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Department of Biochemistry (F.A.F.), Carmel Medical Center, and the Rappaport Family Institute for Research in the Medical Sciences, Faculty of Medicine, Technion-Israel Institute of Technology, Haifa 34362, Israel; Department of Molecular Biology and Pharmacology (D.B.-M., M.P., I.B.), Washington University School of Medicine, St. Louis, Missouri 63110; and Division of Reproductive Medicine (S.Y., A.J.W.H.), Department of Gynecology and Obstetrics, Stanford University Medical Center, Stanford, California 94305
Address all correspondence and requests for reprints to: Dr. Fuad A. Fares, Department of Biochemistry, Carmel Medical Center, Haifa 34362, Israel. E-mail: biochem{at}actcom.co.il
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-subunit
noncovalently linked to a hormone specific ß-subunit. Assembly of
- and ß-subunits is essential for hormone-specific
posttranslational modifications, receptor binding, and bioactivity.
Structure-function studies of TSH and gonadotropins using site-directed
mutagenesis can often affect folding, assembly, and secretion of the
hormone. To circumvent these difficulties, recently, the gonadotropin
heterodimers were converted to single chains. Here we converted the
hTSH heterodimer to a biologically active single chain by genetically
fusing the amino terminal end of the common -subunit to the carboxyl
terminal end of hTSHß in the presence or absence of hCGß carboxyl
terminal peptide (CTP), which was used as a linker. Wild-type hTSH and
the single chains were expressed in Chinese hamster ovary (CHO) cells,
and they were efficiently secreted. Although the secretion rate of the
single chain was 3-fold higher than that of hTSH wild-type. Moreover,
the secretion of the single chain in the presence of the CTP linker was
dramatically increased. On the other hand, receptor binding and
in vitro bioactivity of the single chains were similar
to that of hTSH wild-type. These data indicate the potential of the
single chain approach to further investigate structure-function
relationships of TSH. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-subunit and
hormone ß-specific subunit (1, 2, 3, 4). Assembly of glycoprotein subunits
is vital to the function of these hormones. The maturation of the
hormone-specific oligosaccharides is dependent on the formation of the
heterodimer complex and only dimers are biologically active (1).
Site-directed mutagenesis has become an important tool for studying the
structure and function of glycoprotein hormones. However, mutations in
either - or ß-subunits can alter the folding and ultimately
inhibit subunit assembly and secretion of the hormone (5, 6, 7). To
overcome these limitations, the genes encoding the common -subunit
and either the hCGß or FSHß subunits were genetically fused. The
resulting polypeptide chains were efficiently secreted and were
biologically active (8, 9, 10, 11). These studies presumed that addition of
the human CGß 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 hFSHß (12) hTSHß (13) or to the
-subunit of hCG (14) did not significantly affect assembly or
in vitro biological activity. Moreover, the in
vivo potency of the chimeras containing CTP were substantially
increased.
Assembly of the hTSHß and -subunit is the rate limiting step in
the production of functional heterodimer (15). Thus, converting hTSH to
a single chain form could increase the biological half-life and expand
the range of TSH structure-function studies. Here we describe the
construction of biologically active hTSH single chains. The chimeras
were constructed by fusing the carboxyl end of the hTSHß subunit to
the N-terminus of the -subunit in the absence or presence
of the CTP sequence. Expression of this chimeric genes in CHO cells
produced a single polypeptide hTSH molecules that were secreted
efficiently and are biologically active. However, the presence of CTP
linker, is important for the maximal expression of a functional single
chain but not for receptor binding and signal transduction in
vitro.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell culture media and reagents were obtained from Biological Industries (Beit Haemek, Israel). G418 was obtained from Sigma Chemical Co. (St. Louis, MO). Rabbit antiserum against hTSH dimer was purchased from Fitzgerald (Concord, MA).
