TSH is a dimeric glycoprotein hormone composed of a common α-subunit noncovalently linked to a hormone-specific β-subunit. Previously, the TSH heterodimer was successfully converted to an active single-chain hormone by genetically fusing α and β genes with [TSHβ- carboxyl-terminal peptide (CTP)-α] or without (TSHβ-α) the CTP of human chorionic gonadotropin β-subunit as a linker. In the present study, TSH variants were expressed in Chinese hamster ovarian cells. The results indicated that TSHβ-α single chain has the highest binding affinity to TSH receptor and the highest in vitro bioactivity. With regard to the in vivo bioactivity, all TSH variants increased the levels of T4 in circulation after 2 and 4 h of treatment. However, the level of T4 after treatment with TSH-wild type was significantly decreased after 6 and 8 h, compared with the levels after treatment with the other TSH variants. TSHβ-α and TSHβ-CTP-α single chains exhibited almost the same bioactivity after 8 h of treatment. Evaluating the half-life of TSH variants, TSHβ-CTP-α single chain revealed the longest half-life in circulation, whereas TSH-wild type exhibited the shortest serum half-life. These findings indicate that TSH single-chain variants with or without CTP as a linker may display conformational structures that increase binding affinity and serum half-life, thereby, suggesting novel attitudes for engineering and constructing superagonists of TSH, which may be used for treating different conditions of defected thyroid gland activity. Other prominent potential clinical use of these variants is in a diagnostic test for metastasis and recurrence of thyroid cancer.
TSH is a glycoprotein synthesized in the thyrotroph cells of the pituitary gland. The hormone shares a common α-subunit with the other glycprotein hormones, FSH, LH, and human chorionic gonadotropin (hCG), and has a specific β-subunit, which is responsible for the unique TSH bioactivity (1, 2). Both subunits are formed and assembled in the same cell to compose an active heterodimer, which is secreted to the circulation. Assembly of the subunits is required for secretion, receptor binding, and activity of the hormone, and in this manner, it is a rate-limiting step (1–3). Therefore, and to overcome the stage of subunits assembly, the hetrodimeric hTSH was previously converted to a single-chain polypeptide (4). Using overlapping PCR, the carboxyl end of TSHβ gene was fused to the amino end of the α gene to form hTSHβ-α single chain. Another TSH single-chain analog containing the carboxyl-terminal peptide (CTP) of hCG β-subunit, as a linker between α- and β-subunits, was formed (TSHβ-CTP-α) (4). The CTP is thought to increase flexibility, hydrophilicity, and stability of the hormone. Fusing CTP to β-subunit of hFSH (5), hTSH (6), to hCG α-subunit (7), to human growth hormone (8), or to human erythropoietin (hEPO) coding sequence (9) did not affect assembly, secretion, or in vitro bioactivity. Similarly, ligation of CTP to single-chain variants of glycoprotein hormones had no effect on bioactivity in vitro (4, 10–14). However, fusing the CTP sequence to the signal sequence of FSH, TSH, hCG, or hEPO resulted in increasing in vivo bioactivity and half-life of gonadotropins (6, 14). Because the CTP sequence of hCG carries four O-linked carbohydrate chains attached to four subsequent serine residues, these carbohydrates are thought to play a possible role in the functions mentioned above and attributed to the CTP (15).
In this context, the common α-subunit carries two N-linked oligosaccharide chains on the positions Asn52 and Asn78, whereas the specific TSH β-subunit contains one asparagine-linked oligosaccharide chain on position 23 (1, 2, 16). The carbohydrate residues are thought to have a role in the biological activity of the hormone with regard to intracellular stability, assembly, and signal transduction (1, 16–18). Indeed, it was shown in a previous study performed in our laboratory that genetically N-deglycosylated single-chain TSH variants had higher affinity to the TSH receptor but lower in vitro and in vivo bioactivity than those of TSH-wild type (WT). Therefore, those variants have a possible potential to serve as TSH antagonists (19).
In the present study, bioactivity and longevity of hTSH single-chain variants were evaluated while comparing with TSH-WT heterodimer.
