Key residues of a major cytochrome P4502D6 epitope are located on the surface of the molecule.

Eukaryotically expressed CYP2D6 is the universal target of liver kidney microsomal Ab type 1 (LKM1) in both type 2 autoimmune hepatitis (AIH) and chronic hepatitis C virus (HCV) infection. In contrast, reactivity to prokaryotically expressed CYP2D6 protein and synthetic peptides is significantly lower in HCV infection than in AIH. The aim of the present study was to characterize LKM1 reactivity against a panel of eukaryotically expressed CYP2D6 constructs in the two conditions. LKM1-positive sera obtained from 16 patients with AIH and 16 with HCV infection were used as probes to perform a complete epitope mapping of CYP2D6. Reactivity to the full-length protein and 16 constructs thereof was determined by radioligand assay. We found that antigenicity is confined to the portion of the molecule C-terminal of aa 193, no reactivity being detectable against the aa sequence 1-193. Reactivity increases stepwise toward the C-terminal in both AIH and HCV, but the frequency of reactivity in the two conditions differs significantly between aa 267-337. To further characterize this region, we introduced a five and a three amino acid swap mutation selected from the homologous regions of CYP2C9 and HCV. This maneuver resulted in a substantial loss of LKM1 binding in both conditions, suggesting that this region contains a major epitope. Molecular modeling revealed that CYP2D6(316-327) is exposed on the surface of the protein, and may represent a key target for the autoantibody. These findings provide an initial characterization of the antigenic constitution of the target of LKM1 in AIH and HCV infection.

Antibodies to conformational epitopes of soluble liver antigen define a severe form of autoimmune liver disease.

Prevalence and clinical relevance of antibodies to soluble liver antigen (tRNP((Ser)Sec)/SLA) in autoimmune hepatitis (AIH) have been investigated using partially purified or prokaryotically expressed antigen. The aim of this study was to improve the detection of anti-tRNP((Ser)Sec)/SLA by establishing an immunoassay that was able to identify antibodies directed to conformational epitopes and to investigate the clinical implication of this autoantibody in autoimmune liver disease. By using eukaryotically expressed tRNP((Ser)Sec)/SLA as target in a radioligand assay (RLA), 81 patients with autoimmune liver disease (AILD) (33 type 1 AIH, 31 type 2 AIH, and 17 autoimmune sclerosing cholantitis [ASC]), 147 pathologic, and 56 healthy controls were investigated. RLA results were compared with those obtained using a commercial enzyme-linked immunosorbent assay (ELISA) and immunoblot. Reactivity to tRNP((Ser)Sec)/SLA was present in 58% of patients with type 1 and type 2 AIH, 41% with ASC, but in only 3 pathologic controls. RLA was similarly disease-specific but remarkably more sensitive than ELISA and immunoblot. A prospective study showed that anti-tRNP((Ser)Sec)/SLA-positive patients run a severe clinical course, having worse histology, needing longer to achieve remission, relapsing and requiring liver transplantation or dying more frequently than anti-tRNP((Ser)Sec)/SLA negative patients. Anti-tRNP((Ser)Sec)/SLA production was favored by the possession of DR3 and A1-B8-DR3 in AIH type 1 and ASC, and prevented by the possession of A2 in all 3 types of AILD, particularly in type 2 AIH. In conclusion, anticonformational tRNP((Ser)Sec)/SLA reactivity is frequent in type 1 and type 2 AIH and ASC, defining patients with a worse prognosis.