Hua G, Zhang R, Bayyareddy K, Adang MJ
<i>Anopheles gambiae</i> alkaline phosphatase is a functional receptor of <i>Bacillus thuringiensis jegathasan</i> Cry11Ba toxin. [JOURNAL ARTICLE]
Biochemistry 2009 Sep 14.
Alkaline phosphatases (ALP) (E.C. 3.1.3.1) isolated from lepidopteran and dipteran species are identified as receptors for Cry1Ac and Cry11Aa toxins, respectively [Jurat-Fuentes and Adang, (2004) <i>Eur. J. Biochem.</i> 7, 3127-3135; Fernandez et al. (2006) <i>Biochemical J.</i> 396, 77-84]. In our study, an alkaline phosphatase cDNA (AgALP1) was cloned from midgut of <i>Anopheles gambiae</i> larvae. The encoded 63-kDa protein has a predicted glycosylphosphatidylinositol (GPI) anchor omega-site (<sup>526</sup>Asp), an N-glycosylation site (<sup>239</sup>Asn-Leu-Thr), and an O-glycosylation site (<sup>312</sup>Ser). AgALP1t was expressed in <i>Escherichia coli</i> and used to prepare antiserum and to analyze AgALP interaction with mosquitocidal Cry11Ba toxin. Anti-AgALP serum localized AgALP to apical brush border in anterior and posterior midgut of larvae and detected a 65-kDa on a blot of brush border membrane vesicle (BBMV) protein prepared from larvae. ALP activity was released from larval BBMV prepared by phosphatidylinositol-specific phospholipase C (PIPLC) treatment, and after separation by 2-dimensional gel electrophoresis (2-DE) and blotting, a chain of doublet spots at 65-kDa was detected by anti-AgALP. A subset of these doublet spots bound Cry11Ba on a re-probed blot. Heterologously expressed AgALP1<sub>t</sub> bound <sup>125</sup>I-Cry11Ba on dot blots and reduced <sup>125</sup>I-Cry11Ba binding to brush border membrane vesicles by 41%, a percentage comparable to unlabeled Cry11Ba and aminopeptidase AgAPN2<sub>t1</sub> peptide. AgALP1<sub>t</sub> binds Cry11Ba toxin at high affinity (23.9 nM) and shares a binding site on Cry11Ba with AgAPN2<sub>t1</sub>. In bioassays against <i>An. gambiae</i> larvae, the presence of AgALP1<sub>t</sub> reduced larval mortality from 78% to 8%. We conclude that AgALP1 is a binding protein and a functional receptor for Cry11Ba toxin.
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