Human Thyrotropin (TSH) Variants Designed by Site-Directed Mutagenesis Block TSH Activity in Vitro and in Vivo
http://www.100md.com
内分泌学杂志 2005年第6期
Department of Molecular Genetics (N.A., R.B.-S., F.F.) and Endocrine Research Unit (R.B.-S., Z.K.), Carmel Medical Center and the Faculty of Medicine, Technion-Israel Institute of Technology, Haifa 34362, Israel
Address all correspondence and requests for reprints to: Dr. Fuad Fares, Department of Molecular Genetics, Carmel Medical Center, Michal 7 Street, Haifa 34362, Israel. E-mail: fares@clalit.org.il.
Abstract
TSH is a heterodimeric glycoprotein hormone synthesized in the pituitary and composed of a specific ?-subunit and a common -subunit shared with FSH, LH, and human chorionic gonadotropin. The heterodimer was previously converted into a biologically active single chain protein by genetic fusion of the genes coding to both subunits in the presence of the carboxy-terminal sequence of human (h) chorionic gonadotropin-? subunit as a linker [hTSH?-carboxyl-terminal peptide (CTP)-]. N-linked carbohydrate-free single-chain TSH variants were constructed by site-directed mutagenesis and overlapping PCR: one devoid of both N-linked oligosaccharide chains on the -subunit (hTSH?-CTP-deg) and the other lacking also the oligosaccharides on the ?-subunit (hTSH?deg-CTP-deg). These variants were expressed in Chinese hamster ovary cells and secreted into the culture media. We have previously reported that the variants block the activities of hTSH and thyroid-stimulating immunoglobulins in cultured human thyroid follicles. In the present study, binding affinity of hTSH variants to hTSH receptor and the localization of the antagonistic effect were examined. Moreover, the effect of these variants on TSH activity was tested in vivo. The results of the present study indicate that the hTSH variants bind to the hTSH receptor with high affinity. Experiments using forskolin also indicated that the N-linked carbohydrate-free TSH single-chain variants inhibit TSH activity at the receptor-binding site and not at a postreceptor level. Moreover, the variants significantly inhibited (about 50%) TSH activity with respect to thyroid hormone secretion in vivo in mice. These variants may offer a novel therapeutic strategy in treating hyperthyroidism.
Introduction
TSH IS A PITUITARY glycoprotein composed of two subunits: the -subunit common to TSH, FSH, LH and human chorionic gonadotropin (hCG), and a specific ?-subunit, which confers TSH bioactivity (1, 2). Human (h)TSH subunits are synthesized in the pituitary thyrotroph cells, assembled, and secreted into the circulation. Assembly of the subunits is the rate-limiting step for secretion, receptor binding, and bioactivity of TSH (1, 2, 3). To overcome this limitation, hTSH was converted to a single-chain polypeptide (4). The carboxyl end of TSH? was fused to the amino end of the -subunit, thus forming hTSH?- single chain. The fusion was also performed in the presence of the carboxyl-terminal peptide (CTP) of the ?-subunit of hCG forming TSH?-CTP-. CTP was used as a linker sequence between the subunits, which are presumed to be required for flexibility, hydrophilicity, and stability. Previous studies have shown that ligation of CTP to hFSH? (5) hTSH? (6), or hCG (7) did not affect assembly, secretion, or in vitro bioactivity (4, 8, 9, 10, 11, 12), but it increased bioactivity and half-life of the hormones in vivo (5, 6, 7). We have shown that secretion of the single peptide chains were significantly higher than that of hTSH wild type (WT) (4). Receptor binding and in vitro bioactivity of the single-peptide chains were similar to that of hTSH-WT (4).
TSH? and TSH subunits contain one or two N-linked oligosaccharide chains, respectively (1, 2). These residues have been shown to play a critical role in assembly, secretion, and bioactivity of hTSH (1, 13). We have shown that deletion of oligosaccharide units from the -subunit resulted in a significant reduction in cAMP formation and T3 secretion from cultured human thyroid follicles (14). Similarly, hTSH single-peptide variants lacking the oligosaccharide chains from -subunit (TSH?-CTP-deg) or ?-subunit as well (TSH?deg-CTP-deg) were less potent than TSH WT (15). Moreover, N-linked carbohydrate-free hTSH single-peptide chains inhibited the in vitro activity of hTSH and human thyroid-stimulating immunoglobulin (TSI), the etiologic factor inducing the most common cause for hyperthyroidism (Graves’ disease), in a dose-dependent manner (15). It was therefore suggested that these variants behave as potential antagonists of hTSH and TSI, which may offer novel therapeutic agents in the treatment of hyperthyroidism and Graves’ disease.
In the present study, we evaluated the affinity of the single-chain hTSH variants to human TSH receptor. Moreover, we examined whether the antagonistic effect of these variants is located at the TSH receptor binding site or at a postreceptor level. In addition, the antagonistic effect of hTSH variants was investigated in vivo.
Materials and Methods
Materials
Purified bovine (b) TSH and T3 were obtained from Sigma (St. Louis MO). G418 was obtained from Gibco (Uxbridge, UK). 125IbTSH was purchased from (Kronus, Boise, ID). 125IcAMP was obtained from Amersham Biosciences UK Ltd. (Little Chalfont, Buckinghamshire, UK). Cell culture media and reagents were obtained from Biological Industries (Beit Haemek, Israel). Rabbit antiserum against hTSH-dimer was purchased from Fitzgerald (Concord, MA). TSI (MRC Research standard B, 65/122) was a gift from the National Institute for Biological Standards and Control (London, UK). Chinese hamster ovary (CHO) cells stably expressing human TSH receptor were kindly supplied by Dr. Basil Rapoport (Cedars-Sinai Medical Center, Los Angeles, CA).
hTSH variants
Using site-directed mutagenesis and overlapping PCR, two N-linked carbohydrate-free single-chain hTSH variants were constructed in our laboratory as described before (15): one devoid of both N-linked oligosaccharide chains on the -subunit (hTSH?-CTP-deg) and the other lacking also the oligosaccharides on the ?-subunit (hTSH?deg-CTP-deg). The chimeric genes coding for both variants were inserted into eukaryotic expression vector (PM2HA) as described before (15).
