Recognition of sphingomyelin by lysenin and lysenin-related proteins

Lysenin is a sphingomyelin (SM)-specific toxin isolated from the coelomic fluid of the earthworm Eisenia foetida. Lysenin comprises a family of proteins together with lysenin-related protein 1 (LRP-1, lysenin 2) and LRP-2 (lysenin 3). In the present study, we characterized LRP-1 and LRP-2 together with lysenin using maltose-binding-protein-tagged recombinant proteins. LRP-2 specifically bound SM and induced hemolysis like lysenin. In contrast the binding and hemolytic activities of LRP-1 were 10 times less than those of lysenin and LRP-2. Lysenin and LRP-2 share 30 common sites of aromatic amino acids. Among them, only one position, phenylalanine 210, is substituted for isoleucine in LRP-1. The activity of LRP-1 was dramatically increased by introducing a single amino acid substitution of isoleucine 210 to phenylalanine, suggesting the importance of this aromatic amino acid in biological activities of lysenin and LRPs. The importance of aromatic amino acids was further indicated by a systematic tryptophan to alanine mutation of lysenin. Lysenin contains six tryptophan residues of which five are conserved in LRP-1 and -2. We showed that the conserved tryptophans but not the nonconserved one were required both in the recognition of SM and in the hemolytic activity of lysenin. Our results suggest the importance of tryptophan in the toxin function likely due to a direct recognition of SM or in maintaining the protein structure.     

Sphingomyelin-rich domains are sites of lysenin oligomerization: Implications for raft studies

Lysenin is a self-assembling, pore-forming toxin which specifically recognizes sphingomyelin. Mutation of tryptophan 20 abolishes lysenin oligomerization and cytolytic activity. We studied the interaction of lysenin WT and W20A with sphingomyelin in membranes of various lipid compositions which, according to atomic force microscopy studies, generated either homo- or heterogeneous sphingomyelin distribution. Liposomes composed of SM/DOPC, SM/DOPC/cholesterol and SM/DPPC/cholesterol could bind the highest amounts of GST-lysenin WT, as shown by surface plasmon resonance analysis. These lipid compositions enhanced the release of carboxyfluorescein from liposomes induced by lysenin WT, pointing to the importance of heterogeneous sphingomyelin distribution for lysenin WT binding and oligomerization. Lysenin W20A bound more weakly to sphingomyelin-containing liposomes than did lysenin WT. The same amounts of lysenin W20A bound to sphingomyelin mixed with either DOPC or DPPC, indicating that the binding was not affected by sphingomyelin distribution in the membranes. The mutant lysenin had a limited ability to penetrate hydrophobic region of the membrane as indicated by measurements of surface pressure changes. When applied to detect sphingomyelin on the cell surface, lysenin W20A formed large conglomerates on the membrane, different from small and regular clusters of lysenin WT. Only lysenin WT recognized sphingomyelin pool affected by formation of raft-based signaling platforms. During fractionation of Triton X-100 cell lysates, SDS-resistant oligomers of lysenin WT associated with membrane fragments insoluble in Triton X-100 while monomers of lysenin W20A partitioned to Triton X-100-soluble membrane fractions. Altogether, the data suggest that oligomerization of lysenin WT is a prerequisite for its docking in raft-related domains.     

Lethal and non-lethal responses of spermatozoa from a wide variety of vertebrates and invertebrates to lysenin, a protein from the coelomic fluid of the earthworm Eisenia foetida