Construction of hTSH single chains
The hTSH single chains were constructed using overlapping PCR
mutagenesis as described (8, 9, 16). The nucleotide sequence encoding
the subunit was inserted in frame at the 3' end of the hTSHß
subunit with (hTSHßCTP) or without (hTSHß) the C-terminal end
of hCGß subunit (Fig. 1
).
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Primer 1: 5'- GTGGGATCAGGGGGATCCTAGATTTCTGAGTTA-3'
Primer 2: 5'-TGAGTCGACATGATAATTCAGTGATTGAAT-3'
Primer 3: 5'-CACATCAGGAGCGACAGAAAATCCTAC-3'
Primer 4: 5'-GGATTTTCTGTCGCTCCTGATGTGCAG-3'
Primer 5: 5'-TGAGTCGACATGATAATTCAGTGATTGAAT-3'
Primer 6: 5'-TGAGGAAGAGGAGACAGAAAATCCTAC-3'
For construction of TSHß single chain, the expression vectors,
pM2TSHß and PM2 were used as a template
DNA for PCR. These expression vectors were used previously to produce
hTSH in transfected CHO cells (17, 18). In the first reaction,
pM2TSHß vector and primers 1 and 2 were
used to generate a fragment which contains exons 2 and 3 of hTSHß
(exon 1 contains nontranslated sequences) and the 5' end of exon 2 of
the -subunit (Fig. 1A
). Primer 1 contains the TSHß 5' end
sequence, which includes a new BamHI site and primer 2
contains the first four codons of the -subunit and a stretch of the
TSHß exon 3 sequence. In the second reaction, PM2
vector and primers 3 and 4 were used to synthesize a product containing
the 3' end of hTSHß exon 3 and exons 2–4 of the -subunit (exon 1
contains nontranslated sequence). Primer 3 contains the sequence
corresponding to the last four codons of the hTSHß and the first five
codons of the human common -subunit, and primer 4 contains some of
the flanking sequence of the exon 4 that also includes a newly
created SalI site. In the third reaction, the two fragments
obtained in reactions 1 and 2, were used as overlapping templates for
an additional PCR step with primers 1 and 4. The resulting construct
contains exon 2 and 3 of hTSHß and exons 2–4 of the human gene
(hTSHß).
The construction of a single hTSHßCTP chain was similar to that of
hTSHß except that the vectors pM2TSHß (18) and
pM2CTP (14) were used as templates. In the first
reaction, pM2TSHß was used as a template for primers 1
and 5 to synthesize the entire TSHß coding sequence (exons 2 and 3)
and a part of the CTP sequence (Fig. 1B). Primer 1 contains the TSHß
5' end sequence and a BamHI site. Primer 5 contains the
first four codons of the hCGßCTP and a stretch of the TSHß exon 3
sequence. In the second reaction, pM2CTP was used as a
template for primers 4 and 6 to generate a product containing the 3'
end of TSHß exon 3 and the CTP sequence. Primer 6 contains the
sequence corresponding to the last four codons of the hTSHß and the
first five codons of hCGßCTP, and primer 4 contains some of the
flanking sequence of the exon 4 that also includes a newly created
SalI site. In the third reaction, the fragments obtained in
reactions 1 and 2 were used as overlapping templates to synthesize a
single hTSHßCTP gene using primers 1 and 4.
The PCR generated constructs were completely sequenced to ensure that
no errors were introduced during the PCR. The
BamHI/SalI fragments containing the TSHß or
TSHßCTP chimeric genes were inserted at the
BamHI/SalI cloning sites of the eukaryotic
expression vector, pM2-HA (8).
DNA Transfection and clone selection
Chinese hamster ovary (CHO) cells were transfected with the
expression vectors pM2TSHß or
pM2TSHßCTP using calcium phosphate precipitation
method as previously described (5). To produce wild-type hTSH,
pM2hTSHß and PM2 plasmids were
cotransfected into CHO cells. Stable clones were selected using 0.25
mg/ml active equivalent of G418. Transfected colonies resistant to G418
were harvested and screened for the expression of hTSH (wild-type and
single chains) by quantitative determination of hTSH in the medium
using a TSH immunoradiometric assay and double antibody RIA.