Materials and Methods
Materials
Purified bovine TSH and T3 were obtained from Sigma (St. Louis, MO). The transfection reagent Metafectin was purchased from Biontex (Martinsried/Planegg, Germany). G418 was obtained from GIBCO (Uxbridge, UK). 125IbTSH was purchased from Kronus (Star, ID). 125IcAMP was obtained from Amersham Biosciences UK Limited (Little Chalfont, Buckinghamshire, UK). Cell culture media and reagents were obtained from Biological Industries (Beit Hemak, Israel). Chinese hamster ovarian (CHO) cells stably expressing hTSH receptor were kindly supplied by Dr. Basil Rapoport (Cedars-Sinai Medical Center, Los Angeles, CA). The eukaryotic expression vector pM2-HA and the antiserum against TSHβ-subunit were kindly supplied by Dr. Irving Boime (Medical School, Washington University, St. Louis, MO).
Human TSH variants
Using overlapping PCR, two of the single-chain hTSH analogs were previously constructed in our laboratory as described before (4). TSH single-chain polypeptides were formed by a direct fusion between α and β coding genes with (TSHβ-CTP-α) or without (TSHβ-α) the CTP of hCG β-subunit as a linker. TSH variants used in this study are shown in Fig. 1. All chimeric constructs were inserted into eukaryotic expression vector (PM2-HA) using BamHI and SalI clooning sites (16).
Schematic presentation of the TSH variants studied. Excluding the TSH-WT, all variants were engineered using overlapping PCR. The single-chain TSH peptides were formed in the absence and in the presence of the CTP linker between subunits.
Cell culture
CHO cells were grown in Ham's F-12 medium containing 100 U/ml of penicillin, 100 mg/ml of streptomycin, 2 mm of glutamine (0.25 mg/ml active G418 was added for selection of stable clones), and 5% of fetal calf serum and maintained in humidified 5% CO2 incubator at 37 C.
DNA transfection and clone selection
Expression vectors coding for hTSH variants were transfected into CHO cells using Metafectin reagent according to manufacturer protocol. Three transfections were performed: cotransfection with PM2-hTSHβ and PM2-hTSHα for expression of hTSH-WT dimmer, transfection with PM2-hTSHβ-CTP-α single chain, and transfection with PM2-hTSHβ-α single chain. For stable clone selection, G418 (0.25 mg/ml) was added to culture medium, and single clones were picked, grown, and selected for secretion of the respective variants.
Media collection
Secreted variants were collected from confluent cells grown in T-75 flasks. Upon confluence, 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-kDa 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 TSH variants in condition media were determined using immunoassay containing double antibody against hTSH: monoclonal anti-TSH antibody against the β-subunit and monoclonal anti-TSH antibody against the TSH dimer, according to the manufacture's instructions (Roche, Mannheim, Germany).
Western blotting
Samples were electrophoresed on nondenaturing 15% SDS-PAGE as described before (19). Gels were allowed to equilibrate for 10 min in 25 mm Tris and 192 mm glycine in 20% (vol/vol) methanol (16). Proteins were transferred to a 0.2-μm pore size nitrocellulose membrane (Sigma) at 250 mA for 3 h, using a Mini Trans-Blot electrophoresis cell (Bio-Rad Laboratories, Richmond, CA) according to the method described in the manual accompanying the unit. The nitrocellulose membrane was blocked with 5% nonfat dry milk for 2 h at room temperature and incubated with TSHβ-subunit antiserum (1:1000 titer) for overnight at 4 C. Three consecutive washes in PBS containing 0.1% Tween (10 min/wash) were performed, and then membrane was incubated with secondary antibody conjugated to horseradish peroxidase (Zymed, San Francisco, CA) for 2 h at room temperature. The nitrocellulose paper was washed three times and, finally, was reacted with enhanced chemiluminescent substrate (Pierce, Rockford, IL) for 5 min, dried with Whatman sheet, and exposed to x-ray film.
Receptor binding
TSH binding assays were performed using CHO cells stably transfected with hTSH receptor (TSHR). Cells were plated in 12-well plates (4 × 105 cells/well) and incubated with 125IbTSH (30,000 cpm/well) with or without varying concentrations of unlabeled TSH variants (0.1–2000 μU/ml) for 16–18 h at 4 C. 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, and 0.25% BSA) and lysed with 1 m NaOH. Lysates were collected into 12 × 75 plastic tubes, and the rates of 125Ibovine TSH replacement by TSH variants were determined using γ-counter (20).
In vitro bioactivity of TSH variants
The bioactivity of hTSH variants was determined by measuring their ability to stimulate cAMP formation from CHO cells expressing hTSH receptor, according to the procedure described previously with modification to CHO cells (21). Cells were plated in 24-well plates (200 × 103 cells/well) and were exposed on the next day to varying concentrations of hTSH variants. 1-Methyl-3-isobutylxanthine (0.5 mm) was added to the medium to prevent cAMP degradation. cAMP was measured by RIA as described previously (21).
