In Vitro Activation of the Rhesus Macaque Myeloid α-Defensin Precursor proRMAD-4 by Neutrophil Serine Proteinases
2008; Elsevier BV; Volume: 283; Issue: 47 Linguagem: Inglês
10.1074/jbc.m805296200
ISSN1083-351X
AutoresKarishma Kamdar, Atsuo Maemoto, Xiaoqing Qu, Steven Young, André J. Ouellette,
Tópico(s)Biochemical and Structural Characterization
Resumoα-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. 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. α-Defensins are effectors of mammalian innate immunity in phagocytic leukocytes of myeloid origin and in small intestine following secretion by epithelial Paneth cells (1Selsted M.E. Ouellette A.J. Nat. Immunol. 2005; 6: 551-557Crossref PubMed Scopus (950) Google Scholar). Myeloid and Paneth cell α-defensins are synthesized as ∼10-kDa pre-propeptides that have canonical signal sequences, acidic proregions, and a 3.5–4.5-kDa mature α-defensin peptide component in the C-terminal moiety of the precursor molecule (1Selsted M.E. Ouellette A.J. Nat. Immunol. 2005; 6: 551-557Crossref PubMed Scopus (950) Google Scholar). Biosynthesis of functional α-defensins requires proteinase-mediated conversion of inactive precursors to membrane-disruptive, bactericidal peptides (2Shirafuji Y. Tanabe H. Satchell D.P. Henschen-Edman A. Wilson C.L. Ouellette A.J. J. Biol. Chem. 2003; 278: 7910-7919Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 3Weeks C.S. Tanabe H. Cummings J.E. Crampton S.P. Sheynis T. Jelinek R. Vanderlick T.K. Cocco M.J. Ouellette A.J. J. Biol. Chem. 2006; 281: 28932-28942Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). In human small bowel, evidence shows that the Paneth cell pro-α-defensin pro-HD5(20–94) is activated after secretion by anionic and meso trypsin isoforms that cleave the precursor at Arg-62 ↓ Ala-63 to produce HD5(62–94), the predominant form of HD5 in the ileum (4Ghosh D. Porter E. Shen B. Lee S.K. Wilk D. Drazba J. Yadav S.P. Crabb J.W. Ganz T. Bevins C.L. Nat. Immunol. 2002; 3: 583-590Crossref PubMed Scopus (360) Google Scholar). By contrast, mouse enteric pro-α-defensins (pro-Crps) are converted to active forms by matrix metalloproteinase-7 (MMP-7), which co-localizes with pro-α-defensins in Paneth cell secretory granules (2Shirafuji Y. Tanabe H. Satchell D.P. Henschen-Edman A. Wilson C.L. Ouellette A.J. J. Biol. Chem. 2003; 278: 7910-7919Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 5Wilson C.L. Ouellette A.J. Satchell D.P. Ayabe T. Lopez-Boado Y.S. Stratman J.L. Hultgren S.J. Matrisian L.M. Parks W.C. Science. 1999; 286: 113-117Crossref PubMed Scopus (912) Google Scholar). Pro-Crps lack in vitro bactericidal activity until the proregion is cleaved by MMP-7 at three sites, including the Crp N terminus (2Shirafuji Y. Tanabe H. Satchell D.P. Henschen-Edman A. Wilson C.L. Ouellette A.J. J. Biol. Chem. 2003; 278: 7910-7919Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 3Weeks C.S. Tanabe H. Cummings J.E. Crampton S.P. Sheynis T. Jelinek R. Vanderlick T.K. Cocco M.J. Ouellette A.J. J. Biol. Chem. 2006; 281: 28932-28942Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar), and the cleavage event at Ser-43 ↓ Ile-44 enables full bactericidal activity and membrane disruptive behavior (2Shirafuji Y. Tanabe H. Satchell D.P. Henschen-Edman A. Wilson C.L. Ouellette A.J. J. Biol. Chem. 2003; 278: 7910-7919Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 3Weeks C.S. Tanabe H. Cummings J.E. Crampton S.P. Sheynis T. Jelinek R. Vanderlick T.K. Cocco M.J. Ouellette A.J. J. Biol. Chem. 2006; 281: 28932-28942Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 6Satchell D.P. Sheynis T. Shirafuji Y. Kolusheva S. Ouellette A.J. Jelinek R. J. Biol. Chem. 2003; 278: 13838-13846Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Post-translational processing of inactive human neutrophil pro-α-defensin 1 (pro-HNP-1) produces major intermediate forms of 75 and 56 amino acids as well as mature HNP-1 (7Ganz T. Nat. Rev. Immunol. 