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Keywords:

  • autoantibodies;
  • asialoglycoprotein receptor;
  • autoimmune hepatitis;
  • antigenic sites

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

The aim of this study was to identify the epitopes recognized by antibodies to the asialoglycoprotein receptor, a specific hepatocyte protein, from sera of patients with autoimmune hepatitis. An ELISA test was used to detect anti-asialoglycoprotein receptor antibodies in the sera of patients with autoimmune hepatitis. Positive sera were tested against the same antigen by slot blot, by Western blot and by immunoprecipitation of the untreated protein and following treatment with β-mercaptoethanol (β-ME) and endoglycosidase F. The mature, unglycosylated and partially glycosylated forms of the asialoglycoprotein receptor synthesized by HepG2 cells were tested against positive patients' sera, as well as the in vitro translated unglycosylated form of the H1 subunit of the receptor. Sera from patients with autoimmune hepatitis recognized equally the native form, as well as the β-ME-modified form, but less well the deglycosylated form of the human mature receptor. No reactivity was found when these sera were tested against the denatured human protein. In addition, neither the unglycosylated H1 subunit nor any of the HepG2-synthesized asialoglycoprotein receptor forms bound to the antibodies. Altogether, these results show that anti-asialoglycoprotein receptor antibodies in the sera of patients with autoimmune hepatitis are directed against conformational structures of the mature hetero-oligomeric form of the human liver protein and that at least some epitopes were located on the extracellular domain of the antigen.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

Liver-specific antigens exposed on the hepatocyte cell surface may play a role in the pathogenesis of autoimmune hepatitis (AIH). The asialoglycoprotein receptor (ASGP-R) is an integral membrane protein of the sinusoidal domain of the hepatocyte plasma membrane. This hetero-oligomeric glycoprotein is mainly exposed on the extracellular side of the cell membrane and is readily accessible to circulating antibodies [ 1]. Autoantibodies against the ASGP-R were frequently found in sera from patients with different types of AIH [ 2[3]–4]. Higher titres of these antibodies were found before the beginning of immunosuppressive treatment [ 3] and they identified patients with a higher frequency of relapse after corticosteroid withdrawal [ 5]. Anti-ASGP-R antibodies were shown to be mainly bound to periportal hepatocytes when infused into the rat portal vein [ 6]. These findings might explain the predominance of periportal inflammation observed in patients with active AIH [ 6]. Altogether, these arguments lead to the speculation that anti-ASGP-R autoantibodies might play a role in the pathogenesis of the AIH.

While anti-ASGP-R antibodies have also been found in other liver diseases, such as acute and chronic viral hepatitis, reactivity to human ASGP-R was more specifically associated with AIH [ 2]. Although differences in epitope specificity may explain these differences in reactivity, the epitopes on the protein recognized by anti-ASGP-R antibodies from AIH patients, however, have not yet been identified. The study of the characteristics of the interaction between ASGP-R and autoantibodies by identification of the antigen epitopes might help to understand the generation of the autoimmune response. If these ASGP-R epitopes are located in the extracellular domain of the protein, a new argument may be developed to support the hypothesis that anti-ASGP-R antibodies might be responsible for the hepatocyte damage. The asialoglycoprotein receptor is composed of two subunits, designated H1 and H2. Each subunit is glycosylated and possesses intramolecular disulphide bridges which contribute to its conformational structure [ 1]. In order to identify the ASGP-R epitopes, antibodies recognizing the human ASGP-R were tested against the mature, the unglycosylated and partially glycosylated protein synthesized by normal human liver and by HepG2 cells. Asialofoetuin (ASF) was bound to the mature ASGP-R to test for modification of the putative antigenic sites by the binding of a specific ligand. The ASGP-R was treated by β-mercaptoethanol (β-ME) to disrupt intramolecular disulphide bridges that might be necessary for the constitution of an epitope. Finally, the unglycosylated H1 subunit of the receptor was synthesized in vitro and tested against the anti-ASGP-R autoantibodies.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

Purification of the ASGP-R

Human liver (50–100 mg) was obtained from the right lobe of a brain dead donor from whom only the left lobe was transplanted. ASGP-R was purified according to the protocol described by Hudgin et al. [ 7], with some modifications. An acetone powder was prepared by homogenization of liver tissue in cold acetone (4 ml/g of tissue). The powder was washed with 100 m M Tris–HCl pH 7.8, 200 m M NaCl and resuspended in 1% Triton X-100, and the pH was then adjusted to 7.8 with 1 M Tris–Cl and CaCl2 added to a final concentration of 0.02 M. The suspension was subjected to affinity chromatography using lactose-agarose beads [ 8]. ASGP-R was eluted using 40 m M ammonium acetate pH 6 buffer. The protein concentration was measured by the method described by Lowry et al. [ 9], and the purity of the protein assessed by SDS (10%)–PAGE.

