Origin of tetrodotoxin and toxification mechanism in tetrodotoxin-bearing animals

泰国人经常食用马蹄蟹，因误食马蹄蟹，尤其是马蹄蟹卵而食物中毒的事件时有发生。马蹄蟹是一种原始节肢动物，目前世界上仅存3属4种。rotundicauda、Tachypleus gigas和tridentatus是东盟马蹄蟹，Limulus polyphemus是美洲马蹄蟹。前两种生活在泰国。致病物种C.rotundicauda在所有的病例报道。负责毒素主要由河豚毒素（TTX）及其相关物质组成，以及作为次要成分的麻痹性贝类毒素（PSP）。螃蟹中毒的机制尚不清楚。因此，我们研究了螃蟹对TTX和PSP的耐受性，因为对毒素具有高耐受性的动物可能含有毒素或具有积累毒素的能力。对于TTX， T.gigas和L.polyphemus表现出几乎相同的相当高的耐受性，最小致死剂量（MLD）为50 ~ 150 MU/20g体重。而圆孔菌则表现出非常高的抗性，大约是其他菌的40倍。对PSP的耐受性，圆尾草和水蛭表现出相当高的耐受性，MLD均在100 ~ 200 MU/20g体重。通过dbab -纤维素处理、硫酸铵分离、Sephadex凝胶过滤、sepphacryl S-200和Cellulofine A-500柱层析，从川河豚（Takifugu niphobles）的血浆中纯化出河豚毒素结合蛋白。最后在BK 0-3000 SL柱上进行I-IPLC纯化，得到的蛋白只有一个蛋白峰。通过SDS-PAGE和质谱分析，估计该蛋白的分子量分别为116000和96000。纯化蛋白的氨基末端氨基酸序列为Ala-Pro-Ser-Pro-？- - - - - - ?- - - - - - ?他- ?-Leu-The-Lys-Rro-Val -。

Horseshoe crabs are often eaten by the people in Thailand, and food poisonings by ingesting them, especially their eggs have sporadically occurred. Horseshoe crabs are primitive arthropods, and only three genera and four species are surviving now in the world. Carcinoscorpius rotundicauda, Tachypleus gigas and T.tridentatus are Asean horseshoe crabs and Limulus polyphemus is an American species. The former two species inhabit in Thailand. The causative species was C.rotundicauda in all the cases reported. The responsible toxin is essentially composed of tetrodotoxin (TTX) and related substances, along with paralytic shellfish toxins (PSP) as minor components. The mechanism of intoxication of the crabs is unhnown. Therefore we examined the torelance of the crabs against TTX as well as PSP, because there is a tendency that an animal with high tolerance to toxins may contain the toxins or have the capability to accumulate them. As to TTX, T.gigas and L.polyphemus exibited almost the same considerable high tolerance, with the minimum lethal dose (MLD) of 50-150 MU/20g of body weight. While C.rotundicauda showed a very high resistance that was about 40 times stronger than the others. As to PSP, C.rotundicauda and L.polyphemus exibited almost the same considerable high tolerance, with MLD of 100-200 MU/20g of body weight.A tetrodotoxin binding protein has been purified from the plasma of the puffer fish kusafugu, Takifugu niphobles, through DBAB-cellulose treatment, ammonium sulfate fractionation, Sephadex gel filtrations, and Sephacryl S-200 and Cellulofine A-500 column chromatography. Final purification by I-IPLC on a BK 0-3000 SL column yielded a protein which showed only a single protein peak. The molecular weight of the protein was estimated to be 116,000 and 96,000 by SDS-PAGE and mass spectrometry, respectively. The amino-terminal amino acid sequence of the purified protein was determined to be Ala-Pro-Ser-Pro-? -? -? -His-? -Leu-The-Lys-Rro-Val-.