Proteoglycans: Structure, Biology and Molecular Interactions 9780824746520, 082474652X

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e ~ i by ~ e ~ Jefferson Medical College Thomas Jef f erson Universit y Philadel~hia,Pennsylvania

M A R C EL

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D EK K ER

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Neither this book nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, m i c r o ~ l ~ i n g , and recording, or by any information storage and retrieval system, without permission in writing from the publisher. Current printing (last digit): 1 0 9 8 7 6 5 4 3 2 1

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All life forms are constructedwith the same modules, distributed in different ways. The living world i s a sort of combinatorial system composed of a finite number of parts, like the product of a gigantic erector set; it i s the result of a ceaseless process of evolutionary tinkering. Of Flies, Mic e , an d Men, Franqois Jacob

I stumbled into the proteoglycan world by accident! When I joined the ~niversity of Washington in Seattle in the mid-l970s, my major goal was to finish the Pathology residency and pursue an academic career in basic science doing “bench research.” Thus, in 1979 I started an NIH-funded fellowship with Tom Wight, the youngest member of the faculty at that time. I was interested in cancer and thus started analyzing the glycosaminoglycan content of human colon carcinoma. Specifically, I was captivated by how the formation of tumor stroma was regulated at both the cellular and molecular levels. I thought that this part of the tumor was overlooked, in spite of its conspicuous presence: in some instances it represents more than 50% of the tumor itself! Proteoglycans comprise a significant proportion of this newly formed stromal tissue. I was very lucky: I spent three wonderful years learning cell biology and proteoglycan biochemist^ and came into contact with outstanding scientists who profoundly influenced my scientific life. I a mvery grateful for this and I would like to thank all the investigators who helped me in this learning process. However, that was a time when extracellular matrix was addressed with epithets such as “amorphous material,’’ “ground substance,’’ or even “slime.’ ’ Twenty years ago, when I gave my first research seminar in Seattle, I had a sparse audience. One of the few attendees, the late Earl Benditt, tried to console me by saying: “If you want to attract a larger audience, never use the word ‘proteoglycan’ in the title of your talk.’ ’ Times were changing, however. Acidic mucopolysaccharides were slowly but progressively addressed by the more “educated” investigators as glycosaminoglycans. It was the era of cesium chloride density iiii

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gradients, and the time when the protein core had to be huge, at least 1200 kDa as evidenced by the prototypic aggregating proteoglycan from cartilage. And indeed cartilage research was overwhelming. I remember the comments of a reviewer of my first paper in the Journal of Biological Chemistry in which we reported a ch on d r oit in as e- s en s it iveprotein core with a molecular weight of -50 kDa isolated from colon: ‘‘This is probably an artifact or more likely represents a degradation product from a larger precursor protein.” It turned out to be decorin! It was the time of the first monoclonal antibodies, of metabolic labeling, and of pulse-chase analyses. Radiosulfate was king. Then came the first Cordon Research Conference on Proteoglycans in 1984, the explosion of knowledge regarding the sugar moieties, the linkage region, the delineation of the various biosynthetic pathways, and the intracellular and extracellular degradation steps. The last decade has become molecular, with the cloning and sequencing of at least 30 proteoglycan-encoding genes, a number that will likely double in the next decade. Current investigations on transcriptional control and the generation of mutant animals deficient in specific proteoglycan genes are revealing a high degree of complexity. Proof of the increasing and widespread interest in this field of biology is the large number of papers on proteoglycans published in the last 15 years, reaching approximately 1S,000. This book was designed to encompass the most recent advances in proteoglycan research centering on specific gene products or families of proteoglycanencoding genes, with the hope of attracting new investigators to this fascinating field of research. Amply illustrated and containing over 1700 bibliographic citations, this book offers a comprehensive and up-to-date collection of reviews on proteoglycans, and their structure, biology, and molecular interactions. It consolidates the latest breakthroughs in this expanding field of research, using molecular, cellular, and animal systems in a single-source reference volume, A detailed description of the content of the book is presented in Chapter l, “Introductory Remarks and Overview .’’ If I had known from the beginning how difficult and, at times, f ~ s t r a t i n g it is to be the editor of a multiauthor book, I probably would have declined the offer. However, the fait accompli is highly rewarding and satisfying, Thus, I would like to first thank all the authors for their excellent cooperation. Special thanks go to Dr. C. C. Clark and Kit Foster for their editing help. I am also inde~tedto the staff of Marcel Dekker for their outstanding job, in particular uarez, Sandra Beberman, and the late Graham Garratt.

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Pref a ce Con tribu tors

Ren ato V.

Il l

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io§ynthe§isof ~hondroitin~ulfatean

, Anna Pla a s, Ron a ld J. ~ i d u r a~, i k kJ.i Good ston e, L e n ~ a rRod t &, and Vin cen t C. Ha sca ll

ar ~tructureand ~nteraction§wit

Peter J. Rou gh ley and Joh n S. Mort

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ola , Varpu ~ a in u la in e n and , ~ arkku 2: Ja lk a n en

Jorg e ~ i l m u and s ~ o w a r H. d Son

R i~ h a ~ L. dStev en s, Da n a ld E. Hu m ph ries, and Gu an g W. Won g

.

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Dieter R.

~i~mermunn

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~ a r ~ aM. r aVertel and Anthon~R a t c l i ~ e

Index

403

Project Staff, Department of Biomedical Engineering, The Cleveland Clinic Foundation, Cleveland, Ohio

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Fellow, Department of Research, Shriners for Children, Tampa, Tampa, Florida

.

Associate Professor, Department of Molecular ronto, and Senior Scientist, Department of Cance Research, Sunnybrools and Women’s College Health Sciences Center, Toronto, Ontario, Canada

es Jules and Doris Stein Research to Prevent Blindness, Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania e

Professor, Cancer Research Campaign Depart iversity of Manchester, and Christie Hospital Trust, Manchester, England ,

.

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Wellcome Trust Center for Cell Matrix University of Manchester, Manchester, England

. Department of Biomedical Engineering, The Cleveland Clinic Foundation, Cleveland, Ohio

ii

viii

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o~tri~~tor~

. Professor and Berry Chair Holder, Depa~mentof Biochemistry and Molecular Biology, College of Medicine, University of South Florida, and Director, Department of Research, Shriners Hospital for Children, Tampa, Tampa, Florida .

Research Scientist, Veterans Affairs Medical

Center, Boston, Massachusetts

. Professor, Department of Pathology, Anatomy and Cell Biology, and Codirector of the Cell Biology Program at the Kimmel Cancer Center, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania

.

Research Scientist, Center for Biotechnology, University of Turku, Turku, Finland Professor, Center for Biotechnology, University of Turku, Turku, Finland

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Department of Pediatrics, University of Turku, Turku, Finland

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Assistant Professor, De~artmentof Bioche~istry and Molecular Biology, College of Medicine, University of South Florida, and Research Fellow, Shriners Hospitals for Children, Tampa, Tampa, Florida Cancer Research Department of Medical Oncology, University of Manchester, and Christie Hospital NHS Trust, Manchester, England e

.

Staff Assistant, Department of Biomedical Engineering, Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, Ohio

.

Scientist, Joint Diseases Laboratory, Shriners Hospital for Children, Montreal, Quebec, Canada

e9 Professor, Department of Biochemistry and Molecular Biology, College of Medicine, University of South Florida, and Senior Investigator, Shriners Hospital for Children, Tampa, Tampa, Florida

Associate Scientist, Department of Research, Shriners Hospital for Children, Tampa, Tampa, Florida e

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. Executive Director for Research, Advanced Tissue Sciences, La Jolla, California

.

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Professor of Medicine, D e p ~ m e nof t Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, Alabama

.

Scientist, Genetics Unit, Shriners Hospital for Children, Montreal, Quebec, Canada

.Se. Department of Medical Biophysics, University of Toronto, and Sunnybrook and Women’s College Health Sciences Center, Toronto, Ontario, Canada

. Associate Professor, Department of Medicine, Harvard Medical School and Brigham and Women’s Hospital, Boston, Massachusetts . Professor, Department of Anatomy and Cellular Biology, Tufts University School of Medicine, Boston, Massachusetts

ertel, . Professor, Department of Cell Biology and Anatomy, Finch University of Health ScienceslThe Chicago Medical School, North Chicago, Illinois

. Department of Medicine, Harvard Medical School and Brigham and Women’s Hospital, Boston, Massachusetts . Associate Professor, Department of Cell Biology, University of Alabama at Birmingham, Birmingha~,Alabama

.

Associate Professor, The Burnham Institute, La

Jolla, California

. University of Zurich and Molecular Biology Laboratory, Department of Pathology, University Hospital, Zurich, Switzerland

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Thomas tho er son Univ~rsi~y, Philad~lphia,P~nnsylvania

Don Quixote said to his squire: “Sancho, I have always heard it said that to do a kindness to clowns is like throwing water into the sea. Had I given to thy advice, I had prevented this misfortune: but, since the thing is done, it is needless to repine; this shal l be a warning to me for the future.”

Don Quixote, Cervantes

Proteoglycans comprise a collection of macromolecules that surround the plasma membrane of a cell and constitute part of the substrate upon which the cells are attached and perfom their major functions. The solid frameworks needed for the structural support and functional organization of these assemblies are made primarily of cross-linked fibers of collagen embedded in a fine meshwork of proteoglycans, and sometimes reinforced by deposited minerals. Proteoglycans are involved in maintaining the transparency of the cornea, the tensile strength of the skin and tendon, the viscoelasticity of blood vessels, the compressive properties of cartilage, and the mineralized matrix of bones. In addition, proteoglycans play key roles as storage depots for growth factors and cytokines, and by virtue of their multifunctional properties, they alter the biology of these factors. It seemed, therefore, timely and appropriate to organize a book on the subject in order to evaluate new information, to focus issues for future studies, and to examine new aspects that have emerged from the characterization of the gene and protein modules and from genetic analyses of mutant animals. The goal of this book is to present recent advances in proteoglycans and their biology to a wide audience, All the authors are active and productive investigators and recognized experts in their fields; but all were enthusiastic about the prospect of writing for such an 1

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audience. The chapters are not review articles per se: They are written specifically for this book and represent the state of the art in the proteoglycan field, with ample references to more historical and technical reviews. The contributors have done an outstanding job in ~ a k i n gtheir chapters up to date, although some of the fields are moving so rapidly that they will certainly have advanced between the completion of the manuscript and the book’s publication. However, the basic concepts certainly will not change within this time frame, and will be more enduring. Chapters l to 3 deal primarily with biosynthetic and structural aspects of chondroitin sulfate, heparan sulfate, and hyaluronan. In Chapter 2, Hascall and coworkers provide a unique historical perspective of the field covering over 100 years from the “~hondroitin~chwefelsa~re~ ’ (chondroitin sulfuric acid) to the modern chondroitin sulfate. They also review the fine structure of this glycosaminoglycan, the linkage region, and the detailed biosynthetic pathways leading to the final product, In Chapter 3, ~ a l l a g h e and r Lyon provide an extensive overview of the biosynthesis and fine structural diversity of heparan sulfate. This chapter also provides an up-to-date analysis of the various molecular interactions between heparan sulfate and growth factors and morphogens, and their roles in regulating cell growth and adhesion. Toole (Chap. 4) provides an exciting discussion of hyaluronan, an extraordinary biopolymer with unusual physical properties, including its binding to several types of proteins (hyaladherins), its regulation of pericellular matrices, and its role in morphogenesis and cancer. oughley and Mort (Chap. 5) provide a comprehensive overview of proteoglycan degradation, focusing in particular on the role of matrix metalloproteinases, currently believed to be the major mediators of extracellular proteoglycan degradation. From this chapter, the reader will gain insights into how glycosidases and sulfatases, as well as reactive oxygen species, affect the catabolism of a variety of proteoglycans whose function is discussed in subsequent chapters. The next three chapters (Chaps. 6-8) focus on the cell surface proteoglycans syndecans and glypicans. Jalkanen and coworkers (Chap. 6) cover the structure and function of the syndecan gene family, with special emphasis on the biology of the prototype syndecan-l. Also covered are emerging areas including the syndecans’ roles in adhesion, in maintaining epithelial cell morphology, in modifying heparin-binding growth factor action and in modulating development, wound healing, and tumorigenesis. The chapter by Couchrnan and Woods (Chap, 7 ) is closely related and covers a rapidly emerging area of syndecan biology that involves the signaling process via its intracellular domain. Syndecans are now taking their place among transl~embraneadhesion receptors, albeit they seem to modulate other adhesion mechanisms rather than operate independently. In Chapter 8, Filmus and Song discuss the structure, function, and expression of this intriguing class of proteoglycans that are linked to the plasma membrane via

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a glycosyl-phosphatidylinositol anchor. They also cover recent data on mutant

animals and the involvement of at least one member of this family in causing a human pathological syndrome. In Chapter 9, Stevens and coworkers review the biology of a unique family of proteoglycans, the serglycins, which are stored in the secretory granules of immune competent cells such as mast cells and basophils. The next six chapters (Chaps. 10- 15) focus on the extracellular proteoglycans, broadly defined as proteoglycans that are secreted into the extracellular space and that can occupy strategic locations such as the pericellular space and the b a s e ~ e n tmembrane. Neame and Kay (Chap. 10) discuss the biology of the small leucine-rich proteoglycans. uch has been done on this subject in the last decade and several knockout animals have been generated. This chapter reviews how protein cores with leucine-rich motifs, unique to this class of proteoglycans, induce specific biological functions. In Chapter l 1, Funderburgh focuses on the corneal proteoglycans and provides an up-to-date overview of their functional roles in maintaining corneal transp~ency. assell and Dunlevy (Chap. 12) review the field of basement ~ e ~ b r a n e proteoglycans. En addition to perlecan, new members of this ~ ~ l t i f a c e t group ed are discussed, including agrin and collagen type XVIIE, two full-time proteoglycans deposited in most basement membranes, The cellular and molecular biology of these three proteoglycans are discussed in detail. Zimmermann (Chap. 13) focuses on versican, a prototype proteoglycan that has diverse structures generated by alternative splicing and multiple functions in atherosclerosis and cancer. In Chapter 14, Vertel and Ratcliffe provide a comprehensive appraisal of the major proteoglycan of cartilage, aggrecan, including intracellular processing, secretion, and pathology, In the last chapter (Chap. IS), Yamaguchi reviews the chondroitin sulfate proteoglycans of the nervous system. This is a rapidly expanding area of research since a large number of proteoglycans are expressed in the central nervous system. No work of this sort can be brought to fruition without the special assistance from our colleagues. I have been fortunate in being able to assemble an outstanding cadre of leading experts in various aspects of the proteoglycan fields, who have given unstintingly of their time to review and discuss every chapter. I have really enjoyed a stimulating dialogue with all of them and learned enormously in this process. There is some overlap between chapters due to discussions of basic processes. While it would have been possible to prune chapters to reduce overlap, it has been my objective to make every chapter stand on its own. Therefore, I believe that this format is an advantage to the reader interested in both a specific

4

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proteoglycan as well as its function in normal and pathological processes. Moreover, different authors have different angles of approach and emphasize different aspects of the same proteoglycan. In editing this book, I have not imposed my own views on terminology or other issues. Thus, different terms or abbreviations are used by different authors to refer to the same family of proteoglycans, the best example being the hyalectans, lecticans, or the aggrecan family of proteoglycans. Another example is the three-dimensional model of decorin proposed by N e m e , a model that differs from that which I proposed. I believe that the reader will benefit from this open dialogue which also reflects the rapid evolution and changes in the field. Now that we have seriously begun to study proteoglycan biology, we have a better idea of the breadth of the impending challenges and the distance we must cover in order to solve them. I hope that this book will help the novice as well as the expert investigators who may find some inspiration to continue their work in this field.

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Institute, The Cleveland Clinic Foundation,

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Cleveland, Ohio

~hriners~ospitalfor Chil~ren,Tampa, ?ampa, Flori~a ~niversityof an chest er, an chest er, ~ngland

na n ~niversityof Alabama at girmingham, ~irmingham,Alabama

Chondroitin sulfate and hyaluronan are members of the glycosaminoglycan family with similar chemical structures, but otherwise they have very different histories, properties, and modes of biosynthesis. Chondroitin sulfate is present in living tissues covalently bound to proteins to form proteoglycans such as aggrecan (see Chap. 14), whereas hyaluronan is present without a core protein and hence is a true glycosaminog~ycan.This chapter focuses on the structure and biosynthesis of these glycosa~inoglycans,while other chapters focus on their biological properties (Chaps. 4 and 14).

In 1884, Krukenberg described a preparation isolated from hyaline cartilage by extraction with dilute alkali at room temperature, the first preparation of a glyco-

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Repeating disaccharide structures for (a) chondroit~nsulfate and ( b) hyaluroof the hexosamines distinguishingglucosamine from galactosamine are circled. The conversion of glucuronic acid to iduronic acid that distinguishes chondroitin sulfate from derrnatan sulfate is indicated by the ar r ow.

nan. The epimeric 4”arbon

saminoglycan (1). In 1889, Morner improved the purity of this material and correctly identi~edthe presence of ester sulfate (2). A pivotal paper in 1891 by ~chmiedebergprovided both a name: “Chondroitinschwefel~aure~ ’ (chondroitin sulfuric acid) derived from chondros (cartilage), and a deduced composition: acetate, sulfate, glucuronic acid, and hexosamine (3). The hexosamine was thought to be chitosamine, the then current name for glucosamine. It would take more than 20 years before Levene and La Forge (1914) would correctly identify the structure of the hexosamine, which they called chondrosamine (now named galactosamine) (4). Chondroitin sulfate i s now known to consist of repeats of the disaccharide structure shown in Figure la. However, actual details of this basic structure emerged slowly [see chap. 4 of Brimacombe and Webber for an excellent review ( 5 ) ] . In 1914, Hebting isolated a disaccharide from acid hydrolysates of chondroi-

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tin sulfate (6). The disaccharide, named chondrosine, is now known to be the desulfated and deacetylated form of the structure shown in Figure la. In 1941, Levene (7) reported structural studies of chondrosine that established the presence of the hexosamine and hexuronic acid. However, he proposed an erroneous structure in which the sugars were reversed (hexosamine-~-l,4-hexuronic acid). This had an important impact on a subsequent, extensive chemical study of chondrosine in Wolfrom’s laboratory, reported in 1952 (8). They interpreted their systernatic series of chemical degradation steps and their characterization of products in terms of Levene’s proposed structure. In 195l, Masamune et al. (9) reported that chondrosine gave a positive reaction in the Elson-Morgan procedure, which could only occur if the hexosamine was at the reducing end, Based on this and on products generated from periodate oxidation, they proposed what turned out to be the correct structure. Definitive proof, however, was provided in the studies of Davidson and Meyer, reported in 1954 and 1955 (10,ll). In the first study (lo),they reduced chondrosine which converted the hexosamine to a hexosaminitol. After subsequent esterification of the carboxyl group, the hexuronic acid was reduced to a hexose that was identified as glucose. Further, the reduced disaccharide was cleaved by a pglucosidase, but not by an a-glucosidase. These results established the presence of glucuronic acid in P-linkage to the hexosamine in chondrosine. The glycosidic linkage was proposed to be 3,3, a detail proven in their second paper (1 1) by systematic degradation of reduced chondrosine with ninhydrin and periodate. As a postscript, in 1960, Wolfrom and Juliano (12) added some additional data to support the structure of chondrosine and reinterpreted the results of their original study (8) in light of the correct structure. The positions of the sulfate groups on the chondroitin sulfate backbone were deduced by Meyer’s group (13) and by Mathews (14). The former study (13) showed that the glucuronic acids in tetrasaccharides derived from chondroitin sulfates were sensitive to P-glucuronidase and hence unsubstituted, thereby indicating that sulfation occurs on the galactosamine residues. Mathews (14) analyzed infrared spectra of chondroitin sulfate A (primarily 4-sulfated) and chondroitin sulfate C (primarily 6-sulfated). The latter showed a spectrum characteristic of the presence of equatorial sulfates, as had been determined by Orr (15) in studies of synthetically sulfated hyaluronan, in which only equatorial substitutions are possible. This established sulfation at the 6 position for chondroitin sulfate C, the only equatorial site available on the galactosamine. Chondroitin sulfate A showed a spectrum indicative of an axial sulfate, establishing sulfation at the 4 position, the only axial site available on the galactosamine. The final piece of the puzzle, the linkage between the GalNAc and the GlcA, was established when the structures of the disaccharide products of the bacterial chondroitinases were determined to have an unsaturated bond between the 4- and 5-positions on the uronic acid moiety ( l 6,179. The cleavage mechanism

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therefore involves the elimination of the glycosidic bond facilitated by removal of a proton from the 5-position during formation of the double bond (see Sect. III), which can only occur if the glucuronic acid is substituted at the 4-position. The ability of p-hexosaminidases to cleave the chondroitin sulfates established that the anomeric bond is p (18) (Fig. la). Because dilute alkali readily released chondroitin sulfate from hyaline cartilage in a form which could be isolated free of protein, the prevailing model for the structure of the cartilage matrix in the 1940s was that it consisted of an ionic association between free chondroitin sulfate chains and collagen (19). However, in 1954 Shatton and Schubert reported that some noncollagenous protein could not be removed when chondroitin sulfate was isolated from aqueous (-pH 7) extracts of homo~enizedcartilage, and coined the name “protein polysaccharide” for their preparations (20). This birth announcement for the proteoglycan we now call aggrecan was met with some skepticism. However, the evidence mounted as Helen Muir (1958) showed that amino acids remained associated with chondroitin sulfate even after extensive digestion of a proteoglycan preparation with papain (21). Serine was the only amino acid remaining in quantities large enough to reach a molar ratio to chondroitin sulfate greater than 1. Therefore, Muir proposed that the chondroitin sulfate chains were linked to serine in the initial, intact preparation. She also noted that the linkage was approximately as labile to alkali as an ester or a lactone. The precise nature of the alkaline cleavage was subsequently determined by Meyer and his collaborators who showed that the chondroitin sulfate chains were linked to serine through a glycosidic bond which is very labile to p-elimination (22) (Fig. 2). The elimination of the glycosidic bond from the p-carbon, facilitated by removal of a proton from the adjacent a-carbon of serine, leaves dehydroalanine and releases the reducing end of the chondroitin sulfate chain. The reduction of the dehydroalanines to form alanine provided a nice proof of the mechanis~.This finally provided an answer for why dilute alkali effectively solubilized chondroitin sulfate from cartilage (1). It also explained the reducing power exhibited by proteoglycans when treated with the alkaline copper reagent, which had led Partridge and coworkers to propose that the reducing ends of the chondroitin sulfate chains were free in the original macromolecule (23). In principle, the reducing end of the chondroitin sulfate chains released by p-elimination can be identified after reduction with borohydride and isolation of the sugar alcohol formed, However, the large size of the chondroitin sulfate chains made this approach difficult. Rod& and collaborators used a different strategy to isolate and characterize the linkage region (24-26). They digested a proteoglycan preparation with both testicular hyaluronidase to remove the bulk of the chondroitin sulfate chains and proteolytic enzymes to remove the bulk of the core protein. The resulting glycopeptide fraction was subjected to mild acid

+

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igure 2 The base-catalyzed P-elimination reaction. See text for details.

hydrolysis and a series of fragments from the linkage region were isolated. One of these was 0-P-xylosyl-serine, establishing the point of linkage. Two of the oligosaccharides (GalP1,3GalP1,4Xyl and GlcAP1,3Gal) established the structure of the linkage region through to the initial GlcA of the repeating disaccharide units in the chain. Subsequent work by Oegema and collaborators (27) showed that up to 80% of the linkage region xylose residues on aggrecan are phosphorylated on the Zposition (see Sec. IV). In 1934, 50 years after the discovery of chondroitin sulfate, the birth of hyaluronan was announced by Meyer and Palmer (28). They purified hyaluronan from the vitreous of the eye and showed that it contained a hexuronic acid and a hexosamine, but no ester sulfate. They proposed “for convenience, the name ‘hyaluronic acid, from hyaloid (vitreous) uronic acid.’ In this case, it would only take - 20 additional years to determine its disaccharide structure (Fig. lb), summarized in 1954 by Weissman and Meyer (29) and reviewed in Brimacornbe and Webber (5). Hyaluronan and chondroitin sulfate have very similar backbone structures with alternating P1,3 and Pl,4 glycosidic linkages from the glucuronic acid and the hexosamine residues, respectively. They differ only as epimers at the 4-position of the hexosamine, equatorial in g l u c o s ~ i n ein hyalurona~,and axial in galactosamine in chondroitin sulfate. However, this difference changes the rather simple, monotonous chemical structure of hyaluronan into the more complex, sulfated forms of chondroitin sulfate.

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Techniques have been developed to determine overall patterns of sulfation and, in particular, the structures at the nonreducing termini of chondroitin sulfate chains. These involve digestion of the chains with bacterial chondroitinase enzymes (16,1'7,30,31) (Fig. 3). These enzymes are eliminases that release the glycoside bond during formation of a double bond between G-5 and C-4 of the hexuronic acid residues. This converts hexuronic acid residues in the interior of the chain to 4,5-unsaturated uronic acids, producing Adisaccharides with free reducing groups on the GalNAc residues. These unsaturated Adisaccharides are unstable, p~ticularlyto mild alkali or heat, but can be stabilized by reduction with borohydride (32) or reductive amination with a fluorescent tag (33). The fluorotagged derivatives are then separated, based on differences in number (03)and location of sulfoesters, and their fluorescence monitored for quantification. This provides an average composition of the interior of chondroitin sulfate chains. The residues at the nonreducing termini are released by the enzyme either as a monosaccharide, CalNAc (usually either 4-sulfated or 4,6-disulfated), or as a disaccharide with a nonreducing terminal, unmodified glucuronic acid. Fluorotagged residues of the CalNAc substituents are readily resolved and usually represent 85-90% of the termini (33). The less abundant saturated d i s a c c h ~ d e sare resolved after selective degradation of the unsaturated Adisaccharides with mercuric ion (34). They carry either a 4-sulfate or a 6-sulfate on the CalNAc moiety. Purified aggrecan samples isolated from the cartilage on the articular surfaces in human knees from different age groups were analyzed by these techniques. The results indicate that the fine structure and nonredu~ingt e r ~ i n ion the chondroitin sulfate chains are constant until the time of skeletal maturity when characteristic transitions occur (33) (Fig. 4). After skeletal maturity new values are reached which remain constant thereafter. Thus the interior disaccharides have nearly equal amounts of 4- and 6-sulfated residues before, and almost only 6sulfated residues after, skeletal maturity . Nonreducing t e r ~ i n adisaccharides ~ reflect the same pattern. However, the predominant GalNAc termini show a distinct pattern that reflects the late appearance of a 6-sulfotransferase that has specificity for 4-sulfated GalNAc termini, converting them to 4,6-disulfated residues. Thus, essentially all nonreducing terminal GalNAc residues are solely 4-sulfated before, while -50% are 4,6-disulfated after skeletal maturity is reached. This distinct sulfotransferase activity lags behind the transition to predomina~tly6-sulfation on CalNAc residues in the interior of the chains, such that samples from individuals in the 14- 18 year range have little 4,6-disulfated GalNAc termini even though 6-sulfation of interior disaccharides has nearly reached its maximum. These analyses of the fine structure of aggrecan chondroitin sulfate are now providing clues as to the phenotypic changes that chondrocytes undergo, not only in tissue development and maturation, but also during the pathogenesis of osteoarthritis with

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ure 4 Comparison of fine structure of chondroitin sulfate on aggrecan isolated from normal human knee cartilage at different ages and osteoarthritic cartilage. Before adolescence, 6-sulfation occurs on only -50% of the disaccharides, while during adolescence and adulthood this value increases to >95%. Before adolescence few, if any, chains have 4,6-disulfated-GalNAc on their nonreducing termini, while after adolescence -50% do. Note that during the adolescent transition, the interior 6-sulfation is at adult levels while 4,6-disulfated-GalNAc on nonreducing termini remains closer to preadolescent values. Interestingly,the values for aggrecan from the osteoarthriticcartilage (OA) are characteristic of the adolescent transition.

the reappearance of chondroitin sulfate fine structure characteristic of the transition period (35).

The proteoglycan, aggrecan, is the major biosynthetic product of chondrocytes. Its large core protein (-220,000 MW), synthesized in the rough endoplasmic reticulum, can account for 5% or more of the total protein synthesized by the

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Different sites for synthesis of chondroitin sulfate and hyaluronan. Entry points for radiolabeled precursors are indicated: amino acids (*AA) into the core protein in the rough endoplasmic reticulum, sulfate (PAP*S) into chondroitinsulfate in the Golgi/ trans Golgi compa~ments,and glucosamine (UDP-*hexNAc) as UDP-GalNAc into chondroitin sulfate in the Golgiltrans Golgi compartment and as UDP-GlcNAc into hyaluronan at the plasma membrane. ( ~ o d i ~ from e d Ref. 70.)

cell (36,37).Many posttranslational modi~cationsare required before the mature ~acromoleculeis secreted into the extracellular matrix (Fig. 5). These include l ) conversion of the high mannose N-linked oligosaccharides added in the rough endoplasmic reticulum to complex forms in the Golgi cisternae; 2) addition of 0-linked oligosaccharides, probably in the trans Golgi cisternae, and their subsequent extension with keratan sulfate chains; 3) initiation of the linkage oligosaccharides that forrn the attachment sites for chondroitin sulfate chains in transition vesicles between the rough endoplasmic reticulum and cis Golgi cisternae, and their completion in subsequent Golgi compartments; and 4) elongation and sulfation of the chondroitin sulfate chains. Cho~droitinsulfate is the dominant component of the completed aggrecan macromolecule, and the general ~ e c h a n i s ~ s

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for its elongation are characteristic of the other sulfated glycosaminoglycans: the heparin/heparan sulfate family and keratan sulfate. This occurs within the lumen of the Golgi/trans Golgi network in a compartment that requires the core protein with 100 assembled linkage oligosaccharides on appropriate serine residues; appropriate enzymes to form the repeating disaccharide backbone and to add sulfoesters; and substrates, nucleotide-sugars and phosphoadenosinephosphosulfate. The linkage oligosaccharide for chondroitin sulfate is initiated by transfer of a xylose from UDP-xylose onto the hydroxyl group of a serine acceptor to form xylosylserine (38-40; see Ref. 41 for review). This occurs during the transfer of the core protein from the rough endoplasmic reticulum to the cis Golgi (42,43) (see Chap. 14). Subsequent growth of the linkage region oligosaccharide and elongation of the chondroitin sulfate chain occurs by sequential addition of sugar residues to the nonreducing terminus of the chain, in contrast to the direction of hyaluronan synthesis (see below). Completion of the linkage oligosaccharide involves the addition of two adjacent galactose units from UDP-galactose. This occurs during migration of the core protein through the Golgi, probably in the cis and/or medial cisternae. This is followed by addition of the initial glucuronic acid from UDP-glucuronic acid, which can be considered the beginning of the disaccharide repeat structure. Each of these additions requires a distinct glycosyltransferase. The addition of the initial acetylga galactosamine from UDP-GalNAc onto the linkage oligosaccharide, like the first glucuronic acid residue, is mediated by a distinct enzyme (44) (Fig. 6). Subsequent chain elongation, then, requires two glycosyltransferase activities to add the alternating residues of glucuronic acid (from UDP-GlcA) and N-acetylgalactosamine (from U D ~ - G a l N ~ cThese ) . activities may be embedded in a single enzyme or in two separate enzymes. Distinct sulfotransferases utilize phosphoadenosinephosphosulfate(PAPS) to add sulfoesters to (1) distinct locations on the backbone structure, usually either the 4- or 6-hydroxyl of the GalNAc residues, and occasionally both; and (2) in some cases on the 6-hydroxyl of 4-sulfated-Gal~Acresidues to produce 4,6-disulfatedGalNAc residues that are on nonreducing termini of chondroitin sulfate chains (Fig. 4). Many cells other than chondrocytes al so have an epimerase that converts a proportion of the D-glucuronic acid residues in the interior of the chain to L-iduronic acid residues by epi~erizationof the S-carbon (Fig. l). By definition, such chains are now referred to as dermatan sulfate, a name indicative of the isolation of such chains originally from skin. In some cases, dermatan sulfate can be further modified by adding sulfoesters onto the 2-hydroxyl of either hexuronic acid. Epimerization and addition of sulfoesters appear to occur concurrent with, or shortly after, elongation, which suggests that the site of chondroitin sulfate/dermatan sulfate synthesis may consist of a multienzyme complex.

-

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Schematic for the antiport mechanism for providing substrates for elongation of chondroitin sulfate chains. A serine site on aggrecan with a completed linkage region oligosaccharide, gal act osyl - gal act osyl - xyl osyl -(G-G-S-) and the first glcA (U), is being elongated by addition of N-acetyl-galactosa~ine(N) and glucuronic acid residues inside the lumen of a Colgi/trans Golgi compartment. Two enzymes (E, and E,) are indicated, but one enzyme may have both glycosyl transferase activities in analogy with hyaluronan synthesis. Enzymes for sulfation (E,) are also present. The antiporters are indicated by solid black circles.

Biosynthesis of chondroitin sulfate requires metabolic energy and reactions to convert the basic precursors, glucose and sulfate, to the substrates used by the assembly enzymes. For UDP-GlcA, this involves phosphorylation of glucose (requires ATP), conversion to UDP-glucose (requires UTP), and oxidation to UDP-GlcA (requires NA ). For glucosamine, this involves transamidation of phosp~orylatedglucose to glucosamine (requires glutamine), acetylation to form GlcNAc (requires acetylCoA), conversion to UDP-GlcNAc (requires UTP), and epimerization to U ~ P - ~ a l N AThe c . formation of PAPS requires 2 ATP, The enzymes, substrates, and cofactors that make these activated substrates are located in a cytosolic c o m ~ a r t ~ e nwhile t, the enzymes that assemble chondroitin sulfate chains operate on the other side of the membrane in the lumen of the olgi network. Cells have accomplished this by developing antiport mechanisms that efficiently transport a substrate, for example, UDP-GalNAc,

1

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into the lumen, while transporting an end product, in this case, UMP, out (45). Under normal conditions, the operation of the antiport transporters maintains the substrates in the lumenal compartment at saturating concentrations, and the concentration of the linkage oligosaccharide acceptors is rate limiting. This is apparent, because cells treated with saturating amounts of P-xylosides, which provide alternative acceptors for chondroitin sulfate chain synthesis, always increase net synthesis of chondroitin sulfate, often several fold (46-48). This suggests effective coupling between production of substrates on the cytosolic side and the antiport transporters. Interestingly, a mutated epithelial MDCK cell line has been shown to have a defective UDP-Gal antiporter with only -2% residual activity (49,50). These cells synthesize chondroitin sulfate and heparan sulfate at levels near the wildtype cell line, but are very deficient in keratan sulfate, glycoprotein, and glycolipid synthesis, all of which require UDP-Gal as substrates for key enzymes. This suggests that the galactosyltransferases involved in synthesis of the linkage region oligosaccharide for chondroitin sulfate and heparan sulfate have higher affinity for the limited amount of substrate available compared with the other galactosyltransferases.

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Biosynthesis of hyaluronan is less well understood than biosynthesis of chondroitin sulfate, but clearly differs in fundamental ways. A variety of data support the mechanism, originally proposed by Prehm (51) (Fig. '7).

1. Biosynthesis occurs directly without a core protein or linkage region oligosaccharide. Hyaluronan isolated from chondrocytes metabolically labeled with radiolabeled amino acids did not contain any covalently associated proteins (52,53). 2. The hyaluronan-synthase resides at or near the plasma membrane. Newly synthesized, high molecular weight hyaluronan synthesized by B6 cells and closely associated with the surface of intact cells was susceptible to digestion with Streptomyces hyaluronidase (54); and analyses of vesicles from homogenized mouse oligodendroglioma cells after partitioning by rate zonal centrifugation showed that hyaluronan synthetic activity most closely correlated with plasma membrane markers (55). 3. Elongation occurs at the reducing end by a 2-site mechanism in which the growing chain remains attached to the UDP moiety of the newly added sugar residue. A particulate membrane fraction from teratocarci-

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that in the ~~P-3H-N-acetylhexosamine pool because of the metabolic pathway that converts glucose to glucosamine (57). Radiolabeled sulfate is routinely used as a precursor for sulfated glycosaminoglycans, in this case chondroitin sulfate. In contrast to labeled glucosamine, metabolic sources of sulfate contribute negligibly to the intracellular sulfate pool (58), and the specific activity of the PAPS substrate rapidly equilibrates with the specific activity of the [3sS]sulfatein the medium. For chondroitin sulfate, then, the incorporation of 35S into the chain provides a direct measure of the mass of chondroitin sulfate synthesized during the labeling period when the specific activity of the [35S]sulfatein the medium is known. When both labeling precursors are used in double labeling experiments, then, the ratio of 3 Hto 35Sin the monosulfated disaccharides derived from cho~droitinasedigests of chondroitin sulfate provides a reliable measure of the specific activity of the 3H in the GalNAc residue, and the ratio of ‘H in hyaluronan to 3H in the chondroitin sulfate provides a measure of the ma s s of hyaluronan synthesized during the labeling period (59).

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Brefeldin A is a fungal metabolite that inhibits nucleotide activation steps required to recruit coat proteins from cytosol to intracellular membranes in the process of forming vesicles. This prevents the formation of vesicles involved in antegrade transport through the cell (60). This causes l ) disassembly of the Golgi complex with redistribution of Golgi components to the endoplasmic reticulum by retrograde transport, 2) complete blockage of transport between the trans Golgi cisternae and the trans Golgi network, and 3) blockage of exocytosis to the extracellular matrix and of the delivery of cell surface proteins to the plasma membrane. Chondroitin sulfate synthesis in chondros~comachondrocytes treated with brefeldin A is rapidly inhibited, to -1% within 30 mins, and returns to -20% above control levels when the reagent is removed (61). Conversely, hyaluronan synthesis, as measured by inco~orationof 3H-glucosamine, remained constant at a level -50% above control for at least 8 hours in the presence of the reagent. This indicates that the site of hyaluronan synthesis is not dependent upon maintaining normal intracellular trafficking. Additional results with brefeldin A suggest further subcompa~mentalization of glycosaminoglycan synthesis within cells. Bovine chondrocytes also show complete loss of chondroitin sulfate synthesis on aggrecan, as well as loss of keratan sulfate on fibro~odulin,a small interstitial proteoglycan (62).

significant amounts of unsulfated chondroitin continued to be synthesized, but exclusively on decorin, another small leucine-rich proteoglycan (see Chap. 10). This indicates that elongation, but not sulfation, of chondroitin on decorin occurs in a Golgi compartment that is unaffected by brefeldin A, in contrast to chondroitin sulfate synthesis on aggrecan. Mouse mastocytoma cells treated with brefeldin A continued to synthesize both heparin and chondroitin sulfate, but with reduced sulfation, shorter chains, and at a rate only - 25% of control (63). Addition of a P-xyloside exogenous acceptor to intact cells increases the net synthesis of chondroitin sulfate, but is unable to restore significant amounts of chondroitin sulfate synthesis in the brefeldin A treated cells. This suggests that chondroitin sulfate chain synthesis on P-xylosides may require transport of the xyloside from an early Golgi c o ~ p ~ t m e nperhaps t, where addition of the galactose residues occurs, to later compartments where chondroitin sulfate elongation can occur, The effects of brefeldin A on chondroiti~sulfate synthesis in chon~rocytes,then, suggest that the flow of the core protein of aggrecan through the antegrade pathway is disrupted at a critical step. Once the reagent is removed, and antegrade flow is restored, the a c c u ~ u l a t ~ofo core ~ protein behind the critical step is released, probably accounting for the overshoot in chondroitin sulfate synthesis (64). Overall, the results with brefeldin A indicate that i n t e ~ p t i o nof normal vesicular flow through the Golgi and trans Golgi c o m p a ~ ~ e nrapidly ts shuts down synthesis of chondroitin sulfate on aggrecan by preventing entry of the core protein acceptor and by disrupting the organization of the enzymes required. They also indicate that the site of hyaluronan synthesis continues to have access to enzymes and substrates in the face of an acute blockage of intracellular transport pathways.

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The effects of permeabili~ationof chondrocytes with staphylococcal hemolysin on chondroitin sulfate and hyaluronan synthesis are even more dramatic (65). This toxin forms heptameric pores (2- 3 nm in diameter) in the plasma membrane which allow small molecues of -2000 Da or smaller to diffuse into and out of the cytosol (Fig. 9). During the first 4 hr after treatment with the toxin, i n t r a ~ ~ l l u lar ATP diffuses into the medium, reducing cytosolic ATP concentration at least ~00-fold,and chondroitin sulfate synthesis decreases to 10%. In contrast, hyaluronan synthesis, using [ 3 H ~ g l u c o s ~ ias n ea precursor, is sustained at a high level, >70% even 24 hr after treatment by which time ATP levels are below detection limits in both medium and cells, and c~ondroitinsulfate synthesis is

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Schematic showing pores formed from heptamers of a-hemolysin in the plasma membrane. See text for details.

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only l-2%. Refeeding during the last 3 hr of the 24-hr treatment increased the ATP content of the permeabilized chondrocytes and hyaluronan synthesis recovered further, to -95% of control, while chondroitin sulfate synthesis increased only to -8%. Interestingly, synthesis of both chondroitin sulfate and hyaluronan in either intact or permeabilized cells was inhibited in a dose-dependent response to increasing concentrations of arsenate, a metabolic inhibitor of ATP synthesis. This indicates that both require a metabolic source of ATP to sustain synthesis. The effects of permeabilization on ~yaluronansynthesis pose a puzzle. the one hand, metabolic ATP is required, while on the other, hyaluronan synthesis continues almost unperturbed when cytosolic levels of ATP are reduced below the levels of the detection assay. The cytosolic UDP-sugar substrates should also diffuse out of the ~ e r m e a ~ i l i z ecells, d and their decreased concentrations can provide an explanation f or the inhibition of chondroitin sulfate synthesis. The antiport m e c h a n i s ~would become inef~cientfor lack of ligands, which in turn would reduce the concentrations of the substrates in the lumen of the chondroitin sulfate synthesis compartment. However, if the cytosolic substrates are in direct contact with the hyaluronan synthase, the >SOO-fold decrease would make the substrate concentrations at the site of hyaluronan synthesis well below the

values reported for the enzyme activity (66). A possible mechanism to account for these results would be functional compartmentalization of the hyaluronan synthesis machinery. If the hyaluronan synthase is part of a multienzyme complex, for example, then products from the various steps required to convert glucose and glucosamine to the UDP-substrates could be direct donors to the next enzyme reaction and not in direct contact with cytosolic pools. The multienzyme complex would have to include access to a source of ATP. The mRNA for hyaluronan synthase-2 has a short half-life (2-3 hr) in some cells; for example, in cumulus cells in the preovulatory follicle and hyaluronan synthesis appears to be regulated at the level of transc~ption(67,68). This suggests that hyaluronan synthase-2 has a short half-life. The fact that hyaluronan synthesis is sustained for long times in brefeldin A-treated or a-hemolysin-permeabilized cells appears inconsistent, if the turnover of hyaluronan synthase-2, the operative enzyme in the chondrocytes used in these experiments, is similarly rapid. In this case, the model would have to include the stability of hyaluronan synthase-:! at the site of synthesis, i.e., interruption of the normal egress and regress of the enzyme from the functional compartment. (See Chap. 4 for a discussion of the hyaluronan synthases.)

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While many details of the biosynthetic mechanisms for hyaluronan and chondroitin sulfate synthesis remain enigmatic, they clearly differ in fundamental ways. Almost certainly they arose through independent evolutionary paths. While chondroitin sulfate is widespread among invertebrates, hyaluronan synthesis may have emerged with the notochords (69). The evolution of cartilages with the use of hyaluronan as a scaffold for aggregating chondroitin sulfate proteoglycans created a highly successful partnership which has entertained many investigators since the discovery of these glycosaminoglycans in the 1880s and 1930s, and will no doubt continue to do so for the foreseeable future.

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1. Krukenberg C W . Die chernischen Bestandtheile des Knorpels. Z Biol 1884; 20:

307-326. orner CT. Chemische Studien iiber den Trachealkno~el.Skand Arch Physiol 1889; 1:210-243. 3. Schrniedeberg 0. ober die chemische Z~~saml~enset~ung des Knorpels. Arch Exptl Pathol Pharmakol 1891: 28:354-404.

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4. Levene PA, LaForge FB. On chondroitin sulfuric acid. J Biol Chem 1914; 18:123130. 5. Brimacombe JS, Webber J M. Mucopolysaccha~des:Chemical Structure, Distribution and Isolation. Vol 2. BBA Library, Elsevier Publishing Co. 1964: chaps. 3, 5. 6. Hebting J. Abbau der Chondroitinschwefelsaure iiber krystallinische Produkte. I. Mitteilung. Chondroitin und salzsaurer Chondrosinathylester.Biochem Z 1914; 63: 353-358. 7. Levene PA, LaForge FB. On chondrosin. J Biol Chem 1941; 140:267-277. 8. Wofrom ML, Madison RK, Cron MJ. The structure of chondrosine and of chondroitinsulfuric acid. J Am Chern Soc 1952; 74: 1491-1494. 9. Masamune, H, Yosizawa Z, Maki M. Biochemical Studies on Carbohydrates: On chondrosin and chondroitinsulfuricacid. Tohoku J Exp Med 1951; 55:47-52. 10. Davidson EA, Meyer K. Structural studies on chondroitin sulfuric acid. I. The nature of chondroitin. J Am Chem Soc 1954; 76:5686-5689. 11. Davidson EA, Meyer K. Structural studies on chondroitin sulfuric acid. 11. The glucuronidic linkage. J Am Chem Soc 1955; 77:4796-4798. 12. Wolfrom ML, Juliano BO. Chondroitin sulfate modifications: I. Carboxy-reduced chondroitin and chondrosine. J Am Chem SOC1960; 82:1673- 1677. 13. Hoffman P, Linker A, Meyer K, The acid mucopolysacchairdes of connectivetissue. III. The sulfate linkage. Biochim Biophys Acta 1958; 30:184- 185. 14. Mathews MB. Isomeric chondroitin sulphates. Nature 1958; 181:421-422. 15. Orr SFD. Infra-red spectroscopicstudies of some polysaccharides.Biochirn Biophys Acta 1954; 14:173-181. 16. Linker A, Hoffman P, Meyer K, Sampson P, Kor n ED. The formation of Llnsaturated disaccharides from mucopolysaccharides and their cleavage to a-keto acid by bacterial enzymes. J Biol Chem 1960; 235:3061-3065. 17. Suzuki S. Isolation of novel disaccharides from chondroitin sulfates. J Biol Chern 1960; 235:3580-3588. 18. Meyer K, Davidson E, Linker A, Hoffman P. The acid mucopolysaccharides of connective tissue. Biochim Biophys Acta 1956; 21:506-5 18. 19. Meyer K, Smyth EM. On glycoproteins VI. The preparation of chondroitinsulfu~c acid. J Biol Chem 1937; 119:507-510. 20. Shatton J, Schubert M. Isolation of mucoprotein from cartilage. J Biol Chem 1954; 2111565-573. 21. Muir H. The nature of the link between protein and carbohydrate of a chondroitin sulphate complex from hyaline cartilage. Biochem J 1958; 69:195-204. 22. Anderson B, Hoffman P, Meyer K. The 0-serine linkage in peptides of chondroitin 4-and 6-sulfate. J Biol Chem 1965; 240:156-167. 23. Partridge SM, Davis HF, Adair GS. The chemistry of connective tissues. Biochem J 1961; 79:15-26. 24. Rodeh L, Armand G. Structure of the chondroitin 4-sulfate-protein linkage region: isolation and characterization of the disaccharide 3-O-~-D-glucuronosyl-D-ga1~ctose. J Biol Chem 1966; 241:65-70. 25. Lindahl U, Rod& L. The chondroitin 4-sulfate-proteinlinkage. J Biol Chem 1966; 24112113-21 19.

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26. Rodin L, Smith R. Structure of the neutral trisaccharide of the chondroitin 4-sulfateprotein linkage region. J Biol Chem 1966; 2415949-5954. 27. Oegerna TR, Kraft EL, Jourdian GW, VanValen TR. Phospho~lationof chondroitin glycans from the Swarm rat chondrosarcoma.J Biol Chem 1984;

28. Meyer K, Palmer JW. The polysaccharide of the vitreous humor. J Biol Chem 1934; 629-634. 29. Weissrnan B, Meyer K. The structure of hyalobiuronic acid from umbilical cord. J Am Chem SOC1954; 76:1753-1757. 30. Yamagata T, Saito H, Habuchi 0, Suzuki S. Purification and propedes of bacterial chondroitinases and chondrosulfatases.J Biol Chem 1968; 243:1523-1535. 31. Saito H, Yamagata T, Suzuki S. Enzymatic methods for the deter~nationof small quantities of isomeric chondroitin sulfates. J Biol Chem 1968; 243:1536--1542. 32. Plaas AWK, Hascall VC, Midura RJ. Ion exchangeHPLC microanalysis of chondroitin sulfate: quantitative derivatization of chondroitin lyase digestion products with 2-aminopyridine. Glycobiology 1996; 6:823-829. 33. Plaas AHK, Wong-Palms S, Roughley PJ, Midura RJ, Hascall VC. Chemical and i ~ u n o l o g i c aassay l of the non-reducing terrninal residues of chondroitin sulfate from human aggrecan. J Biol Chem 1997; 272:20603-20610. 34. Ludwigs U, Elgavish A, Esko JD, Meezan E, Rodin L. Reaction of unsaturated uronic acid residues with mercuric salts. Biochem J 1987; 45:795-804. 35. Plaas AHK, West LA, Wong-Palms S, Nelson FRT. Glycosaminoglycan sulfation in human osteoarth~tis.Disease-related alterations at the non-reducing termini of chondroitin and derrnatan sulfate. J Biol Chern 1998; 273:12642-12649. 36. Kimura JH, Hardingham TE, Hascall VC. Assembly of newly synthesizedproteoglycan and link protein into aggregates in cultures of c h o n ~ o s ~ c o m chondrocytes. a J Biol Chem 1980; 255:7134-7143. 37. Morales TI, Hascall VC. Effects of interleu~n-land lipopolysaccharides on protein and carbohydratemetabolism in bovine articularcartilage or gan cultures. Connective Tiss Res 1989; 19:255-275. 38. Grebner EE, Wall CW, Neufeld EF. Glycosylation of serine residues by a uridine diphosphate-xy1ose:proteinxylosyltransferase from mouse mastocytoma. Arch Biochem Biophys 1966; 116391-398, 39. Robinson HC, Telser A, Dorfrnan A. Studies on the biosynthesis of the linkage region of chondroitin-sulfateprotein complex. Proc Natl Acad Sci USA 1966; 56: 1859- 1864. 40. Baker JR, Rod6n L, Stool~llerAC. Biosynthesisof chondroitin sulfate proteoglycan: xylosyl transfer to Smith-degraded cartilage proteoglycan and other exogenous acceptors. J Biol Chem 197-2;247:3838-3847. 41 Rod& L. St~ctureand metabolism of connective tissue proteoglycans. In Lennarz WJ, ed. Biochemistry of Glycoproteins and Proteoglycans.New York: Plenum Press, 1980:267-371. 42. Vertel BM, Walters LM, Flay N, Keams AE, SchwartzNB. ~ylosylationis an endoplasmic reticulum to Golgi event. J Biol Chem 1993; 268:11105-1 1112. 43 Lohrnander LS. Shinomura T. Hascall VC, Kimura JH. Xvlosvl transfer to the core

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44.

45.

46. 47

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48. 49.

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52. 53. 54.

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57.

58.

protein precursor of the rat chondrosarcoma proteoglycan. J Biol Chem 1989; 264: 18775- 18780. Rohrmann K, Niemann R, Buddecke E. Two N- acet yl gal act osami nyl t r ansf e~ases are involved in the biosynthesis of chondroitin sulfate. Eur J Biochem 1985; 148:463469. Hirschberg CB, Robbins PW, Abeijon C. Transporters of nucleotide sugars, ATP, and nucleotide sulfate in the endoplasmicreticulum and Golgi apparatus, Annu Rev Biochem 1998; 67:49-69. Okayama M,Kimata K, Suzuki S. The influence of p-nitrophenyl-P-xylosideon the synthesis of proteochondroitin sulfate by slices of chick cartilage. J Biochern (Tokyo) 1973; 74:1069-1073. Robinson HC, Brett MJ, Tralaggan PJ, Lowther D, Okayama M. The effect of Dxylose, beta-D-xylosides and beta-D-galactosides on chondroitin sulphate biosynthesis in embryonic chicken cartilage. Biochem J 1975; 148:25-34. Schwartz NB.Regulation of chondroitin sulfate synthesis. Effect of P-xylosides on synthesis of chondroitin sulfate proteoglycan, chondroitin sulfate chains, and core protein. J Biol Chem 1977; 252:6316-6321. Brandli Ab', Hansson GC, Rodriquez-Boulan E, Sirnons K. A polarized epithelial cell mutant deficient in translocation of IJDP-galactose into the Golgi complex. J Biol Chem 1988; 263:16283-16290. Toma L, Pinhal MA, Dietrich CP, Nader HB, Hirschberg CB. Transport of IJDPgalactose into the Golgi lumen regulates the biosynthesis of proteoglycans. J Biol Chem 1996; 271:3897-3901. Prehm P. Synthesisof hyaluronate in differentiatedtera~oc~cinoma cells; characterization of the synthase. Biochem J 1983; 211:191-198. Mason RM,Kimura JH, Hascall VC. Biosynthesis of hyaluronic acid in cultures of chondrocytes from the Swam rat chondrosarcoma.J Biol Chern 1982; 257:22362245. Mason RNI, D'Arville C, Kimura JH, Hascall VC. Absence of covalently linked core protein from newly synthesized hyaluronate. Biochem J 1982; 207:445-457, Prehrn P. Hyaluronate is synthesized at plasma membranes. Biochern J 1984; 220: 597-600, Philipson LH, Schwartz NB. Subcellular localization of hyaluronate synthetase in oligodendroglioma cells. J Biol Chem 1984; 259:5017-5023. Su D, Robyt JF. Dete~inationof the number of sucrose and acceptor binding sites for Leuconostoc mesenteroides B-512FM dextransucrase, and the c o n ~ ~ a t i of on the two-site mechanism for dextran synthesis. Arch Biochem Biophys 1994; 308: 47 1-476. Yanagishita M, Salustri A, Hascall VC. Determination of the specific activity of hexosamine precursors by analysisof doublelabeled disaccharidesfrom chondroitinase digestion of chondroitin/dermatan sulfate. Methods in Enzymology 1989; 179: 435-455. Imai V, Yanagishita M, Hascall VC. Effect of sulfate depletion on utilization of cysteine as a metabolic source of sulfate in rat ovarian granulosa cells. Arch Biochem Biophys 1994; 312:392-400.

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59. Salustri A, Yanagishita M, Underhill CB, Laurent TC, Hascall VC. Localization and synthesis of hyaluronic acid in the cumulus cells and mural granulosa cells of the preovulatory follicle. Dev Biol 1992; l 5 1:541-55 1. 60. Klausner RD, Donaldson JG, Lippincott-Schwa~zJ. Brefeldin A: insights into the control of membrane traffic and organelle structure. J Cell Biol 1992; 116:10711080. 61. Calabro A, Hascall VC. Differential effects of brefeldin A on chondroitin sulfate and hyaluronan synthesis in rat chondrosarcoma cells. J Biol Chem 1994; 269: 22764-22770. 62, Wong-Palms S, Plaas AHK. Glycosaminoglycan addition to proteoglycans by articular chondrocytes- evidencefor core protein-specific pathways. Arch Biochern Biophys 1995; 3191383-392. 63. Uhlin-Hansen L, Kusche-Gullberg M, Eriksson I, Kjellgn L. Mouse rnastocytoma cells synthesize undersulfated heparin and chondroitin sulfate in the presence of brefeldin A. J Biol Chem 1997; 272:3200-3206. 64. Calabro A, Hascall VC. Effects of brefeldin A on aggrecan core protein synthesis and maturation in rat chondrosarcoma cells. J Biol Chem 1994; 269:22764-22770. 65. Goodstone NJ, Hascall VC, Calabro A. Differentialeffects ~ ~ S t a ~ ~ y l o c o ca ucreu uss a-hemolysin on the synthesis of hyaluronan and chondroitin sulfate by rat chondrosarcoma chondrocytes. Arch Biochern Biophys 1998; 350:26-35. 66. Ng KF, Schwartz NB. Solubilizationand partial p u ~ ~ c a t i of o nhyaluronate synthetase from oligodendroglioma cells. J Biol Chem 1989; 264: 11776-11783. 67. Fulop C, Salustri A, Hascall VC. Coding sequence of a hyaluronan synthase homologue expressed during expansion of the mouse cumulus-oocyte complex. Arch Biochem Biophys 1997; 337:261-266. 68. Tirone E, D’Alessandris C, Hascall VC, Siracusa G, Salustri A. Hyaluronan synthesis by mouse cumulus cells is regulated by interactions between FSH (or EGF) and a soluble oocyte factor (or TGFP1). J Biol Chem 1997; 272:4787-4794. 69. Mathews MB. Connective Tissue: Macromolecular Structure and Evolution. New York: Verlag, 1975:138-139. 70. Wight TN, Heinegird DK, Hascall VC. Proteoglycans: structure and function. In: Hay ED, ed. Cell Biology of Extracellular Matrix, 2d ed. New York: Plenum Press, 1991:45-78.

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University of anc chest er and ~hristie~ospital~~S Trust, an chest er, England

Heparan sulfate proteoglycans (HSPGs) are found in all mammalian organs and tissues which invariably produce more than one HSPG species (1-3). normally consist of two to three HS chains positioned in close proximity to each other along protein cores that direct newly synthesized PGs to cell surfaces or to the extracellular matrix (ECM) (4). The major cell membrane HSPGs are the transmembrane syndecans and the GPI-anchored glypicans (1,2), whereas in the ECNI, especially in basement membranes, the multidomain perlecan and the agrin core proteins are the main HS-bearing species (5,6). Other forms of membrane HSPG include betaglycan, which is unusual in that it contains only one HS chain (7) and the "3-isoform of CD44 present on keratinocytes and activated monocytes (8). Experimentally, HS chains have been shown to interact with a wide and rather bewildering variety of proteins (e.g., growth factors, chemokines, ECM proteins, enzymes, and enzyme inhibitors), and some of these interactions are mediated by specific intrachain sequences (9,lO). Such specificities are assumed to be indicative of significant biological relevance and this is indeed the case for antithro~binand both basic and acidic fibroblast growth factors (b- and aFGF) which are activated by binding to appropriate sequences in HS (1l), The heparan sulfates are a family of related glycosaminoglycans, distinct in structure from the chemically related heparin (12), which encompass a wide

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range of species with common characteristics but also with fine structural variations that impart selectivity and/or specificity to HS-protein interactions (12,13). Although there may be an element of randomness in these variations, there is a significant degree of controlled diversification which is most apparent in the consistent differences in structure of HS from different cell types (14,lS). The celltype related variability most probably arises through a complex interplay of the PG core protein expression pattern in a particular cell and the cellular concentrations and activities of the complex system of enzymes involved in HS biosynthesis. What is clear is that no single HSPG core protein directs the synthesis of its own unique HS species. For example, syndecan- 1 contains different HS structures when isolated from different cells and tissues (16,17). However, it is not known whether there are minor but consistent variations in the structure of HS on different HSPGs produced in the same cell. Another issue which has received little attention is the degree of functional integration between the protein and HS components of HSPGs. I n f o ~ a t i o nis beginning to emerge on the distinct properties of HSPG core proteins, especially the interactions and signaling roles of the cytoplasmic domains of the syndecans (see Chap. 4), but at the present time it is unclear whether these interactions are influenced by protein ligand binding to its constituent HS chains.

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eparan sulfate is composed of alternating a-linked N-acetyl- or N-sulfo"~lucosamine (GlcNAc or ClcNS03-) and alp-linked hexuronic acid (HexA); the complexity of the chain arises because the HexA. is present in two isomeric forms, glucuronate (GlcA) and its C-S epimer iduronate (IdoA), and because of the variable presence of ester-linked sulfates (O-sulfates) at C-6 of the amino sugars and

1 Biosynthesis of heparan sulfate. The precursor of HS is a nonsulfated polysaccharide called heparan composed of GlcNAc-1-4 GlcA repeat sequences. Conversion of heparan to HS (step 1) begins with the conversion of GlcNAc to GlcNS03 by a dual action N-deacetylase/N~sulfotransferase,This is followed by epimerizationof GlcA to IdoA (step 2) and 0-sulfation of IdoA at C-2 (step 3). Finally the GlcNSO, is sulfttted at C-6 and C-3. These steps are catalyzed by specific epimerase'and 0-sulfotransferase enzymes. At each step, only a fraction of potential substrates is modified. In HS about 50% of GlcNAc residues are converted to GlcNSO, and the trisulfated disaccharide formed after step 4 normally comprises less than 10% of total disaccharides.3-0-sulfation of GlcNS03(step 5) is a relatively rare modification and it can occur independently of 6-0-sulfation of the same residue.

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at C-2 of the iduronate residues. Occasionally C-2 of GlcA and C-3 of GlcNSQ,are also Q-sulfated, and the latter is an essential constituent of the antithrombin binding site in HS and heparin (10). HS is initially synthesized directly on primed core proteins [i.e., proteins in which the protein-glycosaminoglycan linkage tetrasaccharide of GlcA - + Gal -+ Gal ”+ Xyl -+ Ser has already been assembled (4)] as a nonsulfated precursor named heparan consisting only of (ClcNAc a1 - +4 GlcA p1 -+ 4) disaccharide repeats, but during chain elongation polymer modifying enzymes begin to convert the precursor heparan to HS (Fig. l). The modi~cationsoccur in an ordered manner and are always incomplete at each stage (18). They begin with the N-deacetylation and N-sulfation of approximately 40-50% of ClcN residues, followed by conversion of ClcA to IdoA and 0-sulfation of iduronates and a ~ i n osugars. All the enzymes acting after the conversion of ClcNAc -+ GlcNS0, require the N-sulfate group for substrate recognition. N-sulfated disaccharide units enriched in IdoA,2S residues largely occur in clusters or domains (S-domains) of 2-819 units in length which can be excised by the enzyme heparinase I11 (heparitinase) (19; Fig. 2). C-6 sulfation of GlcNS0,- occurs both in S-domains and in sections that flank the S-domains where N-sulfated and N-acetylated disaccharides occur in alternate or mixed sequences. GlcNAc residues are also often C-6 sulfated in these sequences (14,15). Thus in HS there is a very close association between N-and Q-sulfate groups. Polymer sulfation is essential for protein recognition and the S-domains, along with their adjacent mixed sequences, are the regions of hyperv~iabi~ity that give the distinctive characteristics to WS species from different cells (l2,l.S). The spacing between the S-domains is reasonably consistent across almost the entire HS family. The intervening sections are made up of both the mixed sequences and repeating N-acetylated disaccharide units that are devoid of Q-sulfate groups (Fig. 2). As will be discussed later, the spatial a~angementof the S-domains is an i ~ p o r t a n feature t of recognition of oligomeric proteins. The one notable exception to the standard molecular design of HS is that from rat liver (21) in which two or three S-domains are positioned distal to the core protein but with little N-acetylated spacing between them (Fig. 2, structure B). Heparin is synthesised by a similar mechanism to HS, but the polymer modifications are significantly more extensive with the majority of the chain consisting of trisulphated disaccharides of structure GlcNSQ3,6S a i -4 IdoA,2S (3,14). A universal characteristic of HS is the presence of a long, unmodified sequence of 10 or so N-acetylated units proximal to the core protein; the first Sdomain i s a further 6 or so disaccharides “downstream” (20,21), What is the purpose of this N-acetylated region? Its conservation in all HS species argues for a key function. One possibility is that N-acetyl-rich regions are more flexible than the S-domains which will adopt a fairly rigid helical structure (22). A flexible sequence should allow the chain greater freedom of movement which

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Models of the domain structuresof two forms of heparan sulfate. (A) ~ o m ~ ~ n form of HS with S-domains spaced along the chain separated by N-acetyl rich sequences of low sulfation which can be degraded by the enzyme heparinase I11 (hepa~tinase).(B) Liver variant in which three S-domains are in close proximity in the distal region of the polymer chain. The short, low sulfated regions between the S-domains are susceptible to heparinase 111. (In both types of EIS an extended N-acetylated sequence is positioned at the reducing end of the chain proximal to the core protein.)

would in turn assist in the “capture” of soluble protein ligands such as growth factors and enable the chain to adapt to the complex architecture of the EC

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The growth, adhesion, and migration of cells are inte~elatedprocesses and all are subject to regulatory mechanisms which involve HSPGs. In general the HSPGs act as membrane coreceptors operating in dual receptor systems together with other cell surface receptors (e.g., the integrins for ECM proteins, tyrosine kinase receptors for growth factors) to regulate the behavior of cells (23). The term dual receptor is used rather loosely because there is no evidence that are physically linked to other receptors such as the two-component receptor system for hemopoietic growth factors or the type I and type IT receptors for TGF-p.

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The influence of membrane HSPGs on the formation of focal adhesions is a good example of close cooperation between an ECM receptor ( asp1 integrin) and a specific HSPG, syndecan-4 (24) (refer to Chap, 4) The impact of HSPGs on cell growth and migration occurs mainly because many growth factors and chemokines (chemoattractants for leukocytes) bind to HS chains and in some important instances this binding is an essential step in their activation of target cells (25). The fibroblast growth factors (FGF), vascular endothelial growth factor (VEG hepatocyte growth factor (HGF), and neurostimulatory molecules such as midkine and HI(-GAM (heparin-binding growth-associated molecule) can all be described as HS-dependent proteins, whereas for the chemokines their binding to HS on cell surfaces and in the ECM is believed to be significant as a mechanism for restricting diffusion and creating relatively high local concentrations of ligand that exceed the threshold for receptor activation. Proteins bound to HS are also rotected from proteolytic attack and this may prolong their biological lifetime. A very extensive literature has built up in recent years on the binding and regulation of growth factors and cytokines by HS. In the following sections we will consider both the saccharide and protein s t ~ c t u r amotifs l that give specificity to these interactions. We will also discuss some of the latest findings in the emerging field of HSPGs in m ~ m a l i a nand ~ r o s o ~development. ~ i Z ~

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1. Reco~nitionProperties

Acidic and basic FCF (aFGFIFGF-1 and bFGFIFGF-2, respectively) are the prototypic members of the FCF family and they both depend on HS for optimum cell signaling and stimulation of cell growth (26). They are quite small (approximately 18 kDa), compact, monomeric proteins and their highly homologous HS binding regions are formed by a composite of discontinuous surface loops that extend from a core of centrally positioned p strands (2’7,28). The HS binding site of bFGF accommodates five sugar residues, including a key IdoA,2S residue (29), and a minimal binding sequence of GlcNS03a1 -4 IdoA(2S) a 1 4 GlcNS03al4 IdoA a 1 -4 ClcNS03 can be deduced from x-ray analysis of cocrystals of heparin saccharides and bFGF (30). The cocrystal structure also revealed that the 60-sulfate groups of heparin were not involved in binding to the growth factor and this is consistent with biochemical data which indicated that the important functional groups are N- and 2-0-sulfates and carboxylates (29,31). A model of the major points of contact between an HS pentasaccharide and amino acid side chains of bFGF is shown in Figure 3, A unique feature of the iduronate residue

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Model of the interaction of bFGF with the ~ n i m aheparan l sulfate pentasaccharide. The model is based on x-ray analysis of cocrystals of heparin saccharides and bFGF (30). The HS binding site in bFGF is formed from approximation discontinuous loops which bring positively charged and polar ami no acid side chains into an appropriate configuration for recog~itionof the negatively charged saccharide helix. High-af~nityinteractions are shown with solid lines and lower-affinity ones with dashed lines. The amino acids that make contact with sugars 1 and 2 form a high-af~nitysubsite. The a ~ a n g e ~ e n t of the amino acids is schematic.

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is its c o n ~ ~ ~ a t i oplasticity, nal and of the three equienergetic conformers identified by NMR,two are selected and stabilized in the s a c c h ~ i d e - b F ~cocrystals, F the IdoA,2S adopting a 'C4chair and the IdoA a 'Soskew boat (30). The ~exibility of the iduronate ring may be an adaptive mechanism which "molds' ' the saccharide to the complex geometry of the protein surface. The overall topography of the HS-binding region is strongly conserved between aFGF and bFGF but with some differences in amino acid composition which may explain why the two growth factors bind to different s a c c h ~ i d estructures, aFGF requiring one or more 6-0-sulfates, in addition to N- and 2-0-sulfates (31; al so see Sec. 3 below). These differences in binding sequence are very significant because they allow cells to independently control their responses to aFCF and bFCF by modulation of HS sulfation patterns. One of the first studies to indicate the importance of HS for FGF regulation come from Harper and Lobb (32) who demonstrated that one of the lysine residues in the saccharide binding site (Lys 118 in aFCF, equivalent to Lys 126 in bFGF) was in a unique chemical environment, as revealed by its high sensitivity to methylatio~.~odificationof Lys 118 in aFGF caused a dramatic fall in heparin affinity and cell growth stimulation and it was predicted that the activity of aFGF

(and by inference bFGF as well) would be significantly enhanced by the presence of HS (32). This prediction has been amply supported by several important studies in which FGF-receptor bearing cells, deficient in HS, were used to show that receptor binding and cell activation by bFGF are dependent on HS (33,34) although some reports indicate that bFGF can act independently when the growth factor is used at high concentrations (35). It is significant that HS, heparin, or selected saccharides derived from them by enzymic scission will form soluble biologically active complexes with bFGF (and aFGF) and bypass the requirement for HS to be present on the cell surface. This property has provided a very useful means of screening saccharides for FGF activation, and although valuable data have been acquired from such screens (see below) there remain large gaps in our knowledge on how membrane HSPGs mediate the activation and delivery of the FGFs to their signaling receptors on the cell surface.

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Active Site for bFGF in Heparan Sulfate

Occupancy of the HS-binding site of bFGF by short saccharides that contain the ~ i n i m a pentasacc~aride l recognition sequence of moderate affinity fails to activate the growth factor (31,36,37).A long S-domain (comprising seven disaccharides) named Oligo-H that contains an internal repeat of five 1doA,2S“-Gl~NS0~ units was isolated from human skin fibroblast HS by treatment with heparinase 111(heparitinase) and bFGF affinity chromatography (38). This saccharide bound with high affinity to bFGF. The N-sulfates and 2-0-sulfates were important for the interaction, and related sequences in which the 2-0-sulfate content was reduced but with a compensating increase in 6-0-sulfates displayed only weak bFCF binding activity. Other work supported the notion that relatively long sequences of the Oligo-H type formed high affinity sites, but curiously these sequences not only failed to elicit a mitogenic response to bFGF but actually inhibited the growth factor (37). It appears that a bFGF-active site in HS must comprise a “core’ ’ of IdoA,2S-GlcNS03 repeats (three of these units in a decasaccharide seems to be the minimum require~ent)substituted with one, or at most two, 6-0sulfate groups (37,39,40). At present the location of these 6-Osulfates along the active site sequence is unknown. Because 6-0-sulfates are not required for binding to bFGF, their role in growth factor activation is unclear. In fact, the mode of action of HS in strongly promoting bFGF activity is not resolved and a number of models have been proposed. The most straightfor~ardone is that appropriate saccharides induce an activating conformational change in bFGF analogous to the way in which heparin and HS activate antit~ombin111(41,42). This analogy can be taken further because in order to accelerate the binding of antithrombin to thrombin the saccharide acts as a catalytic template, and to do this the pentasaccharide must be extended by six additional sulfated disaccharide units which bind the thrombin (43). This has the effect of bringing the protease and its inhibitor into

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close proximity, and when the protein-protein complex has formed the saccharide dissociates from it. The action of bFGF requires it to also bind to another protein, the FGF-receptor (FGFR), and the need for a longer saccharide than a minimal pentarner suggests that the active site incorporates two subsites, one for bFGF and one for the FGFR (Fig. 4a) ; the 6-0-sulfates could be part of the FGFR subsite

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Proposed mechanisms of interaction of heparan sulfate with bFGF and FGFR. The active site for bFGF in HS is an extended S-domain of 10 or more sugar residues. The active sites may function to bring bFGF and its receptor into close proximity or they may create an “active” dimeric complex of bFGF with two monomers disposed in a “cis” or “trans” co~~guration, the latter in an antiparallel orientation.

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(37). The FGFRs contain a conserved positively charged region in their ectodomains that binds to HS and heparin and this makes the template idea an attractive one (44). A different perspective on the template concept stems from the fact that the active site sequences comprised of 10 or more sugars could accommodate two bFGF molecules and thereby create a dimeric ligand for presentation to the FGFRs. FGF dimers should, in principle, facilitate receptor dimerization which is essential for signal transduction in receptor tyrosine kinases. ~xperimentalevidence using different techniques including PAGE, dynamic light scattering, and NMR all clearly show that bFGF dimers form in the presence of N- and 0 sulfated saccharides, but the concentrations of protein used in these studies are higher than what is required to induce cell growth (45,46). In models based on these studies, the general consensus is that the bFGF dimers form by the close association of monomers on the same side of the saccharide (i.e., “cis”-dimers). In contrast a recent x-ray analysis of cocrystals of a heparin decasaccharide and aFGF revealed what was claimed to be biologically active dimers in a “trans” orientation (47) (Fig. 4b). These dimers lacked a protein-protein interface and the receptor-binding regions were directed away from the saccharide chain. It will be important to determine whether the dimer symmetry in these models is compatible with high-affinity binding and dimerization of the FGFRs. It might be expected if dimerization is necessary for the activation of low solution concentrations of bFGF that binding of the monomer to HS would show positive cooperativity (i.e., binding of one bFGF molecule to HS would increase the probability of binding a second one in close proximity) but there is no evidence for this (29). Springer and colleagues have used protein structure-based mutagenesis and molecular modelling to study the nature of the bFGF signaling complex (48). Their analysis suggested that bFGF is active as an HS-bound monomer with two receptor binding interfaces that interact with separate receptors in a similar way to the dimerization of growth hormone receptors by the monomeric ligand (49). In the Springer model HS is essential to maintain a stable ternary protein complex. Recent studies from our laboratory (49a) using covalent protein-saccharide crosslinking favor the view that bFGF bound to HS is active as a monomer. There are a number of other issues that need to be considered in the context of signaling by bFGF and other members of the FGF family. The cognate receptors, the FGFRs, are a complex group of proteins encoded by four separate, alternatively spliced genes which give rise to many isoforms that vary in their ligand binding properties, and kinase domains (50). In view of this complexity there may be more than one mechanism of activation (51). The type of membrane HSPG involved in FGF binding may also have an important bearing on activation. Syndecans- 1, - 2, and -4 and glypican-l potentiate bFGF activity when coexpressed with FGFR-1 in K562 cells (52), but a surprising and still unexplained result is that the soluble ectodomains of these HSPGs, purified from lung fibro-

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blast cell cultures, inhibit bFGF, whereas a preparation of perlecan from the same cells was a potent activator ( 53) .The syndecan-1 ectodomain from epithelial cells was also shown to be an inhibitor of bFGF even though it could be demonstrated that its high molecular weight S-domains (releasable by heparinase 111) were strong activators (54). These findings suggest that the location of active site sequences in HSPGs is important for cell activation and that steric constraints prevent bFGF bound to the soluble ectodomains from engaging in productive interactions with the FGFRs. In principle, shedding of HSPGs from cell surfaces could be a mechanism for suppressing cell responses to bFGF. Syndecan- 1 ectodomains are released into wound fluids (54), but this may be a specialized response to injury and there are no reports so far of significant syndecan shedding in normal tissues.

3. Activation of aFGF: Comparison with bFGF

d?GF binds heparin and HS with weaker affinity than bFGF ( 23) although, like bFGF, the aFGF monomer interacts with five sugar residues in the heparin chain (55).It was mentioned earlier that in contrast to bFGF, aFGF appears to recognize 4-0-sulfate groups in complementary HS/heparin~bindingsequences. The two growth factors are similar in that activity is induced most effectively by N-and 0-sulfated saccharides of 10 or more sugars in length, but they can be distinguished on the basis of their sensitivity to selective desulfation of heparins ( 32, 37) .The molecular details of these differential responses are rather vague and poorly understood. However, they have biological relevance because they enable cells to respond independently to the growth factors. An example of this occurs in the developing murine nervous system in the period of transition from growth to differentiation of neural progenitors. During this time the progenitors switch their responsiveness from bFGF to aFGF (56) and this appears to be due to a change in the structure of cell surface HS rather than an alteration in FGF expression ( 57) .Another example of cells producing HS with distinct binding and activation properties has emerged from studies on mammary cell cultures which produce two populations of HS chains, and whereas both are able to activate aFGF only one induces a mitogenic response to bFGF (58). It is interesting that the inactive species has a high affinity for bFGF (212-30 nM Kd)and after treatment with heparinase 111, and release of the S-domains, the preparation acquires the capacity to activate bFGF (58). This finding again emphasizes the importance of analyzing the structure of whole HS chains, including the positioning of the growth factor binding sites and details of their adjacent mixed sequences. These regions contain an intermediate level of sulfation, including an appreciable proportion of 6-0-sulfate groups (1S), and they could influence the activating potential of HS-FGF complexes possibly through interference with the receptor docking mechanism.

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Hepatocyte growth factorIscatter factor (HGFISF) is a member of the plasminogen-related growth factor family, together with macrophage-stimulating protein P, also known as HGF-like protein). Both are known to bind to heparin, but the GAG-binding properties of HGFISF have been investigated in detail. /SF is a paracrine growth, motility, and morphogenic factor secreted by mesenchymal cells and active primarily upon epithelial and endothelial cells, but also hemopoietic progenitor cells. It appears to have a major role in organ development in the embryo and organ regeneration and wound healing in the adult. HGFISF is secreted as a single chain proform of -90 quently proteolytically cleaved at a single peptide bond giving rise to a disulfidelinked heterodimer, cornprised of a -60 kDa a-chain and a -30 IDa P-chain. These two chains are structurally highly dissimilar. The a chain possesses a highly disulfide-bonded modular structure, with an N-terminal hairpin loop and four Kringle domains, whilst the P-chain resembles an inactive serine protease domain of plasminogen. Deletion mutations within HGFISF have implicated the N-terminal hairpin loop of the a-chain as being primarily responsible for GAG binding, with a possible secondary interaction within the second mingle domain (59). Recent NMR analysis of the a~ino-terminaldomain of HGFISF (60) has revealed that the hairpin loop is comprised of a two-turn a-helix (residues 6775) followed by two P-strands (residues 86-90 and 95-99). A cluster of basic residues, cornprising R73, R76, and K78, occurs within the hairpin loop and spatially close to a second cluster, comprising K60, K62, and K63, which is just outside it. Deletions of both R73 and R76 result in a 50-fold reduction in heparin affinity (61) suggesting that these may be particularly important residues. Howwithin another basic cluster in the hairpin loop, comprising K91, , also have significant though lesser effects (61) suggesting that the GAG-binding site may involve quite an extensive array of positively charged residues. The GAG-binding properties of HGFISF are pa~icularlyinteresting and clearly distinct from those of bFGF. HGFISF binds strongly to the sulfated domains of H§ (62,63) with a Kd of 0.2-3 nM (64), but uniquely this interaction does not require the presence of N-sulphate groups, and indeed HGFISF also binds to dermatan sulfate with a Kd of 20 nM (65). This suggests that HGFI SF recognizes a core sequence of ~IdoA-HexNR(S)]~~, in which the presence of iduronates is critical but the identity of the hexosamine (GlcN or GalN) and its N~substituent(NAc or NS03) is not (62,65). Also, hexosamine 0-sulfation (at 6-6 in H§ and C-4 in D§) appears to be important, but not 2-0-sulfation of IdoA, which is nevertheless tolerated. The GAG binding site on HGFISF appears to accom~odatea minimum hexasaccharide sequence (62), though a precise binding sequence remains to be determined.

Cells surface GAGs are clearly involved in the optimal expression of the biological activity of HGFISF, though their mechanism of action remains unclear. Though there are some parallels with bFGF, there are also some notable differences. Cells which are naturally, or as a result of mutation, depleted in H G ~ / S F binding GAGs have a reduced response to HGF/SF (66,67), and indeed chloratetreated cells, which completely lack sulfated GAG expression, lose all responsiveness to HGFISF (67a). In the latter case exogenous GAG chains cannot functionally replace the loss of cell surface PG, in contrast to the known behavior of bFGF under similar conditions, and implying a very specific role for intact, cell suxface-associated PG. It has also been reported that the binding of HGFISF to its specific cell surface tyrosine kinase receptor may not in itself be GAG-dependent (61,68,69), suggesting that the potentiating activity of GAGs may lie elsewhere in receptor activation. For example, it has been suggested that they may be important in promoting the dimerization of HGFISF and thus the necessary dimerization of ligand-bound receptors (66,67,69).

In addition to monomeric proteins which possess a single HS-binding site, there are a large number of proteins with two or more such sites by virtue of their natural occurrence as either noncovalent (e.g., interferon-y, midkine, and many chemokines) or covalent (e.g., PDGF, VEGF, and TGF-P) oligomers (primarily dimers). By contrast, the abundant extracellular matrix protein fibronectin is not only a disulfide-bonded dimer, but each monomer possesses two independent and st~cturallydistinctive HS-binding sites. Interestingly, there is no common primary sequence or structural motif which defines a HS-binding site in these proteins, and indeed the ways in which these sites have evolved, and their likely biological roles, are as diverse as the different types of proteins themselves.

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These include interferon-y, the midkine/HB-GA~group, and the structurally distinct a-and P-chemokine subfamilies. l.

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IFN-y is secreted by T-cells and acts upon seven spanner (or serpentine) receptors present on most types of cell. The IFN-y monomer comprises a core of six ahelices together with an extended, unfolded sequence at the C-terminal region which plays an important role in the biology of IFN-y. The active dimer forms by an antiparallel interlocking of two monomers, giving rise to a compact globular

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structure from which the flexible C-termini extend at opposite sides (70). The symmetry of the dimer enables it to bind two IFN-y receptors with high affinity (KDof 0.26 mM).Affinity for HS resides solely in the extended C-termini (residues 125-143) in which two clusters of basic amino acids occur, I2%TC and 137RCRR14~, named C 1 and C2, respectively, with the C 1 sequence contributing most to the interaction (71). The C l region is also directly involved in binding to the IFN-y receptor. Complexation with HS, as well as inhibiting clearance of the protein from the circulation, also appears to protect the protease-sensitive C terminal region of IFN-y and this helps to maintain its receptor binding activity (72). A consequence of the disposition of the HS-binding sites at opposite sides of' the molecule is that the respective binding site on the HS chain is potentially quite large. Experimentally, a protected oligosacch~ideof 10 ld la (equivalent to -20 disaccharides) was isolated from a HS-IFN-y complex after extensive digestion with heparinase I11 (73). Structural analysis of this protected sequence revealed that each protein monomer interacted within adjacent S-domains of the S, with the connecting N-acetylated domain bridging the monomers (but still protected from the heparinase I11 enzyme either by steric exclusion or by additional interactions with the protein). Because the binding regions for HS and receptor overlap in IFN-y, the protein-HS complex is inactive (74). The release of IFN-y, from what may be high local concentrations sequestered by HS, will be determined by the dynamic equilibrium between the bound and free forms, and release may be accentuated by conditions in the microenvironment that affect protein conformation.

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The chemokines are cell migration factors, acting principally on leukocytes, that stimulate movement of different populations of cells from blood to tissues, and their activities are enhanced at sites of tissue injury and in~ammation.Two subfamilies, the a- and P-chemokines, are recognized based on whether a pair of N-terminal cysteines are separated by a single amino acid (CXC) or are contiguous (CC). Platelet factor 4 (PF4) and interleu~in-8(IL-8) are members of the a-chemokine (or CXC chemokine) subfamily and possess very similar monomeric threedimensional structures, in which an ~ - t e ~ i n P-strand-like al structure runs into a core comprised of a three-stranded a n t i p ~ a l ~P-sheet el across which lies a Cterminal a-helix (75-77). In PF4 the side chains of two pairs of almost c~ntiguous IIKK66)protrude from the same exposed face of the a-helix, IL-8 on the other hand possesses a longer, more interrupted basic sequence 54KENWV~ R V V E ~ F L ~in6the * )equivalent a-helix, which is primarily r~sponsiblefor HS/heparin binding (78). In both proteins the monomers self-associate edge-toedge in an antiparallel orientation giving rise to a flattened dimer (Le., two anti-

parallel a-helices lying on top of a six-stranded P-sheet), though in the case of PF4 two such dimers subsequently stack P-sheet to P-sheet, but slightly staggered, to give an asymmetric t e t r a ~ e rThese . noncovalent multimerizations position the basic clusters on one face only of the IL-8 dimer, but on both faces of the PF4 tetramer forming a circle of positive charge, in such a way as to potentially form more extended HS-binding surfaces, In IL-8 it has been suggested that an extended HS sequence could bind parallel to one monomer a-helix, loop around and then bind to the adjacent ahelix of the other ~ o n o m e in r an overall horseshoe fashion (79). From the known dimensions of the IL-8 dimer, a HS sequence of approximately 11- 12 disaccharide units could be accommodated, which would be composed of two interactive S-domains of 5-6 saccharides in length, bridged by a looped i n t e r ~ N-ace~l tylated domain of 6- 7 disaccharide repeats (79). The overall symmetry of this model of HS binding has the advantage that it preserves the same polarity of HS chain interaction on both monomers (Fig. S). The x-ray crystallographic structure of PF4 has revealed that two arginines within each N-terminal strand are also brought into close apposition to the lysine clusters (80), and indeed NMR analyses of heparin-PF4 complexes implicate both arginine and lysine residues in the interaction (8 1). It has been proposed that a HS chain could encircle the PF4 tetramer, but in this case pe~endicularlycrossing each of the four a-helices. From the dimensions of the 3-D structure of the tetramer it was estimated that a heparin sequence of 17 disaccharides in length would be the minimum required to encircle the molecule forming salt bridges with all the relevant basic residues (82). Experimentally it was found that PF4 would protect "21 disaccharides of HS sequence from digestion with the heparinase I11 enzyme (83). In common with IFN-y, further analysis revealed that the extended PF4 binding site spanned two adjacent S-domains in the HS chain, with each dimer face occupying a single S-domain, and the intervening N-acetylated domain looping round the edge of the dimer-dimer interface (83). Deletion of the basic C-terminal sequence in IL-8 eliminates heparin bimding though without affecting receptor binding and activation (84). In both IL-8 and PF4, HS binding is probably utilized primarily for sequestration and retention of the protein near to the site of its release. Interestingly, the members of the P-chemokine (or CC-chernokine) subfamily (e.g., MIP-la, MP-1P, and RANTES), which are structurally distinct from the a-che~okinesbut also bind HSlheparin, do so by use of a different structural motif, Interaction occurs primarily around a basic sequence, "KRNR4*, within a solvent exposed p-turn between adjacent P-strands, where three critical arginine residues (R46, R48, and Rl8) and a less critical lysine ( K 4 3 are brought wi~hin close proximity to one another (all four residues being absolutely conserved across the subfamily) (85). Since there are additional, but nonconserved, basic residues in RANTES (i.e., R22) and MIP-1P (i.e., K19 and R22) which are proxi-

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Assisted dimerization of IL-8by heparan sulfate. IL-8 will form dimers spontaneously but the rate of formation may be accelerated by monomers interacting with correctly spaced S-domainsin a head-to-tail arrangement.The flexible “spacer sequence” between the S-domainsallows the chain to fold and bring the monomers into close antiparallel apposition. In the IL-8 dimers the monomers bind to HS with the same polarity. Preformed IL-8 dimers may also interact directly with HS to produce the same type of protein GAG complex illustrated in this figure.

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mal to the putative binding site (85), it is possible that there may be discernible differences in affinity and/or sequence specificity of HS-binding across these chemokines (86). Although HS may facilitate dimerization (and thereby receptor activation) at very low chemokine concentrations (86), chemokine mutants with defective HS binding appear to be unaffected in their biological activity in vitro (87). The function of a HS-binding site may be primarily to rapidly sequester the chemokine at its site of release, thereby setting up a steep and relatively stable gradient of immobilized chemoattractant for leukocytes in an otherwise unfavorable environment of fluid flow where a soluble gradient would be unstable and subject to rapid dispersal.

3. ~ i d ~ and i nH ~

Midkine and H B - ~ ~(h~parin-binding M growth associated molecule or pleiotrophin) comprise a small family of growth and differentiation factors which are characterized by being rich in lysines and fully conserved cysteines which together lnalse up - 25% of the total amino acids. They are neurotrophic factors although midkine also acts on other cells (e.g., endothelial cells) and high levels of midkine expression in neuronal malignancies correlate with poor prognosis, possibly due to its proangio~enicaction. HB-GAM was isolated from rat brain as a neurite outgrowth promoting protein and its expression in brain coincides with the stage of axonal outgrowth (88). Midkine is by far the better characterized of these two factors in terms of its 3- Dstructure and heparin-binding characteristics. It is simply comprised of two internally disulfide-bonded domains, of similar size and structure, connected by a short hinge. Both domains possess three antiparallel P-strands connected by two loops (one of which is significantly larger in the C-terminal d o ~ a i n )with extended N-and C-termini. Though these proteins are abnormally rich in lysines, with three easily discernible basic regions present within the primary amino acid sequence, the central one of these appears to possess most of the intrinsic heparinbinding activity of the protein (89). NMR analyses of the solution structure of midkine, and the changes consequent upon heparin binding, implicate^ two basic clusters close together on one face of the m o n o ~ e r(90). The critical sequence was *6KKAR89, which is present within the flexible larger loop of the C-terminal domain. This loop is bent toward the adjacent P-sheet where three other basic residues K79, R81 , and R102 are al so solvent exposed; the two clusters thereby contribute to a relatively large confo~ationalHS-binding site. Site-directed mutagenesis of various basic residues has implicated R81 as being a critical residue for heparin binding, with K86 and K87 having weaker contributions (91). Midkine dimerizes noncovalently, in a sy~metricalhead-to-head fashion, a phenomenon which is promoted by heparin/HS, presumably due to the extended

GAG-binding site this forms on one face of the elongated dimer. The interval of positive charges on LyslArg residues in this fused heparin~bindingsite closely matches the spacing of sulfate groups in the heparin helix (90). A heparin oligosaccharide of 6 disaccharides in length will interact with a monomer, but 10 disaccharides are required to promote dimerization. In vitro, at least, midkine dimerization can be covalently stabilized by transglut~inaseacting upon the susceptible Q95 residue on one monomer, which in the intact protein is brought into close proximity to the acceptor residue K63 on the other partner monomer (90). As transglutaminase action promotes biological activity (92), and mutation of the heparin-binding site reduces it (91), it would appear that HS binding, by inducing the dimerization of midkine, may be essential for receptor dimerization/ activation. Since there is -50% sequence identity between HB-GAM and midkine, including conservation of all the basic residues implicated in HS-binding to midkine, except for R89, it is likely that the HB-GAM monomer possesses a very similar tertiary s t ~ c t u r eand HS-binding site, though maybe with a discernible difference in affinity and/or specificity due to the single arginine deficit. The case of midkine is very revealing, in that for a protein so rich in basic amino acid residues, HS-binding appears to be determined by such a small and specific proportion of these which form a c o n f o ~ a t i o n a lsite with the correct exposure, orientation, and spacing of the charged groups. The bioactivity of midkine is compromised when cells are depleted of H and inhibited by heparin fragments greater than 10 disaccharides in length (93). The apparent requirement for cell surface HS and lack of biological activity of mid~ne-heparincomplexes suggests that HSPGs may somehow participate in the cell-signaling mechanism. This situation is reminiscent of HGF which also has a strong dependence on cell surface HS for optimal activity. In common with midkine, HB-GAM combines with heparin in solution to form complexes that are biologically inert, and hep nase 111 (heparitinase) treatment of neuronal cells suppresses their response to GAM (94,95), Syndecan-3 (N-syndecan) isolated from rat brain binds with affinity to H ~ - ~ A M and it may be the receptor on neuronal cells that mediates neurite outgrowth independently of other more conventional signaling receptors (94). This may be achieved by increasing the phosphorylation of c-src and cortactin which are two components of a mixed protein assembly that can be identified as associated with the cytoplasmic domain of syndecan-3 (96). H -GAM binds to an extended heparin sequence of 10 disaccharides or more (another property shared with midkine) with an element of selectivity in its recognition of sulfate groups (95).

