Structure-Activity Determinants in Paneth Cell α-Defensins
2004; Elsevier BV; Volume: 279; Issue: 12 Linguagem: Inglês
10.1074/jbc.m310251200
ISSN1083-351X
AutoresHiroki Tanabe, Xiaoqing Qu, Colby S. Weeks, Jason E. Cummings, Sofiya Kolusheva, Kevin B. Walsh, Raz Jelinek, T. Kyle Vanderlick, Michael E. Selsted, André J. Ouellette,
Tópico(s)Biochemical and Structural Characterization
ResumoPaneth cells secrete microbicidal enteric α-defensins into the small intestinal lumen, and cryptdin-4 (Crp4) is the most bactericidal of the mouse α-defensin peptides in vitro. Here, site-directed Arg to Asp mutations in Crp4 have been shown to attenuate or eliminate microbicidal activity against all of the bacterial species tested regardless of the Arg residue position. R31D/R32D charge-reversal mutagenesis at the C terminus and mutations at R16D/R18D, R16D/R24D, and R18D/R24D in the Crp4 polypeptide chain eliminated in vitro bactericidal activity, blocked peptide-membrane interactions, as well as Crp4-mediated membrane vesicle disruption. Lys for Arg charge-neutral substitutions in (R16K/R18K)-Crp4 did not alter the bactericidal activity relative to Crp4, showing that bactericidal activity appears not to require the guanidinium side chain of Arg at those two positions. Partial restoration of (R31D/R32D)-Crp4 bactericidal activity occurred when an electropositive Arg for Gly substitution was introduced at the peptide N terminus and the (G1R/R31D/R32D)-Crp4 peptide exhibited intermediate membrane binding capability. Also, the loss of peptide bactericidal activity in (G1D/R31D/R32D)-Crp4 and (R16D/R24D)-Crp4 mutants corresponded with diminished phospholipid vesicle disruptive activity. Fluorophore leakage from anionic phospholipid vesicles induced by the charge-reversal variants was negligible relative to Crp4 and lower than that induced by pro-Crp4, the inactive Crp4 precursor. Thus, Arg residues function as determinants of Crp4 bactericidal activity by facilitating or enabling target cell membrane disruption. The role of the Arg residues, however, was surprisingly independent of their position in the polypeptide chain. Paneth cells secrete microbicidal enteric α-defensins into the small intestinal lumen, and cryptdin-4 (Crp4) is the most bactericidal of the mouse α-defensin peptides in vitro. Here, site-directed Arg to Asp mutations in Crp4 have been shown to attenuate or eliminate microbicidal activity against all of the bacterial species tested regardless of the Arg residue position. R31D/R32D charge-reversal mutagenesis at the C terminus and mutations at R16D/R18D, R16D/R24D, and R18D/R24D in the Crp4 polypeptide chain eliminated in vitro bactericidal activity, blocked peptide-membrane interactions, as well as Crp4-mediated membrane vesicle disruption. Lys for Arg charge-neutral substitutions in (R16K/R18K)-Crp4 did not alter the bactericidal activity relative to Crp4, showing that bactericidal activity appears not to require the guanidinium side chain of Arg at those two positions. Partial restoration of (R31D/R32D)-Crp4 bactericidal activity occurred when an electropositive Arg for Gly substitution was introduced at the peptide N terminus and the (G1R/R31D/R32D)-Crp4 peptide exhibited intermediate membrane binding capability. Also, the loss of peptide bactericidal activity in (G1D/R31D/R32D)-Crp4 and (R16D/R24D)-Crp4 mutants corresponded with diminished phospholipid vesicle disruptive activity. Fluorophore leakage from anionic phospholipid vesicles induced by the charge-reversal variants was negligible relative to Crp4 and lower than that induced by pro-Crp4, the inactive Crp4 precursor. Thus, Arg residues function as determinants of Crp4 bactericidal activity by facilitating or enabling target cell membrane disruption. The role of the Arg residues, however, was surprisingly independent of their position in the polypeptide chain. Paneth cells at the base of the crypts of Lieberkühn in the small intestine secrete apically oriented granules as components of innate immunity. The secretory granules are discharged in response to cholinergic stimulation or exposure to bacteria or their antigens (1Satoh Y. Cell Tissue Res. 1988; 251: 87-93Crossref PubMed Scopus (42) Google Scholar, 2Satoh Y. Ishikawa K. Ono K. Vollrath L. Digestion. 