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• W2052148977 abstract "We have examined the interactions of the six known rabbit neutrophil defensin antimicrobial peptides with large unilamellar vesicles (LUV) made from various lipid mixtures based on the lipid composition of Escherichia coli membranes. We find that the permeabilization of LUV made from E. coliwhole lipid extracts differs dramatically from that of single-component LUV made from palmitoyl-oleoyl-phosphatidylglycerol (POPG). Specifically, defensins NP-1, NP-2, NP-3A, NP-3B, and a natural mixture of the six defensins cause fast nonpreferential leakage of high molecular weight dextrans as well as the low molecular weight fluorophore/quencher pair 8-aminonapthalene-1,3,6 trisulfonic acid (ANTS)/p-xylene-bis-pyridinium bromide (DPX) from E. coli whole lipid LUV through large, transient membrane lesions. In contrast, release of ANTS/DPX from POPG LUV induced by the defensins is slow and graded with preference for DPX (Hristova, K., Selsted, M. E., and White, S. H. (1996) Biochemistry35, 11888–11894). Interestingly, defensins NP-4 and NP-5 alone do not induce leakage from E. coli whole lipid LUV, whereas only NP-4 is ineffective with POPG LUV. Examination of the sequences of the six defensins suggests that the inactivity of NP-4 and NP-5 may be due to their lower net positive charge and/or the substitution of a Thr for the Arg or Lys that follows the fourth Cys residue. We found the presence of three major lipid components of E. coli whole lipid to be essential for creation of the large lesions observed in LUV: phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin. Cardiolipin appears to play a key role because no leakage can be induced when only phosphatidylglycerol and phosphatidylethanolamine are present. These results indicate the importance of membrane lipid composition in the permeabilization of cell membranes by rabbit defensins. We have examined the interactions of the six known rabbit neutrophil defensin antimicrobial peptides with large unilamellar vesicles (LUV) made from various lipid mixtures based on the lipid composition of Escherichia coli membranes. We find that the permeabilization of LUV made from E. coliwhole lipid extracts differs dramatically from that of single-component LUV made from palmitoyl-oleoyl-phosphatidylglycerol (POPG). Specifically, defensins NP-1, NP-2, NP-3A, NP-3B, and a natural mixture of the six defensins cause fast nonpreferential leakage of high molecular weight dextrans as well as the low molecular weight fluorophore/quencher pair 8-aminonapthalene-1,3,6 trisulfonic acid (ANTS)/p-xylene-bis-pyridinium bromide (DPX) from E. coli whole lipid LUV through large, transient membrane lesions. In contrast, release of ANTS/DPX from POPG LUV induced by the defensins is slow and graded with preference for DPX (Hristova, K., Selsted, M. E., and White, S. H. (1996) Biochemistry35, 11888–11894). Interestingly, defensins NP-4 and NP-5 alone do not induce leakage from E. coli whole lipid LUV, whereas only NP-4 is ineffective with POPG LUV. Examination of the sequences of the six defensins suggests that the inactivity of NP-4 and NP-5 may be due to their lower net positive charge and/or the substitution of a Thr for the Arg or Lys that follows the fourth Cys residue. We found the presence of three major lipid components of E. coli whole lipid to be essential for creation of the large lesions observed in LUV: phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin. Cardiolipin appears to play a key role because no leakage can be induced when only phosphatidylglycerol and phosphatidylethanolamine are present. These results indicate the importance of membrane lipid composition in the permeabilization of cell membranes by rabbit defensins. Neutrophil defensins have been isolated from several species, including humans (1Selsted M.E. Harwig S.S.L. Ganz T. Schilling J.W. Lehrer R.I. J. Clin. Invest. 