Acetylcholinesterases from the Disease Vectors Aedes aegypti and Anopheles gambiae: Functional Characterization and Comparisons with Vertebrate Orthologues

Mosquitoes of the Anopheles (An.) and Aedes (Ae.) genus are principal vectors of human diseases including malaria, dengue and yellow fever. Insecticide-based vector control is an established and important way of preventing transmission of such infections. Currently used insecticides can efficiently control mosquito populations, but there are growing concerns about emerging resistance, off-target toxicity and their ability to alter ecosystems. A potential target for the development of insecticides with reduced off-target toxicity is the cholinergic enzyme acetylcholinesterase (AChE). Herein, we report cloning, baculoviral expression and functional characterization of the wild-type AChE genes (ace-1) from An. gambiae and Ae. aegypti, including a naturally occurring insecticide-resistant (G119S) mutant of An. gambiae. Using enzymatic digestion and liquid chromatography-tandem mass spectrometry we found that the secreted proteins were post-translationally modified. The Michaelis-Menten constants and turnover numbers of the mosquito enzymes were lower than those of the orthologous AChEs from Mus musculus and Homo sapiens. We also found that the G119S substitution reduced the turnover rate of substrates and the potency of selected covalent inhibitors. Furthermore, non-covalent inhibitors were less sensitive to the G119S substitution and differentiate the mosquito enzymes from corresponding vertebrate enzymes. Our findings indicate that it may be possible to develop selective non-covalent inhibitors that effectively target both the wild-type and insecticide resistant mutants of mosquito AChE.

按蚊属（Anopheles, An.）和伊蚊属（Aedes, Ae.）蚊虫是疟疾、登革热及黄热病等人类疾病的主要传播媒介。基于杀虫剂的媒介生物防控是阻断此类传染病传播的成熟且重要手段。当前使用的杀虫剂可高效控制蚊媒种群，但日益引发人们对其引发的抗药性、脱靶毒性以及改变生态系统能力的担忧。开发低脱靶毒性杀虫剂的潜在靶点之一是胆碱能酶乙酰胆碱酯酶（acetylcholinesterase, AChE）。本研究报道了冈比亚按蚊（An. gambiae）与埃及伊蚊（Ae. aegypti）野生型乙酰胆碱酯酶基因（ace-1）的克隆、杆状病毒表达及功能表征，其中包含冈比亚按蚊天然存在的杀虫剂抗性突变体（G119S）。通过酶解与液相色谱-串联质谱分析，我们发现分泌蛋白发生了翻译后修饰。蚊源乙酰胆碱酯酶的米氏常数（Michaelis-Menten constant）与周转数（turnover number）均低于小家鼠（Mus musculus）与智人（Homo sapiens）的同源乙酰胆碱酯酶。我们还发现，G119S位点突变降低了底物周转速率以及所选共价抑制剂的抑制效力。此外，非共价抑制剂对G119S突变的敏感性更低，且可区分蚊源酶与相应脊椎动物来源的酶。本研究结果表明，有望开发出可同时有效靶向蚊源野生型及杀虫剂抗性突变型乙酰胆碱酯酶的选择性非共价抑制剂。