Bacillus thuringiensis Cry1Ac and Cry1Fa δ-endotoxin binding to a novel 110 kDa aminopeptidase in Heliothis virescens is not N-acetylgalactosamine mediated
Bacillus thuringiensis Cry1Ac and Cry1Fa δ-endotoxin binding to a novel 110 kDa aminopeptidase in Heliothis virescens is not N-acetylgalactosamine mediated
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DOI：
10.1016/s0965-1748(01)00038-8
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发表时间：
2001-07-26
影响因子：
3.8
通讯作者：
Adang, MJ
中科院分区：
文献类型：
--
作者：
Banks, DJ;Jurat-Fuentes, JL;Adang, MJ
我们确定苏云金芽孢杆菌CrylAc和CrylFa δ-内毒素识别来自烟芽夜蛾的刷状缘膜囊泡（BBMV）中相同的110、120和170 kDa氨肽酶N（APN）分子。110 kDa的蛋白质，以前没有被确定为APN，含有一个变体APN的共识序列相同的Helicoverpa punctigera APN 2中发现的。PCR扩增H.基于该序列和保守的APN基序构建的virescens cDNA产生了0.9kb的产物，与H. punctigera APN 2. Western印迹显示，110 kDa的分子不被大豆凝集素识别，表明GalNAc的情况下。具有改变的GalNAc结合口袋的I-125标记的CrylAc结构域III突变体（（509）QNR（511）-AAA）（Lee等人，应用环境。Microbiol. 65（1999）4513）显示与120 kDa APN的结合消失，与170 kDa APN的结合减少，与110 kDa APN的结合增强。高碘酸处理的H.还用I-125标记的-CrylAc和（509）QNR（511）-AAA毒素探测绿僵菌BBMV印迹。这两种毒素仍然识别110 kDa APN和一个> 210 kDa的分子，可能是一种钙粘蛋白样蛋白。此外，I-125-（509）QNR（511）-AAA识别高碘酸盐处理的170 kDa APN。结果表明，110 kDa APN不同于其他Cryl毒素结合APN，并且可能是第一个描述的不含GalNAc的CrylAc结合APN。(C)2001爱思唯尔科技有限公司。保留所有权利。
We determined that Bacillus thuringiensis CrylAc and CrylFa delta -endotoxins recognize the same 110, 120 and 170 kDa aminopeptidase N (APN) molecules in brush border membrane vesicles (BBMV) from Heliothis virescens. The 110 kDa protein, not previously identified as an APN, contained a variant APN consensus sequence identical to that found in Helicoverpa punctigera APN 2. PCR amplification of H. virescens cDNA based on this sequence and a conserved APN motif yielded a 0.9 kb product that has 89% sequence homology with H. punctigera APN 2. Western blots revealed that the 110 kDa molecule was not recognized by soybean agglutinin, indicating the absence of GalNAc. A I-125 labeled-CrylAc domain III mutant ((509)QNR(511)-AAA) that has an altered GalNAc binding pocket (Lee et al., Appl. Environ. Microbiol. 65 (1999) 4513) showed abolished binding to the 120 APN, reduced binding to the 170 kDa APN, and enhanced binding to the 110 kDa APN. Periodate treated H. virescens BBMV blots were also probed with I-125-labeled-CrylAc and (509)QNR(511)-AAA toxins. Both toxins still recognized the 110 kDa APN and a > 210 kDa molecule which may be a cadherin-like protein. Additionally, I-125-(509)QNR(511)-AAA recognized periodate treated 170 kDa APN. Results indicate that the 110 kDa APN is distinct from other Cryl toxin binding APNs and may be the first described CrylAc-binding APN that does not contain GalNAc. (C) 2001 Elsevier Science Ltd. All rights reserved.