2003; 3: 710-720Crossref PubMed Scopus (2343) Google Scholar), but the convertases that mediate pro-α-defensin activation in primate pro-myelocytes remain unknown. Rhesus macaque myeloid α-defensins (RMADs) 3The abbreviations used are: RMAD, rhesus myeloid α-defensin; NE, human neutrophil elastase; CG, human cathepsin G; P3, human proteinase 3; MALDI-TOF-MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; RP-HPLC, reverse-phase high performance liquid chromatography; AU-PAGE, acid-urea polyacrylamide gel electrophoresis; PIPES, 1,4-piperazinediethanesulfonic acid; AMU, atomic mass unit. 3The abbreviations used are: RMAD, rhesus myeloid α-defensin; NE, human neutrophil elastase; CG, human cathepsin G; P3, human proteinase 3; MALDI-TOF-MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; RP-HPLC, reverse-phase high performance liquid chromatography; AU-PAGE, acid-urea polyacrylamide gel electrophoresis; PIPES, 1,4-piperazinediethanesulfonic acid; AMU, atomic mass unit. are broad spectrum bactericidal peptides (8Tanabe H. Ouellette A.J. Cocco M.J. Robinson Jr., W.E. J. Virol. 2004; 78: 11622-11631Crossref PubMed Scopus (42) Google Scholar, 9Tanabe H. Yuan J. Zaragoza M.M. Dandekar S. Henschen-Edman A. Selsted M.E. Ouellette A.J. Infect. Immun. 2004; 72: 1470-1478Crossref PubMed Scopus (40) Google Scholar, 10Tang Y.Q. Yuan J. Miller C.J. Selsted M.E. Infect. Immun. 1999; 67: 6139-6144Crossref PubMed Google Scholar). RMADs 1–3 and 8 have similar primary structures that resemble human neutrophil HNPs 1–3, but RMADs 4/5 and 6/7 are very different from HNP-1, yet differ from each other only by a S28F polymorphism (10Tang Y.Q. Yuan J. Miller C.J. Selsted M.E. Infect. Immun. 1999; 67: 6139-6144Crossref PubMed Google Scholar). proRMAD primary structures deduced from cDNA sequences predict that RMADs 4 and 5 and RMADs 6 and 7 arise by alternative post-translational processing, consistent with the N-terminal Arg in RMADs 4 and 6. Although RMAD-3 and RMAD-4 are both highly bactericidal in vitro, RMAD-4 has anti-HIV activity that RMAD-3 lacks (8Tanabe H. Ouellette A.J. Cocco M.J. Robinson Jr., W.E. J. Virol. 2004; 78: 11622-11631Crossref PubMed Scopus (42) Google Scholar). The activating proteinases of human and mouse Paneth cell pro-α-defensins co-localize with their substrates in secretory granules (1Selsted M.E. Ouellette A.J. Nat. Immunol. 2005; 6: 551-557Crossref PubMed Scopus (950) Google Scholar, 11Ouellette A.J. Curr. Top. Microbiol. Immunol. 2006; 306: 1-25PubMed Google Scholar), suggesting that rhesus myeloid pro-α-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 (12Bainton D.F. Ullyot J.L. Farquhar M.G. J. Exp. Med. 1971; 134: 907-934Crossref PubMed Scopus (558) Google Scholar). Myeloid pro-α-defensins are synthesized and accumulate coincidentally with neutrophil elastase (NE), cathepsin G (CG), or proteinase-3 (P3) serine proteases in pro-myelocytes during myelopoiesis and are localized to the azurophil granules (13Borregaard N. Sorensen O.E. Theilgaard-Monch K. Trends Immunol. 