Nascimento Nathaly Alexandre do, Ferreira Lígia Maria, Romão Tatiany Patrícia, Correia Darleide Maria da Conceição, Vasconcelos Crhisllane Rafaele Dos Santos, Rezende Antônio Mauro, Costa Samara Graciane, Genta Fernando Ariel, de-Melo-Neto Osvaldo Pompílio, Silva-Filha Maria Helena Neves Lobo
Department of Entomology, Centro de Pesquisas Aggeu Magalhães/FIOCRUZ, Recife, PE 50740-465, Brazil.
Department of Microbiology, Centro de Pesquisas Aggeu Magalhães/FIOCRUZ, Recife, PE 50740-465, Brazil.
Insect Biochem Mol Biol. 2017 Feb;81:62-71. doi: 10.1016/j.ibmb.2016.12.009. Epub 2016 Dec 23.
Cqm1 and Aam1 are α-glucosidases (EC 3.2.1.20) expressed in Culex quinquefasciatus and Aedes aegypti larvae midgut, respectively. These orthologs share high sequence similarity but while Cqm1 acts as a receptor for the Binary (Bin) insecticidal toxin from Lysinibacillus sphaericus, Aam1 does not bind the toxin, rendering Ae. aegypti refractory to this bacterium. Aam1 is heavily glycosylated, contrasting to Cqm1, but little is known regarding how glycosylation impacts on its function. This study aimed to compare the N-glycosylation patterns and the catalytic activities of Aam1 and Cqm1. Mutant proteins were generated where predicted Aam1 N-glycosylation sites (N-PGS) were either inserted into Cqm1 or abrogated in Aam1. The mutants validated four N-PGS which were found to localize externally on the Aam1 structure. These Aam1 and Cqm1 mutants maintained their Bin binding properties, confirming that glycosylation has no role in this interaction. The α-glucosidase activity of both proteins was next investigated, with Aam1 having a remarkably higher catalytic efficiency, influenced by changes in glycosylation. Molecular dynamics showed that glycosylated and nonglycosylated Aam1 models displayed distinct patterns that could influence their catalytic activity. Differential N-glycosylation may then be associated with higher catalytic efficiency in Aam1, enhancing the functional diversity of related orthologs.
Cqm1和Aam1分别是在致倦库蚊和埃及伊蚊幼虫中肠表达的α-葡萄糖苷酶（EC 3.2.1.20）。这些直系同源物具有高度的序列相似性，但Cqm1作为球形赖氨酸芽孢杆菌二元（Bin）杀虫毒素的受体，而Aam1不结合该毒素，使埃及伊蚊对这种细菌具有抗性。与Cqm1不同，Aam1高度糖基化，但关于糖基化如何影响其功能知之甚少。本研究旨在比较Aam1和Cqm1的N-糖基化模式和催化活性。生成了突变蛋白，其中预测的Aam1 N-糖基化位点（N-PGS）要么插入Cqm1中，要么在Aam1中被消除。这些突变体验证了四个N-PGS，发现它们位于Aam1结构的外部。这些Aam1和Cqm1突变体保持了它们的Bin结合特性，证实糖基化在这种相互作用中不起作用。接下来研究了两种蛋白质的α-葡萄糖苷酶活性，Aam1具有显著更高的催化效率，这受到糖基化变化的影响。分子动力学表明，糖基化和非糖基化的Aam1模型显示出不同的模式，可能影响它们的催化活性。差异N-糖基化可能与Aam1中更高的催化效率相关，增强了相关直系同源物的功能多样性。
