INTERACTION OF EARTHWORM HEMOLYSIN WITH LIPID-MEMBRANES REQUIRES SPHINGOLIPIDS

Lytic activity in the coelomic fluid of earthworm (Eisenia fetida feti da) has been ascribed to eiseniapore, a hemolytic protein of 38 kDa. S ince receptors for eiseniapore on target cell membranes are not known, we used lipid vesicles of various composition to determine whether sp ecific lipids may serve as receptors, Lytic activity of eiseniapore wa s probed by the relief of fluorescence dequenching from the fluorophor e 8-aminonaphthalene-1,3,6-trisulfonic acid originally incorporated in to the vesicle lumen as a complex with p-xylene-bis-pyridinium bromide , Hemolysin binds to and disturbs the lipid bilayer only when distinct sphingolipids consisting of a hydrophilic head group as phosphorylcho line or galactosyl as well as the ceramide backbone, e,g. sphingomyeli n, are present. Cholesterol enhances eiseniapore lytic activity toward sphingomyelin-containing vesicles probably due to interaction with sp hingomyelin. Leakage of vesicles was most efficient when the lipid com position resembled that of the outer leaflet of human erythrocytes. Pr esumably, an oligomeric protein pore formed by six monomers is respons ible for leakage of sphingomyelin-containing vesicles. The secondary s tructure of eiseniapore did not change upon binding to lipid membranes . The lytic activity of eiseniapore was completely abolished after its denaturation or after preincubation with polyclonal antibodies. Our r esults suggest that the presence of specific sphingolipids is sufficie nt to mediate lytic activity of eiseniapore, This action contributes t o our understanding of earthworm immune responses.