Matrix metalloproteinase-7 activation of mouse paneth cell pro-α-defensins: SER⁴³ ↓ ILE⁴⁴ proteolysis enables membrane-disruptive activity

Small intestinal Paneth cells secrete α-defensin microbicidal peptides as mediators of innate enteric immunity. In mice, production of mature Paneth cell α-defensins, termed cryptdins (Crps), requires proteolytic activation of inactive precursors (pro-Crps) by the convertase matrix metalloproteinase-7. Proteolysis of mouse (pro-Crp4)^20-92 produces the specific cleavage intermediates pro-Crp4^44-92, pro-Crp4 ^54-92, and pro-Crp4^59-92. To identify which cleavage event enables bactericidal activity, recombinant pro-Crp4-processing intermediates were purified to homogeneity and assayed for bactericidal peptide activity. The in vitro bactericidal activities of pro-Crp4-processing intermediates were very similar to fully processed Crp4, contrasting the lack of bactericidal and membrane-disruptive activity shown by pro-Crp4^20-92. Thus, cleavage of pro-Crp4^20-92 at Ser⁴³ ↓ Ile⁴⁴ is sufficient to activate bactericidal activity, and amino acids in the pro-Crp4^20-43 of the proregion maintain the precursor in an inactive state. Because cationic Arg residues are determinants of Crp4 bactericidal peptide activity, we hypothesized that Asp and Glu residues in pro-Crp4 ^20-43 neutralize Crp4 Arg side chains in pro-Crp4^20-92. Therefore, a pro-Crp4^20-92 variant with Gly substitutions at all pro-Crp4^20-43 Asp and Glu positions ((DE/G)-pro-Crp4) was prepared, and it was bactericidal and lysed phospholipid vesicles under conditions where native pro-Crp4^20-92 lacks activity. These findings show that MMP-7 proteolysis of pro-Crp4^20-92 at Ser⁴³ ↓ Ile ⁴⁴ converts inactive precursors to bactericidal forms by removal of covalently associated, inhibitory acidic amino acids from proximity with the Crp4 component of the molecule.