1986; 34: 115-121Crossref PubMed Scopus (34) Google Scholar, 3Ayabe T. Satchell D.P. Wilson C.L. Parks W.C. Selsted M.E. Ouellette A.J. Nat. Immunol. 2000; 1: 113-118Crossref PubMed Scopus (826) Google Scholar), and they contain several antimicrobial peptides and proteins (4Porter E.M. Bevins C.L. Ghosh D. Ganz T. Cell Mol. Life Sci. 2002; 59: 156-170Crossref PubMed Scopus (320) Google Scholar) including lysozyme (5Geyer G. Acta Histochem. 1973; 45: 126-132PubMed Google Scholar, 6Peeters T. Vantrappen G. Gut. 1975; 16: 553-558Crossref PubMed Scopus (129) Google Scholar, 7Cross M. 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For example, mouse α-defensins, termed cryptdins (Crps), 1The abbreviations used are: Crp, cryptdin; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; %CR, percent colorimetric response; PIPES, 1,4-piperazinediethanesulfonic acid; CFU, colony forming units; PDA, polydiacetylene; LUV, large unilamellar phospholipid vesicles; ANTS, 8-aminonaphthalene-1,3,6-trisulfonic acid. 1The abbreviations used are: Crp, cryptdin; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; %CR, percent colorimetric response; PIPES, 1,4-piperazinediethanesulfonic acid; CFU, colony forming units; PDA, polydiacetylene; LUV, large unilamellar phospholipid vesicles; ANTS, 8-aminonaphthalene-1,3,6-trisulfonic acid. account for ∼70% of the bactericidal peptide activity in secretions elicited from Paneth cells (3Ayabe T. Satchell D.P. Wilson C.L. Parks W.C. Selsted M.E. Ouellette A.J. Nat. Immunol. 2000; 1: 113-118Crossref PubMed Scopus (826) Google Scholar) and Crp4 is the most potent of the known mouse α-defensin peptides (16Ouellette A.J. Hsieh M.M. Nosek M.T. Cano-Gauci D.F. Huttner K.M. Buick R.N. Selsted M.E. Infect. Immun. 1994; 62: 5040-5047Crossref PubMed Google Scholar, 17Selsted M.E. Miller S.I. Henschen A.H. Ouellette A.J. J. Cell Biol. 1992; 118: 929-936Crossref PubMed Scopus (278) Google Scholar). Also, Crps became implicated as components of mouse innate enteric immunity in vivo when mice that lacked matrix metalloproteinase-7, the pro-Crp-activating enzyme, were shown to have impaired host defense against oral infections (18Wilson 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 (903) Google Scholar, 19Ayabe 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 (155) Google Scholar). Remarkably, mice transgenic for human Paneth cell α-defensin HD5 expressed the minigene specifically in Paneth cells and they were immune to oral infection by high doses of Salmonella enterica serovar Typhimurium (S. typhimurium) (20Salzman N.H. Ghosh D. Huttner K.M. Paterson Y. Bevins C.L. Nature. 2003; 422: 522-526Crossref PubMed Scopus (634) Google Scholar). Accordingly, an understanding of structure-activity relationships in α-defensins will improve the understanding of mucosal innate immune mechanisms. To investigate the role of α-defensin primary structure in innate immunity, amino acid substitutions that alter charge were introduced into the Crp4 peptide and tested for effects on microbicidal activity and on Crp4-membrane interactions. Regardless of the site of mutagenesis, charge-reversal substitutions at Arg positions altered Crp4 bactericidal activity profoundly