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The Journal of Immunology, 2002, 169: 277-285.
Copyright © 2002 by The American Association of Immunologists

Key Residues of a Major Cytochrome P4502D6 Epitope Are Located on the Surface of the Molecule1

Yun Ma*, Mark G. Thomas{dagger}, Manabu Okamoto*, Dimitrios P. Bogdanos*, Sylvia Nagl{ddagger}, Nanda Kerkar§, Agnel R. Lopes*, Luigi Muratori, Marco Lenzi, Francesco B. Bianchi, Giorgina Mieli-Vergani§ and Diego Vergani2,*

* Institute of Hepatology, {dagger} Department of Biology, Centre for Genetic Anthropology, and {ddagger} Department of Biochemistry and Molecular Biology, Bloomsbury Centre for Structural Biology, University College London, London, United Kingdom; § Institute of Liver Studies, King’s College Hospital, London, United Kingdom; and Department of Internal Medicine, Cardioangiology, and Hepatology, University of Bologna, Bologna, Italy


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Eukaryotically expressed CYP2D6 is the universal target of liver kidney microsomal Ab type 1 (LKM1) in both type 2 autoimmune hepatitis (AIH) and chronic hepatitis C virus (HCV) infection. In contrast, reactivity to prokaryotically expressed CYP2D6 protein and synthetic peptides is significantly lower in HCV infection than in AIH. The aim of the present study was to characterize LKM1 reactivity against a panel of eukaryotically expressed CYP2D6 constructs in the two conditions. LKM1-positive sera obtained from 16 patients with AIH and 16 with HCV infection were used as probes to perform a complete epitope mapping of CYP2D6. Reactivity to the full-length protein and 16 constructs thereof was determined by radioligand assay. We found that antigenicity is confined to the portion of the molecule C-terminal of aa 193, no reactivity being detectable against the aa sequence 1–193. Reactivity increases stepwise toward the C-terminal in both AIH and HCV, but the frequency of reactivity in the two conditions differs significantly between aa 267–337. To further characterize this region, we introduced a five and a three amino acid swap mutation selected from the homologous regions of CYP2C9 and HCV. This maneuver resulted in a substantial loss of LKM1 binding in both conditions, suggesting that this region contains a major epitope. Molecular modeling revealed that CYP2D6316–327 is exposed on the surface of the protein, and may represent a key target for the autoantibody. These findings provide an initial characterization of the antigenic constitution of the target of LKM1 in AIH and HCV infection.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Liver kidney microsomal Ab type 1 (LKM1)3 was originally described as the serological hallmark of autoimmune hepatitis (AIH) type 2 (1, 2, 3, 4, 5), and was later reported to be present in up to 10% of patients with chronic hepatitis C virus (HCV) infection (6, 7). In the two conditions, LKM1 gives an identical immunofluorescence pattern, recognizes the same antigenic target, namely the liver enzyme cytochrome P4502D6 (CYP2D6) (8, 9) and similarly blocks its in vitro catalytic activity (10, 11, 12). There are also differences: in AIH, LKM1 reacts equally well with prokaryotically and eukaryotically expressed CYP2D6 (13), but only with eukaryotic CYP2D6 in HCV infection (14, 15, 16, 17); in AIH, LKM1 recognizes the linear CYP2D6196–218, CYP2D6254–271, and CYP2D6321–351 sequences as immunodominant epitopes but not in HCV infection (12, 16, 17).

Epitope mapping is an important step in understanding the mechanisms triggering autoimmunity and in providing guidance for designing immunomodulatory therapy (18, 19). Epitope mapping comprises two major approaches: linear and conformational, the former using prokaryotically expressed proteins or synthetic peptides as target Ags, the latter using eukaryotically expressed proteins. To date, linear epitope mapping of CYP2D6 has been partially performed with either a limited number of synthetic CYP2D6 peptides or by the use of constructs prokaryotically expressed. Although Yamamoto et al. (16) identified four linear epitopes, CYP2D6254–271, CYP2D6321–351, CYP2D6373–389, and CYP2D6410–429 recognized by LKM1 in AIH, Dalekos et al. (20) showed that in HCV infection, anti-CYP2D6-positive sera recognize two truncated CYP2D6 proteins (aa 250–494 and 321–494) expressed in Escherichia coli.

B cell epitopes, unlike T cell epitopes, are usually conformational, i.e., highly dependent on the three-dimensional structure of the protein (21, 22, 23, 24, 25, 26). The presence of conformational epitopes on CYP2D6 was first suggested by Duclos-Vallee et al. (10) who were unable to reverse inhibition of the CYP2D6 enzymatic activity by preincubation of LKM1-positive sera with synthetic peptides spanning known linear epitopes. The presence of conformational epitopes was confirmed by our group and by Yamomoto et al. (14, 15). Sera from patients with AIH reacted with linear peptides, and prokaryotic and eukaryotic full-length protein, while those from patients with HCV infection reacted only with the eukaryotically expressed, conformationally intact CYP2D6, leading the two groups to conclude that LKM1 from HCV-infected patients preferentially reacts with conformational epitopes (14, 15).

