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

Dmitri Rozanov, Alex Y. Strongin

Research output: Contribution to journalArticle

41 Citations (Scopus)

Abstract

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 ↓ Y112, thus autocatalytically generating the mature MT1-MMP enzyme with an N terminus starting at Tyr112. The mature enzyme undergoes further autocatalysis to the two distinct intermediates (N terminus at Trp119 and at Asn130) and, next, to the three inactive ectodomain forms (N terminus at Thr222, at Gly284, and at Thr299). 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.

Original languageEnglish (US)
Pages (from-to)8257-8260
Number of pages4
JournalJournal of Biological Chemistry
Volume278
Issue number10
DOIs
StatePublished - Mar 7 2003
Externally publishedYes

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Proprotein Convertases
Matrix Metalloproteinase 14
Enzyme Precursors
Pichia
Chemical activation
Peptides
Furin
Cells
Peptide Hydrolases
Neoplasms
Enzymes
Proteolysis
Liquid chromatography
Liquid Chromatography
Tumors

ASJC Scopus subject areas

  • Biochemistry

Cite this

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title = "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",
abstract = "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 ↓ Y112, thus autocatalytically generating the mature MT1-MMP enzyme with an N terminus starting at Tyr112. The mature enzyme undergoes further autocatalysis to the two distinct intermediates (N terminus at Trp119 and at Asn130) and, next, to the three inactive ectodomain forms (N terminus at Thr222, at Gly284, and at Thr299). 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.",
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T2 - Expression of the latent zymogen in Pichia pastoris, autolytic activation, and the peptide sequence of the cleavage forms

AU - Rozanov, Dmitri

AU - Strongin, Alex Y.

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N2 - 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 ↓ Y112, thus autocatalytically generating the mature MT1-MMP enzyme with an N terminus starting at Tyr112. The mature enzyme undergoes further autocatalysis to the two distinct intermediates (N terminus at Trp119 and at Asn130) and, next, to the three inactive ectodomain forms (N terminus at Thr222, at Gly284, and at Thr299). 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.

AB - 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 ↓ Y112, thus autocatalytically generating the mature MT1-MMP enzyme with an N terminus starting at Tyr112. The mature enzyme undergoes further autocatalysis to the two distinct intermediates (N terminus at Trp119 and at Asn130) and, next, to the three inactive ectodomain forms (N terminus at Thr222, at Gly284, and at Thr299). 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.

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