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Epitope analysis of the thyrotropin receptor, 19971
Molecular and Cellular Endocrinology 128 (1997) 11 – 18
At The Cutting Edge
Epitope analysis of the thyrotropin receptor, 19971 Shinji Kosugi *, Hideo Sugawa, Toru Mori Department of Laboratory Medicine, Kyoto Uni6ersity School of Medicine, Room 223, First Clinical Research Building, 54 -Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606 -01, Japan Received 13 December 1996; accepted 22 January 1997
Keywords: Site-directed mutagenesis; Chimeric receptor; Synthetic peptide; Graves’ disease; Determinant spreading
1. Introduction By extensive mutagenesis studies of the N-terminal and C-terminal regions of the extracellular domain of the thyrotropin receptor (TSHR), which are non-homologous with gonadotropin receptors, we have localized thyroid-stimulating antibody (TSAb) epitopes in residues 30–33, 34– 37, 40, 42 – 45, 52 – 56 and 58 –61 in the N-terminal [1,2] and TSBAb (thyroid-stimulating blocking antibody) epitopes in residues 301, 385 and 390, in the C-terminal region of the extracellular domain, [1,3] which are also high affinity binding sites for TSH [4,5]. The N-terminal region also contributes to TSH binding [2]. Residues 303 – 382, which are flanked by two Cys residues, can be deleted without functional loss of the TSHR [4]. However, this region is highly immunogenic, and we have shown that IgGs from Graves’ patients significantly bound peptides from this region and speculated that it thus contributed to triggering the autoimmune response [4,6]. Fig. 1 schematically represents the TSHR structure and critical determinants for binding of TSH, TSAb and TSBAb. In the present review, we summarize data from laboratories other than our own; previous reviews [7,8] have provided detailed summaries and canvassed the implications of our studies. Although there are still some * Corresponding author. Fax: + 81 75 7513233; e-mail: [email protected] 1 Note: All residue numbers are counted from the methionine start site. All TSHR peptides are designated by the corresponding N- and C-terminal residue numbers.
conflicting findings, there appears to be a consensus on the following points, as outlined in the following sections.
2. The N-terminal region (especially residues 22–61) is the site of the major TSAb epitopes in the TSH receptor We have identified residues 30–33, 34–37, 40, 42–45, 52–56 and 58–61 as important for TSAb activity, which does not imply that other residues are not important. No functional substitution mutants of residues 38, 39, 41, 46–51, 57, 62, 75 and 76 were obtained, so that these residues could not be evaluated and it remains possible that these residues are also involved in TSAb/ TSH interaction. We assume that linear and/or conformational epitope(s) in residues 30–65 are critical for TSAb interaction, and although a consensus of the precise location of TSAb major epitope(s) has not been reached, there is a body of evidence supporting these observations, as follows.
2.1. Mutagenesis studies Nagayama et al. [9] reported that TSHR with substitutions at residues 1–170 lost TSAb activity. Further, Nagayama et al. [10] and Jaume et al. [11] showed that half of Graves’ IgGs lost TSAb activity in TSHR with substitutions at residues 25–30. Tahara et al. [12] demonstrated that 67% of Graves’ IgGs showed a loss
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Fig. 1. Putative membrane topology and important determinants for TSH, TSAb and TSBAb binding on the TSHR. Closed diamonds (residues 22 – 61) indicate TSH/TSAb binding sites. Three large closed circles (Cys-301, Tyr-385 and Cys-390) are critical residues forTSH/TSBAb binding. Small closed circles (residues 317–366) and hexagons indicate a full deletable region (residues 303 – 382).
S. Kosugi et al. / Molecular and Cellular Endocrinology 128 (1997) 11–18
of TSAb activity in TSHR with substitutions at residues 8–165, but not in mutants with substitutions at residues 90–165. Kim et al. [13] reported that TSAb activity for 85% of Graves’ IgGs was lost in TSHR with substitutions at residues 9 – 165.
2.2. Peptide binding Murakami et al. [14] showed 125I-labeled peptide 32–56 bound to Graves’ IgGs. Ikeda et al. [15] demonstrated that some Graves’ sera but not Hashimoto’s sera bound peptide 29 – 57 by enzyme linked immunosorbent assay (ELISA). Sugawa et al. [16] showed that peptide 31–50 absorbed TSAb activity of Graves’ IgGs.
2.3. Peptide immunization Endo et al. [17] immunized rabbits with peptide 29–57, and sera from the immunized rabbits showed significant TSAb activity. Matsui et al. [18] immunized rats with five different TSHR peptides; TSAb activities were highest in rats immunized with peptide 22–50. Murakami et al. [19] reported TSAb activity in sera from rabbits immunized with peptide 32 – 56.
