Ciba Foundation Symposium 124

FUNCTIONS OF THE

PROTEOGLYCANS

A Wiley - lnterscience Publication

1986 JOHN WlLEY &SONS

Chtchester New York Brisbane Toronto Singapore

-~

FUNCTIONS OF THE

PROTEOGLYCANS

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Ciba Foundation Symposium 124

FUNCTIONS OF THE

PROTEOGLYCANS

A Wiley - lnterscience Publication

1986 JOHN WlLEY &SONS

Chtchester New York Brisbane Toronto Singapore

0 Ciba Foundation 1986

All rights reserved

No part of this book may be reproduced by any means, or transmitted, or translated into a machine language without the written permission of the publisher.

ISBN 0 471 91090 2

Suggested series entry for library catalogues: Ciba Foundation Symposia

Ciba Foundation Symposium 124 x + 299 pages, 64 figures, 23 tables

Library of Congress Cataloging-in-Publication Data: Functions of the proteoglycans

(Ciba Foundation symposium ; 124) Editors: David Evered (organizer) and Julie Whelan. 'Symposium on Functions of the Proteoglycans, held

at the Ciba Foundation, London, 14-16 January 1986'- Contents p.

Includes indexes. 1. Proteoglycans-Ph ysiological effect-Congresses,

I . Evered, David. 11. Whelan, Julie. 111. Symposium on Functions of the Proteoglycans (1986 : Ciba Foundation). IV. Series. [DNLM: 1 Proteoglycans- physiology-congresses. W1 C161F v.124 / QU 55 F979 19861 QP552. P73F86 1986 6 12'. 015754 8 6 1 8886

ISBN 0 471 91090 2

British Library Cataloguing in Publication Data: Functions of the proteoglycans. - Ciba

Foundation symposium ; 124) 1. Proteoglycans I. Series 574.19'245 QP552.P73

ISBN 0 471 91090 2

Printed and bound in Great Britain.

Contents

Symposium on Functions of the Proteoglycans, held at the Ciba Foundation,

The topic for this symposium was proposed by Professor Klaus E. Kuettner London, 1 4 1 6 January 1986

Editors: David Evered (Organizer) and Julie Whelan

V. C. Hascall Introduction 1

T. C. Laurent and J. R. E. Fraser The properties and turnover of hyaluronan 9 Discussion 24

T. E. Hardingham, M. Beardmore-Gray, D. G. Dunham and A. Ratcliffe Cartilage proteoglycans 30 Discussion 39

L. C. Rosenberg, H. U. Choi, A. R. Poole, K. Lewandowska and L. A. Culp Biological roles of dermatan sulphate proteoglycans 47 Discussion 61

D. Heinegird, A. Franzen, E. Hedbom and Y. Sommarin Common 69 structures of the core proteins of interstitial proteoglycans

Discussion 82

H. Kresse, J. Glossl, W. Hoppe, U. Rauch and E. Quentin Biosynthesis and processing of proteodermatan sulphate Discussion 97

89

J. E. Scott Proteoglycan-collagen interactions 104 Discussion 11 7

L.-A. Fransson, 1. Carlstedt, L. Coster and A. Malmstrom The functions of the heparan sulphate proteoglycans 125 Discussion 137

vi CONTENTS

M. Hook, A. Woods, S. Johansson, L. Kjellh and J. R. Couchman Functions of proteoglycans at the cell surface Discussion 157

143

L. A. Culp, J. Laterra, M. W. Lark, R. J. Beyth and S. L. Tobey Heparan sulphate proteoglycan as mediator of some adhesive responses and cytoskeletal reorganization of cells on fibronectin matrices: independent versus cooperative functions 158 Discussion 178

General discussion I An epithelial cell-surface proteoglycan 184

M. Paulsson, S. Fujiwara, M. Dziadek, R. Timpl, G. Pejler, G. Backstrom, U. Lindahl and J. Engel Structure and function of basement membrane proteoglycans 189 Discussion 200

J. R. Hassell, D. M. Noonan, S. R. Ledbetter and G. W. Laurie Biosynthesis and structure of the basement membrane proteoglycan containing heparan sulphate side-chains 204 Discussion 214

General discussion I1 Characterization and immunolocalization of glomerular basement membrane proteoglycans 223

T. N. Wight, M. G. Kinsella, M. W. Lark and S. Potter-Perigo Vascular cell proteoglycans: evidence for metabolic modulation Discussion 253

241

E. Ruoslahti, M. Bourdon and T. Krusius Molecular cloning of proteoglycan core proteins 260 Discussion 266

R. L. Stevens cells 272 Discussion 280

Secretory granule proteoglycans of mast cells and natural killer

V. C. Hascall Chairman’s summing-up 286

Index of contributors 289

Subject index 291

Participants

P. M. Bartold (Ciba Foundation Bursar) Department of Pathology, The University of Adelaide, PO Box 498, GPO, Adelaide 5001, South Australia

M. T. Bayliss The Mathilda and Terence Kennedy Institute of Rheumatology, 6 Bute Gardens, Hammersmith, London W6 7DW, UK

M. Bernfield Department of Pediatrics, School of Medicine, Stanford University, Stanford, California 94305, USA

A. I. Caplan Department of Biology, Case Western Reserve University, Cleveland, Ohio 44106, USA

B. Caterson Department of Biochemistry, School of Medicine, Medical Center, West Virginia University, Morgantown, West Virginia 26506, USA

L. A. Culp School of Medicine, Department of Molecular Biology and Microbiology, Case Western Reserve University, Cleveland, Ohio 44106, USA

M. G. Farquhar Department of Cell Biology, Yale University School of Medicine, Sterling Hall of Medicine, PO Box 3333, New Haven, Connecticut 0651G3002, USA

L.-,&. Fransson Department of Physiological Chemistry, University of Lund, Box 94, S-221 00 Lund, Sweden

J. T. Gallagher Department of Medical Oncology, University of Manchester, Cancer Research Campaign, Christie Hospital & Holt Radium Institute, Wilmslow Road, Manchester M20 9BX, UK

T. E. Hardingham Division of Biochemistry, The Mathilda and Terence Kennedy Institute of Rheumatology, 6 Bute Gardens, Hammersmith, London W6 7DW, UK

vii

PARTICIPANTS ...

