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• W2003405170 abstract "Clostridium perfringens beta toxin is known to be the primary pathogenic factor of necrotic enteritis in the type C strains that produce beta toxin. The toxin was purified from the supernatant fluid of a culture controlled at pH 7.5 using an anti-alpha toxin affinity column (1Sakurai J. Duncan C.L. Infect. Immun. 1977; 8: 741-745Crossref Google Scholar) or Toyopearl HW 60 column (2Sakurai J. Fujii Y. Toxicon. 1987; 25: 1301-1310Crossref PubMed Scopus (37) Google Scholar). Beta toxin possesses lethal, dermonecrotic, and pressor activities (3Sakurai J. Nagahama M. Ochi S. J. Toxicol. Toxin Rev. 1997; 16: 195-214Crossref Scopus (40) Google Scholar). Recently, we also reported that the plasma extravasation induced by beta toxin in mouse skin is mediated via a mechanism involving tachykinin NK1 receptors (4Nagahama M. Morimitsu S. Kihara A. Akita M. Setsu K. Sakurai J. Br. J. Pharmacol. 2003; 138: 23-30Crossref PubMed Scopus (24) Google Scholar). By using an oligonucleotide probe designed on the basis of the N-terminal sequence of the purified toxin, the beta toxin gene was isolated from C. perfringens type B (5Hunter S.E. Brown J.E. Oyston P.C. Sakurai J. Titball R.W. Infect. Immun. 1993; 61: 3958-3965Crossref PubMed Google Scholar). The deduced amino acid sequence of beta toxin was found to have significant homology with that of Staphylococcus aureus alpha toxin (28% similarity), the A and B components of gamma toxin (22 and 28% similarity, respectively), and the S and F components of leukocidin (17 and 28%, respectively) (5Hunter S.E. Brown J.E. Oyston P.C. Sakurai J. Titball R.W. Infect. Immun. 1993; 61: 3958-3965Crossref PubMed Google Scholar). The alpha toxin is known to form an oligomer in biological membranes and artificial membranes (6Tomita T. Watanabe M. Yasuda T. J. Biol. Chem. 1992; 267: 13391-13397Abstract Full Text PDF PubMed Google Scholar, 7Valeva A. Weisser A. Walker B. Kehoe M. Bayley H. Bhakdi S. Palmer M. EMBO J. 1996; 15: 1857-1864Crossref PubMed Scopus (118) Google Scholar, 8Montoya M. Gouaux E. Biochim. Biophys. Acta. 2003; 1609: 19-27Crossref PubMed Scopus (95) Google Scholar). The alpha toxin is the prototype of a family of these toxins with membrane-damaging activity. On the basis of crystallographic findings of the alpha toxin, the structure of the oligomer of the alpha toxin is divided into four domains. First, the cap of the oligomer is composed of seven β-sandwiches and the N-terminal region. Second, the stem domain forms a transmembrane channel. Third, the rim domain protrudes from the underside of the oligomer, participates in some protomer-protomer interactions, and interacts with the lipid bilayer. Fourth, the triangle region participates in a crucial protomer-protomer interaction (9Song L. Hobaugh M.R. Shustak C. Cheley S. Bayley H. Gouaux J.E. Science. 1996; 274: 1859-1866Crossref PubMed Scopus (1963) Google Scholar). Beta toxin has regions corresponding to the cap, the triangle region, the rim domain, and the stem domain of alpha toxin, according to the conserved amino acid sequences of the family (5Hunter S.E. Brown J.E. Oyston P.C. Sakurai J. Titball R.W. Infect. Immun. 1993; 61: 3958-3965Crossref PubMed Google Scholar). Beta toxin has only one cysteine residue at position 265 (5Hunter S.E. Brown J.E. Oyston P.C. Sakurai J. Titball R.W. Infect. Immun. 1993; 61: 3958-3965Crossref PubMed Google Scholar). Furthermore, the toxin is inactivated by thiol group modifying reagents (10Sakurai J. Fujii Y. Matsuura M. Microbiol. Immunol. 1980; 24: 595-601Crossref PubMed Scopus (19) Google Scholar, 11Sakurai J. Fujii Y. Nagahama M. Toxicon. 