Antimitochondrial antibody recognition and structural integrity of the inner lipoyl domain of the E2 subunit of pyruvate dehydrogenase complex

Jinjun Wang, Madhu S. Budamagunta, John C Voss, Mark J. Kurth, Kit Lam, Ling Lu, Thomas P. Kenny, Christopher Bowlus, Kentaro Kikuchi, Ross L. Coppel, Aftab A. Ansari, M. Eric Gershwin, Patrick S Leung

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

Antimitochondrial autoantibodies (AMAs), the serological hallmark of primary biliary cirrhosis, are directed against the lipoyl domain of the E2 subunit of pyruvate dehydrogenase (PDC-E2). However, comprehensive analysis of the amino acid residues of PDC-E2 lipoyl β-sheet with AMA specificity is lacking. In this study, we postulated that specific residues within the lipoyl domain are critical to AMA recognition by maintaining conformational integrity. We systematically replaced each of 19 residue peptides of the inner lipoyl domain with alanine and analyzed these mutants for reactivities against 60 primary biliary cirrhosis and 103 control sera. Based on these data, we then constructed mutants with two, three, or four replacements and, in addition, probed the structure of the substituted domains using thiol-specific spin labeling and electron paramagnetic resonance (EPR) of a 5Ile→Ala and 12Ile→Ala double mutant. Single alanine replacement at 5Ile, 12Ile, and 15Glu significantly reduced AMA recognition. In addition, mutants with two, three, or four replacements at 5Ile, 12Ile, and 15Glu reduced AMA reactivity even further. Indeed, EPR reveals a highly flexible structure within the 5Ile and 12Ile double-alanine mutant. Autoreactivity is largely focused on specific residues in the PDC-E2 lipoyl domain critical in maintaining the lipoyl loop conformation necessary for AMA recognition. Collectively, the AMA binding studies and EPR analysis demonstrate the necessity of the lipoyl β-sheet structural conformation in anti-PDC-E2 recognition.

Original languageEnglish (US)
Pages (from-to)2126-2133
Number of pages8
JournalJournal of Immunology
Volume191
Issue number5
DOIs
StatePublished - Sep 1 2013

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Dihydrolipoyllysine-Residue Acetyltransferase
Autoantibodies
Antibodies
Electron Spin Resonance Spectroscopy
Alanine
Biliary Liver Cirrhosis
Pyruvic Acid
Sulfhydryl Compounds
Oxidoreductases
Amino Acids
Peptides

ASJC Scopus subject areas

  • Immunology

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Antimitochondrial antibody recognition and structural integrity of the inner lipoyl domain of the E2 subunit of pyruvate dehydrogenase complex. / Wang, Jinjun; Budamagunta, Madhu S.; Voss, John C; Kurth, Mark J.; Lam, Kit; Lu, Ling; Kenny, Thomas P.; Bowlus, Christopher; Kikuchi, Kentaro; Coppel, Ross L.; Ansari, Aftab A.; Gershwin, M. Eric; Leung, Patrick S.

In: Journal of Immunology, Vol. 191, No. 5, 01.09.2013, p. 2126-2133.

Research output: Contribution to journalArticle

Wang, Jinjun ; Budamagunta, Madhu S. ; Voss, John C ; Kurth, Mark J. ; Lam, Kit ; Lu, Ling ; Kenny, Thomas P. ; Bowlus, Christopher ; Kikuchi, Kentaro ; Coppel, Ross L. ; Ansari, Aftab A. ; Gershwin, M. Eric ; Leung, Patrick S. / Antimitochondrial antibody recognition and structural integrity of the inner lipoyl domain of the E2 subunit of pyruvate dehydrogenase complex. In: Journal of Immunology. 2013 ; Vol. 191, No. 5. pp. 2126-2133.
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abstract = "Antimitochondrial autoantibodies (AMAs), the serological hallmark of primary biliary cirrhosis, are directed against the lipoyl domain of the E2 subunit of pyruvate dehydrogenase (PDC-E2). However, comprehensive analysis of the amino acid residues of PDC-E2 lipoyl β-sheet with AMA specificity is lacking. In this study, we postulated that specific residues within the lipoyl domain are critical to AMA recognition by maintaining conformational integrity. We systematically replaced each of 19 residue peptides of the inner lipoyl domain with alanine and analyzed these mutants for reactivities against 60 primary biliary cirrhosis and 103 control sera. Based on these data, we then constructed mutants with two, three, or four replacements and, in addition, probed the structure of the substituted domains using thiol-specific spin labeling and electron paramagnetic resonance (EPR) of a 5Ile→Ala and 12Ile→Ala double mutant. Single alanine replacement at 5Ile, 12Ile, and 15Glu significantly reduced AMA recognition. In addition, mutants with two, three, or four replacements at 5Ile, 12Ile, and 15Glu reduced AMA reactivity even further. Indeed, EPR reveals a highly flexible structure within the 5Ile and 12Ile double-alanine mutant. Autoreactivity is largely focused on specific residues in the PDC-E2 lipoyl domain critical in maintaining the lipoyl loop conformation necessary for AMA recognition. Collectively, the AMA binding studies and EPR analysis demonstrate the necessity of the lipoyl β-sheet structural conformation in anti-PDC-E2 recognition.",
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AU - Budamagunta, Madhu S.

