydrophilic carbohydrate-rich N-terminal sequence exposed at the outer surface of the membrane and a hydrophobic section which interacts with the lipid bilayer (Hargrave, 1977; Saari, 1974; Pober and Stryer, 1975; Worthington, 1973).

26

J. STURGESS,

M. MOSCARELLO, AND H. SCHACHTER

A detailed three-dimensional electron microscope study of the purple membrane of Halobacterium halobium was presented recently by Henderson and Unwin (1975). The purple membrane functions as a light-driven hydrogen ion pump involved in photosynthesis. It has a MW of 26,000, and retinal is covalently linked to each protein molecule in a 1 : 1 ratio, imparting the characteristic purple color. It is often referred to as bacterial rhodopsin. A three-dimensional map of this molecule at a 7 A resolution showed that there were seven rods which were a-helices and extended perpendicularly through most of the width of the membrane. The overall dimensions of the protein were calculated as 25 x 35 x 45 A, with the longest dimension perpendicular to the plane of the membrane. Lipid molecules fill the spaces between the rods, forming a mosaic. Bacterial rhodopsin appears to be an example of an intrinsic membrane protein but is not a glycoprotein. C. Epiglycanin

Codington et al. (1975a,b) have described a high-MW glycoprotein present in the plasma membrane of the murine tumor cell line TA3Ha which they have called epiglycanin. Glycopeptide can be released from epiglycanin by mild proteolysis of intact TA3 cells. The oligosaccharide is of the Ser(Thr)-GalNActype and contains sialic acid, galactose, and GalNAc in the molar ratio 1 : 4 :2. Epiglycanin reacts with Vicia graminea lectin, indicating structural features in common with human blood-group-N substance (Springer et al., 1972). D. Murine Histocompatibility Antigens

Histocompatibility antigens are a complex series of proteins present on the surface of lymphocytes and of probably all nucleated cells (see Letarte, this volume). The genetic regions controlling the expression of histocompatibility antigens are called HL-A (human leukocyte antigens) in humans and H-2 in mice. The products of the H-2 genes are undoubtedly glycoproteins (Nathenson and Muramatsu, 1971), and the products of the HL-A genes are probably also glycoproteins. The oligosaccharide prosthetic groups of the H-2 antigens have been investigated by Nathenson and co-workers (Nathenson and Muramatsu, 1971; Nathenson and Cullen, 1974). A glycopeptide has been isolated from proteolytic digests of purified H-2 antigen, and a partial oligosaccharide structure has been reported on the basis of glycosidase digestion. This structure is of the Asn-GlcNAc N-kcetyllactosamine type

STRUCTURE A N D BlOSYNTHESlS OF M E M B R A N E GLYCOPROTEINS

27

(Fig. 2) and has two or more sialic acid residues per mole and a fucose residue attached to the most internal GlcNAc residue. The position of the fucose residue was determined by digestion of the glycopeptide with an endo-p-N-acetylglucosaminidasefrom Diplococcus pneumoniae. The latter enzyme is of great interest because it apparently can cleave about 80% of the oligosaccharide from intact H-2 glycoprotein (Nathenson and Cullen, 1974); the resulting protein retains all of its H-2 antigenic activity. The current model for the H-2 antigen indicates that it is a protein of about 45,000 MW carrying one or two oligosaccharide units exposed on the external surface of the cell and anchored in the lipid bilayer by a hydrophobic polypeptide of about 3000-6000 MW. E. Ashwell's Mammalian Lectin

In a brilliant series of studies, Ashwell and co-workers (Ashwell and Morell, 1974; Kawasaki and Ashwell, 1976a,b) showed that mammalian hepatocytes carry in their plasma membranes a glycoprotein which acts as a specific binding protein for glycoproteins with exposed terminal galactosyl residues. Thus a variety of sialidase-treated plasma gl ycoproteins are cleared from the circulation by binding to this liver membrane protein followed by endocytosis and proteolysis within the hepatocyte. The liver binding protein has been isolated from rabbit liver and shown to be an intrinsic membrane glycoprotein composed of two subunits with MWs of 48,000 and 40,000, respectively. These subunits tend to aggregate into large complexes in aqueous solution (Kawasaki and Ashwell, 1976a). Pronase digestion of the

GLYCOPEPTIDE I GLYCOPEPTIDE n FIG.4. Oligosaccharides present on a mammalian lectin (asialoglycoprotein-bind-

ing protein) isolated from rabbit liver by Kawasaki and Ashwell (1976b).

28


isolated glycoprotein resulted in quantitative recovery of the carbohydrate moieties which were shown to consist of two glycopeptides of varying composition (Fig. 4). Both glycopeptides are of the AsnGlcNAc type, but it should be noted that one of these structures has one less mannose residue than the typical core structure shown in Fig. 2. Whether or not deiriation from this core structure will prove to be typical of membrane glycoproteins cannot be assessed at our present level of knowledge. The mammalian liver binding protein can be considered a lectin, since it recognizes specific carbohydrate structures and since it can agglutinate red celIs and stimulate mitogenesis in desialylated T lymphocytes (Novogrodsky and Ashwell, 1977). It has recently been shown (Pricer and Ashwell, 1976) that the binding protein is not localized to the plasma membrane of liver cells but occurs in Golgi membranes and other intracellular membranes. F. The LETS Glycoprotein

The large external transformation-sensitive (LETS) glycoprotein is a cell surface glycoprotein present on normal fibroblasts and myoblasts, but on the surfaces of transformed cell lines it is reduced in amount or absent (Hynes, 1976). It has not been established that this glycoprotein is an intrinsic membrane protein, and no detailed information on its carbohydrate composition is available. Hunt and Brown (1975)have, however, reported a similar surface glycoprotein which is present on mouse L cells during the GI phase of the cell cycle (Hunt et al., 1975) and which spans the L-cell plasma membrane, indicating it to be an intrinsic membrane glycoprotein. G. Enveloped Viruses

Enveloped viruses (Compans and Kemp, this volume) derive their membrane envelopes from their host cells. The peptide moieties of viral membrane proteins are products of the viral genome, but the oligosaccharide moieties of viral membrane glycoproteins are assembled by the glycosyltransferases of the host cell. The infection of mammalian cell lines by enveloped viruses therefore offers a unique system for studying the biosynthesis of specific membrane glycoproteins. In recognition of this fact, many groups have recently studied the structures of various enveloped virus membrane glycoproteins. VSV has a single intrinsic membrane glycoprotein (G protein); this protein has two oligosaccharide groups per mole, and both of these groups have a


29

typical fucose and sialic acid-containing Asn-GlcNAc structure of the N-acetyllactosamine type (Fig. 2) (Etchison and Summers, 1977; Reading et al., 1977). Sindbis virus has two membrane glycoproteins, El and Ef,each of which has two different oligosaccharide groups (Sefton and Keegstra, 1974), one an Asn-GlcNAc group containing only core sugars (mannose and GlcNAc) and the other a fucose- and sialic acid-containing Asn-GlcNAc structure of the N-acetyllactosamine type (Fig. 2). H. Cytochrome b5 and Cytochrome b, Reductase

Fatty acid desaturation takes place on the membranes of the endoplasmic reticulum. The first two components catalyzing the desaturation reaction are NADH-cytochrome b, reductase and cytochrome b,. Both components may be glycoproteins (Ozols, 1972), although this has not been firmly established. Amino acid sequence studies on cytochrome b, revealed the sequence Asn-His-Ser at positions 22 to 24 in the human liver enzyme; this may represent the point of attachment of the oligosaccharide to the polypeptide chain. These studies were done on the enzyme released from liver microsomes by mild proteolysis, which had a MW of 11,000-13,000, A detergent-solubilized preparation was obtained by Spatz and Strittmatter (1971), and the MW of this preparation was estimated at 16,600, considerably higher than that of the preparation released by proteolysis. In the absence of detergent, the water-soluble enzyme aggregated, giving a MW of about 120,000. Trypsin treatment of the detergent-solubilized enzyme yielded several peptides (Spatz and Strittmatter, 1971).One of MW 12,000 was enzymically active and showed no tendency to aggregate in water. Another peptide of apparent MW 5000 represented the major hydrophobic peptide. Amino acid analysis showed that this fragment contained 49% nonpolar residues as compared to 29% for the active fragment. The hydrophobic peptide sequence appears to b e at the C-terminus of the molecule and probably serves to anchor the protein in the membrane. Microsomal cytochrome b, reductase has many properties similar to those described for cytochrome b,. The MW is approximately 33,000, higher than that of cytochrome b, (16,600). Although a quantitative sugar analysis is not available, the purified enzyme stains positively for carbohydrate and is probably a glycoprotein. It appears to have a hydrophobic domain (similar to that of cytochrome b,) b y which it is inserted into the membrane.

30


Both cytochrome b, and cytochrome b, reductase have been purified and appear to carry out their catalytic activities in the absence of lipid. However, a lipid requirement for the enzymes is not ruled out. Rogers and Strittmatter (1973) found that electron transport was reduced by more than 80% on the removal of lipid. After restoration of the original phospholipid/protein ratio by incubating the particles in the presence of liposomes (prepared from microsomal lipids), the rate of cytochrome b, reduction was fully restored. More recently, the desaturase system was reconstituted from the isolated components by combining cytochrome b, reductase, cytochrome b,, the desaturase, stearyl-CoA, NADH, oxygen, and lipid, resulting in an active system (Strittmatter et aZ., 1974). Cytochrome b, can be considered an integral membrane protein. The relatively high content of hydrophobic amino acids, especially in the hydrophobic tail of the detergent-solubilized protein (Spatz and Strittmatter, 1971), and its ability to associate with lipids to restore activity, are characteristic of integral membrane proteins. Recent data indicate that cytochrome b, contains two globular domains (Visser et aZ., 1975). One of the domains carries the electron transport site. The function of the other domain is to attach the molecule to the membrane. These two domains are joined by a link region, 30-40 A long, which may be flexible. Therefore the relatively flexible electron transport site is joined to the hydrophobic region anchored in the membrane by a flexible link region. Since these studies were carried out in deoxycholate micelles, one cannot say that cytochrome b, under these conditions is identical with the protein in a natural membrane. Although the complete sequence of cytochrome b, is not known, the C-terminal end (Ozols, 1974) is Glu-Asp-COOH, imparting three negative charges to this end of the molecule; this segment probably cannot lie within the hydrocarbon region of the bilayer, suggesting that the molecule traverses the whole width of the bilayer. However, an alternative model presented recently by Depierre and Dallner (1975) suggests that most of the protein protrudes into the hydrophilic environment on the cytoplasmic side of the membrane, while the hydrophobic portion traverses only half the width of the bilayer.

I. Glycoproteino of Myelin

Myelin is composed of 20-25% protein and 75-80% lipid. The protein composition of myelin is relatively simple (compared to that of other membranes) in that approximately 70% of the total protein is


31

made up of two protein fractions, the basic protein and proteolipid.

The basic protein has potent biological activity, eliciting experimental allergic encephalomyelitis at a dose of a few micrograms. The sequence has been established, and considerable data are available on the secondary and tertiary structures. The proteolipid protein fraction consists of several proteins which are soluble in chloroform-methanol. Lipophilin, which constitutes a large proportion of the proteolipid fraction, has been isolated and purified. Basic protein is discussed in this section, although it is not a glycoprotein, because it is a natural acceptor of N-acetyl-D-galactosamine in the presence of a submaxillary gland N-acetylgalactosaminyltransferase.Lipophilin is an intrinsic membrane protein reported to contain carbohydrate. Basic protein was first described by Kies (1965) as the encephalitogenic protein of myelin; i.e., the injection of a small amount (a few micrograms) into a guinea pig elicited a fatal disease characterized by hind limb paralysis, weight loss, and death. Eylar et al. (1971), after several years of study, reported the complete amino acid sequence. Basic protein has a MW of 18,400 (Eylar, 1972) and is characterized by a relatively large number of basic amino acids (lysine plus histidine plus arginine comprise 25% of the. total residues). The basic residues are located at random in the molecule, and there is no obvious hydrophobic region. A proline-rich segment, Pro-Arg-Thr-Pro-Pro-Pro-Ser, surrounds threonine 98, resulting in a sharp bend in the molecule. Although the primary structure is well established, the secondary structure is still in dispute. The proline-rich region mentioned above places severe constraints on the molecule and creates a hairpin turn in the molecule at this site. Early studies (Kies et al., 1965; Palmer and Dawson, 1969; Chao and Einstein, 1970) showed no a-helical or p structures by circular dichroism (CD). In a more recent study, Epand et al. (1974) showed that the protein had a nonrandom structure in solution, despite the fact that it showed no a-helical or p structure. Intrinsic viscosity studies confirmed the axial ratio of 1 : 10 reported earlier (Eylar and Thompson, 1969). The radius of gyration, calculated from low-angle x-ray scattering measurements (Epand et al., 1974), was found to be 39 k 2 A. The model proposed by Epand et al. (1974) was that of a prolate ellipsoid of dimensions 150 x 15 A. Further support for a highly structured molecule was obtained from surface tension measurements (Moscarello et d.,1974), CD and proton magnetic resonance (PMR) changes in myelin basic protein conformation under specific conditions (Liebes et al., 1975), and 13Cnuclear magnetic resonance (NMR) studies (Chapman and Moore, 1976) showing highly structured regions especially near residues 85 to 116. It is interesting

32


that the region including residues 81 to 118 has been found constant in myelin basic proteins from humans (Carnegie, 1971), cows (Eylar, 1970), and rats (Dunkley and Carnegie, 1974), suggesting some special function for this region. Threonine 98 has been shown to be the site of glycosylation with UDP-N-acetyl-D-galactosamine and an enzyme from the submaxillary gland (Hagopian and Eylar, 1969b). The only other protein of several tested which was an acceptor in this system was the deglycosylated natural salivary gland protein. In a further in vitro study using UDPN-[’4C]acetyl-D-galactosamineand a purified enzyme from bovine submaxillary gland, over 80% of the radioactivity was recovered on threonine 98 and was shown to be in 0-glycosidic linkage (Hagopian et al., 1971). The significance of this finding is not clear. It has been suggested that the basic protein may be transiently glycosylated during its synthesis and insertion into the membrane. The carbohydrate moiety may play a recognition role, initiating contact between different layers of the myelin sheath and functioning as an “organizer” (Carnegie, 1971; Hughes, 1976). In a tightly compacted structure such as myelin, there would be no room for the oligosaccharide chain, thus it is not surprising that it might subsequently be removed from the protein. J. Proteolipid Protein Fraction of Myelin

A hydrophobic protein has been isolated, purified, and characterized from the chloroform-methanol-soluble fraction of human myelin (Gagnon et al., 1971; Moscarelloet al., 1973). It has several interesting structural features. The hydrophobic residues account for about 6264% of the total residues. Two moles of fatty acid are present per mole of protein (assuming a MW of 28,000); these are esterified directly to the peptide backbone and are not present as a phospholipid or sphingolipid (Folch-Pi and Stoffyn, 1972). Analysis revealed the presence of small amounts of carbohydrate, and therefore it may be a glycoprotein. The protein has been called lipophilin. Attempts to elucidate the primary sequence have met with difficulty, largely because of the insoluble nature of the protein and its cleavage products. Nussbaum et al. (1974) have reported the N-terminal sequence of the performic acid-oxidized rat brain protein to be Gly-Leu-Leu-Glu-Cys SO3-CysSO3-Ala-Arg-CysSO,-Leu -Val-GIy -AlaPro-Phe-Ala-X-Leu-Val-Ala-. Although the N-terminal sequence for the human protein is similar, these data must be interpreted with caution since the sequenator yields were low, often less than 15%.


33

In contrast to the basic protein of myelin, which appears to have a fairly rigid secondary structure, lipophilin (formerly called N-2, MOScarello et al., 1973) is conformationally flexible. It can be obtained in water-soluble form in an a-helical or p conformation, depending on the method used to solubilize the protein. The MW of the a-helical form was 86,000, that of the p structure was 500,000, and a monomer MW of 24,000-28,000 was found by equilibrium ultracentrifugation. K. General Comments on Membrane Protein Structure

Although generalizations about membrane protein structure are based on limited data (Table I), a pattern is emerging which supports the concept of a fluid mosaic membrane as detailed by Singer and NicOlson (1972). Integral membrane proteins appear to be amphipathic molecules containing a hydrophobic sequence which interacts strongly with the lipid core of the bilayer. Oligosaccharide groups are clustered near one end of the molecule (probably the N-terminal end), and this hydrophilic portion is exposed on the external side of the plasma membrane or at the inner face (opposite the cytoplasmic face) of intracellular membranes; thus carbohydrate shows an asymmetrical distribution across the membrane bilayer. The oligosaccharide groups studied to date indicate that integral membrane glycoproteins may contain Asn-GlcNAc oligosaccharides of both the oligomannoside and N-acetyllactosamine type (Fig. 2), as well as Ser(Thr)-GalNAc oligosaccharides; these structures do not appear to be significantly different from the oligosaccharides described in secretory glycoproteins, although the possible occurrence of glucose residues in membrane glycoproteins requires further study (Behrens et al., 1973; R. G. Spiro et al., 1976; M. J. Spiro et al., 1976a,b). It should be pointed out that, while Table I lists only membrane glycoproteins, integral membrane proteins have been isolated which do not contain carbohydrate, e.g., the ATPase of sarcoplasmic reticulum (MacLennan, 1975), halophilic bacterial rhodopsin (Oesterhelt and Stoeckenius, 1971), and possibly other proteins. Excluded from the above discussion were acid mucopolysaccharides (glycosaminoglycans),although there is strong evidence for the presence of these molecules at the surface of cultured cells (Kraemer, 1971; Hynes, 1976).These molecules are presently thought to be components of the glycocalyx and therefore more closely associated with peripheral than with integral proteins; however, their topology and function at the cell surface have not been adequately studied.

34

J. STURGESS, M. MOSCARELLO, A N D H . SCHACHTER

111.

MODEL MEMBRANE SYSTEMS

Protein-lipid interactions are difficult to study in natural membranes which possess many different kinds of lipids and proteins. The use of model membranes in which well-characterized isolated proteins are incorporated into lipid vesicles made from well-defined lipids provides us with an experimental model in which to study these protein-lipid interactions. These studies may provide information useful for understanding the incorporation of glycoproteins into membranes in uivo. A large number of proteins has been incorporated into lipid vesicles, and a list of these has been compiled by Tyrell et al. (1976). In addition to membrane glycoproteins, the list includes other proteins such as bovine serum albumin and immunoglobulin (nonmembrane proteins) and synthetic polypeptides such as poly-i-lysine. Some of these are not incorporated into the lipid bilayer but instead interact at the surface of the vesicle. Our discussion here is limited to naturally occurring membrane glycoproteins. Table I lists the properties of some membrane glycoproteins. Two points are worth emphasizing: (1)Where the data are available, the percent of hydrophobic residues (threonine, alanine, proline, tyrosine, valine, methionine, leucine, isoleucine, tryptophan, phenylalanine) is quite high, being 50% or greater in some cases; most soluble glycoproteins contain 20-40% hydrophobic residues, while membrane glycoproteins contain 30-60%; (2) The MWs are relatively low (25,000-50,000). An exception to this rule is the erythrocyte glycoprotein (band 111) of MW 90,000. Although undoubtedly an intrinsic membrane glycoprotein, its relatively high MW must be related to the fact that a considerable portion of it protrudes from both the inner and outer surfaces of the erythrocyte membrane. The membrane portion of this glycoprotein has been estimated to have a MW of 60,000 (Jenkins and Tanner, 1977). Since it is reported to traverse the membrane twice, forming a loop, each arm of this loop has a MW of approximately 30,000. Similarly, the very large size of epiglycanin is due to the fact that it is primarily external to the cell surface. Recent evidence suggests that a polypeptide of about 30,000 MW can span about 60 A of bilayer (Brady et al., 1978). The large percentage of apolar residues in these proteins is no doubt related to the need of the protein to present a large number of hydrophobic groups for interaction with the lipid hydrocarbon chains within the bilayer. An intimate association between protein and lipid described as boundary lipid (Boggs et al., 1976)or lipid annulus (Warren et d . , 1975) has been demonstrated in several cases.


35

In some cases, membrane glycoprotein has been shown to possess more than 60% a-helix, e.g., when lipophilin was incorporated into liposomes made of dipalmitoyl phosphatidylcholine (DPPC) (Cockle et al., 1978).X-ray diffraction studies on similar vesicles showed that the thickness of the bilayer was not increased by the presence of the protein, indicating that it was not protruding beyond the surface of the vesicle (Rand et nl., 1976). Surface-labeling studies with a nonpenetrating reagent, 4,4'-diisothiocyanoditritiostilbene disulfonate have been interpreted as indicative that lipophilin traverses the entire width of the bilayer (Wood et nl., 1978). It should be pointed out that proteins lacking carbohydrates can be incorporated into lipid vesicles in vitro (Tyrell et al., 1976) and that there are naturally occurring integral membrane proteins which are not glycoproteins, e.g., the ATPase of sarcoplasmic reticulum (MacLennan, 1975) and the bacterial rhodopsin discussed in the previous section. Thus carbohydrate is probably not required for the stabilization of integral protein within the bilayer. Carbohydrate may be needed during the insertion of protein into the bilayer but may subsequently be removed; there is, however, no evidence for the latter hypothesis. It appears likely that carbohydrate plays its most essential role when it is on the surface of the cell and not during the insertion of glycoprotein into the membrane. Rhodopsin was recombined into egg phosphatidylcholine bilayers, and details of the interactions were studied with electron spin resonance (ESR) and freeze-fracture techniques (Hong and Hubbell, 1972). By incorporating a spin label into bilayers with and without rhodopsin, the effect of the protein was found to inhibit segmental motion of the hydrocarbon chains. The order parameter (SJ, which is a measure of the fluidity of the bilayer, increased as the mole fraction of rhodopsin increased, supporting the contention that the protein was incorporated into the bilayer. Freeze-fracture electron microscopy revealed the presence of numerous particles on the fracture surfaces, suggesting that rhodopsin was buried deep inside the bilayer. More recently, extensive model membrane studies have been carried out with the myelin glycoprotein lipophilin. Because the protein is very hydrophobic, it should be readily incorporated into the lipid bilayer. Freeze-fracture electron microscopy showed that lipophilin was readily incorporated into lipid vesicles (Vail et al., 1974). The possibility of using this model system to study lipid-protein interactions was evident. Several studies were reported by Papahadjopoulos et al. (1975a,b) on the effect of the protein on the ultrastructure, lipid phase transitions and permeability of phospholipid vesicles. Lipophilin was

36

J. STURGESS, M. MOSCARELLO, A N D H. SCHACHTER

found to bind strongly to phospholipids, irrespective of surface charges, the presence of cholesterol, or double bonds in the fatty acyl chains. Large, closed, multilamellar vesicles were formed with a buoyant density intermediate between that of pure lipid and protein. The presence of the protein in the vesicles increased their permeability to zzNa+by two to three orders of magnitude. Differential scanning calorimetry indicated that the presence of the protein had no effect on the lipid phase transition from solid to liquid crystalline. However, the enthalpy of transition decreased as the amount of protein in the vesicles was increased from 0 to 53%, but there was no change in the midpoint temperature. When the concentration of protein was 50% by weight, the enthalpy was approximately one-half that of the pure lipid. Details of the interaction between lipophilin and lipids have been provided in two recent studies. In one study (Boggs et al., 1976), spin labels were used to study the microenvironment of the protein. Lipophilin was inserted into phosphatidylcholine vesicles and studied with fatty acid spin labels. Two distinct components were present in the spectrum. One was immobilized, presumably as a result of the presence of boundary lipid around the protein, and the second was indicative of anisotropic motion, similar to the spectrum for phosphatidylcholine vesicles. Lipophilin was found to increase the order parameter linearly with increasing concentration of protein. The phase transition temperature measured with 'TEMPO (2,2,6,6-tetramethyl piperidine- 1-oxyl)was not changed, in agreement with the differential scanning calorimetry data (Papahadjopoulos et al., 1975a,b). The presence of lipophilin in the lipid bilayer was found to induce lipid phase separation (Boggs et al., 1977). Differential scanning calorimetry was used to study the effect of the protein on the transition temperature of a mixture of phosphatidylserine (PS) and DPPC. PS melts at 8°C and DPPC at 43"C, while the mixture melts at an intermediate temperature depending upon the PS/DPPC ratio. The higher the PS concentration in the mixture, the lower the melting temperature. Addition of the protein to the PS/DPPC mixture at all ratios raised the melting temperature, which could only be explained by the removal of PS from the mixture, resulting in a lipid mixture with a lower proportion of PS. It was concluded that lipophilin bound preferentially to PS, removing it from the mixture and leaving a lipid phase with a higher proportion of DPPC. The binding of certain classes of lipids to the boundary layer of intrinsic membrane proteins may be one way in which asymmetry can be induced and maintained in a membrane. Many membrane glyco-


37

proteins have specific enzymic activities. Lipids are known to influence the activity of enzymes. Therefore a specific lipid environment around a membrane-bound enzyme can be maintained by lipid phase separation (discussed above). The existence of different lipid environments surrounding some of the glycosyltransferases of Golgi membranes have been described (Mitranic et al., 1976, 1978) for galactosyl- and sialyltransferases on the basis of a different sensitivity to temperature and drugs such as colchicine. When these glycosyltransferases become available in a pure form, it will be interesting to use them as protein components in model membrane system studies. The principles of lipid-protein interactions derived from the study of model membranes are directly applicable to the more complex systems in naturally occurring membranes. IV.

THE GLYCOSYLATION REACTION

Glycosyltransferases are enzymes which transfer monosaccharides or oligosaccharides from activated derivatives to various acceptors. The activated glycose donors are usually phosphorylated derivatives in which the glycose moiety is connected through the anomeric carbon by either a phosphate or a pyrophosphate linkage to a nucleoside moiety (to form a nucleotide sugar or nucleotide oligosaccharide) or to a polyprenol lipid. A glycosyltransferase can catalyze the formation of O-glycosidic linkages between two sugar moieties or between a sugar and a hydroxyamino acid such as serine or threonine, or it can form an N-glycosidic linkage between N-acetylglucosamine and asparagine. Glycoprotein synthesis depends on a variety of complex factors such as the availability of glycose donors, the substrate specificities of glycosyltransferases, and the arrangement of glycosyltransferases within the cell’s endomembrane system. The following section deals with the synthesis of nucleotide sugars and lipid intermediates and with the utilization of these glycose donors b y glycosyltransferases. Much of the information presented is not specific for membrane glycoproteins but has relevance for all glycoproteins. A. Nueleotide Sugar Formation

The pathways of nucleotide sugar formation from D-ghCOSe are outlined in Fig. 5 (Feingold, 1972; Schachter and Roden, 1973). The following discussion deals only with aspects of interest to the problem of glycoprotein synthesis.

38


GL-6-P

I

- -

>..

Glc-1-P

UDP-Glc

Mannose

UDP-Gal

1

Fructose-6-P-Man-6-P

(I-

r -

Mon-1-P

G4cN- 6- P-

Glucosomine

GlcNAC-6-P-

GlcNAc

I

1 Gal-1-P t -Galactose

GDP- Man

-

-GDP;Fuc

I

1

! I-

FuCtl-P Fucose

GlcNAC-1-P

I I uOP-GolNAc f GalNPC- 1-P

UDP-GlcNAc-ManNAc

t

t

Golocrosomtne

ManNAc-6-P

I

N-ocetylneurorntnlc acid-9-P

1

N-acetylneuramlnlc ocld

1

CMP-N-ocelylneuramlnIc acid

FIG.5. Pathways of nucleotide sugar synthesis.

