Distinct epitopes on formiminotransferase cyclodeaminase induce autoimmune liver cytosol antibody type 1

Distinct epitopes on formiminotransferase cyclodeaminase induce autoimmune liver cytosol antibody type 1

Distinct Epitopes on Formiminotransferase Cyclodeaminase Induce Autoimmune Liver Cytosol Antibody Type 1 LUIGI MURATORI,1 ELIZABETH SZTUL,2 PAOLO MURA...

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Distinct Epitopes on Formiminotransferase Cyclodeaminase Induce Autoimmune Liver Cytosol Antibody Type 1 LUIGI MURATORI,1 ELIZABETH SZTUL,2 PAOLO MURATORI,1 YA-SHENG GAO,2 ALESSANDRO RIPALTI,3 CRISTINA PONTI,1 MARCO LENZI,1 MARIA PAOLA LANDINI,3 AND FRANCESCO B. BIANCHI1

Liver cytosol antibody type 1 (LC1) is regarded as a serologic marker of type 2 autoimmune hepatitis, in addition to liver kidney microsomal antibody type 1. Among 38 patients with type 2 autoimmune hepatitis, 23 were positive for LC1 antibodies. The antigen recognized by LC1 has been identified as a liver-specific 58-kd metabolic enzyme named formiminotransferase cyclodeaminase (FTCD). All 23 LC1positive sera immunoprecipitated rat FTCD, and 22 gave an identity reaction with rat FTCD by immunodiffusion. No reaction was observed with sera from 10 patients with type 1 autoimmune hepatitis, 10 with primary biliary cirrhosis, 10 with chronic hepatitis C, and 10 healthy controls. By Western immunoblotting all 23 LC1-positive sera and all the controls tested negative, suggesting that all the antigenic epitopes were destroyed by denaturation. FTCD is a bifunctional protein composed of distinct globular FT and CD domains connected by a short linker. To identify epitopes that trigger the LC1 autoimmune response, we tested LC1 antibodies against FTCD constructs encoding the N-terminal FT domain (amino acids 1-339), or the C-terminal CD domain (amino acids 332-541). Of 20 sera positive against full-length FTCD, 8 (40%) recognized the FT domain and the CD domain, 7 (35%) recognized only the FT domain, and 5 (25%) did not recognize either construct. No sera reacted with only the CD domain. These data indicate that multiple regions of FTCD trigger the LC1 autoimmune response, and that LC1 reactivity is mainly directed to conformation-sensitive epitopes located in the FT region of FTCD. (HEPATOLOGY 2001;34:494-501.) Liver cytosol antibody type 1 (LC1) has been first described, alone or in association with liver kidney microsomal antibody type 1 (LKM1), in patients with type 2 autoimmune

Abbreviations: LC1, liver cytosol antibody type 1; LKM1, liver/kidney microsomal antibody type 1; cDNA, complementary DNA; FTCD, formiminotransferase cyclodeaminase; PBS, phosphate-buffered saline; SDS, sodium dodecyl sulfate; ME, mercaptoethanol; PAGE, polyacrylamide gel electrophoresis; BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay. From the 1Departments of Internal Medicine, Cardioangiology, Hepatology; the 3Department of Clinical and Experimental Medicine, Section of Microbiology, Alma Mater Studiorum, University of Bologna, Bologna, Italy; and the 2Department of Cell Biology, University of Alabama at Birmingham, Birmingham, AL. Received April 9, 2001; accepted June 21, 2001. Address reprint requests to: Luigi Muratori, M.D., Ph.D., Department of Internal Medicine, Cardioangiology, Hepatology, Alma Mater Studiorum, University of Bologna, Policlinico S. Orsola, via Massarenti, 9, 40138 Bologna, Italy. E-mail: [email protected]; fax: (39) 051-340877. Copyright © 2001 by the American Association for the Study of Liver Diseases. 0270-9139/01/3403-0008$35.00/0 doi:10.1053/jhep.2001.27179