Vlll

V. C. Hascall (Chairman) National Institute of Dental Research, National Institutes of Health, Building 30, Room 106, Bethesda, Maryland 20892, USA

J. R. Hassell Laboratory of Developmental Biology and Anomalies, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20892, USA

D. Heinegird Department of Physiological Chemistry, University of Lund, PO Box 94, S-22100 Lund, Sweden

M. Hook Diabetes Research & Training Center/Atherosclerosis Research Unit, The University of Alabama in Birmingham, University Station, Birmingham, Alabama 35294, USA

H. Kresse Physiologisch-Chemisches Institut, Westfalische Wilhelms- Universitat, Waldeyerstrasse 15, D-4400 Munster, Federal Republic of Germany

K. E. Kuettner Department of Biochemistry, Rush-Presbyterian-St Luke's Medical Center, 1753 West Congress Parkway, Chicago, Illinois 60612, USA

T. C. Laurent University of Uppsala, Department of Medical & Physiological Chemistry, Biomedicinska Centrum, Box 575, S-75123 Uppsala, Sweden

R. M. Mason Department of Biochemistry, Charing Cross & Westminster Medical School, Fulham Palace Road, London W6 8RF, UK

M. Paulsson' Department of Connective Tissue Research, Max-Planck- Institut fur Biochemie, Am Klopferspitz Ma, D-8033 Martinsried bei Munchen, Federal Republic of Germany

C. H. Pearson Department of Oral Biology, 6076 Dentistry/Pharmacy Center, University of Alberta, Edmonton, Canada T6G 2NS

A. R. Poole Joint Diseases Laboratory, Shriners Hospital for Crippled Children (Quebec) IRC., 1529 Cedar Avenue, Montreal, Quebec, Canada H3G 1A6

* Present address: Department of Biophysical Chemistry, Biozentrum der Universitat Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland.

PARTICIPANTS ix

L. C. Rosenberg Orthopedic & Connective Tissue Research Laboratories, Montefiore Medical Center, 111 East 210th Street, The Bronx, New York 10467, USA

E. Ruoslahti La Jolla Cancer Research Foundation, 10901 North Torrey Pines Road, La Jolla, California 92037, USA

A,-M. Saamanen (Ciba Foundation Bursar) Department of Anatomy, University of Kuopio, PO Box 6, 70211 Kuopio, Finland

J. E. Scott Department of Biochemistry, Chemistry Building, University of Manchester, Manchester MI3 9PL, UK

R. L. Stevens Department of Rheumatology & Immunology, Harvard Medical School, Brigham and Women’s Hospital, The Seeley G. Mudd Building, Room 628,250 Longwood Avenue, Boston, Massachusetts 02115, USA

S. Suzuki Faculty of Science, Department of Chemistry, Nagoya University, Chikusa, Nagoya, 464, Japan

J. D. Termine Bone Research Branch, National Institute of Dental Research, National Institutes of Health, Building 30, Room 106, Bethesda, Maryland 20892, USA

T. N. Wight Department of Pathology SM-30, University of Washington School of Medicine, Seattle, Washington 98195, USA

Introduction

VINCENT C. HASCALL

National institute of Dental Research, National Institutes of Health, Building 30, Room 106, Bethesda, Maryland 20892, USA

1986 Functions of the proteoglycans. Wiley, Chichester (Ciba Foundation Symposium 124) p 1-8

Proteoglycans were first discovered by Shatton and Schubert, who reported in 1954 that chondroitin sulphate isolated from cartilage after extraction with solutions of potassium chloride contained firmly bound, non-collagenous pro- tein. This paper followed more than half a century of work defining the chemistry and properties of the free glycosaminoglycan, chondroitin sulphate, usually isolated from cartilage after alkali treatment or autolysis of the tissue. It also began to dispel the prevailing model for the structure of cartilage proposed earlier by Karl Meyer and his co-workers (Meyer et a1 1937), namely that it consisted of free chondroitin sulphate chains in ionic complex with collagen. These early roots of proteoglycan research were described by Partridge and Davis in a paper entitled ‘The presence in cartilage of a complex containing chondroitin sulphate combined with a non-collagenous protein’, presented in a Ciba Foundation Symposium on ‘Chemistry and Biology of Mucopolysacchar- ides’ in 1958. Typically for these symposia, Dr Partridge’s presentation was followed by a lively discussion which, in this case, began with a question by Professor A. Neuberger concerning some recent unpublished work by Dr Helen Muir, and similar questions on work in Professor Muir’s laboratory will no doubt enliven this symposium. (We are most unfortunate that she is pre- vented by illness from taking part in our meeting.) As I hope my introductory comments will show, it is timely, almost 30 years later, to devote an entire Ciba Foundation Symposium to these fascinating macromolecules. On behalf of all the participants I want to thank the Ciba Foundation for this opportunity to assess some of the current progress at the frontiers of proteoglycan research. I

1

2

C

HASCALL

FIG. 1. Schematic diagram of cartilage proteoglycan aggregate. In this and all the subsequent figures the solid projecting lines represent chondroitiddermatan sulphate chains; the broken lines, heparan sulphate or heparin chains; wiggly lines, keratan sulphate; the solid circles, 0-linked oligosaccharides and, in cartilage, the linkage structure for the keratan sulphate chains; and the forks, N-linked oligosaccharides and, in the cornea, the linkage structure for keratan sulphate. LP, link protein. HA, hyalur- onic acid.

shall now briefly introduce the cast of proteoglycan characters that will enter- tain us throughout this symposium.

Proteoglycans, like those who investigate them, are a rapidly expanding and heterogeneous family. More properly, proteoglycans constitute several fami- lies. While they share the common, diagnostic characteristic of having one or more covalently bound glycosaminoglycan chains, their core proteins, often buried in a morass of complex carbohydrate structures, provide the key for classifying them and for understanding many of their chemical, metabolic and biological properties. It will become clear from the presentations that many distinct gene families are utilized as core proteins by cells and that within each family there is likely to be a complex theme and variation.

The symposium begins with hyaluronic acid, a deceptively simple and wide-

INTRODUCTION 3

spread glycosaminoglycan involved in numerous biological processes, includ- ing a critical role in the aggregation of cartilage proteoglycans (Fig. 1). Hyalur- onic acid appears to be the only glycosaminoglycan that is synthesized without a covalent attachment to a core protein. Hence it must be given a separate, though well-deserved status as an honorary proteoglycan, in the context of this symposium (see Professor Laurent’s chapter).

