©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Decorin-Type I Collagen Interaction
PRESENCE OF SEPARATE CORE PROTEIN-BINDING DOMAINS (*)

Elke Schönherr (§) , Heinz Hausser , Lesley Beavan (¶) , Hans Kresse

From the (1) Institute of Physiological Chemistry and Pathobiochemistry, University of Münster, Waldeyerstrasse 15, D-48149 Münster, Federal Republic of Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

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.


INTRODUCTION

Decorin, a small dermatan sulfate proteoglycan, is a ubiquitous component of extracellular matrices (1, 2) where it is preferentially found in association with collagen fibrils (3, 4, 5, 6, 7) . 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 (5, 6, 8) . Some additional interactions may be mediated by the dermatan sulfate side chain (9) . As decorin binds to the surface of collagen fibrils, the lateral assembly of individual triple helical collagen molecules is delayed (10, 11) , and the diameter of the fibrils is decreased (12) . However, only moderately low dissociation constants on the order of 10 M have been reported for the complex between type I collagen and decorin (13, 14) , and in vitro studies suggested a preferential binding of decorin to type VI collagen (15) . Nevertheless, mediating molecules like type VI collagen are not required to interpret the data on the interaction between decorin and fibrillar collagens (9) .

In addition to collagen binding, the core protein of decorin interacts with other components of the extracellular matrix (16, 17, 18) , with transforming growth factor- (19) , and with a receptor required for its endocytosis (20) . 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 (21, 22) . The single glycosaminoglycan chain of decorin is linked with serine 4 of the mature core protein (23) . 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 (24) , but the inhibition of fibrillogenesis was abolished by a disulfide bond reduction (25) . 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 (26) . 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.


EXPERIMENTAL PROCEDURES

Preparation of Proteoglycans

Reference (wild-type) decorin was purified to about 95% purity from the secretions of cultured human skin fibroblasts as described (27) . 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. [S]Sulfate-labeled decorin was prepared analogously after incubating the cells for 72 h in the presence of 1.48 MBq/ml [S]sulfate (carrier-free) (Amersham-Buchler, Braunschweig, Germany) and 4% (v/v) fetal calf serum that had been dialyzed against 0.15 M NaCl.

Expression and Purification of Recombinant Decorin Core Protein Fragments

Clone D6, which contains the coding region of human decorin core protein, has been described previously (28) . 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 (21) but has been found more recently (29) . The clone was used to prepare the following recombinant peptides (see 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 DH5. 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.

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 INVF` (Invitrogen) as described (30) . Bacteria were then collected and treated with 10 ml of 6 M guanidine hydrochloride, 0.5 M NaCl, and 20 m M sodium phosphate, pH 7.8/100 ml of original suspension. For purification, 2 ml of a nickel-nitriloacetic acid agarose gel (Qiagen, Hilden, Germany) were put into a spin column and equilibrated with 8 M urea and 0.5 M NaCl in 20 m M sodium phosphate, pH 7.8 (buffer A). Solubilized proteins were loaded onto the column by gravity in two 5-ml portions. The column was washed by centrifugation (1000 g) using 7-ml portions of buffer A until the Awas below 0.01. Nine washing steps were usually required. Further washings were by gravity using 8 M urea, 0.5 M NaCl, 0.05% Tween 80 (Sigma) in 20 m M sodium phosphate, pH 6.5 (buffer B) until the Awas below 0.01 (about 50 ml). Elution was achieved by applying 6 1 ml of 8 M urea, 0.5 M NaCl, and 0.05% Tween 80 in 20 m M sodium phosphate, pH 4.0. The four fractions with the highest Awere pooled, diluted with buffer B to an Aof about 0.03, made 0.05% with BSA() , and dialyzed for 48 h at 4 °C against 20 times the sample volume of 1 M urea, 2 m M glutathione (reduced), 0.02 m M glutathione (oxidized), 0.005% Tween 80 in 50 m M Tris-HCl, pH 8.0. After dialysis overnight against 0.2 PBS, insoluble proteins were removed by centrifugation, and 1.5-ml aliquots of the supernatant were lyophilized and reconstituted with 300 µl of HO by brief sonication.

When indicated, much smaller quantities of BSA were added to the renaturing solution. After dialysis, urea was added to a final concentration of 8 M, and the purification and renaturation protocol was repeated, but this time in the presence of the standard concentration of BSA.

Radioactively labeled proteins were obtained after incubation with [S]sulfate exactly as described (30) .

