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Original Studies |
FIRS Laboratories, RSR Ltd. (Y.O., J.S., S.R., M.M., A.K., J.F., B.R.S.), Parc-Ty-Glas, Llanishen, Cardiff, United Kingdom CF4 5DU; and the Department of Medicine, University of Wales College of Medicine (Y.O., S.R., M.M., J.F., B.R.S.), Heath Park, Cardiff, United Kingdom CF4 4XN
Address all correspondence and requests for reprints to: Dr. B. Rees Smith, FIRS Laboratories, RSR Ltd., Parc Ty Glas, Llanishen, Cardiff, United Kingdom CF4 5DU.
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Abstract |
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During peptide chain glycosylation, high mannose-type sugar residues are attached first and then modified by the formation of complex type structures to form the mature glycoprotein. Our data suggest that in the case of the TSH receptor, this type of posttranslational processing has an important role in forming the TSH-binding site.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The human TSHR consists of a polypeptide chain of 764 amino acids with a calculated molecular mass of 84 kDa (2, 3, 4, 5, 6). The part of the ECD between amino acids 317 and 366 is particularly susceptible to proteolysis, and cleavage of the receptor in this region gives rise to a two-subunit structure in which the ECD (A subunit) and transmembrane domain (B subunit) are linked only by a disulfide bridge(s) (1, 7, 8).
When the human TSHR is expressed in CHO-K1 cells, Western blotting shows the receptor running as a 100-kDa/120-kDa doublet (1, 7, 8, 9, 10), and we now describe a study of sugar residues in these two forms of the receptor. In particular, the extent of glycosylation and types of sugar residues involved have been investigated using glycosidases and lectins. Furthermore, the relationship between TSHR glycosylation and TSH binding was studied after separation of the 100- and 120-kDa TSHR species using lectin affinity chromatography.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Six- to 8-week-old BALB/c mice were immunized with 50 µg glutathione-S-transferase-TSHR fusion proteins, and monoclonal antibodies were produced as described previously (10).
Preparation of detergent-solubilized recombinant human TSHR
Production of a eukaryotic cell line (CHO-K1) expressing the full-length TSHR (4 x 105 functional receptors/cell, as assessed by Scatchard analysis of TSH binding) was as previously described (10, 11). CHO-K1 cells were grown to confluence for 4 days in 175-cm2 flasks, and the cells were washed with Dulbecco’s phosphate-buffered saline without calcium and magnesium ions (Life Technologies) and scraped into 10 mL ice-cold 50 mmol/L NaCl and 10 mmol/L Tris-HCl, pH 7.5, containing 1 mmol/L phenylmethylsulfonylfluoride (buffer A). The cells were centrifuged at 1,000 x g for 5 min at 4 C, the pellet was resuspended in 1 mL buffer A and homogenized with a glass homogenizer on ice. This homogenate was then centrifuged at 12,000 x g for 30 min at 4 C, resuspended in 1 mL ice-cold buffer A containing 1% Triton X-100, homogenized, and centrifuged at 90,000 x g for 2 h at 4 C, and the supernatant was aliquoted and stored at -70 C. Scatchard analysis indicated that the solubilized preparations contained 2.4 µg TSHR/mL (11). Solubilized preparations contained 1.8 µg TSHR/mg total protein as assessed by the method of Bradford (12) (reagents from Bio-Rad Laboratories, Inc., Hemel Hempstead, UK).
Affinity purification of recombinant human TSHR
TSHR antibody IgGs were purified from ascitic fluid using affinity chromotography on Prosep A (Bioprocessing, Consett, UK) according to the manufacturer’s instructions. Twenty milligrams of purified 3F3 IgG (10) were coupled to 1 g cyanogen bromide-activated Sepharose 4B (Pharmacia Biotech, St. Albans, UK) according to the manufacturer’s instructions. Solubilized TSHR was loaded onto the affinity column in 150 mmol/L Tris-HCl (pH 8.3), 50 mmol/L NaCl, and 1% Triton X-100; eluted with 0.1 mol/L glycine/NaOH (pH 11), 500 mmol/L NaCl, and 0.1% Triton X-100; and dialyzed against 10 mmol/L Tris-HCl (pH 7.5), 50 mmol/L NaCl, and 0.1% Triton X-100. The peak fractions were pooled and stored in aliquots at -70 C for deglycosylation and lectin studies. The concentration of TSHR in these preparations was about 300 µg/mg protein (as assessed from Coomassie blue-stained SDS-polyacrylamide gels and Bradford assay).