Next, our attention focuses on the major structural proteoglyan from cartil- age: giant and exceptionally complex, this macromolecule is often considered as the proteoglycan. It has the longest history and has revealed its many secrets begrudgingly and only after painstaking efforts by numerous investigators. Its core protein is almost 400000 in molecular weight‘(M,) as a primary translation product, and it is ultimately substituted with up to 10 times this mass in complex carbohydrate structures: chondroitin sulphate chains, keratan sulphate chains, and 0- and N-linked oligosaccharides (Fig. 1). Electron microscopy has re- vealed that the N-terminal portion of the polypeptide contains two globular domains. One of these carries a binding site for hyaluronic acid, a feature that may be diagnostic for the different core proteins which constitute this family. In some cases, as in immature cartilage, these proteoglycans also appear to have a C-terminal globular domain (see Professor Heinegird’s chapter). This is par- ticularly pertinent because antibodies in polyclonal antisera against the core protein which recognize this region have been used successfully in two labor- atories (John Hassell, see discussion p 86, 270; Marvin Tanzer, personal communication) to identify cDNA clones in expression vectors that code for this region. These cDNA ‘toeholds’ cover only a small portion of the polypeptide, and much work needs to be done even to define the sequence of the remaining 3-4 x lo3 amino acids in the protein.

Another family prominently featured in the symposium is that of the small, interstitial proteoglycans which frequently contain dermatan sulphate (Fig. 2). These molecules can be considered as ‘minimal’ proteoglycans because they usually contain only one chain, the minimum number required to define a proteoglycan. They have core proteins of approximately 40000 M , which are substituted with N-linked oligosaccharides and in some cases 0-linked oligo- saccharides. They are found in all connective tissues with fibrillar collagen matrices, probably closely associated with specific sites along the collagen fibrils, as Professor John Scott will tell us in his chapter. Limited amino acid sequence analysis (Pearson et a1 1983, Oldberg et al 1981) and cDNA clones (see John Termine, discussion p 266, and Dr Ruoslahti’s chapter) are now available for core proteins of several members of this family. Therefore, we can expect a rapid expansion of information and a greater understanding of the properties of these proteoglycans in the near future. Another small proteogly- can, perhaps unique, is found in cornea. This proteoglycan is a modified glycoprotein with N-linked oligosaccharides providing the linkage for the keratan sulphate chains (Fig. 2), and it appears to be essential for the mainte-

4 HASCALL

FIG. 2. Schematic diagrams of (left) the dermatan sulphate proteoglycan characteristic of interstitial connective tissues and (right) the keratan sulphate proteoglycan from cornea. The electron micrograph is a section from cornea stained with Cupromeronic blue to identify the proteoglycans in the tissue. (Micrograph courtesy of Professor J. E. Scott.)

nance of the collagen fibril organization of the corneal stroma that is required for transparency (Hassell e t a1 1984). The dye, Cupromeronic blue, in com- bination with selective enzyme degradation, has been used to localize the dermatan sulphate proteoglycan and the keratan sulphate proteoglycan along the collagen fibrils in cornea (see the chapter by Professor Scott).

Cell surface-associated proteoglycans form a current ‘hot’ area of research that is well represented in this symposium. This ensemble of proteoglycans comes in a wide variety of shapes and sizes, with heparan sulphate, chondroi td dermatan sulphate, or both, on a variety of core proteins (Fig. 3 ) . Monoclonal

INTRODUCTION 5

- - -__ - - _ - - ---%- --/ x- -.. ..: ..... ... ..... . . . . . . . . . . . . . . .

FIG. 3. Schematic diagrams of cell-surface proteoglycans characteristic of (from left to right) the chondroitin/dermatan sulphate proteoglycan on melanoma cells and on ovarian granulosa cells; the heparan sulphate proteoglycan on ovarian granulosa cells; the heparan sulphate and chondroitin/dermatan sulphate hybrid proteoglycan on mam- mary epithelial cells; and the heparan sulphate proteoglycan on hepatocytes.

antibodies have identified core protein precursors as biosynthetic products in two cases: one of about M , 240000 which gives rise to a chondroitiddermatan sulphate proteoglycan characteristic of the cell surface of melanoma cells (Bumol & Reisfeld 1982), and one of about M , 55000 which gives rise to a hybrid proteoglycan that contains both heparan sulphate and chondroitinl dermatan sulphate chains, and resides on the surface of mammary epithelial cells, as Dr Bernfield will discuss (p 184). Representative heparan sulphate proteoglycans have core proteins ranging from about M , 250000 for proteogly- cans on the surface of ovarian granulosa cells (M. Yanagishita & V.C. Hascall, unpublished) to about M , 30000 for proteoglycans on the surface of hepato- cytes (Kjellen et al 1981). An unusual heparan sulphate proteoglycan with two disulphide-linked polypeptide chains, each of M , 90000, which appears to be a modified form of the transferrin receptor, is found on skin fibroblasts (see Dr Fransson’s chapter). All these proteoglycans are characterized by having an intercalated region of their core protein which may span the cell membrane and interact with elements of the microfibrillar cytoskeleton of the cell, as Dr Hook and Dr Culp will be discussing in their chapters. Exactly how many core protein families are represented in this large, general group is unknown. I t is already clear that they have a great diversity of biological functions, ranging from the structural organization of cell membranes to involvement in specialized cell- cell contacts (as in neuromuscular synapses; Anderson & Fambrough 1983), to mediating cell-substratum attachment (see Dr Culp’s chapter), as well as possibly providing anti-thrombogenic surfaces on vascular endothelial cells (R.D. Rosenberg, personal communication).

Basement membrane proteoglycans are directly involved in such critical biological processes as the determination of the selective permeability prop- erties of glomerular filtration (Farquhar 1981) and the morphogenesis of

6 HASCALL

FIG. 4. Schematic diagrams of basement membrane proteoglycans typical of (left) the EHS murine basement membrane tumour and (right) the basement membrane of kidney glomeruli. The electron micrograph of the glomerular basement membrane was provided by Dr Gordon Laurie.

lobulated glands (Bernfield et al 1984). As with cell surface proteoglycans, basement membrane proteoglycans have diverse sizes and structures, with core proteins ranging from almost 400000 in M, for the large heparan sulphate proteoglycan isolated from the mouse EHS basement membrane tumour (de- scribed here by Dr Paulsson and by Dr Hassell), to about 20000 for the heparan sulphate proteoglycan in the glomerular basement membrane (Fig. 4); and they can contain different types of glycosaminoglycan chains as well. It is likely that the different core proteins represented in this general group can interact selectively with other basement membrane macromolecules to form the highly organized networks characteristic of these tissues. A close examination of the morphology of the highly specialized glomerular basement membrane (Far- quhar 1981; Fig. 4) reveals the potential complexity that proteoglycans in very small regions might have. Cell-surface proteoglycans as well as basement membrane proteoglycans may be present. Those surfaces on both the base- ment membrane and the endothelial cells that face the capillary lumen have to be anti-thrombogenic, whereas other surfaces do not. Cells on both sides are closely attached to the basement membrane which itself has a highly uniform structure, and ultimately regulates the size and charge properties of the filtered

INTRODUCTION 7

FIG. 5 . Schematic diagrams of the small proteoglycans characteristic of (left) the heparin proteoglycan in storage granules of mast cells, and (r ight) the rat yolk sac tumour. The general structure of the core protein of the latter as deduced from the cDNA is also shown.

molecules. Proteoglycans are quite likely to be involved in all of these proces- ses-antithrombogenic activity, cell attachment, maintenance of basement membrane structure, and selectivity of filtration.

