Matrix metalloproteinases (MMP) are a group of 8 highly homologous enzymes which share a number of structural and functional characteristics. Each of these MMPs are derived from a basic three- domain MMP prototype either by addition or deletion of domains: (i) an 80-100 residue propeptide which endows the enzyme with catalytic latency, (ii) a 140-160 domain which contains the intact catalytic machinery including a highly conserved Zn-binding site, and (iii) a 200 residue C- terminal domain preceded by a """"""""hinge-region"""""""" which varies in length from enzyme to enzyme. Previous studies in our laboratory have suggested that the substrate specificity of these enzymes is encoded in part by a large, 200 residue C-terminal domain; this domain may be responsible for the ability of fibroblast and polymorphonuclear collagenases (F-CL and PMN- CL, respectively) to cleave interstitial collagens (types I-III), a property not shared by other MMPs. Deletion of this domain, or of the only disulfide bond within this domain, abolishes the ability of the enzyme to cleave collagen, but does not significantly interfere with the ability of the enzyme to cleave unfolded polypeptide substrates such as beta-casein. The overall goal of project 6 is to understand how the various domains of F-CL and other MMPs encode activation pathways, proteolytic function and specificity of substrate use. Our previous studies began to elucidate domains and amino acids important for activation of the latent enzyme, for the cleavage reaction, and for determining substrate specificity. Our focus is on F-CL, PMN-CL and stromelysin-1 (SL-1), since they share 40 to 50% amino acid sequence homology, and yet show different substrate specificities and cleavage rates on different substrates. F-CL and PMN-CL both cleave interstitial collagens but exhibit considerable kinetic differences; SL-1 has a somewhat broader range of substrate specificity but lacks the ability to cleave collagen. We will determine whether these differences are encoded entirely in the C-terminal domain III or rather are a characteristic of the catalytic machinery itself. Domains I and II are important for maintaining F-CL (and probably all MMPs) in an inactive form. Either proteolytic cleavage at sites within domain I or disruption of the cys(73) interaction with the active site is sufficient to activate the enzyme and cause removal of domain I, probably through a series of cleaved intermediates. The analysis of our C73S mutant in the alpha-2M capture assay showed that this altered protein was not fully active, suggesting that other regions within domain I are probably also important for maintaining latency, consistent with the results of others. The other regions important for maintenance of latency and for cleavages important for activation will be mapped by mutagenesis of F-CL.
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