Table_1_Anti-Ro60 Seropositivity Determines Anti-Ro52 Epitope Mapping in Patients With Systemic Sclerosis.DOCX

Epitope mapping of anti-Ro52 antibodies (Abs) has been extensively studied in patients with Sjögren's syndrome (SjS) and systemic lupus erythematosus (SLE). Comprehensive epitope mapping in systemic sclerosis (SSc), where anti-Ro52 antibodies are also frequently detected, has not been performed. The aim of the present study was to fully characterize Ro52 epitopes in anti-Ro52-positive SSc using Ro52 fragments spanning the full antigen. Further analysis was made according to anti-Ro60 status. Epitope mapping was performed in 43 anti-Ro52-positive SSc patients. Seventy eight anti-Ro52-positive pathological controls, including 20 patients with SjS, 28 patients with SLE, 15 patients with dermatomyositis (DM), and 15 patients with primary biliary cholangitis (PBC), and 20 anti-Ro52-negative healthy individuals as normal controls were also tested. Five recombinant Ro52 fragments [Ro52-1 (aa 1-127), Ro52-2 (aa 125-268), Ro52-3 (aa 268-475), Ro52-4 (aa 57-180), and Ro52-5 (aa 181-320) were used to test reactivity by line-immunoassay and in house ELISA. Anti-Ro60 reactivity was tested by ELISA. All anti-Ro52 positive sera reacted with Ro52-2; none recognized Ro52-3. Antibodies against Ro52-1 were less frequently found in SSc than in SjS/SLE (11.6 vs. 41.7%, p = 0.001); and antibodies against Ro52-4 were less frequently found in SSc than in SjS/SLE (27.9 vs. 50%, p = 0.03). In SSc patients, reactivity against Ro52-1 was more frequent in anti-Ro52+/anti-Ro60+ than in anti-Ro52+/anti-Ro60-patients (33.3 vs. 0%, p = 0.003). In this comprehensive analysis of Ro52 epitope mapping in SSc, the coiled coil domain remains the predominant epitope on Ro52. Contrary to SjS and SLE, patients with SSc fail to identify epitopic regions within the N-terminus of the protein, especially if they lack con-current anti-Ro60 reactivity.

抗Ro52抗体的表位作图（epitope mapping）已在干燥综合征（Sjögren's syndrome, SjS）与系统性红斑狼疮（systemic lupus erythematosus, SLE）患者中得到广泛研究。但在抗Ro52抗体亦频繁检出的系统性硬化症（systemic sclerosis, SSc）中，尚未开展全面的表位作图研究。本研究旨在利用覆盖Ro52全抗原的重组片段，全面鉴定抗Ro52阳性SSc患者体内的Ro52表位，并依据抗Ro60抗体状态进行分层分析。本研究共纳入43例抗Ro52阳性SSc患者，同时设置78例抗Ro52阳性病理对照（含20例干燥综合征患者、28例系统性红斑狼疮患者、15例皮肌炎（dermatomyositis, DM）患者及15例原发性胆汁性胆管炎（primary biliary cholangitis, PBC）患者），并以20例抗Ro52阴性健康个体作为正常对照纳入检测。本研究采用5个重组Ro52片段[Ro52-1（氨基酸1-127位，aa 1-127）、Ro52-2（aa 125-268）、Ro52-3（aa 268-475）、Ro52-4（aa 57-180）及Ro52-5（aa 181-320）]，通过线免疫印迹法及实验室自制酶联免疫吸附试验（ELISA）检测抗体反应性；同时采用ELISA检测抗Ro60抗体反应性。所有抗Ro52阳性血清均可与Ro52-2发生特异性结合，无血清识别Ro52-3。与干燥综合征/SLE患者相比，SSc患者中抗Ro52-1抗体阳性率更低（11.6% vs 41.7%，p=0.001）；抗Ro52-4抗体阳性率亦显著降低（27.9% vs 50%，p=0.03）。在SSc患者中，抗Ro52+/抗Ro60+亚组的抗Ro52-1抗体阳性率显著高于抗Ro52+/抗Ro60-亚组（33.3% vs 0%，p=0.003）。本项针对SSc患者Ro52表位作图的全面分析显示，卷曲螺旋结构域（coiled coil domain）仍是Ro52上的主要优势表位。与干燥综合征及SLE患者不同，SSc患者难以识别该蛋白N端（N-terminus）的表位区域，尤其是在同时缺乏抗Ro60抗体反应性的患者中。