1985; 76: 1436-1439Crossref PubMed Scopus (409) Google Scholar, 2Wilde C.G. Griffith J.E. Marra M.N. Snable J.L. Scott R.W. J. Biol. Chem. 1989; 264: 11200-11203Abstract Full Text PDF PubMed Google Scholar) and rabbits (3Selsted M.E. Brown D.M. DeLange R.J. Harwig S.S.L. Lehrer R.I. J. Biol. Chem. 1985; 260: 4579-4584Abstract Full Text PDF PubMed Google Scholar). They are small (M r = ∼4000) cationic antimicrobial peptides that act primarily by permeabilizing the cell membranes of a wide variety of microbes (4Lichtenstein A. Ganz T. Selsted M.E. Lehrer R.I. Blood. 1986; 68: 1407-1410Crossref PubMed Google Scholar, 5Lehrer R.I. Szklarek D. Ganz T. Selsted M.E. Infect. Immun. 1985; 49: 207-211Crossref PubMed Google Scholar, 6Lehrer R.I. Barton A. Daher K.A. Harwig S.S.L. Ganz T. Selsted M.E. J. Clin. Invest. 1989; 84: 553-561Crossref PubMed Scopus (590) Google Scholar). Neutrophil defensins have 29–34 amino acids, are rich in arginine (4–10 per molecule), and have three disulfide bonds that stabilize a rigid β-sheet structure (see Ref. 7White S.H. Wimley W.C. Selsted M.E. Curr. Opin. Struct. Biol. 1995; 5: 521-527Crossref PubMed Scopus (382) Google Scholarfor a review). The amino acid sequences of the six rabbit and four human neutrophil defensins are presented in Fig.1 (residues are numbered according to the NP-1 sequence). Although neutrophil defensins kill bacteria, fungi, and enveloped viruses effectively, they are tolerated at high concentrations inside membrane-bound neutrophil granules. The suggestion has thus been made that antimicrobial peptide selectivity is determined by differences in the lipid compositions of the target and host membranes (8Tytler E.M. Anantharamaiah G.M. Walker D.E. Mishra V.K. Palgunachari M.N. Segrest J.P. Biochemistry. 1995; 34: 4393-4401Crossref PubMed Scopus (119) Google Scholar). Available evidence supports the hypothesis that the principal mode of action of neutrophil defensins involves physical perturbation and permeabilization of the membranes of the target organisms (6Lehrer R.I. Barton A. Daher K.A. Harwig S.S.L. Ganz T. Selsted M.E. J. Clin. Invest. 1989; 84: 553-561Crossref PubMed Scopus (590) Google Scholar, 9Kagan B.L. Selsted M.E. Ganz T. Lehrer R.I. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 210-214Crossref PubMed Scopus (451) Google Scholar, 10Lehrer R.I. Daher K.A. Ganz T. Selsted M.E. J. Virol. 1985; 54: 467-472Crossref PubMed Google Scholar). Therefore, studies of defensin-induced permeabilization of model membranes provide valuable insight to their permeabilization of biological membranes (11Wimley W.C. Selsted M.E. White S.H. Protein Sci. 1994; 3: 1362-1373Crossref PubMed Scopus (336) Google Scholar, 12Hristova K. Selsted M.E. White S.H. Biochemistry. 1996; 35: 11888-11894Crossref PubMed Scopus (82) Google Scholar). An important observation is that neutrophil defensins isolated from different species vary in their mode of action. Human neutrophil defensin HNP-2 forms large multimeric pores in large unilamellar vesicles (LUV) 1The abbreviations used are: LUV, extruded unilamellar vesicles of 100-nm diameter; POPE, 1-palmitoyl-2-oleoyl phosphatidylethanolamine; POPG, 1-palmitoyl-2-oleoyl phosphatidylglycerol; POPC, 1palmitoyl-2-oleoyl phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; diPG, di-phosphatidylglycerol (cardiolipin);E. coli whole lipid, chloroform-methanol extract of lyophilized E. coli cells; ANTS, 8-aminonapthalene-1,3,6 trisulfonic acid, M r = 427.33; DPX,p-xylene-bis-pyridinium bromide, M r = 422.16; HNP, human neutrophil peptide (human defensin) NP, rabbit neutrophil peptide (rabbit defensin); FD-4, fluorescein isothiocyanate-labeled dextran, M r 4,400; FD-20, fluorescein isothiocyanate-labeled dextran, M r18,900; FD-70, fluorescein isothiocyanate-labeled dextran,M r 50,700; NBD-PE, 7-nitro-benzoxadiazole phosphatidylethanolamine; HPLC, high pressure liquid chromatography.formed from POPG, causing the vesicle contents to be released in an all-or-none manner (11Wimley W.C. Selsted M.E. White S.H. Protein Sci. 1994; 3: 1362-1373Crossref PubMed Scopus (336) Google Scholar). Rabbit neutrophil defensins cause graded leakage (12Hristova K. Selsted M.E. White S.H. Biochemistry. 