Studies of Alpha-Defensins and their Precursors in Rhesus Macaques

Mammalian alpha-defensins mediate innate immunity primarily in phagolysosomes of neutrophils and in the intestine following secretion by small intestinal Paneth cells. alpha-Defensins are ∼ 4 kDa, cationic and amphipathic peptides with broad-spectrum bactericidal activities. Structurally, they consist of a three-stranded beta-sheet structure that is established by three invariantly paired disulfide bonds. Despite having highly diverse primary structures, alpha-defensins retain conserved biochemical features that include a canonical Arg-Glu salt bridge, a conserved Gly residue at CysIII+8, and high Arg content relative to Lys. In vitro, alpha-defensins are microbicidal against gram-positive and gram-negative bacteria, fungi, spirochetes, protozoa, and enveloped viruses.;Cationic amino acids are essential to alpha-defensin bactericidal activity. Curiously, although Arg and Lys have equivalent electropositive charge at neutral pH, alpha-defensins contain an average of nine Arg per Lys residue. To investigate the role of high alpha-defensin Arg content, all Arg residues in mouse Paneth cell alpha-defensin cryptdin-4 (Crp4) and rhesus myeloid alpha-defensin-4 (RMAD-4) were replaced with Lys to prepare (R/K)-Crp4 and (R/K)-RMAD-4. Lys for Arg replacements in Crp4 attenuated bactericidal activity, slowed the kinetics of E. coli ML35 cell permeabilization, and (R/K)-Crp4 required longer exposure times to reduce E. coli cell survival. In marked contrast, Lys substitutions in RMAD-4 improved microbicidal activity against certain bacteria and permeabilized E. coli more effectively. Therefore, Arg→Lys substitutions attenuated activity in Crp4 but not RMAD-4, and the functional consequences of Arg→Lys replacements in alpha-defensins are dependent on peptide primary structure. In addition, the bactericidal effects of (R/K)-Crp4 and (R/K)-RMAD-4 were more sensitive to inhibition by NaCl than the native peptides, suggesting that the high Arg content of alpha-defensins may be under selection to confer superior microbicidal function under physiologic conditions.;Although alpha-defensins are diverse in primary structure as activated mature peptides, their genes are translated into precursors containing highly conserved proregions which inactivate microbicidal activity. In both mice and humans, alpha-defensin precursors are inactive until the N-terminal proregion is released from the peptide by their respective convertase. In contrast, the processing states of alpha-defensins differ between mice and humans, with mice containing activated forms within their tissue and humans only the inactive precursor.;To investigate the rhesus macaque enteric alpha-defensin (RED) biology and peptide diversity, ileal mucosal protein and mRNAs were characterized. Analyses of rhesus ileum alpha-defensin cDNAs and alpha-defensin-coding genomic sequences identified fourteen diverse RED cDNAs as well as seven 2-exon alpha-defensin genes that were not represented in the cDNA population. Interestingly, several of the isolated ileal cDNAs and genes encode deduced rhesus alpha-defensins with mutations at canonical alpha-defensin residue positions. In protein extracts from macaque ileum, rhesus alpha-defensins occur predominantly as unprocessed proREDs with mature REDs detectable only at negligible levels. In vitro, natural and recombinant proREDs lack bactericidal peptide activity until converted into bactericidal forms by trypsin. Consistent with these data, mature RED peptides were detected and isolated from colonic luminal contents, and two peptides were identified as RED-14 and RED-15 by N-terminal sequencing. Thus, the processing and activation of Paneth cell alpha-defensins in macaque ileum resembles post-secretory activation of pro-alpha-defensins released into the small intestinal lumen by human Paneth cells. In contrast to humans, however, macaque Paneth cells secrete a markedly more diverse population of alpha-defensins than the two peptides released by human Paneth cells, and the molecules are evident in the large bowel lumen, far from their small intestinal site of secretion.