1. UDP-GALACTOSE Glucose can be converted to UDP-galactose as indicated in Fig. 5. The intact animal, the perfused liver, and thyroid slices have been used to study the incorporation of [14Clglucose into glycoprotein (Schachter and Rod&, 1973), and radioactivity was detected in protein-bound galactose and other sugars. When intact liver cells were exposed to [14C]galactose,appreciable radioactivity was found in protein-bound mannose and hexosamine, as well as in protein-bound galactose (Sarcione, 1964; Richmond, 1965; Moscarello et d.,1972). These experiments demonstrate the in vivo interconversion of glucose and galactose and the conversion of exogenous galactose to nucleotide sugars other than UDP-galactose.

2. GDP-MANNOSE GDP-mannose can be formed from D-glUCOSe via fructose 6-phosphate, mannose 6-phosphate, and mannose 1-phosphate (Fig. 5), the final enzyme in the pathway being GDP-mannose pyrophosphorylase. Exogenous D-mannose can also enter the metabolic scheme (Fig. 5 ) via mannose 6-phosphate and give rise to GDP-mannose as well as to other nucleotide sugars. Thus Mitranic and Moscarello (1972) injected D-[2-3H]mannoseinto rats and studied the incorporation of radioactivity into plasma glycoproteins; they recovered over 80% of the total protein-bound radioactivity in sugars other than mannose, namely, ga-


39

lactose, fucose, sialic acid, and hexosamine. This extensive conversion of mannose to other monosaccharides in rat liver may not occur in all tissues (Whur et al., 1969).

3. GDP-FUCOSE GDP-fucose can be formed from Dglucose via GDP-mannose or from exogenous L-fucose via fucose kinase and GDP-fucose pyrophosphorylase (Fig. 5 ) . Parenterally administered [14Clfucoseis an excellent precursor of glycoprotein and, unlike monosaccharides such as glucose, galactose, mannose, glucosamine, and galactosamine, fucose is not converted to other monosaccharides (Coffey et al., 1964; Bekesi and Winzler, 1967; Bocci and Winzler, 1969; Sturgess et al., 1973; Shull and Miller, 1960). This finding indicates that the conversion of GDP-mannose to GDP-fucose is not reversible (Fig. 5).

4. UDP-N-ACETYLGLUCOSAMINE AND UDP-N-ACETYLGALACTOSAMINE UDP-N-acetylglucosamine and UDP-N-acetylgalactosamine can both be formed from D-glucose as shown in Fig. 5. The first specific step in hexosamine formation is the conversion of fructose 6-phosphate to glucosamine 6-phosphate7 a process catalyzed by two separate enzymes, ~-glutamine:~-fi-uctose-6-phosphate amidotransferase and glucosamine-6-phosphate deaminase [2-amino-2-deoxy-D-glucose-6-phosphate ketol isomerase (deaminating)]. The amidotransferase reaction is irreversible and is probably the major pathway of hexosamine formation. Inhibition of the amidotransferase by the injection of a glutamine analog (duazomycin A) into rats reduced the level of UDP-N-acetylglucosamine in liver by 85% and caused a 5070% inhibition of glycoprotein synthesis by liver (Bates et ul., 1966; Bates and Handschumacher, 1969); both effects could be prevented by administration of exogenous glucosamine, indicating that duazomycin A did not interfere with the incorporation of glucosamine into glycoprotein but blocked the conversion of glucose to hexosamine via the amidotransferase. The amidotransferase, the first step in hexosamine formation, is subject to feedback control by the end product, UDP-N-acetylglucosamine (Kornfeld et al., 1964; Kornfeld, 1967; Mazlen et d., 1969; Ellis and Sommar, 1971; Miyagi and Tsuiki, 1971; Trujillo and Gan, 1973). UDP-N-acetylglucosamine is formed from N-acetylglucosamine 1phosphate by a pyrophosphorylase, and UDP-N-acetylgalactosamine

40


is formed from UDP-N-acetylglucosamine by a 4-epimerase (Fig. 5). N-Acetylglucosamine l-phosphate can be derived from . glucose or from exogenous glucosamine (Fig. 5). Radioactive glucosamine is an excellent precursor for studying glycoprotein synthesis in intact cells; since ~-glutamine:~-fructose-6-phosphate amidotransferase is irreversible, glucosamine is not glycogenic and is not converted to hexoses (McGarrahan and Maley, 1962) although, as expected from the scheme shown in Fig. 5, the administration of radioactive glucosamine to intact cells leads to the appearance of radioactivity in proteinbound sialic acid, as well as in protein-bound glucosamine (Lawford and Schachter, 1966).The in vivo epimerization of UDP-N-acetylglucosamine to UDP-N-acetylgalactosamine in liver is not appreciable (Shetlar et al., 1964; Spiro, 1959; Robinson et al., 1964; MacBeth et al., 1965),presumably because the equilibrium favors formation of the glucosamine derivative. White et al. (1965) injected ~-[l-'~C]galactosamine into rats and recovered most of the plasma protein-bound radioactivity in N-acetylglucosamine and N-acetylgalactosamine; the ratio of labeled glucosamine to labeled galactosamine in serum glycoprotein varied between 6 : l and 19 : l, indicating that most of the exogenous galactosamine underwent epimerization prior to incorporation into protein. The exogenous galactosamine appears to be utilized in rat liver primarily by the same pathway as galactose (Maley et al., 1968), and UDP-N-acetylgalactosamine is synthetized from N-acetylgalactosamine l-phosphate by a pyrophosphorylase (Fig. 5).

5. CMP-SIALICACID The CMP-sialic acids are formed by a series of reactions which originate from N-acetylmannosamine (Fig. 5). The latter compound may be formed from UDP-N-acetylglucosamine b y UDP-N-acetylglucosamine 2-epimerase or from N-acetylglucosamine by N-acetylglucosamine 2-epimerase. N-Acetylmannosamine can be converted to N-acetylneuraminic acid either directly by an aldol condensation with pyruvate (catalyzed by N-acetylneuraminic acid aldolase) or by the series of reactions shown in Fig. 5. The latter route is believed to be the biosynthetic path, while the aldolase is primarily a degradative enzyme in viva Thus N-acetylmannosamine is first phosphorylated to the 6-phosphate which undergoes condensation with phosphoenolpyruvate to form N-acetylneuraminic acid 9-phosphate; cleavage of the phosphate ester results in the formation of N-acetylneuraminic acid. The final step in the activation of sialic acid to CMP-sialic acid is catalyzed by CMP-sialic acid synthetase (CTP:N-acetylneuraminate


41

cytidyltransferase). The formation of N-acetylmannosamine from UDP-N-acetylglucosamine is subject to feedback inhibition by the final product, CMP-N-acetylneuraminic acid (Kornfeld et al., 1964; Kikuchi and Tsuiki, 1973). Several studies have appeared indicating that CMP-sialic acid synthetase is a nuclear enzyme (Kean, 1970; Kean and Brunner, 1971; Van Dijk et al., 1973; Van den Eijnden, 1973). Since sialic acid incorporation into glycoproteins and glycolipids appears to be a function of the Golgi apparatus, it is not clear why CMP-sialic acid should be synthesized in the nucleus. Sialic acids are acetyl and glycolyl derivatives of neuraminic acid and are widely distributed in the animal kingdom (mammals, birds, fish, and echinoderms) and in algae and bacteria (Warren, 1963; Cabezas, 1973). The major naturally occurring sialic acids are N-acetylneuraminic acid, N-glycolylneuraminic acid, N-acetyl-4-O-acetylneuraminic acid, N-acetyl-7-O-acetylneuraminic acid, N-acetyl9-0-acetylneuraminic acid, and N-acetyl-7,9-di-O-acetyl-neuraminic acid. Human tissues contain mainly N-acetylneuraminic acid with only trace amounts of other sialic acids, but many tissues of other mammalian species contain appreciable amounts of N-glycolylneuraminic acid, N,O-diacetylneuraminic acid, and triacetylneuraminic acid. Schauer’s group (Schauer et ul., 1974; Buscher et al., 1974; Kamerling et al., 1975; Schauer, 1973; Jancik and Schauer, 1974) has developed gas-liquid and thin-layer chromatographic techniques for resolving the different sialic acids and has undertaken a detailed study of the biosynthesis of the different sialic acids and their incorporation into macromolecules. N-Acetylneuraminic acid is the precursor of N-glycolylneuraminic acid, the N-acetyl-mono-0-acetylneuraminicacids, and t!!e N-acetyloligo-O-acetylneuraminic acids. N-Acetylneuraminate is converted to N-glycolylneuraminate by the enzyme N-acetylneuraminate, ascorbate, or NADPH:oxygen oxidoreductase (N-acetyl-hydroxylating), first described in cell-free preparations of porcine submaxillary gland (Schauer, 1970~).Schauer (1970a,b) also demonstrated the presence in equine submaxillary glands of N-acety1neuraminate:acetyl-CoA4O-acetyltransferase, and in bovine submaxillary glands of N-acetylneuraminate:acetyl-CoA 7- and/or 9-O-acetyltransferase(s) which convert N-acetylneuraminate to the corresponding N-acetyl-mono-0acetylneuraminic acids; the latter enzyme(s) also forms N-acetyl-7,9di-O-acetylneuraminic acid (Schauer and Wember, 1971). These enzymes are membrane-bound and can hydroxylate or O-acetylate either free N-acetylneuraminate before its incorporation into macromole-

42


cules or N-acetylneuraminate glycosidically linked to membranebound glycoproteins (Schauer and Wember, 1970, 1971). B. Polyprenol Phosphate Sugar Formation

One of the most important discoveries in the field of oligosaccharide synthesis has been the role of long-chain isoprenyl alcohols as intermediates in glycosyl transfer reactions. It is now well-established (Strominger et al., 1972; Lennarz and Scher, 1972) that phosphorylated derivatives of a C,, polyisoprenoid alcohol are involved in the biosynthesis of microbial cell wall peptidoglycan, lipopolysaccharide, mannans, and other polysaccharides; polyprenols are also involved in the synthesis of plant oligosaccharides. Two different reaction sequences have been described (Fig. 6). In type 1,oligosaccharide is assembled while attached to polyprenol by a pyrophosphate bridge and is then transferred to the growing macromolecule; in this type of mechanism, it is interesting to note that UMP is released. In the type 2 mechanism, polyprenol monophosphate merely acts as an intermediate in the transfer of a single monosaccharide from nucleotide sugar.

1. DOLICHOL PHOSPHATEMONOSACCHARIDES A family of long-chain isoprenyl alcohols, the dolichols, containing 16 to 22 five-carbon isoprene units, with the first unit saturated, has been described in animal tissues (Burgos et al., 1963; Butterworth and TYPE 1 MECHANISM UW-A

UMP

Lipid-P

UDP

Lipid-P-P-A

x

Lipid-P-P-A-B . .....etc - .....

UDP-B

....2

Liptd-P-P-A-B

____._

....Z

R-A-B

x

R-H

Li0id-P

P

TYPE 2 MECHANISM UDP-A

UOP

Lipid-P

Lipid-P-A

11': 1

Lipid-P-B

or

UDP-B .. ...etc. - . ..

R-A-B

FIG.6. Mechanisms by which polyisoprenol lipids are involved in complex carbohydrate assembly. A,B . . Z, monosaccharides; P, a monophosphate group; R-H, the acceptor (e.g., a peptide) to which the oligosaccharide becomes attached.

.

STRUCTURE A N D BIOSYNTHESIS OF M E M B R A N E G L Y C O P R O T E I N S

43

Hemming, 1968; Gough and Hemming, 1970), but until recently the function of dolichols in anim'al tissues was unknown. The work of Leloir's group (Behrens and Leloir, 1970; Behrens et al., 1971a,b, 1973; Parodi et al., 1972a,b) has shown that various dolichol monophosphate monosaccharides are synthesized in mammalian tissues and has indicated that these compounds are intermediates in polysaccharide and possibly glycoprotein assembly. Many other laboratories have followed this lead and are now engaged in mammalian polyprenol research. The first strong evidence for the formation of glycosylated polyprenol phosphates in mammalian tissues came from Leloir's group. In their first report (Behrens and Leloir, 1970), lipid was prepared from pork liver and tentatively identified as dolichol monophosphate by comparison with a standard preparation of this compound; rat liver microsomes were shown to catalyze the transfer of glucose from UDP glucose to this lipid to form a compound with the properties to be expected for dolichol monophosphate glucose (type 2 mechanism, Fig. 6). Further, dolichol monophosphate prepared by the chemical phosphorylation of highly purified dolichol also acted as a glucose acceptor in the enzyme reaction. The transferase required Mg2+for activity and was stimulated by Triton X-100 or deoxycholate. In a later paper, Behrens et al. (1971b) showed that microsomes from rat liver and brain catalyzed the formation not only of dolichol phosphate glucose from UDP-glucose but also of dolichol phosphate N-acetylglucosamine from UDP-N-acetylglucosamine and dolichol phosphate mannose from GDP-mannose; the nature of the phosphate bridge (monophosphate or pyrophosphate, Fig. 6) was not established. Both natural dolichol monophosphate and synthetic dolichol monophosphate served as exogenous acceptors in these reactions. No glycosyl transfer to dolichol monophosphate was observed with UDP-galactose, UDPN-acetylgalactosamine, or ADP-glucose. Ghalambor et al. (1974) showed that calf pancreas microsomes catalyzed the incorporation of N-acetylglucosamine from UDP-N-acetylglucosamine into endogenous lipid to form a product identified as dolichyl pyrophosphate N acetylglucosamine (P1-2-acetamido-2-deoxy-D-glucosyl P2-dolichyl pyrophosphate). The enzymic reaction required Mn2+or Mg2+,was inhibited by EDTA and by Triton X-100, but was stimulated by the chaotropic agent potassium thiocyanate. The product was identified by chromatographic and chemical comparisons with synthetic dolichyl pyrophosphate N-acetylglucosamine; both compounds released N-acetylglucosamine on acid hydrolysis (0.1 M hydrochloric acid at 100°C for 10 minutes) and N-acetylglucosamine l-phosphate on alka-

44


line hydrolysis (0.2M sodium hydroxide at 68°C for 1 hour) or on treatment with phosphodiesterase. The formation of lipid product was stimulated by the addition of exogenous dolichyl monophosphate. The evidence supported an earlier contention of Molnar et al. (1971) that a type 1 mechanism (Fig. 6) was involved in the transfer of N-acetylglucosamine to endogenous lipid. Molnar (1974) showed that the formation of lipid pyrophosphate N-acetylglucosamine by rat liver rough-surfaced microsomes is a reversible reaction; UDP-N-acetylglucosamine can be generated by the addition of UMP to glycosylated lipid, and UMP inhibits the formation of glycosylated lipid. 2. DOLICHOLPYROPHOSPHATE N,N'-DIACETYLCHITOBIOSE As indicated in Fig. 6, a type 1 mechanism in bacterial systems has usually been associated with the assembly of an oligosaccharide attached to a polyprenol by a pyrophosphate linkage; this also appears to be the case in mammalian systems. Thus Leloir et al. (1973) found that incubation of rat liver microsomes with exogenous dolichyl monophosphate and UDP N-[14Clacetylglucosamineresulted not only in the formation of radioactive dolichyl pyrophosphate N-acetylglucosamine but also of radioactive dolichyl pyrophosphate N,N'-diacetylchitobiose [P1-2-acetamido-2-deoxy-O-~-~glucopyranosyl-( 1-+ 4)-2acetamido-2-deoxy-~-glucosylPz-dolichylpyrophosphate 11. Mannose residues can be incorporated into dolichyl pyrophosphate N,N'-diacetylchitobiose (Hsu et al., 1974; Lucas et al., 1975; Lucas and Waechter, 1974) and a structure is assembled analogous to the mannose and N-acetylglucosamine-containingcores of Asn-GlcNAc-type prosthetic groups.

3. DOLICHOLD-MANNOPYRANOSYL PHOSPHATE

The most extensive investigations in this area have been carried out with the mannose incorporation systems, and the role of mannosylated polyprenol phosphates as intermediates in mammalian glycoprotein assembly appears well established. The pig liver system has been studied in detail by Hemming's group (Richards et al., 1971, 1972; Richards and Hemming, 1972; Hemming, 1973).Pig liver microsomes catalyze the transfer of mannose from GDP-mannose to endogenous lipid acceptor, and this reaction is stimulated by adding exogenous dolichol phosphate. A large-scale incubation using endogenous lipid acceptor was carried out with microsomes from 10 kg pig liver (Evans and Hemming, 1973), and highly purified l4C-.1abeledmannolipid was


45

isolated by a combination of mild alkaline saponification to destroy glycerolipids, chromatography on silicic acid, DEAE-cellulose and Kieselguhr columns, and preparative thin-layer chromatography. This mannolipid was compared with synthetic dolichyl a-D-mannopyranosyl phosphate (Warren and Jeanloz, 1973a,b), and the two compounds showed identical infrared (IR) and NMR spectra, chromatographic mobilities on thin-layer chromatography in two solvent systems, lability to mild acid, stability to mild alkali, and resistance to catalytic hydrogenation; the last-mentioned finding indicates that the endogenous lipid has a saturated terminal isoprene unit, like dolichol, and is not an allylic alcohol phosphate like the bacterial C,, polyprenol phosphate. Mass spectrometry could not be carried out on either dolichol phosphate or dolichol phosphate mannose, presumably because these compounds are not sufficiently volatile; dolichol gives a good mass spectrum, as do allylic polyprenol phosphates which dephosphorylate readily in the spectrometer (Evans and Hemming, 1973). Similar studies have been carried out with microsomes from myeloma tumor and bovine liver (Baynes et al., 1973), microsomes from hen oviduct and bovine thyroid (Waechter et al., 1973), and microsomes from calf pancreas (Tkacz et al., 1973, 1974). In all cases, divalent cation-dependent mannose transfer from GDP-mannose to an endogenous lipid acceptor was observed. The mannolipid released mannose on mild acid hydrolysis, was stable to saponification with mild alkali, but released varying amounts of mannose l-phosphate on strong alkaline hydrolysis (0.1N sodium hydroxide in 90% ethanol at 80"-100°C for 10 minutes). Baynes et al. (1973) identified their lipid component as a dolichol by mass spectrometry after strong alkaline hydrolysis; the other mannolipid preparations were identified as dolichol monophosphate mannose by chromatographic comparisons with synthetic dolichyl a-D-mannopyranosyl phosphate. Since the bridge between mannose and dolichol appears to be monophosphate in all these systems, it can be concluded that the transfer reaction has a type 2 mechanism (Fig. 6). Further proof of this is the finding that GDP strongly inhibits mannolipid formation when added prior to the start of the reaction and causes loss of mannose from mannolipid when added after initiation of the reaction; GMP has no such effect. The reversibility of the reaction is also indicated by the rapid and extensive transfer of mannose from mannolipid to GDP to form GDP-mannose (Richards et al., 1972; Richards and Hemming, 1972; Baynes et al., 1973; Waechter et al., 1973). Exogenous dolichol phosphate stimulates mannose transfer to lipid in these systems. Other polyprenol phosphates were tested as exogenous acceptors in the calf

46


pancreas system (Tkacz et al., 1973, 1974), and it was found that ficaprenyl phosphate (an allylic polyprenol with 11 isoprene units) and solanesyl phosphate (an allylic poIyprenol with 9 isoprene units) stimulated mannose transfer almost as effectively as dolichyl phosphate (a nonallylic polyprenol with 19 isoprene units). Farnesyl phosphate and citronellyl phosphate (3 and 2 isoprene units, respectively) had no stimulatory effect; retinyl phosphate (4 isoprene units) had a slight stimulatory effect. It was shown that the phosphates of ficaprenol, solanesol, and retinol acted as acceptors for mannose and did not act by stimulating incorporation into endogenous dolichol phosphate. Although there is overwhelming evidence implicating the dolichols as the major endogenous polyprenols involved in mammalian glycosyl transfer reactions, several reports have appeared suggesting a role for retinyl phosphate (DeLuca et al., 1973; Barr and DeLuca, 1974). C. Dolichol Pyrophorphate Oligosaccharides and the Assembly of Asn-GlcNAc Core Oligosaccharide

Initiation of the synthesis of an Asn-GlcNAc-type prosthetic group requires the attachment of an N-acetylglucosamine residue to an asparagine (or aspartic acid) residue in a polypeptide chain. RNase from bovine pancreas exists in at least four forms with identical amino acid sequences; RNase A is carbohydrate-free, whereas RNase B has a single polysaccharide prosthetic group attached to residue 34 (Tarentino et al., 1970).Since this residue is asparagine in RNase A, it is believed that asparagine rather than aspartic acid is the acceptor for N acetylglucosamine. A similar conclusion was drawn from studies comparing the incorporation of L3H]asparticacid and [3H]asparagine into rat serum glycoproteins (Kohno and Yamashina, 1973). There are presently two hypotheses for initiation of the AsnGlcNAc prosthetic group; i.e., either N-acetylglucosamine is incorporated directly into the polypeptide backbone from UDP-N-acetylglucosamine (Marshall, 1974; Khalkhali and Marshall, 1975) or the oligosaccharide core is preassembled while attached to dolichol by a pyrophosphate bridge and is subsequently transferred to the polypeptide (type 1 mechanism, Fig. 6). A large body of amino acid sequence data is now available on glycoproteins carrying Asn-GlcNAc-type prosthetic groups (Hunt and Dayhoff, 1970; Marshall, 1972, 1974).The sequence at the linkage region is always Asn-X-Ser(Thr), where X can be almost any amino acid. However, this tripeptide sequence does not invariably result in glycosylation, since many proteins with this sequence remain unglycosylated even in organs capable of making the Asn-GlcNAc linkage, e.g.,

STRUCTURE A N D BIOSYNTHESIS OF M E M B R A N E G L Y C O P R O T E I N S

47

RNase A in bovine pancreas. The factors which control oligosaccharide initiation are not known, but conformational constraints are undoubtedly involved. 1. DOLICHOLPYROPHOSPHATE OLIGOSACCHAFUDE

In the earlier work from Leloir's laboratory (Behrens and Leloir, 1970; Behrens et al., 1971b), it was reported that dolichol monophosphate glucose could transfer glucose to an endogenous acceptor to form a trichloroacetic acid-insoluble product which was not extracted with chloroform-methanol(2 : 1, v/v). This was at first assumed to be a glycoprotein but, in a most important development (Behrens et al., 1971a; Parodi et al., 1972b), it was subsequently shown that the product was in fact a dolichol pyrophosphate oligosaccharide; this latter material could be solubilized by chloroform-methanol-water (1: 1: 0.3, v/v). This experimental device was quickly applied by other laboratories, and it is now established that mannose also undergoes a similar series of reactions, namely, transfer from GDP-mannose to dolichol monophosphate to form dolichol monophosphate mannose, and transfer from this intermediate to an oligosaccharide attached to lipid (probably dolichol) by a pyrophosphate bridge (Behrens et al., 1973; Waechter et al., 1973; Hsu et al., 1974). Dolichol pyrophosphate oligosaccharides have solubility properties which allow their separation from both dolichol monophosphate monosaccharides and glycoprotein. They are insoluble in water, in aqueous ethanol, in chloroform-methanol(2 : 1, v/v), and in trichloroacetic acid, and fractionate with protein in the insoluble interphase obtained after extraction by the procedure of Folch et al. (1957); they are soluble in dimethyl sulfoxide, 6 M pyridine acetate (pH 4.4), chloroformmethanol-water (1: 1:0.3, v/v), and aqueous detergent solutions. The compounds can be purified by chromatography on DEAE-cellulose with chloroform-methanol-water (1: 1:0.3, v/v) as solvent, using a linear gradient of ammonium formate; dolichol monophosphate monosaccharides are eluted from this column well before lipid pyrophosphate monosaccharides and lipid pyrophosphate oligosaccharides (Behrens et al., 1971a). Mild acid methanolysis (0.1 N hydrochloric acid in methanol at 30°C for 60 minutes) released uncharged oligosaccharides from both [''C]glucose- and [14C]mannose-labeledlipid pyrophosphate oligosaccharides (Behrens et al., 1971a, 1973); the MWs indicated that the mannose-labeled oligosaccharides had from 5 to 18 monosaccharide units, and that the glucose-labeled oligosaccharides were somewhat larger (about 20 monosaccharide units). Mild acid hydrolysis or methanolysis released dolichol monophosphate from a

48


large-scale preparation of glucose-containing lipid pyrophosphate oligosaccharide from rat liver (Parodi et al., 19728); a compound believed to be dolichol pyrophosphate was also released by these treatments. Lipid pyrophosphate oligosaccharide is stable to saponification with mild alkali but can be decomposed by strong alkali treatment; treatment with 10% aqueous ammonia at 100°C for 3 hours released an oligosaccharide with a negative charge due to a phosphate ester (Behrens et al., 1971a). Alkaline hydrolysis of the products of acid methanolysis of lipid pyrophosphate oligosaccharides led to the appearance of positively charged substances believed to be due to the deacetylation of N-acetylhexosamine residues (Parodi et al., 1973; Behrens et al., 1973). ASSEMBLY 2. INITIATION OF Asn-GlcNAc OLIGOSACCHARIDE

The major unresolved question in this problem concerns the role that dolichol phosphate monosaccharides and dolichol pyrophosphate oligosaccharides play in glycoprotein synthesis. It appears to be reasonably well-established in several systems that radioactivity can be transferred either directly or indirectly from GDP-[l4C1mannose,dolichol monophosphate [14C]mannose,dolichol pyrophosphate [l4C1glucose-oligosaccharide and dolichol pyrophosphate [14Clmannose-oligosaccharide to endogenous acceptors to form glycoproteins (Parodi et al., 1972a; Behrens et al., 1973; Hsu et al., 1974; Waechter et al., 1973; Baynes et ul., 1973). For example, Behrens et al. (1973) have reported that rat liver microsomes catalyze the transfer of oligosaccharide from dolichol pyrophosphate [14C]mann~~e-oligosaccharides to endogenous protein acceptors; this product released radioactive glycopeptides on proteolysis or after alkaline hydrolysis. Behrens et al. (1973) have therefore proposed a scheme for the biosynthesis of the core of Asn-GlcNAc-type prosthetic groups: UDP-GlcNAc + dolichol-P 4 UMP + dolichol-P-P-GlcNAc UDP-GlcNAc + dolichol-P-P-GlcNAc + UDP + dolichol-P-P-GlcNAc-GlcNAc (GDP-Man and/or dolichol-P-Man), + dolichol-P-P-(GlcNAc), (GDP and/or dolichol-P), + dolichol-P-P-(GlcNAc),-(Man), (dolichol-P-Glc), + dolichol-P-P-(GlcNAc),-(Man), + (dolichol-P), + dolichol-P-P-(GlcNAc),-(Man),-(Glc), --f

It has been shown (Parodi et al., 1972a; Behrens et al., 1973) that both

dolichol-P-P-(GlcNAc),-(Man), and dolichol-P-P-(GlcNAc),-(Man),(Glc), can transfer their oligosaccharides to endogenous protein acceptors (see type 1 mechanism, Fig. 6).