Lysenin, a novel protein that we isolated from the coelomic fluid of the earthworm Eisenia foetida, binds specifically to sphingomyelin (SM) among various phospholipids found in cell membranes, and causes cytolysis. The plasma membrane of mammalian spermatozoa is known to contain SM at relatively high levels and we therefore examined the effects of lysenin on the spermatozoa of various animals. Lysenin had lethal effects on spermatozoa of 5 of 33 species of invertebrates tested and on spermatozoa of 30 of 39 species of vertebrates. We postulated that plasma membranes of the spermatozoa of most invertebrates might not contain SM whereas those of most vertebrate species might contain SM. These possibilities were supported by our failure to detect SM chemically in the testes of three species of invertebrates, in none of which spermatozoa responded to lysenin. In contrast, we detected SM in the testes of all 25 vertebrate species examined, irrespective of a negative or positive response of spermatozoa to lysenin. None of the six species of Protista examined was affected by lysenin. Our survey suggests that, in general, the spermatozoa of animals can be grouped into two categories, invertebrate and vertebrate, depending on the absence or presence of SM in their plasma membrane. The incorporation of SM into spermatozoa seems first to have occurred in protochordates during the course of evolution. Discussions about the exceptional responses to lysenin observed in the spermatozoa of five species of invertebrates and of nine species of vertebrates are made from phylogenetic and reproductive viewpoints. J. Exp. Zool. 286:538-549, 2000.     

Cholesterol and lipid/protein ratio control the oligomerization of a sphingomyelin-specific toxin, lysenin

Lysenin is a pore-forming toxin that specifically binds sphingomyelin (SM). The binding of the toxin to the membrane is accompanied by the oligomerization of the protein, leading to pore formation. The interaction of lysenin with SM is affected by the presence of other lipids found in the plasma membrane. Although a previous study showed that SM/cholesterol liposomes were 10,000 times more effective than SM liposomes in inhibiting lysenin-induced hemolysis (Yamaji, A., Sekizawa, Y., Emoto, K., Sakuraba, H., Inoue, K., Kobayashi, H., and Umeda, M. (1998) J. Biol. Chem. 273, 5300-5306), the role of cholesterol is not precisely clarified. In the present study, we examined the effects of the presence of cholesterol in the SM membrane on the inhibition of hemolysis, the binding of lysenin to SM, and the oligomerization of lysenin. The addition of cholesterol to SM liposomes dramatically inhibited lysenin-induced hemolysis as described previously. However, the presence of cholesterol did not affect the binding of lysenin to SM liposomes. The oligomerization of lysenin was facilitated by the presence of cholesterol in SM liposomes. The oligomerization of lysenin was also dependent on the SM/lysenin ratio, that is, the amount of lysenin oligomer was increased with the decrease in the SM/lysenin ratio. When the SM/lysenin molar ratio was high, lysenin associated with the membrane as a monomer, which was able to transfer to the erythrocyte membrane. Our results indicate that both cholesterol and the SM/lysenin ratio control the amount of lysenin monomer via altering the state of protein oligomerization, thus affecting hemolysis.     

A Lipid-Specific Toxin Reveals Heterogeneity of Sphingomyelin-Containing Membranes

Little is known about the heterogenous organization of lipids in biological membranes. Sphingomyelin (SM) is a major plasma membrane lipid that forms lipid domains together with cholesterol and glycolipids. Using SM-specific toxin, lysenin, we showed that in cultured epithelial cells the accessibility of the toxin to SM is different between apical and basolateral membranes. Apical membranes are highly enriched with glycolipids. The inhibitory role of glycolipids in the binding of lysenin to SM was confirmed by comparing the glycolipid-deficient mutant melanoma cell line with its parent cell. Model membrane experiments indicated that glycolipid altered the local density of SM so that the affinity of the lipid for lysenin was decreased. Our results indicate that lysenin recognizes the heterogenous organization of SM in biomembranes and that the organization of SM differs between different cell types and between different membrane domains within the same cell. Isothermal titration calorimetry suggests that lysenin binding to SM is presumably the result of a SM-lysenin complex formation of specific stoichiometry, thus supporting the idea of the existence of small condensed lipid complexes consisting of just a few lipid molecules in living cells.     