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 (22). Animals (six per group) were injected IP (200 μl/injection volume) with TSH variants (2 mU/mouse). 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, 6, and 8 h after injection. Blood T4 concentrations were determined by RIA with TT4 RIA Human Neonatal T4 kit (DPC, Los Angeles, CA).
Metabolic clearance rate
The metabolic clearance of hTSH variants was determined in New Zealand White rabbits. The endogenous TSH was suppressed by administering T3 in drinking water (10 mg/liter) for 6 d. Animals were injected with TSH analogs (1.5 mU/animal) through the lateral vein of the ear. Blood samples were collected at various time intervals 10 min, 20 min, 30 min, 1 h, 1.5 h, and 2 h. The sera were separated by centrifugation, and TSH levels were measured using immunoassay with a double antibody against TSHβ-subunit and hTSH dimer (Roche).
All the in vivo experiments were conducted in accordance with the rules of the animal ethics designated by Technion Committee for the Supervision of Animal Experiments.
Statistical analysis
Each experiment was repeated at least three times, and results were expressed as the mean ± sem. Statistical analysis of the data were performed using Student's t test and ANOVA. P < 0.05 was considered significant.
Results
Secretion of hTSH variants from transfected cells was confirmed by SDS-PAGE under nondenaturing conditions and Western blot analysis using anti-hTSHβ antibody. Figure 2 shows that the variant containing CTP, TSHβ-CTP-α single chain (lane 3) was electrically less mobile than TSH-WT and TSHβ-α single chain (lanes 1 and 2). This is due to the addition of 28 amino acids of the CTP with the attached four O-linked oligosaccharide chains.
Expression, secretion, and analysis of the engineered thytropin variants obtained from cultured media of stably transfected CHO cells. Proteins were separated on 15% polyacrylamide SDS-PAGE under nondenaturing conditions and transferred to nitrocellulose-PAGE. The blotting was performed with anti-hTSHβ polyclonal antibody.
Receptor binding assays (Fig. 3) revealed that all TSH variants bind to hTSH receptor with high affinity and the displacement of 125IbTSH occurred in a dose-dependent manner. Interestingly, the displacement of 125IbTSH by the single-chain TSHβ-α occurred at lower concentrations comparing with the displacement by the other constructs. The IC50 values of TSH-WT dimmer, TSHβ-CTP-α single chain, and TSHβ-α single chain are 200, 100, and 10 μU/ml, respectively.
Receptor binding assays of hTSH variants to hTSH receptor expressed by CHO cells. The cells were incubated with 125IbTSH and with varying concentrations of the TSH variants. The results are presented as the percentage from the maximal binding of the isotope at the different concentrations of the variants. The data shown in the displacement curves are the mean ± sd of three experiments. Student's t test was performed, P < 0.05 was considered significant.
The in vitro biological activity of TSH variants was also determined using CHO cells expressing hTSH receptor, by measuring the amounts of cAMP formation (Fig. 4). The results indicated that TSHβ-α single chain was the most active construct, resulting in cAMP formation of approximately 3-fold more than that of hTSH-WT and of approximately 1.4-fold more than that of TSHβ-CTP-α. Therefore, hTSH-WT had the lowest activity with regard to cAMP inducement, compared with the single-chain variants.
In vitro bioactivity of hTSH variants. CHO cells stably expressing hTSHR were exposed to ascending concentrations (5, 25, 50, and 100 μU/ml) of hTSH variants, for 24 h at 37 C. cAMP formation was measured as described before. Each curve is the mean ± sd of four replicates and is a representative experiment from a series of three different experiments.
The biological activity of the TSH variants in vivo was determined by measuring the level of T4 in mice circulation (Fig. 4). The measurements were performed at 2, 4, 6, and 8 h after treatment with each variant. It was shown that T4 levels in the circulation, 2 h after treatment with TSH variants, were similar. However, after 4 h of treatment with TSH-WT, the level of T4 decreased, and after 8 h, it was not detectable (Fig. 4A). In contrast, T4 levels in the serum were sustained until 8 h after injection with single-chain constructs (Fig. 4, B and C).
