Characterization and regulation of MT1‐MMP cell surface‐associated activity

MT1‐MMP was stably expressed and a cell‐based FRET assay developed to quantify activity towards synthetic collagen‐model triple‐helices. Activity measurements were performed using a series of membrane‐anchored MT1‐MMP mutants and compared directly with those of soluble MT1‐MMP. Cell surface localization of MT1‐MMP was found to restrict substrate binding and protein coupled motions for catalysis. Small molecule and triple‐helical transition state analog MMP inhibitors were found to function similarly in solution and at the cell surface.

Abstract

Quantitative assessment of MT1‐MMP cell surface‐associated proteolytic activity remains undefined. Presently, MT1‐MMP was stably expressed and a cell‐based FRET assay developed to quantify activity towards synthetic collagen‐model triple‐helices. To estimate the importance of cell surface localization and specific structural domains on MT1‐MMP proteolysis, activity measurements were performed using a series of membrane‐anchored MT1‐MMP mutants and compared directly with those of soluble MT1‐MMP. MT1‐MMP activity (k_cat/K_M) on the cell surface was 4.8‐fold lower compared with soluble MT1‐MMP, with the effect largely manifested in k_cat. Deletion of the MT1‐MMP cytoplasmic tail enhanced cell surface activity, with both k_cat and K_M values affected, while deletion of the hemopexin‐like domain negatively impacted K_M and increased k_cat. Overall, cell surface localization of MT1‐MMP restricts substrate binding and protein coupled motions (based on changes in both k_cat and K_M) for catalysis. Comparison of soluble and cell surface‐bound MT2‐MMP revealed 12.9‐fold lower activity on the cell surface. The cell‐based assay was utilized for small molecule and triple‐helical transition state analog MMP inhibitors, which were found to function similarly in solution and at the cell surface. These studies provide the first quantitative assessments of MT1‐MMP activity and inhibition in the native cellular environment of the enzyme.

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