49

Although this work most elegantly proved the role of dolichol intermediates in glycoprotein synthesis, it is not clear whether the above mechanism is applicable to all Asn-GlcNAc-type prosthetic groups. Several laboratories are accordingly engaged in studying the role of dolichols in the synthesis of specific glycoproteins such as immunoglobulin light chain (Baynes et al., 1973; Hsu et al., 1974) and hen oviduct glycoproteins (Waechter et nl., 1973). CELLSYSTEM 3. PLASMA Baynes et al. (1973) and Hsu et ul. (1974) carried out studies with the plasma cell tumor MOPC46B which secretes a K-type immunoglobulin light chain with a single Asn-GlcNAc-type oligosaccharide prosthetic group containing four mannose, three N-acetylglucosamine, four galactose, two fucose, and two sialic acid residues. Dolichol monophosphate mannose (mannolipid) synthesis can be distinguished from the transfer of mannose from mannolipid to glycoproteins by the use of EDTA; the former reaction requires a divalent cation, whereas the latter does not. Since EDTA inhibits transfer of mannose from GDP-mannose to glycoprotein, but not transfer of mannose from mannolipid to glycoprotein, it can be concluded that the latter reaction proceeds directly and not through conversion of mannolipid to GDP-mannose. Dolichol pyrophosphate oligosaccharide has been isolated from the plasmacytoma MOPC-46B (Hsu et al., 1974). A Con A-Sepharose column was used to fractionate dolichol monophosphate mannose from dolichol pyrophosphate oligosaccharide; the former compound passed through unretarded, whereas the latter compound absorbed and was eluted with a-methylmannoside. The structure of this compound has been tentatively characterized as dolichol-P-P-(GlcNAc),-(Man),. Kinetic evidence indicated formation of the dolichol pyrophosphate oligosaccharide from dolichol monophosphate mannose with subsequent transfer of the entire oligosaccharide to endogenous protein. The nature of the glycoprotein product formed in plasmacytoma is not known; the protein is membrane-bound and is solubilized by detergent. However, it is interesting that 10-20% of the plasmacytoma glycoprotein product reacted with antiserum to MOPC-46B light chain.

4. HENOVIDUCTSYSTEM Lennarz and co-workers (Waechter et al., 1973; Lucas et al., 1975; Chen et al., 1975; Pless and Lennarz, 1975) have done extensive stud-

50


ies on the role of dolichol intermediates in glycoprotein synthesis by hen oviduct. This tissue also carries out the series of reactions indicated above for rat liver and plasmacytoma. Hen oviduct synthesizes a dolichol pyrophosphate oligosaccharide with the structure (a-Man),Manal-4GlcNAc~l-4GlcNAc-pyrophosphate-dolichol. Direct transfer of radioactive oligosaccharide from this compound to endogenous glycoprotein has been demonstrated (Lucas et al., 1975).The final glycoprotein product could be digePced with proteases; however, the protein did not react with antiovalbumin antiserum and was strongly membrane-bound. The membrane-bound glycoprotein product was shown to contain the same oligosaccharide structure, (a-Man),ManPl4GlcNAca14GlcNAc, as the dolichol pyrophosphate oligosaccharide precursor. Studies have now been carried out on various other tissues; the isolation of dolichol pyrophosphate oligosaccharides from these tissues has provided strong evidence for existence of the biosynthetic pathway outlined in Fig. 7 (see reviews by Lennarz, 1975; Behrens, 1974; Heath et al., 1974; Waechter and Lennarz, 1976). However, this scheme is still tentative, because all the reactions have not been demonstrated in enzyme preparations from a single tissue source; further, none of the enzymes have been isolated and purified. 5. ANOMERICCONFIGURATION

It should be pointed out that every glycosylation reaction in the assembly of dolichol pyrophosphate oligosaccharide is believed to proceed with an inversion of configuration (Fig. 7). The nucleotide sugars required for glycosylations are all a-linked, except for GDP-L-fucose

8 ManeGlcNPcLGLcNPcdP-P-~l~~M~n~=~an~~lcNAc~G,~~~eP-P-~l

S ~ - R

&%%+Dd-p-p

I I

(Ma~=ManLGkNAc+GLcNA&ASN

FIG. 7. The role of dolichol intermediates in glycoprotein biosynthesis. Do], Dolichol; P, a monophosphate group.


51

which is p-linked. Herscovics et al. (1974) and Tkacz and Herscovics (1975) showed that calf pancreas microsomes and human lymphocyte homogenates catalyze the transfer of mannose from GDP-a-mannose to dolichol monophosphate to form dolichol P-Dmannopyranosyl phosphate. The transfer of mannose from GDP-a-mannose to dolichol pyrophosphate N,N’-diacetylchitobiose has been demonstrated with hen oviduct and rat liver microsomes (Levy et al., 1974); the product was tentatively identified as dolichol pyrophosphate P-mannosylN,N’-diacetylchitobiose. Dolichol monophosphate P-mannose could not serve as mannose donor for the synthesis of this dolichol pyrophosphate trisaccharide but could serve as a mannose donor for the further addition of a-linked mannose residues to form larger dolichol pyrophosphate oligosaccharides (Fig. 7). Similar findings were reported for human lymphocyte membranes (Wedgwood et al., 1974). Chen and Lennarz (1976) have reported the synthesis of dolichol pyrophosphate P-mannosyl-N,N‘-diacetylchitobiose by hen oviduct membrane and the transfer of trisaccharide from this lipid to endogenous protein acceptors. Herscovics et al. (1977a) showed that calf pancreas microsomes can effect the transfer of a-linked mannose residues from dolichyl P-D-[14C]mann~pyran~syl phosphate to dolichol pyrophosphate oligosaccharide without prior conversion to GDP-mannose. Thus it appears that GDP-a-mannose is the direct precursor of Plinked mannose residues and dolichyl P-D-mannopyranosyl phosphate is the direct precursor of a-linked mannose residues (Fig. 7). ARE ASSEMBLED BY THE 6. WHAT GLYCOPROTEINS DOLICHOLPATHWAY?

The transfer of oligosaccharide from dolichoI pyrophosphate oligosaccharide to endogenous protein acceptors has been demonstrated in several systems. The linkage of oligosaccharide to protein is stable to mild alkali, indicating that the linkage is probably of the Asn-GlcNAc type, but this has not been established (Waechter and Lennarz, 1976). The nature of the endogenous protein acceptors is not known. In all cases, the protein products are tightly bound to membrane. Further, the protein products could not be identified with the major secretory products of the tissues under study (e.g., immunoglobulin in the case of plasmacytoma, and ovalbumin in the case of hen oviduct). Finally, glucose has consistently been found in dolichol pyrophosphate oligosaccharides synthesized by rat liver (Parodi et al., 1972a; Behrens et al., 1973), calf thyroid (M. J. Spiro et al., 1976a; R. G. Spiro et al., 1976), calf kidney cortex, calf thymus and hen oviduct (M. J. Spiro et

52


al., 1976b), and calf pancreas (Herscovics et al., 1977a,b). Slices from kidney, oviduct, thymus, and thyroid synthesize these glucose-containing, lipid-bound oligosaccharides, while cell-free systems from plasmacytoma and oviduct make smaller lipid-bound oligosaccharides free of glucose; cell-free systems from rat liver (Behrens et al., 1973) and calf pancreas (Herscovics et al., 1977a,b) have been reported to synthesize glucose-containing lipid oligosaccharides. These various findings have indicated that the dolichol pathway (Fig. 7) may be involved in the biosynthesis of membrane-bound glycoproteins but not of secretory glycoproteins. The suggestion that novel glucose-containing glycoproteins may occur in membranes (M. J. Spiro et al., 1976a) has added weight to this hypothesis. Recent data, however, have indicated that the dolichol pathway may apply to both secreted and membrane-bound glycoproteins of the Asn-GlcNAc type. In fact, the biosynthetic scheme shown in Fig. 1 probably applies to both secreted and membrane-bound glycoproteins (Schachter, 1974a,b); the secreted protein, lacking a hydrophobic central section, presumably passes through the endoplasmic reticulum membrane into the intravesicular space, while the membrane protein becomes attached to membrane as indicated in Fig. 1. The dolichol pathway probably becomes operative either while the peptide is still attached to the ribosome or shortly after its release from the ribosome (see Section V). Earlier studies failed to demonstrate the transfer of oligosaccharide from dolichol pyrophosphate oligosaccharide to exogenous protein acceptors derived from secreted proteins such as ovalbumin; however, recent reports have indicated that such transfer can be demonstrated provided the exogenous acceptor is first unfolded by disulfide bond cleavage (Pless, 1976; Struck et al., 1977). Further evidence that the dolichol pathway is involved in ovalbumin synthesis was provided by Struck and Lennarz (1977), who showed that the antibiotic tunicamycin, which inhibits the formation of dolichol pyrophosphate N-acetylglucosamine (Tkacz and Lampen, 1975; Takatsuki et al., 1975), allows normal synthesis by hen oviduct slices of carbohydrate-free ovalbumin but inhibits the incorporation of all carbohydrate into ovalbumin. It is not clear what controls transfer of the oligosaccharide from a lipid oligosaccharide to protein; Chen and Lennarz (1976) showed that a unit as small as a trisaccharide can be transferred to protein. Since the oligosaccharide moieties of the larger dolichol pyrophosphate oligosaccharides resemble the oligomannoside structure (Fig. 2), it is reasonable to suggest that the latter are completely preassembled on lipid prior to incorporation into protein. Adamany and Spiro


53

(1975a,b), however, showed that al-2-linked mannose residues can be incorporated, one at a time, into exogenous glycopeptide acceptors using dolichol phosphate P-mannose as a donor (type 2 mechanism, Fig. 6). This suggests that further elongation of oligomannoside-type oligosaccharides can occur after their transfer from lipid intermediates to protein. While it appears likely that the core of N-acetyllactosamine-type oligosaccharides (Fig. 2) is assembled by the dolichol pathway (Fig. 7), elongation by the addition of N-acetylglucosamine, fucose, galactose, and sialic acid residues occurs by the addition of one sugar at a time to the glycoprotein (see Section IV,D); this process occurs primarily in the Golgi complex (see Section V). D. Elongation of N -AcetyIlactosamine-type 01igosaccharides

Four sugars are involved in the elongation of N-acetyllactosaminetype oligosaccharides, i.e., N-acetylglucosamine, fucose, galactose, and sialic acid (Fig. 2). The glycosyltransferases involved in these reactions have been reviewed (Schachter and Roden, 1973; Schachter, 1974a,b; Spiro et al., 1974); the following sections review more recent work on these enzymes.

1. N-ACETYLGLUCOSAMINYLTWSFERASES A glycoprotein N-acetylglucosaminyltransferasehas been described in goat colostrum (Johnston et al., 1966, 1973), rat liver (Johnston et al., 1973; Schachter et al., 1970),various other rat tissues (Johnston et al., 1973), guinea pig liver (Bosmann, 1970), pig liver (Hudgin and Schachter, 1 9 7 1 ~and ) ~ rat, human, and pig serum (Mookerjea et al., 1971,1972; Hudgin and Schachter, 1971~). The enzyme catalyzes the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to various glycoprotein acceptors containing prosthetic groups with WDmannose residues at the nonreducing ends, e.g., a,-acid glycoprotein and fetuin pretreated with sialidase, P-galactosidase and P-N-acetylglucosaminidase, and native ovalbumin and RNase B. There is a large variation in the V,,, achieved with these various acceptors (Johnston et al., 1973), suggesting that more than one N-acetylglucosaminyltransferase is present in the crude enzyme preparations that have been studied. The liver enzyme is strongly membrane-bound; solubilization was achieved with Triton X-100 or acetone treatment (Bosmann, 1970; ) ~ subsequent purification has so far Hudgin and Schachter, 1 9 7 1 ~but

54


not been possible. The serum and colostrum enzymes are nonsedimentable even in the absence of detergent, but only a 200-fold purification of the colostrum enzyme has been achieved (Johnston et al., 1973). The enzyme requires Mn2+for activity; other divalent cations (Mg+, C$+) are not as effective as stimulants (Bosmann, 1970; Hudgin and Schachter, 1971~). Recent work (Stanley et al., 1975; Narasimhan et al., 1977) has shown that this N-acetylglucosaminyltransferase activity is due to at least two separate enzymes, GlcNAc transferases I and 11. GlcNAc transferase I attaches GlcNAc in Pl-2 linkage to the Manal-S(Man-al,6-)-Man~1-4GlcNAc~1-4GlcNAc-Asn core of Asn-GlcNAc glycopeptides. This enzyme is deleted in a lectin-resistant mutant of Chinese hamster ovary cells. GlcNAc transferase I1 attaches GlcNAc in Pl-2 linkage to the product of GlcNAc transferase I (Fig. 8); it is fully active in the GlcNAc transferase I-deficient Chinese hamster ovary cell line. GlcNAc transferase I action is essential for elongation; if this enzyme is absent, GlcNAc addition cannot occur and, consequently, fucose, galactose, and sialic acid addition cannot occur (Fig. 8).Thus the GlcNAc transferase I-deficient Chinese hamster ovary cell line cannot elongate the Asn-GlcNAc core, thereby explaining the inability of these cells to bind several lectins.

,

P Dol

Gn-F -A:"-

I

G: Gn-F -Ak-

FIG. 8. Elongation of an N-acetyllactosamine-type oligosaccharide. The (Man),(GlcNAc), core strucure is presumably transferred from dolichol pyrophosphate oligosaccharide to a peptide acceptor and subsequently elongated by stepwise addition of sugars in the Golgi complex. The enzymes catalyzing these reactions are N-acetylglucosaminyltransferases (GlcNAc-Tr), fucosyltransferase (Fuc-Tr), galactosyltransferase (Gal-Tr),and sialyltransferases (Sialyl-Tr). See text for additional comments.


55

GlcNAc transferase I can act on Mana1-3Man/31-4GlcNAc and larger oligosaccharides, glycopeptides, and glycoproteins containing this trisaccharide sequence. GlcNAc transferase I1 acts only on the branched structure Man-(GlcNAc-Man)-Man-GlcNAc-R, where R is H or a glycopeptide or glycoprotein. Low-MW compounds such as methyl a-mannopyranoside are ineffective acceptors. 2. FUCOSYLTRANSFERASE Fucose has been found at only one position in Asn-GlcNAc glycoproteins, i.e., attached to the most internal GlcNAc residue (Fig. 2). Fucose has not been conclusively shown attached to the galactose residue of Asn-GlcNAc glycopeptides, although amino acid-free AsnGlcNAc-type oligosaccharides with a Fucal-2Gal sequence have been isolated from the tissues of a fucosidosis patient (Tsay et al., 1976). Two glycoprotein fucosyltransferase activities have been described in pig liver (Jabbal and Schachter, 1971) and human serum (Munro and Schachter, 1973) which transfer fucose from GDP-fucose to sialidase-treated a,-acid glycoprotein and sialidase-, P-galactosidase-treated a,-acid glycoprotein, respectively. The former activity attaches fucose to a terminal P-galactoside residue, and the human serum enzyme is the blood group H-dependent a-2-fucosyltransferase. The transferase acting on sialidase-, P-galactosidase-treated alacid glycoprotein has recently been shown to transfer fucose to the asparagine-linked GlcNAc residue (Fig. 8) (Wilson et al., 1976). The transfer of fucose to the most internal GlcNAc residue requires the prior incorporation of at least one GlcNAc residue in Pl-2 linkage to a mannose residue at the core oligosaccharide (Fig. 8).

3. GALACTOSYLTRANSFERASES Galactose appears in only a single location in Asn-GlcNAc-type oligosaccharides, namely, linked to N-acetylglucosamine either as the terminal or penultimate (to sialic acid) residue at the nonreducing end (Fig. 2). Many tissues are capable of transferring galactose from UDPgalactose to either free N-acetylglucosamine to form N-acetyllactosamine or to gl ycoproteins with a P-N-acetylglucosaminide nonreducing terminus, e.g., @,-acidglycoprotein or fetuin pretreated with sialidase and 0-galactosidase. The linkages synthesized by the glycoprotein galactosyltransferases have not been characterized and, since G a l p l + 4GlcNAc and Galj31+ 6GlcNAc linkages have been described in Asn-GlcNAc-type oligosaccharides, more than a single ga-

56


lactosyltransferase may contribute to the incorporation of galactose into glycoprotein acceptors. Asn-GlcNAc-type glycoprotein galactosyltransferase is tightly bound to membrane within the cell but exists in a soluble form in milk, colostrum, and serum (see Schachter and Rod&, 1973, for references). The milk galactosyltransferase is in fact equivalent to the A protein, one of the two components of lactose synthetase (Brew et al., 1968). The A protein by itself has a very low affinity for glucose, although it can synthesize lactose at very high glucose concentrations; in the presence of the B protein (a-lactalbumin),however, the affinity for glucose is greatly increased and lactose synthesis occurs readily. Since a-lactalbumin occurs only in mammary gland, lactose synthesis is confined to this organ; the function of the A protein in other tissues is believed to be in the biosynthesis of Asn-GlcNAc-type glycoproteins. It is interesting that the galactosyltransferase in liver, serum, and other tissues can make lactose provided exogenous a-lactalbumin is provided (Hudgin and Schachter, 1971b; Fitzgerald et al., 1971); it is not, however, certain that the milk A protein and the various membrane-bound galactosyltransferases are identical proteins. Bovine and human milk A protein has been purified to homogeneity by classic methods (Fitzgerald et al., 1970) and by affinity chromatography with an a-lactalbumin-Sepharose column (Trayer and Hill, 1971; Andrews, 1970; Khatra et al., 1974). The purified enzyme catalyzes the incorporation of galactose into N-acetylglucosamine as well as into oligosaccharides and glycoproteins with a nonreducing p-Nacetylglucosaminide terminus, indicating that these activities are catalyzed by a single enzyme. Although a-lactalbumin under some conditions inhibits transfer to N-acetylglucosamine (Kitchen and Andrews, 1972), there is no effect on galactose incorporation into oligomers of N-acetylglucosamine and into glycoproteins (Schanbacher and Ebner, 1970). The pure enzyme requires Mn2+for activity. Detailed kinetic studies have been carried out on the milk transferase in the presence and absence of a-lactalbumin (Morrison and Ebner, 1971a,b; Khatra et al., 1974), and orders of binding to enzyme have been suggested for monosaccharide, UDP galactose, Mn2+,UDP, disaccharide product, and a-lactalbumin. This galactosyltransferase has been demonstrated in goat colostrum, rat liver, and other rat tissues (McGuire et al., 1965; Schachter et al., 1970; Carlson et al., 1973a), pig liver and serum (Hudgin and Schachter, 1971b), rat serum (Wagner and Cynkin, 1971), human serum (Kim et al., 1972a,b), amniotic fluid (Nelson et al., 1974), cerebrospinal fluid (Den et al., 1970), mouse mastocytoma (Helting and Erbing, 1973), thyroid (Spiro and Spiro, 1968b), human saliva (Naku-


57

mura et al., 1974; Nakumura and Tsunemitsu, 1975a,b), and human urine (Chester, 1974). The A protein is a glycoprotein (Lehman et al., 1975) which exists in milk in at least two forms (MWs 58,000 and 42,000); the smaller proteins are proteolytic products, and a single protein (MW 51,000) can be obtained from bovine colostrum which contains protease inhibitors (Powell and Brew, 1974). The enzyme requires a sulfhydryl group (Magee and Ebner, 1974; Kitchen and Andrews, 1974). Its interaction with a-lactalbumin has been studied fluorimetrically (Prieels and Barel, 1975), and its interaction with UDP-galactose by CD (Geren et al., 1975). 4. SIALYLTWSFERASES Like galactose, sialic acid also occurs in only a single position in Asn-GlcNAc-type prosthetic groups, namely, attached to galactose at the nonreducing terminus (Fig. 2). However, the linkages to galactose may be a 2 + 2, a2 + 3, a2 + 4, and a 2 4 6, and a family of several different sialyltransferases is probably involved in the synthesis of Asn-GlcNAc-type glycoproteins. These sialyltransferases are assayed by measuring the incorporation of sialic acid from CMP-sialic acid into low-MW acceptors such as lactose or N-acetyllactosamine and into gl ycoproteins with P-galactoside termini such as fetuin or a,-acid glycoprotein pretreated with sialidase; these activities have been described in rat mammary gland (Carlson et al., 1973b), goat, bovine, and human colostrum (Bartholomew et al., 1973), rat liver (Schachter et al., 1970; Bernacki and Bosmann, 1973), pig liver and serum (Hudgin and Schachter, 1971a),human serum (Kim et al., 1971; Mookerjea et al., 1972), and thyroid (Spiro and Spiro, 1968a). The same enzyme probably acts on both low- and high-MW acceptors (Hudgin and Schachter, 1971a; Bartholomew et al., 1973). Rat mammary gland sialyltransferase is strongly membrane-bound and could not be solubilized with detergents. There was no requirement for metal. The crude microsomal enzyme utilized either CMPN-acetylneuraminic acid or CMP-N-glycolylneuraminic acid as sialic acid donor with lactose as acceptor. Only compounds with P-galactoside termini were effective acceptors; a large number of other compounds, including several a-galactosides, were ineffective. The finding that the mammary gland enzyme was relatively inactive toward high-MW acceptors (Carlson et al., 197313) is probably due to the fact that no detergent was present in the assay; detergent is known to be required for optimal sialyltransferase activity with glycoprotein acceptors (Schachter et al., 1970). Goat, bovine, and human colostrum contain a soluble sialyltrans-

58


ferase which transferred sialic acid from CMP sialic acid to low- and high-MW /3-galactosides (Bartholomew et al., 1973). The bovine colostrum enzyme has recently been purified 440,000-fold (Paulson et al., 1977a) by the use of affinity chromatography using CDP as a ligand. The colostrum enzyme differs from the mammary gland enzyme in two important respects: (1)the rat mammary gland enzyme showed approximately equal activity with the following acceptors: Galpl + 4GlcNAc, GalP1 + SGlcNAc, Gal/31+ GGlcNAc, and Gal/3l+ 4Glc; the colostrum enzyme (and the pig liver enzyme, Hudgin and Schachter, 1971a) showed a marked preference for Gal/31+.4GlcNAc (the requirement for Galpl + 4GlcNAc was absolute in the case of the pure enzyme, Paulson et al., 1977b); (2) the mammary gland enzyme made predominantly the a2 + 3 linkage, while the pure bovine colostrum enzyme synthesized only the a2 + 6 linkage. The first observation illustrates the important point that, although the terminal sugar of the acceptor is the major factor controlling transferase specificity, internal sugars can also influence transferase activities. The second observation shows that different positional isomers of the same two sugars are synthesized by separate transferases; similar conclusions were drawn from a study of sialyltransferases during embryological development (Hudgin and Schachter, 1972). Considerations of this type are the basis of the one linkage-one enzyme hypothesis (Schachter and Roden, 1973). E. Assembly of Ser(1hr)-GalNAc Oligosaccharides

Ser(Thr)-GalNAc oligosaccharide assembly probably does not involve lipid intermediates and occurs by the sequential addition of one sugar at a time to the growing glycoprotein. The process is illustrated using salivary gland mucins as examples. Ovine and porcine submaxillary mucins (OSM and PSM) have been thoroughly characterized, and all the glycosyltransferases required for assembly of their oligosaccharide prosthetic groups have been described (Schachter and Roden, 1973; McGuire, 1970; Schachter et al., 1971; Carlson et al., 1973c; McGuire and Roseman, 1967). Figure 9 summarizes the biosynthetic path for the major oligosaccharide prosthetic groups of OSM and PSM and indicates that five different glycosyltransferases are involved. Since these glycosyltransferases have not been purified to homogeneity, the possibility of a lipid intermediate in monosaccharide transfer from nucleotide sugar to glycoprotein (type 2 mechanism, Fig. 6) has not been completely ruled out.

59


“

R - G GolNAvSA

R-O-GolNAc I

GP-Fur

R-O-GalNAc

Gbi

-

“cr._NcI Got-Fuc

R- O- G o i N k I 001- Fuc

GoINAc

,,

I?

I R-O-GolNAc-SA

Gbi-Fuc (Kil -,PSM

,

(1,

R ~ Ga:NAc. o ~

I

9

Gai-Fuc GolNAc IA.1 -PSM

FIG. 9. Biosynthesis of OSM and PSM. Question marks designate pathways not directly tested but which could exist because of the presence of the above oligosaccharides in PSM (Carlson, 1968). (A-)-PSM and (A+)-PSM refer to mucins lacking or carrying the human blood-group-A determinant, i.e., GalNAca l-B[Fucu 1-2]Gal-.

The first step in the assembly process is the attachment of N-acetylgalactosamine to an hydroxyamino acid in the peptide backbone (McCuire and Roseman, 1967; McCuire, 1970). The polypeptide N acetylgalactosaminyltransferase is present in particulate form in mammalian submaxillary glands and has been partly purified. The polypeptide acceptor is prepared by treating OSM sequentially with sialidase and a-N-acetylgalactosaminidaseto remove the disaccharide prosthetic groups. The enzyme has a high specificity for this acceptor; a large number of other compounds was ineffective, and pronase digestion of the carbohydrate-free OSM destroyed its acceptor activity. This high degree of specificity for a particular high-MW polypeptide is shown by all the known transferases involved in synthesizing linkages between monosaccharides and amino acids. Bovine submaxillary gland has a similar N-acetylgalactosaminyltransferase(Hagopian and Eylar, 1968a,b, 1969a,b) which transfers N-acetylgalactosamine to carbohydrate-free bovine submaxillary mucin; this enzyme is also very specific for its polypeptide acceptor. Hagopian et al. (1971) showed, however, that the enzyme can transfer N-acetylgalactosamine to a specific threonine residue in a basic protein isolated from bovine myelin; this myelin protein is not a glycoprotein but presumably accepts N acetylgalactosamine because of an amino acid sequence similar to the polypeptide core of submaxillary mucins. Comparison of amino acid sequences near Ser(Thr)-GalNAc linkage regions (Marshall, 1972; Hill, 1976; Hill et al., 1977a,b) has not clarified the specificity requirements for the transferase.

