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

基本信息

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

项目摘要

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，岸蟹 Hemigrapsus sanguineus 对血腔内注射的河豚毒素 (TTX) 具有抗药性，尽管其中未检测到含量的 TTX。本研究项目的目的是阐明血蟹 H. sanguineus 对 TTX 高抗性的机制。分析通过 Sepharose 6B 凝胶过滤显示血淋巴中含有 TTX 结合蛋白，摩尔重量为>2000k 和 400k。其中一种 TTX 结合蛋白（摩尔重量为 40Ok）被纯化，通过使用 500k 摩尔重量截止滤膜进行超滤来监测 TTX 对小鼠致死活性的中和作用。、ConA-Sepharose柱上的凝集素亲和层析和TSKgel G3000柱上的凝胶过滤HPLC得到由此获得的蛋白质。在非还原条件下，TTX 结合蛋白的分子量约为 82k，在还原条件下约为 72k 和 82k，表明其均一性。 … 更多 e TTX 结合蛋白由至少两个亚基组成，这两个亚基似乎对 TTX 没有中和作用。TTX 结合蛋白是一种具有 pI 的酸性糖蛋白3.5，富含天冬氨酸和谷氨酸，但缺乏色氨酸，含有6%的还原糖和12%的氨基糖。TTX结合蛋白选择性地结合TTX，对TTX的中和能力为6.7小鼠单位/毫克蛋白。然而，用酶（胰蛋白酶、蛋白酶 XIV、淀粉酶和淀粉葡萄糖苷酶）和化学洗涤剂（1% SDS、 1% 二硫苏糖醇、8M 尿素和 6M 盐酸胍），表明蛋白质和糖部分都参与了与 TTX 的结合，以及 TTX 结合蛋白的空间构象的重要性。此外，中和活性被 pre 显着抑制。 -TTX结合蛋白与金属离子(Cu ^ 2+ 、Cr ^ 6+ 和Zn ^ 2+ )的孵育。 TTX 结合蛋白可能在无毒岸蟹 H. sanguineus 的血淋巴中充当金属载体蛋白。