hepatitis.1,2 Only sporadically is it detected in patients with chronic hepatitis C virus infection, usually in association with LKM1.3,4 Among the different autoantibodies deemed as serologic markers of autoimmune hepatitis,5 LC1 is particularly intriguing because it targets a liver-specific antigen.1,2 In addition, serum LC1 concentrations appear to fluctuate in parallel with aminotransferase levels, an observation that suggests a possible role of LC1 autoreactivity in the pathogenic mechanisms leading to hepatocyte injury.6 In a recent report, Lapierre et al. after screening a complementary DNA (cDNA) library of HepG2 cells with an LC1positive serum isolated a clone with high sequence homology to pig formiminotransferase cyclodeaminase (FTCD), and showed that a construct encoding the C-terminal portion of FTCD can be recognized by LC1 antibodies.7 FTCD is a mammalian metabolic enzyme involved in the conversion of histidine to glutamic acid,8 and is most highly expressed in the liver. FTCD is bifunctional and is composed of distinct FT and CD domains connected by a short linker. The FT activity transfers a formimino group from N-formimino-L-glutamic acid to tetrahydrofolate to generate glutamic acid and 5formiminotetrahydrofolate, and the CD activity then converts the 5-formiminotetrahydrofolate to 5,10-methenyl tetrahydrofolate and ammonia. Native FTCD is an octamer with 8 identical subunits arranged in a planar ring.8 FTCD is a soluble cytosolic protein, but in cells appears to be preferentially associated with the cytosolic side of Golgi membranes.9-11 In addition, FTCD appears to be a dynamic component of the Golgi, and cycles between the Golgi and earlier compartments of secretory pathways.9 In this report, we show that a human liver cytosol protein immunoprecipitated with a pool of LC1-positive autoimmune sera is FTCD, and that LC1 sera recognize distinct epitopes on FTCD. We used full-length recombinant rat FTCD as antigenic substratum for immunodiffusion, immunoblotting, and immunoprecipitation experiments with a large series of LC1positive and LC1-negative sera obtained from patients with type 2 autoimmune hepatitis. We show that LC1-positive sera recognize conformation-specific epitopes preferentially localized to the FT domain of FTCD. PATIENTS AND METHODS Patients and Sera. Sera were obtained from 38 patients with type 2 autoimmune hepatitis diagnosed according to internationally agreed12 and recently revised criteria,5 and classified on the basis of the autoantibody profile as LC1 positive, LKM1/LC1 positive, and LKM1 positive (Table 1). Female sex was predominant (31 patients out of 38, 81.5%), median age at onset was 12 years (range, 2-53), and median alanine transaminase level was 11 times the upper normal limits (range, 2-80). LC1 positivity was assessed, when isolated,

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TABLE 1. Reactivity to Human and Rat FTCD in 38 Patients With Type 2 Autoimmune Hepatitis Pt.

Autoantibody Pattern (IFL)

LC1 (CIE)

Anti–58-kd (W-IB)

Anti-FTCD (ID)

Anti-FTCD (IP)

Anti-FT (ELISA)

Anti-CD (ELISA)

Anti-FT 1-182 (ELISA)

Anti-FT 176-339 (ELISA)

21 22 29 30 25 27 32 33 34 36 9 11 12 13 2 6 7 31 37 23 24 10 35 28 26 38 1 3 4 5 8 14 15 16 17 18 19 20

LC1 LC1 LC1 LC1 LC1 LC1 LC1 LC1 LC1 LC1 LC1 LC1 LC1 LC1 LC1 LC1 LC1 LKM1/LC1 LKM1/LC1 LKM1/LC1 LKM1/LC1 LKM1/LC1 LKM1/LC1 LKM1 LKM1 LKM1 LKM1 LKM1 LKM1 LKM1 LKM1 LKM1 LKM1 LKM1 LKM1 LKM1 LKM1 LKM1

Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg

Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg

Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Neg Pos Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg

Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg

Pos Pos Pos Pos ND Neg Pos Neg Pos ND Pos ND Pos Neg Neg Pos Pos Pos Neg Pos Pos Pos Pos Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg

Neg Neg Pos Pos ND Neg Neg Neg Neg ND Pos ND Pos Neg Neg Neg Neg Pos Neg Pos Neg Pos Pos Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg

Neg Neg Pos Neg Neg Neg Neg Neg Neg Neg ND Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg ND Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg ND Neg Neg

Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg ND Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg ND Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg ND Neg Neg

NOTE. Thirty-eight patients with type 2 autoimmune hepatitis are grouped based on presence of autoantibodies against LKM1 and/or LC1, as defined by indirect immunofluorescence. Presence of LC1 reactivity was confirmed by appearance of precipitin lines in counterimmunoelectrophoresis against human liver cytosol and by detection of a 58-kd band in Western immunoblotting with human liver cytosol. The ability of the various sera to recognize recombinant rat FTCD (anti-FTCD) was tested by immunodiffusion and immunoprecipitation. Reactivity with the formiminotransferase domain (anti-FT), the cyclodeaminase domain (anti-CD) of FTCD, and with 2 formiminotransferase subdomains (anti-FT 1-182 and anti-FT 176-339) was tested by ELISA. Abbreviations: IFL, indirect immunofluorescence; CIE, counterimmunoelectrofluoresis; W-IB, Western immunoblotting; ID, immunodiffusion; IP, immunoprecipitation; Pos, positive; Neg, negative; ND, not determined.

