Decorin-Type I Collagen Interaction

Abstract

Interactions between the core protein of the small dermatan sulfate proteoglycan decorin and type I collagen have been considered to influence the kinetics of collagen fibrillogenesis and the diameter of and the distance between the fibrils. A variety of recombinant core protein fragments were expressed in Escherichia coli, extracted from inclusion bodies, and renatured in the presence of bovine serum albumin, which was essential for obtaining functional activity. A recombinant protein lacking the first 14 amino acids of the mature core protein (P15-329) interacted with reconstituted type I collagen fibrils and inhibited collagen fibrillogenesis almost as efficiently as intact decorin purified from fibroblast secretions under non-denaturing conditions. Peptides comprising amino acids 15-183 (P15-183) and 185-329 (P185-329) were able to compete for the binding of wild-type decorin, with P15-183 being more active than P185-329. Several other peptides were much less effective. Binding studies using radioactively labeled peptides P15-183 and P185-329 gave direct evidence for the independent binding of both peptides. Peptides 15-183 and 15-125 had the capability of inhibiting collagen fibrillogenesis, whereas peptide 185-329 was inactive. These data suggest (i) that there are at least two separate binding domains for the interaction between decorin core protein and type I collagen and (ii) that binding is not necessarily correlated with an alteration of collagen fibrillogenesis. Interactions between the core protein of the small dermatan sulfate proteoglycan decorin and type I collagen have been considered to influence the kinetics of collagen fibrillogenesis and the diameter of and the distance between the fibrils. A variety of recombinant core protein fragments were expressed in Escherichia coli, extracted from inclusion bodies, and renatured in the presence of bovine serum albumin, which was essential for obtaining functional activity. A recombinant protein lacking the first 14 amino acids of the mature core protein (P15-329) interacted with reconstituted type I collagen fibrils and inhibited collagen fibrillogenesis almost as efficiently as intact decorin purified from fibroblast secretions under non-denaturing conditions. Peptides comprising amino acids 15-183 (P15-183) and 185-329 (P185-329) were able to compete for the binding of wild-type decorin, with P15-183 being more active than P185-329. Several other peptides were much less effective. Binding studies using radioactively labeled peptides P15-183 and P185-329 gave direct evidence for the independent binding of both peptides. Peptides 15-183 and 15-125 had the capability of inhibiting collagen fibrillogenesis, whereas peptide 185-329 was inactive. These data suggest (i) that there are at least two separate binding domains for the interaction between decorin core protein and type I collagen and (ii) that binding is not necessarily correlated with an alteration of collagen fibrillogenesis. Decorin, a small dermatan sulfate proteoglycan, is a ubiquitous component of extracellular matrices (1Voss B. Glössl J. Cully Z. Kresse H. J. Histochem. Cytochem. 1986; 34: 1013-1019Crossref PubMed Scopus (61) Google Scholar, 2Bianco P. Fisher L.W. Young M.F. Termine D.J. Gehron Robey P. J. Histochem. Cytochem. 1990; 38: 1549-1563Crossref PubMed Scopus (545) Google Scholar) where it is preferentially found in association with collagen fibrils (3Scott J.E. Orford C.R. Biochem. J. 1981; 197: 213-216Crossref PubMed Scopus (332) Google Scholar, 4Van Kuppevelt T.H.M.S.M. Rutten T.L.M. Kuyper C.M.A. Histochem. J. 1987; 19: 520-526Crossref PubMed Scopus (47) Google Scholar, 5Scott J.E. Biochem. J. 1988; 252: 313-323Crossref PubMed Scopus (548) Google Scholar, 6Pringle G.A. Dodd C.M. J. Histochem. Cytochem. 1990; 38: 1405-1411Crossref PubMed Scopus (130) Google Scholar, 7Fleischmajer R. Fisher L.W. MacDonald E.D. Jacobs Jr., L. Perlish J.S. Termine J.D. J. Struct. Biol. 1991; 106: 82-90Crossref PubMed Scopus (151) Google Scholar). Evidence has been provided that triple helical type I collagen possesses a specific decorin core protein-binding site at the d-band in each D period (5Scott J.E. Biochem. J. 1988; 252: 313-323Crossref PubMed Scopus (548) Google Scholar, 6Pringle G.A. Dodd C.M. J. Histochem. Cytochem. 