Binding to Reconstituted Type I Collagen Fibrils

Acid-soluble type I collagen from calf skin (Sigma, Deisenhofen, Germany) was dissolved in 17 m M acetic acid (4.4 mg/ml), neutralized by adding 2 volumes of 18 m M sodium phosphate, pH 7.4, containing 0.14 M NaCl (PBS), and incubated for 30 min at 37 °C. The fibrils formed were collected by centrifugation, suspended in 300 µl of 5% (w/v) BSA in PBS/mg of collagen, and dispersed by ultrasonication. In most experiments, recombinant peptides were tested in a competition assay. Wild-type [S]sulfate-labeled decorin (about 8,000 cpm) was mixed with 0.17 mg of reconstituted collagen fibrils and the respective peptides in PBS to give a final volume of 600 µl and a final concentration of 0.875% BSA. Controls were incubated without collagen. After 5 h at 37 °C under constant mixing, the pellet was collected by centrifugation (10,000 g for 10 min) and washed 3 times with PBS. Bound decorin was quantitated after solubilizing the fibrils with 50 µl of 1 M NaOH. All assays were done in duplicate or triplicate. S-Labeled recombinant peptides were assayed analogously. Their quantity was estimated from the protein content of larger non-radioactive samples prepared in parallel.

Fibrillogenesis Assay

Collagen was prepared from bovine tendon, and collagen fibrillogenesis was monitored spectrophotometrically exactly as described (10) . Appropriate mixtures of collagen (100 µg) and either decorin or decorin peptides in a final volume of 1 ml were made on ice and warmed up to 37 °C. The turbidity at 400 nm was measured in 5-min intervals.

Other Methods

SDS-polyacrylamide gel electrophoresis followed by Western blotting was done as described earlier (27) . The polyclonal rabbit antiserum against deglycosylated human decorin (31) was found to react with all recombinant peptides used in this study, but the number of reactive epitopes of the individual peptides was not determined. For the quantification of recombinant proteins, standard concentrations of BSA (1-30 µg) and samples were separated on a 15% SDS-polyacrylamide gel and transferred to nitrocellulose membranes. They were stained with 0.1% Amido Black 10B in methanol/acetic acid/HO (9/2/9, v/v/v) and destained in methanol/acetic acid/HO (45/1/4, v/v/v). Stained bands were cut out, eluted with 300 µl of 25 m M NaOH and 0.04 m M EDTA in 50% aqueous ethanol, and quantitated at 630 nm.


RESULTS

Expression of Recombinant Decorin Peptides

A variety of recombinant decorin peptides (Table I) were expressed as fusion proteins in E. coli strain INVF`. These constructs allowed an easy preparation to more than 95% purity by a single chromatographic step as judged by SDS-polyacrylamide gel electrophoresis of the peptides prepared in the absence of BSA. Previous attempts to purify peptides directly expressed in bacteria had failed. Proteins solubilized with 8 M urea could not be purified in a single step by chromatography on either DEAE- or CM-Trisacryl. Chromatography of proteins solubilized with 4 M guanidine hydrochloride on octyl-Sepharose (32) was hampered by low recovery. Purification of P15-329 from 100 ml of bacteria typically yielded 5 mg of peptide. After renaturation in the presence of BSA, about 800 µg of peptide (with a range of 500-1400 µg in seven separate purifications) were obtained in soluble form. Their quantity was determined after SDS-polyacrylamide gel electrophoresis (Fig. 2), electroblotting, and staining of the respective bands with Amido Black. Because the strong denaturing agent 4 M guanidine hydrochloride had to be used to extract the peptide from inclusion bodies, the following criteria for successful renaturation were used. P15-329 becomes insoluble after reduction and alkylation, indicating that solubility depends on the formation of disulfide bonds. As functional tests, the inhibition of binding of wild-type decorin to type I collagen fibrils and to the endocytosis receptor (not shown) was employed. Successful renaturation required the presence of sufficient quantities of BSA (Fig. 3). When only 10 µg/ml BSA were included in the renaturing buffer, the resulting product failed to compete with wild-type decorin for binding to collagen fibrils. However, refolding to an active state occurred even after further treatment with 8 M urea provided that sufficient quantities of BSA were present during renaturation. BSA was therefore considered to act as a chaperone and to enable the proper folding of P15-329. The experiment also demonstrates that solubility is not a sufficient criterion for the functionality of P15-329. It had recently been reported that decorin core protein, functionally active with respect to transforming growth factor- binding, could be obtained as a fusion protein with E. coli maltose-binding protein (33) . Use of this strategy, however, yields a fusion protein twice the size of the wild-type core protein.