The single-giant unilamellar vesicle method reveals lysenin-induced pore formation in lipid membranes containing sphingomyelin

Lysenin is a sphingomyelin (SM)-binding pore-forming toxin. To reveal the interaction of lysenin with lipid membranes, we investigated lysenin-induced membrane permeation of a fluorescent probe, calcein, through dioleoylphosphatidylcholine(DOPC)/SM, DOPC/SM/cholesterol(chol), and SM/chol membranes, using the single-giant unilamellar vesicle (GUV) method. The results clearly show that lysenin formed pores in all the membranes, through which membrane permeation of calcein occurred without disruption of GUVs. The membrane permeation began stochastically, and the membrane permeability coefficient increased over time to reach a maximum, steady value, Ps, which persisted for a long time(100--500 s), indicating that the pore concentration increases over time and finally reaches its steady value, NP s . The Ps values increased as the SM/lysenin ratio decreased, and at low concentrations of lysenin, the Ps values of SM/DOPC/chol (42/30/28)GUVs were much larger than those of SM/DOPC (58/42) GUVs. The dependence of Ps on the SM/lysenin ratio for these membranes was almost the same as that of the fraction of sodium dodecyl sulfate (SDS)-resistant lysenin oligomers, indicating that NP s increases as the SDS-resistant oligomer fraction increases. On the other hand, lysenin formed pores in GUVs of SM/chol(60/40) membrane, which is in a homogeneous liquid-ordered phase, indicating that the phase boundary is not necessary for pore formation. The Ps values of SM/chol (60/40) GUVs were smaller than those of SM/DOPC/chol (42/30/28) GUVs even though the SDS-resistant oligomer fractions were similar for both membranes, suggesting that not all of the oligomers can convert into a pore. On the basis of these results, we discuss the elementary processes of lysenin-induced pore formation.     

Damage to the gills,skin and other tissues by lysenin and the coelomic fluid of the earthworm Eisenia foetida in two teleosts,Tanichthys albonubes and Oreochromis mossambicus

Lysenin is a 33-kDa protein found in the coelomic fluid (CF) of the earthworm Eisenia foetida. Purified lysenin binds specifically to sphingomyelin (SM). In the present studies, we found that the white cloud mountain minnow Tanichthys albonubes and the Mozambique tilapia Oreochromis mossambicus died in solutions of lysenin (at concentrations above 2.5 microg/ml) and CF (0.6%, v/v) within 2 h. The gills of both species of fish were damaged similarly by lysenin and by CF. Most gill lamellae became irregularly bent or curled, with swelling of the epithelial cells of the lamellae. Red blood cells in the lamellar vascular sinuses, in the central venous sinuses, and in the blood vessels of the entire body became swollen and lysed, choking the sinuses. Epithelial cells in the skin were also damaged. When fish of both species were treated with lysenin or CF that had been incubated with SM-liposomes, they did not die. Their behavior remained normal and there was no damage to any cells or tissues. These findings suggest that SM might be involved in the lethal effects of lysenin and CF. It is likely that purified lysenin and lysenin in CF bound to SM in the cell membranes of the tissues mentioned above, damaging the cells. The presence of SM in the gills and skin was confirmed, supporting this hypothesis. The damage to gills and hemolysis might have resulted in lethal respiratory problems. Damage to the skin might disturb the exchange of ions through the skin, hastening death. Damage by lysenin and CF to epithelial cells of the cornea and the wall of the oral cavity was also recognized, but there was no such damage to the intestine.     

Limonoid compounds inhibit sphingomyelin biosynthesis by preventing CERT protein-dependent extraction of ceramides from the endoplasmic reticulum