DNA transfection and clone selection
CHO cells were transfected with the expression vectors coding for the hTSH variants (PM2TSH?-CTP-deg and PM2TSH?deg-CTP-deg) using the calcium phosphate precipitation method (16). Stable clones were selected by growing the cells in a G418-containing medium (0.25 mg/ml). Transfected clones were screened for the expression of hTSH variants by Western blotting.
Cell culture
Transfected CHO clones were maintained in Ham’s F-12 medium containing 100 U/ml penicillin, 100 mg/ml streptomycin, 2 mM glutamine, 0.25 mg/ml active G418, and 5% fetal calf serum at 37 C in humidified 5% CO2 incubator.
For hormone collection, stable clones expressing TSH WT and mutants were grown to confluency in T-75 flasks. Cells were washed twice with PBS, and medium was replaced by serum-free medium. Media were collected every 24 h, clarified by centrifugation, and concentrated through 10-Da selective membrane, using slow centriprep concentrator (Amicon Corp., Danvers, MA) followed by additional concentration into small volumes with viva-20 spin columns (Biological Industries). Concentrations of the TSH variants in concentrated condition medium were determined using immunoradiometric assay and a double antibody (Diagnostic Products Corp., Los Angeles, CA).
Western blotting
Samples were electrophoresed on nondenaturing 15% sodium dodecyl sulfate-polyacrylamide gels as described before (17). Gels were allowed to equilibrate for 10 min in 25 mM Tris and 192 mM glycine in 20% (vol/vol) methanol (14). Proteins were transferred to a 0.2 μm pore size nitrocellulose membrane (Sigma) at 250 mA for 3 h, using a minitransblot electrophoresis cell (Bio-Rad Laboratories, Richmond, CA) according to the method described in the manual accompanying the unit. The nitrocellulose membrane was incubated in 5% nonfat dry milk for 2 h at room temperature. The membrane was incubated with ?-subunit antiserum (1:1000 titer) overnight at 4 C followed by three consecutive washes in PBS containing 0.1% Tween 20 (10 min/wash). The membrane was incubated with secondary antibody conjugated to horseradish peroxidase (Zymed, San Francisco, CA) for 2 h at room temperature followed by three washes. Finally, the nitrocellulose paper was reacted with enhanced chemiluminescent substrate (Pierce, Rockford, IL) for 5 min, dried on a filter paper (Maidstone, UK), and exposed to x-ray film.
Receptor binding
TSH binding assays were performed using CHO cells stably transfected with hTSH receptor. Cells were plated in 12-well plates (4 x 105 cells/well) and incubated with 125IbTSH (30,000 cpm/well) with or without varying concentrations of unlabeled variants (0.1–2,000 μU/ml) for 16–18 h at 4 C. The cells were then washed three times with cold Krebs Ringer buffer (280 mM sucrose, 3 mM CaCl2, 20 mM HEPES, 5 mM KCl, 1 mM MgSO4, 7 mM NaHCO3, 0.25% BSA) and lysed with 1 M NaOH. Lysates were collected into 12 x 75 plastic tubes, and the rates of 125Ibovine TSH replacement by TSH variants were determined using -counter (18).
In vitro bioactivity of TSH variants
The bioactivity of hTSH variants was determined by measuring their ability to stimulate cAMP formation from cultured human thyroid follicles as described previously (19). Cells were plated in 24-well plates (200 x 103 cells/well) with or without varying concentrations of hTSH variants or forskolin. At the start of the culture period, 1-methyl-3-isobutylxanthine (0.5 mM) was added to the medium. cAMP was measured by RIA as described previously (19).
In vivo bioactivity of TSH variants
Male BALB/c mice (25–30 g weight) were fed with a diet of normal rodent chow and supplied with drinking water containing 3 μg/ml (4.46 μM) T3 for 4 d for endogenous TSH suppression (20). Animals (five per group) were injected ip (200 μl/injection volume) with bovine TSH and/or TSH variants. The control group consisted of animals injected with 200 μl of the maintaining medium. Blood spots from mice tails were blotted to a paper filter at 0, 4, and 8 h after injection. Blood T4 concentrations were determined by RIA with human neonatal T4 kit (Diagnostic Products Corp., Los Angeles, CA).
Statistical analysis
Each experiment was repeated at least three times, and results were expressed as the mean ± SEM. Statistical analysis of the data was performed using Student’s t test and ANOVA. P < 0.05 was considered significant.
Results
hTSH variant genes, WT, and mutants were inserted into PM2HA, a eukaryotic expression vector, and transfected into CHO cells. Clones resistant to G418 and which secreted hTSH variants were selected. Concentrations of TSH variants in the condition media were measured using TSH RIA and a double antibody.