by indirect immunofluorescence and validated by counterimmunoelectrophoresis and Western immunoblotting with human liver cytosol.4 LKM1 positivity was assessed by indirect immunofluorescence and validated by Western immunoblotting with human native and recombinant CYP2D6.13 Isolated LC1 was present in 17 (45%) patients, both LKM1 and LC1 in 6 (16%) patients, and isolated LKM1 in 15 (39%) patients. As controls, we tested 10 patients with antinuclear and/or anti–smooth muscle positive type 1 autoimmune hepatitis, 10 patients with anti–mitochondrial positive primary biliary cirrhosis, 10 patients with chronic hepatitis C, and 10 healthy normal subjects. Isolation and Purification of LC1 Antigen. Human liver cytosolic extract was prepared by differential centrifugation as originally described,1 using normal parenchyma from a partial hepatectomy surrounding a secondary liver lesion. A pool of 4 LC1-positive sera obtained from patients with autoimmune hepatitis was used as probe. LC1 antigen was isolated by counterimmunoelectrophoresis,

as previously described.4 The LC1-specific precipitin lines were extensively washed in ice-cold phosphate buffered saline (PBS), the antigen-antibody complex was excised from the agarose and boiled in reducing lysis buffer (62.5 mmol/L Tris-HCl pH 6.8, 10% glycerol, 2% sodium dodecyl sulfate [SDS], 5% mercaptoethanol [ME]) for 10 minutes. The denatured antigen-antibody complex was run on a 7.5% SDS polyacrylamide gel electrophoresis (PAGE), transblotted onto nitrocellulose sheet, blocked with PBS containing 3% bovine serum albumin (BSA), and probed with a pool of LC1-positive sera at 1:200 dilution. Peroxidase conjugate rabbit anti-human IgG (dilution 1:1,000; Dako, Copenhagen, Denmark) was used as secondary antibody. To isolate LC1 antigen sufficient for protein sequencing, preparative SDS-PAGE gels were run with the bulk of the LC1-LC1 antigen complex prepared by counterimmunoelectrophoresis. Protein bands were visualized by negatively staining the gels with 0.3 mol/L CuCl2, and the 58-kd polypeptide was excised, electroeluted in SDS running buffer, and extensively dialyzed against PBS. Purified

496 MURATORI ET AL. LC1 antigen was treated with trypsin and several of the resulting peptides were subjected to sequence analysis. Immunodiffusion. Immunodiffusion was performed according to the Ouchterlony double immunodiffusion method. Agarose (0.5% wt/vol) was poured onto Petri plates, a 5-mm-diameter central well and 6 wells 2 mm in diameter and 4 mm apart (measured between circumferences) were cut. In the central well 50 ␮L of Escherichia coli lysates containing recombinant rat FTCD (protein concentration, 4 mg/mL) was placed. In some experiments, lysates expressing ␤-galactosidase were placed in central wells. In each of the other 6 wells 10 ␮L of undiluted serum was placed. Plates were incubated at 4°C in a humidified chamber and were examined daily for 3 days. Sera giving precipitin lines were reassessed near an LC1-positive control serum to confirm the identity reaction. Western Immunoblotting. Ten micrograms per lane of lysates of E. coli containing recombinant rat FTCD were separated in 7.5% SDSPAGE minigels (Mini-Protean II System; Bio-Rad Laboratories, Richmond, CA) and transblotted onto nitrocellulose filters. After transblotting, filters were incubated in blocking solution (Tris-buffered saline containing 3% BSA) for 1 hour at room temperature. The filters were then cut into strips, and each strip was incubated overnight at 4°C with serum samples at the final dilution of 1:100. After incubation, the strips were washed 3 times in Tris-buffered saline containing 0.1% Tween 20, and were subsequently incubated for 2 hours at room temperature in blocking solution containing peroxidase conjugate rabbit anti-human IgG (dilution 1:1,000) (Dako, Copenhagen, Denmark) as secondary antibody. After further washing, the colorimetric reaction was developed with 4-chloro-1-naphtol for 10 minutes at room temperature. Immunoprecipitation. Two microliters of test serum was incubated overnight at 4°C with 50 ␮L of E. coli lysates containing recombinant rat FTCD in RIPA buffer. After adding 150 ␮L of Pansorbin Cells (Calbiochem, La Jolla, CA), each vial was incubated for 2 hours on ice and mixed regularly. The vials were centrifuged at 3,000g for 5 minutes and the pellet was washed 5 times with RIPA buffer. After the last washing step, the pellet was resuspended and boiled in 200 ␮L of reducing lysis buffer (62.5 mmol/L Tris-HCl pH 6.8, 10% glycerol, 2% SDS, 5% ME) for 10 minutes, centrifuged for 30 seconds in a microfuge at room temperature, and 2 ␮L of the supernatant were loaded per each lane in a 7.5% SDS-PAGE minigel. After running and transblotting the gel on a nitrocellulose sheet, the filter was blocked with PBS containing 3% BSA for 1 hour at room temperature. The nitrocellulose was then incubated for 2 hours with a monoclonal antibody raised against rat FTCD (monoclonal anti-Golgi 58-kd protein; Sigma ImmunoChemicals, St. Louis, MO) diluted 1:1,000 in blocking solution. The secondary antibody was peroxidase-conjugated goat anti-mouse Ig (Dako) diluted 1:2,000. The colorimetric reaction was developed with 4-chloro-1-naphtol for 10 minutes at room temperature. Adsorption of Anti-FTCD Reactivity. Ten microliters of an LC1-positive serum was incubated overnight at 4°C with 1 mL of E. coli lysates containing recombinant rat FTCD in RIPA buffer, or with 1 mL of control E. coli lysates not expressing rat FTCD. After incubation, the vials were centrifuged at 3,000g for 10 minutes, and the supernatant was used undiluted (final dilution 1:100) for indirect immunofluorescence on rat liver sections and for Western immunoblotting of human liver cytosol, as previously described.4 Molecular Cloning and Expression of Rat FTCD. A rat liver Lambda ZAP (Statagene, La Jolla, CA) cDNA expression library was screened with a monoclonal anti-FTCD antibody (monoclonal anti-Golgi 58K protein; Sigma ImmunoChemical14). Positive clones were isolated, characterized, and sequenced.9 Sequences homologous to pig FTCD sequence were identified in GenBank using the BLAST program. The complete nucleotide sequence of rat FTCD is available at the accession number AF079233 from GeneBankTM/EMBL Data Bank. The open reading frame of rat FTCD is 1,623 base pairs and encodes a polypeptide of 541 amino acids with a calculated molecular weight of 58.9 kd.