1992; 30: 323-330Crossref PubMed Scopus (8) Google Scholar). It therefore was speculated that the Cys residue is important in the lethal activity of the toxin. However, Steinthorsdottir et al. (12Steinthorsdottir V. Fridriksdottir V. Gunnarsson E. Andresson O.S. FEMS Microbiol. Lett. 1998; 158: 17-23Crossref PubMed Google Scholar) reported that replacement of the Cys residue did not affect the toxin activity. Furthermore, we also reported that the replacement of Cys-265 with alanine did not affect the activity of beta toxin and resulted in thiol group reagents having no effect on the activity (13Nagahama M. Kihara A. Miyawaki T. Mukai M. Sakaguchi Y. Ochi S. Sakurai J. Biochim. Biophys. Acta. 1999; 1454: 97-105Crossref PubMed Scopus (25) Google Scholar). The primary amino acid sequence surrounding Cys-265 in the C terminus of beta toxin (positions 255 to 276) is homologous to that at positions 245-267 in the C terminus of the alpha toxin (a conserved 11-amino acid sequence). Thus it appears that Cys-265 in beta toxin corresponds to Asp-255 in the alpha toxin. Walker and Bayley (14Walker B. Bayley H. J. Biol. Chem. 1995; 270: 23065-23071Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar) reported that treatment of D254C and D255C (variant toxins of the alpha toxin) with sulfhydryl-modifying reagent, 4′-acetamido-4-((iodoacetyl)amino)stilbene-2,2′-disulfonate, resulted in a significant reduction or complete loss of binding, oligomer formation, and hemolytic activity, suggesting that the C terminus containing Asp-254 and -255 of the alpha toxin is implicated in binding to cells. Y266A and L268G surrounding Cys-265 in beta toxin showed no lethal activity and did not inhibit lethal activity of the toxin, suggesting that Tyr-266 and Leu-268 play a role in binding to the receptor (13Nagahama M. Kihara A. Miyawaki T. Mukai M. Sakaguchi Y. Ochi S. Sakurai J. Biochim. Biophys. Acta. 1999; 1454: 97-105Crossref PubMed Scopus (25) Google Scholar). These observations suggest that the characteristics of beta toxin resemble those of the alpha toxin. Steinthorsdottir et al. (15Steinthorsdottir V. Halldorsson H. Andresson O.S. Microb. Pathog. 2000; 28: 45-50Crossref PubMed Scopus (66) Google Scholar) showed that beta toxin formed oligomeric complexes on the membranes of human umbilical vein endothelial cells and induced the release of arachidonic acid and inositol from these cells. Shatursky et al. (16Shatursky O. Bayles R. Rogers M. Jost B.H. Songer J.G. Tweten R.K. Infect. Immun. 2000; 68: 5546-5551Crossref PubMed Scopus (70) Google Scholar) hypothesized that the lethal action of beta toxin is based on the formation of cation-selective pores in the lipid bilayers. However, little is known about the precise mechanism of formation of oligomers of beta toxin and the relationship between the biological activities and oligomer formation of the toxin. Direct evidence concerning the formation of the membrane pore and biological activities is lacking due to the absence of a susceptible cell line for in vitro studies of beta toxin activity. In this study, we attempted to find cell lines that are susceptible to beta toxin. We demonstrate the binding of the toxin in cell and oligomer formation of the toxin in biological membranes. Furthermore, we report here a relationship between the biological activities and oligomer formation of the toxin. Materials—Methyl-β-cyclodextrin (MβCD), 1The abbreviations used are: MβCD, methyl-β-cyclodextrin; CO, cholesterol oxidase; CF, 5(6)-carboxyfluorescein; DOPC, dioleolyl-l-α-phosphatidylcholine; MQAE, N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide; BTD, beta toxin derivative; GST, glutathione S-transferase; LDH, lactate dehydrogenase; HBSS, Hanks' balanced