The symposium ends by considering the proteoglycan family with the smal- lest known core proteins having M , values of about 10OOGthat is, only one-fortieth of the size of the cartilage proteoglycan core protein considered at the beginning. Gram for gram (since Britain has gone metric), these ‘macho’ proteoglycans can pack more glycosaminoglycan chains per length of core protein than any other proteoglycan. They are characterized by a long string of serine-glycine repeats (Fig. 5 ) with the hydroxyl groups of serine residues providing points of attachment for the chains. This family is typified by the storage granule proteoglycans found in mast cells (see Dr Stevens’ chapter) and it is represented by a chondroitinldermatan sulphate proteoglycan isolated from rat yolk sac tumour cells for which a cDNA clone covering the coded region of the core protein has been prepared (see Dr Ruoslahti’s chapter). It has now been suggested that this family may include the primary gene product responsible for the biological clock in Drosophila melanogaster, on the basis of a similar, long threonine-glycine repeat sequence in the protein coded by the per gene (Shin et a1 1985). Recent work in the laboratory of Dr Michael Rosbash at Brandeis University has provided evidence that the ultimate biosynthetic product coded for is indeed a proteoglycan (unpublished). The development of this fascinating research problem was described in a recent commentary in Science (Kolata 1985) which included an unorthodox, but reasonable, definition of proteoglycans: ‘to their surprise, they found that similar sequences are in proteoglycans. These are mysterious proteins that are

8 HASCALL

poorly understood’. Perhaps the major purpose of this symposium is to show that some 30 years after their discovery these ‘Rodney Dangerfield’ macro- molecules are beginning to earn more respect and to be better understood; and that the next few years are likely to dispel some, but not all, of their mysteries.

REFERENCES

Anderson MJ, Fambrough DM 1983 Aggregates of acetylcholine receptors are associ- ated with plaques of a basal lamina heparan sulphate proteoglycan on the surface of skeletal muscle fibers. J Cell Biol 97:1396-1411

Bernfield M, Banerjee SD, Koda JE, Rapraeger AC 1984 Remodeling of the basement membrane as a mechanism of morphogenetic tissue interaction. In: Trelstad RL (ed) The role of extracellular matrix in development. Alan R. Liss, New York, p 545-572

Bumol TF, Reisfeld RA 1982 Unique glycoprotein-proteoglycan complex defined by monoclonal antibody on human melanoma cells. Proc Natl Acad Sci USA 79:1245- 1249

Farquhar MG 1981 The glomerular basement membrane: a selective macromolecular filter. In: Hay ED (ed) Cell biology of extracellular matrix. Plenum Publishing, New York, p 335-378

Hassell J , Hascall VC, Ledbetter S, Caterson B, Thonar E, Nakazawa K, Krachmer J 1984 Corneal proteoglycan biosynthesis and macular corneal dystrophy. In: Shef- field JB, Hilfer SR (eds) The cell and developmental biology of the eye, heredity and visual development. Springer-Verlag, New York, p 101-1 14

KjellCn L, Pettersson I , Hook M 1981 Cell-surface heparan sulphate: an intercalated membrane proteoglycan. Proc Natl Acad Sci USA 78:5371-5375

Kolata G 1985 Genes and biological clocks. Science (Wash DC) 230: 1151-1 152 Meyer K, Palmer JW, Smyth EM 1937 On glycoproteins: V. Protein complexes of

chondroitinsulfuric acid. J Biol Chem 119:501-510 Oldberg A, Hayman EG, Ruoslahti E 1981 Isolation of a chondroitin sulfate proteogly-

can from a rat yolk sac tumor and immunochemical demonstration of its surface localization. J Biol Chem 256: 10847-10852

Partridge SM, Davis HF 1958 The presence in cartilage of a complex containing chondroitin sulphate combined with a non-collagenous protein. In: Chemistry and biology of mucopolysaccharides. Churchill, London and Little, Brown, Boston (Ciba Found Symp) p 93-110

Pearson CH, Winterbottom N, Fackre DS, Scott PG, Carpenter MR 1983 The NH,- terminal amino acid sequence of bovine skin proteodermatan sulfate. J Biol Chem 258: 15 101 - 15 104

Shatton J , Schubert M 1954 Isolation of a mucoprotein from cartilage. J Biol Chem

Shin HS, Bargiello TA, Clark BT, Jackson FR, Young MW 1985 An unusual coding sequence from a Drosophifa clock gene is conserved in vertebrates. Nature (Lond) 3 17: 445-448

2111565-573

The properties and turnover of hyalu ronan TORVARD C. LAURENT* and J. ROBERT E. FRASER?

*Department of Medical and Physiological Chemistry, University of Uppsala, Biomedical Center, Box 575, S-75123 Uppsala, Sweden and +Department of Medicine, University of Melbourne, Royal Melbourne Hospital Post Office, Victoria 3050, Australia

Abstract. Hyaluronan (HA) was discovered over 50 years ago but its metabolism and cellular interactions have only recently received detailed attention. HA is synthesized in the plasma membrane by addition of monosaccharides to the reducing terminal. In tissues, it occurs bound to plasma membranes, aggregated with other macromolecules, or as free polysaccharide. Tissue HA enters the bloodstream in significant amounts through the lymph and is rapidly absorbed via a receptor into liver endothelial cells, where degradation follows. HA levels in serum are normally 10-100 pgA, but can be elevated in cirrhosis, rheumatoid arthritis and scleroderma, due either to impaired hepatic uptake or to increased production. Studies on aqueous humour, middle ear secretion, amniotic fluid, lung lavage fluid, urine, skin diseases and cancer have identified other causes of deranged HA metabolism.

HA can be visualized on some cell surfaces as a coating impermeable to particulate material. Specific HA binding occurs on lymphoma cell lines, lung macrophages and SV-3T3 cells but, except in synthesis or uptake, the significance of membrane-associated HA is incompletely understood. It has been reported to activate macrophages and granulocytes, protect cells, control cell migration, and cooperate with intercellular matrix in cell detachment; it also plays a central role in growth control, cellular differentiation and tissue morphogenesis.

