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Abstract
Several recent investigations have demonstrated that matrix metalloproteinase-2 (MMP-2) binds to the cell surface and undergoes zymogen activation via a plasma membrane-associated activity. The purpose of this study was to determine if association of MMP-2 with the plasma membrane also modulates the catalytic efficiency of the active enzyme. Using density gradient centrifugation, we isolated the plasma membrane fractions of two ovarian adenocarcinoma cell lines, DOV 13 and OVCA' 432, previously described either to express MMP-2 or to express no gelatinolytic metalloproteinases, respectively. While DOV 13 cells contained plasma membrane-associated MMP-2 and OVCA 432 did not, both cell types were able to bind exogenous MMP-2. Furthermore, plasma membrane fractions from these cells significantly enhanced the rate of cleavage of [14C]gelatin I substrate by both MMP-2 tissue inhibitor of metalloproteinases-2 (TIMP-2) complex (2.5-8-fold) and TIMP-2-free MMP-2 (5.9-fold). This stimulatory activity was dose-dependent, soluble in Triton X-100, and abolished by trypsin treatment of the membranes, but was stable to heat treatment. Plasma membrane stimulation of MMP-2 resulted in a 3.8-4.6-fold increase in the catalytic efficiency of gelatinolysis. These data suggest that, in addition to promoting zymogen activation, cell surface binding of MMP-2 may regulate enzyme activity by increasing the rate of substrate cleavage. Via this mechanism, tumor cell types that do not express MMPs (such as OVCA 432) nevertheless may be able to utilize exogenous MMP-2 to mediate proteolysis associated with invasion and metastasis.
Highlights
From the Department of Pathology, Duke University Medical Center, Durham, North Carolina 27710 and the Wepartment of Obstetrics and Gynecology, Northwestern University Medical School, Chicago, Illinois 60611
Several recent investigations have demonstrated that matrix metalloproteinase-2 (MMP-2) binds to the cell surface and undergoes zymogen activation via a plasma membrane-associated activity
Plasma membrane fractions from these cells significantly enhanced the rate of cleavage of [ 14C]gela t in I substrate by both matrix metalloproteinases (MMPs)·2·tissue inhibitor of metalloproteinases-2 (TIMP-2) complex (2.5-8-fold) and TIMP-2-free MMP-2 (5.9-fold)
Summary
Plasma membrane stimulation ofMMP-2 resulted in a 3.8-4.6-fold increase in the catalytic efficiency of gelatinolysis These data suggest that, in addition to promoting zymogen activation, cell surface binding ofMMP-2 may regulate enzyme activity by increasing the rate of substrate cleavage. Via this mechanism, tumor cell types that do not express MMPs (such as OVCA 432) may be able to utilize exogenous MMP·2 to mediate proteolysis associated with invasion and metastasis. The current study is designed to assess the effect of membrane association on the catalytic activity of active MMP-2 To address this question, we have prepared the plasma membrane fractions of two ovarian adenocarcinoma cell lines, DOV 13 and OVCA 432, which either express MMP-2 or express no gelatinolytic metalloproteinases, respectively (19). Our data demonstrate that the catalytic activity of MMP-2 is enhanced by plasma membrane association, suggesting a mechanism by which the activity of MMPs may be regulated
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