Using Western blot analysis and TSH antiserum after nondenaturing, SDS-PAGE allowed detecting the secretion of TSH variants in the media (electrophoresis under nondenaturing conditions prevents separation of hTSH subunits). The results indicated that the N-linked carbohydrate-free mutant TSH variants TSH?-CTP-deg (Fig. 1, lane 2) and TSH?deg-CTP-deg (Fig. 1, lane 3) migrated faster than TSH WT (Fig. 1, lane 1) due to lower content of oligosaccharide chains. On the other hand, TSH?-CTP- single chain migrated slower (Fig. 1, lane 4) due to the addition of O-linked oligosaccharides to the CTP.
FIG. 1. Expression and secretion of hTSH variants in a CHO cell system detected by gel electrophoresis and Western blotting. Media samples from transfected CHO cells were separated under nondenaturing conditions on 15% polyacrylamide SDS-PAGE. After protein transfer, the nitrocellulose PAGE was exposed to polyclonal antibody against hTSH?-subunit. Molecular mass markers are indicated by arrowheads in kilodaltons.
Receptor binding assays were performed using CHO cells expressing the hTSH receptor. Receptor binding of human dimeric hTSH WT as well as N-linked carbohydrate-free single-chain variants (TSH?-CTP-deg and TSH?deg-CTP-deg) displaced 125I bTSH in a dose-dependent manner (P < 0.05) (Fig. 2). Interestingly, the displacement by N-linked carbohydrate-free single-chain constructs (TSH?-CTP-deg and TSH?deg-CTP-deg) occurred with lower concentrations, compared with hTSH dimer WT. The IC50s detected for dimeric hTSH-WT, TSH?deg-CTP-deg, and TSH?-CTP-deg were 200, 100, and 18 μU/ml, respectively. The results thus indicated that N-linked carbohydrate-free variants bind with high affinity to hTSH receptor, with the highest affinity belonging to the TSH?-CTP-deg variant (Fig. 2).
FIG. 2. Receptor binding of hTSH variants to human TSH receptor. Stable transfected CHO cells expressing the hTSH receptor were incubated with 125I bTSH in the presence of varying concentrations of hTSH variants. Displacement curves show the percentage of maximal binding of the isotope at each concentration of unlabeled sample. Data shown are the mean ± SD of three experiments.
To determine whether the antagonistic effects of hTSH variants are at the receptor-binding site or a postreceptor level, the effects of hTSH variants on forskolin activity were tested. Forskolin, as expected, increased cAMP levels because it is known to activate adenylate cyclase (AC) (Fig. 3, A and B). The cAMP levels induced by both N-linked carbohydrate-free variants were extremely low. Surprisingly, combination of these variants with forskolin resulted in a synergistic effect with respect to cAMP levels. Similarly, cAMP levels increased synergistically (2- to 7-fold) after combined treatment of forskolin with the hTSH-WT and TSI (Fig. 4, A and B).
FIG. 3. Biological activity of hTSH variants in the presence or absence of forskolin. cAMP formation was measured after exposure of human thyroid follicles for 7 d at 37 C to different concentrations (10, 50, 100 μU/ml) of hTSH?CTP-deg (A) and hTSH?degCTP-deg (B) in the presence or absence of forskolin (Fsk). Each graph is a representative experiment from a series of three different experiments. Each bar represents the mean ± SD of four replicates. *, P < 0.05; ***, P < 0.005, with respect to Fsk.
FIG. 4. Biological activity of hTSH and TSI in the presence or absence of forskolin. cAMP formation was measured after exposure of human thyroid follicles for 7 d at 37 C to different concentrations (10, 50, 100 μU/ml) of hTSH (A) and TSI (0.5, 0.75, 1.5 mU/ml) (B) in the presence or absence of forskolin (Fsk). Each graph is a representative experiment from a series of three different experiments. Each bar represents the mean ± SD of four replicates. **, P < 0.01; ***, P < 0.005, with respect to Fsk.
The effects of hTSH variants were also examined in vivo in mice. Preliminary experiments indicated that treatment of the animals with T3 for 4 d suppressed TSH levels resulting in undetectable T4 levels in the circulation. Treatment of these animals with bTSH-induced T4 secretion in a time-dependent manner, with maximal levels at 4–8 h (data not shown). The blood T4 levels induced by hTSH N-linked carbohydrate-free variants (TSH?deg-CTP-deg, and TSH?-CTP-deg) were significantly lower than that induced by bTSH (P < 0.01) (Figs. 5 and 6). Moreover, treatment with bTSH together with the N-linked carbohydrate-free variants (TSH?-CTP-deg, or TSH?deg-CTP-deg) decreased significantly the bTSH-induced T4 levels up to about 50% (P < 0.05) (Figs. 5 and 6).
FIG. 5. Biological activity of hTSH?CTP-deg and bTSH in vivo. Mice were fed with diet of normal rodent chow and supplied with drinking water containing 3 μg/ml T3 for 4 d to suppress endogenous TSH. Groups (five animals per group) were injected as follows: bTSH (20 mU /mouse) (A); hTSH?CTP-deg (2 mU/mouse) (B); hTSH?CTP-deg (2 mU/mouse) + bTSH (20 mU/mouse); control animals (200 μl of culture medium) (C). D, In the latter group, the T4 levels were undetectable. Bars represent mean ± SD values of two experiments. *, P < 0.05; **, P < 0.01, with respect to bTSH.
FIG. 6. Biological activity experiments of hTSH?degCTP-deg and TSH in vivo. Mice were fed with diet of normal rodent chow and supplied with drinking water containing 3 μg/ml T3 for 4 d to suppress endogenous TSH. Groups (five animals per group) were injected as the following: bTSH (20 mU/mouse) (A); hTSH?degCTP-deg (2 mU/mouse) (B); hTSH?degCTP-deg (2 mU/mouse) + bTSH (20 mU/mouse) (C); control animals (200 μl of culture medium) (D). In the latter group, the T4 levels were undetectable. Bars represent mean ± SD values of two experiments. *, P < 0.05; **, P < 0.01, with respect to bTSH.