2007; 28: 340-345Abstract Full Text Full Text PDF PubMed Scopus (512) Google Scholar) as shown by gene expression profiling and proteomics analyses of neutrophil subcellular fractions (14Cowland J.B. Borregaard N. J. Leukoc Biol. 1999; 66: 989-995Crossref PubMed Scopus (197) Google Scholar, 15Arnljots K. Sorensen O. Lollike K. Borregaard N. Leukemia. 1998; 12: 1789-1795Crossref PubMed Scopus (43) Google Scholar). Accordingly, we reasoned that the co-localizing serine proteinases could function as convertases for myeloid pro-α-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, RMAD-4(62–94). The structural features of proRMAD-4(20–94) that maintain it in an inactive state have been investigated. Preparation of Recombinant α-Defensin Peptides and Variants—Recombinant RMAD-4(62–94) and precursors were expressed in Escherichia coli as N-terminal 6×-histidine-tagged fusion proteins from the EcoRI and SalI sites of the pET28a expression vector (Novagen, Inc. Madison, WI) as described (2Shirafuji Y. Tanabe H. Satchell D.P. Henschen-Edman A. Wilson C.L. Ouellette A.J. J. Biol. Chem. 2003; 278: 7910-7919Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 9Tanabe H. Yuan J. Zaragoza M.M. Dandekar S. Henschen-Edman A. Selsted M.E. Ouellette A.J. Infect. Immun. 2004; 72: 1470-1478Crossref PubMed Scopus (40) Google Scholar, 16Tanabe H. Qu X. Weeks C.S. Cummings J.E. Kolusheva S. Walsh K.B. Jelinek R. Vanderlick T.K. Selsted M.E. Ouellette A.J. J. Biol. Chem. 2004; 279: 11976-11983Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 17Maemoto A. Qu X. Rosengren K.J. Tanabe H. Henschen-Edman A. Craik D.J. Ouellette A.J. J. Biol. Chem. 2004; 279: 44188-44196Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). The following native and variant rhesus macaque myeloid α-defensins RMAD-4(62–94) (10Tang Y.Q. Yuan J. Miller C.J. Selsted M.E. Infect. Immun. 1999; 67: 6139-6144Crossref PubMed Google Scholar), (C4/11/31/32A)-RMAD-4(62–94), and (6C/A)-RMAD-4(62–94) were prepared. The natural pET-28a cloning primers for RMAD-4(62–94) are pET-RMAD-4-F (5′-ACA CAC GAA TTC ATG AGA CGC ACC TGC CGT) with pET-RMAD-4-R (5′-ACA CAC GTC GAC TCA TCA GCG ACA GCA GAG ACT) (9Tanabe H. Yuan J. Zaragoza M.M. Dandekar S. Henschen-Edman A. Selsted M.E. Ouellette A.J. Infect. Immun. 2004; 72: 1470-1478Crossref PubMed Scopus (40) Google Scholar, 16Tanabe H. Qu X. Weeks C.S. Cummings J.E. Kolusheva S. Walsh K.B. Jelinek R. Vanderlick T.K. Selsted M.E. Ouellette A.J. J. Biol. Chem. 2004; 279: 11976-11983Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 18Aarbiou J. Ertmann M. van Wetering S. van Noort P. Rook D. Rabe K.F. Litvinov S.V. van Krieken J.H. de Boer W.I. Hiemstra P.S. J. Leukoc Biol. 2002; 72: 167-174PubMed Google Scholar). Rhesus pro-α-defensins were prepared as follows: The proRMAD-4(20–94) coding sequence was amplified using forward primer 5′-ATA TAG AAT TCA TGA AGT CAC TCC AGG AAA CAG C (EcoRI-M proRMAD-4(20–94)) paired with reverse primer 3′-AAG TCA GAG ACG ACA GCG ACT CAG CTG ATA TA (3′-SalIproRMAD-4(20–94)). Ala for Cys substitutions were introduced using the following primers in PCR from corresponding positions to those described above to mutagenize an existing pET28a-RMAD-4(62–94) cDNA clone (16Tanabe H. Qu X. Weeks C.S. Cummings J.E. Kolusheva S. Walsh K.B. Jelinek R. Vanderlick T.K. Selsted M.E. Ouellette A.J. J. Biol. Chem. 2004; 279: 11976-11983Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Primers used to mutagenize RMAD-4(62–94) included pET-RMAD-4-C4AC6A-F (5′-ACA CAC GAA TTC ATG AGA CGC ACC GCA CGT GCT), pET-RMAD-4-C31AC32A-R (5′-ACA CAC GTC GAC TCA TCA GCG AGC TGC GAG ACT), RMAD-4-C11A-F (5′-TTT GGC CGT