Deglycosylation of TSHRs and SDS-PAGE followed by Western blotting
Endoglycosidase H (Endo H), N-glycosidase F (PNGase
F), and neuraminidase were obtained from Boehringer Mannheim (Lewes, UK). Twenty-five microliters of either
solubilized (
60 ng) or affinity-purified (500 ng) TSHR were
incubated for 16 h at room temperature in the enzyme buffer, with
or without enzyme. Deglycosylation reactions were carried out according
to the manufacturer’s instructions using Endo H (1 mU/reaction) in 50
µL 25 mmol/L sodium acetate (pH 5.5), PNGase F (1 mU/reaction) in 50
µL 50 mmol/L sodium phosphate (pH 6.0), 25 mmol/L ethylenediamine
tetraacetate, 0.05% SDS, 0.5% ß-mercaptoethanol, 0.5%
Triton-X-100, and neuraminidase (200 mU/reaction) in 50 µL 50 mmol/L
sodium acetate (pH 5.5) containing 2 mmol/L CaCl2.
After incubation, the samples were mixed with an equal volume of
SDS-PAGE sample buffer [4% SDS, 20% glycerol, 100 mmol/L Tris-HCl
(pH 6.8), and 0.002% bromophenol blue] plus dithiothreitol (10
mmol/L), heated to 100 C for 3 min, electrophoresed on 9% acrylamide
gels (SDS-PAGE) (13), and blotted onto nitrocellulose (Schleicher & Schuell, Inc., UK Ltd., London, UK). Western blotting analysis
was carried out according to the method of Birk and Koepsell (14). The
membranes were blocked using 1 mg/mL polyvinyl alcohol in
phosphate-buffered saline (Sigma Chemical Co., Poole, UK)
and developed using antimouse horseradish peroxidase conjugate followed
by enhanced chemiluminescence reagents (Amersham, Little
Chalfont, UK).
Lectin analysis
The biotin-labeled lectins Aleuria aurantia (AAL), Canavalia
ensiformis (Con A), Dolichos biflorus (DBA), Datura stramonium (DSL),
Griffonia simplicifolia (GSL), Galanthus nivalis (GNL), Lotus
tetragonobus (LTL), Lycopersion esculentum (LEL), Maackia amurensis
(MALII), Samuccus nigra (SNA), Solanum tuberosum (STL), and wheat-germ
agglutinin (WGA) were purchased from Vector Laboratories, Inc. (Peterborough, UK; Table 1
).
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In some experiments, digoxigenin-labeled lectins GNL, MALII,
Samuccus nigra, and DSL were used according to the manufacturer’s
instructions and developed using the DIG Glycan Differentiation Kit
(Boehringer Mannheim; Table 1
).
GNL affinity chromatography
3F3 affinity-purified receptor (2 mL) was loaded on a 1-mL
column of agarose-bound GNL (Vector Laboratories, Inc.) in
binding buffer [150 mmol/L Tris-HCl (pH 8.3), 50 mmol/L NaCl, and
0.1% Triton X-100], eluted with 0.1 mol/L
-methyl-D-mannoside (Sigma Chemical Co.) in
binding buffer, and dialyzed against 10 mmol/L Tris-HCl (pH 7.5), 50
mmol/L NaCl, and 0.1% Triton X-100. The load material, wash fractions,
and eluted fractions were tested for TSH binding (see below for method)
and were analyzed by SDS-PAGE on 9% polyacrylamide gels followed by
Western blotting.
DSL affinity chromatography
3F3 affinity-purified receptor (2 mL) was loaded onto a 1-mL column of agarose-bound DSL (Vector Laboratories, Inc.) in binding buffer 2 [10 mmol/L Tris-HCl (pH 7.5), 50 mmol/L NaCl, and 0.1% Triton X-100], eluted with 20% chitin hydrolysate (Vector Laboratories, Inc.) in binding buffer 2, and dialyzed against this buffer. The load material, wash fractions, and eluted fractions were then analyzed as described for the GNL affinity chromatography studies.