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One clone in pBluescript vector encoding the full length rat FTCD, was used to transfect E. coli cells and 1 ampicillin-resistant colony was selected for expression of the fusion protein. After growing in liquid culture medium, expression of recombinant ␤-galactosidase FTCD was induced by adding 5 mmol/L isopropyl [beta]-Dthiogalactoside (IPTG). Cells were then collected, washed 3 times in PBS buffer then resuspended in RIPA buffer (150 mmol/L NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mmol/L Tris pH 8.0) and sonicated. Lysates were analysed on a 10% SDS-PAGE. Construction, Expression, and Purification of FT and CD Domains, and of FT (1-181) and FT (176-339) Fragments. To express FT domain (amino

acid 1-339), CD domain (amino acid 332-541), FT fragment spanning amino acids 1-181, and FT fragment spanning amino acids 176-339, the appropriate cDNA sequences were amplified by polymerase chain reaction with the full-length FTCD-pBluescript as the template, and the cDNA fragments were cloned into pET-21b vector (Novagen, Madison, WI). All fragments were 6-His tagged at the C-termini. All constructs were expressed in BL21 (DE3) cells after induction with 0.5 mmol/L IPTG for 2 to 3 hours at 30°C. Cells were harvested by centrifugation at 2,000g for 15 minutes and resuspended in PBS containing 5 mmol/L 2-ME, 5% glycerol, and 0.5 mmol/L phenylmethylsulfonyl fluoride. Cells were sonicated and centrifuged at 20,000g for 15 minutes. To isolate FT, CD, and FT (1-181), supernatants were supplemented with NaCl to 0.3 mol/L and imidazole to 5 mmol/L, and were incubated with Ni-NTA beads (Qiagen, Valencia, CA) for 2 hours at room temperature. The beads were washed 5 times with PBS containing 5 mmol/L 2-ME, 5% glycerol, 0.5 mmol/L phenylmethylsulfonyl fluoride, 0.3 mol/L NaCl, and 5 mmol/L imidazole. The fragments were eluted with the above washing solution containing 170 mmol/L imidazole. To isolate FT (176-339), the 2,000g pellets of the cell lysate were solubilized with the above washing solution also containing 6 mol/L urea. This solution was centrifuged at 20,000g for 15 minutes and the resulting supernatant was incubated with NiNTA beads for 2 hours at room temperature. The beads were washed 5 times with the same solution containing urea and the protein fragment was eluted with the washing solution containing 220 mmol/L imidazole. To remove urea the protein fragment was dialyzed against the above washing solution with slowly decreasing concentrations of urea and then with the washing solution without urea. Enzyme-Linked Immunosorbent Assay. Purified FT, CD, FT (1-181), and FT (176-339) fragments were diluted in carbonate coating buffer (0.05 mol/L Tris-HCl, 0.15 mol/L NaCl, 5 mmol/L CaCl2, pH 7.4). One hundred microliters per well, corresponding to 0.5 ng of purified antigen, were pipetted in 96-well enzyme-linked immunosorbent assay (ELISA) plates and incubated overnight at 4°C. After extensive washings with PBS-Tween, the plates were blocked with PBS containing 3% BSA for 1 hour at room temperature. One hundred microliters per well of serum diluted 1:500 in PBS/BSA was added in duplicate and incubated overnight at 4°C. Wells were rinsed off with washing buffer and