1990; 38: 1405-1411Crossref PubMed Scopus (130) Google Scholar, 8Uldbjerg N. Danielsen C.C. Biochem. J. 1988; 251: 643-648Crossref PubMed Scopus (70) Google Scholar). Some additional interactions may be mediated by the dermatan sulfate side chain (9Hedbom E. Heineg D. J. Biol. Chem. 1993; 268: 27307-27312Abstract Full Text PDF PubMed Google Scholar). As decorin binds to the surface of collagen fibrils, the lateral assembly of individual triple helical collagen molecules is delayed (10Vogel K.G. Paulsson M. Heineg D. Biochem. J. 1984; 223: 587-597Crossref PubMed Scopus (704) Google Scholar, 11Rosenberg L. Tang L.H. Choi H.U. Johnson T. Scott J.E. Dermatan Sulphate Proteoglycans. Portland Press, London1993: 225-239Google Scholar), and the diameter of the fibrils is decreased (12Vogel K.G. Trotter J.A. Collagen Relat. Res. 1987; 7: 105-114Crossref PubMed Scopus (278) Google Scholar). However, only moderately low dissociation constants on the order of 10−8 M have been reported for the complex between type I collagen and decorin (13Brown D.C. Vogel K.G. Matrix. 1989; 9: 468-478Crossref PubMed Scopus (158) Google Scholar, 14Hedbom E. Heineg D. J. Biol. Chem. 1989; 264: 6898-6905Abstract Full Text PDF PubMed Google Scholar), and in vitro studies suggested a preferential binding of decorin to type VI collagen (15Bidanset D.J. Guidry C. Rosenberg L.C. Choi H.U. Timpl R. Hook M. J. Biol. Chem. 1992; 267: 5250-5256Abstract Full Text PDF PubMed Google Scholar). Nevertheless, mediating molecules like type VI collagen are not required to interpret the data on the interaction between decorin and fibrillar collagens (9Hedbom E. Heineg D. J. Biol. Chem. 1993; 268: 27307-27312Abstract Full Text PDF PubMed Google Scholar). In addition to collagen binding, the core protein of decorin interacts with other components of the extracellular matrix (16Schmidt G. Robenek H. Harrach B. Glössl J. Nolte V. Hörmann H. Richter H. Kresse H. J. Cell Biol. 1987; 104: 1683-1691Crossref PubMed Scopus (130) Google Scholar, 17Lewandowska K. Choi H.U. Rosenberg L.C. Zardi L. Culp L.A. J. Cell Biol. 1987; 105: 1443-1454Crossref PubMed Scopus (147) Google Scholar, 18Winnemöller M. Schön P. Vischer P. Kresse H. Eur. J. Cell Biol. 1992; 59: 47-55PubMed Google Scholar), with transforming growth factor-β (19Yamaguchi Y. Mann D.M. Ruoslahti E. Nature. 1990; 346: 281-284Crossref PubMed Scopus (1299) Google Scholar), and with a receptor required for its endocytosis (20Glössl J. Schubert-Prinz R. Gregory J.D. Damle S.P. von Figura K. Kresse H. Biochem. J. 1983; 215: 295-301Crossref PubMed Scopus (32) Google Scholar). Decorin belongs to a proteoglycan family that is characterized by core proteins of about 40 kDa that contain 10-12 homologous, leucine-rich repeats of about 25 amino acids in their central sequence and that have disulfide loops located at conserved positions near the N and C termini (21Krusius T. Ruoslahti E. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 7683-7687Crossref PubMed Scopus (415) Google Scholar, 22Day A.A. Ramis C.I. Fisher L.W. Gehron Robey P. Termine J.D. Young M.F. Nucleic Acids Res. 1986; 14: 9861-9876Crossref PubMed Scopus (26) Google Scholar). The single glycosaminoglycan chain of decorin is linked with serine 4 of the mature core protein (23Chopra R.K. Pearson C.H. Pringle G.A. Fackre D.S. Scott P.G. Biochem. J. 1985; 232: 277-279Crossref PubMed Scopus (97) Google Scholar). Previously, it had been attempted to characterize the collagen-binding domain of decorin in detail. Chemical and enzymatic degradation studies indicated that the 17-amino acid N-terminal peptide including the glycosaminoglycan chain was not required for the interaction of the proteoglycan with a collagen matrix (24Vogel K.G. Koob T.J. Fisher L.W. Biochem. Biophys. Res. Commun. 1987; 148: 658-663Crossref PubMed Scopus (57) Google Scholar), but the inhibition of fibrillogenesis was abolished by a disulfide bond reduction (25Scott P.G. Winterbottom N. Dodd C.M. Edwards E. Pearson C.H. Biochem. Biophys. Res. Commun. 1986; 138: 1348-1354Crossref PubMed Scopus (65) Google Scholar). Further studies came to the conclusion that neither the N-terminal half nor the central leucine-rich repeats of the core protein can, by itself, interact fully with fibrillar collagen (26Pogány G. Vogel K.G. Biochem. Biophys. Res. Commun. 