Keywords: Anti-M2, Epitope mapping, E2-subunit, Pyruvate dehydrog

Keywords: Anti-M2, Epitope mapping, E2-subunit, Pyruvate dehydrogenase complex, Inner lipoyl domain, Active site, Catalytic domain, Primary biliary cirrhosis INTRODUCTION Antimitochondrial Axitinib melanoma antibodies (AMA) are one of the most important criteria for the diagnosis of primary biliary cirrhosis (PBC), a chronic cholestatic liver disease of unknown etiology, which affects mainly middle-aged women and leads to a destruction of small bile ducts. Their target antigen named M2[1,2] is attached to the inner mitochondrial membrane[3] and consists of five components[4], which have been identified in the following years on molecular bases as subunits of the 2-oxo acid dehydrogenase complex of the inner mitochondrial membrane: the pyruvate dehydrogenase complex (PDC), the 2-oxoglutarate dehydrogenase complex and the branched-chain 2-oxo acid dehydrogenase complexes[5-7].

Each complex comprises multiple copies of three component enzymes termed E1 (a thiamine pyrophosphate-dependent decarboxylase), E2 (a coenzyme A-dependent decarboxylase), and E3 (a dihydrolipoyl dehydrogenase). The major M2-antigen which is recognized by nearly 90% of PBC sera is the E2 component of PDC. The immunodominant epitope within PDC-E2 has been mapped to the region associated with the inner lipoyl domain wherein a lysine residue binds the lipoic cofactor for the enzyme with a minimum of 75 residues necessary for characteristic antibody recognition[6,8,9] suggesting that a conformational autoepitope may be primarily recognized[9]. A minor epitope is associated with the outer lipoyl domain[6,10].

Hitherto, epitope mapping with PBC sera has linked AMA-reactivity to the highly conserved amino acids surrounding the Dacomitinib lipoyl-lysine K173, particularly linear peptides AEIETDKATIGFEVQEEG (corresponding to aa 167-184 within the human PDC-E2 but primarily labeled aa 81-100[6] according to its location within a subclone pRMIT-603 used to identify the immunodominant epitope) or LLAEIETDKATIGF (165-178)[11]. It was shown that these short peptides absorbed most reactivity with PBC sera by enzyme-linked immunosorbent assay (ELISA), albeit only at a serum dilution of 1:80 000[6]. Binding of lipoic acid cofactor to K173 has been discussed to be necessary for antibody reaction[10,12]. Until now, the large fragment of the catalytic domain of PDC-E2 (aa 331-560) has been regarded as ��immunologically silent�� due to negative results in several studies[6,9,12-14]. In the present study we used peptides spanning the whole PDC-E2 enzyme, and it will be shown that sera from PBC patients recognize epitopes within the catalytic domain in an even higher incidence than those in the inner lipoyl domain. MATERIALS AND METHODS Patients Sera from 95 patients with clinically well defined PBC were analyzed.

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