2.4. Immunization of mice with extracellular domain of the TSHR Valse et al. [20] immunized mice with recombinant TSHR extracellular domain (ECD) from both an E. Coli and a baculovirus expression system. All sera from BALB/c and CBA/J mice recognized peptide 22–41 but had no TBII (thyrotropin-binding inhibitory immunoglobulin) activity. Gattadahalli et al. [21] immunized mice with TSHR ECD to produce a panel of monoclonal antibodies to the TSHR; one of the antibodies was recognized by peptide 22 – 30, but had no TBII activity.
3. The C-terminal region of the extracellular domain of the TSH receptor (especially Cy-301, Tyr-385 and Cys-390) is the site of the major TSBAb/TBII epitopes in the TSHR
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3.2. Peptide immunization Ohmori et al. [22] reported that chickens immunized with peptide 372–392 produced both TSBAb and TBII activity, but no TSAb activity. Murakami et al. [19] showed that rabbits immunized with peptide 352–378 had TSBAb activity, and Dallas et al. [23] that rabbit antibodies against peptide 367–386 had both TBII and TSBAb.
3.3. ECD immunization Dallas et al. [24] immunized rabbits with baculovirusgenerated ECD of the TSHR producing an antibody which showed TBII activity reversed by preincubation with peptides 292–311, 367–386 and 377–397. Antibodies affinity-purified with these peptides showed TBII activity, indicating the importance of these regions for binding of TSH and blocking antibodies. These observations are thus in close agreement with our mutagenesis studies identifying Cys-301, Tyr-385 and Cys-390 as critical for binding of TSH and blocking antibodies. Wagle et al. [25] similarly immunized BALB/cJ and SJL/J mice with baculovirus-generated ECD of the TSHR, and showed that addition of peptide 367–386 significantly reduced TBII activity of antisera from both groups. 4. Major TSH-binding sites are located at the C-terminus, but the N-terminus also contributes to TSH binding Ohmori et al. [22] showed that preincubation of TSH with peptide 35–50 dose-dependently decreased cAMP accumulation in thyroid cells. Attasi et al. [26] reported that [125I]TSH bound to peptide 32–50. Morris et al. [27] showed that TSH bound to peptides 16–35 and 286–305. Nagayama et al. [9] reported that residues 1–260 also contributed TSH binding using TSHR/ lutropin-choriogonadotropin receptor (LH-CGR) chimeras. 5. The C-terminal region (residues 303–382) can be deleted without functional loss, but some antibodies bind to this region, indicating its importance for immunogenicity
3.1. Mutagenesis
5.1. Mutagenesis studies
Nagayama et al. [9] showed that TSHR with substitutions at residues 260 – 418 decreased the TSH-cAMP response 7-fold in sensitivity, but retained TSAb activity to a similar extent to wild-type (WT). The TBII activity of sera from idiopathic myxedema was markedly decreased with this mutant TSHR.
It is clear that a region in the C-terminal of the TSHR extracellular domain of the TSHR can be deleted without functional loss. Thus, Wasthworth et al. [28] demonstrated that TSHR with deletion of residues 317–366 completely retained TSH binding, and TSH- and TSAb-stimulated cAMP responses.
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5.2. Peptide binding
6.1. Peptide binding
Mori et al. [29] reported that peptide 353–363 did not bind TSH but bound TSAb of Graves’ sera, and that preincubation with peptide decreased TSAb activity. Takai et al. [30] demonstrated that Graves’ sera bound peptide 352 – 366 on ELISA. Ikeda et al. [15] reported that some Graves’ sera but not Hashimoto’s sera bound peptide 359 – 371 on ELISA. Murakami et al. [31] showed that iodinated peptide 352 – 378 bound 83% of IgGs from Hashimoto’s patients without TBII activity, and a small proportion of IgGs from Graves’ patients. Ueda et al. [32] reported that two of 100 Graves’ IgGs had TSAb enhancing activity on addition of peptide 354–367, and also that there were antibodies which bound to regions 354 – 357 and 364 –367 in a small population of Graves’ patients. Further, Ueda et al. [33] and Sugawa et al. [16] showed that TSAb activity of some Graves’ IgGs was absorbed by peptides 338–353 and/or 354 – 367.
Sugawa et al. [16] showed that peptides 91–119 absorbed TSAb activity of Graves’ IgGs. Piraphatdist et al. [36] reported that peptide 123–131 did not bind TSH, but that preincubation of peptide with patients’ sera caused a decrease in TBII activity of TBII positive sera and an increase in TSAb activity of some Graves’ sera, concluding that the peptide thus bound TBII without TSAb activity. Ikeda et al. [15] showed that some Graves’ but no Hashimoto’s IgGs bound peptide 172–202 on ELISA. Morris et al. [27], using a series of 26 overlapping peptides comprising the entire extracellular domain and the three extracellular loops, tested the inhibition of [125I]TSH binding to thyroid membranes. They showed that TSH bound displaceably to peptide 256–275 with a Kd of 3× 10 − 5 M. Further, Morris et al. [37], using the same series of peptides, demonstrated that Graves’ IgGs bound peptide 181– 200 on ELISA.
