A new epitope of CYP2D6 recognized by liver kidney microsomal autoantibody from japanese patients with autoimmune hepatitis.
Liver-kidney microsomal antibodies type 1 (LKM-1) are a diagnostic marker for autoimmune hepatitis type II (AIH-II). However, LKM autoantibodies are also detected in a small percentage of patients with chronic hepatitis C. The autoantigen to anti-LKM-1 has been identified to be CYP2D6. To identify the specific antigenic site of CYP2D6 for LKM-1 serum, we established an ELISA with peptides spanning the entire sequence of CYP2D6. Human CYP2D6 containing histidine tag was expressed in Escherichia coli. Purified CYP2D6 was digested by lysyl endopeptidase. The linker including the histidine tag has a lysine residue in its C-terminal and can be removed by digestion. Digested peptides were separated by reversed-phase HPLC and coated on ELISA plates chemically with glutaraldehyde. The immunoreactivity of two LKM-1-positive sera (HCV-negative) and five HCV-positive sera from Japanese patients was investigated with the plates. These sera recognized peptides 1-146, 181-214, 246-281, 284-391, and 412-429. The peptide 1-146 was recognized by LKM-1-positive sera but not HCV-positive sera and is a new epitope found in this study. Seven short peptides spanning peptide 1-146 were synthesized and ELISAs were conducted with these peptides. However, two sera recognized none of these peptides, suggesting that two LKM-1-positive sera recognize the conformational immunogenic site of peptide 1-146.
Imaoka
Susumu
S
Nanobiotechnology Research Center and Department of Bioscience, School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda 669-1337, Japan. imaoka@ksc.kwansei.ac.jp
Obata
Noriko
N
Hiroi
Toyoko
T
Osada-Oka
Mayuko
M
Hara
Rie
R
Nishiguchi
Shuhei
S
Funae
Yoshihiko
Y
D044466:Asian People; D001323:Autoantibodies / Q000276:immunology*; D002851:Chromatography, High Pressure Liquid; D019389:Cytochrome P-450 CYP2D6 / Q000276:immunology* / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D019693:Hepatitis, Autoimmune / Q000276:immunology* / Q000378:metabolism; D006801:Humans
D001323:Autoantibodies; D000939:Epitopes; C060729:anti-liver kidney microsome antibody; D019389:Cytochrome P-450 CYP2D6