Department of Neuroimmunology, Max-Planck-Institute of Neurobiology, Planegg/Martinsried, Germany.
J Immunol. 2010 Jul 1;185(1):387-99. doi: 10.4049/jimmunol.0903887. Epub 2010 Jun 7.
Anti-neutrophil cytoplasmic Abs (cANCAs) against conformational epitopes of proteinase 3 (PR3) are regarded as an important pathogenic marker in Wegener's granulomatosis (WG). Although the three-dimensional structure of PR3 is known, binding sites of mAbs and cANCAs have not been mapped to date. Competitive binding and biosensor experiments suggested the existence of four nonoverlapping areas on the PR3 surface. In this paper, we present an approach to identify these discontinuous surface regions that cannot be mimicked by linear peptides. The very few surface substitutions found in closely related PR3 homologs from primates, which were further varied by the construction of functional human-gibbon hybrids, resulted in the differential loss of three Ab binding sites, two of which were mapped to the N-terminal beta-barrel and one to the linker segment connecting the N- and C-terminal barrels of PR3. The sera from WG patients differed in their binding to gibbon PR3 and the gibbon-human PR3 hybrid, and could be divided into two groups with similar or significantly reduced binding to gibbon PR3. Binding of almost all sera to PR3-alpha1-protease inhibitor (alpha1-PI) complexes was even more reduced and often absent, indicating that major antigenic determinants overlap with the active site surface on PR3 that associates with alpha1-PI. Similarly, the mouse mAbs CLB12.8 and 6A6 also did not react with gibbon PR3 and PR3-alpha1-PI complexes. Our data strongly suggest that cANCAs from WG patients at least in part recognize similar surface structures as do mouse mAbs and compete with the binding of alpha1-PI to PR3.
抗中性粒细胞胞质抗体（cANCAs）针对蛋白酶 3（PR3）的构象表位被认为是 Wegener 肉芽肿（WG）中的一个重要致病标志物。尽管 PR3 的三维结构已经为人所知，但 mAbs 和 cANCAs 的结合位点迄今尚未被定位。竞争性结合和生物传感器实验表明，PR3 表面存在四个非重叠区域。在本文中，我们提出了一种识别这些不能被线性肽模拟的不连续表面区域的方法。在灵长类动物的 PR3 同源物中发现的极少数表面取代，进一步通过构建功能性人-长臂猿杂种进行了改变，导致三个 Ab 结合位点的差异丧失，其中两个定位在 N 端β桶，一个定位在连接 PR3 的 N 端和 C 端桶的连接片段。WG 患者的血清在与长臂猿 PR3 和长臂猿-人 PR3 杂种的结合方面存在差异，可以分为两组，与长臂猿 PR3 的结合相似或明显减少。几乎所有血清与 PR3-α1-蛋白酶抑制剂（α1-PI）复合物的结合也被进一步减少，并且经常缺失，这表明主要抗原决定簇与 PR3 上与 α1-PI 相关的活性位点表面重叠。同样，小鼠 mAbs CLB12.8 和 6A6 也不与长臂猿 PR3 和 PR3-α1-PI 复合物反应。我们的数据强烈表明，WG 患者的 cANCAs 至少部分识别与小鼠 mAbs 相同的表面结构，并与 α1-PI 与 PR3 的结合竞争。
