Csepregi Antal, Obermayer-Straub Petra, Kneip Susanne, Kayser Anne, Loges Stephanie, Schmidt Eleonore, Nemesánszky Elemér, Szalay Ferenc, Manns Michael P, Strassburg Christian P
Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Carl-Neuberg-Street 1, 30625 Hannover, Germany.
Clin Dev Immunol. 2003 Jun-Dec;10(2-4):173-81. doi: 10.1080/10446670310001642159.
Antimitochondrial antibodies (AMA) which recognize pyruvate acetyltransferase (PDC-E2) represent a highly diagnostic feature of primary biliary cirrhosis (PBC). The analysis of immunofluorescence (IF)-AMA-positive sera in PBC patients indicates a conformational epitope located within the lipoyl binding domain of bovine branched-chain acyltransferase (BCKADC-E2) alone or in combination with AMA directed against PDC-E2 the significance of which is presently unclear. In the present study, immunoreactivities and disease associations of AMA against BCKADC-E2 were analyzed. B-cell autoepitopes on BCKADC-E2 were mapped by immunoprecipitation assay.
Sera of 96 IF-AMA-positive patients with serological evidence of anti-BCKADC-E2 alone (n = 26), anti-PDC-E2 alone (n = 15), and both anti-BCKADC-E2 and anti-PDC-E2 (n = 55) were analyzed by Western blot and ELISA in addition to an analysis of B cell autoepitopes on BCKADC-E2 by immunoprecipitation using in vitro translated, unmodified human proteins. Ninety-four patients without IF-AMA [blood donors (n = 30), rheumatoid arthritis (n = 40), autoimmune hepatitis (AIH)(n = 10) and primary sclerosing cholangitis (PSC) (n = 14) served as controls.
Eighty of 81 (99%) sera positive for BCKADC-E2 recognized the full length, mature protein, while only 2/10 AIH sera and none of the other controls showed reactivity. Of the 68 PBC sera 58 (85%) recognized the N-terminus consisting of aa 1-144 representing the lipoyl domain. Surprisingly, C-terminal sequences (aa 143-421) were recognized by 46 out of 68 sera (68%). Three PBC sera reacted with the C-terminus only. Only 1/7 serum from patients with an "overlap syndrome of PBC and AIH" was reactive with C-terminal sequences.
Our analysis of BCKADC-E2-positive PBC sera identified a novel B cell epitope on the C-terminal part of the human protein. Our data indicate that a distinct subset of AMA recognize sequence(s) on BCKADC-E2 which located outside of the lipoyl binding domain. The absence of immunoreactivity against C-terminal sequences may serve as a marker differentiating patients with PBC and overlap syndrome of PBC with AIH.
识别丙酮酸乙酰转移酶（PDC-E2）的抗线粒体抗体（AMA）是原发性胆汁性肝硬化（PBC）的一项高度诊断特征。对PBC患者免疫荧光（IF）-AMA阳性血清的分析表明，位于牛支链酰基转移酶（BCKADC-E2）硫辛酰结合域内的构象表位单独存在，或与针对PDC-E2的AMA共同存在，其意义目前尚不清楚。在本研究中，分析了针对BCKADC-E2的AMA的免疫反应性及与疾病的关联。通过免疫沉淀试验对BCKADC-E2上的B细胞自身表位进行定位。
除了使用体外翻译的未修饰人蛋白通过免疫沉淀对BCKADC-E2上的B细胞自身表位进行分析外，还通过蛋白质印迹法和酶联免疫吸附测定法（ELISA）对96例IF-AMA阳性患者的血清进行了分析，这些患者血清学证据显示单独抗BCKADC-E2（n = 26）、单独抗PDC-E2（n = 15）以及同时抗BCKADC-E2和抗PDC-E2（n = 55）。94例无IF-AMA的患者[献血者（n = 30）、类风湿关节炎（n = 40）、自身免疫性肝炎（AIH）（n = 10）和原发性硬化性胆管炎（PSC）（n = 14）]作为对照。
81例BCKADC-E2阳性血清中的80例（99%）识别全长成熟蛋白，而10例AIH血清中只有2例显示反应性，其他对照均无反应性。68例PBC血清中的58例（85%）识别由1至144位氨基酸组成的代表硫辛酰域的N端。令人惊讶的是，68例血清中的46例（68%）识别C端序列（143至421位氨基酸）。3例PBC血清仅与C端反应。“PBC与AIH重叠综合征”患者的7例血清中只有1例与C端序列反应。
我们对BCKADC-E2阳性PBC血清的分析在人蛋白的C端部分鉴定出一个新的B细胞表位。我们的数据表明，AMA的一个独特亚群识别位于硫辛酰结合域外的BCKADC-E2上的序列。对C端序列无免疫反应性可能作为区分PBC患者和PBC与AIH重叠综合征患者的一个标志物。
