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Thyrotropin (TSH) Receptor Autoantibodies Do Not Appear to Bind to the TSH Receptor Produced in an in Vitro Transcription/Translation System

FIRS Laboratories, RSR Ltd. (L.P., J.F.S., M.P., R.K., J.S., Y.O., J.F., B.R.S.), Llanishen, Cardiff, United Kingdom CF4 5DU; the Department of Surgery, Railway District Hospital, (D.J.), Poznan, Poland; and the Department of Medicine, University of Wales College of Medicine (M.P., R.K., J.S., Y.O., J.F., B.R.S.), Cardiff, United Kingdom

Address all correspondence and requests for reprints to: Dr. Bernard Rees Smith, FIRS Laboratories, Parc Ty Glas, Llanishen, Cardiff, United Kingdom CF4 5DU.

	Abstract

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An in vitro transcription/translation (TnT) system was used to produce ³⁵S-labeled full-length TSH receptor (TSHR) and TSHR extracellular domain (TSHRex). The interaction of the labeled proteins with TSHR autoantibodies in Graves’ sera was then studied using an immunoprecipitation assay. In the assay, ³⁵S-labeled TSHR or TSHRex were incubated with test sera, and any immune complexes formed were precipitated with protein A-Sepharose (in the case of mouse monoclonal antibodies, antimouse IgG-agarose was used). Rabbit antibodies to the TSHR and a mouse monoclonal antibody precipitated as much as 50% of the ³⁵S-labeled TSHR preparations compared with about 2% for normal rabbit serum and 4% for a control monoclonal antibody. However, none of 34 Graves’ sera (TSHR autoantibody levels ranging from 14–95% inhibition of [¹²⁵I]TSH binding) were able specifically to immunoprecipitate ³⁵S-labeled TSHR or TSHRex. These negative findings were confirmed by analysis of the immunoprecipitates on SDS-PAGE followed by autoradiography. Our results indicate that the TnT system is not useful for producing labeled TSHR preparations that can bind TSHR autoantibodies well. This is in contrast to TnT produced ³⁵S-labeled glutamic acid decarboxylase, thyroid peroxidase, and 21-hydroxylase, which react well with their respective autoantibodies. One main difference between these 3 autoantigens and the TSHR is that the receptor is highly glycosylated, and this extensive glycosylation may be of critical importance for correct folding of the receptor. Consequently, the inability of the TnT system to glycosylate proteins could explain in part why TnT-produced ³⁵S-labeled TSHR and TSHRex do not bind TSHR autoantibodies.

	Introduction

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GRAVES’ DISEASE is characterized by the presence of autoantibodies to the TSH receptor (TSHR) (1), and a number of methods are currently available to detect TSHR autoantibodies (TRAb). One of the most widely used is an inhibition assay, in which autoantibodies compete with ¹²⁵I-labeled TSH to bind to solubilized TSHR (1, 2). In addition, different bioassay systems have been developed in which the biological activity of TRAb can be measured in thyroid cell cultures (1). Also, recombinant TSHRs expressed in mammalian cells can be used successfully in [¹²⁵I]TSH binding inhibition assays and bioassays (1, 3, 4).

There is compelling evidence that TRAb and TSH interact with complex conformational binding sites on the TSHR (1, 5). Other autoantigens known to have conformational epitopes, such as glutamic acid decarboxylase (GAD₆₅) (6, 7), thyroid peroxidase (TPO) (8), and steroid 21-hydroxylase (21-OH) (9) can be produced labeled with ³⁵S in an in vitro transcription/translation (TnT) system, and the resulting proteins bind well to their respective autoantibodies in immunoprecipitation assays (IPAs). Furthermore, a recent report suggests that TnT-produced ³⁵S-labeled TSHR can bind TRAb (10), and we now describe our unsuccessful attempts to confirm this potentially important observation.

	Materials and Methods

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Serum samples

Serum samples were obtained from 34 Graves’ patients in whom disease diagnosis was based on clinical, immunological, and biochemical grounds. TRAb levels ranged from 14–95% of [¹²⁵I]TSH binding inhibition (assay reagents from RSR, Cardiff, UK). Normal pooled sera were obtained by pooling samples from 20 healthy blood donors. A rabbit polyclonal antibody and a mouse monoclonal antibody raised to an 800-bp extracellular fragment of TSHR complementary DNA (cDNA) expressed as a fusion protein with glutathione-S-transferase in Escherichia coli (RSR) were used in each assay as positive controls (11).