incubated with peroxidase-conjugated rabbit anti-human IgG (Dako) diluted 1:4,000 for 1 hour at room temperature. The colorimetric reaction was developed with o-phenylenediamine dihydrochloride for 15 minutes at room temperature, and absorbance was measured at 450 nm. The cutoff was calculated as the mean ⫾ 3 standard deviations of the absorbance values given by 40 LC1-negative controls. RESULTS The Antigen Recognized by LC1 Is FTCD. To identify the LC1 antigen, human liver cytosol and a pool of 4 LC1-positive sera obtained from patients with autoimmune hepatitis were used in counterimmunoelectrophoresis. LC1-specific precipitin lines were detected with each LC1-positive serum (Fig. 1). The material was recovered from the gel and the antigenantibody complexes were analyzed by SDS-PAGE. As shown in Fig. 2, lane B, 2 bands were visible in a gel stained with Coomassie blue: a 58-kd polypeptide corresponding to the

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FIG. 1. Counterimmunoelectrophoresis with human liver cytosol. LC1negative sera (wells 1-3), LC1-positive sera (wells 4-7), and a reference LC1positive serum (well 8) were analyzed against human cytosolic proteins. Immunoprecipitin lines of identity (arrowheads) are evident in wells 4 through 8.

FIG. 4. LC1 immunofluorescence on rat liver. LC1 shows a typical uneven staining of the liver lobule with minimal staining of the cellular layers around the central veins (the juxtavenous hepatocytes). Hepatocyte cytoplasm, but not nuclei, is stained. Within some hepatocytes, a perinuclear punctuate staining similar to the localization of hepatic Golgi can be seen (arrows).

FIG. 2. Purification of LC1 antigen. Left panel: Coomassie blue–stained gel. Human liver cytosolic proteins (lane A), LC1 antigen-antibody complex (lane B), and purified LC1 antigen (lane C) were analyzed by SDS-PAGE. Right panel: LC1 Western immunoblotting. A gel analogous to that in the left panel was transferred to nitrocellulose filter. The filter was incubated with a pool of LC1-positive sera, followed by a peroxidase conjugated secondary antibody. A major 58-kd protein is recovered as the LC1 antigen and is recognized by LC1 sera. In lanes B and B1 IgG heavy chains (52-kd) from the LC1 antigen-antibody complex are present and recognized by the secondary antibody.

FIG. 3. Sequence homology between peptide 1 and human FTCD. Sequence alignment between peptide 1 and amino acids 319 to 331 of human FTCD. Identical amino acids are boxed on gray background.

FIG. 5. Expression of recombinant rat FTCD. Bacteria were induced to express recombinant rat FTCD, lysed, and the lysate was analyzed by SDSPAGE. Coomassie blue–stained gel (lane A) shows a major 70-kd band of recombinant FTCD-␤-galactosidase fusion protein (arrow). Analogous gel was transferred to nitrocellulose and immunoblotted with monoclonal antibodies against rat FTCD. The recombinant protein is recognized by antiFTCD antibodies (lane A1).

FIG. 6. LC1-positive sera recognize recombinant rat FTCD by immunodiffusion. Recombinant rat FTCD was placed in the central well. LC1-positive sera were placed in wells 1 to 3 whereas LC1-negative sera were placed in wells 4 to 6. A precipitin line of identity was obtained with LC1-positive sera.

