【作者】 容明强；

【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， 2011， 博士

【摘要】 Jingzhaotoxin-Ⅲ(JZTX-Ⅲ)是从我国特有的敬钊缨毛蛛中分离得到的一种多肽毒素,它能够选择性的作用于大鼠心肌细胞并使心肌钠离子通道的激活往去极化方向漂移。我们采用酵母发酵系统成功的表达了JZTX-Ⅲ以及其18个突变体,其中除F7A表达量太低外其他的突变体表达量约为4 mg/L。表达的JZTX-Ⅲ以及突变体的CD图谱和天然JZTX-Ⅲ的基本一致,因此CD谱检测证明表达的毒素以及突变体并没有发生结构的改变。膜片钳检测发现表达的JZTX-Ⅲ能够选择性的抑制Nav1.5电流(IC50为348.0±67.9 nM),同时毒素能使Nav1.5往去极化方向漂移约+13 mV但不改变钠通道的稳态失活。因此,表达的JZTX-Ⅲ和天然的JZTX-Ⅲ在结构和功能上都保持一致。检测毒素突变体和Nav1.5的亲和力发现,两个酸性残基(Aspl.Glu3)和一个暴露在毒素表面由色氨酸构成的疏水性斑片(Trp8.Trp9.Trp28.Trp29.Trp30)是JZTX-Ⅲ和Nav1.5作用的关键残基,另外一个碱性残基Arg13突变成Glu后使毒素和通道的亲和力增强了十倍。对Nav1.5 DII S3-S4连接环上的氨基酸残基进行了丙氨酸扫描后,发现Nav1.5的突变体S799A.R800A. L804A能显著的降低JZTX-Ⅲ和Nav1.5的亲和力,这些氨基酸残基可能是JZTX-Ⅲ结合钠通道的关键残基。将Nav1.5和其他钠通道亚型的DIIS3-S4氨基酸序列对比,发现R800为Nav1.5所特有的氨基酸。Nav1.7上相应的位点D816突变成D816R增强了JZTX-Ⅲ和Nav1.7的亲和力,Nav1.5突变体R800D使毒素和通道的亲和力降低约72倍。JZTX-Ⅲ通过酸性氨基酸残基和Nav1.5 DII S3-S4链接环上的碱性氨基酸残基相互作用,这种作用机制不同于以往的毒素,因此JZTX-Ⅲ可以作为毒素和钠通道结构和功能研究的工具试剂。Huwentoxin-Ⅳ(MHWTX-Ⅳ)修饰体是从虎纹捕鸟蛛粗毒中分离得到的一种发生翻译后修饰的毒素。质谱检测表明mHWTX-Ⅳ分子量比天然HWTX-Ⅳ的分子量少18 Da。采用质谱进一步的分析毒素的氨基酸序列,发现HWTX-Ⅳ的N端谷氨酸脱水环化形成焦谷氨酸导致分子量减少18 Da。全细胞膜片钳显示,MHWTX-Ⅳ选择性的抑制大鼠DRG神经元上的TTX-S钠离子通道(IC50～54.16±7.3 nM),而对大鼠DRG细胞的TTX-R钠离子通道以及其他的电压门控离子通道都没有影响。和天然HWTX-Ⅳ一样,HWTX-Ⅳ修饰体不改变TTX-S钠离子通道的I-V曲线、电导曲线以及稳态失活曲线。HWTX-IV抑制的钠离子通道在高电压刺激下与钠离子通道解离,钠电流基本完全恢复,但被HWTX-Ⅳ修饰体抑制的钠电流在高电压(+200 mv,500 ms)刺激下依然没有恢复,证明HWTX-Ⅳ修饰体在高电压的刺激下与钠离子通道依然紧密结合。这有可能是谷氨酸环化形成焦谷氨酸后导致毒素缺失一个负电荷,从而使毒素和钠通道上的负电荷残基的结合更加紧密。这是第一次报道在蜘蛛毒素中有焦谷氨酸的存在,并且第一次证明焦谷氨酸能够增强HWTX-Ⅳ修饰体和钠离子通道的结合。JZTX-34是敬钊缨毛蛛中分离得到一种新的多肽毒素,由于该毒素在粗度中的含量较小,因此采用酿酒酵母表达该毒素。JZTX-34含有35个氨基酸残基其中包括三对半胱氨酸形成二硫键,推测其可能为ICK结构模体的多肽毒素。JZTX-34能选择性作用于大鼠DRG细胞上的TTX-S钠通道(IC50～85 nM),但是对TTX-R钠通道以及钙通道和钾通道无明显抑制作用。JZTX-34能选择性抑制大鼠DRG细胞上的TTX-S钠电流并使钠通道稳态失活往去极化方向漂移10 mV,但是毒素却不改变钠通道的激活。JZTX-34在高电压刺激(+120 mV)下与钠通道发生解离,抑制消除。因此,我们推测JZTX-34是一个位点4的毒素可能结合钠通道的电压敏感元件。虎纹捕鸟蛛(Ornithoctonus huwena)是分布于我国南方毒性最强的蜘蛛之一,其毒液中含有大量的多肽毒素能够快速的杀死猎物。HWTX-Ⅰ和HWTX-Ⅰ是虎纹捕鸟蛛粗毒中两种最丰富的成分,HWTX-Ⅰ是一个双功能活性分子既能抑制大鼠DRG神经元TTX-S钠通道(IC50～57.40±1.42 nM)又能专一抑制N-型钙通道(IC50～91 nM),但是10μM HWTX-Ⅱ对大鼠DRG神经元细胞上钠离子通道和钙离子通道均无明显作用。HWTX-Ⅱ和HWTX-Ⅰ混合后共同作用于TTX-S钠通道与N-型钙通道其活性分别增加10倍和6倍。进一步研究表明,HWTX-Ⅱ不仅增加HWTX-Ⅰ对TTX-S钠通道的抑制活性,也能使HHWTX-Ⅰ结合钠离子通道的速度提高约2倍,但是该毒素不改变HWTX-Ⅰ和钠通道以及钙通道的作用方式。HWTX-Ⅱ和HWTX-Ⅰ的协同作用显著的增强了HWTX-Ⅰ的活性,也揭示了毒液中毒性较弱分子的一个新功能。更多还原

