Tetrodotoxin-binding protein in the haemolymph of shore crab Hemigrapsus sanguineus

滨蟹 Hemigrapsus sanguineus 血淋巴中的河豚毒素结合蛋白

• 批准号: 11660201
• 负责人: NAGASHIMA Yuji
• 金额: $ 2.24万
• 依托单位: Tokyo University of Fisheries
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11660201/
• 关键词: tetrodotoxin-binding protein; tetrodotoxin; shore crab; haemolymph; neutralizing activity; metal ions; イソガニ; タンパク質; 精製

A shore crab Hemigrapsus sanguineus is reported to be resistant to tetrodotoxin (TTX) administrated intrahemocoelically, although it contains no detectable amount of TTX.This research project was carried out to clarify the mechanism of high resistance of the crab H.sanguineus to TTX.Analysis by gel filtration on Sepharose 6B revealed that the haemolymph contained TTX-binding proteins with mol.wt.of >2000k and 4OOk. One of the TTX-binding proteins (mol.wt.of 40Ok) was purified, monitoring the neutralizing effect against the lethal activity of TTX to mice, by ultrafiltration using a 500k mol.wt.cut-off filter membrane, lectin affinity chromatography on a ConA-Sepharose column, and gel filtration HPLC on a TSKgel G3000 column. The protein thus obtained gave only one band in native-PAGE, confirming its homogeneity. The mol.wt.of the TTX-binding protein was estimated to be about 82k under non-reducing conditions and about 72k and 82k under reducing conditions by SDS-PAGE, indicating that th … More e TTX-binding protein was composed of at least two subunits which appeared to have no neutralizing effect to TTX.The TTX-binding protein was an acidic gycoprotein with pI 3.5, abundant in Asp and Glu but absent in Trp, and contained 6% reducing sugar and 12% of amino-sugar.The TTX-binding protein selectively bound to TTX, with a neutralizing ability to TTX of 6.7 mouse unit/mg protein. However, its neutralizing activity was almost lost by treatments with enzymes (trypsin, protease XIV, amylase and amyloglucosidase) and chemical detergents (1% SDS, 1% dithiothreitol, 8M urea and 6M guanidine hydrochloride), suggesting the involvement of both proteineous and sugar moieties in the binding to TTX and the importance of the steric conformation of the TTX-binding protein. Moreover, the neutralizing activity was significantly inhibited by pre-incubation of the TTX-binding protein with metal ions (Cu^<2+>, Cr^<6+> and Zn^<2+>). It is possible that the TTX-binding protein functions as a metal carrier protein in the haemolymph of the non-toxic shore crab H.sanguineus. Less

据报道，一种滨蟹（Hemigrapsus sanguineus）对经腹腔注射的河豚毒素（TTX）具有耐药性，尽管它不含可检测到的TTX。本研究项目旨在阐明血螯蟹对TTX高抗性的机制。Sepharose 6B凝胶过滤分析发现，血淋巴中含有ttx结合蛋白，分子量为bb0, 2000k和4OOk。ttx结合蛋白之一（mol.wt.）用500k mol.wt超滤，对TTX对小鼠的致死活性进行中和作用监测。截止过滤膜，在ConA-Sepharose柱上进行凝集素亲和层析，在TSKgel G3000柱上进行凝胶过滤高效液相色谱。由此获得的蛋白在原生page中只有一个条带，证实了其同质性。通过SDS-PAGE估计，TTX结合蛋白在非还原条件下的分子量约为82k，在还原条件下的分子量约为72k和82k，表明TTX结合蛋白至少由两个亚基组成，这些亚基似乎对TTX没有中和作用。ttx结合蛋白是一种pI为3.5的酸性糖蛋白，含有丰富的Asp和Glu，缺乏Trp，含有6%的还原糖和12%的氨基糖。TTX结合蛋白选择性结合TTX，对TTX的中和能力为6.7小鼠单位/mg蛋白。然而，在酶（胰蛋白酶、蛋白酶XIV、淀粉酶和淀粉葡糖苷酶）和化学洗涤剂（1% SDS、1%二硫苏糖醇、8M尿素和6M盐酸胍）处理下，其中和活性几乎丧失，这表明TTX结合蛋白中既有蛋白质部分，也有糖部分，以及TTX结合蛋白的立体构象的重要性。此外，ttx结合蛋白与金属离子（Cu^<2+>, Cr^<6+>和Zn^<2+>）预孵育显著抑制了中和活性。ttx结合蛋白可能在无毒滨蟹血淋巴中起金属载体蛋白的作用。少