Otvos L, Hoffmann R, Xiang Z Q, O I, Deng H, Wysocka M, Pease A M, Rogers M E, Blaszczyk-Thurin M, Ertl H C
The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA.
Biochim Biophys Acta. 1998 Sep 16;1404(3):457-74. doi: 10.1016/s0167-4889(98)00087-1.
Mutations of the gene encoding the tumor suppressor protein p53 are the most common molecular alterations of cancer cells found in about half of all human tumors. Mutations which cluster in well-defined hot spots change the structure of the protein thus affecting its ability to bind to DNA. Post-translational modifications, primarily phosphorylation, might also influence how p53 binds to DNA or folds to its active tetrameric form. However, the lack of appropriate biochemical markers to characterize the status of phosphorylation in different cell types and in cells at different stages of tumor progression has prohibited such investigations. To generate a sensitive and phosphorylation-specific monoclonal antibody (mAb), we chemically synthesized the C-terminal 23 amino acid stretch of human p53 in a double-phosphorylated form. The peptide 371-393, carrying phosphate groups on Ser378 and Ser392, was co-synthesized with a turn-inducing spacer and peptide 31D, an immunodominant T-helper cell epitope in mice of the H-2k haplotype. After immunization and fusion of splenocytes with myeloma cells, a number of mAbs were obtained, from which mAb p53-18 emerged as a highly sensitive reagent. By enzyme-linked immunosorbent assay, p53-18, a mAb of the IgM isotype, recognized phosphorylated p53, expressed in insect cells infected with a recombinant baculovirus but not p53 expressed in Escherichia coli. Moreover, murine p53 from insect cells could be immune purified with mAb p53-18. Mass spectrometry following tryptic digestion of the purified protein and liquid chromatography of the fragments verified the presence of phosphate groups at both Ser375 and Ser389. From the corresponding human protein fragments, mAb p53-18 bound to the immunizing peptide phosphorylated on Ser378 and on Ser392, but failed to cross-react with the unphosphorylated peptide, or peptides phosphorylated individually on either Ser378 or Ser392. The binding to the unphosphorylated peptide could be restored, however, if the peptide conformation was stabilized to that of an alpha-helix. The immunogenic nature of the multiphosphorylated C-terminus of p53 is indicated by the finding that human sera, mostly from cancer patients, preferentially recognized the double-phosphorylated peptide over the monophosphorylated or unphosphorylated analogs. Antibody p53-18 appears to be a highly useful biochemical marker to detect low levels of p53 protein in different tissues, and to be a key tool to characterize the phosphorylation status of the C-terminus of p53 protein originated from various sources.
编码肿瘤抑制蛋白p53的基因发生突变是癌细胞中最常见的分子改变，在大约一半的人类肿瘤中都能发现。聚集在明确热点区域的突变会改变蛋白质的结构，从而影响其与DNA结合的能力。翻译后修饰，主要是磷酸化，也可能影响p53与DNA的结合方式或折叠成其活性四聚体形式。然而，缺乏合适的生化标志物来表征不同细胞类型以及肿瘤进展不同阶段细胞中的磷酸化状态，阻碍了此类研究。为了生成一种敏感且磷酸化特异性的单克隆抗体（mAb），我们以双磷酸化形式化学合成了人p53的C末端23个氨基酸片段。携带Ser378和Ser392磷酸基团的肽371 - 393与一个诱导转角的间隔区以及肽31D（H - 2k单倍型小鼠中的免疫显性T辅助细胞表位）共同合成。在脾细胞与骨髓瘤细胞免疫和融合后，获得了多种单克隆抗体，其中mAb p53 - 18成为一种高度敏感的试剂。通过酶联免疫吸附测定，IgM同种型的单克隆抗体p53 - 18识别在感染重组杆状病毒的昆虫细胞中表达的磷酸化p53，但不识别在大肠杆菌中表达的p53。此外，来自昆虫细胞的鼠p53可用单克隆抗体p53 - 18进行免疫纯化。对纯化蛋白进行胰蛋白酶消化后的质谱分析以及片段的液相色谱分析证实了Ser375和Ser389处均存在磷酸基团。从相应的人蛋白片段来看，单克隆抗体p53 - 18与在Ser378和Ser392上磷酸化的免疫原性肽结合，但不与未磷酸化的肽或仅在Ser378或Ser392上磷酸化的肽发生交叉反应。然而，如果肽的构象稳定为α - 螺旋构象，则与未磷酸化肽的结合可以恢复。p53多磷酸化C末端的免疫原性体现在以下发现中：大多数来自癌症患者的人血清优先识别双磷酸化肽而非单磷酸化或未磷酸化的类似物。抗体p53 - 18似乎是检测不同组织中低水平p53蛋白的非常有用的生化标志物，并且是表征源自各种来源的p53蛋白C末端磷酸化状态的关键工具。