5.3. Peptide immunization Endo et al. [34] immunized rabbits with peptide 341–370, and the antisera showed significant TSAb activities. Ohmori et al. [22] reported that immunization of chickens with peptide 341 – 358 produced TSBAb and TBII activity but no TSAb activity.
5.4. ECD immunization Dallas et al. [35] showed that antisera from rabbits immunized with baculovirus-generated ECD of the TSHR reacted with peptide 352 – 388, and they identified a dominant epitope 357 – 372 in this region. Further, Dallas et al. [23] demonstrated that rabbit antibodies against peptide 357 – 372 had TSBAb but no TBII.
6. Studies on the middle region of the extracellular domain of the TSHR Most epitope analyses and mutagenesis studies have focused on the N-terminal and C-terminal regions of the extracellular domain of the TSHR, where homology with related gonadotropin receptors is very low. The importance of the middle region of the TSHR extracellular domain, which has high homology with gonadotropin receptors, might thus have been inadequately studied. However, no specific portions in the middle region have been identified by various experimental procedures in different laboratories. A summary of the findings obtained for this region follows.
6.2. Peptide immunization Endo et al. [35] showed TSAb activity in sera of rabbits immunized with peptide 172–202.
7. Studies of the exoplasmic loops. All portions of the TSH receptor on the outer surface of the cell can be immunogenic. In this aspect, Endo et al. [38] reported that immunization with peptides corresponding to the three extracellular loops induced TSAb. One might speculate that the binding of antibodies to extracellular loops connecting bundles of transmembrane helix that directly interact with Gproteins may cause a conformational change, to disrupt the interhelical bonds maintaining the inactive state of the receptor. In this context, it needs to be determined if antibodies against LHR peptide also cause activation of LHR, and it is uncertain if such antibodies are really produced in Graves’ patients. However, Morris et al. [37], using the same series of peptides as described above, showed Graves’ IgGs bound peptide 629–639 corresponding to the 3rd exoplasmic loop on ELISA, and that TSAb activity was enriched by affinity purification using the peptide. In contrast, Ohmori et al. [22] reported that immunization of chicken with peptide 649–661 produced TSBAb and TBII activity, but no TSAb activity.
8. Epitope spreading of the TSHR The new concept of determinant spreading (epitope spreading) has been highlighted as a possibly important
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initiator of autoimmune disorders [39 – 47]. The fundamental concept is that repetitive immunological stress with a highly immunodominant epitope induces aberrant recognition of the cryptic epitope(s) of an autoantigen and thus induces autoimmunity. As described above, polyclonal TSHR antibodies are detected in the sera of patients with autoimmune TSHR diseases [10,16,33,48,49]; autoimmune thyroid disease is thought to be an oligoclonal phenomenon, and there is no clear explanation for the simultaneous appearance of these autoantibodies. Determinant spreading is one reasonable explanation for the oligoclonal disorders in autoimmune thyroid disease.
8.1. T-cell epitope spreading Davies et al. [50] studied the T-cell receptor (TcR) V gene families of intrathyroidal T-cells from patients with autoimmune thyroid diseases. The restricted usage of TcR Va and Vb gene families was marked early in the onset of Graves’ disease, but Vb restriction was lost in patients with long-term disease. In contrast, the usage of the TcR V gene family was poorly restricted in Hashimoto’s thyroiditis with a long natural history.
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recognized by representative monoclonal antibodies from the three groups were assayed using 30 overlapping peptides, and residues 91–119 and 261–280 were frequently recognized. One of the TSAbs bound to two regions (residues 190–213 and 287–305) of the TSHR in addition to its immunizing region. The TSAb activity shown by the monoclonal antibody was not eliminated in Cos-7 cells expressing TSHR with residues 339–367 deleted. These data also support the pathological importance of the concept of ‘determinant (epitope) spreading’ in initiating autoimmune thyroid disease; that is, repeated immune challenge by the immunodominant epitope induces the spreading of the recognition to intramolecular epitopes of TSHR thus producing an oligoclonal autoimmune disorder. During such spreading, some antibodies will acquire biological activities against thyrocytes, orbital tissues or fibroblasts and thus initiate autoimmunity. As noted above, there are many reports of TSHR epitopes, but it is important to review them from the viewpoint of: (i) immunodominance (their role in initiating thyroidal autoimmunity); (ii) as determinants of clinical symptoms; and (iii) in the modulation of autoimmunity by anti-idiotype antibody and following medication with anti-thyroidal drugs.
