中国科学院昆明动物研究所机构知识库

蜈蚣是一种产毒动物，也是一类典型的依靠毒腺和毒液系统进行捕食和防御的节肢动物。蜈蚣毒液成分繁杂，包含多种活性成分，其中各种毒素分子的作用靶点不尽相同，功能各异，因此研究蜈蚣毒素并挖掘其功能，已成为产毒动物研究的一个重要分支。已有研究结果表明，多种蜈蚣毒素的作用靶点为离子通道，且有一定的高效性和专一性。在此基础上，我们拟借助间脚蜈蚣多肽毒素与离子通道之间的相互作用关系，研究毒素与通道的结构功能以及发挥功能的生物学意义。同时，也以离子通道作为工具，研究通道与毒素相互作用的分子机制，了解蜈蚣毒液的成分和功能，为药物筛选和新药的改造研发，提供一种新的工作思路。海南间脚蜈蚣主要分布在中国海南省，目前对其毒液系统的研究成果较少。因此，研究和发掘间脚蜈蚣毒液系统中的活性分子，仍然是蜈蚣毒液研究工作的一个重要方向。本论文以海南间脚蜈蚣毒液系统为研究对象，筛选有生物学活性的毒素分子并对其展开研究。按照“电脉冲刺激采集毒液—葡聚糖凝胶色谱层析分离蜈蚣粗毒—活性初测—HPLC-C18制备柱粗略分离活性成分—活性再测试—HPLC-C18分析柱精细分离活性成分—活性终测—MALDI-TOF质谱鉴定纯度和分子量—Edman降解测其序列”等步骤，从间脚蜈蚣毒液中分离纯化得到一种新型的多肽毒素分子，MALDI-TOF质谱鉴定其荷质比为3698，即其分子量为3698Da，对这一纯品进行Edman降解测序，得其氨基酸序列：AESKGCSAERVKHCLERKCASPCCRHGKCHCGCK。测序结果表明，这一多肽毒素含有34个氨基酸残基，包含八个半胱氨酸，两两配对形成四对二硫键，将其命名为AK34。据其氨基酸序列设计引物构建间脚蜈蚣的cDNA文库，克隆得到能够正确编码AK34的cDNA序列，并成功表达出活性和功能与天然AK34相同的成熟多肽。BLAST比对结果表明，目前尚未出现任何与AK34同源的蛋白，即AK34是一种新型多肽毒素。多肽分子AK34仅含有34个氨基酸残基，却包含8个半胱氨酸，形成四对二硫键。因蛋白分子中每对二硫键的还原动力学不尽相同，借助变性剂盐酸胍和还原剂TCEP，对多肽分子AK34中4对二硫键进行不同程度的还原，通过对还原产物进行烷基化修饰测序和烷基化修饰后酶切二级质谱两种方法，进行AK34的二硫键解析定位。结果表明，AK34分子中的四对二硫键配对方式为：Cys6-Cys14、Cys19-Cys31、Cys23-Cys33、Cys24-Cys29。这种低能耗、高密度的二硫键组织形式，对维系这一多肽的核心结构和生物学功能有着重要的作用，且这一特点在其他已有记录的蛋白中尚未发现。成功解析这一新型多肽毒素的二级结构，对其功能的研究有着重要意义。在成功揭示毒素多肽AK34的一级和二级结构之后，我们通过膜片钳电生理实验研究AK34分子的生物学活性，结果表明500μM的AK34与10μM辣椒素，或3mM2-APB都可完全激活TRPV1通道，说明AK34是一种天然存在的TRPV1离子通道激活剂。且AK34对TRPV1通道的激活具有浓度依赖性，通过Hill函数拟合得到多肽毒素AK34对TRPV1离子通道的半效激活常数为EC50=202.8±49.1μM。了解了多肽毒素AK34的作用靶点及其作用效果之后，我们还展开了间脚蜈蚣毒液酸性微环境对毒素分子功能发挥的意义研究。间脚蜈蚣毒液的pH值为6.2，呈弱酸性，毒液中的各种活性成分均处于酸性微环境中。通过设计不同PH值的实验组，我们发现，pH=6.2时，AK34对TRPV1离子通道的半效激活常数为EC50=50±19μM，较之于中性环境，EC50明显降低，表明酸性环境可以增强AK34与TRPV1离子通道的亲和力，为蜈蚣毒素功能发挥提供有利条件。同时，AK34对TRPV1通道的激活存在着明显的脱敏现象，这一结果表明，AK34对TRPV1通道的激活极有可能采取的是与通道热激活途径相同或者相关的方式，这对我们研究毒素AK34发挥作用的分子机制和解析TRPV1离子通道热激活途径尚未解密的相关影响因素，有着重要的启发作用。本课题研究取得了以下四个方面的进展：1）从间脚蜈蚣毒液中分离得到一种靶向作用于TRPV1离子通道的新型毒素分子AK34，AK34与任何已知蛋白都没有序列同源性；2）多肽毒素AK34是首个被发现的包含四对二硫键的蜈蚣毒素分子，这种高效低耗的二硫键组织形式，对维系这一多肽的核心结构和生物学功能有着重要的意义；3）AK34对TRPV1通道激活同时具有浓度依赖性和PH依赖性，且在低pH条件下能高效激活TRPV1；4）AK34对TRPV1通道的激活存在脱敏现象，表明AK34激活TRPV1通道极有可能是通过通道热激活途径发挥作用。本论文的研究意义包含以下四点：1）通过分离纯化得到一种新型毒素分子AK34，AK34为首个含有四对二硫键的蜈蚣毒素，且与其他已知蛋白都没有序列同源性，丰富了我们对产毒动物毒液系统和毒素分子多态性的认识；2）多肽毒素AK34能在低pH条件下高效激活TRPV1通道，进一步支持酸性环境优化“毒素-受体”结合的理论，说明酸碱微环境等因素的变化可能也是毒液系统进化的一个重要策略；3）TRPV1离子通道是一种重要的疼痛信号感受器，毒素分子AK34能够靶向作用TRPV1通道，这为蜈蚣叮咬引发的疼痛反应机理研究开辟了一条新路径，也为蜈蚣叮咬后止痛药品的研发提供了一种新思路；4）AK34激活TRPV1通道存在脱敏现象，启发我们探究该毒素是否通过通道的热激活途径发挥作用。综上所述，本论文以间脚蜈蚣多肽毒素AK34的结构功能解析为研究重点，揭示了这一多肽毒素复杂的分子结构和生物学功能，也论证了毒液酸性环境可以优化“毒素-受体”结合的理论。激活后脱敏的现象则启发我们进一步深入探究该毒素是否通过通道的热激活途径发挥作用。此外，根据蜈蚣多肽毒素的作用靶点进行止痛药物和治疗药物的研发，或是以毒素分子为探针，靶向作用离子通道来研究通道结构功能，都是新时期研究产毒动物毒液系统的题中之义。

Centipede is one kind of toxicogenic animal, and a typical arthropod which relies on the venom gland and venom system for predation and defense. The ingredient of Centipede venom is very complex, including a variety of different toxins, those toxins have different molecular targets and different functions, so study the centipede toxin and excavate its function, has become an important branch of the toxin-producing animal research. Previous studies have shown that the target of many polypeptide toxins in centipede are ion channels, and the interaction between toxin and ion channel is efficient and specific. On the basis of those results, we intend to use the interaction of polypeptide toxin and ion channel, to study the structure and function of both toxin and channel, and the biological significance of them. At the same time, we also use ion channel as a tool, to study the molecular mechanism of the interaction between channel and toxin, furthermore, to understand the composition and function of the centipede venom. Those work will provide a new thought for drug screening, the research