N-glycosylation influences the catalytic activity of mosquito α-glucosidases associated with susceptibility or refractoriness to Lysinibacillus sphaericus.

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.

Avaliação da alfa-glicosidase Cqm1 de Culex quinquefasciatus e o impacto de suas mutações na fisiologia de larvas / Evaluation of a-glucosidase Cqm1 from Culex quinquefasciatus and the impact of its mutations on larvae physiology

Lysinibacillus sphaericus (Lsp) e uma bacteria entomopatogena que produz a toxina Binaria (Bin) com atividade larvicida para culicideos. A sua acao em Culex quinquefasciatus depende da ligacao da toxina Bin a alfa-glicosidase (Aglu) Cqm1, que atua como receptor no epitelio intestinal de larvas. Na colonia R2362, foram caracterizados dois alelos de resistencia ao Lsp: cqm1REC e cqm1REC-2, cujas mutacoes impedem a expressao da Aglu Cqm1. O objetivo deste trabalho foi avaliar a atividade catalitica da Cqm1 e comparar a atividade alfa-glicosidase e o desenvolvimento pre-imaginal de larvas de individuos susceptiveis (S) e resistentes (R) para cada alelo. Para isto, foram avaliados os seguintes parametros: atividade catalitica da Cqm1 recombinante; padrao de transcricao de outras Aglus paralogas a Cqm1; atividade de Aglus nativas em larvas; sobrevivencia de individuos frente a diferentes dietas. A Aglu Cqm1 mostrou atividade enzimatica otima a 37o C, pH 7,5-8,0 e utilizando o substrato sintetico pNalfaG. A atividade alfa-glicosidase total em larvas S e R foi similar, apesar da ausencia de expressao da Cqm1 nas larvas R. A investigacao in silico revelou 18 proteinas paralogas a Cqm1 e, dentre 11 investigadas, nove sao expressas em larvas S e R. A analise quantitativa de tres paralogas demonstrou que duas tem um padrao de transcricao mais elevado em larvas resistentes, sugerindo a existencia de um mecanismo de compensacao de expressao de alfa-glicosidases. O desenvolvimento pre-imaginal de larvas S foi decrescente nas seguintes dietas: racao de gatos, racao de peixes, leite desnatado, extrato de levedura e sacarose. De uma forma global, a taxa de sobrevivencia de larvas R foi inferior a S em todas as dietas testadas. Os dados obtidos mostram que as mutacoes ligadas aos alelos cqm1REC e cqm1REC-2 nao parecem impactar a atividade Aglu nas larvas e que o custo biologico observado poderia estar relacionado a outros genes e vias metabolicas.

Non conserved residues between Cqm1 and Aam1 mosquito α-glucosidases are critical for the capacity of Cqm1 to bind the Binary toxin from Lysinibacillus sphaericus.

The Binary (Bin) toxin from the entomopathogenic bacterium Lysinibacillus sphaericus acts on larvae of the culicid Culex quinquefasciatus through its binding to Cqm1, a midgut-bound α-glucosidase. Specific binding by the BinB subunit to the Cqm1 receptor is essential for toxicity however the toxin is unable to bind to the Cqm1 ortholog from the refractory species Aedes aegypti (Aam1). Here, to investigate the molecular basis for the interaction between Cqm1 and BinB, recombinant Cqm1 and Aam1 were first expressed as soluble forms in Sf9 cells. The two proteins were found to display the same glycosilation patterns and BinB binding properties as the native α-glucosidases. Chimeric constructs were then generated through the exchange of reciprocal fragments between the corresponding cqm1 and aam1 cDNAs. Subsequent expression and binding experiments defined a Cqm1 segment encompassing residues S129 and A312 as critical for the interaction with BinB. Through site directed mutagenesis experiments, replacing specific sets of residues from Cqm1 with those of Aam1, the 159GG160 doublet was required for this interaction. Molecular modeling mapped these residues to an exposed loop within the Cqm1's structure, compatible with a target site for BinB and providing a possible explanation for its lack of binding to Aam1.