In vitro activation of the rhesus macaque myeloid alpha-defensin precursor proRMAD-4 by neutrophil serine proteinases.
Alpha-defensins are mammalian antimicrobial peptides expressed mainly by cells of myeloid lineage or small intestinal Paneth cells. The peptides are converted from inactive 8.5-kDa precursors to membrane-disruptive forms by post-translational proteolytic events. Because rhesus myeloid pro-alpha-defensin-4 (proRMAD-4((20-94))) lacks bactericidal peptide activity in vitro, we tested whether neutrophil azurophil granule serine proteinases, human neutrophil elastase (NE), cathepsin G (CG), and proteinase-3 (P3) have in vitro convertase activity. Only NE cleaved proRMAD-4((20-94)) at the native RMAD-4 N terminus to produce fully processed, bactericidal RMAD-4((62-94)). The final CG cleavage product was RMAD-4((55-94)), and P3 produced both RMAD-4((55-94)) and RMAD-4(57-94). Nevertheless, NE, CG, and P3 digests of proRMAD4 and purified RMAD-4((62-94)), RMAD-4((55-94)), and RMAD-4(57-94) peptides had equivalent in vitro bactericidal activities. Bactericidal peptide activity assays of proRMAD-4((20-94)) variants containing complete charge-neutralizing D/E to N/Q or D/E to A substitutions showed that (DE/NQ)-proRMAD-4((20-94)) and (DE/A)-proRMAD-4((20-94)) were as active as mature RMAD-4((62-94)). Therefore, proregion Asp and Glu side chains inhibit the RMAD-4 component of full-length proRMAD-4((20-94)), perhaps by a combination of charge-neutralizing and hydrogen-bonding interactions. Although native RMAD-4((62-94)) resists NE, CG, and P3 proteolysis completely, RMAD-4((62-94)) variants with disulfide pairing disruptions or lacking disulfide bonds were degraded extensively, evidence that the disulfide array protects the alpha-defensin moiety from degradation by the myeloid converting enzymes. These in vitro analyses support the conclusion that rhesus macaque myeloid pro-alpha-defensins are converted to active forms by serine proteinases that co-localize in azurophil granules.
Kamdar
Karishma
K
Department of Pathology & Laboratory Medicine, School of Medicine, College of Health Sciences, University of California, Irvine, California 92697-4800, USA.
Maemoto
Atsuo
A
Qu
Xiaoqing
X
Young
Steven K
SK
Ouellette
André J
AJ
D000595:Amino Acid Sequence; D000818:Animals; D056649:Cathepsin G; D002403:Cathepsins / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry; D004789:Enzyme Activation; D006801:Humans; D066298:In Vitro Techniques; D008253:Macaca mulatta; D051379:Mice; D008969:Molecular Sequence Data; D053146:Myeloblastin / Q000378:metabolism; D009504:Neutrophils / Q000201:enzymology*; D011498:Protein Precursors; D011994:Recombinant Proteins / Q000737:chemistry; D012697:Serine Endopeptidases / Q000378:metabolism*; D023084:alpha-Defensins / Q000737:chemistry* / Q000378:metabolism*
D004220:Disulfides; D011498:Protein Precursors; D011994:Recombinant Proteins; D023084:alpha-Defensins; D002403:Cathepsins; D012697:Serine Endopeptidases; C534094:CTSG protein, human; D056649:Cathepsin G; C534096:Ctsg protein, mouse; D053146:Myeloblastin
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