To perform conformational epitope mapping of CYP2D6, we decided to use two complementary strategies: first, to express a full-length and a series of truncated CYP2D6 proteins in a eukaryotic system and second, to characterize the identified epitope by mutagenesis introducing two homologous regions into CYP2D6 under the conditions in which the major antigenic regions remain structurally intact (22, 23). To further characterize the epitope, we localize the epitopes within the CYP2D6 protein by modeling locally on the homologous region of published structure of Bacillus megaterium P450 BM-3 (27, 28).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Construction of truncated CYP2D6 fragments

A complete cDNA of human CYP2D6 (kindly provided by Prof. U. Meyer, University of Basel, Basel, Switzerland) was subcloned into the BamHI and KpnI sites of plasmid pSP72 in vitro transcription vector (Promega, Southampton, U.K.) downstream to a SP6 polymerase promoter. The subcloning was performed using a rapid ligation kit according to the manufacturer’s instructions (Roche Diagnostics, Lewes, U.K.). The recombinant plasmid DNA was amplified through transformation of competent E. coli (DH5a; Life Technologies, Paisley, U.K.) and then extracted and purified with a plasmid DNA Miniprep kit (Qiagen, Crawley, U.K.) and finally used as a template to construct truncated CYP2D6 fragments.

A total of 16 CYP2D6 constructs are shown in Fig. 1Go. They are divided into three groups (A–C) and were obtained using different methods. Group A (N-terminal region) and group C (C-terminal region) fragments were constructed by digestion of the template with different combinations of restriction endonucleases, which enabled maintenance of the reading frame. Group B fragments were built by removing nuclear bases at the 3' end of the full cDNA using Bal31 nuclease. The expected sizes of the constructs were first examined by Agarose MS (molecular screening agarose; Roche Diagnostics) gel electrophoresis and then their sequences were confirmed by nucleotide sequencing analysis (dye terminator) on an ABI377 sequencer (PerkinElmer, Foster City, CA).



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  		FIGURE 1. Strategy for epitope mapping of CYP2D6 and summary of reactivity. The first dotted line () represents 1567 bp, the full-length CYP2D6 cDNA. Four restriction enzymes, NarI, BstEII, BspEI, and Bsu3, were used to construct truncated fragments, and their digestion sites on CYP2D6 are shown. The second dotted line () represents the full-length CYP2D6 (497 aa) protein. The subsequent lines represent three groups of truncated CYP2D6 proteins. Their lengths are indicated by amino acid numbers. A1 to A3 () were created by NarI partial digestion, C1-C4 (solid lines) were created by different combinations of restriction enzyme digestion, such as C4 by NarI at 79 and 580 sites and re-ligation; C1-C3 were created using C4 as a template and then were digested by BstEII, BspEI, and Bsu3, respectively. B1-B9 () were created by removing bases at the 3' end with nuclease Bal31 digestion. The frequency of reactivity against each construct is shown in brackets followed by the number of sera reacted; a–c, frequency of reactivity in AIH compared with that in HCV infection; p = 0.03, 0.05, and 0.06, respectively.

 
N-terminal region fragments CYP2D61–79 (A1), CYP2D61–190 (A2), and CYP2D61–580 (A3) were obtained by partial digestion with restriction endonuclease enzyme NarI, which has three digestion sites on the CYP2D6 molecule at positions 79, 190, and 580. Partial digestion was applied so that the enzyme would cut only on one or two of the three digestion sites in each reaction. This task was achieved by using different quantities of enzyme and reaction times. NarI digestion products were further digested with ClaI, which is located downstream of the KpnI site within vector pSP72 and creates a NarI compatible end with that digested by NarI. The aim of this step was to delete CYP2D6 sequence regions 79–1567, 190-1567, and 580-1567 and then to re-ligate the upstream and downstream regions of the DNA template. Re-ligation was also performed with a rapid ligation kit (Roche Diagnostics).

The second group of truncated fragments (B1 to B9) was constructed using full-length CYP2D61–1567 as a template that remained continuous with vector at the 5' end and with reducing lengths at the 3' end by removing nuclear bases using Bal31 nuclease according to the manufacturer’s instructions (Amersham Pharmacia Biotech, Little Chalfont, U.K.). The 3' end of CYP2D6 was then rendered blunt by Klenow reaction according to the manufacturer’s instructions (Promega). A series of truncated fragments was finally released by 5' BamHI digestion and subcloned into BamHI and EcoRV sites of a new pSP72 vector.

