A 106-kDa aminopeptidase is a putative receptor for Bacillus thuringiensis Cry11Ba toxin in the mosquito Anopheles gambiae.
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.
Zhang
Rui
R
Department of Entomology and Biochemistry, University of Georgia, Athens, Georgia 30602-2603, USA.
Hua
Gang
G
Andacht
Tracy M
TM
Adang
Michael J
MJ
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
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