【作者】 肖玉成；

【导师】 梁宋平；

【作者基本信息】 湖南师范大学 ， 生物化学与分子生物学， 2004， 博士

【摘要】 虎纹捕鸟蛛（Selenocosmia huwena Wang [=Orni thoctonus huwena（Wang）]）、海南捕鸟蛛（Selenocosmia hainana Liang[=Ornithoctonus hainana（Liang）]）和雷氏大疣蛛（Macrothele raveni）是在我国南方发现的具有很强毒性的蜘蛛新种，其粗毒能使昆虫和小型脊椎动物致死。本项研究采用膜片钳技术在NG108-15细胞上首先检验了这三种蜘蛛的粗毒对电压门控钠通道和延迟整流钾通道的影响。结果表明，三种蜘蛛粗毒对外向延迟整流钾通道没有明显作用，但对TTX敏感型钠通道表现出较强的浓度依从性抑制效应，半有效抑制浓度(IC₅₀)分别为：3.4、1.8和11.0mg／L。值得注意的是，三种粗毒抑制钠通道的方式均与其它已知蜘蛛钠通道毒素不同，都不能影响通道的激活与失活时间，表明在这些蜘蛛粗毒中极有可能含有未曾报导过的新型钠通道毒素。 通过阳离子交换和反相高效液相色谱柱层析（HPLC），进一步从海南捕鸟蛛粗毒中分离获得了一种新的神经毒素，命名为海南捕鸟蛛毒素-Ⅴ（hainantoxin-Ⅴ，HNTX-Ⅴ）。同时还利用膜片钳或双电极杆电压钳技术鉴定了该毒素以及其它三种海南捕鸟蛛毒素（包括海南捕鸟蛛毒素-Ⅰ（HNTX-Ⅰ）、海南捕鸟蛛毒素-Ⅲ（HNTX-Ⅲ）和海南捕鸟蛛毒素-Ⅳ（HNTX-Ⅳ））对离子通道的影响特征。四种海南捕鸟蛛毒素均是多肽类钠通道毒素，由33或35个氨基酸残基组成，其中6个Cys以Ⅰ-Ⅳ、Ⅱ-Ⅴ、Ⅲ-Ⅵ的方式配对形成3对二硫键。HNTX-Ⅴ是HNTX-Ⅳ的一种天然突变体，两者之间只有一个残基（Ser20Ala）不同。三维结构测定和同源模建结果表明四者都属于抑制剂胱氨酸结（ICK）模体蛋白家族。四种HNTX均能抑制TTX敏感型钠通道的激活，但不影响TTX不敏感型钠通道。HNTX对TTX敏感型钠通道亚型的选择性不一样。HNTX Ⅲ-Ⅴ能完全抑制大鼠背根神经节（DRG）细胞TTX敏感型钠通道，IC₅₀分别是1.1、44.6和46.8 nM。但HNTX-Ⅰ对DRG细胞钠通道无影响，却能抑制不表达于DRG细胞上的rNa_v1.2／β1和昆虫钠通道para／tipE（外源表达于非洲爪蟾卵母细胞上），IC₅₀分别是68.0和4.3μM。HNTX Ⅲ-Ⅴ还能使DRG细胞TTX敏感型钠通道的稳态失活曲线向超极化方向漂移约10 mV。同粗毒水平实验结果一样，中英文摘要四利，}一六以都不能延缓钠通道的失活时间，也没有漂移电压一「包流（l一V） 狡系曲线。特别是HNTX一工H和HNTX一IV（1 pM）还能完全阻断位点3毒素枷K{（.类。一蝎毒素，来自于东亚钳蝎But力:Is二rtensz’ Karsch粗毒）延缓的失活电流，表明海南捕鸟蛛毒素不是位点3毒素，其影响钠通道的机制应该有别于6一atraCotoxinS和协一agatoXinS等蜘蛛毒素。我们进 步推测HNTX在钠通道上的结合位点是位点l。通过固相化学合成的方法，将日NTX一丁v进行了四个点位突变（Ser12Ala、Arg26Ala、LyS27Ala和八以29A}的。其‘一1，，突变体LyS27Ala和Arg29Ala抑制钠通道的能力只有天然毒素的1/100，而Arg26Ala和serlZAla与天然毒素相差不大，证明}”一“和八r厂’应该是海南捕鸟蛛毒素结合钠通道的关键残基。 敬车l}缨毛蛛（凸ilobl妞‘加￡j’z’n那为、）是最近在我国海南省发现的 ，蜘蛛新种。通过阳离子交换和/或反相HPLC，我们从其粗毒中得到了两种一丰度较高的组分，并命名为敬钊缨毛蛛毒素一I（JZTX一I）和敬钊缨毛蛛一心索一日l（二仪Tx一工H）。MALD工一TOF质谱鉴定和Edman降解测序结果表明:、!/下X一I的分子量为3675.6 Da，含有33个氨基酸残基;而JZTX一工H的分广员为:份1 9.4 Da，含有36个残基。两毒素中均包含6个CyS。结合TCIi尸部分还原修饰、氨基酸序列测定和多酶酶解分析法等技术，鉴定出这6个C、、能够形成3对二硫键，连接方式均为卜工V、工工一V、工工卜V工。我们还采川〔、l）X八末端快速扩增（RACE）法获得了它们的CDNA序列，并由此推测出、J/’f\一l和健限可工I的前体分别含有62和63个氨基酸残基。经分析发现!Z拭11!前体中含有一不常见的蛋白酶酶切位点（一X一Ser一），而不是 丫八:、g一，_且其间隔蛋白区序列只含有5个残基，是目前蜘蛛毒素前体‘扫员脚的·个。!漠片钳实验结果表明JZTX一I和JZTX一IH都是电压门控钠通迈，扮素，子{!对钠通道亲和力与亚型的选择性不一致。JzTX江是日前首次报道的一源于蜘蛛粗毒的类Q一毒素，能抑制棉铃虫幼虫神经元细胞钠通道、少、鼠D既细胞‘I’TX敏感型钠通道和心肌细胞钠通道的快速失活，工C:‘，分别为8.86林M、0.99刚和31.6 nM，对三种细胞_<sub>匕钠电流的峰值大小都无明!IJ娜洲lJ，也不改变通道I一V曲线和稳态失活特征，但能加速D尺G细胞卜通道的火活恢复速率。而有意思的是，JZTX一IH在粗毒中可能存在异构现象，因为在分离过程，}，，该毒素具有两个不同的吸收峰，_且彼此功能明显不户丰丫尽，’i’{:两种‘!Z‘fX一工H都能选择性地作用于心肌细胞竹X不敏感’(口钠通中英文摘要道，但一者为抑制电流的快速失活相，与JZTX一工类似;而另一者为抑制电流激活，10刚几乎可以完全抑制位点3毒素引起的电流失活部分。IC印分别为12.8哪和0.38哪。我们进一步推测:JZTX一工和JZTX一工H（抑制通道失活）应该是位点3毒素，而JZTX一IH(抑制通?更多还原