60


There is a branch point in the biosynthetic pathway (Fig. 9) after the first N-acetylgalactosamine is incorporated. If galactose is incorporated before sialic acid, the pathway proceeds toward the synthesis of complex oligosaccharide side chains (the major components of PSM); if sialic acid is incorporated before galactose, galactose cannot be incorporated and assembly stops at the disaccharide stage (the major component of OSM). The sialyl- and galactosyltransferases responsible for these two reactions are present in both ovine and porcine submaxillary glands, but there is relatively little galactosyltransferase activity in ovine glands (McGuire, 1970; Schachter et al., 1971; Carlson et al., 1973~). Ovine glands therefore make predominantly the disaccharide side chain, whereas porcine glands make both disaccharide and larger oligosaccharide side chains. The key enzyme in this control process is the galactosyltransferase (Schachter et al., 1971). This enzyme is strongly bound to membrane, is activated by Triton X-100, has an absolute requirement for Mn2+, and incorporates galactose into linkage with the terminal N-acetylgalactosamine of sialidase-treated OSM probably in pl 3 linkage. The galactosyltransferase will not act if the terminal N-acetylgalactosamine of the acceptor is substituted with a sialic acid residue (Fig. 9). Both galactosyl- and sialyltransferases compete for the same substrate (Fig. 9), namely, N-acetylgalactosamine attached to the polypeptide core of the mucin. Sialyltransferases capable of transferring sialic acid from CMP-sialic acid to sialidase-treated ovine, bovine, and porcine submaxillary mucins have been described in ovine, bovine, and porcine submaxillary glands (Carlson et al., 1973~). The product formed by the sheep enzyme with CMP-N-acetylneuraminic acid as donor was shown to be N-acetylneuraminyl-(2 -+6)-N-acetylgalactosamine peptide, the same linkage found in naturally occurring OSM. A variety of low-MW compounds either with or without terminal N acetylgalactosaminide residues were ineffective as acceptors. Carlson et al. (1973~) found that the sheep enzyme could use either CMP-Nacetylneuraminic acid or CMP-N-glycolylneuraminic acid as sialic acid donors. Schauer and Wember (1973) examined the latter point more carefully and found that the sialyltransferases of bovine, porcine, and equine submaxillary glands transferred N-acetyl-, N-glycolyl, N-acetyl-7(or g)-O-acetyl-, and N-acetyl-4-O-acetylneuraminic acids from their respective CMP-glycosides to endogenous acceptors at similar rates; all four nucleotide sugars gave the same pH optima and K, values, and there was competition between nucleotide sugars for a common enzyme active site. Thus the mucin sialyltransferases are not specific for the acyl groups attached to neuraminic acid; the

STRUCTURE A N D BIOSYNTHESIS OF M E M B R A N E GLYCOPROTEINS

61

different ratios of various sialic acids found in salivary mucus glycoproteins from different species apparently depend neither on CMPsialic acid synthetase nor on sialyltransferases, but on the activities of the oxidoreductase and acetyltransferases which modify N-acetylneuraminic acid. If galactose is incorporated into protein-bound N-acetylgalactosamine before sialic acid (Fig. 9), further growth of the oligosaccharide can occur. A fucosyltransferase is present in porcine submaxillary gland capable of transferring fucose from GDP-fucose to both high and low-MW acceptors with terminal galactose residues, e.g., sialidasetreated a,-acid glycoprotein, lactose, and the P l + 3, /3l +. 4, and P 1 4 6 isomers of galactopyranosyl-N-acetylglucosamine(McGuire, 1970); the enzyme can also transfer fucose to the terminal galactose residue of the product of the galactosyltransferase reaction (Fig. 9) obtained either from a large-scale galactosyltransferase incubation or by treating blood group A-negative PSM with 1 N hydrochloric acid at 70°C for 2 hours to remove terminal fucose and sialic acid residues. The final enzyme in the synthetic scheme (Fig. 9) occurs only in pigs genetically capable of making an antigen similar to the human blood-group-A antigen. This porcine submaxillary gland N-acetylgalactosaminyltransferase converts blood group A-negative PSM to blood group A-positive PSM (McGuire, 1970; Schwyzer and Hill, 1977a,b) and has the same substrate specificities as the blood group A-dependent N-acetylgalactosaminyltransferase present in human tissues; the enzyme will incorporate N-acetylgalactosamine in a1 -+ 3 linkage to a terminal P-galactoside residue of both low- and high-MW acceptors, provided the terminal galactose has a fucose residue attached in a1 + 2 linkage. The blood group enzymes are not discussed in this chapter. V.

SUBCELLULAR SITES OF GLYCOSYLATION

Figure 1 illustrates a model for membrane glycoprotein biogenesis. Data on the subcellular localization of the various glycosylation reactions support this scheme (Schachter and Rod&, 1973; Schachter, 1974a,b), and some of this evidence is reviewed in this section. A. Autoradiographic Evidence

With the use of radioactively labeled carbohydrate precursors, considerable information has been gained about the site of glycosylation

62


of macromolecules and the turnover of glycoproteins. Monitoring the fate of radioactivity has involved biochemical studies on subcellular membrane fractions (Schachter, 1974a,b) and electron microscope autoradiographic studies to localize the site of radioactivity. The latter results must be interpreted with caution, since the technique lacks specificity for one type of macromolecule, since the precursor may be metabolized prior to incorporation, and since incorporation may not occur at one specific site of the macromolecule. Early autoradiographic studies showed that radioactive carbohydrate precursors produced grains initially in the cytoplasm and later at the cell surface; this was direct evidence that each cell is the source of its own surface material (Ito, 1969). Since that time, autoradiographic techniques have been used extensively to study the sites of assembly of secretory glycoproteins and of membrane-associated glycoproteins (Neutra and Leblond, 1966; Bennett et d.,1974). Labeling of cell surface glycoproteins has been demonstrated using radioactive carbohydrates as precursors and, in general, the pattern of labeling is similar in several cell types. For instance, in the duodenum, incorporation of mannose is observed diffusely in the cytoplasm at 10 minutes and at the cell surface after approximately 5 hours. Fucose appears within 2 minutes in the Golgi complex and within 20 minutes at the apical and lateral cell surfaces, where it remains for up to 30 hours. Galactose appears within 10 minutes in the Golgi complex and at 30 minutes at the apical cell surface (Leblond and Bennett, 1974). From such data, it appears that the elaboration of cell surface glycoprotein follows a pathway similar to that of soluble secretory glycoproteins and also follows a similar time sequence. It should be noted that these techniques do not distinguish membrane glycoproteins from secretory products which remain associated with the cell surface. The incorporation of glucosamine and mannose residues occurs at the level of the rough endoplasmic reticulum and also within the Golgi complex (Sturgess et d.,1972; Moscarello et d.,1972). The interpretation of studies with mannose is subject to some dispute, since it is readily metabolized to other glycosyl residues (Mitranic and Moscarello, 1972). The addition of galactose, fucose, and sialic acid residues occurs exclusively within the Golgi complex. These findings support the idea that the mannose- and glucosamine-containing core (Fig. 2) is assembled in the rough-surfaced endoplasmic reticulum, while elongation (the addition of N-acetylglucosamine, galactose, fucose, and sialic acid) occurs almost entirely in the Golgi’apparatus. The cisternae and tubular network of the hepatocyte Golgi complex (Fig. 10)are the earliest sites of assembly of carbohydrates into macromolecules, and labeled macromolecules are then transferred to se-


63

FIG. 10. Transmission electron micrograph of rat liver showing the characteristic cisternae (C), vesicles (V) representing the tubular network, and larger secretory vesicles of the Golgi complex. The Golgi complex is bounded by microtubules (M) and polarized so that its secretory or trans face (SF)is adjacent to the plasma membrane (PM). The tubular network is presumed to be the site of giycosylation of macromolecules and is a site which may show extensive proliferation following perturbation of the Golgi complex functions. x 56,000.

cretory vesicles which migrate to the cell surface (Sturgess et al., 1973);radioactivity which remains associated with the hepatocyte cell surface for prolonged time intervals has been attributed to the labeling of integral membrane glycoproteins. Few studies of glycoprotein biosynthesis have focused on the labeling of specific membrane glycoproteins, an approach essential in characterizing the biosynthetic site and turnover of these molecules. With rhodopsin, specific labeling experiments have been carried out, and these indicate that assembly occurs in the Golgi complex, with subsequent transfer to plasma membrane (Bok et al., 1974; Papermaster et al., 1975). Rhodopsin offers many advantages in studying this problem. It forms 80-90% of the protein of rod outer segments and is readily purified. The biosyn-

64


thesis of rhodopsin has been studied in frog retina using radioactive amino acids (Papermaster et d.,1975). Incorporation of label was rapid, and radioactivity was transferred from retinal subcellular fractions to rod outer segments after 2 hours. Immunochemical analysis showed that the newly synthesized protein was membrane-bound upon completion of synthesis. A soluble form was not detected. Darkadapted retinas were incubated in vitro in the presence of [3Hlglucosamine (Bok et al., 1974). Radioactivity appeared early over the ribosomes, in the Golgi apparatus after 20 minutes and, after 2 hours, in the base of the outer segment where it was inserted into the disk membrane. It was concluded that rhodopsin was fully glycosylated prior to its assembly into the disk membrane. On the basis of x-ray diffraction analysis, Worthington (1973) concluded that the nonpolar parts of rhodopsin reside in the low-density hydrocarbon chain region and that the polar parts may protrude a short distance from the membrane surface. Support for the conclusion that rhodopsin is probably buried deeply in the membrane comes from studies on model membranes (see Section 111). B. Subcellular localization of Glycosyltrunsferases

The subcellular localization of glycosyltransferases has been reviewed (Schachter, 1974a,b). The evidence is extensive that the Golgi complex is the major site of the transferases involved in elongation of N-acetyllactosamine-type oligosaccharides, i.e., the N-acetylglucosaminyl-, fucosyl-, galactosyl-, and sialyltransferases discussed in Section IV,D. Table I1 shows supportive data obtained from rat liver (Munro et al., 1975). The extensive literature on the cell surface localization of glycosyltransferases (Shur and Roth, 1975; Culp, this volume) has been obtained primarily with cultured cells; further, the relative contributions of the Golgi complex and plasma membrane to cellular glycosyltransferase activity were usually not assessed in these studies. Rat liver plasma membrane is essentially devoid of glycosyltransferase activity (Munro et al., 1975). Thus, evidence suggests that the Golgi apparatus is the main site of oligosaccharide elongation. The role of cell surface glycosyltransferases either in glycosylation reactions or in cell-cell interactions remains controversial. C. The Role of the Ribosome

Initial stages of peptide assembly occur at the ribosomal level, and there is considerable evidence that membrane glycoproteins, des-

66


tined for the cell surface, are synthesized on bound ribosomes (Siekevitz, 1972; Morrison and Lodish, 1975; Bergeron et al., 1975). In contrast, it has been suggested that nonglycosylated membrane proteins, destined for the cytoplasmic face of the endoplasmic reticulum or plasmalemma, are synthesized by free ribosomes (Autuori et al., 1975a,b; Elhammer et al., 1975; Svensson et al., 1976; Lodish, 1973; Ito and Sato, 1969). The membrane glycoproteins of the endoplasmic reticulum present a special biosynthetic problem, since glycosylation is completed in the Golgi complex, and there must be a mechanism for transporting these molecules from the Golgi apparatus back to the endoplasmic reticulum. To explain the transfer of proteins across membranes, Blobel and Dobberstein (1975a,b) have proposed the “signal” hypothesis for which supporting data have been reviewed previously (Blobel and Sabatini, 1971). The hypothesis is based on the presence of a sequence of signal codons on mRNAs whose translation products are destined for transfer across a membrane. Translation of the signal codons results in a unique sequence of amino acids at the N-terminal end of the nascent peptide chain. The assembly of peptides begins on free ribosomes, at which stage the membrane can recognize the N-terminal end with its signal sequence of hydrophobic amino acids 10 to 40 amino acid residues long (Fig. 1).Attachment of the ribosomes to membrane receptor protein occurs only if the signal is present, whereas other peptides remain free in the cytoplasm. As a result of membrane-associated proteolysis, the N-terminal end of the peptide is removed (Milstein et al., 1972; Blobel and Dobberstein, 1975a). The protein may then become an integral part of the membrane or be released into the “soluble” compartment of the endoplasmic reticulum as a secretory protein. The signal hypothesis is based primarily on work with secretory proteins such as pancreatic enzymes and immunoglobulins (Blobel and Dobberstein, 1975a,b; Schechter et al., 1974). It explains why secretory proteins are translated on membrane-bound ribosomes and how a soluble hydrophilic protein crosses the lipid bilayer from the cytoplasmic side of the endoplasmic reticulum membrane to the intravesicular side. It seems highly probable that intrinsic membrane glycoproteins, which must also traverse the lipid bilayer (at least partly) during translation, are also translated on membrane-bound ribosomes and that the nascent peptides of these proteins have signal sequences at their N-terminal ends (Fig. 1).There is strong evidence that all glycoproteins are translated on membrane-bound ribosomes (see Schachter, 1974a,b, for reviews), but evidence suggesting a signal sequence for membrane glycoproteins has only recently become avail-


67

able. It has been shown that the mRNAs coding for the membrane glycoproteins of enveloped animal viruses (such as VSV and Sindbis virus) are translated solely on membrane-bound ribosomes (Morrison and Lodish, 1975; Wirth et al., 1977; Toneguzzo and Ghosh, 1975); nonglycoprotein components of these viruses are 'translated on free ribosomes. Sindbis virus has three structural proteins, two envelope glycoproteins (E, and E,), and a nonglycosylated core protein. El and E, are inserted into the plasma membrane of infected cells as integral membrane proteins and become part of the viral envelope as the virus buds through the plasma membrane. All three structural proteins are translated from a single mRNA using a single initiation site. Wirth et al. (1977) found that all three proteins were translated on membranebound ribosomes; however, El and E, are vectorially transported through the endoplasmic reticulum membrane (they sediment with the membrane and become unavailable to proteases), while the core protein is released to the cytoplasmic side of the membrane. These findings show that neither the mRNA nor the ribosome can determine the segregation of nascent protein to either the cytoplasmic or intravesicular side of the membrane. Rather, the peptide itself, presumably by means of some sort of signal sequence, directs the binding of the ribosome to the membrane and subsequent vectorial discharge through the lipid bilayer (Fig. 1). In rat liver and in plasmacytomas, a small amount of N-acetylglucosamine has been shown to be incorporated into nascent peptide still attached to ribosomes (Lawford and Schachter, 1966; Molnar and Sy, 1967; Sherr and Uhr, 1969; Cowan and Robinson, 1970). In a recent reinvestigation of this point, Kiely et al. (1976) showed that both glucosamine and mannose were present on nascent ovalbumin chains still bound to ribosomes by tRNA. It is likely that this incorporation represents only a small fraction of the total and that most glycosylation is a postribosomal event (Schachter, 1974a,b; Jamieson, 1977).

VI.

MEMBRANE BlOGENESlS

On the basis of present evidence, it is assumed that the biosynthetic mechanisms for secretory glycoproteins and structural glycoproteins of cell membranes are very similar. Common features are found in the soluble, secreted proteins and the membrane glycoproteins in terms of their overall composition and the assembly of oligosaccharide sequences. There is no evidence to date indicating that the mode of biosynthesis is different. The role of the rough- and smooth-surfaced endoplasmic reticulum

68


and Golgi complex in the biosynthesis and intracellular transport of secretory glycoproteins makes it difficult to distinguish glycoproteins destined for secretion from those which are to become an integral part of the membrane, presumably by incorporation into the lipid bilayer through specialized hydrophobic peptide sequences (Fig. 1).At the present time, it is assumed that membrane glycoproteins are synthesized on the rough endoplasmic reticulum and then transported through the endomembrane system to the Golgi complex and then to the final membrane site (Figs. 1 and 11). Other membrane components, presumably lacking carbohydrate, may be made on free ribosomes in the cytoplasm and may migrate through the cytosol as soluble components until they are incorporated into a membrane structure (Bretscher, 1973; Bretscher and Raff, 1975). Glycosyltransferase enzymes are tightly bound to membranes of the endoplasmic reticulum and the Golgi complex (Schachter, 1974a,b), and the glycosylations catalyzed by these enzymes take place predominantly at the internal surface of the cisternae and within the Golgi vesicles (Nicolson and Singer, 1971; Hirano et al., 1972). Thus glycosylation is a membranebound phenomenon, and the available evidence indicates that all glycoproteins must pass through the endomembrane system (Fig. 1). Bretscher (1973) has suggested that glycosylation may occur at the plasma membrane to "lock" into the membrane proteins that migrate to it through the cytosol. This seems unlikely for at least two reasons: (1) Cells such as liver lack plasma membrane glycosyltransferases (Munro et al., 1975); (2) model membrane studies (Section 111) indicate that carbohydrate is not essential for the insertion of a hydrophobic protein into the membrane. Rather, nonglycosylated proteins that migrate to the plasma membrane through the cytosol probably bind to sites on the membranes to become peripheral membrane proteins at the cytoplasmic face (Rothman and Lenard, 1977). The difficulty in distinguishing secretory and membrane proteins at early stages of their biosynthesis suggests that it is best to study membrane glycoprotein synthesis in a nonsecretory tissue (e.g., retinal rod cells which make rhodopsin). Rat liver microsomes contain membrane-associated glycoproteins which can be released only by detergents, but these glycoproteins contain fewer sugars than glycoproteins discharged into the lumen of the microsomal vesicles (Redman and Cherian, 1972); it is therefore not clear whether these membrane glycoproteins are destined to be membrane glycoproteins 'firmly integrated into the membrane by hydrophobic interactions or are unfinished polypeptide moieties of future soluble secretory glycoproteins which are still attached to the membrane. Similarly, in the Golgi com-


69

FIG. 11. Transmission electron micrograph of rat liver showing the Golgi complex and endoplasmic reticulum involved in membrane biogenesis. The Golgi complex (G) is polarized with one side associated with the formation of secretory products. This surface is commonly directed toward the plasma membrane. In membrane biogenesis, the flow of membrane is thought to occur from the nuclear envelope (N), to the rough endoplasmic reticulum (RER), and by transition vesicles (arrows) to the Golgi complex; at this site, membrane differentiation occurs, and membrane is transferred to the cell surface (PM) via small and large secretory vesicles (SV). x 19,000.

plex of rat liver, glycoproteins destined for secretion remain firmly bound to the membrane during early stages of assembly (Moscarello et al., 1972), so that it is difficult to dissociate precursors of soluble glycoproteins from integral membrane glycoproteins. This problem is a complicating factor in many of the studies that

70


bear on the intracellular migration of membrane glycoproteins from the endoplasmic reticulum to the plasma membrane and to other intracellular membrane sites. A. Biogenerir of Plasma Membranes

The transport of secretory (and presumably membrane) proteins from their site of assembly in the rough endoplasmic reticulum to the Golgi complex is an energy-dependent process that is blocked by respiratory inhibitors (Jamieson and Palade, 1968).The transfer of membrane has been characterized morphologically by the appearance of transition vesicles between the rough endoplasmic reticulum and the periphery of the Golgi complex (Fig. 11).These vesicles may arise by degranulation of rough endoplasmic reticulum (Morre et al., 1970), possibly accompanied by insertion of new protein, or by lateral diffusion of membrane protein. An alternative mechanism may involve the growth of membrane from a restricted area of the rough endoplasmic reticulum (Claude, 1970), in which case the individual membrane components may be synthesized and assembled at this site. The transitional elements (smooth-surfaced vesicles) present between the rough endoplasmic reticulum and Golgi complex appear to have distinctive properties. Beadlike structures have been identified at the base of the transition vesicles by staining with bismuth. These structures, which are 10-12 nm in diameter and are probably protein in nature (Locke and Huie, 1976), are exclusive to this intracellular site and may function in membrane biogenesis. 1. THEROLE OF THE GOLGICOMPLEX

The Golgi complex has been assigned a role in the differentiation and distribution of membrane as well as of secretory products. While some phospholipid assembly occurs in the Golgi complex (Chang et al., 1977), it is dependent on the rough endoplasmic reticulum for its protein. Following glycosylation, secretory glycoproteins are transferred from the Golgi complex to the cell surface primarily by the release of secretory vesicles and their fusion with the plasma membrane (Jamieson and Palade, 1971); the same vesicles probably also carry out the transport of membrane glycoproteins. Membrane fusion is followed by restricted transfer and/or recycling of membrane components (Fig. 1).Alternatively, membrane glycoproteins may be transferred to the plasma membrane (or to intracellular membranes) in


71

individual cytoplasmic glycoprotein-lipid complexes which are wholly assimilated into the membrane bilayer. The Golgi complex is highly developed in secretory cells, where it is involved in the biosynthesis and secretion of macromolecules. In monolayer cultures, where cell surfaces are very extensive, a welldeveloped Golgi complex is observed even when secretory activity is low (Fig. 12).In hepatocyte cultures (Odashimaet al., 1976),the Golgi complex shows particularly elaborate networks of tubules (Fig. 13)far more extensive than those observed in isolated hepatocytes. Although these cells continue to secrete plasma glycoproteins in culture, the levels are low, indicating a possible role of the Golgi complex in the differentiation of membrane destined for the cell surface. At the cell surface, the carbohydrate portion of the glycoprotein molecule is exposed at the outer surface of the plasma membrane. In contrast, the carbohydrate portions of glycoproteins membrane-bound in the Golgi complex are predominantly situated at the intracisternal surface of the membrane. Palade and his co-workers demonstrated that, although enzymically and chemically Golgi membranes showed distinct differences from plasma membranes, there was a gradation of

FIG. 12. Transmission electron micrograph of human skin fibroblast showing characteristic cisternae (C) of the Golgi complex and distinctive tubular network (arrows). Secretory vesicles and vacuoles appear to arise from this network and from the segregation of products within the cistemae. x 19,OOO.

72


FIG.13. Transmission electron micrograph of rat hepatocyte in monolayer culture showing elaborate Golgi complex (arrows) characteristic of these rapidly proliferating cells. The Golgi complex exhibits a large membrane surface with numerous cisternae (C) and elaborate tubular networks (arrows). In these cells which secrete serum glycoproteins in small amounts, the extensive Golgi network suggests a direct relationship between the Golgi complex and the extensive cell surfaces. x 3000.

composition. For example, 5'-nucleotidase is concentrated at both the external surface of the liver plasma membrane and at the inner surface of the Golgi secretory vesicle (Farquhar et al., 1974). Similarly, insulin-binding glycoprotein (Bergeron et al., 1973) and polypeptide hormone receptors (Bergeron and Posner, 1975) are present in rat liver Golgi membranes and plasma membranes, a relationship presumed to be biogenetic rather than functional. Current hypotheses indicate that vesicles arising from the Golgi complex are transported to the cell surface, fuse with the plasma membrane, and thus transfer structural proteins into the cell surface. Carbohydrate residues originally intracisternal in the Golgi complex would then be externalized (Fig. 1). With the continuous contribution of membrane components to the cell surface, a balancing mechanism must exist to maintain its normal area. Selected proteins and glycoproteins may congregate by lateral diffusion followed by internalization of membrane vesicles by endocytosis; or internalization of individual proteins may occur in a lipid


73

complex. Degradation and shedding of membrane glycoproteins on the external surface of the cell is also a feature of this mechanism. 2. “MEMBRANEFLOW”AND “MEMBRANE SHUTTLE”

In secretory systems, exportable proteins are transported through the cell along a discontinuous pathway, including a number of membrane-bound compartments (rough endoplasmic reticulum, Golgi complex, secretion granules). The products are segregated within granules and discharged into the extracellular space following fusion of the granule membrane with the plasma membrane. There may be no permanent connection of the membrane compartments, but there remains a functional connection via a fusion-fission system, with specific or exclusive membrane interactions. These interactions are controlled, since the peculiar structure and composition of the different membrane systems are preserved throughout the process. This fact of chemically discrete intracellular membrane compartments is important in understanding the two main hypotheses proposed to explain the synthesis and circulation of membrane in the cell-the membrane flow hypothesis and the membrane shuttle hypothesis. In the membrane flow model (Morre et aZ., 1974), sequential and unidirectional flow of membranes and their contents from the nuclear envelope to the cell surface is coupled with irreversible membrane differentiation in both membrane composition and organization. This model indicates that secretions and membrane move together. In invertebrate cells, morphological studies suggest that the membrane flow hypothesis may be an adequate explanation of membrane biogenesis (Morr6 et aZ., 1974). However, in vertebrate cells, the system is more complex, and the membrane flow hypothesis does not account for certain membrane characteristics. First, the turnover of microsoma1 and plasma membrane components is slower than that of intravesicular materials; e.g., in the rat pancreas, the turnover of secretory protein ranges between 10 and 20 hours, whereas the half-life of specific membrane proteins varies from 3 to 28 days (Meldolesi, 1974a,b). Second, the membrane flow hypothesis does not account for the heterogeneity of turnover rates among specific membrane proteins. The rates of synthesis and assembly of membrane proteins vary and can be selectively modified by pharmacological agents (Schimke, 1974). For example, phenobarbital and 3-methylcholanthrene both cause the synthesis of selected endoplasmic reticulum components; each therefore results in a different pattern of induction of membrane protein. The induction of enzymes in the endoplasmic reticulum is associated

74


with changes in membrane morphology and proliferation (Conney, 1967; Schimke, 1973). Thus the transport of secretory proteins and membrane biogenesis are regulated on different time scales. I n contrast, membrane flow implies simultaneous movement, relocation, and degradation of entire membrane domains. However, membrane flow may occur to a limited extent, provided that it is slower than the flow of secretion and that it is coupled with extensive modification of the transported membrane in its new compartment. The shuttle hypothesis provides an alternative mechanism for membrane biogenesis in which the intracellular transport of membrane components occurs by a process of fusion, fission, and recycling of membranes. In this process the identity of the membrane types is preserved. It is not clear whether complete membrane patches or specific macromolecules migrate from one compartment to another, since the shuttle vehicles have not been characterized. The theory implies as yet unexplained membrane recognition phenomena during fusion and fission, to regulate the specific interactions of membranes in a precise and controlled manner. 3. NONRANDOMTOPOGRAPHY OF INTEGRAL PROTEINS In formulating a model for membrane biogenesis, allowances must be made for the nonrandom topography of integral membrane proteins and glycoproteins. I n most epithelial cells, the membrane glycoproteins vary in distribution in apical, basal, and lateral plasma membranes. For example, the apical membrane of the pancreas closely resembles zymogen granule membrane and not other regions of the plasma membrane (DeCamilli et al., 1976), and membrane receptors of the hepatocyte are confined to specific regions of the plasma membrane. This variation in distribution of membrane macromolecules at the cell surface indicates that a mechanism must exist to segregate the integral proteins and to restrict their lateral movement or free diffusion (Figs. 14 and 15).The segregation of membrane proteins may be determined by specific recognition phenomena during the fusion of vesicles and plasma membrane such that selected membrane components become integrated into specific regions of the plasma membrane. Barriers to the movement of membrane glycoproteins, may include tight junctions, since the dissociation of junction complexes in epithelial cells is accompanied by lateral diffusion of surface carbohydrates (Pisam and Repoche, 1976). Recent evidence indicates that a similar segregation of membrane


75

FIG. 14. Freeze-fracture replica of plasma membranes from rat liver. The fracture plane traverses the hydrophobic interior of the membrane, exposing the protoplasmic face (PF)which represents the hydrophobic aspect of the inner leaflet, and the exoplasmic face (EF) which represents the hydrophobic aspect of the outer leaflet. Numerous particles, presumably intramembrane proteins, are observed on the PF, and fewer on the EF. The distribution is random except for specialized regions of the membrane, such as gap junctions, where the particles have an ordered distribution. X49,OOO.

components may occur within the Golgi complex (Fig. 16), since there is significant variation in the distribution of membrane-bound enzymes and glycoproteins. In the liver Golgi complex, for example, cytochemical studies indicate that the distribution of enzymes such as 5’-nucleotidase is nonrandom, being restricted to certain classes of secretory vesicles (Farquhar et al., 1974). The specific activity of 5’-nucleotidase in Golgi fractions is intermediate between those of the endoplasmic reticulum and plasma membrane (Bergeron et d.,1975),

76


FIG.15. Electron micrographs showing the distribution of anionic binding sites on the plasma membrane, using cationic fenitin as a marker (arrows). Cationic ferritin binds randomly to the inner cytoplasmic surface of the plasma membrane and to associated material. The close proximity of the ferritin particles to the inner membrane surfaces is observed particularly at sites of tight junctions. x 41,000.

but localization of the enzyme varies within the organelle. Reaction product from the enzyme appears on the inside of the very low density lipoprotein-filled secretory granules and on the outside of the cisternal elements. Functional specialization within the Golgi complex is also reflected in the morphological heterogeneity of this organelle and by the variation in distribution of anionic sites on the membrane surfaces (Abe et al., 1976). Based on the binding of cationic ferritin (Fig. 17), the density of anionic sites (presumably sialic acid residues) is greatest on the tubular network. The cisternae show few anionic sites, but those that are present are asymmetrically distributed with a high density on convex surfaces and a low density on concave surfaces (Fig. 17). The restriction on density of binding sites indicates membrane differentiation within the Golgi complex. The localization of specific sites indicates that, despite the fluidity of membrane components, the integral proteins are not free to diffuse within the plane of the membrane. The distribution of sites for binding the lectin Con A to the Golgi complex also varies, with most binding sites restricted to localized areas of the tubular network (Abe et al., 1977). These observations


77

FIG. 16. Freeze-fracture replica of the Golgi complex showing the hydrophobic aspects of the membranes. The intramembrane particles presumably include the membrane-bound enzymes of the glycosyltransferase system. The cistemae (C) and tubular networks (T) of the Golgi complex show mainly a random distribution of particles, with some linear arrays. The number of particles varies on successive cistemae in the Golgi complex, and they are more numerous on the PF than on the E F face (see Fig. 14). The sites of formation of tubules and vesicles (V) are sites for the contribution of membranes to the cell surface. x 115,000.

provide additional evidence that the segregation of membrane glycoproteins occurs in the hepatocyte Golgi complex and that there may be functional specialization among its component structures (Sturgess et al., 1974).Membrane specialization may not necessarily be accompanied b y the segregation of different secretory macromolecules in different vesicles, since Kraehenbuhl et al. (1977) used immunocytochemical localization of different pancreatic secretory proteins to show that all Golgi vesicles appeared to contain the same complement of secretory proteins. Segregation of components at the cell surface may result from selec-

78


FIG.17. Electron micrograph showing distribution of anionic binding sites on membranes of a Golgi complex isolated from rat liver. Cationic ferritin binds mainly to small vesicles and to the tubular network and, with increasing concentration, binding is observed on convex surfaces of the cisternae (C, arrows). Binding is not observed on the concave surfaces, indicating the polarity of the membrane system within this organelle. X84,OOO.

tive incorporation of membrane components. For instance, the fusion of membranes may involve a specific recognition phenomenon. In Tetrahyrnena the fusion of mucocyst granules with the plasma membrane is preceded by reorganization of intramemhrane particles into specific patterns (Satir et al., 1973).The transfer of membrane protein following fusion may not be random. Evidence for this has been presented by freeze-fracture studies on the pancreas, where fusion of secretory granules with the plasmalemma does not result in a random mixing of membrane components (DeCamilli et al., 1976).


