title: Antimitochondrial antibody recognition and structural integrity of the inner lipoyl domain of the E2 subunit of pyruvate dehydrogenase complex.
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 (5)Ile→Ala and (12)Ile→Ala double mutant. Single alanine replacement at (5)Ile, (12)Ile, and (15)Glu significantly reduced AMA recognition. In addition, mutants with two, three, or four replacements at (5)Ile, (12)Ile, and (15)Glu reduced AMA reactivity even further. Indeed, EPR reveals a highly flexible structure within the (5)Ile and (12)Ile 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.
authors.affiliation: Division of Rheumatology, Allergy and Clinical Immunology, University of California Davis, Davis, CA 95616, USA.
authors.forename: Jinjun
authors.initials: J
authors.lastname: Wang
authors.forename: Madhu S
authors.initials: MS
authors.lastname: Budamagunta
authors.forename: John C
authors.initials: JC
authors.lastname: Voss
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Kurth
authors.forename: Kit S
authors.initials: KS
authors.lastname: Lam
authors.forename: Ling
authors.initials: L
authors.lastname: Lu
authors.forename: Thomas P
authors.initials: TP
authors.lastname: Kenny
authors.forename: Christopher
authors.initials: C
authors.lastname: Bowlus
authors.forename: Kentaro
authors.initials: K
authors.lastname: Kikuchi
authors.forename: Ross L
authors.initials: RL
authors.lastname: Coppel
authors.forename: Aftab A
authors.initials: AA
authors.lastname: Ansari
authors.forename: M Eric
authors.initials: ME
authors.lastname: Gershwin
authors.forename: Patrick S C
authors.initials: PS
authors.lastname: Leung
mesh_terms: D000595:Amino Acid Sequence; D000918:Antibody Specificity; D001323:Autoantibodies / Q000276:immunology*; D001324:Autoantigens / Q000276:immunology*; D051047:Dihydrolipoyllysine-Residue Acetyltransferase / Q000276:immunology*; D004578:Electron Spin Resonance Spectroscopy; D006801:Humans; D008105:Liver Cirrhosis, Biliary; D008928:Mitochondria / Q000201:enzymology / Q000276:immunology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary
chemical_list: D001323:Autoantibodies; D001324:Autoantigens; D051047:Dihydrolipoyllysine-Residue Acetyltransferase
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