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摘要
Alkaline phosphatases (ALPs, EC 3131) isolated from lepidopteran and dipteran species are identified as receptors for Cry1Ac an 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, ail alkaline phosphatase cDNA (AgALP1) was cloned from the midgut of Anopheles gambiae larvae. The encoded 63 kDa protein has a predicted glycosylphosphatidylinositol (GPI) anchor omega-site ((526)Asp), an N-glycosylation Site ((239)Asn-Leu-Thr), and an O-glycosylation Site ((312)Ser). AgALP1, 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 Wit 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 it reprobed blot. Heterologously expressed AgALPl(t) bound [(125)I]Cry11Ba on dot blots and reduced the level of binding of [(125)I]Cry11Ba to brush border membrane vesicles by 41%, a percentage comparable to that of unlabeled Cry11Ba and aminopeptidase AgAPN2(t1) peptide. AgALPl(t) binds Cry11Ba toxin with it high affinity (23.9 nM) and shares a binding site on Cry11Ba with AgAPN2(t1). In bioassays against An. gambiae larvae, the presence of AgALPl(t) reduced larval mortality from 78 to 8%. We conclude that AgALP1 is a binding protein and a functional receptor for Cry11Ba toxin.
- 出版日期2009-10-20
