Decorin Binds Fibrinogen in a Zn 2+ -dependent Interaction ∗

We have previously shown that decorin, a member of the small leucine-rich proteoglycan family of extracellular matrix proteoglycans/glycoproteins is a Zn(2+) metalloprotein at physiological Zn(2+) concentrations (Yang, V. W-C., LaBrenz, S. R., Rosenberg, L. C., McQuillan, D., and Höök, M. (1999) J. Biol. Chem. 274, 12454-12460). We now report that the decorin proteoglycan binds fibrinogen in the presence of Zn(2+). The fibrinogen-binding site is located in the N-terminal domain of the decorin core protein and a 45-amino acid peptide representing this domain binds to the fibrinogen D fragment with an apparent K(D) of 1.7 x 10(-6) m, as determined from fluorescence polarization data. Furthermore, we show that Zn(2+) promotes the self-association of decorin. The N-terminal domain of the core protein also mediates this activity. The results of solid-phase binding assays and gel filtration chromatography suggest that the N-terminal domain of decorin, when present at low micromolar concentrations, forms an oligomer in a Zn(2+)-dependent manner. Thus, Zn(2+) appears to play a pivotal role in the interactions and biological function of decorin.


INTRODUCTION
Decorin is a member of the small leucine-rich proteoglycan (SLRP) family of related glycoproteins found in mammalian extracellular matrices (ECMs) (2). The core proteins of SLRPs are similar in structural organization and size, ranging from 35 to 42 kDa. The dominant structural feature is a central domain containing 6-10 leucine-rich repeats (LRRs) that is flanked by N-terminal and C-terminal regions with conserved Cys residues (3). The LRR motif has been found in a number of proteins of diverse origin and function and varies in length from 20-29 amino acids (4). Protein crystal structure analysis of the RNAse inhibitor, internalin B, and glycoprotein 1B suggests that each LRR motif forms a β-strand/turn/helix, giving protein segments composed of tandem repeats of LRRs an arch-like shape (5)(6)(7). The SLRPs contain 24 amino acid long LRRs and may also assume an arch-shaped structure as proposed by molecular modeling studies of decorin (8).
SLRPs may be subgrouped based on gene organization, amino acid sequence similarity, the number of LRRs in the central domain, and the spacing of Cys residues in 4 well as the glycoprotein PRELP, are the members of subgroup II (10 LRRs). Subgroup III (6 LRRs) includes epiphycan and osteoglycin, which may be substituted with dermatan sulfate/chondroitin sulfate and keratan sulfate polysaccharides, respectively, and the glycoprotein opticin (11). The structure of a twelfth member, chondroadherin, differs sufficiently from those of other SLRPs and perhaps should be assigned to a separate subgroup. The spacing of 4 Cys residues in the N-terminal sequence of the members of subgroups I, II and III is Cx 3 CxCx 6 C, Cx 3 CxCx 9 C, and Cx 2 CxCx 6 C, respectively.
One theme in SLRP function appears to be the regulation of extracellular matrix architecture. Decorin, biglycan, fibromodulin and lumican reportedly interact with collagens and influence collagen fibrillogenesis in in vitro assays (12)(13)(14)(15)(16). Analyses of transgenic mice generated with targeted inactivation of individual SLRP genes has revealed that the loss of function of each SLRP resulted in a mild phenotype characterized by the abnormal morphology of specific ECMs. Decorin or fibromodulindeficient mice exhibit collagen fiber defects in tendon, while decorin or lumican-deficient mice have fragile skin, possibly due to abnormal collagen fibers in the dermis (17)(18)(19).
Lumican-deficient mice also display abnormally thickened collagen fibers in the cornea.
Recent studies suggest that the phenotype of mice deficient in both decorin and biglycan is more severe than those observed in cases where decorin or biglycan genes were individually inactivated (20). Taken together, these results indicate a certain degree of functional redundancy among the different SLRP members.
SLRPs may differentially affect cell behavior by modulating growth factor activity or by influencing the interactions of cell surface receptors with matrix components.