79

4. ROLE OF THE CYTOSKELETON The control of integral membrane protein and glycoprotein distribution and mobility may depend on a cytoskeletal structure, involving linked microfilaments and microtubules (Nicolson, 1976). Evidence has been presented which indicates that microfilaments and microtubules are linked to integral membrane proteins (Poste et al., 1975), that the movement of intramembrane particles, observed by freezefracture, is influenced by the agents cytochalasin and colchicine, and that perturbation of the membrane bilayer with local anesthetics can be attributed to a reversible disruption of membrane-associated microfilaments and microtubules (Nicolson, 1976). Capping phenomena involving lateral movement of membrane proteins often occur at the cell surface over the region of the Golgi complex (De Petris, 1975); this may be related to the process of membrane turnover, with interaction of intracellular and plasma membranes through the cytoskeletal system. High-resolution studies with the scanning electron microscope provide a new approach to examining the topography of membrane surfaces. Recent work with plasma membrane and Golgi fractions isolated from rat liver (Sturgess and Moscarello, 1976) demonstrate the asymmetry of the plasma membrane (Fig. 18). The outer surface is apparently smooth, with occasional arrays of globular units. The inner surface has a reticulate appearance and is associated with numerous fine filaments and smooth-surfaced vesicles (Fig. 19). The Golgi membranes are characterized by stacks of superimposed plates which form a dome-shaped complex with cisternae and tubular networks (Fig. 20). Individual Golgi complexes are interconnected by smooth-surfaced tubules. 5.

KINETICS OF VIRAL GLYCOPROTEIN MIGRATION THROUGH THE HOSTCELL

To establish the concepts of membrane biogenesis outlined above, it would be most desirable to follow the kinetics of migration of specific and well-characterized integral membrane glycoprotein precursors through the endomembrane system of the cell. There are two general approaches to this type of experiment. Radioactive precursors can be administered, and the kinetics of incorporation and subsequent migration of the tracer can be followed either by autoradiography (Section V,A) or by subcellular fractionation and subsequent biochemical analysis. These approaches have previously been used primarily on secretory glycoproteins (Schachter, 1974a,b; Schachter and Roden,

80


FIG.18. Scanning electron micrograph of plasma membrane isolated from rat liver, freeze-dried, and gold-coated. The plasma membrane shows two distinct surfaces: The outer surface (E) appears relatively smooth except for occasional linear arrays of large globular particles (arrows) which may be related to specialized regions of the cell surface; and the inner surface (I) adjacent to the cytoplasm has a rougher, reticulate structure. X37,OOO.

1973). Suitable systems for the study of membrane glycoprotein biosynthesis by these methods are limited, but the incorporation of rhodopsin into retinal rod outer segment membranes or of glycoproteins into viral envelopes offers excellent opportunities for experimentation. As mentioned earlier, enveloped viruses such as VSV obtain their membrane envelopes by budding through the plasma membrane of the infected cell. VSV RNA codes for a single glycoprotein polypeptide; glycosylation of this peptide is carried out by the infected cell’s endomembrane machinery. The VSV glycoprotein (G protein) is incorporated into the infected cell’s plasma membrane and eventually becomes a component of the virus envelope. Atkinson et al. (1976) using radioactive fucose, and Knipe et al. (1977a) using radioactive methionine, followed the kinetics of VSV G-protein synthesis and showed a lag of 20 minutes between the completion of glycosylation and the appearance of glycoprotein at the cell surface; nonglycosyl-


81

FIG. 19. Detail of the inner surface of the plasma membrane showing the reticulate appearance. Long, filamentous structures (arrows)and smooth membranes are observed in association with this surface. x 72,000.

ated viral membrane protein (M protein) appears at the plasma membrane within 5 minutes. The extra time is presumably required for the processing of G protein in the host cell’s endomembrane system, while nonglycosylated M protein is quickly transferred from free ribosomes through the cytosol to the plasma membrane (Knipe et al., 1977b), where it rapidly becomes incorporated into the viral envelope; M protein is believed to be a peripheral membrane protein on the cytoplasmic side of the membrane. The study of enveloped viruses offers yet another advantage, namely, the availability of temperature-sensitive viral mutants defecOne such tive in the synthesis of viral proteins (Knipe et al., 1977~). mutant can make G polypeptide, can incorporate certain sugars into this peptide, but cannot add sialic acid (and possibly other sugars); the defect is in the G-protein peptide sequence, since the presence of normal virus in the infected cells cannot correct the defective synthesis. The partially glycosylated G-protein precursor accumulates in the

82


FIG.20. Scanning electron micrograph of Golgi complexes isolated from rat liver, freeze-dried, and gold-coated. The Golgi complex appears as a stack of platelike structures, each with a central saccule or cisterna (C) continuous with a network of fine tubules (arrows).Individual Golgi complexes appear to be interconnected through long, smooth-surfaced tubules. x 37,500.

rough endoplasmic reticulum (Knipe et al., 1977d). There are at least two interpretations of this interesting observation. The defect in the peptide may prevent proper initial glycosylation such that elongation by the addition of external sugars (Fig. 8) is not possible; this hypothesis requires that defective initial glycosylation prevent movement from the rough endoplasmic reticulum to the Golgi apparatus. A more likely theory is that the primary defect is inability to move to the Golgi apparatus for elongation. The two theories could be distinguished by testing the ability of isolated Golgi apparatus to elongate in vitro G precursor purified from cells infected with mutant virus; such experiments have not been carried out. The inability of abnormal G-protein precursor to migrate to the Golgi apparatus and plasma membrane in a normal endomembrane system seems to argue against the membrane flow hypothesis of membrane biogenesis; obviously passive transport


83

of membrane G protein by membrane movement does not occur in cells infected with mutant virus, and some property of the G protein must be required for it to be transported in the endomembrane system. B. Biogenesis of lntmcellular Membranes

There is considerable evidence that glycoproteins are integral components of intracellular membranes. Analyzing the content of sialic acid in ceIl membranes, Click et (12. (1971) demonstrated high levels in plasma membranes, but low levels were present in nuclei and mitochondria. In fact, a low sialic acid content is characteristic of intracelM a r membranes except for those of lysosomes, where the level approvhes that of the plasma membrane. Intracellular membranes also contain protein-bound mannose, galactose, and glucosamine but lack galactosamine (Bergman and Dallner, 1976). 1. ENDOPLASMIC RETICULUM Glycoproteins of the endoplasmic reticulum are stable membrane components and can be distinguished from secretory glycoproteins. It has been suggested that the protein framework of the endoplasmic reticulum is more rigid than that of the plasma membrane, since it retains its form after lipid extraction, and lipid components can be reintroduced to restore enzyme activity (Trump et al., 1970), suggesting that the membrane organization may vary from that at the cell surface. Current evidence suggests that the final stages of membrane glycoprotein assembly occur primarily in the Golgi complex, so that either the complete glycoprotein or an oligosaccharide chain has to be transported from the Golgi complex to the endoplasmic reticulum. The possibility that some glycosylation may occur in other subcellular membranes has not been eliminated. The transport of membrane glycoproteins between intracellular compartments may be mediated by membrane vesicles which fuse to permit the lateral diffusion of integral proteins. An alternative theory is that an unstable cytoplasmic complex is formed between lipid and membrane glycoprotein to create a hydrophobic environment for the protein component; glycoprotein molecules can thus be incorporated into the endoplasmic reticulum membrane as a lipoprotein complex (Svennson et al., 1976).According to Autuori et al. (1975a,b), cytoplasmic sialoproteins, labeled with [3Hlgluc~~amine and [14Clleucine,can

84


be incorporated into microsomal membrane when incubated in vitro with rough microsomes; they cannot be removed by subsequent washing with detergent. These investigators conclude that membrane glycoproteins are transferred from the Golgi complex via cytoplasmic pools into microsomes.

2. MITOCHONDRIA Mitochondria have some capacity to synthesize and glycosylate proteins. Incorporation of glucose, mannose, and galactose occurs in rat liver and brain mitochondria (Bosmann, 1971) and, when radioactively labeled sugar nucleotides are incubated with intact mitochondria, radioactivity is incorporated into acid-precipitable glycoproteins. Autonomous mitochondria1 protein synthesis assembles a total of 4 glycoproteins out of a complete set of at least 15 proteins and 8 gycoproteins. The glycosylation reactions carried out by mitochondria utilize protein moieties made on mitochondrial ribosomes. In vivo, other glycosylation reactions may be carried out by mitochondria on proteins made elsewhere in the cell. It is possible that the activation of nucleotide sugars can take place in the mitochondrial matrix. The incorporation of sugar into mitochondrial glycoproteins is associated with the inner membrane rather than the outer one. Other evidence has shown that the proteins of the outer membranes are synthesized in the cytoplasm, while mitochondrial protein synthesis is concerned with the components of the inner membrane and the cristae. Inhibitors such as chloramphenicol block the incorporation of amino acids and sugars into mitochondrial gl ycoproteins, whereas cycloheximide has no effect.

3. NUCLEI The origin of the nuclear envelope glycoproteins is unknown. It has been assumed that glycoproteins of the nuclear membrane result from polypeptide synthesis and glycosylation in the endoplasmic reticulum and Golgi complex, respectively, with a subsequent exchange between these compartments. The possibility has been raised, however, that CMP-sialic acid synthesis may occur within the nucleus (Van Dijk et al., 1973). In the membrane flow hypothesis of membrane biogenesis, the nuclear envelope releases vesicles which fuse with rough endoplasmic reticulum. If this is the case, then it would be of interest to confirm whether glycosylation mechanisms are significant in the nucleus and


a5

whether they contribute to the biosynthesis of integral membrane glycoprotein in the nuclear envelope and in the endoplasmic reticulum.

4. SECRETORYVESICLES The membranes of the secretory vesicles originate from segregation of membrane from specialized regions of the Golgi complex. They may have a protein composition different from that of the other smooth membranes of the Golgi complex (Palade, 1975) and similar to that of the plasma membrane (Hodson and Brenchley, 1976). Major glycoproteins of the zymogen granule membranes are similar to those of the apical cell membrane of the pancreatic acinar cell but are absent or present in trace amounts only in lysosomal and mitochondria1 membranes. REFERENCES Abe, H., Moscarello, M. A., and Sturgess, J. M. (1976). The distribution of anionic sites on the surface of the Golgi complex. J. Cell Biol. 71,973-979. Abe, H., Young, M. E. M., Moscarello, M. A., and Sturgess, J. M. (1977). Electron microscopic studies of concanavalin A binding to membranes of the Colgi complex. Cyto-

bios 17, 7-15. Adamany, A. M., and Spiro, R. G. (1975a). Glycoprotein biosynthesis: Studies on thyroid mannosyltransferases. I. Action on glycopeptides and simple glycosides. j .

Biol. Chem. 250,2830-2841. Adamany, A. M., and Spiro, R. G. (1975b). Glycoprotein biosynthesis: Studies on thyroid mannosyltransferases. 11. Characterization of a polyisoprenyl mannosyl phosphate and evaluation of its intermediary role in the glycosylation of exogenous acceptors. J . Biol. Chem. 250,2842-2854. Andrews, P. (1970). Purification of lactose synthetase A protein from human milk and demonstration of its interaction with a-lactalbumin. FEBS Lett. 9,297-300. Arima, T., and Spiro, R. G. (1972). Studies on the carbohydrate units of thyroglobulin: Structure of the carbohydrate units of thyroglobulin. Structure of the mannose Nacetylglucosamine containing unit (unit A) of the human and calf pancreasj. Biol.

Chem. 247, 1836-1848. Arima, T., Spiro, M. J., and Spiro, R. G. (1972). Studies on the carbohydrate units of thyroglobulin: Evaluation of their microheterogeneity in the human and calf proteins. ]. Biol. Chem. 247, 1825-1835. Ashwell, G., and Morel], A. C. (1974). The role of surface carbohydrates in the hepatic recognition and transport of circulating glycoproteins. Adv. Enzymol. Relat. Areus M o l . Biol. 41, 99-128. Atkinson, P. H., Moyer, S. A., and Summers, D. F. (1976).Assembly of vesicular stomatitis virus glycoprotein and matrix protein into Hela cell plasma membranes.J. Mol.

Biol. 102,613-631. Autuori, F., Svensson, H., and Dallner, G. (1975a). Biogenesis of microsomal membrane glycoproteins in rat liver. I. Presence of glycoproteins in microsomes and cytosol. J .

Cell Biol. 67,687-699. Autuori, F., Svensson, H., and Dallner, G. (1975b). Biogenesis of microsomal mem-

86


brane glycoproteins in rat liver. 11. Purification of soluble glycoproteins and their incorporation into microsomal membranes. J . Cell Biol. 67,700-714. Barr, R. M., and DeLuca, L. M. (1974). The in vivo incorporation of mannose, retinol and mevalonic acid into phospholipids of hamster liver. Biochem. Biophys. Res. Commun. 60,355-363. Bartholomew, B. A., Jourdian, G. W., and Roseman, S. (1973). The sialic acids, XV. Transfer of sialic acid to glycoproteins by a sialyltransferase from co1ostrum.J. Biol. Chem. 248,5751-5762. Bates, C. J., and Handschumacher, R. E. (1969). Inactivation and resynthesis of glucosamine-6-phosphate synthetase after treatment with glutamine analogues. Adv. Enzyme Regul. 7, 183-204. Bates, C. J., Adams, W. R., and Handschumacher, R. E. (1966). Control of the formation of UDP-N-acetylhexosamine and glycoprotein synthesis in rat liver,J. Biol. Chem. 241,1705-1712. Baynes, J. W., Hsu, A. G., and Heath, E. C. (1973). The role of mannosyl-phosphoryl-dihydropolyisoprenol in the synthesis of mammalian glycoproteins. J . Biol. Chem. 248,5693-5704. Behrens, N. H. (1974). Polyprenol sugars and glycoprotein synthesis. In “Biology and Chemistry of Eucaryotic Cell Surfaces” (E. Y. C. Lee and E. E. Smith, eds.), pp. 159-180. Academic Press, New York. Behrens, N. H., and Leloir, L. F. (1970). Dolichol monophosphate glucose: An intermediate in glucose transfer in liver. Proc. Natl. Acad. Sci. U.S.A. 66, 153-159. Behrens, N. H., Parodi, A. J., and Leloir, L. L. (1971a). Glucose transfer from dolichol monophosphate glucose: The product formed with endogenous microsomal acceptor. Proc. Natl. Acad. Sci. U.S.A. 68,2857-2860. Behrens, N. H., Parodi, A. J., Leloir, L. F., and Krisman, C. R. (1971b). The role of dolichol monophosphate in sugar transfer. Arch. Biochem. Biophys. 143,375-383. Behrens, N. H., Carminatti, H., Staneloni, R. J., Leloir, L. F., and Cantarella, A. I. (1973). Formation of lipid-bound oligosaccharides containing mannose: Their role in glycoprotein synthesis. Proc. Natl. Acad. Sci. U.S.A. 70,3390-3394. Bekesi, J. G., and Winder, R. J. (1967).The metabolism of plasma glycoproteins: Studies on the incorporation of ~-fucose-l-’~C into tissue and serum in the normal rat.J. Biol. Chem. 242,3837-3879. Benedetti, E. L., and Emmelot, P. (1967). Studies on plasma membranes. IV. Ultrastructurd localisation and content of sialic acid in plasma membranes isolated from rat liver and hepatoma. J . Cell Sci. 2,499-512. Bennett, G., Leblond, C. P., and Haddad, A. (1974). Migration of glycoprotein from the Golgi apparatus to the surface of various cell types as shown by radioautography after labelled fucose injection into rats.]. Cell Biol. 60,258-284. Bergeron, J. J., and Posner, B. I. (1975). Polypeptide hormone receptors in the Golgi apparatus of rat hepatocytes. J . Cell Biol. 67, 59a. Bergeron, J. J., Evans, W. H., and Geschwind, I. I. (1973).Insulin binding to rat liver Golgi fracti0ns.J. Cell Biol. 59, 771-6. Bergeron, J. J., Berridge, M. V., and Evans, W. H. (1975).Biogenesis of plasmalemmal glycoproteins: Intracelluiar site of synthesis of mouse liver plasmalemmal 5’-nucleotidase as determined by the subcellular location of messenger RNA coding for 5‘-nucleotidase. Biochim. Biophys. Acta 407,325-337. Bergman, A., and Dallner, G. (1976). Distribution of protein-bound sugar residues in microsomal subfractions and Golgi membranes. Biochim. Biophys. Acta 433,496508.


87

Bernacki, R. J., and Bosmann, H. B. (1973). Activity of a rat liver glycoprotein sialyltransferase utilizing desialyzed prothrombin as an acceptor in normal and hypothrombinemic animals. Eur. J . Biochem. 33,49-58. Blobel, G., and Dobberstein, B. (1975a).Transfer of proteins across membranes. I. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma. J . Cell Biol. 67,835851. Blobel, G., and Dobberstein, B. (1975b). Transfer of proteins across membranes. 2. Reconstitution of functional rough microsomes from heterologous comp0nents.J. Cell Biol. 67,852-862. Blobel, G., and Sabatini, D. D. (1971). Ribosome-membrane interaction in eukaryotic cells. Biomembranes 2, 193-195. Bocci, V., and Winzler, R. J. (1969). Metabolism of ~ - f u c o s e - l - * and ~ C of fucose glycoproteins in the rat. Am. J . Physiol. 216, 1337-1342. Boggs, J. M., Vail, W. J., and Moscarello, M. A. (1976). Preparation and properties of vesicles of a purified myelin hydrophobic protein and phospholipid: A spin label study. Biochim. Biophys. Acta 448,517-530. Boggs, J. M., Wood, D. D., Moscarello, M. A., and Papahadjopoulos, D. (1977). Lipid phase separation induced by a hydrophobic protein in phosphatidylserine-phosphatidylcholine vesicles. Biochemistry 16,2325-2329. Bok, D., Basinger, S. F., and Hall, M. 0. (1974).Autoradiographic and radiobiochemical studies on the incorporation of [6-3HlGlucosamine into frog rhodopsin. Exp. Eye Res. 18, 225-240. Bosmann, H. B. (1970). Glycoprotein biosynthesis: Purification and properties of glycoprotein N-acetylglucosaminyltransferasesfrom guinea pig liver utilizing endogenous and exogenous acceptors. Eur. J . Biochem. 1 4 , 3 3 4 0 . Bosmann, H. B. (1971). Identification of the products of autonomous mitochondrial protein and glycoprotein synthesis. Nature (London),New Biol. 234,54-56. Brady, G , Papahadjopoulos, D., Moscarello M. A. (1978). In press. Bretscher, M. S. (1973). Membrane structure: Some general principles. Membranes are asymmetric lipid bilayers in which cytoplasmically synthesized proteins are dissolved. Science 181,622-629. Bretscher, M. S., and Raff, M. C. (1975). Mammalian plasma membranes. Nature (London) 2 5 8 , 4 3 4 9 . Brew, K., Vanaman, T. C., and Hill, R. L. (1968). T h e role of a-lactaalbumin and the A protein in lactose synthetase: A unique mechanism for the control of a biological reaction. Proc. Natl. Acad. Sci. U S A . 59, 491-497. Brostoff, S., and Eylar, E. H. (1971). Localization of methylated arginine in the A 1 Protein from myelin. Proc. Natl. Acad. Sci. U.S.A. 68,765. Burgos, J., Hemming, F. W., Pennock, J. F., and Morton, R. A. (1963). Dolichol: A naturally-occurring C,,, isoprenoid alcohol. Biochem. J . 88,470-482. Buscher, H. P., Casals-Stenzel, J., and Schauer, R. (1974).New sialic acids: Identificaacids and N-acety1-O-glycoloylneuraminic tion of N-glycoloyl-0-acetylneuraminic acids by improved methods for detection of N-acyl and 0-acyl groups and by gas-liquid chromatography. Eur. J . Biochem. 50, 71-82. Butterworth, P. H. W., and Hemming, F. W. (1968).Intracellular distribution of the free and esterified forms of dolichol in pig liver. Arch. Biochem. Bioph!/s. 128,503-508. Cabezas, J. A. (1973). The type of naturally occurring sialic acids. Reu. Esp. Fisiol. 29, 307-322. Carafoli, E., and Sottocasa, G. (1974).The Ca++transport system of the mitochondrial

88


membrane and the problem of the Ca++carrier. lnt. Congr. Biochem.,Proc. Honour Britton Chance, 9th. Carlson, D. M. (1968).Structures and immunochemical properties of oligosaccharides isolated from submaxillary mucins. J . Biol. Chem. 243,616-626. Carlson, D. M., David, J., and Rutter, W. J. (1973a). Galactosyltransferase activities in pancreas, liver and gut of developing rat. Arch. Biochem. Biophys. 157,605-612. Carlson, D. M., Jourdian, G. W., and Roseman, S. (197313).The sialic acids. XIV. Synthesis of sialyl-lactose by a sialyltransferase from rat mammary g1and.J. Biol. Chem. 248,5742-5750. Carlson, D. M., McGuire, E. J., Jourdian, G. W., and Roseman, S. (1973~).The sialic acids. XVI. Isolation of a mucin sialyltransferase from sheep submaxillary gland.]. Biol. Chem. 248,5763-5773. Carnegie, P. R. (1971).Amino acid sequence of the encephalitogenic basic protein from human myelin. Biochem. J . 123,5747. Chang, P., Moscarello, M. A., and Sturgess, J. M. (1977). Biosynthesis of phosphatidylerthanolamine by the Golgi complex of rat liver in uitro. Experienta 33, 11361137. Chao, L. P., and Einstein, E. R. (1970). Physical properties of the bovine encephalitogenic protein: Molecular weight and conformation. J . Neurochem. 17, 1121-1 132. Chapman, B. E., and Moore, W. J. (1976).Conformation of myelin basic protein in aqueous solution from nuclear magnetic resonance spectroscopy. Biochem. Biophys. Res. Commun. 73,758-766. Chen, W. W., and Lennarz, W. J. (1976).Participation of a trisaccharide lipid in glycosylation of oviduct membrane glycoprotein. J . Biol. Chem. 251,7802-7809. Chen, W. W., Lennarz, W. J., Tarentino, A. L., and Maley, F. (1975).A lipid-linked oligosaccharide intermediate in glycoprotein synthesis in oviduct: Structural studies on the oligosaccharide chain.J. Biol. Chem. 250,7006-7013. Chester, M. A. (1974). The occurrence of a P-galactosyltransferase in normal human urine. FEBS Lett. 46, 59-62. Claude, A. (1970).Growth and differentiation of cytoplasmic membranes in the course of lipoprotein granule synthesis in the hepatic cell. I. Elaboration of elements of the Golgi complex. J . Cell Biol. 47,745-766. Cleve, H., Hamaguchi, H., and Hutteroth, T. (1972).Preparation and further characterization of the MN glycoprotein of human erythrocyte membranes./. E r p . Med. 136, 1140- 1156. Cockle, S . , Epand, R. M., Boggs, J. M., and Moscarello, M. A. (1978). Circular dichroism studies on lipid-protein complexes of a hydrophobic myelin protein. Biochemistry 17,624-629. Codington, J. F., Linsley, K. B., Jeanloz, R. W., Irimura, T., and Osawa, T. (1975a). Immunochemical and chemical investigations of the structure of glycoprotein fragments obtained from epiglycanin, a glycoprotein at the surface of the TA3-Ha cancer cell. Carbohyilr. Res. 40, 171. Codington, J. F., Cooper, A. G., Brown, M. C., and Jeanloz, R. W. (1975b).Evidence that the major cell surface glycoprotein of the TA3-Ha carcinoma contains the Vicia graminea receptor sites. Biochemistry 14,855-859. Coffey, J. W., Miller, 0. N., and Sellinger, 0. Z. (1964). The metabolism of L-fucose in the rat. J . Biol. Chem. 239,4011-4017. Conney, A. (1967). Pharmacological implications of microsomal enzyme induction. Pharmacol. Reu. 19,317-366. Cowan, N. J.,and Robinson, G. B. (1970). The ribosomal incorporation of hexosamine into glycoprotein in a mouse myeloma. FEBS Lett. 8,6-8.


