Lysenin, a Novel Sphingomyelin-specific Binding Protein

Abstract

Lysenin, a novel 41-kDa protein purified from coelomic fluid of the earthworm Eisenia foetida, induced erythrocyte lysis. Preincubation of lysenin with vesicles containing sphingomyelin inhibited lysenin-induced hemolysis completely, whereas vesicles containing phospholipids other than sphingomyelin showed no inhibitory activity, suggesting that lysenin bound specifically to sphingomyelin on erythrocyte membranes. The specific binding of lysenin to sphingomyelin was confirmed by enzyme-linked immunosorbent assay, TLC immunostaining, and liposome lysis assay. In these assays, lysenin bound specifically to sphingomyelin and did not show any cross-reaction with other phospholipids including sphingomyelin analogs such as sphingosine, ceramide, and sphingosylphosphorylcholine, indicating that it recognized a precise molecular structure of sphingomyelin. Kinetic analysis of the lysenin-sphingomyelin interaction by surface plasmon resonance measurements using BIAcore TM system showed that lysenin associated with membrane surfaces composed of sphingomyelin (k on ‫؍‬ 3.2 ؋ 10 4 M ؊1 s ؊1 ) and dissociated extremely slowly (k off ‫؍‬ 1.7 ؋ 10 ؊4 s ؊1 ), giving a low dissociation constant (K D ‫؍‬ 5.3 ؋ 10 ؊9 M). Incorporation of cholesterol into the sphingomyelin membrane significantly increased the total amount of lysenin bound to the membrane, whereas it did not change the kinetic parameters of the lysenin-membrane interaction, suggesting that lysenin specifically recognized sphingomyelin and cholesterol incorporation changed the topological distribution of sphingomyelin in the membranes, thereby increasing the accessibility of sphingomyelin to lysenin. Immunofluorescence staining of fibroblasts derived from a patient with Niemann-Pick disease type A showed that lysenin stained the surfaces of the fibroblasts uniformly, whereas intense lysosomal staining was observed when the cells were permeabilized by digitonin treatment. Preincubation of lysenin with vesicles containing sphingomyelin abolished lysenin immunostaining. This study demonstrated that lysenin bound specifically to sphingomyelin on cellular membranes and should be a useful tool to probe the molecular motion and function of sphingomyelin in biological membranes.Sphingomyelin, N-acylsphingosine-1-phosphorylcholine, is a major lipid constituent of animal cell membranes, accounting for up to 60 mol % of the total phospholipid content of some cells, such as sheep erythrocytes (1). In addition to its role as a structural component of membranes, recent studies have suggested the roles of sphingomyelin in various intracellular signaling pathways (2-6). A sphingomyelin cycle has been proposed in which extracellular agents such as tumor necrosis factor ␣, ␥-interferon, and interleukin 1 activate sphingomyelinase, resulting in the production of ceramide which serves as a second messenger mediating the action of these extracellular ligands (4 -6). Sphingomyelin was also proposed to be enriched in an organized membrane domain, caveolae, which contains cholesterol, glyco...

Lipid Polarity Is Maintained in Absence of Tight Junctions

“…that lysenin recognizes other properties of sphingomyelin-containing membranes. Previous studies have shown that the interaction of lysenin and sphingomyelin is affected by the presence of other lipids (23,24). The mixing of glycosphingolipid and sphingomyelin hinders the formation of clusters of sphingomyelin alone and inhibits the binding of lysenin (24).…”

“…The mixing of glycosphingolipid and sphingomyelin hinders the formation of clusters of sphingomyelin alone and inhibits the binding of lysenin (24). We have previously shown that sphingomyelin/cholesterol liposomes were 10,000 times more effective than liposomes of sphingomyelin alone in inhibiting lysenin-induced hemolysis (23,25).…”

“…Cells-As a tool for visualization of the distribution of sphingomyelin in the cellular membrane, we previously characterized lysenin as a toxin that specifically bound to sphingomyelin (23,24). In this study, to visualize the distribution of sphingomyelin in epithelial cells, we stained cultured epithelial cells using lysenin.…”

Expression Cloning of a Human cDNA Restoring Sphingomyelin Synthesis and Cell Growth in Sphingomyelin Synthase-defective Lymphoid Cells

“…Lysenin is reported as an SM-direct cytolysin purified from the earthworm (27), for which binding to SM causes poring of the plasma membrane and subsequent cell death (22, 23, 25). …”

Intracerebellar Transplantation of Neural Stem Cells into Mice with Neurodegeneration Improves Neuronal Networks with Functional Synaptic Transmission

“…For sphingomyelin stain, we used lysenin which specifically binds to the sphingomyelin, anti-lysenin antiserum, Alexa Fluor 568 (diluted 1:1,000; Molecular Probes) were used [25,30]. For filipin stain, the sections were incubated with 50 g/ml filipin in 1% BSA in PBS for 2 hr at room temperature after blocking with 1% BSA in PBS for 1 hr.…”