Sera

Sera from 26 children with AIH type 1 and from 36 children with AIH type 2 were tested by ELISA for anti-ASGP-R antibodies. The sera were obtained before beginning immunosuppressive treatment in these patients. Criteria used for diagnosis of AIH were those described by the International Autoimmune Hepatitis Group [ 11]. Ten normal control sera were used to define non-specific binding. Two positive control sera were used; one was a rabbit polyclonal antibody against a peptide sequence of the C-terminal region of the H1 subunit of the ASGP-R (kindly provided by H. Lodish, MIT), and the other a human serum tested positive for anti-ASGP-R by solid-phase enzyme immunoassay elsewhere (kindly provided by M. Manns, Hannover, Germany).

ELISA test for anti-ASGP-R

Purified human ASGP-R was diluted to a final concentration of 1 μg/ml of PBS pH 7.4 and 100 μl of this solution were placed in each well of a 96-well polyvinyl plate and then dried overnight at 60°C. Blocking, incubations, washing and development were performed as described [ 12]. Sera dilutions tested were between 1:100 and 1:6400. The ELISA test was considered positive when the optical density (OD) measured was more than twice the mean value for OD of the 10 control sera. An individual serum titre was defined as the last dilution for which the value for OD was positive.

Slot blot and Western blot analysis

Purified ASGP-R was loaded on a 10% SDS–PAGE and then transferred to a nitrocellulose filter [ 13], or applied directly to a nitrocellulose paper using a slot blot apparatus. ASGP-R (1 μg) was loaded on each SDS–PAGE lane or on each well for slot blot. Blocking, incubations, washing and development were performed as previously described [ 14]. Dilutions used were of 1:100 for patients, normal sera and human positive control serum. Rabbit polyclonal antibody was used at a dilution of 1:1000.

HepG2 cell culture and labelling

HepG2 cells were obtained from ATCC (Rockville, MD), and maintained in minimum essential medium (MEM) containing Earle's salts, non-essential amino acids, glutamine, 10% fetal calf serum (FCS) and streptomycin/penicillin. Cultures were made in six-well plates and maintained at 37°C in an atmosphere with 5% CO2. For labelling, cells (≈ 1 × 106) were rinsed with PBS and incubated for 30 min at 37°C with MEM without cysteine. Later, this medium was replaced by fresh MEM with 200 μCi/ml of 35S-cysteine and cells were maintained at 37°C for another 30 min. Pulse–chase was made by replacing the radioactive medium by MEM with unlabelled cysteine at 500 times higher concentration than the radioactive amino acid. Incubation was continued for 90 min at 37°C. To avoid glycosylation of the ASGP-R, HepG2 cells were incubated with tunicamycin at a final concentration of 3 μg/ml in MEM, for 6 h before and during the pulse and chase periods [ 15]. To inhibit transport from the endoplasmic reticulum to the Golgi apparatus and maturation of the ASGP-R, brefeldin and monensin were used at concentrations of 18 μM and 2 μM, respectively. Incubations with these drugs were made for 1 h before and during pulse and chase periods [ 15]. At the end of these periods, cells were resuspended in 500 μl of the following buffer: 10 m M NaCl, 10 m M Tris–HCl pH 7.4, 1.5 m M MgCl2, 1% sodium deoxycholate, 1% Nonidet P-40. The ASGP-R was then immunoprecipitated from the suspension (see below).

125I labelling of purified ASGP-R

Preparation of the 125I-labelled ASGP-R was made by the chloramine-T method [ 10]. Specific activity of 125I-labelled ASGP-R was between 3.4 and 6.7 × 103 ct/min per μg of protein. The 125I-labelled ASGP-R was subjected to electrophoretic analysis in a 10% SDS–PAGE, to show the purity of the isolated protein ( Fig. 2).

image

Figure 2. and 5 μg) and other molecular weight markers (MW) were run as controls.

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In vitro transcription and translation of H1 subunit of ASGP-R

The cDNA in the pGEM coding for the H1 subunit of ASGP-R (kindly provided by H. Lodish) was transcribed and translated in vitro using the Riboprobe system (SP6) and rabbit reticulocyte lysate (nuclease-free) kits following the instructions from the manufacturer, Promega (Madison, WI). The translated protein was immunoprecipitated as described below. Yields of 3–6 μg of RNA/μg of linearized plasmid DNA were obtained. In vitro translation of 2 μg of ASGP-R subunit H1 RNA was made in presence of 10 mCi/ml of 35S-methionine.

