Department of Entomology, Centro de Pesquisas Aggeu Magalhães/FIOCRUZ, Av. Moraes Rego s/n, Cidade Universitária, Recife-PE 50670-420, Brazil.
Insect Biochem Mol Biol. 2010 Aug;40(8):604-10. doi: 10.1016/j.ibmb.2010.05.007. Epub 2010 Jun 2.
Aedes aegypti larvae are refractory to the insecticidal binary (Bin) toxin from Bacillus sphaericus, which is not able to bind to its target tissue in the larval midgut. In contrast, Culex pipiens larvae are highly susceptible to that toxin, which targets its midgut brush border membranes (BBMF) through the binding of the BinB subunit to specific receptors, the Cpm1/Cqm1 membrane-bound α-glucosidases. The identification of an Ae. aegypti gene encoding a Cpm1/Cqm1 orthologue, here named Aam1, led to the major goal of this study which was to investigate its expression. The aam1 transcript was found in larvae and adults from Ae. aegypti and a ≈73-kDa protein was recognized by an anti-Cqm1 antibody in midgut BBMF. The Aam1 protein displayed α-glucosidase activity and localized to the midgut epithelium, bound through a GPI anchor, similarly to Cpm1/Cqm1. However, no binding of native Aam1 was observed to the recombinant BinB subunit. Treatment of both proteins with endoglycosidase led to changes in the molecular weight of Aam1, but not Cqm1, implying that the former was glycosylated. The findings from this work rule out lack of receptors in larval stages, or its expression as soluble proteins, as a reason for Ae. aegypti refractoriness to Bin toxin.
埃及伊蚊幼虫对球形芽孢杆菌的杀虫二元毒素(Bin)具有抗性,该毒素无法与幼虫中肠结合其靶组织。相比之下,库蚊幼虫对该毒素高度敏感,该毒素通过 BinB 亚基与特定受体(Cpm1/Cqm1 膜结合α-葡萄糖苷酶)结合,靶向其中肠刷状缘膜(BBMF)。埃及伊蚊编码 Cpm1/Cqm1 同源物的基因 Aam1 的鉴定,促使我们开展了本研究,主要目的是研究其表达情况。在埃及伊蚊幼虫和成虫中均发现了 aam1 转录本,且抗 Cqm1 抗体在中肠 BBMF 中识别出约 73kDa 的蛋白。Aam1 蛋白具有α-葡萄糖苷酶活性,定位于中肠上皮细胞,通过 GPI 锚与细胞结合,类似于 Cpm1/Cqm1。然而,没有观察到天然 Aam1 与重组 BinB 亚基结合。两种蛋白经内切糖苷酶处理后,Aam1 的分子量发生变化,但 Cqm1 不变,这意味着前者发生了糖基化。本研究结果排除了幼虫阶段缺乏受体或其作为可溶性蛋白表达作为埃及伊蚊对 Bin 毒素产生抗性的原因。