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

Abstract

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.

Fluorescence Techniques Using Dehydroergosterol to Study Cholesterol Trafficking

“…This same conclusion was reached following subsequent video imaging studies involving methyl-β-cyclodextrin complexes using a pulse chase method and the cell lines Hep G2, J774, and TRVb1 (211) where artifactual enhancement of the fluorescent emission was the result of rough surface topology and cell protrusions (211). However, other studies using filipin, which labels all cholesterol in the plasma membrane without differentiating between sterol-rich or sterol-poor, did not reveal any enhancement in intensity as a result of plasma membrane ruffling or tubule sizing issues (212,213).…”

Association of Helicobacter pylori Vacuolating Toxin (VacA) with Lipid Rafts

“…Two pore-forming toxins, aerolysin and Clostridium septicum alpha toxin, each bind to GPI-anchored proteins, which are enriched in lipid raft microdomains of the plasma membrane (57,66). Other pore-forming toxins, such as perfringolysin, bind to cholesterol components of lipid rafts (43). It has been proposed that lipid rafts serve as concentrating platforms to promote oligomerization of these toxins on the cell surface, a process that is required for membrane channel formation (57).…”

“…Several bacterial, viral, and parasitic pathogens seem to use rafts as a site for gaining entry into mammalian cells (40,41). In addition, certain bacterial toxins, including aerolysin, perfringolysin O, cholera toxin, and tetanus toxin, utilize rafts as either a site for high affinity binding and oligomerization on the surface of cells or as a site for internalization into host cells (42)(43)(44)(45)(46). A recent study reported that treatment of HEp-2 cells with phosphatidylinositol-specific phospholipase C (PI-PLC), an agent that removes GPIanchored proteins from the cell surface, inhibited the capacity of VacA to induce cell vacuolation (31).…”

Bacterial protein toxins and lipids: pore formation or toxin entry into cells

“…It is proposed that the high affinity of PFO and also of the cholesterolbinding cytotoxins (K d = 10 nM) for the cholesterol receptor is involved in concentrating the toxin in cholesterol molecules organized into arcs on the target membrane, promoting oligomerization and membrane insertion (Rossjohn et al, 1997). Cholesterol is clustered in lipid membrane microdomains or rafts, and PFO is a useful tool to identify the membrane rafts (Waheed et al, 2001). …”