Membrane type-1 matrix metalloproteinase functions as a proprotein self-convertase. Expression of the latent zymogen in Pichia pastoris, autolytic activation, and the peptide sequence of the cleavage forms.

An understanding of the regulatory mechanisms that control the activity of membrane type-1 matrix metalloproteinase (MT1-MMP), a key proteinase in tumor cell invasion, is essential for the design of potent and safe anti-cancer therapies. A unique proteolytic pathway regulates MT1-MMP at cancer cell surfaces. The abundance of proteolytic enzymes in cancer cells makes it difficult to identify the autocatalytic events in this pathway. To identify these events, a soluble form of MT1-MMP, lacking the C-terminal transmembrane and cytoplasmic domains, was expressed in Pichia pastoris. Following secretion, the latent zymogen and active enzyme were each purified from media by fast protein liquid chromatography. Trace amounts of active MT1-MMP induced activation of the zymogen and its self-proteolysis. This autocatalytic processing generated six main forms of MT1-MMP, each of which was subjected to the N-terminal microsequencing to identify the cleavage sites. Our data indicate that MT1-MMP functions as a self-convertase and is capable of cleaving its own prodomain at the furin cleavage motif RRKR downward arrow Y(112), thus autocatalytically generating the mature MT1-MMP enzyme with an N terminus starting at Tyr(112). The mature enzyme undergoes further autocatalysis to the two distinct intermediates (N terminus at Trp(119) and at Asn(130)) and, next, to the three inactive ectodomain forms (N terminus at Thr(222), at Gly(284), and at Thr(299)). These findings provide, for the first time, a structural basis for understanding the unconventional mechanisms of MT1-MMP activation and regulation. Finally, our data strongly imply that MT1-MMP is a likely substitute for the general proprotein convertase activity of furin-like proteinases, especially in furin-deficient cancer cells.