天然药物功能蛋白质学科组（赖仞）知识库

蜈蚣是一种古老且捕食能力强的产毒动物。尽管它们作为传统中药材使用了几个世纪，但相比于其他产毒动物毒液（蛇毒、蜘蛛毒、蝎毒等）研究，蜈蚣毒液的研究起步较晚。近年来，生物大分子结构与功能研究逐渐成为生命科学领域的研究前沿，而获得高效调控其结构和功能的配体就成为了日益增长的需求。蜈蚣的毒液成分经历了数十亿年的进化，可能成为获得高亲和力的新型多肽毒素的分子资源库。本课题组在前期对蜈蚣毒液成分的研究已经取得了一些进展，揭示了蜈蚣的毒液成分中含有极其丰富的多肽类毒素。这些多肽毒素富含新型的二硫键构型及更为新颖的高级结构，其中一些多肽毒素已经被证明具有对生物大分子的高亲和力，例如SsTx，BmP01和RhTx等。这些多肽毒素对生物大分子结构和功能的高效调节频繁参与了蜈蚣的捕食、防御以及竞争。综上，这些特性表明了蜈蚣毒液是一种高效调控生物大分子结构功能的新型多肽资源库，其中更多的分子资源还有待进一步挖掘和研究。本论文以中国广东地区的红龙蜈蚣（Scolopendra subspinipes deaani china）毒液作为研究对象，旨在从该毒液成分中获得高效调控生物大分子的新型多肽毒素。该蜈蚣具有强壮的鳌枝，其毒液腺每次能分泌10-20 μL具有极高生物活性的毒液。本论文系首次对红龙蜈蚣毒液成分进行分离纯化和功能研究。按照“电刺激法采集毒液—Sephadex G50葡聚糖凝胶色谱分离蜈蚣粗毒—以分子量最小的4号峰作为研究对象—RP-HPLC C18制备柱分离4号峰—活性检测—RP-HPLC C18分析柱分离纯化活性成分—活性终测—MALDI-TOF质谱鉴定纯度和分子量—Edman降解法测序结合转录组筛查”等步骤。获得一个暂未研究报道的新型多肽毒素FfTx，经BLAST序列比对发现，FfTx是一种具有新型结构的多肽毒素，与已知的多肽毒素并无序列同源性。FfTx成熟肽分子量为3729.706 Da，氨基酸序列为SGAHWHLEKMCQQNCNYMEGCKNYRFCRGKI，共31个氨基酸，其中含有4个半胱氨酸，形成1-3、2-4的二硫键配对方式。电生理实验数据表明，FfTx具有较强的电压门控钠离子通道亚型专一性，其抑制电压门控钠离子通道NaV1.5的半效抑制浓度为50 μM。相比于其他的电压门控钠离子通道亚型，FfTx对NaV1.5的选择性在10倍以上。我们进一步探索了FfTx对NaV1.5门控动力学的影响，实验结果表明，FfTx并不影响NaV1.5的稳态激活和稳态失活曲线。因此，我们推测FfTx不影响电压门控钠通道的电压敏感元件，可能以结合NaV1.5孔区的方式阻断其离子流动。基于前期的研究基础，本论文进一步探讨了红龙蜈蚣毒素FfTx在其他几种蜈蚣（少棘蜈蚣Scolopendra subspinipes mutilans、巨人蜈蚣Scolopendra subspinipes Vietnam和间脚蜈蚣Scolopendra subspinipes subspinipes）毒液中的分布情况。通过转录组数据分析，我们发现在巨人蜈蚣转录组中含有FfTx的同源多肽毒素，在少棘蜈蚣和间脚蜈蚣转录组中含有与FfTx仅一个氨基酸差异的同源多肽毒素。这一结果说明FfTx或其同源毒素在蜈蚣类动物中是一种保守存在的多肽毒素；结合FfTx与NaV1.5高效相互作用的特征，该毒素可能是蜈蚣生存策略的重要物质基础。我们推测，当蜈蚣进行捕食和防御时，通过毒鳌将其毒液快速注射到天敌或猎物的体内，活性多肽毒素FfTx或FfTx的类似物会快速抑制其天敌或猎物的电压门控钠离子通道（如NaV1.5和NaV1.7）。由于这些电压门控钠离子通道与心脏功能及疼痛传导的重要联系，FfTx可能引起天敌或猎物的心脏功能障碍同时消除捕食叮咬过程中的痛感，从而达到捕食和防御的目的。本论文以红龙蜈蚣毒液为研究对象，运用分子生物学、生物化学、电生理学等整合生物学研究手段，1）说明了红龙蜈蚣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；2）发现了新型多肽毒素FfTx；3）揭示了多肽毒素FfTx与NaV1.5的相互作用；4）提示FfTx或其同源毒素是多种蜈蚣叮咬引发心脏节律失调等临床症状或其捕食的潜在策略分子。在未来的研究工作中，我们将基于目前的研究结果，1）进一步探索红龙蜈蚣毒素FfTx与电压门控钠离子通道相互作用的机制；2）进一步深入挖掘红龙蜈蚣毒液成分，以期获得更多的分子探针和候选药物分子。 

Centipedes are one of the oldest and most successful venomous terrestrial predators. Despite their use for centuries in traditional medicine, centipede venom study lag behind compared to other venomous animals like snake venom、spider venom and scorpion venom. Recently, studying structures and functions of biological macromolecules has gradually become the research frontier in the field of life sciences，moreover, the acquisition of ligands that efficiently regulate their structures and functions has increased in demand. The venom components of centipedes have evolved for over billions years and may become a molecular resource bank of novel peptide toxins with high affinity. For years, our lab has made progress in the study of centipedes’ venom components, revealing that they constitute diverse peptide toxins. These peptide toxins are rich in novel disulfide bond configurations and more novel high-level structures, some of which have been shown to have high affinity for bio-macromolecules, for example, SsTx, BmP01 and RhTx. Regulating efficiently the structures and functions of bio-macromolecules by these peptide toxins is frequently involved in the predation, defense and competition of centipedes. In summary, these characteristics indicate that centipedes’ venom is novel polypeptides resources that efficiently regulate the structural functions of biological macromolecules hence more molecular resources should be further explored and studied in the future. In the present study, we used venom of the Scolopendra subspinipes deaani china with an aim of obtaining novel polypeptide toxin that efficiently regulates biological macromolecules from the venom component. This centipede has strong poisonous fangs, and its venom gland secretes 10-20 microliters of venom