Location of the Bombyx mori aminopeptidase N type 1 binding site on Bacillus thuringiensis Cry1Aa toxin
Location of the Bombyx mori aminopeptidase N type 1 binding site on Bacillus thuringiensis Cry1Aa toxin
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DOI：
10.1128/aem.71.7.3966-3977.2005
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发表时间：
2005-07-01
影响因子：
4.4
通讯作者：
Sato, R
中科院分区：
文献类型：
--
作者：
Atsumi, S;Mizuno, E;Sato, R
我们分析了家蚕Cry 1Aa毒素上Cry 1Aa受体的结合位点，115-kDa氨肽酶N 1型（BmAPN 1）（K. Nakanishi，K. Yaoi，Y. Nagino，H. Hara，M. Kitami，S. Atsumi，N. Miura和R. Sato，FEBS Lett. 519：215-220，2002），通过使用阻断结合位点和受体之间的结合的单克隆抗体（MAb）。首先，我们制备了一系列抗Cry 1Aa的单克隆抗体，并获得了两种能够阻断Cry 1Aa与BmAPN 1结合的单克隆抗体，即单克隆抗体2C 2和1B 10（阻断单克隆抗体）。MAb 2C 2的Fab片段的表位与BmAPN 1结合位点重叠，而MAb 1B 10的Fab片段的表位不重叠，但靠近结合位点。使用三种表位作图方法，我们确定了Cry 1Aa上阻断单克隆抗体的两个候选表位。我们构建了两个Cry 1Aa毒素突变体，通过在毒素表面的两个候选表位中的每一个上取代半胱氨酸，并且在每个半胱氨酸取代处引入小阻断分子N-（9-吖啶基）马来酰亚胺（NAM）以确定真正的表位。具有与Cys 582结合的NAM的Cry 1Aa突变体不结合两种阻断性MAb中的任一种，表明每种阻断性MAb的真正表位位于含有Val 582的位点，其也由（STLRVN 513）-S-508和（582）VFTLSAHV（589）组成。这些结果表明，BmAPN 1结合位点与MAb 2C 2阻断的区域重叠，该区域接近但排除了Cry 1Aa毒素结构域III上MAb 2C 2的实际表位。我们还讨论了Cry 1Aa毒素作为BmAPN 1结合的新候选位点的另一个领域。
We analyzed the binding site on Cry1Aa toxin for the Cry1Aa receptor in Bombyx mori, 115-kDa aminopeptidase N type 1 (BmAPN1) (K. Nakanishi, K. Yaoi, Y. Nagino, H. Hara, M. Kitami, S. Atsumi, N. Miura, and R. Sato, FEBS Lett. 519:215-220, 2002), by using monoclonal antibodies (MAbs) that block binding between the binding site and the receptor. First, we produced a series of MAbs against Cry1Aa and obtained two MAbs, MAbs 2C2 and 1B10, that were capable of blocking the binding between Cry1Aa and BmAPN1 (blocking MAbs). The epitope of the Fab fragments of MAb 2C2 overlapped the BmAPN1 binding site, whereas the epitope of the Fab fragments of MAb 1B10 did not overlap but was located close to the binding site. Using three approaches for epitope mapping, we identified two candidate epitopes for the blocking MAbs on Cry1Aa. We constructed two Cry1Aa toxin mutants by substituting a cysteine on the toxin surface at each of the two candidate epitopes, and the small blocking molecule N-(9-acridinyl)maleimide (NAM) was introduced at each cysteine substitution to determine the true epitope. The Cry1Aa mutant with NAM bound to Cys582 did not bind either of the two blocking MAbs, suggesting that the true epitope for each of the blocking MAbs was located at the site containing Val582, which also consisted of (STLRVN513)-S-508 and (582)VFTLSAHV(589). These results indicated that the BmAPN1 binding site overlapped part of the region blocked by MAb 2C2 that was close to but excluded the actual epitope of MAb 2C2 on domain III of Cry1Aa toxin. We also discuss another area on Cry1Aa toxin as a new candidate site for BmAPN1 binding.