Characterization of the autoantibody responses to recombinant E3 binding protein (Protein X) of pyruvate dehydrogenase in primary biliary cirrhosis

Autoantibodies to the pyruvate dehydrogenase complex (PDC) are present in t he serum of more than 95% of patients with primary biliary cirrhosis (PBC), the major epitope being the inner lipoyl domain of the E2 component. Immun oblotting suggests a similar prevalence of antibodies to a tightly associat ed lipoic acid-containing protein, E3 binding protein (E3BP). Attempts to r esolve E3BP from E2 have been unsuccessful, restricting study of the nature and significance of antibody responses to the individual proteins. In part icular, it is unclear (1) whether there is true cross-reactivity between E3 BP and E2 and, if so, which is the originating response and (2) whether aut oantibodies preferentially bind a lipoylated epitope on E3BP as is the case with PDC-E2. In this study, complementary DNAs encoding rE2, full-length r E3BP, its single lipoyl domain (rLip), and core domain (rE3BPCore) were clo ned, and the proteins were expressed in Escherichia coli. Sera from 47 PBC patients were studied by immunoblotting and enzyme-linked immunosorbent ass ay (ELISA) against rE2, rE3BP, rE3BPCore, and both unlipoylated (U) and lip oylated (L) rLip. All sera were reactive by ELISA to some degree with all r ecombinant proteins except rE3BPCore, to which only 6 of 47 showed any reac tivity. Significant correlations (P <.0001) were observed when comparing ab sorbance values for rE3BP with both rLip (U) (r = 0.793) and (L) (r = 0.963 ). The mean absorbance for rLip (U, 0.26 +/- 0.05) was, however, significan tly lower than the absorbance for rLip (L) (0.78 +/- 0.12; P <.0001). After probing by immunoblotting and elution of antibodies from rE2 and rE3BP, su bsequent reprobing against the components in whole PDC revealed true cross- reactivity. In summary, the response to E3BP is primarily directed against the lipoylated domain of the protein. It still remains unclear, however, wh ether the initial breakdown of tolerance is to E2 or E3BP.