Cloning and epitope mapping of Cry11Aa-binding sites in the Cry11Aa-receptor alkaline phosphatase from Aedes aegypti.
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.
Fernandez
Luisa E
LE
Instituto de Biotecnología, Universidad Nacional Autónoma de México, Apdo, postal 510-3, Cuernavaca 62250, Morelos, Mexico.
Martinez-Anaya
Claudia
C
Lira
Erandi
E
Chen
Jianwu
J
Evans
Amy
A
Hernández-Martínez
Salvador
S
Lanz-Mendoza
Humberto
H
Bravo
Alejandra
A
Gill
Sarjeet S
SS
Soberón
Mario
M
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
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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