Proregion anionic amino acids mediate inhibition of mouse pro‐α‐defensin bactericidal activity

Abstract

Mouse Paneth cell α‐defensins are activated by proteolysis of inactive precursors, e.g., proCrp4 to bactericidal forms by the convertase MMP‐7. The activating step cleaves 24 residues, including 9 acidic amino acids that collectively block the membrane disruptive behavior of the Crp4 peptide (J Biol. Chem 281:28932–28943 2006). This inhibitory mechanism has been investigated further to identify whether specific cluster(s) of anionic amino acids in proCrp4(20–43) are preferentially responsible for blocking bactericidal activity and membrane disruption. To test this hypothesis, sets of Asp and Glu in proCrp4(20–43) were mutagenized to Gly. The recombinant proCrp4 variant peptides were expressed in E. coli, purified by HPLC to homogeneity and then assayed for differential effects of bactericidal inactivity. Generally, Gly for Glu/Asp replacements produced additive effects with respect to both bactericidal activity and membrane disruption of live E. coli ML35, regardless of their position. These findings support the conclusion that Asp and Glu residues contribute additively via intramolecular interactions to neutralize Crp4 membrane disruptive mechanisms. Supported by NIH grants DK044632 and AI059346