Immunoprecipitation of ASGP-R

Immunoprecipitation of ASGP-R was made from 200 μl of HepG2 cell suspension and from 1.2 × 106 ct/min of the 125I-labelled protein, diluted with four volumes of 190 m M NaCl, 50 m M Tris–Cl pH 7.4, 6 m M EDTA and 2.5% Triton X-100. Rabbit serum (2 μl) and 10 μl of human serum were added to the immunoprecipitation test tube, and the samples were incubated at 4°C overnight. Sera used in the immunoprecipitation reaction were those positive by ELISA at titres between 1:400 and 1:800. Negative and positive controls were described previously. Immunocomplexes were precipitated by adding protein A Sepharose (20 μl of swollen beads) to the solution and incubating for 2 h at room temperature. The immunoprecipitate was analysed by 10% SDS–PAGE and fluorography. The film obtained was scanned in an UltroScan SL-Laser densitometer apparatus from Pharmacia-LKB (Bromma, Sweden), and the area under each peak compared between rabbit serum and sera from patients with AIH. The same procedure was used to immunoprecipitate the H1 subunit translated in vitro.

In order to determine whether occupation of the ASGP-R by the specific ligand ASFO modifies recognition of the receptor by antibody, immunoprecipitations were carried out with 0.1 μg of 125I-labelled or unlabelled ASFO. In other experiments modifications of ASGP-R were made prior to immunoprecipitation. Dissociation of disulphide bridges was achieved by incubating 0.1 μg of 125I-ASGP-R with 5 m Mβ-ME for 10 min at room temperature, followed by evaporation of free β-ME. Deglycosylated receptor was prepared by incubating 0.1 μg of ASGP-R with 2500 U of endoglycosidase F from New England Biolabs (Ontario, Canada) which cleaves between the innermost (Glc NAc) and asparagine residues of high mannose, hybrid and complex oligosaccharides from N-linked glycoproteins. In these experiments the ct/min from immunoprecipitated untreated or treated ASGP-R were measured in the gamma counter before loading on the electrophoresis gel.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

ELISA test

The ELISA test was positive in 65.4% and 44.5% of sera from children with AIH types 1 and 2, respectively. Mean titres of positive sera were 1:500 (range 1:100–1:1600) for AIH type 1 and 1:800 (range 1:100– > 1:6400) for AIH type 2. Rabbit serum titres were > 1:6400. The OD median (range) and mean ( ± s.d.) obtained with anti-ASGP-R+ sera from patients with AIH (types 1 and 2 combined) are represented in Fig. 1a,b, respectively. Individual ODs are shown in Fig. 1c.

image

Figure 1. 00. (a) Median (range). (b) Mean (± s.d.). (c) Individual measurements of optical density (OD) obtained.

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Slot blot and Western blot analysis

The purified ASGP-R showed a molecular mass of 46–48 kD in 10% SDS–PAGE ( Fig. 2). All of the sera positive by ELISA test recognized the native form of the protein when tested by slot blot ( Fig. 3). None of them, however, recognized the ASGP-R denatured form in Western blot analysis as carried out in our laboratory and at the dilutions used in this study ( Fig. 3).

image

Figure 3. . A representative result from Western blot and slot blot testing of purified human asialoglycoprotein receptor (ASGP-R) against sera from patients with autoimmune hepatitis (AIH). Rabbit serum recognizes the receptor in its native (slot blot) and its denatured (Western blot) forms; the AIH patients' sera were only positive in the slot blot test (this serum was previously tested positive by ELISA using the same antigen).

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Immunoprecipitation of the ASGP-R from HepG2 cells

None of the sera from patients with AIH immunoprecipitated the ASGP-R in its native form as synthesized by HepG2 cells ( Fig. 4). This negative result was observed when sera were tested against the mature protein (from untreated cells), the unglycosylated protein cells treated with tunicamycin, and the partially glycosylated forms (cells treated with brefeldin and monensin) (results not shown). These different forms of the ASGP-R, however, were immunoprecipitated by the rabbit polyclonal antibody against a linear epitope of the H1 subunit of the protein. The mature protein of 46 kD as well as a partially glycosylated form were immunoprecipitated from untreated cells. The unglycosylated form of the H1 subunit (34 kD) was immunoprecipitated from cells treated with tunicamycin. A partially glycosylated protein (40 kD) was immunoprecipitated from brefeldin- and monensin-treated cells. The bands observed when solubilized radioactively labelled untreated and drug-treated HepG2 proteins were immunoprecipitated by LKM1 or SMA were considered as non-specific because the same bands appeared whether anti-ASGP-R-positive or -negative sera were used. In addition, the band appearing at 38–40 kD in the LKM1+ samples was considered to be non-specific since this band remained unaltered after tunicamycin treatment and did not correspond with the bands immunoprecipitated by rabbit serum.