Discussion
The present study indicates that N-linked carbohydrate-free single-peptide chain variants of hTSH bind to hTSH receptor with high affinity. These variants reduced the in vitro bioactivity of TSH via inhibition at the TSH receptor-binding site. Moreover, the variants reduced TSH bioactivity (thyroid hormone secretion) in vivo. The role of carbohydrate chains bound to glycoprotein hormones has been widely investigated. Chemical removal of N-linked carbohydrate chains from hCG (21) and LH (22) was shown to decrease biological activity. In addition, the bioactivities of carbohydrate-free dimeric FSH variants, designed by site-directed mutagenesis, were markedly reduced (23). FSH and hCG synthesized in mutant CHO cells with a defect in the processing of N-linked carbohydrates were inactive in vivo (24, 25). Deletion of either or both oligosaccharides on the TSH- subunit did not prevent the dimerization and secretion of the formed TSH variants but resulted in a decreased activity of carbohydrate-free TSH dimer in vitro (14). These findings suggest a critical role of carbohydrates in the function of glycoprotein hormones.
Site-directed mutagenesis may affect assembly of - and ?-subunits and possibly secretion of the dimeric hormone. To overcome this limitation, human TSH heterodimer was converted into a biologically active single-peptide chain. This was performed by gene fusion of the TSH subunits genes with or without the CTP as a linker between the subunits. Both variants were efficiently secreted from CHO cells, bound with high affinity to the TSH receptor, and were even more active than the normal TSH dimer (4). Removal of N-linked carbohydrates by site-directed mutagenesis from the -subunit (TSH?-CTP-deg) and also from the ?-subunit (hTSH?deg-CTP-deg) resulted in a significant decrease in bioactivity in vitro (15). Competitive experiments between the N-linked carbohydrate-free variants and hTSH, as well as TSI, showed inhibition of TSH bioactivity by the variants (15). These findings led to the concept that the N-linked carbohydrate-free variants may serve as TSH antagonists.
To reveal the mechanism of the inhibition of hTSH and TSI by mutant TSH variants, receptor-binding assays were conducted. The finding that the N-linked carbohydrate-free TSH single chains bind to hTSH receptor with higher affinity than dimeric TSH is in correlation with our previous report that TSH?-CTP-deg had the highest potency in inhibiting hTSH and TSI activities (15). However, this is in contrast to the report that dimeric FSH devoid of carbohydrate chains on the -subunit alone has lower receptor binding potency than FSH dimer devoid of all carbohydrate chains on - and ?-subunits (23).
The results thus indicated that the mutated TSH single-chain polypeptides behave as hTSH antagonist by competing at the receptor level. The question as to whether the inhibitory effect also takes place at a postreceptor level was addressed. The surprising synergistic effects of the TSH variants together with forskolin on AC activity, in addition to the findings of the receptor binding assays, demonstrate that the N-linked carbohydrate-free hTSH variants inhibit TSH at the level of the receptor-binding site and not at a postreceptor level.
The mechanism of such synergism, however, is not clear and to the best of our knowledge has not been reported yet. Forskolin is known to bind to the pseudocatalytic site on the C2 domain of AC, which also contains the binding site of GS (26, 27). Because forskolin has been reported to have the ability to potentiate GS-mediated activation of AC (28, 29, 30, 31) and amplify the response to weak or partial agonists of TSH (32), this may tentatively provide a hypothetical explanation for the synergistic effect of TSH variants with forskolin.
The antagonistic effects of N-linked carbohydrate-free TSH variants previously reported in vitro (15) were also studied in vivo in mice. Although the potency of bTSH to activate TSH receptor in mice is known to be higher than that of hTSH (20), the N-linked carbohydrate-free TSH single chains were able to decrease the bTSH-induced thyroid hormone formation by about 50%.
The data thus show that the N-linked carbohydrate-free TSH variants, despite slight agonistic activity, behave as partial TSH antagonists in vitro and in vivo by binding to the TSH receptor with high affinity. These findings may open new horizons regarding the treatment of hyperthyroidism. The treatment of hyperthyroidism has essentially remained unchanged for the past 30 yr and includes the use of radioactive [131I] iodine, surgery, and antithyroid drugs, such as propylthiouracil and methimazole, which inhibit thyroid hormone synthesis. Each approach has its own intrinsic limitations and/or side effects (33, 34). Propylthiouracil and methimazole act slowly and can take up to 6–8 wk to fully deplete the thyroid gland of intrathyroidal stores of iodinated thyroglobulin, during which time hyperthyroidism can have severe consequences in certain individuals. Radiochemical destruction of thyroid tissue by 131I may require 4–6 months to be fully effective and may worsen ophthalmopathy. Surgical thyroidectomy is invasive and is usually preceded with antithyroid drugs to prevent life-threatening complications, such as thyroid storm. These observations emphasize the need for the development of TSH and TSI antagonists, which may overcome the limitations and disadvantages of the traditional treatments of hyperthyroidism.
Further studies are, of course, needed to evaluate the in vivo potency of the hTSH antagonists developed in our laboratory and determine the optimal doses for achieving maximal effects in blocking TSH and TSI activities. Moreover, some difficulties might be faced when implementing such treatment, such as parenteral administration, short half-life, and costs. The potential risk of hypothyroidism would also have to be considered. We believe that the strategy of constructing mutated single-chain TSH variants could lead to the development of a novel strategy in treating hyperthyroidism.
