Functional expression in insect cells of glycosylphosphatidylinositol-linked alkaline phosphatase from Aedes aegypti larval midgut: a Bacillus thuringiensis Cry4Ba toxin receptor.
Autor(es): Dechklar Manasave; Tiewsiri Kasorn; Angsuthanasombat Chanan; Pootanakit Kusol
Resumo: Bacillus thuringiensis produces insecticidal crystal (Cry) proteins which bind to cell surface receptors on the brush border membrane of susceptible midgut larvae. The toxin-receptor interaction generates pores in midgut epithelial cells resulting in cell lysis. Here, a cDNA encoding membrane-bound alkaline phosphatase from Aedes aegypti (Aa-mALP) midgut larvae, based on the sequence identity hit to Bombyx mori membrane-bound ALP, was amplified by RT-PCR and transiently expressed in Spodoptera frugiperda (Sf9) insect cells as a 58-kDa membrane-bound protein via the baculovirus expression system and confirmed by digestion with phosphatidylinositol-specific phospholipase C and LC-MS/MS analysis. Immunolocalization results showed that Cry4Ba is able to bind to only Sf9 cells-expressing Aa-mALP. Moreover, these cells were shown to undergo cell lysis in the presence of 100 ?g/ml trypsin-treated toxin. Finally, trypan blue exclusion assay also demonstrated an increase in cell death in recombinant cells treated with Cry4Ba. Overall results indicated that Aa-mALP protein was responsible for mediating Cry4Ba toxicity against Sf9 cells, suggesting its role as a receptor for Cry4Ba toxin in A. aegypti mosquito larvae.
Imprenta: Insect Biochemistry and Molecular Biology, v. 41, n. 3, p. 159-166
Identificador do objeto digital: 10.1016/j.ibmb.2010.11.006
Descritores: Aedes aegypti - Cell ; Aedes aegypti - Molecular Structure ; Aedes aegypti - Pathogenesis ; Aedes aegypti - Proteins ; Aedes aegypti - RT-PCR
Data de publicação: 2011