8.2. B-cell epitope spreading Vlase et al. [20] induced the intramolecular determinant spreading phenomenon of B-cell epitope using an animal model. They used recombinant ECD of the TSHR to immunize CBA/J mice and serially analyzed the epitope recognized by the antisera using a range of peptides. Initially, the antisera only recognized peptide 21–41. Repeated immunization with the antigen induced a new recognition toward the carboxyl-terminal of the extracellular domain of the TSHR. As detailed above, the TSH receptor specific region, especially the C-terminal of the extracellular domain, is highly immunogenic [14,29], but the biological significance of this region remains paradoxical. Antibodies raised to peptides in this region can stimulate the thyroid or inhibit TSH binding [22,34,51]. Furthermore, 80% of Graves’ patients and many with Hashimoto’s thyroiditis possess Igs or autoantibodies that bind peptides in this region [6,29,47,52]. Site-directed mutagenesis studies, however, clearly show no significant effect on cAMP induction by TSH or TSAb by deleting the region [1,4]. Sugawa et al. [53] repeated immunization of mice with peptide 354 – 367 and established monoclonal antibodies from BALB/c mice. The antibodies were selected by specific binding to the immunized peptide, but the monoclonal antibodies so obtained were not uniform, and were classified into three groups: (i) TSAb-positive; (ii) TSBAb-positive; and (iii) having no significant effect on cAMP production by the thyrocyte. Epitopes
9. Relationship between immunogenicity of the deletable region and the subunit structure of the TSHR There is accumulating and convincing evidence that the TSHR consists of 2-subunits [54–56], an N-terminal A-subunit and a C-terminal B-subunit connected by a disulphide bond, as predicted by Smith et al. [57] before the cloning of the TSHR cloning. As proposed in our previous reviews [7,8], from the similar behavior of mutations of Cys-301 and Cys-390, we assume these Cys residues form a disulphide bond bridge. From the size of the TSHR fragment, the cleavage site should thus be in the deletable region. Although the noncleaved form of the receptor is fully functional [58], TSHR probably exists and operates as a 2-subunit structure in the thyroid gland [8]. Our current hypothesis is that the immunogenic ‘C-peptide’ is released into the blood stream during the cleavage process, and triggers the autoimmune response against the TSHR by ‘epitope spreading’ (Fig. 2).
Acknowledgements This work was supported in part by grants-in-aid from the Japanese Ministry of Education (Nos. 0644128, 06671024, 07671129, 07557353 and 08671152), Mochida Foundation for Medical and Pharmaceutical Research, Kowa Foundation for Life Science, Shimizu
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Fig. 2. Schematic model of TSHR processing, subunit structure, and ‘‘epitope spreading’’. Seven transmembrane helices are eliminated.
Foundation for the Promotion of Immunology Research, Kyoto University Foundation, Inamori Foundation, Kurozumi Foundation, and Clinical Pathology Research Foundation of Japan (S.K.), and SRF for biomedical research (T.M.). We are grateful to Aiko Tamada for her excellent technical assistance, and to Eriko Ito for graphics.
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[43] Mohapatra, S. S. (1994) Determinant spreading: implications in allergic disorders. Immunol. Today 15, 596–597. [44] Lehmann, P. V., Sercarz, E.E., Forsthuber, T., Dayan, C.M. and Gammon, G. (1994) Determinant spreading and the dynamics of the autoimmune T-cell repertoire. Immunol. Today 15, 89 – 90. [45] McRae, B.L., Vanderlugt, C.L., Dal-Canto, M.C. and Miller, D.S.. (1995) Functional evidence for epitope spreading in the relapsing pathology of experimental autoimmune encephalomyelitis. J. Exp. Med. 182, 75–85. [46] Elson, C.J., Barker, R.N., Thompson, S.J. and Williams, N.A. (1995) Immunologically ignorant autoreactive T-cells, epitope spreading and repertoire limitation. Immunol. Today 16, 71 – 76. [47] James, J.A., Gross, T., Scofield, R.H. and Harley, J.B. (1995) Immunoglobulin epitope spreading and autoimmune disease after peptide immunization: Sm B/B%-derived PPPGMRPP and PPPGIRGP induce spliceosome autoimmunity. J. Exp. Med. 181, 453 – 461. [48] Li, H., Akamizu, T., Okuda, J., Sugawa, H., Matsuda, F., Tsubata, T. and Mori, T. (1995) Isolation of Epstein-Barr-virustransformed 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. [49] Okuda, J., Akamizu, T., Sugawa, H., Matsuda, F., Li, H. and 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. [50] Davies, T.F. (1992) Preferential use of T-cell receptor V genes in human autoimmune thyroid disease. Autoimmunity 13, 11 – 16. [51] Sakata, S., Ogawa, T., Matsui, I., Manshourl, T. and Atassi, M.Z. (1992) Biological activities against synthetic human thyro-