1996; 35: 11888-11894Crossref PubMed Scopus (82) Google Scholar). Despite high sequence identity and three-dimensional structural similarity of monomers, HNP-2 exists as dimers in aqueous solution, whereas rabbit defensins are always monomeric. This suggests that the lack of aqueous dimerization by rabbit neutrophil defensins leads to a fundamental difference in defensin assembly on the membrane surface and thus in the mechanism of permeabilization (11Wimley W.C. Selsted M.E. White S.H. Protein Sci. 1994; 3: 1362-1373Crossref PubMed Scopus (336) Google Scholar, 12Hristova K. Selsted M.E. White S.H. Biochemistry. 1996; 35: 11888-11894Crossref PubMed Scopus (82) Google Scholar). Another important observation is that the lipid composition of the LUV is important; small additions of neutral lipids (1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) or 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE)) to POPG LUV eliminate leakage even though defensins can bind to the mixed bilayers (12Hristova K. Selsted M.E. White S.H. Biochemistry. 1996; 35: 11888-11894Crossref PubMed Scopus (82) Google Scholar). This indicates that lipid composition can strongly influence membrane permeabilization by neutrophil defensins. A third observation of importance is that small differences in monomer sequence can have large effects on membrane permeabilization (12Hristova K. Selsted M.E. White S.H. Biochemistry. 1996; 35: 11888-11894Crossref PubMed Scopus (82) Google Scholar). In the present study, our goal was to examine in greater detail the lipid requirements for rabbit neutrophil defensin action using defensin concentrations that are well within the normal physiological range (13Ganz T. Selsted M.E. Lehrer R.I. Eur. J. Hematol. 1990; 44: 1-8Crossref PubMed Scopus (268) Google Scholar). We began with an examination of the interactions of the six known rabbit neutrophil defensins, individually and as the “natural” mixture, with LUV made from whole lipid extracts of Escherichia coli membranes. The lipid content varies among strains but typically is about 20–50% charged lipid (PG and diphosphatidylglycerol (cardiolipin, diPG) and about 50–80% neutral lipid (PE) (14Randle C.L. Albro P.W. Dittmer J.C. Biochim. Biophys. Acta. 1969; 187: 214-220Crossref PubMed Scopus (84) Google Scholar). We found that defensins NP-1, NP-2, NP-3A, NP-3B, and the natural mixture NP-1–5 induced membrane permeabilization, probably through large scale transient lesions. NP-4 and NP-5 alone had no effect on membrane permeability but did have synergistic effects when used in combination with other defensins. Under the same experimental conditions, bilayer permeabilization of POPG vesicles was fundamentally different as indicated by leakage that had a preference to DPX (12Hristova K. Selsted M.E. White S.H. Biochemistry. 