image

Figure 4. . Immunoprecipitation of 35S-cysteine-labelled asialoglycoprotein receptor (ASGP-R) synthesized by HepG2 cells. Rabbit antibodies immunoprecipitated the protein from untreated cells, as well as from cells treated by tunicamycin, brefeldin or monensin. None of the sera from patients with autoimmune hepatitis (AIH) immunoprecipitated either the mature or the unglycosylated receptor. Bands observed in lines obtained by immunoprecipitation with AIH patients' sera were considered non-specific.

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Immunoprecipitation of the H1 subunit

None of the sera from AIH patients, that recognized the ASGP-R in slot blot and ELISA tests, immunoprecipitated the H1 subunit of the receptor (results not shown).

Immunoprecipitation of 125I-labelled ASGP-R

All the sera from AIH patients immunoprecipitated the 125I-labelled ASGP-R purified from human liver in its native form ( Fig. 5). The relative amounts of the labelled ASGP-R immunoprecipitated by the rabbit antibody were greater than that immunoprecipitated by the sera from AIH patients. This result was also observed when a positive control serum from an adult patient with AIH was tested ( Fig. 4). Results of film scanning showed that between 6.6 and 33.3 times more 125I-ASGP-R was immunoprecipitated by each μl of rabbit serum than for each μl of anti-ASGP-R+ AIH patients' sera.

image

Figure 5. sera from an adult patient with autoimmune hepatitis (AIH) was kindly provided by Dr M. Manns).

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Occupation of ASGP-R by the specific ligand ASF did not alter the capacity of human antibody against the ASGP-R to recognize the antigen ( Fig. 6). Pretreatment of ASGP-R with β-ME decreased by half the amount of receptor immunoprecipitated by the rabbit anti-ASGP-R serum ( Fig. 7, lanes b and c), but slightly increased the amount immunoprecipitated by anti-ASGP-R serum from the AIH patient ( Fig. 7, lanes e and f).

image

Figure 6. . Autoantibodies against human liver asialoglycoprotein receptor (ASGP-R) are able to immunoprecipitate the protein, even when the binding site of the receptor is occupied by a specific ligand. 125I-labelled (lanes b and c) or unlabelled ASGP-R (lane a) was incubated with 125I-labelled (lanes a and c) or unlabelled asialofoetuin (ASF) (lane b). The complex was immunoprecipitated by anti-ASGP-R+ sera from patients with autoimmune hepatitis (AIH). 125I-labelled ASGP-R, without preincubation with ASF, and immunoprecipitated by serum of a patient with AIH, served as a control (lane d).

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image

Figure 7. . Untreated or treated 125I-asialoglycoprotein receptor (ASGP-R) was immunoprecipitated by rabbit (lanes b, c, d) or human (lanes e, f, g) antibodies. Lane a, 1.2 × 106 ct/min of ASGP-R, rabbit anti-ASGP-R immunoprecipitated; lane b, 1.2 × 105 ct/min of untreated protein; lane c, 0.5 × 105 ct/min after β-mercaptoethanol (β-ME) treatment; lane d, 0.5 × 105 ct/min after endoglycosidase F (Endo F) treatment; autoimmune hepatitis (AIH) patient serum immunoprecipitated; lane e, 2.1 × 104 ct/min of untreated ASGP-R; lane f, 2.9 × 104 ct/min of β-ME-treated protein; lane g, 1.2 × 104 ct/min endoglycosidase F-treated protein. Untreated ASGP-R and β-ME-treated ASGP-R denoted by open arrowhead; Endo F-treated by filled arrowhead. A high molecular weight band (○) was considered to be the partially deaggregated ASGP-R complex.

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In contrast, AIH patient serum immunoprecipitated a lower amount of labelled ASGP-R (1.2 × 104 ct/min) when it was treated with endoglycosidase F before incubation with the serum ( Fig. 7, lane g).

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

The anti-ASGP-R immune response in patients with AIH may explain the hepatocyte lysis observed in this disease, especially in cells located in the periportal region [ 6]. A more precise knowledge of ASGP-R epitopes could lead to a better understanding of the mechanisms responsible for the generation of this specific B cell response and may shed new light on the possible pathogenic role of anti-ASGP-R in the inflammatory destruction of the liver observed in patients with AIH.

