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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.

辣根毒素(MTX)最早被发现是导致雪卡特拉的毒素之一，雪卡特拉是一种因摄入珊瑚鱼而引起的海鲜中毒。甲氨蝶呤具有极强的生物活性；其毒性特别值得注意，因为它的LD_&50&GT；(50 ng/kg，小鼠ip。)只有几种蛋白质类毒素超过。MTX引起几乎所有细胞和组织中的钙离子内流，这种细胞内钙浓度的升高会导致次要事件，例如，磷脂酰肌醇的分解、花生四烯酸的释放、肌肉收缩以及多巴胺、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细胞内钙内流。它们的抑制力大小顺序为Gm1(IC_lt；50&gt；，约2μM)和Gt；Gm3(约5μM)和Gt；asialo-GM1(约20μM)。Gm1(3μM)可完全阻断甲氨蝶呤(30 NM)诱导的人红细胞膜钙离子内流。当C6细胞用衣霉素(一种抑制N-连接糖基化的抗生素)或刀豆蛋白A(一种对细胞表面低聚糖表现出高亲和力的凝集素)预处理时，MTX诱导的钙内流显著增强。这表明，用衣霉素去除细胞表面的低聚糖或用刀豆蛋白A封端质膜上的糖链可以增强MTX的作用。用Hatanaka开发的代次津-生物素标记试剂进行了MTX结合蛋白的光亲和标记实验。加入MTX抑制剂Brivetoxin-B可消除2D电泳上的一些斑点，与2 KDa相对应。