Evidence That Clustered Phosphocholine Head Groups Serve as Sites for Binding and Assembly of an Oligomeric Protein Pore

Abstract

High susceptibility of rabbit erythrocytes toward the poreforming action of staphylococcal α-toxin correlates with the presence of saturable, high affinity binding sites. All efforts to identify a protein or glycolipid receptor have failed, and the fact that liposomes composed solely of phosphatidylcholine are efficiently permeabilized adds to the enigma. A novel concept is advanced here to explain the puzzle. We propose that low affinity binding moieties can assume the role of high affinity binding sites due to their spatial arrangement in the membrane. Evidence is presented that phosphocholine head groups of sphingomyelin, clustered in sphingomyelin-cholesterol microdomains, serve this function for α-toxin. Clustering is required so that oligomerization, which is prerequisite for stable attachment of the toxin to the membrane, can efficiently occur. Outside these clusters, binding to phosphocholine is too transient for toxin monomers to find each other. The principle of membrane targeting in the absence of any genuine, high affinity receptor may also underlie the assembly of other lipid-inserted oligomers including cytotoxic peptides, protein toxins, and immune effector molecules. High susceptibility of rabbit erythrocytes toward the poreforming action of staphylococcal α-toxin correlates with the presence of saturable, high affinity binding sites. All efforts to identify a protein or glycolipid receptor have failed, and the fact that liposomes composed solely of phosphatidylcholine are efficiently permeabilized adds to the enigma. A novel concept is advanced here to explain the puzzle. We propose that low affinity binding moieties can assume the role of high affinity binding sites due to their spatial arrangement in the membrane. Evidence is presented that phosphocholine head groups of sphingomyelin, clustered in sphingomyelin-cholesterol microdomains, serve this function for α-toxin. Clustering is required so that oligomerization, which is prerequisite for stable attachment of the toxin to the membrane, can efficiently occur. Outside these clusters, binding to phosphocholine is too transient for toxin monomers to find each other. The principle of membrane targeting in the absence of any genuine, high affinity receptor may also underlie the assembly of other lipid-inserted oligomers including cytotoxic peptides, protein toxins, and immune effector molecules. Staphylococcal α-toxin, archetype of a pore-forming cytolysin, is secreted as a water-soluble, 34-kDa monomer that binds to target membranes and then oligomerizes to form membrane-inserted heptameric channels (1Bayley H. J. Cell. Biochem. 1994; 56: 177-182Crossref PubMed Scopus (32) Google Scholar, 2Bhakdi S. Tranum-Jensen J. Microbiol. Rev. 1991; 55: 733-751Crossref PubMed Google Scholar). The three-dimensional structure of the monomer can be deduced from the recently solved structure of the related staphylococcal LukF leukocidin (3Olson R. Nariya H. Yokota K. Kamio Y. Gouaux E. Nat. Struct. Biol. 1999; 6: 134-140Crossref PubMed Scopus (203) Google Scholar), and the structure of the heptamer is also known (4Song L. Hobaugh M.R. Shustak C. Cheley S. Bayley H. Gouaux J.E. Science. 1996; 274: 1859-1866Crossref PubMed Scopus (1946) Google Scholar). Data obtained by Bayley and co-workers (1Bayley H. J. Cell. Biochem. 1994; 56: 177-182Crossref PubMed Scopus (32) Google Scholar, 6Walker B. Braha O. Cheley S. Bayley H. Chem. Biol. 1995; 2: 99-105Abstract Full Text PDF PubMed Scopus (118) Google Scholar, 7Walker B. Krishnasastry M. Zorn L. Bayley H. J. Biol. Chem. 1992; 267: 21782-21786Abstract Full Text PDF PubMed Google Scholar) and ourselves (2Bhakdi S. Tranum-Jensen J. Microbiol. Rev. 1991; 55: 733-751Crossref PubMed Google Scholar, 5Valeva A. Palmer M. Bhakdi S. Biochemistry. 