The metabolic clearance of TSH variants was evaluated, after iv injection of rabbits with TSH constructs, by measuring their concentrations in the serum, using an immunoradiometric assay containing anti-TSH double antibody against hTSH. The initial value of hTSH in circulation before exposure to TSH variants was undetectable. Because of the technical procedure and time needed for dispersal of the substances in circulation, the first measurements after injection were carried out at 10 min after injection and were referred to as the initial concentrations of 100%. The results are presented as the percentage of the initial serum concentrations of TSH variants (Fig. 5).
Bioactivity of the hTSH variants in vivo. BALB/c male mice were fed with normal rodent chow diet supplied with drinking water containing 3 μg/ml T3 for 5 d to suppress endogenous TSH. T4 levels at zero time were not detectable. Animals were divided into four groups (six per group) and injected as follows: 1) hTSH-WT (2 mU/mouse) (A); 2) TSHβ-α single chain (2 mU/mouse) (B); 3) TSHβ-CTP-α single chain (2 mU/mouse) (C); and 4) control group (200 μl of culture medium). In the last group, no T4 were detected during the experiment. Bars are mean ± sd values. Student's t test was performed; **, P < 0.01; ***, P < 0.001 with respect to the amount of T4 at 2 h.
The results indicated that the levels of TSH single-chain variants in serum were significantly (P < 0.01) higher than that of TSH-WT after 2 h. These data suggest that the mechanism of TSH metabolic clearance is affected by the conversion of the heterodimeric structure TSH to a single-chain polypeptide and by the presence or absence of CTP (Fig. 6).
Metabolic clearance of hTSH variants as determined in male rabbits. Rabbits were fed with normal diet and supplied with drinking water containing T3 (10 mg/liter) for 5 d to suppress endogenous TSH activity. Collection of blood samples of 1 ml was performed from animals before treatment indicating no detectable TSH levels. Human TSH compounds were injected into the animals (1.5 mU/animal) through the lateral vein of the ear. Blood samples were collected at 10, 20, 30, 60, 90, and 120 min after injection. Sera were separated by centrifugation, and TSH levels were measured using immunoradiometric assay, which detects hTSH. Due to the technical procedure, the concentrations of TSH variants at 10 min after treatment were defined as 100%. The values on the y-axis are the percentage of the remaining TSH variant in circulation with regard to the defined 100% in each animal. One-way ANOVA test was performed (followed by post hoc comparison Tukey HSD, P < 0.05). a, A, α, and Α refer to TSH-WT, whereas other symbols refer to relevant variants different from each other and/or from TSH-WT. At the 120-min time point, Kruskal-Wallis was preformed, because results did not distribute normally (P < 0.003), showing significant differences among all variants.
Discussion
In this study, we intended to reevaluate the role of CTP linker and the single-chain structure of engineered TSH variants in the in vitro and in vivo bioactivity and in serum stability. These parameters were assayed while comparing between the TSH-WT and single-chain TSH constructs with or without the CTP linker. All variants, including TSH-WT, were expressed in CHO cells and were efficiently secreted into the culture medium. Due to the content of O-linked heavy oligosaccharides, the migration of TSH variant containing the CTP linker was slower than that of the constructs lacking it.
The TSHβ-α single chain showed the highest in vitro activity, followed by the TSHβ-CTP-α single chain and then, finally, by TSH-WT. The IC50 of TSHβ-α single chain was 10- and 20-fold greater than that of TSHβ-CTP-α and TSH-WT, respectively. Thus, the biological activity seems to be significantly correlated with binding affinity of the analogs to TSHR, the higher affinity is the greater bioactivity of the variant.
With regard to the binding affinity, it was reported by Grossmann et al. (14) that both TSH dimmers, TSH-WT, TSHβ-CTP/α, and TSHβ-CTP-α single chain, have similar affinities to TSH receptor. In addition, it was published by Joshi et al. (6) that TSH-WT and TSHβ-CTP-α dimer, expressed in FRTL-5 cells, had about the same activity in vitro. The same results were also obtained by Grossmann et al. (14), who reported similar cAMP induction in vitro by TSH-WT, TSHβ-CTP-α, and TSHβ-CTP-α single chain. We assume that the differences between our current results and the previous published findings may be attributed to that the previous receptor-binding assays were performed with porcine (14) thyroid membranes and not with hTSH receptor. Because the TSH variants are of human kind, their binding characteristics to procine TSH receptor or to hTSH receptor on extracted membranes may be different from binding to hTSH receptor on entire CHO cells. The same explanation may stand also for the differences in the in vitro biological activities. In the previous works, the cAMP assays were carried out in thyroid FRTL-5 cells of the green monkey (4, 6, 14) or CHO-JPOG (14), whereas our assays were performed in CHO cells stably expressing hTSH receptor.
