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

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 DN A encoding its catalytic domain (CD) from a breast carcinoma cDNA library. Human membrane type 4 MMP CD (MT4-MMPCD) protein, expressed as inclusion bo dies in Escherichia coli, was purified to homogeneity and refolded in the p resence 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 m odest efficiency, it catalyzed with much higher efficiency the hydrolysis o f a pro-tumor necrosis factor-ct converting enzyme synthetic substrate; Mca -PLAQAV-DpaR-SSSR-NH2. Catalytic efficiency with the pro-tumor necrosis fac tor-a: converting enzyme substrate was maximal at pH 7.4 and was modulated by three ionizable enzyme groups (pK(a3) = 6.2, pK(a2) = 8.3, and pK(a1) = 10.6). MT4-MMPCD cleaved gelatin but was inactive toward type I collagen, t ype IV collagen, fibronectin, and laminin. Like all known MT-MMPs, MT4-MMPC D was also able to activate 72-kDa progelatinase A to its 68-kDa form. EDTA , 1,10-phenanthroline, reference hydroxamic acid MMP inhibitors, tissue inh ibitor of metalloproteinases-l, and tissue inhibitor of metalloproteinases- a all potently blocked MT4-MMPCD enzymatic activity. MT4-MMP is, therefore, a competent Zn2+-dependent MMP with unique specificity among synthetic sub strates and the capability to both degrade gelatin and activate progelatina se A.