1997; 36: 13298-13304Crossref PubMed Scopus (86) Google Scholar) have led to a consensus model of pore assembly. Membrane-bound monomers interact with each other to form homotypic oligomeric prepores that initially lack permeabilizing activity. Early prepores are stable in non-denaturing detergents but unstable in SDS. Cooperative conformational changes transform the early to the late prepore, which resists dissociation in SDS at 25 °C. The transmembrane channel is formed when the pore-forming amino acid sequence finally inserts into the bilayer to create an amphipathic membrane-spanning β-barrel (8Valeva A. Palmer M. Hilgert K. Kehoe M. Bhakdi S. Biochim. Biophys. Acta. 1995; 1236: 213-218Crossref PubMed Scopus (32) Google Scholar, 9Valeva A. Weisser A. Walker B. Kehoe M. Bayley H. Bhakdi S. Palmer M. EMBO J. 1996; 15: 1857-1864Crossref PubMed Scopus (118) Google Scholar, 10Walker B. Krishnasastry M. Bayley H. J. Biol. Chem. 1993; 268: 5285-5292Abstract Full Text PDF PubMed Google Scholar, 11Ward R.J. Palmer M. Leonard K. Bhakdi S. Biochemistry. 1994; 33: 7477-7484Crossref PubMed Scopus (46) Google Scholar). One major unresolved question relates to the molecular nature of the physiologically relevant binding site on target cells. This issue is surrounded by paradoxes. Mammalian cells differ widely in their susceptibility to permeabilization by α-toxin, classic extremes being represented by rabbit and human erythrocytes. The former are lysed by nanomolar concentrations of the toxin, whereas lysis of human erythrocytes first occurs at 200-fold higher concentrations. Binding studies have revealed the existence of high affinity, saturable interaction sites on rabbit cells that are absent on human erythrocytes. However, both cell types additionally have low affinity binding sites, which are responsible for lysis of the "resistant" human cells at high toxin concentrations (12Hildebrand A. Pohl M. Bhakdi S. J. Biol. Chem. 1991; 266: 17195-17200Abstract Full Text PDF PubMed Google Scholar). All efforts to identify a classic receptor on susceptible target cells have failed. The issue is further confounded by the fact that liposomes and lipid bilayers composed solely of phosphatidylcholine are highly susceptible to permeabilization by α-toxin (13Ikigai H. Nakae T. J. Biol. Chem. 1987; 262: 2156-2160Abstract Full Text PDF PubMed Google Scholar). Hence, a protein or glycolipid receptor is not required for the toxin to bind to membranes and form channels in the first place. Watanabe et al. (14Watanabe M. Tomita T. Yasuda T. Biochim. Biophys. Acta. 1987; 898: 257-265Crossref PubMed Scopus (55) Google Scholar) have considered that phosphatidylcholine might be the ligand for α-toxin. This suggestion derived from the observation that phosphocholine could inhibit toxin-dependent permeabilization of liposomes, albeit only at very high concentrations of ≥20 mm. According to the crystal structure of leukocidin, members of this toxin family indeed possess a putative binding site for phosphocholine (3Olson R. Nariya H. Yokota K. Kamio Y. Gouaux E. Nat. Struct. Biol. 1999; 6: 134-140Crossref PubMed Scopus (203) Google Scholar). Thus, we are faced with another paradox that a molecule destined to interact with α-toxin apparently does so with an affinity that is so low as to render a biological relevance improbable. Here, a concept is advanced that provides an explanation for the past findings. It is proposed that, by clustering, low affinity binding moieties can assume the role of high affinity "receptors" because