C-terminal region fragments CYP2D61–79/580–927 (C1), CYP2D61–79/580–977 (C2), CYP2D61–79/580-1174 (C3), and CYP2D61–79/580-1567 (C4) were constructed through two steps. First, C4 was constructed by NarI partial digestion to delete CYP2D79–580 and then followed by re-ligation of the upstream and downstream regions of the template DNA. Second, C1 to C3 were constructed with the use of C4 as a template. Following initial digestion with EcoRV (location within vector pSP72), C4 was digested by BstEII (partial digestion), BspEI (complete digestion), and Bsu36I (partial digestion) respectively to build up fragments CYP2D6580–927 (C1), CYP2D6580–977 (C2), and CYP2D6580–1174 (C3).

Production of chimeric constructs and in vitro mutagenesis procedures

Based on preliminary results, CYP2D6316–327 was selected as a potential immunodominant epitope, and its sequence was replaced by homologous regions from cytochrome P4502C9 (CYP2C9, GenBank accession no. P11712) and HCV genome polyprotein 1A (GenBank accession no. P26664) to generate chimeric constructs CYP2D6/2C9 and CYP2D6/HCV, respectively (Fig. 2Go). The homologous regions between CYP2D6, CYP2C9, and HCV proteins were identified by using the protein database local homology search tool BLAST2 program (http://www.ncbi.nlm.nih.gov/blast/b12seq/b12.html). rCYP2D61–1567 (full-length) and CYP2D61–1275 (B8) in the pSP72 vector (Promega) were used as templates. A two-step strategy was applied: the first step was to produce chimeric fragments by PCR and the second was to subclone them into a new pSP72 vector. Each 50-µl PCR consisted of 35 cycles of denaturation for 30 s at 94°C, annealing for 30 s at 55°C, and extension at 72°C for 1 min in an automated thermal cycler (Roche Diagnostics) with the use of Super TAQ polymerase (HT Biotechnology, Cambridge, U.K.) and a TaqStart Ab (Clontech Laboratories, Basingstoke, U.K.). A 5 U/µl solution of Taq and a 7-µmol solution of TaqStart Ab were mixed in a ratio of 2:1 before setting up the PCR. The reaction was initiated by a 5-min incubation at 94°C and was completed with a 30-min extension at 72°C.



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  		FIGURE 2. Homologous regions between CYP2D6316–327, CYP2C9309–320, and HCV794–801. Shaded boxes correspond to CYP2D6/2C9 and CYP2D6/HCV point mutants. Vertical bars indicate boundaries for swap mutants. Highly conservative amino acid differences are shown in bold.

 
Two pairs of internal oligonucleotide primers that overlapped by 10–11 nucleotide bases and were engineered with endonuclease restriction sites were used to introduce five amino acid substitutions of CYP2C9 (alanine, leucine, leucine, lysine, and glutamic acid with corresponding CYP2D6 glycine (317), methionine (321), isoleucine (322), leucine (323), and aspartic acid (326)) and three amino acid substitutions of HCV protein (proline, leucine, and leucine of HCV protein with corresponding CYP2D6 317, 321 and 322). Combining the use of two flanking primers spanning the 5' and the 3' ends of multiple cloning site of the vector pSP72, the two overlapping fragments (for each construct) carrying the mutations were amplified and joined by AflII (CYP2C9 mutation) or AatII (HCV mutation) cleavage and ligation. Following BamHI and EcoRV cleavage at the 5' and 3' ends, respectively, the final chimeric products were subcloned into corresponding sites of vector pSP72.

The rDNA was amplified by transforming competent E. coli (DH5a; Life Technologies), which was cultured on agar plates using ampicillin resistance for selection of potential clones. The correct chimeric constructs were first checked by restriction enzyme digestion and finally confirmed by DNA sequencing analysis using an ABI377 sequencer (PerkinElmer).

The primers used were (restriction enzyme sites underlined): CYP2C9 mutating primers, an AflII sense primer: 5'-TGC TTA AGC ATC CGG AGG TGC AGC GCC GTG T-3', and an AflII anti-sense primer: 5'-TGC TTA AGC AAG AGC AGG AGG GCC CAG GCC-3'; HCV mutating primers, an AatII sense primer: 5'-CGG ACG TCC AGC GCC GTG TCC AAC AG-3', and an AatII antisense primer 5'-TGG ACG TCC GGA TGT AGA AGC AAG AGC AGG AGG GGC CAG G-3'; flanking primers, a sense primer: 5'-GAA CTC GAG CAG CTG AAG CTT GCA TG-3', and an antisense primer: 5'-CGA CTC ACT ATA GGG AGA CCG GCA GAT-3'.

