| Sea anemones are rich sources of biologically active peptides and proteins, including neurotoxins, enzymes and cytolysins. Actinoporins are 20 kDa cytolytic proteins that interact with sphingomyelin in cellular and artificial membranes found in sea anemone. Sea anemone sting is a common biological injury. Therefore, it is necessary to isolate, purify and biologically characterized the sea anemone toxin from China's coastal. It can also provide a theoretical basis for effective protection and treatment of the sea anemone stings, and may provide the lead compounds for new drug research and development. This work is focused on the isolation, identification and biological characterization of cytolysin derived from the Sea anemones Stichodactyla gigantean from South China Sea. The main contents and results are as follows:1. Isolation and identification of Gigantoxin-4. Cation-exchange chromatography on Capto S column and Gel-filtration chromatography on Superdex 75 column were applied for the separation and two peaks with hemolytic activity was obtained. SDS-PAGE electrophoresis showed that this new actinoporin had a molecular mass about 19 kDa, and possessed a high hemolytic activity against human erythrocytes (HA50= 40 ng/mL).Its partial and N-terminal amino acid sequences of the purified peaks were both determined by Nano-LC MS/MS and Edman degradation. The result indicated that it was highly homologous to Cytolysin-3 (HMg III) from H. magnifica, RTX-A from Radianthus macrodactylus, and Sticholysin-1 (St I) and Sticholysin-2 (St II) from S. helianthus.Its isoelectric point was about 9.3. We named this protein as Gigantoxin-4.2. Physical and chemical factors on hemolytic activity of Gigantoxin-4. The concentration of Gigantoxin-4 that lysed 50% erythrocytes was 40 ng/mL. The temperature, pH, storage time, freezing and thawing times, the metal ions and other factors on the hemolytic activity of Gigantoxin-4 were also studied, the results showed that the Gigantoxin-4 had good stability. Mg2+ and K+ (5 mmol/L)could promote hemolysis of Gigantoxin-4, however, Ca2+, Zn2+, Mn2+, Ba2+, Cu2+ inhibited the hemolysis of Gigantoxin-4. We also found that the various components of membrane lipid showed different potential interactions with Gigantoxin-4. SUV composed of pure PC showed no significant effect on hemolytic activity. The hemolytic activity of Gigantoxin-4 was inhibited significantly by preincubation with SUV composed of SM/Cho or SM/Cho/PC, while no significant inhibitory effect of PC lipids on the hemolytic activity of Gigantoxin-4 was observed.3. The biological activity of Gigantoxin-4 in vitro and in vivo. Our work confirmed that Gigantoxin-4 induced leakage of calcein wrapped in LUV. No leakage of calcein was observed when LUV composed of pure PC was used. However, calcein leak was detectable when LUV composed of SM: Cho: PC was used, indicating that Gigantoxin-4 had a high affinity to LUV composed of SM and Cho. We also found that Gigantoxin-4 had a similar activity of sphingomyelinase by the hydrolysis of specific substrates, TNPAL-SM. This finding may explain the cytolytic activity and SM specificity of Gigantoxin-4, but the conclusion needs further experiments to confirm. MTT assay showed that the sea anemone cytolysin Gigantoxin-4 had cytotoxic effects on various tumor cells including MCF-7, SW-1990, etc (IC50 1.71, 1.32 ). Acute toxicity test showed LD50 of Gigantoxin-4 was about 73.6μg/kg by tail vein injection into mice, and the toxic effect was rapid and intense. Another important finding of the present study is that administration of a high concentration of Gigantoxin- eprimary factor causing death of the rat. Histological analysis also confirmed that the lung was the most seriously injured organ after Gigantoxin-4 administration. In comparison, rats can live tens of minutes to hours after low-dose Gigantoxin-4 injectioalthough final cardiovascular failure was inevitable. Biochemical analysis showed that plasma levels of liver and heart related enzymes were increased significantly, indicating that the toxic effect of Gigantoxin-4 was systemic.In conclusion, in the present study, we report the the isolation, purification and biological characterization of a new actinoporin toxin from the sea anemone Stichodactyla gigantean belonging to the actinoporin family. In addition, we also tried to clarify interactions between lipid vesicles and Gigantoxin-4. The toxic effect of Gigantoxin-4 in vivo was also studied by direct i.v. injection of Gigantoxin-4 in anaesthetized Sprague-Dawley rats. It probably will be, at least partly, valuable as a tool for researching protein membrane interactions and cardiovascular pharmacology.With the growing concern of marine resources, more sea anemone cytolysin will be found, and their structural and functional characteristics will be more in-depth studied. It helps to reasonable and adequate use of our abundant resources with extensive and in-depth study of sea anemone toxin. It is important for the development of new drugs and the treatment of sea anemone stings measures.
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