they render rapid toxin oligomerization possible. In the absence of clustering, binding of toxin monomers will be too transient to support oligomerization at low concentrations of toxin. Phosphocholine head groups fulfill this function in the case ofα-toxin, and their clustering on susceptible cells occurs through the association of sphingomyelin with cholesterol. Sphingomyelin-cholesterol-rich microdomains represent operational units in which clusters of proteins and lipids coordinately exert their functions (15Brown D.A. London E. Annu. Rev. Cell Dev. Biol. 1998; 14: 111-136Crossref PubMed Scopus (2551) Google Scholar, 16Harder T. Simons K. Curr. Opin. Cell Biol. 1997; 9: 534-542Crossref PubMed Scopus (717) Google Scholar, 17Simons K. Ikonen E. Nature. 1997; 387: 569-572Crossref PubMed Scopus (8113) Google Scholar). Interaction with clustered low affinity binding sites may underlie the physiological action of other oligomerizing toxins and effector molecules of the immune system. Materials—Egg yolk phosphatidylcholine (PC), 2The abbreviations used are: PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine; SM, sphingomyelin; FCS, fetal calf serum; DMEM, Dulbecco's modified Eagle's medium; CHO, Chinese hamster ovary; HBSS, Hanks' balanced salt solution. egg yolk phosphatidylethanolamine(PE), brain sphingomyelin(SM), ceramide, and cholesterol were obtained from Sigma (Deisenhofen, Germany). Production of wild-type α-toxin and the active mutant G130C and labeling of the latter with fluorescein-5-maleimide or with biotin-maleimide was as described (18Palmer M. Weller U. Messner M. Bhakdi S. J. Biol. Chem. 1993; 268: 11963-11967Abstract Full Text PDF PubMed Google Scholar). Carrier-free Na125I was from Amersham Biosciences (Freiburg, Germany), and wild type α-toxin was radioiodinated as described (12Hildebrand A. Pohl M. Bhakdi S. J. Biol. Chem. 1991; 266: 17195-17200Abstract Full Text PDF PubMed Google Scholar). [3H]Choline chloride was from PerkinElmer Life Sciences (Cologne, Germany). Kits for determining ATP and cholesterol were from Roche Diagnostics (Mannheim, Germany). Cell Culture—THP-1 cells were grown in RPMI 1640 medium (Invitrogen), supplemented with 10% fetal calf serum (FCS), 2 mm glutamine, streptomycin (100 μg/ml), and penicillin (100 μg/ml). Jurkat cells were grown in Iscove's modified Dulbecco's medium (Invitrogen), supplemented with 10% FCS, 20 mm Hepes, streptomycin (100 μg/ml), and penicillin (100 μg/ml). Human skin fibroblast cultures were established from surgical foreskin specimens from patients. Pieces of skin were minced and cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% FCS, 2 mm glutamine, and penicillin-streptomycin for 2–3 weeks. Cells were passaged several times in 250-ml tissue culture flasks (Greiner). Cultures between the fourth and eighth passages were used in the experiments. CHO cells (LY-B and LY-B/cLCB1) were cultivated as described (19Hanada K. Hara T. Fukasawa M. Yamaji A. Umeda M. Nishijima M. J. Biol. Chem. 1998; 273: 33787-33794Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 20Hanada K. Nishijima M. Kiso M. Hasegawa A. Fujita S. Ogawa T. Akamatsu Y. J. Biol. Chem. 1992; 267: 23527-23533Abstract Full Text PDF PubMed Google Scholar). The mutant cells LY-B completely lacked de novo synthesis of sphingolipids. Transfection of these cells with a recombinant plasmid expressing cLCB1 (LY-B/cLCB1) complemented the defect (19Hanada K. Hara T. Fukasawa M. Yamaji A. Umeda M. Nishijima M. J. Biol. Chem. 1998; 273: 33787-33794Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 20Hanada K. Nishijima M. Kiso M. Hasegawa A. Fujita S. Ogawa T. Akamatsu Y. J. Biol. Chem. 1992; 267: 23527-23533Abstract Full Text PDF PubMed