In Vitro Activation of the Rhesus Macaque Myeloid α-Defensin Precursor proRMAD-4 by Neutrophil Serine Proteinases

Kamdar, Karishma; Maemoto, Atsuo; Qu, Xiaoqing; Young, Steven

doi:10.1074/jbc.m805296200

Cited by 19 publications

(39 citation statements)

References 31 publications

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“…In this respect, Crp4 and RMAD-4 (rhesus myeloid ␣-defensin-4) differ markedly with regard to charge distribution and the orientation of hydrophobic side chains along the peptide fold. Still, neutralizing anionic amino acids in the proregion of pro-RMAD-4-(20 -94) have the same activating effects as those described here for pro-Crp4-(20 -92) (36). Possibly, the biochemistry of the HNP precursor may have a unique structural context, leaving the question open until additional molecules have been characterized.…”

Section: Discussionmentioning

confidence: 63%

Anionic Amino Acids near the Pro-α-defensin N Terminus Mediate Inhibition of Bactericidal Activity in Mouse Pro-cryptdin-4

Figueredo

Weeks

Young

2009

Journal of Biological Chemistry

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In the small bowel, Paneth cells at the base of the crypts of Lieberkühn secrete ␣-defensins and additional antimicrobial peptides at high levels in response to cholinergic stimulation and when exposed to bacterial antigens (1-4). Paneth cell ␣-defensins show broad spectrum antimicrobial activities and constitute the majority of bactericidal peptide activity in Paneth cell secretions (2, 5-7). The release of Paneth cell products into the crypt lumen is inferred to protect mitotically active crypt cells from colonization by potential pathogens and to confer protection from enteric infection (2, 8 -10). The most compelling evidence for a Paneth cell role in enteric innate immunity is evident from studies of mice transgenic for a human Paneth cell ␣-defensin, HD-5, which are completely immune to infection and systemic disease from orally administered Salmonella enterica serovar typhimurium (11).The biosynthesis of ␣-defensins requires post-translational activation by lineage-specific proteinases (12, 13). Although the enzymes that mediate pro-␣-defensin processing in myeloid and epithelial cells differ, the overall processing schemes are the same. Both myeloid and Paneth cell ␣-defensins derive from ϳ10-kDa prepropeptides that contain canonical signal sequences, electronegative proregions, and a 3.5-4-kDa mature ␣-defensin peptide in the C-terminal portion of the precursor (13-16). Pro-␣-defensin processing in mouse Paneth cells is catalyzed by matrix metalloproteinase-7 (MMP-7) 3 and takes place intracellularly and prior to secretion (17,18). In mouse small intestinal epithelium, only Paneth cells express MMP-7 as components of dense core secretory granules (12), and the bactericidal activity of mouse Paneth cell ␣-defensins depends completely on activation of 8.4-kDa pro-Crps by MMP-7-catalyzed proteolysis (12, 18). MMP-7 gene disruption ablates pro-Crp processing such that mature, activated Crp peptides are absent from the small intestine, and innate immunity to oral bacterial infection is impaired in MMP-7-null mice (12).

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Section: Discussionmentioning

confidence: 63%

Anionic Amino Acids near the Pro-α-defensin N Terminus Mediate Inhibition of Bactericidal Activity in Mouse Pro-cryptdin-4

Figueredo

Weeks

Young

2009

Journal of Biological Chemistry

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“…Recombinant proteins were expressed and purified as His 6 -tagged fusion proteins as described previously (34,37). Expression of recombinant fusion proteins was induced by adjusting exponentially growing E. coli BL21-CodonPlus (DE3)-RIL cells with 0.1 mM isopropyl-␤-D-1-thiogalactopyranoside and incubating them at 37°C for 6 h in Terrific broth as described in earlier reports (14,34,37). Cells were lysed by sonication in 6 M guanidineHCl-100 mM Tris (pH 8.0), and the suspension was clarified by centrifugation.…”

Section: Methodsmentioning

confidence: 99%

“…Samples of proCrp4 and (R/K)-proCrp4 (11 g) and of Crp4 and (R/K)-Crp4 (5 g) were incubated with or without 0.5 molar equivalents of MMP-7 in 1 mM HEPES, 15 mM NaCl, and 0.5 mM CaCl 2 (pH 7.4) for 18 h at 37°C. Similarly, samples of proCrp4 (12 g) and Crp4, (R/K)-Crp4, RMAD-4, and (R/K)-RMAD-4 (6 g) were incubated with human neutrophil elastase in 50 mM Tris-150 mM NaCl (pH 7.5) at 37°C for 2 h at a substrate:enzyme ratio of 40:1 (14). Twelve micrograms of proCrp4 and 6 g of Crp4, (R/K)-Crp4, RMAD-4, and (R/K)-RMAD-4 were incubated with bovine pancreatic trypsin (Sigma-Aldrich, St. Louis, MO) in 50 mM ammonium bicarbonate at a 1:50 proteinase-to-protein ratio for 2 h. Peptide sensitivity to proteolysis was assayed by Coomassie blue staining of peptide digests following AU-PAGE separations (14,20,34).…”