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LC1 antigen and a thick 52-kd band corresponding to the heavy chains of IgGs. The 58-kd protein was isolated from preparative SDS-PAGE and reanalyzed. As shown in Fig. 2, lane C, a homogenous preparation of the 58-kd protein was obtained. To ensure that the 58-kd protein was the LC1 antigen, an analogous gel was transferred onto nitrocellulose filter and probed with a pool of LC1-positive sera. As shown in Fig. 2, lanes B1 and C1, the 58-kd protein was recognized by the LC1-positive sera, confirming that it is the LC1 antigen present in human liver cytosolic preparation (lane A1). The purified LC1 antigen was subjected to trypsin digestion, and several of the resulting peptides were sequenced by Dr. Suzanne C. Perry (Protein Service Laboratory, Biotechnology Department, University of British Columbia, Vancouver, BC, Canada). Of the several internal peptides obtained after trypsin digestion, only 2 were successfully sequenced, because of the low amount (in the range of 3-6 pmol) of available protein. The sequences of the peptides were: ERIDEYLVPIRGE (peptide 1) and QDPRGDSFFI (peptide 2). Ten of 13 consecutive amino acids of peptide 1 were identical to the amino acid sequence 319 to 330 of human FTCD (Fig. 3), a liver cytosolic protein recently identified as an autoantigen of LC1 reactivity.7 Peptide 2 showed only a modest degree of homology with human FTCD and no significant homology to other sequences in the data bank.

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the 29 sera reacted with the recombinant protein by Western immunoblotting (data not shown), indicating that the relevant epitopes were destroyed during denaturation before SDSPAGE. To determine whether FTCD is the major antigen recognized by LC1 antibodies, patient sera were presorbed with recombinant FTCD and then retested by immunofluorescence and Western immunoblotting. Five different LC1-positive sera were tested. After presorption with recombinant rat FTCD, but not after control presorption, the sera no longer gave the typical “zonation” pattern in immunofluorescence experiments on rat liver sections, and no longer reacted with a 58-kd human liver cytosolic protein in Western immunoblotting (data not shown). Distinct FTCD Epitopes Induce Autoimmune LC1 Antibodies.

FTCD is a bifunctional enzyme composed of 2 globular regions: an N-terminal FT domain consisting of amino acids 1 to

LC1-Positive Sera Recognize Conformational Epitopes on Rat FTCD. Rat liver sections are the usual substratum for LC1

detection by indirect immunofluorescence with a characteristic staining of periportal hepatocytes but limited staining of the hepatocyte layers around the central veins. In agreement, LC1-positive sera gave a diffuse cytosolic staining in most rat hepatocytes (Fig. 4). In addition, in some cells, punctate structures with morphology and localization consistent with Golgi complexes15 were seen. The positive reaction of human LC1-positive sera with rat tissue indicates that human LC1 antibodies recognize rat FTCD. We therefore cloned full-length rat FTCD and tested LC1-positive and control sera for anti-FTCD reactivity. Rat FTCD was expressed as a ␤-galactosidase fusion protein in bacteria. Coomassie blue–stained gel shows the induced protein as a dominant band of approximately 70 kd, consisting of 7 ␤-galactosidase residues at the NH2 terminus followed by the 541 amino acids coding for full-length rat FTCD, and 100 additional ␤-galactosidase residues at the COOH-terminus (Fig. 5, lane A). After transblotting to a nitrocellulose filter, a monoclonal anti-FTCD antibody reacts with the recombinant fusion protein, confirming its identity (Fig. 5, lane A⬘). All LC1 sera were tested against the recombinant rat FTCD fusion protein by immunodiffusion, immunoprecipitation, and Western immunoblotting. A precipitin line of identity was obtained with 22 (95%) of the 23 LC1-positive sera with type 2 autoimmune hepatitis (representative analyses are shown in Fig. 6). No precipitin lines were observed when sera were tested with E. coli lysates expressing ␤-galactosidase alone, confirming that LC1 antibodies react against the FTCD portion of the FTCD–␤-galactosidase fusion protein. Immunoprecipitation experiments with recombinant rat FTCD fusion protein showed that all 29 LC1-positive sera, including 1 negative by immunodiffusion, immunoprecipitated the recombinant polypeptide (representative analyses are shown in Fig. 7, lanes 1-4). Sera with isolated LKM1 and control sera did not immunoprecipitate FTCD (representative analyses are shown in Fig. 7, lanes 5-7). Interestingly, none of

FIG. 7. LC1-positive sera immunoprecipitate recombinant rat FTCD. Top panel: LC1-positive sera (lanes 1-4) and LC1-negative sera (lanes 5-7) were used to immunoprecipitate recombinant rat FTCD. The precipitates were analyzed by SDS-PAGE and a silver stained gel is shown. A 70-kd FTCD–␤galactosidase fusion protein was immunoprecipitated by LC1-positive sera (arrowheads), but not by LC1-negative sera. Bottom panel: gel analogous to that in top panel was transferred to nitrocellulose and Western blotted with monoclonal anti-FTCD antibodies. The immunoprecipitated protein in lanes 11-41 was recognized by the anti-FTCD antibody.

