Dubel L, Tanaka A, Leung P S, Van de Water J, Coppel R, Roche T, Johanet C, Motokawa Y, Ansari A, Gershwin M E
Department of Microbiology, Monash University, Victoria, Australia.
Hepatology. 1999 Apr;29(4):1013-8. doi: 10.1002/hep.510290403.
Primary biliary cirrhosis (PBC) is an autoimmune liver disease characterized by the presence of antimitochondrial antibodies (AMA) directed primarily against the E2 subunits of the pyruvate dehydrogenase complex, the branched chain 2-oxo-acid dehydrogenase complex, the 2-oxoglutarate dehydrogenase complex, as well as the dihydrolipoamide dehydrogenase-binding protein (E3BP) of pyruvate dehydrogenase complex. The autoantibody response to each E2 subunit is directed to the lipoic acid binding domain. However, hitherto, the epitope recognized by autoantibodies to E3BP has not been mapped. In this study, we have taken advantage of the recently available full-length human E3BP complementary DNA (cDNA) to map this epitope. In addition, another lipoic binding protein, the H-protein of the glycine cleavage complex, was also studied as a potential autoantigen recognized by AMA. Firstly, the sequence corresponding to the lipoic domain of E3BP (E3BP-LD) was amplified by polymerase chain reaction and recombinant protein and then purified. Immunoreactivity of 45 PBC sera (and 52 control sera) against the purified recombinant E3BP-LD was analyzed by enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Secondly, reactivity of PBC sera was similarly analyzed by immunoblotting against H-protein. It is interesting that preabsorption of patient sera with the lipoic acid binding domain of E3BP completely removed all reactivity with the entire protein by immunoblotting analysis, suggesting that autoantibodies to E3BP are directed solely to its lipoic acid binding domain. Fifty-three percent of PBC sera reacted with E3BP-LD, with the majority of the response being of the immunoglobulin G (IgG) isotype (95%). Surprisingly, there was little IgM response to the E3BP-LD suggesting that the immune response was secondary because of determinant spreading. In contrast, H-protein does not appear to possess (or expose) autoepitopes recognized by PBC sera. This observation is consistent with structural data on this moiety.
原发性胆汁性肝硬化（PBC）是一种自身免疫性肝病，其特征是存在主要针对丙酮酸脱氢酶复合体E2亚基、支链2-氧代酸脱氢酶复合体、2-氧代戊二酸脱氢酶复合体以及丙酮酸脱氢酶复合体的二氢硫辛酰胺脱氢酶结合蛋白（E3BP）的抗线粒体抗体（AMA）。对每个E2亚基的自身抗体反应针对的是硫辛酸结合域。然而，迄今为止，E3BP自身抗体识别的表位尚未定位。在本研究中，我们利用最近获得的全长人E3BP互补DNA（cDNA）来定位该表位。此外，还研究了另一种硫辛酸结合蛋白——甘氨酸裂解复合体的H蛋白，作为AMA识别的潜在自身抗原。首先，通过聚合酶链反应扩增与E3BP硫辛酸域（E3BP-LD）对应的序列，然后纯化重组蛋白。通过酶联免疫吸附测定（ELISA）和免疫印迹分析45份PBC血清（和52份对照血清）对纯化的重组E3BP-LD的免疫反应性。其次，通过对H蛋白的免疫印迹类似地分析PBC血清的反应性。有趣的是，用E3BP的硫辛酸结合域预先吸收患者血清后，通过免疫印迹分析完全消除了与整个蛋白的所有反应性，这表明针对E3BP的自身抗体仅针对其硫辛酸结合域。53%的PBC血清与E3BP-LD反应，大多数反应为免疫球蛋白G（IgG）同种型（95%）。令人惊讶的是，对E3BP-LD几乎没有IgM反应，这表明免疫反应是由于决定簇扩展而继发的。相比之下，H蛋白似乎不具有（或暴露）PBC血清识别的自身表位。这一观察结果与该部分的结构数据一致。