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tropin receptor peptides representing thyrotropin binding regions. Biochem. Biophys. Res. Commun. 182, 1369–1375. Nagy, E.V., Burch, H.B., Lukes, Y.G., Car, F.E., Kosugi, S., Kohn L.D. and Burman. K.D. (1993) Immunogenicity of a unique region of the human thyrotropin receptor. J. Endocrinol. Invest. 16, 485 – 493. Sugawa, H., Ueda, T., Zhang, M., Kosugi, S., Ueda, M. and Mori, T. (1995) Determinant spreading shown by monoclonal thyroid stimulating antibody produced against a TSH receptor peptide. Thyroid 5, S5. Loosfelt, H., Pichon, C., Jolivet, A., Misrahi, M., Caillou, B., Jamous, M., Vannier, B. and Milgrom, E. (1992) 2-subunit structure of the human thyrotropin receptor. Proc. Natl. Acad. Sci. USA 89, 3765 – 3769. Misrahi, M., Ghinea, N., Sar, S., Saunier, B., Jolivet, A., Loosfelt, H., Gerutti, M., Gevauchelle, G. and Milgrom, E. (1994) Processing of the precursors of the human thyroid-stimulating hormone receptor in various eukaryotic cells (human thyrocytes, transfected L cells and baculovirus-infected insect cells. Eur. J. Biochem. 222, 711 – 719. Couet, J., Sar, S., Jolivet, A., Hai, M.-T.V., Milgrom, E. and Misrahi, M. (1996) Shedding of human thyrotropin receptor ectodomain: involvement of a matrix metalloprotease. J. Biol. Chem. 271, 4545 – 4552. Smith, B.R., McLachlan, S.M. and Furmaniak, J. (1988) Autoantibodies to the thyrotropin receptor. Endocr. Rev. 9, 106– 121. Chazenbalk, G.D., McLachlan, S.M., Nagayama, Y. and Rapoport, B. (1996) Is receptor cleavage into 2-subunits necessary for thyrotropin action. Biochem. Biophys. Res. Commun. 225, 479– 484.
At The Cutting Edge
Epitope analysis of the thyrotropin receptor, 19971 Shinji Kosugi *, Hideo Sugawa, Toru Mori Department of Laboratory Medicine, Kyoto Uni6ersity School of Medicine, Room 223, First Clinical Research Building, 54 -Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606 -01, Japan Received 13 December 1996; accepted 22 January 1997
Keywords: Site-directed mutagenesis; Chimeric receptor; Synthetic peptide; Graves’ disease; Determinant spreading
1. Introduction By extensive mutagenesis studies of the N-terminal and C-terminal regions of the extracellular domain of the thyrotropin receptor (TSHR), which are non-homologous with gonadotropin receptors, we have localized thyroid-stimulating antibody (TSAb) epitopes in residues 30–33, 34– 37, 40, 42 – 45, 52 – 56 and 58 –61 in the N-terminal [1,2] and TSBAb (thyroid-stimulating blocking antibody) epitopes in residues 301, 385 and 390, in the C-terminal region of the extracellular domain, [1,3] which are also high affinity binding sites for TSH [4,5]. The N-terminal region also contributes to TSH binding [2]. Residues 303 – 382, which are flanked by two Cys residues, can be deleted without functional loss of the TSHR [4]. However, this region is highly immunogenic, and we have shown that IgGs from Graves’ patients significantly bound peptides from this region and speculated that it thus contributed to triggering the autoimmune response [4,6]. Fig. 1 schematically represents the TSHR structure and critical determinants for binding of TSH, TSAb and TSBAb. In the present review, we summarize data from laboratories other than our own; previous reviews [7,8] have provided detailed summaries and canvassed the implications of our studies. Although there are still some * Corresponding author. Fax: + 81 75 7513233; e-mail: [email protected] 1 Note: All residue numbers are counted from the methionine start site. All TSHR peptides are designated by the corresponding N- and C-terminal residue numbers.
conflicting findings, there appears to be a consensus on the following points, as outlined in the following sections.
2. The N-terminal region (especially residues 22–61) is the site of the major TSAb epitopes in the TSH receptor We have identified residues 30–33, 34–37, 40, 42–45, 52–56 and 58–61 as important for TSAb activity, which does not imply that other residues are not important. No functional substitution mutants of residues 38, 39, 41, 46–51, 57, 62, 75 and 76 were obtained, so that these residues could not be evaluated and it remains possible that these residues are also involved in TSAb/ TSH interaction. We assume that linear and/or conformational epitope(s) in residues 30–65 are critical for TSAb interaction, and although a consensus of the precise location of TSAb major epitope(s) has not been reached, there is a body of evidence supporting these observations, as follows.
