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 omega-site ((526)Asp), an N-glycosylation site ((239)Asn-Leu-Thr), and an O-glycosylation site ((312)Ser). AgALP1(t) 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 AgALP1(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. AgALP1(t) binds Cry11Ba toxin with a high affinity (23.9 nM) and shares a binding site on Cry11Ba with AgAPN2(t1). In bioassays against An. gambiae larvae, the presence of AgALP1(t) reduced larval mortality from 78 to 8%. We conclude that AgALP1 is a binding protein and a functional receptor for Cry11Ba toxin.
Hua
Gang
G
Department of Entomology, University of Georgia,Athens, Georgia 30602-2603, USA.
Zhang
Rui
R
Bayyareddy
Krishnareddy
K
Adang
Michael J
MJ
D000469:Alkaline Phosphatase / Q000378:metabolism*; D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000378:metabolism; D000818:Animals; D000852:Anopheles / Q000201:enzymology* / Q000235:genetics; D001413:Bacillus thuringiensis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001427:Bacterial Toxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D003001:Cloning, Molecular; D019295:Computational Biology; D017124:Conserved Sequence; D017931:DNA Primers; D004175:Diptera; D004731:Endotoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006460:Hemolysin Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007915:Lepidoptera; D008969:Molecular Sequence Data; D010572:Pest Control, Biological; D016133:Polymerase Chain Reaction
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; C115468:CRY11BB protein, Bacillus thuringiensis; D017931:DNA Primers; D004731:Endotoxins; D006460:Hemolysin Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D000469:Alkaline Phosphatase; D000626:Aminopeptidases