GCC TTC AGG CGT), RMAD-4-C11A-R (5′-ACG CCT GAA GGC ACG GCC AAA), RMAD-4-C21A-F (5′-TCT GGG AGT GCT AAC ATC AAT), RMAD-4-C21-R (5′-ATT GAT GTT AGC ACT CCC AGA), and pET-RMAD-4-C4A-F (5′-ACA CAC GAA TTC ATG AGA CGC ACC GCA CGT TGC). To test for the functional role of acidic amino acid positions in the proRMAD-4(20–94) proregion, all Asp codons in proRMAD-4(20–61), wherein residues 20–61 form the proregion of proRMAD-4(20–94) were substituted with Asn codons, and all Glu codons were substituted with Gln. Numbering from the initiating methionine and as reported previously (3Weeks C.S. Tanabe H. Cummings J.E. Crampton S.P. Sheynis T. Jelinek R. Vanderlick T.K. Cocco M.J. Ouellette A.J. J. Biol. Chem. 2006; 281: 28932-28942Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 17Maemoto A. Qu X. Rosengren K.J. Tanabe H. Henschen-Edman A. Craik D.J. Ouellette A.J. J. Biol. Chem. 2004; 279: 44188-44196Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 19Rosengren K.J. Daly N.L. Fornander L.M. Jonsson L.M. Shirafuji Y. Qu X. Vogel H.J. Ouellette A.J. Craik D.J. J. Biol. Chem. 2006; 281: 28068-28078Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar), a series of mutagenizing PCR reactions were to prepare (E24Q/D27N/D28N/E33Q/E37Q/D38/N/D39N/D41N/E47Q/E48Q)-proRMAD-4(20–94), termed (DE/NQ)-proRMAD-4(20–94). Native proRMAD-4(20–94) cDNA served as template in the first amplification reaction using RMAD-4(62–94) (5′-CTT GCT GTC TCC TTT CAA CAA AAT GGA CTC TCT-3′) and RMAD-4-Stop-SalI-R (5′-CGC GTC GAC TCA TCA GCG ACA GCA GAG ACT GA-3′) as forward and reverse primers, respectively. Successive PCR reactions were used to extend mutagenesis toward the 5′-end of proRMAD-4(20–94). The RMAD-4-Stop-SalI reverse primer was used as the reverse primer in all PCR reactions. The forward primers for the subsequent PCR reactions were RMAD-4(37–94) (5′-CAA AAT AAT CAG AAT CTT GCT GTC TCC TTT CAA, RMAD-4(32–94) (5′-CAG CAA CAG CCT GGG CAA AAT AAT CAG AAT CTT), RMAD-4(27–94) (5′-AAT AAT GCT GCA AC CCA GCA ACA GCC TGG GCA G), RMAD-4(22–94) (5′-CTC CAG CAA ACA GCT AAT AA TGC TGC AAC CCA G-3′), RMAD-4(20–94) (5′-AAG TCA CTC CAG CAA ACA GCT AAT AAT GCT GCA-3′). The forward primer, Start D,E-N,Q proRMAD-4 (5′-CGC GAA TTC ATG AAG TCA CTC CAG CAA ACA GCT-3′) was paired with the reverse primer RMAD-4-Stop-SalI in the final PCR reaction. To prepare (E24A/D27A/D28A/E33A/E37A/D38A/D39A/D41A/E47A/E49A)-proRMAD-4(20–94), termed (DE/A)-proRMAD-4(20–94)), Asp and Glu codons in proRMAD-4(20–61) were changed to Ala codons as described above using the following mutagenizing primers. The forward primers used to prepare (DE/A)-proRMAD-4(20–94) were proRMAD4 D,E-A(42–94) (5′-CTT GCT GTC TCC TTT GCA GCT AAT GGA CTC TCT ACT), proRMAD-4(20–94) D,E-A(35–94) (5′-CAG CCT GGG GCA GCT GCA CAG GCA CTT GCT GTC), proRMAD-4(20–94) D,E-A(29–94) (5′-GCT GCA ACC CAG GCA CAG CCT GGG), proRMAD-4(20–94) D,E-A(20–94) (5′-AAG TCA CTC CAG GAA ACA GCT GAT GAC GCT GCA ACC). Primer proRMAD-4(20–94) D,E-A Start F (5′-CGC GAA TTC ATG AAG TCA CTC CAG GCA ACA-3′) was paired with reverse primer RMAD-4-Stop-SalI in the final PCR reaction. The order of PCR-based mutagenesis reactions and their design followed a scheme used previously to mutagenize Cys residue positions in mouse Crp4 (17Maemoto A. Qu X. Rosengren K.J. Tanabe H. Henschen-Edman A. Craik D.J. Ouellette A.J. J. Biol. Chem. 2004; 279: 44188-44196Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Briefly, in PCR reaction 1, a mutant forward primer containing the mutated codon flanked by three natural codons was paired