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Transfection of the decorin cDNA into CHO cells results in the over-expression of the proteoglycan and correlates with reduced proliferation, presumably due to an inhibition of TGF-β signaling (21). Decorin has also been reported to inhibit the growth of certain cancer cell types by a mechanism thought to involve an interaction between decorin and the EGF receptor (22). The ability of SLRPs to affect cell-matrix interactions can involve SLRP-ECM or SLRP-integrin interactions. Decorin has been shown to interfere with the adhesion of mammalian cells to substrates composed of fibronectin and thrombospondin, and in a glycosaminoglycan-dependent manner, decorin inhibits cell migration on fibronectin and collagen matrices (23)(24)(25)(26).

Zn 2+ Enhances Decorin Binding to Matrix Macromolecules
In a previous study, we discovered that Zn 2+ binding to decorin could alter the conformation of the N-terminal segment of the protein. To explore the possibility that the presence of Zn 2+ affects the interaction of decorin with other ECM molecules, a solid phase binding assay was conducted in the presence of excess Zn 2+ or EDTA (Fig. 1).
In these studies, we used a recombinant form of the decorin proteoglycan presumably retaining it's native conformation since it was secreted by a mammalian cell line infected with recombinant vaccinia viruses and isolated without the use of chaotropic or denaturing agents. Decorin was labeled with biotin and incubated in microtiter wells coated with type I, type II, type IV or type V collagen, fibronectin, fibrinogen or ovalbumin. The results of these experiments show that in the presence of the chelating agent EDTA, the amounts of decorin bound to the different ECM proteins were marginally higher than the amounts bound to ovalbumin. However, the addition of Zn 2+ appears to greatly stimulate the binding of decorin to these ECM proteins, with the possible exception of type II collagen. The inclusion of Zn 2+ did not result in a substantial increase in decorin binding to the control protein ovalbumin. Since microtiter plate wells coated with fibrinogen bound the highest amount of decorin in the presence of Zn 2+ and since this interaction previously has not been reported, we chose to further characterize the binding of decorin to fibrinogen as an example of Zn 2+ -dependent decorin binding to ECM molecules.

Localization of the Fibrinogen-Binding Site on Decorin
To identify the domain in decorin that mediates binding to fibrinogen, we examined different components of decorin for their ability to inhibit the binding of biotin-labeled decorin proteoglycan to fibrinogen coated microtiter wells. Initial experiments showed that the intact proteoglycan and the core protein were equally efficient inhibitors in this experiment (data not shown) suggesting that the fibrinogen-binding site is located to the core protein.
The binding of decorin to fibrinogen shows a strong Zn 2+ -dependence. Previously, we localized the Zn 2+ -binding site to the N-terminal segment of the decorin core protein.
Therefore, we explored the possibility that this domain also contains the fibrinogenbinding site ( Fig. 2A). Initially, we examined a recombinant form of the N-terminal domain of decorin expressed in E. coli and presented as a maltose-binding protein fusion (MBPDcnNTD) for the ability to inhibit the binding of biotin-labeled decorin proteoglycan to adsorbed fibrinogen. Indeed, MBPDcnNTD inhibits the binding of biotin-labeled decorin proteoglycan in a concentration-dependent manner. Essentially complete inhibition was seen at concentrations of MBPDcnNTD greater than or equal to To confirm that the fibrinogen binding activity is due to the decorin-derived component of MBPDcnNTD, we examined the inhibitory activity of the decorin Nterminal domain (DcnNTD) in the absence of a fusion partner (Fig. 2B). We expressed the 45 amino acid residue DcnNTD as a recombinant glutathione S-transferase (GST) fusion protein with a thrombin cleavage site located in a linker peptide between GST and DcnNTD. After thrombin digestion of GST-DcnNTD, DcnNTD was initially purified under reducing conditions and subsequently isolated in the presence of Zn 2+ . This Zn 2+charged peptide was then examined for the ability to inhibit the binding of biotin-labeled proteoglycan to fibrinogen. We observed dose-dependent, complete inhibition of decorin proteoglycan binding to fibrinogen by the isolated decorin peptide, with halfmaximal inhibition observed above 1 µM DcnNTD and complete inhibition achieved at 5 µM DcnNTD. From these experiments, we conclude that the fibrinogen-binding site on decorin is located in the N-terminal domain of the core protein. In fact, we subsequently showed in direct binding assays that DcnNTD binds to fibrinogen (see below).

