| 其他摘要 | 有毒哺乳动物非常稀少，目前报道的有毒哺乳动物仅有懒猴、鸭嘴兽、北美短尾鼩鼱和其它少数几种鼩鼱，对这些有毒哺乳动物的毒素的研究也是非常的少。本实验中我们研究了云南黑齿鼩鼱（Blarinella griselda）的毒性，并发现其和北美短尾鼩鼱一样具有毒素，实验发现北美短尾鼩鼱毒腺（颌下腺）的匀浆液可以麻痹昆虫并对小鼠具有毒理学作用。我们构建了黑齿鼩鼱（B. griselda）颌下腺的cDNA文库，并从cDNA文库中筛选出一条毒素序列，命名为BTX。通过Blast比对发现BPTX是含有两个结构域的双头Kazal型丝氨酸蛋白酶抑制剂。随后，我们构建了BTX的原核表达载体，并在大肠杆菌Rosetta（DE3）中成功表达。本实验通过对BTX的活性筛选发现BTX作为丝氨酸蛋白酶抑制剂可以在体外抑制胰蛋白酶（trypsin）、弹性蛋白酶（elastase）、纤溶酶（plasmin），这和已报导的丝氨酸蛋白酶抑制剂性质相符。但是进一步活性研究发现BTX能够显著增强凝血因子凝血酶（thrombin）、FXIIa的活性，这与我们所了解的丝氨酸蛋白酶的性质有很大区别。目前的研究还未见完整的能够增强凝血酶（thrombin）和FXIIa活性的动物毒素来源的丝氨酸蛋白酶抑制剂的报道，唯一相关的报道是水蛭素C末端多肽hirugen。hirugen可以增强凝血酶（thrombin）水解底物的能力，而全长水蛭素却抑制凝血酶的活性。本实验通过三维结构模拟BPTX和hirugen分别同凝血酶（thrombin）的结合发现BTX、hirugen都能结合在凝血酶exositeI位点的富含精氨酸的区域，且与thrombin的结合位点序列相似度很高，关键残基的相似度达90%。BTX和凝血因子FXIIa的分子对接分析结果也显示BTX能结合到凝血因子FXIIa中与凝血酶类似的富含精氨酸区域，该区域同样靠近底物口袋，且通过模拟发现BTX作用于该区域可以明显减少底物口袋的位阻，更有利于底物的结合。随后，通过血栓动物模型发现BTX在角叉菜胶诱导的小鼠尾部血栓模型和小鼠尾部出血时间模型上都表现出促凝的作用，这可以说明BTX可能是与凝血相关的毒素。最近的报道显示中性粒细胞弹性蛋白酶与疼痛相关，而外源弹性蛋白酶抑制剂，特别是毒素来源的弹性蛋白酶抑制剂是否具有镇痛作用尚不明确，而我们的相关的疼痛模型实验发现BTX能够起到抑制炎症性疼痛的作用。为了更好的研究BTX的结构与功能，我们通过原核表达得到了BTX的两个结构域BTX1、BTX2纯品。随后，我们对上述两个结构域的活性筛选发现BTX的两个结构域具有不同功能：BTX1体外可以抑制胰蛋白酶（trypsin）、纤溶酶（plasmin），而BTX2仅可以抑制弹性蛋白酶（elastase）。但是二者在增强凝血酶（thrombin）和FXIIa的活性上是一致的。通过相关血栓模型和镇痛模型实验发现BTX1、BTX2都可以促进血液凝固，而只有BTX2可抑制炎症性疼痛。BTX1和BTX2增强凝血酶和FXIIa上的活性相似，说明这两个结构域在结构上可能有某种共性，与上述BPTX的模拟一样，我们也模拟了两个结构域与凝血酶和FXIIa的结合。根据模拟结果我们分析了结合相关的关键氨基酸残基，构建并表达了BTX1-Asp11和BTX2-Glu27两个突变体，对两个突变体的活性验证结果表明BTX1-Asp11和BTX2-Glu27两个突变体还分别保留着抑制胰蛋白酶和弹性蛋白酶的活性，但是两个突变体在增强凝血酶thrombin和凝血因子FXIIa的活性上分别与BTX1和BTX2相比有所减弱，这说明我们模拟的结合区域中的氨基酸残基确实会对这种活性有影响，后续的实验我们将继续寻找并突变其它关键氨基酸残基。对黑齿鼩鼱毒素的研究加深了我们对有毒哺乳动物生存策略的认识。BTX作为丝氨酸蛋白酶抑制剂，表现出了多样的活性，我们的研究首次发现BPTX通过抑制纤溶系统的纤溶酶，同时促进凝血系统的凝血酶和FXIIa的活性来促进凝血。这种作用机制丰富我们对蛋白酶抑制剂与血液系统相互作用的认识，为诠释蛋白酶抑制剂的结构与功能的开辟了一条新的途径。; Venomous mammals are rare. So far, the reported venomous mammals only include loris, platypus, North American short-tailed shrew and few other shrews. In the reported poisonous mammals, the studies of their toxins are also limited. In this study, we investigated the toxicity of Blarinella griselda which is distributed in South Yunnan and is a closely related to the venomous North American short-tailed shrew. Its homogenate of submandibular gland could paralyze and kill insects and is toxic to mice. Furthermore, we identified a toxic component form its submandibular gland. We constructed cDNA library of the submandibular gland of B.griselda, and screened a sequence named BTX from the cDNA library. Blast showed that BTX was a Kazal-type serine protease inhibitor contained two Kazal domain. Then, we constructed the prokaryotic expression vector for BTX and successfully expressed it by the induction of IPTG in the E. coli Rosetta (DE3). Through activity screening, we found BPTX was a serine protease inhibitor which inhibited trypsin, elastase and plasmin. That accords with the properties of some reported serine protease inhibitors. However, further activity characterization showed that BTX significantly enhanced the hydrolyzing ability of thrombin and coagulation factor FXIIa (FXIIa) to their substrates. This activity is different from what we have known about Kazal-type serine protease inhibitors. It has not been reported