Abstract
Small intestinal Paneth cells secrete alpha-defensin microbicidal peptides as mediators of innate enteric immunity. In mice, production of mature Paneth cell alpha-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 Ser(43) downward arrowIle(44) 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 Ser(43) downward arrowIle(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.
Highlights
Small intestinal Paneth cells secrete ␣-defensin microbicidal peptides as mediators of innate enteric immunity
A pro-Crp420–92 variant with Gly substitutions at all pro-Crp420–43 Asp and Glu positions ((DE/G)-pro-Crp4) was prepared, and it was bactericidal and lysed phospholipid vesicles under conditions where native pro-Crp420–92 lacks activity. These findings show that matrix metalloproteinase-7 (MMP-7) proteolysis of pro-Crp420–92 at Ser432Ile44 converts inactive precursors to bactericidal forms by removal of covalently associated, inhibitory acidic amino acids from proximity with the Crp4 component of the molecule
The pro-Crp4processing intermediates were purified to homogeneity by C18 reversed-phase high-pressure liquid chromatography, which was verified by AU-PAGE analysis (Fig. 1B), and peptide masses were confirmed by MALDI-TOF mass spectrometry
Summary
Small intestinal Paneth cells secrete ␣-defensin microbicidal peptides as mediators of innate enteric immunity. Crp4, contrasting the lack of bactericidal and membrane-disruptive activity shown by pro-Crp420–92. Disulfide connectivities by NMR spectrometry (Fig. 2), ruling Membrane Binding and Disruption by Pro-Crp4-processing out the possibility that peptide bactericidal activities could Intermediates—The mechanisms of pro-Crp444–92 and proresult from disrupted ␣-defensin cysteine pairings.
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