A new epitope of CYP2D6 recognized by liver kidney microsomal autoantibody from Japanese patients with autoimmune hepatitis
A new epitope of CYP2D6 recognized by liver kidney microsomal autoantibody from Japanese patients with autoimmune hepatitis
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DOI：
10.1248/bpb.28.2240
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发表时间：
2005-12-01
影响因子：
2
通讯作者：
Funae, Y
中科院分区：
文献类型：
--
作者：
Imaoka, S;Obata, N;Funae, Y
肝肾微粒体抗体1型（LKM-1）是自身免疫性肝炎11型（AIH-II）的诊断标志物。然而，在一小部分慢性丙型肝炎患者中也检测到LKM自身抗体。抗LKM-1的自身抗原已被鉴定为CYP2D6。为了鉴定CYP2D6对LKM-1血清的特异性抗原位点，我们建立了一种跨越CYP2D6全序列的肽的ELISA。在大肠杆菌中表达含组氨酸标签的人CYP2D6。用赖氨酰内肽酶消化纯化的CYP2D6。包括组氨酸标签的接头在其C末端具有赖氨酸残基，并且可以通过消化去除。通过反相HPLC分离消化的肽，并用戊二醛化学包被在ELISA板上。用该板研究了来自日本患者的2份LKM-1阳性血清（HCV阴性）和5份HCV阳性血清的免疫反应性。这些血清识别肽1 - 146、181 - 214、246 - 281、284 - 391和412 - 429。肽段1 - 146能被LKM-1阳性血清识别，但不能被HCV阳性血清识别，是本研究发现的一个新的表位。合成了跨越肽1 - 146的七个短肽，并用这些肽进行ELISA。然而，两种血清不识别这些肽，表明两种LKM-1阳性血清识别肽1 - 146的构象免疫原性位点。
Liver-kidney microsomal antibodies type 1 (LKM-1) are a diagnostic marker for autoimmune hepatitis type 11 (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.