1996; 35: 11888-11894Crossref PubMed Scopus (82) Google Scholar) and was slower. We then examined the permeabilization by NP-2 and NP-5 of LUV composed of various combinations of the three major E. coli lipid components, POPE, POPG, and cardiolipin, and confirmed the strong influence of lipid composition on the activity of rabbit neutrophil defensins. Cardiolipin was found to be especially important, although all three lipids must be present to mimic E. coliwhole lipid. Further, we identified amino acid residues in the sequence of the six rabbit defensins that may be crucial for the observed activity. Our results indicate that permeabilization of microbial membranes is likely to be extremely sensitive to lipid composition, as well as to the composition of the defensin mixture. E. coli whole lipid extracts as PE from E. coli ∼50%, lot 41H8362, and fluorescein-labeled dextrans FD-4, FD-20, and FD-70 were purchased from Sigma. The lipid composition of the extract was analyzed by Avanti Polar Lipids using normal phase HPLC with evaporative light scattering detection. Consistent with other studies (14Randle C.L. Albro P.W. Dittmer J.C. Biochim. Biophys. Acta. 1969; 187: 214-220Crossref PubMed Scopus (84) Google Scholar, 15Kanemasa Y. Yoshioka T. Ichikawa H. Takai K. Acta Med. Okayama. 1971; 25: 255-260PubMed Google Scholar, 16Kanemasa Y. Akamatsu Y. Nojima S. Biochim. Biophys. Acta. 1967; 144: 382-390Crossref PubMed Scopus (85) Google Scholar, 17Ames G.F. J. Bacteriol. 1968; 95: 833-843Crossref PubMed Google Scholar), the three major components of the extract are PE (78%), diPG (14.4%), and PG (4.7%). The preparation also contained minor components identified as phosphatidylcholine (1.2%), phosphatidylserine (0.8%), and lyso-PE (0.9%). NBD- and rhodamine-labeled lipids were obtained from Avanti Polar Lipids (Birmingham, AL). ANTS and DPX were obtained from Molecular Probes (Eugene, OR). Water was glass distilled. The buffer composition was 10 mm HEPES, 50 mm KCl, 1 mm EDTA, 3 mm NaN3, pH 7.0. About 70 mg of lipid was combined in chloroform that was subsequently removed under a stream of argon. About 1 ml of buffer containing ANTS/DPX or dextran was added to the dry lipids, and the suspension was frozen and thawed 10 times to assure maximum entrapment prior to extrusion (11Wimley W.C. Selsted M.E. White S.H. Protein Sci. 1994; 3: 1362-1373Crossref PubMed Scopus (336) Google Scholar). The final lipid concentration was typically 100 mm. A stock solution of LUV of approximately 0.1 μm in diameter was formed by extrusion under N2 pressure through Nucleopore polycarbonate membranes (11Wimley W.C. Selsted M.E. White S.H. Protein Sci. 1994; 3: 1362-1373Crossref PubMed Scopus (336) Google Scholar,18Mayer L.D. Hope M.J. Cullis P.R. Biochim. Biophys. Acta. 1986; 858: 161-168Crossref PubMed Scopus (1576) Google Scholar). The concentrations of solutes used were 9 mm ANTS, 25 mm DPX, 8 mg/ml FD-4, 20 mg/ml FD-20, and 40 mg/ml FD-70. Assuming that the concentration inside the vesicles equaled the initial concentration, we estimated entrapment of about 200–300 dextrans/vesicle; entrapment of ANTS/DPX should be about an order of magnitude higher. The total KCl concentration in ANTS/DPX-containing vesicles was adjusted so that the entrapped solutions had the same osmolarity as the external 50 mm KCl buffer. Unencapsulated ANTS and DPX were separated from encapsulated material using Sephadex G-100 packed in a 2.5-ml Pasteur pipette. Untrapped FD-4, FD-20, and FD-70 were removed on a 45 × 1.3-cm Sephadex G-100 column run at approximately 1 ml/min. Typically, about 50 μl of the vesicle stock solution (above) was applied to the column and eluted with several milliliters of buffer. The fraction of the effluent collected (about 0.8 ml) had a lipid concentration of about 14 mm. After separation, nonfluorescent dextran of approximately the same molecular weight was added externally to the FD-4, FD-20, and FD-70 liposomal suspensions of E. colilipids to eliminate any osmotic stress on the vesicles. No detectable leakage of any solutes in the absence of defensin was observed during periods of time corresponding to the duration of the experiments. Fluorescence spectroscopy was performed using