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 (De lta(249-451) procollagenase-3) have been stably expressed in myeloma c ells and purified, The truncated proenzyme could be processed by amino -phenylmercuric acetate via a short lived intermediate form (N-termina l 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-te rminal domain, The specific activities of both collagenase-3 and Delta (249-451) collagenase-3 were found to be similar using two quenched fl uorescent substrates, but Delta(249-451) collagenase-3 failed to cleav e native triple helical collagens (types I and II) into characteristic one- and three-quarter fragments, It was noted, however, that the bet a 1,2(I) chains of type I collagen were susceptible to Delta(249-451) collagenase-3, which indicates that the catalytic domain displays telo peptidase activity, thereby generating alpha 1,2(I) chains that are sl ightly shorter than those in native type I collagen, It can be conclud ed that the C-terminal domain is only essential for the triple helicas e activity of collagenase-3, Binding of procollagenase-3 and active co llagenase-3 to type I collagen is mediated by the C-terminal domain, B oth collagenase-3 and Delta(249-451) collagenase-3 hydrolyzed the larg e tenascin C isoform, fibronectin, recombinant fibronectin fragments, and type IV, IX, X, and XIV collagens; thus, these events were indepen dent from C-terminal domain inter actions, In contrast, the minor cart ilage type XI collagen was resistant to cleavage, Kinetic analysis of the mechanism of inhibition of wild-type and <Delta(249-451) collagena se-3 by wild-type and mutant tissue inhibitors of metalloproteinase (T IMPs) revealed that the association rates for complex formation were i nfluenced 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.TI MP-2/C.TIMP-1 by a factor of up to 33, In contrast, the association ra tes for complex formation with TIMP-2 and N.TIMP-1/C.TIMP-2 were only marginally affected by C-terminal domain interactions.