To identify novel inhibitors of sphingomyelin (SM) metabolism, a new and selective high throughput microscopy-based screening based on the toxicity of the SM-specific toxin, lysenin, was developed. Out of a library of 2011 natural compounds, the limonoid, 3-chloro-8β-hydroxycarapin-3,8-hemiacetal (CHC), rendered cells resistant to lysenin by decreasing cell surface SM. CHC treatment selectively inhibited the de novo biosynthesis of SM without affecting glycolipid and glycerophospholipid biosynthesis. Pretreatment with brefeldin A abolished the limonoid-induced inhibition of SM synthesis suggesting that the transport of ceramide (Cer) from the endoplasmic reticulum to the Golgi apparatus is affected. Unlike the Cer transporter (CERT) inhibitor HPA-12, CHC did not change the transport of a fluorescent short chain Cer analog to the Golgi apparatus or the formation of fluorescent and short chain SM from the corresponding Cer. Nevertheless, CHC inhibited the conversion of de novo synthesized Cer to SM. We show that CHC specifically inhibited the CERT-mediated extraction of Cer from the endoplasmic reticulum membranes in vitro. Subsequent biochemical screening of 21 limonoids revealed that some of them, such as 8β-hydroxycarapin-3,8-hemiacetal and gedunin, which exhibits anti-cancer activity, inhibited SM biosynthesis and CERT-mediated extraction of Cer from membranes. Model membrane studies suggest that 8β-hydroxycarapin-3,8-hemiacetal reduced the miscibility of Cer with membrane lipids and thus induced the formation of Cer-rich membrane domains. Our study shows that certain limonoids are novel inhibitors of SM biosynthesis and suggests that some biological activities of these limonoids are related to their effect on the ceramide metabolism.     

Real-Time Visualization of Assembling of a Sphingomyelin-Specific Toxin on Planar Lipid Membranes

Pore-forming toxins (PFTs) are soluble proteins that can oligomerize on the cell membrane and induce cell death by membrane insertion. PFT oligomers sometimes form hexagonal close-packed (hcp) structures on the membrane. Here, we show the assembling of the sphingomyelin (SM)-binding PFT, lysenin, into an hcp structure after oligomerization on SM/cholesterol membrane. This process was monitored by high-speed atomic force microscopy. Hcp assembly was driven by reorganization of lysenin oligomers such as association/dissociation and rapid diffusion along the membrane. Besides rapid association/dissociation of oligomers, the height change for some oligomers, possibly resulting from conformational changes in lysenin, could also be visualized. After the entire membrane surface was covered with a well-ordered oligomer lattice, the lysenin molecules were firmly bound on the membrane and the oligomers neither dissociated nor diffused. Our results reveal the dynamic nature of the oligomers of a lipid-binding toxin during the formation of an hcp structure. Visualization of this dynamic process is essential for the elucidation of the assembling mechanism of some PFTs that can form ordered structures on the membrane.     

Real-Time Visualization of Assembling of a Sphingomyelin-Specific Toxin on Planar Lipid Membranes

Pore-forming toxins (PFTs) are soluble proteins that can oligomerize on the cell membrane and induce cell death by membrane insertion. PFT oligomers sometimes form hexagonal close-packed (hcp) structures on the membrane. Here, we show the assembling of the sphingomyelin (SM)-binding PFT, lysenin, into an hcp structure after oligomerization on SM/cholesterol membrane. This process was monitored by high-speed atomic force microscopy. Hcp assembly was driven by reorganization of lysenin oligomers such as association/dissociation and rapid diffusion along the membrane. Besides rapid association/dissociation of oligomers, the height change for some oligomers, possibly resulting from conformational changes in lysenin, could also be visualized. After the entire membrane surface was covered with a well-ordered oligomer lattice, the lysenin molecules were firmly bound on the membrane and the oligomers neither dissociated nor diffused. Our results reveal the dynamic nature of the oligomers of a lipid-binding toxin during the formation of an hcp structure. Visualization of this dynamic process is essential for the elucidation of the assembling mechanism of some PFTs that can form ordered structures on the membrane.     

Imaging local sphingomyelin-rich domains in the plasma membrane using specific probes and advanced microscopy

Sphingomyelin (SM) is one of the major lipids in the mammalian plasma membrane. Multiple lines of evidence suggest that SM plays at least two functional roles in the cell, as a reservoir of lipid second messengers and as a platform for signaling molecules. To understand the molecular organization and dynamics of the SM-rich membrane domains, new approaches have been developed utilizing newly characterized specific SM-binding probes and state-of-the-art microscopy techniques. The toxic protein from the sea anemone, equinatoxin II, has been characterized as a specific probe for SM. The cytolytic protein from the earthworm, lysenin, has also been used as a SM-specific probe for the analysis of the heterogeneity of SM-rich membrane domains. Recently, using a non-toxic form of lysenin, we showed the spatial and temporal localization of SM in the plasma membrane by confocal and super-resolution microscopy. New microscopy techniques have also been introduced by other groups to help visualize membrane lipid domains. Here we review the most recent studies on imaging the SM-rich domains in biological membranes. This article is part of a Special Issue entitled New Frontiers in Sphingolipid Biology.     