either a SPEX Fluorolog fluorimeter that was upgraded and interfaced to a computer by OLIS, Inc. (Jefferson, GA) or a SLM AMINCO 8100 spectrophotometer (Rochester, NY). Excitation and emission wavelengths were as follows: ANTS (SPEX): excitation, 360 nm (slit, 20 nm), and emission, 515 nm (slit, 50 nm); NBD-PE (SPEX): excitation, 465 nm: (slit, 8 nm), and emission, 530 nm (slit, 8 nm); FD-20 (SPEX): excitation, 495 (slit, 8 nm), and emission, 525 (slit, 8 nm); and FD-4 and FD-70 (SLM AMINCO): excitation, 495 (slit, 1 nm), and emission, 525 (slit, 4 nm). Right angle geometry and no reference signal were used. Emission spectra covering the range of the fluorescence spectra of the fluorophores were collected from samples with and without vesicles to assure that light scattering made no contribution to the fluorescence signals of the fluorophores. We found in all cases that the scattering peak made no significant contribution to the fluorescence signals at the emission wavelengths used. Vesicles containing ANTS/DPX, FD-4, FD-20, and FD-70 at about 400 μm were placed into 1 × 0.2-cm quartz cuvette (volume, 0.5 ml), and the fluorescence increase due to leakage and subsequent dilution of quenched dye was measured after the addition of defensin. Data are presented in terms of fractional fluorescence f F = (F− F initial)/(F max −F initial), where F is the measured fluorescence, F initial is the initial quenched fluorescence, and F max is the fluorescence corresponding to 100% leakage as established by the addition of 0.4% Triton X-100. In the absence of leakage, a fluorescenceF initial = QF max is observed, where Q is the quenching factor (about 0.14–0.2 for E. coli whole lipid/ANTS/DPX vesicles, 0.2 for E. coli whole lipid/FD-4, 0.5 for E. coli whole lipid/FD-20, and 0.6 for E. coli whole lipid/FD-70 vesicles). Note that Q = 0 corresponds to complete quenching, whereas Q = 1 corresponds to no quenching. There are two general mechanisms of leakage. It can be a graded process in which all vesicles release portions of their contents, or it can be an all-or-none process in which some vesicles lose all of their contents while others lose none. One can distinguish between these two possibilities using the so-called fluorescence requenching method, which is based on the idea that the ANTS molecules inside and outside the vesicles show different susceptibility to quenching with externally added DPX (11Wimley W.C. Selsted M.E. White S.H. Protein Sci. 1994; 3: 1362-1373Crossref PubMed Scopus (336) Google Scholar, 12Hristova K. Selsted M.E. White S.H. Biochemistry. 1996; 35: 11888-11894Crossref PubMed Scopus (82) Google Scholar, 19Ladokhin A.S. Wimley W.C. White S.H. Biophys. J. 1995; 69: 1964-1971Abstract Full Text PDF PubMed Scopus (127) Google Scholar, 20Ladokhin A.S. Wimley W.C. Hristova K. White S.H. Methods Enzymol. 1997; 278: 474-486Crossref PubMed Scopus (60) Google Scholar). In brief, one measures the dependence of ANTS quenching inside the vesicles Q in as a function of the fraction of ANTS that has leaked out of the vesicles. If Q inis independent of the fraction of ANTS that has leaked out, then the leakage is all-or-none. If Q in increases withf out then the leakage is graded. For graded release, Q in depends onf out as follows (19Ladokhin A.S. Wimley W.C. White S.H. Biophys. J. 1995; 69: 1964-1971Abstract Full Text PDF PubMed Scopus (127) Google Scholar),Qin={(1+Kd[DPX]0(1-fout)a).