Engineering a Potential Antagonist of Human Thyrotropin and Thyroid-stimulating Antibody*

Fuad A. FaresDagger §, Flonia LeviDagger , Abraham Z. Reznick, and Zaki Kraiem||

From the Departments of Dagger  Biochemistry and Molecular Genetics and  Anatomy and Cell Biology and the || Endocrine Research Unit, Carmel Medical Center and the Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, 34362 Israel

Received for publication, September 9, 2000, and in revised form, November 14, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Thyrotropin (TSH) and the gonadotropins (FSH, LH, hCG) are a family of heterodimeric glycoprotein hormones composed of two noncovalently linked subunits, alpha  and beta . We have recently converted the hTSH heterodimer to a biologically active single chain (hTSHbeta ·CTPalpha ) by fusing the common alpha -subunit to the C-terminal end of the hTSH beta -subunit in the presence of a ~30-amino acid peptide from hCGbeta (CTP) as a linker. The hTSHbeta ·CTPalpha single chain was used to investigate the role of the N-linked oligosaccharides of alpha - and beta -subunits in the secretion and function of hTSH. Using overlapping PCR mutagenesis, two deglycosylated variants were prepared: one lacking both oligosaccharide chains on the alpha -subunit (hTSHbeta ·CTPalpha 1+2) and the other lacking the oligosaccharide chain on the beta -subunit (hTSHbeta ·CTPalpha (deg)). The single chain variants were expressed in CHO cells and were secreted into the medium. hTSH variants lacking the oligosaccharide chains were less potent than hTSHbeta ·CTPalpha wild-type with respect to cAMP formation and thyroid hormone secretion in cultured human thyroid follicles. Both deglycosylated variants competed with hTSH in a dose-dependent manner. The hTSHbeta ·CTPalpha 1+2 variant blocked cAMP formation and thyroid hormone secretion stimulated by hTSH as well as by the antibody, thyroid-stimulating immunoglobulins, responsible for the most common cause of hyperthyroidism, Graves disease. Thus, this variant behaves as a potential antagonist, offering a novel therapeutic strategy in the treatment of thyrotoxicosis caused by Graves' disease and TSH-secreting pituitary adenoma.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Thyrotropin (TSH)1 is a member of the glycoprotein hormone family, which includes lutropin (LH), follitropin (FSH), and human chorionic gonadotropin (hCG). These are heterodimers composed of two noncovalent-linked subunits, a common alpha -subunit and a hormone-specific beta -subunit (1, 2). Assembly of glycoprotein subunits is vital to the function of these hormones. The alpha - and beta -subunits contain one (TSHbeta and LHbeta ) or two (alpha , FSHbeta , and hCGbeta ) asparagine-linked (N-linked) oligosaccharides (1, 2). These residues have been shown to play a role in determining the biological activity of glycoprotein hormones, including the maintenance of intracellular stability, assembly, secretion, signal transduction, and modulation of plasma half-life (1, 3). Deglycosylation of glycoprotein hormones have been utilized using chemical or enzymatic treatments, but these however cannot discriminate between individual sites, are nonspecific, and provide only completely deglycosylated hormones.

Site-directed mutagenesis has become an important tool for studying the structure and function of glycoprotein hormones. However, mutations in either alpha - or beta -subunits can alter the folding and ultimately inhibit subunit assembly and secretion of the hormone (4-6). To overcome these limitations, the genes encoding the common alpha -subunit and either the hCG beta -, FSH beta -, or TSH beta -subunits have been genetically fused. The resulting polypeptide chains were efficiently secreted and were biologically active (7-12). These studies presumed that addition of the human CGbeta C-terminal peptide (CTP) as a linker sequence between the subunits would be required for flexibility, hydrophilicity, stability, and successful expression of the single chain forms. The CTP contains several proline and serine residues and thus lacks significant secondary structure. This may permit the appropriate interactions between the subunits. In addition, previous studies showed that ligation of the CTP to hFSHbeta (13), hTSHbeta (14), or hCGalpha (15) did not significantly affect assembly or in vitro biological activity, but was important for the in vivo potency of the chimeras.

