©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Decorin-binding Sites for Collagen Type I Are Mainly Located in Leucine-rich Repeats 45 (*)

(Received for publication, March 13, 1995; and in revised form, May 26, 1995)

Liz Svensson Dick Heineg Oldberg (§)

From the Department of Cell and Molecular Biology, University of Lund, P. O. Box 94, S-221 00 Lund, Sweden

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Decorin and biglycan are structurally related interstitial proteoglycans synthesized in connective tissues like skin, tendon, and cartilage. Despite the conspicuous sequence similarities, where about 55% of the amino acid residues in decorin and biglycan are located in identical positions, the two proteoglycans show differences in their interaction with collagen. Decorin binds to collagen type I, whereas biglycan in several assay systems shows no affinity for this collagen type. Here we have made use of these structural similarities and affinity differences in studies of the collagen binding properties of decorin. Recombinant biglycan/decorin chimeras were produced in mammalian cells and analyzed for their capacity to bind collagen. In the chimeras, biglycan contributes sequences crucial for synthesis and export from the mammalian cells, and decorin provides potential collagen-binding properties. By using this approach we show that decorin binds to the collagen primarily via leucine-rich repeats 4-5 composed of some 40 amino acid residues. Proteoglycan chimeras containing decorin sequences from the N terminus to leucine-rich repeat 3 or sequences from leucine-rich repeat 6 to the C terminus do not show any detectable binding to collagen. A proteoglycan chimera containing decorin leucine-rich repeats 4-5 flanked by biglycan sequences binds to collagen. However, this chimera binds to collagen with somewhat lower affinity than wild type decorin, suggesting that additional low affinity binding sites may be located in other parts of decorin. Alternatively, the conformation of the collagen binding leucine-rich repeats 4-5 are different in decorin and in the biglycan/decorin chimera, leading to a lower collagen affinity for the latter.


INTRODUCTION

Decorin (1) belongs to a family of structurally related, extracellular matrix proteoglycans and glycoproteins which also include biglycan(2) , fibromodulin(3) , lumican(4) , and chondroadherin(5) . The primary structures of these proteins are homologous and can be divided into three domains. The N-terminal domain shows the least homology between the proteins and contains four cysteine residues of which two are known to be involved in intrachain disulfide bonds(6) . This domain is substituted with glycosaminoglycan chains in decorin and biglycan or tyrosine sulfate residues in fibromodulin and probably lumican(4, 7) . Chondroadherin essentially lacks this domain. The substitutions give this part of the core proteins distinct polyanionic properties. The C-terminal domain contains two cystein residues which form a loop stabilized by an intrachain disulfide bond. The sequence homologies are most pronounced in the central domain which constitutes 70-80% of the core protein and is composed of some 10 repeats of about 25 residues with preferentially leucine residues in conserved positions. Similar leucine-rich repeats (LRR) (^1)exist in a large number of proteins and have in some cases been implicated in protein-protein interactions. An intracellular ribonuclease inhibitor has been demonstrated to bind and inactivate ribonuclease via specific regions in such a leucine-rich repeat domain(8) . The three-dimensional structure of the ribonuclease inhibitor has been determined by x-ray crystallography(8) . The repeats are composed of alternating alpha-helices and beta-sheets, which are arranged in a coil structure. The alpha-helices and beta-sheets are parallel and stabilize the coil structure via lateral interactions. The leucine-rich repeats in the extracellular matrix proteoglycans, although shorter, are homologous to those of the ribonuclease inhibitor. Therefore, the proteins may have a similar three-dimensional structure.

Decorin and fibromodulin bind to fibrillar collagen type I and II (9, 10, 11) . Both glycoproteins delay fibril formation in vitro and also appear to affect the dimensions of the fibrils. Interestingly, decorin and fibromodulin bind to different sites in the collagen type I fibril, with dissociation constants of 16 and 35 nM, respectively(11) . In contrast, biglycan does not bind to collagen type I or II, despite the sequence similarities (10) . Immunostaining of tissues show, as can be expected from the binding specificities, that decorin also in vivo is associated with collagen fibers in the interstitial matrix, whereas biglycan is located in structures around cells(12) .