with a reverse cloning primer, and PCR reaction 2 paired a primer that was the reverse-complement of the mutant forward primer with a forward cloning primer. Samples of purified products from reactions 1 and 2 were combined as templates in PCR reaction 3, using the forward and reverse cloning primers as amplimers, and these putative mutant proRMAD-4 products were cloned in pCR-2.1 TOPO and verified by DNA sequencing. Products were subcloned into SalI and EcoRI sites in pET28a plasmid DNA (Novagen, Inc., Madison, WI), and transformed into E. coli BL21 (DE3)-CodonPlus-RIL cells (Stratagene) for recombinant expression (3Weeks C.S. Tanabe H. Cummings J.E. Crampton S.P. Sheynis T. Jelinek R. Vanderlick T.K. Cocco M.J. Ouellette A.J. J. Biol. Chem. 2006; 281: 28932-28942Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 16Tanabe H. Qu X. Weeks C.S. Cummings J.E. Kolusheva S. Walsh K.B. Jelinek R. Vanderlick T.K. Selsted M.E. Ouellette A.J. J. Biol. Chem. 2004; 279: 11976-11983Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 17Maemoto A. Qu X. Rosengren K.J. Tanabe H. Henschen-Edman A. Craik D.J. Ouellette A.J. J. Biol. Chem. 2004; 279: 44188-44196Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Purification of Recombinant Proteins—Recombinant proteins were expressed at 37 °C in Terrific Broth medium by induction with 0.1 mm isopropyl-β-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 (2Shirafuji Y. Tanabe H. Satchell D.P. Henschen-Edman A. Wilson C.L. Ouellette A.J. J. Biol. Chem. 2003; 278: 7910-7919Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 6Satchell D.P. Sheynis T. Shirafuji Y. Kolusheva S. Ouellette A.J. Jelinek R. J. Biol. Chem. 2003; 278: 13838-13846Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 20Ayabe T. Satchell D.P. Pesendorfer P. Tanabe H. Wilson C.L. Hagen S.J. Ouellette A.J. J. Biol. Chem. 2002; 277: 5219-5228Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar), and His-tagged fusion peptides were purified as described (8Tanabe H. Ouellette A.J. Cocco M.J. Robinson Jr., W.E. J. Virol. 2004; 78: 11622-11631Crossref PubMed Scopus (42) Google Scholar, 16Tanabe H. Qu X. Weeks C.S. Cummings J.E. Kolusheva S. Walsh K.B. Jelinek R. Vanderlick T.K. Selsted M.E. Ouellette A.J. J. Biol. Chem. 2004; 279: 11976-11983Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 17Maemoto A. Qu X. Rosengren K.J. Tanabe H. Henschen-Edman A. Craik D.J. Ouellette A.J. J. Biol. Chem. 2004; 279: 44188-44196Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). 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), (21Selsted M.E. Genet. Eng. (N Y). 1993; 15: 131-147Crossref PubMed Scopus (32) Google Scholar) and analytical C18 RP-HPLC at 230 nm. Bactericidal Peptide Assays—Recombinant peptides were tested for microbicidal activity against E. coli ML35, Salmonella enterica serovar Typhimurium (S. typhimurium) ΔphoP, wild-type S. typhimurium strains CS022, JSG210, and 14082 (from Dr. Samuel I. Miller, University of Washington), Vibrio cholerae, Staphylococcus aureus 710a, and Listeria monocytogenes 104035 (22Lehrer R.I. Barton A. Ganz T. J. Immunol. Methods. 