1986 Functions of the proteoglycans. Wiley, Chichester (Ciba Foundation Sym- posium 124) p 9-29

Hyaluronic acid, discovered by Meyer and Palmer in 1934, is a linear polysac- charide built from repeating disaccharide units consisting of D-glucuronic acid and N-acetyl-D-glucosamine linked by fi( 1+3) and p( 1+4) glycosidic link- ages, respectively. A t physiological pH the carboxyl groups are completely dissociated and the compound is therefore referred to as hyaluronate. I t was recently proposed at an international symposium in St Tropez (unpublished, 1985) that, in conformity with the nomenclature for polysaccharides, the name should be hyaluronan, if no special reference is made to the acid or salt forms. We will in the following use the abbreviation H A .

HA has not been shown to be covalently linked to protein, and therefore has been referred to as an ‘honorary proteoglycan’ by the organizers of this

9

10 LAURENT & FRASER

symposium. It is usually found in tissues as a polydisperse, high molecular weight expanded coil structure (Laurent 1970). The voluminous structure is presumably due to an inherent stiffness in the chain because of hydrogen bonding between the sugar residues. Even at low concentrations the individual chains entangle and form a continuous network in the solution, which gives the system interesting properties, such as a pronounced viscoelasticity. The rheological properties of HA solutions have been utilized clinically in the recently developed technique of viscosurgery (Balazs 1985).

Early work on the structure and physical-chemical properties of HA (Lau- rent 1970) and possible physiological functions of the polymer, related to the properties of the chain networks, has been reviewed (Comper & Laurent 1978). In this chapter we shall briefly summarize the more recent work on the metabolism of the polysaccharide with special emphasis on studies on the catabolism of HA carried out in our own laboratories. We shall also discuss some recent work on the interaction between HA and cell surfaces.

TABLE 1.

Tissue or fluid Concentration (mgll) Reference

Concentration of hyaluronan in tissues and tissue fluids

H u m a n umbilical cord Rooster comb H u m a n synovial fluid Bovine nasal cartilage H u m a n vitreous body H u m a n dermis Rabbi t muscle Rabbi t brain Rabbi t kidney: renal papillae

Sheep lymph H u m a n thoracic lymph H u m a n aqueous humour Human amniotic fluid: at 16 weeks

Human urine H u m a n serum

renal cortex

a t term

4100 7500

1420-3600 1200

140-338 200 27 65

250 4

0.2-50 8.5-18

1.14 20 1

0.1-0.5 0.01-0.1

References: (1) Meyer FA et a1 1977 Biochem J 161:285-291. (2) Szirmai JA 1966 In: Balazs EA, Jeanloz RW (eds) The amino sugars. Academic Press, New York & London, IIB:129-154. (3) Sundblad L 1965 Ibid. IIA:229-250. (4) Laurent UBG, Tengblad A 1980 Anal Biochem 109:38&394. ( 5 ) Balazs EA 1965 In: The amino sugars. IIA:401-460. (6) Pearce RH, Grimmer BJ 1972 J Invest Dermatol58:347-361. (7) Farber SJ, van Praag D 1970 Biochim Biophys Acta 208:219-226. (8) van Praag D et a1 1972 Biochim Biophys Acta 273:14%156. (9) Laurent UBG, Laurent TC 1981 Biochem Int 2:195-199. (10) Tengblad A et a1 1986 Biochem J , 236:521-525. (11) Laurent UBG 1983 Arch Ophthalmol 101:129-130. (12) Dahl L et a1 1983 Biochem Med 30:28&283. (13) Laurent TC, Lilja K, Brunnberg L, unpublished. (14) Engstrom-Laurent A et a1 1985 Scand J Clin Lab Invest 45:497-504. (15) Delpech B et a1 1985 Anal Biochem 149:555-565.

PROPERTIES AND TURNOVER OF HYALURONAN 11

Detection and distribution of HA

New specific and sensitive methods for quantitative analyses, utilizing proteins with affinity for HA, have been developed (Laurent & Tengblad 1980, Delpech et a1 1985). These have made it possible to analyse small samples of tissues and body fluids. H A has been detected in every tissue and fluid investigated, but in varying amounts (Table 1.) It is notable that the concentra- tion in human serum can now be determined routinely (Engstrom-Laurent et a1 1985a, Deipech et a1 1985). The normal level measured by two independent techniques is in the range of 10-100 pg/l with a mean value in the range of 20-40

There are also new methods of detecting HA in tissue sections and tissue cultures. An early technique (Clarris & Fraser 1968) which has recently been revived (see Table 5 ) is based on the ability of a HA network to exclude particles. If erythrocytes or other non-motile particles are added to a culture of fibroblasts one can distinguish a zone around the cells, which cannot be easily penetrated (Fig. 1). This zone can be abolished by specific hyaluronidases and

FIG. 1. Pericellular gels of synovial fibroblasts outlined by heat-killed Saccharomyces cerevisiae (mean diameter 4.2 pm). A. Three hours after addition of yeast suspension. The irregular width of the investments is due in part to cellular movements and changes in shape within the gel boundaries. B. Same field, 30 minutes after addition of Strepto- myces hyaluronidase. Within 20 minutes, yeast cells began to spread randomly across the cellular zones. Bar = 200 pm.

12 LAURENT & FRASER

thus does contain HA. By time-lapse photography it is possible to demonstrate that the coat can be shed by the fibroblasts when they move in the culture. Other recent methods of demonstrating HA are based on the affinity of fluorescent proteins for HA. Knudson & Toole (1985) have used the core protein of the cartilage proteoglycan as a probe and Girard et a1 (1984) utilize the protein hyaluronectin.

In summary, HA has been discovered in tissues as a coat attached to cell surfaces, as part of larger molecular structures such as in aggregates with cartilage proteoglycans (see other contributions in this volume), or as a free polysaccharide, e.g. in synovial fluid or the vitreous body.

TABLE 2.

