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The Journal of Clinical Endocrinology & Metabolism Vol. 84, No. 4 1391-1397
Copyright © 1999 by The Endocrine Society

Original Studies

Expression of the Functional Extracellular Domain of Human Thyrotropin Receptor Using a Vaccinia Virus System: Its Purification and Analysis of Autoantibody Binding1

Mi Hwa Lee, June Young Park, Bo Youn Cho and Chi-Bom Chae

Department of Life Science and Biotechnology Research Center, Pohang University of Science and Technology, Pohang 790–784; and the Department of Internal Medicine, Seoul National University College of Medicine (B.Y.C.), Seoul 110–744, Korea

Address all correspondence and requests for reprints to: Dr. Chi-Bom Chae, Department of Life Science and Biotechnology Research Center, Pohang University of Science and Technology, Pohang 790–784, Korea. E-mail: cbchae{at}postech.ac.kr


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
We produced substantial amount of the extracellular domain of the human TSH receptor (TSHRE) that has a tag of six histidines at C-terminus as a soluble form in the human cell line HeLa using a vaccinia virus system. By sequential nickel-chelating and lentil lectin column chromatography, TSHRE was purified to about 70% purity, with the recovery of around 0.1–0.2 mg TSHRE/L culture (5 x 108 cells/liter culture). The purified TSHRE interacted with TSH as well as Graves’ autoantibodies to TSHR. However, the affinity of TSHRE for TSH was much lower than that of intact TSHR. The IC50 value for inhibition of TSH-dependent cAMP synthesis by TSHRE was about 10-8 mol/L. Most importantly, the purified TSHRE inhibited the binding of the IgG of Graves’ patients to thyroid membrane. About 1 µg/mL (2 x 10-8 mol/L) TSHRE neutralized most of the autoantibody activity of patients’ sera tested in the TSH binding inhibitory immunoglobulin (TBII) assay. Moreover, this protein neutralized thyroid stimulatory antibody-induced cAMP synthesis with an IC50 of 1 x 10-9 mol/L and completely at 0.5–1 µg/mL (1–2 x 10-8 mol/L). In the simple enzyme-linked immunosorbent assay, the TSHRE immobilized on the wells coated with nickel showed significantly higher binding with the IgGs from Graves’ patients than in those from normal individuals. This autoantibody-reactive TSHRE will be useful for further studies on the diagnosis, pathogenesis, and the development of therapy of Graves’ disease.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
GRAVES’ disease is one of the most common diseases among autoimmune thyroid disease, caused by the continuous stimulation of TSH receptor (TSHR) by autoimmune antibodies (TSAbs) leading to hyperthyroidism (1, 2, 3). TSAbs are thought to be heterogeneous and have several different epitopes on the TSHR (3, 4, 5, 6). Based on many epitope-mapping studies, the epitopes of TSAbs appear to exist in the large extracellular domain of TSHR and are critically dependent on the discontinuous, three-dimensional conformational structure of TSHR (7, 8, 9, 10). To understand the pathogenesis and to develop convenient diagnostic and therapeutic methods for Graves’ disease, the availability of large amounts of functional TSHR is essential.

Since the complementary DNA of TSHR has been cloned, a large effort has been made to generate TSHR in many expression systems, including bacteria (11, 12, 13, 14), insect cells (15, 16, 17, 18), and mammalian cells (19, 20, 21, 22, 23, 24). The extracellular domain of TSH receptor (TSHRE) expressed in bacteria or insect cells seems to be mostly insoluble and have some problems, such as incomplete glycosylation, and there are conflicting results on the recognition of the receptor by TSAb. The expression of functional TSHR in mammalian cells has been achieved in the form of membrane-associated receptor (19, 20, 21, 22, 23, 24). Although TSHRE expressed stably in mammalian cells is functional in terms of TSH and autoantibody binding, the expression level has been just marginally higher than that of the TSHR on thyroid cells. Very recently, some groups produced a substantial amount of functional TSHRE, either as truncated forms (down to 261 residues from 418 residues) (25) or as the anchored forms on the cell surface by tagging with the glycosylphosphatidylinositol anchor (26) or by fusion with the cytoplasmic region of CD8 (27). The soluble form of TSHRE was released from the cell surface by cleaving the junction between TSHRE and CD8 with a protease (27).