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These include platelet-derived growth factor ( P ~ ~ F vascular ), ~ndothelial growth factor (VEGF), and transforming growth factor-P (TGF-P), all of which

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are constitutively dimeric due to the presence of interchain disulfide linkages. They all belong to a broad family of proteins which, though functionally distinct, possess the common structural motif known as the cysteine knot. Overall VEGF and PDGF are the most similar, with TGF-P being much less related, and indeed these simila~itiesand differences also extend to their modes of dimerization and the structures of their respective HS-binding sites. A common difference from all the noncovalent oligomeric proteins already discussed is that all three of these proteins can occur as isoforms, some of which do not possess HS-binding sites, either by virtue of alternative exon splicing (i.e., PDGF and VEGF) or by mutational divergence (i.e., TGF-P). 1

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Platelet-de~vedgrowth factor (PDGF) occurs as both homo- and hetero-dimers of two distinct, but highly homologous chains, A and B. These are further complicated by the fact that, as a result of aiternative splicing, the A chain can exist in one of two forms: a long fom, AL, which possesses exon 6, and a short form, As, which does not. r ere as, in con~ast,there is only one mature form of the B chain, the pr form possesses an exon 6 insert similar to the AL chain, but upon cleavage of pro-C-te~inalsequence this is lost and the mature B chain rese~blesthe As chain, Though the two forms of exon 6 present in the AL and pro-B chains differ, in both cases they encode for a highly basic sequence, namely, ~ R E S G K K R K R ~ R LinK AL and R V ~ R P P K G ~ R K FinKpro-€3, which are present toward the C-termini of the secreted proteins. Truncation of the C-termini with loss of the basic sequence effectively destroys the heparin/HS affinity of both forms (97). The 3-D structure of the mature €3 chain monomer is known, and comprises two long and highly twisted antiparallel pairs of P-strands, connected by three solvent-exposed loops, with free Nand C-termini (98). Dimers form by a slightly staggered, antiparallel side-to-side association stabilized by two disulfide bridges, giving rise to an elongated molecule, each pole of which is comprised of the solvent-exposed loops and the free C-teminus (which in the case of the AL and pro-B chains will extend even further from the bulk of the molecule) (98). Evidence suggests that the total positive charge on these extended C-termini is of more importance than specific residues (99), which may explain the lack of sequence conservation between the basic sequences in the two forms of exon 6. Analyses of HS/heparin binding to PDCF-AL suggest that interaction is mediated by very highly sulfated domains, of a minimum of 3-4 disaccharide units in length, which possess N-, 2- Q- and , 6-Q-sulfate groups (1 00). ~ n t ~ r e s t ingly, both cho~droitinand dermatan sulfates also have signi~cant,though lower, af~nities(97,100). This less discriminating GAG specificity than is seen with many other proteins may reflect the usage of an extended, linear, and unstructured highly basic sequence in GF-AL,rather than a precise conformational disposition of a fewer number of basic residues.

Functionally, the possession of GAG-binding properties appears not to be essential for the biological activity of the PDGF isoforms, though it may be of importance for the differential localization of specific isoforms. For example, the GAG-binding sites act as retention signals such that those isoforms which possess them remain bound to the cell surface or pericellular matrix, via proteoglycan interactions, where they may function as local autocrine/juxtacrine factors (99,100,102). By contrast, those isoforms which do not possess GAG binding sites are freely diffusible and act over larger distances.

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2. Vascular Endothelial Growth Factor

The vascular endothelial growth factor (VEGF) family is structurally complex with five potential monomer isoforms (l 21, 145, 165, 189 and 206 amino acids long) occurring by alternative splicing, each of which then forms disulfidebonded homodimers. VEGF acts on tyrosine kinase receptors expressed mainly on endothelial cells. Whereas the shortest isoforrn (VEGFI2])does not bind to HS/heparin, VEGF,4sand the most abundant isoform VEGF165do bind, but with lower affinity than the two largest, and mostly matrix-associated, isoforrns (VEGFlg9and VEGFzo6).As with PDGF, the HS-binding activity resides in the variable C-terminal region, rather than in the more structurally conserved Nterminal region which contains the cysteine knot motif. Plasmin digestion of the VE6F165dimer releases monomeric C-terminal domains (residues 1l l - 165) which possess all the HS/heparin-binding activity of the original dimer (103). The relative affinities of VEGF isoforms for HSlheparin correlates with the presence of either one or both together of the alternatively spliced exons 6 and 7, suggesting that each exon possesses an independent binding site (which must be structurally different as the exons encode for dissimilar structures). At present it is not known which of the basic residues in these two exons mediates HS interaction. However, whereas exon 7 has a scattering of basic residues throughout, exon 6 contains a highly basic consecutive sequence, " 9 R G K G ~ G ~ ~ ~ R K K S R 1 3 which displays a considerable degree of conservation with the equivalent basic region of the alternatively spliced exon 6 of the PDGF-AL chain. Indeed there i s absolute identity of the pentapeptide sequence KRKRK, and the next amino acid is also basic, being arginine in PDGF and lysine in VEGF. ~nfortunately? as yet, there is no available i n f o ~ a t i o non the 3-D structure of these variable C-terminal regions of VEGF. However, from the crystal structure of the receptorbinding domain (residues 8- 109) of VEGF (104), and the overall homology relationship between VEGF and PDGF, it is expected that the mode of VEGF dimerization will be very similar. S appears to potentiate the binding of the relevant isoforms of VE6F to the KDR ( f l k- l )receptor (103,105,106), though not to the alternative flt-l receptor (103). As large oligosaccharides of >10 disaccharides are required for this potentiation (107), whilst smaller ones can even be inhibitory, it again suggests

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that the most effective oligosaccharides are those which are able to span both of the relatively distant S-binding sites on the VEGF dimer. HS-binding also appears to impart other properties to the relevant VEGF isoforms. As with PDGF the relative affinities for HS determines the diffusibility or retention at the cell surface, or in the matrix, of the different isoforms. In addition, the ability of HS to block the binding and inactivation of VEGF by macroglobulin (108) may also be of great importance in retaining localized VEGF activity close to the site of release. 3. T r ~ n s f o r ~ irnow^ ~ ^

Transforming growth factor-p (TGF- P)is much more distantly related to PDGF and VEGF. There are three mammalian isoforms (pl , p2, and p3) which predorninantly form disulfide-bonded homodimers. Unlike FDGF and VEGF these isoforms do not differ in size, but whilst TCF-p1 and - p2 possess affinity for h e p ~ n / H S(109,l lo)?TGF-p3 does not (1 10). Comparisons of the primary sequences of the three isoforrns identifies only two amino acid positions (26 and 60) which are occupied by basic residues in both the heparin/HS-binding p1 and p2 isofoms, but are not conserved as such in P3 and may therefore be candidate critical residues for HS/heparin binding. Also, K26 is present within a basic peptide sequence which, when expressed as a free peptide, was able to inhibit TGFP1 binding to heparin (109). X-ray crystallographic ( l 11) and solution N (1 12) studies of the P1 and P2 isoforms have revealed that residue 26 (Lys in p1 and Arg in p2) is fully solvent exposed and present within the most positively charged surface on the monomer, in combination with five other basic residues ( , K37, and RIK94). TGF-P dimerizes in an antiparallel facet a large interface area. This places the two positively charged ~ o n o m domains ~r on the same face, but at opposite poles, of the dimer with a separation distance of approximately 6 nrn. In contrast to candidate basic residue K/R60, which is also a neutral res unlikely to contribute to a heparin/HS-binding site as it is only partially solvent exposed and situated on the periphery of the monomer-monomer interface on the opposite face of the dimer. TGF-p1 and - p2appear to have a selectivity for binding to heparin or highly species [e.g., the HS frorn liver (21)] and, becaus? of the spacing of the two putative binding sites in the dimer (approximately 60 A) , two S-domains (or one extended one) will be required at a minimum spacing of -7 disacch~ides for optimal binding (1 10). S does not appear to be required for binding of TGFP to its receptor. However, HS affinity may be vitally important in regulating the bioavailability of different isoforms. The ability of, for example, TGF-p1 from activated platelets to bind cell surface HS close to its site of release may be irnpo~antin localizing activity to a wound site, protecting it from de~radation and in generating an immobilized gradient of chemoattraction for mast cells.

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Importantly, binding of TGF-P1 and -P2 to HS also inhibits the sequestration and inactivation of the growth factor by macroglobulin (1lo), in a manner analogous to that seen with VEGF.

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Many growth factors, including heparin/HS-binding growth factors, affect cell differentiation and migration, and it is not surprising that HS is essential for normal developmental processes in multicellular organisms. We discussed earlier the importance of HS fine structure in controlling the actions of aFGF and bFGF during neuron differentiation in the developing mouse brain (Sec. IV.A.3). The syndecan and glypican HSPGs are expressed in distinct spatial and temporal patterns in the mammalian embryo (1,2,113,114) and although no published work has yet appeared on the effect on embryonic growth of eliminating the function of individual HSPG core protein genes, a role in cell proliferation is indicated for the glypican-3 gene which is mutated in the human overgrowth syndrome, Simpson-~olabi-Behmel(see Chap, 8). Immunostaining with specific antibodies has revealed that HS chains are prominent components of the hamster embryo with a striking increase of synthesis in mesenchymal tissues undergoing active morphogenesis (1 15). It is not known whether this elevated synthesis of HS is linked to a differential increase in production of a specific PG core protein. The importance of normal HS synthesis in m a m ~ a l i a nembryogenesis was revealed by the findings of Beddington and coworkers who deleted the C-terminal region of the murine HS 2-0-sulfotransferase (HS2ST) gene using gene trap technology (116). This resulted in the synthesis of a fusion protein containing th terminal region of HS2ST together with resistance and reporter genes ( l 16). cause part of the HS2ST gene is still expressed, enzyme function (see Ref. may not be completely eliminated in the homozygous mutants. Nevertheless, the associated phenotype is severe and the mutant mice die shortly after birth as a consequence of renal agenesis due to failure of branching of the inductive ureteric bud; ocular and skeletal defects were also observed. The renal deficiency concurs with observations in organ culture that ureteric bud growth and branching are blocked by agents that deplete cells of glycosaminoglycans (1 18). Although the critical failure in cell signaling in the HS2ST mutants has not been determined, it seems probable that the growth and inductive activities of HS-dependent growth factors will be involved. A candidate protein effector for kidney specification is a member of the Wnt family of morphogens, Wnt-l 1, which is located bud tips and is dependent on proteoglycans for its sustained expression sic FGF may also affect kidney development because it is synthesized

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in the ureteric bud and can induce condensation of cultured metanephric mesenchyme in the absence of inducting tissue (120). Insufficiency of HS2ST in cultured Chinese hamster ovary cells leads to secondary modifications in N- and 60-sulfation of HS, and to a decrease in HS catabolism (121). Changes in structure of cell surface HS in the HS2ST mice could result in aberrant reciprocal interactions between cells and the ECM in addition to effects on growth factor regulation.

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~ r o s o ~is ~perhaps i Z ~ the best understood ~ulticellularorganism from a develop-

mental viewpoint and it has been subject to extensive mo~hologicaland genetic analysis ( l 22). ~ r o s o ~ ~is iaZreasonable a model for vertebrate development because of the evolutionary conservation of mechanis~sof cell growth, adhesion, and signal transduction. Genetic screens have elucidated important roles for HS and HSPGs in the signaling pathways of two key growth factors and morphogens, named Wingless (Wg) and Decapentaplegic (DPP); these are ~ r o s u ~ ~counteriZa parts of the mammalian Wnt and TGF-P families, respectively, of growth regulatory molecules (123), and their corresponding receptor systems are similar in ~ r u s u ~and ~ m i ~~ m a a l i a norganisms. Mutations in the w g gene cause develo~mentaldefects. These include abnormalities in the segmented embryonic cuticle brought about by the absence of short-range interactions of Wg and defects in wing axis formation that depend on long-range effects (124,125). In the embryonic cuticle the areas of Wg synthesis are delineated by a positive feedback loop with the Hedgehog protein which is synthesized in adjacent segments (Fig. 6); the reciprocal interactions between Wg and Hedgehog proteins lead to the cuticular pattern of alternate smooth and spiked segments. In Wg-mutant embryos the smooth segments are missing and a “lawn of denticles’’ covers the cuticle surface (126). Deficiencies in long-range signaling of Wg result in the failure of wing development from the imaginal disc. The dorsoventral axis of the disc is the origin of the wing and it is identifiable as a stripe of ~g-producingcells across the disc surface. Cells in the remainder of the disc respond to a Wg gradient so that those most distal to the source form the wing tip and those closest to it form the hinge region. The anterioposterior axis of the wing is established by DPP which acts in a complementa~manner with Wg to define the ‘coordinates of wing geometry (123,125). Genetic evidence of a role for glycosaminoglycans (GAG) as mediators of Wg activity came from identification of the ska or suga~Zessgene which encodes the enzyme ~ ~ P - g l u c odehydrogenase se ( ~ ~ P - responsible ~ D ~ ) for the synthesis of the GAG precursor, ~ ~ P - g l u c u r o nacid i c (124127,l28). Mutations in the ska gene cause developmental defects that are very similar to the Wg mutants. Although the biochemical effects of ska gene mutations will affect all the glucuro-

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Heparan sulfate proteoglycans (HSPG) in reception of the Wingless (Wg) signal in the ~ ~ o cuticle: ~ o reciprocal ~ ~ i interactions l ~ with the Hedgehog (Hg) protein. Wingless and Hedgehog are pair-rule genes and they determine cell fate in the ~ r o ~ u ~ ? ~ cuticle (122). Wingless is secreted as a soluble protein and is captured by the S-domains of a membrane HSPG on an adjacent cell. HS chains transfer Wingless to its receptor Frizzled which conveys a signal for Hedgehog protein synthesis. Hedgehog is secreted and diffuses to its receptor Patched; this prevents Patched from blocking a signal from the protein Smoothened which maintainsthe synthesis of Wingless. The model is speculative with regard to the mode of action and type of HSPG involved in mediating the activity of Wingless, although there is some evidenceof a role for the Dally HSPG (6~1-anchored) in Wingless signaling (see text for details). In addition to facilitated transfer, Wingless may undergo an activatingconformationalchange when bound to an appropriate site on HS.

nate-containing GAGS (i.e., HS, chondroitin/dermata~sulfates and hyaluronic phenotype is believed to arise from an HS deficit. S binding protein, it requires HS to activate cell injection of embryos with heparinase enzymes gives c~aracteristicw g- lsk a phenotypes (124,129). More compelling evidence for the involvement of

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the W g phenotype has recently emerged from ~ r u s o ~ ~mutants iZa lacking Nd eacet ylas e/ ~ - s u lfo t r an s fer asactivity. e This enzyme acts specifically in the biosynthesis pathway and enzyme-deficient embryos reproduce the Wg mutant phenotype (130). The mechanism of action of HS in mediating Wg function is unclear and the ideas discussed in earlier sections of this chapter on the regulation of other HS-dependent growth factors are broadly applicable to Wg regulation. Wg may undergo an activating conformational change when bound to HS. There is also a strong possibility that HS is necessary for the creation of a stable gradient of Wg in the imaginal disc. Membrane HSPGs are also likely to be essential for reception of Wg and its efficient transfer to its receptor. The receptor for Wg is named Frizzled, a serpentine type receptor which does not appear to form dimers in the cell membrane ( l 3 l). Thus there may be no requirement for HS to induce dimerization of the Wg protein unless the receptor itself is triggered more efficiently by dimers compared to monomers. The severity of the ska mutant phenotype is only partially offset by overexpression of Wg (12’7) and this suggests that PGs do more than simply restrict Wg diffusion, One possible way in which HSPGs could have the dual effects of Wg gradient stabilization and morphogenetic induction would be for the diffusion rate of Wg from its point of release to be determined by an even distribution of abundant, low-affinity HS across the induction zone, with the mo~hogenetic the Wg concentration and the expression of higher affinity, species on the target cells. he importance of a PG core protein in ~ r o ~ ~ osignaling ~ ~ i Zhas a become apparent from genetic screens for mutants with abnormalities in the pattern of cell division in the developing nervous system. In two functionally linked precursor cell populations, one which forms the ommatidia (clusters of photoreceptors) of the eye, and the other (so-called lamina precursors) which develops into neuronus which make synaptic contacts with photoreceptor axons, the early synchronized cell divisions are d i s ~ p t e ddue to a delay in entry into subtle abnormalities are due to a tion in a gene named ~aZZy(division abnormally delayed) which encodes a anchored protein which is highly homologous to mammalian glypican (132). Other defects in ally mutants include malformation of antennae, genitalia, wing margins, and cuticle. The nature of th a b n o ~ a l i t i e smimic those of Wg and ska mutants. Dally is glycanated by but the Dally mutations investigated so far do not completely eliminate e., they are hypomorphs) (133). ntly, evidence has been presented that Dally is involved in the recepP. The data to support this idea have come from reciprocal genetic studies which revealed an enhancement of the Dally phenotype by mutations that partially inactivate DPP and the suppression by Dally mutants of mo~hological defects caused by ectopic PP synthesis in the wing (134).

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approaches have also suggested a role for Dally in Wg signaling (133). It remains to be formally established that Dally interacts through its HS chains with the DPP and Wg proteins, There may also be i m p o ~ a n tinteractions of the Dally core proteins with external regulatory molecules. The degree of overlap between ~ r o s ~ syndecan ~ ~ i Zand ~ Dally in recognition and activation of growth factors has not been studied, but the existence of the striking Dally phenotypes indicates that normal levels of syndecan HSPG cannot adequately compensate for deficiencies in Dally. ~ r o s u ~ genetics ~ i Z ~has made a big impact on HSPG research and it is illuminating mechanisms and interactions that are relevant to mammalian systems. Further analysis of ~ r o s o ~mutants, ~ i Z ~especially those which affect the polymer modifying enzymes of HS biosynthesis, should lead to new and significant developments on the functions of HS and on its mode of action in the modulation of signaling networks and cellular interactions that underpin the development of complex organisms. *

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119. Kispert A, Vainio S, Shen L, Rowitch DH, McMahon AP. Proteoglycans are required for maintenance of Wnt-l l expressionin the ureter tips. Development 1996; 122:3627-3637. 120. Peratoni AO, Dove LF, K~avanovaI. Basic ~broblastgrowth factor can mediate the early inductive events in renal development. Proc Natl Acad Sci USA 1995; 92:4696-4700. 121. Bai XM, Bame KJ, Habuchi H, Kimata K, Esko JD. Turnover of heparan sulphate depends on 2-0-sulphation of uronic acid. J Biol Chem 1997; 272:23172-23 179. 122. Gilbert SF. The genetics of axis specification in D ~ u s o ~ ~ In: iZ ~ u .e v e l o ~ m eBiolnt ogy 1997. Sunderland, Mass.: Sinquer Associated (Pub), 1997:543-590. ~ ~ Nature la 123. Lecuit T, Cohen SM. Proximal-distal axis formation in the D ~ o s u ~ leg. 1997; 388~139-145. 124. Cumber~edgeS, Reichsman F. Glycosaminoglycans and Wnts: just a spoonful of sugar helps the signal go down. TIGG 1997; 13:421-423. 125. Neumann GJ, Cohen SM. Long-range action of Wingless organises the dors Z u Development 1997; 124:871-880. tral axis of the D r o s o ~ ~ ~wing. 126. Hacker U,Perrimon N. Com~onentsof the Wnt signalli~gpatllway: ~ytos~eletal and cell membrane interactions and signal transduction.In: Cowin P, Klymkowsky MW, eds. Austin, Tex.: Landes Bioscience 1997. ~ i l ~ gene modulates Wing127. Hacker U,Xinhua L, Perrimon N. The ~ r o s u ~sugarless less signalling and encodes an enzyme involved in polysaccharide biosynthesis. Development 1997; 124:3565-3573. ~ n~ a n o u k i aAS. ~l 128. Binari RC, Stavely BE, Johnson WA, Godvarti R, S a i s i s e ~ a r R, Genetic evidence that heparin-like glycosaminoglycans are involved in wingless signalling. De~elopment1997; 124:2623-2632. 129. Reichsman F, Smith L, Cumber~edgeS. Glycosaminoglycalls can modulate extracellular localisationof the wingless protein and promote signal transduction. J Cell Biol 1996; 135:819-827. 130. Perrimon N, Lin X, Hacker U,Michelson A. Role of heparan sulphate proteoglycans in Drosophila melanogaster growth factor signalling. In: Lander A, Nakato H, Selleck SB, TurnbLlll JE, Coat h C, eds. Cell Surface Proteoglycans in Signalling and Development. Strasbourg, France: Human Frontiers of Science Programme. Workshop VI 1999:157-162. 131. Bhanot P, Brink M, Samos CH, Hsieh JC, Want Y, Macke JP, Andrew D, Nathans J, Nasse R. A new member of the frizzled family from D ~ o s o ~ ~functions ila as a Wingless receptor. Nature 1996; 382:225-230. 132. Nakato H, Futch TA, Selleck SB. The division abnormally delayed (dally) gene: a putative integral membrane proteoglycan required for cell division patte~ingdur~~~u. ing postemb~onicd~velopmentof the nervous system in D ~ o s o ~~evelopment 1992; 121~3687-3702. o ~ ~ gene, ~ Z division ~ abnormally 133. Selleck SB. Genetic studies of a D ~ u ~ glypican delayed (Dally). In: Lander A, Nakato H, Selleck SB, Turnbull JE, Coath C, eds. Cell Surface Proteoglycans in Signalling and Development. Strasbourg, France: Human Frontiers of Science Programme. Workshop VI 1999:176- 181. 134. Jackson SM, Nakato H, Sugiura M, Jannazi A, Oakes R, Kaluza V, Golden C, Selleck SB. Dally, a D r o s u ~ ~ iglypican, la controls cellular responses to the TGFP-related mo~hogenDPP. Development 1997; 124:4113-4 120.

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Tufts ~niversity~ ~ h o of o l~edicine,Boston, ~assachusetts

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Almost everything about hyaluronan is unusual; yet its extraordinary attributes derive from a chemical composition that is very simple. Hyaluronan is a unifomly repetitive, linear glycosaminoglycan (GAG) composed of disacch~ides of glucuronic acid and N-acetylglucosamine: [ -p(1,4)-GlcUA-P(1,3)-GlcNAc-], ending on the tissue source, the polymer usually consists of 2,000harides, giving rise to molecular weights ranging from IO6 to lo7 Da and extended len ths of 2- 25 m. Even its name lends itself to mind-nul~bing . . (Cheryl Knudson, personal communication). Unlike other GAGs, however, hyaluronan contains no sulfated groups or epimerized uronic acid residues; it is synthesized at the plasma membrane rather than in the Golgi; it is most likely elongated at the reducing rather than nonreducing terminus; it is huge-not only in molecular weight but most notably in the space it occupies in solution; it exhibits exceptional physical properties; it is uniquely important in regulating cell behavior. Finally, hyaluronan is not covalently linked to protein or synthesized on a protein backbone; thus it is not a proteoglycan and so you might ask what it is doing in this book. Well the truth is that, as the mightiest of the GAGs, hyaluronan is so beloved to the ~ r o t e o g l y c a ~ community that it has been bestowed the title of ~ o ~ oProteoglycan-whatr ~ ~ y ever that means!

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The carbohydrate structure of hyaluronan. (a) Repeating disaccharide of byaluronan: [ --p( 1,4)- GlcUA- P(l ,3)-GlcNAc-],?.Within the polymer each disaccharide is rotated 180" with respect to its neighbors, thus producing a twofold helix with the acetamido and carboxyl side groups projecting from alternatingfaces of the polymer with each rotation (see Fig. 3b). (b) Space-filling model of a hyaluronan pentasaccbaride, showing a hydrophobic patch of eight CH groups ( + signs). The hydrophobic patch extends across appro xi mat el^ three sugar residues on alternating faces of the polymer (see Fig. 3c). Arrows, hydrogen bonds between acetamido and carboxyl groups. [Panel (b) is from Ref. 152.1

Synthesis of hyaluronan is different from that of other GAGS in at least four respects (for further discussion see Chap. 2 of this volume). First, the initial step

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in synthesis of most GAGS is linkage of xylose to a serine residue within a core protein. This is usually followed by addition of several other l~onosaccharide moieties, namely, gal act ose- gal act ose- Gl cUA, prior to repetitive, alternate addition of the two monosaccharides characteristic of the particular CA acetylgalactosamine and GlclJA for chondroitin sulfate or GlcNAc for heparan sulfate. Unlike other GAGs, however, hyaluronan synthesis does not occur on a protein core. The precise mechanism of initiation of hyaluronan synthesis is not known but may simply be linkage of one of the UDP-sugar precursors, i.e., U~P-GlcNAcor UDP-GlcUA, to the reducing end of the second sugar within the active site of hyaluronan synthase, by the same mechanism as described for elongation below. The second difference between synthesis of hyaluronan and other GAGS is that, whereas most GAGs are elongated by transfer of monosaccharides from nucleotide derivatives to the nonreducing end of the growing polymer, hyaluronan is most likely elongated at the reducing end (1,2) (reviewed in Ref, 3 and Chap. 2 of this volume). In this mechanism, it is proposed that UI) to the monosaccharide positioned at the reducing end of the growing chain. The next U~~-monosaccharide substrate is then added to the reducing end, concomitant with release of the previous reducing terminal UDP. Presumably the two lJDP-monosacc~arideswould bind to separate sites within hyaluronan synthase and the growing chain would alternate between these two sites. Initiation could occur by binding of one lJ~P-monosaccharidesubstrate to the other at one of these two sites. Third, unlike most other GAGs, no convincing evidence has been published to indicate that the repeating disaccharide, GlcUA-GlcNAc, is modified during or subsequent to formation of the hyaluronan polymer. Most other GAGs are modified, e.g., by various patterns of 0-sulfation, by deacetylation and N-sulfation of CIcNAc, and/or by epimerization of GlclJA to iduronic acid. The fourth difference is that synthesis of GAGS other than hyaluronan is initiated within the endoplasmic reticulum and completed in the Golgi apparatus; hyaluronan synthesis is unique in that it takes place at the cytoplasmic face of the plasma membrane. Incorporation of the two monosaccharide precursors of hyaluronan, i.e., GlcNAc and GlcUA, into the forming polymer is catalyzed by members of a family of multipass plasma membrane proteins, termed hyaluronan synthases (Fig. 2a) . ~oncomitantwith elongation of the hyaluronan polymer, it is extruded through the plasma membrane (43) (Fig. 2b), but the mechanism whereby this occurs and the nature of pore or chaperone proteins that may be involved are not known. Characterization of hyaluronan synthases has progressed very rapidly over the past several years (reviewed in Ref. 3) due in great part to characterization, cloning, and expression of the gene, termed Has, from bacteria. Several pathogenic bacteria, e.g., group A or C streptococci, produce protective capsules of

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Hyaluronan synthases. (a) Proposed membrane topology of the hyaluronan synthases (HAS). Hydropathy plots suggest the arrangement shown for bacterial and vertebrate synthases. The central cytoplasmic domain, between MD2 and MD3, is believed to include the catalytic sites of the enzymes and i s highly conserved across most bacterial and eukaryotic species. The circled C indicates a cysteine residue present in synthases characterized to date. Eukaryotic synthases are larger than bacterial, especially at the Cterminal end where two additional, putative ~embrane-spanningdomains occur. MD, membrane domain. (b) Hypothetical model for extrusion of hyaluronan. Hyaluronan is extruded through the plasma membrane concomitant with elongation and may be retained at the cell surface via sustained, transmembrane interaction with hyaluronan synthase. It is not yet known whether extrusion requires additional pore-forming proteins (indicated by X). Y- indicates putative regulatory proteins that may coordinate synthase activity with other cellular activities. [Panel (a) is from Ref. 3.1

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hyaluronan that enhance their virulence. An operon involved in hyaluronan synthesis was characterized in group A streptococcus and the Has gene was cloned by transposon inter~ptionof this operon in S. ~ y o ~ e (6,7). ~ e s After expression, the encoded protein was shown to synthesize high molecular weight hyaluronan, thus establishing that a single protein, encoded by the Has gene, is responsible for both of the glycosyltransferase activities required for synthesis of hyaluronan (8). A similar H as gene from an encapsulated group C streptococcus, S. e ~ ~ i s i ~ iEis, has now been chara~terized(9). Interestingly, however, a Has gene with little homology to the above genes has been cloned from ~ ~ s t ~ ~ ~r e~ l ZZ ta o ~ ~ ~ a , another pathogenic encapsulated bacterium (l 0). Several groups have recently been successful in cloning and characterizing three separate genes for vertebrate h y ~ u r o n a nsynthases. The first vertebrate Has gene to be characterized, the murine Hasl gene, was identified by expression cloning wherein cDNAs were screened for hyalLlronan production and their ability to generate hyaluronan-dependent pericellular matrices or “coats” on transfection into cells deficient in hyaluronan synthesis (1 1); shortly thereafter, two gr oups characterized cDNAs for human Has1 (l2,13). Three other groups cloned hyaluronan synthases in the mouse (14,15) and human (16) that proved to be distinct from Ha s1 and were named Has2. A third gene, Has3, has now been characterized (l7,18). Another interesting development in this area was the discovery that Xenopus DG42, a gene active during Xenopus gastrulation, is a hyaluronan synthase (19) and the orthologue of murine and human has^ (18). The three Has genes are located on separate chromosomes (201, expressed in different patterns in the adult and during development ( l @, and presumably subject to different regulatory in~uences.Detailed studies of regulation of the different Has genes during specific physiological and developmental events should provide valuable insights into the various functions of hyaluronan. Of special interest will be the results of detailed analyses of mouse “knockouts,” currently in progress in several laboratories, in which the various Ha s genes have been inactivated. Results obtained so far indicate that the Has2 knockout is an embryonic lethal in which several major developmental defects occur, whereas H a s l and Has3 knockouts show no grossly abnormal he no type ( A. Spicer and J. M c ~ o n a l d , personal co~munication). As stated above, the hyal~~ronan synthases are multipass plasma membrane proteins that catalyze the synthesis of hyaluronan. Figure 2a shows a diagram of the putative a~angementof hyaluronan synthases in the plasma membrane. The hyaluronan synthases have a large, highly conserved, cytoplasmic region that presumably includes binding sites for UDP-GlcNAc and U D ~ - ~ l cand ~A the two glycosyltransferase active sites required for synthesis of the hyaluronan chain. Possible hyaluronan-binding motifs are also present in this region (3). The vertebrate synthases are significantly larger than the bacterial enzymes due mainly to additional carboxy t e ~ i n a transmembrane l domains (Fig. 2a). These

latter regions may be important in regulation of hyaluronan synthesis and secretion by cytokines and other factors (21-23), or in coordination of hyaluronan synthesis with other cellular activities, e.g., proliferation (24) and migration (2527). Transfection of cells deficient in hyaluronan production with cDNAs for hyaluronan synthases leads not only to hyaluronan synthesis but also to secretion and pericellular matrix assembly (1 1,l8). It is not yet clear whether other macromolecules besides synthase itself are required for secretion or matrix assembly under these circumstances. Surprisingly, hyaluronan synthase with sequence similarities to the other Ha s genes has recently been discovered in the virus, PCBV-1, that infects the green algae, C~ ZoreZZ~ (28). Infection of chlorella cells with PG synthesis of high molecular weight hyaluronan which becomes localized to the external surface of the algae. Numerous strains of the virus give rise to expression of hyaluronan in these algae but its function therein is not yet known. The Ha s genes exhibit significant homologies with other polysaccharideforming enzymes, e.g., chitin synthases and cellulose synthases (3,18). However, it is not yet established whether these enzyme families form a superfamily evolved from a common ancestral gene or whether they have evolved independently (18).

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The conformation of hyaluronan in aqueous solution is a gently undulating, tape or ribbonlike, twofold helix which forms as a result of 180" rotations between alternating disaccharides (29) (Fig. 3a,b). Although, overall, hya~uronanis highly hydrophilic9 it also has repeating hydrophobic patches of approximately three sugars in length (Fig. lb), These are arrayed along the two flat sides of the tapelike polymer, with sequential patches alternating between the two sides. The patches most likely form hydrophobic bonds between corresponding regions of neighboring hyaluronan chains aligned in an antiparallel fashion. In addition, hydrogen bonds would occur between the apposing acetamido and carboxyl groups within these antiparallel regions (30-32) (Fig. 3c). Since these hydrophobic and hydrogen bonds would form on both sides of the hyaluro~anpolymer, higher-order aggregates can assemble. Using rotary shadowing electron microscopy, high molecular weight hyaluronan has been shown to form virtually infinite networks, even at concentrations as low as 1 p g/ m L. Presumably these networks form via antiparallel interactions among numerous neighboring hyaluronan molecules. Thus, as the concentration of hyaluronan increases, the branches within the network become thicker (3 1,32). In addition, interactions with other tissue components in vivo would add further ordered complexity to the ~eshwor~-forming

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Models depicting the polymeric structure and interaction of hyaluronan molecules. (a), (b) Projections, at right angles to each other, showing the twofold helix that a hyaluronan molecule adopts in aqueous solution. The dotted lines in (b) represent hydrogen bonds; the circle-square pairs joined by dotted lines represent water bridges between acetamido groups and carboxyls. (c) Proposed mode of interaction between two antiparallel hyaluronan molecules in which hydrophobic patches (see Fig. lb) are apposed and acetamido and carboxyl groups are within hydrogen bonding distance. Shading, hydrophobic patches; circles, acetamido groups; squares, carboxyl groups. (From Ref. 152.)

properties of hyaluronan. On the other hand, some of these interactions, e.g., with proteoglycans such as aggrecan and versican, would cause hyaluronan molecules to become extended and separated (see below), and would most likely block network-forming self-interactions. High molecular weight hyaluronan in very dilute saline solution occupies an enormous domain wherein the mass of hyaluronan itself is 0.1% or less and solvent occupies the remaining volume of this domain (33,34). As the hyaluronan concentration is increased, the interactions described in the previous paragraph

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would occur at greater and greater frequency. These properties give rise to high viscosities at concentrations of 0.5 mglmL or more, as found in many tissues such as synovial fluid, umbilical cord, and skin. However, the viscosity of hyaluronan solutions is highly dependent on flow rates; i.e., high shear causes a reversible decrease in viscosity. This viscoelastic property of hyaluronan may be important in soft tissue lubrication. The rheological and network-forming properties of hyaluronan also contribute to water homeostasis, tissue hydration, and transport o f macromolecules within tissues (33,34), and form the basis of current clinical applications of hyaluronan, e.g., stabilizing eye tissues during surgery, reducing soft tissue adhesions, and alleviating joint problems (35-37).

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yaluronan binds to a wide variety of proteins, termed hyaladherins. Two wellcharacterized groups of hyaladherins are: ( l ) s t ~ c t u r a hyaluronan-binding l proteins of the extracellular matrix, such as link protein and the aggregating proteoglycans, and (2) cell surface h y a l u r o n ~receptors, such as CD44 and the receptor for hyaluronic acid-mediated motility (RHAMM). Hyalurona~receptors are discussed in more detail in the next section, The aggregating proteoglycans, i.e., aggrecan, versican, brevican, and neu~ocan( al so known as the hyalectan or lectican family of proteoglycans) have been reviewed extensively elsewhere (e.g., 38,39) and in C~apters13 to 15 of this volume, Other more general aspects of hyaladherins are discussed in this section. Most well-characterized hyaladherins have st~cturallysimilar hyaluro~anbinding domains with sequence homologies of 30-4076. These domains, sometimes termed link modules or proteoglycan tandem repeats, form disulfidebonded loops and, in many hyaladherins, two modules are arranged in tandem array. Two link modules form the hyaluronan-binding region of link proteins and the aggregating proteoglycans (Refs. 38, 39; Chaps, 13 to 15 in this volume), whereas only a single-link module is found in the hyaluronan-binding domains of CD44 (40) and TSG-6 (41). In CD44, mutatio~of a single arginine residue within this module (Arg41 to Ala in human) causes virtually complete loss of hyal~~ronan-binding capacity (42). ~ u t a t i o n sin other basic amino acids, both within and outside the link module, also cause significant decreases in hyaluronan binding (42,43). Recent s t ~ c t u r a work l demons~atesthat link modules have a very similar c o n f o ~ a t i o nto that of the ca~bohydrate-bindingregion in C-type selectins despite the absence of sequence homology (43-45). Some hyaladherins, notably RHAMM, do not have link modules. mutation and sequence-swapping studies with RHAMM have revealed a possible

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hyaluronan-binding motif which is present, not only within R ~ A M but ~ ,also within or adjacent to the link modules of several of the hyaladherins described in the previous paragraph, e.g., link protein and CD44 (46). The proposed motif is B(X7)B, where B is arginine or lysine and X is any nonacidic amino acid. ~ariationsof this motif, e.g., B(X,)B, also bind to hyaluronan with signi~cant affinity, and clearly clustering of basic amino acids within and around the motif is the key feature that determines binding (46). Several other hyaluronan-binding proteins lack domains with homolo~yto link modules but contain B(X7)B and related sequences. Examples are ICAM (47), hyaluronan synthases (3), mammalian hyaluronidase (48), Cdc37, a hyaluronan-binding cell cycle regulatory protein (49), and P- 32, a hyaluronan-binding protein that associates with splicing factors (50). Although clusters of basic amino acids clearly contribute to hyaluronan binding in several hyaladherins, other structural features, e.g., glycosylation and conformational effects, are also involved (51). An unusual hyaluronan-binding protein is inter-a-trypsin inhibitor ( 52) . This serum protein is composed of a light chain, also known as bikunin, and two heavy chains which are covalently cross-bridged by chondroitin sulfate. ~ y a l u r o nan can replace chondroitin sulfate by transeste~~cation or bind noncovalently to the heavy chains. This interaction has been shown to bind hyaluronan to cell surfaces (53) and to participate in forming a matrix around the oocyte during ovulation (54).

Early work on embryonic development and tissue repair suggested strongly that hyaluronan influences cell behavior (55). Consequently, evidence was sought and obtained for the presence of hyaluronan-binding proteins on the surface of cells ( 55, 56) .Subsequent investigations led to the full molecular characterization of two classes of cell surface hyaluronan receptors, namely, CD44 (57) and RH AM^ (58). N u ~ e r o u reviews s (e.g., Refs. 40,51,59-61) have been published that amply describe work on the structures and functions of CD44 and R H ~ M M . This work as well as more recent developments are summarized here. CD44 is a widely distributed cell s u ~ a c eglycoprotein that is encoded by a single gene but expressed as numerous isoforms as a result of alternative splicing. The simplest and the most widespread isoform is termed the standard isoform of CD44 (CD44s; also often termed the hematopoietic isoforrn or CD44H). CD44s contains transmembrane, cytoplasmic, and extracellular regions that are common to all membrane-bound isoforms of CD44 (Fig. 4). The extracellular region of CD44s includes two major domains. One of these domains, at the

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arnino-te~inus,is a link module and is the presumed binding site for hyaluronan; although binding is subject to numerous positive and negative influences from other regions of the molecule (see below). The second part of the extracellular region, the so-called membrane-pro~imaldomain, lies between the hyaluronanbinding and transmembrane domains. Various co~binationsof the products of 9-1 l variant exons can be spliced into a single position within the membraneproximal domain (see Fig. 4) to give rise to the numerous variant isofoms of CD44 (62,63). Some splicing events result in isoforrns that lack transmembrane and cytoplasmic domains, thus yielding secreted, soluble CD44 (63). soluble CD44 can also arise via shedding from the cell surface, apparently as a proteolytic scission (64,65). aluronan binding is dependent on many structural features of C addition to the presence of the link module and clusters of basic amino acids discussed in the section above. Among these are glycosylation, alternative splicing, dirnerization, clustering in the plasma membrane, and integrity of the cytoplasmic domain (reviewed in Ref. 51). However, few of these effects are completely understood and several apparently contradictory studies have been published on their roles. Also, many of these influences are dependent on experimental conditions or cellular context (51,59).Recent work has somewhat clarified the effects of glycosylation, especially with respect to the role of terminal sialic acid residues on N-linked oligosaccharides (66). These residues greatly reduce hyaluronan binding affinity, probably providing an important negative regulatory rnodification that may explain some of the large differences in hyaluronan-binding capacity of different CD44-bearing cells (59). It is al so likely that variations in hyaluronan-bind in^ affinity of CD44 variants are largely due to introduction of additional glycosylation sites, including sites for GAG chain addition (5 1,6668). Although this recent work is encouraging, detailed structures of the carbohydrate sidechains of CD44 and their physiological contributio~sto hyaluronan binding have not yet been determined.

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Standard (hematopoietic)form of the hyaluronan receptor, CD44.The standard form o f CD44 is composed of extracellular,transmembrane?and cytoplasmic regions. The extracellularregion contains a hyaluronan-binding domain (solid black) that is homologous to the link modules of several other hyal~ronan-bindingproteins; two clusters of basic amino acids implicated in binding (42,46)are expanded. All membrane forms of CD44 contain these regions. The arrow indicates the site of insertion of additional sequences encoded by variant exons. Insertion of multiple variant sequences can result in isoforrns that are as much as twice the size of standard CD44.Solid circles, potential Nglycosylation sites; open circles, potential O-glycosylation sites; diamonds, potential sites for GAG attachment; circled PS,potential phosphorylation sites. (From Ref. 153.)

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Since cell surface hyaluronan-CD44 interactions mediate many cellular effects of hyaluronan, the biochemical ~echanismsby which these interactions are transduced into intracellular signals that bring about these effects are now being intensely studied by several groups. Despite this activity, however, there is little consensus with respect to the signaling pathways initiated by hyaluronan-CD44 interaction. A few of the many promising observations made by a variety of research groups using various cellular systems are that, in T lymphocytes, hyaluronan-CD44 interaction causes an increase in intracellul~Ca2+,clustering of CD44 in the membrane and accu~ulationof ankyrin beneath it (69,70); that, again in T lymphocytes, CD44 interacts with the tyrosine ~nase,,p56lCk, leading to increased phospho~lationof ZAP-70 and other intracellular proteins (71); that, in ovarian carcinoma cells, CD44 binds to the erbB-21neu oncogene product and stimulates its kinase activity as well as cell growth on interaction with hyaluronan (72); that, in endothelial cells (73) and macrophages (74), interaction of low molecular weight hyaluronan with CD44 leads to expression of various genes involved in growth and in~ammation,respectively. Also, evidence has been published documenting that the cytoplasmic tail of CD44 interacts with proteins associated with the cytoskeleton, especially ankyrin (75) and the ezrin-radixin-moesin family (76). However, it still remains to be seen in what ways these or other agents mediate the cellular effects of hya~uronan-C~44 interactions. Less work has been published on RHAMM than CD44 but what has appeared is provocative and potentially interesting. Alternative splicing generates several isoforms of RHAMM, including intracellular and cell surface isoforms (61). Hyaluronan apparently initiates locomotion of ras-transformed fibroblasts via RHA~M-inducedtyrosine kinase activity, and a very rapid response to hyaluronan-RHAMM interaction is transient phosphorylation of ~ 1 2 in5concert ~ ~ ~ with turnover of focal adhesions (26). These events do not occur if HA-RHAMM interaction is suppressed, leading to stabilization of focal contacts and loss of motility (77). Treatment of fibroblasts with a soluble form of HAM^ causes cells to arrest in 62/M, due to inhibition of Cdc2 and cyclin B1 expression, and inhibits fibrosarcoma growth and metastasis in vivo (78). On the other hand, overexpression of cell surface RH AM^ by cDNA transfection of fibroblasts can lead to malignant cell behavior (77).

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Several cell types exhibit highly hydrated, hyaluronan-dependent, pericellular matrices or “coats” (56,79). In culture, these matrices cannot be analyzed readily by conventional light microscopy. However, the coats can be visualized indirectly

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~yaluronan-dependentpericellularmatrices. A-D: Large pericellular matrices surroundi~grat fibrosarcoma cells ( A;Ref. 154) and chick embryo chondrocytes(C; Ref. 80) are visualized by their ability to exclude particles (fixed red blood cells are used here). The matrices are removed by treatment with hyaluronidase (B, D). E-F: yobl lasts (E; Ref. 106) also exhibit large pericellular matrices that are destroyed by hyaluronidase treatment (not shown); these pericellular matrices are lost during myoblast fusion (F).

by exclusion of particles and are usually 5-10 pm in thickness (Fig. S), These ~ e r i c e l l umatrices l~ provide the milieu in which numerous cellular activities take place and in~uencethe behavior of cells in many circumstanc~s.For e ~ a m ~ l e , during tissue formation or remodeling, such matrices would provide a highly ~ydrated,Auid pericellular environment in which assembly of other matrix corn-

ponents and presentation of growth and differentiation factors could readily occur without interference from the highly structured fibrous matrix usually found in fully differentiated tissues. In some cases, such as in cartilage, the pericellular matrix is a unique structural component that protects the cells and contributes to the characteristic properties of the differentiated tissue. Chondrocytes exhibit particularly prominent pericellular matrices (79-82). Their function and assembly have been studied extensively and shown to be dependent on three features (79,81), First, their integrity is dependent on hyaluronan. Thus treatment of cells exhibiting pericellular matrices with hyaluronanspecific hyaluronidase destroys their structure (Fig. 5A, C versus R, D). Second, the assembly and density of chondrocyte pericellular matrix depend on a specific interaction of hyaluronan with the proteoglycan, aggrecan. Third, hyaluronan must be tethered to the cell surface. Tethering of hyaluro~anto different cell types can occur by two independent mechanisms, i.e., by transmembrane interaction of “nascent” hyaluronan with hyaluronan synthase (see Fig. 2b), or by binding to specific hyaluronan receptors, e.g., CD44, on the cell surface. In the case of chondrocytes, tethering is mediated mainly by interaction of hyaluronan with CD44 (81 3 2 ). Chondrocyte pericellular matrix can be reconstituted on living or fixed, matrix-free chondrocytes by addition of hyaluronan and aggrecan (81). A similar pericellular matrix can also be assembled on other cells that lack a pericellular matrix but possess hyaluronan receptors (83). Analysis of the motion of gold particles attached to hyaluronan or aggrecan in the pericellular matrix of chondrocytes has clearly illustrated several features of these matrices (84) (Fig. 6). First, individual tethered hyaluronan molecules can extend as far as -10 pm from the cell surface. Second, extension of hyaluronan molecules from the cell surface depends on interaction with aggrecan. Third, hyaluronan-aggrecan complexes move in a restricted, cone-shaped area

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Model of chondrocyte pericellular matrix. The de Gennes model for polymers grafted at an interface (85) is adapted to the structure of chondrocyte pericellular matrix (84). (a) When hyaluronan (HA) alone is bound to the cell surface it collapses in a “mushroom” configuration, as predicted for grafted polymers at low density. In this configuration pericellular matrix is not evident. (bj When aggrecan interacts with hyaluronan, the resulting negative charge repulsion between GAG side-chains simulates the effect of increased polymer density in the de Gennes model. This causes the hyaluronan-aggrecan complexes to extend out from the cell surface, forming a “brush” (85) which can be visualized as pericellular matrix. Theoretically, the same effect could be achieved at very high hyaluronan concentration at the cell surface. (c) Tethered motion (arrows) in the latter configuration excludes large particles, such as red blood cells. The size of particle excluded will depend on polymer density, e.g., additional exogenous aggrecan causes exclusion of smaller particles (86). (From Ref. 84.)

consistent with tethering of hyaluronan to the cell surface. Fourth, such motion would exclude large, but not small, particles from the pericellular zone (84). This behavior of pericellular hyaluronan-aggrecan complexes fits well with the predicted behavior of polymers tethered to a surface at high density (85).The highproteoglycan concentration in cartilage would increase the density of the pericellular matrix (84,86) and thus increase resistance to compression, a fundamental characteristic of cartilage, Interactions between CD44, hyaluronan, and other hyaluronan-binding proteoglycans, e.g., versican, neurocan, and brevican, would be expected to promote coat formation around cells other than chondrocytes as has been shown to occur in the case of neural crest (79) and glial cells (87). As explained above, tethering of hyaluronan to the chondrocyte surface is usually mediated by interaction with the hyaluronan receptor, CD44. However, in several other cells that exhibit pericellular matrices, hyaluronan is more likely to be in the process of extrusion across the plasma membrane and thus may be tethered by sustained attachment to hyaluronan synthase or associated proteins on the cytoplasmic face of the plasma membrane (see Fig. 2b). Indeed, transfection of cells lacking pericellular matrices with hyaluronan synthase is in some cases sufficient to induce matrix formation (1 1,124). Also, in several cell types it has been shown that pericellular hyaluronan is tethered to the cell surface but is not attached to receptors (88-90). However, it is not clear whether hyaluronan tethered to synthase produces a pericellular matrix without binding to proteoglycan. Theoretically, if a high enough density of hyaluronan molecules were tethered at the cell surface, they would form an extended array or “brush” that would protrude outward from the cell surface (85) and form a continuous network via self-interactions (31,32); this arran~ementmay then be sufficient to form such a matrix. It is clear, however, that addition of proteoglycans favors pericellular matrix formation by increasing its density and thus possibly its stability (84,86), Inclusion of other hyaluronan-binding proteins such as inter-~-trypsininhibitor and TSC-6 may also stabilize pericellular matrices by cross-linking hyaluronan chains (54,91). Some pathogenic bacteria have hyaluronan capsules that may also be tethered via sustained interaction of “nascent” hyaluronan with hyaluronan synthase. These capsules would represent a primitive form of pericellular matrix which, in bacteria, acts to facilitate bacterial invasion by inhibit in^ phagocytosis (92) andlor by promoting adhesion to host tissues (93).

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The pericellular matrices surrounding migrating and proliferating cells, during m o ~ h o ~ e n e s of i s embryonic organs and during regeneration and healing, are

enriched in hyaluronan and resemble the pericellular matrices described in the previous section. Striking examples of such hyaluronan-rich matrices occur: around mesenchymal cells invading the primary corneal stroma; around neural crest cells traveling from the neural tube to form peripheral ganglia; around somite cells encompassing the notochord to form vertebrae; around cushion cells migrating from the endocardium toward the myocardium during formation of heart valves; around proliferating and migrating cells during brain development; and around prolif~ratingmesenchymal cells during embryonic limb development, salamander limb regeneration, tendon regeneration, and fetal wound repair (55,56,79). An important way in which hyaluronan-rich matrix would promote cell proliferation is by provision of a hydrated pericellular zone that facilitates cell rounding during mitosis. Hyaluronan synthase activity has been shown to fluctuate with the cell cycle and to peak at mitosis (24,94,95). Thus extrusion of hyaluronan onto the cell surface at mitosis would create a hydrated microenvironment that promotes partial detachment and rounding of the dividing cells. In support of this idea, inhibition of hyaluronan synthesis has been shown to lead to cell cycle arrest at mitosis, just before cell rounding and detachment (24). It is also possible that intracellular hyaluronan-binding proteins, e.g., the cell cycle r e ~ u l a t ofactor, ~ Cdc37 (49), and an intracellular form of R H A (61), ~ ~may be involved in regulation of these events. With respect to cell migration and invasion, hyaluronan-enriched matrices create hydrated pathways that sedarate cellular or fibrous barriers to penetration by the invading cells (55,56).In addition, modulation of the density of these matrices by variation in the degree of binding of proteoglycans to pericellular h y a l u r o n ~may regulate cessation of migration (79). For example, proteoglycans act as barriers to neural crest cell migration and neurite outgrowth in vitro and in vivo (96). In the case of neural crest cells this inhibition is dependent on interaction of proteoglycan with cell surface hyaluronan (96), which would in turn increase the density of the pericellular matrix (84,86), thus transforming the pericellul~matrix from conducive to inhibitory for cell migration (79). In addition to providing a suitable hydrated milieu, interaction of hyal~lronanwith its cell surface receptors may al so initiate signal promote cell movement or ~roliferation.Several investigators that cell movement in vitro is promoted in the presence of hyaluronan (25-27,9~), that invasion into three-dimensio~alcollagen gels is dependent on hyaluronan synthesis ( ~ 2 , ~ 8 , 9 9and ) , that cell movement is inhibited as a result of hyaluronan or blocking binding of hyaluronan to its receptors, CD44 or (22,25,97-99). Interaction of hyaluronan with R ~ stimulates A tyrosine ~ ~ phospho~lationof several proteins, includin~a key component of focal adbesions, p12YAA", resulting in ~egulationof focal adhesion turnover and promotion 6). ~nteractionof hyaluronan with cell surface CD44 stimulates cell migration in some tumor cell types, e.g., glioma and melanoma cells (25,99).

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However, stimulation of migration of ras-transformed cells, which express high levels of both RHANIM and CD44 receptors, was found to be due to hyaluronanRHANINI rather than hyaluronan-C~44interaction in these cells (97). Thus it seems that interaction of hyaluronan with either receptor can stimulate cell movement, but their relative importance may depend on the cell type or other physiological factors. Similarly, interaction of hyaluronan with CD44 or RHANINI appears to stimulate signaling pathways involved in cell proliferation in different cell types (72,78). Nevertheless, the details of these putative pathways are by no means clear for either cell proliferation or locomotion. Studies carried out on embryonic limb development provide an illustrative example of the way in which modulation of pericellular hyaluronan concentration and organization influences several of the events leading to differentiation in vivo; the results of these studies are summarized below and in Fig. 7. First, the volume of hyaluronan-rich matrix separating cells at different stages of limb development closely parallels their differentiation (80). Early limb mesodermal cells are surrounded and separated by extensive, hyaluronan-enriched matrix in vivo and express voluminous, hyaluronan-enriched, hyaluronan-dependent, pericellular matrices in culture (80). At this stage pericellular ~ y a ~ u r o n aappears n to be tethered to the cell via transmembrane interaction with hyaluronan synthase (see Fig. 2b) since it is retained at the cell surface in a nonreceptor mediated manner (88). The pericellular matrix maintains separation of early mesodermal cells ( l 00), consistent with the predicted behavior of surface-associated polymers, i.e., polymer molecules on apposing surfaces do not interdigitate due to their constant motion (see Fig. 6) (84,85). This hydrated pericellular matrix would facilitate proliferation and migration of early mesenchymal precursors of limb tissues in the manner described in the preceding paragraphs. This has been demonstrated directly in the case of muscle differentiation, where it has been shown that exposure of myoblasts to hyaluronan supports proliferation and migration but inhibits differentiation (101,102). Subsequent to the early stage of limb development described in the previous paragraph the mesoderm condenses; i.e., the intercellular matrix decreases in volume, at sites of future cartilage and muscle differentiation. This is paralleled by loss of ability of the mesodermal cells to form hydrated pericellular matrices in culture. During this condensation, much of the hyaluronan is rerrioved from the intercellular matrix, thus accounting for the decreased intercellular volume (80). owever, cell surface hyaluronan is now retained via interaction with receptors that appear on the mesodermal cells at this stage (88). This cell surface hyaluronan interacts multivalently with receptors on adjacent cells, thus cross-bridging them within the condensate (100) (Fig. 7b). Cross-bridging occurs in an analogous fashion to that previously shown for aggregation of various cell lines in culture (SS,.%). Similar events comprise an early step in mesoderma~condensation in other developing tissues also, e.g., skin and teeth (103-105).

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Modulation of hyaluronan-cell interactions during mesodermal condensation and chondrocyte differentiationin the embryonic limb. (a) Early mesodermal cell precursors are separated by extensive hyaluronan-rich matrix; hyaluronan i s most likely retained at the cell- sur f ace by tran~membraneinteraction with hyaluronan synthase. (b) During mesodermal condensation,hyaluronan-binding sites are expressed and mediate initial aggregation via hyaluronan cross-bridging:hyaluronan binding may also mediate endocytic inte~alizationof hyaluronan. (c) During cartilage differentiation,hyaluronan-binding sites ( 0 4 4 ) retain hyaluronan-aggrecan complexes in the pericellular matrix. (Adapted from Ref. 155.) ~ u r t h e rdifferentiation of condensed limb mesoderm to cartilage is accompanied by extensive matrix formation in vivo and recovery of the ability to form extensive hyaluronan-dependent pericellular matrices in culture (80). In these matrices, however, ~ y a l u r o ~ aisntethered to the cell surface by interaction with CD44 and the proteoglycan concentration i s much higher than in the matrices su~oundingearly mesodermal cells (80-82?88). Thus, the pericellular matrix of differentiate^ chondrocytes would be denser than that of mesodermal cells (84,86), r e ~ e ~ t its i nstructural ~ rather than morphogenetic role. With respect to muscle differentiation? mononucleated yobl lasts are also initially separated from each other by a hyaluronan-rich matrix in vivo and ex-

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press pericellular matrices in culture (106). However, during the processes of condensation and fusion to give myotubes these coats are lost (Fig. 5E, F), thus allowing the interactions required for these cells to differentiate (106). During long bone differentiation, lacunae surrounding hypertrophic chondrocytes are highly enriched in hyaluronan (107), and the swelling pressure exerted by this hyaluronan causes expansion of lacunae as bone growth occurs (108). Subsequently, in the zone of erosion, the hyaluronan within these lacunae is removed via CD44-mediated endocytosis (107). CD44-mediated i n t e ~ a l i z a t i oof~ hyaluronan is an. important step in differentiation of several other tissues also, e.g., skin (103,104) and lung (109). Another interesting system in which hyaluronan plays an important role is expansion of the cumulus oophorus during ovulation. In response to folliclestimulating hormone and a factor produced by the oocyte, hyaluronan synthesis by the cumulus cells surrounding the oocyte increases dramatically (1 10). A stable, gel-like matrix is formed between the cumulus cells due to cross-linking of hyaluronan by inter-a-trypsin inhibitor and TSC-6 (54,91), This matrix is responsible for the integrity of the cumulus cell-oocyte complex which is required for protection and transport of the oocyte during ovulation, entry into the oviduct, and fertilization (1 11). Sperm-associated hyaluronidases allow penetration of this matrix at fertilization (1 12). Recent studies indicate that hyaluronan is important in branching morphogenesis of epithelial organs (113) and in formation of blood vessels (1 14,115). In these cases it is again probable that hyaluronan provides a hydrated milieu that favors proliferation and movement of epithelial and endothelial cells at sites of branch extension. A hyaluronan-enriched pericellular milieu is also important in control of normal cellular behavior in the mature organism. For example, hyaluronan surrounds individual cells within many complex epithelia, e.g., the epidermis and esophagus, and may facilitate m o v e ~ e n tof cells between epithelia strata as they differentiate (1 16). Also, extravasation of lymphocytes at sites of i~flammationinvolves adhesive interactions between CD44 on the lymphocyte surface and hyaluronan on the endothelial cell surface; expression of hyaluronan on the latter cells is induced by proin~ammatorycytokines such as TNFa and IL-1p, and by bacterial lipopolysaccharide (23).

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malignant solid tumors contain elevated levels of hyaluronan (1 17,118). levels of hyaluronan expression correlate with poor differentiation in human ductal breast carcinomas (1 19) and with poor survival rates in human colorectal adenoc~cinomas(1120).

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~nrichmentof hyaluronan in tumors is due to increased production by tumor cells themselves or to interactions between tumor cells and surrounding stromal cells that induce increased production by the latter. For example, elevated hyaluronan production by mouse mammary carcinoma (l2 1) and melanoma cells (122) correlates with metastatic capacity. Also, however, interaction of several types of malignant tumor cells with stromal cells upregulates stromal production of hyaluronan (l 17,123- 125). Thus hyaluronan accumulation occurs at the interface of tumor invasion into host tissues in various tumor types (l 17,126, 127). Direct experimental evidence has been obtained implicating hyaluronan in tumor progression for murine melanoma and breast carcinomas (128-13 l ), However, in the case of the rat pancreatic carcinoma cell line, BSp73AS, metastasis appears to be hyaluronan-in.dep~ndent(132). Hyaluronan receptors have been widely implicated in tumorigenesis but their involvement al so varies. Current evidence suggests that CD44-mediated events can enhance (l33- 135) or inhibit (136,l 37) t umor progression in different types of tumors. In addition, targeted d i s ~ p t i o nof CD44 that results in loss of hyaluronan binding in MDAY-D2 murine lymphosarcoma cells has no apparent effect on their growth and metastasis in vivo (138). Although soluble forms of CD44 have been used as antagoni§ts of lymphoma, melanoma, and carcinoma progression (128- 130,139), a soluble hyaluronan-binding fragment of brevican promotes glioma invasion in vivo (140). As discussed previously, overexpression of cell surface RHAMM causes fibroblasts to become tumorigenic (77) and treatment of fibroblasts with a soluble form of T HAM^ causes inhibition of ~brosarcomagrowth and metastasis in vivo (78). However, the extent to which ~ H A is~involved M in progression of different tumor types is not known. Although considerable evidence points to an important role for hyaluronancell interactions in tumor progression, the nature and breadth of their involvement, and the mechanisms whereby they act, are still unclear. Among the most likely reasons for some of the apparent contradictions mentioned in the previous paragraph is the varying importance of hyaluronan and its receptors in different tissue and tumor types (see above) and at different stages of progression (141,142). Another potentially important issue that may cause confusion is that hyaluronan-receptor interactions may lead to different downstream pathways under different circumstances. Whereas these i~teractionsmay directly influence various intracellular signaling pathways important for cell behavior, it is also quite likely, in. the case of CD44, that binding of hyaluronan would lead to internalization andlor degradation. In this regard it is significant that some tunlor cells have been shown to exhibit elevated levels of hyaluronidase (143,144) or ability to internalize and degrade hyaluronan (129,145). On the other hand, at least one of the several hyaluronidase genes appears to correspond to a previously mapped

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tumor suppressor (146). Finally, other adhesive macromolecules, e.g., selectins, could fulfill potentially redundant functions with respect to CD44 (43-45), and macromolecules such as polysialylated glycoproteins may serve overlapping roles with hyaluronan (14’7). Despite these caveats, it is abundantly clear that different interventions aimed at perturbing events involving hyaluronan and its receptors lead to impressive inhibition of several tumor types in vivo (128331,148-151), and that some of these approaches may form the- basis of new therapies.

As stated at the beg~nningof this chapter, hyaluronan bas extraordinary s t ~ c t u r a l and functional properties despite its very simple, repetitive composition. So, not only the chemical structure of hyaluronan but also the study of its fascinating characteristics and roles goes on an’on an’on an’on an’on. It is now fully apparent that hyaluronan is a central player in the physical and chemical properties of pericellular milieu as well as extracellular matrices. The networ~-forming,viscoelastic, and charge characteristics of hyaluronan are fundamental to the biomechanical properties and homeostasis of many tissues, and one of the most active fields in current hyaluronan research is exploitation of these characteristics in treatment of tissue adhesions and arthritic conditions. Also, specific interactions of hyaluronan with proteins and proteoglycans have long been known to be intrinsic to the structural properties of extracellular matrices. Now, however, it is also appreciated that hyaluronan is a crucial pericellular and cell surface constituent that, through interactions with other macromolecules, participates i~portantlyin regulation of cell behavior during numerous morphogenetic, restorative, and pathological processes. Research in these areas is proceeding at an enormous pace, Of special interest is ongoing work on ( l ) the part played by h~aluronansynthases in regulating the properties of pericellular milieu during dynamic cellular processes, and the coordination between synthase activity and the intracellular signaling pathways g o v e ~ i n gthese processes; (2) the importance of hyaluronan polymer size in various physiolo~ical involvement of cell surface hyaluronan receptors, such as CD44 , in transmitting signals between the extracellular and intracellular c o m p ~ m e n t sand , the role of these signals in cell behavior; and (4) manipulahyaluronan-cell interactions as therapeutic interventions in disease. ay the current high level of interest in hyaluronan and the fascinating new discoveries regarding its structure and functions continue to go on an’on an’on an’on an’on!!

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1. Prehm P. Synthesis of hyaluronate in differentiatedteratocarcinoma cells. Biochem

J 1983; 211:191-198. 2, Asplund T, Brinck J, Suzuki M, Briskin MJ, Heldin P. Characterization of hyaluronan synthase from a human glioma cell line. Biochim Biophys Acta 1998; 1380~377-388. 3. Weigel PH, Hascall VC, Tammi M. Hyaluronan synthases.J Biol Chem 1997;272: 13997- 14000. 4. Prehrn P. Hyaluronate is synthesized at plasma membranes. Biochem J 1984; 220: 597-600. 5. Philipson LH, Schwartz NB. Subcellular localization of hyaluronate synthetase in oligodendroglioma cells. J Biol Chern 1984; 2595017-5023. 6. DeAngelis PL, Papaconstantinou J, Weigel PH. Isolation of a Streptococc~spyogenes gene locus that directs hyaluronan biosynthesis in acapsular mutants and in heterologous bacteria. J Biol Chem 1993; 268:14568-14571. 7. DeAngelis PL, Papaconstantinou J, Weigel pH. Molecular cloning, identification, and sequence of the hyaluronan synthase gene from group A Streptococcus pyogenes. J Biol Chern 1993; 268:19181-19184. 8. DeAngelis PL, Weigel pH. Immunochemical confirmation of the primary structure of streptococcalhyaluronan synthase and synthesis of high molecular weight product by the recombinant enzyme. Biochemistry 1994; 33:9033-9039. 9. Kumari K, Weigel PH. Molecular cloning, expression, and characterizationof the authentic hyaluronan synthase from group C Streptococcus e ~ ~ ~ s J~ Biol ~ ~ Z Chem 1997; 272:32539-32546. I O. DeAngelis PL, Jing W, Drake RR, Achyuthan AM. Identification and molecular cloning of a unique hyaluronan synthase from ~ ~ s t e u r e l~Z a~ Z t o c Ji ~Biol a . Chem 1998; 273:8454--8458. 11. Itano N, Kimata K. Expression cloning and molecular characterization of HAS protein, a eukaryotic hyaluronan synthase. J Biol Chem 1996; 271:9875-9878, 12. Itano N, Kimata K. Molecular cloning of human hyaluronan synthase. Biochem Biophys Res C o m u n 1996; 222916-820. 13. Shyjan AM, Heldin P, Butcher EC, Yoshino T, Briskin MJ. Functional cloning of the cDNA for a human hyaluronan synthase. J Biol Chem 1996; 271:2339523399. 14. Spicer AP, Augustine ML, McDonald JA. Molecular cloning and ch~acterization of a putative mouse hyaluronan synthase. J Biol Chem 1996; 271:23400-23406. 15. Fuliip C, Salustri A, Hascall VC. Coding sequence of a hyaluronan synthasehomologue expressed during expansion of the mouse cumulus-oocyte complex. Arch Biochem Biophys 1997; 337:261-266. 16. Watanabe K, Yamugwchi Y. Molecular identification of a putative human hyaluronan synthase. J Biol Chem 1996; 271 :22945-22948. 17. Spicer AP, Olson JS, McDonald JA. Molecular cloning and characterization of a cDNA encoding the third putative m a ~ a l i a hyaluronan n synthase. J Biol Chem 1997; 272:8957-8961.

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18. Spicer AP, McDonald JA. Characterizationand molecular evolution of a vertebrate hyaluronan synthase gene family. J Biol Chem 1998; 273:1923-1932. 19. Pummill PE, Achyuthan AM, DeAngelis PL. Enzymological characterization of recombinant Xenopus DG42, a vertebrate hyaluronan synthase.J Biol Chem 1998; 273:4976-498 1. 20. Spicer AP, Seldin MF, Olsen AS, Brown N, Wells DE, Doggett NA, Itano N, Kimata K, Inazawa J, McDonald JA. Chromosomal localization of the human and mouse hyaluronan synthase genes. Genornics 1997; 41:493-497. 21. Heldin P, Asplund T, Ytterberg D, Thelin S, Laurent TC. Characterization of the molecular mechanism involved in the activation of hyaluronan synthetaseby platelet-derived growth factor in human mesothelial cells. Biochem J 1992; 283:165170. 22. Ellis I, Banyard J, Schor SL. Differential response of fetal and adult fibroblasts to cytokines:cell ~ g r a t i o nand hyaluronan synthesis. Development 1997; 124:15931600, 23. Mohalnadzadeh M, DeGrendele H, Arizpe H, Estess P, Siegelman M. F r o i n ~ a ~ matory stimuli regulate endothelial hyaluronan expression and CD44/HA-dependent primary adhesion. J Clin Invest 1998; 10~:9?-108. 24. Brecht, M, Mayer U, Schlosser E, Prehm P. Increased hyaluronate synthesis is required for fibroblast detachment and mitosis. Biochem J 1986; 239:445-450. 25. Thomas L, Byers HR, Vink J, Stamenkovic1. CD44H regulates tumor cell migration on hyaluronate-coated substrate. J Cell Biol 1992; 118:971-977. 26. Hall CL, Wang C, Lange LA, Turley EA. Hyaluronan and the hyaluronan receptor RHAMM promote focal adhesion turnover and transient tyrosine kinase activity, J Cell Biol 1994; 126575-588. 27. Savani RC, Wang C, Yang B, Zhang S, Kinsella MG, Wight TN, Stern R, Nance DM, Turley EA. Migration of bovine aortic smooth muscle cells after wounding injury. The role of hyaluronan and RHAMM. J Clin Invest 1995; 95:1158-1168. 28. DeAngelis PL, Jing W, Graves MV, Burbank DE, Van Etten JL. Hyaluronan synthase of Chlorella virus PBCV-l. Science 1997; 278:1800-1 803. 29. Scott JE. Chemical morphology of hyaluronan. In: Laurent TC, ed. The Chemistry, Biology and Medical Applications of Hyaluronan and Its Derivatives. London: Portland Press, 1998:7-15. 30. Heatley F, Scott JE. A water molecule participates in the secondary structure of hyaluronan. Biochem J 1988; 254:489-493. 31. Scott JE, Cummings C, Brass A, Chen Y. Secondary and tertiary structures of hyaluronan in aqueous solution, investigatedby rotary shadowing-electron microscopy and computer simulation. Biochem J 1991; 274:699-705. 32. Mikelsaar R-H. Scott JE. Molecular modelling of secondary and tertiary structures of hyaluronan, compared with electron microscopy and NMR data. Possible sheets and tubular structures in aqueous solution. GlycoconjugateJ 1994; 11:65-71. 33. Comper WD, Laurent TC. Physiological function of connective tissue polysaccharides. Physiol Rev 1978; 58:255-315. 34. Laurent TC, Fraser JRE. Hyaluronan. FASEB J 1992; 6:2397-2404, 35. Balazs EA, Denlinger JL. Clinical uses of hyaluronan. Ciba Found Symp 1989; 143:265-275. i

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36. Bertolami CN, Gay T, Clark GT, Rendell J, Shetty V, Liu C, Swann DA. Use of sodium hyaluronate in treating temporomandibular joint disorders: a randomized, double-blind, placebo-controlled clinical trial. J Oral Maxillofac Surg 1993; 5 1: 232-242. 37. Burns JW, Skinner K, Colt MJ, Burgess L, Rose R, Diamond MP, A hyaluronate based gel for the prevention of postsurgical adhesions: evaluation in two animal species. Fertil Steril 1996; 66:8 14-82 1. 38. Hardingham TE. Cartilage: Aggrecan-link protein-hyaluronan aggregates. Glycoforum: Science of hyaluronan: http://www.glycoforurn.gr.jp 39. Iozzo, RV. Matrix proteoglycans: from molecular design to cellular function. Annu Rev Biochem 1998; 67:609-652. 40. Sherman L, Sleeman J, Herrlich P, Ponta H. Hyaluronate receptors: key players in growth,differentiation,migration and tumor progression. Curr Opin Cell Biol 1994; 6~726-733. 41. Lee TH, Wisniewski H-G, VilEek J. A novel secretory tumor necrosis factor-inducible protein (TSG-6) is a member of the family of hyaluronate binding proteins, closely related to the adhesion receptor CD44. J Cell Biol 1992; 116:545-557. 42. Peach RJ, Hollenbaugh D, Stamenkovic I, Aruffo A. Identification of hyaluronic acid binding sites in the extracellular domain of CD44. J Cell Biol 1993; 122:257264. 43 Bajorath J, Greenfield B, Munro SB, Day AJ, Aruffo A. Identification of CD44 residues important for hyaluronan binding and delineation of the binding site. J Biol Chem 1998; 273:338-343. 44. Kohda D, Morton CJ, Parkar AA, Hatanaka H, Inagaki FM, Campbell ID, Day AJ. Solution structure of the link module: a hyaluronan-binding domain involved in ex~acellularmatrix stability and cell migration. Cell 1996; 86:767-775. 4s. Brissett NC, Perking SJ. The protein fold of the hyaluronate-binding proteoglycan tandem repeat domain of link protein, aggrecan and CD44 is similar to that of the C-type lectin s u p e ~ a ~ lFEBS y . Lett 1996; 388:211-216. 4.6. Yang B, Yang BL, Savani RC, Turley EA. Identification of a common hyaluronan binding motif in the hyaluronan binding proteins RHAMM, CD44 and link protein. EMBO J 1994; 13:286-296. 47. McCourt PAC, Ek B, Forsberg N, Gustafson S. Intercellular adhesion rnolecule1 is a cell surface receptor for hyaluronan. J Biol Chem 1994; 269:30081-30084. 48. Zhu L, Hope TJ, Hall J, Davies A, Stern M, Muller-Eberhard U, Stern R, Parslow TG. Molecular cloning of a mammalian hyaluronidase reveals identity with hemopexin, a serum herne-binding protein. J Biol Chern 1994; 269:32092-32097. 49. Grammatikakis, N, ~rammatikakisA, Yoneda M, Yu Q, Banerjee SD, Toole BP. A novel glycosarninoglycan-binding protein is the vertebratehomologue of the cell cycle control protein, Cdc37. J Biol Chem 199s; 270:16198- 1620s. 50. Deb TB, Datta K. Molecular cloning of human fibroblast hyaluronic acid-binding protein confirms its identity with I?-32, a protein co-purified with splicing factor SF2. J Biol Chem 1996; 271:2206-2212. 51, Kincade PW, Zheng Z, Katoh S, Hanson L. The importance of cellular environment to function of the CD44 matrix receptor. Curr Opin Cell Biol 1997; 9535-642. 52, Huang L, Yoneda M, Kimata K. A serum-derived hyaluronan-associated protein (I

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(SHAP) is the heavy chain of the inter a-trypsin inhibitor. J Biol Chem 1993; 268: 26725-26730. Yoneda M, Suzuki S, Kimata K. Hyaluronic acid associated with the surfaces of cdtured fibroblasts is linked to a serum-derived 85-kDa protein. J Biol Chem 1990; 265~5247-5257. Chen L, Mao SJT, McLean LR, Powers RW, Larsen WJ. Proteins of the inter-atrypsin inhibitor family stabilize the cumulus extracellular matrix through their direct binding with hyaluronic acid. J Biol Chem 1994; 269:28282-28287. Toole BP. Glycosaminoglycans in morphogenesis. In: Hay E, ed. Cell Biology of Extracellular Matrix. New York: Plenum Press, 1981:259-294. Took BP. Proteoglycans and hyaluronan in morphogenesis and differentiation.In: Hay E, ed. Cell Biology of Extracellular Matrix, 2d ed. New York: Plenum Press, 1991:305-341. Aruffo A, StamenkovicI, Melnick M, Underhill CB, Seed B. CD44 is the principal cell surface receptor for hyaluronate. Cell 1990; 61: 1303- 1313. Hardwick C, Hoare K, Owens R, Hohn HP, Hook M, Cripps V, Austen L, Nance DM, Turley EA. Molecular cloning of a novel hyaluronan receptor that mediates tumor cell motility. J Cell Biol 1992; 117:1343-1350. Lesley J, Hyman R, Kincade PW. CD44 and its interaction with extracellular matrix. Adv Immunol 1993; 54:271-335. Borland G, Ross JA, GUYK. Forms and functions of CD44. Immunology 1998; 93~139-148. Entwistle J, Hall CL, Turley EA. HA receptors: regulators of signalling to the cytoskeleton. J Cell Biochem 1996; 61:569-577. Screaton GR, Bell MV, Bell JI, Jackson DC. The identification of a new alternative exon with highly restricted tissue expression in transcripts encoding the mouse Pgp-1 (CD44) homing receptor. J Biol Chem 1993; 268:12235-12238. Yu Q, Toole BP. A new alternatively spliced exon between v9 and v10 provides a molecular basis for synthesis of soluble CD44. J Biol Chem 1996; 271:2060320607. Katoh S, McCarthy JB, Kincade PW. Characterization of soluble CD44 in the circulation of mice. J Immunol 1994; 153:3440-3449. Bazil V, StromingerJL. Metalloprotease and serine protease are involved in cleavage of CD43, CD44, and CD16 from stimulated human granulocytes. J Imrnunol 1994; 152:3314-1322. Skelton TP, Zeng C, Nocks A, Stamenkovic I. Glycosylation provides both stimulatory and inhibitory effects on cell surface and soluble CD44 binding to hyaluronan. J Cell Biol 1998; 140:431-446. Sleeman J, Rudy W, Hofmann M, Moll J, Herrlich P, Ponta H. Regulated clustering of variant CD44 proteins increases their hyaluronate binding capacity. J Cell Biol 1996; 135~1139-1150. Jackson DC, Bell JI, Dickinson R, Timans J, Shields J, Whittle N. Proteoglycan forms of the lymphocyte homing receptor CD44 are alternatively spliced variants containing the v3 exon. J Cell Biol 1995; 128:673-685. Bourguignon LYW, Lokeshwar VB, Chen X, Kerrick WGL. Hyaluronic acid-in-

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duced lymphocyte signal tranduction and HA receptor (GP85/CD44)-cytoskeleton interaction. J Immunol 1993: 15 l :6634-6644. Galluzzo E, Albi N, Fiorucci S, Merigiola C, Ruggeri L, Tosti A, Grossi CE, Velardi A. Involvement of CD44 variant isoforms in hyaluronate adhesion by human activated T cells. Eur J Immunol 1995; 25:2932-2939. Taher TEI, Smit L, Griffioen AW, Schilder-To1EJM, Borst J, Pals ST. Signaling through CD44 is mediated by tyrosine kinases. J Biol Chem 1996; 271:2863-2867. Bourguignon LYW, Zhu H, Chu A, Iida N, Zhang L, Hung M-C. Interaction between the adhesion receptor, CD44, and the oncogene product pS8SFiER2, promotes human ovarian tumor cell activation. J Biol Chem 1997; 272:27913-27918. Deed R, Rooney P, Krrmar P, Norton JD, Smith J, Freemont AJ, Kumar S. Earlyresponse gene signalling is induced by angiogenic oligosaccharidesof hyaluronan in endothelial cells. Inhibition by non-angiogenic,high-molecular-weighthyaluronan. Int J Cancer 1997; 71:25 1-256. McKee CM, Penno MB, Cowman M, Burdick MD, Strieter RM, Bao C, Noble PW. Hyaluronan (HA) fragments induce chemokine gene expression in alveolar macrophages. J Clin Invest 1996; 98:2403-2413. Zhu D, Bourguignon LY. The ankyrin-binding domain of CD44s is involved in regulating hyaluronic acid-mediated functions and prostate tumor cell transformation. Cell Motil Cytoskel 1998; 39:209-222. Yonernura S, Hirao M, Doi Y, Takahashi N, Kondo T, Tsukita S, Tsukita S. Ezrin/ radixin/moesin (ERM) proteins bind to a positively charged amino acid cluster in the juxta-membranecytoplasmic domain of CD44, CD43, and ICAM-2. J Cell Biol 1998; 140~885-895. Hall CL, Yang B, Yang X, Zhang S, Turley NI, Samuel S, Lange LA, Wang C, Curpen GD, Savani RC, Greenberg AH, Turley EA. Overexpression of the hyaluronan receptor RHAMM is transforming and is also required for H-ras transformation. Cell 1995; 82:19-28. Mohapatra S, Yang X, Wright JA, Turley EA, Greenberg AH. Soluble hyaluronan receptor RHAMM induces mitotic arrest by suppressing Cdc2 and cyclin B l expression. J Exp Med 1996; 183:1663-1668. Knudson CB, Knudson W. Hyaluronan-binding proteins in development, tissue homeostasis, and disease. FASEB J 1993; 7:1233-1241. Knudson CB, Toole BP. Changes in the pericellular matrix during differentiation of limb bud mesoderm. Dev Biol 1985; 112:308-318. Knudson CB. Hyaluronan receptor-directed assembly of chondrocyte pericellular matrix. J Cell Biol 1993; 120:825-834. Knudson W, Aguiar DJ, Hua Q, Knudson CB. CD44-anchored hyaluronan-rich pericellular matrices: an ultrast~cturaland biochemical analysis. Exp Cell Res 1996; 228:216-228. Knudson W, Bartnik E, Knudson CB. Assembly of pericellular matrices by COS7 cells transfected with CD44 l~mphocyte-homingreceptor genes. Proc Natl Acad Sci USA 1993; 90:4003-4007. Lee GM, Johnstone B, Jacobson K, Caterson B. The dynamic structure of the pericellular matrix on living cells. J Cell Biol 1993; 123:1899-1907.

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Schrager HM, Alberti S, Cywes C, Dougherty GJ, Wessels MR. Hyaluronic acid capsule modulates M protein-mediated adherence and acts as a ligand for attachment of group A Stre~tococc~s to CD44 on human keratinocytes. J Clin Invest

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95

*

activity of isolated plasma-mel~branefractions of cultured human skin fibroblasts. Biochem J 1986; 237:333- 342. Matuoka K, Namba M, Mitsui Y. Hyaluronate synthetase inhibition by normal and ~ansformedhuman fibroblasts during growth reduction. J Cell Biol 1987; 104:

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Inhibitory effects of PG-Hlaggrecan and PG-M/versican on avian neural crest cell migration. FASEB J 1996; 10:293- 301. Turley EA, Austen L,, Moore D, Hoare K. transformed cells express both CD44 and RHAMM hyaluronan receptors: only RHAMM is essential for hyaluronanpromoted locomotion. Exp Cell Res 1993; 207:277- 282. Boudreau N, Turley E, Rabinovitch M. Fibronectin, hyaluronan, and a hyaluronan binding protein contributeto increased ductus arteriosus smooth muscle cell migration. Dev Biol 1991; 143:235- 247. Koochekpour S, Pil~ingtonGJ, Merzak A. Hyaluronic acid/C~44Hinteraction induces cell detachment and stimulates migration and invasion of human glioma cells in vitro. Int J Cancer 1995; 63:450- 454. Maleski MP, Knudson CB. Hyaluronan-mediated aggre~ationof limb bud mesen-

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zyxwvu zyxw zy zyxwv zyxwvuts zyxwvu chyme and mesenchymal condensation during chondrogenesis. Exp Cell Res 1996; 224:55-66. Kujawa MJ”, Pechak DG, Fiszman MY, Caplan AI. Hyaluronic acid bonded to cell culture surfaces inhibits the program of myogenesis, Dev Biol 1986; 113:lO-16. Krenn V, Brand-Saberi B, Wachtler F. Hyaluronic acid influences the migration of myoblasts within the avian embryonic wing bud. Am J Anat 1991; 192:400406. Underhill CB. Hyaluronan is inversely correlated with the expression of CD44 in the dermal condensation of the embryonic hair follicle. J Invest Dermatol 1993; 101:820-826. Kaya G, Rodriguez I, Jorcano JL, Vassalli P, Stamenkovic I. Selective suppression of CD44 in keratinocytes of mice bearing an antisense CD44 transgene driven by a tissue-specific promoter disrupts hyaluronate metabolism in the skin and impairs keratinocyte proliferation. Genes Dev 1997; 11:996- 1007. Yu Q, Toole BP. Common pattern of CD44 isofoms is expressed in morphogenetically active epithelia. Dev Dynamics 1997; 208: 1-10. Orkin RW, Knudson W, Toole BP. Loss of hyaluronate-dependent coat during myoblast fusion. Dev Biol 1985; 107:527-530. Pavasant P, Shizari TM, Underhill CB. Distribution of hyaluronan in the epiphysial growth plate: turnover by CD44-expressing osteoprogenitorcells. J Cell Sci 1994; 107:2669-2677. Pavasant P, Shizari T, Underhill CB. Hyaluronan contributes to the enlargement of hype~rophiclacunae in the growth plate. J Cell Sci 1996; 109:327-334. Underhill CB, Nguyen HA, Shizari M, Culty M. CD44 positive macrophages take up hyalu~onanduring lung development. Dev Biol 1993; 155:324-336. Tirone E, D’Alessandris C, Hascall VC, Siracusa G, Salustri A. Hyaluronan synthesis by mouse cumuluscells is regulated by interactionsbetween follicle-stimulating hormone (or epidermal growth factor) and a soluble oocyte factor (or transfo~ing growth factor beta l). J Biol Chem 1997; 2724787-4794. Chen L, Russell PT, Larsen WJ. Functional significance of cumulus expansion in the mouse: roles for the preovulato~synthesis of hyaluronic acid within the cumulus mass. Mol Reprod Dev 1993; 34:87-93. Myles DG, Primakoff P. Why did the sperm cross the cumulus? To get to the oocyte. Functions of the sperm surface proteins PH-20 and fertilin in arriving at, and fusing with, the egg. Biol Reprod 1997; 56:320-327. Gakunga P, Frost G, Shuster S, Cunha G, Formby B, Stern R. Hyaluronan is a prerequisite for ductal branching morphogenesis. Development 1997; 124:39873997. Banerjee SD, Toole BP. ~yaluronan-bindingprotein in endothelialcell morphogenesis. J Cell Biol 1992; 119:643-652. MontesanoR, Kumar S, Orci L, Pepper MS. Synergisticeffect of hyaluronan oligosaccharides and vascular endothelial growth factor on angiogenesis in vitro. Lab Invest 1996; 75:249-262. Tammi R, Tarnmi M, Hyaluronan in the epidermis. Glycoforum:Science of hyaluronan. h t ~ :/ / www.glyco fo r u m .gr .jp

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117. Knudson W, Biswas C, Li XQ, Nemec RE, Toole BP. The role and regulation of tumour-associatedhyaluronan. Ciba Found Symp 1989; 143:1.50-1.59. 118. Knudson W. Tumor-associated hyaluronan: providing an extracellular matrix that facilitates invasion. Am J Pathol 1996; 148: 1721-1726. 119. Auvinen PK, Parkkinen JJ, Johansson RT, Agren UM, Tammi RH, Eskelinen MJ, Kosma V-M. Expression of hyaluronan in benign and malignant breast lesions. Int J Cancer 1997; 74:477-481. 120. Ropponen K, Tammi M, Parkkinen J, Eskelinen M, Tarnmi R, Lipponen P, Agren U, Alhava E, Ko s ma V-M. Tumor cell-associated hyaluronan as an unfavorable prognostic factor in colorectal cancer. Cancer Res 1998; .58:342-347. 121. ISirnata K, Honma Y, Okayama M, Oguri K, Hozumi M, Suzuki S. Increased synthesis of hyaluronic acid by mouse mammary carcinoma cell variants with high metastatic potential. Cancer Res 1983; 43: 1347-1 3.54. 122. Zhang L, Underhill CB, Chen L. Hyaluronan on the surface of tumor cells is correlated with metastatic behavior. Cancer Res 1995; .55:428-433. 123. Toole BP, Biswas C, Gross J. Hyaluronate and invasivenessof the rabbit V2 carcinoma. Proc Natl Acad Aci USA 1979; 766299-6303. 124. Knudson W, Biswas C, Toole BP. Interactions between human tumor cells and fibroblasts stimulate hyaluronate synthesis. Proc Natl Acad Sci USA 1984; 81: 6767-677 1. 12.5. Asplund T, Versnel MA, Laurent TC, Heldin P. Human mesothelioma cells produce factors that stirnulate the production of hyaluronan by mesothelial cells and fibroblasts. Cancer Res 1993; .53:388--392. 126. Wang C, Tarnmi M, Guo H, Tammi R. Hyaluronan distribution in the normal epithelium of esophagus, stomach, and colon and their cancers. Am J Pathol 1996; 148:1861-1869. 127. Yea T-K, Nagy JA, Yeo K-T, Dvorak HF, Toole BP. Increased hyaluronan at sites of attachment to mesentery by CD44-positivemouse ovarian and breast tumor cells. Am J Path01 1996; 148~1733-1740. 128. Bartolazzi A, Peach R, Aruffo A, StamenkovicI. Interaction between CD44 and hyaluronate is directly implicated in the regulation of tumor development. J Exp Med 1994; 18053-66. 129. Yu Q, Toole BP, Stamenkovic I. Induction of apoptosis of metastatic mammary carcinoma cells in vivo by disruption of tumor cell surface CD44 function. J Exp Med 1997; 186:198.5-1996. 130. Peterson RIM, Yu Q, Stamenkovic I, Toole BP. Perturbation of hyaluronan interactions prevents murine ma~maryascites tumorigenesis in vivo. Submitted. 131. Zeng C, Toole BP, Kinney SD, Kuo J-W, Stamenkovic I. Inhibition of tumor growth in vivo by hyaluronan oligomers. Int J Cancer 1998: 77:396-401. 132. Sleeman JP, Arming S, Moll JF, Hekele A, Rudy W, Sherman LS, Kreil G, Ponta H, Herrlich P. Hyaluronate-independent metastatic behavior of CD44 variantexpressing pancreatic carcinoma cells. Cancer Res 1996; 56:3134-3 14l. 133. Sy MS, Guo Y-J, Starnenkovic I. Distinct effects of two CD44 isoforms on tumor growth in vivo. J Exp Med 1991; 174:8.59-866. 134. gun the^ U, Hofmann M, Rudy W, Reber S, Zoller M, Haussman I, Matzku S,

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Wenzel A, Ponta H, Herrlich P. A new variant of glycoprotein CD44 confers metastatic potential to rat carcinoma cells. Cell 1991; 65:13-24. Iida N. Bourguignon LY. Coexpression of CD44 variant (vl0/ex14) and CD44S in human mammary epithelial cells promotes tumorigenesis. J Cell Physiol 1997; 171~152-160. Takahashi K, Stamenkovic I, Cutler M, Saya H, Tanabe KK. CD44 hyaluronate binding influences growth kinetics and tumorigenicity of human colon carcinomas. Oncogene 1995; 11:2223-2232. Schmits R, Filmus J, Gerwin N, Senaldi G, Kiefer F, Kundig T, Wakeham A, Shahinian A, Catzavelos C, Rak J, Furlonger C, Zakarian A, Sirnard JJL, Ohashi PS, Paige CJ, Gutierrez-Ramos JC, Mak TW. CD44 regulates hematopoietic progenitor distribution, granuloma formation, and tumorigenicity. Blood 1997; 90: 2217-2233. Driessens MH, Stroeken PJ, Erena NF, van der Valk MA, van Rijthoven EA, Roos E. Targeted disruption of CD44 in MDAY-D2 lymphosarcoma cells has no effect on subcutaneous growth or metastatic capacity. J Cell Biol 1995; 131:1849-1 855. Sy MS, Guo Y-J, StamenkovicI. Inhibition of tumor growth in vivo with a soluble CD44-immunoglobulin fusion protein. J Exp Med 1992; 176:623-627. Zhang H, Kelly G, Zerillo C, Jaworski DM, Hockfield S. Expression of a cleaved brain-specific extracellular matrix protein mediates glioma cell invasion in vivo. J Neurosci 1998; 18:2370-2376. Kogerman P, Sy MS, Culp LA. Over-expression of human CD44s in murine 3T3 cells: selection against during primary tumorigenesis and selection for during micrometastasis. Clin Exp Metastasis 1998; 16:83-93. Soukka T, Salrni M, Joensuu H, Hakkinen L, Sointu P, Koulu L, Kalimo K, Klemi P, Grenman R, Jalkanen S. Regulation of CD44v6-containing isoforms during proliferation of normal and malignant epithelial cells. Cancer Res 1997; 57:22812289. Liu D, Pearlman E, Diaconu E, Guo K, Mori H, Haqqi T, Markowitz S, Willson J, Sy M-S. Expression of hyaluronidase by tumor cells induces angiogenesis in v i v o . Proc Natl Acad Sci USA 1996; 93:7832-7837. Bertrand P, Girard N, Duval C, d'Anjou J, Chauzy C, Menard J, Delpech €3. Increased hyaluronidase levels in breast tumor metastases. Int J Cancer 1997; 73: 327-33 1. Culty M, Shizari M, Thompson EW, Underhill CB. Binding and degradation of hyaluronan by human breast cancer cell lines expressing different forms of CD44: correlation with invasive potential. J Cell Physiol 1994; 160:275-286. Cosoka TB, Frost GI, Heng HH, Scherer SW, Mohapatra G, Stern R. The hyaluronidase gene HYALI maps to chromosome 3p21.2-p21.3 in human and 9Fl-F2 in mouse, a conserved candidate tumor suppressor locus. Genornics 1998; 48:6370. Scheidegger EP, Lackie PM, Papay J, Roth J. In vi t r o and i n v i v o growth of clonal sublines of human small cell lung carcinoma is modulated by polysialic acid of the neural cell adhesion molecule. Lab Invest 1994; 70:95-306. Guo Y, Ma J, Wang J, Che X, Narula J, Bigby M, Wu M, Sy M-S. Inhibition of

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147. 148.

