Structural determinants of procryptdin recognition and cleavage by matrix metalloproteinase-7

The bactericidal activity of mouse Paneth cell alpha-defensins, or cryptdins, is dependent on processing of cryptdin precursors (pro-Crps) by matrix metallo-proteinase-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 vitro bactericidal activity to the level of the mature Crp4 peptide by cleaving pro-Crp4 at Ser43 down arrow Ile(44) and Ala(53) down arrow Leu(54) in the proregion and near the Crp4 peptide NH2 terminus between Ser(58) down arrow Leu(59). Because the Crp4 NH2 terminus occurs at Gly(61), not Leu(59), AMP-7 is necessary but insufficient to complete the processing of Crp4. Crp activating proteolysis at S58 down arrow L59 was unaffected by 144S/144D or L54S/L54D loss-of-function mutations in pro-Crp4, and a (L59S)-pro-CC mutant was cleaved normally at Ser(43) down arrow Val(44) and Ser(53) down arrow Leu(54) sites but not at the peptide NH2 terminus. C57BL/6 mice contain an abundant (L59S)-Crp4 mutant peptide with Leu(54) at its NH2 terminus resulting from Ala(53) down arrow Leu(54) cleavage and loss-of-function at the Ser(58) down arrow Ser(59) cleavage site. Thus, a-defensins resulting from mutations at AMP-7 cleavage sites exist in mouse populations. A pro-CC substrate containing both L54S and L59S mutations resisted cleavage at Ser(43) down arrow Val(44) completely, showing that cleavage at one or both downstream sites must precede proteolysis at Ser(43) down arrow Val(44). These findings show that NHdP-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.