1992; 189: 165-172Crossref PubMed Scopus (38) Google Scholar). In the present study we will describe that at least two domains of decorin core protein are able to bind to fibrillar collagen and that binding does not necessarily correlate with an inhibition of fibrillogenesis. Reference (wild-type) decorin was purified to about 95% purity from the secretions of cultured human skin fibroblasts as described (27Hausser H. Hoppe W. Rauch U. Kresse H. Biochem. J. 1989; 263: 137-142Crossref PubMed Scopus (42) Google Scholar). Briefly, the preparation included sequential anion exchange chromatographies in the presence of protease inhibitors on a DEAE-Trisacryl M column (Serva, Heidelberg, Germany) and on a Bio-Gel TSK DEAE-5 PW high pressure liquid chromatography column (Bio-Rad). Use of urea and of other denaturing agents was avoided. [35S]Sulfate-labeled decorin was prepared analogously after incubating the cells for 72 h in the presence of 1.48 MBq/ml [35S]sulfate (carrier-free) (Amersham-Buchler, Braunschweig, Germany) and 4% (v/v) fetal calf serum that had been dialyzed against 0.15 M NaCl. Clone D6, which contains the coding region of human decorin core protein, has been described previously (28Beavan L.A. Quentin-Hoffmann E. Schönherr E. Snigula F. Leroy J.G. Kresse H. J. Biol. Chem. 1993; 268: 9856-9862Abstract Full Text PDF PubMed Google Scholar). In the context of the present investigation, it is noteworthy that this clone contains a HpaI site in the untranslated downstream region (position 1383) that had not been described in the original publication of decorin cDNA (21Krusius T. Ruoslahti E. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 7683-7687Crossref PubMed Scopus (415) Google Scholar) but has been found more recently (29Danielson 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 clone was used to prepare the following recombinant peptides (see Table I and Fig. 1). P15-329 was cloned into the BamHI/ EcoRI restriction site of the expression vector pRSET A (Invitrogen, San Diego, CA). P15-125 was obtained as follows. An EcoRI/ AlwNI fragment of the cDNA was made blunt ended at the AlwNI site and cloned into the EcoRI/ BamHI site of pGEM-4Z (Promega, Heidelberg), which was propagated in Escherichia coli strain DH5α. This procedure generated a stop codon 3′ of leucine 125. The plasmid was digested with BstUI and HindIII and subcloned into the BamHI/ HindIII site of pRSET A. To obtain the P15-183 construct, the cDNA was treated with EcoRI and BsmI, and additionally with HpaI, to distinguish between the two EcoRI sites 5′ and 3′ of the BsmI site. The EcoRI/ BsmI fragment was cloned into the EcoRI/ KpnI site of pGEM-4Z, thus generating the codons for Arg-Tyr-Pro-Gly-Ile-Leu-Stop 3′ of threonine 183, and subcloned into pRSET A as described for P15-125. A cDNA for P33-160 was obtained by digesting the cDNA with EcoRI/ DdeI. Further subcloning was as described for P15-125 except that BstUI was replaced by TaqI for digestion of the pGEM-4Z cloning intermediate. P125-230 was obtained as follows. A HincII digest of the cDNA was cloned into the BamHI site of pGEM-4Z. The orientation of the insert was determined with HinfI before the vector was digested with AlwNI/ HindIII. This fragment was then ligated into the BamHI/ HindIII site of pRSET C (Invitrogen), thereby creating a leucine and a stop codon 3′ of the valine 230 codon. P154-329 was obtained analogously like P15-329, except that the cDNA was treated with BanI/ EcoRI and subcloned into pRSET B (Invitrogen). For the generation of P185-329 a BsmI/ EcoRI fragment of the cDNA was treated with AlwNI for removal of the 5′ sequence and subcloned into pRSET A as in the case of P15-329.Table I:Recombinant decorin peptidesFIG. 1Schematic structure of mature human decorin core protein and restriction sites of decorin cDNA. In A, the consensus sequence Leu-Xaa-Xaa-Leu-Xaa-Leu-Xaa-Xaa-Asn-Xaa-(Leu/Ile)-(Ser/Thr)-Xaa-(Val/Ile) is boxed. Numerals above the boxes give the degree of conservation of the 7 specified residues without (first numeral) or with (second numeral) taking into account conservative exchanges. Small numerals indicate the number of amino acids between the boxed units and the position of the first amino acid of these units, respectively. The glycosaminoglycan attachment site is indicated by a large arrow, and cysteine residues are shown by smaller arrows. In B, the cDNA is related to the exon boundaries. The restriction sites are indicated by dashed lines.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The three pRSET vectors contain a T7 RNA polymerase promoter, a hexahistidine metal-binding domain, and an enterokinase cleavage site at the 5′ end of the polycloning site. The plasmids were used to express recombinant proteins in E. coli strain INVαF′ (Invitrogen) as described (30Schönherr E. Witsch-Prehm P. Harrach B. Robenek H. Rauterberg J. Kresse H. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). were then and treated with of M M and M of original For of a acid Germany) were into a column and with M urea and M in M proteins were the column by in two The column was by using of A the was were Further were by using M M in M the was was by of M M and in M The with the with B to an of about made with used serum and dialyzed for h at against the of M M M in M against proteins were by and of the were and reconstituted with of by bovine serum much smaller of were to the urea was to a of and the and was but this in the presence of the of labeled proteins were obtained after with [35S]sulfate as described (30Schönherr E. Witsch-Prehm P. Harrach B. Robenek H. Rauterberg J. Kresse H. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). type I collagen from calf skin Germany) was in M acid by of M M and for at The fibrils were by in of in of and by In recombinant peptides were in a decorin was with of reconstituted collagen fibrils and the peptides in to give a of and a of were without h at under the was by for and with decorin was after the fibrils with of M were in or recombinant peptides were was from the protein of prepared in Collagen was prepared from bovine and collagen fibrillogenesis was as described (10Vogel K.G. Paulsson M. Heineg D. Biochem. J. 1984; 223: 587-597Crossref PubMed Scopus (704) Google Scholar). of collagen and decorin or decorin peptides in a of were made on and to The at was in by was as described (27Hausser H. Hoppe W. Rauch U. Kresse H. Biochem. J. 1989; 263: 137-142Crossref PubMed Scopus (42) Google Scholar). The against human decorin P. R. Kresse H. 1992; PubMed Scopus Google Scholar) was found to with recombinant peptides used in this but the number of of the individual peptides was not For the of recombinant of and were on a and to were with in and in were with of 25 M and M in and at A variety of recombinant decorin peptides were expressed as proteins in E. coli strain These an preparation to more than 95% purity by a single as by of the peptides prepared in the of to peptides expressed in had with M urea not be purified in a single by chromatography on or of proteins with 4 M on H.U. Johnson S. Tang Rosenberg L. J. Biol. Chem. 1989; 264: Full Text PDF PubMed Google Scholar) was by low of P15-329 from of of in the presence of about of peptide a of in separate were obtained in was determined after and of the with the denaturing 4 M had to be used to the peptide from inclusion bodies, the following for were P15-329 after reduction and that on the of disulfide As functional the inhibition of binding of wild-type decorin to type I collagen fibrils and to the endocytosis receptor was required the presence of of only were included in the the to compete with wild-type decorin for binding to collagen fibrils. However, to an active after with M urea provided that of were present was considered to as a and to the of The that is not a for the of had recently been reported that decorin core protein, active with to transforming growth factor-β binding, be obtained as a protein with E. coli protein A. M. Heineg D. Ruoslahti E. Biochem. J. PubMed Scopus Google Scholar). Use of this a protein the of the wild-type core of for of recombinant decorin P15-329 was the procedure in the presence of of 40 of the peptides were used in A as a of wild-type decorin In the of of the to In B the as in A was treated with M urea in B and renatured in the presence of of to collagen binding, as in A. The indicate the of three the procedure and in Large Image Figure ViewerDownload Hi-res image Download (PPT) A between wild-type decorin and P15-329 for the inhibition of binding of decorin to collagen indicated that the required for inhibition were of the order of the required for the recombinant peptide were In the of P15-329 was and than that of wild-type The specific between P15-329 and decorin for collagen binding be shown by and of using a is shown in Fig. that P15-329 does not in the of collagen fibrils and that there is between the two for In a of the influence of the recombinant peptide and of decorin on the kinetics of was fibrillogenesis, decorin has to interact with the fibrils, and there be in the of is in Fig. A that P15-329 is as as decorin in the of the which a of decorin and P15-329 on the diameter reduction of the collagen fibrils. A was in the presence of The procedure described above was used to a variety of recombinant decorin peptides that were to and as as a central region 1). of the was to be and to be as a it a single cysteine the of which the central other peptides a number of cysteine residues and were to be of disulfide In of this these peptides reduction and the peptides were used in a the were obtained for the P15-329, and for which contains the first located leucine-rich repeats in addition to the N-terminal region and Fig. A N-terminal required at least three for inhibition independent two located P154-329 and and leucine-rich were able to with collagen binding of wild-type decorin, but the located peptide P125-230 