In this report we have used proteoglycan chimeras composed of biglycan and decorin to characterize the decorin binding to collagen.


MATERIALS AND METHODS

Human biglycan (2) and bovine decorin (13) cDNA in pKS II (Stratagene, La Jolla, CA) were used to make cDNA's encoding proteoglycan chimeras composed of both decorin and biglycan.

Big3dec was constructed by StyI digestion of biglycan cDNA, which cleaves at nucleotide 653 in the biglycan sequence. Decorin cDNA was obtained by PCR, using the primer GCCCAAGGCTGTGTTCAATGGATTGAACC containing a StyI site and the T7 primer GTAATACGACTCACTATAGGGC. The PCR product was digested with StyI and ligated into the pKS II vector containing the biglycan cDNA StyI fragment. Big3dec contains biglycan sequences to amino acid residue 175 and decorin sequences from residue 168. Due to the oligonucleotide primer sequence serine 169 in the decorin sequence is mutated to an alanine residue.

To construct Big5dec cDNA, decorin cDNA in the pKS II vector was used as a template for PCR amplification using a primer GCCCCGGGTTGCTGACACAAATATAACTACC with a SmaI site and the T7 primer. The PCR product was digested with SmaI and XbaI (in the polylinker) and ligated into pKS II. Biglycan cDNA was digested with AviII, which cleaves at nucleotide 769, and KpnI, which cleaves in the polylinker of pKS II. This fragment was then ligated into the pKS II vector containing the decorin fragment, which was opened by digestion with SmaI and KpnI. This construct has a shift in the biglycan amino acid sequence at amino acid residue 213 to decorin at residue 207. Due to the primer sequence isoleucine 208 in the decorin sequence is mutated to a valine residue.

Dec3big was constructed by PCR amplification of the decorin cDNA using a 3` primer CCCAAGCTTGGTGATCTCGTTCTCATG containing a HindIII site together with the T3 primer ATTAACCCTCACTAAAG. The PCR product was digested with HindIII and EcoRI (in the polylinker) and ligated into pGEM 7Z (Promega, Madison, WI). The biglycan fragment was obtained by PCR amplification using a 5` primer CCCAAGCTTCCCAAGGGAGTGTTCAGC with a HindIII site together with the T7 primer. This PCR product was digested with HindIII and BamHI and ligated into the vector containing the Dec3 PCR fragment. This proteoglycan chimera is composed of decorin sequences to amino acid residue 166 and biglycan from residue 175. Due to the primer sequence the valine residue at position 166 in decorin is replaced by a leucine residue.

To construct Big3dec5big cDNA, we used the Big3dec as a PCR template to obtain the 5` part of the cDNA. A primer, CGGGATCCTAGTTATATTTGTGTCAGC, containing a BamHI site was used as a 3`-5` primer together with the T3 primer. The PCR product was digested with BamHI and EcoRI and ligated into the pKS II vector. A PCR fragment of biglycan obtained by using a 5`-3` primer CGGGATCCCCAAAGACCTCCC containing a BamHI site, and the T7 primer was digested with BamHI and ligated into the vector containing the Big3dec PCR fragment. The Big3dec5big proteoglycan is thus composed of decorin, amino acid residues 168-213, flanked by biglycan sequences. Due to the primer sequences serine 169 in the decorin sequence is mutated to an alanine residue, and an arginine residue will replace threonine 222 in the biglycan sequence.

PCR amplifications were performed for 30 cycles in a thermal cycler using Taq DNA polymerase (Boehringer Mannheim, Germany), 95 °C for 30 s, 55 °C for 45 s, and 72 °C for 120 s. The PCR products were subjected to nucleotide sequence analysis (3) to confirm their correct sequence.