In vitro-coupled transcription and translation of CYP2D6

Full-length, truncated, or mutant rCYP2D6 DNA were used as templates to express protein eukaryotically by an in vitro transcription/translation system using a TNT-coupled reticulocyte lysate kit (Promega) as described previously (14). In brief, 1.0 µg of plasmid DNA was incubated at 30°C for 2 h in a 50-µl mixture containing 25 µl rabbit reticulocyte, 1 µl RNA polymerase (40 U/µl), 1 µl amino acid mixture (minus methionine) (1 mM), RNasin inhibitor (20 U/µl), and [35S]methionine (10 Ci/ml) (Amersham Pharmacia Biotech). The translation product was then evaluated by incubating 1 µl of the reaction mixture with 1 M NaOH/2% H2O2, precipitated with 25% trichloroacetic acid and was then harvested onto a Whatman GF/A glass fiber filter (Waterman, Maidstone, U.K.) for radioactive measurement in a MicroBeta counter (EG & G, Milton Keynes, U.K.) to evaluate the efficiency of the reaction.

Radioimmunoprecipitation assay for detection of Abs to CYP2D6 protein

Sera from patients were diluted 1/50 and incubated in immunoprecipitation buffer (20 mM Tris/150 mM NaCl, 0.15% Tween, 0.1% aprotinin, 10 mM benzamidine, 0.1% BSA) at 4°C overnight with a 10,000 cpm aliquot of recombinant protein. The Ab-bound protein was separated from free Ag by the addition of protein A-Sepharose (Pharmacia Biotech, St. Albans, U.K.) and incubated for 1 h at 4°C. The total immunoprecipitation mixture was then transferred into 96-well filtration plates with 0.45-mm filters (multiscreen durapore HVPP membranes; Millipore, Bedford, MA). The immunocomplex bound Sepharose beads were washed 10 times with washing buffer (20 mM Tris/150 mM NaCl, 0.1% Tween 20, 0.1% BSA) and then fixed to dried multiwell filters by MeltiLex (Millipore). The radioactivity of the precipitated protein was counted on multiwell filters in a MicroBeta count (EG & G).

All test sera were assessed in duplicate. A value was considered positive when the cpm was >1050, this representing 3 SD above the mean in 57 normal controls against the full-length protein. Cut-off point for truncated proteins was calculated transferring into each assay 20 normal control samples.

cpm due to reactivity against wild type minus cut-off cpm was assigned a value of 100%. Reduction of reactivity to the mutants (cpm minus cut-off) was expressed as a percentage of the value against wild type.

The intraassay coefficient of variation, calculated on 30 occasions, ranged between 4 and 6% against the full-length CYP2D6 protein. The interassay coefficient of variation, calculated on 80 occasions, ranged between 9 and 16%. To control for intra and interassay variation, one strong positive and five negative sera were transferred in each assay, and were tested in quadruplicate.

Inhibition studies

Inhibition of reactivity to 35S-labeled CYP2D6 was investigated using four competitors: 1) full-length CYP2D6 expressed in a eukaryotic system (Gentest, Woburn, MA); 2) truncated CYP2D61–375 (construct B8); 3) CYP2D61–375/CYP2C9310–320 mutant; and 4) CYP2D61–375/HCV794–801 mutant.

Two sera, one from a patient with AIH (No. 3, Table IGo) and the other from a patient with HCV infection (No. 1, Table IIGo), were selected for the inhibition studies, because they reacted with, and were inhibited by, all the constructs used in the inhibition assay.


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  		Table I. Reactivity to full-length and truncated CYP2D6 proteins in patients with autoimmune hepatitis

 

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  		Table II. Reactivity to full-length and truncated CYP2D6 proteins in patients with HCV infection

 
Fifty-microliter aliquots of the two sera diluted at 1/200 were incubated at room temperature for 4 h with one of the following: 1) 3.0 µg of rCYP2D6 protein (~0.03 µg purified CYP2D6; Gentest) or 2) 2 µl (~0.06 µg) of individual truncated/mutant CYP2D6 protein. The quantities of the Ags used in the inhibition assays and serum dilution were determined from preliminary experiments. Ab binding was then conducted under identical conditions to those described above. Percent inhibition was calculated as 100 x (1 - (inhibited cpm/uninhibited cpm)).

Three-dimensional modeling of CYP2D6

The Swiss-Model, Swiss-Pdb Viewer (29) (both from http://www.expasy.ch/swissmod/SWISS-MODEL.html), and RasMol 2.6 (http://www.umass.edu/microbio/rasmol/) programs were used for investigating and analyzing the derived CYP2D6 protein structure.

LKM1

LKM1 was investigated by indirect immunofluorescence on 5-µm cryostat sections of rat livers, kidneys, and stomachs at the initial dilution of 1/10 in PBS (30). Positive sera were double-diluted to extinction.