In a previous report from our laboratory, we constructed a recombinant vaccinia virus containing the extracellular domain (amino acids 1–414) of human TSH receptor and expressed the receptor in HeLa cells (28). In this system, only about 50% of the receptor was in a soluble form, and it was difficult to purify the receptor reproducibly to an extent amenable for the detection of Graves’ autoantibodies by enzyme-linked immunosorbent assay (ELISA). Here we report a recombinant vaccinia virus system that allows the production of a substantial amount of TSHRE mostly as a soluble form, and also easy purification of TSHRE by affinity chromatography. Investigation of the biological activities of the TSHRE expressed in HeLa cells revealed that this recombinant protein maintains the biological activities in terms of interactions with TSH as well as Graves’ autoantibodies, although the affinity of TSHRE for TSH is much lower than that of intact TSHR on thyroid membranes. Most importantly, the purified TSHRE can neutralize the action of Graves’ autoantibodies. TSHRE immobilized on the wells coated with nickel showed a significantly higher level of interaction with the IgGs from Graves’ patients than with IgGs from normal individuals in the simple ELISA. Therefore, this autoantibody-reactive TSHRE will be useful for further studies on the diagnosis, pathogenesis, and development of therapy of Graves’ disease.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Generation of recombinant vaccinia virus

Recombinant vaccinia virus transfer vector pSC11-TSHRE was constructed by inserting the human TSHR gene corresponding to amino acids 1–414 into the SmaI site of vaccinia recombinant vector pSC11 (29). The TSHRE gene fragment was copied from the full-length human TSHR complementary DNA (from Dr. Kaxuo Tahara, University of Chiba, Chiba, Japan) by PCR with the following primers: forward primer, 5'-CGggatccATGAGGCCGGCGGAC-3'; and reverse primer, 5'-CGgaattcTTA(ATG)6agatctGTAGCCCATTATGTCTTC-3'. The reverse primer coded for amino acid 414, followed by six histidine codons for the histidine tag, and a stop codon.

To generate a recombinant vaccinia virus, African green monkey kidney (CV-1) cells were infected with wild-type vaccinia virus as previously described (29) and transfected with pSC11-TSHRE using Lipofectamine reagent (Life Technologies, Inc.). The virus resulting from the infection/transfection was harvested and selected as previously described (29). A recombinant vaccinia virus expressing TSHRE was selected by propagation in HuTK-143B cells in Eagle’s MEM containing 5-bromodeoxyuridine and X-Gal (5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside). Blue plaques showing ß-galactosidase activity were picked, and the resulting recombinant virus was amplified in HuTK-143B cells.

Expression and purification of TSHRE protein

HeLa S3 cells were grown in spinner MEM with 6% horse serum. A 1-L suspension culture of HeLa cells (5 x 108) was infected at a multiplicity of infection of 10 plaque-forming units/cell with the recombinant vaccinia virus encoding TSHRE and harvested at 24 h postinfection. Harvested cells were suspended in lysis buffer [20 mmol/L Tris (pH 7.4), 0.1 mol/L KCl, and 1 mmol/L phenylmethylsulfonylfluoride] and sonicated with a Branson sonifier (model 450; Branson Ultrasonic Corp., Danbury, CT). The supernatant fraction was loaded on a 5-mL nitrilotriacetic acid (Ni-NTA) column equilibrated with 20 mmol/L Tris (pH 7.4), 0.1 mol/L KCl, and 20 mmol/L imidazole. The bound proteins were eluted with buffer containing 20 mmol/L Tris (pH 7.4), 0.1 mol/L KCl, and 0.2 mol/L imidazole. Positive fractions, confirmed by immunoblotting, were pooled and applied to a 2-mL lentil lectin-Sepharose 4B column (Pharmacia Biotech, Piscatway, NJ) equilibrated with buffer containing 20 mmol/L Tris (pH 7.4) and 0.1 mol/L KCl. Elution was carried out with 0.2 mol/L {alpha}-methyl-D-mannoside in the same buffer. The protein samples were subjected to the immunoblot analysis to monitor TSHRE protein.

Immunoblotting and enzymatic deglycosylation of TSHRE protein

Proteins were separated on 8% SDS-PAGE and stained with Coomassie brilliant blue G250. For immunoblot analysis, proteins were transferred to nitrocellulose membrane and incubated with rabbit anti-His6 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or rabbit anti-TSHR peptide (amino acids 352–362) antibody (28). TSHRE was detected using the enhanced chemiluminescence system (Amersham).

