Autoantibodies to BCOADC-E2 in Patients With Primary Biliary Cirrhosis Recognize a Conformational Epitope PATRICK S. C. LEUNG,' DAVID T. Cm?AbiG,' R. M.m W~JN,~ zyxw SANGHOON CHA,' DEAN J. DANNEII, zy ' AFTAB ANSAKI,' Ross L. cOk>i)E12,' zyxwvut AND M. ERIC GERSHWIN' Primary biliary cirrhosis (PBC)is an autoimmune dis- ease of liver associated with a unique serologic response to mitochondrial autoantigens. Many of the autoanti- gens recognized by autoantibodies in PBC are members of the 2-0x0-aciddehydrogenase complex. The two major autoantigens are the E2 component of the pyruvate de- hydrogenase complex (PDC-E2)and the E2 component of the branched chain 2-0x0-acid dehydrogenase com- plex (BCOADC-E2). The autoantibody response to PDC- E2 has been mapped to one immunodominant epitope, which consists of both linear and conformational compo- nents. The presence of a single immunodominant epi- tope in PDC-E2 is unusual when contrasted to the im- mune response to autoantigens in other human autoimmune diseases. We have mapped the epitope rec- ognized by antimitochondrial autoantibodies (AMA) spe- cific to BCOADC-E2 in patients with PBC by taking advantage of the full-length bovine BCOADC-E2 comple- mentary DNA (cDNA)and a series of expression clones spanning the entire molecule. Reactivity to the various expression clones was studied by immunoblotting, en- zyme-linked immunosorbent assay (ELISA), as well as selective absorption of patient sera by expressed protein fragments. Autoantibodies to BCOADC-E2 map within peptides spanning amino acid residues 1 to 227 of the mature protein; our data demonstrate that the epitope is dependent on conformation and includes the lipoic Abbreviations: AhlA, antimitocliondnal antibodies: PM', primary hiliary PDC-E2. pyruvate dehydrogenase complex E2: H('OAIX-EZ. chain 2-oxo-acid dehydrogenase complex E2; cDNA. complenientary zyxwvuts DNA: IPTG. isopropylthiogalactosid~; SDS-PAGE, sodium dodticyl siilrate piilyacrylamidr gel clectrophoresis: ELISA. enzyme-linked iinnlui1~isorbent assay From the 'Division of lilieumatology. N l e r ~ and Clinical Ininiunology. University of Chlifornia. Ihvis. CA; 'Departnient or Riochemistry, Ilnivcrsity of Texas, Southwestern Medical Center, Dallas. TX: "Departnicmt of' Genetics and Molecular Medicine. Emory IJni ity School of lqedicine, Atlanta. GA; the "Department of Patholob!, Emor nivrrsity. Atlanta. GA: and the ',De- partment of Microbiology, hlonash University. Clayton. Victoria 3168. Auslra- lia. Received November 28. 1994 This study was supported by NIH grants DI< 39.588 and A1 31535 and the Liver Tissue Procurcwicnl and Distribution Systeni Grant 1-DK-6-227.1 Dr Cha's current address is the Ilepartnicnt or Riology and Technology. Kangweon National University. ('hoonchwn, South Korea. Address reprint requests ti): Patrick S. ('. Leung, Phl). Division dRheuiii;l- toloCy. r'dlwgy and Clinical Immunolomv. Lniwrsity of ('alifornia at Davis, Ti3 192. School of Medicine, Davis~ (:A 95616 Copyright c ' ~ 19116 by the American Association for the Study or 1,iv.t.r Diseases. 02iO-9 139/95/2202-00 11)$3.00/0 acid binding region. However, only the full-length clone (amino acid residue 1 to 421) is sufficient to remove all detectable BCOADC-E2 reactivity. Moreover, the ab- sence of lipoic acid on the recombinant polypeptides used in this study indicates that antibody binding to BCOADC-E2 is not dependent on the presence of lipoic acid. (HEPATOLOGY 1995; 22:505-513.) Antimitochondrial autoantibodies (AVA) have been long recognized as a characteristic feature of primary biliary cirrhosis (PBC).' The major autoantigens recog- nized by these autoantibodies are members of the 2-0x0-acid dehydrogenase comples, including the py- ruvate dehydrogenase complex E2 (PDC-EB 1, the branched chain 2-0x0-acid dehydrogenase complex E2 ( BCOADC-E2 1, the 2-0x0-glutarate dehydrogenase complex E2, the E l subunits of the pyruvate dehydro- genase complex and protein X of PDC. As for many autoantigens, the structure and antigenicity of the ma- jor PBC autoantigens are conserved among different species.'." Of these autoantigens, PDC-E2 is most com- monly recognized by serum antibodies of patients with PBC, and much of the research in PBC has focused on determining its reactivity with AMA. Sera from pa- tients with PBC inhibit catalytic function of PDC-E2,".' and the immunodominant epitope has been localized to a stretch of 93 amino acids within the inner lipoic acid domain of the enzyme? However, in addition to antibodies to PDC-E2, approximately 60(Z of patients with PBC are also immunoreactive with BCOADC-E2. Indeed, in some studies, there are 10% to 20% of pa- tients with PBC who react to BCOADC-EB but have no detectable reactivity to PDC-E2."".' The BCOADC complex catalyzes the oxidative decar- boxylation of alpha keto acids derived by transamina- tion of the branched-chain amino acids, valine, leucine, and isoleucine.9This complex consists of three catalytic components: a branched-chain 2-0x0-acid decarboxyl- ase (El), a dihydrolipoyl transacylase tE2), and a dihy- drolipoyl dehydrogenase iE3).'" Of these components, autoantibodies in PBC are limited to the dihydrolipoyl transacylase or BCOADC-E2. The complementary DNA (cDNA) of both the human and bovine BCOADC- E2 have been cloned,"" and primary structures de- rived from sequence cDNA demonstrate that the ma- ture BCOADC-E2 is similar to PDC-E2 in having a lipoic acid binding domain, dihydrolipoyl dehydroge- 505