天然药物功能蛋白质学科组（赖仞）知识库

摘要蜘蛛是一种古老的产毒动物。蜘蛛毒液通常含有强烈的神经毒素。因此蜘蛛叮咬会导致严重的后果，表现出严重的临床症状。例如肌肉痉挛、唾液分泌、出汗、胸前压迫、高血压、焦虑、精神兴奋和剧痛。死亡很少发生，但偶尔发生肺水肿和心功能不全。多种蜘蛛多肽毒素被认为是离子通道的潜在调节剂，这些具有生物活性的多肽被认为是重要的药理工具。本研究的目的是在间斑寇蛛（Latrodectus tredecimguttatus）毒液低分子量的多肽中寻找可作用于电压门控钠离子通道的神经毒素多肽，并阐明其生理活性和作用机制。我们从哈萨克斯坦西南地区捕获了间斑寇蛛，这是一种黑寡妇蜘蛛，它们性格凶猛，富于攻击性。使用凝胶过滤色谱和反相高压液相色谱进行分离纯化发现其毒液中富含可作用于多种离子通道的多肽神经毒素。使用膜片钳技术对纯化后的组分进一步进行功能筛选。我们发现，黑寡妇蜘蛛毒液中存在两种电压门控钠离子通道NaV1.5的抑制剂Ltre-1和Ltre-2。使用Edamn降解法测定了Ltre-1的一级结构序列，通过转录组文库筛选得到了编码Ltre-1的cDNA序列。使用MALDI-TOF质谱鉴定了Ltre-1和Ltre-2的纯度和分子量。其中Ltre-2由32个氨基酸残基构成，其序列为IICGRKPSDTMCSSKGCVLINAESRRNCEKSL。电压门控钠离子通道是所有动物中电信号的主要启动键，而电信号则是神经活动和肌肉收缩等一系列生理过程的控制基础。在人体中，一共有九种已知的电压门控钠离子通道亚型，在不同的器官和生理过程中发挥作用。其中，NaV1.5在人类心肌细胞中起着至关重要的作用，它控制心肌细胞钠离子的流入，引导心肌细胞动作电位的快速去极化，是许多心脏疾病的重要治疗靶点。因此对黑寡妇蜘蛛毒素的进一步研究将有助于更好地理解电压门控钠通的的工作机制，解释黑寡妇蜘蛛咬伤后引起的临床症状，为黑寡妇蜘蛛咬伤提供备选的治疗药物。本论文以一种黑寡妇蜘蛛—间斑寇蛛为研究对象，运用生物化学、分子生物学、蛋白质组学、电生理学等研究手段，说明了间斑寇蛛毒液是一个具有较大挖掘潜力和研究价值的新型多肽资源库。通过一系列的分离纯化，发现了两个新型多肽毒素Ltre-1和Ltre-2，并初步检测了多肽毒素Ltre-1对NaV1.5的作用效力。提示了Ltre-1和Ltre-2是蜘蛛捕食和防御掠食者的分子基础。基于这些研究结果，在未来的研究工作中，我们拟进行以下工作：1、进一步研究蜘蛛毒素Ltre-1和Ltre-2与电压门控钠离子通道NaV1.5互作的方式；2、进一步挖掘间斑寇蛛毒液中的神经多肽组分，以期获得更多的通道分子探针。以上工作有助于揭示钠通道的工作原理，指导以钠通道为靶点的药物设计开发，对相关通道疾病的治疗具有重要的现实意义。关键词：间斑寇蛛Latrodectus tredecimguttatus，黑寡妇蜘蛛，Ltre-1、Ltre-2，电压门控钠离子通道。

AbstractSpider venom contains a wide repertoire of pharmacologically active compounds, and in the case of some spider species bite, toxins from spider venom can play a fatal role for humans as well as other organisms. Among all the spiders, one could say the bite of Latrodectus tredecimguttatus, known as Black Widow spider, is very dangerous and can even lead to tragic consequences. Following a bite, the toxin gets into the lymphatic system of a person inducing pain in the stomach within a short time and subsequently illicit severe symptoms, such as muscle cramps, spasms, motor unrest, salivation, sweating, precordial oppression, hypertension, board-like abdomen, oliguria, anxiety, mental excitation, and agonizing pain. Death occurs rarely but it is generally due to pulmonary edema and cardiac insufficiency.Studying the chemical composition of Black Widow venom and determination of the key substances acting on various receptors of victim organism, like ion channels, GPCR, GAPA and membrane systems are significant to provide a broad understanding of the mechanism between toxins and ion channels. Especially, voltage-gated sodium channels are responsible for propagating action potentials in excitable cells. NaV1.5 plays a crucial role in the human cardiac muscle, where it enhances the influx of sodium ions via the cell membrane, causing the fast depolarization phase of the cardiac action potential. It is also an important therapeutic target for heart disorders. Various venom-derived peptides have been observed as potential modulators of sodium channels, and these biologically active peptides are an abundant source for pharmacological tools. The aim of this study was to determine the low molecular weight peptides in the venom of the Kazakhstan Black Widow spider (L. tredecimguttatus) and also to describe their physiological potency and channel subtype selectivity. A noble peptide modulator of the human channel NaV1.5 was isolated and identified as Ltre-2. The average molecular mass of the isolated toxin was 3.5 kDa, and the peptide sequence was determined as IICGRKPSDTMCSSKGCVLINAESRRNCEKSL.The spiders (L. tredecimguttatus) were captured from the South and West regions of Kazakhstan. Venom was extracted to find noble neurotoxins and determine their activity on ion channels. Gel filtration chromatographic technique along with reverse-phase high-pressure liquid chromatography (R-P HPLC) was used for extensive purification. The next step was the functional screening of the purified components applying patch clamp electrophysiology. The functional screening revealed the presence of several ion channel modulators in Black Widow spider venom. Subsequently, MALDI-TOF and Edman degradation were applied to determine the molecular weight and peptide sequence. Determination of the peptide sequence allowed us to deduce toxin sequences and establish a sequence similarity with other similar toxins.Further studies of Black widow spider toxins will help to better understand the structure-functional relationships, identification of binding sites on modulated ion channels and also explain the relationship between venom envenomation and symptoms. Key words: Latrodectus tredecimguttatus spider, Ltre-1, Ltre-2, ion channel, black widow spider.