Lipoylated and unlipoylated domains of human PDC-E2 as autoantigens in primary biliary cirrhosis: significance of lipoate attachment.
Approximately 95% of patients with primary biliary cirrhosis have antimitochondrial antibodies against the E2 component of the pyruvate dehydrogenase complex (E2p). Immunodominant sites on E2p have been localized to the inner lipoyl domain, which serves as a covalent attachment site for the essential cofactor, lipoic acid. However, it is not clear whether the presence of lipoic acid is necessary for autoimmune recognition of human E2p. To facilitate further studies on the inner lipoyl domain and to assess the importance of lipoic acid in antibody binding, we used the previously cloned human E2p cDNA in the construction and high-level expression in Escherichia coli of a subgene encoding the domain. Purification and analysis of the gene product revealed that both lipoylated and unlipoylated forms of the intact domain are generated. Immunoblotting, enzyme-linked immunosorbent assay inhibition experiments and antibody affinity measurements using isolated lipoylated and unlipoylated domains demonstrated that the presence of the lipoyl residue is crucial for effective recognition by primary biliary cirrhosis patients' autoantibodies, which have a higher relative affinity for the lipoylated form. Contrary to some previous suggestions, these results indicate that antibodies in primary biliary cirrhosis patients' sera bind most effectively to a unique peptide-cofactor conformation in the lipoyl domain of the human E2p polypeptide. Moreover, the availability of large amounts of human lipoyl domain will permit further studies into the role of the antigen (if any) in disease pathogenesis.
Quinn
J
J
Department of Biochemistry and Genetics, Medical School, University of Newcastle upon Tyne, United Kingdom.
Diamond
A G
AG
Palmer
J M
JM
Bassendine
M F
MF
James
O F
OF
Yeaman
S J
SJ
D000123:Acetyltransferases / Q000276:immunology*; D000937:Antigen-Antibody Reactions; D001323:Autoantibodies / Q000276:immunology; D001324:Autoantigens / Q000276:immunology*; D051047:Dihydrolipoyllysine-Residue Acetyltransferase; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology; D006801:Humans; D015151:Immunoblotting; D008105:Liver Cirrhosis, Biliary / Q000276:immunology*; D011768:Pyruvate Dehydrogenase Complex / Q000276:immunology*; D008063:Thioctic Acid / Q000276:immunology*
D001323:Autoantibodies; D001324:Autoantigens; D000939:Epitopes; D011768:Pyruvate Dehydrogenase Complex; D008063:Thioctic Acid; D000123:Acetyltransferases; D051047:Dihydrolipoyllysine-Residue Acetyltransferase