Vulfius Catherine A, Kasheverov Igor E, Kryukova Elena V, Spirova Ekaterina N, Shelukhina Irina V, Starkov Vladislav G, Andreeva Tatyana V, Faure Grazyna, Zouridakis Marios, Tsetlin Victor I, Utkin Yuri N
Institute of Cell Biophysics, Russian Academy of Sciences, Pushchino, Moscow Region, Russia.
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia.
PLoS One. 2017 Oct 12;12(10):e0186206. doi: 10.1371/journal.pone.0186206. eCollection 2017.
Phospholipases A2 (PLA2s) are enzymes found throughout the animal kingdom. They hydrolyze phospholipids in the sn-2 position producing lysophospholipids and unsaturated fatty acids, agents that can damage membranes. PLA2s from snake venoms have numerous toxic effects, not all of which can be explained by phospholipid hydrolysis, and each enzyme has a specific effect. We have earlier demonstrated the capability of several snake venom PLA2s with different enzymatic, cytotoxic, anticoagulant and antiproliferative properties, to decrease acetylcholine-induced currents in Lymnaea stagnalis neurons, and to compete with α-bungarotoxin for binding to nicotinic acetylcholine receptors (nAChRs) and acetylcholine binding protein. Since nAChRs are implicated in postsynaptic and presynaptic activities, in this work we probe those PLA2s known to have strong presynaptic effects, namely β-bungarotoxin from Bungarus multicinctus and crotoxin from Crotalus durissus terrificus. We also wished to explore whether mammalian PLA2s interact with nAChRs, and have examined non-toxic PLA2 from porcine pancreas. It was found that porcine pancreatic PLA2 and presynaptic β-bungarotoxin blocked currents mediated by nAChRs in Lymnaea neurons with IC50s of 2.5 and 4.8 μM, respectively. Crotoxin competed with radioactive α-bungarotoxin for binding to Torpedo and human α7 nAChRs and to the acetylcholine binding protein. Pancreatic PLA2 interacted similarly with these targets; moreover, it inhibited radioactive α-bungarotoxin binding to the water-soluble extracellular domain of human α9 nAChR, and blocked acetylcholine induced currents in human α9α10 nAChRs heterologously expressed in Xenopus oocytes. These and our earlier results show that all snake PLA2s, including presynaptically active crotoxin and β-bungarotoxin, as well as mammalian pancreatic PLA2, interact with nAChRs. The data obtained suggest that this interaction may be a general property of all PLA2s, which should be proved by further experiments.
磷脂酶A2（PLA2s）是在整个动物界都能发现的酶。它们水解sn-2位的磷脂，产生溶血磷脂和不饱和脂肪酸，这些物质会损害细胞膜。蛇毒中的PLA2s具有多种毒性作用，并非所有作用都能用磷脂水解来解释，而且每种酶都有特定的作用。我们之前已经证明，几种具有不同酶活性、细胞毒性、抗凝和抗增殖特性的蛇毒PLA2s能够降低椎实螺神经元中乙酰胆碱诱导的电流，并与α-银环蛇毒素竞争结合烟碱型乙酰胆碱受体（nAChRs）和乙酰胆碱结合蛋白。由于nAChRs参与突触后和突触前活动，在这项研究中，我们探究了那些已知具有强烈突触前作用的PLA2s，即多环眼镜蛇的β-银环蛇毒素和三色矛头蝮的响尾蛇毒素。我们还希望探究哺乳动物的PLA2s是否与nAChRs相互作用，并研究了猪胰腺的无毒PLA2。结果发现，猪胰腺PLA2和突触前β-银环蛇毒素阻断了椎实螺神经元中由nAChRs介导的电流，其半数抑制浓度（IC50）分别为2.5和4.8 μM。响尾蛇毒素与放射性α-银环蛇毒素竞争结合电鳐和人α7 nAChRs以及乙酰胆碱结合蛋白。胰腺PLA2与这些靶点的相互作用方式类似；此外，它抑制放射性α-银环蛇毒素与人α9 nAChR水溶性细胞外结构域的结合，并阻断非洲爪蟾卵母细胞中异源表达的人α9α10 nAChRs中乙酰胆碱诱导的电流。这些结果以及我们之前的结果表明，所有蛇类PLA2s，包括具有突触前活性的响尾蛇毒素和β-银环蛇毒素，以及哺乳动物胰腺PLA2，都与nAChRs相互作用。所获得的数据表明，这种相互作用可能是所有PLA2s的普遍特性，这有待进一步实验证实。