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154. 155.

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human melanoma growth and metastasis i n v i v o by anti-CD44 monoclonal antibody. Cancer Res 1994; 54:1561-1565. Zahalka MA, 'Okon E, Gosslar U, Holzrnann B, Naor D. Lymph node (but not spleen) invasion by murine lymphoma is both 0 4 4 - and hyaluronate-de~endent. J Immunol 1995; 154:5345-5355. Strobe1T, Swanson L, Cannistra SA, In v i v o inhibition of CD44 limits intra-abdominal spread of a human ovarian cancer xenograft i n nude mice: a novel role for CD44 in the process of peritoneal implantation. Cancer Res 1997; 57:1228-1232. Zawadzki V, Perschl A, Rose1 M,Hekele A, Zoller M. Blockade of l~etastasis formation by CD44-receptor globulin. Int J Cancer 1998; 75:919-924. Scott JE. Secondary and tertiary structuresof hyaluronan in aqueous solution. Some biological consequences. Glycoforum: Science of hyaluronan. http://www.glycoforum.gr.jp Lesley J. Hyaluronan binding function of CD44. In: Laurent TC, ed. The chemist^, Biology and Medical Applications of Hyaluronan and Its Derivatives. London: Portland Press, 1998:123-1 34. Underhill CB, Toole BP. Transformation-dependent loss of the hy~luronate-containing coats of cultured cells. J Cell Physiol 1982; 110:123-128. Toole BP, Munaim SI, Welles S, Knudson CB. Hyaluronate-cell inte~~ctions and growth factor regulation of hyaluronate synthesis during limb development. Ciba Found. Symp. 1989; 143:138-145.

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The composite nature of proteoglycans renders them susceptible to attack by several types of degradative agents, both enzymatic and nonenzymatic in nature. The protein cores serve as substrates for an extensive array of proteases, while the glycosaminoglycan side chains are subject to sulfatase and glycosidase attack. In addition, both the protein and carbohydrate c o ~ p o n e n t are s sensitive to reactions with reactive oxygen species, which may result in both polymer modification and cleavage. These agents may cause the catabolism of proteoglycans in both physiological and pathological settings. For example, during normal growth and development cartilage is resorbed and replaced by bone during endochondral ossification. In such a physiological process, the degradation of proteoglycans occurs under careful control. By contrast, in conditions such as arthritis, tumor invasion and metastasis, and the mucopolysaccharidoses, the degradation of proteoglycans, or the lack of it, represents an uncontrolled and important aspect of disease pathology. While some connective tissue macromolecules, particularly the collagens, are relatively resistant to proteolytic attack and require the action of specialized proteases, most proteoglycans appear to be inherently susceptible to degradation by many proteases. The presence of the highly sulfated glycosaminoglycan side chains results in extension of the protein backbone to which they are attached, and renders these regions accessible to protease binding and chain cleavage, at least in vitro. As would be expected, the closely folded nature of any globular domains devoid of glycosaminoglycan chains represents more of a challenge to proteases, so that these regions are generally more resistant to protease digestion. This may result in the accumulation of such globular domains during normal

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turnover. Susceptibility to proteolytic attack is further complicated, in vivo, by the interaction of proteoglycans with other tissue components, which can hamper accessibility of the proteases to their substrates, and so promote the accumulation of partial degradation products. Proteoglycan degradation is usually initiated in the extracellular environment where proteases cleave the protein core. In addition, particularly under inflammatory conditions, reactive oxygen species, released by cells such as poly~~orphonuclearleukocytes and macrophages, can cause both protein and polysaccharide cleavage within the extracellular matrix. This may be a major mechanism of hyaluronan depolymerization. The enzymatic degradation of glycosaminoglycans in the extracellular environment is not a common event. Further degradation of proteoglycans occurs intracellularly within the lysosomes, where a complete repertoire of proteases, sulfatases, and glycosidases is present to convert both the protein and polysaccharide components into their constituent units.

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A bewildering array of proteolytic enzymes (proteases, proteinases) is present in cells and tissues. These have been tabulated and classified by Rawlings and Barrett as the Merops database (http://www.bi.bbsrc.ac.~~k/merops/merops.htm). While the original protease classification system proposed by Hartley in 1960 (1) requires slight refinement based on recent findings, its simple approach of dividing these enzymes into four classes (aspartic, serine, cysteine, and metallo) based on their catalytic mechanism is still valid and widely used (Table 1). The catalytic activity of the aspartic proteases is based on the presence of two active site aspartic acid residues, which accounts for the acidic pH optimum of these enzymes. While the lysosomal aspartic protease, cathepsin D, is probably respon-

Proteases Capable of Degrading Proteoglycans in Tissues Intra~ellular

Cytosol Lysosomes Specialized granules

Cell surface Extracellular

Tissue Plasma

Calpains Cathepsin B, cathepsin D, cathepsin K, cathepsin L, cathepsin S Cathepsin G, elastase, chymases, tryptases MT-”PS ADAMs “Aggrecanase” MMPs, plasminogen activators Plasmin

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sible for a considerable proportion of the proteolysis of endocytosed proteoglycans, it is unlikely that enzymes in this class play any role in extracellular matrix degradation, Serine proteases, where the active site serine residue is responsible for nucleophilic attack on the peptide bond, represent the most abundant class. These enzymes are active at neutral pH and mediate many noma1 extracellular events, such as blood coagulation and clot lysis. The polymo~honuclearleukocyte elastase and cathepsin G, the mast cell chymases and tryptases, and blood plasmin and thrombin are several examples of serine proteases that may play a role in proteoglycan degradation. In the cysteine proteases an active site cysteine residue is used as the catalytic nucleophile. These enzymes have a somewhat acid pH optimum and some (cathepsins l3 and L) show a built-in self-inactivation mechanism on exposure to neutral pH. A large group of these enzymes is found within lysosomes, but there is a remarkable variability in their distribution among different cell types. For example, while cathepsin l3 is found in most cells, cathepsin K is almost uniquely restricted to the osteoclast (2). While lysosomal cysteine proteases are n o ~ a l l yassociated with intracellular events, evidence for an extracellular role for several of these enzymes has been presented. In addition, it has been proposed that calpain, a calcium-dependent cysteine protease normally found on the cytoplasmic face of the cell membrane, may play a role in proteoglycan degradation outside the cell (3). The metal~oproteasesare currently believed to be the major mediators of extracellular proteoglycan degradation (4). In this protease class a metal ion, almost exclusively Zn2+,is chelated into the active site and activates the water molecule that will add to the substrate peptide bond under attack. The MMPs represent a large family of structurally related ~ultidomainmetalloproteases that are active at neutral pH. The presence of domains in addition to that carrying the protease active site provides these enzymes with additional deter~inantsto bind large substrates. The MMPs are found in the extracellular matrix of most tissues and have been shown to degrade a variety of proteoglycans. A subfamily of membrane type (MT) MMPs have been described recently (5). These enzymes are cell-associated due to the presence of a membrane-spanning domain and appear to play a role in the activation of the other MMPs outside the cell. Distantly related to the MMPs are a newly described family of metalloproteases termed ADAMs (a disintegrin and metalloprotease) (6). These enzymes also consist of several domains with different functions. In addition to the Zn2+-dependentprotease domain, the ADAMs contain an integrin-binding domain as well as a membrane spanning region that localizes the enzyme to the outer cell surface, This functional combination results in a protease that is able to bind to and disrupt molecules close to the cell surface in a very directed fashion. Very recently a subclass of the ADAM metalloprotease family has been described, whose members do not contain a transmembrane domain but possess one or more thrornbospondinlike domains. The members of this family are termed ADAMTS proteases.

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Protease Inhibitors Capable of Preventing Proteoglycan Degradation Inhibitor Specificity

Representative Proteases

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Aspartic proteases

Cathepsin D

Serine proteases Cysteine proteases Metalloproteases

Elastase, plasmin Cathepsin B, cathepsin L

General

Inhibitors

"PS ADAMs Most proteases

?

Serpins Cystatins TIMPs ?

~2-macroglobulin

At any one time the proteolytic potential in mammalian tissue is massive and if left uncontrolled would cause its complete destruction. Several control mechanisms prevent this from happening. Firstly, proteases are synthesized as inactive precursors allowing them to pass harmlessly through the biosynthetic machine^ of the cell. The presence of an N-terminal pro-region blocks substrate access to the active site. Removal of these pro-regions occurs by inter- or intramolecular proteolytic cleavage releasing the mature forms of the protease. Under normal conditions processing steps are restricted to sites where protease action is required. As a further control mechanism, essentially every family of proteases has its own family of inhibitors, which bind to and neutralize their cognate proteases (Table 2). Synthesis of both the proteases and their inhibitors is under the control of various growth factors and cytokines, so that a fine functional balance can normally be maintained.

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While proteases show both intra- and extracellular distributions, in mammals most glycosaminoglycan degradation occurs inside the cell, where an extensive array of lysosomal glycosidases and sulfatases disassemble the different sulfated polysacch~idechains. These enzymes have been the subject of extensive studies, since mutations in them result in lysosomal storage diseases, the mucopolysaccharidoses. However, evidence for extracellular heparan sulfate-degrading activity in mammalian systems has accumulated for many years. This has cul~inated in the recent cloning and characterization of an endoglycosidic heparanase (7). There is also evidence for at least two extracellular forms of hyaluronidase ( 8,9). ~lycosaminoglycan-degrading enzymes are also secreted from many prokaryotes, where they appear to provide nutrients for these organisms following the digestion of animal tissue. These enzymes have proven extremely useful as tools

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for the analysis of glycosaminoglycans. An important distinction between these enzymes and their mammalian counterparts is their catalytic mechanism. Whereas the ~ a m m a l i a nenzymes are hydrolases cleaving the glycoside bond by the addition of water, the bacterial enzymes are commonly lyases removing a water molecule and in the process forming an unsaturated uronate residue ( l o) ,

All cells release reactive oxygen species due to spillage from the mitochondria. However some cell types, notably the polymo~honuclearleukocyte and macrophage, contain enzyme systems for the production of hydrogen peroxide, hypochlorous acid, and superoxide, p r i ~ ~ ifor l ythe killing of microorganisms. These species and the transiently produced hydroxyl radical, generated as a result of the Fenton reaction from hydrogen peroxide in the presence of transition metal ions, can react with both the protein and polysaccharide components of proteoglycans. Some of these reactions can result in chain cleavage (1 l). Another reactive oxygen species is nitric oxide, a metabolic regulator produced by many cells that is short-lived under normal ~hysiologicalconditions. An important breakdown product of nitric oxide is nitrous acid, which can cause cleavage of glycosaminoglycans containing nonacetylated hexosamine residues. Thus depolymerization of heparan sulfate has been observed in the presence of nitric oxide (12). Such degradation would be detri~entalto the properties of tissues that rely on heparan sulfate-proteog~ycansfor their function.

More studies have been conducted into the catabolism of aggrecan than any other proteoglycan. Such studies have been facilitated by the high abundance of aggrecan in hyaline cartilage and intervertebral disc and by the retention of some degradation products within these tissues over long periods of time. This latter property is due to the anchoring of one end of the aggrecan protein core within the extracellular matrix via its interaction with hyaluronan. As a consequence, any proteolytic cleavage along the protein core ,will result in one fragment that remains localized within the tissue and one that has the potential to be lost by diffusion. Over time the former type of degradation product accumulates within the extracellular matrix. This phenomenon accounts for the increased structural heteroge~eityand decreased average size exhibited by cartilage aggrecan with creasing age of the individual, together with the continued ability of the rnolecules to interact with hyaluronan (13). The accumulation of such degradation products is aided by the relative resistance of the amino terminal hyaluronanbinding (61) domain to proteolysis, and the abundance of this domain as an

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isolated entity increases with age (14,15). Proteolysis also accounts for the relative deficiency of intact aggrecan molecules possessing a carboxyl terminal globular (63) domain on their protein cores, even in immature cartilage (15). With the exception of the G1 domain, the aggrecan protein core exhibits little resistance to proteolysis and is susceptible to cleavage by most, if not all, proteases, though the extent and rate of cleavage may vary considerably. One of the preferred sites of cleavage resides in the interglobular domain (IGD) between the 6 1 domain and the adjacent G2 domain (Fig. l), and in vitro analyses have identified the precise cleavage site for a variety of physiologically relevant proteases (16- 19). Perhaps surprisingly, all matrix metalloproteinases so far studied (including stromelysins, matrilysin, gelatinases, and collagenases) cleave at a common site (. . . PEN-FFG . .) near the amino teminus of the IGD. In addition, several of the matrix metalloproteinase (matrilysin and collagenases) cleave at a second, less favorable site (. . . SED-LVV . . .) close to the carboxyl teminus of the IGD. All other proteases studied to date (including cathepsin B, plasmin, elastase and urokinase) have been shown to cleave at distinct sites within the region bounded by the matrix metalloproteinase cleavage sites. Collagenase-3

Schematic representation of the aggrecan protein core and the known sites for proteolytic cleavage. The aggrecan protein core is depicted as three globular domains (61, G2, and G3) possessing disulfide bonds with extended intervening domains. The interglobular domain (IGD) between the G1 and G2 domains is expanded to show where a variety of proteinases are able to cleave. The identity of the proteinases cleaving at a given site are listed together with the three amino acid residues (in single letter code) at either side of the point of cleavage (indicated by a vertical arrow).

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(MMP13) and stromelysin-l (MMP3) also cleave at minor sites within this central IGD region. That for collagenase-3 is of note because it follows a proline residue (. . . VKP-VFE . . .), which is unusual for most proteases. Aggrecan degradation has also been studied extensively in cartilage and chondrocyte culture systems, in which degradation has been induced by interleukin-1 (IL-l). Initial studies revealed that cleavage within the IGD was a preferred action of the proteolytic activity being Stimulated, and resulted in the retention of G1 domains within the tissue and the release of fragments bearing the G2 and G3 domains into the culture m e d i u ~(20). Such studies confirm the inability of the G2 and G3 domains to confer strong matrix-binding properties, and help explain the absence of such fragments in aging articular cartilage, where they are presumably lost into the synovial fluid. In view of the known ability of IL1 to stimulate the production of pro-matrix metalloproteinases, it was quite surprising when amino acid sequence analysis revealed that cleavage was occurring at a different site (. . . EGE-ARC . , .) (21). This site was distinct, as cleavage following a glutamate residue is a rare occurrence in previously characterized mamma~ianproteases. The activity responsible for such cleavage was termed “aggrecanase.” Additional sites have now been described throughout the central glycosaminoglycan-binding domain of the aggrecan protein core that also occur following glutamate and are al so presumably due to the action of aggrecanase (22). Moreover, such cleavage does not require the presence of IL-l and proceeds in unstimulated cultures (23), suggesting that aggrecanase may be responsible for aggrecan catabolism not only under conditions of inflammation but also during normal turnover. Aggrecanase has also been demonstrated to be responsible for the degradation of aggrecan induced by culture in the presence of retinoic acid (24,25) or tumor necrosis factor-a (TNFaj (26). Until recently aggrecanase has defied purification and full characterization, in part due to the absence of a convenient specific assay system and in part due to its apparent absence from the medium of cultured cells. This latter property has led to the suggestion that aggrecanase may be a cell- or tissue-associated protease (26). However, the development of a recombinant protein substrate for aggrecanase has allowed aggrecanase activity to be detected in the medium of rat chondrosarcoma cells stimulated with retinoic acid (2’7). Under extreme conditions neutrophil collagenase (MNIP8) is able to cleave at the requisite site within the aggrecan IGD (28). It is now appreciated, however, that this collagenase is not responsible for the cleavages attributed to aggrecanase (29j, as a proteinase inhibitor specific for “33 did not prevent endogenous aggrecanase action in cartilage organ culture. Recently, a protease with aggrecanase activity has been purified from large-scale preparations of culture medium from bovine cartilage stimulated with IL-l (30). Peptide sequence data enabled the cloning of the human analogue of this enzyme, which was shown to be a member of the ADAMTS family. This enzyme has been designated ADAMTS4 and the recombinant form

was shown to cleave aggrecan at all of the sites attributed to aggrecanase. Subsequently, another member of the ADAMTS family, ADAMTS l 1, has been cloned and shown to possess aggrecanase activity (31). The differential contribution of these enzymes to aggrecan degradation, and the mechanisms by which their activities are regulated, remain to be established. Evidence for the involvement of aggrecanase in the catabolism of aggrecan in vivo has been obtained via the analysis of fragments present in synovial fluid. terminal amino acid sequencing of the fragments revealed that cleavage at the aggrecanase site within the aggrecan ICD is a common event in joint injury, inflammatoryjoint disease, and osteoarthritis (32). Such data can give the impression that aggrecanase is the only proteinase involved in cartilage c a t a b o l i s ~in vivo, and that matrix metalloproteinases are not involved. However, one must bear in mind that the aggrecanase cleavage site occurs carboxyl terminal to that of the matrix metalloproteinases, so that whenever both enzyme systems are acting together the product of metalloproteinase cleavage can be converted to that of aggrecanase cleavage. The relatively small ICD fragment that results from such conversion is not amenable to recovery in the presence of the large glycosaminoglycan-containing fragment, While such a scenario would also result in the accumulation of C1 domains t e ~ i n a t i n gin the matrix metalloproteinase site, the analysis of such products from cartilage extracts is not amenable to analysis by terminal sequencing. These difficulties were overcome by the development of antineoepitope antibodies that specifically recognize the new amino terminal sequences created upon protease cleavage of a peptide bond. Moreover, the technique is applicable to both amino and carboxyl terminal analyses. Initially, it was thought that a monoclonal approach was needed to generate the requisite antibody specificity (33), but it is now apparent that a polyclonal approach can also be used (34). Such reagents have now been used to demonstrate that aggrecan fragments arising from cleavage at the sites of action of matrix metalloprot~inases are indeed present in the synovial fluid of arthritic patients (35). They have also been used to demonstrate the C1 domains of aggrecan, arising from cleavage at the site of action of both the matrix metalloproteinases and aggrecanase, in the extracellular matrix of both normal and arthritic articular cartilage (36) and intervertebral disc (37). Thus aggrecan degradation has been shown to be mediated by both matrix metalloproteinases and aggrecanase, though the degree to which the two systems operate varies with both age and tissue (37).

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Aggrecan does not occur in isolation within the extracellular matrix, but exists in the form of proteoglycan aggregates, in which many aggrecan molecules inter-

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act with a central filament of hyaluronan with each interaction being stabilized by the presence of a link protein. Degradation of aggrecan in the proteoglycan aggregates does not occur alone, and the link proteins and hyaluronan also show evidence of degradation throughout life (13). Link protein shares considerable structural homology with the G1 domain of aggrecan and is also highly resistant to proteolysis when participating in proteoglycan aggregates. However, proteolytic modi~cationdoes occur within the amino terminal region of the protein, though this does not seem to impair the function of the molecule. This results in the modified link proteins ~ccumulatingwithin the tissue matrix, and both the abundance and degree of link protein ~odification have been observed to increase with age in both articular cartilage (38) and intervertebral disc (39). In vitro, most proteases are able to cleave within the amino terminal region of link protein to yield characteristic fragments (40), and in vivo three cleavage sites have been identified (Fig. 2). The most amino terrninal in vivo site has been identified throughout life and corresponds to the site of action of matrix metallop~oteinases (. . AIH-IQA . . .) (4 1). The origin of the more carboxyl terminal sites (. . . HIQ-AEN . . . and . . . NGP-HLL . . .) are less clear. The former is compatible with the action of either cathepsin €3 or cathepsin G, while the latter shows the unusual feature of cleavage following a proline residue and is reminiscent of the minor site of action of collagenase-3 in the aggrecan IGD domain, Neoepitope

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Schematic representation of link protein and the known sites for proteolytic cleavage. The link protein is depicted with three disulfide-bondeddomains (analogousto the G1 domain of aggrecan) and an amino terrninal domain. The amino terminal domain is expanded to show the sites where proteolytic cleavage occurs in vivo and where a variety of proteinases are able to cleave in vitro when the link protein participates in a proteoglycan aggregate. The identity of the proteinases cleaving at a given site are listed together with the three amino acid residues (in single letter code) at either side of the point of cleavage (indicated by a vertical arrow).

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analysis has been used to demonstrate that the abundance of the amino terminal cleavage site decreases with age as that of the more carboxyl terminal sites increases (42). In the adult, additional cleavage sites appear within the more central region of link protein but disulfide bonds maintain the integrity of the molecule (38,40). No proteolytic agents have yet been found to duplicate cleavage at these sites in vitro. In articular cartilage, the size of hyaluronan has been shown to decrease with age, even though there is no change in the size of the newly synthesized hyaluronan that can be made by the chondrocytes at different ages (43).Thus, the hyaluronan is presumably undergoing degradation. Since extracellular hyaluronidases have yet to be identified in connective tissues, a glycosidase-mediated degradation pathway seems unlikely. However, some reactive oxygen species are known to depolymerize hyaluronan and could represent the source of degradation. While superoxide radicals and hydrogen peroxide do not induce depolymerization, the presence of trace amounts of iron results in the generation of hydroxyl radicals which are an effective degradative agent (44). In addition, the reaction of superoxide radicals with nitric oxide generates peroxynitrite radicals, which can al so cause hyaluronan depolymerization (45). Whether such pathways could operate in cartilage by design or only as the result of an i n ~ a m l ~ a t o rinsult y remains to be established. It is al so important to note that even though hyaluronan depolymerization is occurring in the cartilage, this process is not extensive with age, This could be due to the presence of low radical fluxes or to the protection afforded by its “coat” of aggrecan GI domains and link proteins when participating in a proteoglycan aggregate. Certainly, a means of limiting hyaluronan degradation is essential if cartilage function is to be maintained, as such damage can result in aggrecan loss without proteolytic cleavage of its protein core. In a similar manner, tight control of proteolysis must be maintained by the chondrocytes if functional i ~ p a i r m e n of t the aggrecan is not to occur by cleavage in its IGD, resulting in the accumulation of the hyaluronan with its GI domain and link protein coat. Such products are relatively stable and would seem to be of little fun~tionalvalue; they can be viewed as being detrimental to the function of the tissue by blocking space into which newly synthesized intact proteoglycan aggregates could be deposited. At present, it is not clear how effectively the extracellular matrix of tissues such as cartilage turnover and maintain their functional properties,

Aggrecan belongs to a family of proteoglycans that share structural and functional homology in their amino and carboxyl terminal globular regions, which has led

to the family being termed hyalectans (46) or lecticans (47). The other family members are versican, neurocan, and brevican, and the presence of their amino terminal hyaluronan-binding (GI) domain allows them to participate in proteoglycan aggregate formation. As with aggrecan, one would predict that the G1 domains would show a relative resistance to proteolysis and might accumulate in the tissues. Such a product has been described for versican, and was originally referred to as glial hyaluronan-binding protein (GHBP) (48) or hyaluronectin (49). Generation of GHBP occurs via cleavage close to the last cysteine residue of the G1 domain (. . . CANATDV-TTT . . .), which in vitro is a site of action for a variety of matrix metalloproteinases (48). Equivalent products have yet to be described for neurocan and brevican. However, cleavage within the central glycosaminoglycan-attachment domains has been described for both molecules. In the case of brevican, processing occurs in the adult at a site following a glutamate residue (. . . ESE-SRG . . .) (50). This bears a striking similarity to the aggrecanase cleavage site within the IGD of aggrecan (. . . EGE-ARG . . .), and strongly suggests a role for this protease in the brain where brevican occurs, as well as in connective tissues rich in aggrecan. In the case of neurocan, cleavage within the brain is also an age-related phenomenon and occurs following a rnethionine residue (. . . VAM-LRA . . .) (5 l,52), though the identity of the protease responsible for the cleavage is not clear. It is interesting to note that both products accumulate in adult brain, and this may be related to the functional activity of the carboxyl terminal lectin homology domain (47). One might also expect versican to exhibit extensive proteolytic processing within its central glycosarninoglycan-binding region, but to date such products have not been characterized and cleavage information i s not available. As with aggrecan, proteolytic degradation of the other hyalectans is likely to modify the properties of tissues in which they occur, as it separates functional domains of the proteoglycans. This may be particularly relevant in a tissue such as brain, which possesses versican, neurocan, and brevican, and where hyalectans and their turnover could be important in regulating both tissue development and function (53).

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Small leucine-rich proteoglycans (SLRPs) form the second major family of proteoglycans present in the extracellular matrix of tissue (46), and at least nine individual family members have now been characterized at the gene level (53). Yet despite the wealth of structural information available on this family, there is relatively little information concerning the proteases involved in their catabolism. In normal tissue the dematan sulfate-proteoglycan, biglycan (DS-PGI), shows the most evidence for proteolytic processing, with cleavages occurring in

the amino terminal region of the protein core, though at present the precise sites at which cleavage is occurring are not known. In both articular cartilage (54) and intervertebral disc (55) the abundance of the proteolytically modified forms of biglycan increases with age, in a manner reminiscent of link protein, and it is tempting to speculate that the same enzymes rnay be involved in the two processes. As the dermatan sulfate chains of biglycan reside at the extreme amino t e r ~ i n u sof the biglycan protein core, the proteolytic processing results in the accu~ulationof nonglycanated forms of biglycan that are devoid of glycosaminoglycan chains. It is not clear whether this deficiency is of any consequence to the function of the molecule. In contrast, the second major dermatan sulfateproteoglycan, decorin (DS-PGII), shows little evidence for proteolytic processing at any age in either cartilage or disc, though some nonglycanated products can be observed in the adult. At least in the disc, proteolytic processing is thought to contribute to this process with cleavage occurring four amino acid residues from the terminus of the decorin protein core (DEAS-GIG . . .) and just following the serine residue to which the single glycosaminoglycan chain is attached. No protease has yet been associated with this event. In contrast to normal cartilage, extensive degradation of the decorin protein core has been observed in arthritic cartilage (56),particularly near the articular surface, suggesting that the proteases involved may originate from the inflamed synovium or synovial fluid. Interestingly, the size of the fragments suggests that cleavage has occurred within the central leucine-rich repeat region of the decorin protein core. While no cleavage site data are available, it is known that matrix metalloproteinases are capable of cleaving within this region in vitro (57). It has also been reported that decorin fragments of a similar size can be extracted from juvenile dermis (58), suggesting that in some tissues such proteolytic processing rnay also be a normal event. Evidence for processing of biglycan within its central region is less forthcoming, but has been de~onstratedin an en~othelialcell culture system (59). The lack of evidence for internal fragmentation of decorin and biglycan in many tissues may relate to an inherent resistance to proteolysis when these molecules fulfill their normal interactions within the tissue, such as with the collagen fibrils. Alternatively, it rnay be due to the clearance of such fragments, as both fibroblasts and chondrocytes are capable of receptor-mediated endocytosis of both decorin and biglycan (60). Decorin and biglycan also share the interesting feature of being secreted as pro-forms, which are then proteolytically modified within the matrix to their mature forms (61). The propeptides are relatively short (14 and 21 m i n o acid residues for decorin and biglycan, respectively) and it is not clear whether they serve any functional role. Hence, at present it is unclear whether this process is a fortuitous or an intended event. In both cases cleavage takes place prior to an aspartate residue (. . . MLE-DEA . . . for decorin, and . . . DECO DEE . . , for bigiycan), and the sequences surrounding the cleavage sites show considerable

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conservation suggesting that a common enzyme is involved in processing. The degree to which processing occurs is both age- and tissue-dependent, with the unprocessed pro-forms being particularly abundant in adult articular cartilage where they can represent up to 20% of the molecules present. In some cell culture systems biglycan has been reported to exist entirely in its pro-form (62), sugesting that processing is not an obligatory event and is independent of proteoglycan synthesis, A third member of the dematan sulfate-proteoglycan family of SLRPs, epiphycan ( ~ S - ~ ~ I has I I )recently , been described, and it may also possess a short propeptide as two amino terminal sequences have been identified (63). The first cleavage follows the signal peptide, and the second occurs 11 amino acid residues later prior to a glutamate residue (. . YNS-ETY , . .). The other four SLRPs are keratan sulfate-proteoglycans. In the case of fibromodulin, lumican, and keratocan the mature protein cores appear to commence immediately following the signal peptides (64-66), and there is little evidence to date for their proteolytic degradation products accumulating in normal tissues. However, it is apparent that proteolytic degradation products of fibromodulin are present in arthritic cartilage, and to a greater extent in joints involved in rheumatoid arthritis than osteoarth~tis(67). As with decorin, the size of the products indicate cleavage occurring within the central leucine-rich repeat region of the protein core, but the precise sites of proteolytic action have yet to be identified. Mimecan is the most recently described member of this family, and proteolytic degradation of the initial gene product is known to occur (68). The corneal form of mimecan lacks the amino terminal portion of the mature mimecan protein core and arises by cleavage prior to an aspartate residue and following a lysine residue (. . . LQK-DET . . .). It has been suggested that cleavage following lysine could be indicative of a serine protease, but it is also worth noting that cleavage prior to aspartate is common with the protease responsible for removing the propeptides from decorin and biglycan. At present it is not clear whether the full-length mature mimecan protein core persists in some tissues. It is also interesting to note that a previously identified leucine-rich repeat protein, osteoglycin, has the same protein sequence as the carboxyl terminal region of mimecan and could therefore represent a proteolytic product. If so, cleavage would also occur following a lysine residue (. . . GIK-ANT . . .), and the same protease could be involved in both events.

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Perlecan is the best-characterized proteoglycan of basement membranes, where it is usually present as a heparan sulfate proteoglycan. The protein core is very large and would appear to be an ideal candidate for proteolytic processing. In-

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deed, it has been suggested that small heparan sulfate proteoglycans that are also present in the basement membrane could represent proteolytic fragments of perlecan (69). Certainly, in vitro the protein core of perlecan is susceptible to cleavage by a variety of proteases, including matrix metalloproteinases and plasmin (71). In addition, proteolytic fragments of the perlecan protein core have been detected in the urine of patients with end stage renal failure (70), presumably arising from degradation within the glomerular basement membrane. Degradation of perlecan has also been implicated with the pathological processes occurring in diabetic nephropathy and tumor metastases (72). In the latter case it is likely that degradation involves not only proteolysis of the perlecan protein core, but also endoglycosidase degradation of the heparan sulfate chains via a heparanase secreted by the metastatic cells (73). It is also possible that under some conditions perlecan could be subject to degradation by free radical mechanisms, such as when neutrophil adherence to the basement membranes occurs (74). The degradation of heparan sulfate by hydroxyl radicals has also been described in an experimental nephrotic syndrome (75). This presumably leads to the impairment of perlecan function in the glomerular basement membrane, leading to defective filtration and proteinuria.

Proteoglycans that form an integral part of the plasma membrane of cells can be divided into two families-those that span the plasma membrane and those that are attached to its outer surface via a glycosyl phosphatidylinositol (GPI) anchor (76). In the latter family proteolytic cleavage of a carboxyl terminal signal peptide is immediately followed by a transa~idationreaction in which the new carboxyl terminus of the proteoglycan protein core is transferred to glycosyl phosphatidylinositol in the membrane of the endoplasmic reticulum. In contrast to the extracellular proteoglycans described previously, the cell surface proteoglycans are subject to rapid turnover with a typical half-life on the cell surface of 3-8 hr (77). Turnover may involve two processes-either direct endocytosis or sheddingfor either family of proteoglycan. Most, if not all, endocytosed proteoglycans are ultimately subjected to complete degradation within lysosomes by a variety of proteinases, glycosidases, and sulfatases. However, the time taken for intracellular degradation can vary for different proteoglycans or cells, depending upon whether the internalized material is transferred directly to lysosomes or whether transfer occurs via intermediate endocytic vacuoles. In rat granulosa cells, the former pathway is utilized for the GPI-anchored proteoglycans, and the latter pathway for the membrane-spanning proteoglycans. The alternative turnover process of shedding is commonly associated with

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proteolytic cleavage in the extracellular domain of the proteoglycans close to the cell surface, though in the case of the GPI-anchored proteoglycans shedding could also involve the action of,phospholipases. The degree to which shedding versus endocytosis are used for turnover varies depending upon the cell type, and it is still not clear how common the process is in vivo. Many of the membrane-spanning proteoglycans, including the syndecans (78) and betaglycan (79), possess a conserved dibasic amino acid sequence near the external face of the plasrna membrane, which is thought to be a potential site for cleavage by a plasmin-like protease so resulting in shedding. Certainly this sequence has been utilized in vitro to allow recovery of the extracellular domain of the proteoglycans following trypsin treatment. In terms of functional consequences, shedding serves to separate the ligand-binding domain of the proteoglycans from the cells and therefore terminates any signaling or cell-attachment property mediated by the proteoglycans.

Serglycin forms the protein core of either heparin or chondroitin sulfate-proteoglycans stored within the secretory granules of hematopoietic cells (80). The protein contains a central region of serine-glycine repeats to which the glycosaminoglycan chains are attached, and which is resistant to proteolysis by known proteases. In contrast, the amino and carboxyl terminal regions of the newly synthesized proteoglycan protein cores are subject to rapid proteolytic degradation in most cells, resulting in storage of the central glycosaminoglycan-bearing region. In mast cells, degranulation in vivo results in the release of low molecular weight heparin, as a consequence of initial proteolysis and subsequent endoglycosidase action (81). At present the identity of this protease is not known, but it would appear to be essential for anticoagulation.

A variety of glycosidases and sulfatases within the lysosomes of cells are able to degrade both hyaluronan (82) and the sulfated glycosaminoglycans (83). In the case of hyaluronan, chondroitin sulfate, and heparan sulfate, initial degradation proceeds by the action of endoglycosidases, whereas such endoglycosidases have yet to be described for keratan sulfate. Further degradation of the glycosaminoglycan fragments occurs by exoglycosidases and sulfatases acting in a sequential manner commencin~from the nonreducing terminus of the fragments. Loss of activity of one of the exoglycosidases or sulfatases results in the impaired degra-

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dation of the glycosaminoglycans, and gives rise to the intracellular accumulation and excessive urinary secretion of partially degraded glycosaminoglycans associated with the mucopolysaccharidoses (83). While the extracellular degradation of glycosa~inoglycansis not a common physiological event, it has been associated with pathological processes, particularly in basement membranes. An endoglycosidase capable of degrading the heparan sulfate chains of basement membrane proteoglycans' has also been associated with the ability of neutrophils to extravasate (84). At present, it is not clear whether the endoglycosidases involved in extracellular degradation of glycosa~inoglycansare distinct from those involved in their intracellular degradation in endosomes or lysosomes.

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While all proteoglycans are subject to proteolytic degradative events in normal tissues, it is not clear whether all these events are directly related to proteoglycan turnover and replacement by new intact molecules. This is particu~arlytrue in the extracellular environment, where some degradation products accumulate and may be viewed as being an impediment to tissue function. Such degradation may be unintentional and more akin to that observed in pathological events. In many cases the identity of the proteases involved in proteoglycan degradation is unknown, as is the contribution made by nonen~ymaticagents such as reactive oxygen species, which could play a major role in some pathological states. Thus, while much is known about what proteoglycan degradation takes place, much less is known about how or why it happens. If the functional consequences of proteoglycan catabolism are to be fully appreciated, these questions will need to be addressed.

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1. Hartley BS. Proteolytic enzymes. Annu Rev Biochern 1960; 29:45-72. 2. Drake FH, Dodds RA, James IE, et al. Cathepsin K, but not cathepsins B, L, or S, is abundantly expressedin human osteoclasts.J Biol Chem 1996; 27 1: 1251l - 125 16. 3. Shimizu K, Hamarnoto T, Hamakubo T et al. Immunohistochemical and biochemical demolistrationof calcium-dependent cysteine proteinase (calpain)in calcifyingcartilage of rats. J Orthop Res 1991; 9:26-36. 4. Woessner JF. The family of matrix metalloproteinases. Ann NY Acad Sci 1994; 737~11-21. 5. Sato H, Takino T, Okada Y, et al. A matrix metalloproteinase expressed on the surface of invasive tumour cells. Nature 1994; 370:61-65. 6. Wolfsberg TG, White J M. ADAMs in fertilization and development. Dev Biol 1996; 180:389-401.

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7. Vlodavsky I, Friedman Y, Elkin M, et al. Mammalian heparanase: gene cloning, expression and function in tumor progression and metastasis. Nat Med 1999; 5:793802. 8. Frost GI, Csoka TB, Won g T, et al. Purification, cloning, and expression of human plasma hyaluronidase. Biochem Biophys Res C o m u n 1997; 236:lO-15. 9. Gmachl M, Sagan S, Ketter S, et al. The human sperm protein PH-20 has hyaluronidase activity. FEBS Lett 1993; 336545-548. 10. Ernst S, Langer R, Cooney CL, et al . Enzymatic degradation of glycosaminoglycans. Crit Rev Biochem Mol Biol 1995; 30:387-444. l l. Roberts CR, Roughley PJ, Mort JS. Human proteoglycan aggregate degradation induced by hydrogen peroxide. Protein fragmentation, amino acid modification, and hyaluronic acid cleavage. Biochem J 1989; 2592305-811. 12. Vilar RE, Ghael D, Li M, et al. Nitric oxide degradation of heparin and heparan sulphate. Biochem J 1997; 324:473-479. 13, Roughley PJ, Mort JS. Ageing and the aggregating proteoglycans of human articular cartilage. Clini Sci 1986; 71:337-344. 14. Roughley PJ, White RJ, Poole AR. Identification of a hyaluronic acid-bindingprotein that interferes with the preparation of high-buoyant-densityproteoglycan aggregates from adult h u ~ a narticular cartilage. Biochem J 1985; 231:129-138. 15. Dudhia J, Davidson M, Wells TM, et al. Age-related changes in the content of the C-terminal region of aggrecan in human articular cartilage. Biochem J 1996; 313: 933-940. 16. Fosang AJ, Neame PJ, Last K, et al. The interglobulardomain of cartilage aggrecan is cleaved by PUMP, gelatinases, and cathepsin B. J Biol Chem 1992; 267:1947019474. 17. Fosang AJ, Last K, Knauper V, et al. Fibroblast and neutrophil collagenasescleave the two sites in the cartilage aggrecan interglobular domain. Biochem J 1993; 295: 273-276. 18. Hardingham TE, Fosang AJ. The structure of aggrecan and its turnover in cartilage. J Rheumatol 1995; 22 Suppl. 43:86-90. 19. Fosang AJ, Last K, Knauper V, et al. Degradation of cartilage aggrecan by collagenase-3 (MMP-13). FEBS Lett 380 1996:17-20. 20. Sandy JD, Boynton RE, Flannery CR. Analysis of the catabolismof agrecan in cartilage explants by quantitation of peptides from the three globular domains. J Biol Chem 1991; 266:8198-8205. 21. Sandy JD, Neame PJ, Boynton RE, et al. Catabolism of aggrecan in cartilage explants. Identi~cationof a major cleavage site within the interglobulardomain. J Biol Chem 1991; 266:8683-8685. 22. Loulakis P, Shrikhande A, Davis G, et al. N-Terminal sequence of proteoglycan fragmentsisolated from medium of interleukin-1-treatedarticular-cartilagecultures. Putative sites of enzymic cleavage. Biochem J 1992; 284589493. 23. Ilic MZ, Handley CJ, Robinson HC, et al. Mechanism of catabolism of aggrecan by articular cartilage. Arch Biochem Biophys 1992; 294:115-122. 24. Lark MW, Gordy JT, Weidner JR, et al. Cell-mediated catabolism of aggrecan.Evidence that cleavage at the “aggrecanase” site is a primary event in proteolysis of the il~terglobulardomain. J Biol Chern 1995; 270:2550-~5~6.

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25. Ilic MZ, Mok MT, Williamson OD, et al. Catabolismof aggrecan by explant cultures of human articular cartilage in the presence of retinoic acid. Arch Biochem Biophys 1995; 322122-30. 26. Buttle DJ, Fowles A, Ilic MZ, et al. “Aggrecanase” activity is implicated in tumour necrosis factor a mediated cartilage aggrecan breakdown but is not detected by an in vitro assay. J Clin Pathol 1997; 50:153-159. 27. Hughes CE, Buttner FH, Eidenmuller B, et al. Utilization of a recombinant substrate rAggl to study the biochemical properties of aggrecanase in cell culture systems. J Biol Chem 1997; 272:20269-20274. 28. Fosang AJ, Last K, Nearne PJ, et al. Neutrophil collagenase (MMP-8) cleaves at the aggrecanase site E373-A374 in the interglobular domain of cartilage aggrecan. Biochem J 1994; 304:347-35 1. 29. Arner EC, Decicco CP, Cherney R et al. Cleavage of native cartilage aggrecan by neutrophil collagenase (MMP-8) is distinct from endogenous cleavage by aggrecanase. J Biol Chem 1997; 272:9294-9299. 30. Tortorella MD, Burn TC, Pratta MA, et al. Purification and cloning of aggrecanase1: a member of the ADAMTS family of proteins. Science 1999; 284:1664--1666. 31. Abbaszade I, Liu RQ, Yang F, et al. Cloning and characterizationof ADAMTSI I, an aggrecanase from the ADAMTS family. J Biol Chern 1999; 274:23443-23450. 32. Lohmander LS, Nearne PJ, Sandy JD. The structureof aggrecan fragments in human synovial fluid. Evidence that aggrecanase mediates cartilage degradation in inflarnrnatory joint disease,joint injury, and osteoarthritis.Arthritis Rheum 1993;36:12141222. 33. Hughes CE, Caterson B, Fosang AJ, et al. Monoclonal antibodies that specifically recognize neoepitope sequences generated by ‘aggrecanase’ and matrix metalloproteinase cleavage of aggrecan: application to catabolism in situ and in v itro. Biochem J 1995; 305~799-804. 34. Lark MW, Williams H, Hoernner LA, et al. Quantification of a matrix metalloproteinase-generated aggrecan G1 fragment using monospecific anti-peptide serum. Biochem J 1995; 307:245-252. 35. Fosang AJ, Last K, Gardiner P, et al. Development of a cleavage-site-specific monoclonal antibody for detecting metalloproteinase-derived aggrecan fragments: detection of fragments in human synovial fluids. Biochem J 1995; 310:337-343. 36. Lark MW, Bayne ET(, Flanagan J, et al. Aggrecan degradation in human cartilage. Evidence for both matrix ~etalloproteinaseand aggrecanase activity in normal, osteoarthritic, and rheumatoid joints. J Clin Invest 1997; 100:93--106. 37. SztrolovicsR, Alini M, Roughley PJ, et al. Aggrecan degradation in human intervertebral disc and articular cartilage. Biochem J 1997; 326:235-241. 38. Mort JS, Poole AR, Roughley PJ. Age-related changes in the structure of proteoglycan link proteins present in normal human articular cartilage. Biochem J 1983; 214: 269-272. 39. Pearce RH, Mathieson JM, Mort JS, et al. Effect of age on the abundance and fragmentation of link protein of the human intervertebral disc. J Orthop Res 1989; 7: 861-867. 40. Nguyen Q, Liu J, Roughley PJ, et al. Link protein as a monitor in situ of endogenous proteolysis in adult human articular cartilage. Biochem J 1991; 278:143-147.

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41. Nguyen Q, Murphy G, Hughes CE, et al . Matrix metalloproteinases cleave at two distinct sites on human cartilage link protein. Biochem J 1993; 295595-598. 42. Hughes CE, Caterson B, White RJ, et al. Monoclonal antibodies recognizing protease-generated neoepitopes from cartilage proteoglycan degradation. Application to studies of human link protein cleavage by stromelysin. J Biol Chem 1992; 267: 16011-16014. 43 Holmes MWA, Bayliss MT, Muir H. Hyaluronic acid in human articular cartilage. Biochem J 1988; 250:435-441. 44. Herkko S. Oxygen derived free radicals and synovial fluid hyaluronate. Ann Rheum Dis 1991; 50:389-392. 45 Li M, Rosenfeld L, Vilar RE, et al . Degradation of hyaluronan by peroxynitrite. Arch Biochern Biophys 1997; 341:245-250. 46. Iozzo RV, Murdoch AD. Proteoglycans of the extracellularenvironment: clues from the gene and protein side offer novel perspectives in molecular diversity and function. FASEB J 1996; 10:598-614. 47. Aspberg A, Mi ur a R, Bourdoulous S, et al. The C-type lectin domains of lecticans, a family of aggregating chondroitin sulfate proteoglycans,bind tenascin-R by proteinprotein interactions independent of carbohydrate moiety. Proc Natl Acad Sci USA 1997; 94:10116-10121. 48. Perides G, Asher RA, Lark MW, et al . Glial hyaluronate-binding protein: a product of metalloproteinase digestion of versican? Biochem J 1995; 312:377-384. 49. Delpech B, Girard N, Olivier A, et al. The origin of hyaluronectin in human tumors. Int J Cancer 1997; 72:942-948. 50. Yamada H, Watanabe K, ShimonakaM, et al. cDNA cloning and the identification of an aggrecanase-likecleavage site in rat brevican. Biochern Biophys Res Commun 1995; 216:957-963. 51. Rauch U,Karthikeyan L, Maurel P, et al . Cloning and primary structure of neurocan, a developmentally regulated, aggregating chondroitin sulfate proteoglycan of brain. J Biol Chem 1992; 267:19536-19547. 52. Matsui F, Watanabe E, Atsuhiko 0. Immunologicalidentification of two proteoglycan fragments derived from neurocan, a brain-specific chondroitinsulfate proteoglycan. ~eurochemInt 1994; 25:425-431. 53. Iozzo RV. Matrix proteoglycans. Annu Rev Biochem 1998; 67:609-652. 54. Roughley PJ, White RJ, Magny M-C, et al . Non-proteoglycan forms of biglycan increase with age in human articular cartilage. Biochem J 1993; 295:421-426. 55. Johnstone B, M ~ ~ o p o u lM, o sNeame P, et al . Identification and ch~acterizationof glycanated and non-glycanated forms of biglycan and decorin in the human intervertebral disc. Biochem J 1993; 292:661-666. 56. Witsch-Prehm P, Miehlke R, Kresse H. Presence of small proteoglycan fragments in normal and arthritic human cartilage. Arthritis Rheum 1992; 35:10421052. 57. Imai K, ~iramatsuA, Fu~ushimaD, et al . Degradation of decorin by matrix metalloproteinases: identification of the cleavage sites, kinetic analyses and transforming growth factor-p1 release. Biochern J 1997; 322:809-814. 58. Garg HG, Burd DAR, Swann DA. Small derrnatan sulfate proteoglycans in human epidermis and dermis. Biomed Res 1989; 10:197-208. *

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59. Kinsella MG, Tsoi CK, Jkvelainen HT, et al. Selective expression and processing of biglycan during ~ g r a t i o nof bovine aortic endothelial cells. The role of endogenous basic fibroblast growth factor. J Biol Chem 1997; 272:318-325. 60. Hausser H, Ober B, Quentin-Hoffmann E. Endocytosis of different members of the small chondroitin/de~atansulfate proteoglycan family. J Biol Chem 1992; 267: 11559-1 1564. 61. Roughley PJ, White RJ, Mort JS. Presence of pro-forms of decorin and biglycan in human articular cartilage. Biochem J 1996; 318:779-784. 62. Marcum JA, Thompson MA. The amino terminal region of a proteochondroitin protein core, secreted by aortic smooth muscle cells, shares sequence homology with the pre-propeptide region of the biglycan protein core from human bone. Biochem Biophys Res Commun 1991; 175:706-712. 63. Johnson HJ, Rosenberg L, Choi HU,et al. Characterization of epiphycan, a small proteoglycan with a leucine-richrepeat protein core. J Biol Chem 1997;272:1870918717. 64. Plaas AHK, Neame PJ, Nivens CM, et al. Identificatio~of the keratan sulfate attachment sites on bovine fibromodulin. J Biol Chem 1990; 265:20634-20640. 65. Funderburgh JL, Funderburgh ML, Mann MM, et al. Arterial lumican. Properties of a corneal-type keratan sulfate proteoglycan from bovine aorta. J Biol Chem 1991; 266~24773-24777. 66. Corpuz LM, Funderburgh JL, Funderburgh NIL, et al. Molecular cloning and tissue dis~ibutionof keratocan. Bovine corneal keratan sulfate proteoglycan 37A. J Biol Chem 1996; 271:9759-9763. 67. Roughley PJ, White RJ, Cs-Szabo G, et al. Changes with age in the structure of fibromodulin in human articular cartilage. OsteoarthritisCartilage 1996;4: 153-161. 68. Funderburgh JL, Corpuz LM, Roth MR, et al. Mimecan, the 25-kDa corneal keratan sulfate proteoglycan, is a product of the gene producing osteoglycin. J Biol Chem 1997; 272:28089-28095. 69. Timpl R. Proteoglycans of basement membranes. Experientia 1993; 49:417-428. 70. Whitelock JM, Murdoch AD, Iozzo RV, et al. The degradation of human endothelial cell-derived perlecan and release of bound basic fibroblast growth factor by stromelysin,collagenase,plasmin and heparanases.J Biol Chem 1996; 271: 1007910086, 71. Oda O., ShinzatoT, Ohbayashi K, et al. Purification and characterizationof perlecan fragment in urine of end-stage renal failure patients. Clin Chim Acta 1996; 255: 119-132. 72. Iozzo RV, Cohen IR Grassel S, et al. The biology of perlecan: the multifaceted heparan sulphate proteoglycan of basement membranes and pericellular matrices, Biochem J 1994; 302:625--639. 73. Vlodavsky I, Korner G, Ishai-Michaeli R, et al. Extracellularmatrix-resident growth factors and enzymes: possible involvement in tumor metastasis and angiogenesis. Cancer Metastasis Rev 1990; 9:203-226. 74. Klebanoff SJ, Kinsella MG, Wight TN. Degradation of endothelial cell matrix heparan sulfate proteoglycan by elastase and the ~yeloperoxidase~H~O~-chloride system. Am J Pathol 1993; 143:907-917. 75. Raats CJI, Bakker MAH, van den Born JHM, et al. Hydroxyl radicals depolymerize

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13

76. 77. 78. 79. 80.

81. 82. 83. 84.

glomerular heparan sulfate in vitro and in experimental nephrotic syndrome. J Biol Chern 1997; 27226734-26741. David C. Integral ~embrane heparan sulfate proteoglycans. FASEB J 1993;7:10231030. Yanagishita M, Hascall VC. Cell surfaceheparan sulfate proteoglycans. J Biol Chem 1992; 26719451-9454. Rapraeger AC. The coordinated regulation of heparan sulfate, syndecans and cell behavior. CUE Opin Cell Biol 1993; 52344-853. Lopez-Casillas F, Cheifetz S, Doody J, et al. Structure and expression of the membrane proteoglycan betaglycan, a component of the TGF-P receptor system. Cell 1991; 67~785-795. Humphries DE, Stevens RL. Regulation of the gene that encodes the peptide core of heparin proteoglycan and other proteoglycans that are stored in the secretory granules of hematopoietic cells. Adv Exp Med Biol 1992; 31359-67. Jacobson KG,Lindahl U. Degradation of heparin proteoglycan in cultured mouse mastocytoma cells. Biochem J 1987; 246:409-415. Laurent TC, Fraser JRE. Hyaluronan. FASEB J 1992; 6:2397-2404. Hopwood JJ, Morris CP. The mucopo~ysacc~aridoses. Diagnosis, molecular genetics and treatment. Mol Biol Med 1990; 7:381-404. Mollinedo F, Nakajima M, Llorens A, et al. Major co-localizationof the extracellular-matrix degradative enzymes heparanase and gelatinase in tertiary granules of human neutrophils. Biochem J 1997; 327:917-923.

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Ever since cells have formed functional groups, e.g., organs, they have been required to build barriers that separate them from each other and from the su~ounding environment. A set of flexible, hydrophilic, and strongly anionic polysaccharides, called glycosaminoglycans (GAGS),can serve this function in many biological barriers such as basement membrane which contains mainly heparan sulfate (HS) proteoglycans. At the cell surfaces, two prototype HS-containing molecule families have been identified. Due to the structural variation of heparan sulfate, these molecules have obtained specific tasks during evolution. They can bind many extracellular effector molecules, like growth factors and extracellular matrix molecules. Although the results of the binding to a cell are not totally understood, circumstantial evidence from the past and more direct evidence from this decade have implied that these binding events can actually lead to very specific responses inside the cell. Syndecans form a group of extracellular effector molecules harboring primarily, but not exclusively, heparan sulfate side chains (1).In this chapter we attempt to relate current syndecan knowledge to its possible function by describing these interaction^. The interactions together with the restricted expression patterns of syndecans strongly argue that in each cell type they may have separate functions. Recent findings of the involvement of syndecans in signaling are described in Chapter 7.

Syndecans are type I transmembrane heparan sulfate proteoglycans ( They contain an extracellular domain or ectodomain, a hydrophobic transmem-

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brane domain, and a short C-terminal cytoplasmic domain. Four m a ~ m a l i a nsyndecan genes have been cloned and sequenced (2-13) (Fig. 1). Their chromosomal localization, exon organization, and sequence relationships with a ~ ~ ~ s o p ~ i syndecan (14) and three Xenopus syndecans (15,16) indicate that the ~ a m m a l i a n syndecan family arose by gene duplication from a single ancestral gene. Syndecans-l and -3 and syndecans-2 and -4 can be considered to form subfamilies based on sequence similarities, GAG attachment sites, and core protein size. The length of the ectodomain varies markedly among family members while the length of the transmembrane and cytoplasmic domains is highly conserved (Fig. 1). The ectodomain contains several Ser-Gly consensus sequences for GAG

Schematic diagram

Name (synonyms)

Syndecan-1

Size of core Size of mRNA protein Species (aa) (kb)

(Syndecan, B-B4, CD 138)

mouse human hamster rat

311 310 309 313

Syndecan-2

human rat

201 21 1

rat chicken

442

human rat chicken

198 202 197

mouse

(Fibroglycan)

Syndecan-3 (N-Syndecan)

Syndecan-4 (Ryudocan, Amphiglycan)

D.~ e ~ a n o g a s t e r syndecan Xenopus syndecan

Cytoplasmic domain Transmembrane domain

2.6, 3.4 2.6, 3.4 2.4, 3.2 2.6, 3.4

~ ~ ~ f

Reference

HS,CS

3

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4 6

HS 2.6, 3.4 l . 1, 2.2, 3.4

HS,CS

2.6 7.0

2 10

7 and 3:

9

2.6 HS 2.6 0.9, 1.3, 2.9

6 11

198

2.7

13

367

2.3, 3.9

HS

8

14

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0 Extracellular domain (ectodomain)

2,4

HS

15

Signal sequence

I

GAG attachment site

A

Predicted protease cleavage site

Thr-Ser-Pro rich region

1 Schematic presentation of the syndecan gene family.

attachment sites and an N-terminal signal peptide required to deliver the ectodomain outside the cell. A try~sin-sensitivesite, through which the ectodomain is cleaved, is located near the transmembrane domain. In syndecans-l and -3, the GAG sites are in two clusters, one near the N-terminus and the other near the plasma membrane. A variable proportion of these attachment sites may be glycanated, and the structural elements of the core protein determine if a site will be glycanated, and if so, whether with heparan or chondroitin sulfate (17,18). The majority of GAG chains added to syndecans are heparan sulfate although syndecan-l (19) and syndecan-4 (20) have been shown to bear chondroitin sulfate (CS) as well. The amount and type of attached GAGs vary depending on cell type and tissue (21,22). Syndecans bind to a variety of extracellular proteins via the GAGs. Other than the GAG attachment sites in syndecan ectodomain sequences are highly variable (Fig. 1).In contrast, the sequence of transmembrane and cytoplasmic domains of syndecans are highly conserved among family members and species. The transmembrane domain links syndecan to the plasma membrane through which it may move ~orizontally.The functions of the cytoplasmic domains have been less obvious but recent studies have revealed new important functions for them. All syndecan core proteins have an identical tetrapeptide sequence EFYA at their C-terminus and four invariant tyrosine as well as one invariant serine reside in the Cytoplasmic domain, Phospho~lationof the cytoplasmic tails has been detected in the serine residue of all syndecans (23-27) and in tyrosine residues of syndecan-l (25). Syndecan-4 phospho~lationsite is localized to S e P 3 which is conserved in all syndecans (27), su~gestingthat this serine may be phospho~latedin all syndecan family members. In addition, Src family tyrosine kinases and their substrates bind a region ( ~ ~ K ~ ~ E G S Y C ) in syndecan-3 Cytoplasmic domain that is also conserved in all syndecans (28). Thus, other syndecans may interact with the cortactin-Src kinase pathway as well. Other regions in the cytoplasmic domain of syndecans are conserved among species but are less homologous among family members. For example, a u n i ~ u e sequence in syndecan-4 cytoplasmic domain binds both protein kinase C alpha (PKCa) (26,29) and phosphatidylinositol 4,s-bisphosphate (PIP2) (30), which promotes syndecan-4 cytoplasmic tail oligome~ization(31) and potentiates protein kinase C activation (26), suggesting a specific role for syndecan-4 at focal contacts, Overall, structural similarities in cytoplasmic domains and diversity of the ectodomains suggests that syndecans were evolved to carry out similar but not identical functions.

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Recent evidence has revealed diverse functions for syndecans (for reviews see Refs. l and 32-34). These functions include anchorage of cells to extracell~l~r

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matrix (ECM) components, maintenance of epithelial morphology, as well as mod~~lation of the activity of several proteases and their inhibitors. Syndecans may also serve as signaling molecules. Importantly, syndecans bind to and modulate the action of heparin-binding growth factors, namely, fibroblast growth factors (FGFs) (Fig. 2). The functions of syndecan family members may be overlapping in many situations, but they are not identical.

A wide variety of extracellular proteins bind heparan sulfate (Table 3). Syndecan-

1 binds cells via its HS chains to a variety of extracellular matrix components and can serve as a matrix receptor. ECM components bound by syndecan include collagen types I, 111, and V (35),fibronectin (36), thrombospondin (37), and tenascin (38). The expression pattern of syndecan-l is also consistent with its role as a matrix receptor. Syndecan-1 polarizes to the basolateral surface of cultured epithelial cells (39) and of simple epithelia (40), and localizes in early embryogenesis to the site of matrix accumulation (41). In addition, syndecans-l and -3 colocalize with tenascin during tooth (42) and limb (43) development, respectively. Furthermore, syndecan-l mediates the binding of B-cells to type I collagen (44). It is expressed on these cells while in contact with matrix, e.g., cells in bone marrow and on differentiated plasma cells in lymphoid tissues but not when the cells are circulating (45). ConSistent with its matrix anchorage function syndecan-l is known to inhibit cell invasion into type I collagen gels (46). To optimally bind ECM molecules and to function as an adhesive molecule syndecan seems to require all the GAG side chains (47). §imultaneously with binding to ECM Components through the GAG chains, the cytoplasmic domain of syndecan can bind to intracellular components. Syntenin, a postsynaptic density protein (PDZ) domain-containing protein, binds the FYA sequence of the cytoplasmic tail of syndecan and affects membrane-cytoskeleton organization (48). Thus, syntenin is a candidate for linking syndecansupported recognition processes to the cytoskeleton and cytoplasmic signaling systems. Syndecan-l expressed in Schwann cells coaligns with actin filaments in response to antibody ligation, a mechanism that is dependent on the third conserved tyrosine in the syndecan-l cytoplasmic domain (49). In addition, syndecan-2 can bind to another Cytoplasmic PDZ protein, CA S ~ / ~ Iof~the - Z guanylate kinase family. Similarly, to syntenin the C A S ~ / ~ 1 may ~ - 2anchor the syndecan-2 cytoplasmic tail to actin filaments (50,51). Besides mediating cellmatrix adhesion syndecan-l can mediate cell-cell adhesion via its H$ chains (52). Syndecan-l can also mediate cell spreading in transfected human lymphoblastoid cells, a process that is independent of H§ or the cytoplasmic domain and that can be inhibited by agents that block actin and microtubule polymeriza-

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Functions of syndecans. Syndecans bind a variety of extracellular proteins through the GAG side chains. These include (1) extracellular matrix (ECM)molecules, (2) growth factors (FGF, fibroblast growth factor-2), and (3) proteases (Ela., elastase; LPL,lipoprotein lipase). Syndecansmay signal either directly by the cytoplasmic domain through adaptor molecules ~syntenin)or by functioning as coreceptors for fibroblast growth factor receptors (FGFR).Depending on the GAG composition and whether syndecans are bound to the plasma membrane (1,2) or whether they are soluble (3), the functions can be different.

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1 Hep~n/HeparanSulfate Binding Proteins

Entactin (nidogen) Fibronectin Laminin

~ r o ~ Factors t h and ~ ~ e m o k i n e s FGF family FGF-l? -2, -4, -5, -7,-8, - 9 EGF family Amphiregulin ~euregulins Heparin-bind~ngEGF-like growth factor HGF (hepatocyte growth factor, scatter factor) Wnt-1 HB-GAM Midkine GM-CSF (granulocyte-macrophage colony sti~ulatingfactor)

Tenascin T~rombospo~din Vitronectin

PDGF isoforrns

TGF-p isoforms

VEGF isoforms

Interferon-~ Interleukins-2, -3, -4, -5, -7, -8 MIP-la, -l p IP- 10

Mac- 1 Neural cell adhesion molecule (N-CAM) Thrombin Tissue plas~inogenactivator ~ e u ~ o p helastase il Cathepsin G

Antithro~binI11 P l a s ~ i n o ~ ea~tivator n inhibitor (PAI-I) Secretory leukoprotease inhibitor (SLPI)

Apolipoprote~nB (Apol3) ~polipoproteinE (ApoE)

Lipoprotein lipase

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Cytomegalovirus simplex virus Human i~munode~ciency virus

Her pes

~

l u s ~f ~ ol c~i p i ~ ~r u~~

tion (53). These data suggest that the core protein of syndecan-l mediates spreading through the formation of a multimolecular signaling complex at the cell surface that stimulates cytoskeletal reorganization. Indeed, binding of fibroblasts and endothelial cells to the extracellular domain of the syndecan-4 core protein suggests an association between the core and other cell surface molecules (54). Syndecan-l may be required to maintain epithelial cell morphology by binding ECM and adjacent cells. Normal murine mammary gland epithelia cells (NMuMG) that were made deficient of syndecan-l by transfection with an antisense syndecan-l expression vector lose their epithelial phenotype and gain a fibroblast-like morphology. These cells show rearranged pl-integrins, markedly reduced E-cadherin expression, and disorganized F-actin filaments (S5). S 115 epithelial carcinoma cells express a fibrob~ast-likephenotype when grown in the presence of steroid hormone testosterone. When transfected with the cDNA of syndecan-1, driven by a steroid-inducible promoter, the epithelial morphology is restored even in the presence of steroids further suggesting a role for syndecanl in the maint~nanceof epithelial morphology (56). En~agementof proteoglycans with other cell surface receptors may be a common adhesion mechanis~,For example, the interaction between cell surface HSPG and fibronectin stimulates focal adhesion formation but only in cooperation with integrins (57,58). Syndecan-4 becomes inserted into the focal adhesions of a number of cell types including fibroblasts, smooth muscle, and endothelial cells (59). The insertion occurs when protein kinase C (PKC) is activated (60). A unique sequence in the central part of the cytoplasmic domain of syndecan-~ can directly activate PKCa and potentiate its activity by phospholipid mediators when the cytoplasmic domain is oligomerized (26,29,61). These data indicate that clustering of syndecan-4 with an unknown ligand, possibly fibronectin, at focal contacts may control adhesion triggered signaling events. In s u m m ~syn, decans can anchor diverse cell types to extracellular matrix, and simultaneously, they can trigger intracellular signaling, thereby contributing to the maintenance of cellular morphology.

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A wide variety of growth factors are known to bind heparin or heparan sulfate

(Table l), including fibroblast growth factor (FGF) family members, hepatocyte growth factor (HGF) (62,63), a splice variant PDGF A chain (64), heparin-binding epidermal growth factor ( H ~ - ~ G (6S), F ) VEGF (66), neuregulins (67), wilzgl l ess (the ~ r o ~ homologue u ~ ~ i of l ~ Wnt- 1 proto-onco~ene)(68), and others. Syndecans-l, -3, and -4 have been shown to specifica~lybind FGF-2 (4,69--72), syndecan-3 binds heparin-binding growth-associated molecule ( H ~ - (28), ~ A ~ )

1

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and syndecan-4 binds midkine, a heparin-binding growth/differentiation factor related to HB-GAM (69). Additional information is presented in Chapter 3. The importance of heparin or more widely expressed heparan sulfate in the action of heparin-binding growth factor signaling was first demonstrated by studies showing that cells lacking heparan sulfate (HS) or H S sulfation caused failure of FGFs to activate FGF-receptor (FGFR) (73-76). Thus, HSPGs were determined to function as coreceptors for FGF-2 (Fig. 2) (77,78). An augmenting effect of H S on signaling has so fa-been demonstrated for FGFs-1, -2, -4, -5, -8, and -9 (79). Several studies have shown the ability of syndecans to promote the action of FGFs. Syndecan-l can bind at the same time ECM components and FGF-2, thereby inducing cell proliferation (72). Syndecans-l , -2, -4 overexpressed in cells that normally express low levels of cell surface HSPGs stimulate FGFR-l signaling (80). Furthermore, the limb outgrowth and mesodermal cell proliferation, a process that is dependent on FCFs (81,82), may also require the presence of syndecan-3 (83). The role of syndecans in FGF action can also be inhibitory. FGF-2-induced cell proliferation is inhibited in NIH3T3 cells overexpressing syndecan-1 either on the cell surface or in a soluble form in culture medium (84.). Also, soluble syndecans-l and - 2 can inhibit FGF-2 binding to its receptor in cell free assays (85). Stimulatory and inhibitory effects of syndecan-l in growth factor triggered cell growth can be explained by different heparan sulfate structures (86). The sulfation of syndecan-l is known to vary depending on the cell type and tissue (21,S?) and the GAGS can be modified on the cell surface by degradation. Furt~ermore,upon shedding, the function of syndecans changes from cell surface coreceptor to a soluble effector that can compete with same ligands. Indeed, soluble ectodomain is an inhibitor of cell growth (84,88) and a recent study has revealed that proteolytic degradation of syndecan-l HScan change it from an FGF inhibitor into a potent activator of FGF (89). HS interactions with heparin-binding growth factors and their receptors provide an attractive mechanism to regulate growth factor actions. Adjustments of cellular responses to growth factors could result from changes in syndecan expression, shedding, and in the fine structure of HSchains attached to syndecans. This could be especially important during development and tissue injury, when rapid changes in cellular responses are required. For example, in the developing neuroepithelium H S structure undergoes a rapid change in growth factor-binding specificity concomitant with the temporal expression of different FGFs (90).

Besides growth factors or cell adhesion molecules syndecans bind other extracellular ligands. These include proteins involved in lipoprotein metabolism, e.g.,

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lipoprotein lipase (LPL), low-density lipoprotein (LDL), and serine proteases and their inhibitors (serpins) (9 1-94). Like the binding of syndecans to other extracellular ligands, these interactions are mediated by H S chains. The activities of some of the proteases and antiproteases can be modified in vitro by heparin, a mast cell-derived GAG. Syndecan is a major cellular source of heparin-like GAG in vivo, and similarly to heparin it can modify protease activities in wound fluids (91>. The metabolism of lipoproteins is partially regulated by HSPGs. HSPGs interact with lipoproteins, apolipoproteins B and E, and with LPL which controls the delivery of fatty acids to tissues. LPL is primarily in the luminal surface of capillary endothelial cells where the enzyme is anchored to HSPGs, mainly syndecan-1 (95). Presumably, LPL bound to syndecan is internalized and translocated to the apical surface of endothelial cells. Syndecan-1 can thus function as a lipoprotein receptor (93). Another class of enzymes regulated by heparin/hep~ansulfate is serine proteases and their inhibitors (serpins). Antithrombin I11 (ATIII), a serpin that inhibits thrombin and other coagulation proteases, binds a heparin-like sequence in heparan sulfate, which dramatically accelerates thrombin/ATIII complex formation (96,97), and heparin has long been clinically used as an anticoagulant. ATIII binding to syndecans on the luminal surfaces of endothelial cells can contribute to the establishment of a nonthrombogenic lining of blood vessels (98). Because only a small proportion of syndecans polarize to luminal surface, other HSPGs may also be involved in producing a nonthrombogenic endothelial luminal surface. Syndecan-l is constitutively released into the culture medium from cell surfaces by the proteolytic cleavage of the ectodomain (99). Soluble syndecanl ectodomain binds neutrophil serine proteases, elastase, and cathepsin G tightly (91). This binding results in reduced affinity of these proteases to their physiological inhibitors al-proteinaseinhibitor (a,-PT), a,-antichymotrypsin (a,-Achy), and squamous cell carcinoma antigen-2 (SCCA2). Purified syndecan- 1 ectodomain protects elastase from inhibition by a,-PI, and cathepsin G from inhibition by a,-Achy and SCCA2. Consistently, both enzymatic degradation of endogenous heparan sulfate and immunodepletion of syndecans-l and -4 from human wound fluids, which also contain proteases and their inhibitors, reduce endogenous elastolytic and chymotryptic activities. Thus, soluble syndecans-l and -4 modulate the proteolytic activity of wound fluid (91). Proteolysis is important for fibrinolysis, growth factor mobilization and activation, cell migration into the wound site, reepithelialization, angiogenesis, and ECNI degradation during wound repair (100). An imbalance of proteases and antiproteases may disturb normal wound repair and capillary morphogenesis which is seen in chronic ulcerous wounds. Soluble syndecans may regulate the

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proteolytic balance in several inflammatory tissue repair processes. The effect of soluble syndecan will depend on the nature of protease inhibitors at the site of interaction.

Cutaneous wound repair and other tissue injuries are situations where the function of syndecans has been extensively studied and where syndecans seem to have multiple functions. The functions of syndecans in cell-ECM and cell-cell anchorage, in modifying protease as well as growth factor activity, are all likely to affect the tissue regeneration process. Normal wound repair involves clot formation, keratinocyte proliferation and migration, neutrophil activation, granulation tissue formation, and generation of new blood vessels (angiogenesis), and requires tightly regulated cooperation between several cell types and soluble factors. The repair process requires regulated protease and growth factor activities. Such control can be provided by heparan sulfate proteoglycans, including syndecans. While inactive in undisturbed tissues, several heparin-binding growth factors, including FGF-2, HB-EGF, and FGF-7, are activated during tissue injury and inflammation (101-103). For example, keratinocytes known to be FCF-2 responsive are in close proximity to matrixassociated FGF-2 on the basement membrane, but parado~ically,do not proliferate or migrate until tissue is disrupted. Because FGF-2 must interact with heparan sulfate to signal via its receptor, syndecan-l , which can promote the action of FGFs but also of other heparin-binding growth factors, has been an obvious candidate to serve this interaction. Indeed, syndecan-l expression is enhanced in keratinocytes at the wound edge and in endothelial cells within newly formed granulation tissue (104). In addition, syndecan-~shedding from cell surfaces is enhanced by growth factors and proteases involved in wound repair and soluble syndecans-1 and -4 are found in acute human wound fluids (91,105). Wound fluids contain a typical syndecanl ~roteoglycansmear that is not detected in human plasma or directed against the cytoplasmic domain of syndecan-l (105). genically active heparin-like fragments, identical in size and mitogenicity to the most potent oligosaccharides derived from syndecan-l HS by heparanase digestion of the syndecan-l ectodomain, are also found in the wound fluids at physiological concen~ations(91). Besides promoting mitogenic effects, soluble syndecan- 1 ectodomains shed from the cell surfaces to the extracellular matrices may inhibit heparin mediated growth factor mitogenicity via its low sulfated domains on the HS chains. However, heparanases released at the site of tissue injury from platelets degrade these inhibitory domains and liberate heparin-like domains that activate FGF-2, thus converting the soluble syndecan-l ectodomain into a highly potent mitogenic

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activator of FGF-2 (89). In this model, FGF-2 inhibition by intact soluble syndecan-l ectodomains and activation by heparin-like domains balances FGF-2 action during tissue injury. This model reconciles previous p~adoxicalfindings of inactive FGF-2 in undisturbed tissues and its activation upon injury, Wound repair has been investigated in a trarisgenic mouse strain that overexpresses syndecan-l. The skin wounds of these mice heal slowly due to excess soluble syndecan-l ( V.~ainulainenet ai., unpublished observation). Thus, soluble syndecan-l ectodomains at the injury site may provide novel physiological control mechanisms for proteolytic activities and growth factor-induced cell proliferation, Taken together, syndecans can function as receptors, coreceptors, and soluble effectors for many hep~in-bindingmolecules (Fig. 2 and Table 1). Because soluble and cell surface syndecans compete for the same ligands, the soluble syndecan ectodomains may function as inhibitors.

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An analysis of each syndecan mRNA level in various mouse cells and tissues has shown that virtually all tissues and cells express at least one syndecan, and some cells and tissues express multiple family members (87). Although the expression of different syndecans is partially overlapping in some cells, each syndecan family member is mainly expressed in a distinct cell-, tissue-, and development-specific pattern, suggesting specific functions for each syndecan. Several epithelia express syndecan-l but not syndecans-2 or -3, whereas neuronal cells contain almost exclusively syndecan-3, and endothelial cells mostly syndecan-4 (87,306,107). Furthermore, skin expresses mainly syndecans-l and -4, kidney mainly syndecan-4, adult brain tissue only syndecan-3, while, for example, the fat cells of liver express all syndecans (1 08). The expression pattern of syndecans changes in response to many situations, including tissue injury or morpholo~ical changes during organ development.

Spatial and temporal changes of syndecan expression occur during embryo development (l ,41,109). Syndecan- l is first detected at the 4-cell stage. At the blastocyst stage, syndecan-l is detected at cell-cell contacts throughout the embryo, and later at the interface of the primitive ectoderm and endoderm, the site of initial matrix accumulation, ~orphogenesisis modified by reciprocal interactions between epithelial derivatives (ectoderm and endoderm) and the mesenchyme. Embryonic syndecan-l expression has been suggested to have a role in this interplay during the development of several organs, including tooth (1 lo), kidney

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(1 1 1), limb (1 12), lung ( l 13), and the optic, vibrissal, nasal and otic tissues (109). Syndecan- 1 expression during the development of these organs shares some common features. In general, the epithelium changing its shape (e.g., forming a bud) transiently loses its cell surface syndecan-1 expression, while the condensing and proliferating mesenchyme around the epithelium starts to express syndecan-l . With further development, the morphologically stable epithelium reexpresses syndecan- 1, while the terminally differentiated mesenchymal cells lose it. Similar findings have been reported for avian syndecan-3. During limb development syndecan-3 is transiently expressed in condensing mesenchyme (9). In the embryo tibia, syndecan-3 i s expressed in prolif~ratingimmature chondrocytes, while differentiated chondrocytes lack the expression ( l 14), suggesting a regulatory role in proliferation during bone development: Furthermore, limb cartilage differentiation can be inhibited with syndecan-3 antibodies in vitro (1 15). Thus, syndecan- 1 and -3 expression in epithelia correlates with epithelial maturation, and in mesenchyme with cell proliferation, consistent with their proposed functions as matrix receptors and growth factor coreceptors. Interestingly, syndecan-3 might also have a role in oligodendrocyte differentiation, since its expression is highly up-regulated during that time of postnatal central nervous system development (106,107,116).

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Consistent with the proposed role of syndecan as an ECM receptor and a modulator of growth factor actions, syndecan-1 expression is regulated during cell differentiation and malignant transformation. For example, in normal tissues syndecan-l i s most abundantly expressed in stratified epithelia where it is localized over the entire surface of suprabasal keratinocytes, whereas basal and the most superficial layers are stained only weakly (40). During skin wound repair, syndecan- 1 expression is increased in proliferating keratinocytes ( l 04,117). However, development of dysplasia, a premalignant condition, in the skin is associated with the loss of syndecan- 1 (1 18). Moreover, the formation of carcinomas is associated with marked reduction in syndecan-l expression. Syndecan-1 gene expression is not totally lost from malignant tumors, but is retained at low levels in neoplasms showing a high degree of differentiation (1 18- 120). In squamous cell carcinomas (SCCs), retained syndecan-l is localized in keratinizing cells around the keratin pearls, but is lost on the actively proliferating cells within the tumor. This pattern of expression suggests that syndecan-l may have a role in keratinocyte differentiation during neoplastic growth. The loss of syndecan-l expression in SCC of the head and neck is associated with a poor clinical outcome (121). In SCCs syndecan-l and E-cadherin show similar expression; both are expressed in well-differentiated cells, while lacking from poorly differentiated ones. Furthermore, they show coordinated expression in mammary epithelial cells genetically manipu-

lated with E-cadherin (122). 0th molecules have been suggested to have a role in the polarization and maintenance of cytoskeleton and cell morphology. The possible role of syndecan-l in malignant transformation and maintenance the epithelial morphology has been studied in vitro using mouse mammary tumor cells (S1 15), renal epithelial cells, and normal mouse mammary cells. The steroid altered phenotype of S1 15 cells can be restored by transfecting the cells with syndecan-l cDNA (56). This effect seems to be mediated solely by the syndecan-l ectodomain since S 3 l 5 cells transfected with a mutant syndecan-l lacking cytoplasmic and transmembrane domains also show benign characteristics (123). Also, overexpression of syndecan-l in transformed human renal epithelial cells causes cells to become more anchorage-dependent and less motile (124). Thus syndecan- 1 expression seems to be required for maintenance of a differentiated epithelial phenotype. A similar concl~~sion was reached from experiments where endogenous syndecan- l expression was suppressed in epithelial cells by transfection with antisense cDNA (55). This changed the cell morphology from cuboidal to fusiform and the cells gained an ability to migrate in collagen gels as well as the ability to grow in an anchorage-independent manner. Related to the growth factor binding activity, FGF-2 binding to HS around microvessels is lost during malignant transformation of breast carcinomas (125). Myeloma provides another example of malignancy where syndecan expression is modified. Similar to plasma cells, several myeloma cells express syndecan which regulates their adhesion to collagen (126). Syndecan is actively shed from myeloma cells and elevated syndecan levels correlate with the tumor volume in patients (127). Soluble syndecan can induce apoptosis and inhibit the growth of these tumor cells and, consistently, increase the survival of mice with myeloma (88). Interestingly, syndecan- l expression may identify Hodgkin’s lymphol~as that originate from E cells ( l 28).

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Both cell surface expression and shedding of syndecans-l and -4 are induced in response to injury. For example, syndecan-l i s induced in aortic neointima in response to a balloon catheter-induced vascular wall injury (129). Also, syndecan-4 is transiently induced in vascular wall injury, whereas the expression of syndecans-2 or -3 do not change (130). Furthermore, induction of myocardial infarction is associated with increased syndecan-l and -4 expression (13 l). The best studied example, however, is dermal wound repair. Syndecan-l is transiently induced in proliferating keratinocytes at the wound edge and in the endothelial cells of the wound bed (104), while syndecan-4 is induced on the fibroblasts that form granulation tissue (1 17). This induction in mesenchymal cells has been shown to be, in part, due to action of neutrophil-derived antirnicrobial peptide PR-39 (1 1’7). Interestingly, syndecan-l is not found at the tip of the

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migrating keratinocyte sheet (104). The induced expression is decreased down to lower levels after re-epithelialization both in keratinocytes and endothelial cells, upp press ion of endothelial syndecan-1 expression has shown to be, in part, due to tumor necrosis factor alpha, the inflammatory cytokine produced by macrophages at the site of injury (132).

A common mechanism of HSPG turnover involves endocytosis and degradation in lysosomes, and a signi~cantfraction of syndecans are removed by this mechanism (133). However, an additional mechanism for removal of syndecans from the cell sudaces is the release of the entire ectodomain into the extracellular space in a process called shedding (99,134). This release is mediated by a proteolytic activity of unknown identity and the precise site of cleavage within the ectodomain is not known. The dibasic sequence adjacent to the plasma membrane attachment site has been considered to be the prime candidate, but shedding of the ~ ~ u s usyndecan ~ ~ i that Z ~ lacks these basic residues (14) suggests that this hypothesis might not be accurate. The importance of this shedding has become of interest after the finding that this shedding is a highly regulated process by growth factors and proteases involved in tissue repair (105), and when the first soluble syndecan ectodomains were found in human derrnal wound fluids (105) and tracheal aspirates of newborn infants undergoing mechanical ventilation (135).

The expression of syndecans is constitutive in many cases, such as the expression of syndecan-l found in resting skin and the expression of syndecan-3 in adult h e ~ tissue t (136). However, in several instances, including organ development and tissue injury, the expression of syndecans is highly regulated, either induced or suppressed. The expression of syndecans can be regulated posttranscriptionally or by transcriptional mechanisms. The regulatory ~ e c h a n i s m shave been most e~tensivelystudied for syndecan- 1.

The expression pattern of syndecan-l during organogenesis and tissue regeneration implies that it might be regulated by soluble factors, supposedly by growth factors. For example, FGFs (FCF-2, FGF-4, FGF-8) are prime candidates for the signal leading to the development of limbs and they may control the epithelial-

mesenchymal interactions at the places where syndecan-l expression is induced (8 1,82,137,138). Likewise, keratinocyte migration during dermal wound healing requires many growth factors including EGF and FGF-7 (101-103,139-141). Several polypeptide growth factors and other soluble effectors can regulate syndecan-l expression (Fig. 3). Treatment of NIH3T3 cells simultaneously with FCF-2 and transforming growth factor-P (TGF-P) increases syndecan-l mRNA and shedding of syndecan1 ectodomain into the culture medium in 24 hr (71). FGF-2 alone can transiently induce syndecan-l expression in NIH3T3 cells at earlier time points (142). Syndecan-l mRNA levels increase several-fold within 4-8 hr after FGF-2 stimulus and then gradually decrease and reach low levels within the next 24 hr. On the MCA3D keratinocyte cell line (143) syndecan-l mRNA i s upregulated severalfold by both EGF and FGF-7. Interestingly, this upregulation is not seen by FGF2, although FGF-2 induces keratinocyte proliferation in these cells (144,l45), indicating that syndecan-l is regulated by different growth factors depending on the cell type. Tumor necrosis factor alpha (TNFa) decreases syndecan-l levels in endothelial cells of wounds and in cultured endot~elialcells (132). Furthermore, during myoblast terminal differentiation syndecan- 1 expression is downregulated by a myogenin- and E- bo^-independent pathway. Its expression in myoblasts is controlled by a proximal region of the promoter that is influenced by FCF-2, TGF-P, and retinoic acid (146). Other examples of growth factor regulation of syndecan-l include suppression of syndecan in B lymphoid cells by interleukin-6 (147) and down-regulation of syndecan-l in S l 15 tumor cells by testosterone (148). Finally, besides regulation of syndecan core protein expression, the GAG chains can be subject to inducible regulation. Both TCF-P and FGFs have been shown to induce attachment of heparan sulfate and chondroitin sulfate side chains onto syndecan ectodomain (149,150). Inducible re~ulationof syndecan family members other than syndecan-l by soluble factors has been reported. For example, syndecan-4 is induced by serum, PDGF and FGF-2 in vascular smooth muscle cells, In contrast to syndecan-l, this regulation is not transcriptional since it is not inhibited by cycloheximide (l30).

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~osttranscriptionalregulation of syndecans can occur by modi~cationof the GAG chains or by shedding syndecan ectodornains from the cell surface, Little is known about other postt~dnscriptionalmechanisms, such as regulation of m RNA stability. Modi~cationof the GAG chains by intracellular enzymes has a major innpact on syndecan functions. First, depending on the cell type, tissue, and syndecan family member involved, either heparan sulfate, chondroitin sulfate, or both

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Regulation of syndecan-l expression. Transcription of the syndecan-l gene can be enhanced by several transcription factors as well as by different mitogens and growth factors. Activation of translation from a stored mRNA pool is enhanced by cyclic adenosine l~onophosphate(CAMP).Shedding of syndecan is enhanced by EGF, and GAG attachment by TGF-P.

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can be attached to the core protein. Second, the length of the GAG side chains can vary in a cell-type specific manner. Third, the composition of the disaccharides within the GAG chains can vary. These modifications determine the affinity of syndecans to heparin-binding molecules, such as fibroblast growth factors. An example of posttranscriptional regulation is seen in peritoneal macrophages. A stored syndecan-1 mRNA pool can be elicited by cyclic adenosine monophosphate (CAMP)-dependent mechanisms to generate cell surface syndecan-l (151). The causes of elevated CAMPcan be several, including the stirnulatory signal triggered by diverse hormones ( l 52). The CAMP-dependent upregulation of syndecan might be restricted to syndecan-l, since syndecan-4 is not regulated in a similar fashion. Another example of posttranscriptional regulation is found in ras-transformed epithelial cells where syndecan-l expression is blocked at the level of translation (153). Enhanced syndecan shedding by phorbol esters (105) resembles that of other membrane proteins, including growth factors, cytokine receptors, and cell adhesion molecules, suggesting a common regulated mechanism for the proteolytic cleavage. Importantly, syndecan-l and -4 shedding is enhanced by proteases (thrombin, plasmin) and growth factors (EGF-family members) involved in tissue injury. Furthermore, there is evidence that syndecan shedding also occurs in vivo, For example, soluble syndecans-l and -4 can be isolated from acute demal wound fluids (10s)and tracheal aspirates (135), and syndecan-3 from an aqueous extraction of neonatal rat brain ( l 16). Furthermore, elevated levels of soluble syndecan-l are found in the serum of multiple myeloma patients (127). These soluble syndecans are not stained on immunoblots with antibodies directed against cytoplasmic domains, consistent with the loss of cytoplasmic domain by proteolysis.

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The syndecan genes are dispersed throughout the mouse and human genomes, but each syndecan gene is linked to one of the four members of the myc oncogene family of transcription factors (2,354-156). The genomic organization of the mammalian syndecans-1 and -4 (157l 59) i s similar to that of the ~ r o s o ~ ~and i ZXenopus u syndecans (Fig. 4) (14,15). Syndecans consist of five exons interrupted by four introns of which the first one is over 17 kb in length. The genes show a strikingly similar exon-intron organization which supports the idea that syndecans arose by gene duplication from a single ancestral gene. Each exon encodes a discrete functional domain: exon l encodes the 5’-untranslated region and signal peptide, exon 2 encodes the N-

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terminal cluster of GAG-attachment sites, exon 3 encodes the ectodomain spacer region, exon 4 encodes the juxtamembrane cluster of GAG-attachment sites and 10 bp of the transmembrane domain, and exon 5 encodes the rest of the transmembrane domain, the cytoplasmic domain, and 3’-untranslated region. The most variable exon in length and sequence is exon 3 which encodes ectodomain region without the conserved GAG-attachment sites. The most conserved exons are the exons 4 and 5 coding for the transmembrane and cytoplasmic domains, respectively. Two alternative polyadenylation sites in the 3’ end of the gene generate two transcripts of different size. The proximal 5‘ region of the syndecan-1 gene harbors several transcription factor binding motifs for both constitutively bound and inducible nuclear proteins (Fig. 4). The binding sites include a TATA-box-like sequence, an antennapedia binding site, an NF- KBbinding site, at least ten AP-2 binding sites and several GTlGC clusters for SPl binding. SPl binding sites are found in many constitutively expressed, housekeeping genes. In normal epithelial murine m a m m ~ gland ( ~ ~ u cells ~ with G ) high constitutive syndecan-l levels, the proximal SP1 binding sites are required and may be sufficient for syndecan-l expression. The TATA-box is not needed in these cells and the transcri~tionis initiated from initiator-like sequences (160) which are also often found in house~eepinggenes. In kidney cells the Wilms’ tumor suppressor gene (WTl), a transcription factor that is necessary for the development of kidney, is known to regulate syndecan-

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l expression. WT1 binds to GC-rich regions in the syndecan-l proximal promoter and activates syndecan-l through these regions (161). Other transcription factors are known to regulate syndecan-1 expression. For example, Blimp- 1, a zinc-finger transcription factor that drives the maturation of B lymphocytes into immunoglobulin-secreting cells, positively regulates syndecan-1 expression in B cells (162). In developing dental mesenchyme Msxl transcription factor may be required for the induced expression of syndecan-l (163). Interestingly, proteases, namely, thrombin, stimulate syndecan expression in vascular smooth muscle cells (164). While the mechanisms of this regulation are unknown, it may provide an interesting regulatory feedback loop for thrombin and antithrombin 111, and thus for blood coagulation regulation. Although the constitutive expression of syndecan-l in epithelia seems to be due to the proximal promoter regulated transcription by SPls (160), genetic mechanisms responsible for the inducible expression have remained less well understood. However, a recent finding of a growth factor-responsive element on the syndecan-l gene has revealed new mechanisms how syndecans may be regulated by growth factors (142). A 280 bp element located at - 10 kb from the syndecan-l translation initiation site mediates the FGF-2 induced activation of syndecan-l in NIH3T3 fibroblasts and has been termed FiRE for (Fig. 4). The element responds to FGF-2 treatment regardless of its orientation and has both constitutively bound and FGF-2 inducible transcription factors. The constitutively bound factors include upstream stimulatory factor- 1 (USF- 1) and an u ~ c h ~ a c t e r i z e46 d kDa protein. The FGF-induced factors are two FodJun complexes, also called activator protein-l (AP-l), and a 50 kDa AP-2 related factor which has been termed FIN-l for FGF-inducible nuclear factor (Fig. 4). Although most of the AP-l-driven promoters are activated by virtually all growth factors, FiRE responds specifically only to selected growth factors. In fibroblasts, it is activated only by FGF-family members and not by serum or several other growth factors, including FGF-7, platelet-derived growth factor BB ( ~ D G F / ~ B ) , EGF, TGF-P, insu~in-likegrowth factor (IGF-I), or interferon gamma (IFN-y), Consistently, it is not activated by some AP-1 inducing chemicals, such as okadaic acid and l ~-~-tetradecanoylphorbol13-acetate (TPA). This suggests that in some special situations syndecan-l expression may be required to be activated solely by FGFs and not other mitogens. Noticeably, the FiRE element seems to be responsible for the upregulation of syndecan- 1 also by FGF-7 and EGF-family members in lceratinocytes and not only by FGF in fibroblasts. Whilst activated by EGF, TGFa and FGF-’7, and FiRE is not activated by FGF-1 or -2 in keratinocytes (144). Furthermore, the FiRE uses a different subset of transc~ption factors and is activated by diverse signaling pathways in fibroblasts and keratinocytes.