For deglycosylation with N-glycosidase F (Boehringer Mannheim, Indianapolis, IN), purified TSHRE was denatured in 1% (wt/vol) SDS and 1% (vol/vol) ß-mercaptoethanol by placing it in boiling water for 3 min. After the addition of reaction buffer [20 mmol/L potassium phosphate, 10 mmol/L ethylenediamine tetraacetate (pH 7.4), 1% (wt/vol) Nonidet P-40, and 1% (vol/vol) ß-mercaptoethanol], the samples were incubated with N-glycosidase F at 37 C for 20 h. The proteins were subjected to SDS-PAGE. One set was stained with Coomassie brilliant blue G250, and the other set was subjected to immunoblot analysis.

Assay for the effect of TSHRE on the synthesis of cAMP induced by TSH

To test the effect of TSHRE on the action of TSH, 1 µU bovine TSH (Sigma Chemical Co., St. Louis, MO) was preincubated with various concentrations of purified TSHRE in 100 µL low sodium hypotonic Hanks’ Balanced Salt Solution containing 0.5 mmol/L 3-isobutyl-1-methylxanthine for 1 h at room temperature and then added to the CHO-TSHR cells (1 x 105) expressing human TSHR (6). After 2-h incubation at 37 C, 150 µL absolute ethanol were added to each well to extract the cAMP inside the cells in culture. The ethanol extract was evaporated to dryness in a Speed-Vac (Savant Instrument Co., Farmingdale, NY). The residues were dissolved in 150 µL 50 mmol/L Tris (pH 7.5) and 4 mmol/L ethylenediamine tetraacetate and assayed for cAMP using a commercially available RIA kit (TRK432, Amersham, Aylesbury, UK).

Assay for the effect on binding of [125I]TSH to thyroid membrane by antibody and TSHRE

The TSH binding inhibitory immunoglobulin (TBII) assay kits were purchased from RSR Ltd. (Pentwyn, Cardiff, UK). The principle of this assay is based on the ability of autoantibodies to compete for [125I]TSH binding to the TSHR solubilized from porcine thyroid membrane (30). To test the interaction of purified TSHRE with autoantibodies, we modified this assay by preincubating serum from Graves’ patients (20 µL) with purified TSHRE (20 µL) for 10 min at room temperature. Solubilized TSHR (25 µL) was then added to the serum/TSHRE mixture and incubated for 15 min, and 50 µL [125I]TSH (~5000 cpm) were added. The complex was precipitated by addition of 1 mL polyethylene glycol, and the precipitated pellet was counted for radioactivity in a {gamma}-scintillation counter.

To determine whether the interaction between [125I]TSH and TSHRE is significant at the concentration used in this assay, the binding of labeled TSH to TSHRE was tested indirectly using a competition assay. Increasing amounts of purified TSHRE protein were incubated with [125I]TSH (~5000 cpm/tube; RSR Ltd.) in 100 µL assay buffer (NaCl-free Hanks’ Balanced Salt Solution and 280 mmol/L sucrose). After incubation for 2 h at room temperature, 25 µL solubilized porcine TSHR (RSR Ltd.) were added, and the mixture was incubated for an additional 2 h at room temperature. Only the complex of [125I]TSH and the solubilized porcine TSHR was precipitated by addition of 1 mL polyethylene glycol, and the precipitated pellet was counted for radioactivity in a {gamma}-scintillation counter. The ability of TSHRE to bind TSH was determined as a percentage of [125I]TSH binding to the solubilized porcine TSHR.

Assay for the effect of TSHRE on the synthesis of cAMP induced by Graves’ autoantibodies

To test the interaction of purified TSHRE with TSAbs specific for Graves’ disease, 100 µg protein-A purified IgG from Graves’ patients were preincubated with the purified TSHRE in 100 µL low sodium, hypotonic HBSS containing 0.5 mmol/L 3-isobutyl-1-methylxanthine for 1 h at room temperature. After the incubation, the reaction mixtures were added to the CHO-TSHR cells (1 x 105) in culture and incubated for 3 h at 37 C. The cAMP synthesized was determined as described above.