Viral tests

Abs to HCV were detected by second-generation ELISA (ELISA II; Chiron, Emeryville, CA) and confirmed by RIBA II (United Biomedical, Hauppage, NY). HCV RNA was detected by nested PCR within the 5' highly conservative noncoding region (Amplicor; Hoffmann La Roche, Basel, Switzerland).

Patients and controls

A total of 32 LKM1-positive patients were studied. Sixteen patients had classical LKM1-positive AIH diagnosed according to international criteria (31). Thirteen were female with a median age of 10 years, ranging from 1 to 20 years. The median LKM1 titer was 1/640, ranging from 1/20 to 1/10,240. Ab to HCV and HCV RNA were negative in all of them. Of the 16 patients, 12 were investigated at diagnosis before starting immunosuppressive treatment, while 4 were on immunosuppression at the time of investigation consisting of prednisolone (0.5–2 mg/kg/day), 3 with and 1 without azathioprine (1–2 mg/kg/day). The other 16 patients had chronic HCV infection. Eleven were female with a median age of 48 years, ranging from 19 to 70 years. The median LKM1 titer was 1/320, ranging from 1/80 to 1/640. All were HCV RNA-positive. Four of the patients had received IFN treatment. The remaining 12 patients were untreated. This project was approved by the Ethics Committees of King’s College Hospital (London, U.K.) and University of Bologna (Bologna, Italy). The project was also approved by the Safety Advisory Unit of University College London (GM Project No. 663).

Statistics

{chi}2 and one-tailed Fischer’s exact tests were used to compare frequencies of reactivity to different constructs in patient groups. The normality of variable distributions was tested using the Kolmogorov-Smirnov goodness-of-fit test. Differences in anti-CYP2D6 levels between patient groups, and between Ab levels to full-length and truncated proteins were analyzed using the Student’s t test. A value of p < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Reactivity to full-length and truncated CYP2D6 proteins

Frequency of reactivity to full-length and truncated CYP2D6 proteins in sera from patients with AIH and HCV infection is shown in Fig. 1Go. Individual readings are shown in Tables IGo and IIGo.

A 100% frequency of reactivity to the full-length CYP2D6 protein was observed in the sera from both LKM1-positive AIH and HCV infection, while no reactivity was observed against the amino-terminal constructs A1, A2, and A3 in the sera from both AIH and HCV infection.

Group B constructs were introduced to define, first, the residues critical for Ab binding and second the regions of differential reactivity between AIH and HCV infection. A total of nine truncated CYP2D6 proteins were expressed, covering the region from starting codon to aa 417. The differences between their lengths ranged from 2 (CYP2D61–265 and CYP2D61–267) to 50 (CYP2D61–267 and CYP2D61–317) aa.

Reactivity to group B constructs showed a stepwise increase among the patients with AIH and HCV infection in terms of frequency (Fig. 1Go) and Ab levels (cpm; mean cpm shown in Tables IGo and IIGo). Reactivity to B1 was observed in 4 of 16 (25%) patients with AIH, significantly more frequent than against A1, A2, and A3 (p = 0.03 for all), and in 1 of 16 (6%) patients with HCV infection. Reactivity to B2 was observed in 38% of patients with AIH and in 31% with HCV infection, respectively. Frequency of reactivity to all group B constructs (B1 to B9) was higher in AIH than in HCV infection. Frequency of reactivity to CYP2D61–327 (B6) and to CYP2D61–337 (B7) was significantly higher in AIH than in HCV infection (13 of 16, 81% vs 7 of 16, 44%, p = 0.03, and 14 of 16, 88% vs 9 of 16, 56%, p = 0.05, respectively). When the length of CYP2D6 protein increased from 317 to 327 aa, the frequency of reactivity increased from 63 to 81% in AIH but remained the same (44%, p = 0.03) in HCV infection.

Differences in only two amino acids could remarkably alter frequency of reactivity which increased from 38 to 50% in patients with AIH and from 31 to 38% in patients with HCV infection when B2 (CYP2D61–265) and B3 (CYP2D61–267) were used as targets.

Four truncated C-terminal constructs, C1-C4, lacking the amino-terminal region 27–192 and increasing in length toward the C terminus, were introduced to investigate further the role of region 27 to 192 on Ab binding. Frequency and levels of Abs increased gradually from C1-C4 (see Tables IGo and IIGo). Reactivity to C1, containing major linear epitopes of CYP2D6, 196–218 and 254–271, respectively (12, 16), was observed in 11 of 16 (69%) patients with AIH and 7 of 16 (44%) patients with HCV infection. Reactivity to C2 was observed in 81 and 50%, respectively (p = 0.06), which was comparable to that against B5, the B construct (without deletion) of similar length. Reactivity to C3, containing the four previously defined linear epitopes, 196–218, 254–271, 321–351, and 373–389, was seen in 14 of 16 (88%) in AIH and 11 of 16 (69%) in HCV infection, which was similar to that against the comparable construct (B8). The frequency of reactivity to construct C4, which contains the full 193–497 sequence, was 100% in both AIH and HCV infection groups.