Acknowledgments
We are grateful to Dr. Basil Rapoport (Cedars-Sinai Medical Center, Los Angeles, CA) for supplying the CHO cells expressing human TSH receptor. We also thank the Israeli Ministry of Science for granting a postdoctoral fellowship (to N.A.).
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Address all correspondence and requests for reprints to: Dr. Fuad Fares, Department of Molecular Genetics, Carmel Medical Center, Michal 7 Street, Haifa 34362, Israel. E-mail: fares@clalit.org.il.
Abstract
TSH is a heterodimeric glycoprotein hormone synthesized in the pituitary and composed of a specific ?-subunit and a common -subunit shared with FSH, LH, and human chorionic gonadotropin. The heterodimer was previously converted into a biologically active single chain protein by genetic fusion of the genes coding to both subunits in the presence of the carboxy-terminal sequence of human (h) chorionic gonadotropin-? subunit as a linker [hTSH?-carboxyl-terminal peptide (CTP)-]. N-linked carbohydrate-free single-chain TSH variants were constructed by site-directed mutagenesis and overlapping PCR: one devoid of both N-linked oligosaccharide chains on the -subunit (hTSH?-CTP-deg) and the other lacking also the oligosaccharides on the ?-subunit (hTSH?deg-CTP-deg). These variants were expressed in Chinese hamster ovary cells and secreted into the culture media. We have previously reported that the variants block the activities of hTSH and thyroid-stimulating immunoglobulins in cultured human thyroid follicles. In the present study, binding affinity of hTSH variants to hTSH receptor and the localization of the antagonistic effect were examined. Moreover, the effect of these variants on TSH activity was tested in vivo. The results of the present study indicate that the hTSH variants bind to the hTSH receptor with high affinity. Experiments using forskolin also indicated that the N-linked carbohydrate-free TSH single-chain variants inhibit TSH activity at the receptor-binding site and not at a postreceptor level. Moreover, the variants significantly inhibited (about 50%) TSH activity with respect to thyroid hormone secretion in vivo in mice. These variants may offer a novel therapeutic strategy in treating hyperthyroidism.
Introduction
TSH IS A PITUITARY glycoprotein composed of two subunits: the -subunit common to TSH, FSH, LH and human chorionic gonadotropin (hCG), and a specific ?-subunit, which confers TSH bioactivity (1, 2). Human (h)TSH subunits are synthesized in the pituitary thyrotroph cells, assembled, and secreted into the circulation. Assembly of the subunits is the rate-limiting step for secretion, receptor binding, and bioactivity of TSH (1, 2, 3). To overcome this limitation, hTSH was converted to a single-chain polypeptide (4). The carboxyl end of TSH? was fused to the amino end of the -subunit, thus forming hTSH?- single chain. The fusion was also performed in the presence of the carboxyl-terminal peptide (CTP) of the ?-subunit of hCG forming TSH?-CTP-. CTP was used as a linker sequence between the subunits, which are presumed to be required for flexibility, hydrophilicity, and stability. Previous studies have shown that ligation of CTP to hFSH? (5) hTSH? (6), or hCG (7) did not affect assembly, secretion, or in vitro bioactivity (4, 8, 9, 10, 11, 12), but it increased bioactivity and half-life of the hormones in vivo (5, 6, 7). We have shown that secretion of the single peptide chains were significantly higher than that of hTSH wild type (WT) (4). Receptor binding and in vitro bioactivity of the single-peptide chains were similar to that of hTSH-WT (4).
TSH? and TSH subunits contain one or two N-linked oligosaccharide chains, respectively (1, 2). These residues have been shown to play a critical role in assembly, secretion, and bioactivity of hTSH (1, 13). We have shown that deletion of oligosaccharide units from the -subunit resulted in a significant reduction in cAMP formation and T3 secretion from cultured human thyroid follicles (14). Similarly, hTSH single-peptide variants lacking the oligosaccharide chains from -subunit (TSH?-CTP-deg) or ?-subunit as well (TSH?deg-CTP-deg) were less potent than TSH WT (15). Moreover, N-linked carbohydrate-free hTSH single-peptide chains inhibited the in vitro activity of hTSH and human thyroid-stimulating immunoglobulin (TSI), the etiologic factor inducing the most common cause for hyperthyroidism (Graves’ disease), in a dose-dependent manner (15). It was therefore suggested that these variants behave as potential antagonists of hTSH and TSI, which may offer novel therapeutic agents in the treatment of hyperthyroidism and Graves’ disease.
In the present study, we evaluated the affinity of the single-chain hTSH variants to human TSH receptor. Moreover, we examined whether the antagonistic effect of these variants is located at the TSH receptor binding site or at a postreceptor level. In addition, the antagonistic effect of hTSH variants was investigated in vivo.
Materials and Methods
Materials
Purified bovine (b) TSH and T3 were obtained from Sigma (St. Louis MO). G418 was obtained from Gibco (Uxbridge, UK). 125IbTSH was purchased from (Kronus, Boise, ID). 125IcAMP was obtained from Amersham Biosciences UK Ltd. (Little Chalfont, Buckinghamshire, UK). Cell culture media and reagents were obtained from Biological Industries (Beit Haemek, Israel). Rabbit antiserum against hTSH-dimer was purchased from Fitzgerald (Concord, MA). TSI (MRC Research standard B, 65/122) was a gift from the National Institute for Biological Standards and Control (London, UK). Chinese hamster ovary (CHO) cells stably expressing human TSH receptor were kindly supplied by Dr. Basil Rapoport (Cedars-Sinai Medical Center, Los Angeles, CA).
hTSH variants
Using site-directed mutagenesis and overlapping PCR, two N-linked carbohydrate-free single-chain hTSH variants were constructed in our laboratory as described before (15): one devoid of both N-linked oligosaccharide chains on the -subunit (hTSH?-CTP-deg) and the other lacking also the oligosaccharides on the ?-subunit (hTSH?deg-CTP-deg). The chimeric genes coding for both variants were inserted into eukaryotic expression vector (PM2HA) as described before (15).