TSH binding assays
TSH binding assays were carried out on duplicate 50-µL
aliquots of 1:8 diluted column fractions that were incubated with 50
µL [125I]TSH [15,000 cpm diluted in 10 mmol/L Tris-HCl
(pH 7.4), 50 mmol/L NaCl, 1 g/L BSA, and 3 mmol/L NaN3] at
37 C for 1 h. TSHR-[125I]TSH complexes were
precipitated by the addition of 2 mL 16% (wt/vol) polyethylene glycol
4000 (RSR Ltd., Cardiff, UK) and 25 µL normal pool serum as
coprecipitant, followed by centrifugation (1,500 x g
for 30 min at 4 C), and the precipitates were counted in a
-counter.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Western blotting analysis of affinity-purified recombinant human
TSHR preparations treated with glycosidases, Endo H, PNGase F, and
neuraminidase is shown in Fig. 1.
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, panel 4) was present as two
bands of 117.0 ± 4.0 kDa (defined as the 120-kDa band) and
99.0 ± 4.0 kDa (defined as the 100-kDa band; mean ±
SD; n = 5). These 120- and 100-kDa bands are referred
to subsequently as the upper and lower (respectively) full-length TSHR
bands. After treatment with Endo H (Fig. 1, panel 1, lane +), TSHR was
visible as two full-length bands of 117.4 ± 4.2 and 83.3 ±
2.8 kDa (mean ± SD; n = 5), whereas TSHR treated
with buffer only (Fig. 1, panel 1, lane -) was present as
two bands of 117.4 ± 4.2 and 99.4 ± 3.6 kDa (mean ±
SD; n = 5). These experiments showed that treatment
with Endo H resulted in a change in mobility of the lower TSHR band of
16 ± 1.9 kDa (mean ± SD; n = 5). The
mobility of the upper band of the TSHR doublet was essentially
unchanged.
TSHR preparations treated with PNGase F (Fig. 1, panel 2, lane +) were
visible as a broad band of 88.0 ± 5kDa (mean ±
SD; n = 5), whereas TSHR treated with buffer only
(Fig. 1, panel 2, lane -) was present as a doublet of 117.4 ±
4.2 and 99.4 ± 3.6 kDa (mean ± SD; n = 5).
Treatment with PNGase F resulted in a change in mobility of 29.2
± 3 kDa (mean ± SD; n = 5) for the upper TSHR
band and 11.2 ± 2.7 kDa (mean ± SD; n = 5)
for the lower TSHR band.
TSHR treated with neuraminidase (Fig. 1, panel 3, lane +) was present
as two bands of 108.5 ± 3.3 and 99.7 ± 3.2 kDa (mean
± SD; n = 5), whereas TSHR incubated with buffer only
(Fig. 1, panel 3, lane -) was present as a doublet of 117.9 ±
3.6 and 99.7 ± 3.2 kDa (mean ± SD; n = 5).
After treatment with neuraminidase, the mobility of the upper band of
the TSHR doublet was decreased by 9.4 ± 1.4 kDa (mean ±
SD; n = 5), whereas the mobility of the lower band was
essentially unchanged.
Lectin blotting analysis
The ability of affinity-purified recombinant human TSHR
preparations to bind different lectins was investigated using lectin
blotting analysis. The reactivity of TSHR with Con A, DSL, and MALII is
shown in Fig. 2. Con A (lane 1) reacted
strongly with the lower TSHR band of 95 ± 4 kDa (mean ±
SD; n = 4) and weakly with the upper 114 ± 6-kDa
(mean ± SD; n = 4) TSHR band. In contrast, DSL
and MALII both reacted only with the upper band of the TSHR doublet, at
115 ± 7 kDa (mean ± SD; n = 7) and
116 ± 2 kDa (mean ± SD; n = 3),
respectively. Neither DSL nor MALII appeared to react with the lower
band of the TSHR doublet. The reactivity of different lectins with the
two full-length TSHR bands is summarized in Table 1.
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). In this
experiment, DSL reaction with the untreated TSHR was evident as a band
at 119 ± 1 kDa (mean ± SD; n = 3; Fig. 3, panel 1). After treatment with Endo H (Fig. 3, panel 2), no shift in
molecular mass was observed. However, after incubation of the TSHR with
neuraminidase, the 119-kDa band reactive with DSL was shifted to
107 ± 2 kDa (mean ± SD; n = 3), a shift of
approximately 12 kDa (Fig. 3, panel 3). In the case of PNGase F (Fig. 3, panel 4), DSL did not react with the TSHR after treatment of the
receptor with this enzyme.
