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 ϳ3 nm. Antibody scanning analysis suggested that the Cterminal 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 rafts on the cell surface are believed to be very important as platforms for various cellular functions. The aim of this study was to know whether defective lipid efflux may influence lipid rafts on the cell surface and their related cellular functions. We investigated macrophages with defective lipid efflux from ATP binding cassette transporter A1-deficient (Abca1-KO) mice. Lipid rafts were evaluated by the following two novel probes: a biotinylated and protease (subtilisin Carlsberg)-nicked derivative of u-toxin and a fluorescein ester of polyethylene glycol-derived cholesterol. Lipid rafts in Abca1-KO macrophages were increased, as demonstrated by both probes. Moreover, activities of nuclear factor kB, mRNA and intracellular distribution, and secretion of tumor necrosis factor-a (TNF-a) were examined after stimulation by lipopolysaccharides (LPSs). LPS-induced responses of the activation of nuclear factor kB and TNF-a were more prompt and accelerated in the Abca1-KO macrophages compared with wild-type macrophages. Modification of lipid rafts by cyclodextrin and nystatin corrected the abnormal response, suggesting an association between the increased lipid rafts and abnormal TNF-a secretion.We report here that Abca1-KO macrophages with defective lipid efflux exhibited increased lipid rafts on the cell surface and accelerated TNF-a secretion. Reverse cholesterol transport (RCT) is one of the major protective systems against atherosclerosis, in which HDL particles play a crucial role as a shuttle carrying cholesterol derived from peripheral tissues to the liver (1). Cholesterol efflux from the cells is the initial step of RCT, in which free apolipoprotein A-I (apoA-I) or small HDLs take up cholesterol from the peripheral cells. We have been trying to elucidate the molecular mechanism for RCT and cholesterol efflux by analyzing the pathophysiology of patients with abnormal HDL metabolism. We have identified molecules involved in cellular cholesterol efflux and apoA-I and HDL binding proteins on macrophages (2-5).Tangier disease (TD) is a model for the impairment of cholesterol efflux from the cells (6, 7). Patients with TD suffer from genetic HDL deficiency, hepatosplenomegaly, orange tonsils, and premature atherosclerosis (8, 9). Many laboratories including ours have reported that mutations in the Abca1 gene lead to defective cholesterol efflux from the cells (10-12). As a result of the mutation(s) in the Abca1 gene, cells from TD patients exhibited a deficiency of apoA-I-mediated cholesterol efflux and a subsequent accumulation of intracellular lipids as lipid droplets, which is closely related to the development of atherosclerosis in this disorder.On the other hand, in the plasma membrane, cholesterol is distributed abundantly in some domain structures Abbreviations: Abca1-KO, ATP binding cassette transporter A1-deficient; apoA-I, apolipoprotein A-I; BCu, biotinylated and protease (subtilisin Carlsberg)-nicked derivative of u-toxin; fPEG-chol, fluorescent polyethylene glycol cholesteryl ether; L...

Hemagglutinin (HA) glycoproteins isolated from influenza virus caused hemolysis and liposome lysis at pH < 6.0. The pH dependence was similar to that of the parent virus. Hemagglutination and hemolysis titers of HA were comparable with those of virus. The time course of hemolysis by HA was somewhat different from that by virus. HA did not cause fusion of erythrocytes in acidic media, in contrast to virus. Both HA and virus, previously incubated at pH < 6.0, lost their low-pH-induced hemolytic activity. Isolated HA formed rosette-like structures at neutral pH, and these aggregated in acidic media. Virus also aggregated in acidic media and its envelope became leaky to negative stain. HA previously incubated at pH < 6.0 became susceptible to trypsin digestion. Both reversible and irreversible structural changes of HA were observed by fluorescence spectroscopy; a reversible change at a pH between neutral and 6.4 and an irreversible one at pH < 6.0. Bromelain-released HA did not cause hemolysis and liposome lysis in acidic media. The precursor form of HA did not have hemolytic activity in acidic media. The similarity in pH dependence indicates that the structural change in HA induced at pH < 6.0 is the cause of activation and inactivation of hemolysis, HA and virus aggregation, and trypsin susceptibility. We propose that the hydrophobic NH2-terminal segment of HA2 is exposed during the structural change and interacts with the target membranes, causing a permeability increase and leading to hemolysis and lysis. The virus-induced hemolysis can be ascribed for the most part to envelope fusion activated in acidic media.Low pH-induced hemolytic activity of influenza virus has recently been observed (1-3). Based on this finding, we proposed an infectious cell entry mechanism of the virus (4), which is essentially the same as that proposed and developed for Semliki Forest virus (5)-i.e., uptake by endocytosis and fusion of endocytosed vesicles with lysosomes, followed by fusion of the virus envelope with the secondary lysosomal membrane because of the low pH. Recently, studies supporting this entry mechanism for influenza virus have been reported (6,7). Hemagglutinin (HA) glycoproteins on the virus envelope were suggested as the molecules responsible for the low pH-induced activity (1, 4). In the present study, we focused on the function of HA isolated from virus in comparison with that of the parent virus. We also studied functions of bromelain-released HA (BHA), which lacks the COOH-terminal hydrophobic anchoring peptide (8), and of the precursor form of HA, HAo. MATERIALS AND METHODSInfluenza virus AOPR8 was grown in embryonated chicken eggs and purified as described (9). Virus was also grown in a monolayer culture of chicken embryo fibroblasts to obtain HAo (4). The virus was suspended by Pipes/saline (135 mM NaCI/10 mM Pipes, pH 7.4) and stored at -80°C.HA was isolated by solubilization with detergent and centrifugation in sucrose density gradients. Virus was treated with Triton X-100 (4 mg/mg of viral protein...

Engagement of the Fas receptor (CD95) initiates multiple signaling pathways that lead to apoptosis, such as the formation of death-inducing signaling complex (DISC), activation of caspase cascades, and the generation of the lipid messenger, ceramide. Sphingomyelin (SM) is a major component of lipid rafts, which are specialized structures that enhance the efficiency of membrane receptor signaling and are a main source of ceramide. However, the functions of SM in Fas-mediated apoptosis have yet to be clearly defined, as the responsible genes have not been identified. After cloning a gene responsible for SM synthesis, SMS1, we established SM synthase–defective WR19L cells transfected with the human Fas gene (WR/Fas-SM(−)), and cells that have been functionally restored by transfection with SMS1 (WR/Fas-SMS1). We show that expression of membrane SM enhances Fas-mediated apoptosis through increasing DISC formation, activation of caspases, efficient translocation of Fas into lipid rafts, and subsequent Fas clustering. Furthermore, WR/Fas-SMS1 cells, but not WR/Fas-SM(−) cells, showed a considerable increase in ceramide generation within lipid rafts upon Fas stimulation. These data suggest that a membrane SM is important for Fas clustering through aggregation of lipid rafts, leading to Fas-mediated apoptosis.