A quantitative comparison between wild-type decorin and P15-329 for the inhibition of binding of [S]sulfate-labeled decorin to collagen indicated that the doses required for half-maximal inhibition (IC) were of the same order of magnitude, although the doses required for the recombinant peptide were always somewhat higher (Fig. 4). In seven different preparations the ICof P15-329 was minimally 1.2-fold and maximally 3.5-fold higher than that of wild-type decorin.

The specific competition between P15-329 and decorin for collagen binding could also be shown by Western blotting and immune staining instead of using a radioactive ligand. It is shown in Fig. 5 that P15-329 does not precipitate during incubation in the absence of collagen fibrils and that there is indeed competition between the two ligands for binding.

In a further set of experiments the influence of the recombinant peptide and of decorin on the kinetics of fibril formation was studied. During fibrillogenesis, decorin has to interact reversibly with the growing fibrils, and there should be no steady state in the quantity of bound decorin. It is seen in Fig. 6 A that P15-329 is equally as efficient as decorin in reducing the turbidity of the fibril suspension, which suggests a similar effect of decorin and P15-329 on the diameter reduction of the collagen fibrils. A prolonged lag phase was also consistently observed in the presence of P15-329.

Existence of Several Distinct Collagen-binding Sites

The procedure described above was used to renature a variety of recombinant decorin peptides that were designed to cover several N- and C-terminal regions as well as a central region (Fig. 1). One of the peptides, P33-160, was anticipated to be biologically inactive and to be employable as a negative control because it contained a single cysteine residue only. With the exception of P125-230, which covered the central cysteine-free region, all other peptides contained a paired number of cysteine residues and were anticipated to be capable of forming disulfide bridges. In support of this expectation, these peptides formed insoluble complexes upon reduction and alkylation.


Figure 1: Schematic 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.



When the different peptides were used in a competition assay, the lowest IClevels were obtained for the ``full-length'' peptide, P15-329, and for P15-183, which contains the first six N-terminally located leucine-rich repeats in addition to the N-terminal cysteine-rich region (Table II and Fig. 7). A shortened N-terminal peptide, P15-125, required at least three times higher doses for half-maximal inhibition (three independent experiments). Unexpectedly, two C-terminally located peptides, P154-329 and P185-329, containing 5 and 6 leucine-rich repeats, respectively, were also able to interfere with collagen binding of wild-type decorin, but the centrally located peptide P125-230 was not. Nevertheless, this peptide exhibited functional activity because it was well suited in interfering with the endocytosis of decorin.()

The interference with decorin binding to collagen by two non-overlapping recombinant peptides prompted us to prepare P15-183 and P185-329 in a radioactively labeled form for use in competition assays. Fig. 8 shows very clearly that the binding of S-labeled P185-329 to collagen fibrils can be best inhibited by the peptide itself and by the full-length peptide P15-329. Very minor inhibition only could be achieved by P15-183.

The situation is less clear for the inhibition of binding of S-labeled P15-183. Although the full-length peptide and P15-183 itself were the best competitors, P185-329 and, to a lesser extent, P125-230 were also able to interfere with binding. This could indicate that the latter two peptides carry a domain interacting with the P15-183 binding site, albeit with lower affinity. Alternatively, the binding of P185-329 affects, by steric hindrance, the binding of P15-183 to an adjacent site, whereas the converse does not occur. Steric hindrance is also a possible explanation for the observation that P15-183 inhibited the binding of native decorin to collagen, whereas the same peptide was not an inhibitor of the interaction between P185-329 and collagen. In the presence of P15-183, an interaction of native decorin via its C-terminal collagen-binding domain should be expected in the absence of steric hindrance.

The radioactively labeled recombinant peptides were used for the determination of dissociation constants. The Kvalue of P15-329 was 3 10mol/liter, an order of magnitude smaller than the Kvalues for both P15-183 and P185-329 (Fig. 9).

Interference with Collagen Fibrillogenesis

Several of the recombinant decorin peptides were tested for their influence on collagen fibrillogenesis. It is shown in Fig. 6 B that P15-183 was approximately as efficient as P15-329 and wild-type decorin in inhibiting the formation of fibrils. The collagen-binding peptide P185-329, however, was ineffective, although all recombinant peptides had an influence on the length of the lag phase preceding fibrillogenesis. It is also noteworthy that P15-125 is well suited for interfering with fibril formation, although its ability to compete with decorin for collagen binding is only moderate. These general findings were corroborated by the analysis of at least two different peptide preparations. Considering the previous finding that the reduction of disulfide bonds of decorin abolishes its capability to inhibit fibrillogenesis (25) , it is tempting to speculate that the N-terminal region of decorin, which contains 4 cysteine residues and is present in P15-183 and in P15-125, is of special importance for this function.


