Department of Interdisciplinary Medicine, Rheumatic and Systemic Autoimmune Diseases Unit, University of Bari Medical School, Bari, Italy.
Department of Biomedical Sciences and Human Oncology, University of Bari Medical School, Bari, Italy.
Clin Exp Immunol. 2023 Apr 7;212(1):32-38. doi: 10.1093/cei/uxad012.
Anti-centromere (ACA) and antimitochondrial antibodies (AMA) are specific for limited-cutaneous systemic sclerosis (lcSSc) and primary biliary cholangitis (PBC), respectively, and can coexist in up to 25 and 30% of SSc and PBC patients. Here, we evaluated whether anti-centromeric protein A (CENP-A) antibodies cross-react with mitochondrial antigens. To this end, sera from two lcSSc patients (pt1 and pt4), one of them (pt4) also affected by PBC, were used as the source of ACA, previously shown to recognize different groups of amino acids (motifs) in the CENP-A region spanning amino acids 1-17 (Ap1-17). Pt1 and pt4 Ap1-17-specific IgG were purified by affinity-chromatography on insolubilized Ap1-17-peptide column and tested by western blotting with nuclear and cytoplasmic protein extract from HeLa cells. Immunoreactive proteins were identified by mass spectrometry and validated by immunodot. The results showed that affinity-purified SSc/PBC pt4 anti-Ap1-17 and not SSc pt1 anti-Ap1-17 Ab, specifically cross-reacted with the E2 component of the mitochondrial pyruvate dehydrogenase complex (PDC-E2), the major mitochondrial autoantigen in PBC. Sequence homology analysis indicated that the motif A-x-x-P-x-A-P recognized by pt4 anti-Ap1-17 IgG and shared by CENP-A and PDC-E2, is also expressed by some members of the Human Herpesvirus family, suggesting that they may trigger the production of these cross-reacting antibodies.
抗着丝点(ACA)和抗线粒体抗体(AMA)分别是局限性皮肤系统性硬化症(lcSSc)和原发性胆汁性胆管炎(PBC)的特异性抗体,在高达 25%和 30%的 SSc 和 PBC 患者中可共存。在这里,我们评估了抗着丝点蛋白 A(CENP-A)抗体是否与线粒体抗原发生交叉反应。为此,我们使用来自两名局限性皮肤系统性硬化症患者(pt1 和 pt4)的血清,其中一名患者(pt4)还患有原发性胆汁性胆管炎,作为 ACA 的来源,先前的研究表明,这些抗体可识别 CENP-A 区域跨越氨基酸 1-17(Ap1-17)的不同氨基酸(基序)组。Pt1 和 pt4 Ap1-17 特异性 IgG 通过固定在 Ap1-17-肽柱上的亲和层析进行纯化,并通过 Western blot 用来自 HeLa 细胞的核和细胞质蛋白提取物进行测试。通过质谱鉴定免疫反应性蛋白,并通过免疫斑点验证。结果表明,亲和纯化的 SSc/PBC pt4 抗 Ap1-17 而非 SSc pt1 抗 Ap1-17 Ab 特异性地与线粒体丙酮酸脱氢酶复合物(PDC-E2)的 E2 成分发生交叉反应,这是 PBC 的主要线粒体自身抗原。序列同源性分析表明,pt4 抗 Ap1-17 IgG 识别的基序 A-x-x-P-x-A-P 与 CENP-A 和 PDC-E2 共享,也被人类疱疹病毒家族的一些成员表达,提示它们可能触发这些发生交叉反应的抗体的产生。