Lysenin-His, a sphingomyelin-recognizing toxin, requires tryptophan 20 for cation-selective channel assembly but not for membrane binding

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.

Purinergic control of lysenin’s transport and voltage-gating properties

“…Lysenin channels exhibit asymmetrical voltage-induced gating at transmembrane voltages within physiological range [42,43,54]. Although this is an ubiquitous feature of voltage-gated ion channels [59], it is not common among PFTs.…”

“…To answer this question, we focused our attention on lysenin, a 297 amino acid PFT extracted from Eisenia foetida, which inserts hexameric pores (∼3 nm diameter) in artificial and natural lipid membranes containing sphingomyelin (SM) [41][42][43][44][45]. Several remarkable features make lysenin an excellent candidate for such studies.…”

“…Several remarkable features make lysenin an excellent candidate for such studies. Lysenin's cytolytic and hemolytic activity has been extensively studied [45,46], and its capability to tamper with the barrier function of artificial lipid bilayers is well-documented [42,43,47,48]. The complete structure of the oligomeric pore inserted into membranes is not yet resolved; however, relatively recent structural data of lysenin interacting with SM in a pre-pore state indicates the existence of a positively charged domain [49] which may promote specific electrostatic interactions with negatively charged adenosine phosphates, similar to the ionotropic P2X receptors [19,50,51].…”

“…The complete structure of the oligomeric pore inserted into membranes is not yet resolved; however, relatively recent structural data of lysenin interacting with SM in a pre-pore state indicates the existence of a positively charged domain [49] which may promote specific electrostatic interactions with negatively charged adenosine phosphates, similar to the ionotropic P2X receptors [19,50,51]. In addition, unlike many other PFTs, lysenin is endowed with regulatory mechanisms by voltage and ligands [41][42][43][52][53][54], which are in fact remarkable features of ion channels. Lysenin channels present voltage regulation manifested as voltage-induced gating at positive transmembrane voltages greater than ∼20 mV [41,54].…”

Controlled Gating of Lysenin Pores

“…The lytic action relies on formation of large conductance channels (water permeable nanopores) in the cell membrane, dramatically disturbing the ion balance and causing cell death [4]. Upon binding to sphingomyelin, lysenin forms hexagonal oligomers in the lipid bilayer that define a large pore (3 nm diameter) in the center of the complex [5,7,9]. Electrophysiological studies have shown that the conductance of lysenin pores in a sphingomyelin-enriched lipid bilayer membrane significantly decreases at positive voltages due to gating [9,10].…”