and the loss of activity correlated directly with quantitative effects on peptide binding to phospholipid vesicles and with peptide-induced vesicular permeabilization. Preparation of Recombinant Crp4 Peptide Variants—Recombinant Crp4 peptides were expressed in Escherichia coli as N-terminal His6-tagged fusion proteins from the EcoRI and SalI sites of the pET28a expression vector (Novagen, Inc., Madison, WI) as described previously (21Shirafuji 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, 22Satchell 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 (101) Google Scholar). The Crp4-coding cDNA sequences were amplified using forward primer [ER1-Met-C4-F], 5′-GCGCGAATTCATCGAGGGAAGGATGGGTTTGTTATGCTATGT-3′, paired with reverse primer [pMALCrp4-R], 5′-ATATATGTCGACTCAGCGACAGCAGAGCGTGTACAATAAATG-3′. N-terminal Variants—To introduce substitutions at the N terminus, the common reverse primer, [pMALCrp4-R], was paired with the following forward primers: (G1D)-Crp4, [ER1-Met-Gly1AspC4-F], 5′-GCGCGAATTCATCGAGGGAAGGATGGACTTGTTATGCTATTGT-3′; (G1V)-Crp4, [ER1-Met-Gly1ValC4-F], 5′-GCGCGAATTCATCGAGGGAAGGATGGTTTTGTTATGCTATTGT-3′; and (G1R)-Crp4, [ER1-Met-Gly1ArgC4-F], 5′-GCGCGAATTCATCGAGGGAAGGATGCGCTTGTTATGCTATTGT-3′, as described previously (23Ouellette A.J. Satchell D.P. Hsieh M.M. Hagen S.J. Selsted M.E. J. Biol. Chem. 2000; 275: 33969-33973Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 24Satchell D.P. Sheynis T. Kolusheva S. Cummings J.E. Vanderlick T.K. Jelinek R. Selsted M.E. Ouellette A.J. Peptides. 2003; 24: 1795-1805Crossref PubMed Scopus (49) Google Scholar). Mutagenesis at Arg Residue Positions—Mutations were introduced into Crp4 molecules by PCR as described previously (21Shirafuji 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). In the first reaction, a mutant forward primer, e.g. Crp4-R16D-F, containing the mutant codon flanked by three natural codons was paired with reverse primer, [pMALCrp4-R], the normal reverse primer at the 3′-end of the desired sequence. In the second reaction, the mutant reverse primer, Crp4-R16D-R, the reverse complement of the mutant forward primer, was paired with the normal forward primer, [ER1-Met-C4-F], at the 5′ end of Crp4. After amplification at 95 °C for 5 min followed by successive cycles at 60 °C for 1 min, 72 °C for 1 min, and 94 °C for 1 min for 40 cycles, samples of purified products from reactions 1 and 2 were combined as templates in PCR in the third reaction using the Crp4 external primers ER1-Met-C4-F and SLpMALCrp4R as amplimers. All of the mutated Crp4 constructs were verified by DNA sequencing, subcloned into pET28a plasmid DNA (Novagen, Inc.), and transformed into E. coli BL21(DE3)-CodonPlus-RIL cells (Stratagene) for recombinant expression. The underlined codons in forward primers denote Met codons introduced upstream of each peptide N terminus to provide a CNBr cleavage site (21Shirafuji 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, 22Satchell 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 (101) Google Scholar, 24Satchell D.P. Sheynis T. Kolusheva S. Cummings J.E. Vanderlick T.K. Jelinek R. Selsted M.E. Ouellette A.J. Peptides. 2003; 24: 1795-1805Crossref PubMed Scopus (49) Google Scholar). The following C-terminal variants of Crp4 were prepared as noted above with the following mutant reverse primers: (R31D/R32D)-Crp4, [3′PDD-Crp4R], 5′-ATATATGTCGACTGTTCAGTCGTCGGGGCAGCAGTACAA-3′; (R31G/R32G)-Crp4, [3′PGG-Crp4R], 5′-ATATATGTCGACTGTTCACCCCCCGGGGCAGCAGTACAA-3′; (R31V/R32V)-Crp4, [3′PVV-Crp4R], 5′-ATATATGTCGACTGTTCAAACAACGGGGCAGCAGTACAA-3′; (ΔR31/R32)-Crp4, [3′PXX-Crp4R], 5′-ATATATGTCGACTGTTCAGGGGCAGCAGTACAA-3′. Similarly, Crp4 variants with C-terminal extensions resembling that of the Crp4(B6b) peptide (21Shirafuji 