Assembly of the hTSH beta - and alpha -subunits is the rate-limiting step in the production of the functional heterodimer (16). Therefore, studying the structure and function of hTSH using site-directed mutagenesis may affect assembly of the subunits and production of functional hormone. Thus, converting hTSH to a single chain form could increase the biological half-life and expand the range of TSH structure-function studies. In the present study, we used site-directed mutagenesis to study the role of the N-linked carbohydrates of the alpha - and/or beta -subunits of the hTSH single peptide chain containing the CTP as a linker between the subunits. The in vitro bioactivity of recombinant hTSH and its derivatives were assayed by cAMP formation and triiodothyronine (T3) secretion in a homologous serum-free culture system of human thyroid follicles (17, 18). Our results indicate that N-linked and O-linked oligosaccharides are not vital for the secretion of hTSH single chain. However, N-linked oligosaccharides are critical for biological activity. Moreover, the deglycosylated variant, hTSHbeta ·CTPalpha 1+2, inhibited the activity of hTSH and thyroid-stimulating immunoglobulins (TSI), which may have clinical implications regarding the treatment of hyperthyroidism.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Enzymes used in the construction of DNA vectors and constructs were purchased from New England BioLabs (Beverly, MA). Oligonucleotides used for chimeric construction were purchased from Genemed Biotechnology (San Francisco, CA). Cell culture media and reagents were obtained from Biological Industries (Beit hemeek, Israel). G418 was obtained from Sigma. Rabbit antiserum against hTSH dimer was purchased from Fitzgerald (Concord, MA). [35S]Cysteine/methionine mix was purchased from Amersham Pharmacia Biotech (Buckinghamshire, UK). hTSH was a gift from the National Hormone and Pituitary Distribution Program, NIDDKD, National Institutes of Health, and hTSI (MRC Research standard B, 65/122) a gift from the National Institute for Biological Standards and Control, London, UK.

Construction of hTSH Single Chain Variants-- The hTSH single chains were constructed using overlapping PCR mutagenesis, as described previously (7, 24). For construction of the hTSHbeta ·CTPalpha 1+2 single peptide chain, which was deglycosylated only on the alpha -subunit, the vectors pM2hTSHbeta ·CTPalpha and pM2alpha 1+2 were used as templates for PCR. The vectors, pM2hTSHbeta ·CTPalpha and pM2alpha 1+2, were prepared in our laboratory as previously described (4, 11). The following oligonucleotides were used for the chimeric construction: primer 1, 5'-GTGGGATCAGGGGGATCCTAGATTTCTGAGTTA-3'; primer 2, 5'-CACATCAGGAGCTTGTGGGAGGATCGG-3'; primer 3, 5'-ATCCTCCCACAAGCTCCTCATGTGCAG-3'; and primer 4, 5'-TGAGTCGACATGATAATTCAGTGATTGAAT-3'. pM2TSHbeta ·CTPalpha was a template for primers 1 and 2 (Fig. 1A). Primer 1 contained the TSHbeta 5'-end sequence, which includes a newly formed BamHI site, and primer 2 contained the first four codons of the alpha -subunit and a stretch of the 3'-end of CTP sequence. Therefore, the newly synthesized fragment contained the entire TSHbeta ·CTP coding sequence and a part of the alpha  sequence. pM2alpha 1+2 was used as a template for primers 3 and 4 to generate a product containing the 3'-end of CTP and the alpha 1+2 fragment. Primer 3 contained the sequence corresponding to the last four C-terminal codons of CTP and the first five codons of the alpha -subunit, and primer 4 contained some of the flanking sequence of the alpha  exon 4 that also included a newly created SalI site. These fragments were used as overlapping templates to synthesize the single hTSHbeta ·CTPalpha 1+2 gene using primers 1 and 4 (Fig. 1A).