Effector Experimental system Effect Reference

Some factors known to influence the biosynthesis of hyaluronan

Hormones Insulin Para thyroid Calcitonin Oestrogen Testosterone Glucocorticoids

Immunological and inflammatory mediators

Interleukin 1 Interferon Connective tissue activating

Monocyte and macrophage

Lipopol ysaccharides

Cyclic AMP Activators of adenylate

cyclase Prostaglandins

Temperature (<37 "C) Decreased extracellular pH Decreased extracellular pH

High cell proliferation Growth factors (PDGF, EGF) Phorbol myristate acetate Interaction with tumour cells

Adenosine

peptides from platelets etc.

factors

Cellular mediators

Environmental factors

Relation to cell proliferation

Miscellaneous factors

Chicken fibroblasts Bone organ culture Embryo calf bone cells Chondrocytes Rooster comb Fibroblasts

Synovial cells Fibroblasts

Fibroblasts

Synovial fibroblasts Fibroblasts

Fibroblasts

Fibroblasts Fibroblasts

Fibroblasts Fibroblasts Chicken embryo cells

Chicken fibroblasts Skin fibroblasts Fibroblasts Fibroblasts

Fibroblasts

Activation Activation Activation Inhibition Activation Inhibition

Activation Activation

Activation

Activation Activation

Activation

Activation Activation

Inhibition Activation Activation

Activation Activation Activation Activation

Inhibition

PROPERTIES AND TURNOVER OF HYALURONAN 13

TABLE 2. ( conf . )

Effector Experimental system

Retinoids Pig epidermis Monensin Rat fibrosarcoma Monensin Chondrosarcoma Sulphated glycosaminoglycans Synovial membrane Endocytosis of undegradable

saccharides Fibroblasts A serum protein Fibroblasts

~ ~~

Effect Reference

Activation (20) Inhibition (21) No effect (22) Activation (23)

Inhibition (24) Activation (25)

References: (1) Moscatelli D , Rubin H 1976 J Cell Physiol 91:79-88. (2) Severson AR et al 1973 Endocrinology 92:1282-1285. (3) Martin TJ et al 1969 Experientia 25:375-376. (4) Mason RM et al 1984 Biochem J 223:401-412. (5) Szirmai JA 1966 In: Balazs EA Jeanloz RW (eds) The amino sugars. Academic Press, New York & London, IIB:12%154. (6) Castor CW 1972 Arthritis Rheum 15:504514. (7) Hamerman D, Wood DD 1984 Proc Soc Exp Biol Med 177:205-210. (8) Yaron M et al 1976 Arthritis Rheum 19:1315-1320. (9) Castor CW 1983 J Rheumatol (suppl 11) 10:5540. (10) Pulkki K et al 1983 Rheumatol Int 3:133-138. (11) Buckingharn RB, Castor CW 1972 J Clin Invest 51:1186-1194. (12) Tomida M et al 1977 Biochem J 162:539-543. (13) Fraser JRE et a1 1979 Ann Rheum Dis 38:287-294. (14) Castor CW, Yaron M 1976 Arch Phys Med Rehab 57:5-9. (15) Clarris BJ et al 1984 Ann Rheum Dis 43:31>319. (16) Moscatelli D, Rubin H 1975 Nature (Lond) 254:6546. (17) Engstrom-Laurent A et al 1985, Ann Rheum Dis 44:614-620. (18) Ullrich SJ. Hawkes SP 1983 Exp Cell Res 148:377-386. (19) Knudson W et a1 1984 Proc Natl Acad Sci USA 81:6767-6771. (20) King IA 1984 Br J Dermatol 110:607408. (21) Goldberg RL, Toole BP 1983 J Biol Chem 258:7041-7046. (22) Mitchell D, Hardingham T 1982 Biochem J 202:249-254. (23) Nishikawa ct al 1985 Arch Biochem Biophys 240:14&153. (24) LeMarshall J et al 1977 Ann Rheum Dis 36:13&138. (25) Tomida M et al 1977 J Cell Physiol 91:323-328.

U D P - GlcNAc - (GlcA - GlcNAC)"

U D P - GlcA P U D P

t U D P - GlCNAc - GlCA - GlcNAC - (GlCA - GlcNAc)n

FIG. 2. Biosynthesis of hyaluronan according to Prehm. HA is synthesized in the plasma membrane. Glucuronic acid (GlcA) and N-acetylglucosamine (GlcNAc) are transferred alternatively as UDP-GlcA and UDP-GlcNAc to the reducing end of HA with the simultaneous release of UDP attached to the chain.

14 LAURENT& FRASER

Biosynthesis

H A is present in the highest concentrations in mesenchymal tissues but most cells seem to be capable of synthesizing the polysaccharide in tissue culture. It has been known for a long time that H A is synthesized from UDP-sugars (RodCn 1980) but it was only recently found that the site of biosynthesis is the plasma membrane (Prehm 1984, Philipson & Schwartz 1984). Furthermore, the elongation of the chain takes place by addition of sugars to the reducing end (Prehm 1983) (see Fig. 2), which contrasts with the formation of proteoglycans (RodCn 1980). The H A synthetase is apparently situated on the inside of the plasma membrane and the chain grows through a pore in the membrane into the extracellular space.

Little is known of the mechanisms controlling the rate of H A biosynthesis. Information on various agents which influence HA synthesis has been collected in Table 2. In general, agents which activate adenylate cyclase seem to activate the production of HA. There is also a correlation between cell proliferation and H A synthesis.

Turnover of circulating HA

The detection of H A in serum raised the question of its origin. An analysis of lymph from sheep and rat indicated that this was the likely source (Laurent & Laurent 1981). A more recent investigation (Tengblad et a1 1986) which also included human fluids showed that lymph contains varying amounts, depend- ing on the site of collection. Especially high concentrations were found in mesenteric lymph. The polymer was polydisperse but contained fractions of high molecular weight ( M , > lo6). A calculation based on the concentration of H A in human thoracic lymph indicated that in the order of 10-100 mg of H A was carried to the general circulation every day.

When trace amounts of radioactive H A were injected intravenously, the tracer had a half-life in the circulation in rabbit and man of 2.5-5.5 minutes (Fraser et a1 1981, 1984). The disappearance from the bloodstream could be blocked in the rabbit by injecting 10 or 20 mg of unlabelled H A before the injection of the tracer. This is evidence of a receptor-mediated uptake of the polysaccharide. Only small amounts of radioactive polymer were found in the urine. However, within half an hour, low M , degradation products from the injected H A ('H,O from the [3H]acetyl group on the polymer) could be detected both in plasma and urine.

The total turnover of H A in the circulation of rabbit and man was calculated from the half-lives and the serum levels in the two species to be 1-2 mg and 1&100 mg per day, respectively (Engstrom-Laurent et a1 1985a), which is in general agreement with the output frcrn the lymph. It is not known how large a part of the total turnover of H A in the body takes this route.

PROPERTIES AND TURNOVER OF HYALURONAN 15

Destination of circulating HA

Already in the first experiments on rabbits (Fraser et a1 1981) it was shown that most of the radioactive material was accumulated in the liver. A minor part was also found in the spleen. Subsequent experiments with whole-body auto- radiography on mice injected with ‘‘C-labelled HA confirmed the accumula- tion in the liver, but small amounts were also found in lymphatic tissues such as spleen, lymph nodes and bone marrow (Fraser et a1 1983).

