Mahler M, Mierau R, Blüthner M
Institute of Molecular Genetics, University of Heidelberg, Germany.
J Mol Med (Berl). 2000;78(8):460-7. doi: 10.1007/s001090000128.
The major targets recognized by anti-centromere autoantibodies are the three centromere-associated proteins (CENPs) A, B, and C, with apparent molecular masses of 19, 80, and 140 kDa, respectively. Previously a major epitope region on the 19-kDa CENP-A antigen was identified by synthesis of a soluble synthetic 15-mer peptide (amino acids 3-17) to be used in enzyme-linked immunosorbent assay and western blot competition assays. However, no systematic experimental scanning for epitope regions on the CENP-A autoantigen has yet been performed. In this study we scanned the complete CENP-A amino acid sequence for epitopes using 19 previously characterized autoimmune-sera. Overlapping peptides 15 amino acids in length and offset by three amino acids were synthesized on activated membranes, covering the whole CENP-A autoantigen. Probing of the membranes with various anti-centromere sera showed that all epitopes are clustered in the N-terminal 45 amino acids. For fine-mapping of this autoreactive region the N-terminus of CENP-A (amino acids 1-45) was scanned again by probing overlapping 15-mer, 12-mer, 10-mer, 8-mer, 7-mer, 6-mer, and 5-mer peptides, all offset by one amino acid, with anti-centromere sera. In this way we localized two epitope core regions within the N-terminal 45 amino acids, one covering amino acids 2-17, recognized by 17 sera, and the other covering amino acids 22-38, recognized by 18 sera. One serum did not react with CENP-A at all. Several sera seem to recognize overlapping individual epitopes within these two epitope core regions. All sera, however, recognize a sequence motif G/A-P-R/S-R-R.
抗着丝粒自身抗体识别的主要靶标是三种着丝粒相关蛋白（CENPs）A、B和C，其表观分子量分别为19 kDa、80 kDa和140 kDa。此前，通过合成一种可溶性合成15肽（氨基酸3 - 17），并将其用于酶联免疫吸附测定和蛋白质印迹竞争测定，确定了19 kDa CENP - A抗原上的一个主要表位区域。然而，尚未对CENP - A自身抗原上的表位区域进行系统的实验扫描。在本研究中，我们使用19份先前鉴定的自身免疫血清扫描了完整的CENP - A氨基酸序列中的表位。在活化膜上合成了长度为15个氨基酸且相互错开三个氨基酸的重叠肽，覆盖了整个CENP - A自身抗原。用各种抗着丝粒血清探测这些膜表明，所有表位都聚集在N端的45个氨基酸中。为了对这个自身反应区域进行精细定位，再次用抗着丝粒血清探测相互错开一个氨基酸的重叠15肽、12肽、10肽、8肽、7肽、6肽和5肽，扫描CENP - A的N端（氨基酸1 - 45）。通过这种方式，我们在N端的45个氨基酸中定位了两个表位核心区域，一个覆盖氨基酸2 - 17，被17份血清识别，另一个覆盖氨基酸22 - 38，被18份血清识别。有一份血清根本不与CENP - A反应。几份血清似乎识别这两个表位核心区域内重叠的单个表位。然而，所有血清都识别一个序列基序G/A - P - R/S - R - R。
