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J. Biol. Chem., Vol. 279, Issue 49, 50949-50955, December 3, 2004

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Use of Molecular Simulation for Mapping Conformational CYP2E1 Epitopes^*

Matteo Vidali, Mats Hidestrand, Erik Eliasson¶, Elisa Mottaran, Emanuela Reale, Roberta Rolla, Giuseppa Occhino, Emanuele Albano||, and Magnus Ingelman-Sundberg

From the Department of Medical Science, University "Amedeo Avogadro" of East Piedmont and Interdipartimental Research Center for Autoimmune Diseases (IRCAD), 28100 Novara Italy, the Division of Molecular Toxicology, Institute of Environmental Medicine, Karolinska Institutet, Stockholm SE-17177, Sweden, and the ^¶Division of Clinical Pharmacology, Department of Laboratory Sciences, Karolinska Institutet, Karolinska University Hospital, Huddinge SE-14186, Sweden

The identification of the epitopes recognized by autoantibodies against cytochrome P450s (CYPs) associated with drug-induced hepatotoxicity is difficult because of their conformational nature. In the present investigation, we used a novel approach based on the analysis of the whole molecule antigenic capacity following single amino acid substitutions to identify the conformational epitopes on CYP2E1. A molecular model of CYP2E1 was generated based on the CYP2C5 crystal structure, and potential motifs for amino acid exchanges were selected by computer simulation in the surface of helices and sheets. Fourteen modified, apparently correctly folded CYP2E1 variants were produced in Escherichia coli and evaluated in immunoprecipitation experiments using sera with anti-CYP2E1 autoreactivity from 10 patients with halothane hepatitis and 12 patients with alcoholic liver disease. Ala substitution of Glu-248 and Lys-251 as well as of Lys-324, Lys-342, Lys-420, and Phe-421 severely decreased or abolished CYP2E1 recognition by the majority of both the halothane hepatitis and alcoholic liver disease sera, whereas the other substitutions had only minor effects. Based on the structural model, these substitutions identified two distinct epitopes on the CYP2E1 surface corresponding to the G-helix and an area formed by juxtaposition of the J' and K'' helices, respectively. The combined use of molecular modeling and single amino acid mutagenesis is thus a useful approach for the characterization of conformational epitopes recognized by autoantibodies.

Received for publication, June 30, 2004 , and in revised form, September 3, 2004.

^* This work was supported by grants from the Regional Government of Piedmont, the Cariplo Foundation (Milan, Italy), and the Swedish Research Council. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^|| To whom correspondence should be addressed: Dept. of Medical Science, University "Amedeo Avogadro" of East Piedmont, Via Solaroli 17, 28100 Novara, Italy. Tel.: 39-0321-660642; Fax: 39-0321-620421; E-mail: albano{at}med.unipmn.it.

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