PROTEIN SYNTHESIS POST-TRANSLATION MODIFICATION AND DEGRADATION
Structural Determinants of Procryptdin Recognition and Cleavage by Matrix Metalloproteinase-7*

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The bactericidal activity of mouse Paneth cell αdefensins, or cryptdins, is dependent on processing of cryptdin precursors (pro-Crps) by matrix metalloproteinase-7 (MMP-7) (Wilson, C. L., Ouellette, A. J., Satchell, D. P., Ayabe, T., Lopez-Boado, Y. S., Stratman, J. L., Hultgren, S. J., Matrisian, L. M., and Parks, W. C. (1999) Science 286, 113–117). To investigate the mechanisms of pro-Crp processing by this enzyme, recombinant pro-Crp4, a His-tagged chimeric pro-Crp (pro-CC), and site-directed mutant precursors of each were digested with MMP-7, and the cleavage products were analyzed by NH2-terminal peptide sequencing. Proteolysis of pro-Crp4 with MMP-7 activated in vitrobactericidal activity to the level of the mature Crp4 peptide by cleaving pro-Crp4 at Ser43↓Ile44 and Ala53↓Leu54 in the proregion and near the Crp4 peptide NH2 terminus between Ser58↓Leu59. Because the Crp4 NH2terminus occurs at Gly61, not Leu59, MMP-7 is necessary but insufficient to complete the processing of Crp4. Crp activating proteolysis at S58↓L59 was unaffected by I44S/I44D or L54S/L54D loss-of-function mutations in pro-Crp4, and a (L59S)-pro-CC mutant was cleaved normally at Ser43↓Val44and Ser53↓Leu54 sites but not at the peptide NH2 terminus. C57BL/6 mice contain an abundant (L59S)-Crp4 mutant peptide with Leu54 at its NH2 terminus resulting from Ala53↓Leu54 cleavage and loss-of-function at the Ser58↓Ser59 cleavage site. Thus, α-defensins resulting from mutations at MMP-7 cleavage sites exist in mouse populations. A pro-CC substrate containing both L54S and L59S mutations resisted cleavage at Ser43↓Val44 completely, showing that cleavage at one or both downstream sites must precede proteolysis at Ser43↓Val44. These findings show that MMP-7 activation of pro-Crps can occur without proteolysis of the proregion, and prosegment fragmentation depends, at least in part, on the release of the Crp peptide from the precursor.

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Published, JBC Papers in Press, December 13, 2002, DOI 10.1074/jbc.M210600200

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This work was supported by National Institutes of Health Grants DK10184 (to D. P. S), DE14040 (to C. L. W.), and DK44632 (to A. J. O.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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Current address: The Dow Chemical Company, Biocides R&D, Buffalo Grove, IL 60089.

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To whom correspondence may be addressed: Division of Allergy and Pulmonary Medicine, Dept. of Pediatrics, Washington University School of Medicine, St. Louis, MO 63110. Tel.: 314-286-2861; Fax: 314-286-2894; E-mail: [email protected].

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To whom correspondence may be addressed: Dept. of Pathology, College of Medicine, University of California, Irvine, CA 92697-4800. Tel.: 949-824-4647; Fax: 949-824-1098; E-mail: [email protected].