that a serine protease inhibitor could enhance the hydrolyzing ability of thrombin or FXIIa, only few articles reported that hirugen, a peptide fragment from C-terminus of hirudin enhanced thrombin’ ability to hydrolyze its substrate, though full length hirudin inhibited the activity of thrombin. In the analysis of molecular docking between thrombin and BTX or hirugen, we found that the docking between BTX and thrombin and that between hirugen and thrombin are similar. BTX or hirugen both binds to exosite I of thrombin, which is rich in Arg residues. The similarity of the key binding residues in BTX and hirugen is 90%. Docking analysis of BTX and FXIIa also showed that BTX could bind to a similar Arg-rich area. This area is closed to the substrate pocket, and the binding of BPTX can significantly reduce the stereo-hindrance effect of substrate binding. Further experiments in both mouse tail thrombosis model induced by carrageen and mouse tail bleeding time model showed BTX could accelerate blood coagulation. Some recent reports illustrated that neutrophil elastase is associated with pain, and we questioned whether animal toxins with elastase– inhibiting activity can inhibit pain. Our experiments in analgesic animal model showed BPTX could inhibit inflammatory pain. In another experiment, we obtained the two domains, BTX1 and BTX2 of BPTX, by prokaryotic expression. The activity screening of two domains of BTX showed that they exerted different functions. BPTX1 inhibited trypsin and plasmin, while BTX2 inhibited elastase. However, both domains had the similar ability to enhance the hydrolysis of substrates by thrombin and FXIIa. Though both BTX1 and BTX2 accelerated blood clotting, only BTX2 inhibited inflammatory pain. Since BTX1 and BTX2 have the similar activity in enhance the hydrolyzing ability of thrombin and FXIIa, the two domains may have some common structure features. We also analyzed the molecular docking between BTX1 and thrombin or FXIIa, and that between BTX2 and thrombin or FXIIa, respectively. According to the key amino acids predicted by the docking, we constructed two mutants, BTX1-Asp11 and BTX2-27 Glu, and expressed them in the E. coli Rosetta (DE3). BPTX1-Asp11 and BTX2-27 Glu could inhibit trypsin and elastase, respectively, but their ability to enhance the activity of thrombin and FXIIa were weaker than that of BTX1 and BTX2. We speculated that the amino acid residues in the vicinity of these residues might also contribute to the activity and we will continue to look for the other key residues. The investigation of toxicity of B.griselda adds to our understanding of the survival strategy of venomous mammals. As a serine protease inhibitor, BTX exerts diverse activities. This work is unprecedented to find that proteinase inhibitor promotes blood coagulation both by inhibiting factors such as plasmin in the fibrinolytic system and enhancing factors such as thrombin and FXIIa in the coagulation system. This mechanism riches our understanding of interaction of protease inhibitors with blood system and opens a door for us to take a new look at the structure-function of protease inhibitors. |
修改评论