Antiidiotypic antibody recognizes an amiloride binding domain within the α subunit of the epithelial Na+ channel
Antiidiotypic antibody recognizes an amiloride binding domain within the α subunit of the epithelial Na+ channel
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DOI：
10.1074/jbc.274.14.9648
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发表时间：
1999-04-02
影响因子：
4.8
通讯作者：
Kleyman, TR
中科院分区：
文献类型：
--
作者：
Kieber-Emmons, T;Lin, CM;Kleyman, TR
我们以前提出了一种抗体（RA6.3）的antidiotypic的方法，其目的是针对上皮Na+通道（ENaC）上的阿米洛利结合域。这种抗体模拟阿米洛利，因为它抑制跨A6细胞单层的跨上皮Na+转运。RA6.3识别衣霉素处理的A6上皮细胞中的72-kDa多肽，与非糖基化非洲爪蟾α ENaC的大小一致。RA6.3特异性识别小鼠和牛ENaC α亚基内的阿米洛利结合结构域。RA6.3的推导的氨基酸序列用于产生抗体的三维模型结构。利用一种新的基于计算机的策略对RA6.3的结合位点进行表位定位。使用程序LUDI通过数据库筛选鉴定可能与RA6.3结合位点相互作用的有机残基。选择的残基以对应于ENaC的α-亚基上的阿米洛利结合结构域内的氨基酸残基的有序线性阵列的方式与抗体对接。跨越该结构域的合成肽抑制RA 6.3与α ENaC的结合。这种分析提供了一种新的方法来开发抗体-抗原相互作用的模型，以及RA6.3结合到α ENaC内的阿米洛利结合结构域的分子视角。
We previously raised an antibody (RA6.3) by an antidiotypic approach which was designed to be directed against an amiloride binding domain on the epithelial Na+ channel (ENaC). This antibody mimicked amiloride in that it inhibited transepithelial Na+ transport across A6 cell monolayers. RA6.3 recognized a 72-kDa polypeptide in A6 epithelia treated with tunicamycin, consistent with the size of nonglycosylated Xenopus laevis alpha ENaC. RA6.3 specifically recognized an amiloride binding domain within the alpha-subunit of mouse and bovine ENaC. The deduced amino acid sequence of RA6.3 was used to generate a three-dimensional model structure of the antibody. The combining site of RA6.3 was epitope mapped using a novel computer-based strategy. Organic residues that potentially interact with the RA6.3 combining site were identified by data base screening using the program LUDI. Selected residues docked to the antibody in a manner corresponding to the ordered linear array of amino acid residues within an amiloride binding domain on the alpha-subunit of ENaC, A synthetic peptide spanning this domain inhibited the binding of RA6.3 to alpha ENaC. This analysis provided a novel approach to develop models of antibody-antigen interaction as well as a molecular perspective of RA6.3 binding to an amiloride binding domain within alpha ENaC.