Cloning and epitope mapping of Cry11Aa-binding sites in the Cry11Aa-receptor alkaline phosphatase from Aedes aegypti.
Cloning and epitope mapping of Cry11Aa-binding sites in the Cry11Aa-receptor alkaline phosphatase from Aedes aegypti.
复制标题
DOI：
10.1021/bi900979b
复制
发表时间：
2009-09-22
期刊：
影响因子：
2.9
通讯作者：
Soberon, Mario
中科院分区：
文献类型：
--
作者：
Fernandez, Luisa E.;Martinez-Anaya, Claudia;Lira, Erandi;Chen, Jianwu;Evans, Amy;Hernandez-Martinez, Salvador;Bravo, Alejandra;Gill, Sarjeet S.;Soberon, Mario
Cry11Aa是苏云金芽孢杆菌以色列毒素中对埃及伊蚊幼虫最具活性的毒素。天哪。埃及伊蚊碱性磷酸酶(ALP)是一种介导毒性的Cry11Aa受体。在此，我们报道了该Ae基因的克隆和功能鉴定。埃及伊蚊Cry11Aa-ALP受体。在三个ALP基因克隆中，重组产生的ALP1亚型可与Cry11Aa和P1BBMV噬菌体结合，该噬菌体可与中肠ALP-Cry11Aa受体特异性结合。抗ALP1抗体抑制了与刷状缘膜囊泡的结合，并抑制了Cry11Aa对体外培养肠道的毒性。通过鉴定Cry11Aa与覆盖ALP1序列的9个重组重叠肽的结合，定位了两个ALP1Cry11Aa结合区(R59-G102和N257-I296)。最后，利用Cry11Aa结构域III的多肽斑点阵列和定点突变，我们发现AlP1R59-G102区域通过结构域II环α-8与Cry11Aa结合，而ALP1N257-I296通过位于β18-β19的结构域III561RVQSQNSGN570与Cry11Aa相互作用。
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 561RVQSQNSGNN570 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.
登录
查看更多内容
影响因子：
--
作者：
Abdullah MA;Valaitis AP;Dean DH
通讯作者：
Dean DH
影响因子：
2.9
作者：
Hua, Gang;Zhang, Rui;Adang, Michael J.
通讯作者：
Adang, Michael J.
影响因子：
5.6
作者：
de Backer, M;McSweeney, S;Hough, E
通讯作者：
Hough, E
影响因子：
3.8
作者：
Jin, XY;Aimanova, K;Gill, SS
通讯作者：
Gill, SS
影响因子：
3.8
作者：
Perera, Omaththage P.;Willis, Jonathan D.;Jurat-Fuentes, Juan L.
通讯作者：
Jurat-Fuentes, Juan L.