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Fig. 1.   Construction of hTSH variants. A, mutant forms of hTSH were engineered using overlapping PCR mutagenesis. B, hTSHbeta ·CTPalpha wild type contains the N-linked glycosylation sites at Asn52 and Asn78 of the alpha -subunit and at Asn23 of the beta -subunit. hTSHbeta ·CTPalpha 1+2 contains one N-linked glycosylation site on Asn23 of the subunit. hTSHbeta ·CTPalpha (deg) has no N-linked glycosylation recognition sites.

To construct the deglycosylated hTSHbeta ·CTPalpha single chain on alpha - and beta -subunits (hTSHbeta ·CTPalpha (deg)), mutant primers 5 and 6 (primer 5, 5'-CAGATGGTGGTGTCGATGGTTAGG-3' and primer 6, 5'-CCTAACCATCGACACCACCATCTG-3') were synthesized for mutagenesis of asparagine (Asn) in position 23 of the beta  sequence to aspartic acid (Asp). The 5'-AAC-3' triplet coding sequence for Asn was converted to the 5'-GAC-3' coding sequence for Asp.

pM2TSHbeta ·CTPalpha 1+2 was used as template DNA, and three PCR reactions were performed for generating the TSHbeta ·CTPalpha (deg) coding sequence. The first reaction included primers 1 and 5, the second reaction included primers 6 and 4, and the final reaction included primers 1 and 4, which resulted in the former fragments containing the mutation in position 23 of the beta -subunit (Fig. 1A).

Construction of Expression Vectors-- The eukaryotic expression vector pM2·HA is an expression vector that contains the ampicillin (AmpR) and the neomycin (NeoR) resistance genes and a strong promoter of the HaMuSV virus, LTR (25, 26). The BamHI/SalI fragments containing the TSHbeta ·CTPalpha 1+2 or TSHbeta ·CTPalpha (deg) chimeric genes were inserted at the BamHI/SalI cloning site of pM2·HA and used for transfection.

DNA Transfection and Clone Selection-- Chinese hamster ovary (CHO) cells (wild-type and/or ldlD), were transfected with pM2hTSHbeta ·CTPalpha , pM2hTSHbeta ·CTPalpha 1+2, or pM2hTSHbeta ·CTPalpha (deg) vectors, according to the calcium phosphate precipitation method (4). Cells were selected for insertion of the plasmid DNA by growth in culture medium containing 0.25 mg/ml of the neomycin analog, G418. Transfected colonies resistant to G418 were harvested and screened for the expression of hTSH variants by metabolic labeling of the cells and immunoprecipitation.

Cell Culture-- CHO cells were maintained in Medium 1 (Ham's F-12 medium supplemented with penicillin (100 units/ml), streptomycin (100 mg/ml), and glutamine (2 mM) containing 5% fetal calf serum), at 37 °C in a humidified 5% CO2 incubator. Transfected clones were maintained in the above culture medium supplemented with 0.25 mg/ml active G418 (Medium II). For hormone collection, cells secreting hTSH variants were plated and grown to confluency in T-75 flasks. Cells were washed twice with serum-free medium and 12 ml of Medium III (Medium I without fetal calf serum) were added. Medium was collected every 24 h, clarified by centrifugation, and concentrated using centriprep concentrators (Amicon, Corp., Danvers, MA). Concentrations of hTSH variants were determined by hTSH immunoradiometric assay and a double antibody radioimmunoassay (Diagnostic Products Corp., Los Angeles, CA). In addition, medium from nontransfected CHO cells was collected as described above and used as control.

Metabolic Labeling-- On day 0, cells were plated into 12-well dishes (350,000 cells/well) in 1 ml of Medium I. For continuous labeling experiments, cells were washed twice with cysteine/methionine-free Medium IV (Medium I supplemented with 5% dialyzed calf serum) and labeled for 5 h in 1 ml of cysteine/methionine-free Medium IV containing 50 µCi/ml [35S]cysteine/methionine mix. For pulse chase experiments, the cells were washed twice and preincubated for 1.5 h with cysteine-free Medium IV, followed by a 20-min pulse-labeling in cysteine/methionine-free Medium IV containing 100 µCi/ml [35S]cysteine/methionine. Pulse-chase experiments using ldlD cells were performed in the presence or absence of 10 µM galactose or 100 µM N-acetyl galactosamine. The labeled cells were then washed twice with Medium IV containing 1 mM unlabeled cysteine/methionine and incubated in this chase medium for the indicated time. Media and cell lysates were prepared, immunoprecipitated using monoclonal antisera against the alpha -subunit and resolved on 15% SDS-polyacrylamide gels as described previously (27).