2.1. Mutagenesis studies Nagayama et al. [9] reported that TSHR with substitutions at residues 1–170 lost TSAb activity. Further, Nagayama et al. [10] and Jaume et al. [11] showed that half of Graves’ IgGs lost TSAb activity in TSHR with substitutions at residues 25–30. Tahara et al. [12] demonstrated that 67% of Graves’ IgGs showed a loss
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Fig. 1. Putative membrane topology and important determinants for TSH, TSAb and TSBAb binding on the TSHR. Closed diamonds (residues 22 – 61) indicate TSH/TSAb binding sites. Three large closed circles (Cys-301, Tyr-385 and Cys-390) are critical residues forTSH/TSBAb binding. Small closed circles (residues 317–366) and hexagons indicate a full deletable region (residues 303 – 382).
S. Kosugi et al. / Molecular and Cellular Endocrinology 128 (1997) 11–18
of TSAb activity in TSHR with substitutions at residues 8–165, but not in mutants with substitutions at residues 90–165. Kim et al. [13] reported that TSAb activity for 85% of Graves’ IgGs was lost in TSHR with substitutions at residues 9 – 165.
2.2. Peptide binding Murakami et al. [14] showed 125I-labeled peptide 32–56 bound to Graves’ IgGs. Ikeda et al. [15] demonstrated that some Graves’ sera but not Hashimoto’s sera bound peptide 29 – 57 by enzyme linked immunosorbent assay (ELISA). Sugawa et al. [16] showed that peptide 31–50 absorbed TSAb activity of Graves’ IgGs.
2.3. Peptide immunization Endo et al. [17] immunized rabbits with peptide 29–57, and sera from the immunized rabbits showed significant TSAb activity. Matsui et al. [18] immunized rats with five different TSHR peptides; TSAb activities were highest in rats immunized with peptide 22–50. Murakami et al. [19] reported TSAb activity in sera from rabbits immunized with peptide 32 – 56.
2.4. Immunization of mice with extracellular domain of the TSHR Valse et al. [20] immunized mice with recombinant TSHR extracellular domain (ECD) from both an E. Coli and a baculovirus expression system. All sera from BALB/c and CBA/J mice recognized peptide 22–41 but had no TBII (thyrotropin-binding inhibitory immunoglobulin) activity. Gattadahalli et al. [21] immunized mice with TSHR ECD to produce a panel of monoclonal antibodies to the TSHR; one of the antibodies was recognized by peptide 22 – 30, but had no TBII activity.
3. The C-terminal region of the extracellular domain of the TSH receptor (especially Cy-301, Tyr-385 and Cys-390) is the site of the major TSBAb/TBII epitopes in the TSHR
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3.2. Peptide immunization Ohmori et al. [22] reported that chickens immunized with peptide 372–392 produced both TSBAb and TBII activity, but no TSAb activity. Murakami et al. [19] showed that rabbits immunized with peptide 352–378 had TSBAb activity, and Dallas et al. [23] that rabbit antibodies against peptide 367–386 had both TBII and TSBAb.
3.3. ECD immunization Dallas et al. [24] immunized rabbits with baculovirusgenerated ECD of the TSHR producing an antibody which showed TBII activity reversed by preincubation with peptides 292–311, 367–386 and 377–397. Antibodies affinity-purified with these peptides showed TBII activity, indicating the importance of these regions for binding of TSH and blocking antibodies. These observations are thus in close agreement with our mutagenesis studies identifying Cys-301, Tyr-385 and Cys-390 as critical for binding of TSH and blocking antibodies. Wagle et al. [25] similarly immunized BALB/cJ and SJL/J mice with baculovirus-generated ECD of the TSHR, and showed that addition of peptide 367–386 significantly reduced TBII activity of antisera from both groups. 4. Major TSH-binding sites are located at the C-terminus, but the N-terminus also contributes to TSH binding Ohmori et al. [22] showed that preincubation of TSH with peptide 35–50 dose-dependently decreased cAMP accumulation in thyroid cells. Attasi et al. [26] reported that [125I]TSH bound to peptide 32–50. Morris et al. [27] showed that TSH bound to peptides 16–35 and 286–305. Nagayama et al. [9] reported that residues 1–260 also contributed TSH binding using TSHR/ lutropin-choriogonadotropin receptor (LH-CGR) chimeras. 5. The C-terminal region (residues 303–382) can be deleted without functional loss, but some antibodies bind to this region, indicating its importance for immunogenicity
3.1. Mutagenesis
5.1. Mutagenesis studies
Nagayama et al. [9] showed that TSHR with substitutions at residues 260 – 418 decreased the TSH-cAMP response 7-fold in sensitivity, but retained TSAb activity to a similar extent to wild-type (WT). The TBII activity of sera from idiopathic myxedema was markedly decreased with this mutant TSHR.
It is clear that a region in the C-terminal of the TSHR extracellular domain of the TSHR can be deleted without functional loss. Thus, Wasthworth et al. [28] demonstrated that TSHR with deletion of residues 317–366 completely retained TSH binding, and TSH- and TSAb-stimulated cAMP responses.
