PMID:12482850

Citation	Shirafuji, Y, Tanabe, H, Satchell, DP, Henschen-Edman, A, Wilson, CL and Ouellette, AJ (2003) Structural determinants of procryptdin recognition and cleavage by matrix metalloproteinase-7. J. Biol. Chem. 278:7910-9
Abstract	The bactericidal activity of mouse Paneth cell alphadefensins, 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 NH(2)-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 Ser(43) downward arrow Ile(44) and Ala(53) downward arrow Leu(54) in the proregion and near the Crp4 peptide NH(2) terminus between Ser(58) downward arrow Leu(59). Because the Crp4 NH(2) terminus occurs at Gly(61), not Leu(59), MMP-7 is necessary but insufficient to complete the processing of Crp4. Crp activating proteolysis at S58 downward arrow 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 Ser(43) downward arrow Val(44) and Ser(53) downward arrow Leu(54) sites but not at the peptide NH(2) terminus. C57BL/6 mice contain an abundant (L59S)-Crp4 mutant peptide with Leu(54) at its NH(2) terminus resulting from Ala(53) downward arrow Leu(54) cleavage and loss-of-function at the Ser(58) downward arrow Ser(59) cleavage site. Thus, alpha-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 Ser(43) downward arrow Val(44) completely, showing that cleavage at one or both downstream sites must precede proteolysis at Ser(43) downward arrow Val(44). 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.
Links	PubMed Online version:10.1074/jbc.M210600200
Keywords	Amino Acid Sequence; Animals; Base Sequence; DNA Primers; Disulfides/chemistry; Hydrolysis; Male; Matrix Metalloproteinase 7/metabolism; Mice; Mice, Inbred C57BL; Mice, Knockout; Molecular Sequence Data; Mutagenesis, Site-Directed; Protein Precursors/chemistry; Protein Precursors/genetics; Protein Precursors/metabolism; Protein Precursors/physiology; Proteins/chemistry; Proteins/genetics; Proteins/metabolism; Proteins/physiology; Recombinant Proteins/chemistry; Recombinant Proteins/genetics; Recombinant Proteins/metabolism; Sequence Homology, Amino Acid; alpha-Defensins
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
MOUSE:Q6GXA3		GO:0051605: protein maturation by peptide bond cleavage	ECO:0000314:		P		Figure 1 shows a gel indicating that pro-Crp4 is cleaved when incubated with MMP7. Additionally, Figure 3 shows that MMP7 activates the bactericidal activity of Crp4.	complete

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