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FIG 8. Structural organization of FTCD. FTCD consists of 2 distinct domains: a globular N-terminal FT domain containing tetrahydrofolate binding site (THF binding) and histidine 82 (His82) believed to be part of the FT active site, and a globular C-terminal CD domain. The 2 regions are bridged by a linker sequence.

324 and a C-terminal CD domain composed of amino acids 334 to 541 (Fig. 8). The 2 domains are bridged by a 10 –amino acid linker sequence. To determine whether LC1 epitopes are clustered or span the entire FTCD molecule, we performed epitope mapping using only the FT or the CD portions of FTCD. Constructs encoding only the FT or the CD domain were engineered and tagged with histidine residues. The proteins were expressed in bacteria and purified to near homogeneity using nickel columns (Fig. 9). Purified proteins were used in ELISA. Twenty LC1-positive autoimmune sera were available for epitope mapping studies. By ELISA, 15 (75%) sera recognized the FT domain, and of those 8 (40%) also reacted with the CD domain, whereas the other 7 did not recognize the CD domain (Table 1). Significantly, there were no sera that recognized only the CD region. Five sera (25%) did not react with either construct. The crystal structure of the FT domain of FTCD has been recently solved.16 FT exists as a dimer, with an N-terminal region protruding from the dimer and the C-terminal region involved in dimer interface formation. To examine which region of FT contains LC1 epitopes, we constructed his-tagged N-terminal subdomain (amino acids 1-181) and his-tagged C-terminal subdomain (amino acids 176-339). The proteins were expressed in bacteria and purified to near homogeneity using nickel columns (Fig. 9). Purified proteins were tested in ELISA. A unique serum (patient 29) that recognized both FT and CD domains also reacted with FT1-181 (Table 1), indicating that this FTCD region contains at least 1 antigenic determinant.

by Western immunoblotting but recognized human FTCD in human liver cytosol using the same technique. The most likely explanation for the differential reactivity is that human and rat FTCDs refold with distinct kinetics after transfer from SDS-PAGE to nitrocellulose, and that the human protein refolds much better than the rat protein. It is likely that LC1positive sera react mainly with conformational epitopes of FTCD that are retained during techniques such as immunofluorescence, immunodiffusion, and immunoprecipitation, but are destroyed during the denaturing conditions of SDSPAGE. It appears that FTCD is the only relevant antigen involved in the LC1 reactivity because LC1-positive sera presorbed with recombinant rat FTCD no longer gave the typical immunofluorescence pattern and did not immunoblot the 58-kd FTCD band in human cytosol. To characterize the autoimmune response to FTCD, we analyzed which regions of the protein elicit LC1 autoreactivity. Based on obtained data, the whole FTCD molecule appears to be immunogenic, with the FT domain preferentially recognized by most LC1-positive patients. The majority of sera (⬎53%) recognized epitopes in both, the globular FT

DISCUSSION

To identify the target antigen of LC1 reactivity, we immunoprecipitated human liver cytosol with autoimmune LC1positive sera. This led to the isolation and the sequencing of a 58-kd protein that showed a high degree of amino acid homology with human, pig, and chicken FTCD. These results confirmed earlier findings by Lapierre et al.,7 and prompted us to search for anti-FTCD antibodies in sera from a number of patients with LC1-positive autoimmune hepatitis. Because the presence of LC1 reactivity is usually assessed by indirect immunofluorescence on rat liver sections, we used full length rat FTCD as the antigenic source for immunodiffusion, immunoprecipitation and Western immunoblotting experiments. All LC1-positive human sera but one gave a precipitin line of identity against rat FTCD by immunodiffusion and all immunoprecipitated rat FTCD. This was expected, based on the fact that the amino acid sequences of rat and human FTCD are highly homologous (Fig. 10), and that the predicted antigenic profiles of the 2 molecules appear comparable (Fig. 11). Surprisingly, all LC1-positive sera failed to react with rat FTCD

FIG. 9. Purified recombinant FT, CD, and FT fragments. Recombinant His-tagged constructs were expressed in bacteria and purified on nickel columns. Coomassie blue–stained gel shows a major 38-kd band of his-FT (lane B), a major 21-kd band of His-FT1-181 (lane C), a major 20-kd band of His-FT 176-339 (lane D), and a major 27-kd band of CD-His (lane E). Molecular weight markers (200, 116, 97, 66, 45, 31, 21, and 14 kd) are shown in lane A.