Tunicamycin Treatment-- Cells were plated into 12-well dishes in 1 ml of Medium I. On the second day, medium was changed with medium I containing 2 µg/ml tunicamycin. Cells were incubated at 37 °C for 1.5 h. At the end of the incubation time, the medium was exchanged with Medium II containing 2 µg/ml tunicamycin and 50 µCi/ml of [35S]cysteine/methionine mix. Further analysis proceeded with metabolic labeling as described above.

In Vitro Bioassay-- The bioactivity of hTSH variants were determined by measuring their ability to stimulate cAMP formation and T3 secretion from cultured human thyroid follicles as described previously (17, 18). Essentially, human thyroid cells were prepared from colloid tissue obtained at thyroidectomy from patients with benign nodules. 200 × 103 plated onto 24-well microtiter plates and incubated with 0.5 ml of serum-free medium (DCCM-1, which contains insulin (1 µg/ml), and antibiotics), in the presence or absence of the hTSH variants and cultured for 7 days at 37 °C, in an atmosphere of 5% CO2 in a water-saturated incubator. For T3 measurements, potassium iodide (0.1 µM) was added to the medium at the start of the culture period. For cAMP measurements, 1-methyl-3-isobutylxanthine (0.5 mM), which inhibits cAMP degradation, was added to the medium. At the end of the culture period, the cAMP and T3 secreted into the medium (concentrations remaining in the cells were negligible) were measured by radioimmunoassay as described previously (17).

Statistical Analysis-- Each experiment was repeated at least three times, and results are presented as the mean ± S.E. of at least three replicate determinations. Statistical analysis of the data was performed using Student's t test and analysis of variance. p < 0.05 was considered significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Oligonucleotide-directed mutagenesis was chosen to examine the functional importance of N-linked oligosaccharides in hTSH single chain (hTSHbeta ·CTPalpha ) bioactivity. The hTSHbeta ·CTPalpha contains three N-linked glycosylation sites: two on the alpha - and one on the beta -subunit (Fig. 1B). Mutagenesis of the Asn in the Asn-X-Thr/Ser recognition sequence for Asn-linked glycosylation is sufficient to prevent transfer of the carbohydrate to the protein (4). The coding sequence of Asn (5'-AAC-3') at positions 52 and 78 of the alpha -subunit was converted to the coding sequence for Asp (5'-GAC-3'). This variant was deglycosylated only on the alpha -subunit (pM2hTSHbeta ·CTPalpha 1+2). To construct deglycosylated variants on the alpha - and beta -subunits (hTSHbeta ·CTPalpha (deg)), the coding sequence for Asn (5'-AAC-3') on the beta -subunit of the hTSHbeta ·CTPalpha 1+2 variant was converted to the coding sequence for Asp (5'-GAC-3') (Fig. 1B). The coding sequence of hTSHbeta ·CTPalpha variants was sequenced to verify the mutations and the absence of other sequence alterations.