Lysenin-His, a sphingomyelin-recognizing toxin, requires tryptophan 20 for cation-selective channel assembly but not for membrane binding

Lysenin is 297 amino acid long toxin derived from the earthworm Eisenia foetida which specifically recognizes sphingomyelin and induces cell lysis. We synthesized lysenin gene supplemented with a polyhistidine tag, subcloned it into the pT7RS plasmid and the recombinant protein was produced in Escherichia coli. In order to obtain lysenin devoid of its lytic activity, the protein was mutated by substitution of tryptophan 20 by alanine. The recombinant mutant lysenin-His did not evoke cell lysis, although it retained the ability to specifically interact with sphingomyelin, as demonstrated by immunofluorescence microscopy and by dot blot lipid overlay and liposome binding assays. We found that the lytic activity of wild-type lysenin-His was correlated with the protein oligomerization during interaction with sphingomyelin-containing membranes and the amount of oligomers was increased with an elevation of sphingomyelin/lysenin ratio. Blue native gel electrophoresis indicated that trimers can be functional units of the protein, however, lysenin hexamers and nanomers were stabilized by chemical cross-linking of the protein and by sodium dodecyl sulfate. When incorporated into planar lipid bilayers, wild type lysenin-His formed cation-selective channels in a sphingomyelin-dependent manner. We characterized the channel activity by establishing its various open/closed states. In contrast, the mutant lysenin-His did not form channels and its correct oligomerization was strongly impaired. Based on these results we suggest that lysenin oligomerizes upon interaction with sphingomyelin in the plasma membrane, forming cation-selective channels. Their activity disturbs the ion balance of the cell, leading eventually to cell lysis.     

Differential Effects of Ceramide and Sphingosine 1-Phosphate on ERM Phosphorylation: PROBING SPHINGOLIPID SIGNALING AT THE OUTER PLASMA MEMBRANE*

ERM proteins are regulated by phosphorylation of the most C-terminal threonine residue, switching them from an activated to an inactivated form. However, little is known about the control of this regulation. Previous work in our group demonstrated that secretion of acid sphingomyelinase acts upstream of ERM dephosphorylation, suggesting the involvement of sphingomyelin (SM) hydrolysis in ERM regulation. To define the role of specific lipids, we employed recombinant bacterial sphingomyelinase (bSMase) as a direct probe of SM metabolism at the plasma membrane. bSMase induced a rapid dose- and time-dependent decrease in ERM dephosphorylation. ERM dephosphorylation was driven by ceramide generation and not by sphingomyelin depletion, as shown using recombinant sphingomyelinase D. The generation of ceramide at the plasma membrane was sufficient for ERM regulation, and no intracellular SM hydrolysis was required, as was visualized using Venus-tagged lysenin probe, which specifically binds SM. Interestingly, hydrolysis of plasma membrane bSMase-induced ceramide using bacterial ceramidase caused ERM hyperphosphorylation and formation of cell surface protrusions. The effects of plasma membrane ceramide hydrolysis were due to sphingosine 1-phosphate formation, as ERM phosphorylation was blocked by an inhibitor of sphingosine kinase and induced by sphingosine 1-phosphate. Taken together, these results demonstrate a new regulatory mechanism of ERM phosphorylation by sphingolipids with opposing actions of ceramide and sphingosine 1-phosphate. The approach also defines a tool kit to probe sphingolipid signaling at the plasma membrane.     