【Abstract】 There are three species of Chinese spiders Selenocosmia huwena Wang [=Ornithoctonus huwena (Wang)], Selenocosmia hainana Liang [=Ornithoctonus hainana (Liang)] and Macrothele raveni distributed in the south of China. Their venoms can kill insects and even some small vertebrates. Under whole-cell patch-clamp recording, we observed the effects of these venoms on tetrodotoxin-sensitive (TTX-S) voltage-gated sodium channels (VGSCs) and delay-rectified potassium channels in undifferentiated NG108-15 cells. It was found that they all had no evident effect on outward delay-rectified potassium currents, while they inhibited TTX-S sodium currents in a dose-dependent mode. Their IC50 values were approximately 3.4 (S. huwena), 1.8 (S, hainana) and 11.0 mg/L (M raveni), respectively. Interestingly, different from other known spider toxins, these spider venoms affected neither channel activation kinetics nor inactivation kinetics, suggesting that there might be some novel sodium channel toxins unreported before.A novel neurotoxic peptide, named hainantoxin-V (HNTX-V), was isolated further from the venom of the Chinese bird spider S. hainana by ion-exchange chromatography and reverse-phase high performance liquid chromatography (rp HPLC). Moreover, we characterized the actions of the novel toxin and other three hainantoxins, including hainantoxin-I (HNTX-I), hainantoxin-III (HNTX-III) and hainantoxin -IV (HNTX-IV), on ion channels expressing in either adult rat dorsal root ganglion (DRG) neurons or Xenopus laevis oocytes using both whole-cell patch-clamp and two-electrode voltage-clamp techniques. Four HNTXs are sodium channel toxins composed of 33-35 residues including six cysteines, which can be linked with three disulfide bonds (I-IV, II-V and III-VI). HNTX-V is a naturally occurring mutant of HNTX-IV for there is only one residue (Ser20Ala) different between them. NMR and homologue modeling analysis indicated that all of them are inhibitor cystine knot (ICK) peptides. Four HNTXs lost to affect tetrodotoxin-resistant(TTX-R) VGSCs in DRG neurons, but they except HNTX-I strongly inhibited the activation of TTX-S VGSCs with the IC50 values of 1.1, 44.6 and 46.8 nM, respectively. HNTX-I was sensitive to rNavl.2/pl and para/tipE, which were not expressed in DRG neurons. HNTX-I blocked the two kinds of VGSCs with IC50 values of 68.0 and 4.3 uM, respectively. Additionally, HNTX III-V caused a 10 mV hyperpolarizing shift in the voltage midpoint of steady-state inactivation. Similar to the crude venom, four HNTXs neither slowed channel inactivation nor shifted the voltage-current relationship of VGSCs. Importantly, HNTX-III and HNTX-IV at 1 uM inhibited the slowing inactivation currents induced by BMK-I (a typical scorpion a-like toxin from the scorpion Buthus martensi Karsch) completely. The results indicate that four HNTXs are novel spider toxins modifying the mammal neural VGSCs through a mechanism quite different from other spider toxins targeting neural receptor site 3, such as 5-atracotoxins and u,-agatoxins. Thus, we assumed that the HNTXs should not be site 3 toxins but belong to the family of site 1 toxins. In order to determine the key residues of HNTXs responsible for binding VGSCs, four mutants of native HNTX-IV (Serl2Ala, Arg26AIa, Lys27Ala and Arg29Ala) were synthesized by solid-phase method. When checked on DRG neurons, the inhibiting abilities for Serl2Ala and Arg26Ala were similar to that for native HNTX-IV, but the IC50 values of Lys27Ala and Arg29Ala are 100 times as big as that of native HNTX-IV. Thus, Lys27 and Arg29 should be important for HNTX to bind VGSCs.Chilobrachys jingzhao was identified recently as a new species of spider in the Hainan province of China. By means of ion-exchange and rp HPLC, we have isolated two main components, denoted jingzhaotoxin-I (JZTX-I) and jingzhaotoxin-III (JZTX-III), from its venom. The molecular weights of two toxins were determined to be 3675.6 and 3919.2 Da by MALDI-TOF mass spectrometry, respectively. JZTX-I is composed of 33 residues while JZTX-III has 36 residues.更多还原