solution; CHO, Chinese hamster ovary; MOPS, 3-(N-morpholino)propanesulfonic acid; TBS, Tris-buffered saline; MDCK, Madin-Darby canine kidney cells. cholesterol, carboxyfluorescein, dioleolyl-l-α-phosphatidylcholine (DOPC), cholesterol oxidase, filipin, nystatin, a protease inhibitor mixture, verapamil, 4-aminopyridine, tetraethylammonium, and quinidine were obtained from Sigma. Polyethylene glycol 200, 300, 400, 600, and 1000 were purchased from Nacalai Tesque (Kyoto, Japan). ω-Conotoxin, charybdotoxin, and margatoxin were purchased from Peptide Institute Inc. (Osaka, Japan). Mouse anti-caveolin-1 and Lyn antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish peroxidase-labeled sheep anti-mouse IgG were purchased from Amersham Biosciences. Dulbecco's modified Eagle's medium and RPMI 1640 medium were purchased from Invitrogen. The fluorescent probes, sodium green and N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE), were obtained from Molecular Probes (Eugene, OR). All other chemicals were of the highest grade available from commercial sources. Beta Toxin and Derivative—The expression and purification of recombinant beta toxin were performed as described previously (13Nagahama M. Kihara A. Miyawaki T. Mukai M. Sakaguchi Y. Ochi S. Sakurai J. Biochim. Biophys. Acta. 1999; 1454: 97-105Crossref PubMed Scopus (25) Google Scholar). To prepare beta toxin derivative (BTD) for labeling of the toxin, plasmid pTB-1 containing the entire beta toxin gene (13Nagahama M. Kihara A. Miyawaki T. Mukai M. Sakaguchi Y. Ochi S. Sakurai J. Biochim. Biophys. Acta. 1999; 1454: 97-105Crossref PubMed Scopus (25) Google Scholar) was used as a template for PCR to add BamHI site and EcoRI site for subcloning into the vector using a forward primer 5′-GGGGATCCAATGATATAGGTAAAAC-3′(BamHI site is underlined) and a reverse primer 5′-GGGTTAACGATATAGGTAAAACTAC-3′ (EcoRI site is underlined). The PCR products digested with BamHI and EcoRI were inserted into pGEX-2TK (Amersham Biosciences). The resultant plasmid, named pGE2BXT, enabled the expression of glutathione S-transferase (GST)-beta toxin derivative (GST-BTD) which contained a pentapeptide recognized by the cyclic AMP-dependent protein kinase. The accuracy of the final DNA construction was confirmed by DNA sequencing. Cells—Cell lines (Vero cells, CHO cells, CHO-K1 cells, COS-7 cells, intestine 407 cells, HeLa cells, MDCK cells, PC12 cells, mouse mastocytoma P-815 cells, and HL 60 cells) were obtained from Riken Cell Bank (Tsukuba, Japan). Vero cells, CHO cells, CHO-K1 cells, COS-7 cells, intestine 407 cells, HeLa cells, and MDCK cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 unit/ml penicillin, 100 μg/ml streptomycin, and 2 mm glutamine. PC12 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% horse serum, 5% fetal bovine serum, 100 unit/ml penicillin, 100 μg/ml streptomycin and 2 mm glutamine. P-815 cells and HL 60 cells were cultured in RPMI 1640 medium containing 20% heat-inactivated fetal bovine serum, 100 unit/ml penicillin, 100 μg/ml streptomycin, and 2 mm glutamine. All incubation steps were carried out at 37 °C in a 5% CO2 atmosphere. Mouse Lethality Test and Cytotoxicity Assay—The lethality of beta toxin and its derivative toward mice was determined as described previously (13Nagahama M. Kihara A. Miyawaki T. Mukai M. Sakaguchi Y. Ochi S. Sakurai J. Biochim. Biophys. Acta. 1999; 1454: 97-105Crossref PubMed Scopus (25) Google Scholar). A group of 6 male ddY mice weighing about 25 g each was injected intravenously with 0.1 ml of the toxin solution, and deaths occurring within 24 h were recorded. For cytotoxicity assays, cells were seeded in 48-well culture plates (1 × 105 cells in 0.2 ml/well) and cultured for 24 