The proteoglycan cDNAs were ligated into the Epstein-Barr virus-based eucaryotic expression vector pCEP 4 (Invitrogen, San Diego, CA). This vector replicates extrachromosomally in primate cells and yields large amounts of recombinant protein(14) . The transcription is driven by the cytomegalovirus promoter, and the polyadenylation signal in constructs containing biglycan sequences in the C-terminal region (biglycan, Dec3big, and Big3dec5big) is provided by the SV40 polyadenylation signal in the vector. Constructs containing decorin sequences in the C-terminal domain (decorin, Big3dec, and Big5dec) utilize the endogenous polyadenylation signal present in the decorin cDNA. Constructs in pCEP 4 were used to transform human HeLa cells by electroporation (Bio-Rad gene pulser, 300V, 500 microfarads), and 48 h later Hygromycin (Calbiochem, San Diego, CA) was added (19 units/ml) to the culture medium of Dulbecco's modified Eagle's medium containing 10% fetal calf serum. After about 2 weeks individual clones were picked and analyzed for proteoglycan expression after incubation in medium containing [S]sulfate. Cells were cultured for 10 h in Dulbecco's modified Eagle's medium containing 0.1 mg/ml bovine serum albumin and 0.1 mCi/ml [S]sulfate, the culture medium was collected, and phenylmethylsulfonyl fluoride added as a protease inhibitor.

Chondroitinase ABC digestion of [S]sulfate-labeled culture medium was performed by adding 1/10 volume 1 M Tris acetate, 0.1 M EDTA, pH 7.3, and 0.2 units/ml chondroitinase ABC (Seikagaku Kogyo Co., Tokyo, Japan). After incubation at 37 °C for 6 h, samples were analyzed by SDS-PAGE.

Collagen binding assays were performed by incubating 5 µl of acid-solubilized bovine skin collagen (Vitrogen 100 Collagen, 3 mg/ml, Celtrix Laboratories, Palo Alto, CA) in 0.1 ml of culture medium from cells radiolabeled with [S]sulfate. After 5 h at 37 °C, the sample was centrifuged for 5 min at 10,000 g, the supernatant was removed, and the pellet washed once with phosphate-buffered saline. The pellet and the supernatant were electrophoresed on a SDS 10% polyacrylamide gel(15) , and the amount of [S]proteoglycan in supernatant and collagen precipitates was determined in a Bio Imaging Analyzer (Fuji Photo Film Co., Japan). In this analysis we measure only the S-labeled proteoglycans corresponding in size to decorin/biglycan.

For the inhibition assays proteoglycan chimeras were used to inhibit the binding of S-labeled proteoglycans. Conditioned HeLa cell culture media were concentrated by Centricon-30 (Amicon Inc., Beverly, MA) filtration. Typically serum-free medium, conditioned for 10-12 h, was concentrated 100-fold. To measure the proteoglycan concentration, samples of proteoglycans were purified by DE52 ion-exchange chromatography. Concentrated culture medium (0.1 ml) was applied to a 0.2-ml DE52 column, which was washed with 0.4 ml of 0.2 M NaCl, 4 M urea, 0.05 M sodium acetate, pH 4, and 1 ml of 0.2 M NaCl, 0.05 M sodium acetate, pH 4, and then eluted with 0.6 ml of 1 M NaCl, 0.05 M sodium acetate, pH 4. The concentration of proteoglycan in the eluate was determined according to Bradford (16) using purified calf articular cartilage decorin as standard. As judged by SDS-polyacrylamide gel electrophoresis, the eluted proteoglycans contained no contaminating proteins which could be stained with Coomassie Brilliant Blue. The HeLa cell lines produced 0.5-1.5 µg of proteoglycans/10^6 cells in 10 h. The inhibition assay contained 90 µl of S-labeled decorin, 5 µl of acid solubilized collagen, and 10 µl of dilutions of inhibitor proteoglycan chimera. Incubation, collection of precipitate, and analysis were as described above for the collagen binding assay.