89

Creswell, P., Turner, M. J., and Strominger, J. L. (1973). Papain solubilized HL-A antigens from cultured human lymphocytes contain two peptide fragments. Proc. Natl. Acad. Sci. U.S.A. 70, 1693-1707. Danon, D. L.,Addstein, V., Marikovsky, Y., and Skutelsky, E. (1972).Use of cationised ferritin as a level of negative charges on cell surfaces.J. Ultrustruct. Res. 38,500-

510. DeCamilli, P., Peluchetti, D., and Meldolesi, J. (1976).Dynamic changes of the hminal plasmalemma in stimulated parotid acinar cells: A freeze fracture study. J . Cell Biol. 70,59-74. DeLuca, L., Maestri, N., Rosso, G., and Wolf, G. (1973).Retinol glycolipids. J. Biol. Chem. 248,641-648. Den, H., Kaufman, B., and Roseman, S. (1970).Properties of some glycosyltransferases in embryonic chicken brain. J. Biol. Chem. 245,6607-6615. D e Petris, S. (1975). Concanavalin A receptors, immunoglobulins, and theta antigen of the lymphocyte surface: Interactions with concanavalin A and with cytoplasmic structures.]. Cell Biol. 65,123-146. Depierre, J. W., and Dallner, G. (1975).Structural aspects of the membrane of the endoplasmic reticulum. Biochim. Biophys. Acta 415,411-472. Dunkley, P. R., and Carnegie, P. R. (1974).Amino acid sequence of the smaller basic protein from rat brain myelin. Biochem. J. 141,243-261. Elhammer, A,, Svensson, H., Autuori, F., and Dallner, G. (1975).Biogenesis of microsoma1 membrane glycoproteins in rat liver. 111. Release of glycoproteins from the Golgi fraction and their transfer to microsomal membranes. J. Cell Biol. 67,715-

724. Ellis, D.B., and Sommar, K. M. (1971).Regulation of ~-glutamine:~-fructose-6-phosphate aminotransferase activity in bovine trachea. Biochim. Biophys. Acta 230,531

-534. Epand, R. M., Moscarello, M. A., Zirenberg, B., and Vail, W. J. (1974).T h e folded conformation of the encephalitogenic protein of the human brain. Biochemistry 13, 1264-1267. Etchison, J. R., and Summers, D. F. (1977).Oligosaccharide structure of the membrane glycoprotein of vesicular stomatitis virus. J. Supramol. Struct., Suppl. 1, 6.New York, N.Y. Evans, P. J., and Hemming, F. W. (1973).The unambiguous characterization of dolichol phosphate mannose as a product of mannosyl transferase in pig liver endoplasmic reticulum. FEBS Lett. 31,335-338. Evans, W. H.,Hood, D. O., and Gurd, J. W. (1973).Purification and properties of a mouse liver plasma membrane glycoprotein hydro1ysing nucleotide pyrophosphate and phosphodiester bonds. Biochem. J . 135,819-826. Eylar, E. H. (1970).Amino acid sequence of the basic protein of the myelin membrane. Proc. Natl. Acad. Sci. U.S.A. 67, 1425. Eylar, E . H. (1972).The structure and immunologic properties of basic proteins of myelin. Ann. N.Y. Acad. Sci. 195,481-491. Eylar, E. H., and Thompson, M. (1969).Allergic encephalomyelitis: The physico-chemical properties of the basic protein encephalitogen from bovine spinal cord. Arch. Biochem. Biophys. 129,468-479. Eylar, E. H., Brostoff, S., Hashim., Caccam, J., and Burnet, P. (1971).T h e basic A, protein of the myelin membrane: The complete amino acid sequence.J. Biol. Chem.

246,5770-5784. Farquhar, M. G., Bergeron, J. J., and Palade, G. E. (1974).Cytochemistry of Golgi fractions prepared from rat liver. J. Cell Biol. 60,8-25.

90


Feingold, D. S. (1972). The role of pyridine nucleotides in interconversions of nucleoside diphosphate sugars. In “Biochemistry of the Glycosidic Linkage” (H. Pontis and R. Piras, eds.), pp. 79-112. Academic Press, New York. Fitzgerald, D. K., Brodbeck, U., Kiyosawa, I., Mawal, R.,Colvin, B., and Ebner, K. E. (1970).a-Lactalbumin and the lactose synthetase reaction.]. Biol. Chem. 245,2103 -2108. Fitzgerald, D. K., McKenzie, L., and Ebner, K. E. (1971).Galactosyl transferase activity in a variety of sources. Biochim. Biophys. Acta 235,425-428. Folch-Pi, J., and Stoffyn, P. J. (1972). Proteolipids from membrane systems. Ann. N.Y. Acad. Sci. 195,86-107. Folch, P. J., Lees, M., and Sloane-Stanley, G. H. (1957). A simple method forthe isolation and purification of total lipids from animal tissues. J . Biol. Chem. 226,497-509. Gagnon, J., Finch, P. R., Wood, D. D., and Moscarello, M. A. (1971).Isolation of a highly purified myelin protein. Biochemistry 10,4756-4763. Gasic, G., and Berwick, L. (1962). Hale stain for acid containing mucins: Adaptation to electron microscopy. J. Cell Biol. 19,223-228. Geren, C. R., Magee, S. C., and Ebner, K. E. (1975). Circular dichroism changes in galactosyltransferase upon substrate binding. Biochemist y 14, 1461-1463. Ghalambor, M. A., Warren, C. D., and Jeanloz, R. W. (1974). Biosynthesis of aPi-2-acetamido-2-deoxy-~glucosylP2-polyisoprenyl pyrophosphate by calf pancreas microsomes. Biochem. Biophys. Res. Commun. 56,407-414. Glick, M. C., Comstock, C. A., Cohen, M. A,, and Warren, L. (1971). Membranes of animal cells. VIII. Distribution of sialic acid hexosamines and sialidase in the L cell. Biochim. Biophys. Acta 233,247-257. Gough, D. P., and Hemming, F. W. (1970).The characterization and stereochemistry of biosynthesis of dolichols in rat liver. Biochem. J. 118, 163-166. Hackenbrock, C. R., and Miller, K. J. (1975). The distribution of anionic sites on the surface of mitochondria1 membranes. J. Cell Biol. 65,615-630. Hagopian, A., and Eylar, E. H. (1968a). Glycoprotein biosynthesis: The basic protein encephalitogen from bovine spinal cord, a receptor for the polypeptidyl: N-acetylgalactosaminyl transferase from bovine submaxillary glands. Arch. Biochem. Biophys. 126,785-794. Hagopian, A., and Eylar, E. H. (1968b). Glycoprotein biosynthesis: Studies on the receptor specificity of the po1ypeptidyl.N-acetylgalactosaminyltransferase from bovine submaxillary glands. Arch. Biochem. Biophys. 128,422-433. Hagopian, A., and Eylar, E. H. (1969a). Glycoprotein biosynthesis: The solubilization of glycosyl transferases from membranes of HeLa cells and bovine submaxillary glycoproteins. Arch. Biochem. Biophys. 129,447-455. Hagopian, A,, and Eylar, E. H. (1969b). Glycoprotein biosynthesis: The purification and characterization of a polypeptide W-acetylgalactosaminyl transferase from bovine submaxillary glands. Arch. Biochem. Biophys. 129,515-524. Hagopian, A., Westall, F. C., Whitehead, J. S., and Eylar, E. H. (1971). Glycosylation of the A1 protein from myelin by a polypeptide N-acetylgalactosaminyl transferase: Identification of the receptor sequence. J . Biol. Chem. 246,2519-2523. Hargrave, P. A. (1977). Molecular properties of rhodopsin. J . Supramol. Struct., Suppl. 1, 3. Heath, E. C., Baynes, J. W., and Hsu, A. F. (1974). The role of mannosyl phosphoryl dihydropolyisoprenol in the synthesis of mammalian glycoproteins. In “Biology and Chemistry of Eucaryotic Cell Surfaces” (E. Y. C. Lee and E. E. Smith, eds.), pp. 181-211. Academic Press, New York.


91

Heller, J. (1968). Structure of visual pigments. I. Purification, molecular weights and composition of bovine visual pigments. Biochemistry 7,2906-2913. Heller, J., and Lawrence, M. A. (1970). Structure of the glycopeptide from bovine visual pigment 500. Biochemistry 9,864-869. Helting, T., and Erbing, B. (1973). Galactosyl transfer in mouse mastocytoma: Purification and properties ofN-acetyllactosamine synthetase. Biochim. Biophys. Acta 293, 94-104. Hemming, F. W. (1973). The formation of sugar derivatives of dolichol phosphate in mammalian systems. Biochem. Soc. Trans. 1, 1029-1033. Henderson, R., and Unwin, P. N. T. (1975). Three-dimensional model of purple membrane obtained by electron microscopy. Nature (London)257,28-32. Herscovics, A., Warren, C. D., Jeanloz, R. W., Wedgwood, J. F., Liu, I. Y., and Strominger, J. L. (1974). Occurrence of a p-D-mannopyranosyl phosphate residue in the polyprenyl mannosyl phosphate formed in calf pancreas microsomes and in human lymphocytes. FEBS Lett. 45,312-317. Herscovics, A., Golovtchenko, A. M., Warren, C. D., Bugge, B., and Jeanloz, R. W. (19774. Mannosyltransferase activity in calf pancreas microsomes. Formation of I4C-labeled lipid-linked oiigosaccharides from GDP-D-[14C]-mannose and panphosphate. J . B i d . Chem. 252, 224creatic dolichyl ~-~-['~C]-mannopyranosyl 234. Herscovics, A., Bugge, B., and Jeanloz, R. W. (19771~).Clucosyltransferase activity in calf pancreas microsomes: Formation of dolichyl D-[14c]-glucosylphosphate and "C-labeled lipid-linked oligosaccharides from U DP-~-[~~C]-glucose.J. Biol. Chem, 252,2271-2277. Hill, H. D., Jr. (1976). Structural characterization of ovine submaxillary mucin. Fed. Proc., Fed. Am. SOC.E x p . Biol. 35, 1443. Hill, H. D., Jr., Reynolds, J. A., and Hill, R. L. (1977a). Purification, composition, molecular weight, and subunit structure of ovine submaxillary mucin.J. Biol. Chem. 252, 3791-3798. Hill, H. D., Jr., Schwyzer, M., Steinman, H. M., and Hill, R. L. (197713).Ovine submaxillary mucin: Primary structure and peptide substrates of UDP-N-acetylgalactosamine:mucin transferase. J . Biol. Chem. 252,3799-3804. Hirano, H., Parkhouse, B., Nicolson, G. L., Lennox, E.S., and Singer, S, J. (1972). Distribution of saccharide residues on membrane fragments from a myeloma-cell homogenate: Its implications for membrane biogenesis. Proc. Natl. Acad. Sci. U S A . 69,2945-2949. Hodson, S., and Brenchley, C . (1976). Similarities of the Golgi apparatus membrane and the plasma membrane in rat liver cel1s.J. Cell Sci. 20, 167-182. Hong, K., and Hubbell, W. L. (1972). Preparation and properties of phospholipid bilayers containing rhodopsin. Proc. Natl. Acad. Sci. U.S.A. 69,2617-2621. Hsu, A. F., Baynes, J. W., and Heath, E. C. (1974).The role ofa dolichol-oligosaccharide as an intermediate in glycoprotein biosynthesis. Proc. Natl. Acad. Sci. U.S.A. 71, 2391-2395. Hudgin, R. L., and Schachter, H. (1971a). Porcine sugar nuc1eotide:glycoprotein glycosyltransferases. I. Blood serum and liver sialyltransferases. Can. J . Biochem. 49, 829-837. Hudgin, R. L., and Schachter, H. (1971b). Porcine sugar nuc1eotide:glycoprotein glycosyltransferases. 11. Blood serum and liver galactosyltransferase. Can. J . Biochem. 49,838-846. Hudgin, R. L., and Schachter, H. (1971~). Porcine sugar nuc1eotide:glycoprotein glyco-

92


syltransferases. 111. Blood serum and liver N-acetylglucosaminyltransferase. Can.J . Biochem. 49,847-852. Hudgin, R. L., and Schachter, H. (1972). Evidence for two CMP-N-acetylneuraminic acid:lactose sialyltransferases in rat, porcine, bovine and human liver. Can.J . Biochem. 50,1024-1028. Hughes, R. C. (1976).“Membrane Glycoproteins, A Review of Structure and Function.” Butterworth, London. Hunt, L. T., and Dayhoff, M. 0. (1970). The occurrence in proteins of the tripeptides Asn-X-Ser and Asn-X-Thr and of bound carbohydrate. Biochem. Biophys. Res. Commun. 39,757-765. Hunt, R. C., and Brown, J. C. (1975). Identification of a high molecular weight transmembrane protein in mouse L cells. J . Mol. Biol. 97,413-422. Hunt, R. C., Gold, E., and Brown, J. C. (1975).Cell cycle dependent exposure of a high molecular weight protein on the surface of mouse L cells. Biochim. Biophys. Actu 413,453-458. Hynes, R. 0. (1976). Cell surface proteins and malignant transformation. Biochim. Biophys. Acta 458, 73-107. Ito, A., and Sato, R. (1969). Proteolytic microdissection of smooth-surfaced vesicles of liver microsomes.J. Cell Biol. 40, 179-189. Ito, S. (1969). Structure and function of the glycocalyx. Fed. Proc., Fed. Am. SOC. Exp. B i d . 28, 12-25. Ito, S., Muramatsu, T., and Kobata, A. (1975). Endo-P-N-acetylglucosaminidasesacting on carbohydrate moieties of glycoproteins: Purification and properties of the two enzymes with different specificities from Clostridium pegringens. Arch. Biochem. Biophys. 171,78-86. Jabbal, I., and Schachter, H. (1971). Pork liver guanosine diphosphate-L-fucose glycoprotein fucosyltransferases. J . Biol. Chem. 246,5154-5161. Jamieson, J. C. (1977). Studies on the site of addition of sialic acid glucosamine to rat a,-acid glycoprotein. Can.J. Biochem. 55,408-414. Jamieson, J. D., and Palade, G. E. (1968). Intracellular transport of secretory proteins in the pancreatic exocrine cell. IV. Metabolic requirements. J . Cell Biol. 39, 589603. Jamieson, J. D., and Palade, G. E. (1971). Synthesis, intracellular transport and discharge of secretory proteins in stimulated pancreatic exocrine ce1ls.J. Cell Biol. 50, 135-158. Jancik, J., and Schauer, R. (1974). Sialic acid-A determinant of the life-time of rabbit erythrocytes. Hoppe-Seyler’s 2. Physiol. Chem. 355,395-400. Jenkins, R. E., and Tanner, M. J. A. (1977). The structure of the major protein of the human erythrocyte membrane. Biochem. J . 161, 139-147. Johnston, I. R., McGuire, E. J., Jourdian, G. W., and Roseman, S. (1966). Incorporation of N-acetyl-D-glucosamine into glycoproteins. J . Biol. Chem. 241, 5735-5737. Johnston, I. R., McGuire, E. J., and Roseman, S. (1973). Sialic acids. XVII. A uridine diphosphate N-acetylg1ucosamine:glycoprotein N-acetylglucosaminyltransferase from goat co1ostrum.J. Biol. Chem. 248,7281-7288. Kamerling, J. P., Vliegenthart. J. F. G.,Versluis, C., and Schauer, R. (1975).Identification of 0-acetylated N-acylneuraminic acids by mass spectrometry. Carbohydr. Res. 41, 7-17. Kawasaki, T., and Ashwell, G. (1976a). Chemical and physical properties of an hepatic membrane protein that specifically binds asialoglycoproteins. J . Biol. Chem. 251, 1296- 1302. Kawasaki, T., and Ashwell, G. (1976b). Carbohydrate stxycture of glycopeptides iso-


93

lated from an hepatic membrane-binding protein specific for asialoglycoproteins.]. Biol. Chem. 251,5292-5299. Kean, E. L. (1970). Nuclear cytidine 5'-monophosphosialic acid synthetase. J. Biol. Chem. 245,2301-2308. Kean, E. L., and Bruner, W. E. (1971). Cytidine 5'-monophosphosialic acid synthetase: Activity and localization in the nuclear fragments of the unfertilized sea urchin egg. Exp. Cell Res. 69,384-392. Khalkhali, Z., and Marshall, R. D. (1975). Glycosylation of rihonuclease A catalysed by rabbit liver extracts. Biochem. 1. 146,299-307. Khatra, B. S., Herries, D. G., and Brew, K. (1974). Some kinetic properties of human milk galactosyl transferase. Eur. J . Biochem. 44, 537-560. Kiely, M. L., McKnight, G. S., and Schimke, R. T. (1976). Studies on the attachment of carbohydrate to ovalbumin nascent chains in hen 0viduct.J. Biol. Chem. 251,54905495. Kies, M. (1965). Chemical studies on an encephalitogenic protein from guinea pig brain. Ann. N.Y. Acad. Sci. 122, 161-170. Kies, M., Thompson, G. E., and Alvord, E. C. (1965).The relationship of myelin proteins to experimental allergic encephalomyelitis. Ann. N.Y. Acad. Sci. 122, 148160. Kikuchi, K., and Tsuiki, S. (1973). Purification and properties of UDP-N-acetylglucosamine 2'-epimerase from rat liver. Biochim. Biophys. Acta 327, 193-206. Kim, Y. S., Perdomo, J., Bella, A., Jr., and Nordberg, J. (1971). Properties of a CMP-Nacetylneuraminic acid:glycoprotein sialyltransferase in human serum and erythrocyte membranes. Biochim. Biophys. Actu 244,505-512. Kim, Y. S., Perdomo, J., and Whitehead, J. S. (1972a). Glycosyltransferases in human blood. I. Galactosyltransferase in human serum and erythrocyte membranes. J . Clin. Invest. 51,2024-2032. Kim, Y. S., Perdomo, J., Whitehead, J. S., and Curtis, K. J. (1972b). Glycosyltransferases in human blood. 11. Study of serum galactosyltransferase andN-acetylgalactosaminyltransferase in patients with liver diseases. J . Clin. Inoest. 51,2033-2039. Kitchen, B. J., and Andrews, P. (1972). The effect of temperature on a reaction catalyzed by lactose synthetase A protein. FEBS Lett. 26,333-335. Kitchen, B. J.,and Andrews, P. (1974). Studies on the thiol group of lactose synthetase A protein from human milk and on the binding of uridine diphosphate galactose to the enzyme. Biochem. J . 141,173-178. Knipe, D. M., Lodish, H. F., and Baltimore, D. (1977a). Localization of two cellular forms of the vesicular stomatitis viral glycoprotein. J. Virol. 21, 1121-1127. Knipe, D. M., Baltimore, D., and Lodish, H. F. (1977h). Separate pathways of maturation of the major structural proteins of vesicular stomatitis virus.1. Virol. 21, 11281139. Analysis of the defects of temperaKnipe, D., Lodish, H. F., and Baltimore, D. (1977~). ture-sensitive mutants of vesicular stomatitis virus: Intracellular degradation of specific viral proteins. J . Virol. 21, 1140-1148. Knipe, D. M., Baltimore, D., and Lodish, H. F. (1977d). Maturation of viral proteins in cells infected with temperature-sensitive mutants of vesicular stomatitis virus. J. Virol. 21, 1149-1158. Kohno, M., and Yamashina, I. (1973). Identification of asparagine as the site of glycosylation in glycoprotein biosynthesis. J. Biochem. (Tokyo)73, 1089-1094, Koide, N., and Muramatsu, T. (1974). Endo-p-N-acetylglucosaminidaseacting on carhohydrate moieties of glycoproteins: Purification and properties of the enzyme from Diplococcus pneumoniae. 1.B i d . Chem. 249,4897-4904.

94


Kornfeld, R. (1967).Studies on L-glutamine D-fructose 6-phosphate amidotransferase. I. Feedback inhibition by UDP-N-acetylglucosamine. J . Biol. Chem. 242,3135. Kornfeld, R., and Kornfeld, S. (1970).The structure of a phytohemagglutinin receptor site from human erythrocytes. J . Biol. Chem. 245,2536-2545. Kornfeld, S., and Kornfeld, R. (1969). Solubilization and partial characterization of a phytohemagglutinin receptor site from human erythrocytes. Proc. Natl. Acad. Sci. U.S.A. 63, 1439-1446. Kornfeld, S., Kornfeld, R., Ndufeld, E. F., and O’Brien, P. J. (1964).The feedback control of sugar nucleotide biosynthesis in liver. Proc. Natl. Acad. Sci. U.S.A.52,371379. Kraehenbuhl, J. P., Racine, L., and Jamieson, J. D. (1977).Immunocytochemical localization of secretory proteins in bovine pancreatic exocrine cells. J . Cell Biol. 72, 406-423. Kraemer, P. M. (1971).Heparan sulfates of cultured cells. I. Membrane-associated and cell sap species in Chinese hamster cells. Biochemistry 10, 1437-1445. Lawford, G. R., and Schachter, H. (1966).Biosynthesis of glycoprotein by liver. The incorporation in oiuo of ‘4C-glucosamine into protein-bound hexosamine and sialic acid of rat liver suhcellular fractions. J . Biol. Chem. 241,5408-5418. Leblond, C. P. (1950).Distribution of periodic acid-reactive carbohydrates in the adult rat. Am. J . Anat. 86, 1-25. Leblond, C. P., and Bennett, G. (1974).Elaboration and turnover of cell goat glycoproteins. In “The Cell Surface in Development” (A. A. Moscona, ed.), p. 29. Wiley, New York. Lehman, E. D., Hudson, B. G., and Ebner, K. E. (1975). Studies on the carbohydrate structure of bovine milk galactosyltransferase. FEBS Lett. 54,65-69. Leloir, L. F., Staneloni, R. J., Carminatti, H., and Behrens, N. H. (1973).The biosynthesis of a N,”-diacetylchitobiose containing lipid by liver microsomes: A probable dolichol pyrophosphate derivative. Biochem. Biophys. Res. Commun. 52, 12851292. Lennarz, W. J. (1975).Lipid linked sugars in glycoprotein synthesis. Science 188,986991. Lennarz, W. J., and Scher, M. G. (1972). Metabolism and function of polyisoprenol sugar intermediates in membrane associated reactions. Biochim. Biophys. Acta 265,417-441. Levy, J. A., Carminatti, H., Cantarella, A. I., Behrens, N. H., Leloir, L. F., and Tabora, E. (1974). Mannose transfer to lipid linked di-N-acetylchitobiose. Biochem. Biophys. Res. Commun. 60, 118-125. Lewis, M. S., Krieg, L. C., and Kirk,W. D. (1974).The molecular weight and detergent binding of bovine rhodopsin. E x p . Eye Res. 18,29-40. Liebes, L. F., Zand, R., and Phillips, W. D. (1975). Solution behaviour, circular dichroism and 220 MHz PMR studies of the bovine myelin basic protein. Biochim. Biophys. Acta 405,27-39. Lloyd, K. O., Kabat, E. A., and Licerio, E. (1968).Immunochemical studies on blood groups. XXXVIII. Structures and activities of oligosaccharides produced by alkaline degradation of blood-group Lewis substance. Proposed structure of the carbohydrate chains of human blood-group A, B, H, Lea, and Leb substances. Biochemistry 7,2976-2990. Locke, M., and Huie, P. (1976).The beads in the Golgi complex-endoplasmic reticulum region. J. Cell Biol. 70,384-394. Lodish, H. F. (1973). Biosynthesis of reticulocyte membrane proteins by membranefree polyribosomes. Proc. Natl. Acad. S c i . U.S.A. 70, 1526-1530.


