The hallmark of rheumatoid arthritis is the progressive destruction of the articular cartilage which ultimately results in loss of joint function and eventual debilitation. Over the years, much information has accumulated which would implicate metalloproteinases in the destruction of cartilage matrix. First, all of these enzymes are fully active at physiological conditions and avidly degrade the major cartilage matrix components including proteoglycans, chondronectin and collagen types II, IX, and XI. Second, these enzymes are increased in the rheumatoid lesion and are produced by inflammatory phagocytes, synovial cells,a nd native chondrocytes. Third, the synthesis of these enzymes is upregulated by inflammatory cytokines which are also present in increased amounts in these tissues. In the past 5 years, the primary structure of the major enzymes of this family has been characterized and striking homologies have been found. In this application, we propose to study certain aspects of the enzymatic function of three members of this family (neutrophil collagenase, 92 kDa gelatinase and stromelysin) and correlate them with the primary structure of the proteins. Our experimental approach will be to construct mutations within the cDNA of these enzymes and express the mutant proteins using eukaryotic and prokaryotic expression vectors. the mutant proteins will be purified by affinity chromatography and functional properties evaluated in specific assay systems. The functional properties will be focused on substrate specificity, divalent cation binding, interaction with the tissue inhibitor of metalloproteinases and identification of the critical carboxylic acid residues. The recombinant mutant enzymes will be assayed for their ability to degrade and/or bind to a panel of substrates known to be degraded by one or more of these enzymes. The mutant proteins will be evaluated for their ability to bind Zn2+ and Ca2+ and divalent cation binding will be correlated with enzymatic activity and substrate binding. In other studies, mutant proteins will be examined for binding to the tissue inhibitor of collagenase (TIMP) and for susceptibility to inhibition by this protein. Finally, our techniques of site-directed mutagenesis will be directed at the putative active site of these enzymes. It is hypothesized that an acidic residue (Glu or Asp) functions as the active base catalyst in these enzymes. Candidate acidic residues in this region will be mutated to identify the essential amino acid(s). These studies will advance our understanding of the action of metalloproteinases to a new level and may lead to therapeutic concepts which would focus on protection of articular tissues from the degradative machinery of the rheumatoid lesion.
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