Decorin and proteoglycan chimera used in the inhibition experiments were purified by ion-exchange chromatography as described above. Decorin purified from calf articular cartilage (17) was used to inhibit the binding of recombinant proteoglycans to collagen. Purified decorin in 4 M guanidine HCl, 0.05 M sodium acetate, pH 5.8 was precipitated by addition of 10 volumes of ethanol, pelleted by centrifugation, dried, and dissolved in phosphate-buffered saline (0.14 M NaCl, 0.01 M sodium phosphate, pH 7.4) prior to use in collagen binding assay, as described (18) .

Scatchard plot analysis (19) was performed using S-labeled proteoglycan chimeras, which were concentrated, quantitated, and analyzed for collagen binding as described above.


RESULTS

To characterize the interaction between decorin and collagen type I, we constructed a number of different cDNAs encoding decorin/biglycan chimeras (Fig. 1). In these cDNA constructs the biglycan, which does not bind to collagen type I, provides sequences for proper biosynthesis and secretion in mammalian cells. The cDNA constructs were used to transform HeLa cells for high level expression, and the resulting proteoglycan chimeras were then characterized by SDS-PAGE before and after digestion with chondroitinase ABC (Fig. 2). The HeLa cell lines transformed with cDNA encoding biglycan, decorin, and proteoglycan chimeras produce S-labeled components of 100-200 kDa representing the small proteoglycans. After chondroitinase ABC digestion, the proteoglycans were reduced in size to 45-55-kDa core proteins, substituted with oligosaccharides and stubs of glycosaminoglycan chains. Variations in carbohydrate substitution presumably explain the observed differences in core protein size. Core proteins containing decorin sequences in the N-terminal region (decorin and Dec3big) are substituted with a single glycosaminoglycan chain and are smaller in size than core proteins containing biglycan sequences in the N-terminal region (biglycan, Big3dec, Big5dec, and Big3dec5big), which are substituted with two glycosaminoglycan chains. The control HeLa cell line transformed with pCEP4 (without insert) does not synthesize detectable amounts of either biglycan or decorin. All HeLa cell lines synthesize large [S]sulfate-labeled components which stay on top of the 10% polyacrylamide gel. These components, which are degraded by chondroitinase ABC, presumably represent large chondroitin sulfate proteoglycans. These large proteoglycans do not bind to collagen type I (not shown) nor do they interfere in the collagen binding assay.


Figure 1: Structure of biglycan/decorin chimeras. The leucine-rich repeats contain biglycan (open box) and decorin (stippled box) sequences. Binding of the proteoglycan chimera to collagen type I is indicated by (+) and nonbinding by(-).




Figure 2: Characterization of proteoglycan chimeras by SDS-PAGE. HeLa-cell lines transformed with the indicated vectors were metabolically labeled with [S]sulfate for 10 h. Samples (100 µl) of the resulting culture media were electrophoresed on a 10% polyacrylamide gel in SDS with or without prior treatment with chondroitinase ABC. Big3Dec5Big (lane 1), Big3Dec5Big digested with chondroitinase ABC (lane 2), Dec3Big (lane 3), Dec3Big digested with chondroitinase ABC (lane 4), Big5Dec (lane 5), Big5Dec digested with chondroitinase ABC (lane 6), Big3Dec (lane 7), Big3Dec digested with chondroitinase ABC (lane 8), decorin (lane 9), decorin digested with chondroitinase ABC (lane 10), biglycan (lane 11), biglycan digested with chondroitinase ABC (lane 12), pCEP4 (lane 13), and pCEP4 digested with chondroitinase ABC (lane 14).



Decorin, biglycan, and the proteoglycan chimeras produced by the HeLa cell lines were analyzed for their binding to collagen type I. The proteoglycan Big3dec, containing decorin sequences from LRR 4 to the C terminus bound to collagen, whereas the proteoglycans Big5dec and Dec3big composed of decorin sequences LRR 6 to the C terminus and N terminus to LRR 3, respectively, did not show any significant binding to collagen (Fig. 3). In addition, the constructs Big1,5dec and Big6dec containing decorin sequences from LRR1,5 to the C terminus and LRR7 to the C terminus, respectively, were analyzed for collagen binding. Big1,5dec bound to collagen whereas Big6dec did not bind (results not shown). These results prompted us to construct the proteoglycan Big3dec5big where LRR 4-5 in biglycan was substituted with decorin sequences. This proteoglycan bound to collagen indicating that major collagen-binding sites are present in decorin LRR 4-5.


