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.