Synergistic Action of Actinoporin Isoforms from the Same Sea Anemone Species Assembled into Functionally Active Heteropores

Abstract

Among the toxic polypeptides secreted in the venom of sea anemones, actinoporins are the pore-forming toxins whose toxic activity relies on the formation of oligomeric pores within biological membranes. Intriguingly, actinoporins appear as multigene families that give rise to many protein isoforms in the same individual displaying high sequence identities but large functional differences. However, the evolutionary advantage of producing such similar isotoxins is not fully understood. Here, using sticholysins I and II (StnI and StnII) from the sea anemone Stichodactyla helianthus, it is shown that actinoporin isoforms can potentiate each other's activity. Through hemolysis and calcein releasing assays, it is revealed that mixtures of StnI and StnII are more lytic than equivalent preparations of the corresponding isolated isoforms. It is then proposed that this synergy is due to the assembly of heteropores because (i) StnI and StnII can be chemically cross-linked at the membrane and (ii) the affinity of sticholysin mixtures for the membrane is increased with respect to any of them acting in isolation, as revealed by isothermal titration calorimetry experiments. These results help us understand the multigene nature of actinoporins and may be extended to other families of toxins that require oligomerization to exert toxicity.

Keywords: equinatoxin; erythrocyte; ion channel; lipid-protein interaction; lysis; oligomerization; pore-forming-toxin; protein cross-linking; sticholysin; toxin.

FIGURE 1.

Left panel, maximum hemolytic rate values (expressed as percentage of hemolysis/s) are represented versus the logarithm of total protein concentration: StnI (white dots), StnII (black dots), and the StnI/StnII (80:20) mixture (black squares). The white squares line was obtained as the arithmetical addition of the rates obtained with the individual proteins for the real concentration of each one in the different mixtures employed. Results shown are the average of four independently performed experiments. Each of these experiments was made in duplicate. Error bars represent ±S.D. Right panel, as a representative example, the hemolytic activity curves of StnI (white dots), StnII (black dots), or a StnI/StnII (80:20) mixture (black squares), at a total protein concentration of 2 nm, are also shown.

FIGURE 2.

Maximum hemolytic rate values (expressed as percentage of hemolysis/s) are represented versus the logarithm of total protein concentration of individual and two different actinoporin mixtures: wild-type StnI and A10PS28P (A) or Y111N (B) StnII mutants. Both panels show the behavior of StnI (white dots), the StnII mutant (black dots), and the StnI/StnII mutant (80:20) mixture (black squares). The white squares line was obtained as the arithmetical addition of the rates obtained with the individual proteins for the real concentration of each one in the different mixtures employed. Results shown are the average of four independently performed experiments. Each of these experiments was made in duplicate. Error bars represent ±S.D.

FIGURE 3.

Left panel, calcein release of maximal rates (expressed as normalized fluorescence intensity increment/s) are represented versus the total protein concentration: StnI (white dots), StnII (black dots), and the StnI/StnII (80:20) mixture (black squares). The white squares line was obtained as the arithmetical addition of the rates obtained with the individual proteins taking into account the real concentration of each one of them in the different mixtures employed. Results shown are the average of three independently performed experiments. Each of these experiments was made in duplicate. Error bars represent ± S.D. Right panel, as a representative example, the calcein leakage traces of StnI (white dots), StnII (black dots), or an StnI/StnII (80:20) mixture (black squares), at a total protein concentration of 5 nm, are also shown.

FIGURE 4.

Immunoblotting detection of 6HStnII previously incubated in the presence, or not, of wild-type StnI, DOPC/SM/Chol (1:1:1) phospholipid vesicles, and/or DSS, as indicated. Proteins were detected using a mouse monoclonal anti-polyhistidine-peroxidase antibody. The amount of 6HStnII loaded was 2.5 pmol. The StnI:6HStnII molar ratio employed was 80:20 in all instances shown. Molecular weight standards (EZ-RUN^TM pre-stained Rec Protein Ladder) were also loaded, and the corresponding molecular masses are indicated in kDa at the left margin.

FIGURE 5.

Coomassie Blue-stained gel (upper panel) and the corresponding immunoblotting detection (lower panel) of 6HStnII titrated with increasing amounts of StnI are shown. The proteins, and also the mixtures assayed, were incubated in the presence, or not, of wild-type StnI, DOPC/SM/Chol (1:1:1) phospholipid vesicles, and/or DSS, as indicated. Proteins were detected using a mouse monoclonal anti-polyhistidine-peroxidase antibody. The amount of 6HStnII loaded was 2.5 pmol in all instances shown. The StnI/6HStnII molar ratio employed is also indicated. Molecular weight standards (EZ-RUN^TM pre-stained Rec Protein Ladder) were also loaded, and the corresponding molecular masses are indicated in kDa at the left margin.

FIGURE 6.

Binding of StnI and StnII to DOPC/SM/Chol (1:1:1) vesicles studied by ITC. Reactant concentrations were those ones shown in Table 1. Binding isotherms were adjusted to a model in which protein membrane binding involves the participation of “n” lipid molecules (30). The c values (c = K_a × P₀) for the graphs shown are within the range 1–1000.

FIGURE 7.

Binding of StnI, StnII, and different StnI/StnII mixtures (molar ratios as indicated) to DOPC/SM/Chol (1:1:1) vesicles studied by ITC. Reactant concentrations for the examples shown are P₀ = 1.5 μm and L₀ = 0.85 mm for all experiments, where P₀ refers to the initial total protein concentration within the calorimeter cell and L₀ is the lipid concentration within the dispensing auto-pipette. Binding isotherms were adjusted to a model in which protein membrane binding involves the participation of “n” lipid molecules (30). The c values (c = K_a × P₀) for the graphs shown were in the 1–1000 range only for the StnI/StnII (0:100), StnI/StnII (99:1), and StnI/StnII (100:0). In the other three thermograms shown the binding affinities were so high that keeping the c values within range involved dilutions below the recommended detection limits of the instrument.

FIGURE 8.

Left panel, maximum hemolytic rate values (expressed as percentage of hemolysis/s) are represented versus the logarithm of total protein concentration of StnI (white dots) and an StnI/StnII (99:1) mixture (black squares). In these experiments the amount of StnII present in all the mixtures was so low that, when assayed in the absence of StnI, its hemolytic activity was undetectable in the time range measured. Results shown are the average of four independently performed experiments. Each of these experiments was made in duplicate. Error bars represent ±S.D. Right panel, as a representative example, the hemolytic activity curves of StnI at 3.96 nm (white dots), StnII at 0.04 nm (black dots), and the corresponding StnI/StnII (99:1) 4 nm mixture (black squares) are also shown.