Prediction of the immunodominant epitope of the pyruvate dehydrogenase complex E2 in primary biliary cirrhosis using phage display

Primary biliary cirrhosis (PBC) is an autoimmune liver disease characterize d by autoantibodies reactive with the pyruvate dehydrogenase complex. A con formational epitope has been mapped to aa 91-227 within the inner lipoyl do main of the E2 subunit (pyruvate dehydrogenase complex E2 (PDC-E2)), We hav e used phage display to further localize this epitope, A random heptapeptid e library was screened using IgG from two patients with PBC, with negative selection using pooled normal IgG, Phage that contained peptide inserts (ph agotopes) selected using PBC sera differed from those selected using IgG fr om patients with RA or polychondritis. Two motifs occurred only among the P BC-selected phagotopes; these were MH (13 sequences, 16 phagotopes) and FV (FVEHTRW, FVEIYSP, FVLPWRI), The phagotopes selected were tested for reacti vity with anti-PDC-E2 affinity purified from four patients with PBC, Phagot opes that contained 1 of 15 different peptide sequences were reactive with one or more of these four anti-PDC-E2 preparations, whereas phagotopes that contained 1of the remaining 28 sequences were negative. The peptides (FVLP WRI, MHLNTPP, MHLTQSP) encoded by three phagotopes that were strongly react ive with all four preparations of anti-PDC-E2 were synthesized. Each of the selected peptides, but not an irrelevant peptide, inhibited the reactivity by ELISA of PBC serum with recombinant PDC-E2 and reduced the inhibition o f the enzyme activity of PDC by a PBC serum. The peptide sequences, along w ith the known NMR structure of the inner lipoyl domain of PDC-E2, allow the prediction of nonsequential residues 131HM132 and 178FEV180 that contribut e to a conformational epitope.