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.

Background: Sphingomyelin synthase (SMS) catalyzes the synthesis of sphingomyelin. Results: Sphingomyelin-deficient cells failed to proliferate in response to transferrin. Transfection of SMS1 enabled these cells to generate sphingomyelin, promoting clathrin-dependent uptake of transferrin and its dependent proliferation. Conclusion: SMS1 is indispensable for transferrin internalization and cell proliferation. Significance: Our findings provide new insights into the role of SMS1 in transferrin biology.

Sphingomyelin plays complex structural and signaling functions in the plasma membrane. Of special interest is that hydrolysis of sphingomyelin to ceramide can modulate dynamics of membrane rafts, which serve as signaling platforms for various receptors. This review is focused on a recently discovered sphingomyelin-binding protein, lysenin, which can be used as a unique probe to trace distribution and turnover of sphingomyelin in cellular membranes. We analyze the primary and secondary structures of lysenin with respect to its interaction with the plasma membrane. The speci¢city of lysenin binding to sphingomyelin, revealed by both biochemical and cytochemical approaches, is discussed.

Despite the role of sphingolipid/cholesterol rafts as signaling platforms for Fc␥ receptor II (Fc␥RII), the mechanism governing translocation of an activated receptor toward the rafts is unknown. We show that at the onset of Fc␥RII cross-linking acid sphingomyelinase is rapidly activated. This enzyme is extruded from intracellular compartments to the cell surface, and concomitantly, exofacially oriented ceramide is produced. Both non-raft and, to a lesser extent, raft sphingomyelin pools were hydrolyzed at the onset of Fc␥RII cross-linking. The time course of ceramide production preceded the recruitment of Fc␥RII to rafts and the receptor phosphorylation. Exogenous C 16 -ceramide facilitated clustering of Fc␥RII and its association with rafts. In contrast, inhibition of acid sphingomyelinase diminished both the ceramide generation and clustering of crosslinked Fc␥RII. Under these conditions, tyrosine phosphorylation of Fc␥RII and receptor-accompanying proteins was also reduced. All the inhibitory effects were bypassed by treatment of cells with exogenous ceramide. These data provide evidence that the generation of cell surface ceramide is a prerequisite for fusion of cross-linked Fc␥RII and rafts, which triggers the receptor tyrosine phosphorylation and signaling.

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We studied an involvement of various cellular ceramide pools in signaling of immunoreceptor FcγII (FcγRII). The cell surface ceramide level was assessed by a technique based on binding of ceramide probes to intact cells. Total cellular ceramide was estimated by radioactive measurements. The activity of sphingomyelinases was measured by NBD-ceramide release while immunoprecipitation and immunoblotting were applied to analyze protein tyrosine phosphorylation. A complex pattern of protein phosphorylation was found to accompany FcγRII activation and the phosphorylation was either diminished by imipramine or increased by B13, modulators of acid sphingomyelinase and acid ceramidase activity. The effects of the drugs on the phosphorylation of FcγRII and NTAL were prominent and correlated with a reduction of the cell surface ceramide production by imipramine and an augmentation of the ceramide generation by B13. The ceramide generation followed activation of acid sphingomyelinase and preceded that of neutral sphingomyelinase. The level of cell surface ceramide was additionally elevated by exogenous bacterial sphingomyelinase, but only at later stages of the receptor activation. The total mass of ceramide was diminished in the course of receptor activation pointing to an engagement of enzymes metabolizing ceramide. The data indicate that FcγRII activates enzymes of the sphingomyelin cycle which affect various sphingomyelin/ceramide pools in a cell.

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