TY - JOUR
T1 - Use of molecular simulation for mapping conformational CYP2E1 epitopes
AU - Vidali, Matteo
AU - Hidestrand, Mats
AU - Eliasson, Erik
AU - Mottaran, Elisa
AU - Reale, Emanuela
AU - Rolla, Roberta
AU - Occhino, Giuseppa
AU - Albano, Emanuele
AU - Ingelman-Sundberg, Magnus
PY - 2004/12/3
Y1 - 2004/12/3
N2 - 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 α helices and β 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.
AB - 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 α helices and β 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.
UR - http://www.scopus.com/inward/record.url?scp=10944223470&partnerID=8YFLogxK
U2 - 10.1074/jbc.M407329200
DO - 10.1074/jbc.M407329200
M3 - Article
SN - 0021-9258
VL - 279
SP - 50949
EP - 50955
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 49
ER -