Figure 3: SDS-PAGE of proteoglycans precipitated with collagen. The indicated S-labeled proteoglycan chimeras were incubated with acid-solubilized collagen at 37 °C for 5 h. The samples were centrifuged, and nonbound proteoglycans in the supernatant (S) and bound to the collagen in the pellet (P) were subjected to SDS-PAGE.



Biglycan, decorin, and the proteoglycan chimeras were analyzed by Scatchard plots to compare their affinity for collagen (Fig. 4). Biglycan, Big5dec, and Dec3big showed very low affinities for collagen, and the low concentrations of these proteoglycans used in the assay precluded an estimation of dissociation constants. Decorin binding to collagen suggested at least two binding sites with dissociation constants of 2 nM and 7 nM. The proteoglycan chimeras Big3dec and Big3dec5big bound with dissociation constants of 3 nM and 4 nM, respectively. These dissociation constants indicated a stronger binding than previously reported by Hedbom and Heineg(11) and also by Brown and Vogel(10) , who used a similar assay procedure. The approximately 10-fold difference in dissociation constants could be due to our use of proteoglycans not exposed to denaturing agents. The collagen precipitation assay is complicated by the binding of proteoglycans to small collagen fibrils remaining in solution after centrifugation, as previously pointed out by Brown and Vogel(10) . Since the proteoglycans bind to the surface of collagen fibrils, the enrichment of large collagen fibrils in the precipitate presumably explains the estimated collagen/proteoglycan molar ratio of 220:1.


Figure 4: Scatchard plot analysis. The indicated S-labeled proteoglycan chimeras were subjected to Scatchard plot analysis. Biglycan, Big5dec, and Dec3big showed very low affinity for collagen type I, whereas decorin, Big3dec, and Big3dec5big bound with high affinity to collagen type I.



Decorin purified from calf articular cartilage was used to inhibit the binding of recombinant decorin, Big3dec and Big3dec5big to collagen (Fig. 5). The purified decorin was an ineffective inhibitor of the binding of recombinant decorin to collagen and more than 1 mg/ml of purified decorin was required for 50% inhibition of Big3dec binding. On the other hand the binding of Big3dec5big was inhibited by 50% at a concentration of 0.06 mg/ml.


Figure 5: Inhibition of proteoglycan chimera binding to collagen by decorin purified from calf articular cartilage. The binding of radiolabeled decorin and proteoglycan chimeras to collagen was inhibited by increasing concentrations of purified calf articular cartilage decorin (open boxes). Recombinant decorin and Big3dec5big were partially purified by ion-exchange chromatography prior to the assay (solid symbols).



We partly purified the recombinant decorin and Big3dec5big to investigate if the radiolabeled culture media, used in the binding assay, contain components that affected the binding. The semi-purified proteoglycans bound to collagen identical to proteoglycans in the culture media, indicating no involvement of other components in the binding (Fig. 5).

To further evaluate the role of additional structures of decorin in the binding to collagen, we performed inhibition experiments where the binding of recombinant decorin was inhibited by the proteoglycan chimeras (Fig. 6). Decorin produced by HeLa cells at a concentration of 0.02 mg/ml inhibited the binding of radiolabeled recombinant decorin by 50%. Big3dec inhibited the binding by 50% at a concentration of 0.2 mg/ml and Big3dec5big at a concentration of about 1 mg/ml. The proteoglycan chimera Big5dec was less efficient as an inhibitor and a concentration of more than 1 mg/ml was required for 50% inhibition.


