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Key Residues of a Major Cytochrome P4502D6 Epitope Are Located on the Surface of the Molecule¹

Yun Ma^*, Mark G. Thomas, Manabu Okamoto^*, Dimitrios P. Bogdanos^*, Sylvia Nagl, Nanda Kerkar, Agnel R. Lopes^*, Luigi Muratori^¶, Marco Lenzi^¶, Francesco B. Bianchi^¶, Giorgina Mieli-Vergani and Diego Vergani^2,*

^* Institute of Hepatology, Department of Biology, Centre for Genetic Anthropology, and Department of Biochemistry and Molecular Biology, Bloomsbury Centre for Structural Biology, University College London, London, United Kingdom; Institute of Liver Studies, King’s College Hospital, London, United Kingdom; and ^¶ Department of Internal Medicine, Cardioangiology, and Hepatology, University of Bologna, Bologna, Italy

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.

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