1988; 108: 153-158Crossref PubMed Scopus (182) Google Scholar). Bacteria growing exponentially in trypticase soy broth at 37 °C, were deposited by centrifugation at 1700 × g for 10 min, washed in 10 mm PIPES (pH 7.4), and resuspended in 10 mm PIPES (pH 7.4) supplemented with 0.01 volumes of trypticase soy broth (2Shirafuji Y. Tanabe H. Satchell D.P. Henschen-Edman A. Wilson C.L. Ouellette A.J. J. Biol. Chem. 2003; 278: 7910-7919Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 6Satchell D.P. Sheynis T. Shirafuji Y. Kolusheva S. Ouellette A.J. Jelinek R. J. Biol. Chem. 2003; 278: 13838-13846Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Bacteria (1–5 × 106 colony-forming units per ml (CFU/ml)) were incubated with test peptides in 50 μl for 1 h in a shaking incubator at 37 °C; then 20-μl samples of incubation mixtures were diluted 1:100 with 10 mm PIPES (pH 7.4), and 50 μl of the diluted samples were plated on trypticase soy agar plates using an Autoplate 4000 (Spiral Biotech Inc., Bethesda, MD). Surviving bacteria were counted as CFU/ml after incubation at 37 °C for 12–18 h. Bactericidal assays performed in Fig. 1B are against a defensin-sensitive strain S. typhimurium ΔphoP whereas the activities shown in Fig. 2B are against three different species of bacteria. Although the peptide concentrations were indicated in μm concentrations in Fig. 1B and as μg/ml in 2B, the data in Figs. 1B and 2B are not discordant.FIGURE 2The major serine protease cleavage products of proRMAD-4(20–94) have equivalent activity. In A, the final azurophil granule serine protease cleavage sites of proRMAD-4(20–94) deduced by MALDI-TOF MS. The native RMAD4(62–94) primary structure is underlined. The major products of proRMAD4(20–94) proteolysis are: NE, RMAD4(62–94) (▾); CG, RMAD4(55–94) (▵); P 3-RMAD4(55–94) and RMAD4(57–94) (▴). B, bactericidal activity of recombinant proRMAD-4(20–94), RMAD-4(62–94), and the final serine proteinase cleavage products against E. coli ML35. Exponentially growing E. coli ML35 (A), S. aureus (B) and V. cholerae (C) were exposed to the peptides at 37 °C in 50 μl of PIPES-TSB buffer for 1 h (see “Experimental Procedures”). Consistent with Fig. 1B, proRMAD-4(20–94) (-•-) lacks bactericidal activity, and native RMAD-4(62–94) (-○-), RMAD4(62–94) (-▾-)RMAD4(55–94), (-▿-), and RMAD4(55–94) +RMAD-4(57–94) (-▪-) are equally bactericidal.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Analyses of proRMAD-4(20–94) and RMAD-4(62–94) in in Vitro Proteolysis—Recombinant RMAD-4(62–94), proRMAD-4(20–94), and disulfide variants digested with NE, P3, or CG (Elastin Products Company, Inc) as described below were analyzed for susceptibility to proteolysis by AU-PAGE, and samples of the proteolytic digests were tested in bactericidal peptide assays and analyzed by MALDI-TOF MS (17Maemoto A. Qu X. Rosengren K.J. Tanabe H. Henschen-Edman A. Craik D.J. Ouellette A.J. J. Biol. Chem. 2004; 279: 44188-44196Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Samples (11 μg) of proRMAD-4(20–94) and disulfide variants and 5-μg samples of RMAD-4(62–94) were incubated with proteinases at 37 °C for 2 h at a substrate to enzyme molar ratio of 4:1 as follows: NE and P3 in 50 mm Tris, 150 mm NaCl (pH 7.5), and CG in the same buffer at pH 8.3. Equimolar quantities of all digests were analyzed by AU-PAGE. The biological effects of enzyme-specific proteolysis of proRMAD-4(20–94) molecules were determined using bactericidal peptide assays as described (9Tanabe H. Yuan J. Zaragoza M.M. Dandekar S. Henschen-Edman A. Selsted M.E. Ouellette A.J. Infect. Immun. 