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5.2. Peptide binding
6.1. Peptide binding
Mori et al. [29] reported that peptide 353–363 did not bind TSH but bound TSAb of Graves’ sera, and that preincubation with peptide decreased TSAb activity. Takai et al. [30] demonstrated that Graves’ sera bound peptide 352 – 366 on ELISA. Ikeda et al. [15] reported that some Graves’ sera but not Hashimoto’s sera bound peptide 359 – 371 on ELISA. Murakami et al. [31] showed that iodinated peptide 352 – 378 bound 83% of IgGs from Hashimoto’s patients without TBII activity, and a small proportion of IgGs from Graves’ patients. Ueda et al. [32] reported that two of 100 Graves’ IgGs had TSAb enhancing activity on addition of peptide 354–367, and also that there were antibodies which bound to regions 354 – 357 and 364 –367 in a small population of Graves’ patients. Further, Ueda et al. [33] and Sugawa et al. [16] showed that TSAb activity of some Graves’ IgGs was absorbed by peptides 338–353 and/or 354 – 367.
Sugawa et al. [16] showed that peptides 91–119 absorbed TSAb activity of Graves’ IgGs. Piraphatdist et al. [36] reported that peptide 123–131 did not bind TSH, but that preincubation of peptide with patients’ sera caused a decrease in TBII activity of TBII positive sera and an increase in TSAb activity of some Graves’ sera, concluding that the peptide thus bound TBII without TSAb activity. Ikeda et al. [15] showed that some Graves’ but no Hashimoto’s IgGs bound peptide 172–202 on ELISA. Morris et al. [27], using a series of 26 overlapping peptides comprising the entire extracellular domain and the three extracellular loops, tested the inhibition of [125I]TSH binding to thyroid membranes. They showed that TSH bound displaceably to peptide 256–275 with a Kd of 3× 10 − 5 M. Further, Morris et al. [37], using the same series of peptides, demonstrated that Graves’ IgGs bound peptide 181– 200 on ELISA.
5.3. Peptide immunization Endo et al. [34] immunized rabbits with peptide 341–370, and the antisera showed significant TSAb activities. Ohmori et al. [22] reported that immunization of chickens with peptide 341 – 358 produced TSBAb and TBII activity but no TSAb activity.
5.4. ECD immunization Dallas et al. [35] showed that antisera from rabbits immunized with baculovirus-generated ECD of the TSHR reacted with peptide 352 – 388, and they identified a dominant epitope 357 – 372 in this region. Further, Dallas et al. [23] demonstrated that rabbit antibodies against peptide 357 – 372 had TSBAb but no TBII.
6. Studies on the middle region of the extracellular domain of the TSHR Most epitope analyses and mutagenesis studies have focused on the N-terminal and C-terminal regions of the extracellular domain of the TSHR, where homology with related gonadotropin receptors is very low. The importance of the middle region of the TSHR extracellular domain, which has high homology with gonadotropin receptors, might thus have been inadequately studied. However, no specific portions in the middle region have been identified by various experimental procedures in different laboratories. A summary of the findings obtained for this region follows.
6.2. Peptide immunization Endo et al. [35] showed TSAb activity in sera of rabbits immunized with peptide 172–202.
7. Studies of the exoplasmic loops. All portions of the TSH receptor on the outer surface of the cell can be immunogenic. In this aspect, Endo et al. [38] reported that immunization with peptides corresponding to the three extracellular loops induced TSAb. One might speculate that the binding of antibodies to extracellular loops connecting bundles of transmembrane helix that directly interact with Gproteins may cause a conformational change, to disrupt the interhelical bonds maintaining the inactive state of the receptor. In this context, it needs to be determined if antibodies against LHR peptide also cause activation of LHR, and it is uncertain if such antibodies are really produced in Graves’ patients. However, Morris et al. [37], using the same series of peptides as described above, showed Graves’ IgGs bound peptide 629–639 corresponding to the 3rd exoplasmic loop on ELISA, and that TSAb activity was enriched by affinity purification using the peptide. In contrast, Ohmori et al. [22] reported that immunization of chicken with peptide 649–661 produced TSBAb and TBII activity, but no TSAb activity.
8. Epitope spreading of the TSHR The new concept of determinant spreading (epitope spreading) has been highlighted as a possibly important
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initiator of autoimmune disorders [39 – 47]. The fundamental concept is that repetitive immunological stress with a highly immunodominant epitope induces aberrant recognition of the cryptic epitope(s) of an autoantigen and thus induces autoimmunity. As described above, polyclonal TSHR antibodies are detected in the sera of patients with autoimmune TSHR diseases [10,16,33,48,49]; autoimmune thyroid disease is thought to be an oligoclonal phenomenon, and there is no clear explanation for the simultaneous appearance of these autoantibodies. Determinant spreading is one reasonable explanation for the oligoclonal disorders in autoimmune thyroid disease.