The TSH variants were also active in vivo. The results indicated that the single-chain constructs had longer effects than the TSH-WT with regard to T4 inducement. This finding suggests a major role of the single-chain structure in the in vivo bioactivity and the long-term effects of the engineered TSH constructs.
Our results in vivo are compatible with those published in other works. The addition of the CTP linker to FSHβ-subunit elongated half-life of the assembled FSH (5). The same effect was achieved also when the CTP was ligated to the hEPO (9). Grossmann et al. (14) reported that the CTP linker elongates the half-life in the circulation by about 2.5-fold when comparing between TSH-WT and TSHβ-CTP-α dimer. The single-chain structure formed by fusion of hTSHβ with TSHα in the presence of the CTP as a linker was found to increase the serum stability by 2- to 6-fold compared with TSHβ-CTP-α dimer and TSH-WT, respectively (14).
The mechanism by which the CTP linker affects biological activity is not clear. The CTP linker is unique in bearing four O-linked oligosaccharide chains attached to four closed Ser residues. Mainly, this glycosylation is posttranslational and even postfolding (23, 24). It is assumed that the O-linked oligosaccharides are not critical for receptor binding and in vitro bioactivity but do have major role in increasing in vivo bioactivity and stability in the circulation (23, 24).
In this study, the TSHβ-α single chain was shown to have higher in vitro activity than the TSHβ-CTP-α single chain. However, both variants had almost the same activity and the same long-term effect in vivo, despite the higher clearance rate of TSHβ-α single chain compared with that of TSHβ-CTP-α single chain. This might be attributed to the higher affinity of the later to the TSH receptor. This assumption is in agreement with the data published by Boime and Ben-Menahem (23), who reported that the conversion of hCG to single-chain peptide increased also the in vitro and not only the in vivo biological activity.
In summary, it seems that fusion of CTP as a linker to TSH coding sequence is not important for binding to hTSH receptor and for the bioactivity in vitro. However, it does have a major role in the in vivo bioactivity and in serum stability of the TSH constructs. In addition, conversion of the dimeric TSH to a single-chain peptide increases the binding affinity of the hormone to its receptor. Additionally, in vitro and in vivo bioactivity and the serum stability of the engineered TSH single chains are increased. Therefore, the strategy of fusing β- and α-subunits of TSH should be adopted when seeking to attenuate the activity and the stability in the serum. The CTP linker may be involved in conformational structures that increase bioactivity and serum half-life, thereby, suggesting novel attitudes for engineering and constructing superagonists or antagonists of TSH. The presence or absence of the CTP linker should be determined according to our goals. For instance, in case a long and a highly active TSH agonist is needed but with rapid clearance, the TSHβ-α single chain might be used. However, when slow clearance is favorable, the TSHβ-CTP-α single chain should be used. These approaches open wide horizons for developing novel and promising TSH analogs, thereby treating different conditions of defected thyroid gland activity. Additionally, the most prominent potential clinical use of long acting hTSH agonist is in a diagnostic test for metastasis and recurrence of thyroid cancer. Existing tests rely on recombinant hTSH to stimulate 131I uptake in patients with differentiated thyroid cancer. One issue regarding the clinical use of hTSH is its rapid clearance from the circulation. Moreover, hTSH binding to the TSHR is a moderate affinity interaction and, therefore, requires high doses of recombinant TSH that must be administered parenterally to be effective. Thus, a hTSH single chain that would have a prolonged half-life in the circulation and an increased biological availability in vivo could alleviate the need of repeated injections of TSH. The clinical use of TSH single chain needs to be established in higher animals and in human clinical trials. However, the fact that the single-chain-engineered variants are biologically active and bind to the receptor with high affinity may indicate that the folding of β- and α-subunits is correct and may not induce formation of antibodies, but this assumption needs to be investigated in future clinical trials.
Acknowledgments
This work was supported by the Israel Science Foundation Grant 617/01 and by the United States-Israel Binational Science Foundation Grant 2009473.
Disclosure Summary: The authors have nothing to disclose.
Abbreviations:
- CHO
Chinese hamster ovarian
- CTP
carboxyl-terminal peptide
- hCG
human chorionic gonadotropin
- hEPO
human erythropoietin
- TSHR
TSH receptor
- WT
wild type.
References