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The expression of syndecan-l in migrating wound keratinocytes suggests that growth factors may regulate syndecan-l also in these cells. Animal studies have revealed that the FiRE element may be responsible for growth factor-induced upregulation of syndecan- l in healing wound keratinocytes. Transgenic mouse strains with a P-galactosidase producing reporter gene (Lac-Z) ligated to FiRE and different promoter regions (either syndecan proximal promoter or thymidine kinase promoter) show that FiRE is activated in vivo specifically in keratinocytes during dermal wound repair and does not require specific proximal promoter sequences. Activation of FiRE occurs only in keratinocytes, but not in dermal or endothelial cells. Furthermore, the activation is seen only during the re-epithelialization phase, It i s first found in the epithelial sheets adjacent to the incision site where the keratinocytes start to migrate toward the wound base and in the remnants of the hair follicle keratinocytes. The activation is robust at the merging epithelium where the two keratinocyte sheets fuse, as well as during the stratification of the epithelium. FiRE activation fades away at the end of re-epithelialization and wound closure without reappearing later, similar to syndecan-l expression. The activation is restricted to migrating keratinocytes. Moreover, the activation of FiRE seems to require both growth factor- and stress-induced signaling pathways, namely, extracellular regulated kinase (ERK) and the p38 K but not, e.g., phophatidylinositol-3 kinase (PI3K) pathway. The activation is blocked by inhibiting either of the two pathways and in resting skin the activation requires both EGF-family members and inducers of stress kinases. Interestingly, the EGF-induced shedding of syndecan-l ectodomain also requires p38 MAPK pathway ( l OS). The expression of syndecan in wounds is regulated by soluble factors also in cell types other than keratinocytes. Tumor necrosis factor alpha (TNFa), which is mainly synthesized by activated macrophages at the site of inflam~ation,decreases syndecan-l levels in endothelial cells of wound granulation tissue as well as in cultured endothelial cells (l 32). Other growth factors and cytokines tested, including IFN-y, IL- l p, TGF-p, FGF-2, and FGF-7, have no effect on the syndecan- l NA levels. Finally, in healing wounds an antimicrobial peptide, PR-39, which is secreted into wound fluids, upregulates syndecans-l and -4 in the dermal fibroblasts (16S), but the mechanisms of this induction are not yet understood.

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1. Bernfield M, Kokenyesi R, Kato M, Hinkes MT, Spring J, Ga llo RL, Lose EJ.

Biology of the syndecans: a family of trans~embraneheparan sulfate proteoglycans. Annu Rev Cell Biol 1992: 8: 365- 393.

2. Marynen P, Cassiman J-J, Van den Berghe H, David G. Partial primary structure of the 48- and 90-kilodalton core proteins of cell surface-associatedheparan sulfate proteoglycans of lung fibroblasts-prediction of an integral membrane domain and evidence for multiple distinct core proteins at the cell surface of human lung fibroblasts. J Biol Chem 1989; 264:7017-7024. 3. Saunders S, Jalkanen M, O'Farrell S, Bernfield M. Molecular cloning of syndecan, an integral membrane proteoglycan. J Cel l Biol 1989; 108:1547-1556. 4. Kiefer MC, Stephans JC, Crawford K, Okino K, Barr PJ. Ligand-affinity cloning and structure of a cell surface heparan sulfate proteoglycan that binds basic fibroblast growth factor. Proc Natl Acad Sci USA 1990; 87:6985-6989. 5. Mali M, Jaakkola P, Arvilommj A-M, Jalkanen M. Sequence of human syndecan indicates a novel gene family of integral membrane proteoglycans. J Biol Chem 1990; 26514884-6889. 6. Kojirna T, Shworak NW, Rosenberg RD. Molecular cloning and expression of two distinct cDNA-encoding heparan sulfate proteoglycan core proteins from a rat endothelial cell line. J Biol Chem 1992; 267:4870-4877. 7. Carey DJ, Evans DM, Stahl RC, Asundi VK, Conner KJ, Garbes P, Cizmeci-Smith G. Molecular cloning and characterizationof N-syndecan, a novel transmembrane heparan sulfate proteoglycan. J Cell Biol 1992; 1 17:191-201. 8. David G, van der Schueren B, Marynen P, Cassiman J-J, van den Berghe H. Molecular cloning of amphiglycan, a novel integral membrane heparan sulfate proteoglycan expressedby epithelial and fibroblastic cells. J Cell Biol 1992; l 18:961969. 9. Gould SE, Upholt WB, Kosher RA. Syndecan 3: a member of the syndecan family of membrane-intercalated proteoglycans that is expressed in high amounts at the onset of chicken limb cartilage differentiation. Proc Natl Acad Sci USA 1992; 89: 3271-3275. 10. Pierce A, Lyon M, Hampson IN, Cowling GJ, Gallagher J. Molecular cloning of the major cell surface heparan sulfate proteoglycan from rat liver. J Biol Chem 1992; 267:l-7. 11. Baciu PC, Acaster C, Goetinck PF. Molecular cloning and genomic organization of chicken syndecan-4. J Biol Chem 1994; 261:696-703. 12. Raulo E, Chernousov MA, Carey DJ, Nolo R, Rauvala H. Isolation of a neuronal cell surface receptor of heparin binding growth-associated nlolecule (HB-GAM). Identification as N-syndecan (syndecan-3).J Biol Chem 1994; 269: 12999- 13004. 13. Tsuzuki S, Kojima T, Katsumi A, Yamazaki T, Sugiura I, Saito H. Molecular cloning, genomic organization, promoter activity, and tissue-specific expression of the mouse ryudocan gene. J Biochem (Tokyo) 1997; 122:17-24. 14. Spring J, Paine-Saunders SE, Hynes RO, Bernfield M. ~ ~ o s osyndecan: p ~ i con~ ~ servation of a cell surface heparan sulfate proteoglycan. h o c Natl Acad Sci USA 1994; 91:3334-3338. 15. Rosenblum ND, Botelho BB, Bernfield M. Expression of a Xenopus counterpart of mammalian syndecan 2 during embryogenesis.Biochem J 1995; 3095976. 16. Tee1 AL, Yost HJ. Embryonic expression patterns of Xenopus syndecans. Mech Dev 1996; 59:115-127.

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decan- 1 expression is down-regulated during myoblast terminal differentiation. Modulation by growth factors and retinoic acid. J Biol Chem 1997; 2721841818424. Sneed TB, Stanley DJ, Young LA, Sanderson R D . Interleu~n-6regulates expression of the syndecan-1proteoglycan on B lymphoid cells. Cell Immunol 1994; 153: 456-467. Leppa S, Harkonen P, Jalkanen M. Steroi~-inducedepithelial-fibroblastic conversion associated with syndecan suppression in S115 mouse m a ~ a r tumor y cells. Cell Regul 1991; 2:1-11. Romaris M, Bassols A, David G, Effect of t r a n s f o ~ growth n ~ factor-beta 1 and basic fibroblast growth factor on the expression of cell surface proteoglycans in human lung fibroblasts. Enhanced glycanation and fibronectin-binding of CD44 proteoglycan, and down-regulation of glypican. Biochem J 1995; 310:7381. Rapraeger A, Transforming growth factor (type p) promotes the addition of chondroitin suflate to the cell surface proteoglycan (syndecan) of mouse mammary epithelial cells. J Cell Biol 1989; 109:2509-2518. Yeaman C, Rapraeger A. Post-transcriptionalregulation of syndecan-l expression by CAMPin peritoneal macrophages. J Cell Biol 1993; 122:941-950. McKnight GS, Cummings DE, Amieux PS, Sikorski MA, Brandon EP, Planas JV, Motamed K, Idzerda RL. Cyclic AMP, PKA, and the physiological regulation of adiposity. Recent Prog Horm Res 1998; 53:139--161. Kirjavainen J, Leppa S, Hynes NE, Jalkanen M. Translationalsuppression of syndecan-l expression in Hams transformed mouse m ~ m a r eptihelial y cells. Mol Biol Cell 1993; 4:849-858. Spring J, Goldberger OA, Jenkins NA, Gilbert DJ, Copeland NG, Bernfield M. Mapping of the syndecan genes in the mouse: linkage with members of the myc gene family. Genomics 1994; 21597-601. Ala-Kapee M, Nevanlinna H, Mali M, Jalkanen M, Schroder J. Localization of gene for human syndecan, an integral membrane proteoglycan and a matrix receptor to chromosome 2, Somatic Cell Molec Genetics 1990; 16:501-505. Kojima T, Inaz aw a J, Takamatsu J, Rosenbery RD, Saito H. Human ryudocan core protein: molecular cloning and characterization of the cDNA, and chromosomal localization of the gene. Biochem Biophys Res Commun 1993; 1990:814-822. Vihinen T, Auvinen P, Alanen-Kurki L, Jalkanen M. Structural organization and genomic sequence of mouse syndecan-l gene. J Biol Chem 1993; 268:1726117269, Hinkes MT, Goldberger OA, Neumann PE, Kokenyesi R, Bernfield M. Organization and promoter activity of the mouse syndecan-l gene. J Biol Chem 1993; 268: 11440-1 1448. Takagi A, Kojima T, Tsuzuki S, Katsumi A, Yamazaki T, Sugiura I, Ha~aguchi M, Saito H. Structural organization and promoter activity of the human ryudocan gene. J Biochem (Tokyo) 1996; 119:979-984. Vihinen T, Maatta A, Jaakkola P, Auvinen P, Jalkanen M. Functional characterization of mouse syndecan-l promoter. J Biol Chem 1996; 271:12532-12541. Cook DM, Hinkes MT, Bernfield M, Rauscher F Jr. Transc~ptionalactivation of

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162. 163. 164. 165.

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the syndecan-l promoter by the Wilms9 tumor protein WT1. Oncogene 1996; 13: 1789-1799. Turner CAJ, Mack DW, Davis MM. Blimp- l, a novel zinc ~nger-cont~ning protein that can drive the maturation of B lymphocytes into i~unoglobulin-secreting cells. Cell 1994; 77:297-306. Chen Y, Bei M, Wo o I, Satokata I, Maas R.Msxl controls inductive signaling in mammalian tooth mor~hogenesis.Development 1996; 1223035-3044. Cizmeci-Smith G, Carey DJ. Thrombin stimulates syndecan-l promoter activity and expression of a form of syndecan-l that binds antithro~bin111 in vascular smooth muscle cells. Arterioscler Thrornb Vasc Biol 1997; 17:2609-2616. Gallo RL, Ono M, Povsic T, Page C, Eriksson E, Klagsbrun M, Bernfield M. Syndecans, cell surface heparan sulfate proteoglycans,are induced by a proline-rich antil~icro~ial peptide from wounds. Proc Natl Acad Sci USA 1994; 91:11035-11039.

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Two features of syndecan core protein structure connect all the members of this family, both vertebrate and invertebrate (l-3). First, all are substituted with glycosaminoglycan chains, these usually being heparan sulfate. Second, their transmembrane and cytoplasmic domains are remarkably homologous. When Drus u ~ ~ i Zand u ~ a ~ ~ a lsyndecans i a n are compared, it is clear that these domains are conserved and indicate important functional roles. Within the ~ a m m a l sthis , conservation of structure is even more apparent (Fig. l). For example, there are no changes in amino acid sequence between human and rodent syndecan-l transmembrane and cytoplasmic domains. The same is true for other syndecans. In contrast, other than the conservation of glycosa~i~oglycan substitution sites, there is distinctly less conservation of ectodomain sequence (1). Apparently, core protein function on the outer face of the membrane is restricted to substitution with glycosaminoglycans, although the topography of these carbohydrates may al so be important. The ectodomains of ~ e ~ and u ~ a~m m ~ a l si a n syndecan-2 core proteins, for example, are equivalent in size (4), perhaps indicating that each syndecan expresses its glycosaminoglycans with a conserved relationship to the cell surface. Closer inspection of the four mammalian syndecans shows that this family can, on protein sequence homology grounds, be separated into two s ~ b f a ~ i l i e s (I). Syndecans-l and -3 are clearly highly related, while syndecans-2 and -4 comprise a second subfamily. In D ~ o ~ o ~ ~atithe Z ucurrent , time, only one syndecan has been identified ( 5) ,perhaps showing that in vertebrates there has been

( a)

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R~KKKDEGSYSLEEPK~~GGA-YQK-PTKQEEFYA R~KKKDEGSYTLEEPKQA-SVT-YQK-PDKQEEFYA R~RKKDEGS~DLGERK-PSS~-YQ~~TK--E R~KKKDEGSYDLG-KK-PI----YK~PT--NEFY c1 I z V c2

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R*BKKDEGSY*L***K*******Y********EFYA R~BKK~EGSY*L***K******Y*K*P****EFYA R~KKK~EGSY*LEEPK~A****Y~KP*KQEEFYA R~BKKDEGSYDLG*BKP****Y*

Sequences of syndecan cytoplasmic domains. (a) Amino acids are denoted by the one-letter code with spaces for alignment. The constant (Cl and C2) and variable ( V)regions of mammalian syndecans are shown. (b) Scheme of conse~ationamong all syndecans, all mammalian syndecans, and between the two pairs of mammalian syndecans that are most homologous. * denotes differences; B, basic residues.

adaptive radiation within the family to serve a distinct series of functions. One clue to cytoplasmic interactions and functions comes from a detailed examination of the cytoplasmic domains of all the syndecans (Fig. 1). The membrane proximal region (Cl) is virtually invariant across all syndecans, and the C-terminal region (the C2 domain) always contains a final EFYA sequence. However, between these two small regions lies a variable (V) region that is different in each syndecan. The most similar are those of syndecans-l and -3, while syndecans-2 and -4 have V regions much different from each other, and from the other two mammalian syndecans. The V regions of ~ r o s u ~ and ~ i lC, u e l e ~ u syndecans ~s are distinct again (3), both from each other, and from all the vertebrate members. Evidence presented below indicates that these distinct V regions do reflect differences in function. In Chapter 6, a role for syndecans as coreceptors was outlined, consistent with much data that show modulatory roles for glycosa~inoglycans,This makes the study of these proteoglycans both interesting and difficult. Since they have the

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potential to alter the interaction of ligands at the cell surface with other families of receptors, syndecans are at a nexus of information reception and forwarding. Their functions are not necessarily restricted to extracellular modulation of ligand binding, such as growth factors or extracellular matrix molecules. They also have the ability to signal independently commensurate both with the conservation of cytoplasmic sequences and with V region differences among syndecan family members. On the other hand, the fact that they seem not to act on their own, but only in conjunction with other receptors, makes the investigation of their functions a more complex task. This is compounded by their transmembrane nature and the s t ~ c t u r aco~plexity l associated with the study of macromolecules having a large heterogeneous carbohydrate component.

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Over the past few years increasing evidence has accumulated for a regulatory function of syndecans in cell adhesion. Much of the earlier data was derived from syndecan-l studies. This syndecan is characteristic of epithelial cells (1- 3), and may influence the expression of an epithelial phenotype in some cases. Antisense cDNA expression in mammary epithelial cells induces a marked change to a fibroblastic phenotype, shown to involve a down-regulation of E-cadherin (6). The relationship between these two receptors is mutual, since decreased expression of E-cadherin in mouse m a ~ epithelial m ~ cells leads to loss of cell surface syndecan- l ('7). No direct interaction has been demonstrated between these molecules, and it is quite possible that this is indirect, perhaps through the catenins and their associated intracellular proteins. The biological significance of this process is indicated by the correlation between loss of syndecan-l and invasive potential of a group of head and neck tumors (8). S i m i l ~ l yexpression , of syndecan-l in ly~phocytesendows the cells with an ability to adhere to, but not invade, collagen gels (9). Here E-cadherin is not a regulator, and these studies point to a connection between cytoskeletal organization and interaction with extracellular matrix. In a third system, Schwann cells transfected with syndecan-l cDNA spread to a much greater degree on planar substrates, and a codistribution of cell surface syndecan-l and the underlying actin cytoskeleton was observed throughout the spreading process (10). once spreading was complete, syndecan-l was not present in specialized focal adhesions, regions of tight cell-matrix adherence at the termini of microfilament bundles. In fact, only very rarely is syndecan- l detected in these adhesions (1 1). Syndecans-2 and -3 have also not been recorded in these structures. In contrast,

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syndecan-4 is frequently found in focal adhesions and its role has been the subject of several studies ( 1 2 ~ 3 ) . yndecan-4 (14,15), is unlike the other mammalian syndecans, not only because it is more widespread and less tissue-restricted than the others, but also because of its association with focal adhesions. This association has been seen in fibroblasts, smooth muscle, epithelial and endothelial cells (12). It is, however, also present in some cells, such as monocytic and lymphocytic lineages, where focal adhesions are not usually detectable in vitro (15). What is its role in cell adhesion? Since focal adhesion formation is an easily recogni~ableend point in the adhesion process of anchorage-dependent cells, this has allowed some elucidation of its role.

Focal adhesions are formed in response to extracellular matrix substrates, whether collagenous or glycoprotein (16). They comprise regions of tight cell-matrix adherence, and represent the termini of actin-containing microfilament bundles (17). While formed readily by many anchorage-dependent cell types on planar substrata, equivalent structures have been seen in many locations in vivo, and notably during wound repair (18). Fibronectin has often been used in tissue culture studies because of its well-understood structure and ease of purification, or recombinant expression, of its domains (19). The major class of receptors for fibronectin and other matrix components are the integrins (20,21), These are a large class of h e t e r o ~ i ~ e rtransmembrane ic glycoproteins, several of which bind fibronectin, and, as a result, become incorporated into focal adhesions (19). Indeed, the consensus i s that focal adhesion formation is absolutely integrin-dependent. ever, studies over a decade ago showed that cells inhibited from exportin own matrix could not form focal adhesions when seeded on substrates comprising only the central region of fibronectin, which interacts with integrins, such as a 5 p l or avp3 (Fig. 2; Ref. 22). One of two further inputs was required. Either the heparin-binding domain (Hep 11) of fibronectin or phorbol ester ~ e a t m e nto t activate protein kinase C (PKC) could provide the nece stimulus for focal adhesion f o ~ a t i o n(22-24). Subsequent analysis of the I I domain of fibronectin identified a particularly active peptide sequence, a ther, that hepasinase I II retreatment of the cells rendered them insensitive to the peptide or the whole ep I1 domain (23). Similarly, PKC inhibi lock focal adhesion formation. n other systems, not only are integrins and ctivation required, but in addition, the C-protein Rho must be activated to complete the adhesion process (24). The latter is now under intense study since it is known to be involved in microfil-

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Syn d ecan - 4 colocaliz ation with in tegrin s at focal adhesions. Im ~ u n o ~ u o r e s cen t labelin g for syn d ecan - 4 ( A)codistributes with that for integrin Dl subunits (B). Ba r = l0 pm.

ament contraction, focal adhesion formation, and also downstream signaling events controlling growth or apoptosis (17,25). Syndecan-4 seems to be the key heparan sulfate proteoglycan in focal adhesion formation, and indeed, its presence in focal adhesions is dependent on activity ( l 3). The connection between this syndecan and PKC is highly unusual, and may not require the involvement of heterotrirneric G-proteins, or of phospholipase-mediated production of the well-known activator, diacylglycerol. The pathway is summarized in Figure 3. Syndecan-4 core protein cytoplasmic domain and PKCa can directly interact, shown by double immuno~uorescencemicroscopy, coirn~unopl.ecipitation,and in vitro assays (27). Moreover, when these molecules interact, the kinase is activated three- to fourfold over control levels whether or not the normal ~hospholipidmediators are included in the assays, In spread fibroblasts, the interaction between syndecan-4 and PKCa requires prior activation of the kinase by, for example, phorbol ester. However, in spreading cells where PKCa is already active (28), the kinase colocalizes with clustering syndecan-4. Thus syndecan-4 may bind PKC and localize it to the site(s) of forrning focal adhesions. Syndecan-4 may not only regulate subcellular locations of PKC but also enhance its activity. The ability of syndecan-4 to do this is apparently dependent on core protein oligomerization (29). This is probably induced

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Scheme of syndecan-4 cytoplasmic domain interactions at focal adhesions. This is not to scale, but represents possible modes of interactions of syndecan-4, syntenin, PKC and PIP2. (Reprinted from Trends in Cell Biology, Vol. 8, Woods A, and Couchman JR, Syndecans: synergistic activators of cell adhesion,pp. 189-192. Copyright 1998, with permission from Elsevier Science.)

by extracellul~ligands, but as discussed below, inositol phospholipids also have a potential role. All syndecans have a tendency to oligomeri~e,which although frustrating in biochemical analysis, might be highly significant in syndecan biology. Carey and coworkers have shown, in a detailed analysis of syndecan-3, that primary sites of homotypic interaction are the transmembrane domain, and the region of the core protein i~mediatelyexternal to it (30). Dimers and higher order oligomers were seen, and it may be that monomeric syndecan core proteins rarely, if ever, are present on the cell surface. Some of these core protein oligomers are sodium dodecyl sulfate-resistant (27,30), a property unrelated to disul~debondowever, dimers are not sufficient for activation of PKC by syndecan-4; order oligomers are required (29). The central V region of this core protein c~toplasmicdomain also has the ability to oligomerize (29), even as synthetic peptides, and the latest data suggest that this can be promoted and stabilized by phosp~atidylinositol4,5bisphosphate (PI4,5P2) (29,3 l ). The key sequence in the V region of syndecan-4 core protein is KKPIUK A; this not only interacts with Since the phospholi~idis itself a modermerges of a ternary signaling complex,

The combination of PI4,5P2 and syndecan-4 cytoplasmic domain more strongly activates PKCa than either agent alone (32). The importance of PI4,5P2 in focal adhesion formation is known (33), and its synthesis is stimulated by integrin ligation (34). The KKPIYKK sequence is also a perfect consensus for binding inositol hexaphosphate (IP6). While preliminary cross-linking experiments do indeed show an interaction with both PI4,5P2 and IP6, the latter will neither promote syndecan-4 core protein oligomerization nor lead to PKC activation (29). Therefore, syndecan-4 control of PKCa localization and activation may itself be regulated by phospholipid metabolism at the cell membrane in response to other ligands such as integrins.

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Nuclear magnetic resonance studies of the V region sequence ~ ~ K K P I Y K K A of syndecan-4 and of its interaction with PI4,5P2 have provided some insight into syndecan cytoplasmic domain dimerization (31). The polypeptides form a compact symmetrical dimer, with interactions involving both the protein backbone at 4V termini, and side chain atoms in their more central regions. The net result i s a twisted clamp structure, schematized in Figure 3. While hydrogen bonding between oxygen of phosphate at the 4 position on the inositol ring and the eNH3 of lys'**(the third of four lysine residues of 4V; residue numbering based on the chicken sequence; Ref. 35)was observed, other phosphates do not seem to be involved. Rather, there are interactions between side chains of the pro ile and lys l ** residues and the anionic head group and fatty acyl groups of the inositol phospholipid. No interactions between backbone atoms of 4V and PIP2 were detectable. Perhaps unexpectedly, the sidechain of the tyr187appears to play no part in either dimeri%ationor interactions with phospholipid (3 1). This is one of four tyrosine residues found in every syndecan cytoplasmic domain in homologous positions (1-3). It, therefore, remains possible that the hydroxyl group of this amino acid is available for interactions with PKC, or for phosphorylation. Consistent with these data, it has been found that substitution of t y P 7 with phenylalanine has the least disruptive effect on 4V oligomerization, but markedly reduces the ability of the 4V peptide to activate PKC (27,29). Tyrosine phosphorylation of syndecan cytoplasmic domains has been reported only once, in syndecan-l, but its role and the identi~cationof which one or more tyrosine residues are involved remain unknown (36). While much is now understood of the role of syndecan-4 cytoplasmic domain, many questions remain. Both PKCa and syndecan-4 become localized to focal adhesions, but precisely why is not known. Presumably, interactions with other focal adhesion plaque components are involved. The requirement for P +

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activity in cell adhesion is also well known, but its substrates and the consequences of their phosphorylation await discovery. It is, however, clear why syndecan-2, the closest homologue to syndecan-4, does not localize to focal adhesions. Its V region is quite dissimilar, and has no PKC activating ability, In fact, er'^^ and ser19*of syndecan-2V are PKC substrates (37), and some relevance for this in tumor cell adhesion has been reported (38).

Work with syndecan-4 points to potentially important interactions in signaling, yet does not indicate how interactions with the actin-associated cytoskeleton may be constructed. In a quite different paradigm, indirect evidence for syndecan-1 Cytoplasmic domain interaction with the cytoskeleton was also recently described. ~nternali~ation and degradation of lipoproteins enriched with the heparinbinding enzyme lipoprotein lipase was enhanced in C H 0 cells transfected with syndecan-l (39). The process was actin cytoskeleton- and tyrosine kinase-dependent. A chimeric construct of Fc receptor ectodomain with transmembrane and cytoplasmic syndecan- l domains was internalized with identical characteristics when challenged with IgG. This indicates that the ~ytoplasmicdomain of syndecan- 1 has key properties for ligand-driven internalization and targeting. ost intriguing was the suggestion that syndecan-~ediatedprocesses involved caveolae rather than coated pits. A recent report demonstrated that the highly conserved FYA region at the C-terminus of syndecan-2 can interact with a novel adaptor protein, syntenin (40). Yeast two-hybrid analysis yielded this protein, which contains two PDZdomains, Once again, syndecan core protein dime~zationis required for interaction, and the intriguing possibility emerges that this adapter may link syndecan core proteins to the cytoskeleton, though as yet, further interactions of syntenin are unknown, Proteins containing PDZ motifs are commonly cell membraneassociated, and are involved in protein-protein linkages, interacting with Cterminal regions of their targets (41). PDZ motifs are found in the tight junction adhesion molecule 20-1, and some guanine exchange factors (GEFs) and GTPase-activating proteins (GAPS), for example (41). This suggests PDZ domains have functions in the regulation of cytoskeleton. Indeed, overexpression of syntenin in C H 0 cells increases spreading, perhaps reflecting such an influence (40). Another recent finding is that the cytoplasmic protein PICK1 contains PDZ motifs which allow it to interact with PKCa (42). It is speculated that this interaction may localize the kinase to the membrane. The parallels with syndecan-4 are striking, not least because both PICK1 and the proteoglycan interact with the catalytic domain of PKC, not its regulatory domain. Further, PICK1 interacts

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with a QSAV sequence unique to PKCa, and it is this isoform of PKC which appears to be involved in focal adhesion formation. However the downstream effectors of syntenin are regulated, it would seem at first sight that it should interact with all syndecans, since the C-terminal EFYA sequence is universal (Fig. I) and includes the invertebrate family members, Interactions between C l domains and cytoplasmic components are now beginning to be reported. When N 18 rat neuroblastoma cells are transfected with syndecan-3 cDNA, neurite outgrowth is enhanced by its ligand, HB-GAM.In addition, the tyrosine kinase c-src, and one of its major substrates, cortactin bound to the C l domain of the transfected syndecan-3 cytoplasmic domain (43). Cortactin is a F-actin-binding protein, associated with motile structures such as the leading edge of fibroblasts, and cytoskeletal regulation rnay be controlled by src kinases (44). This provides the first tantalizing evidence that cytoskeletal proteins can associate with syndecans, which has long been predicted from earlier studies (1,10,45,46).

Syndecans are now taking their place among transmembrane adhesion receptors, albeit they seem to modulate other adhesion mechanisms rather than operate independently. Instances of regulation of integrin- and cadherin-mediated adhesion by syndecans are now recorded. However, while functioning synergistically with other receptors, distinct signaling pathways involving the cytoplasmic domains of the syndecan core proteins are now emerging. Considering syndecan cytoplasmic domain structures, it is probable that the central V region of each syndecan cytoplasmic domain functions to affect one or more distinct signaling pathway(s), in keeping with their unique sequences. The constant (Cl and C2) ank king sequences probably have interactions common to all syndecans, the C2 interaction with syntenin being one such case. It is remarkable that such small cytoplasmic domains are capable of interacting with downstream effectors. Just as with the integrins, multiple direct and indirect interactions may emerge, representing exciting challenges for the future. Syndecans have been implicated in growth factor binding, and rnay have important roles in activating, clustering, and presenting them to high-affinity receptors (see Chap. 3; Refs. l , 3, 4’7). Once again, the prospect is for syndecans to function as “coreceptors,” but as with integrin-based adhesion, there are potential opportunities for independent signaling through syndecan core proteins. Very recent data show that basic fibroblast growth factor, known to bind the heparan sulfate chains of many proteoglycans, rnay also down-regulate phosphorylation of serI7*within the C1 domain of syndecan-4 (chicken sequence number-

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ing). This is the only cytoplasmic serine residue in the syndecan-4 core protein, and may be phospho~latedby novel forms of PKC (nPKC), rather than the conventional forms (cPKC) associating with its V region (48). The functional significance of this phosphorylation/dephospho~lationis not yet clear, but it may affect homooligomerization of the core protein. This is a highly complex field of con ti nu in^ investigation, of s i ~ n i ~ c a n cfor e development, tissue repair, and tumor biology. Last, several puzzles remain c o n c ~ r n i nthe ~ role of syndecan ectodomains. In the few cases where heparan sulfate fine structure from more than one syndecan have been compared within the same cell line, no striking differences between populations have been noted. Moreover, many syndecans bind the same ligands through their glycosa~inoglycanchains. Despite this, however, there must be specificity yet to be discrimi~ated.For example, syndecan-2 is punctate or diffuse over the cell surface of fibroblasts, while in the same cells syndecan-4 is localized to focal adhesions (12), despite glycosa~inoglycaninteractio~swith the same matrix substrate. Additionally, it might be expected that overexpression of syndecan-2 may lead to competition with syndecan-4 and a decrease in focal adhesion formation. This does not, in fact, happen, arguing that there may be subtle differences in fine structure between heparan sulfate chains from different syndecans (49). Alte~ately,since the core proteins are of differing sizes, the relationship of the glycosaminoglycans to the cell surface has thus far been underappreciated as a key factor in their biological activity. These issues await further work, not least the advent of heparan sulfate sequencing techniques that should be available soon.

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Some of the work described here was supported by ~ I grants H JRC and DK 54605 to AW, and a development and feasibility S The Multipu~oseA ~ h r i t i sand ~ u s c u l o s ~ e l e tDisease al Research Center grant (AR 20614) to AW.

1. Rernfield M, Kokenyesi R, Kato M, Hinkes MT, Spring J, Callo RL, Lose EJ. Biol-

ogy of the syndecans. Amu Rev Cell Rio1 1992; 8:365-393. 2. Couchman JR, Woods A. Structure and biology of pericellular proteoglycans. In: Roberts DD, Mecharn RP, eds. Cell Surface and ExtracellularClycoconjugates.New York: Academic Press, 1993:33-82.

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3. Carey DJ. Syndecans: multifunctional cell-surface co-receptors. Biochem J 199’7; 327:1-16. 4. Rosenblum ND, Botelho BB, Bernfield M. Expression of a Xen opu s counterpart of mammalian syndecan 2 during embryogenesis. Biochern J 1995; 309:69-76. ~ i Z ~ conserva5. Spring J, Paine-Saunders S, Hynes R, Bernfield, M.~ ~ o s o psyndecan: tion of a cell-surface heparan sulfate proteoglycan. Proc Natl Acad Sci USA 1994; 9113334-3338. 6. Kato M, Saunders S, Nguyen H, Bernfield M. Loss of cell surface syndecan-1 causes epithelia to trans for^ into anchorage-independent mesenchyme-like cells. Mol Biol Cell 1995; 6559-576. 7. Leppa S, Vleminckx K, Van Roy F, Jalkanen M. Syndecan-1 expression in mammar y epithelial tumor cells is E-cadherin-dependent. J Cell Sci 1996; 109:13931403. 8. Inki P, Joensuu H, Grknman R, Klemi P, Jalkanen M. Association between syndecan-1 expression and clinical outcome in squamous cell carcinoma of the head and neck. Br J Cancer 1994; 70:319-323. 9. Liebersbach BF, SandersonRD. Expression of syndecan-l inhibits cell invasion into type I collagen. J Biol Chem 1994; 269:20013-20019. 10. Carey DJ, Stahl RC, Ciz~eci-SmithG, Asundi, VK. Syndecan-1 expressed in Schwann cells causes morphological transforrnation and cytoskeletal reorganization and associates with actin during cell spreading. J Cell Biol 1994; 124:161- 170. 11. ~amagataM, Saga S, Kat0 M, Bernfield M, Kimata K. Selective dist~butionsof proteoglycans and their ligands in pericellular matrix of cultured fibroblasts. J. Cell Sci 1993; 106:55-65. 12. Woods A, Couchman JR. Syndecan 4 heparan sulfate proteoglycan is a selectively enriched and widespread focal adhesion component. Mol Biol Cell 1994; 5:183192. 13. Baciu PC, Goetinck PF. Protein kinase C regulates the rec~itmentof syndecan-4 into focal contacts. Mol Biol Cell 1995; 6:1503-1513. 14. Kojima T, Shworak NW, Rosenberg RD. Molecular cloning and expression of two distinct cDNA-encoding core proteins from a rat endothelial cell line. J Biol Chem 1992; 267:4870-4877. 15. David G, van der SchuerenB, Marynen P, Cassiman JJ, Van den Berghe H. Molecular cloning of amphiglycan,a novel integral membrane heparan sulfate proteoglycan expressed by epithelial and fibroblastic cells. J Cell Biol 1992; 118:961-969. 16. Woods A, Couchman JR. Focal adhesions and cell-matrixinteractions.Collagen Re1 Res 1988; 8:155--182. 17. Burridge K, Chrzanowska-~odnickaM, Focal adhesions, contractility, and signaling. Annu Rev Cell Dev Biol 1996; 12:463--519. 18. Singer 11, Kawka DW, Kazazis DM, Clark RAF. In vivo co-distribution of fibronectin and actin fibers in granulation tissue: immuno~uorescenceand electron microscope studies of the fibronexus at the myofibroblast surface. J Cell Biol 1984; 98: 2091-2106. 19. Hynes RO. Fibronectins. New York: Springer-Verlag, 1992. 20. Yamada KM, Miyamoto S. Integrin tra~smembranesignaling and cytoskeletal control. Curr Opin Cell Biol 1995; 7:681-689.

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21. Juliafno RL, Haskill S. Signal transduction from the extracellular matrix. J Cell Biol 1993; 120:577-585. 22. Woods A, Couchman JR, Johansson S, Hook M. Adhesion and cytoskeletal organisation of fibroblasts in response to fibronectin fragments. EMBO J 1986; 5:665670. 23. Woods A, McCarthy JB, Furcht LT, Couchman J.R. A synthetic peptide from the COOH-terminal hepa~n-bindingdomain of fibronectinpromotes focal adhesion formation. Mol Biol Cell 1993; 4:605-613. 24. Woods A, Couchman JR. Protein kinase C involvement in focal adhesion formation. J Cell Sci 1992; 101:277-290. 25. Defilippi P, Venturino M, Gulino D, Duperray A, Boquet P, Fiorentini C, Volpe G, Palmieri M, Silengo L, Tarone G. Dissection of pathways implicated in integrinmediated actin cytoskeleton assembly. J Biol Chem 1997; 272:21726-21734. 26. Nobes CD, Hall A. Rho, Rac,.andCdc42 GTPases regulate the assembly ofmultimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia. Cell 1995; 8153-62. 27. Oh E-S, Woods A, Couchman JR. Syndecan-4 proteoglycan regulates the distribution and activity of protein kinase C. J Biol Chem 1997; 272:8133-8136. 28. Vuori K, Ruoslahti E. Activation of protein kinase C precedes a5p1 integrin-mediated spreading on fibronectin. J Biol Chem 1993; 268:21459-21462. 29. Oh E-S, Woods A, Couchman JR. Multimerization of the cytoplasmic domain of syndecan-4is required for its ability to activate protein kinase C. J Biol Chem 1997; 272:11805--11811. 30. Asundi VK, Carey DJ. Self-associationof N-syndecan (syndecan-3) core protein is mediated by a novel structural motif in the transmembrane domain and ectodomain flanking region. J Biol Chem 1995; 270:26404-26410. 31. Lee D, Oh E-S, Woods A, Couchman JR, Lee W. Solution structure of a syndecan4 cytoplasmicdomain and its interaction with phosphatidylinositol 4,5-bisphosphate. J Biol Chem 1998; 273:13022- 13029. 32. Oh E-S, Woods A, Lim S-T, Thiebert AW, Couchman JR. Syndecan-4proteoglycan cytoplasmic domain and phosphatidylinositol4,5-bisphosphate coordinately regulate protein kinase C activity. J Biol Chem 1998; 273:10624- 10629. 33. Gilrnore AP, Burridge K. Regulation of vinculin binding to talin and actin by phosphatidylinositol-4,5-bisphosphate. Nature 1996; 381 :531-535. 34. McNamee HP, Ingber DE, SchwartzMA. Adhesion to fibronectin stimulatesinositol lipid synthesis and enhances PDGF-induced inositol lipid breakdown. J Cell Biol 1993; 121~673-678. 35. Baciu PC, Acaster C, Goetinck PF. Molecular cloning and genomic organization of chicken syndecan-4. J Biol Chem 1994; 269:696-703. 36. Reiland J, Ott VL, Lebakken CS, Yeaman C, McCarthy J, Rapraeger AC. Pervanadate inactivationof intracellular kinases leads to tyrosine phosphorylation and shedding of syndecan-1. Biochem J 1996; 319:39-47. 37. Oh E-S, Couchman JR, Woods A. Serine phosphorylation of syndecan-2proteoglycan cytoplasmic domain. Arch Biochem Biophys 1997; 344:67-74. 38. Itano N, Oguri K, Nagayasu Y, Kusano Y, Nakanishi H, David G, Okayama M. Phosphorylation of a membrane-intercalated proteoglycan, syndecan-2, expressed

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39.

40. 41. 42. 43

I)

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44.

45.

46.

47.

in a stroma-inducing clone from a mouse Lewis lung carcinoma. Biochem J 1996; 315~925-930. Fuki IV, Kuhn K34, Lomazov IR, Rothman VL, Tuszynski GP, Iozzo RV, Swenson TL, Fisher EA, Williams KJ. The syndecan family of proteoglycans. Novel receptors mediating internalization of atherogenic lipoproteins in vitro. J Clin Invest 1997; 100:1611-1622. Grootjans JJ, Z i ~ e r m a n nP, Reekrnans G, Smets A, Degeest G, Durr J, David G. Syntenin, a PDZ protein that binds syndecan cytoplasmic domains. Proc Natl Acad Sci USA 1997; 94:13683-13688. Ponting CP, Phillips C, Davies KE, Blake DJ. PDZ domains: targeting signalling molecules to sub-membranous sites. BioEssays 1997; 19:469-479. StaudingerJ, Lu J, Olson EN. Specific interaction of the PDZ domain protein PICK1 with the COOH terminus of protein kinase C-a. J Biol Chem 1997; 272:3201932024. Kinnunen T, Kaksonen M, Saarinen J, Kalkinnen N, Peng HB, Rauvala H. Cortactinl Src-kinase signalling pathway is involved in ~-syndec~-dependent neurite outgrowth. J Biol Chem 1998; 273:10702-10708. Thomas SM,Soriano P, Imarnoto A. Specific and redundant roles of Src and Fyn in organizing the cytoskeleton. Nature 1995; 376:267-27 1. Carey DJ, Stahl RC, Tucker B, Bendt KA, Cizrneci-Smith G. ~ggregation-induced association of syndecan-1 with microfilamentsmediated by the cytoplasmic domain. Exp Cell Res 1994; 214:12-21. Carey DJ, Bendt KM, Stahl RC. The cytoplasmic domain of syndecan-l is required for cytoskeleton association but not detergent insolubility. J Biol Chem 1996; 271: 15253-15260. David G. Integral membrane heparan sulfate proteoglycans.FASEB J 1993;7: 10231030.

48. Horowitz A, Simons NI. Regulation of syndecan-4 phosphorylation in vivo. J Biol Chem 1998; 273:10914-10918. 49. Longley RL, Woods A, Fleetwood A, Cowling GJ, Gallagher JT, Couchman JR. Control of morphology, cytoskeletonand migration by syndecan-4.J Cell Sci 1999; 112:3421-3431.

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Unjversity of ~ o r o n and t ~ ~ ~ n n y ~ and r o~ o ~o ~ eCo/ n /’ ege ~~ e a ~ t ~ ~ ci en cesenter, Toronto, ~n t ar i o Canada ,

The name ~ Z y ~ identifies i c ~ ~ a family of heparan sulfate proteoglycans (HSPGs) that are linked to the exocytoplasmic surface of the plasma membrane through a covalent glycosyl-phosphatidylinositol (GPI) anchor (l). Proteins anchored in this manner are said to be “glypiated” (2). The possible existence of a distinct population of glypiated cell surface HSPGs (as opposed to the transmembrane HSPGs or those peripherally associated) was recognized about a decade ago (3,4). These initial experiments showed that a fraction of the cell surface HSPGs could be released by treating the cells with phosphatidylinositol-specific phospholi~ase C (PI-PLC). Although alternative e~planationscould be given for the presenc~ of such phospholipase C-labile proteoglycans (e. g. , a glypiated isoform of a transmembrane proteo~lycan),the subsequent cloning and characterization of a protein with its own discrete open reading frame left no doubts as to the distinctiveness of this population of glypiated HSPGs (5). Five m a ~ m a l i a nglypicans and a single ~ r u s u ~gene ~ i (Z~~ZZy ~ have ) been identified to date (Fig. 1) (5-1 l ), Outside of the N-terminal secretory signal peptide and the putative hydro~hobiccarboxyl-te~inalsignal sequence needed for glypiation, which all members carry by definition, other striking features of this family of HSPGs include the presence of two to five consensus sites for the insertion of glycosami~oglycan(GAG) chains near the C-termini, and a significant conservation of their primary polypeptide sequences (Fig. 1). among these conserved amino acids is the presence of 14 cysteine residues which are believed to form intramole~ulardisul~debonds, giving all glypicans a conserved three-dimensional structure.

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N

linker domain

C

( 19% )

(36%)

(23%)

(6 %) (23%)

V

N

C

N”

C

N

C

N

C

Glypican domains (percent similarity)

zy Glypican-1 (GPCl, Glypican)

quail, mouse, rat, human

Glypican- 2 (GPC2, Cerebroglyca rat

Giypican- 3

(GPC3,OCR, MXR7)

rat, human

Glypican- 4 (GPC4,K- glypican) mouse

Giypican- 5

C

N-

(GPCS) human

N Structure of glypicans: Schematic illustration of glypican family members and their domains. Vertical bars indicate the predicted positions of the glycosaminoglycan (GAG) substitutions. Open triangles denote potential N-glycosylation sites, while filled triangles identify the putative cleavage sites described in the literature. Numbers within parentheses indicate the percent similarity in the primary polypeptide sequence of a given domain across all mammalian glypican family members, as predicted by the CLUSTAL multiple alignment software. LP, leader peptide; UR, unconserved region; GAG, GAG domain; GPI, signal peptide for glypiation.

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Although functional studies on glypicans are at an early stage, it is reasonable to speculate that their ability to carry GAG chains, and the conserved threedimensional structure of the protein cores, may allow them to play multiple functions in a cell-type specific manner. In this chapter we summarize our current knowledge of the structure, expression, and function of this still poorly understood proteoglycan family.

Y

One of the unifying structural features of the glypican family is the presence of a GP1 anchor. The process by which proteins are covalently modified by a phospholipid anchor, otherwise termed “glypiation,” occurs post translation all^ within the endoplasmic reticulum (2) (Fig. 2 4 . Initially, the protein is translo-

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X,

=

0-C-

I1

-

& = @ CH2- CH,-

~@W“H2- CH2- CH,- @

I

a1+2

Man-Man

UI+b\

[C161

NW,

Man -GlcNH,--0

GPI- PLC

GPI- PLD

Biochemical structure and attachment of the glycosylphosphatidylinosito~ (GPI) anchor. (a) Posttranslational glypiation of proteins in the endoplasmic reticulum (ER). Proteins to be glypiated are initially anchored to the ER membrane through a short hydrophobic segment of their GP1 signal peptide. A hypothetical transamidase with endoproteolytic activity then cleaves the protein just upstream from the membrane anchoring domain and covalently attaches the newly generated carboxyl terminus to an “acceptor” amino group present in the preassembled GP1 anchor. (b) Structure of mammalian GP1 anchors. GP1 anchors generally consist of a phosphatidylinositolphosphoglyceride whose inositol head group has been modified by a simple glycan moiety consisting of an Nacetylglucosamine and three mannose residues. A phosphoethanolamine group present on the terminal mannose residue serves as the acceptor site for protein attachment.Branching phosphoethanola~inesubstitutions may also be found on the first and second mannose residues. Other variations to the general GP1 anchor structure include the acylation of the myo-inositol ring. Cleavage sites for the bacterial phosphotidylinositol-specificphospholipase C (GPI-PLC) and eukaryotic phospholipase D (GPI-PLD) are indicated. Man, mannose; GlcNH,, N-acetylglucosamine.

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cated across the endoplasmic reticulum membrane, much like its transmembrane counterparts, and is anchored by a short hydrophobic sequence of 10-12 amino acids found at the C-terminus. This hydrophobic sequence, however, is inadequate to serve as a permanent protein anchor. It is instead cleaved by a putative transamidase on the luminal side of the endoplasmic reticulum, 10-12 amino acid residues upstream from the hydrophobic sequence (12). The newly generated carboxyl terminus is then immediately covalently linked to a preassembled GP1 anchor through an amide bond (12). The functional significance of attaching proteins through a lipid anchor has not been clearly established, but several possible roles have been suggested. One of them is to target proteins to specific microdomains within the cell membrane called “rafts” (13,14). These rafts are highly enriched with sphingolipids, cholesterol, Src family kinases, G proteins, and molecules involved in Ca2+influx (13). The t ~ g e t i n gof glypicans to these membrane domains may therefore facilitate interactions with specific signaling molecules (15). Moreover, since rafts are located in the- apical surface of polarized epithelial cells, the addition of a GP1 anchor to proteins can also serve as a mechanism for apical targeting (16). Another possible function of GP1 anchors is to provide a system of regulated release of proteins to the extracellular environment that is not based on protease activity. ~ a m m a l i a cells n produce a GPI-specific phospholipase D capable of releasing GPI-linked proteins, including glypicans, from the cell surface (17) (Figure 2%). Lastly, it has also been proposed that GPI-anchorage renders proteins susceptible to unique endocytic pathways that allow them to be recycled to the cell surface (18).

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Although there has been some speculation that a subpopulation of glypican core proteins may exist that lacks any GAG chains (19), it is unclear whether such nonglycanated forms are present on the cell surface. Thus, glypicans can be considered, at this time, as “full-time” cell surface proteoglycans. To date, heparan sulfate (HS) has been the only type of GAG chain found in endogenously expressed glypicans (5,7,8,10,20). Recent in vitro biochemical characterization of GPC5 in transfected cells indicates, however, that the insertion of chondroitin sulfate (CS) chains is also possible (1 l). It remains to be seen whether CS is also attached to glypican core proteins in vivo, and whether other glypican family members, when expressed in the appropriate cellular context, can also carry CS chains. Unlike syndecans, where the insertion sites for GAG chains can be found along the whole core protein @l), the glycanation sites in glypicans appear to be restricted to the last 50 amino acids of the mature glypican core protein, just upstream from the point of membrane insertion (Fig. l). Typically, these 50

icans

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amino acid domains contain clusters of two to five potential Ser-Gly glycanation sequences surrounded by one or more acidic residues. It has previously been shown*that such motifs preferentially support the attachment of HS chains (22). Although monomeric Ser-Gly motifs are also present in the N-terminal region of certain glypicans, no GAG chain insertions at these sites have been reported (23).

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Based on the deduced amino acid sequence of glypican family members, the predicted size of the mature glypican core protein (e.g., excluding the N-terminal signal sequence and C-terminal glypiation sequence) is approximately 500 amino acids in length. Although discrepancies exist between the predicted and observed molecular weights, probably due to varying degrees of N- and/or O-glycosylations (Fig. l), all glypican core proteins fall between the 55-70 kDa range following heparitinase digestion (5,7,8,10,11,20). The degree of peptide sequence identity between the different glypicans is moderate (from 20 to 45% Fig. 3a), but the conserved positions of the 14 cysteine residues raise the possibility that the three-dimensional structure of all glypicans is similar. Outside of the GAG domain, the mature glypican core proteins can be further subdivided into two structural domains: the linker region, and an N-terminal globular domain (Fig. 1). This is based largely on the recent identificatio~ of an internal proteolytic cleavage site in three glypican family members (8,20,24), A similar proteolytic event appears to be present in a fourth member (10). Interestingly, the 30-40 kDa cleavage product that is subsequently generated from the N - t e ~ i n u sof the glypican core protein remains attached to its Gterminal half through one or more disulfide bridges (8,20). If we take into account potential N- or 0-glycosylations, it would appear that the reported cleavage sites map to the same region within the primary polypeptide sequence, at the proposed globular domain/~inkerjunction (Fig. l).However, no striking sequence similarities exist in this region of the core proteins that would implicate any one particular protease. The globular domains of glypicans contain 1l of the 14 conserved cysteine residues, and exhibit a sequence similarity of approximately 36%. The linker regions, on the other hand, while carrying the remaining three conserved cysteine residues, show a lower level of sequence similarity (23%). Notably, they contain a contiguous region of 30 amino acids just downs~eamfrom the proteolytic cleavage site which lacks any significant sequence similarity among the various glypican family members (Figure 3b). This may suggest the possible existence of a ligand “specificity” pocket in this domain,

PC1

PC2

21 (41)

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23 (42)

Sequence comparison across the glypican family. ( a) Sequence comparison between mammalian glypican core proteins as predicted by the ALIGN global alignment software.Frank numbers denote percent identities between peptide sequences, while those enclosed in parentheses indicate the percent similarities. Rodent and human homologues exhibit identities and/or similarities of greater than 90%, and thus the numbers can be readily applied to known sequences from either species. (b) Amino acid sequence alignment of the non-conserved region of the linker domain as predicted by the CLUSTAL multiple alignment software. Prefixes: r, rat; m, mouse; h, human.

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Studies on the gene structure of rat G PC l, human G PC3, and human G PC4 have been recently reported (25-27). G PC3 is a very large gene spanning more than 500 kb and containing 8 exons (26). Rat G PCl also contains 8 exons, but is only approximately 15 kb long (25). G PC4, on the other hand, contains 9 exons, Interestingly, this gene is located on chromosome Xq26, in close proximity to CPC3 (27). The 5' flanking regulatory regions of rat G PC l, and human, rat, and mouse GPC3 have been sequenced (25,26,28). They all lack a TATA box and contain several SP l binding sites. A fragment containing 1.4 kb of the S' regulatory region

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of the rat GPC3 promoter has been tested in transient expression assays (28). This fragment was able to drive the expression of a reporter gene in intestinal epithelial cells and in fibroblasts. Since GPC3 is not expressed normally in fibroblasts, this result suggests that DNA regulatory sequences that regulate cell typespecific expression of GPC3 are located outside of the 1.4 kb 5’ fragment tested. A 2.9 kb fragment of the rat GPCI 5’ region has also been tested in transient expression assays. This fragment was able to drive expression of a reporter gene in Schwann cells and fibroblasts, which normally express GPCl endogenously (25).

Overall, the studies published to date indicate that GPC 1 and CPC4 are expressed in a large number of embryonic and adult tissues (27,29-31). GPC2 and GPC5, on the other hand, display a more restricted pattern of expression. GPC2 is only present in the developing nervous system (7), while GPC5 expression is restricted primarily to the nervous system, limb, and kidney (1 1). GPC3 is widely expressed during development but its expression in the adult is down-regulated in most tissues (32; Z. M.Wong, and J. Filmus, unpublished observations). Of note, although GPCl is found in cell lines derived from connective tissue, in vivo expression in such tissue has not been detected, suggesting that its expression can be deregulated in vitro (29). With regard to the expression of glypicans in pathological conditions, it has been recently reported that GPCl is overexpressed in a large proportion of human pancreatic cancers (33), and it has been suggested that GPCl plays a role in the responses of pancreatic tumor cells to certain mitogenic stimuli (see below). Another interesting observation with regard to changes in the expression of glypicans during malignant t r a ~ s f o r ~ a t i ohas n been reported in hepatocarcinomas. GPC3 expression is detected in 75% of these tumors but not in normal liver (34). Since GPC3 is highly expressed in developing liver, it seems that this glypican behaves as an oncofetal marker in this tissue, and it has been proposed that it could serve as a sensitive early tumor marker for hepatocellula~carcinomas (34).

A mounting body of evidence accumulated during the last few years, indicates that cell sudace HSPGs regulate the cellular response to physiological concentra-

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tions of the so-called hep~in-bindinggrowth factors (35,36), To explain these results it has been proposed that certain membrane-bound HSPGs can act as coreceptors for these factors, facilitating the fornation of ligand dimers, and the consequent oligomerization of their high-affinity signaling receptors (37). Heparin can also interact directly with the fibroblast growth factor (FGF) receptor, and, conse~uently,the existence of a tripartite complex between FGF, its high-affinity receptor, and HSPGs has been proposed (38). Most of the studies on the role of HSPGs in growth factor response have not identified the HSPGs involved in these interactions. There is some indication, however, that, at least in some cell types, glypicans do indeed bind to FGF-2 and other hep~in-bindinggrowth factors (17,39), and that they can stimulate signaling of these growth factors in cultured cells (33). For example, primary endothelial cells treated with phosphatidylinositol-specific phos~holipaseC (PIPLC) release FGF-2 conjugated with an HSPG (40), and the down-regulation of 1expression significantly inhibits the response of human pancreatic cancer cell lines to FGF-2 and heparin-binding EGF (33). Interestingly, the effect of glypicans on growth factor activity may not always be stimulatory, since it has been shown that GPCl can inhibit the activation of keratinocyte growth factor GF) receptor by KGF in rat myoblasts (41). In addition to binding to heparin-binding growth factors, glypicans can also interact with molecules involved in cell adhesion and migration. For example, GPCl is able to interact with laminin and influence process extension in ~ c h w a n n cells (42). Although these cells do not seem to constitutively express GPCl in vivo (29), it is possible that they synthesize GPCl following injury. Interactions of GPCl with the amyloid precursor protein (APP), a molecule that stimulates neurite outgrowth, have also been reported (43). Although the in vivo function of this interaction is not known, experiments with cultured cells indicate that GPCl is capable of inhibiting APP-induced neurite outgrowth. All reported direct interactions of glypicans to date have been associated with the GAG chains. It has been speculated, however, that the core proteins could have GAG-independent functions (7). This is based on the observation that the protein core sequences of glypicans are highly conserved through evolution (27,44-46), and that the insertion sites for the GAG chains are only found near the C-terminus. In fact, we have recently generated evidence indicating that GPC3 can elicit certain cellular effects in the absence of its GAG chains (19). In this context, it is important to note that the GAG chains may have functions other than serving as the primary ligand binding site. It has recently been reported that the apical sorting of GPCl is inversely related to its heparan sulfate content, suggesti~gthat the level of glycanation may play a role in the apical~basolateral sorting of glypicans (23). The first studies providing information on the function of glypicans in vivo have been performed in ~ r u s o ~ where ~ i l ~the, generation of ~ ~mutants 1 1has~

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demonstrated that this glypican is required for the control of cell division in the developing visual system, and morphogenesis of the eye, wing, antenna, and genitalia (9). In addition, it has been shown that ~ ~controls 2 1 cellular ~ responses to Decapentaplegic, a TGF-P-related morphogen, although the biochemical basis for this is still unclear (4’7). An important cont~butionto the functional studies of glypicans has been provided by the discovery that GPC3 i s mutated in patients with the SimpsonGolabi-~ehmelsyndrome (SGBS) (45). This is an X-linked disorder that is characterized by pre- and postnatal overgrowth, and a broad spectrum of clinical manifestations including a distinct facial appearance (Fig. 4), ~acroglossia,cleft palate, syndactyly, ~olydactyly,supernumerary nipples, cystic and dysplastic kidneys, congenital heart defects, rib and vertebral abnormalities, um~ilical/inguinal hernias, muscular hypotonia, and increased risk for the development of Wilms’ tumors, As many as 50% of affected males die neonatally, although the causes

Coarse facial features of an SGBS patient.

17

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of this high mortality remain unknown (48-51). Most of the GPC3 mutations in SGBS patients are microdeletions encompassing only different exons of GPC3 . Given the lack of correlation between patient phenotypes and the location of GPC3 deletions, it has been proposed that SGBS is caused by a nonfunctiona~ GPC3 protein, with additional genetic factors responsible for the intra- and interfamilial phenotypic variation (49). Some of the anatomic abnormalities of SGBS patients suggest that GPC3 is involved in the regulation of growth and cell survival during the development and morphogenesis of certain tissues (49). Corroborating evidence derives from experiments from our laboratory indicating that GPC3 can induce apoptosis or growth inhibition in a cell line-specific manner. In particular, we have demonstrated that GPC3 can induce apoptosis in cell lines derived from mesothelioma and breast cancer (19), and growth inhibition in a cell line derived from renal collecting ducts (J. Filmus, unpublished observations). The ability of GPC3 to induce apoptosis seems to be related to its capacity to regulate the activity of survival factors. SGBS shares some clinical features with the Beckwith-Wiedemann Syndrome (BWS), another overgrowth s y n d r o ~ ethat is characterized by several dysmorphisms and a high risk of developing pediatric tumors (52). Since overexpression of insulin-like growth factor-:! (IGF2) is thought to be one of the contributing factors to BWS (53),it has been proposed that GPC3 is a negati;e regulator of TGF2, and that the loss-of-function mutations of CPC3 are equivalent to overexpression of IGF2 (45). It is important to note, however, that despite the significant overlap between BWS and SGBS, there are also obvious differences (54). This suggests that even if the proposed effect of GPC3 on IGF2 signaling is confirmed, GPC3 may have other effectors. Furthermore, given the complexity of the SGBS phenotype, it is also conceivable that GPC3 effectors are cell-type specific. This cell-type specificity could be due to the fact that the size and sequence of the GAG chains added to proteoglycans can vary in different cell types, thereby altering the affinity for potential ligands (55). We have recently generated GPC3-de~cient(CPC3-/-) mice by homologous recombination (56). These mice display some of the phenotypic features of SGBS, including developmental overgrowth, perinatal death, respiratory infections, bone abnormalities, and cystic and dysplastic kidneys (Fig. 5). Newborn GPC3-deficient mice are -30% bigger than wild-type littermates, and the heterozygotes show an intermediate level of overgrowth, In the particular case of the kidneys, we have demonstrated that cyst formation is the result of hyperproliferation of the ureteric bud/collecting system cells during development. The lungs of the mutant mice also display abnormalities at time of birth, with the airways containing an admixture of stranding mucus and sloughed epithelial cells. This may have contributed to the perinatal death and increased susceptibility of GPC3-I- mice to respiratory infections.

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A b n o ~ a lrenal development in GPC3-/- embryos. Kidney sections from 4day-old wildtype (top) and GPG3-null (bottom) littermates.

The bone abnormalities observed in the GPC3-I- mice were restricted to the jaw and the palate, with a propo~ionof knockout mice showing incomplete closure of the palate and mandibular hypoplasia. Interestingly, developmental overgrowth of similar ~ a ~ n i t u dtoethat observed in GPC3-I- mice has been reported in IGF2 receptor (IGF2R)-deficient mice (57,58),The IGF2R is a well-characterized negative regulator of IGF2 (59). It binds IGF2 and downregulates its activity by endocytosis and degradation. Thus, the IGF2~-deficientmice display an increase in circulating IGF2. In the case of the GPC3-I- embryos, however, we have not detected any significant alteration in circulating IGF2 (56). Furthermore, we have been unable, as of yet, to detect a direct interaction between GPC3 and ~ e c o ~ b i n a IGF2 nt (60). It can be concluded, therefore, that if GPC3 inhibits IGF2 signaling, it

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does so by a mechanism that is fundamentally different than that used by the IGF2R. Finally, an ongoing controversy with regard to the localization of HSPGs is also having an impact in our understanding of the role of glypicans, This controversy arises from contradicting results with regard to the locali~ationseveral of HSPGs in the nucleus (61,62). In particular, a recent publication has reported the finding of GPCl in the nuclei of neurons from the rat central nervous system (24). The authors of this report speculated that GPCl is involved in the regulation of cell division, but clearly more work is required to confirm the physiological relevance of nuclear GPC 1.

TI

Our current knowledge of glypicans is still very limited, p ~ t i c u l ~ with l y regard to their function, In this respect it is clear that functional studies of these molecules will require a combination of in vivo and in vitro approaches, since, given the complex structure of glypicans, and the different potential functions that have been proposed, it is unlikely that a single approach will provide a full understanding of this problem. Despite our limited knowledge of the function of glypicans, it is becoming increasingly apparent that these molecules play an important modulato~yfunction in the control of cell proliferation. The involvement of GPC3 in an overgrowth syndrome, the phenotype of ~ ~ Z mutants, Zy and the recently reported role of GPC 1 in the control of cellular responses to growth factors are examples of the emerging evidence supporting a role of glypicans in the regulation of cellular proliferation. Furthermore, these examples illustrate the pathological consequences that mutation or disregulation of glypicans can generate. It will not be surprising if other cases of the involvement of glypicans in human pathologies are found in the near future.

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David G. Integral membrane heparan sulfate proteoglycans. FASEB J 1993;7:10231030. Low MC. The glycosyl- p h osp h atid ylin sitolanchor of membrane proteins. Biochim Biophys Acta 1989; 988:427-454. Ishihara M, Fedarko NS, Conrad HE. Involvementof phosphatidyli~ositioland insulin in the coordinate regulation of protoheparan sulfate ~etabolismand hepatocyte growth. J Biol Chem 1987; 262:4708-4716. Carey DJ, Evans DM. Membrane ancho~ngof heparan sulfate proteoglycans by

5. 6.

7. 8. 9.

10. 11.

12. 13. 14. 15. 16. 17.

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20.

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phosphatidylinositoland kinetics of synthesisof peripheral and detergent-so~ubilized proteoglycans in Schwann cells. J Cell Biol 1989; 108:1891-1897. David G, Lories V, Decock B, Marynen P, Cassiman J, Van Den Berghe H. Molecular cloning of a phosphatidylinositol-anchoredmembrane heparan sulfate proteoglycan from human lung fibroblasts. J Cell Biol 1990; 111:3165-3176. Filmus J, Church J, Buick W. Isolation of a cDNA corresponding to a developmentally regulated transcript in rat intestine. Mol Cell Biol 1988; 8:4243-4249. Stipp CS, Litwac ED, Lander AD. Cerebroglycan: an integral ~embraneheparan sulfate proteoglycan that is unique to the developing nervous system and expressed specifically during neuronal differentiation. J Cell Biol 1994; 124:149- 160. Watanabe K, Yamada H, Yamaguchi Y. K-glypican: a novel GPI-linked heparan sulfate proteoglycan that is highly expressed in developing brain and kidney. J Cell Biol 1995; 130:1207-1218. Nakato H, Futch TA, Selleck SB. The division abnormally delayed ( ~ ~ Zgene: ~y ) a putative integral membrane proteoglycan required for cell division patterning during postemb~onicdevelopment of the nervous system in ~ r o s o p ~ iDevelopment Z~ . 1995; 121~3687-3702. Veugelers M, Vemeesch J, Reekmans G, Steinfeld R, Marynen P, David G. Characterization of glypican-5 and chromosomal localizationof human GPC5, a new member of the glypican gene family. Genomics 1997; 40:24-30. Saunders S, Paine-Saunders S, Lander AD. Expression of the cell surface proteoglycan glypican-5 is developmentally regulated in kidney, limb, and brain. Dev Biol 1997; 190:78-93. Udenfriend S, Kodukula K. How glycosylphosphatidylinositol-anchoredmembrane proteins are made. Annu Rev Biochem 1995; 64:563-91:563-591. Simons K, Ikonen E. Functional rafts in cell membranes. Nature 1997; 387:569572. Friedrichson T, Kurzchalia T. Microdomains of GPI-anchored proteins in living cells revealed by crosslinking. Nature 1998; 394:802-805. Casey PJ. Protein lipidation in cell signaling. Science 1995; 268:221-225. Brown DA, Rose JK. Sorting of GPI-anchored proteins to glycolipid-enriched membrane sub~omainsduring transport to the apical cell surface. Cell 1992; 68:533544. Brunner G, Metz CN, Nguyen H, Gabrilove J, Patel SR, Davitz MA, Riflcin DB, Wilson EL. An endogenous glycosy1phosphatidylinositol-speci~cphospholipase D releases basic fibroblast growth factor-heparan sulfate proteoglycan complexes from human bone marrow cultures. Blood 1994; 83:2115-2125. Fransson LA, Edgren G, Havsmark B, Schmidtchen A. Recycling of a glycosylphosphatidylinositol-anchoredheparan sulphate proteoglycan (glypican) in skin fibroblasts. Glycobiol 1995; 5:407-415. Duenas Gonzalez A, Kaya M, Shi W, Song H, Testa JR, Penn LZ, Filmus J. OCI-5I GPC3, a glypican encoded by a gene that is mutated in the Simpson-Golabi-~ehmel overgrowth syndrome, induces apoptosis in a cell line-specific manner. S Cell 1998; 141:1407-1414. Filmus J, Shi W, Wong ZM, Wong MJ. Identification of a new membrane-bound heparan sulfate proteoglycan. Biochem J 1995; 311:561-565.

21. BernfieldM, Kokenyesi R, Kato M, Hinkes MT, Spring J, Gallo RL, Lose EJ. Biology of the syndecans: a family of transmembrane heparan sulfate proteoglycans. Annu Rev Cell Biol 1992; 8:365-393. 22. Zhang L, David G, Esko JD. Repetitive Ser-Gly sequences enhance heparan sulfate assembly in proteoglycans. J Biol Chem 1997; 27027127-27135. 23. Mertens G, Van den Schueren B, Van Den Berghe H, David G. Heparan sulfate expression in polarized epithelial cells: the apical sorting of glypican (GPI-anchored proteoglycan) i s inversely related to its heparan sulfate content. J Cell Biol 1996; 132:487-497. 24. Liang YH, M,, Roughley PJ, Margolis RK, Margolis RU. Glypican and biglycan in the nuclei of neurons and glioma cells: presence of functional nuclear localization signals and dynamic changes in glypican during the cell cycle. J Cell Biol 1997; 139~851-864. 25. Asundi VK, Keister BF, Carey DJ. Organization,5”flanking sequence and promoter activity of the rat GPCl gene. Gene 1998; 206:255-261. 26. Huber R, Crisponi L, Mazzarella R, Chen CN, Su U,Shizuya H, Chen EY, Cao A, Pilia G. Analysis of exonlintron structure and 400 kb of genomic sequence surrounding the 5”promoter and 3”terminal ends of the human glypican 3 (GPC3) gene. Genornics 1997; 45:48--58. 27. Veugelers M, Verrneesch J, Watanabe K, Ymaguchi Y, MarynenP, David G. GPC4,the gene for human K-glypican, flanks GPC3 on Xq26: deletion of the GPC3-GPC4 gene cluster in one family with Simpson-Golabi-Behmelsyndrome.Genomics 1998;53:1-1 1. 28. Li M, Pullano R, Yang HL, Lee HK, Miyamoto NG, Filmus J, Buick RN. Transcriptional regulation of OCI-Slglypican 3: elongation control of confluence-dependent induction. Oncogene 1997; 15:1535-1544. 29. Litwack ED, Ivins JK, Kurnbasar A, Paine-Saunders S, Stipp CS, Lander AD. Expression of the heparan sulfate proteoglycan glypican-1 in the developing rodent. Dev Dyn 1998; 211:72-87. 30. Litwack ED, Stipp CS, Kurnbasar A, Lander AD. Neuronal expression of glypican, a cell surface glycosylphosphatidylinositol-anchoredheparan sulfate proteoglycan, in the adult rat nervous system. J Neurosci 1994; 14:3713-3724. 31. Asundi VK, Keister BF, Stahl RC, Carey DJ. Developmental and cell-type-specific expression of cell surface heparan sulfate proteoglycans in the rat heart. Exp Cell Res 1997; 230:145-153. 32. Li M, Choo B, Wong Z-M, Filmus J, Buick RN. Expression of OCI-5lGlyican 3 during intestinal morphogenesis: regulation by cell shape in intestinal epithelial cells. Exp Cell Res 1997; 235:3-12. 33. Kleef J, Ishiwata T, Kumbasar A, Friess H, Buchler MW, Lander AD, Korc M. The cell surface heparan sulfate proteoglycan glypican-l regulates growth factor in pancreatic carcinoma cells and is overexpressed in human pancreatic cancer. J Clin Invest 1998; 102:1662-1673. 34. Hsu HC, Cheng W, Lai PL. Cloning and expression of a developmentally regulated transcript MXR7 in hepatocellul~carcinoma: biological significance and ternporospatial distribution. Cancer Res 1997; 57:5179-5 184. 35 Ruoslahti E, Yamaguchi Y. Proteoglycans as modulators of growth factor activities. Cell 1991; 64:867-869.

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36. Rapraeger AC, Guimond S, Krufka A, Olwin BB. Regulation by heparan sulfate in ~broblastgrowth factor signaling. Meth Enzym 1994; 245:219-240. 37. Digabriele AD, Lax l, Ghen DT, Svahn CM, Jaya M, Schlessinger J, Hendrickson UTA. Structure of a heparin-linked biologically active dimer of fibroblast growth factor. Nature 1998; 393:812-817. 38. Kan M, Wang F, Xu J, Crabb JW, Hou J, McKeehan WL. An essential heparinbinding domain in the fibroblast growth factor receptor kinase. Science 1993; 259: 1918-1921. 39. Steinfeld R, Van Den Berghe H, David G. Stimulation of fibroblast growth factor receptor-1 occupancy and signaling by cell surface-associatedsyndecans and glypican. J Cell Biol 1996; 133:405-416. 40. Bashkin P, Neufeld G, Gitay-Goren H, Vlodavsky I. Release of cell surface-associated basic fibroblast growth factor by glycosylphosphatidylinositol-specific phospholipase C. J Cell Physiol 1992; 151:126-137. 41. Bonneh-Barkay D, Shlissel M, Berman B, Shaoul E, Adrnon A, Vlodavsky I, Carey DJ, Asundi VK, Reich-Slotky R, Ron D. Identification of glypican as a dual modulator of the biological activity of ~broblastgrowth fqctors. J Biol Chern 1997; 272: 12415-12421. 42. Carey DJ, Stahl RC, Asundi VK, Tucker B. Processing and subcellular distribution of the Schwann cell lipid-anchored heparan sulfate proteoglycan and identificatio~ as glypican. Exp Cell Res 1993; 208:10-18. 43. ~illiamsonTG, Mok SS, Henry A, Cappai R, Lander AD, Nurcornbe V, Beyreuther K, Masters CL, Small DH. Secreted glypican binds to the amyloid precursor protein of Alzheimers Disease (APP) and inhibits APP-induced neurite outgrowth. J Biol Chem 1996; 271:31215-31221, 44. Niu S, Bahl JJ, Adarnson C, Morkin E. Structure, regulation and function of avian glypican. J Mol Cell Cardiol 1998; 30537-550. 45 Pilia G, HL~ghes-BenzieRM, MacKenzie A, Baybayan P, Chen EY, Huber R, Neri G, Cao A, Forabosco A, SchlessingerD. Mutations in GPC3, a glypican gene, cause the Sirnpson-Golabi-Behmel overgrowth syndrome. Nature Genet 1996; 12:241247. 46. Karthikeyan L, Maurel P, Rauch U, Margolis RK, Margolis RU. Cloning of a major heparan sulfate proteoglycan from brain and identification as the rat form of glypican. Biochern Biophys Res C o m u n 1992; 188:395-401. 47. Jackson SM, Nakato H, Sugiura M, Jannuzi A, Oakes R, Kaluza V, Golden C, Selleck SB. dally, a Drosophila glypican, controls cellular responses to the TGF-betarelated rnorphogen Dpp.Development 1997; 124:4113-4 120. 48. Garganta CL, Bodurtha JN. Report of another family with Simpson-~olabi-Behrnel syndrome and a review of the literature. Am J Med Genet 1992; 44: 129-135. 49. Hughes-Benzie RM, Pilia G, Xuan JY, Hunter AGW, Chen E, Golabi M, Hurst JA, Kobori J, Maryrnee K, Pagon RA, Punnett HH, Schelley S, Tolmie JL, Wohlferd MM, Grossrnan T, Schlessinger D, Mackenzie AE. Sirnpson-Golabi-Behmel Syndrome: genotype/phenot~peanalysis of I8 affected inales from 7 unrelated families. Am J Med Genet 1996; 66:227-234. 50. Behmel A, Plochl E, Rosenkranz W. A new X-linked dysplasia gigantism syndrome: identical with the Simpson dysplasia syndrome? Hum Genet 1984; 67:409-413. *

51. Neri G, Gurrieri F, Zanni G, Lin A. Clinical and molecular aspects of the SimpsonGolabi-Behmelsyndrome. Am J Med Genet 1998; 79:279-283. 52. Weng EY, Mortier GR, G r ~ a m JM. Beckwith-Wiedemann Syndrome. Clin Pediat 1995; 317-326. 53. Weksberg R, Squire JA. Molecular biology of Beckwith-~iedemannsyndrome. Med Pediatric Oncol 1997; 27:462-469. 54. Weksberg R, Squire JA, Templeton DM. Glypicans: a growing trend. Nature Genet 1996; 12~225-227. 55. Sanderson RD, Bernfield M. Molecular polymo~hismof a cell surface proteoglycan: distinct structures on simple and stratified epithelia. Proc Natl Acad Sci USA 1988; 85:9562-9566. 56. Cano-Gauci DF, Yang H, McKerlie C, Choo B, Shi W, Pullano R, Piscione TD, Soon S, Grisaru S, Lockwood G, SedlackovaL, Tanswell AK, M& TW, Yeger H, Rosenblum N, Filmus J. Glypican~3-deficientmice exhibit the overgrowth and renal abnormalities typical of the Simpson-Golabi-Behmel syndrome. J Cell Biol 146: 255-264. 57. Lau MM, Stewart CE, Liu Z, Bhatt H, Rotwein P, Stewart CL. Loss of the imprinted IGF2lcation-independentmannose 6-phosphate receptor results in fetal overgrowth and perinatal lethality. Genes Dev 1994; 8:2953-2963. 58. Wang ZQ, Fung MR, Barlow DP, Wagner EF. Regulation of embryonic growth and lysosomal targeting by the imprinted Igf2lMpr gene. Nature 1994; 372:464-467. 59. Ludwig T, Eggenschwiler J, Fisher P, D’Ercole AJ, Davenport ML, Efstratiadis A. Mouse mutants lacking the type 2 IGF receptor (IGF2R) are rescued from perinatal lethality in Igf2 and Igflr null backgrounds. Dev Biol 1996; 177:517-535. 60. Song HH, Shi W, Filmus J. OCI-5lrat glypican-3binds to fibroblast growth factor2 but not to insulin-like growth factor-2. J Biol Chem 1997; 272:7574-7577. 61. Hiscock DR, Yanagishita M, Hascall VC. Nuclear localization of glycosaminoglycans in rat ovarian granulosa cells. J Biol Chem 1994; 269:4539-4546. 62. Fedarko NS, Conrad HE. A unique heparan sulfate in the nuclei of hepatocytes: structural changes with the growth state of the cells. J Cell Biol 1986; 102:587599.

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Ha~ ar~ d e d i c aSchool l and Brigham and ~ o m e n Hospital, ~ s Boston, ~ assachuset t s Veterans A~ a i r ~ s e d i ~Center, al Boston, ~ a s s a c h u s e ~ s

Serglycin proteoglycans (PGs) are localized in the secretory granules of mast cells (MGs), natural killer cells, cytotoxic T lymphocytes, monocytes, macrophages, basophils, eosinophils, neutrophils, and platelets. These PGs are characterized by their uniquely protease-resistant, Ser and Gly rich protein cores and by their covalently attached, highly sulfated glycosaminoglycans (GAGs). The most negatively charged GAGs in the body are often bound to serglycin PGs, and these intracellular PGs appear to play an essential role in the packaging of positively charged proteases and other biologically active proteins in specific molar ratios in the granules of various immune cells. Because the gene that encodes the protein core of serglycin PGs is expressed early during the differentiation of hematopoietic cells and because serglycin PGs interact with so many granule constitue~ts,the regulation of this particular gene and the modification of its translated protein core appear to be extremely important in cellular immunity.

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In 1878, Ehrlich (1) described the presence of a small number of granulated cells in nearly all mammalian tissues that contained large amounts of an undefin~d, negatively charged molecule which avidly bound cationic dyes. Due to the wellfed nature of these cells, Ehrlich called them MCs. The novel metachromatic property of the secretory granules of cutaneous MGs is now known to be due primarily to the presence of a heparin-containing serglycin PC at a concentration of -25 pglcell (2-7). Whereas in the late 1970s it was believed that PGs must reside either on the surface of cells or in extracellular matrices, subcellular fractionation of MC lysates, immunologic activation studies of whole cells (2,8,9), and electron microscopy/~-raydispersion analysis of the elements (i.e., sulfur and calcium) in MC granules (10) established that serglycin PGs preferentially reside in the cytoplasmic secretory granules. Enerback (1 1) first noted that the granules of the subpopulation of MCs that increases in number in the jejunal mucosa and epithelium of helminth-infected rats and mice differ from the granules of cutaneous MCs in their ability to bind safranin 0 . Subsequent biochemical studies revealed that these (12,13) and related (6,14,15) MCs store in their secretory granules serglycin PGs that have unusual chondroitin sulfates (see Chap. 3) rather than heparin. Heparin has more sulfates per disaccharide than any chondroitin sulfate, including those in mucosal MCs. Thus, cutaneous MCs bind safranin more tightly than jejunal because their serglycin PGs are more negatively charged. Although, it was once thought that only MCs express serglycin PGs, it is now apparent that cytotoxic T lymphocytes/natural killer cells ( 1 6 4 9 ) , macrophages (20,21), eosinophils (22,23), neutrophils (24,25), basophils (26-29), and platelets (30,31) also store CSin their secretory granules. Human lung MCs tend to express homotypic serglycin PGs with either chondroitin sulfate or heparin chains (32,33), but these MCs also can store in the same granule hybrid PGs with very different types of GAGS attached to the common protein core,

The development of density-gradient techniques to purify large numbers of heparin-containing MCs to near homogeneity from the rat peritoneal cavity (2) and the development of in vitro techniques to generate large numbers of chondroitin su~fate-containingMCs ( m ~ M M C sfrom ) mouse bone marrow progenitors (6,34) finally gave investigators enough MCs to begin to characterize serglycin PCs at

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the biochemical and molecular levels and to study the regulation of transcription of the serglycin gene. Unlike cell surface and matrix PGs, the mature intracellular PGs isolated from varied rat and mouse MCs cannot be cleaved by trypsin, chymotrypsin, Pronase, collagenase, pepsin, V8 protease, proteinase K, clostripain, or papain (3,6,35-37). Based on these and other observations, it was initially thought that heparin is not synthesized onto a protein core backbone. Nevertheless, Lindahl and coworkers noted (38) that at least some of the heparin chains isolated from pig mucosa were covalently linked at their reducing ends to Ser. The subsequent discovery that radiolabeled Ser and Gly could be inco~orated by rat peritoneal MCs into macromolecular heparin and that the resulting radiolabeled product could be broken down into smaller f r a g ~ e n t safter sodium hydroxide treatment under conditions known to disrupt the 0-linked glycosidic bond between Xyl and Ser finally established that heparin is initially synthesized as a PG (2). Rat MC heparin PGs vary in size but have an average molecular mass of 750 m a (2,3,5). These PGs usually have approximately seven chains that are each -100 kDa attached to a protein core that is 24), which if in the same configuration as RNAse inhibitor would constitute more than a complete circle. For example, chaoptin has 41 consecutive LRRs (34). Buchanan and Gay argue that an all P-sheet structure is substantially more likely than the a-helices that separate LRRs in RNAse inhibitor, and the circular dichroism data (6) favors this theory. Circular dichroism analysis of decorin and biglycan indicate that both have approximately 12% a-helix, considerably less than that present in RNAse inhibitor (6). Both decorin and biglycan have 14% P-turn structure, which is consistent with a p-turn for each LRR. It is our opinion that the parts of the SL are equivalent to the a-helices in RNAse inhibitor ma y have reduced a-helical content. Every third repeat has little or no a-helical content, as they are 3 amino acids shorter than the two preceding repeats. The overall structure has a degree of curvature, but it is considerably less pronounced than the horseshoe shape of NAse inhibitor. The model in Figure 3 may, therefore, be even less curved. The truth, however, will appear only when a crystal structure has been obtained.

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Two types of glycosaminoglycan chain are found on SLRPs: chondroitin/de~atan sulfate and keratan sulfate. Dermatan sulfate (DS) is a form of chondroitin

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A model for the structure of decorin, based on RNAse inhibitor. View (a) shows the overall archlike ~ a n g e m e noft the repeat motifs, with a-helices on the convex face and P-sheets on the concave face. The N-terminal of the protein core is shown at the top and is where the glycosaminoglycan chain(s) are attached. As every third repeat is 3 amino acids shorter, the helices in these repeats are likely to have a less pronounced character. The positions of the N-linked oligosaccharidesin decorin are shown by “N’ ’ and the position of the C - t e ~ i n adisulfide l bond is shown as a line at the bottom. The larger “Ns” are those that are also found in biglycan. View (b) is a side view of a single repeat, showing the positions of the conserved hydrophobic residues, as well as the conserved asparagine that are found in the consensus sequence of L-X-X-L-X-L-)(-X-N-X-L. The asparagines in each repeat would be on the face that is towards the viewer in the lefthand frame. View (c) is a space-filling model of the protein core C-terminal to the first four cysteines. The conserved asparagines in each repeat are shown in dark gray. The Nterminal is at the top right and the C-terminal is at the bottom. Note that circular dichroism spectra for decorin and biglycan suggest less a-helical s ~ c t u r eThis . would have the effect of considerably reducing the overall curvature of the molecule.

sulfate (CS) in which the glucuronic acid has been epimerized to iduronic acid during transport to the cell surface. Keratan sulfate (KS) chains on SLRPs are sulfated forms of polylactosamines which have in their turn been attached to Nlinked oligosaccharides. The KS-chains of SLRPs are thus of the KS-I type. Both of these GAG chains are highly variable, ranging from little or no epimerization to extensive epimerization in the case of dermatan sulfate and from little or no sulfation to extensive sulfation in the case of keratan sulfate. The degree and site of sulfation on chondroitin sulfateldermatan sulfate chains can also vary. Keratan sulfate chains are based on repeating units of-4GlcNAcpl3Galp1-. Sulfation is generally on C-6 of the N-acetylglucosamine, while additional sulfate may occur on the C-6 of galactose. The structures of the keratan sulfate chains on bovine ~ i c u l a (35) r and tracheal (36) fibromodulin have been analyzed by NMR. It is likely that the primary driving force for interactions between GAG chains is hydrophobic and that this interaction is opposed by repulsive ionic forces (37). The orientation and location of sulfates is thus of critical importance, as shown by rotary shadowing/electron microscopy (37). It is important to realize that in many cases the extracellular matrix is somewhat underhydrated. This is particularly true in cartilage. There is therefore considerable thermodyna~icpressure on GAGS to interact with each other rather than be fully hydrated.

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There are two distinct structural elements in SLRPs, the protein core and the carbohydrate. Both of these should be considered to be functionally i ~ p o r t a n t structures. To date, most analyses have been at the level of amino acid sequence or carbohydrate composition. Neither of these are particularly helpful for analysis of functional significance. The protein core needs to be analyzed at the level of tertiary structure, while glycosaminoglycan chains should be analyzed at least at the level of sequence. The structure of the protein core could be analyzed reasonably straightforwardly, given enough material. Circular dichroism has been used to determine the relative amounts of secondary structure of decorin and biglycan produced in eukaryotic cells (6). Both molecules have significant amounts of P-sheet and less a-helix. There are subtle differences in the circular dichroism spectrum of the native proteoglycans with and without glycosaminoglycan chains (R. J. McQuillan, personal communication). Primarily because of technical difficulties, little infor~ationis available on GAG structures. There are many steps to the biosynthesis of the GAG chains

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and a host of points at which substantial variation can be introduced. Thus the keratan sulfate chains can be modified by varying the length of the polylactosamine chain and further modified by varying the amount of sulfation. Even greater variation is possible with the chondroitin/dermatan sulfate GAG chains. In addition to variation in the length of the chain and in the degree of sulfation, the position of sulfation varies between the 2- , 4-, and 6-positions. Dermatan sulfate is chondroitir? sulfate in which varying amounts of the glucuronic acid have been epimerized to iduronic acid. Further variation is therefore possible by adjusting the degree and localization of epimerization. Analysis of chondroitin sulfate by rotary shadowing and electron microscopy showed that chondroitin-6-sulfate can form aggregated meshlike structures in solution, but chondroitin-4-sulfate did not (3’7j. Dermatan-~-s~llfate can aggregate better than 4-sulfated chondroitin sulfate (38). Thus the interplay between type of sulfation and degree of epimerization can modulate the degree to which CS and DS chains interact with each other and with water.

The role of decorin in collagen fibril assembly has been extensively studied. It seems probable that the ratio of decorin to collagen controls fibril diameter so that increased amounts of decorin result in smaller fibrils, although this is likely to be further modified by the degree to which the GAG side chains are hydrated and the degree to which they enable adjacent collagen fibrils to interact. Decorin may also play a role in epithelial/mesenchymal interactions during organ development and shaping (39). Similar roles may be found for other SLRPs. TGF-P has been shown to bind to decorin in a specific and saturable manner (40). Recombinant decorin, biglycan, and fibromodulin expressed in E. coEi have also been shown to bind to TGF-P (41). However, a problem with production of PSin E. coli is that they may not fold well. Hildebrand et al. found that only one-tenth of the expressed proteins had high-affi~itybinding sites for TGF-P (dissociation constant of 1-20 nM) whereas all of the expressed proteoglycan had low-affinity binding sites (50-200 nM) (41). This suggests that not all proteins expressed in prokaryotic systems are folded in the same way.

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Decorin and biglycan were first identified as distinct molecular entities with differing termini in bovine bone (4,42j. Two similar molecules were also shown

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to be present in cartilage and were called PG-I (the larger of the two) and PGI1 (8).Protein (20) and cDNA (43,44) sequence analysis showed that these proteoglycans were distinct, although clearly related to each other. Decorin and biglycan have N-terminal domains which contain chondroitin/dermatan sulfate GAG chains. Biglycan, as its name implies, generally has two GAG chains, whereas decorin, in general, only has one. The GAG chain is attached to the serine in a Ser-Gly pair near the N-terminal. Between the signal sequence and the N-terminal aspartate, a pro-peptide is found in both proteoglycans (43-45), the majority of which is removed intracellularly (45). The removal of this region may not be a necessary event for molecular maturation, as no similar event occurs in fibromodulin or lumican and may simply reflect the population of proteases that are present (46). However, the presence of the pro-form does appear to be necessary for development of full-length glycosaminoglycan chains, at least in decorin (47). As decorin, at least, matures in a different Golgi compartment from aggrecan or fibromodulin (48), the role of the propeptide rnay be in intracellular trafficking. Antibodies against the pro-forms of decorin and biglycan show unequivocally that these forms are present in significant amounts in cartilage (46), in spite of the finding that the majority of material that is isolated is the fully processed form (49). Decorin and biglycan have similar gene structures and rnay have arisen through gene duplication (44,SO). Based on phylogeny analysis (Fig. 2), this would have been substantially prior to divergence between the cartilaginous and the jawed fishes. A noteworthy feature of biglycan is that it is considerably more conserved among species than decorin. Conserved residues are generally involved in protein folding or protein-ligand interactions, whereas residues that are exposed to solvent are under less evolutionary pressure. Thus, a molecule with an exceptionally high degree of amino acid conservation might be expected to have a large percentage of its surface involved in interactions with other macromolecules.

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Retinoic acid, a known morphogen, has opposite effects on decorin and biglycan. Retinoic acid reduces the transcription of the biglycan gene. In contrast, retinoic acid has no effect on transcription of the decorin gene but appears to stabilize decorin mRNA (51). Thus, on treatment of cultured chondrocytes with retinoic acid, decorin levels increase whereas biglycan levels fall. As discussed below, many regulatory molecules have opposite effects on these two proteoglycans, further suggesting that they are evolutionarily a long way removed from each other.

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Decorin can exist as a pro-form in articular cartilage (46). Decorin has one de~atan/chondroitinsulfate glycosaminoglycan chain, except in chicken (52) where two sites are utilized. The second site in chicken does not have the typical acidic residue within two amino acids and the first site is Gly-Ser, rather than the more common Ser-Gly, suggesting that the process of attaching glycosaminoglycans is more complex than a simple consensus sequence. Chick corneal decorin can also contain keratan sulfate (53). The gene for decorin has 8 exons and in humans the first exon (which does not code for protein) has two alternative forms, Ia and Ib (50). It is unclear whether this alternative splice has a regulatory role, The mouse decorin gene does not possess exon Ib (39). The majority of the promoter lies in a 140 bp region on the 5’ side of the first exon. However, an additional region of about 150 bp of homopurine/homopyrimidine elements has significant promoter activity and, by S1 nuclease mapping, appears to have a propensity to form singlestranded DNA (54). The promoter region also contains two TNF-a response elements which are involved in down-regulation of decorin transcription (55). l

Decorin, Cell- CycleControl, and Gene Re ~ ~ l a t i o n

~ a m a ~ u cet h ial. (40) showed that medium from cells transfected with decorin suppressed thymidine inco~oration.They further showed that immobilized decorin could bind labeled TGF-P with an estimated dissociation constant of l . S X M. Initially, it was thought that decorin binding to TCF-P was the primary cause of reduced cell growth in culture. Infusion of decorin into rats with glomeruloneph~tis,a disease apparently caused by excessive TGF-P in the kidney, has the same effect as infusion with anti-TGF-P antibodies (56). Decorin was shown to inhibit growth of transfected Chinese hamster ovary cells that had been stably transfected with a decorin-containing vector (40). Evidence suggests that decorin plays a direct and important role in control of the cell cycle. While some effects may be indirect and result from decorin binding to both collagen and TGF-P, and thus sequester in^ TGF-P in the extracellul~ matrix (40), it is clear that this is not the whole story. Decorin expression is upregulated in confluent cells. A diagram of the elements of this process that are understood is shown in Figure 5. Decorin core protein directly suppresses growth of cells. These effects are consistently associated with induction of p21 (54), a protein that complexes with cyclins and reduces cyc~in-depend~nt kinase activity, thus blocking cell division (57). If decorin synthesis is suppressed with antisense FWA, then cell division resumes (54). he mechanism by which decorin exerts its in~uenceon the cell cycle may, at least in part, be via the epidermal growth factor (ECF) receptor, in that the ECF receptor becomes phosphorylated and the MAP kinaselp21 cascade is acti-

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zyxwvutsrq Decorin sequestered by binding to collagen

Thinner collagen fibrils

transcri tion

Retinoic acid Cell confluence

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~hosphorylation o EGF receptor

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Cytosolic Ca*+ increases

The regulation of decorin and its relationship to the cell cycle control, The diagram indicates what is known about decorin gene regulation and its effect on cells and their extracellular matrix, as described in the text. TNF-a and TGF-P both downregulate decorin, but by different mechanisms. Pecorin core protein has a direct effect on cells, probably through phospho~lationof a receptor and activation of the mitogen-activated protein (MAP) kinase pathway, resulting in upregulation of p21. Outside the cell, increased decorin production will inhibit collagen fibrillogenesis, resulting in thinner collagen fibrils.

vated (58). A specific inhibitor of EGF-receptor kinase (AG1478), or downregulation of the EGF receptor, blocks the effects of decorin on the cell cycle. This effect is not found with biglycan (59). In these experiments, both decorin and biglycan were produced in a eukaryotic system and can therefore be considered to be in their native configurations. TNF-a, a mediator of inflam~ationand remodeling of connective tissue which promotes reentry of confluent, gro~th-arrestedfibroblasts into the cell cycle, reduces decorin expression (SS). This effect can be enhanced by the addition of TCF-P, suggesting that the two cytokines regulate decorin through different

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mechanisms (55). TNF-a regulates the decorin gene through the promoter, whereas TGF-P regulates the gene by posttranscriptional events in both fibroblasts (55) and chondrocytes (60). Progression of human colon carcinoma is associated with hypomethylation of the decorin gene (61). Decorin levels are reduced in hype~rophicscar, consistent with the overgrowth of this tissue (62) while overall levels, and the ratio of biglycan to decorin, are elevated (63). In contrast, retinoic acid upregulates decorin mRNA, most likely by stabilizing the message rather than by increasing transcription (51). In cartilage, mRNA for decorin also increases with age (60) consistent with the quiescent state of the cells in this tissue.

