Stephen C W, Helminen P, Lane D P
Department of Biochemistry, University of Dundee, Scotland, UK.
J Mol Biol. 1995 Apr 21;248(1):58-78. doi: 10.1006/jmbi.1995.0202.
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 detailed 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 can 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.
我们之前描述了利用一个随机六肽噬菌体展示文库来确定和定位单克隆抗体PAb240所识别的人类肿瘤抑制蛋白p53上的表位。在此，我们将这些结果扩展至另外8种抗p53单克隆抗体以及另外两个分别展示12肽和20肽的文库。首先，我们表明从12肽和20肽文库中筛选PAb240结合克隆所得到的结果与筛选6肽文库所获得的结果基本相同。其次，我们利用6肽和12肽文库来确定6种抗体（DO-1、DO-2、DO-7、Bp53-11、Bp53-12和Bp53-19）的详细特异性图谱，这些抗体识别p53相同的短且高度免疫原性的N端片段。最后，我们利用所有这三个文库来揭示PAb421和PAb122这两种单克隆抗体通过变构激活p53的序列特异性DNA结合的独特机制，它们与相同的带正电荷的C端片段特异性反应。在每种情况下，通过探测一系列代表p53一级序列的重叠合成肽，证实了从所选序列推断出的表位位置。这些结果强调了筛选随机肽文库（而非抗原衍生肽文库）对表位作图的影响；具体而言：（1）所选序列的比较揭示了单个残基对结合能和特异性的贡献；（2）交叉反应可导致确定一个与免疫原性表位相关但又不同的共有序列；（3）非免疫原同源“模拟表位”序列的分离揭示了离散的、替代性的配体结构。PAb421和PAb122的结果提供了这样的例子，即从12肽和20肽文库中筛选会导致免疫原同源序列的分离，而从6肽文库中筛选则要么得不到结合克隆（PAb122），要么仅得到与原始抗原无明显同源性的“模拟表位”序列（PAb421）。此外，PAb421的结果表明线性表位可能比之前认为的更长，并且可能是形式上不连续的，由显示混杂相对定位的独立接触基序组成。
