The Biology of the Small Leucine-rich Proteoglycans (1999)

small leucine-rich proteoglycan leucine-rich repeat glycosaminoglycan transforming growth factor β epidermal growth factor EGF receptor the cyclin-dependent kinase inhibitor p21 WAF1/CIP1 discoidin domain receptor If one of the keys to biology is protein structure, then nature is an efficient operator, because it adopts a number of structurally related proteins to perform functions as diverse as maintaining the mineralized matrix of bones and teeth, the transparency of the cornea, the tensile strength of the skin and tendon, and the viscoelasticity of blood vessels. Proteoglycans play key roles in all of these fundamental biological processes and behave as potent effectors of cellular pathways. The past decade has witnessed an explosion of knowledge in the proteoglycan world, with significant advances in the genetics and cell biology of these complex macromolecules. This minireview describes recent advances in the biology of the small leucine-rich proteoglycan (SLRP)1 gene family with special emphasis on the biology of the archetype proteoglycan decorin. The focus is on the “functional network” created by these molecules in tissues, on genetic evidence for their functional roles during ontogeny, and on their activities as modulators of complex pathological processes such as fibrosis and cancer growth. Other more extensive reviews may serve to fill the gaps in this one (1Iozzo R.V. Crit. Rev. Biochem. Mol. Biol. 1997; 32: 141-174Crossref PubMed Scopus (453) Google Scholar, 2Iozzo R.V. Annu. Rev. Biochem. 1998; 67: 609-652Crossref PubMed Scopus (1341) Google Scholar, 3Hocking A.M. Shinomura T. McQuillan D.J. Matrix Biol. 1998; 17: 1-19Crossref PubMed Scopus (414) Google Scholar, 4Neame P.J. Kay C.J. Iozzo R.V. Proteoglycans: Structure, Biology and Molecular Interactions. Marcel Dekker, Inc., New York, NY1999Google Scholar). The SLRP gene family comprises at least nine members that, though structurally related, have evolved from different genes, have acquired unique functions, and have undergone a significant degree of structural sophistication (Fig. 1). They can be synthesized as either glycoproteins containing N-linked oligosaccharides or as proteoglycans containing chondroitin/dermatan sulfate or keratan sulfate chains. They can also contain Tyr sulfation, undergo proteolytic processing, and contain a pre-core that is cleaved under certain conditions and with advancing age. Moreover, the promoter architecture of various SLRP genes is quite distinctive, and this contributes to their differential tissue expression (5Iozzo R.V. Danielson K.G. Prog. Nucleic Acid Res. Mol. Biol. 1999; 62: 19-53Crossref PubMed Scopus (26) Google Scholar). Three classes of SLRPs can be easily identified based on several parameters including their evolutionary protein conservation, the presence of a distinct cysteine-rich cluster in the N-terminal region, the number of the leucine-rich repeats (LRR), and their genomic organization (Fig. 1). This group includes decorin (6Krusius T. Ruoslahti E. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 7683-7687Crossref PubMed Scopus (414) Google Scholar) and biglycan (7Fisher L.W. Termine J.D. Young M.F. J. Biol. Chem. 1989; 264: 4571-4576Abstract Full Text PDF PubMed Google Scholar), which show the highest homology (∼57% identity) and are the only SLRP members that contain a pro-peptide. The pro-peptide is highly conserved across species and may function as a recognition signal for xylosyltransferase, the first enzyme involved in the synthesis of glycosaminoglycan (GAG) chains. These proteoglycans contain an N-terminal domain that is usually substituted with either one (decorin) or two (biglycan) chondroitin/dermatan sulfate side chains, leading to pronounced polyanionic properties. The most salient feature of decorin and biglycan is the presence of 10 LRRs (see below) flanked by cysteine-rich regions (Fig. 1). We previously identified a pattern of amino acid spacing among the four N-terminal Cys residues and predicted that this spacing would be characteristic of each class of SLRPs (1Iozzo R.V. Crit. Rev. Biochem. Mol. Biol. 1997; 32: 141-174Crossref PubMed Scopus (453) Google Scholar). Not only is the spacing of the Cys residues conserved within each subfamily but also the nature of the intervening amino acids is maintained. For example, class I has an N-terminal Cys consensus sequence that is unique (CX 3CXCX 6C) and different from the other two classes (Fig. 1). Another notable feature of class I members is that they are both encoded by genes composed of eight exons with intron/exon junctions in highly conserved positions (8Fisher L.W. Heegaard A.