Cell culture
The clones were maintained in medium 1 (Ham’s F-12 medium
supplemented with penicillin (100 U/ml), streptomycin (100 g/ml) and
glutamine (2 mM) containing 5% FCS and 0.125 mg/ml G418 at
37 C in humidified atmosphere of 5% CO2. For hormone
collection, cells secreting hTSH wild-type or single chains were plated
and grown to confluency into T-75 flasks. Cells were washed twice with
serum-free medium and 12 ml of medium II (medium I without FCS and
G418) were added. Medium were collected every 24 h, clarified by
centrifugation and concentrated using centriprep concentrators (Amicon
Corp., Danvers, MA). Concentrations of hTSH and its variants were
determined by hTSH immunoradiometric assay and a double antibody RIA
and immunological activity was expressed as international units
(IU).
Western blot analysis
For Western blot analysis, samples of concentrated medium (10
µIU) were electrophoresed on a reducing or nonreducing 15%
SDS-polyacrylamide gels by the method of Laemmli (19). After SDS-PAGE,
the gels were allowed to equilibrate for 10 min in 25 mM
Tris and 192 mM glycine in 20% (vol/vol) methanol as
previously described (20). The protein was transferred to 0.2 µm pore
size nitrocellulose (Sigma) at 100 V for 2 h using a Bio-Rad Mini
Trans-Blot electrophoresis cell (Bio-Rad Laboratories, Richmond, CA),
according to the method described in the manual accompanying the unit.
The proteins were detected using the Western-Light Plus
Chemiluminescent Detection System (Tropix, Bedford, MA) and hTSH
antisera according to the methods described in the manual accompanying
the system.
Metabolic labeling
On day 0, cells were plated into 12-well dishes (350,000 cells
per well) in 1 ml medium I. For pulse chase studies, the cells were
washed twice with cystein-free medium III (medium I supplemented with
5% dialyzed calf serum) and preincubated for 1.5 h with
cystein-free medium III, followed by a 20-min pulse-labeling in
cysteine-free medium III containing 100 µCi/ml
[35S]cysteine. The cells were then chased by washing
twice with medium III containing 1 mM unlabeled cysteine
and incubated in this chase medium for the indicated times. Media and
cell lysates were prepared, immunoprecipitated using polyclonal
antiserum against the human -subunit, and resolved on 15%
SDS-polyacrylamide gels (21, 22).
In vitro bioassay and radioligand receptor assay
The in vitro bioactivity of TSH and its derivatives
were assayed by their ability to stimulate cAMP in FRTL-5 cells. FRTL-5
cells were purchased from the American Type Culture Collection (ATCC)
(Rockville, MD). These cells are clonal rat thyroid cells that are
dependent on TSH for their growth and used as a convenient method to
assay the in vitro bioactivity of TSH (20, 23). Briefly, the
cells were maintained in Coon’s modified Ham’s F12 supplemented with
bovine TSH (bTSH) and growth factors. After maintaining 6–8 days in
the absence of bTSH, cells were incubated with different concentrations
of the TSH preparations, diluted in hypotonic HBSS containing 40
mM HEPES, 4 mM isobutylmethylxanthine and 4
mg/ml BSA for 1 h at 37 C. The amount of cAMP released into the
supernatant was measured by a RIA.
For the radioligand receptor assay, hTSH was iodinated by the lactoperoxidase method (24). Human thyroid biopsies were homogenized in ice-cold Dulbecco’s PBS containing 0.1% BSA. The homogenate was centrifuge at 20,000 x g for 30 min at 4 C. The crude membrane preparation was incubated at 37 C with [125I]hTSH (100,000 cpm) in the presence or absence of TSH derivatives for 1 h.
