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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.

Antimitochondrial autoantibodies are present in sera from close to 95% of patients with primary biliary cirrhosis. The so-called primary biliary cirrhosis-specific antigen, named M2, was found to be associated with an enzyme complex of the inner mitochondrial membrane and, more precisely, with the E2 component, dihydrolipoamide acetyltransferase, of the pyruvate dehydrogenase complex. We recently established that an immunodominant epitope recognized in direct enzyme-linked immunosorbent assay by primary biliary cirrhosis M2+ sera, but not by non-primary biliary cirrhosis M2+ sera, could be mimicked by a synthetic peptide encompassing residues 167-184 of the E2 component and associated with lipoic acid. This fragment is present in the natural inner lipoyl-binding site of the human enzyme, and the presence of lipoic acid located on lysine 173 was found to be essential to allow IgG antibody binding. In this study we have improved the enzyme-linked immunosorbent assay test based on the synthetic peptide-lipoic acid conjugate by using a multiple antigen peptide system containing eight copies of the peptide as antigen. This approach avoids the use of a peptide conjugated to a carrier protein and was found to be particularly efficient because 23 of 27 primary biliary cirrhosis M2+ sera (85%) could be identified. A multiple antigen peptide without lipoic acid was not recognized by primary biliary cirrhosis antibodies. The peptide used in the multiple antigen peptide construction was a short 13-mer peptide encompassing a highly conserved sequence present in both the outer (residues 40-52) and the inner (residues 167-179) lipoyl-binding sites of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

In normal individuals there is an adaptive immune response to a foreign antigen in which antibodies of increasing affinity are produced with time. This is not always true of an autoimmune response. However, because only a limited number of autoantigens have been cloned or purified, this issue has not been studied well. In primary biliary cirrhosis the predominant manifestation of autoimmunity is antimitochondrial antibodies that react with dihydrolipoamide transacetylase. The availability of recombinant dihydrolipoamide transacetylase and the development of a rapid and reproducible enzyme-linked immunosorbent assay for autoantibodies has allowed us to address the affinity of autoantibodies using thiocyanate inhibition. Thiocyanate is a chaotropic compound known to inhibit antigen-antibody binding in a concentration-dependent manner. We used this property to inhibit the binding by enzyme-linked immunosorbent assay of human recombinant dihydrolipoamide transacetylase with serum autoantibodies from 55 patients with primary biliary cirrhosis. The relative affinity and serum autoantibody titers were then compared with the histological stage of the liver biopsy sample. Interestingly, we found a considerable heterogeneity of relative affinities. These relative affinities did not correlate with the histological stage or the serum titer of antimitochondrial antibodies. However, the ability of serum autoantibodies to inhibit intact primary biliary cirrhosis enzyme activity was found to correlate highly (R2 = 0.751) with the relative affinity. Thus there are profound differences between patients with respect to qualitative expression of autoantibodies. The significance of this data will be unclear until more is determined regarding the nature of the epitope that drives T cells and leads to B-cell responses.

The availability of recombinant mitochondrial autoantigens may permit the experimental study of the pathophysiology of primary biliary cirrhosis. Previously, we demonstrated that high-titer antibodies to the 74 kD mitochondrial autoantigen dihydrolipoamide acetyltransferase could be generated when BALB/c mice were immunized with purified recombinant protein. Based on these data, we attempted an 8-month study to induce antibodies and liver dysfunction by immunizing AKR/J, C3H/J and CBA/HeJ mice as well as rats, guinea pigs, rabbits and rhesus monkeys with purified recombinant human dihydrolipoamide acetyltransferase. Antibodies to dihydrolipoamide acetyltransferase were readily induced and detected in all species of experimental animals with species and strain differences in the titer of the responses. Of particular interest, rabbits and guinea pigs produced antibodies which were specifically reactive with the functional site of dihydrolipoamide acetyltransferase, whereas the other strains and species produced antibodies to other epitopes on the molecule. Finally, similar to data on humans with primary biliary cirrhosis, the pyruvate dehydrogenase enzyme pathway was inhibited in the presence of immunized animal sera. These data imply that features other than simply an antibody response to mitochondrial enzymes are required for the development of primary biliary cirrhosis. Further studies will be necessary to determine the mechanisms by which mitochondrial proteins elicit an immune response.

Antimitochondrial autoantibodies are characteristically present in sera of patients with primary biliary cirrhosis. The antimitochondrial autoantibodies recognize four major antigens from beef heart mitochondria at relative molecular weights of 74, 56, 52 and 48 kD. In the present study, we report that the 56 kD antigen is the protein X of pyruvate dehydrogenase complex and that it possesses cross-reactive antimitochondrial autoantibody epitope(s) with the 74 kD antigen, the acetyltransferase (E2) of the pyruvate dehydrogenase complex. This was demonstrated by comparing the specificities of primary biliary cirrhosis sera with a protein X-specific rabbit antiserum and by absorbing primary biliary cirrhosis sera with recombinant pyruvate dehydrogenase-E2 fusion protein. In the two-dimensional gel analysis, primary biliary cirrhosis sera and protein X-specific rabbit antiserum reacted to the same two isoelectric point polypeptides at 56 kD molecular weight. The absorption of primary biliary cirrhosis sera with the human recombinant pyruvate dehydrogenase-E2 removed reactivity toward both the 74 and 56 kD antigens. Furthermore, analysis of 82 antimitochondrial autoantibody-positive primary biliary cirrhosis sera by immunoblotting did not reveal any sera which reacted solely against either the 74 or 56 kD antigen. Finally, primary biliary cirrhosis sera recognized protein X from human, bovine and porcine sources but not protein X from rat or mouse origin. The identification of protein X as another major target of the autoimmune response in primary biliary cirrhosis suggests that the pyruvate dehydrogenase complex may have a central role in the induction of this enigmatic disease.