The matrix metalloproteinase stromelysin-3 (ST3 or MMP11) was initially identified as a breast carcinoma associated protease and has since been shown to be highly expressed in diverse carcinomas and in developmental processes that involve extensive cell death (apoptosis) and tissue remodeling. Unlike other MMPs, purified ST3 has little activity toward known extracellular matrix (ECM) proteins in vitro but cleaves strongly a few non-ECM, extracellular proteins, including human α1-proteinase inhibitor (α1-PI). To investigate the possibility of α1-PI as a conserved physiological substrate for ST3 during vertebrate development, we analyzed the ability of Xenopus laevis ST3 catalytic domain to cleave frog α1-PI. Surprisingly, we found the ST3 failed to recognize the site in α1-PI equivalent to the major cleavage site in human α1-PI by mammalian ST3. Sequence and mutagenic analysis revealed that multiple substitutions at P2-P3' positions between human and Xenopus α1-PI contributed to the inability of Xenopus α1-PI to be cleaved by ST3. Our studies showed that (A)(G/A)(A)(M)(F/A)(L) (P3-P3') as a preferred cleavage site for ST3. We further demonstrated that mutations in the cleavage sites affected cleavage by ST3 differently from several other MMPs. These findings, together with earlier reports on ST3, showed that ST3 has distinct substrate specificities compared to other MMPs. Our results further suggest that α1-PI is unlikely to be a physiological substrate for ST3, at least with regard to evolutionarily conserved developmental processes.

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