Figure 6: Collagen fibril formation in the presence of decorin ( DCN) and recombinant peptides.




DISCUSSION

In this study, recombinant decorin core protein fragments were analyzed for their interaction with type I collagen. All the peptides had to be extracted from inclusion bodies by a strong denaturing agent and had to be renatured. This was possible only when BSA was present as a chaperone protein during the renaturation process. The renatured protein P15-329 exhibited a similar, albeit quantitatively not exactly the same, collagen binding activity as wild-type decorin. A strong argument for the general success of the renaturing procedure is the observation that a protein that did not bind to collagen could be converted to a species capable of interacting with collagen, even after additional denaturation with urea, provided that the appropriate conditions for renaturation were employed. The presence of large quantities of BSA, however, precluded the use of physicochemical methods like measurements of CD spectra for an evaluation of the renaturation process. CD spectra of N-glycan-free decorin showed a broad minimum around 212 nm in contrast to the sharper minimum seen for intact decorin (34) . These data were explained as an indication of aggregate formation, but it was also demonstrated that oligosaccharide-free decorin retained its ability to bind to collagen fibrils and to retard collagen fibrillogenesis in vitro.

An inherent difficulty of the use of protein fragments is the fact that negative results of an assay could be due either to the absence of the active structure within the partial sequence or to improper renaturation. In addition to focusing on the full-length peptide P15-329, we focused our studies on the peptides P15-125, P15-183, P185-329, and P125-230. P125-230 was almost completely inactive with regard to collagen binding. However, other than the full-length peptide, it was the most efficient inhibitor of decorin endocytosis.Thus, all four of the peptides were consistently found to be active in at least one assay system. The segment of the core protein represented by the recombinant peptide was therefore considered to possess at least a cryptic site for the respective interaction.

A surprising diversity of the interacting domains of decorin core protein is the main result of our studies. Separate high affinity binding sites to collagen were observed for P15-183 and P185-329. P15-125 was especially active in the inhibition of collagen fibrillogenesis, whereas higher doses were required to compete with decorin binding to preformed collagen fibrils when compared with P15-183. This could suggest that P15-125 contains a lower affinity binding site. The existence of a low affinity binding site has been proposed previously (13) . A recent preliminary report also arrives at the conclusion that decorin core protein contains at least two functional domains that are involved in the interaction with the collagen-like molecule C1q (35) .

The existence of two high affinity binding sites may be difficult to understand when one considers the globular structure, determined by rotary shadowing, of the relatively small core protein (36) . However, the crystal structure of ribonuclease inhibitor, which, like decorin, is a protein with leucine-rich repeats, showed that this protein has the non-globular shape of a horseshoe and represents a new class of / protein folds (37) . Provided that decorin core proteins exhibit an analogous structure, the data presented in this study are compatible with the assumption that the surface area formed by the fifth and/or sixth of the leucine-rich repeats, which are not contained within P15-125 (see Fig. 1), and a repeat from the C-terminal half of the molecule are involved in the binding to collagen fibrils. The flexible N-terminal part of the molecule with its two disulfide bridges is contained within both P15-125 and P15-183 and is considered to be involved in fibrillogenesis. Further studies, however, are needed to elucidate the interacting structures in greater detail.

  
Table: Recombinant decorin peptides


  
Table: Inhibition of decorin-type I collagen interaction by recombinant decorin peptides

Inhibition of binding of [S]sulfate-labeled wild-type decorin to collagen fibrils by recombinant peptides was measured as described in the legend of Fig. 7.



FOOTNOTES

*
This work was supported by the Deutsche Forschungsgemeinschaft (SFB 310, Project B2) and by the Fonds der Chemischen Industrie. 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: Inst. of Physiological Chemistry and Pathobiochemistry, University of Münster, Waldeyerstr. 15, D-48149 Münster, Germany. Tel.: 49-251-835584; Fax: 49-251-835596.

Present address: Dept. of Biochemistry, Charing Cross and Westminster Medical School, University of London, Fulham Palace Rd., London W6 8RF, Great Britain.

The abbreviations used are: BSA, bovine serum albumin; PBS, phosphate-buffered saline.

E. Schönherr, H. Hausser, L. Beavan, and H. Kresse, unpublished results.


ACKNOWLEDGEMENTS

We are indebted to M. Bahl for skillful technical assistance.


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