(1+Ka[DPX]0(1-fout)a)}-1Equation 1 where [DPX]0 is the initial DPX concentration inside the vesicles, and α is the selectivity defined as the ratio of the rates of release of ANTS and DPX. The constantK d is the dynamic quenching constant, andK a is the association constant for the ANTS/DPX nonfluorescent complex. They were determined previously to be 50m−1 and 490 m−1, respectively (19Ladokhin A.S. Wimley W.C. White S.H. Biophys. J. 1995; 69: 1964-1971Abstract Full Text PDF PubMed Scopus (127) Google Scholar). The selectivity α is determined by fitting this equation to the experimental data using standard nonlinear least squares methods (19Ladokhin A.S. Wimley W.C. White S.H. Biophys. J. 1995; 69: 1964-1971Abstract Full Text PDF PubMed Scopus (127) Google Scholar). The extent of aggregation of 400 μm vesicles of E. coli whole lipid by rabbit defensins was assayed by measuring the total concentration of defensin and lipid and the concentrations remaining in solution after centrifugation of the lipid/defensin mixture in a table top centrifuge (1000 × g) for 10 min. The pellet was resuspended in buffer and also assayed. The lipid concentration was determined by fluorescence measurements using E. coli whole lipid vesicles doped with 1 mol% NBD-PE. Peptide concentration was assayed by HPLC (21Wimley W.C. White S.H. Anal. Biochem. 1993; 213: 213-217Crossref PubMed Scopus (43) Google Scholar). A 50 μm solution of E. coliwhole lipid LUV containing 1 mol% of both NBD-PE and rhodamine-PE were diluted with a 10-fold excess of unlabeled E. coli whole lipid vesicles, and the defensin was added to the mixture (22Struck D.K. Hoekstra D. Pagano R.E. Biochemistry. 1981; 20: 4093-4099Crossref PubMed Scopus (1142) Google Scholar). The fluorescence increase due to dilution of the quencher was monitored and quantitated as fractional fluorescence f F = (F final −F initial)/(F max −F initial), where F finalis the final plateau level of fluorescence,F initial is the initial quenched fluorescence, and F max is the fluorescence of 550 μm E. coli whole lipid LUV suspensions doped with 0.1 mol% of both NBD-PE and rhodamine-PE. Mixing of vesicle contents upon fusion was assayed by mixing equal amounts of ANTS- and DPX-containing LUV at 400 μm total concentration and monitoring ANTS fluorescence after the addition of defensins (23Ellens H. Bentz J. Szoka F.C. Biochemistry. 1984; 23: 1532-1538Crossref PubMed Scopus (429) Google Scholar). The leakage of low molecular weight markers fromE. coli whole lipid LUV was examined using the dye/quencher pair ANTS/DPX as described under “Experimental Procedures.” Shown in Fig. 2 are the fractional fluorescence changes that occur following the addition of 30 μg/ml of NP-2, NP-5, or the natural mixture NP-1–5 corresponding to the naturally occurring ratio of the six rabbit defensins (26% NP-1, 17% NP-2, 6% NP-3A, 6% NP-3B, 15% NP-4, and 30% NP-5). The zero level in Fig. 2 corresponds to zero leakage, and the maximum level corresponds to complete release of ANTS/DPX. The results show that both NP-2 and NP-1–5 induce leakage of contents of E. coli whole lipid vesicles and that NP-5 does not. NP-1, NP-3A, and NP-3B behaved similarly to NP-2, whereas NP-4, like NP-5, caused no leakage (data not shown). For both NP-2 and NP-1–5, a fast initial fluorescence signal increase followed by a slower one is observed. Although particular fluorescence levels are the same as for POPG LUV (12Hristova K. Selsted M.E. White S.H. Biochemistry. 1996; 35: 11888-11894Crossref PubMed Scopus (82) Google Scholar), the kinetics of the increase are much faster (5–10 min versus several hours for POPG). However, we observed subtle differences in the kinetics of release. For instance, as seen in Fig. 2, NP-2 causes a faster fluorescence increase toward a plateau level than NP-1–5. Fig. 3 shows the fractional fluorescence increase f F caused by leakage from E. coli whole lipid vesicles induced by NP-2 and NP-1–5 as a function of defensin concentration. The measurements were made in the plateau region (see Fig. 2) approximately 30 min after defensin addition. f F is a sigmoidal function of defensin concentration, indicating defensin cooperativity. The results for NP-2 and NP-1–5 presented in Fig. 3 were obtained using a single LUV preparation; the exact shape varied somewhat between preparations. Also shown in Fig. 3 are measurements of increases inf F caused by leakage from various mixed lipid vesicles induced by NP-2 (see below). In our previous study of the interactions of rabbit neutrophil defensins with POPG LUV (12Hristova K. Selsted M.E. White S.H. Biochemistry. 