An ELISA test was set up using well characterized negative and positive control sera. This test using purified human ASGP-R detected the presence of antibodies against this protein in the sera of patients with AIH types 1 and 2. These results were confirmed by slot blot analysis, where the protein was also tested in its native form. However, when both subunits of the protein were submitted to an electrophoresis in an SDS–PAGE and tested in denatured form by Western blot, no reactivity was observed with any of the sera, under the conditions used in this study ( Fig. 2). Subsequently sera were tested for reactivity against the native forms of each subunit and the mature form of the receptor. Two models were used: (i) the in vitro synthesis of the H1 subunit; and (ii) the protein forms at different stages of their biosynthetic pathway in HepG2 cells. In both models immunoprecipitation of radioactive labelled ASGP-R was performed due its greater sensitivity than Western blot analysis. The ASGP-R is a hetero-oligomeric molecule and its formation requires the association of the H1 and the H2 subunits [ 1, 16, 17]. The H1 subunit synthesized in human liver has a faster electrophoretic mobility than this subunit in HepG2 cells. Because the peptide sequence is the same in both cellular types, this difference is most probably due to the carbohydrate chains [ 16]. More importantly, the ratio of H1 and H2 subunits in the mature heteromeric ASGP-R is different in human liver and HepG2 cells [ 16]. In this study several methods were used to modify the structure of the receptor in order to determine which are the components critical for its recognition by circulating antibodies. The unglycosylated H1 subunit alone, synthesized in vitro from the cDNA, or the unglycosylated H1 and H2 subunits, synthesized by the HepG2 cells treated with tunicamycin, were not immunoprecipitated by anti-ASGP-R+ sera, showing that a partial or complete glycosylation may be necessary for the reaction of the anti-ASGP-R with the protein. In addition, none of the sera tested recognized the ASGP-R from HepG2 cells, either in its partially glycosylated form (brefeldin- and monensin-treated cells) or in its mature form, probably because of the characteristics of the carbohydrate chains. Although one might propose that the particular H1/H2 ratio in the ASGP-R from HepG2 cells modified putative conformational epitope(s), from our findings we speculate that the conformation of the heteromeric molecule and the carbohydrate chains could be part of conformational epitopes on the ASGP-R against which the autoimmune response has been directed.

The only proof from these results that AIH patients' sera react with the human ASGP-R were the ELISA and the slot blot tests. These techniques, however, do not definitively exclude a cross-reactivity against a non-specific contaminant undetected by methods used to determine the purity of the protein preparation. The ASGP-R preparation was labelled with 125I and analysed in a 10% SDS–PAGE ( Fig. 7). Rabbit anti-ASGP-R and AIH patients' sera immunoprecipitated a protein of the same size ( Fig. 5). The relative amount of 125I-labelled human liver ASGP-R immunoprecipitated by AIH patients' sera was lower than that immunoprecipitated by the rabbit serum. Higher titres of anti-ASGP-R antibodies in rabbit serum than in AIH patients' sera might explain these differences.

Having determined that: (i) anti-ASGP-R antibodies present in the sera of patients with AIH are mainly directed against conformational epitopes; (ii) that hetero-oligomeric complexes in the mature form are necessary for recognition of the ASGP-R by the autoantibodies; and (iii) that carbohydrate chains probably form part of the epitope site(s), further experiments were performed to confirm these results and to determine whether the ligand binding site forms part of the epitope and whether a modification of mature human ASGP-R conformational structure affects recognition by anti-ASGP-R antibodies. After binding of the specific ligand ASF, anti-ASGP-R antibodies still recognized the receptor, suggesting that most epitopes recognized by anti-ASGP-R antibodies are located outside the ligand binding site. Modification of conformational structure by β-ME treatment did not conclusively alter antibody recognition of the receptor ( Fig. 6). However, deglycosylation of the human mature receptor reduced the binding of ASGP-R antibodies ( Fig. 7g), suggesting that carbohydrate chains located in the extracellular domain of ASGP-R form at least part of the epitopes. The lack of a similar reduction following endoglycosidase treatment when testing with rabbit anti-ASGP-R serum is not surprising given that this antibody is directed to a peptide sequence in the C-terminal region of the ASGP-R.

In conclusion, this study shows that ASGP-R antibodies in sera of patients with AIH are directed against conformational epitopes of the mature hetero-oligomeric form of the human receptor and that at least some of the epitopes are located on the extracellular domain of the antigen and are readily accessible to circulating autoantibodies.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

This work was supported by an MRC grant to F.A. O.H. is the recipient of a fellowship from the Canadian Liver Foundation.

References

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References
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