The role of the c-terminal domain of human collagenase-3 (MMP-13) in the activation of procollagenase-3, substrate specificity, and tissue inhibitor of metalloproteinase interaction

Recombinant human procollagenase-3 and a C-terminal truncated form (Delta(249-451) procollagenase-3) have been stably expressed in myeloma cells and purified, The truncated proenzyme could be processed by amino-phenylmercuric acetate via a short lived intermediate form (N-terminal Leu(58)) to the final active form (N-terminal Tyr(85)), The kinetics of activation were not affected by removal of the hemopexin-like C-terminal domain, The specific activities of both collagenase-3 and Delta(249-451) collagenase-3 were found to be similar using two quenched fluorescent substrates, but Delta(249-451) collagenase-3 failed to cleave native triple helical collagens (types I and II) into characteristic one- and three-quarter fragments, It was noted, however, that the beta 1,2(I) chains of type I collagen were susceptible to Delta(249-451) collagenase-3, which indicates that the catalytic domain displays telopeptidase activity, thereby generating alpha 1,2(I) chains that are slightly shorter than those in native type I collagen, It can be concluded that the C-terminal domain is only essential for the triple helicase activity of collagenase-3, Binding of procollagenase-3 and active collagenase-3 to type I collagen is mediated by the C-terminal domain, Both collagenase-3 and Delta(249-451) collagenase-3 hydrolyzed the large tenascin C isoform, fibronectin, recombinant fibronectin fragments, and type IV, IX, X, and XIV collagens; thus, these events were independent from C-terminal domain inter actions, In contrast, the minor cartilage type XI collagen was resistant to cleavage, Kinetic analysis of the mechanism of inhibition of wild-type and <Delta(249-451) collagenase-3 by wild-type and mutant tissue inhibitors of metalloproteinase (TIMPs) revealed that the association rates for complex formation were influenced by both Nand C-terminal domain interactions, The C-terminal domain of wild-type collagenase-3 promoted increased association rates with the full-length inhibitors TIMP-1 and TIMP-3 and the hybrid N.TIMP-2/C.TIMP-1 by a factor of up to 33, In contrast, the association rates for complex formation with TIMP-2 and N.TIMP-1/C.TIMP-2 were only marginally affected by C-terminal domain interactions.