Statistical analysis
Each experiment was repeated at least three times and results
are presented as the mean ± SEM of more than three
replicate determinations. Statistical analysis was done using
Student’s t test and P < 0.05 was
considered as significant different form corresponding controls.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, or
hTSHßCTP were selected. Assembly of - and ß-subunits and the
secretion of hTSH variants were assessed by Western blot analysis under
nonreducing conditions using hTSH-specific antiserum which recognizes
hTSH dimer. One major form of the heterodimeric hTSH wild-type was seen
(Fig. 2A, lane 2). For the hTSHß
single chain devoid of linker, it appeared as two bands (lane 3) where
the mobility of the lower band is similar to that of TSH-WT. The upper
band may reflect some aggregation of the hTSHß chimera. By
contrast, the single chain constructed with the linker exhibited high
molecular weight material of one major band (Fig. 2A, lane 4). When the
proteins were resolved in reduced polyacrylamide gels only a single
band of both single chains was detected (Fig. 2B). The difference in
mobility between hTSHßCTP (Fig. 2B, lane 2) and hTSHß (Fig. 2B, lane 3) is due to the additional protein sequence and to the
O-linked oligosaccharides on the CTP (12, 14). This is
consistent with previous data that the O-linked
glycosylation sites of the CTP are preserved even though the sequence
is fused to a different subunits of the glycoprotein hormone family
(12, 14).
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, or
hTSHßCTP were secreted approximately 45+9, 130+15 and 355+50
µIU/106 cells/24 h, respectively. Thus, the secretion of
hTSH single peptide chain is significantly enhanced compared with that
of wild-type (P < 0.05). Moreover, the presence of a
linker between the subunits, dramatically enhances the total secretion
of the single chain.
The secretion kinetics of the single chain were determined by
pulse-chase analysis (Fig. 3). Both
chains are released quantitatively and the secretion kinetics of
hTSHßCTP is comparable to that of hTSHß. The secretion of the
single chain devoid of a linker is prolonged compared with the single
chain with the linker (t
= 240 min vs.
90 min); its delayed secretion is reflected by increases in the
intracellular content. It is curious that in some experiments the
signal of TSHßCTP in the medium is reduced after 24 h of
chase. While it is not clear why this occurs, it does not appear to be
the result of degradation (Sato, A., and I. Boime). These data show
that a linker is required for efficient secretion presumably by
altering the folding at the junction point between the and ß
domains.
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). The hTSH
wild-type and the single chains were examined for their ability to
stimulate cAMP from rat FRTL-5 thyroid culture cells. Treatment of the
cells with increasing concentrations of hTSH wild-type or single chains
(1–100 nM) resulted in a similar dose-dependent increase
in cAMP formation (Fig. 5). These results
indicate that hTSH-WT and the hTSH single chains exhibited similar
binding affinity and signal transduction activity in
vitro.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ß. Our results are
consistent with earlier studies demonstrating that hCG and hFSH can be
synthesized as biologically active single chains (8, 9, 10, 11). Thus, the
noncovalent heterodimeric structure is not critical for the function of
the glycoprotein hormone family.
Secretion of hTSH single chains from stable clones was comparable with
hTSH wild-type. Two major forms of hTSHß appeared in the media.
The lower band was similar to that of hTSH wild-type where the upper
band may reflect some aggregation of the single chain. The secreted
form of the CTP-linker migrated slower than the wild-type and the
hTSHß forms. This shift reflect the addition of the
O-linked carbohydrates associated with the CTP (12, 14).