2,

Decorin Binding to Collagen

The control of collagen fibril growth is not simple. The mixture of collagen subtypes found in a fibril can modify its growth, but here we are primarily concerned with proteoglycans. The protein core of decorin binds to collagen types I and I1 (64) and inhibits precipitation of collagen fibrils from solution (65). Reduction of disulfide bonds or thermal denaturation of decorin destroys this effect (22). The incorporation of decorin into collagen fibrils results in collagen with increased tensile strength (66). Stretching of collagen fibrils which have incorporated decorin results in loss of decorin (66), implying that some or all of the decorin is not covalently cross-linked to the collagen. It has been suggested that decorin can bind to two parallel collagen chains, stabilizing the positions of the fibrils with respect to each other (67). Decorin inhibits the contraction of collagen gels (68), possibly by partially immobilizing the collagen fibrils through selfassociation of the dematan sulfate side chains. Decorin isolated from fibrous tissues has shorter DS-PG chains. Fibrous tissues have thicker collagen fibrils. It has been suggested that it i s the GAG chain length which controls fiber diameter (69). It has also been suggested that the degree of epirnerization and/or the ratio of CS-PG and HA to DS-PG is a controlling factor in collagen fibril diameter, in that fetal tissue, which has a higher level of CS-PG and HA, has thinner fibrils (70). The DS-PC chains of decorin bind to collagen type XIV in a saturabje manner (71) and may provide an additional level of complexity in the fibril surface. Decorin binds to different sites on collagen types I and I1 from fibromodulin, with a dissociation constant of between 16 X M (72) and 7 X M (29). In an elegant series of experiments involving domain swapping between biglycan (which does not bind to type I collagen) and decorin, Svensson et ai. showed that the region between residues 168 and 213, in the center of the protein, was p ~ i c u l a r l yimportant for collagen binding in decorin (19). This corresponds to LRR repeats 4 and 5. Mutagenesis of a residue (glutamate -+ lysine) that is likely to be facing into the concave side of the putative archlike structure largely

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reduces the ability of recombinant decorin to bind to collagen (73) and confirms the domain-swapping experiments. In contrast, the latter authors showed that removal of repeat 6 has no effect. It has been proposed that decorin could accommodate a single collagen triple helix within its concave surface (30).

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3. Decorin Knockouts

Decorin core protein has been shown to bind to the NC3 domain of collagen type XIV in the I?-terminal fibronectin type I1 repeat (74). An additional site toward the C-terminal of this domain also interacted with the glycosa~inoglycan chain. A dramatic demonstration of the importance of decorin in forming an ordered extracellular matrix has been shown by targeted disruption of the decorin gene in mice (75). Phenotypically, mice that are homozygous for the decorin null mutation have fragile skin which tears easily. Collagen fibrils in these mice are of uneven thickness along their length, althoug~the average thickness is similar to that in wild-type animals (75). These results suggest that while collagen is responsible for short-range tensile strength, both tensile strength over longer ranges (longer than a collagen fibril) and collagen fibril assembly are at least partly regulated by decorin.

4. Decorin Bin~ingto Other Tissue Components

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Decorin has been implicated in cell binding; it appears to inhibit binding of cells to the cell-adhesion domain of fibronectin and this appears to be due to decorin binding to fibronectin, rather than decorin binding to cells (76). Decorin binds to C l q, part of the first component of the complement activation pathway (6,77), and therefore may play a role in the i n ~ a m m a t oprocess. ~ Decorin has been implicated in the ability of the pathogenic spirochete Borrelia ~ u r g ~ o ~(Lyme e r i disease) to bind to collagen (78); however, the decorin used for this had been prepared using chaotropic agents, and so caution should be applied to the results until more is understood about the structural stability of SLRPs in chaotropic solvents.

Biglycan was first identified as one of the two major small proteoglycans of bone and was shown to be closely related to decorin (44). Biglycan has a different distribution from decorin, and in addition to being found in cartilage and bone (42), is found in endot~elialcells and smooth muscle cells (79,80), It is frequently associated with the cell surface, or with the pericellular matrix. In approximately physiological conditions, and with proteoglycan concentration >l mglmL, biglycan self-associates into dimers. On addition of zinc at

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5 mM, biglycan forms hexamers, and both this and the dimerization appear to be properties of the core protein (81). Sedimentation velocity esperiments in strongly chaotropic solvent (6 M guanidine HCl) give an average molecular weight of 93 W a (81). A s is the case for decorin, biglycan has a signal peptide of 19 residues which precedes a propeptide sequence of 21 residues. The propeptide-containing form is more prevalent in articular cartilage, although this varies to a greater extent than is the case for decorin (46). The highest ratio of propeptide to mature protein core occurs in the nucleus pulposus. Biglycan can undergo further proteolytic cleavage to remove the N-terminal glycosaminoglycan chains (46,82), resulting in a nonproteoglycan form of the molecule. Similarly to decorin, the biglycan gene has 8 exons and, in humans, is approximately 8 kb (1 l). Full promoter activity has been demonstrated in the 78 bp 5’ of the first exon (83). Regulation of biglycan in chondrocytes is generally opposite to that of decorin; mRNA for biglycan is more abundant in young individuals and is reduced in adult articular cartilage (60). However, TNF-a down-regulates biglycan gene expression via the biglycan promoter, probably through a similar mechanism to that for decorin (83). iglycan, Cell-Cycle Control, and Gene R e g ~ ~ a t i o ~

Unlike decorin, biglycan does not inhibit cell growth (54) and does not affect calcium levels in carcinoma cells (59). Biglycan was up-regulated at the edges of epithelial monolayers that were disrupted by “wounding” and this upregulation is similar to that induced by treatment with basic-FGF (FGF2). In both experimental systems, a proteolytic fragment of biglycan can be found in the medium (84). This fragment represents the ~ - t e r ~ i ntwo-thirds al of biglycan. This proteolytic degradation would represent a cleavage of the ~RR-containingregion. Biglycan biosynthesis in tendon is enhanced by cyclic compression, mimicking the process that occurs as tendon moves over a pulley (85). This process is similar to that occurring as a result of TGF-p treatm~ntin the same tissue (86,87). The biglycan gene i s on the X-chromosome and therefore might be expected to be affected by X-inacti~atio~. This is a mechani~mthat ensures consistent transcription of a gene regardless of the number of copies. c, the expression level of biglycan correlates with the number of copie gene, indicating that biglycan escapes inactivation (88). Rodent-human hybrid cells show that the actual gene does undergo inactivation. The explanation is therefore that biglycan transcription is regulated by a gene that does not undergo Xinactivation and is associated with genes involved in regulation of stature (88).

inding to Collagen iglycan has been reported to be unable to bind to fibrillar collagens. However, combin in ant radiolabeled biglycan produced in a eukaryotic system binds to mi-

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crotiter wells coated with collagen types I, 11, 111, V and VI approximately as well as decorin (5). The biglycan-type I collagen interaction has a dissociation constant 100-fold higher than the decorin/type I collagen interaction, which has a dissociation constant of 7 X 20"O h4 (29). The association between biglycan and collagen type I is likely mediated by its glycosaminoglycan chains and is inhibited by high concentrations (> 30 mM) of phosphate or sulfate, in contrast to the interaction between decorin and collagen, which is independent of phosphate concentration (64). ~ n f o l d i n gwith chaotropes abolishes the secondary structure, as determined by circular dichroism and this unfolding largely abolishes the binding to type V collagen (5).

iglycan ~nockouts

Biglycan-deficient mice are phenotypically normal at birth, but three months after birth show reduced growth and reduced bone mass (89). The mutant mice showed reduced cortical bone thickness and al so reduced trabecular structure. The size changes are reminiscent of Turner syndrome, an X-linked disorder characterized by short stature, although it is probable that biglycan is only one of the genes associated with this disorder (88).

i n ~ i n gto Other Tissue ~ o ~ ~ o n ~ n t s As is the case for decorin, biglycan al so binds to Clq, and therefore may play a role in inflammation (5). The highly conserved nature of the biglycan sequence compared to other SLRPs (Fig. 2) suggests that a substantial portion of its surface may potentially be occupied by various interactions.

Keratocan and lumican were first identified as major proteoglycans of the cornea (90) (dealt with in more detail in Chap. 1l),while fibromodulin was first identified as a major glycoprotein in cartilage. Al l four members of this family share a tyrosine-rich N-terminal domain and have several N-linked oligosaccharides attached to the globular domain of their protein cores. The N-linked oligosaccharides may be converted into lactosatnines and sulfated to result in N-linked KS chains. The W-terminal domain varies in length, in the order fibromodulin > lumican >keratocan.

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Fib~omodulinwas first identified as a 59 kDa glycoprotein found in cartilage. Like decorin, it wa s capable of inhibiting collagen fibrillogenesis (91). It was

subsequently sequenced and found to be structurally related to decorin and biglycan (92). It is possible that fibromodulin, like decorin, binds TGF-P (41). In fibromodulin, and probably in the other members of this subfamily, some of the tyrosine residues in the N-terminal domain are sulfated (93). There are three consensus sequences for tyrosine sulfation (tyrosine followed by an acidic group) in this region. Fibrornodulin binds to a different site from decorin on collagens I and TI with a dissociation constant of 35 X 10-9M (72). It is found in the gap region (D band), and in cartilage, has the highest surface density on collagen near the joint surface and in the interterritorial matrix, with the lowest level in the pericellular matrix (94). ~ibromodulinis primarily found as a sulfated proteoglycan in immature cartilage, whereas in mature articular cartilage it is a smaller glycoprotein, lacking polyla~tosal~ine-modified N-linked oligosacc~arides(95). The keratan sulfate chains in bovine articular (35) and tracheal cartilage (35) differ, those in articular cartilage containing fucose linked a 1-3 to N-acetyl glucosamine and also containing a (2-6)-linked neurarninic acid as a capping group on the end of the chain. The capping group on the end of the chain in tracheal cartilage is invariably a(23)-linked N-acetylneuraminic acid. The GAGs are short, being 8-9 disaccharides long in articular cartilage and 5-7 disaccharides long in tracheal cartilage.

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Lumican was originally detected in bovine cornea (96) and has been characterized from chicken (97) and bovine cornea (98) and subsequently from human noncorneal tissue (15). The genes for mouse (99) and chick (100) lumican are 8- 10 kb and consist of 3 exons (Fig. 3). mRNA coding for lurnican has been found in a variety of tissues, but is scarce or absent in brain, liver, and spleen (101). Lumican is essentially absent in mouse early embryonic development prior to day '7, but subsequently appears in a variety of organ systems with the highest level in heart and eye (99). During corneal development lumican is initially found in a nonsulfated, polylactosamine-bearing form. The appearance of sulfated proteoglycans is correlated with development of corneal transparency at about day 15 (102), suggesting that the sulfated GAGs , together with the lumican core protein, are necessary for closely regulating collagen fibril diameter and density within the extracellular matrix (99). Most, but not all, mice that are homozygous for a lumican null mutation lose their corneal transparency after about 10 weeks, indicating that lumican is a major factor in maintenance, but not initiation, of transparency (103). In cartilage, lumican m RNA and protein are considerably more abundant in the adult than in juveniles. As is the case for fibromodulin, the mature protein exists primarily as a proteoglycan in juveniles, whereas in adults, it is primarily

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a glycoprotein (15). Lumican binds to type I collagen and appears to limit lateral fibril growth and inhibit fibrillogenesis in a similar way to decorin and fibromodulin. The fibrils that are precipitable from a mixture of lurnican and collagen monomeric triple helices appear to contain proteoglycan at a ratio of l molecule of lumican to every 10 collagen triple helices (Hassell and ~ c ~ u i l l apersonal n, communication). This is a ratio that would suggest that in the presence of lumican, fibrils could be very narrow. However the relative roles of SLRPs and “minor” fibril-forming collagens, which can also impede lateral collagen fibril growth, are unclear. The sites in both chick lumican and keratocan at which the N-linked oligosaccharides are converted into keratan sulfate chains have been identified and correspond to the first, third, and fourth N-linkage consensus sequences (104). Two consensus sequences for tyrosine sulfation are found in the N-terminal domain ( 105).

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1. L u ~ i c ~ Knockouts n

Absence of lumican has a striking effect on the tensile strength of skin. Mice that are homozygous for a lurnican null mutation have skin that has lost 86% of its strength (103). This is concomitant with collagen fibrils that are irregular in diameter and reminiscent of the fibrils seen in mice deficient in decorin.

Keratocan was originally isolated as a keratan sulfate proteoglycan from the cornea (106). Northern blotting and immunohistochemistry shows that it is most abundant in cornea and sclera, although it is detectable in skin, ligament, cartilage, arteries, and striated muscle, where it exists as a glycoprotein rather than a proteoglycan (107). At the time of writing, the human sequence has not been determined, although a fetal retina expressed sequence tag from the Washington ~ n i v e r s i t y / ~ e r cEST k project (Table 1) is similar to bovine keratocan and may be the human equivalent. The sites in chick keratocan at which the N-linked oligosaccharides are converted into keratan sulfate chains have been identified (see Sec. VII. B.)

Osteoadherin was isolated as a keratan sulfate proteoglycan from bone (108). Osteoadherin has a tyrosine-rich N-terminal, which is likely to have sulfated tyrosines, as is typical of its family (109). It al so contains two other novel features; a pair of cysteines on LRR groups 5 and 6, placed adjacent to each other on an RNAse inhibitor-based model, and a 60-amino acid C-terminal extension which

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contains a group of 22 amino acids that are 60% acidic residues (aspartate and glutamate). The adjacent cysteines would be in an ideal position for a disulfide bond, although isolation of osteoadherin required reducing conditions, suggesting that it may be disulfide-bonded to other matrix components, possibly osteonectin (109). Osteoadherin appears to be involved in cell-matrix adhesion and binds to the osteoblast integrin a v P3(108).

The epiphycan family has fewer (seven) LRRs than other SLRPs. The pattern of repeats differs from the (long-long-short)3pattern seen in, for example, decorin and biglycan and is a long-long-sho~-long-short-long-longpattern (2 l). Members of this family also have a substantially larger and structural1y very heterogeneous N-terminal domain.

Osteoglycin is the smallest of the SLRPs, but it has the largest number of names. Osteoglycin was first isolated (1 10) and characterized (l 1l ) as an osteoinductive factor from bovine bone. It originally appeared to induce ectopic bone formation and was also thought to be restricted to bone lineage cells (1 12). Subsequently, it was realized that trace amounts of growth factors were likely to be causing bone formation, and the name was changed to osteoglycin. The human form has been named mirnecan, as it is small and elusive (the allusion is to Mime, the unfortunate dwarf in Wagner’s ring cycle) (101). Osteoglycin has been shown by ~ o ~ h e analysis rn to follow the sarne wide distribution as l u ~ i c a nwith , the exception that it is not very abundant in muscle or myocardium, and it is more abundant in the ligamentum nuchae (101).

PG-Lb (Lb indicates low buoyant density in cesium chloride gradients) was first isolated from developing chick limbs as a chondroitin/dermatan sulfate proteoglycan, and was histologically associated with the developing ~iddiaphysisjust prior to the chondrocytes becoming hypertrophic (1 13). It continued to be associated with flattened, prehypertrophic ~hondrocytest h ~ u g h o u tlimb growth, but was not found in adult tissue. This proteoglycan therefore appears to be associated with specific developmental changes in the developing limb matrix. PG-Lb has

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also been named epiphycan, due its isolation from the epiphyses of the developing bovine skeleton (21). The structure of PG-Lb has been determined in chickens (1 14), mice (11 3 , humans (g), and cattle (21). Chick PG-Lb has been estimated to have GAG chains totaling 52,000 Da. This implies that there is either one large GAG chain or two smaller chains (113). Similar results were found with bovine P~-Lb/epiphycan

(21).

The role of PG-Lb is unclear. However, if the assumption is made that one feature of the SLRPs is that they form a concave surface which binds another macromolecule, then the fact that members of the epiphycan family have fewer repeats suggests that they bind their ligand with less affinity than the SL have more LRR repeats. A weaker interaction may result in the construction of a matrix that is more amenable to remodeling. In the case of PG-Lb, this is a tempting hypothesis, as the epiphysis is completely remodeled and replaced by bone. However, the argument could equally well be made that PG-Lb prevents remodeling and that its loss is one of the events that permits chondrocytes to become hypertrophic.

Are there other SLRPs waiting to be discovered? While it is dangerous to make predictions of this sort, it is probably reasonable to assume that at least in mammals, the structures of representative members of the three classes of SLRPs are complete. The length of time for which the members of these families appear to have been diverging suggests that to find new structures it will be necessary to analyze the s ~ c t u r e of s SLRPs in, for example, cartilaginous fishes. It is probable that other, distantly related molecules could be found in invertebrates, as has occurred with the collagens. It is clear that decorin is an important molecule in both matrix organization and in cell cycle control. It is becoming clear that biglycan has a similarly complex function. Lumican is also clearly important in regulation of collagen fibril diameter. It would then be surprising if other SLRPs did not have similarly complex roles, providing large numbers of subtle variations to the or~anizationof tissues. In this regard, the SLRPs are similar to growth factor families or embers of the MAP kinase cascade; many variations on a theme with different regulation and regulatory properties resulting in both common and unique responses to a given stimulus. A critical question is why there is so much apparent redundancy. The absence of decorin or lumican alone disrupts collagen fibrils in skin, and yet is not lethal. Why are both proteoglycans necessary? Are they both present at levels low

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enough to absolutely require the other proteoglycan? Would, therefore, decorin overexpression compensate for lumican absence, or vice versa? The role of decorin in cell cycle control, while likely important, is not essential. If this is the case for other SLRPs, it will make the analysis of their respective roles quite difficult. It is likely that within the near future new roles for SLRPs will be found. However, one aspect of the properties of these molecules that is going to continue to be difficult to analyze, is the precise way in which they organize the extracellular matrix. When large molecules with periodic repeats, such as collagen fibrils, have other molecules associated with them, the aggregate property of the whole structure can be based on large numbers of weak interactions. It is probable that many of the properties of the extracellular matrix, such as its flexibility combined with strength, rely on these types of interactions. These interactions are difficult to define using standard laboratory tools, as it will be difficult to demonstrate specificity and saturability, and yet these interactions are every bit as real as the high-af~nityinteractions that are usually analyzed. These complex interactions also make it possible for an individual molecule, such as a proteoglycan, to move through the extracellular matrix relatively easily. Once the proteoglycan has bound to an individual site (for example, the surface of a collagen fibril), additional interactions will inhibit its release, resulting in relatively tight binding to the matrix and a stable large-scale structure. The development of eukaryotic systems for producing large amounts of recombinant proteins ( 5, 6) makes it likely that we will soon have crystal structures for some of the SLRPs. Comparison of their surface properties will enable rational experiments to be performed to test the location of their binding sites for other macromolecules. It will also be possible to analyze their dissociation constants for other macromolecules and perhaps to develop a mathematical model for matrix formation. However, the examples of decorin, lurnican, and fibrornodulin, which all bind to collagen but at different sites, suggests that few simple explanations will be forthcoming.

This work was supported by grants from the Shriners of North America.

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1. I ozzo RV, Murdoch AD. Proteoglycans of the extracellular environment: clues

from the gene and protein side offer novel perspectives in molecular diversity and function. FASEB J 1996; 10598- 614.

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tion, chromosome location, and expression of a 55-kDa matrix protein (PRELP) of human articular cartilage. Genornics 1996; 38:109-117. 132. Grover J, Chen X-N, Korenberg JR, Roughley PJ. The structure and chromosome location of the human chondroadherin gene. Genomics 1997; 45:379-385. 133. Landgren C, Beier DR, Fassler R, Heinegird D, Sommarin Y. The mouse chondroadherin gene: characterization and chromosomal localization. Genomics 1998; 47:84-91.

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University of ~ittsburghSchool of ~edicineiPittsburghi Pennsylva~ia

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The cornea transmits almost all incident light to the interior of the eye, ,serving as the first optical component in the visual system of vertebrate organisms and simultaneously providing a tough physical barrier to the external environment. This unique combination of physical properties exemplifies the diversity of extracellular matrix function. The obvious corneal role in light transmittance and the accessibility of the cornea to observation and experimental manipulation has led to an extensive body of research focused on the structure and function of the corneal extracellular matrix and its individual molecular components, The fact that corneal scming contributes s i g n i ~ c a ~ t ltoy blindness worldwide adds an important rationale for these investigations into matrix function. A s might be expected, corneal extracellular matrix exhibits a number of specialized features. This is particularl~true with respect to the corneal proteoglycans, which differ markedly from those of other interstitial connective tissues and include a unique class of molecules. This chapter s u ~ m ~ i z the e s historical development of our knowledge of the corneal proteoglycans and reviews recent progress in the determination of the molecular structure and biological roles of these interesting molecules.

The cornea ( as shown in Fig. 1) is a relatively simple tissue, consisting of three cellular layers. The external surface is covered by a stratified squamous epithelium supported by a well-developed basement membrane. Posterior to the epithe-

Structure of the cornea. This drawing illustrates features visible in the cross section of a human cornea prepared by standard histology. The stroma has been reduced in thickness for purposes of illustration.

lium is the stroma, a tissue comprising 90% of corneal thickness in most organisms. The anterior portion of the stroma in some species (including primates) consists of a narrow layer of disorganized, acellular tissue known as Bowman’s layer. Posterior to Bowman’s layer is a highly organized connective tissue sparsely populated with mesenchymal cells known as keratocytes. The posterior stromal boundary is a basement membrane known as Descemet’s membrane covered by a single epithelial layer, the “corneal endothelium,” with its apical surface oriented toward the aqueous humor.

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1. Stromal Lamellae

The ultrastructure of the corneal stroma is characterized by an elegantly regular organization. This tissue is composed of 20- 70 acellular collagenous lamellae, each consisting of tightly packed collagen fibrils embedded in a hydrated matrix of glycoproteins and proteoglycans. As illustrated in Figure 2, within each lamella all of the collagen fibrils are parallel and tightly packed. The directional orientation of fibrils differs between adjacent lamellae. Ends of fibrils are not apparent; thus each collagen fibril may extend the entire width of the cornea. This arrangement ofinelastic fibers embedded in a viscous matrix generates a composite structure with extremely high tensile strength, endowing the cornea with a remarkable toughness.

Collagen fibril orientation in stromal lamellae. This drawing presents features typical in electron micrographs of human corneas. Portions of two layers (lamellae) of the stroma show the uniform, parallel, close packing of collagen fibrils and the alteration of fibril orientation between two adjacent layers.

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The relationship between the corneal lamellar structure and corneal strength is exemplified by the properties of several man-made structural materials utilizing a similar composite a~angementof fibers of high tensile strength in a viscous or friable matrix such as fiberglass and reinforced concrete. Such composite materials display strength to weight ratios many-fold greater than solid materials. The diameter of the stromal collagen fibrils is smaller than fibrillar collagen in other interstitial connective tissues and extremely regular in its size distribution (l). Fibrillar collagen in the stroma consists primarily of types I and V (2). The stroma i s also very rich in type VI non fib rill^ collagen, which may constitute as much as 40% of the total collagen in this tissue (3). The keratocytes, flattened mesenchymal cells of neural crest origin, lie sandwiched between stromal lamellae. These cells make up only about 4% of the stromal volume and are the source of the molecular components of the stroma. 2. Corneal Tr an ~ ~ ar en cy

The remarkable transparency of the cornea to light is dependent on the unique ultrast~ctureof the stromal connective tissue. The index of refraction of corneal

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collagen differs from that of the hydrated matrix in which the fibril are embedded, creating a heterogeneous optical material that would, under most circumstances, cause scattering of incident light and an opalescent appearance (4). The fact that light is not scattered in the stroma is a result of the regular diameter and very tight packing of the collagen fibrils. These features provide a crystal-like structure in which scattered light is canceled by means of destructive interference (5-9). Recent comp~rativestudies have shown that the most highly conserved pr~perty of the stroma in a number of vertebrate species is the ratio between fibrillar and extrafibrillar volumes (10). This ratio depends on stromal hydration, a dynamic property of the tissue controlled by a metabolic pump function of the corneal endothelium. If the endothelium is damaged or metabolically poisoned, the hydrophilic extrafibrillar matrix rapidly absorbs water resulting in rapid swelling of the cornea to as much as 10-fold its normal thickness (1 1,12). The volume of collagen fibrils, on the other hand, is not sensitive to changes in hydration (13). Stromal edema disrupts the volume ratio between collagen fibrils and the extrafibrillar matrix, eliminating the tight, highly ordered fibril spacing, thereby producing a loss of transparency. Corneal transparency, therefore, is the result of a dynamic balance between water removed by the endothelium and passive inflow caused by the hydro~hi~ic nature of the stromal matrix. This hydrophilic character is a direct function of the stromal proteoglycans,

The most abundant proteoglycans of the cornea are members of the expanding family known as small leucine-rich proteoglycans (SLRPs) (34). In the cornea, the tissue-specific molecular features of these molecules have long engendered speculation that they play a major role in maintenance of corneal transparency. Consequently, this group of molecules has been the focus of a large number of studies over a period of more than six decades. These molecules have historically been grouped according to their glycosaminoglycan chains into keratan sulfate and dermatan sulfate proteoglycans (KSPGs and DSPGs). Although much recent research on these proteoglycans has focused on the core proteins and their genes, the biological properties of these molecules can be understood only in terms of both the proteins and their u n i ~ u eglycosylation (see also Chap. l o).

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1. Structure In 1939 Suzuki isolated a unique glycosamino~lycanfrom the cornea that contained galactose (15). This material was independen~lyisolated and characterized by Karl Meyer in 1953, who named it keratosulfate (16). The presence of a neutral

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sugar (galactose) in place of uronic acid distinguishes keratan sulfate from other glycosaminoglycans (see also Chap. 2). Keratan sulfate is a linear polymer of Gal(P1-4)GlcNAc (i.e., N-acetyllactosamine) polymerized via a P-glycosidic bond to the 3-position of the galactose. Sulfate is esterified to the 6-carbon of glucosamine and less frequently to the 6-carbon of galactose (17,18). Corneal keratan sulfate chains vary in length from 10-50 disaccharide moieties (19). Structural domains have been identified in porcine corneal keratan sulfate in the distribution of the sulfate along the chain. The 4-6 ~-acetyllactosaminedisaccharide moieties nearest the protein are unsulfated and the distal 10- 12 disaccharides are sulfated only on the glucosamine. The nonreducing termini of the keratan sulfate chains constitute a domain of disulfated disaccharides that varies from 2 to 20 moieties (20). Specific sequences of the sulfated components of the keratan sulfate chain have not been identified chemically; as is the case for heparan sulfate, however, the existence of specific sequence elements in keratan sulfate is implied by antigenicity studies. Several monoclonal antibodies to keratan sulfate chains have been developed that recognize unique epitopes consisting of patterns of sulfated sugars within the keratan sulfate chains (21). Several studies have demonstrated tissuespecific and developmental regulation in the expression of these epitopes (2225). Both sulfated and nonsulfated regions of keratan sulfate exhibit biological functions; however, it is yet to be determined whether any of the structures identified as epitopes recognized by anti-keratan sulfate monoclonal antibodies represents a bio~ogicallyactive domain in these molecules. The reducing end of the keratan sulfate chain is linked to asparagine of the core protein via a mannose-containing oligosaccharide identical to the biantennary, N-linked oligosaccharides typical of glycoproteins (26-32). Removal of keratan sulfate chains from the linkage region with endo-P-galactosidase leaves sialic acid associated with the linkage oligosaccharide, indicating that some or all of the keratan sulfate chains are attached to an oligosaccharide in which the second arm terminates in a short nonsulfated chain capped by sialic acid (27). Stuhlsatz and coworkers used m e t h y l a t i o n / ~ ~analysis C of porcine corneal keratan sulfate to characterize linkage oligosaccharides after sequential degradation with exoglycosidases (31). This study showed the branch linked to the 6-position of the core mannose to be extended with keratan sulfate and the 3-position branch to be truncated with sialic (Fig. 3A). An earlier study by Ziegler and Mersmann reported that the man nos id as^ inhibitor swainsonine did not block incorporation of mannose into corneal keratan sulfate (33). This inhibitor blocks processing of the 6-linked arm of the tri-mannose core and inhibits extension of po1y-~-acetyllactosaminein many cells, Lack of complete inhibition by swainsonine of corneal keratan sulfate synthesis thus presents the possibility that some keratan sulfate chains extend the 3-linked arm of the mannose oligosaccharide in addition to modifying the 6-linked arm, ac-

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Keratan sulfate glycoforrns. The oligosaccharidelinking corneal keratan sulfate to protein is shown schematically according to the model of Stuhlsatz et al. (31). Asn in $e protein on the left (not shown) is linked to keratan sulfate poly-N-acetyllactosamine (rectangles) via an oligosaccharideconsisting of N-acetylglucosamine (open circles), galactose (filled circles), mannose (filled squares), and sialic acid (open diamonds). Unsulfated N-acetyllactosamine disaccharidesare open rectangles; sulfation on only the N-acetylglucosamine is represented by a light gray fill; and sulfation on both glucosamine and galactose is designated by dark gray. (A) Typical corneal keratan sulfate; (B) keratan sulfate secreted by cultured bovine keratocytes(69);(C) glycosylation of the KSPG protein lurnican purified from bovine aorta (78).

cording to the Stuhlsatz model. Tai and coworkers carried out careful analyses of the molecular weight and sugar composition of keratan sulfate-plus-linkage region saccharides released intact from core proteins using the endoglycosidase N-~lycanase.Their data also support the likelihood of bi-antennary extension of keratan sulfate chains on (at least some of the) linkage oligosaccharides (34). We labeled protein-bound oligosaccharides with %Lgalactose using galactosyltransferase following removal of the keratan sulfate chains with endo-pgalactosidase (35). This procedure labeled stubs of linkage regions that had been extended with keratan sulfate. Sialic acid residues were also labeled on the proteins using mild periodate oxidation followed by reduction with N,B3H4.Tryptic peptides were isolated from the labeled core proteins and separated by reversephase HPLC. This analysis showed some peptides containing attach~entsites for both ~ e ~ a t sulfate an and sialic acid (as per the Stuhlsatz model in Fig. 3), whereas others contained only keratan sulfate attachment sites. These findings support the idea that some specific sites on the core proteins bear N-linked oligosaccharides with two keratan sulfate chains (bi-antennary extension), but other sites are modified with oligosaccharides with one keratan sulfate chain with the second m of

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the linkage oligosaccharide terminating in sialic acid (mono-antennary extension). The distribution of the mono- and bi-antennary keratan sulfate glycans may, therefore, be a function of the amino acid sequence of the core protein. Recent analyses by Nieduszynski and coworkers have shown the nonreducing terminus of corneal keratan sulfate to be modified with specialized carbohydrate moieties (34,36,3'7),These elegant studies documented a variety of capping structures in both human and bovine corneal keratan sulfates. In bovine keratan sulfate, neuraminic acids were the most abundant capping structures, followed by (a-3)Cal and GalNAc(S04) in relative abundance. Human corneal keratan sulfate in a similar study lacked the a-Gal moieties. Fucose was also present in a minority of the capping structures. The presence of capping on these chains may afford specific biologic roles to these molecules or could serve as chain terminators during biosynthesis.

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2. Tissue ~ i s t r i ~ u t i o n Keratan sulfate was originally discovered in and named for the cornea; however, soon after its naming a similar sulfated polymer was isolated from cartilaginous tissues. When monoclonal antibodies to keratan sulfate became available, it became apparent that this material was present in most animal tissues (38). In addition to its typical interstitial tissue localization, keratan sulfate is also associated with cell surface and intracellular compartments. In many tissues keratan sulfate appears to be developmentally regulated (39). A quantitative study using two different anti-keratan sulfate monoclonal antibodies showed that on a weight basis, keratan sulfate in the cornea was almost 10-fold more abundant than in cartilage and several orders of magnitude more abundant than in most other tissues (38). The remarkable concentration of this glycosaminoglycan in cornea argues for a specialized function in the corneal stroma. In addition to high abundance, corneal keratan sulfate displays several tissue-specific structural features, most notably N-linkage to protein. In cartilage, keratan sulfate is linked to serine or threonine via a galactosamine-contain in^ ester linkage similar to that found in mucins (40). The keratan sulfate of phosphacan in nervous tissue is ~ - l i n k e dthrough mannose to serine (41). These noncorneal linkage structures appear to be limited to the specific proteins on which the keratan sulfate chains are located. Corneal keratan sulfate, on the other hand, is linked by a chemical structure present in a large number of widely distributed glycoproteins; thus cornea may possess a tissue-specific mechanism for initiation of keratan sulfate elongation. The keratan sulfate termination in cornea differs from that in cartilage as well. About 70% of corneal chains terminate with neuraminic acid, the remainder with PGalNAc and a-Gal. Cartilage keratan sulfate, on the other hand, contains a fucosylated neuraminic acid not present in corneal keratan sulfate (34,36),

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3. Biosynthesis

The oligosaccharides linking keratan sulfate to core proteins closely resemble those common in many glycoproteins. Biosynthesis of these oligosaccharides involves formation of a high-mannose, lipid-linked intermediate in a process sensitive to the antibiotics tunicamycin and deo~ynojirimycin(33,412). These drugs also abolish biosynthesis of corneal keratan sulfate, demon st rat in^ that the linkage of keratan sulfate to protein involves f o ~ a t i o n sof lipid-linked, and highmannose i n t e ~ e d i a t e scommon in dozens of non-keratan sulfate-containing glycoproteins. Little is known as to the mechanism by which a subset of the I?-linked oligosaccharides in the KSPG proteins is selected to serve as sites of keratan sulfate polymerization, Extension of the keratan sulfate chain at these sites occurs via the action of glycosyltransferase activities that alternately add galactose and ~-acetylglucosamineto the growing keratan sulfate terization of corneal galactose transfer activity shows the enzyme involved to resemble the ~-galactosyltransferaseabundant in serum and milk as a component of lactose synthase (43). In many tissues ~-galactosyltransfe~ase is considered a “housekeeping” gene and is expressed at levels independent of cell activity. In cornea, however, expression of ~-galactosyltransferaseis upregulated during development and is maintained at unusually high levels in adult corneal cells (44). Interestingly, ~-~alactosyltransferase continues to be expressed at high levels by corneal cells in culture that have lost keratan sulfate synthesis (43). The ~-acetylglucosaminyltransferaseactivity of cornea has not been extensively characterized, A recent study reported a widely distributed enzyme that appears to be universally involved in the synthesis of linear poly-I?-acetyllactosamine structures, and therefore a likely candidate for the transferase involved in keratan sulfate synthesis (45). Sulfation of corneal keratan sulfate appears to be carried out by at least two separate enzymes (46). Recent studies have identified and cloned two sulfotransferase enzymes that modify keratan sulfate (47,48). One of these proteins adds sulfate to ~-acetylgalactosaminemoieties of chondroitin sulfate and also to galactose moieties in keratan sulfate. The second enzyme also catalyzes transfer of sulfate to galactose of keratan sulfate but does not use chondroitin sulfate as an acceptor. Messenger RNA for the keratan sulfate-specific sulfotransferase shows a restricted localization to brain and cornea. It would therefore appear likely that this keratan sulfate-specific transferase represents an enzyme involved in corneal keratan sulfate biosynthesis, Presumably a separate enzyme is responsible for transfer of sulfate to the glucosamine residues in keratan sulfate; however, description of such an enzyme has not been reported. Keratan sulfate chain length correlates with sulfation levels, suggesting that during synthesis chain elongation and sulfation are coordinated. This hypothesis is supported by biosynthetic studies with cell-free corneal extracts that showed a coordinate change in the V ,,,, of both elongation and sulfation activities with

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respect to keratan sulfate chain length. These studies suggest that accumulation of disulfated disaccharides at the nonreducing terrninal of keratan sulfate tends to slow elongation leading to chain termination (49,SO).

4.

Synthesis of Keratan Sulfate Proteo~~ycans

One feature of corneal keratan sulfate biosynthesis that has long perplexed researchers in attempts to understand metabolism of this molecule is the inability to detect keratan sulfate synthesis in cultured corneal cells. The original observation by Conrad and Dorfman that synthesis of keratan sulfate could be detected in isolated chick keratocytes for only a few hours after the start of culture (51)has been corroborated in numerous studies (52-67). Keratocytes maintained under standard cell culture conditions (i.e., monolayer cultures containing sera) assume a fibroblastic morphology and divide rapidly. Under these conditions typically less than 2% of the secreted glycosaminoglyca~is keratan sulfate. Hassell and coworkers, using human keratocytes in culture, reported that the cells failed to express the core proteins to which keratan sulfate is normally linked (66). We were able to show that intact rabbit cornea continued secretion of keratan sulfate for extended periods in serum-free culture media (68) and more recently documented that monolayer cultures of bovine keratocytes secrete KSPG proteins when maintained in culture medium with reduced serum (69). Analyses of the KSPG made in these cultures revealed the KSPC proteins to be modified with truncated keratan sulfate chains with reduced sulfation. A model comparing the keratan sulfate made by these fibroblastic cells in vitro with that from intact cornea is shown in Figure 3B. This study demonstrated that continuous cultures of bovine keratocytes retain expression of KSPG core proteins and at least some level of the enzymes necessary for construction of a fully sulfated keratan sulfate molecule. Primary cultures of bovine keratocytes maintained under quiescent conditions in reduced serum appear to secrete fully glycosylated KSPG molecules (70). These experiments with bovine keratocytes suggest that down-regulation of keratan sulfate biosynthesis in vitro is primarily the result of reduced chain elongation and sulfation of keratan sulfate and may be a response to specific cytokines in serum. The alteration in KSPG structure that is seen in culture appears similar to the changes in KSPG that occur during wound healing and in chronic corneal diseases (discussed below).

The second major class of proteoglycans in the stroma is modified with dematan sulfate (see also Chap. 2). These glycosaminoglycans were described by Meyer and originally identified as chondroitin sulfate. The observation that 10-15% of the uronic acid moieties in this polymer are iduronic acid has led to its classification as dematan sulfate (7 1).In normal cornea dermatan sulfate contains less than

one sulfate group for each disaccharide; thus these glycosaminoglycan chains are considerably less sulfated than the keratan sulfate molecules that typically contain 1.0-1.5 sulfates per disaccharide (72). As discussed below, corneas respond to a variety of different pathological conditions by accumulation of dermatan sulfate more highly sulfated and iduronate-rich than normal corneal dermatan sulfate. Because of the ubiquitous distribution of dermatan and chondroitin sulfates, few studies have addressed corneal biosynthesis of these molecules. Balduini and coworkers have suggested that the relatively low abundance of dermatan sulfate compared to keratan sulfate in the cornea results from competition for UDPglucose that, due to limited stromal oxygen, is poorly converted to uronic acid (73,74). This hypothesis, however, cannot account for the high keratan/dematan ratios achieved in recent in vitro experiments carried out in the presence of unrestricted oxygen. As with keratan sulfate, it seems likely that expression of specific enzymes involved in elongation and sulfation of the dermatan sulfate polymer may be the factors determining the specialized dermatan sulfate structure of noma1 and pathological corneas.

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Efforts to characterize the proteins attached to keratan sulfate have been greatly facilitated by the development of endo-glycosidases specific for keratan sulfate. Treatment of keratan sulfate proteoglycans with endo-P-galactosidase or keratanase was initially found to generate proteins of several sizes (75). We showed, using tryptic peptide mapping, that at least two proteins were covalently linked to keratan sulfate in KSPC preparations from bovine cornea (76). In a subsequent study, three unique proteins were isolated from bovine KSPC using a combination of chromatographic and electrophoretic techniques (35). Protein sites of keratan sulfate attachment were identified by hydrolysis of keratan sulfate with endoP-galactosidase and labeling of the protein-associated oligosaccharide “stubs” with galactosyltransferase and tritiated galactose. The labeled proteins were subjected to tryptic digest and peptides separated by reverse-phase chromatography. This analysis showed unique tryptic maps for each of the three proteins as well as a unique pattern of glycosylation for each (35). Antibody against intact bovine corneal KSPC identified KSPG proteins in numerous tissues (38), but size and charge of the noncorneal KSPC proteins suggested sulfated keratan sulfate chains to be absent on these molecules (77). Analysis of a purified KSPG from aorta demonstrated N-linked oligosaccharides modified with nonsulfated p oly- N- acet yllact os am in e,6-8 disaccharides in length (78) (Fig. 3C). The molecular forms assumed by keratan sulfate illustrated in Figure 3 demonstrate the variable glycosylation exhibited by these molecules. The widespread presence of the nonsulfated form of the KSPG proteins leads to the conclusion that these proteins are common extracellular glycoproteins that

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in the cornea receive unique tissue-specific glycosylation consisting of elongation and sulfation of PT-linked oligosaccharides normally present on these proteins. In the lens the transparent crystalline proteins result from overexpression of cornmon housekeeping enzymes, a phenomenon known as “gene sharing” (79). The KSPGs seem to exhibit a similar phenomenon in the cornea. Unlike lens crystallines, however, the overexpression of KSPGs in the cornea is coupled with a tissue-specific glycosylation of these proteins providing a new functionality to the molecule. Cloning and analysis of the cDNAs for KSPG proteins have provided a wealth of information about the structure and potential functions for these molecules. The first KSPG was cloned from a chicken corneal cDNA library and named lumican (80). Subsequently, this cDNA has been cloned from variety of mammalian species. The amino acid sequence of lumican shows it to be a member of the small leucine-rich proteoglycan (SLRP) family of genes (14), most closely related to fibromodulin (Fig. 4). In agreement with immunohistochemical studies, lumican mRNA transcripts are present in many tissues. Lumican mRNA is most

Small leucine-richproteoglycans (SLRPs) of the cornea. The nine most closely related members of the SLRP family are grouped according to amino acid sequence similarity using the unweighted pair group method with arithmetic mean (UPGMA) (189). Horizontal lines linking the proteins provide a quantitativeestimate of sequence similarity. Roman numerals show subfamilies as designated by Iozzo (103). Proteins abundant in the corneal stroma are underlined.

abundant in cornea but also highly expressed in sclera, dermis, striated muscle, and intestine. During mouse development, lumican mRNA was only detectable after E7 as interstitial connective tissue begins to develop (81). In the mouse eye, both cornea and sclera express high levels of lumican mRNA, beginning during the formation of the cornea around E12. Unlike mRNA for collagen type I, lumican mRNA is expressed at high levels in adult corneal stroma, suggesting a rapid turnover of this protein compared with collagen. The location and number of keratan sulfate chains that modify lumican is a focus of current inquiry. A study using transfer of labeled galactose to sites from which keratan sulfate had been removed with endo-~-~alactosidase demonstrated a single major labeled tryptic peptide in bovine lumican and two weakly labeled components (35). More recently, attachment sites were investigated by determination of the amino acid sequence of keratan sulfate-linked tryptic peptides from chicken corneal KSPG (82). This experimental approach demonstrated three of the consensus glycosylation sites to carry keratan sulfate chains (Fig. 5) . Each of the sites is located in the central region of the protein, adjacent to one of the 11 leucine-rich repeats that constitute the major structural feature of these proteins, The experimentally determined locations of keratan sulfate, according to the protein folding models developed for decorin and RNAse inhibitor (see Chap. l o) , place the keratan sulfate chains protruding from the "exterior', of the bow-shaped molecule, supporting the hypothesis that the concave side of the

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Features of the keratan sulfate core proteins. This cartoon maps structural domains in the deduced amino acid sequences of the three corneal KSPCs.Leucine-rich repeats are shown as boxes. Dotted boxes indicate absence of 1 of the 7 conserved amino acids in the domain. Circles denote consensus sites for tyrosine sulfation. Dashed boxes below the line show potential cysteine-cysteinebonds. Arrows designate consensus sequence for ~-glycosylation.A star above an arrow indicatesthe experimentally determined presence of keratan sulfate in chicken KSPG (82). The star in parentheses shows the hypothesized location of the keratan sulfate chain in bovine mirnecan (87). (Modified from Kef. 87.)

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protein interacts with collagen fibrils, whereas the glycosaminoglycan chains protrude from the convex side (83). Although keratan sulfate can modify lumican at multiple sites, no study has yet determined if these sites can be occupied simultaneously, thus providing more than one KSPG chain per protein molecule. cDNA for a second corneal KSPG was obtained by screening a bovine keratocyte library with degenerate oligonucleotides based on reverse translation of KSPG amino acid sequence. This protein (originally designated 37A, now named keratocan) has a size and amino acid composition very similar to lumican (84). Its amino acid sequence placed it as a member of the SLRPs gene family with close similarity to fibromodulin, lumican, and to a nonproteoglycan member of the family PRELP (85) (Fig, 4). Northern lotting with keratocan cDNA and Western blotting using peptide antisera showed keratocan distribution to be much more limited than that of lumican (84). In adult bovine tissues keratocan was most abundant in cornea and sclera but could be detected in greatly reduced amounts in several other tissues. Like lumican, keratocan was found only in the proteoglycan form in cornea. Scleral keratocan was smaller and more homogeneous in size than corneal keratocan, suggesting modification of the protein with short, unsulfated oligo-N-acetyllactosamin~ rather than keratan sulfate (84). Amino acid sequencing of keratan sulfate-linked peptides from chicken corneal keratocan identified three unique attachment sites (82) (see Fig. 5). As with lumican, the keratan sulfate attachment sites in keratocan fall in the central portion of the molecule between leucine-rich repeats, supporting the hypothesis that keratan sulfate chains protrude from the external, convex side of the folded protein. During mouse development, keratocan transcripts were detected by in situ hy~ridizationin periocular mesenchymal tissue on day 12, immediately before formation of the corneal stroma (86). During population of this tissue with new keratocytes, keratocan expression was observed in both cornea and sclera. After birth, however, keratocan expression in the mouse was limited to the cornea (86). Keratocan, therefore, appears to be the most cornea-specific of the KSPGs, and in the adult mouse keratocan expression appears to be limited to keratocytes of the corneal stroma. Keratocan is the first gene identified with a clearly stromaspecific pattern of expression. Identification of the keratocan promoter may, therefore, be useful in future construction of transgenic mice with ectopic gene expression targeted to the corneal stroma. The third KSPG (originally designated KSPG25) was identified using BLAST searches of GenBank data with amino acid sequence obtained from bovine corneal KSPG25 (87). Eleven of the 13 amino acids determined to be present at the N-terminus of the 25 kDa bovine KSPG core protein were found in the interior of an open reading frame of a cDNA coding for a protein isolated from bone, This protein was originally named osteoinductive factor (OIF) and later renamed osteoglycin (88). A s shown in Figure 6, osteoglycin is a 12 kDa protein comprising the G-terminal 35% of an open reading frame coding a potential 34

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Relationship of products of the mimecan gene. This diagram presents the relationship between two experimentally isolated proteins, osteoglycin (88), and KSPG25 (87) and the full length product of the cDNA, secreted by keratocytes, mimecan. The 5’terminus of the cDNA and the N-termini of the proteins are shown on the left. (Modified from Ref. 87.)

kDa mature protein. The N-terminus of the KSPG25 protein starts 28 residues closer to the beginning of the open reading frame and includes the osteoglycin sequence. The N-termini of both proteins occur immediately C-terminal to lysine or arginine residues, suggesting that these proteins may be proteolytic fragments of a full length translation product of cDNA. Pulse-labeling studies with cultured keratocytes showed that, indeed, a full-length protein is the primary translation product of this cDNA. This gene and its full-length product were named mimecan in reference to the deceitful dwarf Mime of Wagnerian legend (87). The rnimecan amino acid sequence exhibits structural features of SLRP proteins but differs significantly from lumican and keratocan. The mimecan protein has an extended N-terminus and only seven leucine-rich repeats, two of which are incomplete. Features in common with lumican and mimecan are the presence of Tyr-S04 consensus sites and N-linked glycosylation sites falling between the leucine-rich repeat regions. Like lumican and keratocan, mimecan ex-

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ists as a KSPG only in the cornea. The distribution of mimecan mRNA and protein is more limited than that of lumican. Mimecan mRNA and protein is abundant in artery, sclera, dermis, and was al so detected in cartilage (87). Northern blotting and RNAse protection assays have identified ~ u l t i p l etranscripts of mimecan mRNA, found to be generated by alternate splicing in the 5”untranslated region and by usage of alternate polyadenylation sites (89). Distribution of the different rnRNA transcripts was tissue specific. At the present time splicing variants have not been identified that alter mimecan protein structure; however, alternative splicing in the 5’-untranslated region could affect translation efficiency of the mRNA. Western blotting showed mimecan in noncorneal tissues with molecular weights different from corneal rnimecan (87). Unlike corneal mimecan, only a minor shift in molecular size was observed after treatment of noncorneal rnimecan with endo-P-galactosidase, demonstrating that, like lumican and keratocan, mimecan is a proteoglycan only in the cornea. Deglycosylated mimecan proteins show different molecular sizes from corneal and noncorneal sources, but the origin of these differences is not yet clear. A monoclonal antibody to mimecan reacted with KSPG molecules from bovine and human corneas but not with mouse or chicken KSPG (87). As shown in Figure 5, one consensus site for N-linked glycosylation is present in bovine rnimecan in a location likely to be modified with keratan sulfate (e.g., between leucine-rich repeats). Interestingly, the amino acid sequence of mouse mimecan lacks a consensus sequence for N-glycosylation at this site, raising the question as to whether murine mimecan can be modified with keratan sulfate. Direct sequence analysis of tryptic peptides attached to chicken corneal keratan sulfate did not identify mimecan as one of the peptides linked to keratan sulfate (82). These findings raise some question as to whether mimecan assumes the role of KSPG in all or only a subset of vertebrate corneas. Fibrornodulin is a keratan sulfate-containing SLRP present in cartilage that was originally reported to be present in cornea using immunoassay techniques (90). No further data have been presented documenting fibromodul~nto be a corneal KSPG; neither has its presence in cornea been definitively ruled out. We are therefore not in a position to terminate the list of proteins that constitute the keratan sulfate proteoglycans of the cornea,

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Initial characterization of the dermatan sulfate-containing proteoglycans of the cornea found a single glycosaminoglycan chain and 1 to 3 N-linked oligosaccharides attached to a core protein of approximately 55 kDa (91,92). Antibodies to decorin and to a lesser extent biglycan reacted with corneal DSPGs; however, only decorin rnRNA has been identified in adult bovine and fetal human corneal

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stroma (93,94). Decorin has been cloned from chicken, rabbit, and bovine corneal mRNA libraries and determined to be the major DSPG in adult vertebrate cornea (80,94-94). As discussed elsewhere in this volume (Chap. 10) decorin is the most prevalent of the SLRPs, abundant in most interstitia1 connective tissues. The glycosylation of decorin in cornea, however, differs from that in many other tissues. Corneal dermatan sulfate chains are relatively low in sulfation and iduronic acid content (9 1,97,98). Interestingly, a portion of corneal decorin in both bovine and adult chicken corneas contains keratan sulfate chains in addition to dermatan sulfate (80,9~).This decorin-associated keratan sulfate appears to be N-linked, similar to other corneal keratan sulfate, and may be presumed to modify one or more of the N-linked oligosaccharides present in decorin in the central portion of the molecule. This unusual decorin modification has not been detected in other tissues. Its presence suggests the in~uenceof the amino acid sequence of the leucine-rich repeats near the N-linked oligosaccharides in selection of sites that receive corneal keratan sulfate. Decorin has clearly been shown to be the primary DSPC in cornea of embryonic chickens and humans and also of slaughter-aged cattle; however, there is some evidence that biglycan may be present in older corneal tissue. A recent study of stromal proteoglycans from adult human corneas identified a 40 kDa DSPG core protein that reacted with antibody to a specific amino acid sequence of human biglycan (99). This reactivity was blocked with a synthetic peptide containing biglycan amino acid sequence, but not with a peptide containing analogous decorin-specific sequence. Human corneal biglycan was found to have a molecular size distribution similar to that of decorin (99). This suggests that corneal biglycan may have only a single dermatan sulfate chain, rather than the two chains originally identified on the biglycan protein, Such “monoglycanated” biglycan proteins are found in other tissues and may, in fact, be common (100,101).

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Figure 4 presents a graphic compar~sonof amino acid sequence similarity among those members of the SLRP family present in cornea. Nine SLRP gene products fall into four groups each with closely related amino acid sequence. One member of each group is found in cornea as major corneal proteoglycan. Within each group the proteins share as much as 40% sequence identity; however, there is relatively little identity among distant members of these proteins. Common features of this group are the leucine-rich repeats and the highly conserved cysteines. A s shown in Figure 5, the keratan sulfate-containing corneal SLRPs all contain at least one consensus site for Tyr sulfation N-terminal to the first cysteine. SLRPs not modified with keratan sulfate (i.e., decorin, biglycan, PRELP, and epiphycan) lack these consensus sites. ~ibromodulinhas been shown to contain sulfated Tyr;

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however, sulfation of the consensus site has yet to be demonstrated for any of the other KSPGs (102). A functional role for this sulfation is unknown, although it has been suggested that the negative charge at the ~ - t e ~ i n of u sthese proteins is functionally analogous to the glycosaminoglycan chains on decorin and biglycan and plays a role in interaction with collagen (103). The correlation of sulfated Tyr with the presence of keratan sulfate on an SLRP appears to be loo%, presenting the possibility that this Tyr-S04modification represents a signal for the addition of lseratan sulfate to an SLRP. These hypotheses both await experimental confirmati0n:~urther discussion of structural features of SLRP genes is presented in Chapter 10 of this volume.

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1. Tissue ~ y ~ r a t i o n

The historical role for the SLRPs of the stroma is in tissue hydration. As described above, corneal transparency depends on dynamic maintenance of a critical level of stromal hydration. Influx of water from tears and aqueous humor is offset by an energy-dependent water transport mechanism of the corneal endothelium. Poisoning of the endothelial pump results in rapid swelling of the stroma to a thickness of up to 10-fold that of the normal tissue. A force of 60 mm Hg is required to counteract stromal swelling (104,105). Studies by Hedbys showed that the stromal glycosaminoglycans are responsible for corneal swelling pressure, and that the majority of the swelling was due to glycosaminoglycan sulfation rather than uronic acid content of the tissue (106). The water present in normal stroma occurs in bound (nonfreezable) and unbound (freezable) states (107). Studies by ~ettelheimusing purified proteoglycans correlated this water binding with the different glycosaminoglyca~s(108). Dermatan sulfate proteoglycans create a tight network due to self-interaction of the derrnatan sulfate chains, resulting in. a low molecular hydration of DSPG but with most of the water tightly bound. KSPG, on the other hand, forms an open network with large hydration capacity but with very little retentive power. At the hydration levels characteristic of normal stroma, dermatan sulfate is fully hydrated but keratan sulfate is only partially hydrated. The high absorptive power of keratan sulfate and its ready reversibility appear to be essential to maintenance of the dynamic stromal hydration levels critical for corneal transparency. The importance in the glycosaminoglyc~balance in the stroma is illustrated indirectly in diseases in which this balance is altered. The mucopolysaccharidoses are classic lysosomal storage disorders in which glycosaminoglycans accumulate in tissues. In cases in which dermatan sulfate accumulates in the cornea, for example, MPS1 (Hurler’s and Scheie’s) syndromes, corneal opacity is an early manifestation of the disease (309,110). Collagen fibrils in Scheie’s

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corneas lack the regular spacing of normal corneas, suggesting that the water associated with the excess dermatan sulfate in the stroma is too tightly bound to be removed by the endothelial pump and remains in the e~trafibrillarcompartment of the stroma, disrupting the critical fibrillar/extrafibrillarvolume ratio required for corneal transparency (111- 114). Macular corneal dystrophy is a heritable disease in which corneal keratan sulfate is undersulfated or lacks sulfate altogether (1 15-1 1’7).Patients with this condition develop corneal opacity in the second decade of life. Recently, a null mutation for lumican in mice was reported to show a 25% reduction in corneal keratan sulfate and exhibited a similar loss in stromal hydration. These mice developed corneal haze after 3-6 months of life (118). Taken together these results strongly support the hypothesis that corneal transparency is dependent on maintenance of abundant keratan sulfate and relatively low amounts of derrnatan sulfate with reduced sulfation,

2. Collagen Fibril Organization

As mentioned above, corneal stroma contains collagen fibrils of remarkably small

and uniform diameters organized in close regular crystal-like arrays in the stromal lamellae. These features are widely thought to be essential for corneal transparency. Stromal collagen fibrils are heterotypic, containing types I and V (2). The abundance of type V collagen in these fibrils has been shown to regulate the fibril diameter (1 19,120). The SLRPs are known to interact with fibrillar collagen in vitro and may act to modulate fibril formation in vivo as well. Both corneal decorin and corneal KSPGs showed a marked effect on fibrillogenesis of soluble corneal collagen in vitro (121). KSPGs were much more effective than decorin on a quantitative basis, and collagen fibrils formed in the presence of KSPGs were much smaller and regular in diameter than the fibers formed in the absence of affecting molecules. Removal of keratan sulfate from KSPGs had no effect on their ability to alter fibrillogenesis. These results have lead to the hypothesis that the abundance of KSPG proteins in the stroma contributes to the uniquely small and regular nature of the corneal collagen fibrils. This hypothesis is supported by recent data generated from null mouse mutants (knockouts) lacking in vivo expression of specific SLRPs. Lumican knockout mice show a significant increase in average fibril diameter and have nonuniform spacing of stromal collagen in conjunction with a loss of corneal transparency after 3-6 months of age (1 18). On the other hand, mice lacking decorin exhibit normal corneal collagen fibril diameters and packing and clear corneas (122) (D. Birk, personal communication). These reports suggest that the individual corneal SLRPs intera~twith collagen with a molecular specificity that in turn influences stromal ultrast~ctura~ morpholo~y.

Even though the corneal KSPGs show indirect (and apparently important) interactions with collagen, direct binding of these proteins has not yet been demonstrated. Other SLRP proteins, decorin, biglycan, and fibromodulin, all do bind directly to fibrillar collagen in vitro with varying affinities (83,90,123,124). Electron micrographic analyses of corneal collagen fibrils suggest direct association in vivo between specific regions of the collagen fibrils and both KSPG and DSPG molecules. Ch on d roitin as e- s en s itiveglycosaminoglycans (i.e., dermatan sulfate) stained with cuprolinic blue dye, can be observed associated with specific features of collagen designated as bands “a” and “c” in the repetitive structure of collagen fibrils. Keratan sulfate molecules, on the other hand, are found in the gap zone (bands “d” or “e”) ( 125,126). Birk and coworkers have demonstrated that collagen fibrils form by association of short directional segments that condense both end-to-end and laterally (227,128). This pattern of fibril growth opens the possibility that the individual SLRPs may exert different influences during collagen fibrillogenesis based on their site of binding. Decorin may act in regulating fibril elongation by binding to ends of the condensing segments, whereas KSPGs may bind at regions that interfere with lateral association of the growing fibrils, thereby regulating fibril diameter (D. Birk, personal communication). This hypothesis would explain the different roles of the two SLRPs suggested by phenotypes of mice lacking decorin and iumican. Cells and tissues from these knockout mutants may allow the design of future experiments confirming roles for individual SLRP proteins in fibrillogenesis.

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3.

Interaction with Other Matrix Molecules

A common feature among virtually all the proteins containing leucine-rich repeats is their interaction with other proteins. The most well-documented feature of corneal SLRPs is the interaction with fibrillar collagen discussed above; however, there are several reports of specific interactions with other molecules as well. One of the most abundant compounds in the corneal stroma is type VI collagen, which may make up as much as 40% of the total collagen in the stroma. It is localized in a dense microfibrillar network that appears to associate with and possibly stabilize fibrillar collagen (3,129,130). Type VI collagen is reported to interact with type I collagen, as well as hyaluronan and several proteoglycans ( l 3 l - 135). Decorin specifically shows high-af~nityinteractions with type VI collagen in vitro (134). Treatment of mouse corneas with ATP causes type VI to form beaded microfilaments that are associated with collagen fibrils at the “a” and “c” bands, similar to the pattern described for the localization of corneal DSPG (132,136). Treatment of the tissue with chondroitinase eliminated the association of type VI with fibrillar collagen. This raises the possibility that a DSPG serves as a linker between fibrillar and microfibrillar (type VI) collagens in the stroma.

Another type of interaction is that of lumican with laminin. A prelimina~ report showed interactions between laminin and the low-sulfate form of lumican obtained from artery (137). Corneal KSPG did not interact with laminin, but removal of keratan sulfate chains with endo-~-galactosidaseallowed reactivity. Of the three corneal KSPGs, only lumican bound laminin. This pattern of reactivity is similar to that of the macrophage cell-surface lumican receptor described below, and may have biological relevance during corneal wound healing in which both laminin and low-sulfate lumican are present in the cornea. 4.

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Mouse macrophages were found to attach and spread rapidly on surfaces coated with low-sulfate lumican (138). As with the interaction with laminin, sulfated corneal KSPG did not support macrophage attachment. KSPG, in fact, was strongly antiadhesive for macrophages. Macrophage attachment was inhibited by chelators of divalent metal ions and by polyanions such as dextran sulfate. These characteristics differ from those of the scavenger receptor and from the fibronectin receptor present on macrophages. Flow cytometry showed that the majority of mouse peritoneal macrophages express the lumican receptor. Neither of the other corneal KSPG proteins nor decorin exhibited interaction with macro~hages similar to that shown by lumican, These results suggest that low-sulfate KSPG present during corneal wound-healing may serve to localize i n f l a ~ ~ a t o cells. ry

In addition to the major SLRPs, there are less abundant proteoglycans in the stroma, proteoglycans associated with basement membranes that border the stroma, as well as cell-associated proteoglycans in the several cell types of the cornea.

1. ~ a s e ~ e ~n et ~ b r a n~reot eogl ycan Immunohistochemical studies have identified typical basement membrane proteoglycans, perlecan, and bamacan in the epithelial basement membrane and in Descemet’s membrane (139). Interestingly, the stromal keratocytes also appear to secrete low levels of extracellular matrix molecules found in basement membranes (66). Laminin, type IV collagen, and perlecan can be detected immunohistochemica~1yin pericellular locations of human corneal stroma and have also been identified as products secreted by keratocytes in culture. These molecules attest to the neural crest ancestry of the keratocytes and suggest the possibility that the pericellular environment of the cells in vivo may more closely resemble that of an epithelium rather than that of interstitial connective tissue fibroblasts. This hypothesis is strengthened by recent observations that keratocytes in vivo

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form a cellular network interconnected via adherens and gap junctions containing cadheren and catenin, proteins important in epithelial cell-cell interactions (140,141).

2.

Collagenous Proteoglycans

In addition to the SLRPs, the long form of type XI1 collagen has been identified in the cornea (142,143). Type XI1 collagen is known as a FACIT collagen (fibril associated collagen with interrupted triple helices) and is found in two forms (long and short) that are produced by alternate mRNA splicing (144). The long form has chondroitin sulfate attached to the noncollagenous domains, and consequently is a proteoglycan (145,146)- Type XI1 was originally identified in the cornea-sclera junction (143) and in developing and regenerating corneal stroma (147-150), but more recently Wessel et al. have demonstrated its presence in normal adult cornea (15 1). Type XI1 associates with the surface of collagen fibrils and is localized at regular intervals (152,153). The presence of type XI1 noncollagenous domains NC-3 and NC-2 stabilizes type I collagen gels against deformation in vitro and increases interaction between cells and these gels (153). Type XI1 also interacts strongly with decorin in vitro (154). It appears that type XI1 collagen cont~ibutesto the presence of chondroitinase-sensitiveglycosaminoglycans located at regular intervals along corneal collagen fibrils described by Scott (155), thought to be important in regulation of interfibrillar spacing. The reported upregulation of type XI1 in healing wounds may possibly be the source of large chondroitinase-sensitive, fibril-associated glycosaminoglycans described in experimental wounds and in human keratoconus corneas (156- 159).

3. Cell Surface Proteoglycans Most epithelial cells contain one or more of the cell membrane-bound syndecan proteoglycans, widespread mediators of cell attachment (see Chap. 6). Syndecanl was recently demonstrated immunohistochemically in rabbit corneal epithelium. Corneal syndecan was downregulated in basal layers of the migrating epithelial layer after abrasion wounds, but returned to all layers of the epithelium after 48 hr. A less well understood cell surface proteoglycan, NG2, has recently been identified on corneal stromal cells (160). This -300 ma membrane-spanning, chondroitin sulfate proteoglycan was originally identified on neural-derived cells, is expressed on a number of cells during early phases of or~anogenesis,and is downregulated later during development (161- 163). NG2 shows strong binding to type V and type VI collagens via protein-protein interactions (13 1,164). In developing chicken cornea, NG2 is expressed by periocular mesenchyme and embryonic stromal cells (160). Later in development, on day 14 after the keratocytes have fully populated the stroma and tissue dehydration begins, NG2 was

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present on only a subset of the keratocytes. Chemical cross-linking studies of cultured keratocytes showed NG2 to be associated with extracellular type VI collagen, suggesting that stromal cells use this proteoglycan as a type VI receptor. The adult corneal stroma is a fibronectin-poor tissue and normal keratocytes lack the a 5 p l integrin that constitutes the major type I collagen and fibronectin receptors; however, the tissue is very rich in type VI and V collagens. The NG2 proteoglycan may, therefore, constitute a major transmembrane attachment molecule in the normal stromal keratocyte. Further investigations will be required to define possible roles of NG2 proteoglycan.

n ~ o t ~ e l iKeratan al Sulfate

Several studies have suggested the presence of keratan sulfate in the corneal endothelium but not in the epithelium. An early study by Hart showed separated corneal endothelium to be capable of synthesis of keratan sulfate (165). Keratan sulfate associated with corneal endothelium was also observed in im~unohistochemical studies with antikeratan sulfate monoclonal antibodies (166). Recently this endothelial keratan sulfate was localized to the surface of corneal endothelial cells using gold-labeled antibodies and scanning electron microscopy (167). The apical exterior surface and cell junctions exhibited uniform labeling that was sensitive to keratanase. The cell-surface keratan sulfate appeared to vary in abundance from one cell to another and was markedly up-regulated on cells migrating at the edge of scrape wounds. Cell-surface keratan sulfate has also been reported on human keratinocytes (39). Dermal keratin proteins carry the keratan sulfate epitopes associated with keratinocytes; however, the protein to which keratan sulfate is bound in corneal endothelium has not yet been determined (168). The unusual cell-surface localization of this keratan sulfate and its regulation in response to cell migration suggests a potentially active biological role.

Embryonic formation of the cornea is initiated by secretion of an acellular layer of connective tissue by the ectoderm that overlays the developing lens. In chickens at day 5 of development this primary stroma contains mostly chondroitin sulfate (169). Shortly before arrival of migrating neural crest-derived cells, the primary stroma swells due to the accumulation of hyaluronan (169). In situ hybridization studies of developing mouse cornea show that the migrating neural crest cells in the periocular mesenchyme express both lumican and keratocan mRNAs before they enter the primary stroma (86,170). In chicken, sulfated epitopes of keratan sulfate are not detected until 12 hr after invasion of this cell population into the primary stroma (25). Subsequently, keratan sulfate accumu-

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lates rapidly in the stroma progressing in a posterior to anterior gradient. This pattern of proteoglycan secretion suggests that a high level of expression of the KSPG core proteins is initiated early in the keratocyte developmental program, but modification of these proteins with keratan sulfate appears to be dependent on their localization in the corneal stroma. KSPG molecules in the stroma become progressively more highly sulfated throughout embryonic development and, in the chicken, this KSPG accumulation and increased sulfation continues after hatching (25). In the mouse, corneal lumican is found primarily in its glycoprotein form at birth and begins to accumulate in the proteoglycan form about the time of eye opening at 10- 14 days (170). Analyses of human corneas suggest a similar increase in sulfated keratan sulfate in the stroma throughout life (171). This postnatal developmental increase in keratan sulfate sulfation may be important for proper physiological function of the adult cornea. Lumican knockout mice which have reduced keratan sulfate initially exhibit clear corneas and only develop haze after 3-6 months of life (1 18). Similarly, humans with macular corneal dystrophy, a condition in which keratan sulfate is missing or ulldersulfated, typically enjoy normal vision until the second or third decade of life (1 15). Proteoglycans containing dermatan andlor chondroitin sulfate exhibit a pattern of developmental expression differing markedly from that of the KSPGs . The derrnatan sulfate of chicken corneas undergoes a modest decrease during embryonic development (172). In human corneas the content of dermatan sulfate remained relatively constant during embryonic development, but the sulfation of the dermatan sulfate molecules decreased after birth, stabilizing around 2 years of age (171). These studies suggest that, unlike keratan sulfate, length and sulfation of dermatan sulfate is not tightly linked to stromal localization or to developmental age. The impo~anceof the stromal environment in maintenance of keratan sulfate biosynthesis is well illustrated by the numerous studies documenting loss of keratan sulfate secretion in vitro. The development-dependent sulfation of KSPG has been attributed to increases in the pool of PAPS, the molecular source of glycosaminoglycan sulfate (273,174). This hypothesis, however, does not account for the selective nature of the increased sulfation of keratan sulfate compared to dermatan sulfate. The recent d e ~ o n s t r a t ~ oofn a keratan sulfate-specific sulfotransferase that is localized to the cornea provides the most likely candidate for a gene involved in the developmentally regulated sulfation of corneal keratan sulfate (47). The physiological function of the decades-long increase in corneal keratan sulfate sulfation is not understood. In the adult cornea, fibrillar collagen is highly cross-linked and there is little synthesis of type I collagen or expression of its m RNA. This suggests the possibility of a dual role for KSPG that is developmentally dependent, During stromal morphogenesis, high levels of the SLRPS influence collagen fibrillogenesis and the packing of the stromal collagen required for transparency. Postnatally, when fibrillogenesis has ceased and the eye is open,

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modification of the same SLRPs with increasingly more highly sulfated keratan sulfate may be necessary for maintenance of stromal hydration.

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In animal wound-healing models, damage to the corneal epithelium and wounds that penetrate into the stroma both result in activation of keratocytes that migrate to the wound site (175). Two weeks after penetrating wounds, hyaluronan and dermatan sulfate are abundant in the newly deposited scar tissue ( 176,177). The relative amount and sulfation of dermatan sulfate is elevated, whereas keratan sulfate is reduced or not detected ('72,178,179). The DSPG molecules in the actively healing wound are heterogeneous in size, with a significant proportion of the DSPG larger than the decorin of normal cornea (72,178). The dermatan sulfate glycosaminoglycan chains in this unusual DSPC were similar in size to those in normal proteoglycans suggesting a dermatan sulfate-linked protein not present in normal tissue (178). After 1year, KSPG in scar tissue was similar in size and sulfation to that of normal tissue, but continued to be reduced in amount compared to the amount of DSPG present (72). The reduction of MSPG in corneal scar tissue has also been demonstrated in immunohistochemical studies using antikeratan sulfate monoclonal antibodies and by the presence of keratanase-sensitive structures stained with cuprolinic blue dye in electron microscopic analyses (157,158,180). Although the DSPGs of the scar tissue exhibit a normal size distribution after the initial period of active healing, the dermatan sulfate glycosaminoglycan chains associated with these molecules are more highly sulfated and contain more iduronic acid than normal for at least 1 year after wounding (72,181,182). This characteristic change in dermatan sulfate was also observed in corneal grafts undergoing rejection and in pathological human corneas with keratitis and lattice degeneration (183,184). The long-term increase in highly sulfated dermatan sulfate correlates with an increased water content of the experimental scar tissue (177). Scarred human corneal tissue typically has undergone years or decades of chronic pathology frequently involving inflammation and edema. In spite of the differences in the timing and development of these corneal scars compared with those generated in acute experimental animal wound-healing models, many of the matrix changes appear to be similar. Staining of pathological human corneas using monoclonal antibodies against keratan sulfate demonstrated a significant decrease in sulfated keratan sulfate in most of the samples examined (185,186). Antibodies to the KSPC core proteins, however, indicated no significant decrease

in the pathological corneas (187). DSPG response in pathological corneas was opposite that of KSPG. Sulfated epitopes of dermatan sulfate were elevated in keratoconus corneas (l56). Analysis of extracts from human corneas with chronic edema, bullous keratopathy, and keratoconus using antibodies against decorin and biglycan showed significant elevation of both of these proteins in the pathological corneas (99). In about half the samples, biglycan was elevated more than sixfold over normal levels. Both decorin and biglycan proteins were associated with dermatan sulfate of increased sulfation. Proteoglycans visualized in the electron microscope using cuprolinic blue dye staining appear as rods or spheres of distinct size and shape usually associated with the collagen fibrils. Keratanase-sensitive proteoglycans localized to the ‘ ‘a’’ and “c’ ’ fibrillar bands and chondroitinase sensitive proteoglycans to the “d” and “e’ ’ bands (125). Two weeks after experimental wounding of rabbit corneas, keratanase-sensitive proteoglycans were absent and the chondroitinase-sensitive material formed unusually large limacine (sluglike) structures (157,158). Such features were also observed in human corneas with keratoconus and with bullous keratopathy (156,188). The timing and localization of these structures correlates well with the appearance of a large DSPG in experimental wounds and the marked elevation of biglycan in human corneas with chronic pathologies. Taken together the data suggest a role for biglycan in stromal pathology. The pathological pattern of proteoglycan biosynthesis appears to be very similar to that observed by keratocytes in cell culture (69). This provides an experimental system in which the cellular environment that stimulates secretion of normal differentiated extracellular matrix by the keratocytes and the signals that induce scar tissue secretion may be defined.

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In spite of intensive research over almost six decades, we still have much to learn about the biosynthesis and expression of the corneal proteoglycans. The mechanism by which sites of keratan sulfate attachment are recognized and the enzymes that polymerize keratan sulfate and add sulfate are currently poorly understood. A problem of perhaps greater complexity is the description of the signaling system that coordinately regulates the large number of enzymes involved in secretion of the characteristic proteoglycans found in normal corneal tissue and the transition to the proteoglycans typical of healing wounds. The most longterm challenge to our understanding is definition of biological roles for the unique proteoglycans of the cornea and the roles of the subtle structural changes that these molecules exhibit in response to development, aging, and external stimuli.

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I wish to thank Gary Conrad, Martha Funderburgh, and Mary Roth for their help in preparing this manuscript and Daniel Funderburgh for artwork. This work was supported by NIH Grants EY09368 (to JLF) and EY00952 (to Gary W. Conrad).

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glycan NG2 binds to collagens V and VI through the central nonglobular domain of its core protein. J Biol Chem 1997; 272:10769-10776. Hart GW. Biosynthesisof glycosa~noglycansby the separated tissues of the embryonic chick cornea. Dev Biol 1978; 62:78-98. Hyldahl L, Aspinall R, Watt FM. Immunolocalization of keratan sulphate in the human embryonic cornea and other human foetal organs. J Cell Sei 1986; 80: 181191. Fullwood NJ, Davies Y, Nieduszynski IA, Marcyniuk B, Ridgway AE, Quantock AJ. Cell surface-associated keratan sulfate on normal and migrating corneal endothelium. Invest Ophthalmol Vis Sci 1996; 37:1256- 1270. Schafer IA, Sorrel1 JM. Human keratinocytescontain keratin filaments that are glycosylated with keratan sulfate. Exp Cell Res 1993; 207:213-219. Toole BP, Trelstad RL. Hyaluronate production and removal during corneal development in the chick. Dev Biol 1971; 26:28-35. Ying S, Shiraishi A, Kao CW, Converse RL, Funderburgh JL, Swiergiel J, Roth MR, Conrad GW, Kao WW, Characterization and expressionof the mouse lumican gene. J Biol Chem 1997; 272:30306-30313. Praus R, Brettschneider I. Glycosaminoglyc~sin embryonic and postnatal human cornea. Oph~almolRes 1975; 7:452-458. Hart GW. Biosynthesisof glycosaminoglycans during corneal development. J Biol Chem 1976; 251:6513-6521. Conrad GW, Woo ML. Synthesis of 3’-phosphoadenosine-5’-phosphosulfate (PAPS) increases during corneal develo~ment,J Biol Chem 1980; 255:3086-3091. Beckenhauer DM, Conrad GW. The effect of thyroxine on transparency and PAPS synthesis in the avian cornea. Dev Biol 1981; 84:225-229. Matsuda H, Smelser GK. Electron microscopy of corneal wound healing. Exp Eye Res 1973; 16:427-442. Cintron C, Kublin CL. Regeneration of corneal tissue. Dev Biol 1977; 61:346357. Cintron C, Schneider H, Kublin C. Corneal scar formation. Exp Eye Res 1973; 17: 25 1-259. Cintron C, Gregory JD,Damle SP, Kublin CL. Biochemical analyses of proteoglycans in rabbit corneal scars. Invest Ophthalmol Vis Sci 1990; 31:1975-1981. Praus R, Dohlman CH. Changes in the biosynthesis of corneal ~lycoaminoglyca~s during wound healing. Exp Eye Res 1969; 8:69-76. Funderburgh JL, Cintron C, Covington HI, Conrad GW. I~unoanalysis of keratan sulfate proteoglycan from corneal scars. Invest Ophthalmol Vis Sci 1988;29: 1116l 124. Funderburgh JL, Chandler JW. Proteoglycans of rabbit corneas with nonperforating wounds. Invest O p h ~ ~ l mVis o l Sci 1989; 30:435-442. Anseth A, Fransson LA. Studies on cornealpolysaccharides. VI. Isolation of derrnatan sulfate from corneal scar tissue. Exp Eye Res 1969; 8:302-309. Anseth A. Studies on corneal polysaccharides,VIII. Changes in the glycosaminoglycans in some human corneal disorders. Exp Eye Res 1969; 8:438-441. Anseth A. Studies on corneal polysaccharides. VII. Changes in glycosaminoglycans in penetrating corneal grafts. Exp Eye Res 1969; 8:310-314.

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Corneal Proteo~lyc~ns

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185. Funderburgh JL, Funderburgh ML, Rodrigues MM, Krachmer JH, Conrad GW.

186. 187. 188. 189.

Altered antigenicity of keratan sulfate proteoglycan in selected corneal diseases. Invest Op~thalrnolVis Sci 1990; 31:419-428. Rodrigues M, Nirankari V, Rajagopalan S, Jones K, Funderburgh J. Clinical and histopathologic changes in the host cornea after epikeratoplasty for keratoconus. Am J Ophthalmol 1992; 114:161-170. Funderbu~~h JL, Panjwani N, Conrad GW, Baurn J. Altered keratan sulfate epitopes in keratoconus. Invest Ophthalmol Vis Sci 1989; 30:2278--2281. Quantock AJ, Meek KM. Proteoglycan distribution in the corneas of individuals with bullous keratopathy. Biochern SOCTrans 1990; 18:958. Nei M. Molecular Evolutionary Genetics. New York: Columbia University Press, 3987:293--298.

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r Children, ?ampa, ?ampa, Florida

College of ~ e d i ~ i nUniversity e, of South Florida, and S~riners~ospital for Children, Tampa, Tampa, ~lorida

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Proteoglycans were first discovered in basement membranes by the presence of their sulfate esters and their ability to be detected with cationic dyes (1,2). The presence of heparan sulfate proteoglycans in basement membranes was observed by the sensitivity of cationic dye staining to treatment with heparitinase or nitrous acid (3). Since these initial studies, major efforts have concentrated on the isolation and puri~cationof basement membrane proteoglycans. The EngelbrethHolm-Swarm (EHS) tumor, which produces other basement membrane molecules such as laminin and collagen IV, was the source used to first isolate a heparan sulfate proteoglycan in large quantities which was subsequently called large lowdensity heparan sulfate proteoglycan (4). Antibodies to this proteoglycan showed it was present in al l basement membranes including al l vascular basement membranes, as well as in many extracellular matrices, and showed the core protein to be 400 kD (5,6). The antibodies were also used to isolate the first cDNA clones (’ 7) which provided the initial characterization of its core protein. This heparan sulfate proteoglycan (HSPG) was then named perlecarz, referring to its rotary shadowed electron microscopy structure which appeared as “beads along a

string” (8). Perlecan is one of the most heavily studied proteoglycans, to date. For reviews see Refs. 9-14. This review will concentrate on the recent findings. Since perlecan is found in all basement membranes and many extracellular matrices, it has a wide range of regulatory controls, binding properties, and interactions. Perlecan’s gene structure is enormously complex and the promoter region has recently been characteri~ed,thus providing a plethora of information about the regulatory control elements which determine the expression of this proteoglycan. Perlecan is not only regulated by cytokines but can also be bound to them, as well as to growth factors, with high affinity. Some of these interactions involve only perlecan’s heparan sulfate side chains, and others involve the core protein. Perlecan also appears to be an early response gene since several studies have reported rapid induction of perlecan expression. Perlecan can act as a growth stimulant for some cell types including cancer cells, and is a potent inhibitor of proliferation for others such as vascular smooth muscle cells. This proteoglycan can act as a coreceptor or prevent other molecules from interacting with theirs. In addition, perlecan has now been shown to bind most of the basement membrane components as well as several extracellular matrix molecules. Recent studies on the nematode perlecan gene have provided striking details on the role of perlecan in myofilament formation and organization as well as on how the basement membrane is formed. Perlecan also plays an important role in the development of p u l ~ o n aintestinal, ~, cartilaginous, muscular, cardiovascular, and organ maturation of mesenchymal tissues. It has recently been shown to have an active role in the pathogenesis of a wide range of diseases, including Alzheimer’s disease, Scrapie, diabetes, arteriosclerosis, and cancer; and it can be essential to tumor growth, involved in the sequestering of cytokineslgrowth factors, induction of angiogenesis for tumor blood supply, and in enhancing metastatic potential. Perlecan is even being targeted in the therapies for some of these diseases. Clearly, the ubiquitous expression of this proteoglycan and its potential diversity through alternative splicing and the attachment of either heparan sulfate or chondroitin sulfate or both types of side chains has led to many recent discoveries and many more are sure to follow.

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Perlecan has been cloned in mouse (7,15), human (16,1?) and C. elegans (18). In the mouse, a 12 kb mRNA codes for a protein core with a deduced M, = 396 k D , a value close to the observed M W = 400 kD of the large low-density proteoglycan, Human perlecan is coded by a 14.35 kb mRNA with a deduced M,= 46’7 Mammalian perlecan core protein contains 5 domains: 1 unique domain at the N-terminal region and 4 domains contain in^ motifs present in other proteins (Fig. 1). Homology between the mouse and human sequences was exten-

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Alternate splice sit

ure 1 Structural domains of perlecan. Perlecan can be divided into five domains based on amino acid homology to other known proteins. Alternative splicing occurs in domain IV, with the human sequence coding for 21 Ig domains and the mouse and nematode coding for 14 Ig domains. The well-defined GAG attachment sequences are located in close proximity to one another in a region unique to perlecan at the N-terminal end of the molecule.

sively examined for each domain (19) and was found to range between 36 and 90%. Two primitive form s of perlecan have been identified, one in C a e n o r ~ a ~ ditis elegans encoded by the unc-52 gene (18) and one in Xenopus Zaevis (partial cDNA, Accession #AJ224485). The C. elegans gene product has an M, = 270 and was found by characterizing nematodes with mutations in the unc-52 or perlecan allele (18). The comparison among mouse, human, and nematode sequences reveals

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the role of alternative splicing among species. The mouse perlecan sequence lacks seven immunoglobulin-lilce modules in domain IV, corresponding to the mid-5th to the mid-12th Ig repeat, that are present in human perlecan (19). The nematode sequence from C. elega n s revealed a truncated form of the perlecan gene product where ~ o m a i nI is altered, having little homology with the mammalian counterparts; domain I11 is lacking a module and domain V is entirely missing (12,18). Alternative splicing of the perlecan gene within species has also been observed for the mouse and nematode species but not for human, although searching for alternative splicing in human was extensive (12). In mouse, the major transcript is 12 kb in both EHS and mouse melanoma M2 mRNA, but after prolonged exposure of Northern blots, two additional bands in the M2 population were found at 13- 14 kb. This study indicates that the alternative splicing of mouse perlecan may be cell- or tissue-specific as well as species-specific (19). One of the most interesting developments in the perlecan field recently is the discovery of a 45 W, apparently alternatively spliced variants of perlecan expressed in a mouse neuroepithelial cell line (20). These cells, named 2.3D, have three message sizes detected by Northern blot analysis with a 96 bp probe corresponding to amino acids 1134-1 165 of perlecan. There are a weak 12.5 kb, moderate 6.5 kb, and strong 3.5 kb sized transcripts when using this perlecan probe. However, Southern analysis indicated that all of these transcripts were generated by a single perlecan gene (20). Another study found several unexpected sized bands during RT-PCR experiments on human and rodent hippocampus tissue in which domain I was being amplified (21). These differences resulted in 100-200 bp changes which would not be detectable changes in Northern analysis of the 12.5 kb construct. These different sized bands were further characterized and appeared to be spliced variants of perlecan (21). Western blot analysis using a polyclonal perlecan antibody to the 2.3D conditioned medium resulted in three perlecan core protein sizes of 400 W, 58 kD,45 kD, and 35 kD (20). However, a monoclonal antibody to perlecan reacted with the 400 kD, 58 kD and partially with the 45 kD bands. The 45 kD band was the major perlecan variant which was expressed as a 290 kD native HSPG and is believed to have a 3.5 lcb message size. This protein was named perlecan-related-molecule(PRM). A polyclonal antibody specific to PRM had a proportion of the antibodies reacting with the heparan sulfate (HS) chains of PRM but not with perlecan. These results suggest that perlecan and PRM have substantially different HS chain co~positionand in support of this, PRM has been shown to have specifically high affinity for FGF-2 (20). The HS chains on PRM also appear to be located throughout the small protein core and not particularly localized to one end of the molecule. The immunolocalization of PRM was extensively examined during neuronal development and showed a different pattern of localization than perlecan, including being uniquely localized to the neuroepithelial cells of the spinal cord. PRM not only intensely stained the basement membrane of various structures of mesodermal and endodermal

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origin but was also found in the developing gut as well as the heart and blood vessels of the vascular system (20). In nematode, three alternative spliced forms of the unc-52 gene were found, all encoding different carboxy-terminal sequences. The larger transcripts code for all or a part of domain IV while the smallest message encodes a protein that completely lacks domain IV (18).

Domain I

Perlecan's structure contains five domains, four of which share homologies with other known proteins (Fig. 1). Mammalian perlecan contains a 21 amino acid signal peptide sequence, followed by a region within domain I that is the only region unique to perlecan. This unique module contains an acidic region followed by three Ser-Gly-Asp sequences located in close proximity to one another and are the primary glycosaminoglycan (GAG) acceptor sites for perlecan (Table 1). Several studies using recombinant domain I had mixed results regarding the ability to express the domain I protein. Recombinant domain I alone is not expressed in some cell lines, including COS-7 which also does not express domain 1/11(22) and the baculovirus insect cell system (23). However, modifications of mouse perlecan domain I, such as using the BM-40 signal peptide and the addition of six His residues at the C-terminal end of domain I, generated domain I proteins capable of being expressed in human embryonic kidney 293 cells (24) and C H 0 K1 (25), respectively. The baculovirus insect cell system demonstrated the unusual finding that domain I/ IIa contained 100%chondroitin sulfate/der~atansulfate (CS/DS) chains (23). This re~ombinantCS-containing protein was capable of binding FGF-2 in a GAG-dependent manner with relatively low affinity. Conversely, other cell systems and studies have shown that perlecan domain I supports both HS and CS chain attachment, but HS attachment is usually favored. Perlecan from the EHS tumor has been shown to have several GAG species attached to its core protein including HS chains only, a hybrid HS and CSIDS and some as a CS/DS species only (26). Additionally, all three potential GAG attachment sites can support either HS or CS/DS attachment and all sites are usually substituted (22,24,25). Glycosylation sites in perlecan domain I have also

le 1 Sequences in Heparan Sulfate Proteoglycans That Can Receive Heparan Sulfate (HS) Chains"

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HS are in bold and acidic residues are

been found where four Thr and one to two Ser residues could receive short branched oligosaccharides (24). Core protein sequences present within perlecan which can enhance or regulate HS versus CSIDS attachment have also been determined. The cluster of acidic residues located N-terminally to the 3 Ser-Gly-Asp sites are the critical determinants for HS attachment (22) (Table 1). Elements that appear to enhance attachment of HS (i.e., when these elements were missing there was an increase in percentage CSlDS attached) include perlecan domains II/JII (25,27), the SEA module of domain I, and the unique N-terminal amino acids 36-5 1 (22). When perlecan domain I I lI I I was replaced with the globular G3 domain from aggrecan, there was a substantial decrease in %HS and subsequent increase in CS attachment (27). These findings indicate that GAG-free domains can influence the utilization and composition of potential GAG attachment sites in domains bearing GAG chains. The rotary shadowed image of perlecan with its beads along a string appearance containing long GAG chains attached at one end of the molecule (8) has sparked much research on the GAG attachment sites in domain I. However, it is worth noting that mouse perlecan does contain an additional eight Ser-Gly-AsplGlu and 30 Ser-Gly sites in the remaining four domains. Although most of the studies involving perlecan’s domain I have concentrated on the GAG attachment sites, the C-terminal end of this domain contains a unique region called an SEA module, originally discovered by computer analysis studies (28). The acronym SEA stands for sperm protein, enterokinase, agrin which are three proteins that share homology in this region. Proteins which contain SEA modules are generally heavily glycosylated and are comprised of multimodule domains shared with other known proteins. ~haracteristicsof SEA modules include a region of homology spanning approximately 80 amino acids with a C-terminal nonhomologous region of about 40 amino acids separating the SEA module from the next downstream module. The predicted secondary structure of the SEA module is successive g-strands interrupted by an a-helix (28). The function of this module is not clear; however, it may aid in enhancing 0-linked glycosylation since this is the main common feature of proteins containing this domain. Evidence to support this theory includes the study in which the SEA domain was deleted from a perlecan domain I lI I lI I I construct where the percentage of HS attach~entdecreased while concurrently the percentage CS increased (22).

