Effect of lipidic factors on membrane cholesterol topology — mode of binding of θ-toxin to cholesterol in liposomes

Ohno‐Iwashita, Yoshiko; Iwamoto, Machiko; Ando, Susumu; Iwashita, Shintaro

doi:10.1016/0005-2736(92)90190-w

Cited by 74 publications

(59 citation statements)

References 29 publications

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“…Cholesterol depletion by one third, which corresponds to a decrease from 33 to 24 mol% of cholesterol in platelets, almost completely abolished BC binding. This indicates that there is a threshold concentration of membrane cholesterol for toxin binding in intact cells, which is consistent with our previous observations with liposomes (14,20). Cholesterol depletion from erythrocytes also gave similar results (data not shown).…”

Section: Resultssupporting

confidence: 92%

“…This difference in staining patterns clearly demonstrates distinct specificity of BC binding to cholesterolrich membrane domains from indiscriminate binding of filipin to cell cholesterol. The critical reduction of cholesterol from the membrane microdomains as observed in platelets would be the cause for complete reduction of BC labeling in A431 cells on cholesterol depletion, because BC binds to cholesterol in cholesterol-rich artificial membranes (14,20). These results further indicate the selectivity of BC binding to membrane microdomains of intact cells.…”

Section: Resultsmentioning

confidence: 72%

“…By using artificial membranes we have demonstrated that -toxin and its derivatives selectively bind to cholesterol in cholesterol-rich membranes (14,20). Based on this we hypothesized that the -toxin and its derivatives could bind to cholesterol-rich microdomains of intact cells.…”

mentioning

confidence: 95%

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Selective binding of perfringolysin O derivative to cholesterol-rich membrane microdomains (rafts)

Waheed

Shimada²,

Heijnen³

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

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207

182

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There is increasing evidence that sphingolipid-and cholesterol-rich microdomains (rafts) exist in the plasma membrane. Specific proteins assemble in these membrane domains and play a role in signal transduction and many other cellular events. Cholesterol depletion causes disassembly of the raft-associated proteins, suggesting an essential role of cholesterol in the structural maintenance and function of rafts. However, no tool has been available for the detection and monitoring of raft cholesterol in living cells. Here we show that a protease-nicked and biotinylated derivative (BC) of perfringolysin O (-toxin) binds selectively to cholesterol-rich microdomains of intact cells, the domains that fulfill the criteria of rafts. We fractionated the homogenates of nontreated and Triton X-100-treated platelets after incubation with BC on a sucrose gradient. BC was predominantly localized in the floating lowdensity fractions (FLDF) where cholesterol, sphingomyelin, and Src family kinases are enriched. Immunoelectron microscopy demonstrated that BC binds to a subpopulation of vesicles in FLDF. Depletion of 35% cholesterol from platelets with cyclodextrin, which accompanied 76% reduction in cholesterol from FLDF, almost completely abolished BC binding to FLDF. The staining patterns of BC and filipin in human epidermoid carcinoma A431 cells with and without cholesterol depletion suggest that BC binds to specific membrane domains on the cell surface, whereas filipin binding is indiscriminate to cell cholesterol. Furthermore, BC binding does not cause any damage to cell membranes, indicating that BC is a useful probe for the detection of membrane rafts in living cells.

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Section: Resultssupporting

confidence: 92%

Section: Resultsmentioning

confidence: 72%

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Selective binding of perfringolysin O derivative to cholesterol-rich membrane microdomains (rafts)

Waheed

Shimada²,

Heijnen³

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

207

182

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“…The proposed model rationalizes the effects of a wide variety of lipids and of variations in the cholesterol concentration (32) under a single common principle. It may also accommodate the previous finding that, at constant lipid composition, VCC is more active in small unilamellar vesicles than in LUV (7), because membrane curvature also preferentially strains the headgroup layer.…”

Section: Discussionmentioning

confidence: 96%

Coupling of Cholesterol and Cone-shaped Lipids in Bilayers Augments Membrane Permeabilization by the Cholesterol-specific Toxins Streptolysin O and Vibrio cholerae Cytolysin

