Yin Xianzhe, Kiriake Aya, Ohta Akira, Kitani Yoichiro, Ishizaki Shoichiro, Nagashima Yuji
Department of Food Science and Technology, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato, Tokyo 108-8477, Japan.
Faculty of Biosciences and Aquaculture, NORD University, Bodø 8049, Norway.
Toxicon. 2017 Sep 15;136:56-66. doi: 10.1016/j.toxicon.2017.06.006. Epub 2017 Jun 23.
Marine pufferfish of the Tetraodontidae family contain high levels of tetrodotoxin (TTX) in the liver and ovary. TTX is suggested to transfer from the liver to the ovary in female pufferfish during maturation. TTX in pufferfish eggs may act as a repellent against predators and as a sexual pheromone to attract male pufferfish. The toxification mechanism of the pufferfish ovary is poorly understood. Here we evaluated the chemical form of TTX and its related substances in the ovary of the panther pufferfish Takifugu pardalis by LC-ESI/MS. TTX and its analogs 4-epi-TTX, 4, 9-anhydroTTX, deoxyTTX, dideoxyTTX, and trideoxyTTX were detected in a low molecular weight fraction by Sephacryl S-400 column chromatography. The finding of an unknown TTX-related substance in a high molecular weight fraction from the Sephacryl S-400 column suggested the occurrence of toxin-binding protein in the ovary. The toxin-binding protein in the ovary was purified by ion-exchange HPLC, gel filtration HPLC, and SDS-PAGE. Amino acid sequencing and cDNA cloning revealed that the toxin-binding protein, TPOBP-10 (Takifugu pardalis ovary toxin-binding protein with a molecular mass of 10 kDa) was homologous with the predicted vitellogenin-1-like protein [Takifugu rubripes] subdomain, a von Willebrand factor type D domain. TPOBP-10 mRNA was highly expressed in the ovary and liver and less in other organs of female individuals based on RT-PCR. These findings reveal a novel function of the vitellogenin subdomain as binding with TTX-related substances, and its involvement in the toxification of the pufferfish ovary.
四齿鲀科的海洋河豚在肝脏和卵巢中含有高浓度的河豚毒素（TTX）。据推测，在雌性河豚成熟过程中，TTX会从肝脏转移至卵巢。河豚鱼卵中的TTX可能起到抵御捕食者的作用，同时作为性信息素吸引雄性河豚。河豚卵巢的毒素形成机制尚不清楚。在此，我们通过液相色谱 - 电喷雾电离质谱法（LC - ESI/MS）评估了豹纹东方鲀（Takifugu pardalis）卵巢中TTX及其相关物质的化学形态。通过Sephacryl S - 400柱色谱法在低分子量组分中检测到了TTX及其类似物4 - 表 - TTX、4,9 - 脱水TTX、脱氧TTX、双脱氧TTX和三脱氧TTX。在Sephacryl S - 400柱的高分子量组分中发现了一种未知的TTX相关物质，这表明卵巢中存在毒素结合蛋白。通过离子交换高效液相色谱法、凝胶过滤高效液相色谱法和十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（SDS - PAGE）对卵巢中的毒素结合蛋白进行了纯化。氨基酸测序和cDNA克隆显示，毒素结合蛋白TPOBP - 10（豹纹东方鲀卵巢毒素结合蛋白，分子量为10 kDa）与预测的类卵黄生成素 - 1蛋白[红鳍东方鲀]亚结构域（一种血管性血友病因子D结构域）同源。基于逆转录 - 聚合酶链反应（RT - PCR），TPOBP - 10 mRNA在雌性个体的卵巢和肝脏中高表达，而在其他器官中表达较少。这些发现揭示了卵黄生成素亚结构域与TTX相关物质结合的新功能，以及它在河豚卵巢毒素形成中的作用。