h at 37 °C. Beta toxin was added to each well and additionally incubated for a further 12 h. After the incubation, the cells in each well were washed with phosphate-buffered saline containing Mg2+ and Ca2+. Cell viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt conversion assay (Promega, Madison, WI). The absorbance was read at 490 nm using an enzyme-linked immunosorbent assay plate reader. Percentage of cell viability was calculated as follows: the mean absorbance value of a toxin group/that of a control. Release of lactate dehydrogenase (LDH) from HL 60 cells was determined with a microplate assay according to the manufacturer's instructions (Roche Applied Science). Ion Assays—HL 60 cells (1 × 106 cells/ml) were inoculated in 6-well plates in Hanks' balanced solution (HBSS). For the K+ efflux assay, the cells were incubated with the toxin at 37 °C for various times. K+ concentrations in the supernatants were determined at the given times with an atomic absorption spectrophotometer (Hitachi Z-8200, Tokyo, Japan) as described previously (17Nagahama M. Nagayasu K. Kobayashi K Sakurai J. Infect. Immun. 2002; 70: 1909-1914Crossref PubMed Scopus (49) Google Scholar). For the Ca2+ influx assay, beta toxin and 5 × 105 cpm of 45Ca2+ (11.92 mCi/mg, PerkinElmer Life Sciences) were added to the cells and incubated at 37 °C for various times. After washing the cells with HBSS three times, the radioactivity in the cells was counted with a scintillation counter (Aloka Co., Tokyo, Japan). For the Na+ and Cl- influx assay, fluorescence probes were used according to the methods described by Petit et al. (18Petit L. Maier E. Gibert M. Popoff M.R. Benz R. J. Biol. Chem. 2001; 276: 15736-15740Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). After incubation of HL 60 cells (1 × 106 cells/ml) with the toxin in HBSS at 37 °C for various times, the cells were washed with 25 mm MOPS (pH 7) containing 150 mm glucose (MOPS/glucose) and lysed with 1% Triton X-100. Each fluorescent probe was added to the lysed cells to a final concentration of 10 μm for sodium green (Na+ assay) and MQAE (Cl- assay). The fluorescence intensity of sodium green and MQAE was measured in a microplate fluorometer MTP-32 (Corona Electric Co., Katsuda, Japan) with the following filters: excitation 485 nm and emission 538 nm for sodium green and excitation 355 nm and emission 475 nm for MQAE. The data are expressed as percents of fluorescence intensity from the toxin-untreated cells (control) (18Petit L. Maier E. Gibert M. Popoff M.R. Benz R. J. Biol. Chem. 2001; 276: 15736-15740Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Estimation of the Functional Diameter of the Membrane Pore—Toxin-induced lysis of HL 60 cells was assayed at 37 °C in HBSS containing polyethylene glycols of different sizes at a concentration equivalent to 40 mOsm. The total osmotic pressure of the extracellular space was adjusted to 295 mOsm by changing the concentrations of NaCl. The values for the hydrodynamic diameters of polyethylene glycols were taken from the reports of Scherrer and Gerhardt (19Scherrer R. Gerhardt P. J. Bacteriol. 1971; 107: 718-735Crossref PubMed Google Scholar) and Sabirov et al. (20Sabirov R.Z. Krasilnikov O.V. Ternovsky V.I. Merzliak P.G. Gen. Physiol. Biophys. 1993; 12: 95-111PubMed Google Scholar), where the hydrodynamic diameters were calculated on the basis of the viscosity of the polyethylene glycol solutions. Preparation of Radiolabeled Toxin Derivatives—Beta toxin was radiolabeled with 125I by the following two methods. The purified toxin (15 μg) was incubated with 36.2 GBq of Na125I (643.8 GBq/mg; PerkinElmer Life Sciences) and IOAD-BEADS iodination reagent (Pierce) in 60 μl of phosphate-buffered saline for 15 min at room temperature. Beta toxin (15 μg) was incubated with 250 μCi of 125I-labeled Bolton-Hunter reagent (74 TBq/mmol; Amersham Biosciences) as described previously (17Nagahama M. Nagayasu K. Kobayashi K Sakurai J. Infect. Immun. 