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Purification and Characterization of Gigantoxin-4, a New Actinoporin from the Sea Anemone Stichodactyla Gigantea

2011; Ivyspring International Publisher; Volume: 7; Issue: 6 Linguagem: Inglês

10.7150/ijbs.7.729

1449-2288

Bo Hu, Wei Guo, Lianghua Wang, Jianguang Wang, Xiaoyu Liu, Binghua Jiao,

Marine Toxins and Detection Methods

A new Cytolysin, termed as Gigantoxin-4, was isolated from the sea anemone Stichodactyla gigantea and found to be highly homologous with Cytolysin-3 (HMg III) from Heteractis magnifica, RTX-A from Radianthus macrodactylus, and Sticholysin-1 (St I) and Sticholysin-2 (St II) from Stichodactyla helianthus (homology 82%, 86%, 82% and 86% respectively). Its 20 N-terminal residues were identified and the full-length cDNA sequence was obtained by reverse transcription-polymerase chain reaction (RT-PCR). Multiple sequence alignments with other Cytolysins of the actinoporin family clearly indicated that Gigantoxin-4 belongs to this protein family. SDS-PAGE electrophoresis showed that this new actinoporin had a molecular mass of about 19 kDa, and possessed a high hemolytic activity to human erythrocytes (HA(50)= 40 ng/ml), which was inhibited by pre-incubation with sphingomyelin (SM) or SM-cholesterol mixtures. Our in vivo experiments showed that Gigantoxin-4 had wide toxicity to the rat cardiovascular system and the respiratory system. A concentration of 30 μg/kg Gigantoxin-4, i.v. produced a positive inotropic effect on the rat heart although final cardiovascular failure was inevitable, and 60 μg/kg Gigantoxin-4 caused respiratory arrest rapidly resulting in rat death. HE staining indicated pathological changes in various organs and tissues after i.v. administration of Gigantoxin-4.