Interaction of Earthworm Hemolysin with Lipid Membranes Requires Sphingolipids

Abstract

Lytic activity in the coelomic fluid of earthworm (Eisenia fetida fetida) has been ascribed to eiseniapore, a hemolytic protein of 38 kDa. Since receptors for eiseniapore on target cell membranes are not known, we used lipid vesicles of various composition to determine whether specific lipids may serve as receptors. Lytic activity of eiseniapore was probed by the relief of fluorescence dequenching from the fluorophore 8-aminonaphthalene-1,3,6-trisulfonic acid originally incorporated into the vesicle lumen as a complex with p-xylenebis-pyridinium bromide. Hemolysin binds to and disturbs the lipid bilayer only when distinct sphingolipids consisting of a hydrophilic head group as phosphorylcholine or galactosyl as well as the ceramide backbone, e.g. sphingomyelin, are present. Cholesterol enhances eiseniapore lytic activity toward sphingomyelin-containing vesicles probably due to interaction with sphingomyelin. Leakage of vesicles was most efficient when the lipid composition resembled that of the outer leaflet of human erythrocytes. Presumably, an oligomeric protein pore formed by six monomers is responsible for leakage of sphingomyelin-containing vesicles. The secondary structure 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 results suggest that the presence of specific sphingolipids is sufficient to mediate lytic activity of eiseniapore. This action contributes to our understanding of earthworm immune responses.

Kinetic Analysis of Binding between Shiga Toxin and Receptor Glycolipid Gb3Cer by Surface Plasmon Resonance

“…Consistently, it has been reported that antibody binding to antigens on liposomes (34) and the interaction of hemolysin with the lipid membrane (35) are enhanced in the presence of cholesterol.…”

Mammalian Cell Mutants Resistant to a Sphingomyelin-directed Cytolysin

“…Another research group has recently found that a cytolysin (named eiseniapore) purified from the earthworm E. foetida recognizes SM (24). Interestingly, eiseniapore binds to both SM and galactosylceramide (24), whereas lysenin shows no significant crossreaction with galactosylceramide (10), and the specific product of the lysenin cDNA binds specifically to SM, 2 suggesting that eiseniapore is a distinct molecule from lysenin. Eiseniapore may be a lysenin-like protein with a broader substrate specificity than lysenin, although confirmation of this possibility should await cDNA cloning of eiseniapore.…”

Redefining cholesterol's role in the mechanism of the cholesterol-dependent cytolysins

“…Linder and Bernheimer (32) observed nearly two decades ago that the cytolytic activity of several pore-forming toxins was decreased by the depletion of membrane cholesterol. Since that time, the presence of membrane cholesterol has been found to enhance pore formation for the sea anenome-derived pore-forming toxin sticholysin II (33), Staphylococcus aureus alpha hemolysin (34), aerolysin (35), the earthworm-derived hemolysin eiseniapore (36), the Escherichia coli hemolysin ClyA (SheA) (37), and the Vibrio cholerae hemolysin (38). One explanation for the common enhancement of pore-formation is that cholesterol may interact with these toxins in such a way as to promote insertion of their transmembrane domains and͞or by the promotion of certain membrane structures (inverted hexagonal phases) (39) that may facilitate the insertion of the transmembrane domains.…”