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) were prepared using the following primers: (Arg-33/Arg-34)-Crp4, [3′-C4-PRRRR], 5′-GCGCGTCGACTCAGCGGCGGCGGCGTGGGCAGCAGTACAAAAATCG-3′;(Δ-Pro-30/Arg-33/Arg-34)-Crp4, [3′-C4-RRRR], 5′-GCGCGTCGACTCAGCGGCGGCGGCGGCAGCAGTACAAAAATCG-3′. The following primers were used to prepare (R16D/R18D)-Crp4 as noted above: [Crp4-R16D/R18D-F], 5′-AGAGGAGAAGACGTTGACGGGACT-3′, and [Crp4-R16D/R18D-R], 5′-AGTCCCGTCAACGTCTTCTCCTCT-3′. Primers [Crp4-R16K/R18K-F], 5′-AGAGGAGAAAAAGTTAAAGGGACT-3′, and [Crp4-R16K/R18K-R], 5′-AGTCCCTTTAACTTTTTCTCCTCT-3′ were used to prepare (R16D/R18D)-Crp4 and (R16K/R18K)-Crp4, respectively, by the same strategy noted above. To prepare (R16D/R24D)-Crp4 and (R18D/R24D)-Crp4 variants, (R24D)-Crp4 was prepared first using [Crp4-R24D-F], 5′-ACTTGTGGAATAGACTTTTTGTA-3′, and [Crp4-R24D-R], 5′-TACAAAAAGTCTATTCCACAAGT-3′ as mutagenizing primers as above. Subsequently, the (R24D)-Crp4 amplification product was used as template to prepare (R16D/R24D)-Crp4 and (R18D/R24D)-Crp4 double mutants with the following respective primer sets: [Crp4-R16D-F], 5′-GGAGAAGACGTTCGTGGGACT-3′; [Crp4-R16D-R], 5′-AGTCCCACGAACGTC TTCTCC-3′; [Crp4-R18D-F], 5′-GGAGAACGAGTTGACGGGACT-3′; and [Crp4-R18D-R], 5′-AGTCCCGTCAACTCGTTCTCC-3′. Purification of Recombinant Crp4 Proteins—Recombinant proteins were expressed at 37 °C in Terrific Broth medium by induction with 0.1 mm isopropyl-β-d-thiogalactopyranoside for 6 h at 37 °C, lysed by sonication in 6 m guanidine-HCl in 100 mm Tris-Cl (pH 8.1), and clarified by centrifugation (21Shirafuji 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, 22Satchell 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 (101) Google Scholar, 24Satchell D.P. Sheynis T. Kolusheva S. Cummings J.E. Vanderlick T.K. Jelinek R. Selsted M.E. Ouellette A.J. Peptides. 2003; 24: 1795-1805Crossref PubMed Scopus (49) Google Scholar). His-tagged Crp4 fusion peptides were purified using nickel-nitrilotriacetic acid (Qiagen) resin affinity chromatography from bacterial cells lysed in 6 m guanidine-HCl, 20 mm Tris-HCl (pH 8.1) as described previously (21Shirafuji 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). After CNBr cleavage, Crp4 peptides were purified by C18 reverse-phase high performance liquid chromatography and quantitated by bicinchoninic acid (Pierce) and 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 Mass Spectroscopy Facility, Department of Chemistry, University of California (Irvine, CA). Bactericidal Peptide Assays—Recombinant peptides were tested for microbicidal activity against E. coli ML35, S. typhimurium (PhoP-), Vibrio cholera, Staphylococcus aureus 710a, and Listeria monocytogenes 104035 (25Lehrer R.I. Barton A. Ganz T. J. Immunol. Methods. 1988; 108: 153-158Crossref PubMed Scopus (180) 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 volume of trypticase soy broth (21Shirafuji 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, 22Satchell 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 (101) Google Scholar). Bacteria (∼5 × 106 CFU/ml) were incubated with test peptides in 50 μl for 1 h in a shaking incubator at 37 °C, and 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 colony forming units per milliliter after incubation at 37 °C for 12-18 h. Peptide Interactions with Phospholipid/Polydiacetylene (PDA) Mixed Vesicles—Crp4 and three mutant peptides with varied Arg charge reversals were investigated for their relative membrane perturbation activities. Colorimetric phospholipid/PDA vesicles were prepared using dimyristoylphosphatidylcholine (Sigma) as described previously (22Satchell 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 (101) Google Scholar). Vesicles consisting of two parts phospholipid