Specific reactivity of recombinant human PDC-E1 alpha in primary biliary cirrhosis

The mitochondrial autoantigens recognized by autoantibodies in patients with primary biliary cirrhosis have been identified as components of related multi-enzyme complexes, including acyltransferases of the pyruvate dehydrogenase complex (PDC), the branched-chain alpha-keto acid dehydrogenase complex (BCODH), the alpha-ketoglutarate dehydrogenase complex (OGDC), protein X and pyruvate dehydrogenase (PDC) E1 alpha and E1 beta. The major autoantigens, PDC-E2, BCODH-E2 and OGDC-E2, share some sequence homology; the epitopes on these antigens appear to be close to, or identical with, the lipoic acid binding site. Furthermore, all three antigens share some structural homology. In contrast, antibodies to PDC-E1 alpha are present in lower titers, and have been more difficult to detect. PDC-E1 alpha also differs from the three major autoantigens in that it lacks any covalently bound lipoic acid. PDC-E1 alpha cannot be purified in large quantities and becomes unstable in the absence of PDC-E1 beta. To address these problems, we have subcloned recombinant human PDC-E1 alpha to pGEX, pGEX is a vector which produces a recombinant polypeptide fused to glutathione S-transferase. The resultant E1 alpha fusion protein is stable and has a low background in immunoassays. Using the recombinant protein, we have developed an ELISA that allows rapid and reproducible quantification of antibodies to human PDC-E1 alpha. Finally, we demonstrate that a major epitope on PDC-E1 alpha is within a 300 amino acid region that contains the enzyme functional sites, namely the phosphorylation site and the TPP binding site.