2004; 72: 1470-1478Crossref PubMed Scopus (40) Google Scholar, 16Tanabe H. Qu X. Weeks C.S. Cummings J.E. Kolusheva S. Walsh K.B. Jelinek R. Vanderlick T.K. Selsted M.E. Ouellette A.J. J. Biol. Chem. 2004; 279: 11976-11983Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 17Maemoto A. Qu X. Rosengren K.J. Tanabe H. Henschen-Edman A. Craik D.J. Ouellette A.J. J. Biol. Chem. 2004; 279: 44188-44196Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). To test for proteinase-mediated activation of bactericidal peptide activity, equimolar quantities (0 to 20 μg/ml) of RMAD-4(62–94), proRMAD-4(20–94), or their variants were incubated for 2 h or 18 h at 37 °C with or without proteinases as noted in individual legends. Subsequently, digests were incubated with exponentially growing bacterial cells (∼1–5 × 106 CFU/ml) for 60 min at 37 °C (2Shirafuji Y. Tanabe H. Satchell D.P. Henschen-Edman A. Wilson C.L. Ouellette A.J. J. Biol. Chem. 2003; 278: 7910-7919Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Samples (20 μl) of each incubation mixture were diluted 1:100 with 10 mm PIPES (pH 7.4) and 50 μl of the diluted, peptide- or digest-exposed bacteria were plated on trypticase soy agar using a Spiral Biotech Autoplate 4000 device (Spiral Biotech Inc., Bethesda, MD). Surviving bacteria were quantitated as CFU/ml on plates after incubation at 37 °C for 12–18 h. Purification of Digestion Products of proRMAD-4(20–94)—40 μg of proRMAD-4(20–94) digested with 1 μg of NE, CG, or P3 and the products of proteolysis were separated and purified using C18-RP HPLC, quantitated by absorbance 230 nm, and molecular masses of digested peptides were determined using MALDI-TOF MS. Peptide homogeneity was assessed by AU-PAGE (21Selsted M.E. Genet. Eng. (N Y). 1993; 15: 131-147Crossref PubMed Scopus (32) Google Scholar). Permeabilization of Live E. coli ML35 Cells—Exponentially growing E. coli ML35 cells were washed and resuspended in 10 mm PIPES (pH 7.4) supplemented with 0.01 volume of trypticase soy broth. Bacteria were exposed in triplicate to RMAD-4(62–94), proRMAD-4(20–94), and (DE/NQ)-proRMAD-4(20–94) in the presence of 2.5 mm ONPG for 2 h at 37 °C. E. coli ML35 express β-galactosidase constitutively but are permease-negative and do not take up ONPG unless permeabilized by external factors, such as defensins. β-Galactosidase hydrolysis was measured at 405 nm on a 96-well Spectra-Max plate spectrophotometer (Molecular Devices, Sunnyvale, CA). In Vitro Activation of the Rhesus Myeloid proRMAD-4-(20–94)— The precursors of mouse Paneth cell α-defensin Crp4 and human neutrophil α-defensins 1–3 (HNP-1–3) lack bactericidal activity until enzymatic conversion to their active forms (2Shirafuji Y. Tanabe H. Satchell D.P. Henschen-Edman A. Wilson C.L. Ouellette A.J. J. Biol. Chem. 2003; 278: 7910-7919Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 3Weeks C.S. Tanabe H. Cummings J.E. Crampton S.P. Sheynis T. Jelinek R. Vanderlick T.K. Cocco M.J. Ouellette A.J. J. Biol. Chem. 2006; 281: 28932-28942Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 23Valore E.V. Ganz T. Blood. 1992; 79: 1538-1544Crossref PubMed Google Scholar, 24Valore E.V. Martin E. Harwig S.S. Ganz T. J. Clin. Investig. 1996; 97: 1624-1629Crossref PubMed Scopus (121) Google Scholar, 25Ganz T. Liu L. Valore E.V. Oren A. Blood. 1993; 82: 641-650Crossref PubMed Google Scholar). To test whether rhesus macaque myeloid pro-α-defensins also require proteolytic conversion from inactive precursors to active forms, the bactericidal peptide activities of recombinant RMAD-4(62–94) and proRMAD-4(20–94) molecules were compared. In contrast to the robust in vitro bactericidal activity of mature RMAD-4(62–94) and consistent with findings for proCrp4 and proHNP-1, equimolar quantities of proRMAD-4(20–94) had no bactericidal effects against varied bacterial cell targets (Fig. 1B). Thus, RMAD-4(62–94) bacteric
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