Abstract
INTRODUCTION: The cadherin G-protein coupled receptor BT-R(3) in the mosquito Anopheles gambiae is a single membrane-spanning α-helical (bitopic) protein that represents the most abundant and functionally diverse group of membrane proteins. Binding of the Cry4B toxin of Bacillus thuringiensis subsp. israelensis (Bti) to BT-R(3) triggers a Mg2+-dependent signalling pathway in the mosquito that involves stimulation of G protein α-subunit, which subsequently launches a coordinated signalling cascade involving Na(+)/K(+)-ATPase. Described in this study is the behaviour of the Cry4B purified active protein toxin in solution relative to its protoxin predecessor produced by Bti as well as identification of the region within BT-R(3) of An. gambiae to which the toxin binds. MATERIALS AND METHODS: The relationship and behaviour of protoxin and toxin were ascertained in vitro by solubility studies in an alkaline environment like that of the mosquito larval midgut. To identify the specific toxin-binding site within BT-R(3), the full-length coding sequence of the bt-r3 gene was amplified and cloned in pENTR/D-TOTO and subcloned in pXINSECT-DEST38 resulting in recombinant pXINSECT-DEST38-bt-r3. Cytotoxicity was analysed using Trichoplusia ni High Five™ insect cells transfected with the pXINSECT-DEST38-bt-r3 plasmid rendering them susceptible to the Cry4B toxin. Truncation mutational analyses, receptor-toxin binding studies and live cell experiments were used to elucidate the toxin-binding site in BT-R(3). RESULTS: The N-terminal half of the Cry4B protoxin was cleaved releasing active Cry4B toxin. The nontoxic C-terminal portion was degraded into small peptide fragments. The receptor BT-R(3) contained a single toxin-binding site--a 106-amino acid polypeptide bounded by Ile1359 and Ser1464 ((1359)IS(1464)) localized in the 11th cadherin repeat of the receptor. CONCLUSIONS: The structural features of the toxin-binding site are critical to the specificity, selectivity and affinity of the active toxin and for the design and development of novel Bti-based biopesticides.