ELISA: a simple method to assess the binding of TSHR autoantibodies to TSHRE

Ni-NTA HisSorb strips (Qiagen, Chatsworth, CA), whose inner surfaces are coated with a spacer bearing a Ni-NTA group, were coated with 100 ng purified TSHRE in 100 µL phosphate-buffered saline (PBS; pH 7.4) overnight at 4 C or for 3 h at room temperature. The wells were blocked for nonspecific binding sites with 5% (wt/vol) milk in PBS for 1 h at room temperature and treated with 100 µg/mL protein A-purified IgG from Graves’ patients in 100 µL blocking buffer for 2 h at room temperature. The wells were washed with PBS containing 0.05% Tween-20 (pH 7.4) between each step. The bound Ig was detected using goat anti-human Ig Fc conjugated with horseradish peroxidase. 2,2'-Azinobis-[3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt was used as a substrate, and the intensity of color developed was determined at 405 nm.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Expression and purification of TSHRE protein using the recombinant vaccinia virus

We constructed a recombinant vaccinia virus containing the extracellular domain (amino acids 1–414) and a six-histidine tag at the C-terminus for ease of purification by inserting the gene into the SmaI site of pSC11 transfer vector. A similar recombinant virus was constructed in a previous report that lacked the six-histidine tag in the TSHRE (28). When this virus was used for infection of HeLa cells, the TSHRE was found to be restricted inside the cell and present in roughly the same proportion in the soluble fraction and as a precipitate. Also, it was difficult to purify the receptor reproducibly.

To assess the expression of TSHRE from the new virus construct, the HeLa S3 cells infected with the recombinant vaccinia virus encoding TSHRE were subjected to SDS-PAGE and immunoblot analysis (Fig. 1Go). Unlike the previous report from our laboratory in which TSHRE without the histidine tag was found in both soluble as well as insoluble fractions in roughly the same proportion, most of the TSHRE with a six-histidine tag at the C-terminus was found in the soluble fraction with a molecular mass of around 63 kDa, as detected by means of anti-TSHR peptide (amino acids 352–362) antibody (28) and antihistidine tag antibody. The reason for the improved solubility of TSHRE is not clear at the present time. It is possible that the presence of the six-histidine tag at the C-terminus aided in the correct folding of the receptor.



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  		Figure 1. Expression of the extracellular region of TSHR in HeLa cells. HeLa cells infected with either recombinant or wild-type vaccinia virus were lysed in 20 mmol/L Tris (pH 7.4), 0.1 mol/L KCl, and 1 mmol/L phenylmethylsulfonylfluoride and centrifuged. The proteins in the supernatant and pellet were separated by SDS-PAGE and subjected to immunoblot analysis with rabbit anti-TSHR peptide (amino acids 352–362) antibody. TSHRE was detected using the enhanced chemiluminescence system (Amersham). Wild-type virus: pellet (1 ) and supernatant (3 ) of 5 x 104 cells. Recombinant virus: pellet (2 ) and supernatant (4 ) of 5 x 104 cells.

 
For further characterization, the TSHRE expressed in HeLa cells was purified by sequential nickel-chelating chromatography and lentil lectin chromatography. Even though a six-histidine tag is added to the carboxyl-terminus without any spacer, the recombinant TSHRE showed affinity for the Ni-NTA resin. The TSHRE from the Ni-NTA column showed approximately 30% purity. Because of the low purity, we employed an additional column, the lentil lectin column. As mentioned earlier, the expressed TSHRE is heavily glycosylated. Based on this property, we attempted to use a lentil lectin column. Lentil lectin binds reversibly to polysaccharides and glycoconjugates containing glucose or mannose type sugars (31). Therefore, we purified TSHRE by sequential nickel-chelating and lentil lectin chromatography. After two-column chromatography, the recovery of TSHRE was 0.1–0.2 mg from a 1-L suspension culture with 60–70% purity, and the purified TSHRE could be visualized by staining with Coomassie blue after SDS-PAGE (Fig. 2Go).



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  		Figure 2. SDS-PAGE and immunoblot of the purified recombinant TSHRE protein. A, Coomassie blue-stained gel; B, immunoblotting of the same fractions containing the same amounts of proteins as in A. Lane 1, Fractions after Ni-NTA column chromatography; lane 2, flow-through after lentil lectin column chromatography; lane 3, fractions eluted from a lentil lectin column. TSHRE proteins transferred from gel to nitrocellulose membrane were probed with rabbit anti-TSHR peptide (amino acids 325–362) using the enhanced chemiluminescence system.

