The matrix metalloproteinase (MMP) family of enzymes has been functionally linked to the remodeling of extracellular matrix manifested by a variety of invasive and migratory cells. The gen structure and enzymatic capabilities of the MMPs have been extensively studied, but the regulation and mechanism of how their zymogen forms are activated in various biological systems remains undefined. In this proposal we will examine the hypothesis that the production by transformed cells of highly activatable TIMP-free 72 kDa progelatinase (MMP-2) is a requisite for the cell- mediated activation of the progelatinase. The testing of the hypothesis will include the addition to cultures that are undergoing transformation, purified TIMP-2 to titer out the endogenously produced TIMP-free progelatinase. The appearance of active 62 kDa gelatinase in these and control cultures will be quantitated. Radiolabeled TIMP-free 72 kDa progelatinase, also will be added to normal and transformed cultures and the appearance of radiolabeled 62 kDa gelatinase will be monitored. How transformed cells generate TIMP-free 72 kDa progelatinase will be explored utilizing our newly cloned cDNAs for chicken TIMP-2 and MMP-2. A possibly unique cellular mechanism for 72 kDa progelatinase activation will also b investigated. We have demonstrated that 62 kDa gelatinase can cleave native triple helical type I collagen int 3/4 and 1/4 fragments at the precise site in collagen cleaved by interstitial collagenase (MMP-1). We propose to initiate a structural and functional analysis of this new triple helicase activity. The kinetic constants, Kcat, Km, will be determined for MMP-2's triple helicase activity and compared to that of MMP-1. The ability of active MMP-2 to hydrolyze native collagen fibrils also will be ascertained. The protein domains in MMP-2 that are involved in the triple helicase activity will be investigated using deletion mutagenesis. A unique monoclonal antibody (mAb 6-6B) that specifically reacts with human MMP-9 and inhibits its catalytic activity will be used to test the involvement of MMP-9 in human tumor cell invasion in vitro and human tumor metastasis in vivo. In addition, the specificity of mAb 6-6B in Western blots (it recognizes zymogen and all activated forms of MMP-9) will be used to determine if pro-MMP-9, which is not activated under non-invading, culture conditions, is activated during the invasion process.
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