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

血吸虫血淋巴对河豚毒素(TTX)具有抗药性，但未检测到河豚毒素(TTX)的存在。本研究旨在阐明血蟹对河豚毒素(TTX)的高抗性机制。通过500kmol.wt.截断滤膜超滤、ConA-Sepharose柱凝集素亲和层析和TSKGel G3000凝胶过滤高效液相色谱分离纯化了一种TTX结合蛋白(分子质量为400Ok)，用于监测TTX对小鼠致死活性的中和作用。所获得的蛋白在自然-PAGE中只有一条带，证实了其同源性。…-PAGE测得TTX结合蛋白在非还原条件下的摩尔质量约为82K，在还原条件下的摩尔质量约为72K和82K。河豚毒素结合蛋白由至少两个似乎对河豚毒素无中和作用的亚基组成。河豚毒素结合蛋白是一种酸性蛋白，等电点为3.5，富含天冬氨酸和谷氨酸，但不含色氨酸，含6%还原糖和12%氨基糖。然而，用酶(胰酶、蛋白酶XIV、淀粉酶和淀粉糖苷酶)和化学洗涤剂(1%十二烷基硫酸钠、1%二硫苏糖醇、8M尿素和6M盐酸胍)处理后，其中和活性几乎丧失，这表明蛋白质和糖基都参与了与TTX的结合，并说明了TTX结合蛋白的空间构象的重要性。此外，TTX结合蛋白与金属离子(Cu^&lt；2+&gt；，Cr^&lt；6+&gt；和Zn^&lt；2+&gt；)预先孵育后，其中和活性明显受到抑制。TTX结合蛋白可能是无毒血蟹血淋巴中的一种金属载体蛋白。较少

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