Cloning and Epitope Mapping of Cry11Aa-Binding Sites in the Cry11Aa-Receptor Alkaline Phosphatase from Aedes aegypti

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• 作者：Luisa E. Fernandez ; Claudia Martinez-Anaya ; Erandi Lira ; Jianwu Chen ; Amy Evans ; Salvador Hernndez-Martnez ; Humberto Lanz-Mendoza ; Alejandra Bravo ; Sarjeet S. Gill ; Mario Sobern
• 刊名：Biochemistry
• 出版年：2009
• 出版时间：September 22, 2009
• 年：2009
• 卷：48
• 期：37
• 页码：8899-8907
• 全文大小：1112K
• 年卷期：v.48,no.37(September 22, 2009)
• ISSN：1520-4995

文摘

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 α-8 while ALP1 N257−I296 interacts with Cry11Aa through domain III p>561p>RVQSQNSGNNp>570p> located in β18-β19. Our results show that Cry11Aa domain II and domain III are involved in the binding with two distinct binding sites in the ALP1 receptor.