In patients with AIH, mean cpm levels against the full-length protein were significantly higher than those against truncated proteins (B1-B7, C1, and C2 shown in Table IGo). In patients with LKM1/HCV infection, mean cpm levels against full-length protein were also higher than those against truncated proteins (B2-B5 and C1, Table IIGo). There were no significant differences in Ab levels between patient groups.

Reactivity to CYP2D6 chimeric mutants

Based on the results obtained by epitope mapping showing that CYP2D6316–327 was able to differentiate between AIH and HCV infection, this sequence was selected for further analysis by introducing site-specific mutations into it. Within this region, CYP2D6317–327 shares 81% (9/11) homology with CYP2C9, including six identical and three conservative amino acids (Fig. 2Go) and CYP2D6316–323 shares 88% (7/8) homology including five identical and two conservative amino acids, with polyprotein 1A of HCV (Fig. 2Go), a viral sequence suggested to act as the molecular mimic capable of inducing tolerance breakdown against CYP2D6 (32). CYP2D6/2C9 and CYP2D6/HCV mutations were introduced into construct B8 (CYP2D61–375) and full-length CYP2D6 thus generating four mutants: 1) CYP2D61–375/CYP2C9310–320; 2) CYP2D61–375/HCV794–801; 3) CYP2D61–497/CYP2C9310–320; and 4) CYP2D61–497/HCV794–801.

Reduction of reactivity to all four mutants was observed in both groups of patients. Frequency of sera showing a significant reduction in binding (cpm), i.e., >= 30% when compared with that against the unmodified polypeptides (B8 and wild-type CYP2D6), was analyzed. Reduction in binding to CYP2D61–375/CYP2C9310–320, CYP2D61–375/HCV794–801, and CYP2D61–497/CYP2C9310–320 (mutants I, II, and III) was seen in 5 of 14 (36%), 10 of 14 (71%), and 11 of 16 (69%) patients with AIH, and in 5 of 11 (45%), 7 of 11 (64%), and 9 of 16 (56%) patients with HCV infection. Although the reduction in reactivity was similar in AIH and HCV patients against mutants I, II, and III, that against mutant IV was significantly greater in AIH (12 of 16, 75%) than in HCV patients (6 of 16, 38%, p = 0.03).

Inhibition studies

Results of inhibition are shown in Fig. 3Go. Four proteins were used as target Ags and also as inhibitors. They were: 1) full-length CYP2D6 protein; 2) construct B8 (CYP2D61–375); 3) mutant I (CYP2D61–375/CYP2C9310–320); and 4) mutant II (CYP2D61–375/HCV794–801). Preincubation of the AIH serum with the full-length protein resulted in 68, 82, 91, and 95% inhibition against full protein, construct B8, and mutants I and II, respectively (Fig. 3GoA). Preincubation with construct B8 led to inhibition against itself and against mutant I, but neither against the full-length protein nor against mutant II. Preincubation with mutant I led to a high-level inhibition against itself and mutant II and a low-level inhibition against construct B8. Similarly, mutant II could reduce Ab binding against itself and also against mutant I (Fig. 3GoA). When the serum from a patient with HCV infection was preincubated with the four competitors, a similar pattern of inhibition was observed (Fig. 3GoB), the only difference being that construct B8 inhibited Ab binding against mutant I to a lesser extent than that seen in AIH serum (35% in HCV serum vs 72% in AIH serum).



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  		FIGURE 3. Inhibition with full-length CYP2D6 protein, CYP2D61–375, CYP2D61–375/CYP2C9310–320, and CYP2D61–375/HCV794–801 mutants. The two sera, one from a patient with AIH and the other from a patient with HCV infection, were preincubated with the inhibitors shown in the x-axis: full-length CYP2D6 ({blacksquare}); CYP2D61–375 (construct B8) (); CYP2C9 mutant (mutant I) (), and HCV mutant (mutant II) ({square}). Percent inhibition is shown on the y-axis (specified under the bars). A, The percent inhibition after preincubation of the serum from a patient with AIH; B, The serum from a patient with HCV infection.

