Anopheles gambiae Alkaline Phosphatase Is a Functional Receptor of Bacillus thuringiensis jegathesan Cry11Ba Toxin

Alkaline phosphatases (ALPs, EC 3.1.3.1) isolated from lepidopteran and dipteran species are identified as receptors for Cry1Ac and Cry11Aa toxins, respectively [Jurat-Fuentes, J. L., and Adang, M. J. (2004) Eur. J. Biochem. 7, 3127−3135; Fernandez, L. E., et al. (2006) Biochem. J. 396, 77−84]. In our study, an alkaline phosphatase cDNA (AgALP1) was cloned from the midgut of Anopheles gambiae larvae. The encoded 63 kDa protein has a predicted glycosylphosphatidylinositol (GPI) anchor ω-site (526Asp), an N-glycosylation site (239Asn-Leu-Thr), and an O-glycosylation site (312Ser). AgALP1t was expressed in Escherichia coli and used to prepare antiserum and to analyze the interaction of AgALP with mosquitocidal Cry11Ba toxin. Anti-AgALP serum localized AgALP to the apical brush border in the anterior and posterior midgut of larvae and detected a 65 kDa species on a blot of brush border membrane vesicles (BBMVs) protein prepared from larvae. ALP activity was released from larval BBMVs prepared by phosphatidylinositol-specific phospholipase C (PIPLC) treatment, and after separation by two-dimensional gel electrophoresis 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 reprobed blot. Heterologously expressed AgALP1t bound [125I]Cry11Ba on dot blots and reduced the level of binding of [125I]Cry11Ba to brush border membrane vesicles by 41%, a percentage comparable to that of unlabeled Cry11Ba and aminopeptidase AgAPN2t1 peptide. AgALP1t binds Cry11Ba toxin with a high affinity (23.9 nM) and shares a binding site on Cry11Ba with AgAPN2t1. In bioassays against An. gambiae larvae, the presence of AgALP1t reduced larval mortality from 78 to 8%. We conclude that AgALP1 is a binding protein and a functional receptor for Cry11Ba toxin.