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Domain I I

Domain I1 of perlecan is homologous to the class A, low-density lipoprotein (LDL) receptor. These receptors contain a repetitive element consisting of six cysteine residues spaced over a -40 amino acid region with conserved acidic and hydrophobic amino acids (7,l 1). This element repeats four times in domain TI of perlecan and is flanked, at the C-terminal end, by a single immunoglo~ulin-

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like module (Ig). One study on domain 11, expressed the four LA and single IG modules of mouse perlecan with the BM-40 signal peptide sequence in human embryonic kidney 293 cells. Results showed that the structure of the recombinant protein resembled that of the native domain based on availability of proteolytic cleavage sites, the circular dichroism spectrum, and rotary shadowing analysis (29). This study also showed that the four LA modules of domain I1 contribute to the rodlike secondary structure of this region and that the protein size variation observed may be due to glycosylation at the six potential 0-linked sites (29). It has also been noted that domain I1 in mammalian perlecan contains the sequence DGSDE which is thought to be responsible for mediating binding to the lowdensity lipoprotein receptor (1 l). Interestingly? the binding and uptake of highdensity lipoprotein (HDL) by hepatocytes has been shown to be dependent on cell surface HSPG (30). Whether perlecan is the cell surface HSPG involved has yet to be shown. It has also been speculated that domain I1 may support entactinl nidogen binding although with a lower affinity than intact perlecan (29). Some of the difficulties in studying this domain may be att~butedto an inability to recombinantly express this domain alone or in conjunction with domain I in several cell systems.

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Domain Ill

Domain I11 of perlecan is comprised of modules with homology to the laminin ECF-like domain (LE) and the laminin B domain (LamB). One LE module followed by a single LamB and three LE modules comprise a structure that is repeated three times to comprise perlecan domain 111. The LE modules are rich in cysteine residues while the LamB lacks cysteine and has a globular structure. Domain 111 contains a potential GAG attachment site; however, studies indicate that this site is unsubstituted (22). Structural studies on the third repeat of recombinant domain I11 expressed in embryonic kidney 293 cells indicated that the LE modules provide a rodlike scaffold while the third LamB module was present as a large 200 amino acid loop or globular region that was more susceptible to protease digestion than the flanking LE domains (3 l), Circular dichroism studies indicated a distinct secondary structure and a mixed alp conformation for the LamB module (31). A series of rat monoclonal antibodies to mouse perlecan indicated that the most antigenic region of perlecan was the third LamB globular domain within domain I11 (32). The LE regions of domain I11 were also found to be antigenic with two antibodies recognizing a similar secondary structure in two different LE modules. All of the antibodies that recognized domain I11 epitopes immunostained all tissues, similarly indicating that^ alterations in epitope availability did not vary in different tissue structures (32). Domain I11 of mouse and nematode perlecan contains an Arg-Gly-Asp or RCD sequence in the second LamB module. However, this sequence, which is

commonly highly conserved and known for binding to integrin receptors, was not conserved in human perlecan. The availability of perlecan RGD sequence to the cell surface and its role in binding integrins is controversial. In one study, recombinant mouse domain I11 was expressed in human fibrosarcoma HT1080 cells and was purified from stably transfected clones using column chromatography in 2 M urea and dialysis in buffered saline (33). Another study generated recombinant clones for each of the three domain I11 repeating units, expressed the products in human embryonic kidney 293 cells and purified the recombinant products without using the denaturants urea or guanidine (34). Both studies determined the integrity of the recombinantly generated products by limited protease digestion and rotary shadowing electron microscopy (33,34). The recombinant product encoding all of domain I11 was found to promote cell attachment at 4065% of the efficiency of laminin (33). The cell attachment could be specifically inhibited by the addition of RGDS peptide or intact perlecan, whereas addition of a scrambled peptide or intact laminin had no effect. These results indicate that mouse perlecan domain I11 can bind the cell surface of mouse mammary NZMT cells in an RGD-dependent manner most likely through an integrin receptor. This is also supported by the finding that perlecan, like fibronectin, can be a focal adhesion component (35) and by studies on integrins. Perlecan has been shown to support endothelial attachment and spreading through both p1 and p3 integrins (36). ~ l t h o u g hcell adhesion was higher to intact perlecan than to the core protein, the core protein was clearly involved in mediating adhesion. In these studies, both p1 and p3 integrins mediated RGD-dependent binding to the perlecan core, most likely domain 111, while only the p1 integrin had substantial RGD-independent activity (36). The second study examined the ability of the individual domain 111 fragments and their Combination to support attachment of several, mainly human, cell lines (34). None of the cell lines adhered to the recombinant domain I11 modules and only six of the nine cell lines could adhere to intact perlecan. The recombinant units did not bind the a5pl and avp3 integrins in solid phase assays, whereas denatured domain 111, unit 2, which contains the RGD sequence, could bind the a5pl integrin, This latter result is perplexing because unlike many of the integrin receptors, a5pl is not promiscuous, binding only fibron~ctin,and does not bind to other R~D-containingextracellular matrix molecules. The findings by Hayashi et al . (36) may explain some of the confusion on the use of RGD in binding to integrins. The requirement may be cell-type-specific, integrinspecific, and species-specific. In summary, these studies indicate that domain I11 of perlecan may support cell surface binding through an RGD-dependent receptor, most likely and p3 integrins, but this interaction may depend on refolding after denaturation, it may be species-specific (mouse perlecan binding to mouse cells) and may be dependent on an intact domain 111. However, perlecan also exhibits substantial RGD-independent integrin binding,

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Domain IV

Perlecan domain TV is comprised solely of immunoglobulin IgG repeats. These repeats align with themselves as well as with the immunoglobulin repeats of neural cell adhesion molecule (N-CAM) (7). There are 14 Ig repeats in mouse and nematode perlecan and 21 Ig repeats in human. Despite the fact that domain IV is the largest domain in perlecan, there have been very few studies addressing the role of this domain. Therefore, the function of domain IV remains conjectural, based mostly on the function of Ig domains in other molecules. It is possible that domain IV is responsible for bomophilic binding of perlecan molecules or other protein-protein interactions. The nematode mutant studies do, however, provide some insight into the role of domain IV. The nematode mutant of perlecan indicates that the organization of the Ig repeats is important for myofilament assembly and in the ~aintenanceof yof filament organization in muscle cells (37).

Domain V

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Domain V is the C-terminus of perlecan and contains three laminin G domainlike modules (LamG) spaced by two EGF-like modules (EG). Domain V has the potential to support GAG attachment since there is one Ser-Gly-Xxx-Gly in the second LamG module, two Ser-Gly-AsplGlu in the third LamG module, and several additional Ser-Gly sequences scattered throughout this domain (l 0 ). Mouse domain V has been expressed recombinantly in human embryonic kidney 293 cells and was found to be secreted in several forms: full-length domain V containing both HS and GSlDS chains, full-length domain V lacking GAG chains, and two domain V fragments due to an endoproteolytic cleavage site between the last EG and LamG modules (38). Interestingly, d o ~ a i nV appears to have different functions depending on the presence of the GAG chains. Several, but not al l , cell lines tested could adhere to perlecan domain V, whereas none of the cell lines could attach to domain V containing GAG chains (38). Cellular adhesion to domain V could be inhibited by the addition of antibodies to p1 integrin, suggesting a direct cell surface receptor interaction (38). appears to play a role in binding to a laminin-entactin complex, entactin alone, and to a lesser extent fibulin-2. Binding of domain V to entactin was mapped to both the central globular domain G2 and C-terminal domain G3 of entactin al though the majority of the adhesion affinity appears to be contributed by the G2 domain (38). Another potential role for domain V may be the end-to-end binding of multiple perlecan molecules. Purified HSPG from the EHS tumor was shown by rotary s h a d o ~ i n gand sedimentation analysis to form dimers and less commonly oligomers at the end opposite the GAG attachment domain, now known as domain V (39). This in vitro self-binding could occur in the presence or absence of GAG chains; however, a large proteolytic fragment of perlecan could not support the assembly of multimers (39).

Fkxlecan has a complex gene structure that spans over 120 kb of genome. There are 94 exons ranging in size from 45 bp to 1.2 kb with an average exon size of only 150 bp that encode for perlecan, revealing one of the most intricate gene structures characterized to date (40; also reviewed in Refs, 1 1, 12). Similar to the protein, the gene structure has homologies with other known genes based on the repeated domains. These findings imply that perlecan evolved from a common ancestor through gene duplication or exon shuffling. Exon 1 contains the 5’ untranslated region and encodes for perlecan’s signal peptide. Domain I is encoded by exons 2-6 and contains the sequence necessary for the three GAG attachment sites within this domain. The first Ser-Gly-Asp attachment sequence resides between exons 2 and 3 where the junctional splicing occurs with the Asp residue. The remaining two Ser-Gly-Asp sequences are encoded within exon 3. Therefore, if exon 3 were to be removed by alternative splicing, then perlecan would lose those three GAG attachment regions and would most likely be expressed as a glycoprotein. Domain I1 is comprised of five exons, where three exons encode for the LDL receptor-like domain and two exons encode for the Ig-like module. There is a high conservation between perlecan domain I1 and the LDL receptor gene structure with all the introns residing in phase I, Conversely, perlecan domain 111, which is encoded by 27 exons, does not share exon conservation with laminin E3 short arm and laminin EGF-like domain. Domain IV contains the most number of exons with 40, The Ig repeats 1- 17 of domain TV have a one module, twoexon gene structure with the first exon being in phase 0 and the second in phase I which is similar to the exon organizational pattern in P- CAMI.This arrangement allows for alternative splicing between Ig domains such as the case among human, mouse, and nematode perlecan. Although the pattern of exon-intron junctions of the Ig domains are not conserved between human and nematode, there is conservation of the multiple combination of the Ig domains which indicates there are functional constraints on domain IV (37). Domain V is encoded by 16 exons and its gene structure has little homology with the laminin G region. Exon 94 is the largest exon in perlecan’s gene structure and it encodes for the Cterminal end of domain V and the entire 3’ untranslated region. I n summary, perlecan has an enormously complex gene structure that has evolved in a manner where domains 11and IV have superior conservation between the exon structure and the internal repeats of the corresponding domains. Conversely, domains I11 and V, which both share homology with laminin, exhibit little gene to proteinrepeat structure conservation. Therefore, the common ancestral gene has undergone extensive rearrangement or exon shuffling as these proteins evolved resulting in two different gene structures ( l 1,12,40).

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Mouse perlecan has been mapped to Chromosome 4 with linkage to the alkaline phosphatase-2 (Akp-2)gene by restriction fragment length variance (41). Human perlecan has been mapped to Chromosome lp36.1 near the telomere by using Southern blot analysis of DNA from human/rodent somatic cell hybrids (42) and by restriction fragment length polymorphism (43). Data based on this and on the linkage of the Akp-2 gene indicate the preservation of a syntenic region between mouse Chromosome 4 and human Chromosome 1. There are few human genetic diseases that have been identified in or near this region of Chromosome 1. However, there are several cancer markers and a progressive disease, SchwartzJampel-Averfeld syndrome, characterized by multiple skeletal and muscle disorders, localized in this region of Chromosome 1. Perlecan’s promoter has been well characterized and was found to lack typical TATA or CAAT boxes but did contain multiple cis-acting elements comrnonly found in housekeeping or growth control related genes (40,44). Approximately 2.5 kb of genomic DNA located 5’ to perlecan exon I was subcloned and sequenced, revealing multiple regulatory elements (44). The region 500 bp immediately upstream from exon 1 has a high G + C content with >80% GC, typical of CpG islands which have been correlated with transcriptional control elements. The proximal promoter region also contains four GC boxes and 15 consensus hexanucleotide binding sites for the Zn finger transcription factor SF1. There are an additional five SP1 sites located within exon 1. Multiple AP-2 motifs were also present, with eight located in the proximal 1.5 kb, two in a distal region of the promoter, and additional AF-2 motifs scattered through the noncoding strand. The proximal promoter also contains three palindromic inverted repeats in close proximity which may potentially form a secondary structure that could influence the regulation of gene expression (44). Other elements found in the perlecan promoter region include several transcriptional factors involved in hematopoiesis such as PEA3 motifs, ETS-l?a PU. 1 box, and GATA-2 motifs, There are two fully conserved CTF-NF- 1 elements which may potentially serve as negative regulators in suppressing gene transcription in a cell specific manner (44). The distal promoter contained a binding site for N F - ~ € 3which are found in the promoter region of almost all growth factors and cytokines. The 5’-flanking region spanning -2.5 kb was tested for the ability to promote transcription in various cells and this region was found to have activity in all cell types examined, which attests to the widespread distribution of perlecan expression (44). A series of deletions within this promoter region revealed that the majority of the activity resides in the region spanning from -461 bp through exon 1, which contains nearly all of the regulatory elements needed to provide full functional promoter activity (44). A TGF-P responsive region was also identified within perlecan’s promoter region located between -461 and -285 bp. Structurally, this responsive element was characterized by internal NF-1 and AF-2 motifs which may

potentially mediate the action of TGF-P (44). In summary, the 5”flanking region of the perlecan gene acts as a functional promoter in several cell types and contains a complex array of cis-acting elements necessary for driving gene expression of constitutively synthesized proteins in a wide variety of tissues.

Regulation of perlecan expression has been examined at the transcriptional, translational, and posttrans~ationallevels revealing a wide range of potential mechanisms that can be used to control the presence of perlecan in a particular tissue, during development or in disease. At the transcriptional level, the SV-40 Large T oncogene was found to induce a decrease in perlecan mRNA synthesis by twoto threefold while concurrently increasing the actin mRNA levels and hyaluronic acid synthesis in a temperature-sensitive SV-40 transformed renal cell line (45). Therefore, the SV-40 oncogene appeared to inhibit transcription of the perlecan gene while stimulating the transc~ptionof the actin gene. Other aspects of the role of perlecan in cancer will be discussed later in this chapter. Previous studies have shown that during the process of differentiation, basement membrane genes are often transcriptionally induced. F9 embryonal carcinoma cells were used to study the effects of differentiation, stimulated by addition of retinoic acid and dibutyryl CAMP,on perlecan transcription, and results showed that in comparison to laminin, the perlecan gene was transcribed more rapidly with maximal induction of mRNA and protein levels being reached before the induction of laminin (46). However, the maximal rate of perlecan protein synthesis was only 1-296 of the maximal rate for laminin (46). The rapid induction of perlecan transcription and translation suggests a control mechanism that is directly responsive to retinoic acid or CAMP and the study on perlecan’s promoter proposed a putative CAMP transduction element (44). Other studies on rat glomerular epithelial cells showed that cAMP and prostaglandins E, and E’, two agents which are known to increase cAMP concentrations, caused a rapid decrease in perlecan message and protein levels (47). Interestingly, the CAMP-mediated decrease in perlecan mRNA in glomerular epithelial cells was as rapid as the increase in perlecan message in embryonal F9 carcinoma cells (46,47). These results emphasize the tissue- or developmentally specific regulations that the same agent can have on perlecan expression. Induction of basement membrane synthesis also plays a critical role in diabetes (discussed in further detail later in the chapter) where an increase in many glomerular basement membrane components is accompanied by a decrease in HSPG content leading to altered ultrafiltration. The role of glucose on the transcription of perlecan has therefore, been examined. Mesangial, glomerular, and smooth muscle cells cultured in 30 mM high glucose medium for 1-7 days exhibit an inhibition of perlecan transcription leading to a decrease in perlecan

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protein synthesis (48,49). The decrease of HS content was also examined and it was determined that, since there was no alteration in the charge density or GAG chain size of the perlecan that was being synthesized, the decrease in HS directly correlated with the decrease in core protein synthesis. These studies also indicate that the effect of glucose on perlecan is not strictly at the transcriptional level and that posttranscriptional regulatory mechanisms also appear to play a role in altering the levels of perlecan (48,49). At the translational level, the C. elegans perlecan gene named ur i c- 52 provides insight into how perlecan message may be regulated. The mec-8 gene encodes for a trans-acting factor that can putatively bind and regulate the alternative splicing of perlecan pre-mRNA (50). These studies suggest that the MEC-8 protein contains two copies of an RNA recognition motif that corresponds to an RNA-binding domain and that MEC-8 rnay block the splice site donor downstream of exon 18. The resulting regulation allows for two populations of perlecan mRNA that lack either exons 16, 17, and 18 or exons 17 and 18. This alternative splicing would then generate a perlecan protein with a truncated domain IV' with Ig repeats 8, 9, and 10 missing or repeats 9 and 10 missing, respectively (18,50). Mutated mec-8 generates a mutant phenotype in C. elegans characterized by body wall muscle defects, as seen in the unc-52 mutants, as well as defects in the function of mechanosensory and chemosensory neurons. The latter defects rnay be the effect of MEC-8 binding to RNA from another gene product or it rnay indicate a neurobiology role for perlecan. Regardless, it is clear that MEC-8 is essential for the generation and accumulation of a population of alternatively spliced perlecan that is necessary for a normal muscle phenotype in C. elegans (50). Another model of translational modification of perlecan is the mouse embryo attachment competence during acquisition. Perlecan expression at the trophectodermal surface appears to be developmentally regulated in blastocysts, functioning mainly in attachment that may be HS chain dependent (51). Perlecan message is present in both day 4 (unhatched) and day 4.5 (hatc~ed)blastocysts, while the perlecan protein is only on the surface of the latter (52). Results showed that the expression of perlecan protein and the subsequent presence of HS is due to a response to estrogen. Therefore, perlecan rnay be subject to tr~nslational regulation through an estrogen-stimulated pathway which is a critical step in the attachment of competent blastocysts to the epithelium of the uterus (52). Another mechanism for perlecan regulation is the rate at which it is turned over in the matrix. Several studies have examined the factors which degrade perlecan, thereby allowing for turnover or release of growth factors from the matrix. In the vasculature, endothelial cells cultured in the presence of thrombin showed a decrease in perlecan core protein expression most likely through the suppression of core protein synthesis (53). Other studies showed that plasmin, stromelysin (MMP-3), and collagenase ("P-13) all degrade perlecan's core protein releasing significant amounts of FGF-2 from human endothelial cell lay-

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ers (54). Interestingly, these proteases had different mechanisms of action. For example, stromelysin cleaved perlecan at many sites, abolishing immunoreactivity to domains I, 111, and V while plasmin appeared to release domain I from perlecan, and hence FGF-2 bound to HS, leaving a 300 kD fragment containing intact domain I11 (54). Collagenase ( ~ ~ P - 1 also 3 ) significantly degraded the core protein of perlecan. Other enzymes which released FGF-2 were heparitinase I and to a lesser extent heparinase I, suggesting that FGF-2 is bound to heavily sulfated regions of the HSchain. The cleavage of perlecan was relatively specific to these proteases in that other MMPs and thrombin did not exhibit an ability to degrade perlecan or release FGF-2 (54). Cytokines and growth factors are responsible for regulating a wide variety of cellular functions including embryogenesis, differentiation, migration, proliferation, and tumorigenesis. They function at extremely low concentrations and bind to very high affinity receptors where they can stimulate a signaling cascade, be internalized, and sometimes relocalize to nucleus to specifically bind to DNA regulatory elements, Like many extracellular matrix and basement membrane molecules, perlecan expression can be regulated by cytokines and growth factors. Interferon-y (Im-y) is a 20-25 kD glycoprotein produced mainly by T lymphocytes and natural killer cells that functions as a potent inhibitor of viral replication and can regulate a variety of immunological activities. Since IFN-y has been shown to exhibit antitumor activity as well, the effect of IFN-y on perlecan gene expression was examined, Results showed that 160 nglml of IFN-y completely growth-arrested human colon carcinoma cells, in a reversible manner, without any evidence of inducing apoptosis (55). Further investigation determined that this cytokine increased the proportion of cells in GI and subsequently decreased the percentage in S and G2 phases of mitosis. Examination of gene expression revealed that transc~ptionalactivity of the perlecan gene was rapidly decreased, within 2 hr, and this activity was not dependent on new protein synthesis (55). This result indicates that perlecan is an early response gene, which is in agreement with other previous studies (46). Further experimentation showed that the region of the promoter responsible for the effects of IFN-7 was located distally and that the transcriptional activator STATl was required for inducing the transcriptional silencing of the perlecan gene. Additional evidence to support the data is that the distal region of the perlecan promoter contains several GAS elements which are elements that bind STAT proteins. Therefore, it appears that perlecan can be transcriptionally regulated by IFN-y through a STATl proteindependent pathway. Transforming growth factor-P (TGF-P) is a 25 kD homodimer that exists in several isoforms and is secreted as a latent, inactive form of the molecule dependent on protease digestion for activation (reviewed in 56). TGF-P is produced in large quantities by platelets, but many cell types produce the growth factor in smaller concentrations. The actions of TGF-P vary depending on cell

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type where it can act as a stimulant or inhibitor of cellular processes. The effect of TGF-P on perlecan expression in human colon carcinoma cells and on mesangial cells has been investigated. TGF-P induced an increase of perlecan message levels, core protein synthesis, and subsequent sulfate incorporation in human colon carcinoma cells (57). The increase in sulfate incorporation was determined to be due to an increase in core protein synthesis and not a result of increased sulfation or number of HS chains attached. The increase in perlecan expression in colon carcinoma cells appears to be at the posttranscriptional level possibly by enhancing mRNA stability (57). TGF-P was also found to increase perlecan core protein production in human synovial cells (58). Similarly, human mesangial cells also increase perlecan expression in response to TGF-P (59). This study found that angiotensin TI induced a decrease in perlecan message levels and an increase in TGF-P mRNA which was directly correlated with the subsequent protein levels for these proteins. When the effects of TGF-P production were blocked using antibodies, the levels of perlecan protein decreased even further to the point where perlecan could not longer be detected (59). Angiotensin 11 was also found to inhibit HS production and N-sulfation in mesangial cells. Perlecan is the major HSPG of vascular basement membranes and its role in the development of the cardiovascular system has been examined. Perlecan has been shown to be a potent growth inhibitor for vascular smooth muscle cells (60,61), directly correlating its expression with the more mature quiescent cells in the blood vessel wall. In the developing rat aorta, perlecan message was found to be undetectable at embryonic days 13-17, present in low levels at embryonic day 18 and at significant levels at embryonic day l 9 and in the adult which directly correlated with the protein levels (61). During aorta development, the tissue matures from a highly replicative state to a fully quiescent, differentiated tissue. As the tissue datures, perlecan expression increases as the replication of the smooth muscle cells decreases. Perlecan mRNA is mostly limited to postreplicative smooth muscle cells, indicating that perlecan may play a role in helping to maintain the quiescent state of the matured blood vessel wall (61). In the developing mouse embryo, perlecan expression was observed in early vasculogenesis, embryonic day 10.5, including the heart, peric~dium,and major blood vessels (62). The observed developmental timing indicates that perlecan may have a role in controlling smooth muscle replication (62) and these observations in perlecan expression are most likely directly related to regulation of growth factor stimulation. Additional evidence of perlecan’s inhibitory activity is revealed by studies on skeletal muscle differentiation. Myoblasts have been shown to express perlecan protein on their cell surface and contain perlecan message (63). Conversely, differentiated myotubes lack perlecan on the cell surface and message for perlecan is decreased during skeletal muscle differentiation. FGF-2 is a strong inhibitor of skeletal muscle differentiation. Therefore, the differentiated myotubes con-

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trol the inhibitory actions of FGF-2 by lowering or removing perlecan from the cell surface so that even if FGF-2 is present, it cannot bind the receptor as an active molecule and signal the cell. Since there is a high concentration of perlecan on the surface of the myoblasts, it is also possible that perlecan acts as an adhesion molecule that is not needed for attachment once the cells have differentiated (63). Studies on the nematode mutants have revealed that perlecan does have a critical role in muscle cell attachment and in development. ~ i s ~ p t i of o nperlecan expression can lead to paralysis and alterations during embryogenesis as well as in later development. Perlecan or the unc-52 gene products were found to be expressed as early as the comma stage of development and are critical for myofilament lattice assembly and muscle attachment as well as the maintenance of myofilament organization (37,64). Since perlecan was found to behave in a cell-autonornous fashion, not spreading beyond the borders of the matrix underlying that cell, the primary function of perlecan was determined to be anchoring the myofilament lattice (64). The distribution of perlecan during pulmonary development has been examined at the newborn and adult levels. Perlecan was found to be a major component of alveolar and airway basement membranes from birth through adulthood and was prominent in pulmonary arteries but only weakly expressed in pulmonary veins (65). Although perlecan expression in fetal lung was not examined, FGF-2 is present in prenatal, postnatal, and adult lungs which suggests an early developmental role for perlecan in the neonate. In intestinal development in the rat, perlecan’s pattern of distribution undergoes a change near birth where the basement membrane expression of perlecan is discontinuous and irregular from the middle to the top of the villi (66). Interestingly, HS was found to be poorly or completely unsulfated in the early fetal intestine (66). In the adult intestine, perlecan expression is similar to that of other basement membrane components where it is found in all the layers ofthe intestine including the subepithelial basement membrane, blood vessels, muscularis mucosae, and around muscle cells (66). In a very comprehensive paper on the deve~opmentalexpression of perlecan in mouse embryogenesis, perlecan expression was found to generally correlate with tissue maturation (62). Perlecan synthesis and deposition varied.greatly between vascular and avascular tissues. Perlecan expression was prominent in the endothelial basement membrane of all vascularized organs such as liver, lung, spleen, pancreas, and kidney. Perlecan was also expressed at critical sites of vasculogenesis, again indicating a role in angiogenesis. Perlecan expression was found to be universally associated with organ maturation and remained high through adult life in endothelial layers and in mesenchymal organs. Perlecan expression also appeared in cartilaginous development, particularly during endochondral ossification where perlecan has a role in osteocartilage development and homeostasis. Perlecan was found to be primarily expressed by proliferating chondrocytes while the surrounding bone was devoid of proteoglycan. Hypertro-

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phic chondrocytes also synthesize perlecan which may even be a developmental marker for stages of cartilage development. However, the protein and message levels of perlecan did not always correspond directly, with protein levels often being higher. This finding may indicate that once perlecan is deposited into the extracellular matrix it has a long half-life and subsequent slow turnover rate (62). In the CNS, perlecan was found only in the choroid plexus of the developing mouse brain (62). However, perlecan expression was found to increase during neuronal differentiation of P19, murine embryonal carcinoma cells (67). The P19 stem cells differentiate into neuronlike cells when stimulated with retinoic acid. The stern cell layer was found to contain mainly CSlDS with only minor levels of perlecan expressed as an HSPG. When the P19 cells were stimulated to differentiate, the primary proteoglycan peak contained perlecan, representing an increase in MSPG production by almost fourfold, with minor levels of CS/DS proteoglycan (67).The increase in perlecan protein was correlated with an increase in its mRNA which showed a 1Zfold increase between neurons and stem cells. Additionally, the HSPG was larger in size in the differentiated cultures compared to the stem cells. Immunostaining revealed perlecan along the cell bodies and processes of the neurons whereas the stem cells lacked this staining pattern (67). The increase in expression of perlecan in differentiated cells coincided with an increase in the expression of P-amyloid precursor protein suggesting that perlecan may play an important role in Alzheimer’s disease (67). The correlations between perlecan and Alzheimer’s disease will be discussed in Section F.

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Basement membranes are formed by interactions among laminin, collagen IV, collagen XVIII, entactin/nidogen, fibulin, agrin, and perlecan, which are most of the major components of basement membrane identified to date. Perlecan has now been shown to bind many of these basement membrane components and recent studies have provided insight into how these matrix molecules may interact to form this complex structure. The binding of mouse perlecan to mouse laminin 1 (comprised of al , Pl, yl chains) appears to be mainly HS-chain-dependent. This interaction involves the E3 fragment of laminin which is the C-terminal globular domain of laminin A ( a l ) chain shown to have heparin binding properties (68). ~ r o s o p h iZ alaminin, comprised of a5, Pl, yl chains, was also found to bind mouse perlecan (69) and, interestingl~,was found to bind perlecan with higher efficiency than mouse perlecan binding to mouse laminin l , This may indicate that perlecan can bind ~ u l t i p l eisoforms of laminin with different affinities. One model of basement membrane does indicate the interaction of perlecan with laminins 1, 6, and 7 (70).The ~ r o s o p h iZ laminin-perlecan a interaction was

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also found to be dependent mainly on the HS chains where treatment of perlecan with heparitinase decreased binding by at least 500-fold (69). The HS-laminin binding was carbohydrate-specific in that the addition of heparin completely inhibited perlecan-laminin binding, whereas the addition of CS had no effect despite the fact that perlecan can be a HSICS hybrid. Using recombinantly expressed domains to map the binding $ofperlecan to laminin, it was found that HS substituted on both domains I and V of perlecan could bind to the laminill A chain g l o b u l ~domain with an affinity of 5-7 nM (71). Since perlecan has the capability of binding laminin at both N- and C-terminal ends of the proteoglycan, it is possible that these two ~ o l e c u l e scould form a linear copolymer in the basement membrane (71). Although the majority of the high-affinity interactions between laminim and perlecan involves the HS chains of perlecan, it has also been reported that the core protein of perlecan is capable of binding laminin possibly through perlecan domain V (38). Perlecan’s HS chains also bind to collagen IV (68). Entactin contains three globular domains separated by two rodlike segments. Perlecan, collagen IV,fibulin, and laminin all bind to entactin, which appears to act as the major linking agent of the basement membrane. Perlecan and collagen IV have been shown to bind to the second globular domain of entactin (G2) although the two molecules do not compete for the same binding site (68,’72). Additionally, the core protein of perlecan, and not the HS chains, appears to be involved in this interaction albeit at a lower affinity than the laminin-perlecan interaction (38,68). Recombinant domain V of perlecan has also been shown to bind to entactin (38). The perlecan core protein to entactin interaction appears to be instigated mainly through entactin 6 2 d o ~ a i nalthough some weaker affinity interactions have been reported with the G3 domain of entactin (38). ~ i ~ u l i nhas - 2 also been reported to interact with both perlecan core protein and recombinant domain V of perlecan, albeit with a much lawer affinity than to l a ~ i n i nor entactin (38). Perlecan can also be found in many extracellular matrices and has also been shown to interact with other extracellular matrix components. Perlecan’s core protein, in addition to its HS chains, has been reported to bind to fibronectin. This i~teractionwas first reported by Isemum et al. (73) who discovered a hybrid HS/DS perlecan isolated from human placenta bound to fib~onectinand laminin but not to collagens I, 11,IV or gelatin. The latter contradictory result may have been due to the tissue specificity of the perlecan isolated. The core protein of perlecan can bind fibronect~n(73) in a dose-d~pendent(74), selective manner under physiological conditions and with a high affinity of Kd = M (75). The interaction between perlecan and fibronectin has been mapped to the second type I11 repeat located C-terminal to the gelatin-collagen binding domain and within the N-terminal region of the 140 kD heparin binding/cell binding fragment of fibronectin (75). A separate study found that perlecan did not bind the 120 kD cell binding domain fragment of fibronectin, support in^ the evidence that

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perlecan binds within the first couple of type I11 repeats (74). Mesangial cell adhesion to fibronectin was found to be inhibited by both intact and core protein of perlecan (74). The source of perlecan used in this study was porcine and it is not yet known if this species of perlecan contains the RGD sequence. However, Gauer et al. (74) found that perlecan strongly increased the inhibitory effect of exogenous RGD peptide, indicating that the antiadhesion effect did not involve a RGD sequence from perlecan, Fibronectin contains 2 heparin-binding domains, one at the N-terminal end of the molecule and another, higher-affi~ityheparinbinding domain located toward the C-terminus, both of which are thought to interact with HSPGs (76). Therefore, it is not surprising that perlecan has also been found to bind fibronectin through its HS chains (68,75). Much is known about how fibronectin interacts with integrins to link cell surfaces to the extracellular matrix and this subsequent interaction with perlecan may function to “pull” the HSPG towards the cell surface. Subsequently, perlecan may be stabilized within the matrix by fibronectin and this interaction may also act to anchor the pericellular matrix to the underlying interstitial matrix (75). Another extracellular matrix molecule that has been shown to interact with perlecan is thro~bospondin.Vascular endothelial cells not only have perlecan on the apical cell surface, a location that was originally observed in colon carcinoma cells (77), but also have perlecan localized as a dense fibrillar network surrounding the cells tightly associated with the subendothelial matrix (78). Thrombospondin l is also localized at the cell surface of endothelial ,cells and the possibility of the two molecules associating was examined, It was found that perlecan could bind thrombospondin 1 in a specific, dose-dependent manner, that this interaction was dependent on perlecan’s HS chains, and that calcium was found to influence the interaction (78). This study also proposed that perlecan was required for binding and organizing thrombospondin 1 at the apical surface of vascular endothelial cells and that this interaction may be involved in receptor mediated endocytosis (78). A novel cell surface binding protein called heparin/HS interacting protein (HIP) has also been shown to colocalize with and apparently bind to perlecan in an HS-dependent manner with high affinity (79). HIP functions as an adhesion molecule for several cell lines including trophoblastic cells and colocalizes in the uterine stroma at regions of invasive cytotrop~oblastsexpressing HIP and in the basal lamina underlying the vascular elements (79). This study indicates that perlecan and HIP appear to interact during cytotrophoblast to uterine matrix interactions in human placentation (79).

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2. Growth F~ct ors/ Cyt ok in~s

The fibroblast growth factors (FGF) are a family of cytokines that are potent regulators of cell proliferation and differentiation. There have been fifteen FGFs

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identified, FGF- 1 through FGF- 15. Like many cytokines, FGFs have a high affinity for heparin but unlike other molecules, they require heparin or HS binding for activation of their receptors. The members of the FGF family that have been shown to require HS for signaling are FGF-1, -2, -4, -5, -8, and - 9 (80). Different FGFs appear to have distinct HS requirements for receptor activation which may reflect different tissue requirements. We have already discussed how different tissues or cells can express perlecan as HS, HSKS hybrid, or CS proteoglycans. Interestingly, FGF binds with higher affinity to perlecan that has only HS chains compared to a HSKS hybrid or a mainly CS form of the proteoglycan (23,81); when perlecan’s GAG chains are cleaved from the core protein they are not as effective in promoting FGF responses as intact proteoglycan (81). This indicates that the core protein of perlecan allows the GAG chains to have a secondary structure more conducive to growth factor binding or that the overall charge density of the three HS chains in close proximity allows for a more efficient binding of FGF to its receptor. Studies have also shown that perlecan is the only HSPG examined that was highly effective in potentiating FGF binding with no significant activity detected for other HSPGs such as syndecan, fibroglycan, or glypican (82). The absoluteness of the perlecan requirement for FGF signaling is exemplified by experiments where the expression of perlecan is null, such as in the C H0 HS-deficient mutants or by the expression of perlecan antisense cDNA. In these studies the cells are not capable of eliciting a response to FGF at all (82,233). There are four FGF receptors which are tyrosine kinases that depend on phosphorylation of the cytoplasmic tail, and like other cytokine receptors have promiscuous binding properties allowing for binding and activation of several FGFs with varying affinities. This latter property of the receptor allows different tissues or cells to have different cellular responses to the various FGFs. Fried1 et al. (80) have shown that FGF-2 and FGF-7 bind to basement membranes from various tissues in strikingly different patterns, emphasizing the different HS requirements for the different FGFs. The FGF-heparin or HS interaction may induce a conformational change in the growth factor which allows binding to the high-affinity receptor. Another possibility is that the HSPG, FGI;, and FGF receptor form a complex that is stabilized by the presence of HS; possibly even 6-0 sulfation of the chains is needed before activation of the receptor takes place (80,84). Control of FGF stimulation can be regulated by three different mechanisms: the expression of the growth factor, the expression of the FGF receptor, and the expression as well as chain Composition of the HSPG, namely perlecan. Perlecan appears to play a significant role in the regulation of FGF stimulation not only through the binding of FGF directly, most likely through the HS chains in domain I, but also in being capable of sequestering the growth factor in the basement membrane (54). In the basement membrane of the cornea, HSPG i s believed to be responsible for sequestering FGF-2, preventing the growth factor from stimulating vascularization of this avascular tissue ( $5) The . colocalization

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of perlecan and FGF-2 and the requirement for these two molecules for angiogenesis suggest a regulatory role in tissue and tumor vascularization (82). The abnormal release of perlecan from the basement membrane could be responsible for corneal vascularization in a variety of ocular diseases or severe injury (85). Conversely, the codistribution of HSPG and FGF-2 in developing first-trimester placenta appears to be pivotal for the growth of the villous tree and therefore essential to the morphological processes in the early stages of gestation (86). This latter study is the first in showing that the codistribution of HSPG and FGF-2 plays a key role in the development of an organ (86). Perlecan can also form a sheath around cells which may prevent FGF from interacting with its receptor or HS on the cell surface. The release or expression of proteases capable of degrading perlecan in the basement membrane is another method that cells can use to free FGF from the matrix during wound healing or in carcinogenesis, respectively. Plasmin, stromelysin (MMP-3), and collagenase (MMP-13), but not thrombin, have all been shown to significantly degrade perlecan’s core protein, thereby releasing significant concentrations of FGF-2 into the matrix (54). Perlecan also acts as a coreceptor for FGF-7 in human colon carcinoma cells and has been shown to bind to FGF-7 in a ~S-independentmanner (87). Clearly, the interactions of perlecan and FGF are extensive, affect many tissues, and are subject to several methods of regulation. Perlecan has al so been shown to interact with other growth factors and cytokines, although these interactions are not as well characterized, Gohring et al. (88) have found that both isoforms of PDGF (platelet derived growth factor) - AAand -BB can bind to perlecan domain 111-2 with moderate affinity, Kd 8 nM. PDGF-BB was also shown to bind domains 1, IV-l, and V but with low affinity, Kd = 34-64 nM. The binding of PDGF to perlecan appears to be specific particularly since domain 111-2 showed binding capabilities, whereas domains 111-1 and 111-3 lacked binding properties despite very similar structures (88). This interaction of the growth factor with perlecan core protein is unique since most all of the interactions reported to date involve the binding to the HS chains of perlecan. PDGF has been shown to be a potent mitogen for several cell types and is an important sti~ulant/chemoattractantduring embryogenesis and wound healing. Therefore, these findings indicate that perlecan in the basement membrane may aid in the storage of PDGF which when released may play a pivotal role in cell-matrix interactions (88). The HSPG component of the EMS tumor, presumably perlecan, appears to be the active factor in binding to IFN-y in a time- and concentration-dependent manner (89). The affinity of IFN-y for HSPG is high at 1.5 nM and specifically binds to HS and heparin, as CS, KS, and DS had no binding capabilities, The carboxy-terminal region of IFN-y, which contains amino acid sequences found in other hep~in-bindingproteins, appears to be the domain that binds the HSPG, as monoclonal antibodies to this region inhibit HS binding (89). This is an indica-

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tion that perlecan may not only bind and sequester IFN-y in the matrix andlor basement membrane, but that most other heparin binding growth factors and cytokines may be stored hidden in the matrix as well. Perlecan has also been shown to bind to transthyretin which is one of two specific proteins involved in the transport of thyroid hormones in plasma (90). Both syndecan and perlecan were found in the medium of HepG2 cells, but only perlecan was capable of binding to a transthyretin affinity column. This form of perlecan contained -20% CS and 80% HS chains; however, the interaction appears to involve the core protein since heparin was found to be incapable of binding the transthyretin-affinity column (90). This interaction between perlecan and transthyretin may have implications in both retinol metabolism and transthyretin-associated amyloidosis (90).

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Perlecan, and more specifically domain 111, has been shown to bind to integrins, pa~icularlyp1 and p3 integrins as was discussed earlier in Section B. One role of the per~ecan-integrininteraction is to adhere the cell to the surrounding matrix. Another role for this interaction has also been examined and that is the role of the integrin in organizing the u l ~ a s t ~ c t u rcomposition al of the basement membrane, Using pl-integrin double knockout of embryonic stem cells to form subcutaneous teratomas has shown that the p1 -integrin is vital to the formation of a normal basement membrane (9 l). The pl-integrin deficient tumors had a 90% decrease in laminin-1 content and a 70% decrease in the entactin content, the latter most likely being the result of undue exposure to proteases. Although the presence of perlecan, fibulin, and fibronectin were unaffected, the remarkable ultrast~ctural differences indicate that matrix interactions were certainly disrupted (91). In C. elegans, the st549 mutant is essentially a perlecan or unc-52 knockout due to a premature stop codon in exon 7. These mutants were previously id~ntifiedby defects in the localization of P-integrin and vinculin in dense bodies and ”lines of developing body wall muscle cells (37,92). This indicates that there is a critical ~erlecan-inte~rin interaction in C. elegans necessary for normal myofilament lattice assembly (37). These studies emphasize that integrin interactions with the basement membrane, including interactions directly with perlecan, are critical for proper formation of the basement membrane ultrast~ctureand can potentially affect the transcription of some basement membrane components (91).

1. ~ ~ u r o ~ a t h o l and o g y~lzheimer’sDisease The role of perlecan in the pathological processes of neurodegenerative diseases has expanded tremendously over the past 10 years. It is now clear that GAG

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7

constituents, in particular those on perlecan, are likely candidates for the causative agents of amyloidosis in Alzheimer's disease (AD) as well as CNS and systemic amyloidosis, This indicates that they are also the most relevant targets for therapeutic approaches to the disease mechanisms. The amyloid plaques that are found mainly in the hippocampus of A patient brain are comprised of a multitude of proteins and proteoglycans whic include perlecan as the major if not only HSPG, P-amyloid protein, apolipoprotein E, P component, ~1-antichymot~psin, complement factors C l q and C3, CS, keratan sulfate, and decorin as the major derrnatan sulfate proteoglycan (93,94). The P-amyloid protein (AP), which has been mapped to human Chromoso~e21, is a 39-43 amino acid peptide that is derived from the larger precursor molecules called amyloid precursor proteins, PPPs, or APP (95,96). Although a range of proteoglycans have been found in the amyloid plaques associated with AD, only HSPG has been found to be immunolocalized to all three major lesions: neuritic plaques, neurofibrilla^ tangles, and cerebrovascular deposits (97). In addition, HSPG has been found to be associated with at least five different amyloidoses which include amyloid A related to inflammation, amylin amyloid in type 2 diabetes, AP in AD, the prion amyloids and prealbumin in familial amyloidotic polyneuropathy (96). The role of perlecan or HSPG within the amyloid plaques or fibrils has been mapped using a range of antibodies which recognize the core protein and/or HS chains leading some of the reports to carefully speculate that HSPG is present but not necessarily perlecan. To date the major HSPG found to be associated with the Ap plaques is perlecan or perlecan-related variants. Castillo et al. (97) developed a novel, rapid purification procedure for isolating perlecan from the EHS tumor. The perlecan isolated by this method lacked detectable contaminating basement membrane components or free GAG, all of which can have a deleterious effect on the induction of fibrillar deposits in the animal model. When this high-purity perlecan was coinfused with Ap into the rat hippocampus, fibrillar A@deposits were formed in 100% of the animals compared to only 60% of the animals forming deposits when Ap alone was infused (97). The use of extremely pure perlecan in the perlecan-AP coinfusion procedure has provided a reliable animal model for the study f AD and possible therapeutic agents. Perlecan has been shown to bind to the proteolytically cleaved AB with two binding affinities; a high-affinjty, low-capacity interaction with I S d in t 10"1-10"2 M range and low-affinity, high-capacity association in the I O- * range (reviewed in Refs. 93, 94). The source of perlecan does not seem to alter the affinity for Ap as endothelial, smooth muscle cell, and EHS tumor perlecan all bind to AP using approsimately the same two binding affinities (93,94). Perlecan also binds to amyloid deposits present within the blood vessels and is responsible for the accumulation of Ap in the cerebrovasculature of AD patients (98). The binding of perlecan to Ap is instigated partially through its H§ chains,

particularly since free HS chains bound to AP prevents perlecan from binding and forming plaques (99); however, maximal binding requires the use of both the core protein and HS chains (93,98). One binding site on AP has been determine6 to be located between H i ~ , ~ - L , y swhich , ~ , is close to the postulated proteolytic cleavage site on Lys,, (98). The other binding site has yet to be determined but may occur between amino acids 29-40 since full-length AP (amino acids 140) has enhanced binding capabilities compared to AP amino acids 1-28 (98). The binding of perlecan to AP is only the initial step in the interactions between this protein and the proteoglycan. By binding to AP, perlecan induces a confor~ationalchange in AP from an a-helical structure to predominantly Ppleated sheets (94). Perlecan was also found to be capable of binding AP regardless of the extent of fibril formation (93). Subsequently, perlecan was found to accelerate the rate of AP fibril formation with perlecan’s capacity to bind AP actually increasing as fibril formation progresses (93). Perlecan also acts to protect the AP amyloid fibrils from proteolytic degradation and subsequent removal (93,94). Perlecan appears to have a role in the earliest stages of amyloid fibrillogenesis since its expression precedes that of detectable amyloid in the mouse model of AA amyloidogenesis (100). These findings indicate that the perlecanAP interactions are the major pathogenic regulators of a multitude of amyloidoses. In AD brain amyloid core containing neuritic plaques and diffuse plaques exist in many regions including the hippoca~pus,amygdala, neocortex, and cerebellum, although the cerebellum contains mainly diffuse amyloid plaques (95). Upon further examination it was determined that the diffuse amyloid plaques of the cerebellum lacked perlecan or HSPG even in the presence of AP and other plaque components, whereas the infrequent amyloid core fibrils did contain perlecan core protein and HS (95). Conversely, both diffuse plaques and amyloid core fibrils in the hippocampus contained perlecan core protein and HS, The presence of perlecan in the hippocampus is responsible for the maintenance and persistence of the amyloid plaques where the absence of perlecan in the diffuse plaques of the cerebellum allows, for the most part, this region of the brain to clear the amyloid from these plaques. This also brings into question which cell types in the brain are responsible for the production and deposition of perlecan. In developing mouse embryo, the neuroepithelium, mesenchymal, and endodermal tissues produce the perlecan-related molecule (PRM) at high levels whereas the intact perlecan seems to be almost absent (20,62). Message levels for perlecan were found to increase over 12-fold in cultured P19 cells that were induced to differentiate into neuronal cells (67). Using the rat model to induce amyloidogenesis by infusion with Ab and perlecan, microglia/macrophages were found to be located primarily at the infusion site (101). In cell culture, microglia produced perlecan as both an HSPG and glycoprotein (101). And RT-PCR has identified perlecan message in both normal human and AD patient microglia cultures (101). Perlecan was immunolocalized to the microglia within rat brain but not

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within human brain despite the fact that the antibody is able to stain perlecan in the amyloid deposits (101). This may indicate that a perlecan isoform such as PRM could be involved or that the available epitopes are blocked or buried. Using primers within domain I of perlecan, RT-PCR was used to determine if the levels of perlecan message increased within the brains of AD patients compared to controls (21). These studies showed that perlecan mRNA levels were not increased in AD compared to normal brains. This implies that the perlecan message levels are at a steady state level and that perlecan may have a lower turnover rate in AD hippocampus, Another explanation is that the AD tissue used was from advanced stages of the disease and this affected the levels of perlecan message (21). Perlecan plays a critical role in the development, formation, and persistence of amyloid plaques in AD brain, and several studies have tried therapeutic measures aimed at targeting HSPG perlecan. Since HS GAG chains alone infused with AD prevented the formation of amyloid plaques in rodent brain, a likely target would be to protect Ap from binding to perlecan. Low molecular weight (MW) forms of heparin were used in an in vitro blood-brain barrier model to ascertain whether these oligosaccharides would cross the blood brain barrier and prevent the deleterious effects that full-length heparin or perlecan can induce ( l 02). This study found that low MW forms of heparin, particularly disaccharides, were effective in crossing the in vitro blood-brain barrier and prevent in^ the binding of heparin to Ap peptide (102). In addition, the low MW forms of heparin did not induce any observed toxic effect. Another study successfully used sulfonates and sulfates to inhibit fibril formation in a mouse model of AA amyloid formation (94). These studies appear promising at these preli~inarystages and certainly warrant further investigation as therapeutic agents for AD and other types of amyloid plaque-induced neurodegenerative diseases.

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2.

Other ~iaq~e- elated Diseases

Perlecan has also been implicated in the formation of amyloid fibrillar plaques related to other, non-Al~heimer’s,diseases. In type 2 diabetes, the presence of amyloid deposits in the pancreatic islets occurs in more than 90% of the patients (103). Arnylin is a 37 amino acid peptide in the islet amyloid, and its fibril formation in the pancreas may cause islet cell dysfunction and cell death. Perlecan has now been shown to bind amylin with moderate affinity in the low6M range and was found to be a potent enhancer of fibril formation in a dose-dependent manner (97). It was also determined that the HS chains were primarily responsible for the proteoglycan-amylin interaction but not totally dependent on it either. This study indicates that perlecan’s ability to bind to amylin enhances amylin fibril formation and appears to play an important pathological role by contributing to the toxicity observed within the pancreatic p cells of type 2 diabetic patients (103).

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Scrapie is a member of a group of fatal transmissible s p o n g i f o encepha~ lopathies in which amyloid plaques are a prominent pathological feature (104). The scrapie plaques are comprised of infection-specific host-coded cell surface sialoglycoprotein or PrP, The disease characteristic of the mouse scrapie mode1 has many similarities to human AD, although the two diseases are biochemically distinct; the AP and PrP amyloid plaques share similar mo~hologicalappearances and Congo red dye binding properties, In scrapie-infected mice, HSPG (both perlecan core protein and HS chains) were found in association with all forms of PrP pathology (104). These studies also indicated that HSPG may be an early marker for infection and may precede the appearance of abnormal PrP. HSPG may also promote the alignment of the fibrillar arrays, thereby enhancing plaque formation and stability, and may be responsible for the conformational change in the amyloidogenic protein from an a-helical structure to a P-pleated sheet (104). Perlecan, and HSPG association with diffuse forms of PrP in neuroanatomically defined target areas, appears to contribute significantly to the pathology of the transmissible neurod~generativedisease, scrapie (104). In summary, HSPG and perlecan have been shown to be critical stimulators in amyloidosis by (1) influencing the secondary structure of amyloid proteins from an a-helical structure to one of predominantly P-pleated sheets, (2) determining the anatomical location of amyloid accumulation, and (3) making a critical contribution to the stability of amyloid fibrils, including protecting them from proteolysis (94). Perlecan/HSPG has been found to be associated with all amyloid plaques in many diseases, including Alzheimer’s disease, infectious prion diseases such as scrapie, and in the pancreatic islet amyloids found in almost all type 2 diabetic patients. Down’s syndrome patients, defined by trisomy 21~hromosome21 which also encodes the amyloid gene, were found to have prominent HS immunoreactivity in neurons as early as one day after birth in the absence of any AP reactivity (94). The studies on the Down’s syndrome patients indicate that the early HS accumulation in neurons may be a primary event leading to the formation of perlecan and Ab plaques. Perlecan core protein and HS chains contribute to the protection and persistence of the amyloid plaques in these diseases and effective therapies will need to target the AP to perlecan interaction in order to be effective.

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3. Cancer

Most tumor cell sources upregulate perlecan in order to enhance responses to FGF andlor other cytokines such as TGF-P. This has been found to be true for breast, colon, liver, lung, and melanomas (12). In human breast carcinomas, both invasive and noninvasive, the epithelial mRNA level was increased for perlecan as well as the other matrix components (105). Tumor and stromal cells had increased mRNA levels for perlecan but the extracellular levels of perlecan were

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not as high as expected possibly due to an increase in protease activity (105). In fact, several proteases such as plasmin and the metalloproteases, stromelysin (MMP-3), and collagenase (MMP-13) have been shown to degrade perlecan and release signi~cantlevels of FGF into the medium (54). In normal breast tissue, the glands are su~oundedby intact basement membrane. Conversely, noninvasive intraductal carcinomas had basement membrane focal defects and duplications while invasive carcinomas had very little intact basement membrane left (105). In liver neoplastic nodules there was a 20-fold increase in GAG content compared to normal liver, and in invasive melanomas perlecan expression was elevated 15fold with abundant perlecan deposition in the pericellular matrix (106,107). In both human melanoma cells and in human breast carcinomas highly elevated levels of perlecan mRNA were associated with invasive potential, and in melanoma cells the enrichment of basement membrane perlecan was directly correlated with metastatic capabilities (105,106). Conversely, in lung cancer and in ~brosarcomacells low levels of perlecan expression were correlated with increased tumor growth and metastatic potential (108,109). These differences are most likely related to the different cellular growth factor requirements for proliferation and differentiation. Perlecan localization in breast tissue was found to be scattered in invasive ductal and papillary carcinomas as well as surrounding the tumor blood vessels (82). Perlecan, in conjunction with FGF-2, is capable of inducing angiogenesis which appears to be the function of the proteoglycan in the latter observation. High levels of perlecan deposition are present in the newly vascularized stroma of breast, colon, and prostate carcinomas, also most likely due to a FGFlperlecan interaction (12). In human primary liver tumors, perlecan and syndecan-3 were present in high concentrations within tumor stromal vessels, suggesting a perlecan-dependent enhancement of angiogenesis (1 10). Blocking perlecan synthesis by stably tra~sfecting hum^ colon carcinomas cells with perlecan antisense cDNA led to the prevention of neoplastic growth, mainly through the inactivation of FGF-7 receptor binding/signaling, inhibition of tumor angiogenesis, and cellular invasion (87). Sharma et al. indicated that perlecan gene expression is essential for the processes that lead to tumor capillarization and that anticancer approaches that implement restrictions on perlecan synthesis may be effective in regulating tumor growth as well as metastatic potential (87). FGF cellular responses have also been postulated to be the mode of action for metastatic liver tumor increase in perlecan synthesis and metastatic cell growth (107). Perlecan is capable of storing FGFs in the basement membrane, and growth factor release by proteolytic degradation may result in a large pool of available FGF capable of stimulating cell growth andlor angiogenesis (54). TGF-P is another cytokine that has been. implemented in accelerating the negative effects of tumor cells. TGF-PI mRNA and protein levels were found to be significantly increased in both tumor and stromal cells of mammary carcinoma which was postulated to be responsible for

the increase in basement membrane components (105). TGF-P was also found to elevate message levels, core protein synthesis, and subsequent sulfate incorporation in human colon carcinoma cells (5’7). The importance of an intact basement membrane, controlled deposition of perlecan in the pericellular matrix which regulates the cellular stimulation by FGF and the maintenance of normal HS chain composition, in particular sulfation content, may be critical to the containment and growth control of a variety of tumors and carcinomas.

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4. Kidney

The kidney glomerulus contains two extracellular matrices, the g l o m e r u l ~basement membrane and the mesangial matrix, which harbor large amounts of proteoglycans as well as collagens and glycoproteins. These matrices are synthesized and maintained by glomerular visceral epithelial cells, glomerular endothelial cells, and the mesangial cells (1 11-1 13). The highly anionic GAGS within these matrices appear to be responsible for maintaining a fixed negative charge at the filtration barrier and provide structural support, in addition to sequestering growth factors and cytokines needed for proliferation and matrix synthesis (1 12,114). Perlecan is one of the HSPGs present within both the glomerular basement membrane and the mesangial matrix (32) and is rapidly turned over with a t1,2 of approximately 3 hr for 95% of the perlecan population (l 15,116). Using a monoclonal antibody raised against recombinant domains I and 11,the immunolocalization of perlecan within the glomerular basement membrane was characterized as being on the endothelial side of the matrix and distributed in a nonhomogeneous manner with many segments of the basement membrane lacking detectable perlecan (1 16). The nature of the cells responsible for synthesizing perlecan is more controversial. Several in vitro studies have shown that a variety of kidney cells produce perlecan. One group used RT-PCRwithin domain I and found that both human mesangial cells and glomerular visceral epithelial cells produce perlecan (1 1I), another group found that rat mesangial cells produce perlecan in culture (1 12), while a third study found that MDCK strains I and 11, representing distal tubule epithelial cells and proximal tubule cells, respectively, synthesized and secreted perlecan mainly basolaterally (1 13). Another in vitro assay showed that angiotensin-I1induced a decrease in perlecan message in mesangial cells and that the concurrent induction of TGF-P expression acted to partially block the inhibitory effects of angiotensin-I1 on perlecan transcription (59). Conversely, one in vivo study found, using in situ hybridization with a domain V probe, that in rat kidney, glomerular cells did not contain perlecan transcripts and that only the papillary tubuli contained mRNA for perlecan (114). It has been difficult to isolate full-length perlecan from the kidney and it has even been proposed that the immunoreactivity observed in the matrix may be a proteolytic fragment of per-

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lecan, synthesized elsewhere, that became trapped in the glomerular basement membrane as part of the filtration process (1 14). These differences between the reports may be due to in vitro versus in vivo analysis since perlecan expression can be induced in several cell types by simply growing the cells in tissue culture. Another explanation is that since different domains of perlecan were being studied, the results may be due to alternative splicing of perlecan or the presence of multiple HSPGs. A polyclonal antibody to HSPG that recognizes a nonperlecan core protein immunostains the glomerular basement membrane in a linear manner and is less widely distributed in various tissues than perlecan (l 16). Another study described a HS/CS proteoglycan hybrid with a core protein of 80 that does not react with either perlecan monoclonal or polyclonal antibodies ( l 1I ). We now know that perlecan is not the only HSPG in glomerular basement membranes: agrin and collagen XVIII are also present and they may play a role in ionic filtration (see the following sections on agrin and collagen

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XVIII) In pat~ologicalprocesses, the glomerular basement membrane undergoes alterations and perlecan synthesis is affected. In diabetes, the glomerular basement membrane thickens with an increase in collagen IV and there is a decrease in overall HSPG content in diabetic animals (48,49). When rat glomerular, mesangial, or smooth muscle cells were cultured in the presence of 30 mM high glucose, there was a marked decrease in perlecan or sulfate incorporation (48,49). In rat glomerular cells the decrease in perlecan synthesis was partially due to a reduction in mRI?A, but transcription and posttranslational controls also appeared to contribute to the reduction of perlecan in the matrix (48). In type I (insulindependent) diabetes, perlecan p o l y m o ~ h i s ~ were s found in the perlecan gene (HSPG2), where results indicated that patients possessing a 250 bp allele had a risk of nephropathy at 2.4 fold those not containing the 250 bp allele (1 17). This is the first report that shows an association of diabetic nephropathy and the gene of a structural glomerular protein (117). I

5. Smooth Muscle

The functions of the extracellular matrix of cardiac and vascular tissues include moderating the intensity of the physical forces such as pressure, stretch, and shear force under both normal and pathological conditions (1 18). Smooth muscle cell replication and matrix deposition have been implicated in the pathogenesis of atherosclerosis and other vascular pathologies. Smooth muscle cells (SMC), the major type of cell in the arterial wall, are normally quiescent. However, during vascular development and under pathological conditions these cells proliferate and produce an abundant extracellular matrix ( l 19). In vitro, SMC plated at low density and induced to proliferate increased their proteoglycan synthesis signifi-

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cantly compared to quiescent cells (120). A dramatic increase in the steady state levels of perlecan, and other matrix molecules, mRNA were also noted (120). The overall changes in proportions of proteoglycans synthesized in proliferating versus quiescent SMC lead to alterations in the affinity of the proteoglycan fraction to low-density lipoprotein, LDL (120). Since there was an increase in HSPGs and perlecan domain I1 is homologous to the LDL receptor, it appears likely that perlecan may be a candidate for binding LDL in the proteoglycan fraction. Perlecan has been shown to negatively regulate the expression of the transcriptional factor Oct-1 which is required for cell replication (119). SMC do not express Oct-l in uninjured, adult vessel wall but Oct-1 transcripts are rapidly induced upon disruption of SMC-matrix interactions. Perlecan was the only matrix component tested shown to have a negative regulatory effect on the transcription of Oct-l . In a proposed model, adult SMC are surrounded by a perlecan-rich growth inhibitory matrix that prevents SMC replication through the negative regulation of the expression of transcriptional factors such as Oct-l (1 19). SMC have been shown to increase matrix production after arterial injury and in response to elevated blood pressure induced by coarctation ( l 18,121). In the rat carotid artery balloon injury model, message levels of perlecan and other proteoglycans were found to increase starting one week after injury and perlecan d N A levels remained two times higher, two and four weeks after injury (121). Perlecan protein was also found to present in the neointima and the media after injury (121). In the rat ~ o ~ c t a t i hype~ensive on rat model, perlecan steady state message levels were found to increase, along with other basement membrane components in the heart and vasculature, and peaked before maximal hypertrophy occurs at five days; however, the levels of perlecan deposited in the matrix remained unchanged ( l 18). Since two glycoproteins, laminin and fibronectin, were found to increase in both message and protein deposition, this indicates that in hypertrophied vasculature, basement membrane deposition appears to be controlled at both. the transcriptional and translational levels (118). Perlecan appears to have a dual role in vascular pathology. Under normal conditions, perlecan acts as a negative regulator of proliferation by preventing the expression of transcriptional factors such as Oct-l required for replication and may even sequester growth factors, thereby preventing the stimulation of SMC growth (1 19). During vascular injury there is increased deposition of matrix and stimu~ationof SMC replication. Perlecan was found to be deposited along with other matrix molecules in both atherosclerotic lesions and restenosis lesions but not in the hyper~ophiedvasculature. However, perlecan is absent from early atherosclerotic lesions but accumulates to higher levels in advanced lesions colocalizing with growth factors and cytokines (122). Whether there are differences in HS chain composition, between normal and vascular pathogenesis, which would change the affinity of perlecan for growth factors and hence the role of the proteoglycan in the matrix, has yet to be determined,

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6.

Liver

Normal adult livers contain only a loose extracellular matrix, and continuous basement membranes are predominantly located around vessels and bile ducts (l 23). Basement membrane components are abundant in the sinusoids of developing livers, then steadily decrease after birth and by adulthood contain a discontinuous basement membrane (123). In normal adult liver, perlecan has been localized to the liver vasculature, bile ducts, sinusoids, and the space of Disse discontinuously interacting with hepatocyte microvilli, indicating a close interaction of hepatocytes with the perlecan basement membrane (123- 126). The cells shown to synthesize perlecan in vivo are vascular and sinusoidal endothelial cells, bile duct cells, and epithelial cells of the Hering canal (123,125). In the developing liver, perlecan is one of the first matrix proteins expressed and perlecan levels were higher in 17- and 19-day-old fetal and neonatal livers than in normal adult (123). In vivo hepatocytes and Ito cells do not synthesize perlecan, but studies in vitro indicate that in cell culture both hepatocytes and Ito cells as well as hepatic stellate cells secrete perlecan (123-126). However, cell-to-cell contacts between hepatocytes and Ito cells appear to be a prerequisite for complete basement membrane formation and hepatocytes have been shown to bind to a perlecan matrix most likely through p1 integrins (123,125). Many chronic liver diseases including hepatic schistosomiasis mansoni, chronic cholestasis, cirrhosis, and necrosis lead to fibrosis during which the deposition of perlecan and other basement membrane components increases dramatically (123,124,126,127). Perlecan has been identified in most of the fibrotic liver diseases as well as liver regeneration where it plays a role through growth factors and cytokines in addition to its increased expression in the matrix (128). Perlecan’s role in the pathogenesis of liver tumors has also been examined. In human primary tumors there was strong expression of perlecan as well as syndecan-3 in tumor stromal vessels and liver tumor cells also had an altered HSPG staining pattern (110). When liver tumors were induced in rats, perlecan was found in a continuous layer in the space of Disse, in malignant nodules, and intracellular staining was found in sinusoidal endothelial cells (125). These studies indicate that perlecan’s role in liver tumorigenesis may be in tumoral angiogenesis and as a growth factor reservoir within the malignant stroma (110).

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7. Cartilage, Bone, and ~ e ~ a t o p o i e tCells ic The bone marrow microenvironment consists of stromal cells, the surrounding extracellular matrix and bound growth factors all of which are essential for controlled proliferation and differentiation of hematopoietic progenitor cells (129). Perlecan is highly expressed in human bone marrow as well as in long-term marrow cultures, thought to mimic hematopoiesis in vitro (129). Perlecan has an antiadhesive effect on unfractionated bone marrow cells and this effect appears

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to involve the core protein since heparitinase treatment did not abolish the antiadhesive effect (129). The multipotential cell line derived from a patient with chronic myelogenous leukemia, K562, produces significant amounts of perlecan and this expression could be increased at least twofold by treatment with turnorpromoting phorbol esters, which differentiates the cells into rnegakaryoblasticlike cells (130). In contrast, HL-60, a promyelocytic cell line, produced periecan levels that were barely detectable while a murine bone marrow stromal cell line MS-5 was found to synthesize 7 different HSPGs, including perlecan which was present at low levels compared to the other proteoglycans produced (130,13 1). Several hematopoietic cell lines including K562, HL-60, and KGla were shown to have antiadhesive behavior when plated on perlecan substrates (129). These studies suggest that perlecan may play a role in the differentiation of cells of lymphoid origin, bind growth factors such as granulocyte/macrophage colony stimulating factor, and provide an antiadhesive surface which appears to be related to co~partmentalizationof the microenvironment ( 129,330). Hyaline cartilages contain specialized connective tissues comprised of chondrocytes surrounded by abundant matrix that is designed to resist compression (132). Aggrecan is the major proteoglycan of cartilage tissues which contains mainly CS chains but also keratan sulfate chains. Perlecan is also located in the matrix of nasal septum hyaline cartilage, the articular surface of the bone and the growth plate of developing bone (132). ~mmunostainingof hyaline cartilage showed that perlecan is primarily deposited and retained in the chondrocyte pericellular matrix (132). Proteoglycan extracts of bovine articular cartilage indicated that perlecan is present as a HS/CS hybrid in this tissue and that the core protein was smaller in size than expected, 260 kD (132). During murine embryogenesis, perlecan expression was found in cartilaginous development especially during endochondral ossification (62). Perlecan was expressed primarily by proliferating chondrocytes, while the su~oundingbone was negative for perlecan. Hypertrophic chondrocytes appeared to be primarily responsible for perlecan production and it could possibly be a developmental marker for this stage of cartilage development (62). Perlecan was also found to be secreted by chondrocytes derived from rat chondrosarcoma, and perlecan functions as an adhesive surface for immortalized rat chondrocytes in vitro (132). The role of perlecan in cartilage appears to be to support development of osteocartilage and homeostasis, provide an adhesive matrix for chondrocytes, interact with other macromolecules to produce the compressed matrix, and bind necessary growth factors (62,132). The synovial-cartilage junction is highly vascularized, in contrast to the avascular cartilage tissue, and is important to the pathology of the joint where i n ~ a ~ m a t o diseases ry such as arthritis cause damage (58). Cultures of normal human synovial cells were shown to contain perlecan mRNA and produce high levels of perlecan as a HSPG (58). The synthesis of perlecan's core protein by

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synovial cells could be upregulated by TGF-P and downregulated by FGF-2 (58). Dodge et al. indicates that perlecan is present in human synovium, its synthesis is controlled by growth factors and cytokines, but whether perlecan has an active role in the pathogenesis of arthritis has yet to be determined (58).

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Agrin was first isolated and identified from extracts of the electric organ of electric ray Torpedo ca Z~ or?z ica as a 150kD polypeptide that causes patches of acetylcholine receptors (AChRs) and acetylcholinesterase (AChE) to form on cultured myotubes (133). AChRs and AChE are concentrated in the synaptic cleft at the neuromuscular junction and the 150 kD polypeptide that caused them to assemble in patches was named agrin, from the Greek “ageirein,” to assemble (1 33,134). cDNA clones to rat ( l 35), chick (136), and ray (137) agrin were subsequently obtained and the deduced sequence from these cDNAs indicated the molecular m ass of the protein to be larger than 200 kD. In separate studies, a HSPG that copurified with NCAM was isolated from chick brain (138). This proteoglycan was initially characterized using antibodies and found to be unrelated to any previously characterized proteoglycan, but using these antibodies to isolate cDNA clones to this proteoglycan yielded cDNA clones that matched agrin (139). Furthermore, regions of the deduced chick sequence were homologous to amino acid sequences obtained from peptides derived from an HSPG isolated from bovine renal tubular basement membranes (140). Concurrently, a primer extension library made for chick agrin yielded cDNA clones that contained an additional 350 bp of 5’ sequence that encoded for a signal peptide and novel N-terminal sequence (141). A,chick agrin cDNA containing the additional 5’ sequence was transfected into COS cells and expressed as an HSPG, while agrin cDNA lacking this 5’ sequence was not produced as a segregated recombinant product (141). These studies now convincingly identify agrin as an HSPG. The initial studies, however, did not recognize agrin as an HSPG. There are several explanations for this. The proteolytic fragments of agrin were biologically active in causing AChRs and AChE to assemble in patches, s o there was little motivation to l ook for larger forms. These biologically active fragments are from the C-terminal half of agrin, and since there are no GAG attachment sites on this region it would not appear to be a proteoglycan. However, even if there was some intact agrin (the proteoglycan) in the preparation, it would run as a Smear on the gel (and therefore be spread or diluted over a large area), and because proteoglycans do not transfer well from the acrylamide gel to the nitrocellulose

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membrane, the proteoglycan form would not be readily detected by these antibodies. Once the GAG side chains are removed by heparitinase or nitrous acid the core protein is readily detected by Western blot (139).

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Agrin consists of a 225 kD core protein containing two or possibly three HS side chains. The core protein is composed of numerous repeating modules (Fig. 2) that are homologous to the modules found in many other matrix proteins?particularly those proteins found in basement membranes such as laminin and perlecan (135-137,28,142). The N-terminal 113 of the core protein has eight follistatinlike modules that are thought to provide protection from proteases (143). The C-terminal 113 has three laminin G modules and three EG modules arranged much like that of the C-terminal region of perlecan. The middle region and the follistatin-rich N-terminal region of agrin has several serine residues that serve as attachment sites for HS chains. Serine residues followed by a glycine residue can serve as attachment sites for both HS and CS but not all Ser-Gly sequences are substituted with GAG chains. Experimental studies, however, indi~aterepetitive Ser-Gly sequences and/or clusters of acid residues near the Ser-Gly appear to favor HS (144,27). These elements are contained in at least one of the sites for GAG attachment in agrin (Table 1). Agrin also has an SEA ~ o d u l ein its core protein and this module in perlecan has been shown to also favor HS assembly (27)). Alternate splicing is an important feature in agrin. There are three alternate splice sites. One site is near the N-terminus (145), between the signal peptide and the follistatin repeats (Fig. 2). The other two alternate splice sites are near the C-terminus of agrin (146,147). The first of these is the A site which is located in the middle laminin G domain module (Fig. 2). The second site (the B site) is located more C-terminally between an EGF module and the C-terminal Laminin G domain module. The B site can contain one of three different sequences (Fig. 2). The length of each of the alternatively spliced sequences in the A and B sites is relativity short, only 4-19 amino acids, but the presence or absence of these sequences determines the biological activity and molecular interactions of agrin (see Sec. D below).

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A 16 kb fragment of a mouse genomic clone for agrin was characte~zedand found to contain all the sequence for mouse agrin, including the C-terminus splice sites, in 36 exons except for the exons that would encode for the signal peptide and the N-terminal splice site (148). Interestingly? the intron/exon str~ctureat

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ure 2 Structuraldomains of agrin. Similarto perlecan, agrin's protein sequence contains homologies to other known proteins. Alternative splicing, which can alter the functional properties of agrin, have been found in three regions, one at the N-terminus and two near the C-terminus. There are also two GAG attachment sites located N-terminal to the SEA domain, that aids in HS addition.

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the 5' end of the gene corresponds to the module or domain structure of the Nterminal region core protein. The first seven follistatin modules are each encoded by a separate exon although the 8th and 9th follistatin modules are divided into 2 exons each. Most of the remaining modules are divided into multiple exons. The alte~ativelyspliced 4 amino acid sequence for the A site is contained in its own exon while the alternatively spliced sequences in the €3 site is in two exons. One exon codes for the €38 sequence of 8 amino acids, the other exon codes for

the B l1 sequence of 1 1 amino acids, and these two exons together code for the I319 sequence. The agrin gene has been localized to human Chromosome l pterp32 and mouse Chromosome 4.

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Agrin is expressed in the extracellular matrices and basement membranes of a variety of tissues including neuronal, muscle, capillary, and kidney (142,149). It is a major HSPC in the glomerular basement membrane of the kidney (1 50,15 l ) where the HS chains of agrin may be involved in ionic filtration. Immunological studies indicate there is more agrin than perlecan in the glomerular basement membrane (150). Heparitinase digestion of agrin isolated from the g l o m e ~ l a r basement membrane revealed core proteins of 170, 105, and 70 D. Antibodies to the ~ - t e r ~ i nregion al of agrin react with these fragments (15 l). Since there are no known splice variants that would produce these size products, it is suggested that these represent proteolytic fragments of the 225 kD core protein and that the HS chains are on the N-terminal half of agrin. This would be consistent with the earlier work (133) that originally identified agrin as a 150 kD polypeptide that is now known, because of its biological activity in causing AChRs and AChE to cluster in patches, to be from the C-terminal half of agrin. While agrin is expressed in a number of different basement membraneproducing tissues only neuronal tissues produce agrin capable of causing AChRs and AChE to cluster. This activity does not appear to be related or due to any proteolytic activation but rather due to the alternate splicing that can occur at sites A and B (Fig. 2) which adds as much as 23 amino acids to the agrin core protein. The four known isoforms generated by alternative splicing are A4BC), A4B8,A4B11 and A4B19, Neuronal tissues expressing agrin containing these alternately spliced sequences include ciliary ganglion (152), developing and adult retina (153), spinal cord and sensory ganglia (154), and the brain (155). The agrin isoforms are also developmentally regulated. For example, the B I and I3 19 isoforms are expressed at day 4 in the mouse spinal cord embryo with B decreasing and disappearing by day 20, while the B8 isoform is first detected on day 14 and increases thereafter (155). This would suggest that the different agrin isoforms each have a different function in the development of the nervous system. The mechanism or factors that control the alternate splicing itself are not understood but it is clearly tissue specific and developmentally regulated.

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Agrin is present in all basement ~ e m b r a n e sas a HSPG and acts as a major structural component that interacts with components of the matrix as well as with the surfaces of cells. The HS side chains present on the terminal half of agrin

bind to the heparin binding site on NCAM, which suggests it may play a role in cell-cell interactions (138,142,156). These HS chains may also interact with the heparin-binding proteins in the basement membrane, like laminin and tibronectin, to stabilize this matrix. The most important binding activity is in the C-terminal half of agrin that contains the three laminin G-like modules and the alternate splice sites A and B. The C-terminal region of agrin binds to HS, via the alternatively spliced 4"mino acid sequence at site A. The C-terminal region also binds to the cell surface receptor dystroglycan (157), but the presence of the ,alternatively spliced sequences at the A and B sites are not required for this binding. Dystroglycan consists of a and p subunits that are derived from the same precursor polypeptide. The a subunit binds to agrin present in the basement membrane and to the p subunit of dystroglycan which traverses the cell membrane. The agrin isoforms function differently in extrasynaptic and synaptic basement membranes. In extrasynaptic basement membranes, p dystroglycan binds to the cytoplasmic protein dystrophin, but in synaptic basement membranes, p dystroglycans binds to the cytoplasmic protein utrophin (see Ref. 142). Perlecan is also found in all basement membranes and there is recent evidence that perlecan also binds to the dystroglycan complex (158). Both agrin and perlecan serve as extracellular anchors for the cell and, in the case of muscle, provide one means of transmitting contractile energy to the extracellular matrix. The agrin in synapses which contains the alternatively spliced sequences in sites A and B also causes, in some unknown way, the AChRs to cluster. This synapse isoform of agrin does not interact directly with the AChRs (158-160). Furthermore, synthetic peptides of the sequences at the B site do not cause AChRs to cluster but antibodies to these peptides block agrin-induced AChRs clustering (153). This would suggest that sequences flanking the alternatively spliced region may also be involved in inducing AChRs clustering and that there is another agrin receptor that interacts specifically with the synapse agrin.

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The most definitive pathology for agrin comes from the agrin null mutant in mice (161). This mutant was produced by deleting a portion of the genomic DNA that contained the alternatively spliced sequences for the B site. This resulted in a loss of the 8.2 kb mRNA for agrin although a minor band of 9.5 kb was detected in the mutant. I~munostainingfor agrin on both Western blot and tissue sections showed a marked reduction in agrin in the mutant. The animals developed normally but died in utero on day 18 or were born dead, and because of this it was not possible to test the absence of agrin on the glomerular filtration function of the kidney. Examination of the muscles, however, showed that AChR clusters were reduced in size and number but total AChR levels in muscle were not reduced. Thus, in the absence of agrin, synapse formation was severely impaired

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but not eliminated. This suggests that there may be other synapse organizers. One possible candidate is the muscle specific receptor tyrosine kinase named MUSK since a null mutant for this gene product resulted in a phenotype similar to the agrin null mutant (162).

All collagen molecules consist of three polypeptide chains containing extensive contiguous repeats of Gly-Xaa-Yaa triplets (where Xaa and Yaa are any amino acids other than Gly) known as collagenous domains. Collagen molecules are named by roman numerals in order of discovery and are essentially divided into two subgroups based on structure. The fibrillar subgroup contains a long, uninterrupted collagenous domain flanked by smaller N- and C-terminal noncollagenous domains. The extensive collagenous domains in these collagens permit their lateral self-association into fibrils. This fibrillar subgroup consists of collagens I111as well as types V and XI. The other subgroup of collagen molecules is known as the nonfibrillar group. They are considerably heterogeneous in structure but have in common the presence of one or more noncollagenous sequences interrupting the collagenous sequence. This nonfibrillar group consists of collagens IV, VI-X, and XII-XIX. The a1 chain of collagen XVIII was simultaneously discovered by two groups screening cDNA libraries using cDNA clones to the a 1 chain of collagen XI1 (163) and to the a1 chain of collagen XI11 (164). The gene for collagen XVIII is on human Chromosome 21 and on mouse C ~ o m o s o ~10e(165). The structure of collagen XVIII consists of l0 noncollagenous domains separated and flanked by l 1 noncol~agenousdomains (Fig. 3). The N- and C-terminal noncollagenous domains are the largest and there is alternate splicing in the N-terminal noncollagenous domain to produce short and long forms of collagen XVIII (166,167). Interestingly, a 20 kD polypeptide known as endostatin, which inhibits endothelial cell proliferation, is a fragment of the C-terminal noncollagenous domain of collagen XVIII (168). Endostatin may serve as a potential inhibitor of angiogenesis and tumor growth. It was recognized early on that collagen XVIII, like collagens IX and XII, contained Ser-Gly sequences that are potential attachment sites for GAG side chains. However, while collagens IX and XI1 were sensitive to chondroitinase ABC, indicating the presence of CS or DS side chains, collagen XVIII was resistant to chondroitinase digestion (165). Collagen XVIII was first shown to be a proteoglycan when a third group, using antibodies to a previously unidentified but unique HSPG present in chick basement membrane, screened a chick yolk sac library and obtained D N A clones that coded for the a1 chain of

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Structural domains of collagen XVIII. Collagen XVIII is a nonfibrillar collagen and its domains are characterized by the spacing of collagenous and noncollagenous regions within the protein. Alternative splicing at the N-terminus produces long and short forms of the human molecule. There are also two GAG attach~entsequences that can contain HS.

collagen XVIII of chick (169). Collagen XVIII appears as a smear at -330 kD in Western blots, and heparitinase digestion or heparin lyase I1 and I11 digestion prior to electrophoresis produced a core protein of 180 kD (169,170). amin in at ion of sequence flanking the potential Ser-Gly attachment sites for GAG shows it to contain the numerous acidic residues characteristic of HSPG (Table 1). Collagen XVIII is a ubiquitous basement membrane component and readily detected in all vascular and epithelial basement membranes, including the glomerular basement membrane. Thus, in addition to agrin and perlecan, collagen XVIII is another HSPG that may be involved in the ionic filtration accomplished by

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the glomerular basement membrane. The alternatively spliced variants of collagen XVIII are, however, expressed differently. The short variant is present primarily in the basement membranes of blood vessels, epithelial-type cells, and in muscle. The long variant, however, was primarily expressed in the liver sinusoids. Each variant has a different signal peptide and promoter, which accounts for its tissue-specific expression (17 1).

Until recently, perlecan was the only basement membrane HSPG known; now agrin and collagen XVIII join this rank. This brings into question whether perlecan has been attributed with functions that belong to agrin or collagen XVIII. Many of the perlecan studies have been carried out using polyclonal antibodies that recognize the core protein and, at least in part, the HS chains. This along with the domain similarities between agrin and perlecan, as well as the discovery of a possible perlecan-related molecule, may challenge the conclusions made with polyclonal antibodies to perlecan. A series of antibodies that recognize specific HS chain st~ctures/sulfationpatterns indicate the kidney has substantial HS chain heterogeneity with different ultrastructural components of the kidney possessing different HS chain compositions (172). This may be due to the differential expression of the three basement membrane HSPGs which clearly needs to be sorted out in kidney as well as in neuropathology. Interestingly, only perlecan and agrin have GAG attachment sequences that are located in close proximity to one another which may indicate enhanced functions for high-affinity binding to the heparin-binding domains of other proteins such as cytokines and growth factors. Future knockout mice for perlecan and collagen XVIII, along with the current agrin knockout mice, will help to determine the function of each proteoglycan in the basement membranes of various tissues and organs. The perlecan mutants in C. eEegans which essentially lead to a perlecan knockout have already provided new discoveries regarding perlecan’s function in the organization and development of myofilaments and basement membranes. Whether agrin and collagen XVIII also contribute to nematode mutants has yet to be discovered but could lead to important findings regarding not only these molecules themselves but also the evolution of these basement membrane components. Interestingly, both agrin and perlecan have been mapped to human Chromosome 1~312-36, which suggests they share similarities in exon shuffling and gene duplication patterns. The future of basement membrane HSPG studies will most likely involve sorting the attributes and contributions of perlecan, agrin, and collagen XVIII to basement membranes, establishing the identity of any new basement membrane HSPG by cDNA cloning and developing an understanding of the role the core protein plays in HS chain diversity.

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Studies on the perlecan null mice (Hspg2 --/ --) have shown that perlecan is essential for the development of cartilage and the cephalic region (173). Forty percent of the null mice died at embryonic day 10.5 due to defective cephalic development, while the remaining 60% of the --/- mice died shortly after birth due to skeletal dysplasia. The cartilage morphology in the perlecan null mice was disrupted with severe disorganization of chondrocytes, reduction in chondrocyte proliferation, a diminished pre~ypertrophiczone and a reduced matrix with disorganized collagen fibrils and proteoglycans. This significant study confirms the critical role of perlecan in the structure of the extracellular matrix and its imperative role in cartilage development.

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zyxwvu zyxwvut Cohn RH, Banerjee SD, Bernfield MR. Basal lamina of embryonic salivary epithelia. J Cell Biol 1977; 73:464-478. Kanwar YS, Farquhar MG. Anionic sites in the glomerular basement membrane: In vivo and in vitro localization to the laminae rarae by cationic probes. J Cell Biol 1979; 81:137-153. Kanwar E'S, Farquhar MG. Presence of heparan sulfate in the glomerular basement membrane. Proc Natl Acad Sci USA 1979; 76:1303-1307. Hassell JR, Robey P, Barrach H, Wilczek J, Rennard S, Martin GR. Isolation of a heparan sulfate-containing proteoglycan from basement membrane. Proc Natl Acad Sci USA 1980; 77:4494-4498. Ledbetter SR, Tyree B, Hassell JR, Horigan EA. Identification of the precursor protein to basement membrane heparan sulfate proteoglycans. J Biol Chem 1985; 260:8106--8113. Murdoch AD, Liu B, Schwarting R, Tuan RS, Iozzo RV. Widespread expressionof perlecan proteoglycan in basement membranes and extracellularmatrices of human tissues as detected by a novel monoclonal antibody against domain I11 and by in situ hybridization. J Histochem Cytochem 1994; 42:239-249. Noonan DM, Fulle A, Valente P, Cai S, Horigan E, Sasaki M, Yamada Y, Hassell JR. The complete sequence of perlecan, a basement membrane heparan sulfate proteoglycan, reveals extensive sieilarity with laminin A chain, low density lipoprotein-receptor, and the neural cell adhesion molecule. J Biol Chem 1991; 266: 22939-22947. Laurie GW, Inoue S, Bing JT, Hassell JR. Visualization of the large heparan sulfate proteoglycan from basement membrane. Am J Anat 1987; 181:320-326. Murdoch AD, Iozzo RV. Perlecan: the multidomain heparan sulfate proteoglycan of basement membrane and extracellular matrix. Virchows Archiv A Pathol Anat 1993; 423~237-242.

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26. Couchman JR, Kapoor R, SthanamM, Wu RR. Perlecan and basement membranechondroitin sulfate proteoglycan (bamacan) are two basement membrane chondroitin/dermatan sulfate proteoglycans in the Enge1bre~-Holm-Swarmtumor matrix. J Biol Chem 1996; 271:9595-9602. 27. Doege K, Chen X, Cornuet PK, Hassell JR. Non-glycosaminoglycan bearing domains of perlecan and aggrecan influence the utilization of sites for heparan and chondroitin sulfate synthesis. Matrix Biol 1997; 16:211-221. 28. Bork P, Patthy L. The SEA module: A new extracellular domain associated with 0-glycosylation. Protein Sci 1995; 4:1421-1425. 29. Costell M, Sasaki T, Mann K, Yamada Y, Timpl R. Structural characterizationof recornbinant domain I1 of the basement membrane proteoglycan perlecan. FEBS Lett 1996; 396:127-133. 30. Ji ZS, Dichek HL, Miranda RD, Mahley RW. Heparan sulfate proteoglycans participate in hepatic lipase and apolipoprotein E-mediated binding and uptake of plasma lipoproteins, including high density lipoproteins. J Biol Chem 1997; 272:312853 1292. 31. Schulze B, Mann K, Battistutta R, Wiedemann H, Timpl R. Structural properties of recombinant domain 111-3of perlecan containing a globular domain inserted into an epide~al-g~owth-factor-like motif. Eur J Biochem 1995; 23 1551-556. 32. Couchman JR, Ljubimov AV, SthanamM, Horchar T, Hassell JR. Antibody mapping and tissue localization of globular and cysteine-rich regions of perlecan domain 111. J Histochern Cytochem 1995; 43:955-963. 33. Chakravarti S, Horchar T, Jefferson B, Laurie GW, Hassell JR. Recombinant domain I11 of perlecan promotes cell attachment through its RGDS sequence. J Biol Chem 1995; 270:404-409. 34. Schulze B, Sas aki T, Costell M, Mann K, Timpl R. Structural and cell-adhesive properties of three recombinant fragmentsderived from perlecan domain 111.Matrix Biol 1996; 15:349-357. 35. Singer 11, Scott S, Kawka DW, Hassell JR. Extracellular matrix fibers containing fibronectin and basement membrane heparan sulfate proteoglycan coalign with focal contacts and microfil~entbundles in stationary fibroblasts. Exp Cell Res 1987; 173:558-571. 36. Hayashi K, Madri JA, Yurchenco PD. Endothelial cells interact with the core protein of basement membrane perlecan through p1 and p3 integrins: an adhesion modulated by glycosaminoglycan. J Cell Biol 1992; 119:945-959. 37. Rogalski TM, Gilchrist EJ, Mullen GP, Moerman DG. Mutations in the unc-52 gene responsible for body wall muscle defects in adult C a e ~ o r ~ elegarts ~ ~ ~ are i ~ located in altematively spliced exons. Genetics 1995; 139:159-169. 38. Brown JC, Sasaki T, Gohring W, Yamada Y, Timpl R. The C-terminal domain V of perlecan promotes p1 integrin-mediated cell adhesion, binds heparin, nidogen and fibulin-2 and can be modified by glycosaminoglycans. Eur J Biochem 1997; 250~39-46. 39. Yurchenco PD, Cheng YS, Ruben GC. Self-assembly of a high molecular weight basement membrane heparan sulfate proteoglycan into dimers and oligomers.J Biol Chem 1987; 262:17688-17676. 40. Gohen I, Grassel S, Murdoch AD, Iozzo RV. Structural characterization of the

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41. 42.

43

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complete human perlecan gene and its promoter. Proc Natl Acad Sci USA 1993; 90:10404-10408. Chakravarti S, Phillips SL, Hassell JR. Assignment of the perlecan (heparan sulfate proteoglycan) gene to mouse chromosome 4. Mamm Genome 1991; 1:270272. Dodge GR, Kovalszky I, Chu ML, Hassell JR, McBride OW, Yi HF, Iozzo RV. Heparan sulfate proteoglycan of human colon: partial molecular cloning, cellular expression, and mapping of the gene (HSPG2) to the short arm of human chromosome 1. Genomics 1991; 10:673-680. Kallunki P, Eddy RL, Byers MG, Kestila M, Shows TB, Tryggvason K. Cloning of the human heparan sulfate proteoglycan core protein, assignment of the gene (HSPG1) to lp36.1-p35 and identification of a BamHl restriction fragment length polymo~hisl~. Genomics 1991; 11~389-396. Iozzo RV, Pillarisetti J, Sharma B, Murdoch AD, Danielson KG, Uitto J, Mauviel A. Structural and functional characterizationof the human perlecan gene promoter. J Biol Chem 1997; 2725219-5228. Piidagnel R, Prii D, Cassingina R, Ronco PM, Lelong B. SV40 Large-T oncogene inhibits transcription of perlecan-related proteoglycans but stimulates hyaluronan synthesis in a temperature-sensitiverenal-tubule principal cell line. J Biol Chem 1994; 269~37469-17476. Chakravarti S, Hassell JR, Phillips SL. Perlecan gene expression precedes laminin gene expression during differentiation of F9 embryonal carcinoma cells. Dev Dyn 1993; 197~107-114. KOCW, Bhandari B, Yee J, Terhune WC, Maldonado R, Kasinath BS. Cyclic AMP regulates basement membrane heparan sulfate proteoglycan, perlecan, metabolism in rat glomerular epithelial cells. Mol Cell Biochem 1996; 162~65-73. Kasinath BS, Grellier P, Choudhury GC, Abboud SL, Regulation of basement membrane heparan sulfate proteoglycan, perlecan, gene expression in glomerular epithelial cells by high glucose medium. J Cell Physiol 1996; 167:I31- 136. Templeton DM, Fan MY. Posttranscriptional effects of glucose on proteoglycan expression in rnesangial cells. Metabolism 1996; 45: 1136-1 146. Lundquist EA, Herman RK, Rogalski TM, Mullen GP, Moerman DG, Shaw JE. The mec-8 gene o f C. elegans encodes a protein with two RNA recognition motifs and regulates alternative splicing of unc-52 transcripts. Development 1996; 122: 1601-1610. Carson DD, Tang JP, Julian J. Heparan sulfate proteoglycan (perlecan) expression by mouse embryos during acquisition of attachment competence. Dev Biol 1993; 155:97-106. Smith SE, French MM, Julian J, Paria BC, Dey SK, Carson DD. Expression of heparan sulfate proteoglycan (perlecan) in the mouse blastocyst is regulated during normal and delayed implantation. Dev Biol 1997; 184:38-47. Fujii N, Kaji T, Akai T, Koizurni F. Thrombin reduces large heparan sulfate proteoglycan molecules in cultured vascular endothelial cell layers through inhibition of core protein synthesis. Thrombosis Res 1997; 88:299-307. Whitelock JM, Murdoch AD, Iozzo RV, Underwood PA. The degradationof human endothelialcell-derivedperlecan and release of bound basic fibroblast growth factor

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49. 50.

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55. 56. 57. 58. 59.

60. 61. 62. 63. 64. 65. 66. 67.

