In vitro activation of pro‐α‐defensins by neutrophil serine proteinases of rhesus macaque

Abstract

α‐Defensins are abundant antimicrobial peptides in the azurophilic granules of phagocytic leukocytes or in the Paneth cells of small intestine. To investigate the mechanisms of myeloid pro‐α‐defensin processing, recombinant pro‐α‐defensin 4 (proRMAD4) was exposed to human neutrophil elastase (HNE), cathepsinG (cG) and proteinase 3 (P3) from azurophil granules and analyzed biochemically and in bacterical peptide assays. ProRMAD4 was converted in vitro to active RMAD4 by these proteases. Only HNE cleaved at the native RMAD4 N‐terminus. cG and P3 activated proRMAD4, but they cleaved at different sites within the proregion. Mutagenesis of acidic proregion amino acids (Asp,Glu) to charge neutral (Asn, Gln) did not significantly alter the bactericidal property of proRMAD4 suggesting H‐bonding interactions also participate in the peptide inhibition. Disulfide variants of proRMAD4 were extensively degraded by HNE, cG and P3 showing that the disulfide array protects the α‐defensin from proteolysis during conversion to the bactericidal form. These data support the hypothesis that azurophil serine proteinases activate macaque myeloid α‐defensins and convert them to active forms. This research was supported by NIH grants DK044632, AI059346, Human Frontiers Science Project