Differential Effect of Membrane Composition on the Pore-Forming Ability of Four Different Sea Anemone Actinoporins

Abstract

Sea anemone actinoporins constitute a protein family of multigene pore-forming toxins (PFT). Equinatoxin II (EqtII), fragaceatoxin C (FraC), and sticholysins I and II (StnI and StnII, respectively), produced by three different sea anemone species, are the only actinoporins whose molecular structures have been studied in depth. These four proteins show high sequence identities and practically coincident three-dimensional structures. However, their pore-forming activity can be quite different depending on the model lipid system employed, a feature that has not been systematically studied before. Therefore, the aim of this work was to evaluate and compare the influence of several distinct membrane conditions on their particular pore-forming behavior. Using a complex model membrane system, such as sheep erythrocytes, StnII showed hemolytic activity much higher than those of the other three actinoporins studied. In lipid model systems, pore-forming ability when assayed against 4:1 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/sphingomyelin (SM) vesicles, with the membrane binding being the rate-limiting step, decreased in the following order: StnI > StnII > EqtII > FraC. When using 1:1:1 DOPC/SM/cholesterol LUVs, the presence of Chol not only enhanced membrane binding affinities by ∼2 orders of magnitude but also revealed how StnII was much faster than the other three actinoporins in producing calcein release. This ability agrees with the proposal that explains this behavior in terms of their high sequence variability along their first 30 N-terminal residues. The influence of interfacial hydrogen bonding in SM- or dihydro-SM-containing bilayers was also shown to be a generalized feature of the four actinoporins studied. It is finally hypothesized that this observed variable ability could be explained as a consequence of their distinct specificities and/or membrane binding affinities. Eventually, this behavior can be modulated by the nature of their natural target membranes or the interaction with not yet characterized isotoxin forms from the same sea anemone species.

Stichodactyla helianthus' de novo transcriptome assembly: Discovery of a new actinoporin isoform

“…Overall, this distribution would be consistent with the fact that actinoporins are not enzymes and therefore they do not show catalytic effects as phospholipases and metalloproteases do. StnII is the most potent actinoporin known from cytolytic assays against red blood cells (Garcia-Linares et al, 2016a). Our present results show it is also the most highly expressed Stn (Table 3) in S. helianthus, potentially explaining the potency of its venom in causing cell lysis.…”

“…This sequence was almost complete in both cases and only lacked a small fragment at the C-terminal edge. However, this missing fragment occurs in a sequence stretch which is also conserved across all the most-well studied actinoporins (Garcia-Linares et al, 2016a).…”

“…2). This feature precluded its complete analysis given that major differences in these proteins primarily accumulate at the Nterminal end (Garcia-Linares et al, 2016a;Ros et al, 2015). However, the sequences recovered as StnIV in the two assemblies were not identical but differed at some positions as well as to StnI and II.…”

“…Stichodactyla helianthus is a Caribbean sea anemone which produces large amounts of two easily detectable actinoporin variants: sticholysin I (StnI) and sticholysin II (StnII) (Lanio et al, 2001). StnII is the most potent actinoporin of those that have been studied with molecular detail (Garcia-Linares et al, 2016a). StnI is approximately four times less active than StnII in terms of hemolytic activity (Monastyrnaya et al, 2010;Garcia-Linares et al, 2016a).…”

“…StnII is the most potent actinoporin of those that have been studied with molecular detail (Garcia-Linares et al, 2016a). StnI is approximately four times less active than StnII in terms of hemolytic activity (Monastyrnaya et al, 2010;Garcia-Linares et al, 2016a). Both isoforms interact with each other to produce a synergistic effect; just trace amount of StnII enhances StnI binding to lipid model vesicles driving a dramatic improvement of hemolytic activity (Rivera- de-Torre et al, 2016).…”