Google Scholar, 21Fukasawa M. Nishijima M. Itabe H. Takano T. Hanada K. J. Biol. Chem. 2000; 275: 34028-34034Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). The sphingomyelin content is reduced to less than 30% following culture of either cell type in sphingomyelin-free medium (19Hanada K. Hara T. Fukasawa M. Yamaji A. Umeda M. Nishijima M. J. Biol. Chem. 1998; 273: 33787-33794Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 20Hanada K. Nishijima M. Kiso M. Hasegawa A. Fujita S. Ogawa T. Akamatsu Y. J. Biol. Chem. 1992; 267: 23527-23533Abstract Full Text PDF PubMed Google Scholar, 21Fukasawa M. Nishijima M. Itabe H. Takano T. Hanada K. J. Biol. Chem. 2000; 275: 34028-34034Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Ham's F-12 medium supplemented with 10% fetal calf serum, penicillin (100 μg/ml), and streptomycin (100 g/ml) was used as the normal culture medium. Nutridoma medium (F-12 medium containing 1% Nutridoma SP medium (Roche Diagnostics) and gentamicin (10 μg/ml)) and Nutridoma BO-medium (Nutridoma medium supplemented with 0.1% FCS and 10 μm sodium oleate/albumin (Sigma)) were used as sphingomyelin-deficient medium. The revertant cells (LY-B/cLCB1) can replenish their sphingomyelin content in Nutridoma BO medium, whereas the mutant cells cannot. There are no appreciable differences in the contents of other major phospholipids or cholesterol in these cells (19Hanada K. Hara T. Fukasawa M. Yamaji A. Umeda M. Nishijima M. J. Biol. Chem. 1998; 273: 33787-33794Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 20Hanada K. Nishijima M. Kiso M. Hasegawa A. Fujita S. Ogawa T. Akamatsu Y. J. Biol. Chem. 1992; 267: 23527-23533Abstract Full Text PDF PubMed Google Scholar, 21Fukasawa M. Nishijima M. Itabe H. Takano T. Hanada K. J. Biol. Chem. 2000; 275: 34028-34034Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). CHO cells were routinely maintained in the normal culture medium in a 5% CO2 atmosphere at 37 °C. For culture in different media, the mutant and revertant cells were seeded into a 100-mm Petri dish containing 5 ml of normal culture medium and incubated at 37 °C for 2 h and washed twice with phosphate-buffered saline. Then cell monolayers were cultured in Nutridoma medium with 10% FCS to normalize their sphingomylin content or in Nutridoma BO-medium or in Nutridoma-medium for 3 days at 37 °C, 5% CO2. Lysis Inhibition by Phosphocholine—α-Toxin was preincubated in a microtiter plate with increasing concentrations of phosphocholine in a total volume of 100 μl of HBSS for 30 min, 22 °C. One-hundred microliters of a rabbit erythrocyte suspension (5 × 108/ml) were then added to each well. After 1 h, 22 °C, the plates were centrifuged, and hemolysis was quantified by measurement of hemoglobin absorption at 412 nm. Cholesterol Depletion—Cholesterol extraction from rabbit erythrocytes was performed using phosphatidylcholine liposomes (22Frenkel E.J. Kuypers F.A. Op den Kamp J.A. Roelofsen B. Ott P. Biochim. Biophys. Acta. 1986; 855: 293-301Crossref PubMed Scopus (18) Google Scholar, 23Giraud F. M'Zali H. Chailley B. Mazet F. Biochim. Biophys. Acta. 1984; 778: 191-200Crossref PubMed Scopus (22) Google Scholar). Rabbit erythrocytes (4 × 108 cells/ml) were incubated with 1 mm phosphatidylcholine liposomes at 37 °C. After 3 h, the cells were sedimented and washed five times with HBSS. Cholesterol extraction from nucleated cells was achieved using methyl-β-cyclodextrin (Sigma). THP-1 cells (4 × 106 cells/ml) were incubated with 10 mm methyl-β-cyclodextrin in DMEM for 30 min, 37 °C, and washed three times with DMEM. Half of the cells were used for binding or spectrofluorometric studies; the other half were washed in HBSS, and a cholesterol determination (Roche Diagnostics) was performed. Labeling of Phosphocholine—Fibroblasts were cultured in the presence of [3H]choline chloride (1 μCi per culture dish) in DMEM with 1% FCS at 37 °C, 5% CO2 for 3 h. Cells were washed three times with DMEM and treated with 0.1 unit/ml Bacillus cereus-sphingomyelinase (Sigma) or 0.1 unit/ml B. cereus-phospholipase C (Calbiochem, Bad Soden, Germany) or with 10 mm methyl-β-cyclodextrin in DMEM. Cell-associated radioactivity and radioactivity in cell supernatants were determined after 30 min, 37 °C. Determination of Sphingomyelin and Phosphatidylcholine—Cells were seeded, incubated in the normal culture medium for 1 day, and after washing with serum-free medium were cultured in Nutridoma medium with 10% FCS or in Nutridoma BO-medium or sphingomyelin-free Nutridoma medium for 3 days at 37 °C. The cells were prelabeled with [3H]choline (1 μCi/ml, overnight; PerkinElmer Life Sciences). At the end of the incubation, cells were washed and harvested by scraping, lipids were extracted with chloroform/methanol (ratio 2:1) (24Folch J. Lees M. Sloane Stanley G.H. J. Biol. Chem. 1957; 226: 497-509Abstract Full Text PDF PubMed Google Scholar), and unlabeled sphingomyelin and phosphatidylcholine were added as internal standards. Phospholipids were separated by thin-layer chromatography on silica 60 gel plates (Merck, Darmstadt, Germany) using chloroform/methanol/acetic acid (65/25/10 v/v) as solvent and stained by iodine vapor, and the spots corresponding to sphingomyelin and phosphatidylcholine were scraped off and counted for radioactivity in a liquid scintillation counter. Liposome Preparation—Lipid films containing 1 mg of total lipid in different molar ratios were dried down from chloroform/methanol (2,1 v/v) under nitrogen and then rehydrated in 1 ml of HBSS, pH 7.5. Liposomes containing sphingomyelin were rehydrated at 45 °C, while the others were rehydrated at room temperature. Liposomes were rapidly frozen (–70 °C acetone bath) and thawed (45 °C water bath) six times. After freeze-thawing, liposomes were passed through a 0.1-μm polycarbonate filter (Nuclepore, Clifton NJ) 10 times, using a Miniextruder (Avanit Polar Lipids, Alabaster, AL). The sphingomyelin liposomes were extruded at 45 °C, and the others were extruded at room temperature. Quantitation of α-Toxin Binding—Binding studies using erythrocytes were performed with the radioiodinated α-toxin as described (12Hildebrand A. Pohl M. Bhakdi S. J. Biol. Chem. 1991; 266: 17195-17200Abstract Full Text PDF PubMed Google Scholar). Fluorescein-labeled G130C α-toxin was used in binding studies with nucleated cells and liposomes. THP-1 cells or CHO cells were incubated with 30 nm fluorescein-labeled G130C α-toxin for 1 h at 37 °C. After washing, cells were resuspended in 1 ml of HBSS, and bound toxin was quantified by spectrofluorometry. Fluorescence emission spectra were recorded in a Spex Fluoromax fluorometer (wavelength, excitation: 488 nm, emission: 500–550 nm, scanning interval: 1 nm). The spectra of appropriate blanks (buffer or cell suspension) were recorded and subtracted from the sample spectra. In the liposome experiments, membrane-bound and free toxin were separated by flotation. Fluorescein-labeled α-toxin was incubated with the liposomes for 2 h, 25 °C, and sucrose was added to 25% (w/v). 