Structural Basis for the Specific Neutralization of Stx2a with a Camelid Single Domain Antibody Fragment.
Structural Basis for the Specific Neutralization of Stx2a with a Camelid Single Domain Antibody Fragment.
复制标题
用骆驼单域抗体片段特异性中和 Stx2a 的结构基础。
DOI：
10.3390/toxins10030108
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发表时间：
2018-03-01
期刊：
影响因子：
4.2
通讯作者：
Muyldermans S
中科院分区：
文献类型：
--
作者：
Bernedo-Navarro RA;Romão E;Yano T;Pinto J;De Greve H;Sterckx YG;Muyldermans S
背景资料：产滋贺毒素大肠杆菌（STEC）是导致腹泻、溶血性尿毒综合征甚至死亡等疾病的病原体的一个子集。滋贺毒素是主要的毒力因子，并分为两组：Stx 1和Stx 2，其中后者更经常与人类的严重病理有关。结果如下：用重组滋贺毒素2a B亚基（rStx 2a B）免疫羊驼，构建了纳米抗体（Nbs）库，以获得多个rStx 2a B特异性Nbs。ELISA和Western blot结果表明，5个Nbs对rStx 2aB具有特异性。Nb 113具有最高的亲和力（9.6 nM），其二价构建体表现出高100倍的功能亲和力。通过X射线晶体学确定了与rStx 2aB复合的Nb 113的结构。Nb 113-rStx 2aB复合物的晶体结构揭示了Nb 113的五个拷贝结合到rStx 2aB五聚体，并且Nb 113表位与Gb 3结合位点重叠，从而为在Vero细胞上观察到的Nb 113中和Stx 2a提供了结构基础。最后，与单价Nb 113相比，串联重复的二价Nb 1132表现出更高的毒素中和能力。结论：对rStx 2aB具有最高亲和力的Nb也是最好的Stx 2a和Stx 2c毒素中和Nb，特别是在二价形式中。该前导Nb通过竞争Gb 3受体来中和Stx 2a。预期二价Nb 1132与血清白蛋白特异性Nb的融合将联合收割机高毒素中和潜力与延长的血液循环结合。
Background: Shiga toxin-producing Escherichia coli (STEC) are a subset of pathogens leading to illnesses such as diarrhea, hemolytic uremic syndrome and even death. The Shiga toxins are the main virulence factors and divided in two groups: Stx1 and Stx2, of which the latter is more frequently associated with severe pathologies in humans. Results: An immune library of nanobodies (Nbs) was constructed after immunizing an alpaca with recombinant Shiga toxin-2a B subunit (rStx2aB), to retrieve multiple rStx2aB-specific Nbs. The specificity of five Nbs towards rStx2aB was confirmed in ELISA and Western blot. Nb113 had the highest affinity (9.6 nM) and its bivalent construct exhibited a 100-fold higher functional affinity. The structure of the Nb113 in complex with rStx2aB was determined via X-ray crystallography. The crystal structure of the Nb113–rStx2aB complex revealed that five copies of Nb113 bind to the rStx2aB pentamer and that the Nb113 epitope overlaps with the Gb3 binding site, thereby providing a structural basis for the neutralization of Stx2a by Nb113 that was observed on Vero cells. Finally, the tandem-repeated, bivalent Nb1132 exhibits a higher toxin neutralization capacity compared to monovalent Nb113. Conclusions: The Nb of highest affinity for rStx2aB is also the best Stx2a and Stx2c toxin neutralizing Nb, especially in a bivalent format. This lead Nb neutralizes Stx2a by competing for the Gb3 receptor. The fusion of the bivalent Nb1132 with a serum albumin specific Nb is expected to combine high toxin neutralization potential with prolonged blood circulation.