Recently, Bentsen et al (1986) catheterized patients with hypertension and made a study of the extraction of circulating HA in different organs. They confirmed that there was a high extraction rate in the liver, but they also found a loss of HA when blood passed through the kidneys. It is possible that this is due to glomerular filtration. HA has a low molecular weight in serum (Teng- blad et a1 1986), probably due to a preferential uptake of the large HA molecules in the liver (J.R.E. Fraser, unpublished).

Intravenously injected HA is taken up mainly by the non-parenchymal cells in the liver (Fraser et a1 1981). When the three main types of rat liver cells (hepatocytes, endothelial cells and Kupffer cells) were separated and grown in tissue culture (Smedsrod & Pertoft 1985), only the liver endothelial cells were

TABLE 3. Some substances reported to be cleared by specific receptors on liver endothelial cells

Substance Reference

Hyaluronan Chondroitin sulphate and its proteoglycans Collagen chains Lipoproteins Acetylated LDL (‘scavenger receptor’) Forrnylated serum albumin (‘scavenger receptor’) Ovalbumin (‘mannose receptor’) Enzymes carrying terminal mannose or N-acetylglucosamine Ceruloplasmin Transferrin Lactoferrin Insulin

References: ( 1 ) Eriksson et al 1983 Exp Cell Res 144:22>228. (2) Smedsrcbd et al 1985 Biochem J 229:63-71. (3) Srnedsrcbd et a1 1985 Biochem J 228:415424. (4) Gustafson S et a1 1985 Biochim Biophys Acta 834:308-315. ( 5 ) Nagelkerke JF et a1 1983 J Biol Chem 258:12221-12227. (6) Blomhoff R et al 1984 J Biol Chem 259:8898-8903. (7) Pitas RE et al 1985 J Cell Biol 100:103-117. (8) Blomhoff R et al 1984 Biochem J 218:81-86. (9) SmedsrGd B et a1 1982 In: Knook DL, Wisse E (eds) Sinusoidal liver cells. Elsevier Biomedical Press. p 263-270. (10) Hubbard AL et al 1979 J Cell B i d 83:4744. (11) Niesen TE et al 1984 J Leukocyte Biol 36:307-320. (12) Kataoka M, Tavassoli M 1984 Exp Cell Res 155:232-240. (13) Soda R, Tavassoli M 1984 Blood 63:27&276. (14) Courtoy PJ et al 1984 Lab Invest 50:329-334. (15) Soda R, Tavassoli M 1983 Exp Cell Res 145:389-395.

16 LAURENT & FRASER

able to bind and metabolize H A (Smedsr~d et a1 1984). It was subsequently shown by electron microscope autoradiography that in vivo the endothelial cells are also completely responsible for the uptake of H A in the liver (Fraser et al 1985).

The liver endothelial cells are apparently important scavenger cells which remove from the circulation various degradation products originating in the connective tissues (Table 3). The cells in lymphoid tissues which accumulate H A have not yet been identified.

Processing of HA in liver endothelial cells

The work on liver endothelial cells in vitro was done on primary cultures containing approximately 200 000 cells/cm2 (Smedsr~d & Pertoft 1985). Cul- tures kept at 7 "C could bind 3H-labelled H A to the cell surface without endocytosing it (Smedsrod et all984). The binding was saturable and could be suppressed by unlabelled HA, indicating a specific receptor for the polymer.

The number of molecules of H A bound to the cells and the dissociation constant of the receptor-HA complex were functions of the molecular weight of the polymer (Laurent et a1 1986). Approximately lo5 H A oligosaccharides ( M , 2000-7000), lo4 molecules of H A of M , 4 x lo5 or lo3 molecules of H A of M , 6.4 x lo6 could bind per cell. This dependence on molecular size was explained by a mutual exclusion of the large coiled molecules from the cell surface. The increasing affinity of H A for the receptor with increasing molecu- lar weight (from K,= 5 x M for an octasaccharide to K,,= 9 x lo-'* M for H A with M , 6.4 x lo6) was mainly attributed to the increased probability of binding large chain molecules with repeated sequences that are recognized by the receptor.

When the molecular weight dependence of binding had been clarified it was possible to show that HA and chondroitin sulphate competed for the same receptor on the liver endothelial cells and that the receptor had a higher affinity for the chondroitin sulphate structure (Laurent et a1 1986).

When H A was added to the liver endothelial cells at 37 "C it was rapidly internalized (Smedsrod et a1 1984). This internalization was mediated by the receptor (Laurent et al 1986) but the process also had features of fluid endocytosis (Smedsrod et a1 1984). At a physiological H A concentration in the medium (50 pg/l), each cell accumulated approximately 0.1 fg of polysacchar- ide ( M , 400 000) per minute. HA was incorporated into lysosomes and degraded. Low M , degradation products could be detected within half an hour both in vivo and in vitro. H A labelled with tritium in the acetyl group was degraded to [3H]acetate in the cultures and HA labelled with I4C in the sugar rings terminated in [I4C]lactate (Smedsrod et a1 1984). These compounds were rapidly metabolized by hepatocytes in v i m and the label from [3H]HA was recovered mainly as tritiated water in vivo (Fraser et a1 1981, 1984).

PROPERTIES AND TURNOVER OF HYALURONAN 17

Pathological serum levels of HA

The elucidation of the turnover of circulating HA has made it possible to search for and explain abnormal serum levels of the compound. In a study of patients with liver disease it was possible to demonstrate that some patients had serum levels of HA up to 100 times the normal value (Engstrom-Laurent et al1985b). The elevated level was strongly correlated to morphological signs of liver cirrhosis. Similar results have also been obtained by B. Delpech and collabor- ators (personal communication). Turnover studies with labelled H A in pa- tients with liver cirrhosis (Fraser et a1 1986) showed that the clearance rate was slower in the patients but that the total amount of HA turned over was approximately the same as in healthy persons. The results thus indicated that the high HA levels in liver cirrhosis were due to a defective clearance of circulating HA in the liver.

Rheumatoid arthritis is another disease in which high levels of serum HA were noted (Engstrom-Laurent & Hallgren 1985). In this disease we found a normal clearance rate (Fraser et a1 1986). Interestingly, the HA level in rheumatoid arthritis was related to physical activity (A. Engstrom-Laurent & R. Hallgren, personal communication). Almost normal values were found when patients woke up in the morning but the level was often ten times higher when the patients had been up for an hour. This elevation was related to the degree of joint inflammation in the patients and also to the degree of morning stiffness. Apparently, HA was synthesized and deposited in the tissues during the night and carried by lymph flow to the general circulation when the patients started to move their joints and muscles in the morning. HA which is accumu- lated in the tissues during rest will presumably immobilize water in the tissues and cause oedema, which could give rise to morning stiffness.