Section: Methodsmentioning

confidence: 99%

Electropositive Charge in α-Defensin Bactericidal Activity: Functional Effects of Lys-for-Arg Substitutions Vary with the Peptide Primary Structure

Llenado¹,

Weeks²,

Cocco³

et al. 2009

Infect Immun

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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.

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“…To test whether positioning of hydrophobic residues as shown for Crp-4 is a general requirement for ␣-defensin bactericidal activity, we tested the effect of modifying hydrophobicity in RMAD-4. RMAD-4 was selected for study, because it has the same overall bactericidal activity (19,28,36) and cationic charge as Crp-4 (ϩ8), but RMAD-4 differs from Crp-4 in primary structure and in the distribution of peptide surface charge (Fig. 2) (19).…”

Section: Resultsmentioning

confidence: 99%

“…Crp-4 and RMAD-4 peptides were prepared as described previously (24)(25)(26)(27)(28). Briefly, recombinant peptides were expressed in E. coli as His 6 -tagged fusion proteins using the pET28a expression vector (Novagen, Inc., Madison, WI) at the EcoRI and SalI sites.…”

Section: Methodsmentioning

confidence: 99%

Hydrophobic Determinants of α-Defensin Bactericidal Activity

Tai

Selsted

2014

Infect Immun

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Mammalian ␣-defensins are approximately 4-to 5-kDa broad-spectrum antimicrobial peptides and abundant granule constituents of neutrophils and small intestinal Paneth cells. The bactericidal activities of amphipathic ␣-defensins depend in part on electropositive charge and on hydrophobic amino acids that enable membrane disruption by interactions with phospholipid acyl chains. Alignment of ␣-defensin primary structures identified conserved hydrophobic residues in the loop formed by the Cys III -Cys V disulfide bond, and we have studied their role by testing the effects of mutagenesis on bactericidal activities. Mouse ␣-defensin 4 (Crp-4) and rhesus myeloid ␣-defensin 4 (RMAD-4) were selected for these studies, because they are highly bactericidal in vitro and have the same overall electropositive charge. Elimination of hydrophobicity by site-directed mutagenesis at those positions in Crp-4 attenuated bactericidal activity markedly. In contrast to native Crp-4, the (I23/F25/L26/G)-Crp-4 variant lacked bactericidal activity against Salmonella enterica serovar Typhimurium and did not permeabilize Escherichia coli ML35 cells as a result of removing aliphatic side chains by Gly substitutions. Ala replacements in (I23/F25/L26/A)-Crp-4 restored activity, evidence that hydrophobicity contributed by Ala methyl R-groups was sufficient for activity. In macaques, neutrophil ␣-defensin RMAD-6 is identical to RMAD-4, except for a F28S difference, and (F28S)-RMAD-4 mutagenesis attenuated RMAD-4 bactericidal activity and E. coli permeabilization. Interestingly, (R31/32D)-Crp-4 lacks activity in these assays despite the presence of the Ile23, Phe25, and Leu26 hydrophobic patch. We infer that electrostatic interactions between cationic ␣-defensin residues and negative charge on bacteria precede interactions between critical hydrophobic residue positions that mediate membrane disruption and bacterial cell killing.

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In Vitro Activation of the Rhesus Macaque Myeloid α-Defensin Precursor proRMAD-4 by Neutrophil Serine Proteinases

Cited by 19 publications

References 31 publications

Anionic Amino Acids near the Pro-α-defensin N Terminus Mediate Inhibition of Bactericidal Activity in Mouse Pro-cryptdin-4

Anionic Amino Acids near the Pro-α-defensin N Terminus Mediate Inhibition of Bactericidal Activity in Mouse Pro-cryptdin-4

Electropositive Charge in α-Defensin Bactericidal Activity: Functional Effects of Lys-for-Arg Substitutions Vary with the Peptide Primary Structure

Hydrophobic Determinants of α-Defensin Bactericidal Activity

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