Crystal structure and activating effect on RyRs of AhV_TL-I, a glycosylated thrombin-like enzyme from Agkistrodon halys snake venom

A snake venom thrombin-like enzyme (SVTLE) from Agkistrodon halys pallas venom was isolated by means of a two-step chromatographic procedure. The purified enzyme, named AhV_TL-I, showed fibrinogenolytic activity against both the A alpha and B beta chains of bovine fibrinogen. Unlike the other SVTLEs, AhV_TL-I has poor esterolytic activity upon BAEE substrate. The N-terminal sequence of AhV_TL-I was determined to be IIGGDEXNINEHRFLVALYT, and the molecular mass was confirmed to 29389.533 Da by MALDI-TOF mass spectrometry. Its complete cDNA and derived amino acid sequence were obtained by RT-PCR. The crystal structure of AhV_TL-I was determined at a resolution of 1.75 . A disaccharide was clearly mapped in the structure, which involved in regulating the esterolytic activity of AhV_TL-I. The presence of the N-glycan deformed the 99-loop, and the resulting steric hindrances hindered the substrates to access the active site. Furthermore, with the carbohydrate moiety, AhV_TL-I could induce mouse thoracic aortic ring contraction with the EC50 of 147 nmol/L. Besides, the vasoconstrictor effects of AhV_TL-I were also independent of the enzymatic activity. The results of [Ca2+](i) measurement showed that the vasoconstrictor effects of AhV_TL-I were attributed to Ca2+ releasing from Ca2+ store. Further studies showed that it was related to the activation of ryanodine receptors (RyRs). These offer new insights into the snake SVTLEs functions and provide a novel pathogenesis of A. halys pallas venom.

通过两步色谱法从蝮蛇（Agkistrodon halys pallas）蛇毒中分离出一种蛇毒类凝血酶（SVTLE）。纯化后的酶被命名为AhV_TL - I，它对牛纤维蛋白原的Aα链和Bβ链均显示出纤维蛋白原溶解活性。与其他蛇毒类凝血酶不同的是，AhV_TL - I对BAEE底物的酯解活性较弱。AhV_TL - I的N端序列经测定为IIGGDEXNINEHRFLVALYT，通过基质辅助激光解吸电离飞行时间质谱（MALDI - TOF）确定其分子量为29389.533 Da。通过逆转录聚合酶链反应（RT - PCR）获得了其完整的cDNA及推导的氨基酸序列。AhV_TL - I的晶体结构在1.75 Å的分辨率下被测定。结构中清晰地定位出一种二糖，它参与调节AhV_TL - I的酯解活性。N - 聚糖的存在使99环变形，由此产生的空间位阻阻碍了底物进入活性位点。此外，由于含有碳水化合物部分，AhV_TL - I能够诱导小鼠胸主动脉环收缩，半数有效浓度（EC50）为147 nmol/L。而且，AhV_TL - I的血管收缩作用也与酶活性无关。细胞内钙离子浓度（[Ca2 +](i)）测量结果表明，AhV_TL - I的血管收缩作用归因于钙离子从钙库中释放。进一步研究表明，这与兰尼碱受体（RyRs）的激活有关。这些研究为蛇毒类凝血酶的功能提供了新的见解，并为蝮蛇蛇毒的发病机制提供了新的依据。