Construction of pTSHR and pTSHRex

cDNA coding for the full-length (TSHR) was placed under the control of the T7 promoter in plasmid pYES2 (Invitrogen, N. V. Leek, Netherlands). cDNA coding for the extracellular fragment of TSHR (TSHRex) starting from amino acid 1 up to amino acid 415 (12) was also cloned into pYES2.

TSHR preparations

TnT (Promega, Southampton, UK) of full-length TSHR and the TSHRex, either unlabeled or labeled with [³⁵S]methionine, was carried out as described previously (9). The TnT reaction mixture was passed through a 0.5 x 16-cm column of Sephadex G-50 (Pharmacia, Biotech, St. Albans, UK) in 150 mmol/L Tris buffer (pH 8.3), 200 mmol/L NaCl, and 10 g/L BSA [hereafter called high salt buffer (HSB)] containing 10 mL/L Tween-20. The unretarded fractions were collected, pooled, and tested for reactivity with TSHR antibodies. In some experiments, the TnT reaction of full-length TSHR was also carried out in the presence of dog pancreatic microsomal membranes (5 µL) as an aid to posttranslational processing of the protein (Promega).

The labeled translated proteins were analyzed by SDS-PAGE on 7% or 9% gels and visualized by autoradiography.

Solubilized native human TSHRs were obtained from human thyroid tissue as described previously (2), using 1% Triton X-100 in 10 mmol/L Tris-HCl (pH 7.5) and 50 mmol/L NaCl. Solubilized recombinant human TSHR were obtained in a similar way from the membranes of Chinese hamster ovary (CHO)-K1 cells, expressing about 4 x 10⁵ receptors/cell (13).

IPA for TRAb

The IPA was carried out in a 96-well filtration plate system, as described previously (9). Briefly, ³⁵S-labeled TSHR or TSHRex (30,000 dpm in 50 µL) were incubated with duplicate aliquots of serum diluted in HSB containing 1% Triton X-100 for 2 h at room temperature. The ³⁵S-labeled antigen-antibody complexes were precipitated with either protein A-Sepharose (Pharmacia; in the case of human and rabbit antibodies to the TSHR) or anti-mouse IgG-agarose (Sigma Chemical Co., Poole, UK; in the case of mouse monoclonal antibody to the TSHR) for 1 h at room temperature. After filtration and washing, the radioactivity of the pellets was counted in a scintillation counter (Packard, Pangebourne, UK).

Similar experiments were performed in which HSB was replaced by 1) low salt buffer [20 mmol/L NaCl, 150 mmol/L Tris (pH 8.3), and 10 mg/mL BSA], 2) HSB diluted 1:10 in water (dilute HSB), and 3) assay buffer [50 mmol/L NaCl, 10 mmol/L Tris (pH 7.5), and 1 mg/mL BSA] containing either 1% Tween-20 or 1% Triton X-100.

Inhibition assays were carried out as described for the IPA, except for a preincubation step in which antibody was incubated with 50-µL serial dilutions (in HSB and 1% Triton X-100) of nonlabeled native or nonlabeled recombinant TSHR preparations.

Analysis of immunoprecipitates by SDS-PAGE

An IPA was carried out as described above, except that it was performed in 1.5-mL tubes. The antigen-antibody complexes were precipitated with solid phase protein A (RSR). After washing, the pellets were resuspended in sample buffer [4% SDS, 20% glycerol, 100 mmol/L Tris-HCl (pH 6.8), and 0.002% bromophenol blue] plus dithiothreitol (10 mmol/L) and heated at 100 C for 3 min. After centrifugation, the supernatants were loaded onto 7% or 9% gels, and electrophoresis was carried out as described by Laemmli (14). After electrophoresis, the gels were stained with Coomassie brilliant blue (Sigma), destained, and prepared for autoradiography.

TSH binding assay

Fifty microliters of nonlabeled TnT produced TSHR preparations were incubated with [¹²⁵I]TSH (30,000 cpm in 100 µL; RSR) with or without the addition of unlabeled TSH (1 mU in 100 µL) for 1 h at 37 C, followed by precipitation with polyethylene glycol and normal pool serum. After centrifugation at 2,400 rpm for 30 min at 4 C and aspiration, the radioactivity of the pellets was counted in a -counter. Native porcine TSHR (RSR) preparations were used as controls.

	Results

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Production of TSHR in the TnT system

Analysis by SDS-PAGE of the ³⁵S-labeled TnT reaction products obtained with TSHR cDNA and TSHRex cDNA is shown in Fig. 1. Two major ³⁵S-labeled protein bands were obtained with full-length TSHR cDNA. Immunoprecipitation of the reaction mixtures with monoclonal and polyclonal antibodies to the TSHR followed by analysis of the precipitates on SDS-PAGE showed that the upper band (mean molecular mass ± SD, 82.1 ± 6.7 kDa; n = 9; determined using 7% acrylamide gels) was ³⁵S-labeled TSHR (data not shown). TSHRex cDNA gave a single major band of 52.3 ± 2.6 kDa (n = 9; determined using 9% acrylamide gels; Fig. 1), and immunoprecipitation with monoclonal and polyclonal antibodies to TSHR showed that this band was TSHRex (Fig. 2).