2002; 70: 1909-1914Crossref PubMed Scopus (49) Google Scholar). Radioiodinated proteins were separated from free iodine by gel filtration on a Sephadex G-25 column (0.75 × 10 cm; Amersham Biosciences), equilibrated with Tris-buffered saline (TBS; 50 mm Tris-HCl buffer (pH 7.4) containing 150 mm NaCl). The specific activity of Na125I-labeled and 125I-Bolton-Hunter reagent-labeled beta toxin was 440 and 560 kcpm/μg protein, respectively. Phosphorylation of BTD with 32P was performed as follows. Approximately 200 μg of GST-BTD was loaded onto a glutathione-Sepharose 4B column (bed volume, 200 μl; Amersham Biosciences), and then phosphorylated using the catalytic subunit of bovine heart protein kinase (Sigma) and [γ-32P]ATP (167 TBq/mmol; ICN Biochemicals, Costa Mesa, CA) according to the protocol recommended by the manufacturer. The specific activity of 32P-labeled GST-BTD was about 32 kcpm/μg protein. 32P-BTD was prepared from 32P-labeled GST-BTD by cleavage at the thrombin-specific site within the C-terminal portion in a GST linker sequence (see “Results”). Sucrose Gradient Fractionation—Separation of lipid rafts was carried out by flotation-centrifugation on a sucrose gradient (21Waheed A.A. Shimada Y. Heijnen H.F. Nakamura M. Inomata M. Hayashi M. Iwashita S. Slot J.W. Ohno-Iwashita Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4926-4931Crossref PubMed Scopus (200) Google Scholar, 22Falguieres T. Mallard F. Baron C. Hanau D. Lingwood C. Goud B. Salamero J. Johannes L. Mol. Biol. Cell. 2001; 12: 2453-2468Crossref PubMed Scopus (235) Google Scholar). HL 60 cells were incubated in fresh medium containing 500 ng/ml 32P-BTD at 37 °C for various times. The cells were rinsed with HBSS and treated or untreated with 1% Triton X-100 for 30 min at 4 °C in HBSS containing the protease inhibitor mixture and sonicated for 20-s pulses (two and six times, respectively) using a tip-type sonicator. The lysates were adjusted to 40% sucrose (w/v), overlaid with 2.4 ml of 36% sucrose and 1.2 ml of 5% sucrose in HBSS, centrifuged at 45,000 rpm (250,000 × g) for 18 h at 4 °C in a SW55 rotor (Beckman Instruments, Palo Alto, CA), and fractionated from the top (0.4 ml each, a total of 10 fractions). The aliquots were subjected to SDS-PAGE and autoradiographed. Immunoblot Analysis of Lipid Raft Marker Proteins—Aliquots of the flotation sucrose gradient fractions were heated in 2% SDS-sample buffer at 99 °C for 3 min. The samples were electrophoresed on an SDS-PAGE gel, followed by transfer to a polyvinylidene difluoride membrane. The membrane was blocked with TBS containing 2% Tween 20 and 5% skim milk and incubated first with the primary antibody in TBS containing 1% skim milk, then with a horseradish peroxidase-conjugated secondary antibody, and finally with an enhanced chemiluminescence analysis kit (Amersham Biosciences). Cholesterol Depletion and Repletion—To remove cholesterol, HL 60 cells (2-4 × 106 cells/ml) were incubated for 1 h at 37 °C in the presence or absence of 10 mm MβCD in HBSS and then washed with HBSS. For the cholesterol repletion experiment (22Falguieres T. Mallard F. Baron C. Hanau D. Lingwood C. Goud B. Salamero J. Johannes L. Mol. Biol. Cell. 2001; 12: 2453-2468Crossref PubMed Scopus (235) Google Scholar), 40 mg of cholesterol was coated on the walls of a glass tube, and 5 ml of 50 mm MβCD in HBSS was added and incubated after sonication for 15 h at 37 °C. The resulting solution (50 mm MβCD/cholesterol) was