Engineering a Potential Antagonist of Human Thyrotropin and
Thyroid-stimulating Antibody*
Fuad A.
Fares
§,
Flonia
Levi
,
Abraham Z.
Reznick¶, and
Zaki
Kraiem
From the Departments of
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 |
Thyrotropin (TSH) and the gonadotropins
(FSH, LH, hCG) are a family of heterodimeric glycoprotein hormones
composed of two noncovalently linked subunits,
and
. We
have recently converted the hTSH heterodimer to a
biologically active single chain (hTSH
·CTP
) by fusing the common
-subunit to the C-terminal end of the
hTSH
-subunit in the presence of a ~30-amino acid
peptide from hCG
(CTP) as a linker. The hTSH
·CTP
single chain was used to investigate the role of the
N-linked oligosaccharides of
- and
-subunits in the
secretion and function of hTSH. Using overlapping PCR
mutagenesis, two deglycosylated variants were prepared: one
lacking both oligosaccharide chains on the
-subunit
(hTSH
·CTP
1+2) and the other lacking the
oligosaccharide chain on the
-subunit (hTSH
·CTP
(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 hTSH
·CTP
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 hTSH
·CTP
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 |
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
-subunit and a hormone-specific
-subunit (1, 2). Assembly of
glycoprotein subunits is vital to the function of these hormones. The
- and
-subunits contain one (TSH
and LH
) or two
(
, FSH
, and hCG
) 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
- or
-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
-subunit
and either the hCG
-, FSH
-, or
TSH
-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 CG
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 hFSH
(13),
hTSH
(14), or hCG
(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
- and
-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
- and/or
-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,
hTSH
·CTP
1+2, inhibited the activity of hTSH and thyroid-stimulating immunoglobulins (TSI), which may have clinical implications regarding the treatment of hyperthyroidism.
 |
EXPERIMENTAL PROCEDURES |
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
hTSH
·CTP
1+2 single peptide chain, which was
deglycosylated only on the
-subunit, the vectors
pM2hTSH
·CTP
and pM2
1+2 were used
as templates for PCR. The vectors,
pM2hTSH
·CTP
and pM2
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'.
pM2TSH
·CTP
was a template for primers 1 and 2 (Fig.
1A). Primer 1 contained the
TSH
5'-end sequence, which includes a newly formed BamHI site, and primer 2 contained the first four codons of
the
-subunit and a stretch of the 3'-end of CTP sequence. Therefore, the newly synthesized fragment contained the entire
TSH
·CTP coding sequence and a part of the
sequence. pM2
1+2
was used as a template for primers 3 and 4 to generate a product
containing the 3'-end of CTP and the
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
-subunit, and primer 4 contained some of the flanking sequence
of the
exon 4 that also included a newly created SalI
site. These fragments were used as overlapping templates to synthesize
the single hTSH
·CTP
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, hTSH ·CTP wild type contains the
N-linked glycosylation sites at Asn52 and
Asn78 of the -subunit and at Asn23 of the
-subunit. hTSH ·CTP 1+2 contains one
N-linked glycosylation site on Asn23 of the
subunit. hTSH ·CTP (deg) has no N-linked glycosylation
recognition sites.
|
|
To construct the deglycosylated hTSH
·CTP
single chain on
-
and
-subunits (hTSH
·CTP
(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
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.
pM2TSH
·CTP
1+2
was used as template DNA, and three PCR reactions were performed for
generating the TSH
·CTP
(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
-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
TSH
·CTP
1+2 or
TSH
·CTP
(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
pM2hTSH
·CTP
,
pM2hTSH
·CTP
1+2,
or
pM2hTSH
·CTP
(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
-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 |
Oligonucleotide-directed mutagenesis was chosen to examine the
functional importance of N-linked oligosaccharides in hTSH single chain (hTSH
·CTP
) bioactivity. The hTSH
·CTP
contains three N-linked glycosylation sites: two on the
-
and one on the
-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
-subunit was converted to the coding sequence for Asp (5'-GAC-3'). This variant was deglycosylated only on the
-subunit (pM2hTSH
·CTP
1+2).
To construct deglycosylated variants on the
- and
-subunits
(hTSH
·CTP
(deg)), the coding sequence for Asn (5'-AAC-3') on
the
-subunit of the hTSH
·CTP
1+2 variant was
converted to the coding sequence for Asp (5'-GAC-3') (Fig. 1B). The coding sequence of hTSH
·CTP
variants was
sequenced to verify the mutations and the absence of other sequence alterations.
Stable clonal cell lines expressing hTSH
·CTP
,
hTSH
·CTP
1+2, and hTSH
·CTP
(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-
antiserum, and the proteins were resolved by SDS-polyacrylamide gel
electrophoresis. Intracellular (lysate) forms of hTSH
·CTP
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
hTSH
·CTP
and hTSH
·CTP
1+2 variants
secreted from cells treated with tunicamycin have, as expected, the
same mobility as hTSH
·CTP
(deg) secreted from treated and
untreated cells (Fig. 2B). This indicates that
N-linked oligosaccharides are present in the
- and
-subunits of hTSH
·CTP
as well as in the
-subunit of
hTSH
·CTP
1+2 and absent in the
hTSH
·CTP
(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
-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-
antiserum (Fig.
3). Whereas the hTSH
·CTP
(Fig.
3A) and hTSH
·CTP
1+2 (Fig. 3B)
were secreted efficiently with a similar t1/2 of
~2 h, the secretion rate of hTSH
·CTP
(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
- and
-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 hTSH ·CTP (A),
hTSH ·CTP 1+2 (B), and
hTSH ·CTP (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 -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 hTSH
·CTP
, the maximal effect of
hTSH
·CTP
1+2 or hTSH
·CTP
(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 hTSH
·CTP
(deg) and to undetectable
amounts after exposure to hTSH
·CTP
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 hTSH
·CTP
1+2 (IC50 = 70 microunits/ml) or hTSH
·CTP
(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 hTSH
·CTP
1+2 (IC50 = 33 microunits/ml) or hTSH
·CTP
(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
hTSH
·CTP
(deg), respectively. 200 microunits/ml of the
variant hTSH
·CTP
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.
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 |
DISCUSSION |
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,
hTSH
·CTP
(deg) reduced the biological activity of hTSH or hTSI,
whereas hTSH
·CTP
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
hTSH
-subunit significantly reduced TSH dimer formation (14). Other
studies indicate that loss of oligosaccharides from the
-subunit
reduced assembly and/or stability of hCG (4). To bypass the problem of
dimerization of deglycosylated subunits, the subunits
and
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
-
and
-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
- and
-subunits (11). Previous studies indicated
that fusing the CTP to the C-terminal end of hFSH
(13, 21), to the
N-terminal of hCG
(15), or to hTSH
(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. hTSH
·CTP
(deg) was more potent than
hTSH
·CTP
1+2, and this may be related to the
difference in conformation of the variants.
The competition experiments indicated that
hTSH
·CTP
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
hTSH
·CTP
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
hTSH
·CTP
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
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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.
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Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.