Stable clonal cell lines expressing hTSHbeta ·CTPalpha , hTSHbeta ·CTPalpha 1+2, and hTSHbeta ·CTPalpha (deg) were selected. Cells were labeled in the presence of [35S]methionine/cysteine mix for 7 h, media and lysates were immunoprecipitated with polyclonal human anti-alpha antiserum, and the proteins were resolved by SDS-polyacrylamide gel electrophoresis. Intracellular (lysate) forms of hTSHbeta ·CTPalpha wild-type and its mutants migrated faster than corresponding extracellular (medium) forms (Fig. 2A). This is because of the differences in terminal processing of the N-linked oligosaccharides and the addition of the O-linked oligosaccharides prior to secretion. The secreted mutant forms migrated faster than wild-type (19). This is because of their lower content of oligosaccharide chains. To confirm this assumption, CHO cells were treated with tunicamycin. Because tunicamycin prevents the addition of N-linked oligosaccharides to the protein, a difference in mobility is expected between proteins secreted from cells treated with tunicamycin compared with those untreated. The results showed that hTSHbeta ·CTPalpha and hTSHbeta ·CTPalpha 1+2 variants secreted from cells treated with tunicamycin have, as expected, the same mobility as hTSHbeta ·CTPalpha (deg) secreted from treated and untreated cells (Fig. 2B). This indicates that N-linked oligosaccharides are present in the alpha - and beta -subunits of hTSHbeta ·CTPalpha as well as in the beta -subunit of hTSHbeta ·CTPalpha 1+2 and absent in the hTSHbeta ·CTPalpha (deg) variant.



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Fig. 2.   Expression of hTSH variants from transfected CHO cells. A, expression of hTSH variants in CHO cells. L and M correspond to lysate (intracellular) and medium (secreted) forms, respectively. B, expression forms of hTSH variants from CHO cells in the presence (+) or absence (-) of tunicamycin. Samples were immunoprecipitated with antiserum against alpha -subunit and were subjected to SDS-polyacrylamide gel electrophoresis. The positions of the molecular mass markers are indicated in kDa.

The secretion kinetics of hTSH variants was determined by pulse-chase analysis and immunoprecipitation with anti-alpha antiserum (Fig. 3). Whereas the hTSHbeta ·CTPalpha (Fig. 3A) and hTSHbeta ·CTPalpha 1+2 (Fig. 3B) were secreted efficiently with a similar t1/2 of ~2 h, the secretion rate of hTSHbeta ·CTPalpha (deg) (Fig. 3C) was significantly slower with a t1/2 of ~17 h. These differences were not because of the O-linked oligosaccharides associated with the CTP, because similar results were detected using lDld cells (results not shown), which had a reversible defect in the synthesis of O-linked oligosaccharides (20). The data thus indicate that conversion of hTSH into a single peptide chain together with CTP as a linker allows the heterodimeric-like configuration of the mutated alpha - and beta -subunits, as shown by secretion of the protein into the medium.



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Fig. 3.   Kinetics of hTSH variants secretion from CHO cells. Cells expressing hTSHbeta ·CTPalpha (A), hTSHbeta ·CTPalpha 1+2 (B), and hTSHbeta ·CTPalpha (deg) (C) were pulse-labeled with 100 µCi/ml of [35S]cysteine for 20 min and chased for the indicated times (h). Lysate and medium samples were immunoprecipitated with antiserum against the alpha -subunit and subjected to SDS-polyacrylamide gel electrophoresis. The positions of the molecular mass markers are indicated in kDa.

The biological activity of hTSH variants was examined by their ability to stimulate cAMP formation and T3 secretion from cultured human thyroid follicles. Treatment of the cells with increasing concentrations (1-100 microunits/ml) of hTSH single chain resulted in a dose-dependent increase in cAMP formation (Fig. 4A) and triidothyronine (T3) secretion (Fig. 4B). The maximal effect on cAMP formation and T3 secretion was seen at concentrations of 50 and 5 microunits/ml, respectively.



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Fig. 4.   In vitro biological activity of hTSH variants. cAMP formation (A) and T3 secretion (B) were measured after exposure of human thyroid follicles for 7 days at 37 °C to different concentrations of hTSH variants. The cAMP and T3 concentrations in the medium were assayed by radioimmunoassay. Each point represents the mean ± S.E. of triplicate culture wells.

Compared with hTSHbeta ·CTPalpha , the maximal effect of hTSHbeta ·CTPalpha 1+2 or hTSHbeta ·CTPalpha (deg) on cAMP formation in thyroid cells was about 8 and 23% (p < 0.001), respectively (Fig. 4A). Similarly, the maximal effect on T3 secretion was 64% (p < 0.001) after exposure to hTSHbeta ·CTPalpha (deg) and to undetectable amounts after exposure to hTSHbeta ·CTPalpha 1+2 (Fig. 4B).

