Cloning and epitope mapping of Cry11Aa-binding sites in the Cry11Aa-receptor alkaline phosphatase from Aedes aegypti
Autor(es): Fernandez, Luisa E.; Martinez-Anaya, Claudia; Lira, Erandi; Chen, Jianwu; Evans, Amy; Hernndez-Martnez, Salvador; Lanz-Mendoza, Humberto; Bravo, Alejandra; Gill, Sarjeet S.; Sobern, Mario
Resumo: 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-*b96) 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 *a-8 while ALP1 N257-*b96 interacts with Cry11Aa through domain III 561RVQSQNSGNN570 located in *b18-*b19. Our results show that Cry11Aa domain II and domain III are involved in the binding with two distinct binding sites in the ALP1 receptor.
Palavras-Chave: Biological control; Recombinants; Antibodies; Toxicants; Receptors; Peptides; Toxicity; Phosphatase; Aquatic insects; Site-directed mutagenesis; Phages; Digestive tract; Alkaline phosphatase; Brush border membranes; Membrane vesicles; Epitope mapping; Midgut; Toxins; Bacillus thuringiensis israelensis; Aedes aegypti
Imprenta: Biochemistry, v. 48, n. 37, p. 8899-8907, 2009.
Descritores: Aedes aegypti - Immunology
Data de publicação: 2009
