help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Akamizu, T.
Right arrow Articles by Nakao, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Akamizu, T.
Right arrow Articles by Nakao, K.
Endocrinology Vol. 140, No. 4 1594-1601
Copyright © 1999 by The Endocrine Society

ARTICLES

Characterization of Recombinant Monoclonal Antithyrotropin Receptor Antibodies (TSHRAbs) Derived from Lymphocytes of Patients with Graves’ Disease: Epitope and Binding Study of Two Stimulatory TSHRAbs1

Takashi Akamizu, Kenji Moriyama, Masako Miura, Misa Saijo, Fumihiko Matsuda and Kazuwa Nakao

Department of Medicine and Clinical Science (T.A., K.M., M.M., M.S., K.N.) and Department of Medical Chemistry (F.M.), Kyoto University Graduate School of Medicine, Kyoto 606-8507, Japan

Address all correspondence and requests for reprints to: Takashi Akamizu, Department of Medicine and Clinical Science, Kyoto University School of Medicine, 54 Shogoin-Kawaharacho, Sakyo-ku, Kyoto 606-8507, Japan. E-mail: akataka{at}kuhp.kyoto-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Anti-TSH receptor autoantibodies (TSHRAbs) are known to be involved in Graves’ disease. To elucidate the molecular mechanism of the pathogenesis of Graves’ disease, we previously isolated and reconstituted the Ig genes of two B cell clones (101–2 and B6B7) producing a monoclonal thyroid-stimulating antibody (TSAb), a stimulating type of TSHRAb, obtained from patients with Graves’ disease. In the present study, we produced a large amount of recombinant monoclonal TSAbs in eukariotic cells using these genes and characterized them. First, we tried to identify their epitopes in the TSHR, by using a panel of mutants of the extracellular domain of the TSH receptor (TSHR). Substantial cell surface expression level of each mutant was confirmed by fluorescence-activated cell sorter analysis using a TSHRAb. Mutations in the N-terminal (but not C-terminal) region of the extracellular domain of TSHR abrogated or reduced TSAb activities of both antibodies, whereas they had opposite effects on TSH activity; cAMP generation by 101–2 significantly decreased in the receptors mutated in amino acids 52–56 and 58–61, and that by B6B7 decreased in amino acids 34–37 and 58–61. Secondly, purified antibodies were radiolabeled and tested for binding to cells expressing high levels of TSHR. Although their affinities were lower than that of TSH, their binding was not displaced by TSH. The antibody binding was not mutually competitive. These findings suggest that these antibodies interact with the N-terminal region of the receptor and transduce a signal through binding sites different from TSH. We believe that this is the first report of the characterization of human monoclonal TSHRAbs on their epitopes and bindings, confirming previous reports using patient sera or murine monoclonal antibodies.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
ANTI-TSH RECEPTOR antibodies (TSHRAb) are involved in the pathogenesis of autoimmune thyroid diseases, Graves’ disease (1, 2) and idiopathic myxedema (3), and have been shown to be responsible for hyperthyroidism in the former disorder and for hypothyroidism in the latter (4). The contrasting expressions of morbid states involving TSHRAbs are based upon their functional heterogeneity: autoantibodies that can mimic TSH actions and stimulate thyroid cells are called thyroid-stimulating antibodies (TSAbs), whereas those that block TSH actions are called TSH receptor blocking antibodies (TSBAbs). Antibodies that inhibit TSH binding to the receptor are called TSH-binding inhibitor Igs (4, 5). To understand the mechanism of the heterogeneity, it is essential to study the interactions between the TSH receptor (TSHR) and the TSHRAb at the molecular level. Cloning of the TSHR gene (5, 6, 7) and the TSHRAb genes (8, 9, 10) has allowed us to study this problem. Indeed, molecular cloning of the TSHR gene provided a breakthrough in the elucidation of the binding sites of TSH and TSHRAb on the TSHR (5, 7). TSAb epitopes exist in the N-terminus of the extracellular domain, and TSBAb epitopes in the C-terminus (4, 5, 7). In addition, there is a possibility that the TSAb and TSBAb epitopes may be in close proximity within the proposed tertiary structure of TSHR (5, 7). Both regions are thought to be involved in TSH binding: TSH seems to bind to a broad area or multiple discontinuous sites of the extracellular domain. These findings obviously demonstrated multiple TSHRAb-binding epitopes, explaining why TSAb and TSBAb have different functions. However, because polyclonal antibodies derived from patient sera that may contain heterogeneous TSHRAbs were used in these studies, the results varied among sera and were not conclusive. To overcome this problem, monoclonal antibodies are needed. Several human monoclonal TSHRAbs have been reported (11, 12, 13, 14), but their epitopes have not been clarified. Although mouse monoclonal antibodies have been generated and their epitopes have been analyzed (15, 16, 17), their epitopes may not be precisely the same as naturally occurring autoantibodies (18). Recently, we tried to isolate and reconstitute variable region complementary DNAs (cDNAs) of both heavy and light chains of IgM and IgG TSHRAbs from several B cell clones producing monoclonal TSHRAbs from lymphocytes of patients with Graves’ disease (8, 9, 19), as well as from those with primary hypothyroidism with potent TSBAbs (10, 20). So far, we succeeded in two clones, 101–2 and B6B7 (9). This allowed us to produce a large amount of monoclonal TSAb protein by genetic engineering.