The Decorin-Binding Site on Fibrinogen
To localize the decorin-binding site on fibrinogen, we first conducted a solid-phase binding assay to determine whether biotin-labeled decorin proteoglycan or MBPDcnNTD recognizes the plasmin generated fibrinogen fragments D or E (Fig. 3).
We found that in the presence of Zn 2+ , both decorin proteins bound to microtiter wells coated with intact fibrinogen or fragment D but not to the E fragment or the control protein ovalbumin. In the presence of EDTA, the binding of decorin to all four proteins by guest on March 24, 2020 http://www.jbc.org/ Downloaded from was minimal. Hence, in a Zn 2+ -dependent fashion, decorin appears to specifically recognize the globular D regions of fibrinogen, mainly consisting of amino acid residues 111-197 of the alpha chain, 134-461 of the beta chain and 88-406 of the gamma chain (35,36).

Characterization of the Decorin-Fibrinogen Interaction
To gain insight on the mechanism of decorin-fibrinogen recognition, we initially wanted to determine the K D for the interaction of decorin proteoglycan with fibrinogen ( Fig. 4A). In the presence of Zn 2+ , fibrinogen or ovalbumin coated microtiter wells were incubated with increasing concentrations of the biotin-labeled proteoglycan until equilibrium was reached. Minimal amounts of decorin were retained in the ovalbumin containing wells. However, decorin bound fibrinogen in a concentration-dependent, saturable manner. Assuming that there are two identical, independent decorin-binding sites on fibrinogen, we analyzed these data utilizing the program DynaFit and obtained an apparent K D of 6.8 X 10 -7 M 2 .
In a previous experiment, we showed that the decorin peptide, DcnNTD, completely inhibits decorin proteoglycan binding to fibrinogen. To directly observe the interaction of DcnNTD with fibrinogen, increasing concentrations of biotin-labeled decorin peptide were allowed to equilibrate with fibrinogen or ovalbumin coated wells in the presence of Zn 2+ (Fig. 4B). Low levels of DcnNTD were detected in the ovalbumin containing wells.
In contrast, the decorin peptide binding to fibrinogen was concentration-dependent and saturable with an apparent K D of 3.0 X 10 -7 M 2 .
We also demonstrated that MBPDcnNTD recognizes both fibrinogen and fragment D in a Zn 2+ -dependent fashion. In this experiment, the concentration-dependent binding of biotin-labeled MBPDcnNTD to adsorbed fibrinogen, fragment D or ovalbumin was observed under equilibrium conditions with Zn 2+ present (Fig. 4C). In an ELISA-type experiment, we also monitored the binding of 0.25 µM fibrinogen to wells coated with either MBPDcnNTD or MBP over time (Fig. 6C). Fibrinogen slowly accumulated on the MBP coated surface in what we think is a nonspecific interaction.
On the other hand, fibrinogen binding to MBPDcnNTD reached a plateau already after approximately 30 min. The results of these time-dependent binding experiments lead us to hypothesize that decorin-fibrinogen interaction involves more than one step.

Zn 2+ Promotes the Self-association of Decorin
Earlier studies by Liu  Initially, we examined the possibility that the 45 amino acid decorin peptide, DcnNTD, self-associates in a Zn 2+ -dependent fashion by gel filtration chromatography (Fig. 7).
The peptide at a concentration of 47 µM in the presence of either Zn 2+ or EDTA with DTT was applied to a Superdex-75 gel filtration column with a reported separation range of 3 to 70 kDa. These chromatograms show that the decorin peptide elutes in a significantly lower volume in the presence of Zn 2+ than in the presence of EDTA with DTT. The Zn 2+ -charged peptide elutes in a broad peak located at 8.5 ml, the same volume required to elute albumin (Table I)