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Use of molecular simulation for mapping conformational CYP2E1 epitopes. J Biol Chem 2004 Dec 03;279(49):50949-55

Date

10/01/2004

Pubmed ID

15456790

DOI

10.1074/jbc.M407329200

Scopus ID

2-s2.0-10944223470   21 Citations

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 alpha helices and beta 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.

Author List

Vidali M, Hidestrand M, Eliasson E, Mottaran E, Reale E, Rolla R, Occhino G, Albano E, Ingelman-Sundberg M

Author

Mats Hidestrand PhD Adjunct Assistant Professor in the Surgery department at Medical College of Wisconsin




MESH terms used to index this publication - Major topics in bold

Anesthetics, Inhalation
Computer Simulation
Crystallography, X-Ray
Cytochrome P-450 CYP2E1
Cytochrome P-450 Enzyme System
Cytochrome P450 Family 2
DNA
DNA Primers
DNA, Complementary
Epitope Mapping
Epitopes
Escherichia coli
Halothane
Hepatitis
Humans
Immunoprecipitation
Liver Diseases, Alcoholic
Lysine
Models, Molecular
Mutagenesis
Mutagenesis, Site-Directed
Mutation
Protein Conformation
Protein Folding
Protein Structure, Secondary
Protein Structure, Tertiary
Recombinant Proteins
Steroid 21-Hydroxylase
jenkins-FCD Prod-411 e00897e83867fcfa48419861683711f8d99adb75