Catalytic activities and substrate specificity of the human membrane type 4 matrix metalloproteinase catalytic domain

Abstract

Membrane type (MT)-matrix metalloproteinases (MMPs) are recently recognized members of the family of Zn2+- and Ca2+-dependent MMPs. To investigate the proteolytic capabilities of human MT4-MMP (i.e. MMP-17), we have cloned DNA encoding its catalytic domain (CD) from a breast carcinoma cDNA library. Human membrane type 4 MMP CD (MT4-MMPCD) protein, expressed as inclusion bodies in Escherichia coli, was purified to homogeneity and refolded in the presence of Zn2+ and Ca2+. While MT4-MMPCD cleaved synthetic MMP substrates Ac-PLG-[2-mercapto-4-methylpentanoyl]-LG-OEt and Mca-PLGL-Dpa-AR-NH2 with modest efficiency, it catalyzed with much higher efficiency the hydrolysis of a pro-tumor necrosis factor-α converting enzyme synthetic substrate, Mca-PLAQAV-Dpa-RSSSR-NH2. Catalytic efficiency with the pro-tumor necrosis factor-α converting enzyme substrate was maximal at pH 7.4 and was modulated by three ionizable enzyme groups (pK(α3) = 6.2, pK(α2) = 8.3, and pK(α1) = 10.6). MT4-MMPCD cleaved gelatin but was inactive toward type I collagen, type IV collagen, fibronectin, and laminin. Like all known MT-MMPs, MT4-MMPCD was also able to activate 72-kDa progelatinase A to its 68-kDa form. EDTA, 1,10-phenanthroline, reference hydroxamic acid MMP inhibitors, tissue inhibitor of metalloproteinases-1, and tissue inhibitor of metalloproteinases-2 all potently blocked MT4-MMPCD enzymatic activity. MT4-MMP is, therefore, a competent Zn2+-dependent MMP with unique specificity among synthetic substrates and the capability to both degrade gelatin and activate progelatinase A.