to three parts PDA were prepared by dissolving the phospholipids and 10,12-tricosadiynoic acid monomer (GFS Chemicals, Powell, OH) together in chloroform/ethanol (1:1), drying in vacuo to constant weight, suspending in H2O, probe-sonicating for 3 min at 70 °C, and incubating overnight. PDA was polymerized by irradiation at 254 nm for 10-20 s, producing suspensions with an intense blue appearance. Peptides (0.2-20 μm) were added to 60 μl of vesicle solutions (0.5 mm total lipid) in 25 mm Tris-HCl (pH 8) and diluted to 1 ml, and spectra were acquired at 28 °C between 400 and 700 nm on a Jasco V-550 spectrophotometer (Jasco Corp., Tokyo, Japan) using a 1-cm optical path cell. Blue-to-red color transitions within the vesicle solutions, defined as the percent colorimetric response (%CR), were calculated as described previously (22Satchell 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 (101) Google Scholar, 26Kolusheva S. Kafri R. Katz M. Jelinek R. J. Am. Chem. Soc. 2001; 123: 417-422Crossref PubMed Scopus (170) Google Scholar). Fluorescence-based Vesicle Leakage Assays of Peptide-Membrane Interactions—Crp4, (R31D/R32D)-Crp4, (G1D/R31D/R32D)-Crp4, (R16D/R24D)-Crp4, and pro-Crp4 were tested for their relative abilities to induce leakage from large unilamellar phospholipid vesicles (LUV) of defined composition. LUV of palmitoyl-oleoyl-phosphatidylglycerol (Avanti Polar Lipids, Birmingham, AL) were loaded with a fluorophore/quencher system (27Smolarsky M. Teitelbaum D. Sela M. Gitler C. J. Immunol. Methods. 1977; 15: 255-265Crossref PubMed Scopus (68) Google Scholar, 28Cummings J.E. Satchell D.P. Shirafuji Y. Ouellette A.J. Vanderlick T.K. Austr. J. Chem. 2003; 56: 1031-1034Crossref Scopus (16) Google Scholar). Aqueous lipid solutions consisting of 17 mm 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS, Molecular Probes, Eugene, OR), 60.5 mm DPX (p-xylene-bis-pyridinium bromide, Molecular Probes), 10 mm HEPES, 31 mm NaCl, and 19.5 mm NaOH (260 mosm/liter, pH 7.4) were vortexed, frozen, and thawed for five cycles and then extruded through 100-nm pore size polycarbonate filters. Vesicles were separated from unencapsulated ANTS/DPX by gel-permeation chromatography with 130 mm NaCl, 10 mm HEPES, and 4.5 mm NaOH (260 mosm/liter, pH 7.4) as column eluant. Vesicular suspensions diluted with eluant buffer to ∼74 μm of total lipid were incubated with peptides at ambient temperature. Time-dependent fluorescence produced by ANTS release was monitored at 520 nm (excitation at 353 nm) as described previously (24Satchell D.P. Sheynis T. Kolusheva S. Cummings J.E. Vanderlick T.K. Jelinek R. Selsted M.E. Ouellette A.J. Peptides. 2003; 24: 1795-1805Crossref PubMed Scopus (49) Google Scholar, 28Cummings J.E. Satchell D.P. Shirafuji Y. Ouellette A.J. Vanderlick T.K. Austr. J. Chem. 2003; 56: 1031-1034Crossref Scopus (16) Google Scholar). The kinetics of vesicular leakage was a function of peptide concentration, and equilibrium was attained ≤4 h. Thus, 4-h values were expressed relative to fluorescence obtained by vesicular solubilization with Triton X-100. Bactericidal Activities of Recombinant Crp4 Variants—Recombinant peptides (Fig. 1A) were prepared using the pET-28 vector system (21Shirafuji 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). All of the peptides were purified to homogeneity by reverse-phase high performance liquid chromatography after chemical cleavage with CNBr, migrating as single entities and as expected relative to native Crp4 and pro-Crp4 molecules (21Shirafuji 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, 24Satchell D.P. Sheynis T. Kolusheva S. Cummings J.E. Vanderlick T.K. Jelinek R. Selsted M.E. Ouellette A.J. Peptides. 