Cytochrome P4502D6193–212: A New Immunodominant Epitope and Target of Virus/Self Cross-Reactivity in Liver Kidney Microsomal Autoantibody Type 1-Positive Liver Disease

Abstract

Cytochrome P4502D6 (CYP2D6), target of liver kidney microsomal autoantibody type 1 (LKM1), characterizes autoimmune hepatitis type 2 (AIH2) but is also found in patients with chronic hepatitis C virus (HCV) infection. To provide a complete linear epitope B cell map of CYP2D6, we tested peptides spanning the entire sequence of CYP2D6. In addition to confirming previously described antigenic sites, we identified four new epitopes (193–212, 238–257, 268–287, and 478–497). CYP2D6193–212 is immunodominant and was the target of 12 of 13 (93%) patients with AIH2 and 5 of 10 (50%) HCV/LKM1-positive patients. Because LKM1 is present in both AIH2 and a viral infection, we tested whether Abs to CYP2D6193–212 arise through cross-reactive immunity between virus and self. We identified a hexameric sequence “RLLDLA” sharing 5 of 6 aa with “RLLDLS” of HCV2985–2990 and all 6 aa with CMV130–135. Of 17 CYP2D6193–212-reactive sera, 11 (7 AIH and 4 HCV) reacted by ELISA with the HCV homologue, 8 (5 AIH and 3 HCV) with the CMV homologue, and 8 (5 AIH and 3 HCV) showed double reactivity. Autoantibody binding to CYP2D6193–212 was inhibited by preincubation with HCV2977–2996 or CMV121–140. Recombinant HCV-nonstructural protein 5 and CMV-UL98 proteins also inhibited Ab binding to CYP2D6193–212. Affinity-purified CYP2D6193–212-specific Ab inhibited the metabolic activity of CYP2D6. The demonstrated similarity and cross-reactivity between CYP2D6193–212 and two unrelated viruses suggests that multiple exposure to viruses mimicking self may represent an important pathway to the development of autoimmunity.

Use of Molecular Simulation for Mapping Conformational CYP2E1 Epitopes

“…Furthermore, it has been shown that some of these epitopes share structural homologies with protein sequences present in type 1 herpes simplex virus and hepatitis C virus (17,21). The use of deleted CYP mutants has also provided important information concerning the structure of different epitopes present in CYP2C9, CYP3A1, and CYP1A2 (15)(16)(17).…”

“…We focused our attention to protruding areas suitable for antibody binding, whereas sequences that were in troughs or not apparently accessible were excluded. Since most of the epitopes so far identified in CYPs largely involve the C-terminal portion of the molecule (15)(16)(17)(18)(19)(20)(21), the sequence between amino acids 200 and 493 was investigated. Preliminary experiments using purified rat or rabbit CYP2E1 showed that human anti-CYP2E1 IgG cross-reacted with the enzyme from these species in enzyme-linked immunosorbent assays (not shown).…”

“…Epitopes in CYP11A (cholesterol side-chain cleavage enzyme), CYP17 (steroid-17␣ hydroxylase), and CYP21A2 (steroid-21␣ hydroxylase) are also recognized by autoantibodies associated with autoimmune polyendocrine syndrome and autoimmune Addison's disease (13,14). For some of these autoantibodies, extensive epitope mapping studies have been performed to get a better understanding of the mechanisms leading to autoimmunity (15)(16)(17)(18)(19)(20)(21). The data so far obtained regarding anti-CYP2D6, anti-CYP2C9, and anti-CYP3A1 autoantibodies show that several linear and conformational epitopes are recognized in the different CYPs (15)(16)(17)(18)(19)(20)(21).…”

Primary biliary cirrhosis is characterized by IgG3 antibodies cross-reactive with the major mitochondrial autoepitope and its Lactobacillus mimic

“…Reaction for a given peptide was considered positive when optical density (OD) test /OD control peptide was 2 or more, this cutoff representing the mean ϩ 5 SD of 135 readings using serum (tested in triplicate) from 15 healthy subjects against the mimicking peptides. 5,15,22,26,28 Inhibition Studies. To investigate whether the simultaneous reactivity to LACDE BGAL 266-280 and human PDC-E2 212-226 was due to cross-reactivity, competition ELISAs were performed, measuring residual IgG3 anti-LACDE BGAL 266-280 or anti-PDC-E2 212-226 reactivity after preincubation respectively with LACDE BGAL 266-280 , PDC-E2 212-226 , purified PDC antigen, control peptide, and control antigen (CYP2D6, Pharmacia & Upjohn) as liquid phase competitors, as previously described.…”

“…To investigate whether the simultaneous reactivity to LACDE BGAL 266-280 and human PDC-E2 212-226 was due to cross-reactivity, competition ELISAs were performed, measuring residual IgG3 anti-LACDE BGAL 266-280 or anti-PDC-E2 212-226 reactivity after preincubation respectively with LACDE BGAL 266-280 , PDC-E2 212-226 , purified PDC antigen, control peptide, and control antigen (CYP2D6, Pharmacia & Upjohn) as liquid phase competitors, as previously described. 5,15,22,26,28,29 Inhibition of anti-ECOLI PDC-E2 231-245 reactivity was measured under conditions similar to those described for LACDE BGAL [266][267][268][269][270][271][272][273][274][275][276][277][278][279][280] .…”