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

α-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-α-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 α-defensin moiety from degradation by the myeloid converting enzymes. These in vitro analyses support the conclusion that rhesus macaque myeloid pro-α-defensins are converted to active forms by serine proteinases that co-localize in azurophil granules.

The activating proteinases of human and mouse Paneth cell pro-␣-defensins co-localize with their substrates in secretory granules (1,11), suggesting that rhesus myeloid pro-␣-defensins may be activated by proteinases that also occur in neutrophil azurophil granules. Human neutrophil granules form sequentially during myeloid cell differentiation with azurophilic, myeloperoxidase-positive granules being the first to appear at the myeloblast and promyelocyte stage of neutrophil development (12). Myeloid pro-␣-defensins are synthesized and accumulate coincidentally with neutrophil elastase (NE), cathepsin G (CG), or proteinase-3 (P3) serine proteases in promyelocytes during myelopoiesis and are localized to the azurophil granules (13) as shown by gene expression profiling and proteomics analyses of neutrophil subcellular fractions (14,15). Accordingly, we reasoned that the co-localizing serine proteinases could function as convertases for myeloid pro-␣-defensins. That hypothesis was tested by exposing recombinant myeloid proRMAD-4 (20 -94) to human NE, CG, or P3 to test for evidence of proteolysis and in vitro activation of RMAD-4 (62-94) bactericidal peptide activity. Each proteinase converted inactive proRMAD-4 (20 -94) to bactericidal peptides, but only NE cleaved the proRMAD-4 (20 -94) molecule at the known RMAD-4 N terminus,  . The structural features of proRMAD-4 (20 -94) that maintain it in an inactive state have been investigated.
Purification of Recombinant Proteins-Recombinant proteins were expressed at 37°C in Terrific Broth medium by induction with 0.1 mM isopropyl-␤-D-1-thiogalactopyranoside for 6 h at 37°C, lysed by sonication in 6 M guanidine-HCl in 100 mM Tris-Cl, pH 8.1, clarified by centrifugation (2,6,20), and His-tagged fusion peptides were purified as described (8,16,17). After CNBr cleavage of fusions, the recombinant peptides were purified by C18 reverse-phase high performance liquid chromatography (RP-HPLC), and quantitated by absorbance at 230 nm. Molecular masses of purified peptides were determined using matrix-assisted laser desorption ionization mode mass spectrometry (Voyager-DE MALDI-TOF, PE-Biosystems, Foster City, CA) in the UCI Physical Sciences Mass Spectroscopy Facility. Peptide homogeneity was confirmed by acidurea polyacrylamide gel electrophoresis (AU-PAGE), (21) and analytical C18 RP-HPLC at 230 nm.
The Major Products of proRMAD-4 (20 -94) Serine Proteolysis Have Equivalent Activity-Because samples of complete in vitro digests of proRMAD-4 (20 -94) with NE, CG, or P3 were bactericidal, the active bactericidal molecules in those digests were characterized by purification of the major serine proteinase cleavage products using C 18 RP-HPLC (see "Experimental Procedures"). Based on the known C 18 elution profiles of proRMAD-4 (20 -94) and RMAD-4 (62-94) , the major RMADcontaining fractions were identified using MALDI-TOF MS. To summarize, the major product purified from NE digests had a mass of 3964.6 AMU, which corresponds precisely to native RMAD-4 (62-94) . Similarly, the major CG digestion product was 4627 AMU, and the two main products of P3 cleavage had masses of 4627 and 4396 AMU. Based on these analyses, the final cleavage events mediated by these proteinases occur at Ala-612Arg-62 for NE, the N terminus of native RMAD-4, at Leu-542Arg-55 for CG, corresponding to  , and at Leu-542Arg-55 and Ala-562Ser-57 for P3, corresponding to RMAD-4 (55-94) and RMAD-4 (57-94) , respectively ( Fig.  2A). Although the NE and CG enzymes have endogenous antimicrobial activity, NE and P3 lack activity under the conditions of these bactericidal peptide assays (supplemental Fig. S1). Similarly, CG has modest bactericidal activity at 10 g/ml, but that concentration is ten times greater than those used in these experiments (supplemental Fig. S1).
Possibly, although Asp and Glu mutagenesis eliminates charge neutralizing effects of carboxyl groups in the proform, hydrogen bonding interactions between Asn and Gln side chains and RMAD-4 basic residues also may contribute to blocking proRMAD-4 (20 -94) bactericidal activity. Alternatively, proregion amino acids other than Asp and Glu may also inhibit proRMAD-4 (20 -94) bactericidal action. These collective results suggest that proRMAD-4 (20 -94) lacks bactericidal activity because proregion Asp and Glu side chains prevent electrostatic RMAD-4 interactions with bacterial cell targets through a combination of charge neutralizing and hydrogen-bonding interactions (3,6,16).

DISCUSSION
The three azurophil granule serine proteases, NE, CG, and P3, cleave and activate proRMAD-4 (20 -94) in vitro. The fact that the human and mouse Paneth cell pro-␣-defensin convertases co-localize with their substrates in secretory granules (4,5), suggested that proRMAD-4 (20 -94) activation also could be mediated by neutrophil proteinases associated with azurophil granules (13). To test this hypothesis, recombinant proRMAD-4 (20 -94) molecules were exposed to NE, CG, or P3 to test for evidence of proteolysis and bactericidal peptide activity. Each enzyme cleaved proRMAD-4 (20 -94) at several different sites within the proregion, at no site within the ␣-defensin moiety, and processing by all three enzymes resulted in activation. Although only NE cleaved proRMAD-4 (20 -94) at the known RMAD-4 peptide N terminus to yield RMAD4 (62-94) ( Fig. 2A), all final processed products of proRMAD-4 (20 -94) activation, RMAD-4 (55-94), RMAD-4 (57-94) , or RMAD-4 (62-94) , have equivalent activities (Fig. 2B). NE and P3 cleave at the N terminus of small aliphatic residues such as Ala, and CG cleaves at the N terminus of Lys. These sites are consistent with their known recognition sites. Unexpectedly, P3 cleaves at Arg in proRMAD-4 perhaps because the tertiary structure of the folded precursor influences sterically an alternative cleavage that P3 is able to recognize (27). Thus, NE, CG, and P3 are individually capable of mediating proRMAD-4 (20 -94) activation to the same extent in vitro even though CG and P3 do not cleave at the RMAD-4 natural N terminus. Because they co-localize with ␣-defensins in neutrophil azurophilic granules, it seems likely that the three serine proteinases would participate coincidentally in pro-␣-defensin activation with NE catalyzing the final Ala-612Arg-62 cleavage event. This suggestion is supported by the fact that CG and P3 intermediates have not been isolated from macaque neutrophils (Tang et al. (10)).