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首页> 外文期刊>Journal of Molecular Biology >CHARACTERISATION OF EPITOPES ON HUMAN P53 USING PHAGE-DISPLAYED PEPTIDE LIBRARIES - INSIGHTS INTO ANTIBODY PEPTIDE INTERACTIONS [Review]
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CHARACTERISATION OF EPITOPES ON HUMAN P53 USING PHAGE-DISPLAYED PEPTIDE LIBRARIES - INSIGHTS INTO ANTIBODY PEPTIDE INTERACTIONS [Review]

机译：利用噬菌体展示的肽库表征人类P53上的表位-抗体肽相互作用的研究[综述]

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摘要

We previously described the use of a phage-displayed library of random hexapeptides to define and localise the epitope on the human tumor suppressor protein p53 recognised by the monoclonal antibody PAb240. Here we have extended these results to a further eight anti-p53 monoclonal antibodies and to two further libraries, which display 12-mer and 20-mer peptides, respectively First, we showed that selection of PAb240 binding clones from the 12-mer and 20-mer libraries gives essentially identical results to those obtained by screening the 6-mer library. Second, we used the 6-mer and 12-mer libraries to define the derailed specificity profiles of six antibodies (DO-1, DO-2, DO-7, Bp53-11, Bp53-12 and Bp53-19), which recognise the same short, highly immunogenic N-terminal segment of p53. Finally, we employed all three libraries to reveal the distinct mechanisms by which PAb421 and PAb122, two monoclonal antibodies that allosterically activate sequence-specific DNA binding by p53, react specifically with the same positively-charged C-terminal segment. In each case the epitope locations inferred from the selected sequences were confirmed by probing an array of overlapping synthetic peptides representing the primary sequence of p53. The results emphasise the consequences for epitope mapping of screening random, as opposed to antigen-derived, peptide libraries; specifically (1) that comparison of selected sequences reveals the contribution of individual residues to binding energy and specificity; (2) that heteroclitic reactions call lead to definition of a consensus that is related to but distinct from the immunising epitope and (3) that isolation of non-immunogen-homologous ''mimotope'' sequences reveals discrete, alternative ligand structures. The results with PAb421 and PAb122 provide examples where, while selection from the 12-mer and 20-mer libraries leads to isolation of immunogen-homologous sequences, selection from the 6-mer library results in the isolation either of no binding clones (PAb122) or solely of ''mimotope'' sequences with no discernible homology to the original antigen (PAb421). In addition the results with PAb421 reveal that linear epitopes can be longer than previously thought and can be formally discontinuous, consisting of independent contact motifs, which show promiscuous relative positioning. [References: 111]
机译：我们先前描述了使用噬菌体展示的随机六肽文库来定义和定位单克隆抗体PAb240识别的人肿瘤抑制蛋白p53上的表位。在这里，我们将这些结果扩展到了另外8种抗p53单克隆抗体和另外2个文库，分别显示了12-mer和20-mer肽。首先，我们显示了从12-mer和20的PAb240结合克隆的选择-mer文库与筛选6-mer文库获得的结果基本相同。其次，我们使用6-mer和12-mer库定义了六种抗体（DO-1，DO-2，DO-7，Bp53-11，Bp53-12和Bp53-19）的脱轨特异性谱。与p53相同的短而高度免疫原性的N末端片段。最后，我们利用这三个文库揭示了不同的机制，PAb421和PAb122这两种单克隆抗体通过p53变构激活序列特异性DNA结合，它们与相同的带正电的C末端片段特异性反应。在每种情况下，通过探测代表p53一级序列的重叠合成肽阵列，可以确定从选定序列推断出的表位位置。结果强调了随机筛选抗原表位库相对于抗原表位作图的结果。具体来说（1）所选序列的比较揭示了各个残基对结合能和特异性的贡献; （2）异源反应引起对与免疫表位有关但与之不同的共识的定义，（3）非免疫原同源“模拟表位”序列的分离揭示了离散的，可替代的配体结构。 PAb421和PAb122的结果提供了一些示例，其中，虽然从12-mer和20-mer文库中进行选择可以分离出免疫原同源序列，但从6-mer文库中进行选择却可以分离出没有结合克隆（PAb122）或只与原始抗原（PAb421）没有明显同源性的“拟表位”序列。另外，PAb421的结果表明线性表位可能比以前认为的更长，并且可能在形式上不连续，由独立的接触基序组成，显示出混杂的相对位置。 [参考：111]

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