Wellcome Centre for Cell-Matrix Research, Faculty of Biology, Medicine and Health, University of Manchester, UK.
Manchester Institute of Biotechnology, University of Manchester, UK.
J Autoimmun. 2020 Jan;106:102308. doi: 10.1016/j.jaut.2019.102308. Epub 2019 Aug 5.
Patients with membranous nephropathy have autoantibodies against PLA2R (up to 80%), or THSD7A (up to 2%). We previously described the immunodominant epitope within PLA2R but epitopes in THSD7A are still unknown. To find anti-THSD7A sera for this study, we screened 1843 sera from biopsy-proven MN patients by ELISA and identified 22 sera as anti-THSD7A positive representing 1.2% of MN cases. Anti-THSD7A positive sera were further characterized by western blotting and slot blotting on THSD7A protein fragments and peptides. Real time interaction analyses and antibodies off-rate could be reliably determined using bio-layer interferometry. A signature motif in the N-terminal domain of THSD7A (T28mer) with sequence homology to the major PLA2R epitope (P28mer) was identified. B-cell epitope prediction analysis and homology modelling revealed this sequence to be antigenic and surface available suggesting it is accessible for the antibody to bind. All ten selected sera bound to the T28mer confirming this sequence as a dominant epitope in THSD7A. Reactivity to this sequence was lost following kallikrein protease cleavage within the predicted epitope. Importantly, cross-reactivity of both PLA2R and THSD7A autoantibodies was observed at the peptide but not the protein level. We propose that this common motif shared by both autoantigens could be an epitope involved in the initial B-cell triggering event in MN.
膜性肾病患者的自身抗体针对 PLA2R（高达 80%）或 THSD7A（高达 2%）。我们之前描述了 PLA2R 中的免疫优势表位，但 THSD7A 中的表位仍不清楚。为了在这项研究中找到抗 THSD7A 血清，我们通过 ELISA 筛选了 1843 份经活检证实的 MN 患者血清，并鉴定出 22 份抗 THSD7A 阳性血清，占 MN 病例的 1.2%。抗 THSD7A 阳性血清进一步通过 Western blot 和 THSD7A 蛋白片段和肽的插槽印迹进行特征描述。使用生物层干涉测量法可以可靠地确定实时相互作用分析和抗体的脱落率。在 THSD7A 的 N 端结构域中鉴定出一个与主要 PLA2R 表位（P28mer）具有序列同源性的特征基序（T28mer）。B 细胞表位预测分析和同源建模表明，该序列具有抗原性和表面可用性，表明它可供抗体结合。从预测的表位中经激肽酶切割后，十种选定的血清均与 T28mer 结合，证实该序列是 THSD7A 中的一个主要表位。在肽而非蛋白水平上观察到 PLA2R 和 THSD7A 自身抗体的交叉反应性。重要的是，这两种自身抗原共有的共同基序可能是 MN 中初始 B 细胞触发事件涉及的表位。