In competition experiments, cells were grown in the presence of submaximal concentrations of normal hTSH (50 microunits/ml) or of hTSI (0.75 milliunits/ml) and different concentrations (5-200 microunits/ml) of deglycosylated variants (Fig. 5). Both deglycosylated variants competed with hTSH in a dose-dependent manner. The cAMP levels induced by submaximal doses of hTSH were decreased in the presence of 200 microunits/ml of hTSHbeta ·CTPalpha 1+2 (IC50 = 70 microunits/ml) or hTSHbeta ·CTPalpha (deg) (IC50 = 158 microunits/ml) by 87 and 66% (p < 0.001), respectively (Fig. 5A). Similarly, the T3 levels induced by submaximal doses of hTSH were decreased in the presence of 200 microunits/ml of hTSHbeta ·CTPalpha 1+2 (IC50 = 33 microunits/ml) or hTSHbeta ·CTPalpha (deg) (IC50 = 135 microunits/ml) by 92 and 87% (p < 0.001), respectively (Fig. 5B). The cAMP formation and T3 secretion induced by submaximal doses of hTSI were decreased by 40% (p < 0.001, IC50 = 2.5 microunits/ml) (Fig. 6A) and 55% (p < 0.001, IC50 = 42 microunits/ml, Fig. 6B) in the presence of 100 microunits/ml of hTSHbeta ·CTPalpha (deg), respectively. 200 microunits/ml of the variant hTSHbeta ·CTPalpha 1+2 reduced cAMP formation by 90% (p < 0.001, IC50 = 58 microunits/ml, Fig. 6A) and completely blocked (p < 0.001, IC50 = 15 microunits/ml) the secretion of T3 (Fig. 6B) induced by submaximal doses of hTSI.



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Fig. 5.   Competitive effect of hTSH variants with hTSH. Cultured thyroid follicles were incubated for 7 days at 37 °C with submaximal doses of hTSH (50 microunits/ml) in the presence of different concentrations of hTSH variants. The cAMP (A) and T3 (B) concentrations in the medium were assayed by radioimmunoassay. Each point represents the mean ± S.E. of triplicate culture wells.



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Fig. 6.   Competitive effect of hTSH variants with hTSI. Cultured thyroid follicles were incubated for 7 days at 37 °C with submaximal doses of hTSI (0.75 milliunits/ml) in the presence of different concentrations of hTSH variants. The cAMP (A) and T3 (B) concentrations in the medium were assayed by radioimmunoassay. Each point represents the mean ± S.E. of triplicate culture wells.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The present study indicates that deglycosylated variants of hTSH single chains are expressed and secreted from CHO cells. These variants are less potent than wild-type hTSH with regard to cAMP formation and T3 secretion in cultured human thyroid follicles. Moreover, hTSHbeta ·CTPalpha (deg) reduced the biological activity of hTSH or hTSI, whereas hTSHbeta ·CTPalpha 1+2 significantly blocked the activity of hTSH and hTSI.

The Asn-linked oligosaccharides have been implicated in several physiologic functions such as maintenance of intracellular stability, secretion, biological activity, and modulation of the plasma half-life (1, 3). Site-directed mutagenesis has become an effective method to study the role of individual carbohydrate chains on multiglycosylated proteins. However, site-directed mutagenesis may affect assembly of heterodimer subunits. It has been reported that mutating the hTSHbeta -subunit significantly reduced TSH dimer formation (14). Other studies indicate that loss of oligosaccharides from the alpha -subunit reduced assembly and/or stability of hCG (4). To bypass the problem of dimerization of deglycosylated subunits, the subunits alpha  and beta  were genetically fused in a single chain hormone. Single chains of hCG (7), hFSH (8), and hTSH (11, 12) retained a biologically active conformation similar to that of the heterodimer (11, 12). Therefore, fusion of alpha - and beta -subunits in a single chain bypasses the assembly of the subunits, which is a rate-limiting step for hormone secretion and bioactivity. It is apparent that despite the single chain structure, correct conformation of the subunits occurs, and the single chains have a normal biological activity (11, 12).