DNA transfection and clone selection
CHO cells were transfected with the expression vectors coding for the hTSH variants (PM2TSH?-CTP-deg and PM2TSH?deg-CTP-deg) using the calcium phosphate precipitation method (16). Stable clones were selected by growing the cells in a G418-containing medium (0.25 mg/ml). Transfected clones were screened for the expression of hTSH variants by Western blotting.
Cell culture
Transfected CHO clones were maintained in Ham’s F-12 medium containing 100 U/ml penicillin, 100 mg/ml streptomycin, 2 mM glutamine, 0.25 mg/ml active G418, and 5% fetal calf serum at 37 C in humidified 5% CO2 incubator.
For hormone collection, stable clones expressing TSH WT and mutants were grown to confluency in T-75 flasks. Cells were washed twice with PBS, and medium was replaced by serum-free medium. Media were collected every 24 h, clarified by centrifugation, and concentrated through 10-Da selective membrane, using slow centriprep concentrator (Amicon Corp., Danvers, MA) followed by additional concentration into small volumes with viva-20 spin columns (Biological Industries). Concentrations of the TSH variants in concentrated condition medium were determined using immunoradiometric assay and a double antibody (Diagnostic Products Corp., Los Angeles, CA).
Western blotting
Samples were electrophoresed on nondenaturing 15% sodium dodecyl sulfate-polyacrylamide gels as described before (17). Gels were allowed to equilibrate for 10 min in 25 mM Tris and 192 mM glycine in 20% (vol/vol) methanol (14). Proteins were transferred to a 0.2 μm pore size nitrocellulose membrane (Sigma) at 250 mA for 3 h, using a minitransblot electrophoresis cell (Bio-Rad Laboratories, Richmond, CA) according to the method described in the manual accompanying the unit. The nitrocellulose membrane was incubated in 5% nonfat dry milk for 2 h at room temperature. The membrane was incubated with ?-subunit antiserum (1:1000 titer) overnight at 4 C followed by three consecutive washes in PBS containing 0.1% Tween 20 (10 min/wash). The membrane was incubated with secondary antibody conjugated to horseradish peroxidase (Zymed, San Francisco, CA) for 2 h at room temperature followed by three washes. Finally, the nitrocellulose paper was reacted with enhanced chemiluminescent substrate (Pierce, Rockford, IL) for 5 min, dried on a filter paper (Maidstone, UK), and exposed to x-ray film.
Receptor binding
TSH binding assays were performed using CHO cells stably transfected with hTSH receptor. Cells were plated in 12-well plates (4 x 105 cells/well) and incubated with 125IbTSH (30,000 cpm/well) with or without varying concentrations of unlabeled variants (0.1–2,000 μU/ml) for 16–18 h at 4 C. The cells were then washed three times with cold Krebs Ringer buffer (280 mM sucrose, 3 mM CaCl2, 20 mM HEPES, 5 mM KCl, 1 mM MgSO4, 7 mM NaHCO3, 0.25% BSA) and lysed with 1 M NaOH. Lysates were collected into 12 x 75 plastic tubes, and the rates of 125Ibovine TSH replacement by TSH variants were determined using -counter (18).
In vitro bioactivity of TSH variants
The bioactivity of hTSH variants was determined by measuring their ability to stimulate cAMP formation from cultured human thyroid follicles as described previously (19). Cells were plated in 24-well plates (200 x 103 cells/well) with or without varying concentrations of hTSH variants or forskolin. At the start of the culture period, 1-methyl-3-isobutylxanthine (0.5 mM) was added to the medium. cAMP was measured by RIA as described previously (19).
In vivo bioactivity of TSH variants
Male BALB/c mice (25–30 g weight) were fed with a diet of normal rodent chow and supplied with drinking water containing 3 μg/ml (4.46 μM) T3 for 4 d for endogenous TSH suppression (20). Animals (five per group) were injected ip (200 μl/injection volume) with bovine TSH and/or TSH variants. The control group consisted of animals injected with 200 μl of the maintaining medium. Blood spots from mice tails were blotted to a paper filter at 0, 4, and 8 h after injection. Blood T4 concentrations were determined by RIA with human neonatal T4 kit (Diagnostic Products Corp., Los Angeles, CA).
Statistical analysis
Each experiment was repeated at least three times, and results were expressed as the mean ± SEM. Statistical analysis of the data was performed using Student’s t test and ANOVA. P < 0.05 was considered significant.
Results
hTSH variant genes, WT, and mutants were inserted into PM2HA, a eukaryotic expression vector, and transfected into CHO cells. Clones resistant to G418 and which secreted hTSH variants were selected. Concentrations of TSH variants in the condition media were measured using TSH RIA and a double antibody.
Using Western blot analysis and TSH antiserum after nondenaturing, SDS-PAGE allowed detecting the secretion of TSH variants in the media (electrophoresis under nondenaturing conditions prevents separation of hTSH subunits). The results indicated that the N-linked carbohydrate-free mutant TSH variants TSH?-CTP-deg (Fig. 1, lane 2) and TSH?deg-CTP-deg (Fig. 1, lane 3) migrated faster than TSH WT (Fig. 1, lane 1) due to lower content of oligosaccharide chains. On the other hand, TSH?-CTP- single chain migrated slower (Fig. 1, lane 4) due to the addition of O-linked oligosaccharides to the CTP.
