Immunoreactivity of porcine heart dihydrolipoamide acetyl- and succinyl-transferases (PDC-E2, OGDC-E2) with primary biliary cirrhosis sera: characterization of the autoantigenic region and effects of enzymatic delipoylation and relipoylation.
Analysis of the primary structure of the lipoyl domain of the dihydrolipoamide acetyltransferase (PDC-E2) component of the porcine pyruvate dehydrogenase complex (PDC) reveals a high degree of homology with M2 antigen and human PDC-E2. The porcine PDC-E2 and the dihydrolipoamide succinyltransferase (OGDC-E2) component of the porcine 2-oxoglutarate dehydrogenase complex (OGDC) were identified as mitochondrial autoantigen with sera from patients with primary biliary cirrhosis (PBC). Immunodominant regions (autoepitopes) on the porcine-PDC-E2 component have been mapped to two regions around Lys-46 (outer lipoyl domain) and Lys-173 (inner lipoyl domain), which contained covalently bound lipoic acid prosthetic group. When these lipoyl domains were cleaved at Asp-45 or Asp-172 with endoproteinase Asp-N, the autoantigenicities of the two domains completely disappeared; this suggested the requirement of Asp-45 or Asp-172 residues for the immunoreaction with PBC sera. In addition, a single 14-amino acid epitope peptide histidine-substituted at Asp-172 did not exhibit competitive inhibition of autoantigen binding. Fragmentation of lipoyl domain of the porcine PDC-E2 by limited proteolysis and BrCN-cleavage after alkylation resulted in rapid loss of autoantigenicity. Enzymatic delipoylation and relipoylation of the complexed and free PDC-E2 and OGDC-E2 components did not influence immunoreactivity with PBC sera.
Koike
K
K
Department of Pathological Biochemistry, Atomic Disease Institute, Nagasaki University School of Medicine, Japan.
Ishibashi
H
H
Koike
M
M
D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D001323:Autoantibodies / Q000097:blood / Q000276:immunology*; D001324:Autoantigens / Q000276:immunology*; D051047:Dihydrolipoyllysine-Residue Acetyltransferase; D006801:Humans; D016056:Immunodominant Epitopes / Q000276:immunology*; D007655:Ketoglutarate Dehydrogenase Complex / Q000276:immunology*; D008105:Liver Cirrhosis, Biliary / Q000097:blood / Q000276:immunology*; D008969:Molecular Sequence Data; D011768:Pyruvate Dehydrogenase Complex / Q000276:immunology*; D013552:Swine
D001323:Autoantibodies; D001324:Autoantigens; D016056:Immunodominant Epitopes; D011768:Pyruvate Dehydrogenase Complex; D007655:Ketoglutarate Dehydrogenase Complex; D051047:Dihydrolipoyllysine-Residue Acetyltransferase