Venom and Toxin Research Programm, Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597.
Biochimie. 2010 Dec;92(12):1854-66. doi: 10.1016/j.biochi.2010.07.012. Epub 2010 Aug 17.
Phospholipase A(2) (PLA(2)), a common toxic component of snake venom, has been implicated in various pharmacological effects. In this study, a basic myotoxic PLA(2), named EcTx-I was isolated from Echis carinatus snake venom by using gel filtration on Superdex G-75, and reverse phase HPLC on C18 and C8 Sepharose columns. PLA(2), EcTx-I was 13,861.72 molecular weight as estimated by MALDI-TOF (15 kD by SDS-PAGE), and consisted of 121 amino acid residues cross-linked by seven disulfide bonds. The N-terminal sequences revealed significant homology with basic myotoxic PLA(2)s from other snake venoms. The purified PLA(2) EcTx-I was evaluated (250 μg/ml) for bactericidal activity of a wide variety of human pathogens against Burkholderia pseudomallei (KHW&TES), Enterobacter aerogenes, Escherichia coli, Proteus vulgaris, Proteus mirabilis, Pseudomonas aeruginosa and Staphylococcus aureus. EcTx-I showed strong antibacterial activity against B. pseudomallei (KHW) and E. aerogenes among the tested bacteria. Other Gram-negative and Gram-positive bacteria showed only a moderate effect. However, the Gram-positive bacterium E. aerogenes failed to show any effect on EcTx-I protein at tested doses. The most significant bacteriostatic and bactericidal effect of EcTx-I was observed at MICs of >15 μg/ml against (B. pseudomallei, KHW) and MICs >30 μg/ml against E. aerogenes. Mechanisms of bactericidal and membrane damaging effects were proved by ultra-structural analysis. EcTx-I was able to induce cytotoxicity on THP-1 cells in vitro as well as lethality in BALB/c mice. EcTx-I also induced mild myotoxic effects on mouse skin, but was devoid of hemolytic effects on human erythrocytes up to 500 μg/ml. It is shown that the toxic effect induced by E. carinatus venom is due to the presence of myotoxic PLA(2) (EcTx-I). The result also corroborates the hypothesis of an association between toxic and enzymatic domains. In conclusion, EcTx-I displays a heparin binding C-terminal region, which is probably responsible for the cytotoxic and bactericidal effects.
磷脂酶 A(2)（PLA(2)）是蛇毒中的一种常见毒性成分，与多种药理学作用有关。在这项研究中，从 Echis carinatus 蛇毒中分离出一种碱性肌肉毒性 PLA(2)，命名为 EcTx-I。通过 Superdex G-75 凝胶过滤和 C18 和 C8 Sepharose 柱反相 HPLC 进行分离。PLA(2)，EcTx-I 的分子量为 13861.72，根据 MALDI-TOF（SDS-PAGE 为 15 kD）估计，由 121 个氨基酸残基通过 7 个二硫键交联而成。N 端序列与其他蛇毒中的碱性肌肉毒性 PLA(2)具有显著同源性。纯化的 PLA(2) EcTx-I 用于评估（250μg/ml）针对广泛的人类病原体对伯克霍尔德氏菌（KHW&TES）、产气肠杆菌、大肠杆菌、普通变形杆菌、奇异变形杆菌、铜绿假单胞菌和金黄色葡萄球菌的杀菌活性。EcTx-I 对测试的细菌中伯克霍尔德氏菌（KHW）和产气肠杆菌具有很强的抗菌活性。其他革兰氏阴性菌和革兰氏阳性菌仅表现出中等作用。然而，革兰氏阳性菌产气肠杆菌在测试剂量下对 EcTx-I 蛋白没有任何作用。EcTx-I 对（B. pseudomallei，KHW）的 MIC 值>15μg/ml 和对产气肠杆菌的 MIC 值>30μg/ml 时，表现出最显著的抑菌和杀菌作用。超微结构分析证明了杀菌和破坏膜的作用机制。EcTx-I 能够在体外诱导 THP-1 细胞的细胞毒性以及 BALB/c 小鼠的致死性。EcTx-I 还在小鼠皮肤上引起轻微的肌肉毒性作用，但在高达 500μg/ml 的浓度下对人红细胞没有溶血作用。结果表明，E. carinatus 毒液引起的毒性作用是由于存在肌肉毒性 PLA(2)（EcTx-I）。结果还证实了毒性和酶结构域之间存在关联的假设。总之，EcTx-I 显示出肝素结合的 C 端区域，这可能是细胞毒性和杀菌作用的原因。