 
Three-dimensional modeling of CYP2D6316–327 and its mutants

CYP2D6316–327, CYP2D6/CYP2C9310–320, and CYP2D6/HCV794–801 mutants were modeled locally on the homologous region in B. megaterium cytochrome P450 BM-3 (27, 28). Their three-dimensional structures are shown in Fig. 4Go. The sequence CYP2D6316–327 is located on the surface of the protein. This epitope displays a hydrophobic patch that is situated between an N-terminal aromatic residue (W316) and a C-terminal histidine (H324). The negatively charged D326 is expected to play an important role in epitope recognition through electrostatic interaction. The amino acid composition of CYP2C9310–320 is very similar with the exception of a bulky positively charged residue (K316) just C-terminal of the hydrophobic patch. This L323K swap (conservative amino acid replacement) may substantially affect Ab binding, because the insertion of a large positively charged residue (K) may interfere with surface complementarity or electrostatic interactions. The structure of HCV794–801 is similar to its corresponding region of CYP2D6316–323, because the hydrophobic patch still remains after introducing M321L and I322L mutations (conservative amino acid replacement). N-terminal amino acid replacement was performed by switching glycine 317 to proline 796.



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  		FIGURE 4. Homology models of epitopes regions. CYP2D6316–327, CYP2D6/CYP2C9310–320, and CYP2D6/HCV794–801 mutants were modeled locally on the homologous region in the B. megaterium cytochrome P450 BM-3 using SWISS-MODEL (PDB code 1FAH.pdb). Epitope residues in CYP2D6 and its CYP2C9 and HCV mutants are displayed in stick mode and colored according to physicochemical properties or individual amino acid identities: orange, aromatic; brown, small; yellow, hydrophobic; cyan, histidine; green, proline; red, negatively charged; blue, positively charged. Amino acids in the CYP2D6 sequence are shown in italics.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Using eukaryotically expressed full-length protein and constructs thereof, we show that CYP2D6 is divided into two main regions with respect to antigenicity. The N-terminal region comprising amino acid 1–193 appears to be devoid of antigenicity, while the C-terminal 193–497 accounts for all LKM1 reactivity. This is true whether the LKM1 positive sera used as probes are derived from patients with AIH or patients with HCV infection. Moreover, we show that the sequence 316–327, which is part of a larger region capable of differentiating LKM1 reactivity in AIH and HCV infection, is exposed on the surface of the molecule.

The choice of using eukaryotically expressed full-length protein and constructs was dictated by the fact that some 70% of HCV/LKM1-positive sera do not react with the prokaryotically expressed molecule (13), indicating that there must be posttranslational modifications of critical importance for recognition of the enzyme by HCV/LKM1-positive sera. In the present study, we were able to confirm that the totality of the LKM1-positive sera from either condition recognize the eukaryotically expressed cytochrome.

The use of a set of three N-terminal constructs A1, A2, and A3 enabled us to show that none of the LKM1-positive sera was reactive with the region comprising aa 1–193. Reactivity increased stepwise from 0 to 100% using constructs increasing in length toward the C terminus. It could be argued that the region 1–193, though not harboring identifiable B cell epitopes, may influence the antigenicity of the molecule, contributing to its tertiary structure. Experiments using the C constructs with the aa 27–192 deletion, do not support this hypothesis. All LKM1-positive sera reacted with construct C4, which contains the entire sequence C-terminal to aa 193, but is devoid of the N-terminal region aa 27–192. This indicates that the removal of 159 aa from the N-terminal 1–192 sequence has no direct or indirect effect on the antigenicity of the whole molecule expressed eukaryotically.

Constructs of differing lengths, with or without the 27–192 deletion, were used to characterize further areas of antigenicity. The finding that constructs B6 1–327(1–327) and C3 193–391(1–26, 193–391) are recognized by four-fifths of the LKM1-positive sera from patients with AIH was expected because both contain the sequence 254–271, the major linear epitope as defined by synthetic peptides (16). Of interest is the finding that one-quarter of the patients with AIH recognize the construct B1 1–225(1–225), which does not contain any of the reported linear epitopes; this indicates that 33 aa C-terminal to position 193 impart antigenicity to the molecule. In keeping with this finding, our group has recently shown that the sequence comprising aa 193–212, expressed as a synthetic peptide, harbors a linear epitope which is recognized by all sera of patients with AIH and by 50% of those with HCV infection. The lower frequency of reactivity against B1, as observed in this study, compared with that using synthetic peptide 193–212 could be due to the fact that a partial unfolding of the molecule is required for this linear epitope to be recognized.

The sequence containing the first 265 aa (B2) is recognized by a similar proportion of sera from patients with AIH (38%) and patients with HCV infection (31%). The frequency of reactivity increases steadily with the C-terminal extension of the molecule in AIH, but not in HCV infection. Thus 80% of sera from patients with AIH react with the construct B6 1–327(1–327) compared with only 44% of those from patients with HCV infection. This significant difference is also detectable using a sequence 10 aa longer (B7) or one containing the 27–192 deletion (C2).