Figure 6: Inhibition of recombinant decorin binding to collagen by proteoglycan chimeras. The binding of recombinant S-labeled decorin was inhibited by increasing concentrations of the indicated proteoglycan chimeras. Decorin (black square), Big3dec (), Big3dec5big (bullet) and Big5dec ().




DISCUSSION

In this investigation we use proteoglycan chimeras composed of decorin and biglycan to characterize the interaction between decorin and collagen type I. The amino acid sequences of these two proteoglycans are highly homologous, and 50-60% of the amino acid residues are located in identical positions. There are two main reasons why we chose to use recombinant biglycan/decorin chimeras produced in mammalian cells to study the interaction of decorin with collagen. First, contrary to decorin, biglycan does not bind to collagen type I in the assay system used, despite the extensive sequence similarities. Second, the use of recombinant native proteoglycans produced by mammalian cells is advantageous to proteoglycans purified from tissues or synthesized by bacteria. The isolation of proteoglycans from tissues or bacteria is achieved by using strong denaturing agents, such as guanidine HCl. Proteoglycans exposed to denaturing agents bind collagen differently, since the collagen binding of decorin synthesized by HeLa cells was not inhibited by decorin purified from cartilage (Fig. 5). On the other hand, recombinant decorin produced by HeLa cells efficiently inhibited the binding of radiolabeled recombinant decorin to collagen (Fig. 6).

The biglycan/decorin chimeras were constructed so that no insertions or deletions were introduced in the amino acid sequence. Such changes in the alpha-beta repeat sequences in most cases lead to intracellular retention of the core proteins (data not shown). This is presumably due to improper tertiary structure and defective folding of the core protein, with ensuing intracellular degradation(20) .

Decorin appears to bind to collagen via more than one binding site acting in concert. Replacement of decorin sequences from the N terminus, with biglycan sequences, results in lower affinity for collagen. Intact decorin binds with higher affinity to collagen than Big3dec, which in turn binds with higher affinity than Big5dec. The Big5dec chimera containing decorin sequences from LRR 5 to the C terminus and Dec3big containing decorin sequences from the N terminus to LRR 3, do not show any apparent affinity for collagen. The Big3dec5big chimera, which contains leucine-rich repeats 4 and 5 from decorin, binds to collagen with a similar affinity as Big3Dec. However, the inhibition experiment shows that Big3dec was a more potent inhibitor of decorin binding to collagen than Big3dec5big. These different affinities may reflect the contribution of decorin sequences located C-terminal of repeat 5 to the binding strength. These binding sites presumably have a low affinity for collagen and are manifested only in cooperation with binding sites in repeats 4-5. Additional sequences located N terminally of repeat 4 may also contribute to the high affinity collagen binding of decorin. Alternatively, the conformation of repeats 4-5 may be different in the biglycan/decorin chimeras as compared with decorin, allowing the higher collagen affinity of decorin. In this case sequences in repeats 4-5 would represent the only collagen-binding sites in decorin, but depend on the correct surroundings for correct conformation and maximal binding strength. The data presented, however, show that the major decorin-binding site or sites for collagen are located in LRR 4-5.

The Big3dec5big chimera appears to bind collagen via a limited number of binding sites, since the binding could be inhibited by decorin purified from cartilage. Presumably the purified decorin, which has been exposed to guanidine HCl, only exposes certain binding sites and/or lacks the conformation needed for high affinity binding. In support, purified decorin neither inhibits the binding of HeLa cell-produced decorin nor does Big3dec, which presumably contains multiple cooperative collagen-binding sites. The detailed specificity of the interaction requires further study. Some information may be gained from comparing the sequences of decorin and biglycan in the binding domain. The two molecules show 56% identity in leucine-rich repeats 4-5. Mutational analysis of this region may give information about the structural requirements for collagen binding.


FOOTNOTES

*
This work was supported by grants from the Swedish Medical Research Council, Anna-Greta Crafoords stiftelse, Axel and Margareta Ax:son Jonson`s stiftelse, and the Medical faculty, Lund University. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed.

(^1)
The abbreviations used are: LRR, leucine-rich repeats; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.