 
Because the predicted molecular mass of TSHRE is around 46 kDa, the expressed TSHRE might be highly glycosylated. To confirm this, we attempted the enzymatic deglycosylation of the purified TSHRE. Deglycosylation of the purified TSHRE using N-glycosidase F, which cleaves high mannose and complex carbohydrate side-chains, caused a reduction in the molecular mass from 63 to 50 kDa, confirming the glycosylated state of the expressed TSHRE (Fig. 3Go).



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  		Figure 3. Enzymatic deglycosylation of the purified recombinant TSHRE protein. Purified TSHRE was treated with N-glycosidase F, and the proteins were analyzed by SDS-PAGE as described in Materials and Methods. Lane 1 shows the partially purified TSHRE before treatment with N-glycosidase F. Lane 3 shows TSHRE treated with N-glycosidase F. In lane 2, the sample was treated in the same way as the sample in lane 2, except no enzyme was added. A, Coomassie blue staining; B, immunoblotting probed with rabbit anti-TSHR peptide (amino acids 325–362).

 
Interaction of recombinant TSHRE with TSH

The binding of TSHRE to TSH was investigated by reduction of the TSH-dependent synthesis of cAMP in the presence of TSHRE. Various concentrations of purified TSHRE were preincubated with 1 µU bovine TSH, and the mixture was added to the culture wells containing CHO-TSHR cells. TSH-induced cAMP synthesis was reduced by TSHRE in a dose-dependent manner, with an IC50 of 1.5 x 10-8 mol/L (Fig. 4Go). About 10 µg/mL (2 x 10-7 mol/L) TSHRE could block TSH-induced cAMP synthesis almost completely. Therefore, it appears that the TSHRE expressed in HeLa cells by infection with the recombinant vaccinia virus can interact with TSH. However, the affinity was much lower than that of intact TSHR on the thyroid membrane (Kd, 10-10-10-11 mol/L) (32, 33). The complete structure of the receptor must be required for the high affinity binding of TSH to its receptor.



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  		Figure 4. Inhibition of the TSH-induced cAMP synthesis by TSHRE. One microunit of bovine TSH (bTSH) was preincubated with various concentrations of TSHRE in 100 µL and then added to CHO-TSHR cells in culture of 48-well plates. After 2-h incubation at 37 C, cAMP was determined as described in Materials and Methods. One microunit of bTSH produced 5.8 ± 0.5 pmol cAMP, and the background value was 1.4 ± 0.5 pmol. The IC50 for TSHRE was about 1.5 x 10-8 mol/L. Similar results were obtained in three independent experiments.

 
We also investigated the effect of TSHRE on the binding of labeled TSH to the solubilized thyroid membrane by incubation of TSHRE with [125I]TSH followed by addition of the solubilized thyroid membrane. The complex of TSH and the solubilized receptor was precipitated by the addition of polyethylene glycol. The TSHRE-[125I]TSH complex is not precipitated by polyethylene glycol. At a concentration below 5 µg/mL (1 x 10-7 mol/L) TSHRE, the interaction between TSHRE and [125I]TSH was not significant. This may seem paradoxical in view of the fact that 5 µg/mL TSH almost completely inhibited the synthesis of cAMP in response to 10 µU/mL TSH (Fig. 4Go). However, the concentration of radioactive TSH used in this binding assay was surprisingly about 100-fold higher (1 mU/mL). At 100 µg/mL, TSHRE inhibited the binding of labeled TSH to the soluble porcine thyroid membrane (Fig. 5Go). The IC50 value for the inhibition of binding of labeled TSH to the solubilized thyroid membrane by TSHRE was about 6 x 10-7 mol/L (30 µg/mL).



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  		Figure 5. Effect of TSHRE on the binding of [125I]TSH to thyroid membrane. Increasing amounts of the purified TSHRE were diluted in 50 µL binding buffer and preincubated with about 5000 cpm [125I]TSH in 50 µL, and the solubilized porcine thyroid membrane was added as described in Materials and Methods. The complex of [125I]TSH and thyroid membrane was precipitated with polyethylene glycol, and the radioactivity in the precipitate was determined. Only [125I]TSH, the solubilized porcine thyroid TSHR complex, was precipitated by the addition of polyethylene glycol. Approximately 2000 cpm [125I]TSH bound to the solubilized porcine thyroid membrane when no TSHRE was added, and there was a background value of about 100 cpm in the absence of the thyroid membrane.