Scleroderma is a third disease which showed moderately increased levels of serum HA (Engstrom-Laurent et a1 198%). Interestingly, the HA level was correlated to the platelet count as well as the plasma level of p- thromboglobulin. The latter is an indicator of release of a-granules from platelets. These granules also contain the platelet-derived growth factor (PDGF), which is a specific growth factor for mesenchymal cells. The action of PDGF could thus explain connective tissue growth in scleroderma. PDGF also stimulated HA synthesis by skin fibroblasts in v i m .

Delpech et a1 (1985) have noticed that many cancer patients have high H A levels in serum. They suggested that this was due to hyaluronidase activity released by the tumours. Another possibility would be that the tumour cells induce HA synthesis in normal fibroblasts (see Table 2).

Turnover of HA in specialized tissues

A number of tissues and tissue fluids have recently been studied with regard to

18 LAURENT & FRASER

TABLE 4. fluids

Some studies on the concentration and turnover of hyaluronan in tissue

Fluid or tissue Investigation Reference A bnormat condition Reference

Vitreous body

Aqueous humour

Middle ear

Synovial lining cells

Amniotic fluid

Tissue fluid from skin Lung lavage fluid

Urine

Concn, M,, and (1, 2) turnover of HA Concn, M,, and (1, 2, 3 ) HA level decreased (4) turnover of HA by cortisone

HA present in middle ( 5 ) ear secretion

Synthesis of HA in (6) Increased synthesis in (6) vitro rheumatoid arthritis Concn, M , of HA (7, 8) No HA in amniotic (8) as function of fluid when fetus had gestational age cystic kidneys Concn of HA (9) Increased levels in (9)

psoriasis No detectable HA (10) Significant levels in (10) in healthy sarcoidosis individuals Daily excretion of (11, 12) Increased excretion (12) HA in Werner’s

syndrome

References: (1) Laurent UBG, Granath K 1983 Exp Eye Res 36:481-492. (2) Laurent UBG, Fraser JRE 1983. Exp Eye Res 36:493-504. (3) Laurent UBG 1981 Exp Eye Res 33:147-155. (4) Laurent UBG 1983 Acta Ophthalmol 61:751-755. (5) Laurent TC et al 1986 J Laryngol Otol 100:135-140. (6) Dahl IMS, Husby G 1985 Ann Rheum Dis 44:647457. (7) Dahl L et a1 1983 Biochem Med 30:28G283. (8) Dahl L et a1 1986 Biochem Med Metab Biol 35:219-236. (9) Lundin A, Engstrom-Laurent A , Michaelsson G , Tengblad A, unpublished. (10) Hallgren R et a1 1985 Br Med J 290:177%1781. (11) Laurent UBG, Laurent TC 1981 Biochem I n t 2:195-199. (12) Laurent TC, Lilja K , Brunnberg L, Engstrom-Laurent A, Laurent UBG, Lundqvist U, Murata K , Ytterberg D, unpublished.

normal levels of HA and in some cases its turnover. In some instances it has also been possible to identify pathological conditions showing abnormal HA levels. Such studies are listed in Table 4.

The eye can be regarded as a model of other connective tissues. The vitreous body represents the intercellular matrix and the aqueous humour, the lymph. Studies on eye tissues have shown that HA disappears from the vitreous body by diffusion and from the anterior chamber by the regular flow of the aqueous humour. In spite of the low concentrations of HA in the aqueous humour (Table 1) the total amount of HA that is turned over in the anterior chamber is larger than that in the vitreous body, indicating a significant synthesis of HA in the anterior segment. This is a demonstration that the HA concentration in a tissue cannot be used as an indicator of the rate of synthesis of HA.

Various types of inflammatory mediators induce HA synthesis (Table 2).

PROPERTIES AND TURNOVEROFHYALURONAN 19

This explains the high H A levels in otitis media, synovitis, psoriatic skin and lung fluid in sarcoidosis (Table 4). It is tempting to suggest that the HA level could be used as an indicator of inflammation.

The H A concentration in human amniotic fluid is in the order of 20 mg/l around the 16th week of gestation and drops to 1 mg/l late in pregnancy (Table 1). Furthermore, the HA has a low M , , which indicates that it originates in the fetus and is excreted through the kidneys. In agreement with this hypothesis it was found that a fetus with defective kidney function lacked amniotic HA. The drop in H A concentration between the 20th and 30th weeks does therefore presumably mirror a change in the metabolism of H A in the fetus.

Although the present data will certainly be followed by many more observa- tions on HA levels in pathological conditions, it is already apparent that HA is an interesting indicator of various clinical conditions.

Interaction of HA with cells

Some investigations that have shown an interaction between cell surfaces and H A are summarized in Table 5. There is now ample evidence that H A can bind to certain cells, although there are apparently different modes of binding. For example, H A synthesized as described by Prehm must be bound to UDP in the reducing terminal and to H A synthetase, which is attached to the cell mem- brane. It is possible, although not proved, that the extensive H A coat around fibroblasts (Fig. 1) could be bound to the synthetase.

A second mode of interaction is via a receptor. The first evidence for the presence of HA-binding proteins on cell surfaces (‘receptors’) was obtained by adding small amounts of H A to cell suspensions. The cells then aggregated. This was demonstrated with lymphoma cells, lung macrophages and virus- transformed cells (SV-3T3 and PY-BHK cells). Subsequently, Underhill and Toole and co-workers purified and characterized the receptor on SV-3T3 cells, and the existence of such receptors has thus been proved.

The effects of a H A layer around the cells have not been explored in detail. Addition of high concentrations of exogenous H A to lymphomyeloid cells inhibits migration, proliferation, phagocytosis and immune responses, pre- sumably through the viscoelasticity of the polysaccharide solution. The exact concentration of H A in the coat on fibroblasts has not been determined but its effective exclusion of erythrocytes would indicate that the concentration is appreciable and that the coat protects the cell from coming into contact with other cells and particulate material more than about 3 pm in diameter. It does not seem to inhibit the fibroblast itself from moving.

The function of H A in differentiation is intriguing. Apparently, the cells can move in embryonic tissues and may be directed in their movement by HA. When H A disappears the cells differentiate into their specialized forms. Thus, myoblasts lose their HA coat when they fuse into myotubes.