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AU - Kurth, Mark J.

AU - Lam, Kit

AU - Lu, Ling

AU - Kenny, Thomas P.

AU - Bowlus, Christopher

AU - Kikuchi, Kentaro

AU - Coppel, Ross L.

AU - Ansari, Aftab A.

AU - Gershwin, M. Eric

AU - Leung, Patrick S

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N2 - Antimitochondrial autoantibodies (AMAs), the serological hallmark of primary biliary cirrhosis, are directed against the lipoyl domain of the E2 subunit of pyruvate dehydrogenase (PDC-E2). However, comprehensive analysis of the amino acid residues of PDC-E2 lipoyl β-sheet with AMA specificity is lacking. In this study, we postulated that specific residues within the lipoyl domain are critical to AMA recognition by maintaining conformational integrity. We systematically replaced each of 19 residue peptides of the inner lipoyl domain with alanine and analyzed these mutants for reactivities against 60 primary biliary cirrhosis and 103 control sera. Based on these data, we then constructed mutants with two, three, or four replacements and, in addition, probed the structure of the substituted domains using thiol-specific spin labeling and electron paramagnetic resonance (EPR) of a 5Ile→Ala and 12Ile→Ala double mutant. Single alanine replacement at 5Ile, 12Ile, and 15Glu significantly reduced AMA recognition. In addition, mutants with two, three, or four replacements at 5Ile, 12Ile, and 15Glu reduced AMA reactivity even further. Indeed, EPR reveals a highly flexible structure within the 5Ile and 12Ile double-alanine mutant. Autoreactivity is largely focused on specific residues in the PDC-E2 lipoyl domain critical in maintaining the lipoyl loop conformation necessary for AMA recognition. Collectively, the AMA binding studies and EPR analysis demonstrate the necessity of the lipoyl β-sheet structural conformation in anti-PDC-E2 recognition.

AB - Antimitochondrial autoantibodies (AMAs), the serological hallmark of primary biliary cirrhosis, are directed against the lipoyl domain of the E2 subunit of pyruvate dehydrogenase (PDC-E2). However, comprehensive analysis of the amino acid residues of PDC-E2 lipoyl β-sheet with AMA specificity is lacking. In this study, we postulated that specific residues within the lipoyl domain are critical to AMA recognition by maintaining conformational integrity. We systematically replaced each of 19 residue peptides of the inner lipoyl domain with alanine and analyzed these mutants for reactivities against 60 primary biliary cirrhosis and 103 control sera. Based on these data, we then constructed mutants with two, three, or four replacements and, in addition, probed the structure of the substituted domains using thiol-specific spin labeling and electron paramagnetic resonance (EPR) of a 5Ile→Ala and 12Ile→Ala double mutant. Single alanine replacement at 5Ile, 12Ile, and 15Glu significantly reduced AMA recognition. In addition, mutants with two, three, or four replacements at 5Ile, 12Ile, and 15Glu reduced AMA reactivity even further. Indeed, EPR reveals a highly flexible structure within the 5Ile and 12Ile double-alanine mutant. Autoreactivity is largely focused on specific residues in the PDC-E2 lipoyl domain critical in maintaining the lipoyl loop conformation necessary for AMA recognition. Collectively, the AMA binding studies and EPR analysis demonstrate the necessity of the lipoyl β-sheet structural conformation in anti-PDC-E2 recognition.

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