High frequency of anti-PDC-E1a/E1b antibodies in sera from primary biliary cirrhosis patients: affinity purification of the mitochondrial M4 antigen predominantly enriches pyruvate dehydrogenase complex E1achains

Objective: Sera from patients with primary biliary cirrhosis (PBC) contain antibodies against intracellular antigens such as denatured epitopes of the pyruvate dehydrogenase complex (PDC) derived from bovine heart mitochondria (M2). In addition, native proteins were detected within the sucrose gradient fractions derived from rat liver mitochondria (M4) only by the complement fixation test but not in Western Blot analysis. Thus, the major objective of this study was to identify these proteins. Methods: IgG fractions of M4-CFT positive (n=5) or negative (n=5) PBC marker sera were used to purify the M4 proteins by affinity chromatography. Proteins were identified by Western blotting, silver staining, and sequence analysis. Further, a cohort of 57 PBC patients was tested for the reactivity to M4 related proteins. Results: Two different AMA-pattern of the marker sera occurred: M4-CFT positive sera could be characterized by Western Blot analysis as PDC-E2+/E1+ while the M4-CFT negative sera were PDC-E2+/E1-. Using the PDC-E2+/E1+ sera for affinity chromatography, the major proteins of the mitochondrial M4 fraction could be identified as the PDC-E1a and E1b subunits. Further, we purified fragments of PDC-E2 and branched-chain 2-oxo acid dehydrogenase complex E2, and an enzyme of the b-oxidation of fatty acids, the enoyl-CoA-hydratase. A clear-cut association between anti-M4 reactivity in the CFT and the reactivity to both PDC subunits could be also documented in the cohort of 57 PBC patients showing anti-PDC-E1a and E1b antibodies in a frequency of 74% and 67%. Conclusion: CFT reactivity against M4 antigens could be preferentially identified as a reaction against PDC-E1a and E1b. In view of the high frequency of antibodies to these subunits, they must be considered as not less important as compared to PDC-E2.