3-ml samples were applied to the bottom of centrifuge tubes and were overlayered with 1 ml of 20% sucrose, 1 ml of 15% sucrose, and 0.2 ml of HBSS. The liposomes were floated to the top of the gradient by centrifugation overnight in an SW50.1 swing-out rotor (150,000 × g, 4 °C), and the lipid-associated fluorescence was analyzed by spectrofluorometry and SDS-PAGE. In the kinetic experiments, liposomes with 40% cholesterol, either 10% PC or 10% SM, and supplemented with 25% of PE and phosphatidylserine (PS) each were mixed with pyrene-labeled toxin (mutant S3C) in a stopped-flow apparatus (SF-61-DX2, HiTech, Salisbury, UK). The excitation wavelength was set to 335 nm. Emitted light was collected employing a filter cutting wavelengths below 455 nm. Experiments were performed at 20 °C in HBSS buffer. Labeling of the toxin was performed as described (25Valeva A. Pongs J. Bhakdi S. Palmer M. Biochim. Biophys. Acta. 1997; 1325: 281-286Crossref PubMed Scopus (36) Google Scholar). Non-denaturing PAGE—The native gel electrophoresis system described previously was used (26Zitzer A. Harris J.R. Kemminer S.E. Zitzer O. Bhakdi S. Muething J. Palmer M. Biochim. Biophys. Acta. 2000; 1509: 264-274Crossref PubMed Scopus (29) Google Scholar), with the exception that electrophoresis buffer contained 25 mm rather than 2.5 mm deoxycholate. In these experiments, a 1% suspension of rabbit erythrocyte ghosts was incubated with 30 nm of biotinylated G130C α-toxin. After 1-, 5-, or 10-min incubation on ice, membranes were pelleted, washed with ice-cold buffer, and solubilized at 4 °C with 5% (v/v) Triton X-100. Unsolubilized material was removed by centrifugation, and supernatants were subjected to non-denaturing PAGE and SDS-PAGE. Gels were subsequently blotted, and α-toxin bands were detected by enhanced chemiluminescence using streptavidin-peroxidase (BM chemiluminescence blotting substrate, Roche Diagnostics). Cholesterol Depletion Selectively Destroys the High Affinity Binding Site for α-Toxin on Rabbit Erythrocytes—Rabbit erythrocytes were suspended in buffer containing 15 mm dextran 4 to prevent hemolysis and depleted of cholesterol by incubation with liposomes (22Frenkel E.J. Kuypers F.A. Op den Kamp J.A. Roelofsen B. Ott P. Biochim. Biophys. Acta. 1986; 855: 293-301Crossref PubMed Scopus (18) Google Scholar, 23Giraud F. M'Zali H. Chailley B. Mazet F. Biochim. Biophys. Acta. 1984; 778: 191-200Crossref PubMed Scopus (22) Google Scholar). This method was employed because rapid depletion with methyl-β-cyclodextrin led to high cellular fragility and spontaneous hemolysis. In of liposomes led to depletion that could be the cells too Cells containing 5% of their cholesterol content could be and used in the binding experiments. were performed as described (12Hildebrand A. Pohl M. Bhakdi S. J. Biol. Chem. 1991; 266: 17195-17200Abstract Full Text PDF PubMed Google Scholar). Cells were incubated with α-toxin in the absence or presence of increasing of unlabeled toxin. in binding of the to native rabbit and binding was by unlabeled toxin at the (12Hildebrand A. Pohl M. Bhakdi S. J. Biol. Chem. 1991; 266: 17195-17200Abstract Full Text PDF PubMed Google Scholar) and revealed the presence of saturable, high affinity binding sites on the cells. the of unlabeled toxin was binding of the This was as previously described (12Hildebrand A. Pohl M. Bhakdi S. J. Biol. Chem. 1991; 266: 17195-17200Abstract Full Text PDF PubMed Google Scholar) and the presence of a of binding sites. rabbit erythrocytes were a completely different of toxin binding was that the previously obtained with human erythrocytes. no binding of the toxin was in the absence or presence of low concentrations of unlabeled toxin. This revealed that the saturable, high affinity binding sites the of unlabeled toxin was binding of the of the low affinity, saturable binding sites. Thus, in rabbit erythrocytes could be to the human (12Hildebrand A. Pohl M. Bhakdi S. J. Biol. Chem. 1991; 266: 17195-17200Abstract Full Text PDF PubMed Google Scholar) through depletion of membrane cholesterol. High Affinity Interaction of α-Toxin with THP-1 Cells by Cholesterol and by the of THP-1 cells were either depleted of cholesterol by incubation with methyl-β-cyclodextrin S. Biochem. J. 1998; PubMed Scopus Google Scholar, G.H. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar), or they were treated with or with C from B. but not B. sphingomyelin in biological membranes Microbiol. Rev. 1993; PubMed Google Scholar). α-Toxin was applied at 30 nm so that binding only high affinity in with methyl-β-cyclodextrin or with led to in α-toxin whereas binding was not by with C. that and methyl-β-cyclodextrin were with [3H]choline to the phosphocholine head in but not of the to the cell Cholesterol that not to of cellular cholesterol. C was was that this which phosphatidylcholine but not sphingomyelin Microbiol. Rev. 1993; PubMed Google Scholar), not appreciable of phosphocholine from the cells This with early that phosphatidylcholine are not to B. C in biological membranes Microbiol. Rev. 1993; PubMed Google of [3H]choline after of cells with and 0.1 10 0.1 in a of toxin binding was by of both and cells incubation with 30 nm α-toxin of ATP for 2 h. However, when toxin were to nm, membrane permeabilization in cells not of Phosphocholine for Binding of α-Toxin to Rabbit lysis by Watanabe et al. (14Watanabe M. Tomita T. Yasuda T. Biochim. Biophys. Acta. 1987; 898: 257-265Crossref PubMed Scopus (55) Google Scholar) employed liposomes as but using susceptible cells have not performed. In the of of α-toxin was performed in the presence of increasing concentrations of It was that phosphocholine binding of α-toxin to the high affinity binding sites, with lysis being at mm of the Binding of α-Toxin to phosphocholine head groups of sphingomyelin might indeed in binding α-toxin, were in CHO mutant cells and revertant cells The of sphingomyelin in these cells are in The sphingomyelin content was normal in both cells following incubation in Sphingomyelin content was reduced by when either cell was cultured in sphingomyelin-free medium. In culture in Nutridoma BO medium led to of sphingomyelin content in the revertant whereas the content in the mutant LY-B cells content of and and medium, in a 4 the toxin binding of toxin bound to revertant or mutant cells following culture in FCS After of sphingomyelin in the revertant cells these bound normal of α-toxin, whereas binding to the mutant cells was In the absence of sphingomyelin both cells reduced toxin binding Sphingomyelin and Cholesterol the Binding Site for α-Toxin in α-toxin was applied at 30 nm to liposomes containing an of PE and with in of either cholesterol or 20 sphingomyelin led to no appreciable toxin However, binding when both cholesterol and sphingomyelin were the latter liposomes were treated with the binding site The role of phosphocholine further from the lack of toxin binding to liposomes supplemented with cholesterol and 20% that the phosphocholine head groups of sphingomyelin represented the toxin binding sites but that they to be clustered through with cholesterol to fulfill their this liposomes containing cholesterol were supplemented with 5 phosphatidylcholine or 5 in only liposomes containing cholesterol sphingomyelin bound phosphatidylcholine was not However, when the phosphatidylcholine was to binding This binding was to no on the presence of cholesterol. We that at the latter the phosphocholine head groups are on the membrane to support toxin oligomerization of cholesterol. Evidence α-Toxin in on the to the that α-toxin might be binding to clustered phosphocholine head and this might the toxin monomers to at the binding sites. rabbit erythrocyte membranes were to 30 nm α-toxin on for or 10 min, and solubilized in Triton X-100. PAGE was performed in to monomers from the