The present study attempted to characterize recombinant monoclonal TSAbs and apply them to investigate the molecular basis of interactions between TSHR and TSHRAb. This is an important step in elucidating the functional heterogeneity of TSHRAb and, eventually, the pathophysiology of autoimmune TSHR diseases or TSHRAb diseases.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Preparation of recombinant TSAbs
Myeloma cells, transfected with cDNA constructs of 101–2 and B6B7 (9), were ip injected into BALB/c mice pretreated with 2,6,10,14-tetramethyl-decanoic acid (Wako Pure Chemical Industries, Ltd., Osaka, Japan). Both constructs were made for IgG1 production. Human IgGs, which were produced in ascites of mice, were purified with Protein A Sepaharose 4 Fast Flow (Pharmacia Inc., Piscataway, NJ) and subjected to various assays. The IgG and protein concentrations of purified samples were determined by the enzyme-linked immunosorbent assay method described previously (8, 9) and the Bio-Rad protein assay (Bio-Rad Laboratories, Inc., Hercules, CA), respectively. The 196–14 clone, which produces recombinant chimeric antibody (IgG1) against ovarian cancer-associated antigen (CA125) (21) not related to the TSHRAb activity, was used as a negative control.

Flow cytometry
Cytofluorographic analysis was performed by a FACScan flow cytometer (Becton Dickinson and Co., Mountain View, CA) using mouse TSHRAb, MCA1281 (Serotec, Oxford, UK), and fluorescein isothiocyanate (FITC)-conjugated antimouse Ig antibody (Amrad Biotech, Boronia, Australia). MCA1281 binds to an epitope at the carboxy terminus of the extracellular domain of the receptor between amino acids 354–359. Approximately 106 cells were harvested by extensive pipetting and incubated with 3 µg MCA1281. After being stained with FITC-antimouse Ig, cells were gated by forward and side scatter. Dead cells were excluded by using propidium iodide or 7-amino-actinomycin D staining and appropriate gating. Dilutions, washings, and incubations were performed in PBS at 4 C.

TSAb assays
TSAb activities were measured using Cos-7 cells (ATCC CRL 1651), transfected with a panel of mutants of rat TSHR cDNAs (Refs. 22, 23 , Fig. 1Go) by the method described previously (24). Forty-five microliters of monoclonal or patient’s IgG samples was incubated with the cells for 2 h. The cAMP levels of supernatants were measured by an RIA kit. TSAb activities were expressed as the percentage of generated cAMP, relative to controls. Controls included measurements of an equal amount of control IgG, 196–14, and normal IgG to increase cAMP levels in the same transfectants; in no case did control or normal IgG cause an increase. All assays were performed in triplicate and, on at least three separate occasions, with different cell cultures. Patient IgGs were prepared for the TSAb assay as described (23), and the concentrations of 1 mg/ml were used for the assay. Bovine TSH was purchased from Sigma Chemical Co. (St Louis, MO).



View larger version (17K):
[in this window]
[in a new window]
  		Figure 1. Schematic representation of five TSHR deletion mutants in the N-terminal region of the receptor (A) and three mutants in the C-terminal region of the extracellular domain of the receptor (B). Mutant receptors are designated as previously described (22 23 ). The amino acids are shown in the single letter code. Abbreviations for the amino acid residues are: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr. The dashes denote the same amino acids as the wild-type rat TSHR sequence, and the boxes designate the regions substituted or deleted. The numbers assigned to the amino acids are those published for the rat TSHR (24 ). Mutants written in bold, the cell surface expressions of which were not remarkably different from that of the wild-type (Fig. 2Go), were used for subsequent TSAb assays (Figs. 3Go and 4Go).

 
Binding study of antibody
Clones 101–2 and -B6B7 were radioiodinated with a low dose of chloramin T (25) or lactoperoxidase (26, 27). For both methods, specific activities of 125I-IgG were calculated to be 60–80 µCi/µg. 125I-TSH, from a TSHR antibody assay kit (Cosmic Co., Tokyo, Japan), was used for TSH binding. Bovine TSH, used for the unlabeled hormone in the binding assay and the cAMP level assay, was purchased from Sigma Chemical Co.

CHO-rTSHR-14 cell was a gift from Dr. T Fujiwara, Otsuka Pharmaceutical Co. Ltd., Tokushima, Japan) and was obtained with the dihydrofolate reductase (dfhr) amplification system. The EcoRI/XbaI fragment of rat TSHR cDNA (-54 to 2341, Ref. 24) was inserted into a pRSVS-dhfr and introduced into CHO DG44 dhfr- cells by electroporation. The surviving clones in the culture medium without hypoxanthine/thymidine were screened for TSH binding. CHO-rTSHR-48 cell clone, which exhibited the highest TSH binding and growth, was used for the amplification, which was achieved by progressively increasing concentrations of methotrexate (MTX). CHO-rTSHR-14 cells, one of clones derived from CHO-rTSHR-48 by MTX amplification, were resistant to 500 nM of MTX and exhibited the highest TSH binding, with an affinity similar to Cos-7 cells transfected with the wild-type rat TSHR cDNA. Because increasing the MTX concentration to more than 500 nM increased the cpm of 125I-TSH binding to the cells but decreased the binding affinity, CHO-rTSHR-14 cells were used for the binding study. Scatchard plots were analyzed with a personal computer using a hyperbolic fitting program or a line-regression program.