 
There are contradictory data in the literature concerning the binding of TSH to the extracellular domain of TSHR in the absence of membranes (11, 14, 15, 16, 17, 26, 27). The TSHRE expressed in HeLa cells without the transmembrane region binds TSH. However, the binding affinity of TSH for TSHRE is much lower than that reported for the intact TSHR (32, 33), presumably due to the absence of the transmembrane domain. The idea that the extracellular domain alone can bind TSH is also supported by the recent report that the glycosylphosphatidylinositol-anchored TSHRE bound TSH with an affinity as high as that for the intact receptor (26). Further studies are still necessary to clarify the role of the transmembrane region and the extracellular loops in ligand binding.

Interaction of recombinant TSHRE with Graves’ autoantibodies

TSHRE as an autoantigen of several autoimmune thyroid diseases, including Graves’ disease, should be capable of binding with autoantibodies for its use in studies on Graves’ disease. The methods currently used for the detection of TSAbs are based on the stimulation of cAMP synthesis in thyroid cells or a RRA (TBII assay) in which the binding of [125I]TSH to detergent-solubilized porcine TSHR is inhibited by the antibody of patients with Graves’ disease. Here, we tested whether the TSHRE expressed in HeLa cells has functional activity in terms of binding to TSAb using these two methods.

To test the functional activity of TSHRE, we used a modified TBII assay in which the purified TSHRE was preincubated with patients’ sera before adding detergent-solubilized porcine thyroid membrane and labeled TSH. Because the interaction of labeled TSH and TSHRE was shown not to be significant at the concentrations of TSHRE used (Fig. 5Go), the recovery of the reduction of binding of labeled TSH with thyroid membrane in the presence of antibodies and TSHRE could be considered an indication of the interaction of TSHRE and autoantibodies. As the concentration of TSHRE was increased, the reduction of binding of labeled TSH to thyroid membrane by autoantibodies was recovered up to 100% (Fig. 6Go). Moreover, most of the patients’ sera tested showed similar results: TSHRE at 1 µg/mL (2 x 10-8 mol/L) neutralized the autoantibody activity in most of the Graves’ patients (Fig. 7Go). Therefore, the purified TSHRE was able to neutralize the binding activity of TSHR autoantibody to detergent-solubilized porcine TSHR.



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  		Figure 6. Neutralization of TSHR autoantibodies by TSHRE in a dose-dependent manner. TSHRE-autoantibody interaction was assessed using the TBII assay, in which the inhibition of TSH binding to thyroid membrane by autoantibodies from a Graves’ patient was reduced by the presence of various concentrations of the purified TSHRE. TSHRE (20 µL) was preincubated with patients’ sera (20 µL) for 10 min at room temperature, and then solubilized TSHR (25 µL) was added to the serum/TSHRE mixture. After 15-min incubation, 50 µL [125I]TSH were added and incubated for 2 h. The complex was precipitated by the addition of 1 mL polyethylene glycol, and the precipitated pellet was counted for radioactivity in a {gamma}-scintillation counter. One microgram per mL TSHRE corresponds to 2 x 10-8 mol/L. All samples were tested in duplicate. GD, Graves’ disease.

 


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  		Figure 7. Neutralization of TBII activity of IgG from several Graves’ patients by TSHRE. Serum from Graves’ patients and purified TSHRE were incubated and mixed with porcine solubilized thyroid membrane and [125I]TSH. The [125I]TSH-membrane complex was precipitated with polyethylene glycol, and radioactivity was determined as described in Materials and Methods. All samples were tested in duplicate, and the entire experiment was repeated with very similar results. HT, Hashimoto’s thyroiditis patient; N, normal individual.

 
Even though the TBII assay has been used as a clinical method to diagnose Graves’ disease, it cannot discriminate the presence of TSAbs from that of thyroid-stimulatory blocking antibodies, which only affect the binding of TSH to the TSHR and have epitopes different from those of TSAbs. To confirm the neutralization activity of TSHRE for TSAbs, we investigated the effect of TSHRE on the cAMP synthesis induced by IgG from Graves’ patients. Purified TSHRE was preincubated with IgG from patients and added to CHO-TSHR cells. The TSAb-induced synthesis of cAMP was reduced in the presence of TSHRE in a dose-dependent manner (Fig. 8Go), and the IC50 value at this condition was around 0.05 µg/mL (1 x 10-9 mol/L). In most of the cases we tested, 2 x 10-8 mol/L (1 µg/mL) TSHRE neutralized the TSAb activity of Graves’ patients completely (Fig. 9Go). In this experiment, the patients’ sera that showed a significant increase in cAMP were tested. Therefore, TSHRE expressed in HeLa cells by the recombinant vaccinia virus maintains the biological activities in terms of interactions with Graves’ autoantibodies.



