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 anti mitochondrial antibodies against the E2 component of the pyruvate dehy drogenase complex (E2p). Immunodominant sites on E2p have been localiz ed to the inner lipoyl domain, which serves as a covalent attachment s ite for the essential cofactor, lipoic acid. However, it is not clear whether the presence of lipoic acid is necessary for autoimmune recogn ition 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 d omain. Purification and analysis of the gene product revealed that bot h lipoylated and unlipoylated forms of the intact domain are generated . Immunoblotting, enzyme-linked immunosorbent assay inhibition experim ents and antibody affinity measurements using isolated lipoylated and unIipoylated domains demonstrated that the presence of the lipoyl resi due is crucial for effective recognition by primary biliary cirrhosis patients' autoantibodies, which have a higher relative affinity for th e lipoylated form. Contrary to some previous suggestions, these result s indicate that antibodies in primary biliary cirrhosis patients' sera bind most effectively to a unique peptide-cofactor conformation in th e lipoyl domain of the human E2p polypeptide. Moreover, the availabili ty of large amounts of human lipoyl domain will permit further studies into the role of the antigen (if any) in disease pathogenesis.