title: A 106-kDa aminopeptidase is a putative receptor for Bacillus thuringiensis Cry11Ba toxin in the mosquito Anopheles gambiae.
abstract: Bacillus thuringiensis (Bt) insecticidal toxins bind to receptors on midgut epithelial cells of susceptible insects, and binding triggers biochemical events that lead to insect mortality. Recently, a 100-kDa aminopeptidase N (APN) was isolated from brush border membrane vesicles (BBMV) of Anopheles quadrimaculatus and shown to bind Cry11Ba toxin with surface plasmon resonance (SPR) detection [Abdullah et al. (2006) BMC Biochem. 7, 16]. In our study, a 106-kDa APN, called AgAPN2, released by phosphatidylinositol-specific phospholipase C (PI-PLC) from Anopheles gambiae BBMV was extracted by Cry11Ba bound to beads. The AgAPN2 cDNA was cloned, and analysis of the predicted AgAPN2 protein revealed a zinc-binding motif (HEIAH), three potential N-glycosylation sites, and a predicted glycosylphosphatidylinositol (GPI) anchor site. Immunohistochemistry localized AgAPN2 to the microvilli of the posterior midgut. A 70-kDa fragment of the 106-kDa APN was expressed in Escherichia coli. When purified, it competitively displaced 125I-Cry11Ba binding to An. gambiae BBMV and bound Cry11Ba on dot blot and microtiter plate binding assays with a calculated K d of 6.4 nM. Notably, this truncated peptide inhibited Cry11Ba toxicity to An. gambiae larvae. These results are evidence that the 106-kDa GPI-anchored APN is a specific binding protein, and a putative midgut receptor, for Bt Cry11Ba toxin.
authors.affiliation: Department of Entomology and Biochemistry, University of Georgia, Athens, Georgia 30602-2603, USA.
authors.forename: Rui
authors.initials: R
authors.lastname: Zhang
authors.forename: Gang
authors.initials: G
authors.lastname: Hua
authors.forename: Tracy M
authors.initials: TM
authors.lastname: Andacht
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Adang
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D000852:Anopheles / Q000235:genetics / Q000378:metabolism*; D001413:Bacillus thuringiensis / Q000235:genetics / Q000378:metabolism*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D018826:CD13 Antigens / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004731:Endotoxins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D006460:Hemolysin Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D007306:Insecticides / Q000378:metabolism*; D007814:Larva / Q000201:enzymology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010802:Phylogeny; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D004731:Endotoxins; D006460:Hemolysin Proteins; D007306:Insecticides; D011994:Recombinant Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D018826:CD13 Antigens