8.1. T-cell epitope spreading Davies et al. [50] studied the T-cell receptor (TcR) V gene families of intrathyroidal T-cells from patients with autoimmune thyroid diseases. The restricted usage of TcR Va and Vb gene families was marked early in the onset of Graves’ disease, but Vb restriction was lost in patients with long-term disease. In contrast, the usage of the TcR V gene family was poorly restricted in Hashimoto’s thyroiditis with a long natural history.
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recognized by representative monoclonal antibodies from the three groups were assayed using 30 overlapping peptides, and residues 91–119 and 261–280 were frequently recognized. One of the TSAbs bound to two regions (residues 190–213 and 287–305) of the TSHR in addition to its immunizing region. The TSAb activity shown by the monoclonal antibody was not eliminated in Cos-7 cells expressing TSHR with residues 339–367 deleted. These data also support the pathological importance of the concept of ‘determinant (epitope) spreading’ in initiating autoimmune thyroid disease; that is, repeated immune challenge by the immunodominant epitope induces the spreading of the recognition to intramolecular epitopes of TSHR thus producing an oligoclonal autoimmune disorder. During such spreading, some antibodies will acquire biological activities against thyrocytes, orbital tissues or fibroblasts and thus initiate autoimmunity. As noted above, there are many reports of TSHR epitopes, but it is important to review them from the viewpoint of: (i) immunodominance (their role in initiating thyroidal autoimmunity); (ii) as determinants of clinical symptoms; and (iii) in the modulation of autoimmunity by anti-idiotype antibody and following medication with anti-thyroidal drugs.
8.2. B-cell epitope spreading Vlase et al. [20] induced the intramolecular determinant spreading phenomenon of B-cell epitope using an animal model. They used recombinant ECD of the TSHR to immunize CBA/J mice and serially analyzed the epitope recognized by the antisera using a range of peptides. Initially, the antisera only recognized peptide 21–41. Repeated immunization with the antigen induced a new recognition toward the carboxyl-terminal of the extracellular domain of the TSHR. As detailed above, the TSH receptor specific region, especially the C-terminal of the extracellular domain, is highly immunogenic [14,29], but the biological significance of this region remains paradoxical. Antibodies raised to peptides in this region can stimulate the thyroid or inhibit TSH binding [22,34,51]. Furthermore, 80% of Graves’ patients and many with Hashimoto’s thyroiditis possess Igs or autoantibodies that bind peptides in this region [6,29,47,52]. Site-directed mutagenesis studies, however, clearly show no significant effect on cAMP induction by TSH or TSAb by deleting the region [1,4]. Sugawa et al. [53] repeated immunization of mice with peptide 354 – 367 and established monoclonal antibodies from BALB/c mice. The antibodies were selected by specific binding to the immunized peptide, but the monoclonal antibodies so obtained were not uniform, and were classified into three groups: (i) TSAb-positive; (ii) TSBAb-positive; and (iii) having no significant effect on cAMP production by the thyrocyte. Epitopes
9. Relationship between immunogenicity of the deletable region and the subunit structure of the TSHR There is accumulating and convincing evidence that the TSHR consists of 2-subunits [54–56], an N-terminal A-subunit and a C-terminal B-subunit connected by a disulphide bond, as predicted by Smith et al. [57] before the cloning of the TSHR cloning. As proposed in our previous reviews [7,8], from the similar behavior of mutations of Cys-301 and Cys-390, we assume these Cys residues form a disulphide bond bridge. From the size of the TSHR fragment, the cleavage site should thus be in the deletable region. Although the noncleaved form of the receptor is fully functional [58], TSHR probably exists and operates as a 2-subunit structure in the thyroid gland [8]. Our current hypothesis is that the immunogenic ‘C-peptide’ is released into the blood stream during the cleavage process, and triggers the autoimmune response against the TSHR by ‘epitope spreading’ (Fig. 2).
Acknowledgements This work was supported in part by grants-in-aid from the Japanese Ministry of Education (Nos. 0644128, 06671024, 07671129, 07557353 and 08671152), Mochida Foundation for Medical and Pharmaceutical Research, Kowa Foundation for Life Science, Shimizu
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Fig. 2. Schematic model of TSHR processing, subunit structure, and ‘‘epitope spreading’’. Seven transmembrane helices are eliminated.
Foundation for the Promotion of Immunology Research, Kyoto University Foundation, Inamori Foundation, Kurozumi Foundation, and Clinical Pathology Research Foundation of Japan (S.K.), and SRF for biomedical research (T.M.). We are grateful to Aiko Tamada for her excellent technical assistance, and to Eriko Ito for graphics.
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