Origin of tetrodotoxin and toxification mechanism in tetrodotoxin-bearing animals

在泰国，人们经常食用马蹄蟹，摄入马蹄蟹引起的食物中毒事件时有发生，尤其是马蹄蟹的卵。马蹄蟹是原始节肢动物，目前世界上仅存3属4种。圆尾蟹(Carcinoscorpius Rotundicauda)、长尾河蟹(Tachypleus Gias)和三齿蟹(T.tridentatus)是东盟的一种马蹄蟹，多刺河蟹是一种美洲种。前两个物种生活在泰国。在所有报告的病例中，致病物种均为圆形尾巴线虫。负责的毒素主要由河豚毒素(TTX)和相关物质组成，以及麻痹性贝类毒素(PSP)作为次要成分。螃蟹中毒的机理尚不清楚。因此，我们研究了螃蟹对TTX和PSP的抵抗力，因为有一种趋势是，对毒素耐受性高的动物可能含有毒素或有能力积累毒素。对河豚毒素的耐受性较高，其最小致死剂量(MLD)为50-150MU/20g体重。而圆尾鱼表现出很高的抵抗力，大约是其他品种的40倍。对于PSP，圆尾河豚和多头河豚几乎都具有相当高的耐受性，MLD为100-200MU/20g体重。河豚血浆经DBAB-纤维素处理、硫酸铵分级、Sephadex凝胶过滤、Sephacryl S-200和Cellulofine A-500柱层析，从河豚血浆中纯化出一种河豚毒素结合蛋白。经I-IPLC在BK 0-3000SL柱上纯化，得到一种只有一个蛋白质峰的蛋白质。经SDS-PAGE和MS分析，该蛋白的相对分子质量分别为116,000和96,000。纯化蛋白的氨基端氨基酸序列为Ala-Pro-Ser-Pro-？-？-His-？-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-.