Oligomerization and pore formation of a sphingomyelin-specific toxin,lysenin

Lysenin is a novel protein derived from coelomic fluid of the earthworm Eisenia foetida, which specifically recognizes sphingomyelin and induces cytolysis. The mechanism underlying lysenin-induced cell lysis has not been clarified. In this report we studied the interaction of lysenin with red blood cells as well as artificial liposomes. Our results showed that lysenin bound membranes and assembled to SDS-resistant oligomers in a sphingomyelin-dependent manner, leading to the formation of pores with a hydrodynamic diameter of approximately 3 nm. Antibody scanning analysis suggested that the C-terminal region of lysenin was exposed, whereas the N-terminal was hidden in the isolated oligomer complex. Differential scanning calorimetry revealed that lysenin interacted with both hydrophilic head group and hydrophobic hydrocarbon tails of sphingomyelin. Oligomerization but not binding was affected by the amide-linked fatty acid composition of sphingomyelin, suggesting the role of membrane fluidity in the oligomerization step.     

Lipid polarity is maintained in absence of tight junctions

The role of tight junctions (TJs) in the establishment and maintenance of lipid polarity in epithelial cells has long been a subject of controversy. We have addressed this issue using lysenin, a toxin derived from earthworms, and an influenza virus labeled with a fluorescent lipid, octadecylrhodamine B (R18). When epithelial cells are stained with lysenin, lysenin selectively binds to their apical membranes. Using an artificial liposome, we demonstrated that lysenin recognizes the membrane domains where sphingomyelins are clustered. Interestingly, lysenin selectively stained the apical membranes of epithelial cells depleted of zonula occludens proteins (ZO-deficient cells), which completely lack TJs. Furthermore, the fluorescent lipid inserted into the apical membrane by fusion with the influenza virus did not diffuse to the lateral membrane in ZO-deficient epithelial cells. This study revealed that sphingomyelin-cluster formation occurs only in the apical membrane and that lipid polarity is maintained even in the absence of TJs.     

Endogenous sphingomyelin segregates into submicrometric domains in the living erythrocyte membrane

We recently reported that trace insertion of exogenous fluorescent (green BODIPY) analogs of sphingomyelin (SM) into living red blood cells (RBCs), partially spread onto coverslips, labels submicrometric domains, visible by confocal microscopy. We here extend this feature to endogenous SM, upon binding of a SM-specific nontoxic (NT) fragment of the earthworm toxin, lysenin, fused to the red monomeric fluorescent protein, mCherry [construct named His-mCherry-NT-lysenin (lysenin*)]. Specificity of lysenin* binding was verified with composition-defined liposomes and by loss of ¹²⁵I-lysenin* binding to erythrocytes upon SM depletion by SMase. The ¹²⁵I-lysenin* binding isotherm indicated saturation at 3.5 × 10⁶ molecules/RBC, i.e., ∼3% of SM coverage. Nonsaturating lysenin* concentration also labeled sub­micrometric domains on the plasma membrane of partially spread erythrocytes, colocalizing with inserted green BODIPY-SM, and abrogated by SMase. Lysenin*-labeled domains were stable in time and space and were regulated by temperature and cholesterol. The abundance, size, positioning, and segregation of lysenin*-labeled domains from other lipids (BODIPY-phosphatidylcholine or -glycosphingolipids) depended on membrane tension. Similar lysenin*-labeled domains were evidenced in RBCs gently suspended in 3D-gel. Taken together, these data demonstrate submicrometric compartmentation of endogenous SM at the membrane of a living cell in vitro, and suggest it may be a genuine feature of erythrocytes in vivo.     

