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Original Studies |
FIRS Laboratories, RSR Ltd. (J.S., Y.O., S.R., A.K., T.R.,
J.B., V.M., S.W., J.F., B.R.S.), Parc Ty Glas, Llanishen, Cardiff CF4
5DU, Wales, United Kingdom; Department of Surgery (D.J.), Railway
District Hospital, Puszczykówko, 62-041 Pozna
,
Poland; Department of Medicine (Y.O., S.R., J.F., B.R.S.),
University of Wales College of Medicine, Heath Park, Cardiff CF4 4XN,
Wales, United Kingdom
Address correspondence and requests for reprints to: Dr. B. Rees Smith, FIRS Laboratories, RSR Ltd., Parc Ty Glas, Llanishen, Cardiff CF4 5DU, Wales, United Kingdom.
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Abstract |
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Introduction |
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There have been extensive studies attempting to localize the binding sites for TSH and TRAbs on the TSHR, but, to date, most of the studies have been inconclusive (3, 4). An ability to study the binding of TRAbs to the TSHR directly (rather than via inhibition of labeled TSH binding or stimulation of cyclic AMP) would be potentially useful in studying the TRAb-TSHR interaction in more detail.
Consequently, we have prepared a monoclonal antibody (MAb) to the C terminus of the TSHR, which is able to bind to the receptor at the same time as TSH or at the same time as TRAbs. This MAb has been labeled with 125I and then used to label the TSHR. We now describe a study of the interaction between the labeled receptor and TRAbs.
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Materials and Methods |
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Expression of the C-terminal end of the porcine TSHR in E. coli as a fusion protein with glutathione S-transferase (GST) was carried out, as described previously (5), using the 3' end of complementary DNA (1809–2295 bp) coding for the last 160 amino acids [77% homologous to the human TSHR C terminus (3)]. This was cloned in-frame with the GST fusion protein in pGEX2T vector (Pharmacia Biotech., St. Albans, UK) (5).
Monoclonal antibody preparation and characterization
Electroeluted pTSHR/GST protein was used to immunize BALB C mice (50 µg per mouse per injection) until the titre of antibody to the TSHR was high. The TSHR antibody level in mouse sera or culture supernatants were tested by immunoprecipitation assay (IPA) based on 35S-labeled TSHR (6). Hybridomas secreting TSHR monoclonal antibodies (MAbs) were prepared, and IgG and (Fab)2 preparations were made from hybridoma cultures, as described previously (5). In addition to the IPA based on 35S-labeled TSHR, the interaction of the TSHR MAbs with the TSHR was assessed by Western blotting and Scatchard analysis, as described previously (5). One MAb, designated 4E31 (which reacted strongly with the TSHR), was selected for further study.
TSHR preparations
Expression of full-length recombinant TSHR in CHO cells and detergent solubilization was as described previously (5), as was the preparation of native porcine solubilized TSHRs (7).
Assay of TSHR antibodies
Antibody activity was assessed in terms of inhibition of TSH binding (7) to porcine TSHRs (reagents from RSR Ltd., Cardiff, UK) and stimulation of cyclic AMP production in isolated porcine thyroid cells (8) (reagents from Yamasa Corporation, Tokyo, Japan). The ability of some sera to act as TSH antagonists (1, 2, 3, 4) by blocking TSH stimulation of cyclic AMP production was also assessed (8, 9).
Serum samples
Sera from patients attending the Department of Surgery and suspected of having Graves’ disease were used in the study. In addition, sera from 20 patients with a clinical diagnosis of Hashimoto’s thyroiditis with high levels of thyroglobulin and/or thyroid peroxidase autoantibodies, but negative for TRAb (all antibodies were tested using kits from RSR Ltd.) were also used. Further control sera included samples from 10 patients with systemic lupus erythematosus (SLE) positive for dsDNA autoantibodies and from 19 healthy blood donors. In addition, sera from five patients with autoimmune thyroiditis and high levels of TRAb with TSH antagonist activity were studied.
Binding of TSHR preparations to 4E31 MAb-coated tubes
4E31 IgG preparations [100 µL of 10 µg/mL in 0.1 M Na2CO3 (pH 9.2)] were coated onto plastic tubes [Maxisorp Star tubes; Nunc (Life Technologies, Inc., Paisley, UK)] by incubation overnight at 4°C. After washing and postcoating (10 mg/mL BSA), the tubes were washed again with assay buffer [10 mM Tris-HCl, 50 mM NaCl, and 1 mg/mL BSA (pH 7.4) containing 1% Triton X-100]. Then, 100 uL solubilized porcine TSHR preparations diluted in assay buffer were added, and the tubes were incubated for 15 min with gentle shaking at room temperature. Next, the TSHR preparations were removed, the tubes were washed with assay buffer, and 50 µL serum or other test material were added. After incubation (30 min) at room temperature with shaking, the tubes were washed again, and 100 µL 125I-TSH (20,000 cpm; RSR Ltd.) were added. The incubation was continued for 1 h, and the tubes were washed and counted for 125I.
Interaction of 125I-labeled MAb with TSHR
Aliquots of 125I-labeled 4E31 (Fab)2 (20 µCi/µg) diluted in assay buffer were incubated for 15 min at room temperature with solubilized porcine TSHR preparations. The 125I-4E31-TSHR complexes (labeled TSHR, 20 µL containing 20,000 cpm) were then incubated (1 h at room temperature) with test and control sera (20 µL), followed by the addition of solid-phase protein A (100 µL; RSR Ltd.). In some experiments, increasing amounts of unlabeled bovine TSH (from RSR Ltd.; 100 U/mg) in assay buffer were incubated with the labeled TSHR (final TSH concentration 0–200 µg/mL) for 15 min at room temperature before adding test and control sera.
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Results |
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IgG isolated from 4E31 MAb (50 µg of IgG) precipitated about 35% of 35S-TSHR (30,000 dpm) compared with 5% precipitation observed with a control MAb to glutamic acid decarboxylase. Furthermore, in Western blotting analysis (reducing conditions), 4E31 antibody (1–10 µg/mL) reacted with both the 100-kDa and the 120-kDa bands of the full-length TSHR, as observed for other TSHR MAbs (5) (data not shown). In addition, Western blotting analysis with GST fused to different segments of the TSHR showed that 4E31 recognized an epitope formed by amino acids 683–764 (data not shown).
Scatchard analysis of the interaction between 125I-labeled (Fab)2 and solubilized porcine TSHR gave an association constant of 5 x 109 molar-1.
Interaction of 125I-labeled TSH with TSHR immobilized on plastic tubes
In the absence of TRAb or unlabeled TSH, about 40% of the
125I-labeled TSH added (20,000 cpm added), bound to the
tubes containing immobilized receptor. This was reduced in a
dose-dependent manner in the presence of increasing amounts of TRAb or
unlabeled TSH. For example, thyroid-stimulating antibody first
international standard 90/672 (NIBSC, Hertfordshire, UK) at 5 mU/mL and
40 mU/mL gave 22 ± 2.5% and 66 ± 2.3% (mean ±
SD, n = 6) inhibition of TSH binding, respectively.
Furthermore, inhibition of labeled TSH binding to the immobilized
receptor by a series of 29 Graves’ sera correlated well with their
ability to inhibit labeled TSH binding to nonimmobilized receptor, as
measured by the polyethylene glycol precipitation method of Southgate
et al. (7) (Pearson’s coefficient, r = 0.97;
P < 0.001) (Fig. 1
). Studies on the
effect of sera from different groups of control patients on inhibition
of labeled TSH binding to immobilized TSHR showed that inhibition of
TSH binding ranged from -2% to +4% for healthy blood donors (n
= 17), from -3.5% to +3% for Hashimoto’s disease (n = 15), and
from -3.8% to 3% for SLE (n = 10).
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When mixtures of 125I-4E31 and TSHR were incubated
with TRAb-positive Graves’ sera, followed by precipitation with
protein A, the radioactivity of the precipitate was between 4% and
25% compared with the radioactivity precipitated by healthy blood
donors of 3–5% (Fig. 2). Fig. 2 also shows the results
of immunoprecipitation of labeled TSHR with Hashimoto sera and dsDNA
autoantibody-positive SLE sera.
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) (mean ± SD for the 49 control sera
= 117 ± 21). Of the 35 Graves’ sera, 31 (89%)
immunoprecipitated more labeled TSHR than any of the 49 control sera
(Fig. 2).
A comparison of the ability of 63 Graves’ sera to immunoprecipitate
labeled TSHR with the ability of the sera to inhibit labeled TSH
binding to detergent-solubilized TSHR is shown in Fig. 3
and Table 1. Overall, there was good agreement between
the two parameters with a Pearson’s correlation coefficient of r
= 0.92 (P < 0.001).
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and Table 1). Fig. 5 and Table 1 show
the relationship between the ability of the same 50 sera to inhibit
labeled TSH binding to the TSHR and to stimulate cyclic AMP production
in the bioassay (r = 0.76; P < 0.001).
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). All 12 of these clearly negative sera were
also negative in the cyclic AMP stimulation and IPAs. One of the sera,
borderline positive for inhibition of TSH binding (12% inhibition),
was positive for stimulation of cyclic AMP (285%) but negative in the
IPA. Twenty of the 50 sera were negative in the immunoprecipitation
assay (Table 1), but 5 of these negative sera were clearly positive by
inhibition of TSH binding assay and 2 more were borderline positive
(12% inhibition of TSH binding). Furthermore, five of the IPA-negative
sera were positive by cyclic AMP stimulation assay.
In terms of stimulation of cyclic AMP production, 20 of the 50 were
negative (Table 1), but 5 of these were positive by both inhibition of
TSH binding and IPAs.
Initial studies with 29 Graves’ sera that were able to
immunoprecipitate labeled TSHR showed that the immunoprecipitation
reaction could be inhibited by unlabeled TSH (final concentration, 200
µg/mL) in all 29 sera investigated. The effects of TSH were
dose-dependent (as shown in Fig. 6, and eight sera with
a range of TSH agonist activities and five sera with blocking (TSH
antagonist) activities were investigated in more detail. The
relationship between serum dilution and immunoprecipitation of labeled
receptor is shown in Fig. 7 and the effects of TSH on
immunoprecipitation in Fig. 8.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In addition to being able to immobilize the TSHR, 4E31 could be labeled with 125I and used to label the TSHR itself. It was then possible to immunoprecipitate the labeled receptor with Graves’ sera containing TRAbs. Comparison between the ability of the sera to immunoprecipitate labeled TSHR and to inhibit 125I-labeled TSH binding was quite closely correlated with an correlation coefficient value of 0.92. This is consistent with the TSHR antibodies present in the sera being directed principally to the same region of the TSHR as the TSH binding site. Alternatively, the close association between immunoprecipitation and inhibition of TSH binding results could be explained by the presence in each serum sample (at similar relative concentrations) of two distinct populations of autoantibodies, one population which interacted with the TSH-binding site and caused inhibition of TSH binding, and one population which was responsible for immunoprecipitation of the receptor. The availability of 125I-labeled TSHR enabled these alternatives to be investigated; in particular, it was possible to study the effects of TSH on the direct binding (as judged by immunoprecipitation) of autoantibodies to labeled TSHR. These investigations showed that TSH inhibited autoantibody binding essentially completely in almost all sera studied, indicating that the antibodies responsible for immunoprecipitation interacted with the TSH binding site. Consequently, the TRAbs in the sera we studied seemed to interact principally with the TSH binding site. This site is known to be formed by three-dimensional folding of the TSHR extracellular domain (3, 4), and the experimental methods we used were unable to distinguish between different stretches of amino acids within the TSH binding site. Our investigations showed that TSH inhibited TRAb direct binding to labeled TSHR whether the sera contained TRAbs with TSH agonist activity (i.e., stimulating antibodies) or TRAbs with TSH antagonist activity (i.e., blocking antibodies). This suggests that the differences between TRAbs with these different activities are quite subtle. Such subtle differences would be consistent with observations on the characteristics of receptor binding by more conventional G protein-linked receptor agonists and antagonists (11, 12, 13, 14). However, our results seem inconsistent with some studies (15) that suggest that autoantibodies with TSH agonist activity bind to different regions of the TSHR than those with TSH antagonist activity.
In particular, it has been proposed that the major functional epitopes for TSHR autoantibodies with TSH agonist activity are on the N-terminal segment of the TSHR extracellular domain, whereas TRAbs with TSH antagonist activity bind to the C-terminal part of this domain (for review, see Ref. 4). If this was the case (i.e., if the epitopes for the two types of autoantibodies were quite distinct), the competition experiments with TSH we describe would be expected to detect some differences between TRAbs with TSH agonist and with TSH antagonist activities.
However, the natural agonist TSH is able to inhibit the ability of TRAbs to bind to the TSHR irrespective of whether the autoantibodies act as TSH agonists or antagonists. Furthermore, both types of TRAbs inhibit TSH binding to the TSHR, and these two independent sets of observations provide compelling evidence that both types of autoantibodies bind to a region of the TSHR closely related to the TSH binding site.
It is difficult to reconcile the different conclusions of the previous studies (4, 15) and the current study, but our use of readily available porcine materials rather than human materials might possibly provide a part explanation. In addition, our immunoprecipitation studies would not be able to detect any TRAbs that might be directed to the intracellular region (amino acids 683–764) of the TSHR, although any such autoantibodies would not be expected to inhibit labeled TSH binding or stimulate isolated thyroid cells.
Our observation that immunoprecipitation of labeled TSHR by 1 of 13
sera shown in Fig. 8 (serum 11) was only partially inhibited by TSH may
reflect the presence of autoantibodies to sites on the TSHR distinct
from the region of the TSH binding site, together with autoantibodies
that bind to the same region as TSH. Alternatively, the partial
inhibition could reflect the presence of autoantibodies to the TSH
binding region, which bound with a particularly high affinity and, as
such, would be difficult to inhibit.
Comparison of inhibition of TSH binding, immunoprecipitation, and stimulation of cyclic AMP assays suggested that in the 50 sera studied in all three assays, the inhibition of TSH binding assay was the most sensitive way to detect TSHR antibodies. One serum however, which was positive by stimulation of cyclic AMP assay, was negative by IPA and only borderline positive (12% inhibition of TSH binding) by inhibition of TSH binding. Five of 50 sera were positive by IPA and inhibition of TSH binding assays, but negative by stimulation of cyclic AMP assay. This may have reflected: 1) the presence of a mixture of TRAbs with TSH agonist and with TSH antagonist activities in the sera; or 2) limitations of assay sensitivity; or 3) the presence of "isolated" receptor binding, TSH binding inhibition, or cyclic AMP stimulation activities (17).
The ability of Graves’ sera to immunoprecipitate labeled TSHR and stimulate cyclic AMP production in isolated thyroid cells correlated significantly (r = 0.58; n = 50). A closer correlation (r = 0.76) was observed when interaction of the same sera with the TSHR was measured in terms of inhibition of labeled TSH binding, rather than direct binding as judged by immunoprecipitation. Consequently, assessment of TRAbs by their ability to inhibit labeled TSH binding to the receptor tends to show better agreement with the stimulation of cyclic AMP assay than direct immunoprecipitation of labeled TSHR, at least with readily available porcine preparations.
Overall, our results indicate that TRAbs bind principally to the same region of the TSHR as TSH itself, and this seems to be the case whether the autoantibodies act as TSH agonists or antagonists.
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Acknowledgments |
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Footnotes |
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Received February 18, 1999.
Revised May 4, 1999.
Accepted July 2, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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;
2, 
; 3, +; 4, •; 5, 
; 6, *; 7, X; 8, 
) of TSH (200 µg/mL). See text and the
legends to Figs. 1