1996; 35: 11888-11894Crossref PubMed Scopus (82) Google Scholar), no synergy was observed between the six peptides despite the occurrence of antimicrobial synergy in vivo (24Lehrer R.I. Szklarek D. Ganz T. Selsted M.E. Infect. Immun. 1986; 52: 902-904Crossref PubMed Google Scholar). In the present case, however, there was significant synergy between NP-5 and a mixture of NP-1, NP-2, NP-3A, NP-3B, and NP-4 (NP-1–4). Fig. 4 shows the kinetics of ANTS/DPX leakage induced by 40 μg/ml NP-1–5 and by 30 μg/ml NP-1–4 mixture containing the same amounts of NP-1, NP-2, NP3A, NP-3B, and NP-4. The data reveal that although NP-5 did not cause leakage by itself (Fig. 2), it enhanced the permeabilizing properties of NP-1–4. Similarly, NP-4 enhanced the permeabilizing properties of a mixture of NP-1, NP-2, NP-3A, NP-3B, and NP-5 (data not shown). The possibility of synergy among other defensin combinations remains to be examined. The mechanism of ANTS/DPX leakage was established by the fluorescence requenching method that has been described in detail elsewhere (see “Experimental Procedures” and Refs. 11Wimley W.C. Selsted M.E. White S.H. Protein Sci. 1994; 3: 1362-1373Crossref PubMed Scopus (336) Google Scholar, 12Hristova K. Selsted M.E. White S.H. Biochemistry. 1996; 35: 11888-11894Crossref PubMed Scopus (82) Google Scholar, and 19Ladokhin A.S. Wimley W.C. White S.H. Biophys. J. 1995; 69: 1964-1971Abstract Full Text PDF PubMed Scopus (127) Google Scholar). Briefly, when fluorescence reaches a plateau after the addition of a particular amount of defensin, DPX is added externally to quench the released ANTS. The remaining unquenchable fluorescence is then due to the ANTS retained in the vesicles (if any). Such measurements allow one to determine the residual internal quenching Q in as a function of the fraction f out of ANTS released in a series of release experiments. As discussed under “Experimental Procedures,” if Q in is independent off out, then release is all-or-none, whereas ifQ in increases with f out, then release is graded. One can further establish in the latter case whether the release is preferential for ANTS or DPX by means of the selectivity parameter α, which is the ratio of DPX to ANTS that leaked out of the vesicles. Fig.5 A shows the results of requenching experiments for release induced by NP-2 from E. coli whole lipid vesicles. The internal quenching,Q in, of ANTS by DPX increases withf out and is above the initial level ofQ in of 0.14. The dotted line in Fig.5 A shows the result expected had the leakage been all-or-none. The solid line in Fig. 5 A is the best fit of the data to Equation 1, which yields a value for α of 1.17 ± 0.24. Similar results were obtained for NP-1–5 as shown in Fig. 5 B (α = 1.13 ± 0.26) and for NP-1, NP-3A, and NP-3B (data not shown). A χ2 test showed that these values are not statistically different from 1.0 (reduced χ2 ∼ 0.87). Thus, the experiments did not distinguish preferential leakage for ANTS or DPX, suggesting that the release of solutes occurs in a nondiscriminative manner. The nonpreferential leakage of ANTS and DPX suggested that the leakage path aperture might be much larger than the characteristic dimensions of ANTS and DPX. We therefore examined the effect of the rabbit defensins on the leakage of high molecular weight dextrans fromE. coli whole lipid vesicles. The leakage of high molecular weight compounds was examined using fluorescein-labeled dextrans (FD-4,M r = 4,400; FD-20, M r = 18,700; and FD-70, M r = 50,700). The possibility of these dextrans binding to E. coli whole lipid liposomes was examined as described elsewhere (25Ladokhin A.S. Selsted M.E. White S.H. Biophys. J. 1997; 72: 1762-1766Abstract Full Text PDF PubMed Scopus (193) Google Scholar). No evidence of binding was found. 