Origin of tetrodotoxin and toxification mechanism in tetrodotoxin-bearing animals

河豚毒素的来源及河豚毒素动物的中毒机制

• 批准号: 09460090
• 负责人: YAMAMORI Kunio
• 金额: $ 4.74万
• 依托单位: Kitasato University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09460090/
• 关键词: tetrodotoxin; saxitoxin; horseshoe crab; tolerance to TTX; tolerance to STX; puffer fish; tetrodotoxin binding protein; TTX結合タンパク質; フグ; 淡水フグ; 蓄積

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-.

泰国民众经常食用鲎，因食用鲎尤其是鲎蛋而引起的食物中毒事件时有发生。鲎是原始节肢动物，目前世界上仅存3属4种。 Carcinoscorpius rotundicauda、Tachypleus gigas 和 T.tridentatus 是东盟鲎，Limulus polyphemus 是美洲物种。前两种栖息在泰国。在所有报告的病例中，致病物种都是圆尾 C.rotundicauda。造成这种毒素的主要成分是河豚毒素（TTX）和相关物质，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，我们检查了螃蟹对TTX和PSP的耐受性，因为对毒素具有高耐受性的动物有可能含有毒素或有能力积累毒素。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50-150MU/20g体重。而C.rotundicauda表现出非常高的抵抗力，比其他的强大约40倍。对于PSP，C.rotundicauda和L.polyphemus表现出几乎相同的高耐受性，MLD为100-200 MU/20g体重。通过DBAB-纤维素处理、硫酸铵分级、Sephadex凝胶从河豚鱼、Takifugu niphobles的血浆中纯化出河豚毒素结合蛋白。 过滤、Sephacryl S-200 和 Cellulofine A-500 柱色谱法。在 BK 0-3000 SL 柱上通过 I-IPLC 最终纯化产生仅显示单个蛋白质峰的蛋白质。通过SDS-PAGE和质谱估计该蛋白质的分子量分别为116,000和96,000。纯化蛋白的氨基末端氨基酸序列确定为Ala-Pro-Ser-Pro-α。 -？ -？ -他的-？ -Leu-The-Lys-Rro-Val-。

项目成果

T.Matsui, K.Yamamori et al.: "Purification and some properties of a tetrodotoxin binding protein from the blood plasma of kusafugu, Takifugu niphobles." Toxicon. (in press). (1999)

T.Matsui、K.Yamamori 等人：“从草东方鲀、泷东方鲀血浆中纯化河豚毒素结合蛋白及其一些特性。”

T.Matsui, K.Yamamori et al.: "Purification and some properties of a tetrodotoxin binding protein from the blood plasma of kusafugu, Takifugu niphobles." Toxicon. 印刷中. (1999)

T.Matsui、K.Yamamori 等人：“草河豚血浆中河豚毒素结合蛋白的纯化和一些特性”，出版中。

T.Matsui,K.Yamamori et al.: "Purification and some properties of a tetrodotoxin binding protein from the blood plasma of kusafugu,Takifugu niphobles." Toxicon. (印刷中). (1999)

T.Matsui、K.Yamamori 等人：“草河豚血浆中河豚毒素结合蛋白的纯化和一些特性”（正在出版）。