To measure IgG binding, CHO-rTSHR-14 cells were plated in 6-well plates (1 x 106 cells/well) and washed twice with binding assay buffer (222 mM sucrose-supplemented NaCl-free HBSS, containing 0.5% BSA and 20 mM HEPES, pH 7.4) and then incubated for 2 h at 22 C in 0.8 ml of the same buffer, containing 2–4 x 104 cpm 125I-101–2 or 125I-B6B7 plus or minus unlabeled IgG, as noted. TSH binding was performed as described (about 6 x 103 cpm 125I-TSH was used) (24). Specific binding was calculated by subtracting values obtained in the presence of 10-6 M IgG or 3.3 x 10-7 TSH. Nonspecific bindings of 125I-IgG and 125I-TSH to these cells were about 4.6–5.4% and 6.1%, respectively. The counts of maximal specific bindings by 101–2, B6B7, and TSH were 1.2–2.4 x 103, 1–2 x 103, and 1.4 x 103 cpm, respectively. All assays were performed in duplicate and on at least three separate occasions with different cell cultures. As a negative control, wild-type CHO DG44 dhfr- cells were used to confirm no specific binding of 125I-101–2, 125I-B6B7, or 125I-TSH. In addition, 125I-196–4 was tested for CHO-rTSHR-14 cells as a negative control.

Immunodetection of receptor proteins
Western blot and immunodetection analyses of CHO-rTSHR-14 cells, CHO-rTSHR-48 cells, and transfected Cos-7 cells with the wild-type of TSHR cDNA were performed as described (28, 29). In brief, 107 cells were harvested by extensive pipetting and homogenized with a Dounce homogenizer (Wheaton Science Products, Millville, NJ). Aliquots (10 mg protein each) of the membrane fractions were subjected to SDS-PAGE. The gel proteins were electroblotted to the nitrocellulose membrane, and the membrane was incubated with 50-fold diluted antibody against a peptide specific for the TSHR (residues 352–366). After washing, [125I]antirabbit IgF(ab')2 (3µCi; Amersham, Little Chalfont, Buckinghamshire, UK) was added to the membrane. Finally, the membrane was subjected to autoradiography.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Production of a large amount of monoclonal B6B7 and 101–2
B6B7 and 101–2 are recombinant monoclonal IgG class TSAbs produced from myeloma cells stably transfected with reconstituted Ig heavy- and light-chain genes (9). The concentrations of human IgGs produced in ascites of mice were approximately 0.5–1.5 mg/ml. For each clone, about 50 ml of ascites of each clone were subjected to protein-A affinity chromatography. The purities and recoveries of IgGs (B6B7 and 101–2) after the chromatography were 40–60% and 50–70%, respectively. The purity of IgG was calculated based upon simultaneous measurements of protein and IgG concentrations of the samples. We speculated that the main contaminant of the human IgG preparations would be murine IgG, because it was present in ascites, especially in bloody acites. Protein A sepharose strongly binds murine IgG2a and IgG2b, as well as human IgG1 and IgG2, whereas it weakly binds human IgG4 and murine IgG1. The measurement of murine IgG concentrations of the preparations by enzyme-linked immunosorbent assay supported the speculation that most of the contaminants were murine IgG. This contamination, however, would not induce artefactual results, because a negative control, 194–14, also had similar contaminants, and sera from wild-type BALB/c mice are known to have no TSHRAbs. More than 10 mg of pure 101–2 and B6B7 preparations were obtained, which allowed us to perform the following studies.

Epitope mapping of monoclonal TSAbs using TSHR mutants
We tested TSAb activities of B6B7 and 101–2 in a panel of TSHR mutants in the N-terminal region of the receptor, and we selected three representative mutants: S34–37, F52–56, and F58–61 (Fig. 1AGo). Corresponding segments of rat gonadotropin receptors or hydrophilic (serine) and hydrophobic (alanine) amino acids were substituted as appropriate (22). All mutants retained TSH-induced cAMP responsiveness, with a loss or reduction in reactivity to patient sera that contained TSAbs. FACS, analysis using TSHRAb, showed that each mutant was substantially expressed on the cell surface of Cos-7 cells transiently transfected with each cDNA construct (Fig. 2Go; D, F, and G). Although the expression level of S34–37 (11.2%) and F58–61 (12.8%) might be slightly lower than that of the wild-type receptor (16.0%), their responses to TSH were not smaller than that of the wild-type (Fig. 3Go, Ref. 22) and subjected to the epitope mapping study. On the other hand, positively stained cells of S30–33 and S42–45, set by the same fluorescence intensity marker as other mutants, were 5.5% and 6.3%, respectively (Fig. 2Go, C and E). Positive cells of Anti-S were 0.9% (Fig. 2BGo), and those of CHO-rTSHR cells (20), which express a low level of recombinant TSHR but are routinely used for TSAb assays in our laboratory, were (at most) 1.4% (Fig. 2KGo). Differing density distributions of proteins on the cell surface have been determined by FACS analysis (30, 31). Thus, FACS analysis indicated that the cell surface expressions of S30–33 and S42–45 significantly existed but were greatly reduced, compared with other mutants. Therefore, S30–33 and S42–45 were omitted from the epitope mapping study to evaluate the result among mutants expressed similarly on the cell surface.



View larger version (43K):
[in this window]
[in a new window]
  		Figure 2. Cell surface expression level of each TSHR mutant (C, D, E, F, G, H, I) or the wild-type (A) in the Cos-7 cells transfected with each cDNA. The DNA construct of each mutant is shown in Fig. 1Go. Cells were stained by using mouse TSHRAb (MCA1281) and FITC-conjugated antimouse Ig antibody and analyzed by a flow cytometer. CHO-rTSHR-14 cells (J) and Cos-7 cells transfected with constructs containing the rat TSHR cDNA insert in the opposite orientation (Anti-S) (B) were also stained as a positive and negative control, respectively. CHO-rTSHR cells (K) express a low level of recombinant TSHR (20 ). Each panel shows the reaction in the absence (fainter line) and the presence (bolder line) of MCA1281. Percentages of positive cells set by the marker (M1) are indicated in the upper right corner as underlined. The experiments were performed on two separate occasions, and representative data are shown.

