Bao L, Varden C E, Zimmer W E, Balczon R
Department of Structural and Cellular Biology, University of South Alabama, Mobile 36688, USA.
Mol Biol Rep. 1998 Mar;25(2):111-9. doi: 10.1023/a:1006814217037.
Characterization of epitope domains of autoantigens is important for deducing the cellular functions of autoantigens and may be important for understanding the autoimmune response. In the reported studies, epitope analysis of the centrosome autoantigen PCM-1 was performed. For these investigations, portion of the PCM-1 cDNA were subcloned into the pMAL expression plasmid, fusion proteins were induced, and aliquots of the extracts were probed by immunoblot analysis using two human autoimmune anticentrosome autoantisera. Immunoblotting identified three individual autoepitopes of 26-40 amino acid residues, amino acids 506-545, 1434-1465, and 1661-1686, within the PCM-1 protein. ELISA assays using non-denatured proteins did not identity any additional autoepitopes in the remainder of the PCM-1 molecule. To analyze the identified autoepitopes further, synthetic peptides were generated that covered each of the three autoepitopes and the synthetic peptides then were probed using the scleroderma sera. Peptides that covered the antigenic regions from amino acids 506-545 and 1434-1465 failed to react with the anticentrosome autoantisera suggesting that overall protein conformation may be important for the formation of those two autoepitopes. Peptides derived from the sequence of the third autoepitope were recognized by autoantibodies present in the anticentrosome autoantisera allowing the identification of the tripeptide KDC as the autoepitope in this region of the PCM-1 molecule. These studies lay the foundation for future investigations of the autoimmune response in scleroderma patients that are producing anticentrosome autoantibodies and should allow an investigation of the cellular role of the PCM-1 protein.
自身抗原表位结构域的鉴定对于推断自身抗原的细胞功能很重要，并且对于理解自身免疫反应可能也很重要。在已报道的研究中，对中心体自身抗原PCM-1进行了表位分析。在这些研究中，将部分PCM-1 cDNA亚克隆到pMAL表达质粒中，诱导融合蛋白表达，然后使用两种人类自身免疫性抗中心体自身抗血清通过免疫印迹分析检测提取物的等分试样。免疫印迹鉴定出PCM-1蛋白中三个分别由26 - 40个氨基酸残基组成的自身表位，即氨基酸506 - 545、1434 - 1465和1661 - 1686。使用非变性蛋白的ELISA分析未在PCM-1分子的其余部分鉴定出任何其他自身表位。为了进一步分析鉴定出的自身表位，合成了覆盖三个自身表位中每一个的合成肽，然后使用硬皮病血清检测这些合成肽。覆盖氨基酸506 - 545和1434 - 1465抗原区域的肽未能与抗中心体自身抗血清反应，这表明整体蛋白质构象对于这两个自身表位的形成可能很重要。源自第三个自身表位序列的肽被抗中心体自身抗血清中存在的自身抗体识别，从而确定三肽KDC为PCM-1分子该区域的自身表位。这些研究为未来对产生抗中心体自身抗体的硬皮病患者自身免疫反应的研究奠定了基础，并且应该有助于对PCM-1蛋白的细胞作用进行研究。