Binding of a pleurotolysin ortholog from Pleurotus eryngii to sphingomyelin and cholesterol-rich membrane domains

A mixture of sphingomyelin (SM) and cholesterol (Chol) exhibits a characteristic lipid raft domain of the cell membranes that provides a platform to which various signal molecules as well as virus and bacterial proteins are recruited. Several proteins capable of specifically binding either SM or Chol have been reported. However, proteins that selectively bind to SM/Chol mixtures are less well characterized. In our screening for proteins specifically binding to SM/Chol liposomes, we identified a novel ortholog of Pleurotus ostreatus, pleurotolysin (Ply)A, from the extract of edible mushroom Pleurotus eryngii, named PlyA2. Enhanced green fluorescent protein (EGFP)-conjugated PlyA2 bound to SM/Chol but not to phosphatidylcholine/Chol liposomes. Cell surface labeling of PlyA2-EGFP was abolished after sphingomyelinase as well as methyl-β-cyclodextrin treatment, removing SM and Chol, respectively, indicating that PlyA2-EGFP specifically binds cell surface SM/Chol rafts. Tryptophan to alanine point mutation of PlyA2 revealed the importance of C-terminal tryptophan residues for SM/Chol binding. Our results indicate that PlyA2-EGFP is a novel protein probe to label SM/Chol lipid domains both in cell and model membranes.     

Molecular determinants of sphingomyelin specificity of a eukaryotic pore-forming toxin

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.     

Toxicity of coelomic fluid of the earthworm Eisenia foetida to vertebrates but not invertebrates: probable role of sphingomyelin

The coelomic fluid (CF) of the earthworm Eisenia foetida exhibits a wide variety of biological activities. We found that the CF was not toxic to 42 species, belonging to seven invertebrate phyla, almost all in aquatic adults and larvae exposed to CF. Eleven teleostean species tested died in 0.2-1% CF mostly between 10 and 120 min and the effects were dose-dependent. Tadpoles of the toad Bufo japonicus formosus died in 0.4-2% CF between 80 and 225 min depending upon size, with larger tadpoles surviving longer. Before dying, all experimental tadpoles developed curled and shrunken tails. The Okinawa tree lizard, soft-shelled turtle, Japanese quail, mouse and rat all died after i.v. injection of CF (above 20 microl/kg). Thus, CF was not toxic to invertebrates, but toxic to vertebrates. After heating, CF lost its toxicity to fish, tadpoles and mice. Both CF and lysenin incubated with sphingomyelin-liposomes (SM-liposomes) were no longer toxic, suggesting the involvement of SM in the toxicity. Lysenin, which is a constituent of CF and known to bind specifically to sphingomyelin, exhibited toxicity similar to that of CF. Thus, lysenin in CF is probably responsible for the toxic effects of CF by binding to SM in vertebrate tissues. The bodies of invertebrates might contain little or no SM, while those of vertebrates do contain SM. The coelomic fluid of the earthworm Pheretima communissima has no toxicity to mouse.     

Sphingomyelin functions as a novel receptor for Helicobacter pylori VacA

The vacuolating cytotoxin (VacA) of the gastric pathogen Helicobacter pylori binds and enters epithelial cells, ultimately resulting in cellular vacuolation. Several host factors have been reported to be important for VacA function, but none of these have been demonstrated to be essential for toxin binding to the plasma membrane. Thus, the identity of cell surface receptors critical for both toxin binding and function has remained elusive. Here, we identify VacA as the first bacterial virulence factor that exploits the important plasma membrane sphingolipid, sphingomyelin (SM), as a cellular receptor. Depletion of plasma membrane SM with sphingomyelinase inhibited VacA-mediated vacuolation and significantly reduced the sensitivity of HeLa cells, as well as several other cell lines, to VacA. Further analysis revealed that SM is critical for VacA interactions with the plasma membrane. Restoring plasma membrane SM in cells previously depleted of SM was sufficient to rescue both toxin vacuolation activity and plasma membrane binding. VacA association with detergent-resistant membranes was inhibited in cells pretreated with SMase C, indicating the importance of SM for VacA association with lipid raft microdomains. Finally, VacA bound to SM in an in vitro ELISA assay in a manner competitively inhibited by lysenin, a known SM-binding protein. Our results suggest a model where VacA may exploit the capacity of SM to preferentially partition into lipid rafts in order to access the raft-associated cellular machinery previously shown to be required for toxin entry into host cells.