per time with extremely high biological activity. Its venom was first isolated and purified with the "electrical stimulation to collect venom—glucan gel chromatography to separate centipede crude venom—chose the smallest molecular weight peak as the research object—RP-HPLC, using C18 preparative column to separate the fourth peak—biological activity test—RP-HPLC, using C18 analytical column to separate and purify the active ingredients—biological activity final test—MALDI-TOF mass spectrometry to identify the purity and molecular weight—Edman degradation sequencing combine transcriptomic ".Obtained a novel peptide toxin named it as FfTx which has not been reported yet，Sequence Alignment by BLAST, FfTx is a polypeptide toxin with a novel structure that has no sequence homology to known polypeptide toxins.The mature peptide molecular weight is 3729.706 Da and its amino acid sequence is SGAHWHLEKMCQQNCNYMEGCKNYRFCRGKI, it consists of 31 amino acid residues, including 4 cysteines to form 1-3, 2-4 disulfide bond pairing mode. Electrophysiological experimental data showed that FfTx has strong subtype specificity, and its half-effect inhibitory concentration of inhibiting voltage-gated sodium channel NaV1.5 is 50 μM. Compared to other voltage-gated sodium channel subtypes, FfTx has a selectivity of more than 10 times for NaV1.5. We further explored the effect of FfTxon NaV1.5 gated kinetics, the experimental results show that FfTx does not affect the steady-state activation and steady-state inactivation curves of NaV1.5. Therefore, we speculate that FfTx does not affect the voltage sensor of the voltage-gated sodium channel and may block its ion flow by binding to the NaV1.5 pore region. Based on the previous research basis, this paper further explored the distribution of FfTx in other centipede venoms. By screening the FfTx sequence in transcriptome of Scolopendra subspinipes Vietnam、Scolopendra subspinipes subspinipes and Scolopendra subspinipes mutilans, we found a homologous toxin in Scolopendra subspinipes Vietnam with a sequence completely similar to FfTx, while the sequence had one amino acid difference for Scolopendra subspinipes subspinipes and Scolopendra subspinipes mutilans. This indicates that FfTx or its homologous toxin is a conserved polypeptide toxin in centipedes; combined with the function of the polypeptide toxin FfTx, this toxin may be a survival strategy molecule in nature. We speculate that, when centipede is preying and defending, the venom is quickly injected into the body of its enemy or prey by poisonous fangs. Consequently, the active peptide toxin FfTx inhibit the voltage-gated sodium channels (NaV1.5、NaV1.7), causing cardiac dysfunction and also eliminating the pain in the process of predation and bite, thus achieving the purpose of predation and defense. The venom of Scolopendra subspinipes deaani china was used as as the research subject. Integrated biological research methods such as molecular biology, biochemistry, and electrophysiology, reveals the following; 1) Scolopendra subspinipes deaani china venom is a new type of peptides resource with great potential for explore and research; 2 FfTx is a novel peptide toxin; 3) The peptide toxin FfTx interacts with NaV1.5; 4) the peptide toxin FfTx is a material basis of the survival strategy. For future research work, we propose the following based on the current results, 1) Exploring further the molecular mechanism of interaction between FfTx and voltage-gated sodium ion channels; 2) Further investigation of the composition of the Scolopendra subspinipes deaani china venom, in order to obtain more molecular probes and candidate drug molecules.