Use of Molecular Simulation for Mapping Conformational CYP2E1 Epitopes

Abstract

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.

Alcohol, oxidative stress and free radical damage

“…In this context, it has been observed that about 40% of patients with advanced ALD have circulating IgG directed against CYP2E1 (Vidali et al 2003). These autoantibodies recognize at least two distinct conformational epitopes present in the C-terminal portion of the CYP2E1 molecule (Vidali et al 2004). One of the mechanisms proposed to explain the formation of anti-CYP autoantibodies postulates that the binding of reactive drug metabolites to CYP promotes a humoral immune response against the modified protein and concomitantly favours the activation of normally-quiescent auto-reactive lymphocytes, which leads to the production of antibodies to the native CYP molecules (Van Pelt et al 1995).…”

Breaking self-tolerance toward cytochrome P4502E1 (CYP2E1) in chronic hepatitis C: Possible role for molecular mimicry

“…The correct sequence of the inserts was confirmed by automated DNA sequencing. Wild-type and mutated CYP2E1 variants were expressed and purified as previously reported [22].…”

Immune mechanisms in alcoholic liver disease

“…Anti-CYP2E1 auto-antibodies from ALD patients are similar to those associated with halothane hepatitis and recognize at least two distinct conformational epitopes in the C-terminal portion of the molecule [78]. These epitopes are located in the molecule surface and account for the recognition by anti-CYP2E1 IgG of CYP2E1 expressed on the outer layer of the hepatocyte plasma membranes [78].…”