was Nevertheless, this peptide functional it was in with the endocytosis of H. L. and H. Table of I collagen interaction by recombinant decorin of binding of wild-type decorin to collagen fibrils by recombinant peptides was as described in the of Fig. of binding of wild-type decorin to collagen fibrils by the recombinant peptides P15-183 and P185-329. data of two separate are of the was Large Image Figure ViewerDownload Hi-res image Download (PPT) of binding of wild-type decorin to collagen fibrils by recombinant peptides was as described in the of Fig. The with decorin binding to collagen by two recombinant peptides to prepare P15-183 and P185-329 in a radioactively labeled for in Fig. that the binding of P185-329 to collagen fibrils be inhibited by the peptide and by the peptide inhibition only be by The is less for the inhibition of binding of the peptide and P15-183 were the P185-329 to a P125-230 were able to with This indicate that the two peptides a domain with the P15-183 binding with the binding of P185-329 by the binding of P15-183 to an whereas the does not is a for the that P15-183 inhibited the binding of decorin to whereas the peptide was not an of the interaction between P185-329 and In the presence of an interaction of decorin its collagen-binding domain be in the of The radioactively labeled recombinant peptides were used for the of dissociation The of P15-329 was an order of smaller than the for both P15-183 and P185-329 Several of the recombinant decorin peptides were for their influence on collagen fibrillogenesis. is shown in Fig. B that P15-183 was as as P15-329 and wild-type decorin in inhibiting the of fibrils. The collagen-binding peptide was recombinant peptides had an influence on the of the fibrillogenesis. is noteworthy that P15-125 is for with its to compete with decorin for collagen binding is only These were by the of at least two peptide the that the reduction of disulfide of decorin its capability to fibrillogenesis (25Scott P.G. Winterbottom N. Dodd C.M. Edwards E. Pearson C.H. Biochem. Biophys. Res. Commun. 1986; 138: 1348-1354Crossref PubMed Scopus (65) Google Scholar), it is to that the N-terminal region of decorin, which contains 4 cysteine residues and is present in P15-183 and in is of for this In this recombinant decorin core protein fragments were for their interaction with type I the peptides had to be extracted from inclusion by a denaturing and had to be This was only was present as a protein the The renatured protein P15-329 a not the collagen binding as wild-type A for the of the procedure is the that a protein that not bind to collagen be to a of with after additional with provided that the for were The presence of large of the of like of for an of the of decorin a in to the for intact decorin P.G. Dodd C.M. Res. 1990; PubMed Scopus Google Scholar). These data were as an of but it was that decorin its to bind to collagen fibrils and to collagen fibrillogenesis in An of the of protein fragments is the that of an be to the of the active structure the sequence or to In addition to on the peptide P15-329, we studies on the peptides and P125-230 was almost with to collagen However, other than the it was the of decorin of the peptides were found to be active in at least The of the core protein by the recombinant peptide was considered to at least a site for the A of the domains of decorin core protein is the of high binding sites to collagen were for P15-183 and P185-329. P15-125 was active in the inhibition of collagen fibrillogenesis, whereas were required to compete with decorin binding to collagen fibrils with This suggest that P15-125 contains a binding site. The of a low binding site has been previously (13Brown D.C. Vogel K.G. Matrix. 1989; 9: 468-478Crossref PubMed Scopus (158) Google Scholar). A at the conclusion that decorin core protein contains at least two functional domains that are in the interaction with the J.A. J. K. Biol. Scholar). The of two high binding sites may be to the determined by of the small core protein M. Paulsson M. A. Heineg D. J. Biol. Chem. 1989; 264: Full Text PDF PubMed Google Scholar). However, the structure of like decorin, is a protein with leucine-rich that this protein has the of a and a of protein B. J. Nature. 1993; PubMed Scopus Google Scholar). that decorin core proteins an the data in this study are with the that the surface by the of the leucine-rich which are not P15-125 (see Fig. and a from the half of the are in the binding to collagen fibrils. The N-terminal of the with its two disulfide is both P15-125 and P15-183 and is considered to be in fibrillogenesis. Further are to the in detail. are to M. for