-M. Vetter U. Vogel W. Just W. Termine J.D. Young M.F. J. Biol. Chem. 1991; 266: 14371-14377Abstract Full Text PDF PubMed Google Scholar, 9Danielson K.G. Fazzio A. Cohen I. Cannizzaro L.A. Eichstetter I. Iozzo R.V. Genomics. 1993; 15: 146-160Crossref PubMed Scopus (90) Google Scholar). The 10 LRRs are encoded by six exons (exons III–VIII). The C-terminal domain is the least studied region and comprises about 50 amino acid residues and two disulfide-linked cysteine residues separated by ∼32 amino acids. This group comprises five members that can be further divided into three distinct subfamilies. Fibromodulin (10Oldberg Å. Antonsson P. Lindblom K. Heinegård D. EMBO J. 1989; 8: 2601-2604Crossref PubMed Scopus (228) Google Scholar) and lumican (11Blochberger T.C. Vergnes J.-P. Hempel J. Hassell J.R. J. Biol. Chem. 1992; 267: 347-352Abstract Full Text PDF PubMed Google Scholar, 12Funderburgh J.L. Funderburgh M.L. Brown S.J. Vergnes J.-P. Hassell J.R. Mann M.M. Conrad G.W. J. Biol. Chem. 1993; 268: 11874-11880Abstract Full Text PDF PubMed Google Scholar) constitute the first subfamily and exhibit ∼48% protein sequence identity; keratocan (13Corpuz L.M. Funderburgh J.L. Funderburgh M.L. Bottomley G.S. Prakash S. Conrad G.W. J. Biol. Chem. 1996; 271: 9759-9763Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar) and PRELP (14Bengtsson E. Neame P.J. Heinegård D. Sommarin Y. J. Biol. Chem. 1995; 270: 25639-25644Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar) constitute the second subfamily with ∼55% protein identity, whereas osteoadherin (15Sommarin Y. Wendel M. Shen Z. Hellman U. Heinegård D. J. Biol. Chem. 1998; 273: 16723-16729Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar) constitutes a distinct subfamily with 37–42% protein identity to the other class II members. All of them share an identical cysteine-rich region consensus just before the LRRs. The assignment of novel SLRPs to various classes, as predicted by the consensus sequence for the N-terminal region, has so far held true because osteoadherin, the latest SLRP member to be cloned (15Sommarin Y. Wendel M. Shen Z. Hellman U. Heinegård D. J. Biol. Chem. 1998; 273: 16723-16729Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar), has the greatest homology to class II SLRPs and indeed contains the predicted consensus (CX 3CXCX 9C) (Fig. 1). In contrast to the N-terminal region of decorin/biglycan, class II members contain clusters of Tyr-sulfate residues that would contribute to the polyanionic nature of the proteoglycan. Class II members are primarily substituted with keratan sulfate chains, and polylactosamine, essentially an unsulfated keratan sulfate, can be found in both fibromodulin (16Plaas A.H.K. Wong-Palms S. J. Biol. Chem. 1993; 268: 26634-26644Abstract Full Text PDF PubMed Google Scholar) and keratocan (13Corpuz L.M. Funderburgh J.L. Funderburgh M.L. Bottomley G.S. Prakash S. Conrad G.W. J. Biol. Chem. 1996; 271: 9759-9763Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). Finally, class II members are encoded by only three exons, with a large central exon encoding nearly all 10 LRRs (Fig. 1). Epiphycan/PG-Lb (17Shinomura T. Kimata K. J. Biol. Chem. 1992; 267: 1265-1270Abstract Full Text PDF PubMed Google Scholar, 18Kurita K. Shinomura T. Ujita M. Zako M. Kida D. Iwata H. Kimata K. Biochem. J. 1996; 318: 909-914Crossref PubMed Scopus (24) Google Scholar, 19Johnson J. Rosenberg L. Choi H.U. Garza S. Höök M. Neame P. J. Biol. Chem. 1997; 272: 18709-18717Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) and mimecan/osteoglycin (20Funderburgh J.L. Corpuz L.M. Roth M.R. Funderburgh M.L. Tasheva E.S. Conrad G.W. J. Biol. Chem. 1997; 272: 28089-28095Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar), which exhibit only ∼40% protein sequence identity, are the two members of this class. These proteoglycans can be distinguished by a unique cysteine-rich region consensus (CX 2CXCX 6C) and by the presence of only six LRRs. In addition, they are encoded by a gene containing seven exons, and the LRRs are encoded by only three exons (exons V–VII). Epiphycan contains either chondroitin sulfate or dermatan sulfate and can be secreted as a glycoprotein. In