2003; 24: 1795-1805Crossref PubMed Scopus (49) Google Scholar). Purity was verified by analytical reverse-phase high performance liquid chromatography (data not shown) and acid-urea-PAGE analyses (Fig. 1B). The molecular masses of individual recombinant peptides were determined by MALDI-TOF mass spectrometry, and they matched the respective theoretical values. Thus, the purified recombinant peptides were homogeneous (Fig. 1B) and their biochemical features were consistent with the modifications introduced to the natural Crp4 molecule (29Selsted M.E. Genet. Eng. 1993; 15: 131-147Crossref PubMed Scopus (32) Google Scholar). As a first step toward investigating structure-activity relationships in Crp4, the in vitro microbicidal activities of Crp4, pro-Crp4, and selected N-terminal Crp4 variants were measured against a panel of bacterial test species (data not shown) (24Satchell D.P. Sheynis T. Kolusheva S. Cummings J.E. Vanderlick T.K. Jelinek R. Selsted M.E. Ouellette A.J. Peptides. 2003; 24: 1795-1805Crossref PubMed Scopus (49) Google Scholar). The overall bactericidal activities of the N-terminal variants differed only slightly over a range of 0.6-10 μg/ml of peptide as observed previously (24Satchell D.P. Sheynis T. Kolusheva S. Cummings J.E. Vanderlick T.K. Jelinek R. Selsted M.E. Ouellette A.J. Peptides. 2003; 24: 1795-1805Crossref PubMed Scopus (49) Google Scholar). In contrast to the bactericidal activities of these mature Crp4 peptide variants, pro-Crp4 lacks microbicidal activity because matrix metalloproteinase-7-mediated proteolysis is required for Crp4 activation (18Wilson 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 (903) Google Scholar, 19Ayabe 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 (155) Google Scholar, 21Shirafuji 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). Although the (G1D)-Crp4 peptide was consistently less active at ≤5 μg/ml of peptide (data not shown), modifications at the Crp4 N terminus had very modest effects on bactericidal activity (24Satchell D.P. Sheynis T. Kolusheva S. Cummings J.E. Vanderlick T.K. Jelinek R. Selsted M.E. Ouellette A.J. Peptides. 2003; 24: 1795-1805Crossref PubMed Scopus (49) Google Scholar). Modifications at the Crp4 C terminus—Because the Cys-1 to Cys-6-disulfide bond common to α-defensins places the Crp4 N and C termini in proximity (13Selsted M.E. Harwig S.S. J. Biol. Chem. 1989; 264: 4003-4007Abstract Full Text PDF PubMed Google Scholar), we prepared a series of Crp4 peptide variants with charge-modified C termini including (R31D/R32D)-Crp4, (R31G/R32G)-Crp4, (R31V/R32V)-Crp4, and (ΔR31/R32)-Crp4 (Fig. 1). Bactericidal activity assays of these variants showed that deleting the two C-terminal Arg residues or converting them to Gly or Val altered peptide activity variably depending on the bacterial target species (Fig. 2, compare B with D). In contrast, however, the R31D/R32D charge-reversal mutation eliminated bactericidal activity, even against the S. typhimurium PhoP-strain (data not shown), which is very sensitive to membrane-active cationic peptides (Fig. 2) (30Fields P.I. Groisman E.A. Heffron F. Science. 1989; 243: 1059-1062Crossref PubMed Scopus (404) Google Scholar, 31Miller S.I. Pulkkinen W.S. Selsted M.E. Mekalanos J.J. Infect. Immun. 