However, the hTSHß CTP single chain with the linker is still
heterogeneous which may reflect differences in the processing of the
N and/or the O-linked carbohydrates. The presence
of these complexes in the hTSHß suggests that absence of the
linker resulted in more denatured form of the protein. Aggregates were
observed when cys mutants of CGß subunit monomers (25, 26) and single
chains (27) were analyzed under nonreduced conditions, and also during
the purification of nature urinary hCG (28, 29). The presence or
absence of hCG carboxy-terminal peptide (CTP) as a linker between the
subunits, did not affect binding affinity and biological activity of
the single chains in vitro, whereas, the intracellular
behavior as manifested by differences in the total secretion was
significantly modified by incorporating the linker. The single chain
containing the linker was efficiently secreted compared with the single
chain lacking the linker. Previous studies indicated that fusing the
CTP to the C-terminal end of hFSHß (12), to the N-terminal
region of hCG -subunit (14) or to hTSHß (13), assembly, secretion
and signal transduction of dimers (-hFSHß-CTP, CTP--hCGß or
hTSHß-) were comparable to that of wild-type. Thus, using the CTP
as a linker permits the -subunit to assume the proper conformation
with the ß sequences. Despite these differences in the secretion
rate, the receptor binding affinities and the extent of cAMP induction
by both analogs are similar to that of hTSH wild-type. This implies
that the conformation for secretion and biological action are not the
same. Moreover, the CTP is required for maximal secretion rate of the
hTSH single chain form.
The conformation of hTSH heterodimer is critical for secretion,
hormone-specific posttranslational modifications and signal
transduction. Previously we showed that cotransfection of the
expression vectors encoding hTSHß and minigenes, resulted in a
formation of functional hTSH heterodimer (17, 18). Posttranslational
processing studies show that hTSHß combines rapidly with the
-subunit resulting in quantitative secretion of dimer into the
medium (18, 30, 31). Using site-directed mutagenesis, it was reported
that mutating hTSHß subunit significantly reduced TSH dimer formation
(13). Thus, conversion of hTSH heterodimer to a single chain form
represents an important model to further investigate structure-function
relationships of human TSH since difficulties in producing mutants
where assembly is rate-limiting can be avoided. This conversion may
increase stability and activity of the hormone that could be important
for in vivo studies.
On the clinical level, this approach could lead to the development of a highly effective, long acting hTSH agonist or antagonist. Human or bovine TSH are used as a diagnostic agent for patients suffering from thyroid carcinoma (29, 32). It was reported that administration of recombinant hTSH in humans is safe and effective in stimulating radioiodine uptake and thyroglobulin secretion (29). One issue regarding the clinical use of hTSH is its rapid clearance from the circulation (13, 32). On the other hand, repeated use of bovine TSH in humans generates neutralizing antibodies (33, 34). Thus a human TSH single chain that would have a prolonged half-life in the circulation and an increased biological availability in vivo could alleviate the need for the bovine hormone and minimize immunologic reactions. In addition, TSH antagonist could offer a novel therapeutic strategy in Graves’ disease, where antibodies directed against the hTSH receptor site in the thyroid cell membrane still has the capacity to stimulate growth and function of the thyroid. In addition, an antagonist could be important in treating hyper TSH-secreting pituitary adenomas that often results in thyrotoxicosis. Previous studies indicated that deleting the N-linked oligosaccharide units on hTSH had no effect on receptor binding, whereas the biological activity was significantly reduced (17, 35, 36). Thus, the availability of human TSH single chain together with a linker sequence and the hTSH deglycosylated forms, may have important implications in designing a hTSH-selective antagonist.
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Acknowledgments |
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Footnotes |
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Received September 26, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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subunit in the secretion
and assembly of human chorionic gonadotropin. J Cell Biol 106:1049–1059 and chorionic
gonadotropin ß subunits in tandem. Proc Natl Acad Sci USA 92:2041–2045 subunit and the follicle-stimulating
hormone ß subunit. J Biol Chem 271:10445–10448 subunit and either the
CGß or FSHß subunit: role of a linker sequence. Mol Cell Endocrinol 125:71–77[CrossRef][Medline]
-subunit:
O-linked glycosylation and in vivo bioactivity of
chimeric hCG. Mol Endocrinol 9:54–63[Abstract]
subunit in human
thyrotropin bioactivity. Endocrinology 137:555–560[Abstract]
-subunit is critical for
secretion and stability of the human thyroyropin ß-subunit. Mol
Endocrinol 2:95–100[Abstract]
. Mol Endocrinol 9:1279–1287[Abstract]
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