【作者】 曾雄智；

【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， 2005， 博士

【摘要】 敬钊缨毛蛛（Chilobrachys jingzhao）是最近在我国海南省发现的一种蜘蛛新种。通过阳离子交换与反相高效液相色谱层析,从该蜘蛛粗毒中纯化到两种新组分,并分别命名为敬钊缨毛蛛毒素-Ⅴ（Jingzhaotoxin-Ⅴ）和敬钊去整合素—Ⅰ（Jingzhaotin-Ⅰ）。MALDI-TOF质谱鉴定和Edman降解测序结果表明:JZTX-Ⅴ的分子量为3605.73Da,含有29个氨基酸残基;而Jingzhaotin-Ⅰ的分子量为3631.42Da,含有34个氨基酸残基。我们还采用cDNA末端快速扩增（RACE）法获得了它们的全长cDNA序列,并由此推测出JZTX-Ⅴ和Jingzhaotin-Ⅰ的前体分别含有83和66个氨基酸残基。为了更好的了解它们的结构与功能的关系,利用同源模建技术模建了敬钊缨毛蛛毒素-Ⅴ和敬钊去整合素—Ⅰ分子的三维结构。Jingzhaotin-Ⅰ是首次报道源于蜘蛛粗毒的一个含有KGD序列的去整合素。该肽拥有抗凝血活性,能够抑制ADP诱导的人血小板的聚集。在较低浓度时它能促进人的血小板聚集,而较高浓度时则抑制血小板聚集,进一步推测它的作用位点可能是血小板糖蛋白受体Ⅱb/Ⅲa。膜片钳和电压钳实验表明,JZTX-Ⅴ能够抑制大鼠背根神经节（DRG）细胞上的TTX不敏感型以及TIX敏感型钠通道,其IC₅₀值分别是19.8和35.4nM。JZTX-Ⅴ对处于静息关闭与失活状态的TTX不敏感型与TTX敏感型钠通道均表现出非常高的亲和性。JZTX-Ⅴ能够抑制外源表达于非洲爪蟾卵母细胞上的Kv4.2钾通道(IC₅₀值为604.2nM),但对表达的Kv1.2、Kv1.3和Kv1.4钾通道却没有影响。JZTX-Ⅴ能够引起TTX不敏感型与TTX敏感型钠通道的起始激活电压或最大峰值电流激活电压分别往去极化方向漂移约10mV,而它们的半数稳态失活电压却往超极化方向漂移约更多还原