 


View larger version (38K):
[in this window]
[in a new window]
  		Figure 3. Ability of B6B7, 101–2, and two different patient IgG preparations to elevate cAMP levels in Cos-7 cells transfected with the N-terminal mutant or wild-type TSHR cDNAs (Fig. 1AGo). Anti-S (see the legend of Fig. 2Go) was also used as a negative control. TSAb activities were expressed as the percentage of generated cAMP, relative to controls (Materials and Methods). *, Statistically significant (P < 0.05) decrease, compared with wild-type activity; bars, mean ± SEM of triplicate cultures. The experiments were performed on three separate occasions, and representative data are shown.

 
cAMP, generated by the monoclonal antibodies of Cos-7 cells transfected with mutant TSHR cDNAs, was measured. The IgG concentrations used were 30 µg/ml and 15 µg/ml for B6B7 and 101–2, respectively, which are optimal, based on dose-dependent curves in the two antibodies. Each dose-response curve of them, using Cos-7 cells transfected with the wild-type of rat TSHR cDNA, showed a very similar pattern to that obtained by using FRTL-5 cells (9). B6B7 gave significant TSAb activities between the IgG concentrations of 15–120 µg/ml, and the activities were maximal around 30 µg/ml. The maximal TSAb activities of B6B7 in three experiments were 167 ± 16% (mean ± SD). The TSAb activities of 101–2 IgG were observed between 15–60 µg/ml. The maximal TSAb activity, given around 15 µg/ml in three experiments, was 198 ± 32% (mean ± SD). The TSAb activities were dose-dependent in both antibodies, and TSAb activity levels decreased at IgG concentrations higher than the maximal point. Control monoclonal IgG, 196–14, a recombinant chimeric antibody against ovarian cancer-associated antigen (CA125), did not show TSAb activity in any mutant TSHRs. As shown in Fig. 3Go, compared with the wild-type, the amount of cAMP generated by B6B7 was significantly decreased in S34–37 and F58–61, but not in F52–56, whereas those by various concentrations of TSH were not decreased. Because B6B7 is a monoclonal antibody that is considered to recognize several amino acid residues, this finding suggests that it interacts with discontinuous regions of the linear amino acid sequence of receptor involving these residues. However, there remains the possibility that antibody bindings may change the conformational structure of some mutants and result in the reduction in activity. An Ig of a patient from whom the lymphocytes B6B7 were derived showed a decreased cAMP response in all five mutants. Diminished cAMP generations in S34–37 and F52–56 are more marked in patient Ig than in B6B7. These findings suggest the existence of TSAbs other than B6B7 in patient 1 serum. The amount of cAMP generated by 101–2 was significantly decreased in F52–56 and F58–61 but not in S34–37. This finding suggests again that 101–2 interacts with the N-terminal region of the receptor involving these residues. Because the Ig of the patient from whom 101–2 was derived had no TSAb activity (9), we could not determine its TSAb epitopes.

Next, we tested TSHR mutated in the C-terminal region of the extracellular domain of the receptor (23, 32). Two mutants, Del 295–306 and 385Q, were used as representatives of mutants of this region (Fig. 1BGo) and expressed on the cell surface in a manner reasonably similar to that of the wild-type receptor (Fig. 2Go, H and I). Because residues 287–404 of the TSHR exhibit little homology to gonadotropin receptors, deletion or mutation, rather than substitution, was thought to be more appropriate in this region. Mutations in this region resulted in the alteration of activities to TSH and polyclonal TSBAbs but not TSAbs (32). In fact, both monoclonal TSAbs and patient antibodies retained TSAb activities in TSHR mutated in the C-terminal region of the extracellular domain, whereas the ability of TSH to increase cAMP levels in these mutants decreased (Fig. 4Go). Compared with polyclonal IgGs, monoclonal IgGs tend to have lower relative activities. This may reflect that the presence of TSBAbs in the polyclonal antibodies partly inhibit their TSAb activities, whereas there are no such effects in monoclonal TSAbs.



View larger version (44K):
[in this window]
[in a new window]
  		Figure 4. Ability of B6B7, 101–2, and two different patient IgG preparations to elevate cAMP levels in Cos-7 cells transfected with the C-terminal mutant or wild-type TSHR cDNAs (Fig. 1BGo). The experimental procedures and the symbols are the same as in Fig. 3Go.

 
The present results are consistent with previous studies that suggested the existence of TSAb epitopes in the N-terminal region of the receptor (5, 7, 22). Mutation in the N-terminal region of the extracellular region of the receptor resulted in alterations of activities to polyclonal TSAbs. Graves’ IgGs showed various patterns and degrees of loss of TSAb activity in the mutants of this region. Some mutants lost TSAb activity with some Igs but not with others. In other mutants, TSAb activity to Graves’ IgGs was decreased but maintained. These findings may be attributable to the existence of heterogeneous TSHRAbs in polyclonal Igs. Heterogeneous TSAbs, which recognize multiple discrete epitopes in the N-terminal of TSHR, may be present in polyclonal patient IgGs. Alternatively, the interactions of TSHRAbs other than TSAbs, such as TSBAbs (which are simultaneously present in polyclonal patient IgGs) might influence TSAb activities. The use of monoclonal antibodies can eliminate these problems, and it demonstrates that epitopes of TSAbs are heterogeneous within the N-terminal of TSHR.

The epitopes of mouse monoclonal antibodies have been mapped (15, 16, 17). They were assigned to be amino acid residues 125–369 (2C11 and 3B12) (15), 22–35 (A10-A11), 402–415 (A7), and 147–228 (A9) (16), and 22–30 (mAb47) and 32–41 (mAb28) (17). Some of them, epitopes of A10-A11 and mAb28, may overlap with those of 101–2 and B6B7. However, none of the monoclonal antibodies exhibited TSAb activity (15, 16) or were tested for the activity (17). In addition, mouse monoclonal antibodies raised experimentally may differ from naturally occurring autoantibodies (18). Therefore, it is impossible to compare our findings with those of mouse antibodies.

