Immunocytochemical study of lysenin, a sphingomyelin-specific binding protein, localized on erythrocyte membrane

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Lysenin is a sphingomyelin-specific binding protein purified from coelomic fluid of the earthworm Eisenia foetida. Although the lysenin is known to induce erythrocyte lysis, its molecular mechanism for lysis is still unknown. In the present study, we examined ultrastructural organization of lysenin reacted with liposome and sheep or human erythrocytes. The liposome and erythrocytes were incubated with lysenin. After fixation, the specimens were negatively stained with aqueous uranyl acetate. For an immunocytochemcal study, MBP (maltose-binding protein)-tagged lysenin was incubated with erythrocytes, fixed, and immunostained with anti-MBP antibody followed by colloidal gold-conjugated secondary antibody. The immunostained specimens were negatively stained as described above. Lysenin formed uniform hexagonal structures on sphingomyelin-containing liposome. On the sheep or human erythrocytes, it formed sheet-like or ring-like aggregates. The immunogold labeling revealed that MBP-lysenin was accumulated on such aggregates. These results suggested that, after binding with sphingomyelin, lysenin formed hexagonal pore complexes and induce the erythrocyte lysis. Sphingomyelin is a major component of lipid rafts. So the lysenin will make a very useful tool to identify or modify the raft structures.
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