1990; 58: 3706-3710Crossref PubMed Google Scholar). As expected, wild-type S. typhimurium and the PhoP-constitutive (PhoPc) CS022 strain were less sensitive to all of the peptides (data not shown). Additional (R31D/R32D)-Crp4 variants with modified N termini were prepared and analyzed including an Arg1 for Gly1 substitution ((G1R/R31D/R32D)-Crp4) and an Asp1 for Gly1 variant ((G1D/R31D/R32D)-Crp4). Under the conditions of the assays, no bactericidal activity was detected with the (G1D/R31D/R32D)-Crp4 peptide but the (G1R/R31D/R32D)-Crp4 activity improved with the added electropositive side chain at the N terminus (Fig. 3). These findings showed that modifications of combined N- and C-terminal charge can modulate Crp4 bactericidal activity. To investigate the effect of added C-terminal Arg residues on Crp4 bactericidal activity, additional modifications were introduced at the Crp4 C terminus. C57/BL6 mice express a Crp4 variant (Crp4(B6b)) that has four Arg residues at its C terminus (21Shirafuji 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), but because Crp4 and Crp4(B6b) differ at several other positions, the specific role of C-terminal charge in these peptides cannot be compared directly. Accordingly, the natural Crp4 C terminus was extended by two Arg residues to place the Crp4-(B6b) C terminus in the context of the Crp4 primary structure. The peptide, (Arg-33/Arg-34)-Crp4, had greater in vitro bactericidal peptide activity than Crp4 against E. coli and wild-type and PhoPc S. typhimurium strains (Fig. 4), suggesting that C-terminal charge could be a determinant of bactericidal activity. However, the Crp4(B6b) molecule lacks the Pro-30 residue present in Crp4 between Cys-29 and Arg-31. Therefore, the effect of the Pro-30 residue was tested by assaying the bactericidal activity of (ΔPro-30/Arg-33/Arg-34)-Crp4, a variant of (Arg-33/Arg-34)-Crp4 from which Pro-30 was deleted (Fig. 1). (ΔPro-30/Arg-33/Arg-34)-Crp4 was less active than Crp4 and (Arg-33/Arg-34)-Crp4 against E. coli and S. typhimurium PhoPc (Fig. 4), and the results in Fig. 4 also are representative of the relative bactericidal activities of these peptides against V. cholera, S. aureus, and L. monocytogenes (data not shown). Thus, addition of Arg residues at the C terminus per se is not sufficient to improve peptide activity in the absence of proline at residue position 30. Because the R31D/R32D C-terminal charge reversal eliminated Crp4 bactericidal activity, the effects of charge alterations at other residue positions in the Crp4 polypeptide chain were studied to test whether the effects were specific to C-terminal modification. Charge Reversal at Arg Positions Result in Loss-of-function—Crp4 mutants with double Asp for Arg substitutions were introduced at three additional Arg pairs, purified, and assayed for bactericidal peptide activity (see "Experimental Procedures"). As was observed for (R31D/R32D)-Crp4, the R16D/R18D, R16D/R24D, and R18D/R24D variants of Crp4 all lacked bactericidal activity under the conditions of these in vitro assays (Fig. 5). Thus, the loss of activity by charge reversal is not a specific effect at the C terminus, because all of the peptides with double charge-reversal mutations were inactive. Perhaps, Asp for Arg substitutions eliminate bactericidal activity by reducing the overall electropositivity of the Crp4 peptide. Nevertheless, it is unlikely that Arg residues at specific positions are required for Crp4 bactericidal activity because all of the mutations introduced resulted in equivalent loss-of-function independent of the position of the replacements. To determine whether a specific requirement exists for the guanidinium group of the Arg side chain in Crp4 or whether charge-neutral substitutions at Arg positions would result in peptides with equivalent bactericidal activity, we prepared (R16K/R18K)-Crp4 (Fig. 1A) and evaluated its antimicrobial activity. Because all of the Crp4 charge-reversal mutants were attenuated similarly including (R16D/R18D)-Crp4, the (R16K/R18K)-Crp4 internal charge-neutral mutant was taken to be representative of such peptide alterations. As stated earlier, the Lys for Arg substituted peptide was purified to homogeneity as i
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