95

Lucas, J. J., and Waechter, C. J. (1974). Structure of an oligosaccharide-lipid intermediate. Fed. Proc., Fed. Am. SOC. E x p . Biol. 33, 1368. Lucas, J. J., Waechter, C. J., and Lennarz, W. J. (1975). The participation of lipid-linked oligosaccharide in synthesis of membrane g1ycoproteins.J. Biol. Chem. 250,19922002. Luft, J. H. (1971). Ruthenium red and violet. 11. Fine structural localisation in animal tissues. Anat. Rec. 171,369-416. MacBeth, R. A. L., Bekesi, J. C., Sugden, E., and Bice, S. (1965).The metabolism of plasma glycoproteins. I. Studies on the rate of incorporation of g l u c ~ s a m i n e - l - ~ ~ C into protein-bound hexosamine and N-acetylneuraminic acid in the normal rat. J . Biol. Chem. 240,3707-3713. MacLennan, D. H. (1975). Resolution of the calcium transport system of sarcoplasmic reticulum. Can. J . Biochem. 53,251-261. McCarrahan, J. F., and Maley, F. (1962). Hexosamine metabolism. I. The metabolism in uioo and oitro of D-glucosamine-l-Cl4 and N-acetyl-D-ghcosamine-l-Cl4in rat liver. J. Biol. Chem. 237, 2458-2465. McGuire, E. J. (1970). Biosynthesis of submaxillary mucins. In “Blood and Tissue Antigens” (D. Aminoff, ed.), pp. 461-478. Academic Press, New York. McGuire, E. J., and Roseman, S. (1967). Enzymatic synthesis of the protein-hexosamine linkage in sheep submaxillary mucin. J. Biol. Chem. 242,3745-3747. McGuire, E. J.. Jourdian, G. W., Carlson, D. M. and Roseman, S. (1965)J. Biol. Chem. 240, PC 4113. Magee, S. C., and Ebner, K. E. (1974). Inactivation of soluble bovine milk galactosyltransferase (lactose synthetase) by sulhydryl reagents and trypsin. J . Biol. Chem. 249,6992-6998. Maley, F., Tarentino, A. L.,McGarrahan, J. F., and DelCiacco, R. (1968). The metabolism of D-galactosamine and N-acetyl-D-galactosaminein rat liver. Biochem. J . 107, 637 -644. Marchesi, V. T., Furthmayr, H., and Tomita, M. (1976). The red cell membrane. Annu. Reo. Biochem. 45,667-698. Marchesi, V. T., Tillack, T. W., Jackson, R. L., Segrest, J. P., and Scott, R. E. (1972). Chemical characterization and surface orientation of the major glycoprotein of the human erythrocyte membrane. Proc. Natl. Acad. Sci. U S A . 69, 1445-1449. Marshall, R. D. (1972). Glycoproteins. Annu. Rev. Biochem. 41,673-702. Marshall, R. D. (1974). The nature and metabolism of the carbohydrate-peptide linkages of glycoproteins. Biochem. SOC. Symp. 40, 17-26. Mazlen, R. G., Muellenberg, C. G., and OBrien, P. J . (1969). The control of L-glutamine:D-fmctose-6-phosphateamidotransferase in bovine retina. Biochim. Biophys. Acts 171,352-354. Meldolesi, J. (1974a). Membranes and membrane surfaces: Dynamics of cytoplasmic membranes in pancreatic acinar cells. Phil. Trans. R. SOC. London, Ser. B 268,3953. Meldolesi, J. (1974h). Secretory mechanisms in pancreatic acinar cells: Role of the cytoplasmic membranes. Ado. Cytophamacol. 2, 71-85. Merrick, J. M., Schifferle, R., Zadarlik, K., and Milgrom, F. (1977). Isolation and partial characterization of the infectious mononucleosis heterophile antigen from bovine erythrocytes. Fed. Proc., Fed. Am. SOC. E x p . Biol. 36,702. Milstein, C., Brownlee, G . G., Harrison, T. M., and Mathews, M. B. (1972). A possible precursor of immunoglobulin light chains. Nature (London), New Biol. 239, 117120. H proteinMitranic, M., and Moscarello, M. A. (1972). The conversion of r n a n n o ~ e - ~to

96

J. STURGESS,

M. MOSCARELLO, AND H. SCHACHTER

bound sialic acid, fucose, galactose and hexosamines in rat serum. Biochem. Biophys. Res. Commun. 47,74-80. Mitranic, M., Sturgess, J. M., and Moscarello, M. A. (1976).The effect of temperature on the galactosyl- and sialyltransferases and on the ultrastructure of Golgi membranes. Biochim. Biophys. Acta 443, 190-197. Mitranic, M., Boggs, J. M., Sturgess, J. M., and Moscarello, M. A. (1978). The effect of colchicine on galactosyl- and sialyltransferases of rat liver Golgi and N-acetylgalactosaminyltransferase from porcine submaxillary gland. Submitted for publication. Miyagi, T., and Tsuiki, S. (1971). Effect of phosphoglucose isomerase and glucose-6phosphate on UDP-N-acetylglucosamine inhibition of L-glutamine:D-fructose-6phosphate aminotransferase. Biochim. Biophys. Acta 250,51-62. Molnar, J. (1974). A proposed pathway of plasma glycoprotein synthesis in the liver. Colloq. Int. CNRS No. 221, 921-935. Molnar, J., and Sy, D. (1967).Attachment of glucosamine to protein at the ribosomal site of rat liver. Biochemistril 6, 1941-1947. Molnar, J., Chao, H., and Ikehara, Y. (1971). Phosphoryl-N-acetylglucosaminetransfer to a lipid acceptor of liver microsomal preparations. Biochim. Biophys. Acta 239, 401-4 10. Montreuil, J. (1975). Recent data on the structure of the carbohydrate moiety of glycoproteins: Metabolic and biological implications. Pure Appl. Chem. 42,431-477. Mookejea, S . , Chow, A., and Hudgin, R. L. (1971). Occurrence of UDP-N-acetylglucosamine:glycoprotein N-acetylglucosaminyltransferase activity in human and rat sera. Can. J . Biochem. 49,297-299. Mookerjea, S., Michaels, M. A., Hudgin, R. L., Moscarello, M. A., Chow, A., and Schachter, H. (1972).The levels of nucleotide-sugar:glycoproteinsialyl- and N-acetylglucosaminyltransferases in normal and pathological human sera. Can. J . Biochem. 50,738-740. Morawiecki, A. (1964). Dissociation of M- and N-group mucoprotein into subunits in detergent solution. Biochim. Biophys. Acta 83,339-347. MorrB, D. J., Mollenhauer, H. H., and Bracker, C. E. (1970). Origin and continuity of Golgi apparatus. In “Results and Problems in Cell Differentiation. Vol. 2: Origin and Continuity of Cell Organelles” (J. Reinert and H. Ursprung, eds.), pp. 82-126. Springer-Verlag, Berlin and New York. Morre, D. J., Keenan, T. W., and Huang, C. M. (1974).Membrane flow and differentiation: Origin of Golgi apparatus membranes from endoplasmic reticulum. Adu. Cytophamacol. 2, 107-125. Morrison, J. F., and Ebner, K. E. (1971a). Studies on galactosyltransferase: Kinetic investigations with N-acetylglucosamine as the galactosyl group acceptor. J . Biol. Chem. 246,3977-3984. Morrison, J. F., and Ebner, K. E. (1971b). Studies on galactosyltransferase: Kinetic investigations with glucose as the galactosyl group accept0r.J. Biol. Chem. 246,39853991. Morrison, T. G., and Lodish, H. F. (1975). Site of synthesis of membrane and nonmembrane proteins of vesicular stomatitis virus. J . Biol. Chem. 250,6955-6962. Moscarello, M. A. (1976). Chemical and physical properties of myelin proteins. Curr. Top. Membr. Transp. 8, 1-28. Moscarello, M. A., Kashuba, L., and Sturgess, J. M. (1972). The incorporation of l4C-Dgalactose and ’H-D-mannose into Golgi fractions of rat liver and into serum. FEBS Lett. 26,87-91.


97

Moscarello, M. A., Cagnon, J., Wood, D. D., Anthony, J., and Epand, R. M. (1973).Conformational flexibility of a-myelin protein. Biochemistry 12,3402-3406. Moscarello, M. A., Katona, E., Neumann, W., and Epand, R. M. (1974).The ordered structure of the encephalitogenic protein from normal human myelin. B i o p h p . Chem. 2,290-295. Munro, J. R., and Schachter, H. (1973). The presence of two GDP-L-fucose:glycoprotein fucosyltransferases in human serum. Arch. Biochem. Biophys. 156,534-542. Munro, J. R., Narasimhan, S., Wetmore, S., Riordan, J. R., and Schachter, H. (1975).Intracellular localization of GDP-L-fucose:glycoprotein and CMP-sialic acid:apolipoprotein glycosyltransferases in rat and pork livers. Arch. Biochem. Biophys. 169, 269-277. Nakamura, R., and Tsunemitsu, A. (19754.Presence of cytidine 5‘-monophospho sialic acid:glycoprotein sialyltransferase in human saliva. J. Dent. Res. 54, 188. Nakamura, R., and Tsunemitsu, A. (1975b).Presence of guanosine diphosphofucose:glycoprotein fucosyltransferase in human saliva. J. Dent. Res. 54,417. Nakamura, R., Watanabe, T., Yasutake, A., and Tsunemitsu, A. (1974).Presence of urigalactosyltransferase in human dine 5’-diphosphogalactose:N-acetylglucosamine saliva. J. Dent. Res. 53,937. Narasimhan, S., Stanley, P., and Schachter, H. (1977).Control of glycoprotein synthesis: Lectin-resistant mutant containing only one of two distinct N-acetylglucosaminyltransferase activities present in wild type Chinese hamster ovary cells. J. Biol. Chem. 252,3926-3933. Nathenson, S. G., and Cullen, S. E . (1974).Biochemical properties and immunochemical-genetic relationships of mouse €I-2alloantigens. Biochim. Biophys. Acta 344, 1-25. Nathenson, S. G., and Muramatsu, T. (1971).Properties of the carbohydrate portion of mouse H2 alloantigens. In “Glycoproteins of Blood Cells and Plasma” ( G . A. Janiieson and T. J. Greenwalt, eds.), pp. 245-262.Lippincott, Philadelphia, Pennsylvania. The occurrence and propNelson, J. D., Jato-Rodriguez, J. J., and Mookejea, S. (1974). erties of soluble UDP-ga1actose:glycoprotein galactosyltransferase in human amniotic fluid. Can. ]. Biochem. 52,42-50. Neutra, M., and Leblond, C. P. (1966).Radioautographic comparison of the uptake of galactose-H3 and glucose-H3 in the Golgi region of various cells secreting glycoproteins or mucopolysaccharides. J. Cell Biol. 30, 137-150. Nicolson, G.L. (1976).Transmembrane control of the receptors on normal and tumour cells. I. Cytoplasmic influence over cell surface components. Biochim. Biophys. Acta 457,57-108. Nicolson, G. L., and Singer, S . J. (1971).Ferritin conjugated plant agglutinins as specific saccharide stains for electron microscopy: Application to saccharides bound to cell membranes. Proc. Natl. Acad. Sci. U.S.A. 68, 942-945. Novogrodsky, A., and Ashwell, G. (1977).Lymphocyte mitogenesis induced by a mammalian liver protein that specifically binds desialyated glycoproteins. Proc. Natl. Acud. Sci. U.S.A.74, 676-678. Nussbaum, J. L., Rouayreng, J. F., Mandel, P., Jolles, J., and Jolles, P. (1974).Isolation and terminal sequence determination of the major rat brain myelin proteolipid P7 apoprotein. Biochem. Biophys. Res. Commun. 57, 1240-1247. Odashima, S., Sturgess, J. M., and Rothstein, A. (1976).Characteristics of adult rat liver cells in culture. I. Morphological and biochemical studies. Cell Tissue Res. 169, 167.

98


Oesterhelt, D., and Stoeckenius, W. (1971). Rhodopsin-like protein from the purple membrane ofHalobacterium halobium. Nature (London),New Biol. 233,149-152. Okumura, T., Lombart, C., and Jamieson, G. A. (1976). Platelet glycococalicin. 11. Purification and characterization. J . Biol. Chem. 251,5950-5955. Osborne, Beverley, H., Sardet, C., and Helenius, A. (1974). Bovine rhodopsin: Characterization of the complex formed with Triton X-100. Eur. J. Biochem. 44,383-390. Ozols, J. (1972). Cytochrome b, from normal human liver: Isolation and partial amino acid sequence. J . Biol. Chem. 247,2242-2245. Ozols, J. (1974). Cytochrome b, from microsomal membranes of equine, bovine, and porcine livers: Isolation and properties of preparations containing the membranous segment. Biochemistry 13,426-434. Palade, G. E. (1975). Intracellular aspects of the process of protein secretion. Science 189,347-349. Palmer, F. B., and Dawson, R. M. C. (1969).The isolation and properties of experimental allergic encephalitogenic protein. Biochem. J . 111,629-636. Papahadjopoulos, D., Vail, W. J., and Moscarello, M. A. (1975a).Interaction of a purified hydrophobic protein from myelin with phospholipid membranes: Studies on ultrastructure, phase transitions and permeability. J . Mernbr. Biol. 22, 143-164. Papahadjopoulos, D., Moscarello, M., Eylar, E. H., and Isac, T. (1975b). The effects of proteins on thermotropic phase transitions of phospholipid membranes. Biochim. Biophys. Acta 401,317-335. Papermaster, D. S., Converse, C. A., and Sill, J. (1975). Membrane biosynthesis in frog retina: Opsin transport in the photoreceptor cell. Biochemistry 14, 1343-1352. Parodi, A. J., Behrens, N. H., Leloir, L. F., and Carminatti, H. (1972a).The role of polyprenol-bound saccharides as intermediates in glycoprotein synthesis in liver. Proc. Natl. Acad. Sci. U.S.A.69, 3268-3272. Parodi, A. J., Behrens, N. H., Leloir, L. F., and Dankert, M. (1972b). Glucose transfer from dolichol monophosphate glucose: The lipid moiety of the endogenous acceptor. Biochim. Biophys. Acta 270,529-536. Parodi, A. J., Staneloni, R., Cantarella, A. I., Leloir, L. F., Behrens, N. H., Carminatti, H., and Levy, J. A. (1973). Further studies on a glycolipid formed from dolichyl-Dglucosyl monophosphate. Carbohydr. Res. 26,393-400. Paulson, J. C., Beranek, W. E., and Hill, R. L. (1977a).Purification ofa sialyltransferase from bovine colostrum by affinity chromatography on CDP-agarose.J . Biol. Chem. 252,2356-2362. Paulson, J. C., Rearick, J. I., and Hill, R. L. (1977b). Enzymatic propeities of p-D-galactoside a-26 sialyltransferase from bovine co1ostrum.J. Biol. Chem. 252,2363-2371. Pisam, M., and Repoche, P. (1976). Renouvellement des glycoproteins de surface: ktude radioautographique de I'epithelium de la vessie de grenouille. J . Microsc. (Paris) 17,261. Plantner, J. J., and Kean, E. L. (1976).The carbohydrate composition of bovine rhodopsin. J. Biol. Chem. 251, 1548-1552. Pless, D. D. (1976). Oligosaccharide-lipid as substrate for glycosylation of exogenous proteins. Fed. Proc., Fed. Am. SOC.E x p . Biol. 35, 1539. Pless, D. D., and Lennarz, W. J. (1975). A lipid-linked oligosaccharide intermediate in glycoprotein synthesis: Characterization of [Man-14C] glycoproteins labeled from [Man-14C]oligosaccharide-lipid and GDP-["C] Man. J. Biol. Chem. 250, 70147019. Pober, J. S., and Stryer, L. (1975). Light dissociates enzymatically cleaved rhodopsin into two different fragments. J . Mol. Biol. 95,477-481.


99

Poste, G., Papahadjopoulos, D., and Jacobson, K. (1975). Local anesthetics increase susceptibility of untransformed cells to agglutination of concanavalin A. Nature (London) 253,552-554. Powell, J. T., and Brew, K. (1974).The preparation and characterization of two forms of bovine galactosyl transferase. Eur. J. Biochem. 48,217-228. Pricer, W. E., Jr., and Ashwell, G. (1976). Subcellular distribution of a mammalian hepatic binding protein specific for asialoglycoproteins. J. Biol. Chem. 251, 75397544. Prieels, J. P., and Barel, A. 0. (1975).A fluorimetric study of the interactions of insolubilized human a-lactalbumin with galactosyl transferase (A protein) and with anti-alactalbumin antibodies. Biochim. Biophys. Acta 393,496-504. Rambourg, A., and Leblond, C. P. (1967). Electron microscope observations on the carbohydrate-rich cell coat present at the surface of cells in the rat. J. Cell Biol. 32, 27-53. Rambourg, A., Neutra, M. M., and Leblond, C. P. (1966). Presence of a “cell coat” rich in carbohydrate at the surface of cells in the rat. Anat. Rec. 154,41-71. Rambourg, A., Hernandez, W., and Leblond, C. P. (1969). Detection of complex carbohydrates in the Colgi apparatus of rat ce1ls.J. Cell Biol. 40,395-414. Rand, P., Papahadjopoulos, D., and Moscarello, M. A. (1976). On the interaction of lecithins both with a purified hydrophobic protein from myelin and with fatty acids and lysolecithin. Biophys J. 16, 192. Reading, C. L., Penhoet, E. E., and Ballou, C. E. (1977). Carbohydrate structure of vesicular stomatitis virus g1ycoprotein.J. Supramol. Struct., Suppl. 1, 7. Redman, C. M., and Cherian, M. G. (1972).The secretory pathways of rat serum glycoproteins and albumin: Localisation of newly formed proteins within the endoplasmic reticu1um.J. Cell Biol. 52, 231-245. Renthal, R., Steinmemann, A., and Stryer, L. (1973).The carbohydrate moiety of rhodopsin: Lecting binding, chemical modification and fluorescence studies. E x p . Eye Res. 17,511-515. Richards, J. B., and Hemming, F. W. (1972). The transfer of mannose from guanosine diphosphate mannose to dolichol phosphate and protein by pig liver endoplasmic reticulum. Biochem. J. 130,77-93. Richards, J. B., Evans, P. J., and Hemming, F. W. (1971).Dolichol phosphates as acceptors of mannose from guanosine diphosphate mannose in liver systems. Bi0chem.J. 124,957-959. Richards, J. B., Evans, P. J., and Hemming, F. W. (1972).Dolichol phosphate and mannose transferase reactions in animal systems. I n “Biochemistry of the Glycosidic Linkage” (R. Piras and H. G. Pontis, eds.), pp. 207-220. Academic Press, New York. Richmond, J. E. (1965). Role of sugar nucleotides in the incorporation of sugars into glycoproteins. Biochemistry 4, 1834-1838. Robinson, G. B., Molnar, J., and Winzler, R. J. (1964). Biosynthesis of glycoproteins. I. Incorporation of gl~cosamine-’~C into liver and plasma proteins of the rat. J. Biol. Chem. 239, 1134-1141. Roden, L. (1970). Biosynthesis of acidic glycosaminoglycans (mucopolysaccharides). In “Metabolic Conjugation and Metabolic Hydrolysis” (W. H. Fishman, ed.), Vol. 2, pp. 345-442. Academic Press, New York. Rogers, M. J., and Strittmatter, P. (1973).Lipid-protein interactions in the reconstitution of the microsomal reduced nicotinamide adenine dinucleotide-cytochrome bs reductase system. ]. Biol. Chem. 248,800-806.

100


Roseman, S. (1970). The synthesis of complex carbohydrates by multiglycosyltransferase systems and their potential function in intercellular adhesion. Chem. Phys. Lipids 5,270-297. Rothman, J. E., and Lenard, J. (1977). Membrane asymmetry. Science 195,743-753. Rovis, L., Anderson, B., Kabat, E. A., Gruezo, F., and Liao, J. (1973). Immunochemical studies on blood groups: Structures of oligosaccharides produced by base-borohydride degradation of human ovarian cyst blood group H, heb and Lea active glycoproteins. Biochemistry 12,5340-5354. Saari, J. C. (1974). The accessibility of bovine rhodopsin in photoreceptor membranes. J. Cell Biol. 63,480-491. Sarcione, E. J. (1964). The initial subcellular site of incorporation of hexoses into liver protein. J. Biol. Chem. 239, 1686-1689. Satir, B. C., Schooley, C., and Satir, P. (1973). Membrane fusion in a model system. J . Cell Biol. 59, 153-176. Schachter, H. (1974a). The subcellular sites of glycosylation. Biochem. SOC. Symp. 40, 57-71. Schachter, H. (1974b). Glycosylation of glycoproteins during intracellular transport of secretory products. Ado. Cytophannacol. 2,207-218. Schachter, H., and Roden, L. (1973). The biosynthesis of animal glycoproteins. I n “Metabolic Conjugation and Metabolic Hydrolysis” (W. H. Fishman, ed.), Vol. 3, pp. 1149. Academic Press, New York. Schachter, H., Jabbal, I., Hudgin, R. L., Pinteric, L., McGuire, E. J., and Roseman, S. (1970). Intracellular localization of liver sugar nucleotide glycoprotein glycosyltransferases in a Golgi-rich fraction. J . Biol. Chem. 245, 1090-1100. Schachter, H., McGuire, E. J., and Roseman, S. (1971). Sialic acids. XIII. A uridine diphosphate D-ga1actose:mucin galactosyltransferase from porcine submaxillary gland. J. Biol. Chem. 246, 5321-5328. Schanbacher, F. L., and Ebner, K. E. (1970). Galactosyltransferase acceptor specificity of the lactose synthetase A protein. J . Biol. Chem. 245, 5057-5061. Schauer, R. (1970a). Biosynthesis of N-acetyl-0-acetylneuraminicacid I. Incorporation of I4C-acetate into slices of the submaxillary salivary glands of ox and horse. HoppeSeyler’s 2.Physiol. Chem. 351,595-602. Schauer, R. (1970b). Biosynthesis of N-acetyl-0-acetylneuraminicacid. 11. Substrate and intracellular localization of the bovine acetyl-coenzyme AN-acetylneuraminate-7- and 8-0-acetyltransferase. Hoppe-Seyler’s Z. Physiol. Chem. 351,783-791. Schauer, R. (19704. Biosynthesis of N-glycoloylneuraminic acid by an ascorbate or NADPH-dependent of N-acetyl hydroxylating “N-acety1neuraminate:oxygenoxidoreductase in homogenates of porcine submaxillary gland. Hoppe-Seyler’s 2.Physiol. Chem. 351,783-791. Schauer, R. (1973).Chemistry and biology of acylneuraminic acids. Angew. Chem. 85, 128- 140. Schauer, R., and Wember, M. (1970). Studies on the subcellular site of the biosynthesis of N-glycoloylneuraminic acid in porcine submaxillary gland. Hoppe-Seyler’s 2. Physiol. Chem. 351,1353-1358. Schauer, R., and Wember, M. (1971). Hydroxylation and 0-acetylneuraminic acid bound to glycoproteins of isolated subcellular membranes from porcine and bovine submaxillary glands. Hoppe-Seyler’s 2.Physiol. Chem. 352, 1282-1290. Schauer, R., and Wember, M. (1973). Studies on the substrate specificity of acylneuraminate cytidyltransferase and sialyltransferase of submandibular glands from cow, pig and horse. Hoppe-Seyler’s 2.Physiol. Chem. 354, 1405-1414.


101

Schauer, R., Buscher, H. P., and Casals-Stenzel, J. (1974). Sialic acids: Their analysis and enzymic modification in relation to the synthesis of submandibular gland glycoproteins. Biochem. SOC.Symp. 40,87-116. Schechter, I., McKean, D. J., Guyer, R., and Terry, W. (1974). Partial amino acid sequence of the precursor of immunoglobulin light chain programmed by messenger RNA in uitro. Science 188, 160-162. Schimke, R. T. (1973). Control of enzyme levels in mammalian tissues. Adu. Enzymol. Relat. Areas Mol. Biol. 37, 135-187. Schimke, R. T. (1974). The synthesis and degradation of membrane proteins. Adu. Cytopharmacol. 2,63-69. Schwyzer, M., and Hill, R. L. (1977a). Porcine A blood group-specific N-acetylgalactosaminyltransferase. I. Purification from porcine submaxillary glands. J. B i d . Chem. 252,2338-2345. Schwyzer, M., and Hill, R. L. (1977b). Porcine A blood group-specific N-acetylgalactosaminyltransferase. 11. Enzymatic properties. J. B i d . Chem. 252,2346-2355. Sefton, B., and Keegstra, K. (1974). Glycoproteins of Sindbis virus: Preliminary characterization of the oligosaccharides. J. Virol. 14, 522-530. Segrest, J. P., Kahne, I., Jackson, R. L., and Marchesi, V. T. (1973). Major glycoprotein of the human erythrocyte membrane: evidence for an amphipathic molecular structure. Arch. Biochem. Biophys. 155, 167-183. Sherr, C. J., and Uhr, J. W. (1969). Immunoglobulin synthesis and secretion. 111. Incorporation of glucosamine into immunoglobulin on polyribosomes. Proc. Natl. Acad. Sci. U.S.A. 64,381-387. Shetlar, M. R., Capps, J. C., and Hem, D. L. (1964). Incorporation of radioactive glucosamine into the serum proteins of intact rats and rabbits. Biochim. Biuphys. Acta 83,93-101. Shull, K . H., and Miller, 0. N. (1960).Formation in uiuo of glycogen by certain intermediates of the lactate-propranediol pathway. J . B i d . Chem. 235, 551-553. Shur, B. D., and Roth, S. (1975). Cell surface glycosyltransferases. Biochim. Biophys. Acta 415,473-512. Siekevitz, P. (1972). The turnover of proteins and the usage of information. J . Theor. B i d . 37,321-333. Singer, S . J. (1974). The molecular organization of membranes. Annu. Reu. Biochem. 43, 805-833. Singer, S . J., and Nicolson, G. L. (1972). The fluid mosaic model of the structure of cell membranes. Science 175,720-731. Spatz, L., and Strithnatter, P. (1971). A form of cytochrome b, that contains an additional hydrophobic sequence of 40 amino acid residues. Proc. Natl. Acad. Sci. U.S.A. 68, 1042-1046. Spiro, R. G. (1959). Studies on the biosynthesis of glucosamine in the intact rat. J . B i d . Chem. 234,742-748. Spiro, M. J., and Spiro, R. G. (1968a). Glycoprotein biosynthesis: Studies on thyroglobulin. Thyroid sialyltransferase. J. B i d . Chem. 243,6520-6528. Spiro, M. J., and Spiro, R. G. (1968b). Glycoprotein biosynthesis: Studies on thyroglobulin. Thyroid galactosyltransferase. J . B i d . Chem. 243,6529-6537. Spiro, M. J., Spiro, R. G., and Bhoyroo, V. D. (1976a). Lipid-saccharide intermediates in glycoprotein biosynthesis. I. Formation of an oligosaccharide-lipid by thyroid slices and evaluation of its role in protein glycosylation. J. Biol. Chem. 251,64006408.

Spiro, M. J., Spiro, R. G., and Bhoyroo, V. D. (197613).Lipid-saccharide intermediates in

102


glycoprotein biosynthcsis. 111. Comparison of oligosaccharide-lipids formed by slices from several tissues.]. Biol. Chem. 251,6420-6425. Spiro, R. G., Spiro, M. J., and Adamany, A. M. (1974).Enzymic assembly of the carbohydrate units of glycoproteins and the role of lipid intermediates. Biochem. S O C . Symp. 40,37-56. Spiro, R. G., Spiro, M. J., and Bhoyroo, V. D. (1976). Lipid-saccharide intermediates in glycoproteinhiosynthesis. 11. Studies on the structure of an oligosaccharide-lipid from thyroid.]. Biol. Chem. 251,6409-6419. Springer, G . F., and Yang, H. J. (1977). Isolation and partial characterization of blood group M- and N-specific glycopeptides and oligosaccharides from human erythrocytes. lmmunochemistry 14,497-502. Springer, G. F., Nagai, Y., and Tegtmeyer, H. (1966). Isolation and properties of human blood group NN and meconium-Vg antigens. Biochemistry 5,3254-3272. Springer, G . F., Codington, J. F., and Jeanloz, R. W. (1972). Surface glycoprotein from a mouse tumour cell as specific inhibitor of antihuman blood group or agglutinin. J . Natl. Cancer Inst. 49, 1469-1470. Stanley, P., Narasimhan, S., Siminovitch, L., and Schachter, H. (1975). Chinese hamster ovary cells selected for resistance to the cytotoxicity of phytohemagglutinin are deficient in a UDP-N-acetylglucosamine-glycoproteinN-acetylglucosaminyltransferase activity. Proc. Natl. Acad. Sci. U.S.A. 72,3323-3327. Steiner, D. F., Kemmler, W., Tager, H. S., and Rubenstein, A. H. (1974). Molecular events taking place during intracellular transport of exportable proteins: The conversion of peptide hormone precursors. Ado. Cytopharmacol. 2, 195-205. Strittmatter, P. (1971). The characterization of interconversion of two conformational states of cytochrome b, reduct0se.J. Biol. Chem. 246, 1017-1074. Strittmatter, P., Spatz, L., Corcoran, D., Rogers, M. J., Setlow, B., and Redline, R. (1974). Purification and properties of rat liver microsomd stearyl coenzyme A desaturase. Proc. Natl. Acad. Sci. U S A . 71,4565-4569. Strominger, J. L., Higashi, Y., Sandermann, H., Stone, K. J., and Willoughby, E. (1972). The role of polyisoprenyl alcohols in the biosynthesis of the peptidoglycan of bacterial cell walls and other complex polysaccharides. In “Biochemistry of the Glycosidic Linkage” (R. Piras and H. C. Pontis, eds.), pp. 135-154. Academic Press, New York. Struck, D. K., and Lennarz, W. J. (1977). Evidence for the participation of saccharidelipids in the synthesis of the oligosaccharide chain of ovalbumin. J . Biol. Chem. 252, 1007-1013. Struck, D. K., Chen, W. W., and Lennarz, W. J. (1977). Enzymatic incorporation of saccharides from saccharide-lipids into endogenous and exogenous protein acceptors. Fed. Proc., Fed. Am. Soc. E x p . Biol. 36,745. Sturgess, J. M., and Moscarello, M. A. (1976). Surface ultrastructure of the Golgi complex. Scanning Electron Microsc. symp., IITtU/1976 7, Part 2, 146. Sturgess, J. M., Mitranic, M. M., and Moscarello, M. A. (1972).The incorporation of Dglucosamine-3H into the Golgi complex from rat liver and into serum glycoproteins. Biochem. Biophys. Res. Commun. 46, 1270-1277. Sturgess, J. M., Minaker, E., Mitranic, M. M., and Moscarello, M. A. (1973). The incorporation of L-fucose into glycoproteins in the Golgi apparatus of rat liver and in serum. Biochim. Biophys. Acta 320, 123-132. Sturgess, J. M., Moscarello, M. A., and Vail, W. J. (1974). The fine structure of membranes of the Golgi complex. Proc. Int. Congr. Electron Microsc., 8th 11, 194. Svensson, H., Elhammer, A,, Autuori, F., and Dallner, G. (1976).Biogenesis of microsoma1 membrane glycoproteins in rat liver. IV. Characteristics of a cytoplasmic lipo-


103

protein having properties of a membrane precursor. Biochim. Biophys. Actu 455, 383-398.