filtered and added back to cholesterol-depleted cells (2 × 106 cells/ml) by incubation for 2 h at 37 °C with the indicated concentrations of MβCD/cholesterol. Cholesterol contents were assayed spectrophotometrically using a diagnostic kit (Cholesterol C-Test, Wako Pure Chemical, Osaka, Japan) Liposomes—DOPC-cholesterol (1:1) liposomes containing carboxyfluorescein (CF) were prepared, and CF release was monitored by a procedure described previously (23Nagahama M. Michiue K. Sakurai J. Biochim. Biophys. Acta. 1996; 1280: 120-126Crossref PubMed Scopus (50) Google Scholar). The binding of 32P-BTD to liposomes was performed as described previously (23Nagahama M. Michiue K. Sakurai J. Biochim. Biophys. Acta. 1996; 1280: 120-126Crossref PubMed Scopus (50) Google Scholar). Morphological Alterations of Cells Induced by Beta Toxin—HL 60 cells were small and round, and displayed distinct dark outlines, as shown in Fig. 1A, when the cells were observed by phase-contrast microscopy. However, when HL 60 cells were incubated with 2.5 μg/ml of beta toxin at 37 °C, the cells began to swell within 1 h, and a large number of cells became swollen and translucent within 2 h, but the blebs did not, as shown in Fig. 1B. At this time, swollen cells with a granular-like nucleus not seen in control cells were observed. However, DNA from cells treated with the toxin did not exhibit laddering classically associated with apoptosis (data not shown), suggesting that changes in the nuclei of cells treated with the toxin do not occur under these conditions. The cells were completely lysed after 6 h of incubation (data not shown). When the cells were incubated with the toxin at concentrations from 0.5 to 10.0 μg/ml at 37 °C for 1 h, swollen cell counts increased in a dose-dependent manner under the conditions (data not shown). The effects of beta toxin on various cell lines (Vero cells, CHO cells, MDCK cells, CHO-K1 cells, COS-7 cells, P-815 cells, PC12 cells, HeLa cells, and intestine 407 cells) were investigated. The toxin had no effect on these cell lines, even when incubated with 50 μg/ml of the toxin at 37 °C for 12 h (data not shown). Our data show that, of the 10 cell lines tested, only the HL 60 cell line was susceptible to beta toxin. Efflux of K + and Lactate Dehydrogenase (LDH) and Influx of Ca 2+ , Na + , and Cl - in the Cells Treated with Beta Toxin—To test whether beta toxin affects the membrane permeability of the cells, we measured the efflux of K+ and LDH from the cells and the influx of Ca2+, Na+, and Cl- into the cells. HL 60 cells were incubated with various concentrations of beta toxin in HBSS at 37 °C. As shown in Fig. 2A, beta toxin at concentrations from 0.5 to 5.0 μg/ml induced efflux of K+ from the cells in a dose- and time-dependent manner. Incubation of the cells in the absence of beta toxin at 37 °C or in the presence of beta toxin at 4 °C induced no efflux of K+ (data not shown). Furthermore, when the cells were incubated with 2.5 μg/ml of beta toxin at 4 °C for 2 h, washed, and incubated at 37 °C for 1 h, efflux of K+ was observed (data not shown). Fig. 2B shows that the toxin (5.0 μg/ml) induced influx of Ca2+, Na+, and Cl- and that these events reached a maximum after1hof incubation at 37 °C. Beta toxin at concentrations below 5.0 μg/ml induced LDH leakage from HL 60 cells in a dose- and time-dependent manner, as shown in Fig. 2A. LDH release induced by 5.0 μg/ml of beta toxin began after 1 h and reached a maximum after 6 h, showing that lysis of the cells began after 1 h of incubation of the cells with the toxin and was complete after 6 h. It therefore is likely that release of K+ and uptake of Ca2+, Na+, and Cl- induced by the toxin increased until lysis of