Abstract
In mouse Paneth cells, alpha-defensins, termed cryptdins (Crps), are activated by matrix metalloproteinase-7-mediated proteolysis of inactive precursors (pro-Crps) to bactericidal forms. The activating cleavage step at Ser(43) downward arrow Ile(44) in mouse pro-Crp4-(20-92) removes nine acidic amino acids that collectively block the membrane-disruptive behavior of the Crp4 moiety of the proform. This inhibitory mechanism has been investigated further to identify whether specific cluster(s) of electronegative amino acids in pro-Crp4-(20-43) are responsible for blocking bactericidal activity and membrane disruption. To test whether specific cluster(s) of electronegative amino acids in pro-Crp4-(20-43) have specific positional effects that block bactericidal peptide activity and membrane disruption, acidic residues positioned at the distal (Asp(20), Asp(26), Glu(27), and Glu(28)), mid (Glu(32) and Glu(33)), and proximal (Glu(37), Glu(38), and Asp(39)) clusters in pro-Crp4-(20-92) were mutagenized, and variants were assayed for differential effects of mutagenesis on bactericidal peptide activity. Substitution of the mid and proximal Asp and Glu clusters with Gly produced additive effects with respect to the induction of both bactericidal activity and membrane permeabilization of live Escherichia coli ML35 cells. In contrast, substitution of distal Glu and Asp residues with Gly or their deletion resulted in pro-Crp4-(20-92) variants with bactericidal and membrane-disruptive activities equal to or greater than that of fully mature Crp4. These findings support the conclusion that the most distal N-terminal anionic residues of pro-Crp4-(20-92) are primarily responsible for blocking Crp4-mediated membrane disruption in the precursor.
Highlights
In the small bowel, Paneth cells at the base of the crypts of Lieberkuhn secrete ␣-defensins and additional antimicrobial peptides at high levels in response to cholinergic stimulation and when exposed to bacterial antigens [1,2,3,4]
To test whether specific cluster(s) of electronegative amino acids in pro-Crp4-(20 – 43) have specific positional effects that block bactericidal peptide activity and membrane disruption, acidic residues positioned at the distal (Asp20, Asp26, Glu27, and Glu28), mid (Glu32 and Glu33), and proximal (Glu37, Glu38, and Asp39) clusters in pro-Crp4-(20 –92) were mutagenized, and variants were assayed for differential effects of mutagenesis on bactericidal peptide activity
Pro-Crp4 requires matrix metalloproteinase-7 (MMP-7)-mediated proteolytic conversion to Crp4, and disruption of disulfide pairings would result in extensive peptide proteolysis [22]
Summary
Paneth cells at the base of the crypts of Lieberkuhn secrete ␣-defensins and additional antimicrobial peptides at high levels in response to cholinergic stimulation and when exposed to bacterial antigens [1,2,3,4]. To test whether specific cluster(s) of electronegative amino acids in pro-Crp4-(20 – 43) have specific positional effects that block bactericidal peptide activity and membrane disruption, acidic residues positioned at the distal (Asp20, Asp26, Glu27, and Glu28), mid (Glu32 and Glu33), and proximal (Glu37, Glu38, and Asp39) clusters in pro-Crp4-(20 –92) were mutagenized, and variants were assayed for differential effects of mutagenesis on bactericidal peptide activity.
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