Previous studies show that membrane-damaging activity of cardiotoxins (CTXs) is associated with their binding with anionic phospholipids on membrane. In order to examine this proposition, studies on the interaction between Taiwan cobra CTXs and zwitterionic phospholipids were conducted in the present study. Although membrane-damaging activity of CTX2 and CTX4 was affected by phospholipid compositions, CTX2 and CTX4 were able to induce membrane permeability of zwitterionic phospholipid vesicles. CTX2 showed a higher membrane-damaging activity toward egg yolk phosphatidylcholine (EYPC), EYPC/egg yolk phosphatidylethanolamine (EYPE) or EYPC/egg yolk phosphatidylglycerol (EYPG) than CTX4 did. Moreover, CTX2- and CTX4-induced membrane permeability of EYPC and EYPC/EYPE vesicles was higher than that of EYPC/EYPG vesicles. CTX2 and CTX4 displayed different membrane-bound mode in binding with EYPC, EYPC/EYPE and EYPC/EYPG vesicles as revealed by N-( fluorescein - 5 - thiocarbamoyl) - 1, 2 - dihexadecanoyl-phosphatidyl ethanolamine fluorescence enhancement, color transformation of phospholipids/polydiacetylene membrane assay and rhodamine-self quenching assay. CTX2 and CTX4 showed a similar binding affinity with phospholipid vesicles, but the reversibility in binding with phospholipid vesicles differed between CTX2 and CTX4. Compared with that of CTX4, membrane-damaging activity of CTX2 was markedly inhibited by anti-CTX antibodies. Taken together with the finding that gross conformation of CTX2 and CTX4 differs, our data suggest that membrane-damaging activity of CTX2 and CTX4 critically depends on their membrane-bound mode and is affected by phospholipid composition. Moreover, the ability of CTX to induce membrane permeability is not exclusively attributed to the binding with anionic phospholipids.

Naja naja atra Cardiotoxins (CTXs), basic polypeptides of 60 amino acid residues adopt a three-fingered loop-folding topology and show cytotoxicity for human tissues in targeting cell membrane. Despite having highly similar sequence, the six CTX isoforms also display different cytotoxic potencies and hemolytic activities. The goal of these studies is to explore the mechanical processes that involved in membrane-damaging activities of CTXs on vesicles composed of different cell membrane components, and to delineate the events that lead to different biological activities of CTXs. The studies were performed by estimating the color transformation of phospholipid/polydiacetylene vesicles and the fluorescence enhancement of fluorescein-labeled phospholipid/protein or fluorescein released from vesicles. It was found that vesicles consisted of unsaturated phospholipids improve membrane-damaging activity of CTXs and adopt a vital membrane-bound conformation of CTXs. In contract, the characteristic of vesicles consisted of saturated phospholipids was against CTXs adopting an essential membrane-damaging structure. It was also found that not only electrostatic force but also hydrophobic force were involved in the interaction between CTXs and membrane. Comparing with phosphatidylcholine-only vesicles, CTXs displayed higher membrane-damaging activity for the sphingomyelin-containing vesicles, and the loop2 region of CTXs play a crucial role for the membrane-damaging activity of sphingomyelin-containing vesicles. Besides, the CTX3 and CTX5 would interact with the H-antigen of blood group O red blood cells, but only the binding of CTX3 with H-antigen reduce its membrane-damaging activity for red blood cells membrane. Moreover, the fusogenicity of CTXs is responsible for the membrane-damaging activity of CTXs toward bacterial membrane-mimicking vesicles. The cardiolipin have the potency to improve the fusogenicity of CTX3, which induced the bactericidal activity toward the cardiolipin-containing bacterium.