This lower reactivity against region 316–337 in HCV/LKM1 as compared with AIH/LKM1 sera, is an intriguing finding. An octameric sequence WGLLLMIL (CYP2D6316–323) contained in this region shares 7 aa identities or conserved substitutions with the octamer WPLLLLLL (HCV794–801) of the HCV polyprotein and was suggested to be a major trigger of HCV-induced anti-CYP2D6 autoimmunity. Our findings would appear to question this notion. However, it is possible for this sequence to be involved in Ag recognition as a CD4 cross-reactive epitope in chronic HCV infection. When we replaced this wild-type sequence within CYP2D6 with the homologous viral sequence, sera from both AIH and HCV infection lost reactivity significantly. Studies of the structure of the HCV region (HCV794–801) corresponding to that of CYP2D6 (CYP2D6316–323) show that they are similar toward the C terminus, the two amino acids swapped being similar in size. The change from glycine 317 to proline 796 may have major consequence, however. Although both glycine and proline are small amino acids, proline may be introducing a "kink" in the epitope helix, leading to a changed geometry between the aromatic W (316) and the hydrophobic patch. In the middle of a helix, the proline ring pushes away the preceding turn of the helix by ~1 Å on that side, producing a bend of ~30° in the helix-axis and also breaking the next hydrogen bond. These changes would readily explain the lost reactivity we observed.

Experiments performed using another swap mutant, where the CYP2D6 sequence was replaced by its homologue from CYP2C9, the target of the autoantibody LKM2 (33), also showed a decreased reactivity by LKM1-positive sera from both patients with AIH or HCV infection. This confirms that the region modified in the swap mutants is part of a major antigenic determinant. Despite the fact that the three locally modeled structures of this region are similar, all containing a hydrophobic patch between an N-terminal aromatic residue (W316) and a C-terminal histidine, the two swap mutants not only react less with LKM1-positive sera, but are also less efficient competitors in the inhibition studies. Though the two swap mutants differ between themselves, the CYP2C9 mutant having a C-terminal amino acid mutation L323K and the HCV mutant an N-terminal switch between glycine 317 and proline 796, they are more similar to each other than to the wild protein and behave similarly in inhibition studies. Taken together, the combinations of all these mutations may rearrange the epitope helix and greatly alter the properties of the epitope region, particular in terms of surface shape and charge, and consequently result in the loss of Ab binding.

Relevant to the present findings is our previous observation that the region spanning 316–337 aa, which we show in the present study to be capable of differentiating LKM1 reactivity in AIH and HCV infection, is the focus of cross-reactive autoimmunity (34). In that report, we showed that CYP2D6321–339 shares extensive homology with carboxypeptidase H (35) 33–51 and 21-hydroxylase (an autoantigen in Addison’s disease; Ref. 36) 307–325 and that the serum of a patient suffering from AIH type 2, insulin-dependent diabetes, and Addison’s disease contained autoantibodies reacting with the CYP2D6 sequence and cross-reacting with the homologous regions of carboxypeptidase H and 21-hydroxylase (34), a finding outlining the key role of the region in the generation of autoimmunity.

The aa sequence 316–327, which is part of the major antigenic determinant described in this study, is exposed on the surface of the protein. This, together with the recent demonstration that the CYP2D6 molecule is expressed on the hepatocyte membrane and is targeted by LKM1 (37, 38), suggests that this sequence may be directly involved in autoantibody mediated liver cell damage, akin to the scenario in myasthenia gravis (39), autoimmune thyroiditis (40), autoimmune thrombocytopenia purpura (41), hemolytic anemia (42), and neonatal systemic lupus erythematosus (43).

The use of sera from two distinct diseases, AIH and chronic HCV infection, sharing the same autoantibody has allowed us to provide an initial characterization of the antigenic constitution of the target. Further studies should aim at establishing human mAbs to CYP2D6 from both clinical conditions to obtain information on the fine antigenic specificity. Moreover, in view of the fact that LKM1 belongs to the IgG class implying a T cell-directed class switch, the role of CD4 cells in the generation of the autoimmune attack should be investigated.


   Acknowledgments
 
We thank Dr. Kaushik Choudhuri for reviewing the manuscript and giving critical comments.


   Footnotes
 
1 This work was supported by a Royal Society (London, U.K.) Dorothy Hodgkin Fellowship (to Y.M.), the Children’s Liver Disease Foundation (Birmingham, U.K.; to D.P.B. and G.M.-V., and Grant NL1720 to Y.M.), and the Children Nationwide Medical Research Trust (London, U.K.; to G.M.-V.). Back

2 Address correspondence and reprint requests to Dr. Diego Vergani, Institute of Hepatology, University College London Medical School, 69–75 Chenies Mews, London WC1E 6HX, U.K. E-mail address: D.Vergani{at}ucl.ac.uk Back

3 Abbreviations used in this paper: LKM1, liver kidney microsomal Ab type 1; AIH, autoimmune hepatitis; HCV, hepatitis C virus. Back

Received for publication March 11, 2002. Accepted for publication April 8, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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