the cells began. The Effect of Polyethylene Glycols on the Events Induced by the Toxin—To test whether the toxin forms functional pores in HL 60 cells, the cells were incubated with beta toxin in the presence of various sizes of polyethylene glycols at 37 °C for 60 min, and the effects of the polyethylene glycols on the toxin-induced K+ efflux, Ca2+ influx, and swelling of cells were investigated (Table I). The toxin induced no swelling of the cells in the presence of polyethylene glycols 600 and 1000 (molecular size of 1.6 and 1.8 nm, respectively), and the toxin induced Ca2+ influx by about 25% of the control under these conditions. Polyethylene glycols with a molecular size of 1.12, 1.16, and 1.36 nm blocked the toxin-induced swelling and Ca2+ influx in a size-dependent manner. However, these polyethylene glycols had no effect on toxin-induced K+ efflux. To investigate whether K+ efflux and Ca2+ influx induced by the toxin are dependent on the activation of K+ and Ca2+ channels, the effect of these channel blockers on these events was tested. Ca2+ channel blockers, verapamil (10 μM), and ω-conotoxin SVIB (2 μm), and K+ channel blockers, 4-aminopyridine (10 mm), tetraethylammonium (60 mm), charybdotoxin (5.0 μm), quinidine (50 μm), and margatoxin (10 nm) had no effect on the toxin-induced swelling and events of the cells (data not shown).Table IEffect of polyethylene glycols with different hydrodynamic diameters on beta toxin-induced actions in HL 60 cellsPolyethylene glycolsK+ effluxaK+ efflux and Ca2+ influx are expressed as a percentage of the data in the absence of polyethylene glycol.Ca2+ influxaK+ efflux and Ca2+ influx are expressed as a percentage of the data in the absence of polyethylene glycol.Cell swellingHydrodynamic diameter of PEG%nmNone101.0 ± 6.4100.5 ± 9.2+ + +−PEG20095.4 ± 5.372.8 ± 13.2+ + +1.12PEG30096.8 ± 4.150.5 ± 8.8bp <0.05.+ + +1.16PEG40095.1 ± 3.831.4 ± 4.9cp <0.01.+ +1.36PEG60094.2 ± 4.925.8 ± 5.2cp <0.01.−1.60PEG100095.0 ± 5.525.4 ± 4.7cp <0.01.−1.80a K+ efflux and Ca2+ influx are expressed as a percentage of the data in the absence of polyethylene glycol.b p <0.05.c p <0.01. Open table in a new tab Binding of Beta Toxin to HL 60 Cells—It has been reported that several toxins such as perfringolysin O (21Waheed A.A. Shimada Y. Heijnen H.F. Nakamura M. Inomata M. Hayashi M. Iwashita S. Slot J.W. Ohno-Iwashita Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4926-4931Crossref PubMed Scopus (200) Google Scholar), C. perfringens epsilon toxin (24Miyata S. Minami J. Tamai E. Matsushita O. Shimamoto S. Okabe A. J. Biol. Chem. 2002; 277: 39463-39468Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar), shiga toxin (25Katagiri Y.U. Mori T. Nakajima H. Katagiri C. Taguchi T. Takeda T. Kiyokawa N. Fujimoto J. J. Biol. Chem. 1999; 274: 35278-35282Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar), tetanus toxin (26Herreros J. Ng T. Schiavo G. Mol. Biol. Cell. 2001; 12: 2947-2960Crossref PubMed Scopus (156) Google Scholar), and Helicobacter pylori vacuolating toxin (27Schraw W. Li Y. McClain M.S. van der Goot F.G. Cover T.L. J. Biol. Chem. 2002; 277: 34642-34650Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar) bind to lipid rafts and that some bacteria and viruses enter cells via lipid rafts (28Duncan M.J. Shin J.S. Abraham S.N. Cell. Microbiol. 