View larger version (76K): [in this window] [in a new window]   Figure 1. Analysis by SDS-PAGE (9%) and autoradiography of TSHR proteins produced labeled with [35S]methionine in in vitro transcription/translation reactions. Lane 1, Products from reaction using full-length TSHR cDNA; lane 2, products from reaction using cDNA coding for TSHRex; lane 3, material from control reaction (plasmid not containing cDNA). Of the two protein bands in lane 1 (82 and 67 kDa), the upper band was identified as representing TSHR, and the 52-kDa protein band in lane 2 was identified as TSHRex by immunoprecipitation with monoclonal and polyclonal antibodies.

View larger version (100K): [in this window] [in a new window]   Figure 2. Analysis by SDS-PAGE (9%) and autoradiography of immunoprecipitates of 35S-labeled TSHRex. Lanes 1 and 2, Immunoprecipitation with rabbit antibody diluted 1:10 and 1:100, respectively. Lane 3, Immunoprecipitation with normal rabbit serum diluted 1:10. Lanes 4–8, Immunoprecipitation with five different Graves’ sera (percentage of [125I]TSH binding inhibition ranged from 15–95%) diluted 1:10. Lanes 9–11, Immunoprecipitation with sera from three different healthy blood donors diluted 1:10. Lanes 12 and 13, Immunoprecipitation with TSHR monoclonal antibody diluted 1:100 and 1:1000, respectively. See text for experimental details.

The rabbit TSHR polyclonal antibody precipitated 46% of the full-length [³⁵S]TSHR preparation at a 1:10 dilution of antibody and 29% at a 1:100 dilution (Table 1). This can be compared to normal rabbit serum, which bound only 2% of the [³⁵S]TSHR (Table 1). Binding of rabbit antibody to the preparation of the ³⁵S-labeled TSHRex was 27% at a 1:10 dilution and 25% at a 1:100 dilution. The mouse monoclonal antibody bound 22% of the ³⁵S-labeled full-length TSHR at a 1:100 dilution and 20% of the [³⁵S]TSHRex at the same dilution.

Various assay conditions were tested, including different buffer components, incubation times, and incubation temperatures.

The TSHR rabbit antibody diluted 1:10 bound about 50% of the ³⁵S-labeled full-length TSHR preparations in all buffers studied (Table 2). The mean binding of 10 Graves’ sera for the different buffers used ranged from 0.4–2.6% compared to 0.6–3.1% for healthy blood donor sera (Table 2).

Analysis of immunoprecipitates by SDS-PAGE

Figure 2 shows analysis of [³⁵S]TSHRex immunoprecipitates by SDS-PAGE and autoradiography. Rabbit antibody to the TSHR and monoclonal antibody to the receptor clearly immunoprecipitated TSHRex (band at 52 kDa), but TRAb-positive Graves’ sera showed the same negative result as healthy blood donor sera.

Inhibition IPA

The specificity of rabbit antibody binding to TSHR produced in the TnT system was analyzed by an inhibition assay using solubilized TSHR preparations isolated from human thyroid tissue or CHO cells expressing full-length recombinant TSHR. Binding of both full-length ³⁵S-labeled TSHR and ³⁵S-labeled TSHRex to rabbit antibody was inhibited by unlabeled recombinant (Fig. 3) and native (data not shown) TSHR.

View larger version (15K): [in this window] [in a new window]   Figure 3. Inhibition of rabbit antibody binding to 35S-labeled TSHR (a) and TSHRex (b) by unlabeled recombinant human TSHR. , Recombinant TSHR expressed in CHO cells solubilized in 1% Triton X-100; , nontransfected CHO cell membranes solubilized in 1% Triton X-100. In the experiments using receptor undiluted, approximately 100 ng receptor were added to each reaction tube.

[¹²⁵I]TSH binding to unlabeled TSHR produced in the TnT system was analyzed. TSHR preparations were incubated with [¹²⁵I]TSH with or without the addition of unlabeled TSH. Binding of [¹²⁵I]TSH to both full-length TSHR and TSHRex was approximately 4% in the absence of unlabeled TSH and approximately 3.5% in the presence of unlabeled TSH (1 mU; data not shown). [¹²⁵I]TSH binding to the control porcine TSHR was about 30% in the absence and 9% in the presence of unlabeled TSH (1 mU), respectively (data not shown).

	Discussion

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Our studies show that both TSHRex and the full-length receptor can be produced labeled with ³⁵S in the TnT system, as reported by Morgenthaler et al. (10).

When analyzed by SDS-PAGE, full-length TSHR ran as a band of 82 kDa, and the extracellular domain ran as a band of 52 kDa. These molecular masses are consistent with the expected nonglycosylated peptide chain molecular masses of full-length TSHR (85 kDa) and TSHRex (50 kDa) (1, 12, 13, 15, 16), as glycosylation of proteins does not usually occur in the TnT system (17). The [³⁵S]TSHR preparations produced in the TnT system bound our rabbit polyclonal TSHR antibody and mouse monoclonal TSHR antibody well (up to 50%) when analyzed by immunoprecipitation assay. This binding was inhibited by the addition of solubilized native and recombinant TSHR, emphasizing the specificity of the reaction.

None of the 34 Graves’ sera we studied specifically immunoprecipitated ³⁵S-labeled TSHR or TSHRex preparations produced in the TnT system despite changing assay conditions, such as different buffer components, incubation times, and temperature. Li et al. (18) used the addition of canine pancreatic microsomes to the TnT system to improve the production of autoantibody-reactive calcium-sensing receptor, and we tried this approach for the TSHR. However, the addition of these microsomes to the TSHR TnT reaction did not result in a preparation that could be immunoprecipitated by Graves’ sera. However, it should be noted that the overall molecular masses of TSHRs produced in the absence and/or presence of microsomal preparations were not different. This could indicate that more complex posttranslational modifications (including glycosylation) were not induced by the microsomes.

Binding of TSHR autoantibodies to the TSHR is known to be dependent on the correct conformational folding of the receptor (1, 19, 20, 21), and the difficulty in demonstrating TRAb binding to TnT-produced TSHR may reflect in part the incorrectly folded nature of the receptor produced in this system. In particular, the inability of the TnT reaction to glycosylate the TSHR may be of critical importance. For example, the ability of TnT-produced GAD₆₅, 21-OH, and TPO to react well with their respective autoantibodies indicates that glycosylation is not important in forming the conformationally dependent autoantibody-binding sites on these three proteins (6, 7, 8, 9). However, the TSHR is heavily glycosylated by comparison with GAD₆₅, 21-OH, and TPO, and the lack of sugar residues may be responsible in part for the inability of the TnT-produced receptor to form a correctly folded TRAb-binding site (20, 22, 23).

We were also unable to show binding of ¹²⁵I-labeled TSH to unlabeled TnT-produced TSHR, and Morgenthaler et al. (10) reported similar results. As the TSH- and TRAb-binding sites on the TSHR are closely related and similarly complex, the inability of TnT-produced TSHR to bind TSH is probably also related to incorrect folding of the receptor, although production of insufficient amounts of receptor could also be an explanation.

Our observations on Graves’ sera are in distinct contrast to the recent study of Morgenthaler et al. (10), which reported that about 70% of Graves’ sera could immunoprecipitate TnT-produced ³⁵S-labeled TSHR and TSHRex. The reasons for this important discrepancy are not clear at present. There are some minor differences between the procedures used in the two studies. For example, in the case of the 50-kDa TSHRex, we used cDNA containing the signal sequence, whereas Morgenthaler et al. used cDNA without the signal sequence. However, this difference could not explain our inability to detect binding to TSHR autoantibodies, as cDNA with the signal sequence was used for the production of full-length TSHR in both studies.

The same in vitro transcription/translation system reagents were also used (supplied by Promega), and the expected amounts of recombinant proteins are in the range of 150–500 ng/50 µL reaction (as detailed in the Promega kit instructions). Monoclonal and rabbit antibodies to the TSHR immunoprecipitated TnT produced ³⁵S-labeled TSHR and TSHRex in both studies, indicating that effective production of the recombinant proteins occurred. It should be noted, however, that in Morgenthaler’s study (10), the appropriate specificity experiments were not carried out. In particular, inhibition studies in which Graves’ sera were preincubated with unlabeled native and/or recombinant TSHR before interaction with labeled TSHR or TSHRex. Consequently, evidence to date suggests that ³⁵S-labeled TSHR preparations produced in the TnT system do not bind well to TSHR autoantibodies.

	Footnotes

 
¹ Recipient of an RSR Fellowship.

² Supported by a grant from the Ministry of Education, Science, and Culture, Japan.

Received November 7, 1996.

Revised December 18, 1996.

Accepted January 1, 1997.

	References

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