Biochemical characteristics of Eiseniapore, a pore-forming protein in the coelomic fluid of earthworms

The cytolytic protein Eiseniapore (38 kDa) from coelomic fluid of the earth worm Eisenia fetida functionally requires sphingomyelin as revealed by usin g mammalian erythrocytes and phospholipid vesicles. The effects of ions, gl ycoproteins and phospholipids were investigated for the two-step Eiseniapor e action mode, binding and pore formation in different assays. Eiseniapore lysis is activated by thiol groups but inhibited by metal ions. Eiseniapore binding to target membranes is inhibited by Eiseniapon-regulating factor, vitronectin, heparin and lysophosphatidylcholine. Ca2+ and Mg2+ were found to be not necessary for membrane binding or lytic activity. Sphingomyelin w as essential for Eiseniapore-induced leakage of liposomes. We describe a cy tolytic protein/toxin in Eiseniapore which differs from the established cla ssification; it can be activated by thiol groups and is inhibited by sphing omyelin. Electron microscopy of erythrocyte membranes confirmed ring-shaped structures (pores) with a central channel with outer (10 nm) and inner (3 nm) diameters as shown previously [Lange, S., Nussler, F., Kauschke, E., Lu tsch, G., Cooper, E.L. & Hemnann, A. (1997) J. Biol. Chem 272, 20 884-20 89 2] using artificial membranes. Functional evidence of pore formation by Eis eniapore was revealed as protection of lysis by carbohydrates occurred at a n effective diameter above 3 nm. From these results, we suggest a plausible explanation for the mechanism by which components of the earthworm's immun e system destroy non-self components.