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. This article is protected by copyright. All rights reserved

Shuai, Z., Wang, J., Badamagunta, M., Choi, J., Yang, G., Zhang, W., et al. (2017). The Fingerprint of Antimitochondrial Antibodies and the Etiology of Primary Biliary Cholangitis. HEPATOLOGY, 65(5), 1670-1682 [10.1002/hep.29059].

The Fingerprint of Antimitochondrial Antibodies and the Etiology of Primary Biliary Cholangitis

Invernizzi, P;
2017

Abstract

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. This article is protected by copyright. All rights reserved
Articolo in rivista - Articolo scientifico
2-octynoic acid; Antimitochondrial antibodies; E2 subunit of the pyruvate dehydrogenase; electron paramagnetic resonance spectroscopy; inner lipoyl domain; intein mediated ligation; primary biliary cholangitis; xenobiotics
English
31-mar-2017
2017
65
5
1670
1682
none
Shuai, Z., Wang, J., Badamagunta, M., Choi, J., Yang, G., Zhang, W., et al. (2017). The Fingerprint of Antimitochondrial Antibodies and the Etiology of Primary Biliary Cholangitis. HEPATOLOGY, 65(5), 1670-1682 [10.1002/hep.29059].
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/141463
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