and development of new drugs. this kind of centipede is mainly distributed in Hainan province, and the research result on its venom system is very little. Therefore, the study and exploration of the active molecules in the centipede venom system is still an important direction of the research work of centipede venom.In this paper, we choose the centipede venom system as the research object, screening for the biological active toxin molecules and then do researches about it. In accordance with the "electrical stimulation to collect venom - glucan gel chromatography to separate centipede crude venom - biological activity preliminary test - HPLC, using C18 column to separate the active ingredients roughly-biological activity test again - HPLC, using C18 column to separate the active ingredients finely - biological activity final test - MALDI-TOF mass spectrometry to identify the purity and molecular weight - Edman degradation sequencing", and then we separate and purify a new kind of peptide toxin from the centipede venom, MALDI-TOF mass spectrometry result shows that the charge-mass ratio of this toxin is 3698, that is to say, its molecular weight is 3698 Da. Edman degradation sequencing its amino acid sequence is: AESKGCSAERVKHCLERKCASPCCRHGKCHCGCK. The sequencing results shows that this toxin contains 34 amino acid residues, containing eight cysteines and four pairs of disulfide bonds, then we named it as AK34. According to its amino acid sequence, we design primers to establish the centipede cDNA library, then we get correct AK34 cDNA sequence and express this protein, which has same activity and function as the natural AK34 mature peptide.BLAST comparison result shows that there has no protein which is homologous with AK34, that is to say, AK34 is a novel polypeptide toxin. AK34 contains only 34 amino acid residues, but contains 8 cysteines, forming four pairs of disulfide bonds. As we known, the reduction dynamics of each pair of disulfide bond is not the same, we use guanidine hydrochloride as denaturant and TCEP as reductant, to reduct four pairs of disulfide bond of AK34,by means of alkylation modification of reduction products, then sequencing, or digest the alkylating product to do the secondary mass spectrometry. Use those two methods to analysis the disulfide bond constitution of AK34. The results show that the four pairs of disulfide bonds of AK34 are cys6-cys14, cys19-cys31, cys23-cys33, cys24-cys29. This kind of low energy, high density of disulfide bond consumption, is critical to maintain the core structure and biological function of AK34, this characteristic hasn’t been found in protein database. Resolving the secondary structure of this new toxin has great significance in the study of AK34.After resolving the primary and secondary structure of AK34, we use the patch clamp electrophysiological experiment to test the biological activity of AK34, the result shows that 500um AK34 and 10um capsaicin, or 3mM 2-APB can activate TRPV1 channel completely, proving that AK34 is a kind of natural existent activator of TRPV1 ion channel. In addition, the activation is concentration dependent and pH dependent. We use the Hill function to deal with the experimental data, then get the semi-effect activation constant EC50, which is 202.8± 49.1μM.After understanding the action target and the effect of AK34, we also carry out a study on the significance of the acidic microenvironment of the centipede venom. The PH value of the centipede venom is 6.2, which is weakly acidic, and all the active components in the venom are in acidic microenvironment. By