FIG. 1. Expression and secretion of hTSH variants in a CHO cell system detected by gel electrophoresis and Western blotting. Media samples from transfected CHO cells were separated under nondenaturing conditions on 15% polyacrylamide SDS-PAGE. After protein transfer, the nitrocellulose PAGE was exposed to polyclonal antibody against hTSH?-subunit. Molecular mass markers are indicated by arrowheads in kilodaltons.
Receptor binding assays were performed using CHO cells expressing the hTSH receptor. Receptor binding of human dimeric hTSH WT as well as N-linked carbohydrate-free single-chain variants (TSH?-CTP-deg and TSH?deg-CTP-deg) displaced 125I bTSH in a dose-dependent manner (P < 0.05) (Fig. 2). Interestingly, the displacement by N-linked carbohydrate-free single-chain constructs (TSH?-CTP-deg and TSH?deg-CTP-deg) occurred with lower concentrations, compared with hTSH dimer WT. The IC50s detected for dimeric hTSH-WT, TSH?deg-CTP-deg, and TSH?-CTP-deg were 200, 100, and 18 μU/ml, respectively. The results thus indicated that N-linked carbohydrate-free variants bind with high affinity to hTSH receptor, with the highest affinity belonging to the TSH?-CTP-deg variant (Fig. 2).
FIG. 2. Receptor binding of hTSH variants to human TSH receptor. Stable transfected CHO cells expressing the hTSH receptor were incubated with 125I bTSH in the presence of varying concentrations of hTSH variants. Displacement curves show the percentage of maximal binding of the isotope at each concentration of unlabeled sample. Data shown are the mean ± SD of three experiments.
To determine whether the antagonistic effects of hTSH variants are at the receptor-binding site or a postreceptor level, the effects of hTSH variants on forskolin activity were tested. Forskolin, as expected, increased cAMP levels because it is known to activate adenylate cyclase (AC) (Fig. 3, A and B). The cAMP levels induced by both N-linked carbohydrate-free variants were extremely low. Surprisingly, combination of these variants with forskolin resulted in a synergistic effect with respect to cAMP levels. Similarly, cAMP levels increased synergistically (2- to 7-fold) after combined treatment of forskolin with the hTSH-WT and TSI (Fig. 4, A and B).
FIG. 3. Biological activity of hTSH variants in the presence or absence of forskolin. cAMP formation was measured after exposure of human thyroid follicles for 7 d at 37 C to different concentrations (10, 50, 100 μU/ml) of hTSH?CTP-deg (A) and hTSH?degCTP-deg (B) in the presence or absence of forskolin (Fsk). Each graph is a representative experiment from a series of three different experiments. Each bar represents the mean ± SD of four replicates. *, P < 0.05; ***, P < 0.005, with respect to Fsk.
FIG. 4. Biological activity of hTSH and TSI in the presence or absence of forskolin. cAMP formation was measured after exposure of human thyroid follicles for 7 d at 37 C to different concentrations (10, 50, 100 μU/ml) of hTSH (A) and TSI (0.5, 0.75, 1.5 mU/ml) (B) in the presence or absence of forskolin (Fsk). Each graph is a representative experiment from a series of three different experiments. Each bar represents the mean ± SD of four replicates. **, P < 0.01; ***, P < 0.005, with respect to Fsk.
The effects of hTSH variants were also examined in vivo in mice. Preliminary experiments indicated that treatment of the animals with T3 for 4 d suppressed TSH levels resulting in undetectable T4 levels in the circulation. Treatment of these animals with bTSH-induced T4 secretion in a time-dependent manner, with maximal levels at 4–8 h (data not shown). The blood T4 levels induced by hTSH N-linked carbohydrate-free variants (TSH?deg-CTP-deg, and TSH?-CTP-deg) were significantly lower than that induced by bTSH (P < 0.01) (Figs. 5 and 6). Moreover, treatment with bTSH together with the N-linked carbohydrate-free variants (TSH?-CTP-deg, or TSH?deg-CTP-deg) decreased significantly the bTSH-induced T4 levels up to about 50% (P < 0.05) (Figs. 5 and 6).
FIG. 5. Biological activity of hTSH?CTP-deg and bTSH in vivo. Mice were fed with diet of normal rodent chow and supplied with drinking water containing 3 μg/ml T3 for 4 d to suppress endogenous TSH. Groups (five animals per group) were injected as follows: bTSH (20 mU /mouse) (A); hTSH?CTP-deg (2 mU/mouse) (B); hTSH?CTP-deg (2 mU/mouse) + bTSH (20 mU/mouse); control animals (200 μl of culture medium) (C). D, In the latter group, the T4 levels were undetectable. Bars represent mean ± SD values of two experiments. *, P < 0.05; **, P < 0.01, with respect to bTSH.
FIG. 6. Biological activity experiments of hTSH?degCTP-deg and TSH in vivo. Mice were fed with diet of normal rodent chow and supplied with drinking water containing 3 μg/ml T3 for 4 d to suppress endogenous TSH. Groups (five animals per group) were injected as the following: bTSH (20 mU/mouse) (A); hTSH?degCTP-deg (2 mU/mouse) (B); hTSH?degCTP-deg (2 mU/mouse) + bTSH (20 mU/mouse) (C); control animals (200 μl of culture medium) (D). In the latter group, the T4 levels were undetectable. Bars represent mean ± SD values of two experiments. *, P < 0.05; **, P < 0.01, with respect to bTSH.
