title: Cloning and epitope mapping of Cry11Aa-binding sites in the Cry11Aa-receptor alkaline phosphatase from Aedes aegypti.
abstract: Cry11Aa is the most active Bacillus thuringiensis israelensis toxin against Aedes aegypti larvae. Ae. aegypti alkaline phosphatase (ALP) was previously identified as a Cry11Aa receptor mediating toxicity. Here we report the cloning and functional characterization of this Ae. aegypti Cry11Aa-ALP receptor. Of three ALP's cDNA clones, the recombinant produced ALP1 isoform was shown to bind Cry11Aa and P1.BBMV peptide phage that specifically binds the midgut ALP-Cry11Aa receptor. An anti-ALP1 antibody inhibited binding to brush border membrane vesicles and toxicity of Cry11Aa in isolated cultured guts. Two ALP1 Cry11Aa binding regions (R59-G102 and N257-I296) were mapped by characterizing binding of Cry11Aa to nine recombinant overlapping peptides covering the ALP1 sequence. Finally, by using a peptide spot array of Cry11Aa domain III and site-directed mutagenesis, we show that the ALP1 R59-G102 region binds Cry11Aa through domain II loop alpha-8 while ALP1 N257-I296 interacts with Cry11Aa through domain III 561RVQSQNSGNN570 located in beta18-beta19. Our results show that Cry11Aa domain II and domain III are involved in the binding with two distinct binding sites in the ALP1 receptor.
authors.affiliation: Instituto de Biotecnología, Universidad Nacional Autónoma de México, Apdo, postal 510-3, Cuernavaca 62250, Morelos, Mexico.
authors.forename: Luisa E
authors.initials: LE
authors.lastname: Fernandez
authors.forename: Claudia
authors.initials: C
authors.lastname: Martinez-Anaya
authors.forename: Erandi
authors.initials: E
authors.lastname: Lira
authors.forename: Jianwu
authors.initials: J
authors.lastname: Chen
authors.forename: Amy
authors.initials: A
authors.lastname: Evans
authors.forename: Salvador
authors.initials: S
authors.lastname: Hernández-Martínez
authors.forename: Humberto
authors.initials: H
authors.lastname: Lanz-Mendoza
authors.forename: Alejandra
authors.initials: A
authors.lastname: Bravo
authors.forename: Sarjeet S
authors.initials: SS
authors.lastname: Gill
authors.forename: Mario
authors.initials: M
authors.lastname: Soberón
mesh_terms: D000330:Aedes / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D001665:Binding Sites; D003001:Cloning, Molecular; D004731:Endotoxins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D018604:Epitope Mapping; D006460:Hemolysin Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D007814:Larva / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D011956:Receptors, Cell Surface / Q000378:metabolism*; D016415:Sequence Alignment
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D004731:Endotoxins; D006460:Hemolysin Proteins; D007527:Isoenzymes; D011956:Receptors, Cell Surface; C030956:insecticidal crystal protein, Bacillus Thuringiensis; C110838:MC14 alkaline phosphatase I; D000469:Alkaline Phosphatase
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