cornea, mimecan is a keratan sulfate proteoglycan (20Funderburgh J.L. Corpuz L.M. Roth M.R. Funderburgh M.L. Tasheva E.S. Conrad G.W. J. Biol. Chem. 1997; 272: 28089-28095Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar) with multiple transcripts generated by alternative polyadenylation and differential splicing (21Tasheva E.S. Corpuz L.M. Funderburgh J.L. Conrad G.W. J. Biol. Chem. 1997; 272: 32551-32556Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). The common central domain, which can constitute up to ∼80% of the protein moiety, is composed of ∼10-fold repeats (with the exception of class III SLRPs) of a 24-amino acid residue LRR with Asn and Leu residues preferentially in conserved positions (LX 2LXLX 2NX(L/I)). If the consensus for the LRRs is interpreted with less stringency (2Iozzo R.V. Annu. Rev. Biochem. 1998; 67: 609-652Crossref PubMed Scopus (1341) Google Scholar), then there could be two additional LRRs flanking either side of the central LRR domain. The LRR is a structural module used in molecular recognition processes as diverse as cell adhesion, signal transduction, DNA repair, and RNA processing. The crystal structure of the ribonuclease inhibitor, a leucine-rich protein with structural homology to decorin, defines a new class of α/β protein folds (22Kobe B. Deisenhofer J. Nature. 1993; 366: 751-756Crossref PubMed Scopus (545) Google Scholar). The non-globular shape of the molecule and the exposed face of the parallel β-sheet could explain why LRRs are used to achieve strong protein/protein interactions. Molecular modeling of decorin (Fig.2 A) has revealed a more open structure than the ribonuclease inhibitor (23Weber I.T. Harrison R.W. Iozzo R.V. J. Biol. Chem. 1996; 271: 31767-31770Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar). The overall dimensions of the arch-shaped decorin, which are similar to those obtained with rotary shadowed electron microscopy (24Scott J.E. Biochemistry. 1996; 35: 8795-8799Crossref PubMed Scopus (215) Google Scholar), allow the interaction with a single triple helix of collagen. The open configuration of decorin allows an extensive binding area and thus the formation of several favorable contact points with biological ligands such as the triple helix of collagen (Fig. 2 A) or the EGF receptor (see below). The overall structure provides a flexible platform that can adjust to specific requirements of a particular interacting molecule. The modeling (25Kajava A.V. J. Mol. Biol. 1998; 277: 519-527Crossref PubMed Scopus (449) Google Scholar) further shows that it is feasible to build horseshoe structures for all the members of the LRR superfamily, including the bacterial proteins with the shortest 20-residue LRRs. Indeed, the superfamily of LRR proteins has been recently divided into six subfamilies typified by distinct lengths (20–29 residues) and consensus sequences (25Kajava A.V. J. Mol. Biol. 1998; 277: 519-527Crossref PubMed Scopus (449) Google Scholar). LRRs from different subfamilies never occur concomitantly within a given LRR protein. Structural modeling provides an explanation for this mutually exclusive relationship; the orientation of the variable non-β structural parts of the LRRs is unique to each subfamily and cannot pack together well, whereas the packing of LRRs from one subfamily allows the formation of a specific hydrogen bond network between neighboring LRRs. Thus, it is likely that other members of the SLRP family would fold in a fashion similar to decorin with β-strands and α-helices parallel to a common axis. Conformational flexibility could be achieved, perhaps, by varying the angle of the protein, which may be more or less open as recently proposed (4Neame P.J. Kay C.J. Iozzo R.V. Proteoglycans: Structure, Biology and Molecular Interactions. Marcel Dekker, Inc., New York, NY1999Google Scholar), or by altering specific amino acid sequences that bestow functional specificity. For example, decorin and biglycan are 57% identical but also 43% different at the protein level! From various studies, it can be concluded that several independent evolutionary paths (for example, note the different genomic organization visà vis the LRR in Fig. 1) converged to produce a similar superhelical fold (26Iozzo R.V. Murdoch A.D. FASEB J. 1996; 10: 598-614Crossref PubMed Scopus (550) Google Scholar). Thus, proteins with LRRs provide a unique solution for a multiplicity of functional activities, and their structural properties appear to be the principal reason for their effectiveness as protein binding motifs (4Neame P.J. Kay C.J. Iozzo R.V. Proteoglycans: Structure, Biology and Molecular Interactions. Marcel Dekker, Inc., New York, NY1999Google Scholar, 22Kobe B. Deisenhofer J. Nature. 