68. 69.

by strornelysin, collagenase, plasmin, and heparanases. J Biol Chem 1996; 271: 10079- 10086. Sharma B, Iozzo RV. Transcriptionalsilencing of perlecan gene expression by interferon-gamma. J Biol Chem 1998; 273:4642-4646. Roberts AB, Sporn MB. The transforming growth factor-8s. In: Sporn MB, Roberts AB, eds. Handbook of Experimental Pharmacology: Peptide Growth Factors and Their Receptors 1. New York: Springer-Verlag, 1990; 95/1:419-472. Dodge GR, Kovalszy I, Hassell JR, Iozzo RV. Transforminggrowth factor-(3alters the expression of heparan sulfate proteoglycan in human colon carcinoma cells. J Biol Chem 1990; 265:18023-1 8029. Dodge GR, Boesler EW, Jimenez SA. Expression of the basement membrane heparan sulfate proteoglycan (perlecan) in human synovium and in cultured human synovial cells. Lab Invest 1995; 73:649-657. van Det NF, Tamsma JT, van den Born J, Verhagen NAM, van den Heuvel LPWJ, Lowik CWGM, Berden JHM, Bruijn JA, Daha MR, van der Woude FJ. Differential effects of angiotensin I1 and transforming growth factor-8 on the production of heparan sulfate proteoglycan by mesangial cells in vitro. J Am SOCNephrol 1996; 7:1015-1023. Benitz WE, Kelley RT, Anderson CM, Lorant DE, Bernfield M. Endothelial heparan sulfate proteoglycan. I. Inhibitory effects on smooth muscle cell proliferation. Am J Resp Cell Molec Biol 1990; 2:13-24. Weiser MCM, Belknap JK, Grieshaber SS, Kinsella MG, Majack RA. Developmental regulation of perlecan gene expressionin aortic smooth muscle cells. Matrix Biol 1996; 15:331-340. Handler M,Yurchenco PD, Iozzo RV. Developmental expression of perlecan during murine embryogenesis. Dev Dyn 1997; 210:130- 145. Larrain J, Alvarez J, Hassell JR, Brandan E. Expression of perlecan, a proteoglycan that binds myogenic inhibitory basic fibroblast growth factor, is down regulated during skeletal muscle differentiation. Exp Cell Res 1997; 234:405-412. Moeman DG, Hutter H, Mullen GP, Schnabel R. Cell autonomous expression of perlecan and plasticity of cell shape in embryonic muscle of C u e n o ~ ~ z eEeu~~~~~s guns. Dev Biol 1996; 173:228-242. Sannes PL, Wang J. Basement membranes and pulmonary development.Exp Lung Res 1997; 23:lOl-108. Simon-Assmann P, Kedinger M, DeArcangelis A, Rousseau V, Simo P. Extracellular matrix components in intestinal development. Experientia 1995; 5 1:883900. Sekiguchi RT, Potter-Perigo S, Braun K, Miller BJ, Ngo C, Fukuchi K, Wight TN, Kimata K, Snow AD. Characterization of proteoglycans synthesized by murine embryonal carcinoma cells (P19) reveals increased expression of perlecan (heparan sulfate proteoglycan) during neuronal differentiation. J Neurosci Res 1994; 38: 670-686. Battaglia C, Mayer U, Aumailley M, Timpl R. Basement-membrane heparan sulfate proteoglycan binds to laminin by its heparan sulfate chains and to nidogen by sites in the protein core. Eur J Biochem 1992; 208:359-366. Mayer U, Mann K, Fessler LI, Fessler JH, Timpl R. ~ ~ o s o plaminin ~ i Z ~binds to

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m a ~ a l i a nnidogen and to heparan sulfate proteoglycan. Eur J Biochem 1997;

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85

Folkman J, Klagsbrun M, Sasse J, Wadzinski M, Ingber D, Vlodavsky I. A Heparin-

ulfate ~ r o t e o ~ l y c a n ins asement ~ e m ~ r a n e s

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100. 101.

102.

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104.

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112.

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glycan gene expressionin rat liver after partial hepatectomy. Biochem Biophys Res Commun 1996; 228:690-694. Klein G, Conzelmann S, Beck S, Timpl R, Muller CA. Perlecan in human bone marrow: a growth-factor~presenting,but anti-adhesive, extracellularmatrix component for hematopoietic cells. Matrix Biol 1994; 14:457-465. Grassel S, Cohen IR, Murdoch AD, Eichstetter I, Iozzo RV. The proteoglycan perlecan is expressed in the erythroleukemia cell line K562 and is upregulated by sodium butyrate and phorbol ester. Mol Cell Biochem 1995; 145:61-68. Drzeniek Z, Siebertz B, Stocker G, Just U, Ostertag W, Greiling H, Haubeck HD. Proteoglycan synthesis in haematopoietic cells: isolation and characterization of heparan sulfate proteoglycans expressed by the bone-marrow stromal cell line MS-S. Biochem J 1997; 327:473-480. SundarRajN, Fite D, Ledbetter S, Chakravarti S, Hassell JR. Perlecan is a component of cartilage matrix and promotes chondrocyte attachment. J Cell Sci 1995; 108:2663-2672. Nitkin RM, Smith MA, Magill C, Fallon JR, Yao Y-MM, Wallace BG, McMahan UJ. Identification of agrin, a synaptic organizing protein from Torpedo electric organ. J Cell Biol 1987; 105:2471-2478. Wallace BG. Agrin-induced specializations contain cytoplasmic, membrane, and extracellular matrix-associated components of the postsynaptic apparatus. J Neurosci 1989; 9:1294-1302. Rupp F, Payan DG, Magill SC, Cowan DM, Scheller RH. Structure and expression of a rat agrin. Neuron 1991; 6:811-823. Tsim KWK, Ruegg MA, Escher G, Kroger S, McMahan UJ. cDNA that encodes active agrin. Neuron 1992; 8:677-689. Smith MA, Magill SC, Rupp F, Yao Y-MM, Schilling JW, Snow P, McMahan UJ. Isolation and characterization of an agrin homologue in the marine ray. Mol Cell Neurosci 1992; 3:406-417. Burg MA, Halfter W, Cole GJ. Analysis of proteoglycan expression in developing chicken brain: characterizationof a heparan sulfate proteoglycan that interacts with the neural cell adhesion molecule. J Neurosci Res 1995; 41:49-64. Tsen G, Halfter W, Kroger S, Cole GJ. Agrin is a heparan sulfate proteoglycan. J Biol Chem 1995; 270:3392-3399. Hagen SG, Michael AF, Butkowski RJ. I~munochemicaland biochemical evidence for distinct basement membrane heparan sulfate proteoglycans.J Biol Chem 1993; 26817261-7269. Denzer AJ, Gesemann M, SchumacherB, Ruegg MA. An a~~ino~terminal extension is required for the secretion of chick agrin and its binding to extracellular matrix. J Cell Biol 1995; 131:1547- 1560, Denzer AJ, Gesemann M, Ruegg MA. Diverse functions of the extracellularmatrix molecule agrin. Neurosci 1996; 8:357-366. Biroc SL, Payan DG, Fisher JM. Isofoms of agrin are widely expressed in the developing rat and may function as protease inhibitors. Brain Res Dev Brain Res 1993; 75~119-129. Zhang L, Esko J. Amino acid determinants that drive heparan sulfate assembly in a proteoglycan. J Biol Chem 1994; 269:19295-19299.

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145. Tsen G, Napier A, Halfter W, Cole GJ. Identification of a novel alternatively spliced agrin d N A that is preferentially expressed in non-neuronal cells. J Biol Chem 1995; 270~15934-15937. 146. Gesemann M, Cavalli V, Denzer AJ, Brancaccio A, Schurnacher B, Ruegg MA. Alternative splicing of agrin alters its binding to heparin, dystroglycan, and the putative agrin receptor. Neuron 1996; 16:755-767. 147. O’Toole JJ, Deyst KA, Bowe MA, Nastuk MA, McKechnie BA, Fallon JR. Alternative splicing of agrin regulates its binding to heparin alpha-dystroglycan,and the cell surface. Proc Natl Acad Sci USA 1996; 93:7369-7374. 148. Rupp F, Ozcelik T, Linial M, Peterson K, Francke U, Scheller R. Structure and chromosomal localization of the mammalian agrin gene, J Neurosci 1992; 12: 3535-3544. 149. Bowe MA, Fallon JR. The role of agrin in synapse formation. Annu Rev Neurosci 1995; 18~443-462. 150. Groffen AJ, Ruegg MA, Dijkman H, van de Velden TJ, Buskens CA, van den Born J, Assmann KJ, Monnens’LA,Veerkamp JH, van den Heuvel LP. Agrin is a major heparan sulfate proteoglycan in the human glomerularbasement membrane. J Histochem Cytochem 1998; 46:19-27. 151. Raats CJI, Bakker MAH, Hoch W, Tamboer WPM, Groffen AJA, van den Heuvel LPWJ, Berden JHM, van den Born J. Differential expression of agrin in renal basement membranes as revealed by domain-specific antibodies. J Biol Chern 1998; 273~17832-17838. 152. Srnith MA, O’Dowd DK. Cell-specific regulation of agrin RNA splicing in the chick ciliary ganglion. Neuron 1994; 12:795-804. 153. Kroger S. Differential distribution of agrin isoforrns in the developing and adult avian retina. Mol Cell Neurosci 1997; 10:149-161. 154. Ma E, Morgan R, Godfrey EW. Agrin mRNA variants are differentially regulated in developing chick embryo spinal cord and sensory ganglia. J Neurobiol 1995; 26~585-597. 155. Cohen NA, Kaufmann WE, Worley PF, Rupp F. Expression of agrin in the developing and adult rat brain. Neurosci 1997; 76581-596. 156. Cole GJ, Halfter W. Agrin: an extracellular matrix heparan sulfate proteoglycan involved in cell interactions and synaptogenesis.Perspectives Dev Neurobiol 1996; 3:359-37 l. 157. Sugiyarna J, Bowen DC, Hall ZW. Dystroglycan binds nerve and muscle agrin. Neuron 1994; 13:103-115. 158. Peng HB, Ali AA, Daggett DF, Rauvala H, Hassell JR, Smalheiser NR. The relationship between perlecan and dystroglycan and its i~plicationin the formation of the neuromuscular junction. Cell Adh C o m u n 1998; 5:475-489. 159. Godfrey EW, Nitkin RM, Wallace BG, Rubin LL, McMahan UJ. Components of Torpedo electric organ and muscle that cause aggregation of acetylcholinereceptors on cultured muscle. J Cell Biol 1984; 99:615-627. 160. Ma J, Nastuk MA, McKechnie BA, Fallon JR. The agrin receptor. Localization in the postsynaptic membrane, interaction with agrin, and relationship to the acetylcholine receptor. J Biol Chem 1993; 268:25108-25117. 161. Gautam M. Noakes PG. Moscoso L, RUDDF, Scheller RH, Merlie JP, Sanes, JR.

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University of Zurich and University ~ospital,Zurich, Switzerland

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Versican ( l ) belongs to the family of large aggregating proteoglycans that has recently been named the family of hyalectans (2) or lecticans (3). Other members of the hyalectan family include aggrecan, neurocan, and brevican. A detailed description of these related proteoglycans is given in Chapters l 4 and 15. All hyalectans have a modular core protein structure and are, with the exception of a brevican splice-variant, extracellular matrix proteoglycans. Hyalectans share highly similar N- and C-terminal globular domains that are separated by clearly distinctive g l y c o s a m i ~ o g l y c a n - c ~ i nmiddle g portions. Versican is, among the hyalectans, probably the most versatile member in regard to structure and tissue distribution. Recent data emerging from in vitro studies suggest that versican modulates cell adhesion, proliferation, and migration and hence plays a central role in tissue development and maintenance, as well as in a number of pathologic processes.

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To date, the entire primary structures of human (l,4), murine (S), bovine (6), and chick versican ( al so known as PG-M) ('7) have been deduced from cDNA clones.

In addition, partial cDNA sequences have been determined for the monkey homologue (8) and for a versican-like axolotl proteoglycan (9). Several versican core proteins have been identified. The structural diversity originates from alternative splicing processes, which generate four splice-variants

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of human (4,lO), mouse (5,lO) and bovine versican (6) (Fig. l). Six versican isofoms may exist in the chicken (l1). All versican splice-forms include at the N - t e ~ i n aend l a link-proteinlike structure with an immunogl~bulin-likeloop and a tandem-repeat domain, and at the C-terminus a set of two EGF-like elements, a C-type lectin domain and a sushi (CRP) module. The differences among the versican splice-variants are found in the central portion of the core proteins. In versican VO, two chondroitin s u l f a t e - c ~ i n g segments, named GAG-a and GAG-P, are present, whereas the smaller V1 and V:! isoforms lack the GAG-a

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or the GAG-P domain, respectively. None of the glycosaminoglycan-carrying modules are included in versican V3. Based on consensus sequences, we have estimated the number of potential chondroitin sulfate attachment sites to be 1723 in human versican VO, 12- 15 in versican VI, and 5-8 in versican V2 (4). Due to the absence of both central domains, versican V3 is most likely devoid of glycosaminoglycan side chains and therefore may not be a proteoglycan (10). For the chick versican homologue (PG-M), evidence for the existence of additional splice-variants has recently been presented ( I 1). Unlike the mammalian counterparts, chick versican contains a stretch of 138 amino acids, the socalled PLUS domain, which is located N-terminal of the GAG-a module (Fig. 1). Origin of this peculiarity in chick versican is an internal alternative 5”splice donor site in the exon that encodes the GAG-a domain, Accordingly, there are two chicken V1 -- and V3--isofoms that lack the PLUS domain in addition to the four V0’- to V3+-variantsthat all contain this sequence portion. Of note is the considerable amino acid similarity of 40% between the versican PLUS and the chick aggrecan keratan sulfate (KS) domain, suggesting that this extra domain may function as a keratan sulfate attachment domain. The calculated molecular masses for human versican core proteins are 370, 262, 180, and 72 kDa for the VO, Vl , V2, and V3 isoforms, respectively. These theoretical values are significantly lower than the core protein sizes deduced from SDS-polyac~lamidegel electrophoresis, where versicans VO, V1 and V2 migrate after chondroitinase ABC digestion around 550,500, and 400 kDa, respectively (4,6). This difference may on one hand originate from a high content of N- and 0-linked oligosaccharides and on the other hand from the very low isoelectric points of the versican core proteins (calculated between 4 and S); this may lead to aberrant gel migration properties as result of a reduced SDS-bill din^. Even more difficult to estimate are the molecular weights of the intact proteoglycan isoforms of versican, due to the large heterogeneity of the glycosaminoglycan side chains. Sizes between 6 X lo5 and 1.5 X lo6 for the intact proteoglycan variants of versican (V2 to VO) are conceivable. No protein chemical data are currently available for the smallest versican V3 splice-form, The glycosaminoglycan side chains of the large versican isoforms isolated from cell and organ cultures differ in size and composition depending upon tissue origin and culture conditions. The chondroitin sulfate chains of versican VO/ V1 from chick limb bud have an average molecular weight of over 60 Id la (12), whereas monkey aortic smooth muscle cells secrete versican molecules with 4045 kDa side chains under standard culturing conditions (13,14), In both versican ~reparations,chondroitin-6-sulfate to chondroitin-4-sulfate ratios of 2 ; 1 have been measured. In electron microscopy, a large proteoglycan from aorta most likely corresponding to versican V1 appears as a rodlike structure carrying several glycosaminoglycan side chains (15). As inferred from the rotary shadowing images,

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the molecule consists of a 237 nm long central portion linked at either end to one globular domain of 11 and 12 nm diameter, respectively. There is a large number of reports describing “two” 40-70 k Da glycoproteins named hyaluronectin ( 16) and glial hyaluronate-binding protein (GHAP) (17). Both of these polypeptides contain sequences that are identical with portions of the N-terminal domain of versican (18,19). Although the molecular weight of the larger hyaluronectin and GHAP peptides are in the range of the calculated size for the versican V3 isoform, it seems more likely that they are proteolytic products of the larger versican V1 splice-variant (20). Finally, proteoglycans that are similar or may even be identical to versican include the Cat-301 antigen (21) and the DSD-l-proteoglycan (22). Since no sequence data are currently available for these proteoglycans, it remains unclear, how they relate to versican.

The versican splice-variants are encoded by a single gene localized on chromosome 5q12-14 in the human (23) and on chromosome 13 in the mouse genome (24). The organization of the gene tightly follows the domain structure of the core proteins (25,26). Both the human and the mouse gene extend over 90-100 kb and are divided into 15 exons. The glycosaminoglycan attachment domains, GAG-a and GAG-P, are each encoded by a giant exon of 3 and 5.3 kb size in the human and 2.9 and 5.2 kb in the mouse gene, respectively. Alternative m ~ ~ A - s p l i c i nofg these exons gives rise to transcripts of about 12 kb size for the V0 isoform, 9 kb for versican VI, 6.5 kb for versican V2, and 3 kb for the smallest V3 variant. Since the 3’ untranslated regions contain several polyadenylation signals (three in the human and four in the mouse gene), the different splice-variants appear as multiple bands closely spaced on Northern blots. The expression of the versican gene (Cspg2) is regulated by a promoter that harbors a typical TATA box and potential binding sites for a number of transcription factors including AP2, CCAAT-bindi~gtransc~ptionfactor, SP1, xenobiotic responsive element-binding factor, CCAAT enhancer binding protein, and CAMPresponsive element-binding protein (25). Experiments with reporter constructs demonstrate that the versican promoter is active in fibroblasts as well as in cells of epithelial origin. Various growth factors influence the expression of versican. TGF-Pl and PDGF up-regulate the versican mRNA and protein levels in aortic smooth muscle cells and affect the size and composition of the chondroitin sulfate side chains (14). The ~DGF-stimu~ation of the versican transcription seems to be mediated by tyrosine kinases, since the effect can be blocked with genistein (27). Marked

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increases in versican expression are al so observed in cultures of skin and gingival fibroblasts upon addition of TGF-P1 (28), whereas an inverse effect on the versican steady state mRNA level has been noted after IL-lP treatment (29). Furthermore, a reduced versican expression has been described for osteoblast-like cells from young human donors in response to IGF-I (30) and for fetal lung fibroblasts exposed to atmospheres with elevated oxygen contents (31).

Versican seems to interact with a number of different ligands. The interactions may either be mediated by specific core protein domains or by the carbohydrate side chains. Best characterized is the interaction between hyaluronan and the Nterminal globular domain of versican with an estimated dissociation constant of 4 X M (32,33).It appears that this complex is further stabilized by link protein in analogy to the hyal ur onan- aggr ecan- l i nkprotein complex in cartilage

(13,15).

The C-terminal end of the versican core protein, which contains a set of domains similar to selectins (l), may interact with carbohydrate andlor protein structures of other extracellular matrix components or cell surface receptors. Bacterial fusion proteins containing the two EGF repeats, the C-type lectin domain, and the sushi-element of versican bind to D-mannose, D-galactose, L-fucose, Nacetyl-D-glucosamine,heparin, and heparan sulfate in a calcium-dependent manner (34). These interactions seem not only to be mediated by the lectin domain, since removal of the sushi-element completely abolishes the binding. The recombinant C-type lectin domain alone isolated from a m a ~ ~ a l i expression an system interacts with tenascin-R from brain extracts (35). This calcium-dependent binding, which originally was reported to be based on a protein-carbohydrate interaction, s e e m rather to be mediated by a protein-protein interaction between the lectin domain of versican and the fibronectin type I11 repeats 3-5 of tenascin-R (36). Whether intact versican also reacts with tenascin-R still needs to be shown. Other potential ligands of versican are fibronectin and collagen I, which bind to isolated chicken versican (PG-M) in solid phase assays (32). These interactions have not been characterized in more detail,

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During early embryonic development versican VOlV1 i s transiently expressed in a number of mo~hogeneticallyactive tissues (12,37-39). Versican VOlVl ap-

pears in chick embryos first in the subectoder~alregion dorso-lateral to the neural tube, in association with the basement membranes of early epithelioid somites and the neural tube, in the early perinotochordal mesenchyme, and in the posterior halves of the sclerotomes. At later stages, the expression shifts to the condensing mesenchyme (Fig. 2) of the developing limb buds and the pelvic girdle precursor, being subsequently found in all prechondrogenic tissues. After initiation of cartilage formation, versican disappears mostly from these areas and is replaced by aggrecan. Versican VO/V1 is also highly expressed in the developing gut, in the heart and the newly formed blood vessels (40; Landolt and Zimmer~ann, unpublished). In the central nervous system, versicans V0 andlor V1 are transiently expressed in the retina (41) and in the optic tectum (42). At the end of histogenesis, a rather wide versican expression pattern similar to the distribution in the adult organism is observed (43). In adult human tissues versicans V1 and/or V0 are mainly found in the loose connective tissues of internal organs and in smooth muscle tissues (particularly blood vessels) (43). To a lesser extent, they are also present in fibrous and

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Immunohistochemical stainings demonstrating the changes of the versican VOlVl dist~butionduring the development of the chick hind limb. At embryonic day 3.5

(stage 20) versican VOlVl is present throughout the limb bud mesenchyme. At E 4 (stage 22), the staining is accentuated in the pelvic girdle precursor forming a transient barrier to axons that have meanwhile reached the versican-free plexus region. At E 4.5-5 (stage 2S), gaps have formed in the pelvic girdle precursor and the axons are now able to innervate the limb. At this time point, versican is completely absent from the presumptive axonal pathways and is restricted to prechondrogenic areas.

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elastic cartilage, in tendon and skeletal muscle, in the dermis and the basal layer of the epidermis, little in the central and peripheral nervous systems, and on the luminal surfaces of some glandular epithelia (43-45). In the elastic tissues a close association of versican VO/ Vl with elastic fibers and laminae was evident (43,45). The versican staining generally mirrored the distribution of elastin, fibulin-l (46), and the microfibril-associated glycoproteins, MAGP (47) and fibrillin-l (48). Notable exceptions: Versican was completely absent from the ~ eand ~elastic^i i ~ ~t e r l~ f af ~ i of ~ normal ~ e muscular arteries and from the elastin-free microfibrillar matrix surrounding the mesangial cells in the kidney glomeruli, Interestingly, proliferating glomerular mesangial cells do synthesize a versican-like proteoglycan in vitro (49). Apart from versicans V0 and VI, a similarly wide expression pattern has been observed for the shortest versican splice-variant V3 by RT-PCR experiments (~ours~zimmermann and zimmermann, unpublished). In contrast, versican V2 is restricted to the central nervous system (4), where it seems to be a predominant component of the mature brain extracellular matrix (6). It is currently unclear how an earlier described versican preparation from brain tissue relates to the different versican isoforms (50).

The suggested functions of aggregating proteoglycans range from structural support of hyaluronan-rich extracellular matrices to modulatory roles in cell adhesion, migration, and proliferation (for a review see Refs. 2, 3, 51-55 and Chaps. 14 and 15 of this book). Although functional data on versican are to date very rudimentary, specific expression patterns and very few in vitro studies point to an inhibitory role of versican splice-variants in cell adhesion and migration processes. The most direct evidence is provided by in vitro experiments of ~ a m a g a t a and coworkers, who have demonstrated that the large versican splice-variants V0 and V1 interfere with the attachment of various cells to collagen I, fibronectin, and laminin (56). The inhibition is apparently mediated by the glycosaminoglycan side chains, since chondroitinase ABC abolishes this effect. Interestingly, versican is completely absent from focal contacts, the actual adhesion sites of cultured fibroblasts (37). Versican V0 also failed to support the adhesion of chicken neural crest cells (5’7).This is in line with our immunohistochemical observations d~monstratinga close correlation between versican expression and the formation of tissues that act as barriers to migratory neural crest cells and outgrowing peripheral axons during embryonic development (38). Moreover, splotch mice carrying a mutation in the Pax3 gene overexpress versican, which

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in turn may be responsible for the defects in neural crest cell migration in this mouse strain (58). In sum, it seems likely that versicans V0 and/or V1 play an active role in the migration-inhibitory function of barrier tissues. In addition to this putative involvement in the regulation of cell adhesion and migration, versican isoforms may also participate in the control of cell proliferation as suggested by the increased expression during keratinocyte and dermal fibroblast proliferation (45). It seems conceivable that a transient increase of versican and hyaluronan secretion during the cytokinesis phase of mitosis leads to the formation of a highly hydrated pericellular coat, which reduces integrin-mediated cell-matrix interaction and induces the rounding up of cells in vitro (45,59). And lastly, as a structural component of elastic tissues, versican may inhibit interactions between the elastic network and the cytoplasmic membrane in order to minimize mechanical stress to the cell and to prevent elastic fibers from sticking to each other and from tangling up after the release of tensile forces.

The accumulation of proteoglycans in the arterial vessel wall has long been considered a key event in the development of atherosclerosis (reviewed in Ref. 60). Only recently the proteoglycans involved in the formation of primary atherosclerotic lesions, restenosis, and transplant arteriopathy have been characterized in more detail (43,6146). Based on these studies, there is growing evidence that versican plays a major role in the pathogenesis. In the normal vascular system, versican is present in the extracellular matrix of all types of blood vessels ranging from the large caliber aorta and vena cava down to the smallest capillaries (43,61). Versican VOIV1 is localized in all three wall layers of veins and elastic arteries (in tim a , m ed ia , and a ~ v e n t it ia ),but it is restricted to the tu n ica a d v e ~ t it iain muscular arteries. In vitro experiments demonstrated that versican is expressed by arterial smooth muscle cells (14) and vascular endothelial cells (67). During the formation of atherosclerotic lesions, the increased versican expression correlates with smooth muscle cell proliferation (62,63,65). Versican VO/V l deposition is predominant in smooth muscle rich regions of diffuse intimal thic~enings,fibrous caps, and zones of loose, myxoid connective tissue, as well as in thrombi and at borders of the lipid-rich cores in advanced disease stages (62,64). The rather dramatic increase of versican deposition during atherogenesis may at least partly be mediated by TGF-p1 (14), since antibodies against this cytokine effectively suppress the intimal hyperplasia and abolish the versican overexpression in a rat model system (68).

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Changes in proteoglycan expression patterns have frequently been linked to the development of malignant tumors (for a review see Ref, 69). Significant increases in chondroitin sulfate proteoglycan and hyaluronan synthesis are usually accompanied by a decrease in cell surface heparan sulfate proteoglycans. Large deposits of chondroitin sulfate proteoglycans probably identical to versicans V0 or V1 are observed in the extracellular matrix of malignant epithelial and nonepithelial tumors, whereas often little versican is encountered in the benign forms (70-72). Exceptions to this “rule” are brain tumors of glial origin, where a downregulation of versican, presumably the V2 splice-variant, has been noted (73). In those tumors, elevated versican VOlVl depositions are restricted to the blood vessels. It appears that versican is in most of the carcinomas secreted by reactive mesenchymal cells rather than by the neoplastic cells themselves. In contrast, various types of sarcoma cells do express the different versican splice-variants. The consequence of the elevated versican levels in the tumor matrix is to date unclear. In light of a putative role of versican in cell adhesion, migration, and proliferation, at least two interpretations are possible: l) The versican deposits form barriers to invading tumor cells in the context of a host defense ~ e c h a n i s m or 2) versican-rich tumor matrices either synthesized by the neoplastic cells themselves or by the admixed mesenchymal cells provide an environment favoring cell growth and locomotion. Whereas the former hypothesis points to the putative bmier function of versican in embryonic development (38,57,58), the latter is supported by the in vitro finding that a partial reversion of the malignant adhesion phenotype of MG63 osteosarcoma cells can be achieved by a 10-fold suppression of the versican expression (74). It is evident that further experiments are needed to understand the role of versican in tumor development and metastasis.

In recent years the primary goal s of versican research have been the elucidation of the core protein structure and the analysis of its expression. Due to the difficulties in isolating biochemical quantities of the intact versican splice-variants, functional aspects have only been addressed marginally. Most of the concepts about the putative role of versican in extracellular matrix structure, in cell adhesion, proliferation, and migration originate from particular expression patterns and are rarely based on cell biological assays. The isolation of recombinant versican isoforms or fragments thereof have been very helpful in preparing core protein-

specific tools and in identifying potential versican ligands. However, proteoglycans isolated from m a ~ a l i a noverexpression systems frequently differ from their ex vivo c o u n t e ~ in ~ snumber, length, and structure of the glycosaminoglycan side chains and hence are only of limited use for ~ n c t i o experiments, n~ for which correct carbohydrate structures may be required. We therefore are making new efforts to isolate intact versican splice-variants from bovine tissues (6) in order to study the involvement of versican in cell adhesion, migration, and proliferation. Alternatively, insights into the function of versicans may be drawn from gene inactivation experiments, Recently, Mjaatvedt and coworkers reported that the complete knockout of the versican gene through random integration of a transgene causes intrauterine death at E 10.5 due to a defect in the early heart development (40,75). Hence, a conditional or splice-variant specific inactivation of the versican gene will be required to study the function of versican in restricted tissues and during specific develo~mentalperiods. Since knockout mice, lacking either the neurocan or the brevican gene, seem not to suffer from immediately obvious i~pairments(76),multiple inactivation of hyalectan genes may be necessary to obtain phenotypic changes in particular tissues, such as the central nervous system. We are confident that the function of versican will prove to be as versatile as its structure.

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This work was supported by grants from the Krebsliga des Kantons Zurich and from the Lydia Hochstrasser Foundation. Special thanks go to Maria Teresa ours-Zimme~ann and Michael Schmalfeldt for helpful comments on the m~uscript.

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35. Aspberg A, Binkert C, Ruoslahti E. The versican C-type lectin domain recognizes the adhesion protein tenascin-R. Proc Natl Acad Sci USA 1995; 92:10590-10594. 36. Aspberg A, Miura R, Bourdoulous S, Shimonaka M, Heinegird D, Schachner M, Ruoslahti E, Yamaguchi Y. The C-type lectin domains of lecticans, a family of aggregating chondroitin sulfate proteoglycans, bind tenascin-R by protein-protein interactions independent of carbohydrate moiety. Proc Natl Acad Sci USA 1997; 94110116-10121. 37. Yamagata M, Saga S, Kato M, Bernfield M, Kirnata K. Selective distributions of proteoglycans and their ligands in pericellular matrix of cultured fibroblasts. Irnplications for their roles in cell-substraturn adhesion. J Cell Sci 1993; 106:55-65. 38. Landolt RM, Vaughan L, Winterhalter KH,Zimermann DR. Versican is selectively expressed in embryonic tissues that act as barriers to neural crest cell migration and axon outgrowth. Development 1995; 121:2303-2312. 39. Shinomura T, Jensen m,Yamagata M, Kimata K, Solursh M. The distribution of mesenchyme proteoglycan (PG”) during wing bud outgrowth. Anat Embryo1 (Berl) 1990; 181:227-233. 40. Mjaatvedt Ch, Yarnamura H, Capehart AA, Turner D, Markwald RR. The cspg2 gene, disrupted in the mutant, i s required for right cardiac chamber and endocardial cushion formation. Dev Biol 1998; 20256-66. 41. Zako M, Shinomura T, Miyaishi 0, Iwaki M, Kimata K. Transient expression of PG-Mlversican, a large chondroitin sulfate proteoglycan in developing chicken retina. J Neuroche~1997; 6932155-2161. 42. Yamagata M, Herman JP, Sanes JR. Lamina-specific expression of adhesion molecules in developing chick optic tectum. J Neurosci 1995; 15:4556-4571. 43 Bode-Lesniewska B, Do~rs-Zimme~ann MT, Odermatt BF, Briner J, Heitz PU, Z i m m e ~ a n nDR. Distribution of the large aggregating proteoglycan versican in adult human tissues. J Histochem Cytochem 1996; 44:303-312. 44. Campbell MA, Tester AM, Handley CJ, Checkley GJ, Chow GL, Cant AE, Winter AD, Cain WE. Characterizationof a large chondroitin sulfate proteoglycan present in bovine collateral ligament. Arch Biochem Biophys 1996; 329:181- 190. 45. Zimmermann DR, Dours-Zimmermann MT, Schubert M, B ~ c k n e r - T u d e ~ aL.n Versican i s expressed in the proliferating zone in the epidermis and in association with the elastic network of the dermis. J Cell Biol 1994; 124:817-825. 46. Roark EF, Keene DR, Haudenschild CC, Godyna S, Little CD, Argraves WS. The association of human fibulin-1 with elastic fibers: an immunohistological,ultrastructural, and RNA study. J Histochem Cytochem 1995; 43:401-411. 47. Kumaratilake JS, Gibson MA, Fanning JC, Cleary EG. The tissue distribution of microfibrilsreacting with a monospecific antibody to MAGP, the major glycoprotein antigen of elastin-associatedmicrofibrils. Eur J Cell Biol 1989; 50: 117-127. 48. Sakai LY, Keene DR, Engvall E. Fibrillin, a new 350-kD glycoprotein, is a component of extracellular microfibrils. J Cell Biol 1986; 103:2499-2509. 49. Thomas GJ, Bayliss MT, Harper K, Mason RM, Davies M. Glomerular mesangial cells in vitro synthesize an aggregating proteoglycan immunologically related to versican. Biochem J 1994; 302:49-56. 50. Perides G, Rahemtulla F, Lane WS, Asher RA, Bignarni A. solation of a large aggregating proteoglycan from human brain. J Biol Chem 1992; 267:23883-23887. *

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51. Margolis RK, Margolis RU. Nervous tissue proteoglycans. Experientia 1993; 49: 429-446. 52. Wight TN, Kinsella MG, Qwarnstrom EE. The role of proteoglycans in cell adhesion, migration and proliferation. Curr Opin Cell Biol 1992; 4:793-801. 53. Esko JD. Genetic analysis of proteoglycan structure, function and metabolism. Curr Opin Cell Biol 1991; 3:805-816. 54. Ruoslahti E. Proteoglycans in cell regulation. J Biol Chem 1989; 264:13369-13372. 55. Margolis RU, Margolis RK. Chondroitin sulfate proteoglycans as mediators of axon growth and pathfinding. Cell Tissue Res 1997; 290:343-348. 56. Yamagata M, Suzuki S, Akiyama SK, Yamada KM, Kimata K. Regulation of cellsubstrate adhesion by proteoglycans immobilized on extracellular substrates. J Biol Chem 1989; 264:8012-8018. 57. Perris R, Perissinotto D, Pettway Z, Bronner-Fraser M, Morgelin M, Kimata K. Inhibitory effects of PG-H/aggrecan and PG-~/versicanon avian neural crest cell migration. FASEB J 1996; 10:293-303. 58. Henderson DJ, Ybot-ConzalezP, Copp AJ. Over-expression of the chondroitin sulphate proteoglycan versican is associated with defective neural crest migration in the pax3 mutant mouse (splotch). Mech Develop 1997; 69:39-51. 59. Brecht M, Mayer U, Schlosser E, Prehm P. Increased hyaluronate synthesis is required for fibroblast detachment and mitosis. Biochem J 1986; 239:445--450. 60. Wight TN. Cell biology of arterial proteoglycans. Arteriosclerosis 1989; 9:l-20. 61. Lark MW, Yeo T-K, Mar H, Lara S, Hellstrom I, Hellstrorn K-E, Wight TN. Arterial chondroitin sulfate proteoglycan: localization with a rnonoclonal antibody. J Histochem Cytochem 1988; 36:1211-1221. e zObrien KD, Ferguson M, Nikkari ST, Alpers CE, Wight TN. Differ62, ~ u t i e ~P, ences in the dis~butionof versican, decorin, and biglycan in atherosclerotic human coronary arteries. Cardiovasc Pathol 1997; 6:271-278. 63. Lin H, Kanda T, Hoshino Y, Takase S, Kobayashi I, Nagai R, McManus BM. Versican, biglycan, and decorin protein expressionpatterns in coronary arteries-analysis of primary and restenotic lesions. Cardiovasc Pathol 1998; 7:31-37. 64. Wight TN, Lara S, Riessen R, LeBaron R, Isner J. Selective deposits of versican in the extracellular matrix of restenotic lesions from human peripheral arteries. Am J Pathol 1997; 151:963-973. 65. Matsuura R, Isaka N, Imanaka YK?Yoshida T, Sakakura T, Nakano T. Deposition of PG-Mlversicanis a major cause of human coronary restenosis after percutaneous transluminal coronary angioplasty. J Pathol 1996; 180:311-3 16. 66. Lin H, Wilson JE, Roberts CR, Horley KJ, Winters GL, Costanzo MR, McManus BM. Biglycan, ~ecorin,and versican protein expression patterns in coronary arteriopathy of human cardiac allograft: distinctnessas compared to native atherosclerosis. J Heart Lung Transplant 1996; 15:1233- 1247. 67. Morita H, Takeuchi T, Suzuki S, Maeda K, Yamada K, Eguchi G, Kimata K. Aortic endothelial cells synthesize a large chondroitin sulphate proteoglycan capable of binding to hyaluronate. Biochem J 1990; 26551-68. 68. Wolf YG, Rasmussen LM, Ruoslahti E. Antibodies against transfor~nggrowth factor-p1 suppress intimal hyperplasia in a rat model. J Clin Invest 1994; 93:11721178.

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69. Iozzo RV. Proteoglycans and neoplasia. Cancer Metastasis Rev 1988; 7:39--SO. 70. Sobue M, Takeuchi J, Yoshida K, Akoa S, Fukatsu T, Nagasaka T, Nakashima N. Isolation and characterization of proteoglycans from human nonepithelial tumors. Cancer Res 1987; 47:160-168. ?l. Sobue M, Nakashima N, Fukatsu T, Nagasaka T, Fukata S, Ohiwa N, Nara Y, Ogura T, Katoh T, Takeuchi J. Production and immunohistochemical characterizationof a monoclonal antibody raised to proteoglycan purified from a human yolk sac tumour. Histochem J 1989; 21:455-460. 72. Nara Y, Kat0 Y, Torii Y, Tsuji Y, Nakagaki S, Goto S, Isobe H, Nakashima N, Takeuchi J. Immunohistochemical localization of extracellular matrix components in human breast tumours with special reference to PG-M/versican. Histochem J 1997; 29121-30. n nDifferential expres73. Paulus W, Baur I, Dours-Zim~ermannMT, Z i ~ e ~ a DR. sion of versican isoforms in brain tumors. J Neuropath01 Exp Neurol 1996;55:528-533. 74. Yamagata M, Kimata K. Repression of a malignant cell-substratumadhesion phenotype by inhibiting the production of the anti-adhesiveproteoglycan, PG-Mlversican. J Cell Sci 1994; 107:2581-2590. 75. Yamamura H, Zhang M, Markwald RR, Mjaatvedt CH. A heart segmental defect in the anterior-posterior axis of a transgenic mutant mouse. Dev Biol 1997; 186: 58-72. 76. Rauch U. Modeling an extracellularenvironmentfor axonal pathfinding and fasciculation in the central nervous system. Cell Tissue Res 1997; 290:349-356.

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Finch University of ~ e a l t hSciencedThe Chicago ~ e d i c aSchool, l ~ o Chicago, ~ h Illinois Advanced Tissue Sciences, La Jolla, California

Aggrecan, the large aggregating proteoglycan of cartilage, is the first proteoglycan to have been identified and characterized in detail. It is one of the most abundant and widely studied of the proteoglycans, and one for which the relationship between structure and biological function is well defined. Aggrecan was first recognized as a protein-polysaccharide complex in intervertebral disc and cartilage (I), and the use of density gradient centrifugation for characterization and purification (2) provided a key for the detailed studies that followed. Physical and biochemical analyses of aggrecan structure (3-6), characterization of aggrecan interactions with hyaluronan and link protein in the formation of aggregates (5,7,8), and visualization under the electron microscope (9,lO) were all major milestones in the development of an understanding of this molecule. Although aggrecan is now well characterized as the large chondroitin sulfate (CS)and keratan sulfate (KS)-containing proteoglycan that forms link protein-stabilized aggregates with hyaluronan in the extracellular matrix (ECM), questions regarding its function and modulation remain. In this chapter, we discuss aggrecan structure, function, biosynthesis, gene organization, and the regulation of aggrecan gene expression. Genetic and degenerative diseases involving aggrecan are also examined.

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Aggrecan is expressed in cartilage at all stages of skeletal development, It is essential to the function of cartilage in the absorption of compressive forces in joints throughout life, and as the template for bone replacement during long bone development. These normal cartilage functions are severely compromised in several genetic diseases where aggrecan is either absent from the ECM or undersulfated, and when it is modified and depleted in diseases such as osteoarthritis. Aggrecan contributes to the biomechanical properties of the intervertebral disc (1 l ), meniscus, and tendon (12,13), but its role in the developing brain and notochord (14) is more mysterious.

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Aggrecan is uniquely structured to fill space by effectively concen~ratingnegative charges (15,16). Enormous aggregates composed of noncovalently interacting aggrecan and hyaluronan, stabilized by link protein, accomplish this task (Figs, 1 and 2). These multimolecular aggregates may have lengths of up to 15 pm and achieve dimensions roughly equivalent to the dimensions of an entire cell. Within an aggregate, each aggrecan molecule has a molecular mass of 1-5 X l o6 Da, with sulfated glycosaminoglycan (GAG) chains and N- and 0-linked oligosaccharides contributing up to 90% of the mass. The 220-250 m a aggrecan core protein is typically modified by the covalent attachment of approximately 100 CS chains, 30 KS chains, and 8-10 shorter N- and 0-linked oligosaccharides, CS and KS chains are long, linear structures; CS chains are composed of 40-50 repeats of the disaccharide GlcUA-GalNAc and KS chains contain 20-25 repeats of the disaccharide Gal-GlcNAc. CS chains are polymerized on linkage regions which have the sequence GlcUA-Gal-Gal-Xyl. The Xyl residue itself is covalently attached to specific Ser residues of the core protein. In contrast, KS chains are attached to asialo branches of 0-oligosaccharides covalently linked to the core protein (see Chap. 2). Carboxyl groups and the extensive 0-sulfate substitutions of the GAG chains provide aggrecan molecules with a large number of negative charges. Link protein-stabilized aggregates formed with hyaluronan become compressed within the network of collagen fibrils, packed to 1110-115 their volume in free solution. The resultant high concentration of packed negative charges per unit of space causes positive counterions and water to be entrapped, endowing cartilage tissues with their characteristic resilience, space-filling capacity, and remarkable hydration. The space-filling capability of aggrecan is best demonstrated by aggrecan molecules and aggregates prepared using molecular spreading methodologies,

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ure 1 Electron microscopy of rotary shadowed aggrecan aggregate and monomers. A native proteoglycan aggregate from the Swarm rat chondrosarcoma was prepared by

mica sandwich squeezing/rotary shadowing and shows the G1 domains of aggrecan and link proteins bound to hyaluronan, with the glycosaminoglycan-substitutedregions of aggrecan extending outward, G2 and G3 domains are also apparent. Aggrecan monomers from bovine nasal cartilage after glycerol spraying/rotary shadowing are shown in the insert. G1, G2, and G3 are indicated by small arrows. Note the globular and extended domains of the molecule, as shown schematicallyin Figure 2. Bar, 0.25 pm. (From Ref. 174.)

improved through the application of glycerol spraylrotary shadowing and mica sandwich/rotary shadowing (1’7,18). In Figure 1, structural features of the interactions of aggrecan and link protein with hyaluronan and the extended GAGsubstituted region of aggrecan are revealed in the large aggregate, with the two N-terminal and one C-terminal globular (G) domains and interglobular (IGD) regions of individual aggrecan molecules highlighted in the insert. Aggrecan is organized into well-defined domains, and these reflect specific st~cture/functionrelationships in the protein and the corresponding exon pattern

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Schematic representation of aggrecan and its domains aligned with the genomic m a p of the human aggrecan gene. The coding exons (filled bars), noncoding exon 1 (open bar), and introns (line) are shown in (a) and aligned with domains of the core protein and the aggrecan proteoglycan in (b) and (c), respectively. Interaction of the G1 domain with link protein and hyaluronan is indicated in (c), as are CS and KS GAG chains, a few N-and 0-linked oligosaccharides,and disulfide bonds. (From Ref. 174.)

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within the aggrecan gene (Fig. 2). The structure of the aggrecan gene has now been described in detail for human, mouse, chicken, and rat (19-23). This knowledge, combined with the characterization of cDNA sequences (24-27), affords a complete picture of the gene and protein organization. Evolutionary interrelationships concerning the origin and divergence of specific domains are suggested now that detailed sequence information is available for a number of species (recently reviewed in Ref. 28). The striking homologies that aggrecan shares with other hyaluronan binding proteoglycans such as versican, neurocan, and brevican have led to consideration of an aggrecan or hyalectan/lectican gene family (see Chaps. 13 and 15). The aggrecan gene is large and complex, composed of 1819 exons ranging from 77 to 4224 bp. Exons 2 to 19 of the human gene encode a protein core of 2,454 amino acids with a calculated mass of 254,379 Da. The coding exons are flanked by noncoding sequences (exon 1 and the 3’ end of exon 19) that, combined with the sequence upstream of exon 1, appear to be involved in the regulation of gene expression (29,30) (see Sec. III.B.l). The entire coding region is contained in over 39 kb of the gene. The organization of exons is strongly related to the specific domains of the protein core. Briefly, G1 is encoded by exons 3-6, the IGD by exon 7, G2 by exons 8-10, and G3 by exons 13- 18 [including alternatively spliced epidermal growth factor (EGF)-like and complement regulatory-like domains]. Exon l 1 codes for the 5‘ half of the KS-rich region, while exon 12 is a pa~icularlylarge exon that encodes the 3’ half of the KS-rich region as well as the entire CS-rich region. The correspondence between exon organization and the protein domains argues strongly for the modular assembly of the aggrecan gene during evolution. The amino terminal G1 domain is very impo~ant,for it forms noncovalent but stable interactions with hyaluronan and link protein, and thereby serves to immobilize the aggrecan molecule in the ECM. It contains an N-terminal loop (A), which shares sequence homology with the immunoglobulin superfamily, and two loops (B and B’), termed proteoglycan tandem repeats (PTRs), which share homology with each other. The G2 domain, of unknown function, bas two homologous PTRs, but lacks the Ig superfamily-homologous N-terminal loop and exhibits no hyaluronan binding (31). The sets of exons coding for the B and B’ loops of G1 and G2 are identical in size and organization. Link protein, which also interacts with hyaluronan (32), contains regions that are homologous to the A, B, and B’ loops of the GI domain (33,34). Other hya~uronan-bindingproteins share partial sequence homologies with these same regions (31), and members of the aggrecan or hyalectan/lectican gene family have nearly identical organization (see Chaps. 13 and 15). A detailed analysis of interactions with hyaluronan, using a battery of recombinant aggrecan protein fragments expressed in a mammalian cell line, suggests the importance of the A loop in hyaluronan binding, acting in a cooperative manner with subdomains B and B’ (35). The data further suggest that carbohydrate modifications of the aggrecan domain facilitate high-

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level hyaluronan binding. In contrast to G1 and G2, the IGD between them is shown to be linear and fairly inflexible in rotary-shadowed preparations. The great sensitivity of the IGD to specific degradative enzymes such as metalloproteinases or “aggrecanase” is notable. Combined with the strategic position of this region, between the G1 domain that immobilizes aggrecan in link-sta~ilized hyaluronan aggregates and the GAG~substitutedregion that confers the functional capability of concentrating charge, the region is implicated as a critical site involved in aggrecan turnover in the ECM and in cartilage degradation (see Chap. 5 for further discussion). The highly conserved C-terminal domain, G3, is particularly intriguing. Sequence homologies of G3 subdomains are shared with EGF, the type C lectins, and the complement E3 regulatory element, The same structural motifs are characteristic of the selectins (36) and several other proteoglycans of the hyalectanf lectican gene family. The selectin homologies strongly suggest a role in carbohydrate or protein recognition. Weak carbohydrate binding has been demonstrated for the lectinlike domain of aggrecan (37,38), which is characterized by a remarkable degree of conserved secondary structure shared with a superfamily of 131 proteins (39). The recent demonstration of calcium-dependent interactions of the lectin domain with tenascin-R through protein rather than carbohydrate moieties expands the possibi~itiesfor potential inter~olecularinteractions (40). Interestingly, both the lectinlike and complement regulatory domains of aggrecan contain basic residues that are atypical of their supe~amiliesand may be related to unique functions (39). Two EGF-like sequences, EGFl and EGF2, have been identified in the aggrecan gene, and both the EGF and complement r e g u l a t o ~protein domains are subject to alternative splicing (41-43). Expression of the EGF2 sequence is relatively uniform among different species, present in about 6% of the aggrecan transcripts, while EGFl expression is significant only in human aggrecan transcripts, at a level of 25%. No age-related variation or physiological significance has been ascribed to the observed alternative splicing. However, by analogy, it is possible that the EGF-like domain is involved in protein interactions (44) or in growth stimulation, as has been demonstrated for the EGF-like modules of ECM molecules such as laminin (45). Recently, the G3 domain of versican was shown to enhance proliferation (46). The alternative splicing of the EGF domains shown for aggrecan is unusual, and thus far has not been demonstrated for the other proteoglycans of the hya~ectican/lectican family. ~ o t a b l ygreater , than half of the mature aggrecan molecules in the cartilage are missing the G3 domain (47). Evidence suggests that G3 is more prevalent in newly synthesized aggrecan molecules, and that the loss of G3 reflects proteolytic processes and the age-related turnover of aggrecan in the ECM (48). These observations are compatible with a role for G3 in stabilizing initial inter-

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actions in the ECN or functioning in intracellular events (see Sec. TI1 for discussion). The long linear stretch between G2 and the C-terminal G3 is the principal region of the molecule substituted with GAG chains. It is the least-conserved region, and varies among species both in the type of substitutions and the organization of its domains. One striking difference is represented in the degree and distribution of KS modifications. A KS domain was first described in bovine aggrecan as a 23 repeat Glu-Pro rich hexapeptide lying just beyond G2 (459, and has been identified since as a 12-fold repeat in human aggrecan (25). When the domain is present, about two thirds of the KS chains are clustered there. In certain aggrecan molecules characterized by minimal KS substitution, such as rat aggrecan, only a few, if any, poorly conserved repeats are detected (19,23), Sequences outside of this domain are also capable of specifying KS addition, insofar as KS substitution has been observed for the CS and IGD regions, and, to a lesser extent, for C1 and G2 in aggrecans that contain the hexapeptide repeats. In addition, chicken aggrecan is well substituted with KS although it has at most a few poorly conserved repeats (discussed in Refs. 28 and 50). In all cases, the CS-rich region is encoded by a single, large exon, and characterized by many Ser-Gly dipeptide repeats that serve as sites for CS attachment. The specific size and organization of the region is variable, and the actual substitution of specific Ser residues may also depend upon the spacing of SerGly pairs and whether or not they follow acidic residues (discussed in Refs, 28 and 50). CS1 and CS2 domains are roughly distinguished by the spacing and clustering of the Ser-Gly repeats. The CS1 region is characterized by a 20 amino acid repeat represented 15 times in rat and 29, times in human aggrecan, while the CS2 region has a more variable 100 amino acid repeat structure with fewer repeats that appears to be the result of repeated amplifications and fusions of a fundamental 10 amino acid sequence. Chicken aggrecan is characterized by a separate 20 amino acid repeat present in the CS2 region, a smaller CS1 region bearing less similarity to other CS 1 regions, and a more significant representation of a third repeat region, CS3. Variability is further suggested by the high frequency of polymorphisms in the CS region recently reported for the human aggrecan gene (51). Thus, aggrecan is an unusually large and complex molecule, with design characteristics that promote a variety of molecular interactions and serve specific functions. It should be emphasized, however, that the aggrecan molecule we have described above exists in a fully extended state in vitro, and that within the physiological context of its native state in the cartilage ECNI, aggrecan often occupies a greatly restricted space. How the GAG chains and protein cores interact under those conditions remains to be determined.

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In articular and other cartilages, aggrecan has a well-defined, mechanical function. The two primary components of the cartilage ECM, apart from water and counterions, are collagen and proteoglycan. The type I1 and minor collagens form a dense meshwork of fibrils and larger fibers, and thereby provide the tensile properties of the tissue. This fibrous network is embedded in a high concentration of aggrecan, which contributes compressive resistance and resilience. The composite material formed from the two components supports the unique biornechanical characteristics of articular cartilage that allow the tissue to withstand significant loads and provide smooth articulation of joints throughout life (for review, see Refs. 52 and 53). Aggrecan complexes become immobilized within the ECM, primarily through steric hindrance involving the collagen network and other matrix molecules. The many negatively charged GAG chains of aggrecan provide a highly polyanionic environment that leads to charge-charge repulsion and the creation of Donnan osmotic pressure. As a result, the collagen network is maintained in a distended and stretched state (54), and the cartilage tissue is characterized by a high level of hydration. Cartilage normally functions under varying degrees of mechanical loading. The biomechanical properties, and underlying enginee~ngprinciples by which cartilage functions, have been described in biphasic (55) and triphasic (56) models. When cartilage is first loaded, the fluid (water and salts) carries the load. With time, the fluid flows, tissue deformation occurs, and the load is transferred from the fluid to the solid matrix (e.g., the collagen and proteoglycan). Aggrecan plays a vital role in these events, evidenced by the fact that loss of the sulfated GAG chains from cartilage reduces its compressive stiffness by over 80% (54). Aggrecan also contributes significantly to the viscoelastic properties of cartilage, in tension and shear (54,57). The compressive resistance of cartilage is regulated in part by its permeability, which ranges from to m4/N.s. As a consequence, a very high drag coefficient exists. Again, the sulfated GAGS of aggrecan contribute greatly to this feature. At high concentrations such as those found in cartilage, aggrecan molecules form networks with significant mechanical strength and energy storage capability (54,58,59). The extent to which aggrecan exhibits these properties depends upon the characteristics of the aggregan complex, including the proportion of aggrecan molecules present as aggregates, link protein stabilization, the size of hyaluronan in the aggregate, and solution concentration. In interactions with collagen, aggrecan reduces collagen network stiffness and apparent viscosity, probably by filling the interfibrillar space and keeping the collagen fibrils apart (60). The aggrecan molecules may also act as a lubricant, allowing the fibrils to slide more easily over each other.

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Aggrecan plays a significant role in the developing skeleton. In the process of endochondral bone formation, cartilage provides the model replaced by bone. The unique qualities of the cartilage ECM, highly hydrated yet with some degree of tensile strength, lend support, and at the same time, create space for the continued proliferation and maturation of chondrocytes. Thus the cartilage model is necessary for directed linear growth in the growth plates of long bones, and later provides a scaffold for the deposition of bone by osteoblasts. As discussed earlier, the structural features of aggrecan promote the concentration of negative charges, which in turn maintains the hydrated state and expanded tissue volume of the cartilage. The critical role of aggrecan in these processes is emphasized by genetic disorders such as n u n o~ eZ iuand cartilage matrix deficiency (cm d ), dwar~sms characterized by the absence of aggrecan in the cartilage ECM (see below). It has been proposed that aggrecan may also function in mineralization in the lower hypertrophic zone of the growth plate by undergoing condensation as calcium binds to aggrecan CS chains during the process of calcification. This concentration of calcium is thought to be part of a pathway leading to an interaction with inorganic phosphate and calcium phosphate precipitation (61). Aggrecan expression, at both the mRNA and protein levels, is tightly correlated with chondrogenesis in vivo and in cell culture. It has been suggested that aggrecan in the ECM contributes to the maintenance of the chondrocyte phenotype early on in cartilage differentiation by interfering with cellular adhesion (14). Aggrecan is likely to have a mechanical function in connective tissues other than cartilage. In tendon, aggrecan is expressed at relatively high levels in fibrocartilage regions subjected to compressional load and is also detected in tensional regions (12,13). While aggrecan isolated from compressed regions is similar to cartilage aggrecan, the aggrecan from tensional regions lacks KS, and, apparently, lacks the G1 domain as. well (13). In bone, a function for aggrecan is suggested by the expression of aggrecan in normal calvarium and by the observation of intramembranous bone defects and increased bone stiffness in nanomelic chickens (62), which lack aggrecan in the ECM (see below). Using the embryonic chicken as a model, screening with the avian aggrecan-specific S103L monoclonal antibody has led to the identification of aggrecan in the notochord and developing brain (reviewed in Ref. 14). In the notochord, aggrecan was detected at embryonic stage 16 (considerably earlier than the onset of chondrogenesis) and remained present during the period of neural crest migration, through the beginning of sclerotome differentiation (63). In light of evidence that the notochord and large CS proteoglycans (including aggrecan) are inhibitory for the migration of neural crest cells, the timing and pattern of aggrecan expression are compatible with a role for aggrecan in creating a notochordal environment that excludes neural crest cells (64,65). In addition, aggrecan serves as a component of the notochordal ECM in conjunction with types TI and IX collagen and hyaluronan to provide structural support to the embryonic

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axis, and perhaps promotes chondrogenesis in the sclerotome. In the brain, aggrecan is synthesized by neurons, subject to developmental regulation. Expression is demonstrated from day 7 to day 19 in the embryo, with a peak for aggrecan mRNA at day 10 and for S 103L immunoreactivity at day 13. The correlation of aggrecan expression with the period of active neuronal migration and the establishment of neuronal nuclei led to speculation that aggrecan may be involved in the mechanism that arrests neuronal migration (14,66). These are reasonable proposals for specific aggrecan functions in the developing notochord and brain, but difficult to verify, because in both cases other CS proteoglycans are also present. The lack of obvious effects of the nanomelic mutation on the development of the peripheral or central nervous system suggests that, in contrast to cartilage, aggrecan function may be redundant or adequately assumed by other CS proteoglycans in the notochord and brain. Several aspects of comparative aggrecan expression are of' note. All evidence indicates that a single aggrecan gene (AGC1) exists in the genome; in humans, the aggrecan gene was localized to chromosome 15q26 by in situ hybridization (67,68), and in mouse, to chromosome 7 (69). The identification of the same molecular defect in aggrecan RNA from the cartilage, notochord, and brain of nanomelic chickens is also consistent with a single gene for aggrecan (63,70). Thus, cellular mechanisms must be in place for the developmental and tissuespecific regulation of aggrecan expression. For the most part, aggrecan mRNA and protein have been shown to increase in parallel, suggesting regulation at the level of transcription. Studies to dissect the transcriptional control elements of the aggrecan gene are currently underway, and are discussed below in Sec, I1I.B. 1. Also of interest are the observations that despite transcription from the same gene, the resulting aggrecan proteoglycans exhibit distinct, tissue-specific s~ucturalproperties. For example, chicken aggrecan is substituted by CS and KS in ca~ilage,is substituted by CS but lacks KS and acquires the HNK-1 epitope (a 3-sulfo~lucuronicacid modification) in notochord, and in brain is substituted by fewer and shorter CS chains and lacks both KS and HNK-1 Nonetheless, the core protein transcripts appear to be identical in each case, with no suggestion of tissue-specific alternative splicing. In bovine tendon, CS and KS substitutions were observed for aggrecan in compressed regions, but aggrecan in tensional regions lacked KS.Together, these data support the view that, in addition to its regulated expression, the aggrecan core protein undergoes tissue-specific post~anslationalprocessing, suggesting that the cellular processing machinery is modulated in a differentiation-specific manner as well. Thus, the complexities of the aggrecan molecule are well characterized with regard to structure/function relationships in cartilage. However, the consideration of aggrecan structure, function, and expression in other tissues and organs has raised new questions, and will most assuredly lead to an expanded view and appreciation of the molecule.

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Our understanding of aggrecan structure/function is enhanced significantly by a consideration of the genetically based and degenerative diseases involving aggrecan. Although no human disease or genetic mutation has been identified that directly affects the aggrecan core protein, two autosomal recessive chondrodysplasias, ~ ~ n o m einZchickens i~ (Fig. 3, see color insert) and cartilage matrix deficiency (cm d ) in mice, result from mutations in the aggrecan gene. The molecular defect of n ~ ~ o ~is ae GZ toi T~ transversion that replaces a Glu with a premature stop codon and leads to the production of a truncated core protein reduced 30% in size, which lacks the C-terminal region (43). In the case of cm d , a 7 base-pair deletion in exon 5 (within the B loop of the G1 domain) results in an ev truncation (69). In each case, aggrecan is absent from the cartilage IEC a consequence, the homozygous recessive embryos have significantly shortened long bones and gross skeletal abnormalities (Fig. 3). The cm d mouse appears not to produce any protein, while nanomelic chickens synthesize small amounts of a truncated core protein that fails to progress through the secretory pathway (see Sec. 111). Both mutations are lethal, most likely due to respiratory difficulties arising from the collapse of defective cartilage rings in the trachea. Recently, late-onset spinal disorders were described in cmd heterozygous mice, suggesting that an error in the aggrecan gene may predispose an individual toward spinal defects (7 l). The ability to concentrate negative charges is central to aggrecan function, and several genetic diseases highlight this important aspect of aggrecan structure/ function. For example, ~ u t a t i o n sin the diastrophic dysplasia sulfate transporter (DTDST), a novel sulfate transporter first identified by positional cloning, give rise to a spectrum of autosomal recessive, short-limbed dwarfisms (72). Diastrophic dysplasia is the mildest disease of the group, recognized at birth by severe club foot deformities and hitchhiker thumbs. Individuals are also afflicted with joint dysplasias and deformities of the spine and outer ear. Atelosteo~enesis type I I is a perinatal lethal disease with similar anomalies, and individuals with achondro~enesislB,the most severe form, are either stillborn or die minutes after birth (73-75). Fibroblasts from patients with DTDST mutations exhibit defects in sulfate transport and the sulfation of proteoglycans. Differences in severity among the three diseases reflect the level of sulfate transporter activity remaining (and the specific mutation in the DTDST gene), with achondrogenesis 1B considered to be the null phenotype with complete loss of function. The brachymo~hic ( ~ ~ / mouse ~ m is) another short-limbed dwarfism characterized by a sulfation deficiency. In this case, however, the defect is the result of reduced quantities of phosphoadenosine phosphosulfate (PAPS), the high-energy sulfate donor used in the sulfation of glycos~inoglycanchains (see Ref. 76 and references therein). The mutation affects catalytic properties of the sulfation activation system by

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altering the function of a novel enzyme coupling mechanism involving a bifunctional sulfurylase kinase (SIC), leading to insufficient levels of PAPS.In a recent report, the defect was identified as a missense mutation in a highly conserved amino acid residue that destroys the kinase activity (77). This study also provides evidence for a family of bifunctional SI( enzymes (with the SK2 gene residing at the bm locus), suggesting that members of this gene family have nonredundant, tissue-specific roles. Overall, mutations that depress components of the metabolic pathway involved in sulfation exert severe effects in cartilage because chondrocytes produce undersulfated aggrecan molecules under conditions of impaired sulfate availability. As a result, negative charge and the level of hydration in the cartilage ECM is reduced. Consequently, the cartilage tissue model occupies a substantially diminished total space and, when replaced by bone, creates a dwarfed skeleton. With respect to degenerative diseases, aggrecan deficiences are a hallmark of the joint diseases osteoarthritis and rheumatoid arthritis. Clinically, the diseases are manifested as joint pain, infla~mation,and loss of motion. ~articularly in osteoarthritis, the major pathological event is degeneration of the articular cartilage, which initiates as roughening and fibrillation and progresses to loss of cartilage exposing the subchondral bone. Since osteoarthritis is diagncised at relatively late stages of the disease process, much of our knowledge about early phases of the disease has come from studying animal models of joint disease. One of the first changes observed in cartilage during the onset of osteoarthritis is a decrease in aggrecan concentration, particularly in the superficial zone of the cartilage (78). However, this decrease in concentration is primarily due to an expansion in the collagen network, resulting in an increase in cartilage volume and decrease in proteoglycan concentration, and not resulting from an abnormally high turnover of aggrecan. Subsequently, the chondrocytes initiate a repair response, involving an increased rate of proteoglycan synthesis (79), but this is accompanied by an increased loss of newly synthesized and established proteoglycan from the cartilage ECM (80-82). The increased catabolism is reflected in increased levels of aggrecan fragments in the synovial fluid of the diseased joint, and it has been suggested that these fragments may serve as bioc~emical markers of early joint disease (83-86). The repair process eventually fails, and results in significant decreases in cartilage aggrecan. Characteristic biochemical changes in the properties of the proteoglycan within the cartilage include increased extractability, decreased relative content of KS to CS, and a reduction in its ability to interact with hyaluronan, The CS chains of aggrecan undergo subtle changes in structure, including increased size. Some of these changes may be detected by monoclonal antibodies (87). The structural changes in newly synthesized aggrecan reflect alterations in posttranslational modi~cationsof the protein core, and the resulting aggrecan molecules share features with the aggrecan synthesized in younger, developing cartilage.

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Figure 3 Skeletal structures of nanomelic (left) and normal (right) day-17 chicken embryos are shown stained with alizarin red and alcian blue (for calcified bone and glycosarninoglycans of the cartilage, respectively). Note the shortened, alizarin red-stained bones of the nanomelic embryo and the absence of alcian blue staining. In contrast, the skeletal structures of the norrnal embryo are stained intensely with both alizarin red and alcian blue. (From Ref. 174.)

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The mechanical properties and overall behavior of the cartilage also change in osteoarthritic cartilage. It is likely that most of the decrease in compressive stiffness of the cartilage can be assigned to the decreased concentration and reduced aggr~gationof aggrecan, but a combination of other biochemical and structural alterations may also be involved. The in~ammatorycomponent of rheumatoid arthritis and related diseases has a direct effect on aggrecan in the articular cartilage. Cytokines such as interleukin (IL)-1 and tumor necrosis factor (TNF) a stimulate chondrocytes to actively and rapidly degrade aggrecan in the cartilage matrix. The primary protease responsible has yet to be identified, although it has already been termed aggrecanase. Other proteases that have the capacity to degrade aggrecan include some of the metalloproteinases. The acute loss of aggrecan from the articular cartilage can be reversed when the inflam~atoryepisode ends. However, the same cytokines also suppress aggrecan synthesis, and since this can occur at cytokine levels five- to tenfold less than those required to induce aggrecan catabolism, the net result may be a long-term chronic effect on the joints, with a depletion of aggrecan from the cartilage.

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The distinguishing structural properties of aggrecan are the result of a series of complex biosynthetic events involving thousands of individual reactions. The elongation and sulfation of GAG chains have been studied for some time, expedited by the ease and sensitivity of sulfate inco~orationand the availability of biochemical procedures for the isolation and characterization of proteoglycans. The investigation of early stages in aggrecan biosynthesis became possible once the core protein was identified and could be followed through subsequent processing (88-92). Because of its strong commitment to these activities, the chondrocyte, with its well-de~nedendoplasmic reticulum (ER) and Golgi apparatus, is an ideal model for aggrecan biosynthesis. The basic features of aggrecan biosynthesis and processing are exemplified by molecules that utilize the constitutive secretory pathway. In these cases, biosynthesis is initiated by translation in the cytosol and translocation of the nascent polypeptide into the lumen of the E through the translocon, via a signal peptide-mediated process (93,94). Continued translation and modification, transport to the cis Golgi and transit through the cis, medial, and trans Golgi compartments are accompanied by progressive processing, and finally, secretion into the extracellular space where interactions with other matrix constituents serve to construct the ECM. If the aggrecan core protein fails to be synthesized or transported through the secretory pathway, or undergoes

abnormal CO- or posttranslational modification, skeletal defects and the compromised cartilage function discussed previously can result. After the nascent aggrecan core protein is translocated to the ER lumen, N-linked oligosaccharides are added cotranslationally to Asn residues from dolichol phosphate intermediates, as is the case for typical glycoproteins (95). The formation of disulfide bonds, folding of the G1, G2, and G3 globular domains, most likely facilitated by chaperones, and initial trimming of the Nlinked oligosaccharides presumably occur before the modified core protein exits the ER (96). Several aspects of aggrecajn processing appear to involve late ER compartments continuous with, but distinct from, the rough ER. Using immunoelectron microscopy, aggrecan precursors were identified in smooth membrane-limited ER subcompartments in chicken chondrocytes (Fig. 4; Ref. 97), and shown to accumulate in these regions under conditions known to slow vesicular transport (98), suggesting that some rate-limitingistep in aggrecan processing takes place there. Other studies, utilizing semipermeabilized chondrocytes in combination with electron microscopic autoradiography and subcellular fractionation, have established that xylose addition, the first step in the formation of CS chains, begins in these late-ER compartments (99). Based on several lines of evidence, including the coordinate expression of aggrecan and xylosyl transferase in chondrocytes, it has been proposed that iylosylation may be a key regulatory step in aggrecan biosynthesis (reviewed in Ref. 100). However, the observation that product expressed from aggrecan domains is secreted from transfected xylosyl transferase-deficient C H 0 cells would suggest that xylosylation is not obligatory for progress through the secretory pathway (101). Studies of nanomelic chondrocytes further demonstrate that exit from the ER or from late ER subcompartments is crucial for continued progress of aggrecan intermediates through the secretory pathway leading to the secretion of aggrecan as a fully glycosylated and sulfated proteoglycan (98,102). As a result of the premature stop codon within the CS2 domain, nanomelic chondrocytes synthesize a truncated core protein that fails to become a proteoglycan. The abnormal core protein is modified by the addition of N-linked oligosaccharides and xylose but it moves no further in the secretory pathway than the late ER (Fig. 4e). The abnormal precursor is not translocated to the Golgi and does not accurnulate in the ER, but, based on pulselchase labeling studies, exhibits a time-dependent loss with characteristics suggesting that the process of ER-associated degradation is involved. Presumably, the truncated core protein is recognized as abnormal by cellular quality control mechanisms that target it for degradation. As currently understood, a misfolded or otherwise abnormal protein (in this case, the truncated nanornelic core protein) is detected as such by a lumenal sensing mechanism and translocated out of the ER to the cytosol via a translocon pore, where it becomes ubiquitinated and degraded by proteasomes (103,104). In this

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ure 4 Intracellular aggrecan immunolocalization in normal and nanomelic chondrocytes. Intracellular compartments of chicken chondrocytes involved in aggrecan biosynthesis were revealed by immunostaining after removal of the EGM by hyaluronidase digestion followed by fixation and pemeabilization. Normal chondrocytes shown in phase (a) were double-stained with polyclonal antibodiesthat recognize aggrecan, and its precursors (b) and with the 5-D-4 monoclonal antibody that recognizes KS (c). Localization was visualized using fluorescence-labeled secondary antibodies. Arrows indicate the perinuclear Golgi regions that stain with both antibodies, while arrowheads indicate pre-Golgi, aggrecan-precursor-containingregions. In (d), ultrastructurallocalizationof the polyclonal aggrecan antibodies using i~munopero~idase reactions demonstrates that the pre-Golgi compartments are smooth-membrane-limited regions of the ER, distinct from, yet continuous with, the rough ER. In nanomelic chondrocytes (e), aggrecan antibodies react only with the late-ER regions and exhibit no staining of the perinuclear Golgi. Ba r for a, b, c, e = 2 pm; for d = 0.5 pm. (From Ref. 174.)

aggrecan first enters the region of the cis Golgi network, subsequently travels through the cis, medial, and trans Golgi, and exits the cell from the region of the trans Golgi network. Several aspects of Golgi-mediated GAG modi~cations are discussed below (see Chap. 2 for additional information). KS chain formation involves the repeated addition of individual monosaccharides, initially at the terminal Gal of asialo branches, to create repeating GalGlcNAc disaccharides. It is likely that chain elongation and sulfation are concurrent events, probably occurring in the late Golgi, since 0-oligosaccharide glycosylation itself occurs there (9 1,114), and KS has been localized immunohistochemically to the late Golgi (Fig. 4c; S. LaFrance and B. Vertel, un~ublished). The addition of KS as a late Golgi process is also supported by the absence of KS on aggrecan molecules synthesized by chondrocytes treated with brefeldin A (115), an inhibitor that prevents anterograde transport through the Golgi complex and leads to the formation of a hybrid ER-Golgi compartment by disassembly of the cis, medial, and trans Golgi and their subsequent fusion with the ER (1 16). Although hexapeptide repeats within the KS domain presumably signal KS substitution, a number of questions remain regarding KS addition. For example, what specifies KS substitution of 0-oli~osaccharidesalong the aggrecan core protein outside of the KS domain? Why does KS substitution of aggrecan increase with age? And why do notochord, brain, and tensional tendon aggrecans lack KS? This last question is intriguing and may have implications for a cell-type selectivity in the organization of the biosynthetic machinery, because it has been established that there is a single gene encoding aggrecan, the mRNAs for both notochord and brain aggrecans (lacking evidence of alternative splicing) correspond completely to aggrecan mRNA in cartilage, where aggrecan is well substituted with KS, and, at least in brain, other KS-containing proteoglycans are synthesized concu~ently (14). In this regard, it is of interest that a single sulfotransferase cloned from and expressed in chicken chondrocytes can function in the 6 - 0 sulfation of both CS and KS (1 17-120), and that a different enzyme identified in a human fetal brain library was found to be expressed in brain and cornea (121). More is known about early events in the formation of CS chains (see also Chap. 2). Syl-substituted serine residues of the aggrecan core protein are further modified by the addition of the linkage sugars GlcUA-Gal-Gal in the early Golgi. Sugars are each added sequentially by specific glycosyltransferases in the cis to medial Golgi (reviewed in Ref. 114). The subsequent addition of GalNAc initiates the polymerization of CS. This is a determinative event, because the same ~ l c ~ A - G a l - G a l - sequence ~yl can also serve as the linkage region for the polymerization of heparan sulfate (HS) chains. Several studies suggest that HS chains are added preferentially when a number of sequence-based criteria are met, and when they are not, CS chains are added, in a sense by default (122,123). In this regard, a recent report presents evidence that the G3 domain of aggrecan (a non-

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GAG~substitutedregion) can enhance CS chain production (124). With respect to the linkage sugars themselves, several atypical modifications have been described. Frequently, Xyl is phosphorylated, and Gal may be sulfated (125- 127). It has been suggested that the modified sugars serve as signals for targeting or trafficking, or perhaps specify an aspect of subsequent biosynthetic processing. The cellular basis for these modifications is not known. Chain elongation and sulfation follow as later Golgi events. The polymerization and Q-sulfation of GluUA-GalNAc disaccharide repeats begin in the medial/trans Golgi, occur rapidly, and are thought to be catalyzed by multienzyme complexes organized within the Golgi membranes (reviewed in Ref. 114). Progress has been made in the characterization of the modifying enzymes with the purification of the glucuronosyl transferase I1 (128), the 6-sulfotransferase (1 17-1 19), and most recently, the 4-sulfotransferase (129). The photoaffinity labeling of specific substrates has been instrumental in the success of some of these efforts (l 18,128). Based on current progress, we can anticipate significant strides in the further cloning and characterization of the enzymes over the next few years. With respect to specific features of the CS chains, the age-related regulation of chain length, decisions regarding the extent and type of 4- or 6-0-sulfation of GalNAc, and the generation of unique epitopes are poorly understood. Recently, methods adapted for the detailed analysis and quantification of GAG chain fine structure have revealed differences in the chain termini of adult as compared to fetal and growth plate cartilage aggrecan CS chains (130). Ultimately, an informative model of proteoglycan biosynthesis must accommodate both standard and unusual features of GAG chain structure. Once fully modified aggrecan is secreted from the cell, it associates with other matrix molecules and becomes assembled into a characteristic ECM. At the cell surface, the pericellular matrix provides a linkage to the extended EGM through hyaluronan synthesized at the plasma membrane (see Chap. 2), mediated by cell surface hyaluronan receptors such as CD44 (13 1,132). In the cartilage ECM, link-stabilized aggregates are formed with hyaluronan, and interact in the extended matrix with the organized network of fibrillar collagens and their associated glycoproteins and proteoglycans. Some studies suggest that the G1 domain of aggrecan must undergo an extracellular c o n f o ~ a t i o n a maturation l before it is able to interact effectively with hyaluronan and link protein (133,134). It has also been suggested that G3 may be involved in stabilizing matrix interactions for newly secreted aggrecan. The process of extracellular assembly and matrix deposition for noncartilage aggrecans is largely unexplored. Although aggregate formation involving hyaluronan and link protein may be involved, unique subsets of ECM components present at significantly different concen~ationsare found in these noncartilage locations, and it is expected that the structural and functional properties of the resultant ECMs will be quite different from those in cartilage.

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The regulation of aggrecan expression is exemplified by its tissue specificity, changes in expression and synthesis during different stages of development, and alterations in concentrations with changes in function (which may vary with time and activity), age, and disease state. The regulation of synthesis can occur at several discrete points, including ~odulationat the levels of transcription, RNA processing, translation, and posttranslational modifications. Indeed, since most of the aggrecan molecule is added posttranslationally, there is great oppo~unity for final ~odificationsof the newly synthesized molecule through the addition of oligosaccharide and GAG chains and sulfation, Regulation of aggrecan concentration also occurs at the level of catabolism, as it has been shown that tissue concentrations of aggrecan reflect not only the rates of synthesis but also the rates of breakdown of aggrecan molecules within the ECM. Although the understanding of aggrecan regulation is still incomplete, it is clear that both anabolic and catabolic processes are responsive to a variety of factors. The sections below describe transcriptional regulation of the aggrecan gene, and the soluble mediators such as growth factors and cytokines, and the mechanical inputs that affect aggrecan metabolism. The mechanisms by which cells (chondrocytes and other cell types) orchestrate aggrecan metabolism remain poorly defined, but the ability to sense so many different types of signals, and consequently coordinate and modify regulation of aggrecan synthesis and breakdown, is remarkable. It is anticipated that within the cell, common intracellular signaling pathways exist to coordinate such disparate cellular influences as growth factors and mechanical load. Studies of aggrecan regulation, p ~ i c u l a r l yat the level of aggrecan synthesis, have utilized several methodologies. Each has limitations that impact the inte~retationof the results. A commonly used method is the measurement of 35S-sulfateincorporation into GAG chains (133, a method that itself does not have an inherent specificity for aggrecan. However, specificity is gained by the experimental systems used, in which aggrecan is the predominant proteoglycan being synthesized. It should be recognized that 35S-sulfatewill be incorporated into the sulfated GAG of any proteoglycan being synthesized, and the data generated will be a summation of all proteoglycan synthesis at that time. Measurements of aggrecan mRNA levels by Northern blotting and polymerase chain reaction (PCR) (1 36) offer excellent specificity. However, aggrecan mRNA expression is not a true measure of aggrecan synthesis, and its interpretation as a representation of aggrecan synthesis is dependent on the absence of significant superimposed posttranslational regulation within the experiment. Although age-related and tissue-specific differences in GAG chains suggest that some regulation is operative at this level, the posttranslational regulation of aggrecan is not a major mechanism used by cells to control aggrecan synthesis. Quantification of secreted aggrecan

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by immunological methods offers specificity (137), but the sensitivity of these methods in some experimental systems can be a significant limitation. 1. Transcriptio~alRegulation of the Aggrecan Gene

Expression of the aggrecan gene is regulated by the 5’ upstream promoter region, and by the 5’ and 3’ untranslated regions (UTRs) that flank the coding sequence for the aggrecan core protein (for further discussion, see Ref. 28). Promoter activity was first documented for the rat aggrecan gene (20). Recently, the chicken and human aggrecan promoters have been characterized in greater detail (29,30). Both lack TATA boxes, as do several other matrix molecules [including perlecan, (138)], but two TATA-like TCTAA sequences defined previously in other promoter regions were identified in the 1800 bp 5’ flanking region of the chicken aggrecan gene. A likely transcription start site spanned by several potential SP-1lAP-2 transcription factor binding sites was identified within the human promoter, while in the chicken three major transcription start sites and several potential cis regulatory elements were found. For the human promoter, significant stimulatory activity appears to reside in the region about 50 bp upstream of exon 1. Sequence analysis revealed the presence of a number of potential transcription factor binding sites, including sites for the ubiquitous SP-1, AP-2, and AP-4 transcription factors. NF- KBsites and cislplatelet-derived growth factor (PDGF)inducible binding sites have been identified, and these may confer responsiveness to cytokines. Also located in the promoter and exon 1 are four shear stress response elements (139). These elements have been identified as sites for DNA binding proteins in the promoters of genes such as PDGF-B and monocyte chemotactic protein- 1, which undergo transcriptional upregulation in endothelial cells during flow-induced shear stress (140,141). While the functional activity of these sites in the aggrecan promoter and gene has not been established, it is attractive to consider these as potentially important sites in aggrecan mechanoregulation. Recently, regulatory functions for the human aggrecan gene have been ascribed to exons 1 and 19, which flank the coding region of the aggrecan gene and encode the 5’ and 3’ UTRs, respectively. Both regions influence transcription; the 5’ UTR stimulates expression substantially, whereas the 3’ UTR inhibits activities of the aggrecan promoter (30). Positive and negative regulatory activities were also reported for the chicken (29). The transcriptional regulation of tissuespecific expression during development has yet to be elucidated.

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ole of Soluble Mediators A variety of growth factors and other cytokines have been shown to modulate aggrecan gene expression by chondrocytes and other cell types (see Table 1 for a summary and specific references). While these activities have been described in experimental studies, in many cases it remains difficult to place this informa-

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Table l'

Factors Affecting Aggrecan Gene Expression and Synthesis

Modulator TGF-P l

Measurement

mRNA, 3sS-sulfate

Effect

Increase

IGF- 1,-2

"S-sulfate

Insulin BMP2

mRNA, 3sS-sulfate "S-sulfate

Increase/ maintain Increase Increase

BMP4 BMP7 IL-I

3sS-sulfate 3sS-sulfate rnRNA, "S-sulfate mRNA 35S-sulfate IllRNA

Increase Increase Decrease Decrease Decrease Decrease

TNF-a

Retinoic acid Interferon-?

Cell

Reference

Chondrocyte

144 145

Meniscus fibroblasts Tendon fibroblasts Chondrocyte Chondrocyte Chondrocyte Chondrocyte Chondrocyte Chondrocyte Chondrocyte Chondrocyte Chondrocyte

146 147 148 148 149 150 145 151 152, 153 154 155 156

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tion into an appropriate physiological context, since the studies may reflect experimental conditions rather than tissue conditions in situ. In general, however, anabolic factors are regarded as being involved in skeletal development and homeostasis, whereas agents such as inflammatory cytokines, which suppress the expression and synthesis of aggrecan, are more likely to be involved in pathologic responses. Skeletal development requires relatively high levels of aggrecan gene expression and synthesis at tissue-specific sites, either transiently or for prolonged periods of time, as part of a coordinated program of ECM synthesis and remodeling. These events are often regulated by soluble mediators. Some of the bone morphogenetic proteins (BMPs; e.g., BMP2, 4, and 7), members of the transforming growth factor (TGF)- Psuperfamily, are known to stimulate aggrecan gene expression and synthesis by chondrocytes, likely to be functioning as a part of the coordinated expression of ECM components during endochondral ossification. Stimulatory agents also include insulin-like growth factors (IGF)-l and -2. Since they have been used for long-term maintenance of cartilage explants, it is reasonable to regard these agents as important in homeostasis. In contrast, the inflammatory cytokines IL-I and TNF a are both powerful inhibitors of aggrecan synthesis, probably involved primarily in transcriptional regulation. They al so induce proteoglycan catabolism in cartilage, acting through a variety of proteases

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(see Chap. 5). The effects on both synthesis and degradation are important in the inflammation-induced degradation of cartilage in diarthrodial joints. Aggrecan synthesis is also responsive to the concurrent exposure of chondrocytes to several soluble mediators. For example, a synergistic stimulatory effect was observed for TGF-p and IGF-I or insulin, above levels achieved when only one agent was used (142,143). Inflammatory cytokines can act synergistically as well. In a study using human chondrocytes, interferon-y and TNF-a added together were able to induce a suppression of aggrecan expression greater than when the agents were used individually. It is of interest that TGF-P has been reported to overcome the suppressive effects of IL-I and retinoic acid. Since IL-I is an important inflammatory mediator and inflammation is an important component of connective tissue injury, this effect of TGF-P on IL-I-mediated suppression of aggrecan synthesis may be a significant reparative event in initiating matrix repair after injury to aggrecan-containing connective tissues. Those agents that modulate synthesis often effect the breakdown of aggrecan in an ECM as well (Table 2). IL-I and TNF-a are potent Stimulators of aggrecan catabolism, and TGF-P and IGF-I have the ability to suppress aggrecan breakdown. As discussed above, some agents, such as IL-I and TNF-a when used in suboptimal doses, can act synergistically, and may in some instances negate the effects of others, as exemplified by the inhibition of IGF-I effects by IL-I or TNF-a. Aggrecan catabolism occurs through the action of one or more proteases, with the primary site of cleavage being within the IGD. This is thought to be an efficient mechanism of breakdown, since a single cleavage event in this region converts aggrecan to a nonaggregating proteoglycan able to be cleared from the ECM with relative ease (see Chap, 5). Overall, increased aggrecan synthesis is associated with decreased breakdown, while suppression of synthesis is associated with increased breakdown, These coordinated activities effectively modulate aggrecan metabolism within a tissue, Since catabolism is effected through specific enzymes, modulation is likely to involve multiple intracellular signaling pathways.

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ble 2 Factors Affecting Aggrecan Catabolism

Modulator

Effect

Cell

Reference

TNF- cx

Increase Increase

Chondrocyte Chondrocyte

Retinoic acid TGF-Pl IGF-I, -11 BMP2 BMP7

Increase Decrease Decrease Decrease Decrease

Chondrocyte Chondrocyte Chondrocyte Chondrocyte Chondrocyte

157 154 l55 158

IL-I

144

159 149 151

recan

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3. Re~ulat ionby ~ech an i calLoading

In tissues and organs that function mechanically (for example, bone, cartilage, ligament, and blood vessel), cells respond to the mechanical environment. In cartilage, aggrecan expression is well known to be sensitive to mechanical forces, and mechanical stimuli are major modulators of aggrecan metabolism. In vivo studies have shown that mechanical loads are vital for the maintenance of aggrecan homeostatis in cartilage. For example, if a joint is immobilized or placed in disuse, thereby removing load from the articular cartilage of that joint, the rate of aggrecan synthesis is decreased and catabolism is increased. The net result is a dramatic decrease in the cartilage proteoglycan levels (82,160,16 1). Importantly, r e ~ o b i l i ~ a t i oofn the joint will result in a reversal of these events to reestablish proteoglycans in the cartilage. When a compressive load is applied to cartilage, there are a number of resultant mechanical effects within the tissue, including increased hydrostatic and osmotic pressures, fluid flow, streaming potential, cell and matrix deformation, and changes in the local fixed charge density. In vitro studies of cartilage explants and isolated chondrocytes have shown that the chondrocytes respond to a number of these factors to alter aggrecan synthesis. Static compressive loading can induce a transient increase in aggrecan gene expression, followed by the suppression of aggrecan synthesis (78,162,163). This increase in gene expression was detected after 1 hr of loading, required only 10 min of loading to induce a response at 1 hr, and was not detectable after 4 hr of loading (30,164). Thus, aggrecan synthesis can be modulated rapidly in chondrocytes by changes in the mechanical environment. Recovery from compressive loading has also been demonstrated in chondrocytes, and in some cases a “rebound” phenomenon has been observed (78). Conversely, cyclic compressive loads (regarded to be more physiologically relevant) can induce a stimulation of aggrecan synthesis. These experimental studies indicate that the aggrecan gene is designed to respond rapidly to the mechanical environment of the chondrocyte. However, the precise mechanical signal, or signals, to which the aggrecan gene is responding is unclear from these studies since the compressive loading of cartilage induces a variety of mechanical events. Shear stress on chondrocytes, induced by fluid flow, is a mechanical input that has been shown to stimulate aggrecan synthesis (165167). Hydrostatic pressure has also been shown to stimulate aggrecan synthesis by chondrocytes (167,168), although probably only to a modest extent (165,169,170). Cell shape and deformation may themselves be major signals, since chondrocytes in cartilage have been shown to deform under load (171), and changing cell shape has been linked to the modulation of chondrocyte activity. For example, when chondrocytes in culture were induced to round up, aggrecan synthesis was stimulated (172). Thus, although mechanical effects have been demonstrated, it remains difficult to separate out the specific signals that operate in the in vivo state.

Less is known about the mechanical modulation of aggrecan synthesis in other tissues. However, it has been shown in tendon that aggrecan expression is higher in regions that are subject to Compression as compared to tensional regions (12,173), and that aggrecan synthesis can be induced in cultured tendons by the application of compressive load (147). These effects clearly implicate mechanical loads as inducers of aggrecan synthesis at these sites, but again, the specific mechanical signals remain difficult to identify.

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Aggrecan was the first proteoglycan to be characterized comprehensively, in part due to its abundance and central importance in cartilage. Its unique structural properties are ideally suited to resist compression and fill space in, adult cartilage, and to participate in the formation of the normal skeleton during development. Although biomechanical and developmental functions in cartilage and other connective tissues are relatively well defined, the presence of aggrecan in notochord and brain during significant developmental windows suggests additional possibilities that are only now beginning to be explored. Normal aggrecan function is underscored by its established involvement in genetic and degenerative musculoskeletal diseases, and it is likely that the regulation of aggrecan synthesis and catabolism will be important in the modulation of many of these disease processes. This understanding will be facilitated by continued progress in the investigation of aggrecan biosynthesis and gene regulation. Overall, interesting new advances are anticipated on several fronts over the next few years.

The authors thank John Keller, Marvin Tanzer, Geetha Sugumaran, and the editor for their constructive comments and suggestions on the manuscript, and thank Matthias Morgelin, Wilmot Valhmu, and Tung-Ling Chen for their generous contributions to Figures 1 and 2. Support of the original research was provided by National Institutes of Health grant DK28433 (BMV).

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1. Shatton J, Schubert M. Isolation of mucoprotein from cartilage. J Biol Chem 1954; 211:565-573.

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136. Re P, Valhmu WB, Vostrejs M, Howell DS, Fischer SG, Ratcliffe A. Quantitative polymerase chain reaction for aggrecan and link protein gene expression in cartilage. Anal Biochem 1995; 225:357-360. 137. Ratcliffe A, Hardingham TE. Cartilage proteoglycan binding region and link protein. Radioimmunoassays and their detection of masked determinantsin aggregates. Biochem J 1983; 213:371-378. 138. Iozzo RV, Pillarisetti J, Sharma B, Murdoch AD, Danielson KG, Uitto J, Mauviel A. Structural and functional characterizationof the human perlecan gene promoter. Transcriptional activation by transforming growth factor-beta via a nuclear factor l-binding element. J Biol Chem 1997; 272:52219-52228. 139. Resnick N, Collins T, Atkinson W, Bonthron DT, Dewey CF, Gimbrone MA. Platelet-derived growth factor B chain promoter contains a cis-acting fluid shear-stressresponsive element. Proc Natl Acad Sci USA 1993; 90:4591-4595. 140. Resnick N, Gimbrone MA. Hemodynamic forces are complex regulators of endothelial gene expression. FASEB J 1995; 9:874--882. 141. Shyy JY, Li YS, Lin MC, Chen W, Yuan S, Usami S, Chien S. Multiple cis-elements mediate shear stress-induced gene expression.J Biomechan 1995; 28: 14511457. 142. Verbruggen G, Malfait AM, Dewulf M, Broddelez C, Veys. Standardization of nutrient media for isolated human articular chondrocytes in gelified agarose suspension culture. Osteoarthritis Cartilage 1995; 3:249-259. 143. Yaeger PC, Masi TL, de Ortiz JL, Binette F, Tub0 R, McPherson JM. Synergistic action of transforming growth factor-beta and insulin-like growth factor-I induces expressionof type I1collagen and aggrecan genes in adult human articularchondrocytes. Exp Cell Res 1997; 237:318-325. 144. Morales TI, Roberts AB. Transforminggrowth factor beta regulates the metabolism of proteoglycans in bovine cartilage organ cultures, J Biol Chem 1988; 263:1282812831. 145. Luyten FP, Chen P, Paralkar V, Reddi AH. Recombinant bone morphogenetic protein-4, transforming growth factor-beta 1, and activin A enhance the cartilage phenotype of articular chondrocytes in vitro. Exp Cell Res 1994; 210:224-229. 146. Collier S, Ghosh P. Effects of transforming growth factor beta on proteoglycan synthesis by cell and explant cultures derived from the knee joint meniscus. Osteoarthritis Cartilage 1995; 3:127-138. 147. Robbins JR, Evanko SP, Vogel KG. Mechanical loading and TGF-P regulate proteoglycan synthesis in tendon. Arch Biochem Biophys 1997; 342:203-211. 148. Luyten FP, Hascall VC, Nissley SP, Morales TI, Reddi AH. Insulin-like growth factors maintain steady-statemetabolism of proteoglycans in bovine articular cartilage explants. Arch Biochem Biophys 1988; 267:416-425. 149. Luyten FP, Yu YM, Yanagishita M, Vukicevic S, Hammonds RG, Reddi AH. Natural bovine osteogenin and recombinant human bone morphogenetic protein-2B are equipotent in the maintenance of proteoglycans in bovine articular cartilage explant cultures. J Biol Chem 1992; 267:3691-3695. 150. Erlacher L, McCartney J, Piek E, ten Dijke P, Yanagishita M, Oppermann H, Luyten FP. Cartilage-derived morphogenetic proteins and osteogenic protein- l differentiallv regulate osteogenesis. J Bone Miner Res 1998: 13:383-392.