Binding study of monoclonal antibodies to TSHR
Next, the binding of B6B7 and 101–2 to the receptor was studied. We first used radiolabeled IgGs and Cos-7 cells transfected with TSHR cDNA for the binding study. The 101–2 and B6B7 were labeled with Na125I, using either a low dose of chloramin T or lactoperoxidase because it was reported that high concentrations of chloramin T alone and high levels of iodination abolished TSHRAb activities (25). However, we detected no significant binding to the Cos-7 cells. This might be attributable to a rather low level of TSHR in the Cos-7 cells plated after the electroporation; the transfection efficiency is, at most, 10–20%; and about half of the cells are dead after the electroporation and are removed at the binding assay. To overcome these limitations, we used CHO-TSHR-14 cells, expressing very high levels of rat TSHR by the dfhr amplification system. By progressively increasing the concentrations of MTX, up to 500 nM, 125I-TSH binding to CHO-TSHR-14 cells per well in the 6-well plate increased to 2.4 times that of unamplified CHO-TSHR-48 cells, and to 4.6 times that of Cos-7 cells transfected with TSHR cDNA (Fig. 5AGo). CHO-TSHR-14 exhibited two component binding sites (Kd1 = 8.3 x 10-10 M and Kd2 = 6.6 x 10-8 M) (Fig. 5BGo), similar to FRTL-5 cells (24). A high level of TSHR protein expression was detected by Western blot analysis; the band around 95 kDa was more abundant than that of Cos-7 cells transfected with TSHR cDNA or CHO-TSHR-48 cells (Fig. 5CGo). Larger molecular mass bands, such as 180 kDa, were not observed in CHO-TSHR-14 or -48 cells. This suggests different processing of TSHR receptor between CHO and Cos-7 cells. In CHO-TSHR-48 and -14 cells, a 95-kDa band seemed to form a 95- to 120-kDa broad band, being characteristic of glycoprotein. The 62-kDa band was more prominent than in the wild-type CHO cells and might be a cleaved fragment of TSHR. However, because its size differed from others (33) and the density did not differ between CHO-TSHR-48 and -14 cells, further study is necessary to elucidate this.



View larger version (21K):
[in this window]
[in a new window]
  		Figure 5. TSH-binding assay and immunodetection analysis for CHO-rTSHR-14 cells. A, The radioactivity of bound 125I-TSH in CHO-rTSHR-48 and -14 cells (about 1 x 106 cells per well), and Cos-7 cells transfected with or without the wild-type TSHR cDNA. About 1 x 106 Cos-7 cells were plated into each well after electroporation; 1.5 x 104 cpm of 125I-TSH was added to the cells cultured in 6-well plates. Closed and open bars, Absence and presence of cold TSH (3.3 x 10-7 M), respectively. Mean values of duplicate determinations are shown. B, Scatchard plots of 125I-TSH binding to CHO-rTSHR-14 cells. The average values of duplicate determinations are shown. In immunodetection analysis (C), the crude membrane fraction of CHO-rTSHR-48 and -14 cells, and Cos-7 cells transfected with TSHR cDNA was run on 4–12% gradient gel. After SDS-PAGE, proteins in the gel were electroblotted and immunodetected using an antibody against a TSHR-specific synthetic peptide (residues 352–366) and 125I-antirabbit IgF(ab')2, as described previously (27 28 ). Autoradiography was performed for 72 h. The left margin assigns molecular mass standards and the right margin indicates 180, 95, and 62 kDa bands with arrows.

 
In CHO-TSHR-14 cells, the binding of 101–2 and B6B7 to the receptor was studied using 125I-IgG labeled with a low dose of chloramin T. Specificity of 125I-101–2 and -B6B7 binding to CHO-TSHR-14 cells were confirmed by comparing these bindings to wild-type DG44 dhfr- cells. No significant 125I-101–2 or -B6B7 binding to wild-type CHO DG44 dhfr- cells was observed: up to 10-6 M of unlabeled IgGs hardly displaced apparent low-125I-IgG binding to wild-type CHO DG44 dhfr- cells. 125I-196–4, radiolabeled with a low dose of chloramin T or lactoperoxidase, did not bind to CHO-rTSHR-14 cells, either (data not shown). As shown in Fig. 6AGo, 101–2 exhibited a low-affinity (yet significant) binding to the receptor (Kd = 1.3 x 10-8 M). To eliminate the possibility that high levels of iodination of IgG by the chloramin T method deteriorate IgG activity, lactoperoxidase was used for iodination. However, very similar binding activities of 101–2 to the cells were observed with both methods (data not shown). The binding of 125I-101–2 to the cells was not inhibited by TSH, B6B7 (Fig. 6AGo), or control IgG (196–14, data not shown). The binding of 125I-TSH to the cells was not inhibited by 101–2 at all, either (data not shown). These findings suggest that the 101–2-binding site is different from that of TSH, because TSH-binding affinity is higher than that of 101–2. Although TSH tended to slightly enhance the binding at high concentrations (Fig. 6AGo), the significance of this is unknown. Because 101–2 epitopes in the N-terminal region of the receptor are not related to TSH binding (22), it is unlikely that TSH directly affects the 101–2-binding sites. Instead, TSH binding may alter a conformational structure of the receptor and facilitate the binding of 101–2 to the receptor.