Takatsuki, A., Kohno, K., and Tamura, G . (1975). Inhibition of hiosynthesis of polyisoprenol sugars in chick embryo microsomes by tuniram-r-i- A-w:- Biol. Chem. 39, 2089-2091.

Tanigari, N., Nakamuro, K., Apella, E., Poulik, M. d.,- - - A :';asman, D. (1973). Identity of the HL-A common portion fragment and human p,-microglobulin. Biochem. Biophys. Res. Commun. 55, 1234-1239. Tarentino, A. L., and Maley, F. (1974). Purification and properties of an endo-@-N-acetylglucosaminidase from Streptomyces griseus. J. Biol. Chem. 249,811-817. Tarentino, A., Plummer, T. H., Jr., and Maley, F. (1970). Studies on the oligosaccharide sequence of ribonuclease B. J. B i d . Chem. 245,4150-4157. Thomas, D. B., and Winzler, R. J. (1969a). Oligosaccharides from M-active sialoglycopeptide of human erythrocytes. Biochem. Biophys. Res. Commun. 35,811-818. Thomas, D. B., and Winzler, R. J. (1969b).Structural studies on human erythrocyte glycoproteins: Alkali-labile oligosaccharides. J. B i d . Chem. 244,5413-5946. Thomas, D. B., and Winzler, R. J. (1971). Structure of glycoproteins of human erythrocytes: Alkali-stable oligosaccharides. Biochem. J. 124, 55-59. Tkacz, J. S., and Herscovics, A. (1975). Ozonolytic cleavage of authentic and pancreatic dolichyl mannopyranosyl phosphates: Determination of sugar configuration in the fragments with a-and p-mannosidases. Biochem. Biophys. Res. Comrnun. 64,1009 -1017.

Tkacz, J . S., and Lampen, J. 0. (1975). Tunicamycin inhibition of polyisoprenyl N-acetylglucosaminyl pyrophosphate formation in calf liver microsomes. Biochem. Biophys. Res. Commun. 65,248-257. Tkacz, J. S., Herscovics, A., Warren, C. D., and Jeanloz, R. W. (1973). Characterization of mannolipids formed in a calf pancreatic microsomal fraction from guanosine diphosphate [I4C]mannose and either endogenous lipid or exogenous polyisoprenyl phosphates. Biochem. SOC. Truns. 1, 1174-1175. Tkacz, J. S., Herscovics, A., Warren, C. D., and Jeanloz, R. W. (1974). Mannosyltransferase activity in calf pancreas microsomes: Formation from guanosine diphosphate-~-['~C mannose ] of a I4C-labeled mannolipid with properties of dolichyl mannopyranosyl phosphate. J. Biol. Chem. 249,6372-6381. Tomita, M., and Marchesi, V. T. (1975). Amino acid sequence and oligosaccharide attachment sites of human erythrocyte glycophorin. Proc. Natl. Acad. Sci. U.S.A. 72, 2964 -2968.

Toneguzzo, F., and Ghosh, H. P. (1975). Cell-free synthesis of vesicular stomatitis virus proteins: Translation of membrane-bound polyribosomal mRNAs. FEBS Lett.50, 369-373.

Trayer, I. P., and Hill, R. L. (1971). The purification and properties of the A protein of lactose synthetase. J. Biol. Chem. 246,6666-6675. Trujillo, J. L., and Can, J. C. (1973). Gycoprotein biosynthesis. VI. Regulation of UDPN-acetyl-D-glucosamine metabolism in bovine thyroid gland slices. Biochim. Biophys. Actu 304,32-41. Trump, B. F., Sultera, S. M., and Byrne, W. L. (1970). Membrane structure: Lipid-protein interactions in microsomal membranes. Proc. Nutl. Acud. Sci. U S A . 66,433440.

Tsay, G. C., Dawson, G., and Sung, S. S. J. (1976). Structure of the accumulating oligosaccharide in fucosidosis. J. Biol. Chem. 251,5852-5859. Tyrell, D. A., Heath, T. D., Colley, C. M., and Ryman, B. E. (1976). New aspects of liposomes. Biochim. Biophys. Actu 457,259-302.

104

’


Vail, W. J,, Papahadjopoulos, D., and Moscarello, M. A. (1974). Interaction of a hydrophobic protein with liposomes: Evidence for particles seen on freeze fracture as being proteins. Biochim. Biophys. Actu 345,463-467. Van den Eijnden, D. H. (1973). Subcellular localization of cytidine 5‘-monophospho-Nacetylneuraminic acid synthetase in calf brain. J . Neurochem. 21,949-958. Van Dijk, W., Ferwerda, W., and Van den Eijnden, D. H. (1973). Subcellular and regional distribution of CMP-N-acetyl neuraminic acid synthetase in calf kidney. Biochim. Biophys. Acta 315,162-175. Visser, L., Robinson, N. C., and Tanford, C. (1975). The two-domain structure of cytochrorne b, in deoxycholate solution. Biochemistry 14, 1194-1199. Waechter, C. J,, and Lennan, W. J. (1976). The role of polyprenol-linked sugars in glycoprotein synthesis. Annu. Rev. Biochem. 45,95-112. Waechter, C. J., Lucas, J. J., and Lennan, W. J. (1973).Membrane glycoproteins. I. Enzymatic synthesis of mannosyl phosphoryl polyisoprenol and its role as a mannosyl donor in glycoprotein synthesis. J . Biol. Chem. 248,7570-7579. Wagner, R. R., and Cynkin, M. A. (1971).Glycoprotein metabolism and UDP-galactose:glycoprotein galactosyltransferase of rat serum. Biochem. Biophys. Res. Commun. 45,57-62. Warren, C. D., and Jeanloz, R. W. (1973a). The characterization of glycolipids derived from long-chain polyprenols: Chemical synthesis of a-Dmannopyranosyl dolichyl phosphate. FEBS Lett. 31,332-334. Warren, C. D., and Jeanloz, R. W. (197313). Chemical synthesis of dolichyl a-Dmannopyranosyl phosphate and citronellyl a-D-mannopyranosyl phosphate. Biochemistry 12,5038-5045. Warren, G. B., Houslay, M. D., Metcalfe, J. C., and Birrall, N. J. M. (1975). Cholesterol is excluded from the phospholipid annulus surrounded by an active calcium transport protein. Nature (London)225,684-687. Warren, L. (1963). The distribution of sialic acids in nature. Comp. Biochem. Physiol. 10, 153-157. Wedgwood, J. F., Warren, C. D., Jeanloz, R. W., and Strominger, J. L. (1974). Enzymatic P*-dolichyl pyrophosphate and its chemical utilization of PI-di-N-acetylchitobiosyl synthesis. Proc. Natl. Acad. Sci. U.S.A. 71, 5022-5026. White, B. N., Shetlar, M. R., Shurley, H. M., and Schilling, J. A. (1965). Incorporation of ~ - [ l - ’ ~ Cgalactosamine l into serum proteins and tissues of the rat. Biochim. Biophys. Actu 101,259-266. Whur, P., Herscovics, A., and Leblond, C. P. (1969).Radioautographic visualization of the incorporation of galactose-3H by rat thyroids in uitro in relation to the stages of thyroglobulin synthesis. J . Cell Biol. 43,289-311. Wilson, J. R., Williams, D., and Schachter, H. (1976). The control of glycoprotein synthesis: N-Acetylglucosamine linkage to a mannose residue as a signal for the attachment of L-fucose to the asparagine-linked N-acetylglucosamine residue of glycopeptide from a,-acid glycoprotein. Biochem. Biophys. Res. Commun. 72,909-916. Winzler, R. J. (1969).A glycoprotein in human erythrocyte membranes. In “Red Cell Membrane” (G. A. Jamieson and T. J. Greenwalt, eds.), pp. 157-171. Lippincott, Philadelphia, Pennsylvania. Winzler, R. J. (1970). Carbohydrates in cell surfaces. Znt. Reu. Cytol. 29,77-125. Winzler, R. J. (1972). Glycoproteins of plasma membranes: Chemistry and function. In “Glycoproteins. Their Composition, Structure and Function,” Part B (A. Gottschalk, ed.), pp. 1268-1293. Elsevier, Amsterdam. Wirth, D. F., Katz, F., Small, B., and Lodish, H. F. (1977).How a single Sindbis virus


105

mRNA directs the synthesis of one soluble protein and two integral membrane glycoproteins. Cell 10,253-263. Wood, D. D., Boggs, J., and Moscarello, M. A. (1978). Labelling of lipophilin in phosphatidylcholine vesicles. In preparation. Worthington, C. R. (1973). X-ray analysis of retinal photoreceptor structure. E x p . Eye Res. 17,487-501.


CURRENT TOPICS IN MEMBRANES AND TRANSPORT. VOLUME

11

Techniques for the Analysis of Membrane Glycoproteins R . L. JULZANO Research Institute The Hospital for Sick Children Toronto, Onturio

I. Introduction. . . . . . . . . . . . . . . . . . 11. Identification of Cell Surface Glycoproteins . . . . . A. Surface Labeling . . . . . . . . . . . . . . B. Membrane Isolation . . . . . . . . . . . . . C. MetabolicLabelingof Membrane Glycoproteins . . . . . 111. Fractionation of Membrane Glycoproteins . . . . . . . . A. Detergents. . . . . . . . . . . . . . . . B. Denaturing Solvents. . . . . . . . . . . . . . C. Fractionation of Membrane Glycoproteins by Column and Polyacrylamide Gel Techniques . . . . . . . . . . D. Lectin Affinity Techniques . . . . . . . . . . . E. Other Approaches to Membrane Glycoprotein Fractionation . IV. Chemical Analysis of Membrane Glycoproteins . . . . . . . A. Preparation of Glycoproteins and Oligosaccharides . . . . B. Analysis of the Polypeptide Portion of Glycoproteins . C. Analysis of Individual Sugars . . . . . . . . . . . D. Determination of the Carbohydrate Structure of Glycoproteins E. MWs of Membrane Glycoproteins . . . . . . . . . V. Genetic Analysis of Membrane Glycoproteins . . . . . . . References . . . . . . . . . . . . . . . . . .

.

.

.

. .

1.

. .

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

. .

. . . .

. . . . . . . . . .

107 108 108

115 116 117 117 119 120 122 128 129 129 130 131 131 132 133 134

INTRODUCTION

This article is intended as a guide to current approaches for the identification, solubilization, fractionation, and chemical analysis of plasma membrane glycoproteins. The material reviewed is confined to studies of glycoproteins emanating from mammalian and avian cells, tumors, and tissues, and does not deal with prokaryotic or lower eukaryotic organisms. Certain highly specialized surface glycoproteins, such as hormone receptors and surface immunoglobulins, have been reviewed elsewhere (Kahn, 1976; Vitetta and Uhr, 1975)and are Copyright @ 19711 by Academic Press. lnc. All rights ofreprodiiction in any form reserved. ISBN 0- 12- 1533I 1-5

107

108

R. 1. JULIANO

not considered here. This article deals primarily with the more recent literature dating from about 1973; for surveys of the earlier literature, the reader is directed to several excellent reviews and treatises on the biochemistry of glycoproteins (Marshall, 1972; Spiro, 197313; Gottschalk, 1972; Cook and Stoddart, 1973; Ginsberg, 1972; Sharon, 1975; Hughes, 1976). Much of the methodology for the analysis of individual sugars and of oligosaccharide sequences is dealt with in the treatises edited by Gottschalk and by Ginsberg in greater detail than is possible here. To reiterate, this article is a guide to current approaches for membrane glycoprotein analysis and reflects the author’s own interests and experience in this area; it is not intended as a comprehensive survey of all techniques which have been applied to the biochemistry of glycoproteins. II. IDENTIFICATION OF CELL SURFACE GLYCOPROTEINS A. Surface Labeling

A nontrivial problem in the analysis of membrane glycoproteins is to discriminate molecules residing on the outer surface of the cell from other glycoprotein species associated with internal elements such as the endoplasmic reticulum, mitochondria, and lysosomes. Since the overall glycoprotein pattern of animal cells is so complex (Wray and Perdue, 1974), there has been widespread use of a very powerful approach to the problem of the cellular localization of proteins and glygoproteins, namely, the employment of surface-labeling reagents. The term “surface label” connotes a type of reagent which reacts covalently with proteins or glycoproteins, but whose size or solubility properties render it unable to enter cells, thus restricting its sites of reaction to the cell periphery. The surface label approach to the analysis of membrane organization has been reviewed previously (Juliano, 1973; Carraway, 1975); nonetheless, it seems appropriate to discuss this technique and its advantages and liabilities within the confines of this article. Basically, two types of surface label reagents are currently in use: (1)low-MW, impermeant molecules which react directly with accessible surface groups, and (2) enzymes which catalyze or promote the transfer of a radiolabel to accessible sites on the cell surface. Carraway (1975) lists seven low-MW membrane-labeling reagents which have been used on animal cells; most of these reagents are bulky anions, moieties known to penetrate cell membranes only very slowly (Roth-

TECHNIQUES FOR THE ANALYSIS OF MEMBRANE GLYCOPROTEINS

109

stein et al., 1976). In most instances these reagents react primarily with exposed amino groups; this is true in the cases or isothiocyanostilbene disulfonic acids (SITS and DIDS), diazotized sulfanilic acid (DASA), formylmethionyl methyl phosphate (FMMP), and trinitrobenzene sulfonate (TNBS), while the photoreactive compound N (4-azido-2-nitrophenyl)-2-amino ethanesulfonate (NAP taurine) has a much broader range of reactivity. Most of these reagents, including DIDS (Cabantchik and Rothstein, 1974), FMMP (Bretscher, 1971), and NAP taurine (Staros and Richards, 1974; Cabantchik et al., 1976), have been used primarily to investigate molecular organization in erythrocytes. Another amino-reactive probe, namely, pyridoxal phosphate-[3H]borohydride, has also been used on enveloped viruses (Rifkin et al., 1972) and on cultured mammalian cells (Juliano and Behar-Bannelier, 1975b; Hunt and Brown, 1974). Some of the problems associated with the use of low-MW probes in studies on complex nucleated cells are considered in the following discussion. 1. LACTOPEROXIDASE

The most widely used surface-labeling reagent is the enzyme lactoperoxidase, which catalyzes the iodination of accessible tyrosine and possibly histidine residues when used in the presence of hydrogen peroxide. The reaction proceeds mainly via an enzyme-bound activated iodine intermediate and requires close contact between the enzyme and its protein substrate (Morrison et d.,1971). The application of the lactoperoxidase technique to the analysis of erythrocyte membrane organization has been reviewed elsewhere (Juliano, 1973; Carraway, 1975). Lactoperoxidase iodination has also been applied to the analysis of surface membrane proteins in lymphoid cells (Marchalonis et al., 1971; Vitetta et al., 1971; Vitetta and Uhr, 1975), normal and transformed fibroblasts (Hynes, 1973; Hogg, 1974; Yamada and Weston, 1974; Teng and Chen, 1975, 1976), platelets (Phillips, 1972; Tanner et al., 1974), adipocytes (Trosper and Levy, 1974), and a variety of cultured and ascitic tumor cells (Shin and Carraway, 1973; Hunt and Brown, 1974; Huang et al., 1973; Mastro et al., 1974; Butters and Hughes, 1975; Juliano and Behar-Bannelier, 1975a,b). In using lactoperoxidase for the analysis of surface glycoproteins in cultured hamster cells (line CHO), we have found it advantageous to employ relatively large amounts of enzyme (40 pg/ml) and radioiodine (0.2-0.5 mCi/ml) and to limit the reaction time to 5 minutes at 37°C or 10-15 minutes at room temperature. This seems to give useful levels of labeling (see Fig. 1)without causing a great deal of cell dam-

R. 1. JULIANO

zoo

100

m

FIG.1. Protein staining patterns and autoradiographs of SDS gels of CHO cell plasma membranes. Membranes from CHO cells labeled with 13'1 by the lactoperoxidase method were run on 5.6% slab gels; the gels were stained for protein and prepared for autoradiography. This approach allows a direct comparison of the staining pattern and labeling pattern. Photographic negatives of the dried and stained gels and of the corresponding autoradiographs were scanned on a densitometer. Corresponding bands are indicated by symbols. (a) Radiograph; (b) coomassie blue stain. (See Juliano and Behar-Bannelier, 1975b.) (Reproduced with the permission of the American Chemical Society.)

age, whereas prolonged exposure to protein-free salt solutions results in a rapid loss of cell viability (Juliano and Behar-Bannelier, 1975a; Juliano, unpublished observations). Lactoperoxidase iodination of cells seems inherently a rather inefficient procedure; typically, about 1%of the total label is incorporated into trichloroacetic acid (TCA)precipitable cell-associated radioactivity (it should be noted that, even after the careful washing of labeled cells, a substantial amount of non-TCA-precipitable cell-associated radioactivity remains). Using the conditions described above, we found a level of labeling in the vicinity of 1 x lo5cpm of lz5Iper milligram of membrane protein to be usual (Juliano and Behar-Bannelier, 1975b), although higher levels


111

have been reported elsewhere (Hynes, 1973; Butters and Hughes, 1975).

2. GALACTOSEOXIDASE The most commonly used approach to labeling the carbohydrate moieties of the cell surface is the galactose o~idase-[~H]borohydride technique (Steck, 1974; Gahmberg and Hakomori, 1973a). In this system, neuraminidase is used to cleave sialic acid from cell surface glycoproteins; this exposes penultimate galactose residues which are then oxidized to aldehydes by the enzyme galactose oxidase. This is followed by reduction with [3H]borohydride, which regenerates hydroxyl groups and introduces a tritium label into surface galactose residues (see Fig. 2). Earlier studies employing galactose oxidase[3H]borohydrideto label erythrocyte membranes suggested that three

FIG.2. An electron microscope autoradiograph of murine leukemia L-1210 cells in which cell surface galactose and N-acetylgalactosamine moieties were isotopically labeled by NaB3H, reduction after neuramindase and galactose oxidase treatment according to Gahmberg and Hakomori (1973a). Note that the preponderance of silver grains is associated with the cell periphery. x 8500. (Courtesy of R. J. Bernacki and C. W. Porter, Department of Experimental Therapeutics, Roswell Park Memorial Institute, Buffalo, New York.)

112

R. 1. JULIANO

to four glycoproteins existed on the cell surface and that they could be detected by Iactoperoxidase labeling as well as by galactose oxidaseinduced labeling (Carraway, 1975). Recently, however, Gahmberg (1976)reported that at least 20 different glycoproteins can be detected on the surface of the human erythrocyte using galactose oxidase-induced labeling coupled with fluorography. The galactose ~xidase-[~H]borohydridelabeling system has also been applied to studies of the surfaces of normal and transformed fibroblasts (Gahmberg and Hakomori, 197313; Critchley, 1974) and of other cultured cells (Hunt and Brown, 1974; Juliano and Behar-Bannelier, 1975a,b). In an interesting recent study, Gahmberg and Hakomori (1975) probed the surface of hamster NIL cells by examining the effect of lectin binding on galactose oxidase-induced labeling. In using galactose oxidase, as in the case of lactoperoxidase, we have found it advantageous to use fairly large amounts of enzyme and isotope and to minimize the period of labeling so as to avoid loss of cell viability (Juliano and Behar-Bannelier, 1975a). A problem peculiar to techniques which employ [3H]borohydride is that this reagent may react with certain proteins without prior enzymic treatment; thus it is necessary to employ careful controls to ensure that it is indeed galactose residues and not other groups on proteins which are being labeled (Gahmberg, 1976). Two interesting variations on the surface labeling of carbohydrate residues have appeared recently. Itaya et al. (1975) used galactose oxidase to generate cell surface aldehyde moieties and then reacted them with [35S]methionine sulfone hydrazide. This approach, which enables one to introduce a very high-specific-activity 35S label into the cell surface, seems worthy of further development. Datta (1974) employed endogenous sialyltransferase activity to catalyze the incorporation of [14C]sialicacid into cell surfaces using [14C]CMP-sialic acid as a donor. Since there seems to be considerable evidence for the existence of cell surface glycosyltransferases in a variety of cells (Porter and Bernacki, 1975), the use of nucleotide sugars for labeling surface carbohydrate moieties may have quite general utility. A potential problem with this approach might be cleavage of the nucleotide sugars by surface glycosidases, followed by uptake and metabolic incorporation of the free sugar by internal cellular components. 3. TRANSGLUTAMINASES

An interesting new approach to cell surface modification is the use of transglutaminase enzymes which catalyze the formation of y-gluta-


113

myl-elysine crosslinks in proteins, or which can b e used to substitute a variety of primary amines onto protein glutamine residues (Dutton and Singer, 1975). Since glutaminases are high-MW entities, the application of these enzymes to intact cells results in cross-linking among surface-exposed proteins and glycoproteins or, in the presence of an excess of a nonpenetrating radioactive or fluorescent amino compound, serves as a surface-labeling system similar to lactoperoxidase. This approach has been most recently applied in studying the intermolecular associations of fibronectin [the large external transformation-sensitive (LETS) protein], a high-MW component of the surface of normal fibroblasts (Keski-Oja et al., 1976). In this case, the blood coagulation factor XIIIa (plasma transglutaminase) was the enzymic reagent employed. 4. IDENTIFICATION OF TRANSMEMBRANE PROTEINS USING SURFACE LABELS Surface labeling with low MW reagents and with lactoperoxidase has been used successfully to demonstrate the transmembrane orientation of certain erythrocyte membrane proteins (reviewed in Carraway, 1975). The general approach has been to label either intact cells or “leaky” membrane vesicles with the surface label reagent, to isolate the component of interest (for example, by cutting the band out of a polyacrylamide gel), and to subject the labeled component to proteolysis and “fingerprint” analyses of its peptides. If the component has a transmembrane orientation, then more peptides should be labeled when the labeling reagent is applied to leaky membranes, where both faces of the membrane are accessible, than when the labeling reagent is applied to intact cells (this is obviously an oversimplification; e.g., see Carraway, 1975). This approach has not been widely applied to cells more complex than erythrocytes and, with one exception, little information is available on the transmembrane orientation of proteins in nucleated animal cells. Hunt and Brown (1975) used lactoperoxidase iodination coupled with proteolytic dissection of “inside-out” membrane vesicles (see Section I1,B) to establish the transmembrane orientation in mouse L cells of a high MW protein which may be similar to fibronectin or the LETS protein.

5. EVALUATION OF SURFACE LABELTECHNIQUES Any surface label study must include an evaluation of the validity of the results. The fact that a particular reagent has been used success-

114

R. L. JULIANO

fully to identify surface macromolecules in one biological system is no guarantee that it will be equally useful in another system. The simplest criterion for deciding whether a particular reagent is truly labeling cell surface components and not labeling internal macromolecules is a comparison of the specific activity of plasma membrane and intracellular proteins after the labeling reaction. In erythrocytes, where it is quite easy to prepare highly purified plasma membranes and where there is an excellent intracellular marker (hemoglobin), the application of this criterion is quite simple. In more complex cell systems where it is more difficult to prepare pure plasma membrane fractions, this aspect of the evaluation is not trivial. Nonetheless, a comparison of the ratio of the activity per milligram of protein in a plasma membrane fraction to that in a cytoplasmic fraction seems to be essential. This ratio should be at least as great as the degree of purification of a known marker enzyme (e.g., Na+,K+-ATPase,or 5’-nucleotidase) in the plasma membrane fraction. One observation which compounds this problem is that some surface label reagents such as lactoperoxidase, for example, seem to prefer to label particulate components rather than soluble components (Juliano and Behar-Bannelier, 1975a). It should be recognized that, if a particular membrane component fails to react with a surface label reagent, this in itself does not constitute evidence that the component is lacking on the outer surface of the cell. In some systems certain outer membrane components are simply unreactive with particular surface labels because of the absence of appropriate reactive moieties or because of shielding phenomena. The best documented example of this is the lack of reactivity of equine erythrocyte glycoprotein with the lactoperoxidase reagent (Carraway et d.,1975). In a similar vein, we observed very substantial differences in the labeling patterns produced by different reagents in cultured hamster cells. Thus, while the major components visualized by lactoperoxidase or by galactose oxidase labeling are above 60,000 apparent MW, the major components visualized by pyridoxal phosphate -[3Hlborohydride labeling are in the 30,000-60,000 MW range (Juliano and Behar-Bannelier, 1975b). It should also be recognized that certain types of reagents may have an unusually high affinity for particular membrane components. Thus, although we originally found SITS and DIDS to be useful surface labels for mammalian cells (Juliano, 1974), we later discovered that in the CHO cell these compounds react primarily with a low-MW entity (

Current Topics in Membranes and Transport. Volume 11: Cell Surface Glycoproteins: Structure, Biosynthesis, and Biological Functions (v. 11)

Current Topics in Membranes and Transport. Volume 11: Cell Surface Glycoproteins: Structure, Biosynthesis, and Biological Functions (v. 11)

Membrane Receptors (Current Topics in Membranes and Transport ; V. 18)

CURR TOPICS IN MEMBRANES & TRANSPORT V15, Volume 15 (Current Topics in Membranes and Transport)

Current Topics in Membranes and Transport, Volume 05

Current Topics in Membranes and Transport, Volume 10

Current Topics in Membranes and Transport, Volume 9

Current Topics in Membranes and Transport, Volume 3

Current Topics in Membranes and Transport, Volume 20

Current Topics in Membranes and Transport, Volume 04

Current Topics in Membranes and Transport, Volume 07

Current Topics in Membranes and Transport, Volume 10

Current Topics in Membranes and Transport, Volume 07

Current Topics in Membranes and Transport, Volume 04

Current Topics in Membranes and Transport, Volume 14

Current Topics in Membranes and Transport, Volume 08

Current Topics in Membranes and Transport, Volume 02

Current Topics in Membranes and Transport, Volume 06

Current Topics in Membranes and Transport, Volume 9

Current Topics in Membranes and Transport, Volume 26 Na+ - H+ Exchange, Intracellular Ph, and Cell Function

Surface volume 2011-11

Current Topics in Membranes & Transport Vol. 25: Regulation of Calcium Transport Across Muscle Membranes (Current Topics in Membranes and Transport) (v. 25)

Cell Biology and Membrane Transport Processes, Volume 41 (Current Topics in Membranes)

Potassium Transport: Physiology and Pathophysiology (Current Topics in Membranes)

Topics in Stereochemistry, Volume 11

Aquaporins (Current Topics in Membranes, Volume 51)

Claudins (Current Topics in Membranes, Volume 65)

Alkaloids: Chemical and Biological Perspectives. Volume 11

Molecular Neurobiology: Endocrine Approaches (Current Topics in Membranes and Transport, V. 31)

ADVANCES IN CATALYSIS VOLUME 11, Volume 11

Current Topics in Membranes and Transport. Volume 11: Cell Surface Glycoproteins: Structure, Biosynthesis, and Biological Functions (v. 11)