Discussion
The present study indicates that N-linked carbohydrate-free single-peptide chain variants of hTSH bind to hTSH receptor with high affinity. These variants reduced the in vitro bioactivity of TSH via inhibition at the TSH receptor-binding site. Moreover, the variants reduced TSH bioactivity (thyroid hormone secretion) in vivo. The role of carbohydrate chains bound to glycoprotein hormones has been widely investigated. Chemical removal of N-linked carbohydrate chains from hCG (21) and LH (22) was shown to decrease biological activity. In addition, the bioactivities of carbohydrate-free dimeric FSH variants, designed by site-directed mutagenesis, were markedly reduced (23). FSH and hCG synthesized in mutant CHO cells with a defect in the processing of N-linked carbohydrates were inactive in vivo (24, 25). Deletion of either or both oligosaccharides on the TSH- subunit did not prevent the dimerization and secretion of the formed TSH variants but resulted in a decreased activity of carbohydrate-free TSH dimer in vitro (14). These findings suggest a critical role of carbohydrates in the function of glycoprotein hormones.
Site-directed mutagenesis may affect assembly of - and ?-subunits and possibly secretion of the dimeric hormone. To overcome this limitation, human TSH heterodimer was converted into a biologically active single-peptide chain. This was performed by gene fusion of the TSH subunits genes with or without the CTP as a linker between the subunits. Both variants were efficiently secreted from CHO cells, bound with high affinity to the TSH receptor, and were even more active than the normal TSH dimer (4). Removal of N-linked carbohydrates by site-directed mutagenesis from the -subunit (TSH?-CTP-deg) and also from the ?-subunit (hTSH?deg-CTP-deg) resulted in a significant decrease in bioactivity in vitro (15). Competitive experiments between the N-linked carbohydrate-free variants and hTSH, as well as TSI, showed inhibition of TSH bioactivity by the variants (15). These findings led to the concept that the N-linked carbohydrate-free variants may serve as TSH antagonists.
To reveal the mechanism of the inhibition of hTSH and TSI by mutant TSH variants, receptor-binding assays were conducted. The finding that the N-linked carbohydrate-free TSH single chains bind to hTSH receptor with higher affinity than dimeric TSH is in correlation with our previous report that TSH?-CTP-deg had the highest potency in inhibiting hTSH and TSI activities (15). However, this is in contrast to the report that dimeric FSH devoid of carbohydrate chains on the -subunit alone has lower receptor binding potency than FSH dimer devoid of all carbohydrate chains on - and ?-subunits (23).
The results thus indicated that the mutated TSH single-chain polypeptides behave as hTSH antagonist by competing at the receptor level. The question as to whether the inhibitory effect also takes place at a postreceptor level was addressed. The surprising synergistic effects of the TSH variants together with forskolin on AC activity, in addition to the findings of the receptor binding assays, demonstrate that the N-linked carbohydrate-free hTSH variants inhibit TSH at the level of the receptor-binding site and not at a postreceptor level.
The mechanism of such synergism, however, is not clear and to the best of our knowledge has not been reported yet. Forskolin is known to bind to the pseudocatalytic site on the C2 domain of AC, which also contains the binding site of GS (26, 27). Because forskolin has been reported to have the ability to potentiate GS-mediated activation of AC (28, 29, 30, 31) and amplify the response to weak or partial agonists of TSH (32), this may tentatively provide a hypothetical explanation for the synergistic effect of TSH variants with forskolin.
The antagonistic effects of N-linked carbohydrate-free TSH variants previously reported in vitro (15) were also studied in vivo in mice. Although the potency of bTSH to activate TSH receptor in mice is known to be higher than that of hTSH (20), the N-linked carbohydrate-free TSH single chains were able to decrease the bTSH-induced thyroid hormone formation by about 50%.
The data thus show that the N-linked carbohydrate-free TSH variants, despite slight agonistic activity, behave as partial TSH antagonists in vitro and in vivo by binding to the TSH receptor with high affinity. These findings may open new horizons regarding the treatment of hyperthyroidism. The treatment of hyperthyroidism has essentially remained unchanged for the past 30 yr and includes the use of radioactive [131I] iodine, surgery, and antithyroid drugs, such as propylthiouracil and methimazole, which inhibit thyroid hormone synthesis. Each approach has its own intrinsic limitations and/or side effects (33, 34). Propylthiouracil and methimazole act slowly and can take up to 6–8 wk to fully deplete the thyroid gland of intrathyroidal stores of iodinated thyroglobulin, during which time hyperthyroidism can have severe consequences in certain individuals. Radiochemical destruction of thyroid tissue by 131I may require 4–6 months to be fully effective and may worsen ophthalmopathy. Surgical thyroidectomy is invasive and is usually preceded with antithyroid drugs to prevent life-threatening complications, such as thyroid storm. These observations emphasize the need for the development of TSH and TSI antagonists, which may overcome the limitations and disadvantages of the traditional treatments of hyperthyroidism.
Further studies are, of course, needed to evaluate the in vivo potency of the hTSH antagonists developed in our laboratory and determine the optimal doses for achieving maximal effects in blocking TSH and TSI activities. Moreover, some difficulties might be faced when implementing such treatment, such as parenteral administration, short half-life, and costs. The potential risk of hypothyroidism would also have to be considered. We believe that the strategy of constructing mutated single-chain TSH variants could lead to the development of a novel strategy in treating hyperthyroidism.
Acknowledgments
We are grateful to Dr. Basil Rapoport (Cedars-Sinai Medical Center, Los Angeles, CA) for supplying the CHO cells expressing human TSH receptor. We also thank the Israeli Ministry of Science for granting a postdoctoral fellowship (to N.A.).
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