1993; 366: 751-756Crossref PubMed Scopus (545) Google Scholar). The evidence favoring protein/protein interactions for the SLRP gene members is overwhelming. It is through these non-covalent, and presumably reversible, binding events that connective tissues are properly assembled. Several SLRPs bind fibrillar collagens including types I, II, III, V, VI, and XIV and inhibit fibril formation in vitro. Although it is clear from fibril-reconstitution experiments that the main information to build periodic fibrils resides in the amino acid sequence of the collagen, several macromolecules can regulate this complex process (27Kadler K.E. Holmes D.F. Trotter J.A. Chapman J.A. Biochem. J. 1996; 316: 1-11Crossref PubMed Scopus (1087) Google Scholar). The kinetics of assembly and the ultimate fibril diameter are modulated by these factors, and both acceleration and inhibition of fibril formation have been reported. In the SLRP case, the overall effects of this interaction include an initial delayed assembly and a final reduction in the average fibril diameter (28Vogel K.G. Trotter J.A. Collagen Relat. Res. 1987; 7: 105-114Crossref PubMed Scopus (277) Google Scholar). Removal of the GAG chain or the N-terminal 17-amino acid residues of the decorin protein does not affect the ability of decorin to inhibit fibrillogenesis (29Vogel K.G. Koob T.J. Fisher L.W. Biochem. Biophys. Res. Commun. 1987; 148: 658-663Crossref PubMed Scopus (57) Google Scholar). However, reduction of disulfide bonds abolishes this interaction, whereas renaturation after exposure to dissociative solvents fails to restore all of the effects of decorin on fibrillogenesis (30Ramamurthy P. Hocking A.M. McQuillan D.J. J. Biol. Chem. 1996; 271: 19578-19584Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Thus, the collagen-regulating activity is mediated by the protein core, likely via the central LRR4–6 (31Svensson L. Heinegård D. Oldberg Å. J. Biol. Chem. 1995; 270: 20712-20716Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 32Schönherr E. Hausser H. Beavan L. Kresse H. J. Biol. Chem. 1995; 270: 8877-8883Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 33Kresse H. Liszio C. Schönherr E. Fisher L.W. J. Biol. Chem. 1997; 272: 18404-18410Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), whereas the GAG chains maintain interfibrillar space by extending outward from the protein core. In the case of fibromodulin, inhibition of fibrillogenesis requires more than one binding site including the C-terminal end of the molecule (34Font B. Eichenberger D. Goldschmidt D. Boutillon M.-M. Hulmes D.J.S. Eur. J. Biochem. 1998; 254: 580-587Crossref PubMed Scopus (50) Google Scholar), in agreement with the proposed model for decorin-collagen interaction (23Weber I.T. Harrison R.W. Iozzo R.V. J. Biol. Chem. 1996; 271: 31767-31770Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar). This connective tissue “cooperation” is evolutionarily conserved and physiologically relevant as exemplified by the diverse phenotypes of knockout animals in which specific class I and II SLRP genes have been disrupted by gene targeting. Decorin null animals show an abnormal skin fragility phenotype caused by a reduced tensile strength (35Danielson K.G. Baribault H. Holmes D.F. Graham H. Kadler K.E. Iozzo R.V. J. Cell Biol. 1997; 136: 729-743Crossref PubMed Scopus (1179) Google Scholar). Close analysis of the dermal collagen provides a structural basis for the skin fragility; the collagen fiber network of the null animals is more loosely packed and exhibits irregular collagen contours (Fig.2 B). This is confirmed by mass mapping of isolated collagen fibrils, which show a pronounced non-uniformity in their axial mass distribution. Thus, skin fragility in these mutant animals could be ascribed to this anomalous collagen network, which could allow for full body development but would lead to a reduced tensile strength with potential complications such as an increased incidence of injury and an abnormal healing process. Targeted disruption of the biglycan gene leads to an osteoporosis-like phenotype (36Xu T. Bianco P. Fisher L.W. Longenecker G. Smith E. Goldstein S. Bonadio J. Boskey A. Heegaard A.