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To separate the two bands forming the TSHR doublet, we used lectin
affinity chromatography. In these experiments, GNL affinity column
fractions were reacted with 3C6 TSHR monoclonal antibody (ascites fluid
diluted 1:100) (10) in Western blotting (Fig. 4). The unfractionated load material was
present as two full-length TSHR bands at 115.0 ± 2.5 and
98.0 ± 0.8 kDa (mean ± SD; n = 4) and some
TSHR A subunit at 51.0 ± 1.0 kDa (mean ± SD;
n = 4). The amounts of A subunit in different TSHR
preparations showed some variations, but the major components observed
were the two full-length receptor bands. The unretarded column
fractions (Fig. 4, lanes W1–W8) contained the upper TSHR band and the
receptor A subunit, whereas the eluted fractions (Fig. 4, lanes E4–E9)
contained the lower band. TSH binding assays were carried out in three
of the four experiments on the unfractionated load material, the
unretarded column fractions, and the eluted fractions. An example of
TSH binding to GNL column fractions is shown in Fig. 5. The TSH-binding activity in the
unretarded column fractions represented 82 ± 17% (mean ±
SD; n = 3) of the TSH-binding activity loaded onto the
column (Table 2).
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The results obtained with DSL affinity chromatography were
opposite those obtained with GNL, as shown in Fig. 6. Unfractionated load material contained
two full-length TSHR bands of 113.0 ± 4.0 and 98.5 ± 1.0
kDa (mean ± SD; n = 5) and a lower molecular
mass band of 50.8 ± 0.4 kDa (mean ± SD; n
= 5) corresponding to the receptor A subunit. The unretarded column
fractions (Fig. 6, lanes W3–W8) contained the lower band (98.5 kDa) of
the TSHR doublet, whereas the eluted fractions (Fig. 6, lanes E4–E10)
contained mainly the upper band of the doublet and the 50-kDa A
subunit. Small amounts of the lower 98-kDa band were also observed in
the eluted fractions. TSH binding assays were carried out in three of
the five experiments on the unfractionated load material, the
unretarded column fractions, and the eluted fractions. An example of
TSH binding by the unretarded column fractions and the eluted fractions
for one DSL column is shown in Fig. 5. The TSH-binding activity in the
unretarded column fractions represented 29 ± 1.2% (mean ±
SD; n = 3) of the TSH-binding activity loaded onto the
column (Table 2). The TSH-binding activity in the unretarded column
fractions of the GNL column was different from that of the DSL column,
as determined using unpaired t test with Welch’s correction
(P < 0.01).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our results show that sugar residues contribute approximately 33 kDa to the overall molecular mass of the TSHR upper band and approximately 16 kDa to the overall molecular mass of the TSHR lower band, as judged by mobility on SDS-PAGE. Furthermore, our studies indicate that the lower TSHR band contains predominantly high mannose-type sugars whereas the upper TSHR band contains mostly complex-type sugars. These results are in agreement with reports from other laboratories using pulse labeling of CHO-K1 cells and L cells (9). In addition, pulse labeling experiments in stably transfected L cells suggested that the lower recombinant TSHR band was the precursor for the upper, fully glycosylated TSHR band (9). This is consistent with the general understanding that during the glycosylation process high mannose-type sugar residues are first attached to glycosylation sites on the peptide chain, and these residues are then modified to form complex-type structures that give rise to final "matured" glycosylated protein (17). Consequently, our studies together with results from other laboratories suggest that the 100-kDa band of the TSHR doublet represents the high mannose precursor, whereas the 120-kDa band represents the mature, fully glycosylated TSHR (15).
