Connective tissue cells have the ability to synthesize and secrete collagenase and at least two other matrix metalloproteases MNP- 2 (""""""""gelatinase"""""""") and MMP-3 (""""""""proteoglycanase"""""""")) that digest various extracellular matrix macromolecules, but these enzymes are secreted from the cells as inactive zymogens. The long-term objectives of the proposed research are to investigate the mechanisms of activation of these zymogens and to identify active enzymes in situ in order to understand their roles in pathological destruction as well as in normal turnover of the connective tissue matrix. Three zymogens (procollagenase, proMMP-2 and proMMP-3) will be purified from human rheumatoid synovial cells in culture by various chromatographic techniques. The primary structure of proMMP-2 will be deduced from the DNA sequence which is complementary to proMMP-2 mRNA. The sequence information is used to investigate the mechanisms of activation of the proenzymes at the sequence level. Various tissue and plasma proteases and 4-aminophenylmercuric acetate will be tested for their abilities to activate proMMP-2 and proMMP-3, and their actions on proenzymes will be characterized by the NH2-terminal sequence analyses of active enzymes. The """"""""cascade"""""""" hypothesis for the accelerated activation of these proenzymes will be also examined by the recombination of zymogens and activated enzymes. Enzymic properties of MMP-2 and MMP-3 will be further investigated by their action on the basement membrane components such as type IV collagen, laminin and eutactin. The substrate specificities of these enzymes are characterized by their action on reduced, carboxymethylated transferrin. Attempts will be made to localize active MMP-3 in the tissues where rapid matrix resorption is occurring. Antibodies specific to the propeptide region of proMMP-3 and monoclonal antibodies specific to the active MMP-3 will be prepared, and used for the dual localization of both active and zymogen forms of MMP-3 in human rheumatoid synovium and articular cartilage, and rabbit postpartum uterus and interleukin 1-treated articular cartilage. Once the detailed knowledge is obtained about the zymogen activation, enzyme specificities, and the sites of activation in the tissue, it may be possible to suggest specific ways to limit the unwanted proteolysis of the extracellular matrix that occurs in a variety of connective tissue diseases.
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