For studying the role of N-linked oligosaccharides on hTSH function, we used the single chain of hTSH that contains the CTP as a linker between alpha - and beta -subunits (11). Previous studies indicated that fusing the CTP to the C-terminal end of hFSHbeta (13, 21), to the N-terminal of hCGalpha (15), or to hTSHbeta (22) does not affect the assembly, secretion, and signal transduction of the respective dimers compared with the wild type. In addition, it was reported that CTP and associated O-linked oligosaccharides in hCG are not important for receptor binding or in vitro signal transduction but are critical for in vivo biological response (23). Moreover, it has been shown that the secretion of the hFSH single chain increased in the presence of CTP as a linker between the subunits (8). The fact that deglycosylated variants of hTSH are secreted efficiently from CHO and ldlD cells (ldlD cells have a reversible defect in synthesizing oligosaccharide chains, data not shown) indicates that N-linked and O-linked oligosaccharides are not vital for the secretion of the hTSH single chain. Therefore, the signal for the secretion of the hormone exists in the single chains itself.

The bioactivity of the variants was examined in an in vitro system of human thyroid follicles cultured in suspension, under serum-free conditions, in which the follicular three-dimensional structure is retained (17, 18). This bioassay has several advantages over the current methods used for testing thyroid biological activity. First, it allows the measurement of T3 secretion, the physiologically relevant hormonal end-point response, which is very seldom measured when testing for thyroid biological activity. Second, the cells are of human origin, which is important in view of wide species variation in thyroid response to TSH agonists. The results indicated that deletion of the N-linked oligosaccharides from the single chain diminished biological activity. However, a difference in bioactivity between the variants was apparent. hTSHbeta ·CTPalpha (deg) was more potent than hTSHbeta ·CTPalpha 1+2, and this may be related to the difference in conformation of the variants.

The competition experiments indicated that hTSHbeta ·CTPalpha 1+2 markedly blocked cAMP formation and T3 secretion induced by hTSH and hTSI. Therefore, this variant can be considered as a potential antagonist to both hTSH as well as hTSI. It is worth noting that we tested, to the best of our knowledge for the first time, not only TSH but TSI as well, i.e. the factor responsible for the most common cause of hyperthyroidism, Graves' disease, thus substantiating considerably the clinical implications of our study. Therefore, the hTSHbeta ·CTPalpha 1+2 variant, behaves as a potential antagonist that may offer a novel therapeutic strategy of thyrotoxicosis because of Graves' disease and TSH-secreting pituitary adenoma. The existence of CTP as a linker between the subunits in the hTSHbeta ·CTPalpha 1+2 may prevent rapid degradation in vivo and increase their half-life in the circulation. The therapeutic efficacy of this analog needs to be established in in vivo studies and clinical trials.


    ACKNOWLEDGEMENTS

We thank Dr. Irving Boime (Washington University, St. Louis, MO) for his constructive comments regarding the manuscript. We would also like to thank Orit Sadeh and Dr. Ronit Heinrich for assistance in the bioassays.


    FOOTNOTES

* This work was supported by United States-Israel Binational Sciences Foundation (BSF) Grant No. 93-00088.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Genetics, Carmel Medical Center, 7 Michal St. 7, Haifa 34362, Israel. Tel.: 972-4-8250407; Fax: 972-4-8343023; E-mail: fares@actcom.co.il.

Published, JBC Papers in Press, November 16, 2000, DOI 10.1074/jbc.M008093200


    ABBREVIATIONS

The abbreviations used are: TSH, thyrotropin; FSH, follitropin; LH, lutropin; hCG, human chorionic gonadotropin; CHO, Chinese hamster ovary cells; TSI, thyroid-stimulating immunoglobulins; CTP, C-terminal peptide, T3, triiodothyronine; PCR, polymerase chain reaction.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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