Abstract
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.