Fibronectin Type II Module Effects on MMP-2 Substrate Interactions

Current evidence implies that the three fibronectin (FN) type II modules, which form the collagen binding domain (CBD) of matrix metalloproteinase-2 (MMP-2), are central to the enzyme function. Therefore, it is feasible that homologous type II modules from other molecules could alter the functions of MMP-2 by occupying CBD binding sites on substrate or other extracellular matrix molecules. Objectives: To characterize whether the FN collagen binding type II modules modify MMP-2 functions. Methods: Prokaryotic expression vectors were constructed to contain DNA sequences encoding for full-length MMP-2 and several FN and MMP-2 segments containing FN type II modules. Recombinant (r) proteins were expressed in E. coli and purified by affinity chromatography. Protein-protein interactions were determined by solid phase assays using biotinylated proteins and by fluorescent spectroscopy with FITC-labeled proteins. Enzymatic activity assays utilized fluorescent labeled gelatin as substrate. Results: All FN-derived constructs, that were either limited to the two type II modules or also contained the flanking type I modules (rI6I7), bound less avidly to gelatin compared to the CBD from MMP-2 (rCBD). The apparent Kds were ~3.7 x 10^-7 and 1.5 x 10^-8 M, respectively. When added simultaneously, a 5-fold molar excess of rI6I7 did not inhibit rCBD binding to gelatin. In comparison, rCBD reduced rI6I7 binding by nearly 80%. Non-functional acetylated rCBD did not alter rI6I7 binding to gelatin indicating a specific competition for closely positioned binding sites. The FN constructs and acetylated rCBD also did not alter MMP-2 binding to gelatin whereas rCBD reduced the MMP-2 binding by up to 70%. Importantly, the FN type II modules had no significant inhibiting effect on the MMP-2 gelatinolytic activity. Conclusions: These results indicate that FN fragments containing type II modules have limited or no effects on MMP-2 activities. (Supported by NIDCR grants DE14236 and 12818).