Sphingomyelin (SM) is abundant in the outer leaflet of the cell plasma membrane, with the ability to concentrate in so-called lipid rafts. These specialized cholesterol-rich microdomains not only are associated with many physiological processes but also are exploited as cell entry points by pathogens and protein toxins. SM binding is thus a widespread and important biochemical function, and here we reveal the molecular basis of SM recognition by the membrane-binding eukaryotic cytolysin equinatoxin II (EqtII). The presence of SM in membranes drastically improves the binding and permeabilizing activity of EqtII. Direct binding assays showed that EqtII specifically binds SM, but not other lipids and, curiously, not even phosphatidylcholine, which presents the same phosphorylcholine headgroup. Analysis of the EqtII interfacial binding site predicts that electrostatic interactions do not play an important role in the membrane interaction and that the two most important residues for sphingomyelin recognition are Trp 112 and Tyr 113 exposed on a large loop. Experiments using site-directed mutagenesis, surface plasmon resonance, lipid monolayer, and liposome permeabilization assays clearly showed that the discrimination between sphingomyelin and phosphatidylcholine occurs in the region directly below the phosphorylcholine headgroup. Because the characteristic features of SM chemistry lie in this subinterfacial region, the recognition mechanism may be generic for all SM-specific proteins. Sphingomyelin (SM)8 is an important eukaryotic membrane lipid, located for the most part in the outer leaflet of the plasma membrane in the form of specialized cholesterol-rich microdomains, so-called lipid rafts (1, 2). Many pathogens and toxic proteins employ lipid rafts to invade cells (3, 4), but currently little is known about the molecular details of the recognition mechanism of the lipid components present in the rafts. In the particular case of SM, the specific recognition occurs even though SM exposes the same phosphorylcholine headgroup as the other abundant lipid, phosphatidylcholine. SM-binding proteins are currently exploited as specific markers for cellular SM (5) and are used to identify other proteins involved in sphingolipid metabolism (6).Actinoporins are extremely potent cytolysins produced exclusively by sea anemones (7,8). They may be used to capture prey, in intraspecific aggression, or in preventing adhesion of other organisms (7, 9). The two most studied representatives are EqtII, isolated from the sea anemone Actinia equina, and sticholysin II (StII) from Stichodactyla helianthus. Actinoporins constitute a family of conserved proteins that cause hemolysis of red blood cells by colloid-osmotic lysis and exhibit cytolytic activity against various cell lines (7, 10 -12). Even the most distant members of the family share more than 60% sequence identity and the available threedimensional structures of EqtII and StII are nearly superimposable (13-15). The structure is composed of a tightly folded ␤-sandwich flanked b...

No abstract

Although sphingomyelin is an important cellular lipid, its subcellular distribution is not precisely known. Here we use a sea anemone cytolysin, equinatoxin II (EqtII), which specifically binds sphingomyelin, as a new marker to detect cellular sphingomyelin. A purified fusion protein composed of EqtII and green fluorescent protein (EqtII-GFP) binds to the SM rich apical membrane of Madin-Darby canine kidney (MDCK) II cells when added exogenously, but not to the SM-free basolateral membrane. When expressed intracellularly within MDCK II cells, EqtII-GFP colocalizes with markers for Golgi apparatus and not with those for nucleus, mitochondria, endoplasmic reticulum or plasma membrane. Colocalization with the Golgi apparatus was confirmed by also using NIH 3T3 fibroblasts. Moreover, EqtII-GFP was enriched in cis-Golgi compartments isolated by gradient ultracentrifugation. The data reveal that EqtII-GFP is a sensitive probe for membrane sphingomyelin, which provides new information on cytosolic exposure, essential to understand its diverse physiological roles.

Membrane rafts are transient and unstable membrane microdomains that are enriched in sphingolipids, cholesterol, and specific proteins. They are involved in intracellular trafficking, signal transduction, pathogen entry, and attachment of various ligands. Increasing experimental evidence on the crucial biological roles of membrane rafts under normal and pathological conditions require new techniques for their structural and functional characterization. In particular, fluorescence-labeled cytolytic proteins that interact specifically with molecules enriched in rafts are of increasing interest. Cholera toxin subunit B interacts specifically with raft-residing ganglioside G(M1), and it has long been the lipid probe of choice for membrane rafts. Recently, four new pore-forming toxins have been proposed as selective raft markers: (i) equinatoxin II, a cytolysin from the sea anemone Actinia equina, which specifically recognizes free and membrane-embedded sphingomyelin; (ii) a truncated non-toxic mutant of a cytolytic protein, lysenin, from the earthworm Eisenia foetida, which specifically recognizes sphingomyelin-enriched membrane domains; (iii) a non-toxic derivative of the cholesterol-dependent cytolysin perfringolysin O, from the bacterium Clostridium perfringens, which selectively binds to membrane domains enriched in cholesterol; and (iv) ostreolysin, from the mushroom Pleurotus ostreatus, which does not bind to a single raft-enriched lipid component, but requires a specific combination of two of the most important raft-residing lipids: sphingomyelin and cholesterol. Nontoxic, raft-binding derivatives of cytolytic proteins have already been successfully used to explore both the structure and function of membrane rafts, and of raft-associated molecules. Here, we review these four new derivatives of pore-forming toxins as new putative markers of these membrane microdomains.

Ostreolysin is a cytolytic protein from the edible oyster mushroom (Pleurotus ostreatus), which recognizes specifically and binds to raft-like sterol-enriched membrane domains that exist in the liquid-ordered phase. Its binding can be abolished by micromolar concentrations of lysophospholipids and fatty acids. The membrane activity of ostreolysin, however, does not completely correlate with the ability of a certain sterol to induce the formation of a liquid-ordered phase, suggesting that the protein requires an additional structural organization of the membrane to exert its activity. The aim of this study was to further characterize the lipid membranes that facilitate ostreolysin binding by analyzing their lipid phase domain structure. Fourier-transformed infrared spectroscopy (FTIR) and electron paramagnetic resonance (EPR) were used to analyze the ordering and dynamics of membrane lipids and the membrane domain structure of a series of unilamellar liposomes prepared by systematically changing the lipid components and their ratios. Our results corroborate the earlier conclusion that the average membrane fluidity of ostreolysin-susceptible liposomes alone cannot account for the membrane activity of the protein. Combined with previous data computer-aided interpretation of EPR spectra strongly suggests that chemical properties of membrane constituents, their specific distribution, and physical characteristics of membrane nanodomains, resulting from the presence of sterol and sphingomyelin (or a highly ordered phospholipid, dipalmitoylphosphatidylcholine), are essential prerequisites for ostreolysin membrane binding and pore-formation.