View larger version (14K):
[in this window]
[in a new window]
  		Figure 6. 125I-101–2 binding to CHO-rTSHR-14 cells. A, 125I-101–2 binding to CHO-rTSHR-14 cells was measured in the presence of various concentrations of unlabeled 101–2 ({blacksquare}), B6B7 ({blacktriangleup}), or TSH (•); B, the Scatchard plot of 125I-101–2 binding described in A. The average values of duplicate determinations are shown. The entire experiment was repeated, with very similar results.

 
B6B7 exhibited one-order lower affinity binding to the receptor than 101–2 (Kd = 1.3 x 10-7 M, Fig. 7Go). Similar results were obtained when 125I-B6B7 was radiolabeled using either chloramin T or lactoperoxidase, although the iodination itself might affect the binding activity of B6B7 to the cells. The binding was not inhibited by TSH, 101–2 (Fig. 7AGo), or control IgG (data not shown). Because the binding epitopes of 101–2 and B6B7 seem to overlap each other in some sites (Fig. 3Go) and 101–2 exhibited higher binding affinity than B6B7 (Fig. 6BGo, 7BGo), it is interesting that 101–2 did not displace B6B7. This suggests that there are marked differences in binding epitopes between them or that the low-affinity binding of B6B7 masks the influence of 101–2 on 125I-B6B7 binding. As with 101–2, TSH and B6B7 did not inhibit their binding each other at all; probably because B6B7 epitopes in the N-terminal region of the receptor are also not related to TSH binding.



View larger version (13K):
[in this window]
[in a new window]
  		Figure 7. 125I-B6B7 binding to CHO-rTSHR-14 cells. A, 125I-B6B7 binding in CHO-rTSHR-14 cells measured in the presence of various concentrations of unlabeled B6B7, 101-2, or TSH; B, the Scatchard plot of 125I-B6B7 binding described in A. The experimental procedure and the symbols are the same as in Fig. 6Go.

 
Both 125I-101–2 and -B6B7 exhibited low-affinity binding to the cells expressing TSHR and were active at fairly high concentrations, microgram-per-milliliter range. McLachlan and Rapoport (18) stated that monoclonal TSHRAbs were unlikely of much lower affinity and should be detected at nanogram-per-milliliter concentrations. It is, however, frequently observed that monoclonal antibodies show a lower affinity and/or biological activity, compared with polyclonal antibodies. Because we analyzed only two TSAbs, it would be premature to generalize the above assumption. There is, however, evidence to support this: 1) site-directed mutagenesis of TSHR revealed that low-affinity binding of TSH to TSHR was sufficient to transduce a full signal (7, 23, 28, 29); 2) the alteration of TSH molecule does not show proportional changes in the relationship between the binding and signal transduction (34); 3) none of the monoclonal TSAbs exhibited TSH-binding inhibitor activities, suggesting TSAbs do not transduce a signal through high-affinity binding sites of TSH (2, 7, 9); and 4) it takes much longer for TSAb to reach a maximum effect than for TSH. In in vitro assay, TSAb activity is usually measured after 2 h of incubation, and TSH after 30 min (35). In in vivo assay, TSAb, which is known as a long-acting thyroid stimulator, is measured after 24 h, and TSH after 1 h (36). The low-affinity binding of TSAb to the receptor may partly explain this slow-acting stimulator effect. Actually, in vivo functional studies, using these monoclonal antibodies, are currently performed.

The present study clearly indicates that monoclonal TSAb-binding epitopes can be distinct from TSH-binding sites. The large quantities of monoclonal TSAb produced in this study allowed us to perform epitope and binding studies of TSAbs. Further, they will be useful for crystallization studies to identify the three-dimensional structure of TSAb, which is essential to elucidate the decisive structure-function relationship. Thus, they may be useful for investigating the molecular basis of the interactions between TSAb and TSHR in autoimmune TSHR diseases or TSHRAb diseases.


   Acknowledgments
 
We thank Dr. Tsutomu Fujiwara (Otsuka Pharmaceutical Co. Ltd.), Miss Sachiko Ueoku, and Miss Hitomi Hiratani for excellent technical assistance. We also thank Drs. Shinji Kosugi and Leonard D Kohn for providing several mutant strains of TSHR, and Mr. Daniel Mrozek for correcting the English.


   Footnotes
 
1 This work was partly supported by grants in aid from the Ministry of Education, Science and Culture of Japan (Nos. 05671922, 07672487, 08557150, and 08671150), the Ryoichi Naito Foundation for Medical Research, the Kurozumi Medical Foundation, the Kato Memorial Trust for Nambyo Research, the Uehara Memorial Foundation, and the Inamori Foundation (to T.A.). This work was presented, in part, at the 70th Annual Meeting of The American Thyroid Association, Colorado Springs, Colorado, 1997 [Thyroid (Suppl 1) 7:S-74 (Abstract 147)]. Back

