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Molecular Mechanism of Action for Particularly Potent Marine Biotoxins.

特别有效的海洋生物毒素的分子作用机制。

基本信息

批准号：
10480148
负责人：
MURATA Michio
金额：
$ 6.02万
依托单位：
Osaka University (1999-2000)The University of Tokyo (1998)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B).
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 2000
项目状态：
已结题

项目摘要

Maitotoxin (MTX) was first discovered as one of the toxins responsible for ciguatera, a seafood poisoning caused by ingestion of coral reef fish. MTX has extremely potent bioactivities ; its toxicity is particularly worth noting, since its LD_<50>(50 ng/kg, mouse ip.) is exceeded only by a few proteinaceous toxins. MTX elicits Ca^<2+> influx in virtually all cells and tissues and this elevation in intracellular calcium concentration leads to secondary events ; e.g., phosphoinositide breakdown, arachidonic acid release, muscle contraction, and secretion of dopamine, norepinephrine, and insulin. Gangliosides GM1 and GM3 strongly inhibited MTX-induced Ca^<2+> influx in C6 cells. Their inhibitory potency was in the order GM1 (IC_<50>, ca. 2 μM)>GM3 (ca. 5 μM)>asialo-GM1 (ca. 20 μM). GM1 (3 μM) completely blocked MTX(30 nM)-induced Ca^<2+> influx in human erythrocyte ghosts. When C6 cells were pretreated with tunicamycin, an antibiotic which inhibits N-linked glycosylation, or concanavalin A, a lectin which exhibits high affinity for cell-surface oligosaccharides, MTX-induced Ca^<2+> influx was significantly potentiated. This suggests that removal of oligosaccharides from the cell surface by tunicamycin or capping of sugar chains on plasma membranes by concanavalin A can potentiate the action of MTX.Photoaffinity labeling experiment to identify MTX-binding protein was carried out with use of a daizirine-biotin conjugated reagent developed by Hatanaka. Some spots on 2D electrophoresis, corresponding to 2 KDa were eliminated by addition of an MTX inhibitor, brevetoxin-B.

Maitotoxin （MTX）最初被发现是导致雪卡毒素的毒素之一，雪卡毒素是一种因摄入珊瑚礁鱼类而引起的海产品中毒。甲氨蝶呤具有极强的生物活性；其毒性特别值得注意，因为其LD_<50>（50 ng/kg，小鼠体重）只有少数蛋白质类毒素超过。MTX在几乎所有细胞和组织中引起Ca^<2+>内流，细胞内钙浓度的升高导致继发性事件；例如，磷酸肌肽分解、花生四烯酸释放、肌肉收缩、多巴胺、去甲肾上腺素和胰岛素的分泌。神经节苷脂GM1和GM3强烈抑制mtx诱导的C6细胞Ca^<2+>内流。它们的抑制效价依次为GM1 （IC_<50>，约为2 μM）、>GM3（约为5 μM）、>asialo-GM1（约为20 μM）。GM1 （3 μM）完全阻断MTX（30 nM）诱导的Ca^<2+>内流。当用tunicamycin（一种抑制n -连接糖基化的抗生素）或concanavalin A（一种对细胞表面寡糖具有高亲和力的凝集素）预处理C6细胞时，mtx诱导的Ca^<2+>内流显著增强。这表明，tunicamycin从细胞表面去除寡糖或concanavalin A在质膜上盖上糖链可以增强MTX的作用。利用Hatanaka开发的daizirine-biotin偶联试剂进行了鉴定mtx结合蛋白的光亲和标记实验。通过添加MTX抑制剂brevetoxin-B，可以消除2D电泳上与2kda对应的一些点。