【Abstract】 Chilobrachys jingzhao was identified recently as a new spider species of the family of Theraphosidae in the Hainan province of China. By means of ion-exchange and reverse phase HPLC, we have isolated two new components, named jingzhaotoxin-V （JZTX-V） and jingzhaotin-I （JZTIN-I）, from its venom. The molecular weights of the two peptides were determined to be 3605.73 and 3631.42 Da by MALDI-TOF mass spectrometry, respectively. JZTX-V is composed of 29 residues while JZTIN-I has 34 residues. By using 3’-RACE and 5’-RACE, the full-length cDNAs were obtained and deduced to encode a 83-residue precursor of JZTX-V and a 66-residue precursor of JZTTN-I. To better understand their structure-function relationships, we constructed two models of JZTX-V and JZTIN-I based on the NMR structures of their homologous toxins respectively. JZTIN-I is a new disintegrin containing the KGD sequence, which is the first disintegrin found in spider venoms. It inhibits platelet aggregation induced by adenosine 5-diphosphate （ADP） in human platelet-rich plasma and possesses anticoagulant activity. JZTIN-I at low concentration facilitated the platelet aggregation, but JZTIN-I restrained the platelet aggregation at high concentration. It is further assumed that the binding site of JZTIN-I on platelets is the platelet glycoprotein IIb/IIIa. JZTX-V inhibits both tetrodotoxin-resistant and tetrodotoxin-sensitive sodium currents in adult rat dorsal root ganglion neurons with IC₅₀ values of 19.8 and 35.4 nM, respectively. Moreover, the toxin exhibits high affinity to both the resting closed and inactivated states of the channels. JZTX-V can inhibits Kv4.2potassium channels expressed in Xenpus Laevis oocytes （IC50 = 604.2 nM）, but has no effects on Kvl.2, Kvl.3 and Kvl.4 expressed in Xenopus laevis oocytes. JZTX-V alters the gating properties of both tetrodotoxin-resistant and tetrodotoxin-sensitive sodium channels by causing a depolarization shift of about 10 mV in the threshold of the initial activated voltage or the active voltage of peak inward currents and a hyperpolarizing shift of about 7 mV in the voltage midpoint of steady-state sodium channel inactivation. Small unilamellar vesicles binding assays show that the partitioning of JZTX-V into lipid bilayer requires negatively charged phospholipids. Tryptophan fluorescence and acrylamide quenching experiments hint that tryptophan residues on the hydrophobic surface of JZTX-V have a reduced exposure to water when the toxin interacts with membranes. Furthermore, JZTX-V exhibits a red edge excitation shift of 5.7 nm when bound to acidic phospholipid membranes. The present studies hinted that when bound to the phospholipid bilayer JZTX-V may be located at the interface between polar headgroups and the hydrophobic phase of the membrane. Mutants R26A-JZTX-V, K27A-JZTX-V, K22A-JZTX-V and R20V-JZTX-V were synthesized by solid-phase Fmoc chemistry, followed by oxidative refolding of purified peptides under the optimal conditions. Human tetrodotoxin-resistant sodium channel was reported to be a potential target for treatment of pain, highlighting the importance of JZTX-V as potential lead for drug development.Using two-dimensional lH NMR techniques, the complete sequence-specific assignments of proton resonance in the !H-NMR spectra of JZTX-I were obtained by analyzing a series oftwo-dimensional spectra, including DQF-COSY, TOCSY and NOESY spectra in H2O and D2O. All the backbone protons except Gln4 and more than 95% of the side-chain protons were identified by daN, d^, dpN and dun connectivities in NOESY spectrum. Furthermore, the secondary structure of JZTX-I was identified from NMR data. It contained mainly a short triple-stranded antiparallel p-sheet consisted of residue Trp7-Cys9, Phe20-Lys23, and Leu28-Trp31. Small unilamellar vesicles binding assays showed that the partitioning of JZTX-I into lipid bilayer does not require negatively charged phospholipids. Tryptophan fluorescence and acrylamide quenching experiments hint that tryptophan residues on the hydrophobic surface of JZTX-I have a reduced exposure to water when the toxin interacts with membranes. Furthermore, JZTX-I exhibits a red edge excitation shift of 8.0 and 7.4 nm respectively when bound to acidic or neutral phospholipid membranes. By contrast, interaction between BMK Ml （a sodium channel site 3 toxin） and lipid vesicles could not observed under the same condition. It is further assumed that the mechanism of JZTX-I may be different from these site 3 toxins that have been reported up to the present.Five neurotoxic peptides, named Raventoxin-I （RVTX-I） Raventoxin-HA （RVTX-HA）, Raventoxin-HB （RVTX-HB）, Raventoxin-IIC （RVTX-HC） and Raventoxin-m （RVTX-III） were isolated from the venom of the spider Macrothele raveni. The molecular weights of these peptides were determined to be 4840.11, 3133.48, 3020.35, 3115.63 and 3286.58 Da by MALDI-TOF mass spectrometry, respectively. These toxins are composed of 43, 34, 33, 34 and 29 residues respectively. Both RVTX-I and RVTX-III are lethal to mice and blockneuromuscular transmission in an isolated mouse phrenic nerve diaphragm preparation. The LD50 of Raventoxin-I in mice is 0.772mg/kg. Moreover, Raventoxin-I could exert an effect of exciting firstly and followed with inhibition on the contraction of mouse diaphragm muscle caused by electrically stimulating phrenic nerve, but Raventoxin-EQ could not. Electrophysiological experiments showed that RVTX-I not only could delay TTX-S sodium channels fast inactivation, but also inhibited the peak inward currents of sodium channels on rat DRG neurons, and that RVTX-III could inhibit high-voltage-activated calcium channels on rat DRG neurons. RVTX-HA, RVTX-IIB and RVTX-IIC are a group of natural mutants. RVTX-IIA contains 6 cysteines stabilized by three intracellular disulfide bridges （I-IV, II-V and ffl-VI）, assigned by partial reduction and sequencing. Raventoxin-IIA and Raventoxin-IIB are lethal to tettigoniidaes （Holochlora nawae Mats et Shiraki,） and their LD50 value in tettigoniidaes are 17.99 nmol/g and 54.95nmol/g, respectively. While Raventoxin-HC completely lacks anti-tettigoniidae activity. The structure-activity relationship indicates that the residue Asp5 may be a key amino acid residue for the anti-insect activity of Raventoxin-IIA, while the residue Leu1 is not.更多还原