2002; 4: 783-791Crossref PubMed Scopus (130) Google Scholar). To test the toxin binding to the cells, the toxin was radiolabeled with Na125I using IODO-BEADS iodination reagent, to modify tyrosine residues, and 125I-labeled Bolton-Hunter reagent, to modify amino group. However, these labeled toxins drastically lost lethal activity in mouse. To resolve this problem, we constructed a GST-beta toxin fusion protein containing a protein kinase recognition site (RRXSV) at the N terminus of the toxin (Fig. 3). The fusion protein was purified, phosphorylated with protein kinase in the presence of [32P]ATP, and cleaved with thrombin. The phosphorylated beta toxin derivative (32P-BTD) showed no loss of lethal activity, compared with that of the wild-type toxin. To investigate the possible interaction of beta toxin with lipid rafts of HL 60 cells, 32P-BTD was incubated with HL 60 cells in RPMI 1640 medium containing 20% heat-inactivated fetal bovine serum at 37 °C for 15 and 30 min, and the cells were treated with 1% Triton X-100 at 4 °C. The Triton X-100-insoluble components were fractionated by sucrose density gradient centrifugation, as described under “Experimental Procedures.” The fractions were subjected to SDS-PAGE analysis and autoradiography. Low density fractions (fractions 3-5) showed three of the labeled bands of about 35 kDa, which is the expected size of monomeric toxin, and of about 191 and 228 kDa, which are the expected sizes of hexameric and heptameric toxins, respectively, as shown in Fig. 4A. These results suggest that these oligomers of beta toxin are associated with the detergent-insoluble fraction. When the cells were incubated with the toxin at 37 °C for 60 min, the detergent-insoluble fractions showed two faint labeled bands at 35 and 191 kDa (Fig. 4A), indicating that the oligomer at 228 kDa disappeared within 1 h. When raft markers in the fractions obtained by sucrose density gradient centrifugation were analyzed using anti-caveolin-1 and anti-Lyn, caveolin-1 and Lyn were only detected in the low density fractions (fractions 3-5) (Fig. 4, C and D). Next, when cholesterol was measured in these fractions, more than 85% of cholesterol was detected in the fractions (fractions 3-5) (Fig. 4B). Therefore, it appears that the fractions (fractions 3-5) are lipid rafts, suggesting that monomers and oligomers of beta toxin are specifically located within lipid rafts in HL 60 cells. No oligomer of the toxin was detected when the toxin was incubated with HL 60 cells at 4 °C, or when the toxin was incubated with MDCK cells, an insensitive cell, under these conditions (data not shown).Fig. 4Sucrose density gradient analysis of 32P-BTD-bound HL 60 cells. A, HL 60 cells were incubated with 32P-BTD (500 ng/ml) in the medium at 37 °C for 15 (a), 30 (b), and 60 (c) min and then extracted with HBSS containing 1% Triton X-100 at 4 °C for 30 min and sonicated. The extracts were brought to 40% sucrose and then loaded at the bottom of a centrifuge tube. After sucrose gradient ultracentrifugation, 0.4-ml gradient fractions were collected from the top of the tubes. The aliquots of gradient fractions were dissolved in 2× SDS sample buffer and incubated at 37 °C for 10 min. Samples were subjected to SDS-PAGE, followed by autoradiography. B, distribution of cholesterol in the sucrose gradient fractions was determined as described under “Experimental Procedures.” The aliquots of gradient fractions were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes. After transfer, the blots were treated with anti-Lyn (C) or anti-caveolin-1 (D). Peroxidase-conjugated second antibodies bound to the membrane were detected by enhanced chemiluminescence as described under “Experimental Procedures.”View Large Image Figure ViewerDownload (PPT) The Effect of Methyl-β-cyclodextrin, Cholesterol Oxidase, and Nystatin on the Cytotoxicity of Beta Toxin—It has been reported (29Kilsdonk E.P.C. Yancey P.G. Stoudt G.W. Bang" @default.
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• W2003405170 date "2003-09-01" @default.
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