-M. Sommer B. Satomura K. Dominguez P. Zhao C. Kulkarni A.B. Robey P.G. Young M.F. Nat. Genet. 1998; 20: 78-82Crossref PubMed Scopus (389) Google Scholar) consistent with the different tissue distribution and collagen binding ability of biglycan. The biglycan null animals show reduced bone mass detectable at 3 months of age that becomes more pronounced with aging. Thus, biglycan acts as a positive regulator of bone formation and bone mass by affecting the cellular processes of bone formation that occur during both development and adult life. Interestingly, mice lacking fibromodulin exhibit an abnormal tendon phenotype (37Svensson L. Aszódi A. Reinholt F.P. Fässler R. Heinegård D. Oldberg Å. J. Biol. Chem. 1999; 274: 9636-9647Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar). In contrast to the decorin null mice, the fibromodulin-deficient animals have collagen fibrils thinner than the wild-type animals as a result of a predominance of a very thin fibril population in an overall similar range of fibril diameters. A significant increase of lumican in connective tissues of the fibromodulin-deficient animals suggests that a coordinate transcriptional or post-transcriptional control for certain SLRP members may be operational in vivo. Disruption of the lumican gene also causes a skin fragility phenotype. In addition, the lumican-deficient animals develop bilateral corneal opacity (38Chakravarti S. Magnuson T. Lass J.H. Jepsen K.J. LaMantia C. Carroll H. J. Cell Biol. 1998; 141: 1277-1286Crossref PubMed Scopus (577) Google Scholar). The structural is of the decorin null animals in that collagen fibrils are However, the lumican null animals show abnormal collagen formation not only in the but also in the The presence of multiple SLRPs in the explain why has been in mice in decorin, or It is that decorin and biglycan not play a significant in corneal transparency because the binding of dermatan sulfate SLRPs and at the and of collagen, in contrast to the keratan sulfate SLRPs and that bind to the a and of collagen J.E. J. 1995; Google Scholar). 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These in binding with the that expression of decorin leads to growth and in and properties of Y. Mann Ruoslahti E. Nature. PubMed Scopus Google Scholar). of decorin growth or inhibition of that the of decorin is the of These initial have been in an model of in which are with in causes a the of matrix in the and matrix and the fibrosis leads to and decorin in T. J.R. Y. Ruoslahti E. Nature. 1992; PubMed Scopus Google Scholar), a pathological process that can be by gene decorin into the of animals Y. K. Y. E. Nat. 1996; PubMed Scopus Google Scholar). The of decorin for several and decorin is increased in and of Y. K. Y. E. Nat. 1996; PubMed Scopus Google Scholar). This provides strong evidence that SLRPs as for essentially an and for other of fibrosis such as those affecting the and the function of SLRPs is an ability to affect cellular For example, expression of decorin the growth of a of The growth is with an of a potent inhibitor of cyclin-dependent kinase activity M. T. B. 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Matrix Biol. 1996; 15: PubMed Scopus Google Scholar). decorin and other SLRP members are with fibrillar collagen, a complex in which interactions in an be in decorin in the could functional interaction with the which in a that the cell In this it is that a knockout of decorin and a shows a between these two genes and an acceleration of R.V. D. McQuillan D.J. T. B. Eichstetter I. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar). lacking both genes show a of development and to within This result that the of decorin is for in a model to cancer and suggests that in decorin and may in the of and lead to a more phenotype. is to be about the biology of the New members are cloned and and additional and are and are at various Although the of SLRP gene knockout mice has the of members in various of connective tissue it has also revealed new of that more of gene promoter may also explain of the and signal in mutant include of the key events and unique through which SLRP members their specific biological that the of the mutant protein with more activities, and that could growth factor activities or collagen binding properties are of the various SLRP proteins to fibrosis or cancer may not be far in the The is I to I. C. and S. for and C. C. and J. L. Funderburgh for of this