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FIG. 10. Sequence alignment of human and rat FTCD. MacVector software (Oxford Molecular Group, Oxford, UK) was used to align and compare human and rat FTCD. In the consensus sequence an asterisk (*) indicates identical amino acids, a dot ( 䡠 ) conserved substitutions. Eighty-five percent identity and 96% similarity is seen between the sequences.

(amino acids 1-339) and the globular CD (amino acids 332541) domains of the protein, indicating that the LC1 response is multiclonal. A smaller proportion (⬍47%) recognized only epitopes within the FT domain. We have mapped 1 antigenic epitope to an FT region spanning amino acids 1-181. Significantly, there were no cases in which only epitopes in the CD region were recognized, suggesting that FT epitopes might be the initial or the major trigger of the autoimmune response. The IgG class of such autoreactivity implies that the process is antigen driven and orchestrated by specific CD4⫹ T cells. Immunity against a single epitope may initiate and expand an autoimmune response against the whole molecule from which the epitope has been derived, a process known as “epitope spreading.17 Such a process has been described with

both T- and B-cell epitopes,18,19 and observed in animal models20 as well as in human autoimmune diseases.21,22 On the basis of the presented data, a B-cell epitope spreading process may be hypothesized also in the development of LC1 autoreactivity. It remains to be investigated how anti-FTCD reactivity arises, and if this event may have some relevance in the pathogenesis of the disease. The latter is suggested by the finding that global levels of circulating LC1 seem to parallel, at least biochemically, the occurrence of liver damage in LC1positive patients with autoimmune hepatitis.6 It is likely that there is a relationship between the B-cell (and possibly T-cell) epitope spreading process and the exacerbations and remissions of autoimmune liver disease.

FIG 11. Antigenicity profiles of human and rat FTCD. MacVector software (Oxford Molecular Group, Oxford, UK) was used to predict antigenic regions of rat and human FTCD. Highly similar overall pattern is evident.

HEPATOLOGY Vol. 34, No. 3, 2001

Although LC1 is only rarely detected in patients with concomitant hepatitis C virus infection,2-4 it will be interesting to see whether in such viral patients the epitopic specificities of LC1 antibodies are similar to those we observed in autoimmune patients. REFERENCES 1. Martini E, Abuaf N, Cavalli F, Durand V, Johanet C, Homberg JC. Antibody to liver cytosol (anti-LC1) in patients with autoimmune chronic active hepatitis type 2. HEPATOLOGY 1988;8:1662-1666. 2. Abuaf N, Johanet C, Chretien P, Martini E, Soulier E, Laperche S, Homberg JC. Characterization of the liver cytosol antigen type 1 reacting with autoantibodies in chronic active hepatitis. HEPATOLOGY 1992;16: 892-898. 3. Lenzi M, Manotti P, Muratori L, Cataleta M, Ballardini G, Cassani F, Bianchi FB. Liver cytosolic 1 antigen-antibody system in type 2 autoimmune hepatitis and hepatitis C virus infection. Gut 1995;36:749-754. 4. Muratori L, Cataleta M, Muratori P, Manotti P, Lenzi M, Cassani F, Bianchi FB. Detection of anti-liver cytosol antibody type 1 (anti-LC1) by immunodiffusion, counterimmunoelectrophoresis and immunoblotting: comparison of different techniques. J Immunol Methods 1995;187:259264. 5. Alvarez F, Berg P, Bianchi F, Bianchi L, Burroughs A, Cancado E, Chapman R, et al. International Autoimmune Hepatitis Group Report: review of criteria for diagnosis of autoimmune hepatitis. J Hepatol 1999;31:929938. 6. Muratori L, Cataleta M, Muratori P, Lenzi M, Bianchi FB. Liver/kidney microsomal antibody type 1 and liver cytosol antibody type 1 concentrations in type 2 autoimmune hepatitis. Gut 1998;42:721-726. 7. Lapierre P, Hajoui O, Homberg JC, Alvarez F. Formiminotransferase cyclodeaminase is an organ-specific autoantigen recognized by sera of patients with autoimmune hepatitis. Gastroenterology 1999;116:643-649. 8. Beaudet R, Mackenzie RE. Formiminotransferase cyclodeaminase from porcine liver. An octomeric enzyme containing bifunctional polypeptides. Biochim Biophys Acta 1976;453:151-161. 9. Gao Y, Alvarez C, Nelson DS, Sztul E. Molecular cloning, characterization, and dynamics of rat formiminotransferase cyclodeaminase, a golgiassociated 58-kDa protein. J Biol Chem 1998;273:33825-33834.

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