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  		Figure 8. Inhibition of the TSAb-induced synthesis of cAMP by TSHRE. IgG of Graves’ patient 7 (1 mg/mL) in low sodium, hypotonic HBSS was preincubated with TSHRE before adding it to the CHO-TSHR cells in culture, and after 3-h incubation at 37 C, cAMP was determined as described in Materials and Methods. The amount of cAMP produced by 1 mg/mL IgG of GD7 was 4.5 ± 0.5 pmol, and the background value was 0.6 ± 0.3 pmol when no IgG was added. The IC50 for TSHRE was about 1 x 10-9 mol/L. All samples were tested in duplicate.

 


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  		Figure 9. Effect of TSHRE on the cAMP synthesis induced by IgG of several patients. The purified TSHRE (100 ng) was preincubated with 100 µg patients’ IgG in 100 µL and added to the CHO-TSHR cells in culture. After 3-h incubation at 37 C, cAMP was determined as described in Materials and Methods. All samples were tested in duplicate, and the entire experiment was repeated with very similar results.

 
ELISA as a simple method to assess the binding of TSHR autoantibodies to TSHRE

Direct interaction between TSHR autoantibodies and TSHRE, confirmed by a modified TBII assay and a cAMP measurement assay, led us to attempt ELISA for assessing the direct binding of TSHR autoantibodies to TSHRE in this simple assay. To reduce the nonspecific binding caused by other proteins present in TSHRE preparations, we used Ni-NTA HisSorb strips whose inner surfaces are coated with a spacer bearing a Ni-NTA group. Histidine-tagged TSHRE bound to the wells and showed the interactions with IgGs from Graves’ patients (Fig. 10Go). Twelve of 13 patients’ IgGs showed relatively higher binding activities with TSHRE than those IgGs from normal individuals and Hashimoto’s patients whose sera contains autoantibodies to thyroid peroxidase and thyroglobulin.



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  		Figure 10. Simple ELISA to assess the binding of TSHR autoantibodies to TSHRE. The purified TSHRE (100 ng) was added to the wells coated with a spacer bearing a Ni-NTA group, and 100 µL IgGs (100 µg/mL) were added to each well as described in Materials and Methods. The interaction between TSHRE and autoantibodies was detected by goat anti-human Ig Fc conjugated to horseradish peroxidase using 2,2'-azinobis-[3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt as a substrate. The same amount of BSA was used as a negative control to coat the wells. The data shown here are the average of three independent experiments.

 
The current diagnostic test used for the presence of Graves’ autoantibodies is the TBII assay, which is based on the inhibition of binding of labeled TSH to thyroid membrane by autoantibodies. This assay method requires the use of radiolabeled TSH. However, if a reliable ELISA method is available, direct binding of TSHR and autoantibodies can be assessed without using radioactive isotopes. Based on our ELISA data, it seems that this direct ELISA can be used for the diagnosis of autoimmune disease against TSHR without using radioisotope-labeled TSH.

In summary, we can produce a substantial amount of the extracellular domain of the TSH receptor using the recombinant vaccinia virus. The purified TSHRE is highly glycosylated and recognized by the autoantibodies for TSH receptor. The TSHRE will be useful for future investigations of mapping of the epitopes for TSHR autoantibodies and of the pathogenesis and development of diagnostic and therapeutic methods for Graves’ disease. Work is in process to increase the yield of TSHRE further by modifying the vaccinia virus system.


   Footnotes
 
1 This work was supported by grants from the Han (Highly Advanced National) Project of Ministry of Health and Welfare and Biotechnology 2000 Program of Ministry of Science and Technology, Korea. Back

Received July 27, 1998.

Revised November 11, 1998.

Accepted January 19, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

  1. Zakarija M, Mckenize JM. 1987 The spectrum and significance of autoantibodies reacting with the thyrotropin receptor. Endocrinol Metab Clin North Am. 16:343–363.[Medline]
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