【Abstract】 Jingzhaotoxin-Ⅲ(JZTX-Ⅲ) is a unique sodium channel gating modifier from the tarantula Chilobrachys jingzhao, which can selectively inhibit the activation of cardiac sodium channel but not neuronal subtypes and cause a depolarization shift about 10 mV in activation. In this study, we showed that JZTX-Ⅲwas efficiently expressed by the secretary pathway in yeast. CD spectra of JZTX-Ⅲmutants and the expressed JZTX-Ⅲalmost overlapped completely with that of the native toxin, indicating that all expressed toxins did not significantly change the secondary structure of JZTX-Ⅲ. Like the native JZTX-Ⅲ, the expressed JZTX-Ⅲinhibited Nav1.5 channel with the IC50 value of 348.0±67.9 nM and cause a depolarization shift about+13 mV in activation. Alanine-scanning analysis indicated that two acidic residues (Asp1, Glu3) and an exposed hydrophobic patch, formed by four Trp residues (8,9,28 and 30), play very important roles in the binding of JZTX-III to Navl.5. JZTX-III docked to Nav1.5 DIIS3-S4 linker. Mutants S799A, R800A and L804A could additively reduce toxin sensitivity of Nav1.5. We also demonstrated that the unique Arg800, not emerging in other sodium channel subtypes, is responsible for JZTX-Ⅲselectively interacting with Nav1.5. The reverse mutants D816R in Nav1.7 greatly increased the sensitivity of the neuronal subtype to JZTX-Ⅲ. Conversely, the mutant R800D in Nav1.5 decreased JZTX-Ⅲ’s IC50 by 72-fold. Therefore, our result indicated that JZTX-Ⅲis a site 4 toxin, but do not possess the same critical residues on sodium channels with other site 4 toxins.A variety of posttranslational modifications of peptides have been found in venomous animal, mostly in cone snails. A novel posttranslational modified spider toxin MHWTX-Ⅳwas purified from Chinese tarantula Ornithoctonus huwen. Mass spectrum showed than the molecular weight of MHWTX-Ⅳwas 18 Da lower than that of the native Huwentoxin-Ⅳ. Sequence analysis by mass spectrum indicated that it was highly similar to spider toxin Huwentoxin-Ⅳexcept that the N-terminal glutamic acid is replaced by pyroglutamic residue. Like the native HWTX-Ⅳ, the modified HWTX-Ⅳ(MHWTX-Ⅳ) could inhibit trodotoxin-resistant VGSCs with an IC50 value of 54.16±7.3 nM in dorsal root ganglion neurons, but not change the activation or inactivation kinetics. However, in contrast to the dissociation of HWTX-Ⅳfrom sodium channel at extreme depolarization, the inhibition of TTX-S sodium channels by MHWTX-Ⅳcould not reversed by even stronger depolarization (+200 mV,500ms). It is possible that the loss of negative charge induced by glutaminyl cyclization makes the modified toxin binding stronger. To our knowledge, this is the first report of pyroglutamic acid residue found in spider toxin and the modification could increase the trap ability to voltage sensor of sodium channel.We have isolated and characterized a novel neurotoxin named JZTX-34 from the tarantula Chilobrachys jingzhao and successfully expressed by yeast. JZTX-34 is a C-terminally amidated peptide composed by 35 amino acid residues including six cysteine residues with three disulfide bridges. JZTX-34 selectively inhibits TTX-Ssodium channels of DRG neurons with the IC50 value of 85 nM, while having no effect on TTX-R sodium channels. JZTX-34 shows no effect on activation of TTX-S sodium channels but causing a depolarization shift about 10 mV on steady-state sodium channel inactivation. The inhibition by JZTX-Ⅲwas completely reversed after a high depolarization (+120 mV). Based on those results, we propose that JZTX-34 may be a site 4 toxin and trap the voltage sensor of sodium channel.The Chinese tarantula Ornithoctonus huwena is one of the most venomous spiders in south China, its venom contains various peptide toxins and can quickly kill prey. Huwentoxin-Ⅰ(HWTX-Ⅰ) and huwentoxin-Ⅱ(HWTX-Ⅱ) are two abundant toxic components in the venom of O. huwena. HWTX-Ⅰinhibits both sodium channel (IC50～57.40±1.42 nM) and calcium channel (IC50～91 nM) of adult rat dorsal root ganglion (DRG) neurons. HWTX-Ⅱshows no effect on both sodium channel and calcium channel. When two toxins were mixed together, the activity of HWTX-Ⅰincreases 10 fold on sodium channel and 6 fold on calcium channel. HWTX-Ⅱnot only increases the activity of HWTX-Ⅰbut also accelerates the binding rate to sodium channel with about 2 fold, while having no effect on activation or inactivation kinetic. These results reveal synergistic interactions among insect and mammalian toxins and imply a new functional role for polypeptides toxins with weak activity.更多还原