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151. Leitman SA, Yanagishita M, Sampath TK. Stimulation of proteoglycan synthesis in explants of porcine articular cartilage by recombinant osteogenicprotein-1 (bone morphogenetic protein-7). J Bone Joint Surg 1997; 79:1132-1137. 152. Bolton MC, Dudia J, Bayliss MT. Quanti~cationof aggrecan and link-protein mRNA in human articular cartilage of different ages by competitive reverse transcriptase-PCR,Biochem J 1996; 3 19:489-498. 153. Tyler JA. Articular cartilage cultured with catabolin (pig interleukin 1) synthesizes a decreased number of normal proteoglycan molecules. Biochem J 1985; 227:869878. 154. SaklavalaJ. Tumour necrosis factor alpha stimulates resorption and inhibits synthesis of proteoglycan in cartilage. Nature 1986; 322:547-549. 155. Campbell IK, Piccoli DS, Roberts MJ, Muirden KD, Hamilton JA. Effects of tumor necrosis factor alpha and beta on resorption of human articular cartilage and production of plasminogen activator by human articular chondrocytes. Arthritis Rheum 1990; 33:542-552. 156. Dodge GR, Diaz A, Sanz-Rodriguez C, ReginatoAM, Jimenez SA. Effects of interferon-gamma and tumor necrosis factor-alphaon the expression of the genes encoding aggrecan, biglycan, and decorin core proteins in cultured human chondrocytes. Arthritis Rheum 1998; 41:274-283. 157. Tyler JA. Chondrocyte-mediated depletion of articular cartilage proteoglycans in vitro. Biochern J 1985; 225:493-507. 158. Buttle J, Handley CJ, Ilic MZ, Saklatvala J, Murata M, Barrett AJ. Inhibition of cartilage proteoglyan release by a specific inactivator of cathepsin B and an inhibitor of matrix metalloproteinases. Evidence for two converging pathways of chondrocyte-mediated proteoglycan degradation.Arthritis Rheum 1993; 36:17091717. 159. Tyler JA. Insulin-like growth factor 1 can decrease degradation and promote synthesis of proteoglycan in cartilage exposed to cytokines. Biochem J 1989; 260: 543-548. 160. Palmoski MJ, Brandt KD. Running inhibits the reversal of atrophic changes in canine knee cartilage after removal of a leg cast. Arthritis Rheum 1981; 24: 13291337. 161. Behrens F, Kraft EL, Oegema TR. Biochemical changes in articular cartilage after joint immobilization by casting or external fixation. J Orthop Res 1989; 7:335343. 162. Gray ML, Pizzanelli AM, Grodzinsky AJ, Lee RC. Mechanical and physiochemical determinantsof the chondrocyte biosynthetic response, J Orthop Res 1988; 6:777792. 163. Sah EL, Kim YJ, Doong JY, Grodzinsky AJ, Plaas AH, Sandy JD. Biosynthetic response of cartilage explants to dynamic compression.J Orthop Res 1989; 7:619636. 164. Bachrach NM, Valhmu \NB, StazzoneE, Ratcliffe A, Lai MW, Mow VC. Changes in proteoglycan synthesis rates of chondrocytes in articular cartilage are associated with the time dependent changes in their mechanical environment. J Biomechan 1995; 28~1561-1569. 165. Buschmann MD, Gluzband YA, Grodzinsky AJ, Hunziker EB. Mechanical com-

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166. 167. 168. 169.

170. 171. 172. 173. 174.

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pression modulates matrix biosynthesis in chondrocyte/agaroseculture. J Cell Sci 1995; 108~1497-1508. Kim YJ, Bonassar LJ, Grodzinsky AJ. The role of cartilage streaming potential, fluid flow and pressure in the stimulation of chondrocyte biosynthesis during dynamic compression. J Cell Sci 1995; 28:1055-1066. Smith RL, Donlon BS, Gupta MK, Mohtai M, Das P, Carter DR, Cooke J, Gibbons G, Hutchinson N, Schurrnan DJ. Effects of fluid-inducedshear on articularchondrocyte morphology and metabolism in vitro. J Orthop Res 1995; 13:824-831. Hall AC, Urban JP, Gehl KA. The effects of hydrostaticpressure on matrix synthesis in articular cartilage. J Orthop Res 1991; 9:1-10. Smith RL, Rusk SF, Ellison BE, Wessells P, Tsuchiya K, Carter DR, Caler WE, Sandell LJ, Schurman DJ. In vitro stimulation of articular chondrocyte M N A and extracellular matrix synthesis by hydrostatic pressure. J Orthop Res 1996; 14:360. Quinn T, Grodzinsky AJ, Buschmann MD, Kim YJ, Hunziker E. Mechanical compression alters proteoglycan deposition and matrix deformation around individual cells in cartilage explants. J Cell Sci 1998; 111:573-583. Guilak F, Ratcliffe A, Mow VC. Chondrocyte deformation and local tissue strain in articular cartilage: a confocal microscopy study. J Orthop Res 1995; 13:410421. Newman P,Watt FM. Influence of cytochalasin-D-induced changes in cell shape on proteoglycan synthesis by cultured articular chondrocytes. Exp Cell Res 1988; 178:199-220. Robbins JR, Vogel KG. Regional expressionof mRNA for proteoglycans and collagen in tendon. Eur J Cell Biol 1994; 64:264-270. Vertel BM. The ins and outs of aggrecan. Trends Cell Biol 1995; 5:458--464.

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The urnh ha^ Institute, La Jolla, Califor~ia

The structure and function of the nervous system is highly dependent on cellular interactions. Indeed, the fundamental unit of brain function, the synapse, is actually a highly specialized example of cell adhesion. Because the function of the adult brain is dependent predominantly on the immensely complex, yet precise geometric network of neurons, its development relies heavily on accurate control of the migration of neurons and their axons, both of which require exquisite regulation of cell-cell and cell-substrate interactions. In other words, both developmental and physiological aspects of the nervous system are heavily dependent on cellular interactions. Interestingly, nervous tissues have a peculiar repertoire of extracellular matrix (ECM) compone~ts,abundant in various types of proteoglycans while lacking most of common ECM proteins such as collagens and fibronectin. Thus it is expected that proteoglycans might play substantial roles in the nervous system. Work done during the past decade has validated this expectation. The nervous system has become one of the most attractive systems among all organs and tissues in studying the functions of proteoglycans. There is now a wealth of data showing that proteoglycans indeed play important roles in the nervous system. Here I review the historical background and recent progress in understanding the function of one of the major classes of proteoglycans, chondroitin sulfate proteaglycans (CSPCs).

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Nervous tissues express many proteoglycans. Herndon and Lander (1) first reported an overall description of proteoglycan expression in the brain. Their approach utilized isolation of a proteoglycan-enriched fraction by DEAE ion exchange chromatography coupled with sequential washing, iodination of core proteins, digestion with heparitinase or chondroitinase, and separation of core proteins by SDS-PAGE. This approach resolved 25 putative proteoglycan core proteins in embryonic and adult rat brain, and demonstrated that the fraction extracted with physiological buffers (‘ ‘soluble fraction”) contained mainly CSPGs, whereas HSPGs were the predominant components in detergent extracts of the membrane fraction (1). These studies have provided a basic overall picture of proteoglycan expression in developing and adult brain. Herndon and Lander (1) reported that among 25 putative proteoglycan core proteins, 16 were CSPGs. In fact, not all of these bands represented distinct GSPG core proteins, since some represent proteolytic fragments of larger CSPG core proteins. Nevertheless, these data first de~onstratedthe wide diversity of CSPG expression in the brain. Thus far six CSPGs [four lecticans (2-5) (see also Chaps. 13 and 14), ~PTPp/phosphacan(6,7), and NG2 (8); see below], whose identities have been confirmed at the molecular level, comprise the majority of the known nervous system CSPGs (Fig. 1). Neuroglycan C (9) and the Alzheimer’s amyloid p protein precursor or “appican” (10) are additional nervous system CSPGs of which cDNAs have been cloned. Testican is a hybrid proteoglycan contain in^ both chondroitin sulfate and heparan sulfate chains originally identified in h u ~ a n testis (1 1). It was later shown to be predominantly expressed in the brain in mice, most notably in postsynaptic regions (12). Other than these CSPGs, there are several reported cases of CSPGs which have significant biological implication, but not yet identi~edat the molecular level. Such CSPGs include Cat-301 (l 3), astrochondrin (14), DSD-l-PG (15), TI antigen (16), and somatoglycan-S (17). Some of them may prove to be identical to known CSPGs.

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Proteoglycans belonging to the aggrecan family represent the largest group of CSPGs expressed in the nervous system. This family has been named lectican (18) or hyalectan (19,20), based on the ~ y ~ l u r o binding ~ a n and lectin domains shared by these CSPGs. There are four members in this family which have been characterized by molecular cloning, namely, aggrecan (2), versican (3), neurocan (4), and brevican (5). [The putative hyaluronan-bin~ingprotein BEHAB (21) is a 5’ partial cDNA of brevican itself (22).] Other than these cloned members, there are several other brain CSPGs which show properties similar to lecticans

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1 Structures of major CSPGs expressed in the nervous system. The N-termini of proteoglycans are drawn toward the left. Abbreviations: Ig, i~unoglobulin;CRP, complement regulatory protein; CAH, carbonic anhydrase; €W 111, fibronectin type 111; PTP, protein tyrosine phosphatase; CM, cell membrane.

(13,16,23,24). These CSPGs have been characterized by biochemical analysis or by monoclonal antibodies, but not yet cloned, Some of these CSPGs may turn out to be novel members of the lectican/hyalectan family. However, no new member of the family has been identified at the molecular level since 1994, and PCR screening with degenerate primers in conserved regions has not yielded any novel cDNAs belonging to this family (S, Suzuki, personal communication), This may indicate that the four members currently known may represent the entire lecticanlhyalectan family.

The protein structure of lecticans is one of the most remarkable in the entire proteoglycan world (Fig. l). The two globular domains, the N-terminal hyaluronan-binding domain and the C-terminal globular domain containing a C-type lectin domain, have attracted a great deal of attention as to their molecular interactions. The configuration of the core protein strongly suggests that these proteoglycans are capable of linking two distinct molecules through the ~ - t e ~ i n and al C-terminal globular domains, respectively. The N-terminal globular domain consists of an immunoglobulin (&)-like loop followed by two link proteinlike tandem repeats. Only aggrecan has a second set of tandem repeats called G2 domain. The Ig-like loop is not highly conserved among members of the lectican/hyalectan family (21-34%), while the tandem repeats are significantly more conserved (57-64%). At the opposite end of the molecule, the C-terminal globular domain consists of one or two copies of an EGF-like repeat, a C-type lectin domain, and a complement regulatory protein (CRP)-like domain. These are the same structural motifs found in selectins, which mediate leukocyte-endothelial adhesion through various carbohydrate ligands (25,26). The EGF-like repeats and the lectin domain show high-sequence similarities among different lecticans (50-70%). Compared with the EGF-like and lectinlike domains, the CRP domains are less well conserved (4 l-44%). In contrast to these globular domains, the central domains of lecticans show little homology with each other. Interspecies homologies within individual lecticans are low [for example, 68% between rat and bovine brevican (27)], The size of this region is also diverse, ranging from 27 1 amino acid residues in rat brevican to 1693 residues in human versican (see also Chap. 13). The central domains lack any significant structural motifs currently known to be relevant to molecular interactions. Instead, they provide attachment sites for chondroitin sulfate chains. Because of the huge difference in the sizes of the central domains, the number of potential glycosaminoglycan attachment sites also varies greatly. The potential numbers of chondroitin sulfate chains attached to the core proteins have been estimated to be -100, -20, -7, and -3, for aggrecan, versican, neurocan, and brevican, respectively (2-5). Brevican not only has the fewest number of potential chondroitin sulfate attachment sites, but it is also a so-called “part-time proteoglycan.” A substantial proportion of brevican molecules present in brain tissue is not substituted with any chondroitin sulfate chains (5,28). Other lecticans are not known to be part-time proteoglycans. There are variants of lecticans generated by alternative splicing. The existence of such variants has been reported for aggrecan (29,30), versican (3 1,32), and brevican (33). Among these, versican and brevican variants are of interest with regard to the nervous system. There are at least four alternatively spliced transcripts for mouse versican. Interestingly, one of the variants named V3, an isoform expressed in both mouse and human brain, lacks the entire central do-

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main; it consists of only the N-terminal and G-terminal globular domains (32). Only one variant has been reported for brevican, but its structure as a glycosylphosphatidylinositol (GP1)-anchored form is unique among various lectican variants (33). The open reading frame of this CPI-anchored variant is identical to the authentic rat brevican cDNA in its N-terminal globular domain and central domain, but diverges at the beginning of the EGF-like domain ending with the signal sequence for the GPI-anchors.

All four lecticans have been shown to be expressed in the nervous system. The expression of versican is ubiquitous, the nervous system being one of its sites of expression (34). Glial hyaluronate binding protein, which was initially characterized as a 60 kDa glia-derived glycoprotein (35,36), later proved to be a truncated form of versican core protein corresponding to its N-terminal hyaluronanbinding domain (37). Aggrecan was initially thought to be cartilage-specific, but a proteoglycan recognized with a monoclonal antibody to cartilage aggrecan has been identified in brain extracts (38,391. The expression of aggrecan mRNA has also been demonstrated in the brain (see Ref. 18). Both neurocan and brevican are expressed in the nervous system in highly specific manners (5,21,33,40,41). Spatiotemporal expression patterns in developing and adult nervous systems have been studied in detail for neurocan and brevican. Western blotting analysis demonstrated that neurocan becomes detectable in rat cerebrum as early as embryonic day 14. (E14) (41), whereas brevican is not detected until postnatal day 0 (PO) (42). Immunohistoche~icalstudies have demonstrated that no substantial amount of brevican is detected in embryonic forebrain, except in developing hippocampus around E17-PO (K, Hagihara and Y, Yamaguchi, unpub~ishedresults). In contrast, neurocan is more actively expressed in developing cerebral cortex, By E16-19, strong staining of neurocan is seen in the marginal zone and subplate (43), and at E20, the staining spreads into the cortical plate (41). Subsequently, the intensity of the staining decreases as the brain matures, and in adult brain, only weak, diffuse staining is observed (41). In developing cerebellum, brevican immunoreactivity occurs after P21 corresponding to the maturation of the internal granular layer. The intensity of the staining increases signi~cantlyfrom P28 to P60 (42). Staining for brevican in the cerebellum is essentially restricted to glomeruli in the internal granular layer, a strategic site in the cerebellar cortex where incoming mossy fiber axons form numerous synapses with cerebellar neurons. No substantial staining i s seen in the molecular layer. In contrast, it has been shown that neurocan immunoreactivity is strongest in the molecular layer in adult rat cerebellum (40). Temporally, the appearance of neurocan in the rat cerebellum occurs significantly earlier than

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that of brevican; strong staining is already seen at P7 (40,44,45). Although the spatiotemporal expression patterns of aggrecan and versican in the developing cerebellum have not been investigated, these observations suggest that the expression of the lectican/hyalectan family CSPGs is differentially regulated. While immunohistochemistry provides highly useful information about spatiotemporal expression patterns, cellular origins of expression cannot be determined by immunohistochemistry alone or even by immunoelectron microscopy, because these CSPGs are secreted molecules. A combination of immunohistochemistry and in situ hybridization, and sometimes analysis of cultured cells, is necessary to fully characterize the cellular origin of molecules in vivo. At the level of primary cultures, both neurons and astrocytes produce neurocan (41), while brevican mRNA is expressed in cerebellar astrocytes but not in cerebellar granule neurons (5). Detailed analysis of cerebellar glomeruli revealed that brevican is localized on the surface of astrocytes which form the neuroglial sheaths of glomeruli (42). In situ hybridization demonstrated that brevican mRNA is detected in these astrocytes. These data indicate that in cerebellar cortex, astrocytes forming neuroglial sheaths synthesize brevican and deposit it on astrocytic surfaces (42). [This is not to say that brevican is glial cell specific. In situ hybridization showed that some neurons in other parts of the brain express brevican mRNA (46; K, Hagihara and Y. Yamaguchi, unpublished result).]. Interestingly, another prominent glial cell type in the cerebellum, the Bergmann glia, does not express brevican. In contrast, neurocan immunoreactivity is detected in two types of cerebellar neurons, namely, granule cells and Purkinje cells (43). Results of in situ hybridization corroborated the expression of neurocan in these cerebellar neurons (47).

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Binding to hyaluronan through the N-terminal domain has been studied mainly for aggrecan in the context of cartilage ECM organization. Few detailed studies have focused on the role of lectican-hyaluronan binding in the context of brain ECM. Among these studies, Rauch et al. (40) showed that neurocan binds hyaluronan. The N-terminal fragment of versican had been known as glial hyaluronate binding protein for some time (3’7) and recombinant protein of the N-terminal versican fragment indeed binds hyaluronan (48). These results suggest that lecticans actually interact with hyaluronan in the brain ECM. The colocalization of some of the lecticans and hyaluronan in a structure called p~rineuronalnets (49) supports this possibility (see below). Molecular interactions of the C-terminal globular domains have recently been elucidated. Aspberg et al. (50) demonstrated that recombinant versican lectin domain binds tenascin-R. This interaction is calcium-dependent, as expected of a carbohydrate-protein interaction mediated by a C-type lectin domain. Ini-

~ h o n ~ r o i tSulfate in ~roteo~lycans in the Nervous System

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tially, it was supposed that the interaction is mediated by carbohydrate chains attached to tenascin-R as in the case of selectins and other C-type lectins (50). However, this proved not to be the case. Like versican, the C-type lectin domain of brevican has been shown to bind tenascin-R, but surprisingly, this interaction is mediated by a protein-protein interaction and does not require any carbohydrates on tenascin-R (51). The lectin domains of versican and other lecticans have also been shown to bind tenascin-R by protein-protein interactions. All these interactions are mediated by fibronectin type I11 domain 3-5 of tenascin-R. Among the lecticans, the brevican lectin has the highest affinity for tenascin-R, and natural brevican coimmunoprecipitates with tenascin-R from adult rat brain extracts (51). Furthermore, brevican colocalizes with tenascin-R in many regions in adult rat brain (52). These results suggest that brevican is a physiologically relevant ligand for tenascin-R. Molecular interactions of the C-type lectin domain of lecticans are probably not limited to tenascin-R. The lectin domains of lecticans have been shown to be able to bind simple carbohydrates and hep~inlheparansulfate (50,53-55). It has been demonstrated that the C-terminal fragment of brevican which contains the lectin domain binds to the surface of glial cell lines and that this cell surface binding is mediated by an unknown cell surface binding molecule(s) different from tenascin-R (42). Recently, it has been demonstrated that this cell surface binding is mediated by a classical lectin-type interaction with sulfated cell surface glycolipids, sulfatides and HNK- 1 reactive sulfoglucuronyl glycolipids (56). Molecular interactions involving neurocan has been extensively studied. Neurocan binds N-CAM (45,57), Ng-CAMlL1 (43, Nr-CAM (58), tenascinC (44,59,60), tenascin-R (58), TAG-llaxonin-1 (61), hepa~n-bindinggrowthassociated molecule (HB-GAM) (58), and amphoterin (58). The binding to HBCAM and amphoterin requires chondroitin sulfate chains on neurocan (58). It has not been examined whether other lecticans also bind this variety of ligands.

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RPTPP is a transmembrane CSPG expressed in the nervous system (Fig. 1). RPTPP (also known as PTPC) was initially cloned as a novel receptor-type protein tyrosine phosphatase (RPTP) by PCR based on sequence homology with tyrosine phosphatases (6,62). Like other RPTPs, RPTPP has two tandemly repeated phosphatase domains, Compared to their cytoplasmic domains, the extracellular domains of RPTPs are structurally diverse, but the majority contains varying nurnbers of im~unoglobulin-likerepeats andlor fibronectin type 111domains. RPTPP does not contain any Ig domains in its extracellular domain. Instead, the extracellular domain of RPTPP contains a unique carbonic anhydrase domain in its Nterminus,

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In 1994, several laboratories independently reported that RPTPP is a CSPG. Maurel et al. (7) cloned a cDNA using a monoclonal antibody which recognizes a soluble CSPG (3F8 proteoglycan). The cDNA turned out to be identical to the extracellular domain of RPTPP. They concluded that 3F8 proteoglycan is a soluble form of RPTPP generated by alternative splicing and named this form “phosphacan.” On the other hand, Shitara et ai. (63) cloned a bovine cDNA by using a polyclonal antibody that recognized ~ u l t i p l ebrain CSPG core proteins (5). This cDNA was subsequently identified as the bovine homologue of RPTPP, suggesting that RPTPP is a CSPG. Antibodies raised against human RPTPP confirmed that essentially all RPTPP molecules expressed in human brain exist in the form of CSPG (63). Barnea et al. (64) demonstrated that RPTPP cDNA transfected into human 293 cells is expressed as a membrane-bound CSPG. Maeda et al. (65) showed that rat brain CSPGs with a 380 W a core protein have protein tyrosine phosphatase activity and identified the CSPG as RPTPP. In addition to chondroitin sulfates, RPTPP/phosphacan contains keratan sulfates (40,66), and it is the major proteoglycan carrying keratan sulfates in chick brain (66). Spatiotemporal expression of RPTPP/phosp~acanin the brain has been studied by in situ hybridization and immunohistoche~istry(43,47,66,67). These studies demonstrated that phosphacan is expressed predominantly in glial cells, but not in neurons. In developing brain, RPTPP/phosphacan is expressed in the radial glia of the ventricular zone. In adult cerebellum, the Golgi epithelial cells express RPTPP/phosphacan, and their Bergrnann glial fibers are strongly stained with these antibodies. The extracellular domain of RPTPP (or phosphacan) has been shown to bind several molecules. Such molecules include I?-CAM (68), Ng-CAM (68), Nr-CAM (58), the GPI-anchored neural cell adhesion molecule contactin (69), tenascin-C (44,64), tenascin-R (58,70), HB-GAM (58,71) and amphoterin (58). It is believed that these molecular interactions are involved in the role of phosphacan to modulate neurite outgrowth (see below).

NC2 is another transmembrane CSPG expressed in the nervous system. NG2 was first identified with antisera directed against immortalized rat neural cell lines and characterized as a putative lineage marker (72), which was later found to be a proteoglycan (73). It was also independently identified as rnelanoma-associated proteoglycan (74,75). The 260 IsDa core protein of NG2 consists of a large extracellular domain, a single transmembrane domain, and a short cytoplasmic domain, and exhibits no significant homologies to any other proteins or proteoglycans (8). The extracellular domain contains nine serine-glycine sequences, two of which are in good agreement with efficient glycosaminoglycan attachment

sequences. Yet NG2 core proteins without glycosaminoglycan chains are detected in rat brain tissues, indicating that NG2 is a part-time proteoglycan. NG2 is expressed in proliferating glial progenitor cells of the 0 2 A lineage (76). 0 2 A progenitor cells are bipotential progenitor cells which generate either oligodendrocytes or stellate ‘‘type 2” astrocytes in vitro (77). When cultured in the presence of serum, 0 2 A progenitor cells differentiate into GFAP-positive type 2 astrocytes. In the absence of serum, 0 2 A cells differentiate into oligodendrocytes. NG2 is expressed in progenitor cells but its expression is lost as cells differentiate into galactosylceramide-positive oligodendrocytes (77). Thus NG2 serves as a marker for 0 2 A progenitor cells. The expression of NC2 is not restricted to the nervous system. Outside the nervous system, NC2 is expressed in skeletal myoblasts, aortic smooth muscle cells, developing cartilage, and brain capillary endothelial cells (78). NG2 is also expressed in a number of melanoma cell lines and tissues, while normal melanocytes express little NG2 (78). NG2 interacts with various ECM and cell surface molecules. It has been shown that NG2 binds type V and type VI collagens (79-81), PDGF~-receptor (82), and an unidentified 280 kDa cell surface protein (83). Among these interactions, the binding to type VI collagen is thought to be relevant to developing cartilage and blood vessels. NG2 has been shown to colocalize with PDGF-a receptor in oligodendrocyte progenitor cells (82) and vascular smooth muscle cells (84).

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A number of earlier studies indicated that CSPGs have inhibitory effects on neurite outgrowth in vitro and that the glycosaminoglycan moieties of these molecules are responsible for these effects (85-88). These studies were done before any of the currently known brain CSPGs were identified or cloned, and therefore the material used in these studies were generally cartilage-derived CSPGs (containing mainly aggrecan) and chondroitin sulfates derived from cartilage or other sources unrelated to nervous tissues. Because of these in vitro results, the distribution of chondroitin sulfates and CSPGs was studied in developing embryos. Using monoclonal antibodies against intact chondroitin sulfate (such as CS-56) or against ch “stubs” generated by chondroitinase digestion (such as 3B3, these studies revealed remarkable distribution patterns for chondroitin sulfates consistent with their in vitro effects. These studies demonstrated that chondroitin sulfates (and keratan sulfates in some cases) are distribute^ in so called “barriers” against axon growth, which are specific regions in the developing nervous system where advancing growth cones do not enter. Such barriers represent repulsive guidance cues crucial for establishing stereotypic neuronal connectivity.

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Oakley and Tosney (89) examined chick and quail embryos in detail and showed that chondroitin 6-sulfate is located in various axon barriers including the posterior sclerotome and perinotochordal mesenchyme? the roof plate of the spinal cord, and the early limb bud. Brittis et al. (90) extended these lines of study and showed that chondroitin sulfates play a crucial role in neuronal differentiation and axonal guidance in vivo. In developing retina, differentiation of retinal ganglion cells occurs first in the area near the optic fissure and proceeds toward the periphery. As ganglion cells commit to differentiation, they extend axons toward the optic fissure, opposite to the direction of the wave of differentiation. Chondroitin sulfates first disappear from the central area of the retina where ganglion cells undergo differentiation, and the regression of chondroitin sulfates correlates well with the progress of ganglion cell differentiation (90). The removal of chondroitin sulfate by chondroitinase treatment of the cultured retina caused the disruption of this ordered differentiation process, resulting in axons extending in all directions. This work indicates that chondroitin sulfate is a key molecule for d e t e ~ i n i n gthe direction of axonal growth in the retina. More recently, the effects of CSPGs on neurite outgrowth have been studied with regard to individual CSPGs using purified CSPG samples and specific antibody probes to respective CSPG (Table l). The picture emerging from these studies is somewhat unexpected and complicated. In the case of lecticans, ag-

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Effects of Individual CSPGs on Neurite Outgro~th CSPG Aggrecan Aggrecan Versican

Active Moiety CSlKS

CS

CS

N/D Neurocan CS Brevican ~PTP~/phosphacan CAH E~TP~/phosphacan CP RPTP~/phosphacan CP CP NG2 DSD-l-PG CS

Effect

Neuron Type

Inhibitory Inhibitory Inhibitory Inhibitory Inhibitory Inhibitory Promoting Promoting No effect Inhibitory Inhibitory Promoting Promoting

Dorsal root ganglion neurons Retinal ganglion neurons Dorsal root ganglion neurons PC12 cells 9-d chick brain cells Cerebellar granule neurons Chick tectal neurons Cortical neurons Thalamic neurons 9-d chick brain cells Cerebellar granule neurons Mesencephalic neurons Hippoca~palneurons

Ref. 88

91 92

45 42 69, 95 94 68 93 15

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Abbreviations: CS, chondroitin sulfate; KS, keratan sulfate; CP, core protein; CAH, carbonic anhydrase domain; N/D:not determined.

grecan (88,91), versican (92), neurocan (45), and brevican (42) have all been shown to be inhibitory to neurite outgrowth, and the inhibitory activity is in the chondroitin sulfate chains (and keratan sulfate chains, in the case of aggrecan) (42,88,91,92). However, there are also reports indicating that core proteins, not chondroitin sulfate chains, are crucial for such repulsive activities. It has been reported that NG2 inhibits neurite outgrowth, but that the inhibitory activity is localized in its core protein (93). RPTPP/phosphacan also affects neurite outgrowth through its core protein, but in this case, the reported effects appear more complicated. Milev et al. (68) showed that pbosphacan on Ng-CAM substrate inhibits neurite outgrowth. On the other hand, Maeda and Noda (94) reported that phosphacan promotes neurite outgrowth from cortical neurons but has no effect on thalamic neurons. In both cases, chondroitinase treatment of the CSPG does not abolish the activity to inhibit or promote neurite outgrowth, indicating that both of these activities are present in the phosphacan core protein. The presence of neurite outgrowth"promoting activity in the core protein has also been demonstrated by studies using recombinant fragments co~tainingthe carbonic anhydrase domain without carrying any chondroitin sulfate chains (69,95). In these cases, the neurite outgrowth promoting activity is thought to be mediated by the binding of the carbonic anhydrase domain to contactin on the neuronal surface. Thus these reports indicate that some CSPG core proteins have their own effects on neurite outgrowth. There is yet another kind of complexity. A CSPG called DSD-l-PG carries a unique chondroitin sulfate/dermatan sulfate hybrid epitope recognized by a monoclonal antibody 473H , DSD-1-PG exhibits neurite outgrowth promoting activity, which is blocked either by 473HD monoclonal antibody or chondroitinase treatment (15). Recently, Nadanaka et al. (96) found that chondroitin sulfate D from shark cartilage has the ability to inhibit the interaction between 473HD and DSD- l-PG. Analysis of the hexasaccharides derived from this chondroitin sulfate revealed that active hexasaccharides share the common structure of HexAal-3GalNAcP1-4(GlcAP 1-3Galf\TA~)~. These observations indicate that we probably need to amend our classical view of the role of CSPGs on neurite outgrowth. A CSPG can affect neurite outgrowth either through its chondroitin sulfate moiety or through its core protein. Chondroitin sulfates are often inhibitory to neurite outgrowth, but can also be stimulatory if certain unique carbohydrate structures are present. We do not know how these complicated activities are orchestrated in vivo. However, observations by Emerling and Lander (97) suggest that developing tissues in vivo, in fact endow such a complicated role to chondroitin sulfates, During the developmental process of the thalamoco~icalpathway, axons of thalamic neurons entering the developing cortex elongate within a layer called the subplate, always avoiding the neighboring layer, the cortical plate. Emerling and Lander (97) used adhesion assays on cortical tissue slices to show that the cortical plate is inhibitory to

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neuronal cell adhesion and neurite outgrowth, while the subplate is stimulatory, consistent with in vivo development. Surprisingly, both of these opposing activities were abrogated by chondroitinase treatment. This result indicates,that chondroitin sulfates in these neighboring layers have opposite activities on axon growth. Thus the regulation of axonal guidance in vivo by CSPGs is determined not only by how cells choose to express certain CSPG core proteins but also by how certain carbohydrate structures are incorporated into chondroitin sulfate chains. Further studies employing genetic manipulation of chondroitin sulfate synthesis will be needed to dissect and define the role of chondroitin sulfates and individual CSPGs in axonal guidance in vivo.

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The problem of axonal regeneration is apparently a special case of the broader problem of axon growth and guidance. Thus it is not surprising that CSPGs are implicated in axonal regeneration, This problem has additional importance because of its clinical implications. The mechanism of axon regeneration is not just a problem of the axons themselves. There is also the involvement of reactive gliosis. Reactive gliosis is a type of tissue repairing process that occurs in the adult brain after trauma and in~anlmation.This process repairs the wound, but at the same time blocks regenerative axonal outgrowth. Rudge and Silver (98) showed that materials produced by scar tissues promote little neurite outgrowth, whereas materials from normal tissues promote it. These results suggest that the inhibition of axonal regeneration in reactive gliosis is, at least in part, due to the expression of inhibitory molecule(s) on the surface of injured tissues. Because of its neurite outgrowth inhibitory activity, chondroitin sulfate has drawn attention as a key component of the inhibitory substance in reactive gliosis. In fact, it has been shown that CSPGs are accumulated in reactive astrocytes in culture (99), in experimental scar tissues in vivo (100,10~),and followil~gthe experimental crush injury of sciatic nerve (102). Furthermore, chondroitinase treatment of these scar or injured tissues significantly improves neurite outgrowth (101,102). These studies suggest that chondroitin sulfates are a key component to hinder regeneration of injured axons. The inhibition of axonal regeneration by chondroitin sulfates has significant clinical implications. Davies et al. (103) have recently shown that when transplanted to adult rat brain, adult rat dorsal root ganglion neurons can frequently survive and extend axons for long distances in white matter. Instances in which transplanted neurons failed to extend axons were correlated precisely with increased production of CSPGs within the ECM at the transplant interface (103). This observation not only supports the notion that chondroitin sulfates are a key Component for inhibition of axonal regeneration in vivo, but also suggests that

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axonal regeneration can be facilitated by removing chondroitin sulfates or neutralizing their inhibitory activity. Identities of CSPG(s) upregulated in reactive gliosis and in inhibitory scar tissues have not been determined. Canning et al. (99) found that in reactive astrocytes in culture a small proteoglycan containing both chondroitin sulfate and heparan sulfate is specifically upregulated. Braunewell et al. (104) showed that human sciatic nerve contains two CSPGs which are likely to correspond with versican and decorin, and that the chondroitin 6-sulfate epitope on the versicanlike CSPG is upregulated during regeneration in mouse sciatic nerve. They suggested that versican-like and decorin-like CSPGs are involved in the regeneration of peripheral nerve.

Histologically, brain tissue is unique in that it does not contain readily discernible, well-defined stromal spaces. Before the 1970s, it was generally accepted among histologists that no appreciable amounts of “classical” stroma or ECM exist in the central nervous system. This notion was later challenged by the discovery of significant extracellular spaces filled with ECM-like materials that were visible when tissues were fixed with agent that preserve proteoglycans and hyaluronan (105). However, the concept of brain ECNI was still not readily accepted due to another unique feature of brain tissue, namely, that adult brain tissues lack most of the common ECM proteins (106-108). These ECM proteins, including fibronectin, laminin, and collagens, are expressed in the brain parenchyma transiently during development, but later become restricted to the basement membranes of blood vessels and meninges in adult brain. In the meantime, it has been shown that brain tissues constitutively express high levels of other types of ECNI molecules, particularly various types of proteoglycans, hyaluronan, and tenascins, suggesting that these molecules may be the core components of the brain ECM. It is ironic to note that the brain ECM had actually been recognized for more than 100 years. In the 1890s, Carnillo Golgi and Santiago Ramon y Cajal recognized the presence of reticular networks on the surface of neuronal cell bodies and proximal dendrites (Fig. 2). This cell surface network, called “perineuronal nets,” is now believed to represent a form of the brain ECM deposited in the space between neurons and the processes of astrocytes (49). Immunohistochemical studies have shown that perineuronal nets contain a unique set of molecules, including CSPGs, tenascin-C, tenascin-R, hyaluronan, HNK- l carbohydrates, and carbohydrates recognized by several lectins (49). Among CSPGs, versican (34), brevican (52), Cat-301 antigen (13), and DSD-1PG (109) have been shown to be present in perineuronal nets. From this list of molecules, it is easy to spot a set of molecules which can bind each other, namely lecticans (CSPGs), tenascin-R, and hyaluronan. This striking colocalization of

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Perineuronal nets. (a)Drawings of perineuronal nets by Ramon y Cajal(l898). (Modified from Ref. 49.) (b) Perineuronal nets of neurons in the deep cerebellar nuclei stained with cationic iron colloid. (c) Hypothetical model of the ECM organization in perineuronal nets. [Photograph (b) was kindly provided by Dr. Takuro Muraka~iof Okayama University School of Medicine, Japan.]

the molecules capable of associating with these types of proteoglycans suggests the possi~ilitythat lecticans form a tertiary complex with tenascin-R and hyaluronan, and that the physiological site for these i~teractionsis perineuronal nets (Fig. 2c). Colocalization of lecticans, tenascin-R, and hyaluronan sheds light on the possible function of perineuronal nets. It is interesting to note that both lecticans

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and tenascin-R are known to be repulsive molecules. Although the role of hyaluronan in neurite outgrowth has not been examined as extensively, there have been indications that hyaluronan is inhibitory to cell adhesion during cell division (1 IO), cell migration (1 1l), and neurite outgrowth (85,87,112). Thus the putative tertiary complex may be super-repulsive to incoming growth cones. This hypothesis is consistent with the observation that neuronal surfaces covered by perineuronal nets are devoid of synaptic contacts ( l 13). It is speculated that one of the roles of perineuronal nets is to present a nonpermissive substrate which blocks the formation of new synaptic contacts (49). The hyaluronan-lectican-tenascinR complex may be instrumental in the inhibition of synaptogenesis, On the other hand, it is also possible that the binding of lectican neutralizes the repulsive activity of tenascin-R, for the lectican binding site and the repulsive site are overlapping on tenascin-R (5 1,114). Whatever net biological effects the hyaluronan-lectican-tenascin-Rcomplex might have on differentiation and neurite outgrowth of neurons, the association of these three molecules is likely to be a basic framework of the brain ECM. The abundance of chondroitin sulfate and hyaluronan implicit in this mode1 of brain ECM is consistent with the unique nature of brain tissues in terms of tumor invasion, It is known that the brain presents a unique environment for invading tumor cells. Although tumor cells do metastasize to the brain, the metastasizing cells are not usually as invasive as they are in the nonneural tissues (1 15). This invasion-resistant property of the brain is shared by cartilage (18), which also has an ECM abundant in hyaluronan and chondroitin sulfate. Thus it appears that the hyaluronan/chondroitin sulfate-rich ECM provides a unique environment for cells that associate with it (18). In this vein, it is interesting to note that Zhang et al. (1 16) have shown that forced expression of the terminal fragment of brevican renders noninvasive glioma cells highly invasive in vivo. This model of brain ECM also has an important implication in our understanding of the developmental regulation of synaptic plasticity. In the developing brain, a huge number of synapses are initially fomed. While the majority of these initial synapses eliminated during the early postnatal period, some of the synapses are stabilized as mature synapses, which are functionally more efficient and less plastic. Hockfield and colleagues (1 17) proposed that this developmental modification of synapses is accompanied, or possibly caused, by the conversion of embryonic type ECM, which is more fluid and occupies larger extracellular spaces, to adult type ECM, which is less fluid and occupies smaller extracellular spaces, Since chondroitin sulfates and hyaluronan are capable of regulating hydrodynamic volume and fluidity of the matrix, the brain ECM based on the kctican-hyaluronan-tenascin- complex seems to fit well with this model. In fact, Cat-301 proteoglycan, the molecule originally implicated in the model (13,118), is likely to be a lectican (24). Thus the conversion between embryonic type ECM and adult type ECM may be achieved, at least in part, by expressing kcticans

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carrying different numbers of chondroitin sulfate chains. A potential advantage of lecticans in this respect might be that they have different capacities for introducing chondroitin sulfates into the tissue while sharing similar abilities to mediate molecular interactions through the N-terminal and C-terminal globular domains. Therefore, by expressing different lecticans, brain tissue may be able to change the fluidity and size of extracellular spaces without altering the basic molecular or~anizationof ECM.

It is probably fair to say that work done during the past decade has largely cornpleted molecular description of proteoglycans expressed in the nervous system, This field will focus on the elucidation of biological functions of these molecules. Yet it may not be easy to define “functions” for each proteoglycan. Thus far, few knockout mice have provided phenotypes informative for defining proteoglycan functions in the nervous system. This is presumably, at least in part, due to functional compensation by similar proteoglycans, or “redundancy.” For example, the molecular interactions of lecticans are substantially redundant; all four lecticans bind the same set of ligands, namely, tenascin-R and sulfated glycolipids through the lectin domain, and hyaluronan through the N-terminal globular domain. Another explanation for the lack of phenotypes might be that some of the proteoglycans exert their effects only in adult brain, so that knockout mice do not show a developmental phenotype. It may be necessary to conduct physiological studies on adult brain function to identify phenotypes due to the disruption of such molecules. Finally, we will need to define the roles of chondroitin sulfate in the brain separately from those of core proteins. One of the int~guingfuture directions is the genetic manipulation of chondroitin sulfate synthesis in mice. This will become possible once transferases involved in chondroitin sulfate synthesis are cloned and characterized.

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I thank Dr. William Stallcup for many helpful suggestions on experi~entsand critical reading of this manuscript, and Dr. Takuro ~ u r a k a for ~ iproviding photographs of perineuronal nets. I also thank all my colleagues who have been involved in the study on brain proteoglycans in my laboratory. Work done in the author’s laboratory and the preparation of the manuscript were supported by National Institutes of Health grants HD 25938 and NS 32717.

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zy zyxwvuts zyxwvu zyxwvu zyxw zyxwvu zyxwvu zyxwv zyxwvuts

76. Raff M, Williams B, Miller R. The in vitro differentiation of a bipotential glial progenitor cell. EMBO J 1984; 103:1857-1864. 77. Stallcup WB, Beasley L. Bipotential glial precursor cells of the optic nerve express the NG2 proteoglycan. J Neurosci 1987; 7:2737-2744. 78. Levine JM, Nishiyama A. The NG2 chon~oitinsulfate proteoglycan: a multifunctional proteoglycan associated with immature cells. Persp Dev Neurobiol 1996; 3: 245-259. 79 StallcupWB,Dahlin K, Healy P. Interaction of the NG2 chondroitin sulfate proteoglycan with type VI collagen. J Cell Biol 1990; 111:3177-3188. 80. Burg MA, Tillet E, Timpl R, Stallcup WB. Binding of the NG2 proteoglycan to type VI collagen and other extracellular matrix molecules. J Biol Chem 1996;271: 26110-261 16. 81. Tillet E, Ruggiero F, Nishiyarna A, Stallcup WB. The membrane-s~anningproteoglycan NG2 binds to collagens V and VI through the central nonglobular domain of its core protein. J Biol Chem 1997; 272:10769-10776. 82. Nishiyama A, Lin XH, Giese N, Heldin CH, Stallcup WB. Interaction between NG2 proteoglycan and PDGFa-receptor on 02A progenitor cells is required for optimal response to PDGF. S Neurosci Res 1996; 43:315-330. 83. Dou CL, Levine JM. Identification of a neuronal cell surface receptor for a growth inhibitory chondroitin sulfate proteoglycan (NG2). J Neurochem 1997; 68:10211030. StallcupWB. P~icipationof the NG2 proteoglycan in rat aortic smooth 84. Grako U, muscle cell responses to platelet-derived growth factor. Exp Cell Res 1995; 21: 231-240. 85. CarbonettoS, Gruver MM, Turner DC. Nerve fiber growth in culture on fibronectin, collagen, and glyco~aminoglycansubstrates. J Neurosci 1983; 3:2324-2335. 86. Akeson R, Warren SL. PC12 adhesion and neurite formation on selected substrates are inhibited by some glycosaminoglycans and a fibronectin-de~vedtripeptide. Exp Cell Res 1986; 162:347-362. 87. Verna JM, Fichard A, Saxod R. Influence of glycosaminoglycans on neurite morphology and outgrowth patterns in vitro. Int J Dev Neurosci 1989; 7:389-399. 88. Snow DM, Lemrnon V, Carrino DA, Caplan AI, Silver J. Sulfated proteoglycans in astroglial barriers inhibit neurite outgrowth in vitro. Exp Neurobiol 1990; 109: 111-130. 89. Oakley RA, Tosney KW,Peanut agglutinin and chondroi~n-6-sulfateare molecular markers for tissues that act as barriers to axon advance in the avian embryo. Dev Biol 1991; 147:187-206. 90. Brittis PA, Canning DR, Silver J. Chondroitin sulfate as a regulator of neuronal patterning in the retina. Science 1992; 225:733-736. 91. Snow DM, WatanabeM, Letourneau PC, Silver J. A chondro~tinsulfate proteoglycan may influence the direction of retinal ganglion cell outgrowth. Development 1991; 11311473-1485. 92. Braunewell KH, Pesheve P, McCarthy JB, Furcht LT, Schmitz B, Schachner M. Functional involvement of sciatic nerve-derived versican- and decorin-like molecules and other chondroitin sulphate proteoglycansin ECM-mediated cell adhesion and neurite outgrowth. Eur J Neurosci 1995; 7:805-814. *

l y c ~ n sin the Nervous System

1

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zyxwvuts zyx zyxwvu zyxwvu zyxwv zyxwvu

93. Dou CL, Levine JM. Inhibition of neurite outgrowth by the NG2 chondroitin sulfate proteoglycan. J Neurosci 1994; 14:7616-7628. 94. Maeda N, Noda M. 6B4 proteoglycan/phosphacanis a repulsive substratum but promotes morphological differentiation of cortical neurons. Development 1996; 122:647-658. 95 Sakurai T, Lustig M, Nativ M, Hemperly JJ, Schlessinger J, Peles E, Grumet M. Induction of neurite outgrowth through contactin and Nr-CAM by extracellular regions of glial receptor tyrosine phosphatase p. J Cell Biol 1997; 136:907-918. 96. Nadanaka S, Clement A, Masayama K, Faissner A, Sugahara K. Characteristic hexasaccharide sequences in octasaccharides derived from shark cartilage chondroitin sulfate D with a neurite outgrowth promoting activity. J Biol Chern 1998; 273:3296-3307. 97. Emerling DE, Lander AD. Inhibitors and promoters of thalamic neuron adhesion and outgrowth in embryonic neocortex: functional association with chondroitin sulfate. Neuron 1996; 17:1089- 1 100. 98. Rudge JS, Silver J. Inhibition of neurite outgrowth on astroglial scars in vitro. J Neurosci 1990; 10:3594-3603. 99. Canning DR, Hoke A, Malemud CJ, Silver J. A potent inhibitor of neurite outgrowth that predominates in the extracellular matrix of reactive gliosis. Int J Dev Neurosci 1996; 14:153-175. 100. McKeon RJ, Schreiber RC, Rudge JS, Silver J. Reduction of neurite outgrowth in a model of glial scarring following CNS injury is correlated with the expression of inhibitory molecules on reactive astrocytes. J Neurosci 1993; 11:3398-3411. 101. McKeon RJ, Hoke A, Silver J. Injury-induced proteoglycans inhibit the potential for laminin-mediated axon growth on astrocytic scars. Exp Neurol 1995; 136:3243 102. Zuo J, Hernandez YJ, Muir D. Chondroitinsulfate proteoglycan with neurite-inhibiting activity is up-regulated following peripheral nerve injury. J Neurobiol 1998; 34:41-54. 103. Davies SJ, Fitch MT, Memberg SP, Hall AK, Raisman G, Silver J. Regeneration of adult axons in white matter tracts of the central nervous system. Nature 1997; 3901680-683. 104. Braunewell KH, Martini R, LeBaron R, Kresse H, Faissner A, Schrnitz B, SchachnerM. Up-regulation of a chondroitin sulphate epitope during regeneration of mouse sciatic nerve: evidence that the i~unoreactivemolecules are related to the chondroitin sulphate proteoglycans decorin and versican. Eur J Neurosci 1995; 7:792--804. 105. Tani E, Ametani T. Extracellular distribution of ruthenium red-positive substance in the cerebral cortex. J Ultrastruct Res 1971; 34:1-14. 106. Carbonetto S. The extracellular matrix of the nervous system. Trends Neurosci 1984; 7:382-387. 107. Rutka JT, Apodaca G, Stern R, Rosenblum M. The extracellular matrix of the central and peripheral nervous systems: structure and function. J Neurosurg 1988; 69: 155-170. 108. Sanes J. Extracellular matrix molecules that influence neural development. Ann Rev Neurosci 1989; 12:491-516. *

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109. Wintergerst ES, Faissner A, Celio MR. The proteoglycan DSD-1-PG occurs in perineuronal nets around parvalbumin-immunoreactive inte~euronsof the rat cerebral cortex. Int J Dev Neurosci 1996; 14:249-255. 110. Brecht M, Mayer U,Schlosser E, Prehm P. Increased hyaluronate synthesis is required for fibroblast detachment and mitosis. Biochem J 1986; 239:445-450. 111. Haddon CM, Lewis JH. Hyaluronan as a propellant for epithelial movement: the development of semicircularcanals in the inner ear of Xenop us. Development 199l ; 112:541--550. 112. Powell EM, Fawcett JW, Celler HM, Proteoglycans provide neurite guidance at an astrocyte boundary. Mol Cell Neurosci 1997; 10:27-42. 113. Bruckner C, Brauer K, Hartig W, Wolff JR, Rickrnann MJ, Derouiche A, Delpech B, Girard N, Oertel W W ,Reichenbach A. Perineuronal nets provide a polyanionic, glia-associatedform of microenvironrnentaround certain neurons in many parts of the rat brain. Glia 1993; 8:183-200. 114. Xiao Z, Taylor J, Montag D, Rougon G, SchachnerM. Distinct effects of recombinant tenascin-R domains in neuronal cell functions and identification of the domain interacting with the neuronal recognition molecule F3/ 11. Eur J Neurosci 1996; 8: 766-782. 115. Paganetti PA, Caroni P, Schwab ME. Glioblastoma infiltration into central nervous tissue in vitro: involvement of a metalloproteinase. J Cell Biol 1988; 107:22812291. 116. Zhang H, Kelly G, Zerillo C, Jaworski D, Hockfield S. Expression of a cleaved brain-specific extracellular matrix protein mediates glioma cell invasion in vivo. J. Neurosci. 1998; 18:2370-2376. 117. Hockfield S, Kalb S, Fryer H. Expression of neural proteoglycans correlates with the acquisition of mature neuronal properties in the mammalian brain. Cold Spring Harbor Syrnp Quant Biol 1990; 55:505--514. 118. Sur M, Frost DO, Hockfield S. Expression of a su~ace-associatedantigen on Ycells in the cat lateral geniculate nucleus is regulated by visual experience. J Neurosci 1988; 8:874--882,

zyxwv zy

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Abnormalities bone, 17 l Acetylation, 15 Acetylcholine receptors (ACh Rs), 307 Acetylcholinesterase (AChE), 307 Acidic fibroblast growth factor(s) (a/FGF/FGF-l), 27, 32, 33, 36, 48 activation of, 37 Acti~-containingmicro~lamentbundles, 150 ADAMs ( a disintegrin and metalloprotease), 95, 99 Adhesion(s) focal, 32 formation, 155 molecule, 290 t r a n s ~ e m ~ r a receptors, ne 155 a/FGF/FGF-l (see also Acidic fibroblast growth factor), 27, 32, 33, 36, 48 activation of, 37 Aggrecan, 8, 10, 12, 67, 68, 74, 97-100, 306, 343377

[Aggrecan] biosynthesis, 355-360 cartilage expression, 350-352 function, 350-352 catabolism of, 97, 100, 364 core protein, 20, 344 family, 380 function, 350-352 gene, 347, 352 organization, 344-349 transcriptional regulation of 361, 362 IGD, 99 processing, 356 regulation of, 36 1-366 mechanical loading, 365366 space-filling capability of, 344 structure, 344-349 mechanical modulation, 366 synthesis, 358, 364 undersulfated molecules, 354 Aggrecanase, 99, 100, 348 Aggregates link-stabilized, 360 proteoglycans, 100- 102 Agrin, 27, 291, 307-312, 308, 310, 311, 314

zyxwvuts zyxwv zyx

Alkaline cleavage, 8 (see al so Glycosaminoglycan chains) al-antichymotrypsin (a,-Achy), 123, 297 al-proteinase inhibitor (a,-PI), 123 a5Pl integrin, 282 (see al so Integrin) a-chemokine(s), 40, 41 a-glucosidase, 7 a-helices, 209 (see al so SLRP) a-helix, 21 1 (see al so SLRP) a-hemolysin, 22 effects on chondroitin sulfate, 20-22 effects on hyaluronan, 20-22 staphylococcal, 20 a-macroglobulin, 47, 48, 96 Alternative splicing, 327 of perlecan, 278 Alu-like repetitive DNA motifs, 18 1 Alzheimer’s disease, 276, 29 1, 296, 300 Amphoterin, 386 Amylin, 299 Amyloid core fibrils, 298 ~brillogenesis,298 plaques, 299, 300 precursor protein (APP), 168 Angiogenesis, 123, 295, 301 tumor, 301 Antennapedia binding site, 132 Antineoepitope antibodies, 100 Antiparallel interaction(s), 66 Antiport mechanism(s), 20 Antithrombin, 27, 34 binding site, 30 Aortic smooth muscle cells, 387 AP-2, 330, 362 binding site, 132 motifs, 285 AP-4, 362 Apolipoprotein E, 297

Apoptosis, 15 1, 170 Appican (amyloid P) precursor, 380 Arg-Gly-Asp (or RGD sequence), 28 1 Arthritis osteoarth~tis,105, 354 rheumatoid, 105, 354 ATP, 15, 20, 21 Axonal guidance, 387, 388 regeneration, 390, 39 1

Basement membrane, 106, 296, 304, 305, 312 component, 3 13 Basic fibroblast growth factor(s) (b/FGF/FGF-2), 27, 32, 33, 34, 35, 37, 38, 39, 48, 220 dimers, 36 b/FGF/FGF-2 [see Basic fibroblast growth factor(s)] Bec~with-Wiedemannsyndrome (BWS), 170 P-amyloid protein, 297 P-chemokine(s), 40, 41 subfamily MIP-la, 41 MIP-1P, 41, 43 Rantes, 41, 43 P-galactosidase, 134 P-galactosyltransferase, 244 P-glucuronidase(s), 7 Betaglycan, 27, 107 P-hexosaminidase(s), 7 P-xylosides, 16, 20 Pl-integrins, 121, 283 (see al so Integrins) Biglycan(s) (DS-PGI), 103, 104, 206, 208, 209, 215, 252, 26 l

zyxwvu

[Biglycan(s)] circular dichroism analysis of, 211, 281 knockouts, 221 nonglycanated forms of, 104 Bikunin, 69 Biopolymer, 66-68 Biosynthesis of chondroitin sulfate, 12- 16 of heparan sulfate, 28-31 of hylauronan, 16- 18 Blimp- 1, 133 Bone marrow multipotential cells, 182 Borohydride, 10 ~ o ~ ~ ~e ul i~a g d o(Lyme ~ e ~disi ease), 219 Bowman’s layer, 238 B r a c h y ~ o ~ h(i ~c m / ~mouse, m ) 353 Brefeldin A, 19, 20, 22, 186, 359 effects on synthesis of chondroitin sulfate, 19-20 effects on synthesis of hyaluronan, 19-20 Brevican, 68, 76, 103, 347, 380, 382, 383, 389 Bullous keratopathy, 26 1

[Carcinoma(s)] ductal breast, 81 mamma^, 8 1 progression of, 8 Cartilage, 74 adult articular, 105 aggrecan expression, 350-352 function, 350-352 arthritic, 105 articular, 101, 102, 104, 355 chondrodysplasias, 353 developing, 387 hyaline, 5, 8 keratan sulfate, 243 large aggregating proteoglycan of, 343 matrix deficiency (cm d mouse), 351, 353 small leucine-rich proteoglycans (SLRPS), 103, 105, 201235, 202, 213, 224, 240 viscoelastic properties, 350 CASWLIN-2, 118 Cat-301 antigen, 330 proteoglycan, 393 Catenins, 149 Cathepsin B, 95, 98 inhibitor, 100 D, 95 G, 95, 123 Cationic dyes, 275 CCAAT enhancer binding protein, 330 CD44 (hyaluronan receptor), 27, 68, 69, 70, 72, 76, 77, 80, 81 Cell(s) attachment, 282 cycle control, 225 deformation, 265 glial, 386

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Cancer(s) human pancreatic, 167 C-type lectin, 328 (see also Versican) domain, 382 Calpain, 95 CAMP (cyclic adenosine monophosphate), 131 dibutyryl, 286 responsive element binding protein, 330 Carcinoma(s) cells P19, murine embryonal, 29 l

index

[Cell(s)] growth regulation of, 3 1-32 migration regulation of, 3 1-32 shape, 365 Cellular immunity, 177 Cellulose synthases, 66 Cerebral cortex, 383 Cerebrovascular deposits, 297 C-5 epimer iduronate (IdoA), 28 Chain(s) elongation, 359 sulfation, 359 Chaperone, 358 protein(s), 63, Chemokine(s), 32, 40-43 a-chemokines, 40, 41 P-chemokines, 40, 4 1 mutants, 43 Chitin synthases, 66 ~ h l o ~ e ~66l a , Cholesterol, 164 Chondrocyte(s), 12, 74 chondros~coma,19 rat cells, 99 differentiated, S26 effects of a-hemolysin, 20-22 effects of brefeldin A, 19-20 hy~ertrophic,80, 29 1 immortalized rat in vitro, 306 nanomelic, 356 ~er~eabilization, 20, 2 1 proliferating immature, 126 Chondrodysplasias, 353 achondrogenesis 1R, 353 atelosteogenesis type 11, 353 diastrophic dysplasia sulfate transporter (DTDST), 353 Chondrogenesis, 35 1, 352

Chondroitin 6-sulfate, 388 sulfate, 5-26, 15, 20, 22, 69, l 17, 184, 329, 343, 389, 390 a-hemolysin, 20-22 biosynthesis, 12- 16 brefeldin A, 19-20 metabolic labeling, 18-19, proteoglycan(s), 335, 380, 384 axon regeneration, 390-391 axonal guidance, 387 membrane-bound, 386 nervous system, 379-402 NG2 trans~embrane,386 proteolytic fragments of, 380 structure, 5-9 sulfate A, 7 sulfate R, 7 unsulfated, 20 Chondroitinase, l 9 bacterial, 10 treatment, 390 Chondrosine, 7 Chymase(s), 188 MC, 188 mMCP-5, 187 Circular dichroism, 22 l analysis, 213, 281 of biglycan, 21 l of decorin, 21 1 Clot lysis, 95 Club foot deformities, 353 Clusters of photoreceptors (ommatidia), 5 S Collagen fibril assembly, 214 fibril organization of cornea, 254-255 gap region (D band), 222 types I, 118, 218, 223, 259, 331, 333 11, 218

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zyxw zyxwvu zyxwvu zyx zy

[Collagen] 111, 118 IV, 291, 292 V, 118, 221, 254 VI, 255 XII, 257 XIV, 219 XVIII, 291, 312-314 Collagenase(s) (MMP 13), 98, 287, 295 -3 (MMP13), 98, 101 Colorectal adenocarcinomas, 8 1 Complement B regulatory element, 348 Compressive load(s), 365 cyclic, 365 static, 365 resistance and resilience, 350 Coreceptors, 155 Cornea, 238, 294 collagen fibril organization, 254 Scheie’s, 254 transparency, 254 Corneal decorin, 252 endothelium, 238 extracellular matrix, 237 keratan sulfate, 241, 243 lamellar, 239 macular corneal dystrophy, 254, 258 pathology and wound healing, 260-261 proteoglycans, 237-273 structure, 237 transparency, 222, 239-240 Cortactin, 44 Covalent dimeric proteins, 44 CRP module, 328 c-src, 44 CS-rich region, 349, 354

CTF-NF- 3, 285 Cumulus oophorus, 80 Cuprolinic blue dye, 255, 260 Cyclic adenosine onop phosphate (CAMP), 129 compression, 220 fibrosis, 358 Cytokine(s), 66 in~ammatory,364

Da lly (division abnormally delayed), 5 1, 52, 169

mutants, 168, 172 Deacetylation, 63, 185 Decapentaplegic (DPP), 49, 5 1, 169 Decorin (DS-PGII), 20, 104, 201, 206, 209, 215, 216, 225, 252, 260, 297 circular dichroism analysis of, 21 1 corneal, 252 disulfide bonds in, 208 hypomethylation of, 2 18 model for, 210 null mutation, 219 Degradative agents, 93 Dehydroalanine, 8 Deoxynojirimycin, 244 (glycosylation inhibitor) Dermatan/chondroitin sulfate glycosaminoglycan chain, 2 16 Dermatan sulfate, 14, 211, 214, 245-246, 259 proteoglycans (DSPGs), 240, 253 Descemet’s membrane, 238, 256 Development intestinal, 290 limb embryonic, 78

[Development] retina, 388 skeletal, 344, 351 Dextran, 17 dextran sucrase, 17 Diabetes, 286 type 11, 300 Differentiated myotubes, 289 Di~eptidylpeptidase I (DPPI), 188 Disaccharide(s), 6, 41 ~ d i s a c c h a ~ d e10 s, N-sulfated, 30 Dornain(s) amino terminal hyaluronan-bind" ing (Cl), 97, 102, 347 c-terminal globular, 291 GI, 98, 99 G2, 98, 99 G3, 99 heparin-binding domain of fibronectin, 150 highly-sulfated, 45 hydrophobic transrnembrane, 115 inte~rin-binding,95 interglobul~,98 polysynaptic density protein (PDZ), 118 Down's syndrome, 300 DPP (decapentaplegic), 49, 5l ~ r o s o ~ ~48-52, ~ l a ,147, 168 chaoptin, 210 membrane receptor Toll, 210 syndecans, 1 16 Dysplasia, 126 Dystroglycan, 3 11

Early response gene, 276 E-cadherin, 149 Ectodornain, 115, 116

EGF (epidermal growth factor), 133, 348 1,348 2,348 inhibitor of (AG1478), 217 like elements, 328 receptor, 2 16 repeat, 382 Elastase, 95, 98, 123 Eliminases, 10 Embryonic cuticle, 49 forebrain, 383 limb development, 78 stem cells, 182 Entactin, 292 Entactin/nidogen (see also Nido~en/entactin),28 1, 29 1 Endo-~-ga1actosidase,242, 246, 248, 251 Endochondral bone formation, 351 ossification, 93, 290, 306 Endocytosis (shedding) direct, 106 Endoglycosidase, 108 N-glycanase, 242 Endothelial cells brain capillary, 387 Engelbreth-Holm-Swarm (EHS) tumor, 275, 279, 283 Epidermal growth factor (EGF), 133, 348 1, 348 2, 348 inhibitor of (AG1478), 217 like elements, 328 receptor, 2 16 Epimerase, 14 Epimerization, 63 C5, 185

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zyxwv zyxwvu zyx

Index

zyxwvuts zyxwvu zyxwvu

Epiphycan (PG-Lb/~S-PGIII),105, 208, 209, 224, 225, 252 Epithelial morphology, 118-121 cell anchorage of, 118-321 maintenance of, 118- 121 ErbB -2lneu oncogene, 72 Ester-linked sulfates (0-sulfates), 28 ETS-1, 285 Exoglycosidases, 107, 24 1 Exopeptidase, 187 m MC- CPA, 187 Extracellular regulated kinase (ERE(), 134

F

~,

[Fibroblast growth factor (FGF)] FGF-4, 128 FGF-7, 129, 133, 134, 294, 296 FGF-8, 128 FGF-family, 133 Fibroblast growth factor-receptor (FGFR), 35, 36, 37, 122 Fibromodulin, 105, 206, 207, 209, 221, 222, 251, 252 Fibronectin, 118, 121, 150, 292, 293, 331, 333 h e ~ ~ i n ~ b i n d idomain ng (HEW), 150 Fibulin, 29 1 -2, 292 FiRE (FGF-inducible response element), 133, 134 Focal adhesion(s), 32 component, 282 formation, 153, 155 Follistatin modules, 309 FoslJun factor, 133 Free radical mechanisms, 106

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F9 embryonal carcinoma cells, 286 F-actin filaments, 118 FGF (fibroblast growth factor), 32, 121, 294 FGF-2, 121, 124, 125, 128, 129, 134, 168, 278, 288, 294, 295 -induced cell proliferation, 122 FGF-4, 128 FGF-7, 129, 133, 134, 294, 296 FGF-8, 128 FGF-family, 133 FGF-inducible Response Element (FiRE), 133 FGFR (fibroblast growth factorreceptor), 35, 36, 37, 122 Fibrinogen, 189 Fibrinolysis, 123 Fibroblast growth factor (FGF), 32, 121, 294 FGF-2, 121, 124, 125, 128, 129, 134, 168, 278, 288, 294, 295 -induced cell proliferation, 122

Frizzled, 5 1

Fructose, 17 Fucosylated neuraminic acid, 243

GAG(s) (glycosaminoglycans), 5, 22, 27, 38, 61, 62, 63, 93, 94, 107-108, 115, 129, 147, 190, 209, 225, 240, 241, 349, 358, 386 - a, 328, 330 -p, 328, 330 attachment domains, 179, 184, 386 binding, 44, 46 chains, 164-165, 168, 214, 246 de~atan/chondroitinsulfate, 216

[GAG(s) (glycosaminoglycans)] elongation, 355 sulfation, 355 -degrading enzymes, 96 Ser-Gly -ASP, 279 consensus sequences for GAG attachment, 116 dipeptide, 349 glycanation sequences, 165 repeat, 184 structure, 2 11-2 13 structure/function relationships, 213 sulfated, 14 Galactosamine, 7 Galactose, 240 GalNAc, 14, 15, 18, 30, 63 Ganglion cells, 388 GATA -2 motifs, 285 motifs, 182 GC boxes, 285 -rich regions, 133 Genistein, 330 Gelatinase(s), 98 Cia1 hyaluroanan-binding protein (GHBP) or hyaluronectin, 103, 330 GlcA (gluc~ronate),15, 17, 28, 30 GlcNS03, 30 GlclJA, 63 Glial cells, 76 proliferating progenitor cells, 387 Globular domains, 93 Glo~erularbasement membrane, 302, 303 Glomeruli cerebellar, 384 Glucuronic acid, 7, 14, 61, 213

Glucosamine, 15 N-sulfo, 28 Glucose, 17, 303 high glucose medium, 286 Glucuronate (GlcA), 28, 30 Glutamine, 15 Glycolipids sulfated, 393 Glycosaminoglycan(s) (GAG), 5, 22, 27, 38, 61, 62, 63, 93, 94, 107-108, 115, 129, 147, 190, 209, 225, 240, 241, 349, 358, 386 - a, 328, 330 attachment domains, 179, 184, 386 -p, 328, 330 binding, 44, 46 chains, 164-165, 168, 214, 246 de~atan/chondroitinsulfate, 216 elongation, 355 sulfation, 355 -degrading enzymes, 96 Ser-Gly -ASP, 279 consensus sequences for GAG attachment, 116 dipeptide, 349 glycanation sequences, 165 repeat, 184 structure, 2 11-2 13 structure/function relationships, 213 sulfated, 14 Glycosidase(s), 94, 96-97, 107 attack, 93 Glycosidic linkage, 7 Glycosyl phosphatiylinositol (GPI) anchor, 106, 161, 162164, 383 -specific phospholipase D,164

zy zyx

zyxwvu zy

Glycosyltransferase(s), 185, 358 Glypiation, 162 Glypican(s), 48, 51, 151-176, 294 core protein, 165- 166 gene organization, 166- 167 glypican- 1, 36 GP1 anchored, 27, 51 3 gene, 48 Golgi, 19, 20 apparatus, 63, 358 cisternae, 13, 355 complex, 358 enzymes, 358 late process, 359 membranes, 360 network, 359 trans, 355, 359 GP1 anchor (Glycosyl phosphatidylinositol), 106, 161, 162- 164, 383 -specific phospholipase D,164 G proteins, 164 Granule cells, 384 Guanylate kinase family, 118

Hematopoietic cells, 107, 178 HepII (heparin-binding domain of fibronectin), 150 Heparanase(s), 106 extracellular, 96 Heparin-binding epidermal growth factor (HB-EGF), 121, 124, 168 Heparin-binding growth-associated molecule (HB-GAM), 32, 43, 44, 121, 155, 385 Heparin-binding growth factor, 121-122 Heparin-binding molecules, 131 Heparan Sulfate (HS), 27-60, 28, 30, 33, 34, 36, 41, 43, 47, 48, 49, 50, 51, 52, 115, 117, 122, 147, 184, 187, 241, 295, 302 binding, 43, 45, 46, 47 biosynthesis of, 28-3 1 chains, 27, 293, 302 depolymerization of, 97 development and differentiation, 48-52 interactions with growth factors, 31-32 molecular structure, 28-3 1 Heparan sulfate proteoglycan(s) (HSPGs), 27, 28, 36, 37, 48, 51, 105, 121, 124, 128, 161, 168, 172, 281, 283, 294, 295, 303, 307, 312, 380 in basement membranes, 275326 cell surface, 167 chain, 41 perlecan, 299 species, 27 type I transmembrane, 115

zy zyxwv zyxwv zyxwv zyxwvut

HAS (hyaluronan synthase), 16, 21,

22, 63, 65, 69 gene, 65, 66 HASl,66 HAS2,65 HAS3, 65 knockout, 65 HB-EGF (heparin-binding epidermal growth factor), 121, 124, 168 HB-GAM (heparin-binding growthassociated molecule), 32, 43,44, 121, 155, 385 Hedgehog protein(s), 49

Heparin, 20, 33, 34, 36, 37, 41, 47, 107, 122, 178, 183, 184, 186 binding domain of fibronectin (HepII), 150 binding growth factors, 168 Heparin/HS -binding growth factors, 48 binding proteins, 50 interacting protein (HIP), 293 Heparinase, 275 I, 288 111, 37, 40 Heparitinase, 306, 308 digestion, 165, 310 I , 288 Hepatocyte growth factor (HGF), 32, 121 Hepatocyte growth factorlscatter factor (HGFISF), 38 Heptameric pores, 20 Heterotrimeric G-proteins, 151 Hexasaccharide sequence, 38 Hexosamine, 7 H e x o s ~ i n i t o l ,7 Hexuronic acid, 7 HGF (hepatocyte growth factor), 32, 121 HGF/SF (hepatocyte growth factor/ scatter factor), 38 Hippocampus developing, 383 Hitchhiker thumbs, 353 Hodgkin’s lymphomas, 127 HS/heparin, 44, 45, 46 HSPG(s) (heparan sulfate proteoglycans), 27, 28, 36, 37, 48, 51, 105, 121, 124, 128, 161, 168, 172, 281, 283, 294, 295, 303, 307, 312, 380 in basement membranes, 275-326

[HSPG(s)I cell surface, 167 chain, 41 perlecan, 299 species, 27 type I transmembrane, 115 HS 2-0-sulfortransferase (HS2ST), 48 Human overgrowth syndrome (Simpson-Golabi-~ehmel),48, 169, 170, 172 Human sciatic nerve, 391 Hyaluronan, 5-26, 16, 17, 21, 22, 61-92, 62, 66, 67, 70, 72, 74, 76, 77, 78, 81, 82, 101, 102, 331, 334, 335, 343, 350, 35 1, 354, 384, 391, 392 a-hemolysin, 20-22 amino terminal-binding (G1) domain, 97 binding motif, 69 proteins, 69, 347, 380 proteoglycans, 102- 103 biosynthesis of, 16- 18 brefeldin A, 19-20 and cancer, 80-82 comp~mentalization,22 depolymerization, 94 metabolic labeling, 18- 19 morphogenesis, 76-80 properties network-forming, 68 rheological, 68 receptor(s), 8 1 cell surface, 69-72 CD44, 27, 68, 69, 70, 72, 74, 77, 78, 80, 81, 82 RHAMM, 68, 69, 72, 77, 78, 81, 82, -rich matrix, 80

zyx zyxwv zyxwv

zyxw zyxwvu zyxwvut zyxwvu zyxwvu

~Hyaluronan] structure, 5-9 synthase, 76, 77 synthase-2, 22 synthesis machinery, 22 tissue remodeling, 76-80 viscoelastic property of, 68 viscosity of solutions, 68, Hyaladherins, 68-69 H y ~ e c t ~ ( s68,103,327,380-383 ), Hyaluronan synthase (HAS), 16, 21, 22, 63, 65, 69 gene, 65, 66 HASl, 66 HAS;?, 65 HAS3, 65 knockout, 65 Hyaluronectin (or glial hyaluronanbinding protein GHBP), 103, 330 Hyaluronidase(s), 17, 81, 82, 96, 102 ~ a ~ m a l i a69 n, streptomyces, 16 Hydrolases, 97 Hydrophobic bonds, 66 Hydrophobic patches, 66 Hydrophobic transmembrane domain, l 15 Hydrostatic pressure, 365 Hydroxyl radicals, 102, 106

IGD, 99 (see also Aggrecan) IGF-I, 363 IGF2 (Insulin-like growth factor-2), 170 IGF2R (IGF2R Insulin-like growth factor-2 receptor), 171, 172 IL-lp, 134 IL-8 (Interleu~n-8),40 dimer, 41 Imaginal disc, 51 Immunoglobulin (IgG) repeats, 283, 347 Insulin-like growth factor-2 (IGF2), 170 Insulin-like growth factor-2 receptor (IGF2R), 171, 172 Integrin(s), 150, 296 pl, 296 p3, 296 binding domain, 95 Inter-a-trypsin, 76, 80 inhibitor, 69 Interferon-y (IFN-y), 39, 41, 134, 288, 295, 296, 364 receptor, 40 Interglobular regions (IGD), 345 Interleukin -1 (IL-l), 99, 100, 355, 363 -3 (IL-3), 184 -6 (IL-6), 129 -8 (IL-8), 40 dimer, 41 Internal granular layer, 383 Intervertebral disc, 101, 104, 344

zyxwv zyxwvu I

ICAM, 69 IdoA ( G 5 epimer iduronate), 28 (see also Iduronate) Iduronate, 33 idoA ( C " epimer iduronate), 28 IFN-y (Interferon-y), 39, 41, 134, 288, 295, 296, 364 receptor, 40

K562 cells, 36 J D R (flk-l) receptor, 46 Keratanase-sensitive proteoglycans, 26 1

Keratan sulfate (KS), 184, 213, 244, 246, 258, 343, 354 biosynthesis of, 244-245 cartilage, 243 chains, 206, 242 corneal, 241, 243 decorin-associated, 252 N-linked, 22 1 proteoglycan(s) (KSPGs), 105, 240, 245-25 1, 247, 248, proteins, 253 synthesis of, 245 tryptic peptide mapping of, 246 reducing end, 241 -specific sulfotransferase, 259 structure, 240-243 tissue distribution, 243 Keratinocytes, 238, 260 growth factor (KGF), 168 migrating, 134 proliferating, 127 Keratocan, 105, 209, 221, 223, 249, 251, 258 Keratoconus, 26 1 Keratocytes, 245, 256, 258 Keratosulfate, 240 chain, 359

Leucine-rich repeat(s) (LRR), 20 1, 210, 21 1, 224, 249, 252, 255 motif, 207, 209 region, 104 Link modules, 68, 69 protein(s), 68, 69, 101, 102, 104, 343, 345 stabilization, 350 Linkage regions, 344 Lipoprotein(s), 123 lipase (LPL), 123 high-density (HDL), 28 1 low-density (LDL), 123, 280, 304 -receptor gene structure, 284 metabolism, 122 LRR(s) (Leucine-rich repeat), 201, 210, 211, 224 (see a lso SLRPs) motif, 207, 209 region, 104 Lumican, 105, 209, 221, 222, 223, 25 1 knockouts, 223 mice, 254 mRNA, 248 Lyme disease (Borrelia burgd orf eri), 219 Lymphoma, 81 Lysosomal storage diseases (mucopolysaccharidoses), 96, 358

zyxwvut zyx zyx

Lacunae, 80 expansion of, 80 Laminin, 256, 291, 333 A(a1) chain, 291 B domain (Lam B), 281 E3 fragment of, 291 EGF-like domain (LE), 281, 284 Lectican(s), 68, 103, 327, 380383 gype C, 348 Lectin-type interaction, 385

zyxwvut Macular corneal dystrophy, 254, 259 Mannose oligosaccharide, 241 Mannosidase, 241

Index

zyxwvuts zyxwv

MAP kinaselp2 1, 21 6 Mast cell(s), 107 chymases, 95 secretory granules of, 177 Mastocytoma cell(s) in mouse, 20 Matrilysin, 98 Matrix rnetalloproteinases (MMPs ), 95, 98, 99, 103, 106 collagenases, 98 gelatinases, 98 inhibitors, 100 matrilysin, 98 membrane type (MT), 95 stromelysins, 98 MC(s), 179 chymases, 188 function of serglycyn, 187 MEC-8, 287 Melanoma(s), 8 1, 301 Meniscus, 344 Mesangial cell, 293 matrix, 302 Mesenchyme, 49 condensing, 332 metanephric, 49 Mesoderm, 78 Mesodermal condensation, 78 Metabolic labeling chondroitin sulfate, 18- 19 hyaluronan, 18- 19 Metalloproteinases, 348 Midkine, 43, 44, 122 Migration factors, 40 neuronal, 352 Mirnecan, 105, 224, 244-251 (se e a lso Osteoglycin) Molecular structure of heparan sulfate, 28-31 Monosaccharide, 63

Mouse brain developing, 48 Multienzyme complex, 20 Myeloma, 127 Myofilament lattice assembly, 290 Myogenin, 129 Myotubes, 80

zy

N-acetyl a-linked, 28 N-acetylgalactosamine, 14, 6 1, 62, 244

N- acet ylglu cos am in ylt r an s f~ r as e,

244 N-CAM, 284, 307, 385, 386 N- d eacetylase/ N- su lfotran sferase,

185 activity, 5 1 N-deacetylation, 30 N-linked glycosylation, 250 N-sulfated disaccharide, 30 groups, 38 N-sulfates, 33 N-sulfation, 30, 185, N-sulfo glucosamine 28, 30 N-terminal domain sequencing, l00 tyrosine-rich, 22 1 Na n om elia , 35 1, 353 NDST, 186 NDST-1, 185, 186, 190 NDST-2, 185, 187, 190 Nervous system developing, 167 Neural crest cells, 76, 77, 333 migration, 77 tube, 332

zyxwvu

Neuregulins, 121 Neurite outgrowth, 388, 389, 390 Neuritic plaques, 297 Neurocan, 68, 76, 103, 347, 380, 383, 384, 389 Neuroepithelial cells, 278 ~eurofibri1larytangles, 297 Neuroglycan C, 380 Neuronal cell adhesion, 390 differentiation, 388 Neurons, 386 Neurotrophic factors, 43 NF-KB binding site, 132, 285, 362 NG2, 257, 258 proteoglycan, 258 melanoma associated, 386 transmembrane CSPG, 386 Ng-CAMILl, 385, 386 Nidogen/entactin (see also Entactin), 281 Nitric oxide depolymeri~ationof, 97 Nitrous acid, 275, 308 NMR analysis, 41 Notochord, 35 1 Nr-CAM, 385, 386

Ommatidia (clusters of photoreceptors), 51 Osteoadherin, 209, 221, 224 Osteoarthritis, 10 Osteoinductive factor, 249 Osteoglycin (see Mimecan), 105, 224, 249-25 1 Osteonectin, 224 0-sulfate(s) (ester-linked sulfates), 28 0-sulfation, 63, 185

zyxw zyxwvut

O-~-xylosy1-serine,9 Oct-l, 304 Oligo-H type, 33 Oligomeric protein(s), 39-48 noncovalent, 39-44 Oligosaccharide(s), 46, 221 linkage, 13, 14 mannose, 24 l N-linked, 13, 241, 242, 252, 344 pol~lactosamine-modified,222 processing of, 358 0-linked, 13, 344, 358

p21, 216 p38 MAPK, 134 Pancreatic p cells, 299

Pasteu~e~la ~ u l t u ~ i d65 a,

PCBV-l, 66 PDGF (platelet-derived growth factor), 39, 44, 45, 46, 47, 295 PDGF A, 121 PDGF-AL, 46 a-receptor, 387 PDZ (Polysynaptic density protein domain), 118 PEA3, 285 Pericellular hyaluronan-aggrecan complexes, 76 Pericellular matrix(s), 65, 72-76, 78 Periodate oxidation, 7 Perlecan, 27, 105-106, 256, 275, 276, 277, 279, 284, 287, 291, 295, 296, 300, 301 gene alternative splicing, 278 homophilic binding of, 283 inhibition of transcription, 286 nematode, 276 polymorphisms, 303 promoter, 285 proteolytic fragment of, 303

[Perlecan] regulation of expression, 286 -related molecule (PRM), 278 synthesis blocking, 301 PF4 (platelet factor 4), 40, 41 PG-Lb, 207 (see also Epiphycan), 225 PG-M (chick versican), 327, 329 Phophatidylinositol3 kinase (PI13K), 134 4,5 biophosphate (PI4,5P2), 152 specific phospholipase C (PIPLC), 161 Phorbol ester, 150, 183 Phosph~adenosinephosphosulfate (PAPS), 14, 15, 19, 185, 259, 353, 358 P~osphocan(RPTPP), 385, 386 Phospholipid(s) inositol, 152, 153 metabolism, 153 Phosphorylation of cytoplasmic tails, 117 of syndecan cytoplasmic domains, 153 Photoreceptor axons, 5 1 PKC (protein kinase C), l21 , 150, 151, 152, 153, 154, 155,

Polylactosamines, 2 13 Polymer sulfation, 30

zyxwvu zyxwvuts zyx zyxwv zyxwv zyxwv zyxwv zyx

156

alpha (PKCa), 117, 121, 151, 153, 154, 155 Plasmin, 95, 98, 106, 288, 295 Plasminogen, 38 Platelet-derived growth factor (PDGF), 39, 44, 45, 46, 47, 295 PDGF A, 121 PDCF-AL, 46 Platelet factor 4 (PF4), 40, 41 PLUS domain (chicken versican), 329

Poly-~-acetyllactosamine,24 l Polysynaptic density protein (PDZ) domain, 118 regions, 380 PRELP, 252 Protease(s), 93, 94-96, 99, 187 aspartic, 94 calpain, 95 cysteine, 95 inhibitors, 96, 189 a2-macroglobulin, 96 cystatins, 96 serpins, 96 TIMPs, 96 positively charged (packaging of), 177 serine, 95, 123, inhibitors, 123 Proteasomes, 356 Protein(s) bone morphogenetic (BMP), 363 chaperone, 63 core, 213 covalent dimeric proteins, 44 heat shock (hsp), 358 hedgehog, 49 hyaladherins, 68 link, 68, 69, 101, 102, 104, 343, 345 oligomeric, 39 serum, 69 Protein kinase C (PKC), 121, 150, 151, 152, 153, 154, 155, 156 alpha (PKCa), 117, 121, 151, 153, 154, 155 Protein polysaccharide, 8 Proteoglycan(s), 67, 74, 77, 82, 94, 121, 391

41

zyxwvutsr zy Index

[Proteoglycan(s)] aggregates, 100-102 link-stabilized, 360 basement membrane, 256-257 Cat-301, 393 catabolism of, 93- l 13 cell surface, 106-107 chondroitin sulfate (CSPG), 335, 380, 384 axon regeneration, 390 axonal guidance, 387 membrane-bound, 386 nervous system, 379-402 proteolytic fragments of, 380 NG2 transmembrane, 386 degradation, 94 dermatan sulfate (DSPG), 240, 253 DSD-1, 330 endocytosed, 95 family of hyalectan, 68 lectican, 68 GPI-anchored, 107 neural cell adhesion molecule contactin, 386 hyaluronan-binding, 102- 103 keratanase-sensitive, 26 1-262 keratan sulfate (KSPG), 105, 240, 247, 248 proteins, 253 synthesis of, 245 trypic peptide mapping of, 246 membrane spanning, 107 NG2, 258, 380 melanoma associated, 386 serglycin, 177-199, 183, 185, 187, 189 distribution, 178 function of, 187- 189 in hematopoietic cells, 178

[Proteoglycan(s)] identification, 178 protein core analysis, 178- 181 signaling and syndecan proteoglycans, 147-159 small leucine-rich, 303, 105, 201-235, 202, 213, 224, 240 gene structure, 206-208 protein, 255, 259 structure of, 206-208 role of, 214 of stroma, 240 stromal functional roles, 253-256 tandem repeats, 68, 347 Proteolysis, l01 control of, 96 Proteolytic degradation products, 105 PU.l box, 285 Purkinje cells, 384

zyxwv Rafts, 164 Reactive astrocytes, 391 gliosis, 390 Reactive oxygen species, 94,97, 102 hydrogen peroxide, 97 hypochlorous acid, 97 hydroxyl radical, 97 nitric oxide, 97 nitrous acid, 97 superoxide, 97 Receptor(s) dimerization, 36, 43 IFN- y, 40 spanner (or serpentine), 39 transmembrane adhesion, 155

zyxw zy zyx zyxwvu zyxwvu zyxwvutsr zyxwvu zyxwv

Reductive amination, 10 Regulation of cell growth, 31-32, 170 of cell migration, 3 1-32 Renal agenesis, 48 Repulsive molecules, 393 Retinoic acid, 99, 100, 215, 286 RGD -dependent binding, 282 sequence (or Arg-Gly-Asp), 281 RHAMM (receptor for hyaluronic acid-mediated motility), 68, 69, 72, 77, 78, 80, 81, 82 RNAse inhibitor, 209, 210 Rotary shadowing analysis, 28 1 electron microscopy, 66, 282 RPTP~/phosphacan,380, 385-386

Scheie’s corneas, 254 Schwann cells, l67 Scrapie, 300 S-domains, 30, 37, 40, 41 SEA (sperm protein, enterokinase, agrin), 280 module, 280 Selectin(s), 82, 348 S. e ~ u i s i ~ i z i65 s, Serglycin(s), 107, 181, 184, 186, 190 gene, 179, 183 genomic organization, 181- 183 transcriptional regulation, 18l 183 posttranscriptional modification of, 183- 187 proteoglycans, 177-199, 183, 185, 189 distribution of 178

[Serglycin(S)] function of, 187- 189 in hematopoietic cells, 178 identification of, 178 protein core analysis, 178- 181 Serine protease(s), 95, 123, 187 inhibitors of (serpins), 123 Serpentine type receptor(s), 5 1 Shear stress, 365 Signal peptide, 220 Simpson-Golabi-Behmel Syndrome (SGBS) (human overgrowth syndrome), 48, 169, 170, 172 Ska (sugarless)gene, 4% Skeletal muscle differe~tiation,289 myoblasts, 387 SLRPs (Small leucine-rich proteoglycans), 103, 105, 201235, 202, 213, 224, 240 gene structure, 206-208 horseshoe shape, 21 l protein, 255, 259 structure of, 206-208 role of, 214 of stroma, 240 6-sulfotransferase, 10 Small leucine-rich proteoglycans (SLRPS), 103, 105, 201235, 202, 213, 224, 240 gene structure, 206-20% protein, 255, 259 structure of, 206-208 role of, 214 of stroma, 240 SPl(s), 133, 362 binding, 132 Spanner (or serpentine) receptors, 39 Sphingolipids, 164 S. pyagenes, 65

zy zyx I ndex

zy zyxwvuts

Src family kinases, 164 STAT1, 288 Stromal lamellae, 238-239, 254 proteoglycans functional roles, 253 Stromelysin(s) (MMP3), 98, 287, 295 -1 (MMP3), 99 Sugarless (ska) gene, 49 Sulfatases, 93, 94, 96-97, 107 Sulfation, 13, 20 polymer, 30 Sulfotransferase(sj, 185 4-, 360 6-, 360 Sulfurylase kinase (SKj, 354 Superoxide radicals, 102 Swainsoni~e inhibitor, 24 1 Syndecan(s), 27, 52, 107, 115-145, 123, 124, 128, 147, 294, 296 C. elegans, 148, 277, 314 cell adhesions, 149- 150 cytoplasmic dimerization, 153154 cytoskeleton connections, 154155 ~rosophila,1 16, 128 ectodomains, 156 emerging directions of, 155- 156 expression of, 125- 128 in development, 125- 126 malignant transformation, 126127 soluble syndecans, 128 tissue injury, 127- 128 focal adhesions, 150- 153 function of, 1 17-125 regulation of expression, 128- 134

[Syndecan(sj] inducible, 128-129 in tissue injury, l34 posttranscriptional, 129- 131 transcriptional, 131- 133 signaling through proteoglycans, 147-159 soluble ectodomains, 125 structure of, 115-1 17 syndecan-1, 37, 48, 118, 121, 122, 124, 125, 126, 127, 131, 133, 134, 149, 154 syndecan-2, 118, 125, 149, 154 syndecan-3, 44, 122, 125, 126, 128, 131, 149, 152, 155 syndecan-4, 32, 121, 125, 126, 150, 151, 152 tyrosine ~hosphorylation of syndecan cytoplasmic domains, 153 Xenopus, 116 Synovial fluid, 100, 104 human cells, 306 Syntenin, 1 18, 154 Systematic amyloidosis, 297

zyxwvu TATA-box, 132, 330, 362 Tenascin, 118, 391 -C, 385 -R, 384, 385, 386, 392, 393, 394 Tendon, 344, 351 Tensile strength, 2 18 Testican, 380 Testosterone, 129 Tetrapeptide sequence EFYA, 117 TCF-P (see also Transforming growth factor-$), 39, 44, 45, 47-48, 49, 129, 133,

[TGF-PI 134, 214, 216, 218, 288, 289, 302, 364 isoforms (p1, p2, p3), 48 responsive region, 285 Thalamoco~icalpathway, 389 3-0-sulfate motif, 187 Thrombin, 34, 95, 295 IATIII, 123 Thrombospondin, 118, 293 Thyrotropin receptor, 21 1 TNF-a, 80, 129, 217, 218, 355, 363, 364 response elements, 216 Transamidase, 164 Transamidation reaction, 106 Transglutaminase, 44 Transforming growth factor-P (TGF-P), 39, 44, 45, 4748, 49, 129, 133, 134, 214, 216, 218, 288, 289, 302, 364 isoforms @l, p2, p3), 48 responsive region, 285 Translocon pore, 356 Transparent crystalline proteins, 247 Transposon, 65 Tryptase(s), 95 mMCP-6, 188 Tryspin-sensitive site, 117 Tyr sulfation, 252 TSG-6, 68, 76, 80 Tumor angiogenesis, 301 vascularization, 295 Tumorigenesis, 305 Tumor necrosis factor-a (TNFa), 80, 99, 128 response elements, 2 16 Tunicamycin, 244 2-O-sulfates, 33

zyx

Tyrosine kinase receptor, 39 MUSK, 3 12 phosphorylation of syndecan cytoplasmic domains, 153 sulfation, 252, 253

Ubiquination, 356 UDP, 63 -galactose, 14, 246 -Gal antiporter, 16 -GalNAc, 14, 15, 18 -GlcA, 15, 17 -GlcNAc, 17, 18, 65 -GlcUA, 65 -glucose, 15 -sugar substrates, 17, 21, 22 UDP-glucose dehydrogenase (UDPGDH), 49 Unc-52, 279, 287 Ureteric bud, 49 inductive, 48 tips, 48 Urokinase, 98 Uronic acid, 9

zyxwvu Vascular basement membranes, 289 endothelial growth factor (VEGF), 39, 44, 45, 4647, 121 pathology, 304 smooth muscle cells, 289 VEGF (vascular endothelial growth factor), 39, 44, 45, 46-47, 121 Versican, 66, 68, 76, 103, 327-341, 347, 380, 389

zy zyxwvut zyxwvu zyx zyxwv zyxwvut Index

[Versican] atherosclerosis, 334 cancer, 335 chick (P6 - M), 327 6 3 domain of, 348 gene knockout, 336 structure, 330-33 1 Viscoelastic property of hyaluronan, 68 Viscosity of hyaluronan solutions, 68 Vitreous, 9

Wg (wingless), 49, 50, 5 1, 52 Wilms tumor repressor gene (WTl), 132, 133

Wing~ess(Wg), 49, 50, 51, 52, 121 Wnt, 49 -11, 48 -family of morphogens, 48

Xenopus

DC,65

gastrulation, 65 syndecans, 116 X-linked disorder, I69 Xylosylserine, 14 Xylosyl transferase-deficient CH0 cells, 356

Zn 2+-dependentprotease, 95