2A. Ladokhin, personal communication. We took advantage of the fact that the fluorescein-dextrans self-quench as indicated by the observation that the concentration dependence of dextran fluorescence in vesicle-free solutions deviates from linearity at high concentrations (data not shown). Self-quenching concentrations of dextrans were therefore entrapped, and defensin-induced leakage was detected by increases in fluorescence due to dextran dilution during leakage. The fractional fluorescence increases off F following additions of NP-2 and NP-1–5 to solutions of 400 μm E. coli whole lipid LUV with entrapped dextrans are plotted in Fig.6 as a function of defensin concentration. All measurements were made in the plateau region of the release kinetics approximately 30–40 min after defensin addition. The zero level corresponds to vesicle fluorescence in the absence of defensins. The maximum level of fluorescence, determined by lysis of the vesicles with Triton X-100 detergent, was assigned a value of 1. For two of the NP-1–5 concentrations, we performed several experiments with different FD-70 vesicle preparations to estimate experimental variability (error bars, Fig. 6 B). Fig. 6 shows that NP-1–5 and NP-2 induce the leakage of all of the dextrans studied. Furthermore, the leakage increases with defensin concentration, up to about 20 μg/ml, and then levels off at about 40–50%, independent of dextran molecular weight. The kinetics of the leakage of ANTS/DPX and FD-20 from E. coli whole lipid vesicles induced by NP-2 are compared in Fig.7. The data show that NP-2-treated vesicles are leaky to both small molecules (ANTS/DPX) and large dextran molecules. Note, however, that although ANTS fluorescence quickly reaches its plateau level of almost 1, the fluorescence of FD-20 levels off at about 0.5 with a slower time course. The kinetics and extent of leakage of the release of FD-4 and FD-70 are very similar to those of FD-20 under similar conditions (data not shown). The data presented in Figs. 6 and 7 reveal that dextran leakage is independent of molecular weight and that it is never complete (Fig. 6). We considered the possibility that the dextran-loaded E. coli whole lipid vesicles are incapable of releasing all of their contents regardless of the leakage mechanism. To examine this possibility, we took advantage of the fact that denatured defensins (reduced disulfide bonds) induce leakage by a different mechanism than native defensins and are far more effective in causing leakage of ANTS/DPX (11Wimley W.C. Selsted M.E. White S.H. Protein Sci. 1994; 3: 1362-1373Crossref PubMed Scopus (336) Google Scholar, 26Fujii G. Selsted M.E. Eisenberg D. Protein Sci. 1993; 2: 1301-1312Crossref PubMed Scopus (148) Google Scholar). The time dependence of the fractional fluorescence increase f F accompanying FD-4 leakage induced by 15 μg/ml reduced NP-2 is shown in Fig.8. Although the increase inf F is" @default.
• W2052148977 created "2016-06-24" @default.
• W2052148977 creator A5000748364 @default.
• W2052148977 creator A5045196992 @default.
• W2052148977 creator A5053094051 @default.
• W2052148977 date "1997-09-01" @default.
• W2052148977 modified "2023-10-03" @default.
• W2052148977 title "Critical Role of Lipid Composition in Membrane Permeabilization by Rabbit Neutrophil Defensins" @default.
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