The fingerprint of antimitochondrial antibodies and the etiology of primary biliary cholangitis.
The fingerprint of antimitochondrial antibodies and the etiology of primary biliary cholangitis.
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DOI：
10.1002/hep.29059
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发表时间：
2017-05
期刊：
影响因子：
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通讯作者：
Gershwin ME
中科院分区：
文献类型：
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作者：
Shuai Z;Wang J;Badamagunta M;Choi J;Yang G;Zhang W;Kenny TP;Guggenheim K;Kurth MJ;Ansari AA;Voss J;Coppel RL;Invernizzi P;Leung PSC;Gershwin ME
识别导致耐受性丧失的环境因素被称为自身免疫的圣杯。我们的工作集中在抗线粒体自身抗体(AMA)对化学异源生物的反应性上，并假设在失去耐受性时，主要线粒体自身抗原PDC-E2内的修饰肽将被免疫学识别。在这里，我们成功地应用内含子技术构建了包含PDC-E2 177-314氨基酸残基的PDC-E2蛋白片段，方法是将跨越177-252残基的重组多肽(PDC-228)与包含PDC-E2 ILD的62个残基的合成肽(PP)连接在一起。我们将这个由内含子构建的片段命名为PPL。重要的是，PPL以及硫辛酸偶联PPL(LA-PPL)和异生2-辛酸偶联PPL(2OA-PPL)都被AMA识别。重要的是，AMA对20A修饰的PDC-E2 ILD多肽骨架具有特异性，不同于与天然脂基化PDC-E2多肽反应的抗体。有趣的是，这种独特的AMA亚组分是IgM亚型，在早期PBC中更占优势，这表明接触类似20OA-PPL的化合物发生在AMA生成的早期。为了了解这种差异识别的结构基础，我们用电子顺磁共振波谱分析了PPL、LA-PPL和20A-PPL，并通过ELISA法、免疫印迹和亲和抗体分析进行了证实。我们证明了与硫辛酸结合时，PDC-E_2 ILD的构象发生了改变。总之，从分子水平上了解外源修饰的PDC-E2的构象对于理解外源修饰和PBC耐受性的丧失是至关重要的，这对于环境化学物质在诱导自身免疫中的作用具有广泛的意义。
The identification of environmental factors that lead to loss of tolerance has been coined the Holy Grail of autoimmunity. Our work has focused on the reactivity of antimitochondrial autoantibodies (AMA) to chemical xenobiotics and has hypothesized that a modified peptide within PDC-E2, the major mitochondrial autoantigen, will have been immunologically recognized at the time of loss of tolerance. Herein we successfully applied intein technology to construct a PDC-E2 protein fragment containing amino acid residues 177–314 of PDC-E2 by joining a recombinant peptide spanning residues 177 to 252 (PDC-228) with a 62 residue synthetic peptide from 253 to 314 (PP), which encompasses PDC-E2 ILD. We named this intein-constructed fragment PPL. Importantly, PPL, as well as lipoic acid conjugated PPL (LA-PPL) and xenobiotic 2-octynoic acid conjugated PPL (2OA-PPL), are recognized by AMA. Of great importance, AMA has specificity for the 2OA modified PDC-E2 ILD peptide backbone distinct from antibodies that react with native lipoylated PDC-E2 peptide. Interestingly, this unique AMA subfraction is of the IgM isotype and more dominant in early stage PBC, suggesting that exposure to 2OA-PPL-like compounds occurs early in the generation of AMA. To understand the structural basis of this differential recognition we analyzed PPL, LA-PPL and 2OA-PPL using electron paramagnetic resonance spectroscopy, with confirmations by ELISA, immunoblotting and affinity antibody analysis. We demonstrate that the conformation of PDC-E2 ILD is altered when conjugated with 2OA, compared to conjugation with lipoic acid. In conclusion a molecular understanding of the conformation of xenobiotic modified PDC-E2 is critical for understanding xenobiotic modification and loss of tolerance in PBC with widespread implications for a role of environmental chemicals in the induction of autoimmunity.