title: Functional expression in insect cells of glycosylphosphatidylinositol-linked alkaline phosphatase from Aedes aegypti larval midgut: a Bacillus thuringiensis Cry4Ba toxin receptor.
abstract: 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 μg/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.
authors.affiliation: Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Nakornpathom 73170, Thailand.
authors.forename: Manasave
authors.initials: M
authors.lastname: Dechklar
authors.forename: Kasorn
authors.initials: K
authors.lastname: Tiewsiri
authors.forename: Chanan
authors.initials: C
authors.lastname: Angsuthanasombat
authors.forename: Kusol
authors.initials: K
authors.lastname: Pootanakit
mesh_terms: D000330:Aedes / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D000469:Alkaline Phosphatase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001413:Bacillus thuringiensis / Q000235:genetics / Q000378:metabolism; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D004731:Endotoxins / Q000235:genetics / Q000378:metabolism; D017261:Glycosylphosphatidylinositols / Q000235:genetics / Q000378:metabolism; D006460:Hemolysin Proteins / Q000235:genetics / Q000378:metabolism; D019476:Insect Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D054801:Phosphoinositide Phospholipase C / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011971:Receptors, Immunologic / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D018411:Spodoptera / Q000378:metabolism
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D004731:Endotoxins; D017261:Glycosylphosphatidylinositols; D006460:Hemolysin Proteins; D019476:Insect Proteins; D011971:Receptors, Immunologic; C048071:endotoxin receptor; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D000469:Alkaline Phosphatase; D054801:Phosphoinositide Phospholipase C