Received July 16, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

  1. Smith BR, McLachlan SM, Furmaniak J 1988 Autoantibodies to the thyrotropin receptor. Endocr Rev 9:106–121[Abstract/Free Full Text]
  2. Akamizu T, Mori T, Nakao K 1997 Pathogenesis of Graves’ disease: Molecular analysis of anti-TSH receptor antibodies. Endocr J 44:633–646[Medline]
  3. Konishi J, Iida Y, Kasagi K, Misaki T, Nakashima T, Endo K, Mori T, Shinpo S, Nohara Y, Matsuura N, Torizuka K 1985 Primary myxedema with thyrotrophin-binding inhibitor immunoglobulin: clinical and laboratory findings in 15 patients. Ann Intern Med 103:26–31
  4. Mori T, Akamizu T, Kosugi S, Sugawa H, Inoue D, Okuda J, Ueda Y 1994 Recent progress in TSH receptor studies with a new concept of "autoimmune TSH receptor disease". Endocr J 41:1–11[Medline]
  5. Akamizu T, Kohn LD, Mori T 1995 Molecular studies on thyrotropin (TSH) receptor and anti-TSH receptor antibodies. Encocr J 42:617–627
  6. Vassart G, Dumont JE 1992 The thyrotropin receptor and the regulation of thyrocyte function and growth. Endocr Rev 13:596–611[Abstract/Free Full Text]
  7. Nagayama Y, Rapoport B 1992 The thyrotropin receptor 25 years after its discovery: new insight after its molecular cloning. Mol Endocrinol 6:145–156[Abstract/Free Full Text]
  8. Shin EK, Akamizu T, Matsuda F, Sugawa H, Fujikura J, Mori T, Honjo T 1994 Variable-regions of immunoglobulin heavy-chain genes encoding anti-thyrotropin (TSH) receptor antibodies of patients with Graves’ disease. J Immunol 152:1485–1492[Abstract]
  9. Akamizu T, Matsuda F, Okuda J, Li H, Kanda H, Watanabe T, Honjo T, Mori T 1996 Molecular analysis of stimulatory anti-thyrotropin receptor antibodies (TSAbs) involved in Graves’ disease: isolation and reconstruction of antibody genes, and production of monoclonal TSAbs. J Immunol 157:3148–3152[Abstract]
  10. Okuda J, Akamizu T, Matsuda F 1995 Variable regions of immunoglobulin genes encoding "blocking type" antithyrotropin receptor antibodies of patients with primary myxedema. Thyroid [Suppl 1] 5:S-210
  11. Valente WA, Vitti P, Yavin Z, Yavin E, Rotella CM, Grollman EF, Toccafondi RS, Kohn LD 1982 Monoclonal antibodies to the thyrotropin receptor: stimulating and blocking antibodies derived from the lymphocytes of patients with Graves’ disease. Proc Natl Acad Sci USA 79:6680–6684[Abstract/Free Full Text]
  12. Yoshida T, Ichikawa Y, Ito K, Homma M 1988 Monoclonal antibodies to the thyrotropin receptor bind to a 56-KDa subunit of the thyrotropin receptor and show heterogeneous bioactivities. J Biol Chem 263:16341–16347[Abstract/Free Full Text]
  13. Baker Jr JR, Saunders NR, Kaulferoch W, Burman KD 1988 Development of a human monoclonal antibody from a Graves’ disease patient that identifies a novel thyroid membrane antigen. J Immunol 140:2593–2599[Abstract]
  14. Morgenthaler NG, Kim MR, Tremble J, Huang GC, Richter W, Gupta M, Scherbaum WA, McGregor AM, Banga JP 1989 Human IgG autoantibodies to the thyrotropin receptor from Epstein-Bar virus transformed B lymphocytes: characterization by immunoprecipitaion with recombinant antigen, and biological activity. J Clin Endocrinol Metab 81:3155–3161[Abstract]
  15. Johnstone AP, Cridland JC, DaCosta CR, Harfst E, Shepherd PS 1994 Monoclonal antibodies that recognized the native human thyrotropin receptor. Mol Cell Endocrinol 105:R1–R9
  16. Nicholson LB, Vlase H, Graves P, Nilsson M, Molne J, Huang GC 1996 Monoclonal antibodies to the human TSH receptor: epitope mapping and binding to the native receptor on the basolateral plasma membrane of thyroid follicular cells. J Mol Endocrinol 16:159–170[Abstract/Free Full Text]
  17. Seetharamaiah GS, Wagle NM, Morris JC, Prabhakar BS 1995 Generation and characterization of monoclonal antibodies to the human thyrotropin (TSH) receptor: antibodies can bind to discrete conformational or linear epitopes and block TSH binding. Endocrinology 136:2817–2824[Abstract]
  18. McLachlan SM, Rapoport B 1996 Editorial: Monoclonal, human autoantibodies to the TSH receptor - the holy grail and why are we looking for it? J Clin Endocrinol Metab 81:3152–3154[CrossRef][Medline]
  19. Li H, Akamizu T, Okuda J, Sugawa H, Matsuda F, Tsubata T, Mori T 1995 Isolation of Epstein-Barr virus-transformed lymphocytes producing IgG class monoclonal antibodies using a magnetic cell separator (MACS): preparation of thyroid-stimulating IgG antibodies from patients with Graves’ disease. Biochem Biophys Res Commun 207:985–993[CrossRef][Medline]
  20. Okuda J, Akamizu T, Sugawa H, Matsuda F, Li H, Mori T 1994 Preparation and characterization of monoclonal antithyrotropin receptor antibodies obtained from peripheral lymphocytes of hypothyroid patients with primary myxedema. J Clin Endocrinol Metab 79:1600–1604[Abstract]
  21. Kanda H, Mori K, Koga H, Taniguchi K, Kobayashi H, Sakahara H, Konishi J, Endo K, Watanabe T 1994 Construction and expression of chimeric antibodies by a simple replacement of heavy and light chain V genes into a single cassette vector. Hybridoma. 13:359–366
  22. Kosugi S, Ban T, Kohn LD 1993 Identification of thyroid-stimulating antibody-specific interaction sites in the N-terminal region of the thyrotropin receptor. Mol Endocrinol 7:114–130[Abstract/Free Full Text]
  23. Kosugi S, Ban T, Akamizu T, Kohn LD 1991 Site-directed mutagenesis of a portion of the extracellular domain of the rat thyrotropin receptor important in autoimmune thyroid disease and nonhomologous with gonadotropin receptors: relationship of functional and immunogenic domains. J Biol Chem 266:19413–19418[Abstract/Free Full Text]
  24. Akamizu T, Ikuyama S, Saji M, Kosugi S, Kozak C, McBride WO, Kohn LD 1990 Cloning, chromosomal assignment, and regulation of the rat thyrotropin receptor: expression of the gene is regulated by thyrotropin, agents that increase cAMP levels, and thyroid autoantibodies. Proc Natl Acad Sci USA 87:5677–5681[Abstract/Free Full Text]
  25. Endo K, Borges M, Amir S, Ingbar SH 1982 Preparation of 125I-labeled receptor-purified Graves’ immunoglobulins: properties of their binding to human thyroid membranes. J Clin Endocrinol Metab 55:566–576[Abstract/Free Full Text]
  26. Miyachi Y, Vaitukaitis JL, Nieschlag E, Liprett MB 1972 Enzymatic radioiodination of gonadotropins. J Clin Endocrinol Metab 34:23–28[Abstract/Free Full Text]
  27. Akamizu T, Mori T, Ishii H, Yokota T, Nakamura H, Imura H 1988 Purification of TSH receptor from porcine thyroid membrane and effect of various protease inhibitors on receptor stability. Endocrinol Jpn 35:275–283[Medline]
  28. Akamizu T, Inoue D, Kosugi S, Ban T, Kohn LD, Imura H, Mori T 1993 Chimeric studies of the extracellular domain of the rat thyrotropin (TSH) receptor: amino acids (268–304) in the TSH receptor are involved in the ligand high affinity binding, but not in TSH receptor-specific signal transduction. Endocr J 40:363–372[Medline]
  29. Akamizu T, Inoue D, Kosugi S, Kohn LD, Mori T 1994 Further studies of amino acids (268–304) in thyrotropin (TSH) - lutropin/chorionic gonadotropin (LH/CG) receptor chimeras: cysteine-301 is important in TSH binding and receptor tertiary structure. Thyroid 4:43–48[Medline]
  30. Shimonkevitz R, Luescher B, Cerottini JC, MacDonald HR 1985 Clonal analysis of cytolytic T lymphocyte-mediated lysis of target cells with inducible antigen expression: correlation between antigen density and requirement for lyt-2/3 function. J Immunol 135:892–899[Abstract]
  31. Lu L, Pelus LM, Broxmeyer HE, Moore MAS, Wachter M, Walker D, Platzer E 1986 Enhancement of the proliferation of human marrow erythroid (BFU-E) progenitor cells by prostaglandin E requires the participation of OKT8-positive T lymphocytes and is associated with the density expression of major histocompatibility complex class II antigens on BFU-E. Blood 68:126–133[Abstract/Free Full Text]
  32. Kosugi S, Ban T, Akamizu T, Kohn LD 1992 Identification of separate determinants on the thyrotropin receptor reactive with Graves’ thyroid-stimulating antibodies and with thyroid-stimulating blocking antibodies in idiopathic myxedema: these determinants have no homologous sequence on gonadotropin receptors. Mol Endocrinol 6:168–180[Abstract/Free Full Text]
  33. Rapoport B, McLachlan SM, Kakinuma A, Chazenbalk GD 1996 Critical relationship between autoantibody recognition and thyrotropin receptor maturation as reflected in the acquisition of complex carbohydrate. J Clin Endocrinol Metab 81:2525–2533[Abstract]
  34. Thotakura NR, Desa RK, Bates LG, Cole ES, Pratt BM, Weintraub BD 1991 Biological activity and metabolic clearance of a recombinant human thyrotropin produced in Chinese hamster ovary cells. Endocrinology 128:341–348[Abstract/Free Full Text]
  35. Kasagi K, Konishi J, Iida Y, Ikekubo K, Mori T, Kuma K, Torizuka K 1982 A new in vitro assay for human thyroid stimulator using cultured thyroid cells: effect of sodium chloride on adenosine 3',5'-monophosphate increase. J Clin Endocrinol Metab 54:108–114[Abstract/Free Full Text]
  36. Florsheim WH, Williams AD, Schonbaum E 1970 On the mechanism of the McKenzie bioassay. Endocrinology 8:881–888



