Mutational analysis of the transin (rat stromelysin) autoinhibitor region demonstrates a role for residues surrounding the 'cysteine switch'

A. J. Park, L. M. Matrisian, A. F. Kells, R. Pearson, Z. Yuan, M. Navre

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91 Scopus citations

Abstract

The family of mammalian extracellular matrix metalloproteases (MMPs) are secreted by cells in an inactive (latent) proenzyme form. A highly conserved amino acid sequence, PRCGVPDV, is found near the COOH-terminal end of the pro-domain of these MMPs and believed to act as an 'autoinhibitor.' Recent studies (Springman, E.B., Angleton, E.L., Birkedal-Hansen, H., and Wart, H.E.V. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 364-368) indicate the Cys of this sequence ligands to the active-site zinc keeping the proenzyme in an inactive state, and mutational analysis (Sanchez-Lopez,R., Nicholson, R., Gesnel, M. C., Matrisian, L.M., and Breathnach, R. (1988) J. Biol. Chem. 263, 11892-11899) suggests that the conserved residues surrounding this Cys are required for latency. We have constructed 16 new site-directed mutations of the PRCGVPDV autoinhibitor region of the MMP transin (rat stromelysin) and tested whether these mutant enzymes are produced in a latent or activated form. We find that the conserved Arg as well as the Cys are essential for maintaining latency. The Cys cannot be replaced by other zinc-liganding amino acids, and the Arg cannot be replaced by Lys. Residues immediately surrounding the Cys are sensitive to even conservative amino acid substitutions. We show that a synthetic peptide PRCGVPDV is capable of acting as a weak inhibitor of transin and that replacement of the Cys with a Ser abolishes inhibition by the peptide. A review of the current knowledge of MMP substrate specificity in combination with these new results suggests that the PRCGVPDV sequence does not inhibit activity by mimicking the known substrates of the protease.

Original languageEnglish
Pages (from-to)1584-1590
Number of pages7
JournalJournal of Biological Chemistry
Volume266
Issue number3
StatePublished - 1991

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