Cationic amino acids contribute to ␣-defensin bactericidal activity. Curiously, although Arg and Lys have equivalent electropositive charges at neutral pH, ␣-defensins contain an average of nine Arg residues per Lys residue. To investigate the role of high ␣-defensin Arg content, all Arg residues in mouse Paneth cell ␣-defensin cryptdin 4 (Crp4) and rhesus myeloid ␣-defensin 4 (RMAD-4) were replaced with Lys to prepare (R/K)-Crp4 and (R/K)-RMAD-4, respectively. Lys-for-Arg replacements in Crp4 attenuated bactericidal activity and slowed the kinetics of Escherichia 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, Arg3Lys substitutions attenuated activity in Crp4 but not in RMAD-4, and the functional consequences of Arg3Lys replacements in ␣-defensins are dependent on the 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 those of the native peptides, suggesting that the high Arg content of ␣-defensins may be under selection to confer superior microbicidal function under physiologic conditions.

Human immunodeficiency virus type 2 (HIV-2) evolved from the zoonotic transmission of simian immunodeficiency virus (SIV) that naturally infects sooty mangabeys found in West Africa. Using sera from HIV-2-infected humans, we discovered that an hypervariable region (the V4 loop) of HIV-2 induces antibody responses only weakly reactive against itself but strongly reactive against analogous sequences from the V4 loop of strains of SIV. Available sequence data indicates that all strains of HIV-2 have large deletions in the V4 region that truncate an immunodominant neutralizing B cell epitope among strains of SIV. Infection of a macaque with a sequenced clone of HIV-2 similarly elicited antibodies that poorly recognized the V4 loop of HIV-2 but readily bound to analogous SIV sequences. Our data are consistent with a scenario whereby a disparate antibody response directed against the V4 loop may have influenced the selective expansion and survival of HIV-2 in humans.

Mammalian α‐defensins are endogenous cationic antimicrobial peptides that contain an average of nine times more Arg than Lys. To investigate the functional role of such high Arg content, Arg residues in the mouse Paneth cell α‐defensin cryptdin‐4 (Crp4) and rhesus myeloid α‐defensin RMAD4 were completely replaced with Lys to prepare (R/K)‐Crp4 and (R/K)‐RMAD4, respectively. Arg mutagenesis of Crp4 attenuated peptide bactericidal activity against several species and the kinetics of (R/K)‐Crp4‐mediated E. coli ML35 cell permeabilization were markedly slower than Crp4, needing a 20 min peptide exposure to detect permeabilization. In contrast to the attenuating effects of Arg to Lys replacements on Crp4, corresponding mutations in RMAD4 improved both microbicidal activity against certain bacterial species and the ability to permeabilize live E. coli. Therefore, although Arg→Lys substitutions in Crp4 delay E. coli cell permeabilization and extend exposure times required to induce bacterial cell death, comparable mutagenesis of RMAD4 does not attenuate either activity. Collectively, these findings are inconsistent with the positive selection of Arg in α‐defensins solely to confer selective advantage through superior antibacterial function. Supported by NIH grants DK044632 and AI059346.

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Paneth cells (PCs) occur in small intestinal crypts from which they secrete α‐defensins at high levels. To investigate rhesus macaque enteric α‐defensin (REDs) diversity, ileal mucosal protein and mRNAs were characterized. Analyses of ileum α‐defensin cDNA and gene sequences identified 20 distinct RED cDNAs and 7 RED genes not represented in the cDNA population. Certain deduced RED proteins coded by abundant mRNAs have mutations at canonical α‐defensin structural features. α‐Defensin proteins isolated from ileum occur predominantly as proREDs with negligible processed REDs detected by acid‐urea PAGE and MALDI‐TOF MS. In vitro, purified natural and recombinant proREDs were activated by trypsin to yield structurally intact, mature RED peptides. Because mature RED peptides are lacking from ileal PCs and mouse PC α‐defensins persist in colonic lumen (J. Biol. Chem. 284: 27848–27856, 2009), colonic luminal contents were assayed for evidence of post‐secretory proRED processing. Mature, intact RED peptides were evident in large bowel lumen, far downstream from their small intestinal site of secretion, and they were identified as α‐defensins by Edman sequencing. The processed state of α‐defensins in rhesus PCs resembles that in human PCs and differs from the mouse PCs, where activated, mature α‐defensins are abundant in PC dense core granules. Supported by NIH grants AI059346 and DK044632.