This article has been cited by other articles:


Home page
 Molecular Cancer TherapeuticsHome page
U. K. Misra, Y. Mowery, S. Kaczowka, and S. V. Pizzo
Ligation of cancer cell surface GRP78 with antibodies directed against its COOH-terminal domain up-regulates p53 activity and promotes apoptosis
Mol. Cancer Ther., May 1, 2009; 8(5): 1350 - 1362.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
C.-R. Chen, S. M. McLachlan, and B. Rapoport
Suppression of Thyrotropin Receptor Constitutive Activity by a Monoclonal Antibody with Inverse Agonist Activity
Endocrinology, May 1, 2007; 148(5): 2375 - 2382.
[Abstract] [Full Text] [PDF]

Home page
 Endocr. Rev.Home page
S. M. McLachlan, Y. Nagayama, and B. Rapoport
Insight into Graves' Hyperthyroidism from Animal Models
Endocr. Rev., October 1, 2005; 26(6): 800 - 832.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Endocrinol. Metab.Home page
C. H. Byun, J. Y. Park, T. Akamizu, and C.-B. Chae
Identification of the Peptides That Inhibit the Function of Human Monoclonal Thyroid-Stimulating Antibodies from Phage-Displayed Peptide Library
J. Clin. Endocrinol. Metab., July 1, 2001; 86(7): 3311 - 3318.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Akamizu, T.
Right arrow Articles by Nakao, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Akamizu, T.
Right arrow Articles by Nakao, K.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals