Isolation, Characterization Of Sea Anemone Toxins From Anthopleura Xanthogrammica In Zhoushan

Anthopleura xanthogrammica is a small sea anemone found abundantly in zhoushanregion and along the east coast line of China. They have many tentacles that incorporateand eliminate substances for metabolism and that contain venom-producing nematocystsallowing them to prey on small crustaceans and defend themselves against predators.These tentacles are well-known sources for the isolation of toxic proteins. Here severalpeptide toxins exhibiting molecular weight ranging from2-5KDa was isolated from thesea anemone AnthopLeura xanthogrammica. Purification of the peptide was achieved bya multistep chromatographic procedure monitoring its strong paralytic activity onridgetailwhite prawn (Exopalaemon carinicauda (Holthuis)), mealworms (Tenebrio molitor) andmouse (Mus musculus).The paralitic or lethalic effect of toxins on ridgetail white prawnand mealworm are very similar, also are more active on ridgetail white prawn andmealworms than mouse. Toxicities against prawn are about200μg/kg. Given the closeevolutionary relationship between crustaceans and insects, these peptides can beconsidered as insecticidal lead compounds in the development of insecticides. It also laidthe foundation for purifying more insecticidal peptide from A. xanthogrammica andstudying its mechanism.To understand the molecular composition and characteristics of sea anomene toxins ofA. xanthogrammica affecting on Na+channel. An excitatory toxin, named AX-1waspurified from A. xanthogrammica by acetone precipitation-high performance liquidchromatography (HPLC). The toxin molecule consists of48amino acids, molecularweight of5018.2Da, with three disulfide bonds. Three-dimensional structure modelingindicates that the structure of AX-1is mainly anti-parallel β-sheets and loops; patchclamp studies have shown that the toxin can inhibit sodium channel inactivation andsignificantly increased sodium channel current in rat dorsal root ganglion cells. Theresults show that AX-1is an excitatory peptide toxin, a potential cardiac peptide drug.In addition, to investigate possible mechanisms of tumor cell apoptosis induced by seaanemone toxin from A. xanthogrammica, toxic fragment with relative molecular massgreater than10000Da from A. xanthogrammica was got. Cytotoxicity assay shows thatthe toxic fragment can inhibit the proliferation of human lung cancer cell line A549, andsmall cell lung cancer H446, and human umbilical vein cell, and their median lethalconcentration are53.24μg/mL at,15.19μg/mL, and69.71μg/mL. This study lays thefoundation for the next step purification of the anti-tumor sea anemone toxins.In summary, by using cell biology, biochemistry, mass spectra etc, we haveinvestigated the insecticidal, antitumor functions of A. xanthogrammica, and its toxicity on Na+channel. These studies laid the foundation for screening and purifying moreinsecticidal and antitumor toxins.