Zitzer

Bittman

Verbicky

et al. 2001

Journal of Biological Chemistry

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Vibrio cholerae cytolysin (VCC) forms oligomeric pores in lipid bilayers containing cholesterol. Membrane permeabilization is inefficient if the sterol is embedded within bilayers prepared from phosphatidylcholine only but is greatly enhanced if the target membrane also contains ceramide. Although the enhancement of VCC action is stereospecific with respect to cholesterol, we show here that no such specificity applies to the two stereocenters in ceramide; all four stereoisomers of ceramide enhanced VCC activity in cholesterol-containing bilayers. A wide variety of ceramide analogs were as effective as D-erythro-ceramide, as was diacylglycerol, suggesting that the effect of ceramide exemplifies a general trend of lipids with a small headgroup to augment the activity of VCC. Incorporation of these cone-shaped lipids into cholesterol-containing bilayers also gave similar effects with streptolysin O, another cholesterol-specific but structurally unrelated cytolysin. In contrast, the activity of staphylococcal ␣-hemolysin, which does not share with the other toxins the requirement for cholesterol, was far less affected by the presence of lipids with a conical shape. The collective data indicate that sphingolipids and glycerolipids do not interact with the cytolysins specifically. Instead, lipids that have a conical molecular shape appear to effect a change in the energetic state of membrane cholesterol that in turn augments the interaction of the sterol with the cholesterol-specific cytolysins.To bacterial pore-forming cytolysins, cholesterol is a logical choice as a target molecule, because it confers specificity for animal as opposed to bacterial cell membranes. The specificity for cholesterol is shared between Vibrio cholerae cytolysin (VCC) 1 (1) and streptolysin O (SLO) (2). Otherwise, these toxins are not related, and the oligomeric pores they form are very different in size and morphology (1, 3). Although with SLO the sterol is already required in the initial event of membrane binding of the monomeric toxin (4), it only comes into play at the stage of oligomerization in the case of VCC (5, 6). When the sterol is incorporated into phosphatidylcholine (PC) bilayers at physiologically realistic concentrations (i.e. up to 40% by mol), these membranes do not become significantly sensitive to VCC. However, it was previously found that membrane susceptibility toward the cytolysin was greatly enhanced by inclusion of ceramide; free ceramide and monohexosyl ceramides proved similarly effective (7). A combined specificity for cholesterol and sphingolipids has previously been shown for the fusion protein of Semliki Forest virus. In that instance, the interaction with ceramide proved to be highly stereoselective (8 -10). Accordingly, we have examined the structural properties of the ceramide molecule responsible for the sensitization of membranes to VCC. To our surprise, no dependence on stereospecific features of ceramide could be detected. Membrane sensitization was readily achieved with a variety of synthetic ceramides...

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“…The probe is derived from a pore-forming cytolysin produced by the pathogenic bacterium Clostridium perfringens (28). This thiol-activated cytolysin, called perfringolysin O ( -toxin), specifically binds to free (unesterified) cholesterol (29)(30)(31)(32) and forms oligomeric pores in the membranes (33). The probe BC was prepared by a twostep procedure.…”

mentioning

confidence: 99%

Biotinylated θ-toxin derivative as a probe to examine intracellular cholesterol-rich domains in normal and Niemann-Pick type C1 cells

Sugii

Reid

Ohgami

et al. 2003

Journal of Lipid Research

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Effect of lipidic factors on membrane cholesterol topology — mode of binding of θ-toxin to cholesterol in liposomes

Cited by 74 publications

References 29 publications

Selective binding of perfringolysin O derivative to cholesterol-rich membrane microdomains (rafts)

Selective binding of perfringolysin O derivative to cholesterol-rich membrane microdomains (rafts)

Coupling of Cholesterol and Cone-shaped Lipids in Bilayers Augments Membrane Permeabilization by the Cholesterol-specific Toxins Streptolysin O and Vibrio cholerae Cytolysin

Biotinylated θ-toxin derivative as a probe to examine intracellular cholesterol-rich domains in normal and Niemann-Pick type C1 cells

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