Journal
INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY
Volume 41, Issue 3, Pages 159-166Publisher
PERGAMON-ELSEVIER SCIENCE LTD
DOI: 10.1016/j.ibmb.2010.11.006
Keywords
Bacillus thuringiensis; Cry delta-endotoxin; Membrane-bound alkaline phosphatase; Spodoptera frugiperda; Baculovirus system; Cytotoxicity assay
Categories
Funding
- BIOTEC
- Thailand Research Fund
- Commission on Higher Education (CHE), Ministry of Education (Thailand)
- CHE
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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 mu 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 SP cells, suggesting its role as a receptor for Cry4Ba toxin in A. aegypti mosquito larvae. (C) 2010 Elsevier Ltd. All rights reserved.
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