designing different PH value as the experimental group, we find that when the environment PH is 6.2, the semi-effect activation constant EC50 is 50±19 μm. Compared with neutral environment, EC50 decreased significantly,this result shows that acidic environment can enhance the affinity of AK34 andTRPV1 ion channel and provide favorable conditions for the centipede toxin function.At the same time, we find that there will have a desensitization phenomenon when AK34 activate TRPV1 channel,that is to say, maybe this activation share the same or relavant pathway with the heat activation of TRPV1 channel. This is an important inspiration for us to study the molecular mechanism of the toxin AK34 and the relevant influencing factors that have not been decrypted in the analysis of the thermal activation pathway of TRPV1 ion channel.This project research has made progress in the following four aspects: 1) separate and purify a new type of toxin from centipede venom, which can target on TRPV1 ion channel, and there has no protein which is homologous with AK34; 2) AK34 is the first time purified centipede toxin which contains four pairs of disulfide bond, This kind of low energy, high density of disulfide bond consumption, is critical to maintain the core structure and biological function of AK34; 3) AK34 can activate TRPV1 channel with both concentration dependence and PH dependence. The activation can be more efficient under low PH conditions; 4)The activation have the desensitization phenomenon, shows that maybe this activation share the same or relevant pathway with the heat activation of TRPV1 channel.The significance of this paper includes the following four points: 1)After separation and purification,we obtain a new type of centipede toxin, AK34 is the first time purified centipede toxin which contains four pairs of disulfide bond and there has no protein which is homologous with AK34, this result enriches our horizon of the enterotoxigenic animal’s venom system and the polymorphism of toxin molecules; 2)AK34 can efficiently activate TRPV1 channel at low PH condition, this result further support the acid environment optimize the combination of the toxin and receptor, we assume that environment factors such as acid or alkali changes,may be another important strategy for the venom system evolution; 3) TRPV1 ion channel is an important pain signal sensor and AK34 can target on TRPV1 exclusively, maybe this result can open a new path to study the pain reaction mechanism of the centipede bite. At the same time,it will provide a new thinking to develop the analgesic drug to deal with centipede biting; 4) The desensitization after AK34 activate TRPV1 channel inspires us to explore whether this toxin play its role through the channel's thermal activation pathway. To sum up, study of the structure and function of centipede toxin AK34 is the point of this project, we reveal the complex molecular structure and biological function of this toxin, then demonstrate the acid environment of venom can optimize the combination of the toxin and receptor. The desensitization after AK34 activate TRPV1 channel inspires us to explore whether this toxin play its role through the channel's thermal activation pathway. By analogy, the specific interaction between toxin and ion channel can make contributions to the development of analgesic drugs and therapeutic drugs, or using toxin molecule as a probe to target on ion channels, to study the structure and function of ion channel. Those are very important to study the venom system of toxicogenic animal in the new period 。