A more detailed analysis of the carbohydrate residues present in the
two bands of the TSHR doublet was carried out using lectin blotting
analysis. The lower band of the TSHR was found to react with Con A,
which is specific for -linked mannose (18) and also reacted with
GNL, which recognizes (1, 2, 3)-linked mannose residues (19). This was
not unexpected, as a wide variety of membrane glycoproteins contain a
core oligosaccharide structure of -linked mannose (17). The upper
band of the TSHR doublet reacted with Aleuria aurantia,
indicating the presence of either fucose-linked [(1, 2, 3, 4, 5, 6)]
N-acetylglucosamine or fucose-linked [(1, 2, 3)]
N-acetylactosamine (20), and with DSL, indicating the
presence of ß(1, 2, 3, 4)-linked N-acetylglucosamine (21). In
addition, the upper TSHR band reacted with both Lycopersion
esculentum and wheat-germ agglutinin, indicating the presence of
N-acetylglucosamine (22, 23) and with MALII, which is known
to bind to carbohydrate structures containing (2, 3)-linked sialic
acid (24). A weak reaction with the upper TSHR band was also observed
with Con A, probably due to a low affinity reaction between this lectin
and N-acetylglucosamine (18). All of the sugar residues
found to be associated with the upper TSHR band are complex-type sugar
residues. Consequently, lectin blotting analyses confirmed that the
lower band of the TSHR doublet contains high mannose-type carbohydrate,
whereas the upper band contains complex-type carbohydrate.
These studies were extended by analysis of the reactivity of biotin-labeled DSL with the TSHR after treatment with different glycosidases. After Endo H treatment, reactivity of the upper band (running at 119 kDa) was unaffected, suggesting that no terminal mannose residues were present in this component of the TSHR doublet. However, after treatment with neuraminidase, DSL reacted with the digested upper TSHR band now running at a lower molecular mass of 107 kDa (showing a difference of approximately 12 kDa from the untreated upper band), confirming the presence of sialic acid containing complex-type carbohydrates. After digestion with PNGase F, the upper band of the TSHR doublet did not bind DSL, probably due to the removal of all sugar residues that were capable of binding to this lectin.
A selection of lectins of different specificities was also tested in blotting analysis with the receptor preparations, but did not show reactivity. This lack of reactivity with the TSHR doublet could be attributed to the absence of the appropriate sugar residues or their inaccessibility to lectin binding (17). Alternatively, the concentration of the TSHR on the Western blot might have been too low to detect binding of the lectins.
Using lectin affinity chromatography we were able to separate the 120- and 100-kDa components of the TSHR doublet. The upper TSHR band (containing complex carbohydrate) bound to the DSL affinity column, whereas the unretarded column fractions contained the lower molecular mass TSHR band (containing high mannose residues). The opposite effect was seen when a GNL affinity column was used, and with this lectin the upper TSHR band (containing complex carbohydrate) was found in the unretarded column fractions, whereas the high mannose-containing lower band was retained by the GNL column. [125I]TSH binding studies revealed that about 80% of the TSH binding activity loaded onto the GNL column was present in the unretarded fractions containing the upper 120-kDa TSHR band. In contrast, only 30% of the TSH-binding activity loaded onto the DSL column was present in the unretarded fractions containing the 100-kDa TSHR high mannose precursor.
The observation that not all of the TSH-binding activity of the TSHR preparations was retarded by the DSL column probably reflected the limitations of the column affinity and/or capacity. This suggestion is supported by Scatchard analysis, which indicated that the affinities of TSHR in the nonretarded fractions from both DSL and GNL columns were similar. The results with the two lectin columns suggest that the 120-kDa band is principally responsible for TSH binding. The inability to detect the 120-kDa band in the unretarded fractions of the DSL column (which showed a small amount of TSH-binding activity) probably reflected limitations in the sensitivity of the Western blotting analysis. Consequently, the acquisition of complex carbohydrate by the TSHR appears to be an important factor in enabling the TSHR to bind TSH. This can be compared with reports that N-linked carbohydrates on the FSH receptor are important for folding the receptor into a conformation allowing FSH binding (25). In contrast, acquisition of complex carbohydrate has not been found important for hormone binding in the case of the LH/CG receptor (26, 27).
Although the DSL affinity column bound the 120-kDa band of the TSHR, attempts to elute the bound receptor with sugars were not very successful. In particular, eluted material did not bind TSH well even though the 120-kDa band was clearly present. The loss of TSH-binding activity was due presumably to unfolding of the receptor during binding to and elution from the lectin column.
An important role for complex carbohydrates in the correct folding of the extracellular domain of the TSHR is consistent at least in part with the inability to demonstrate TSH binding to TSHR produced in expression systems other than mammalian cells. For example TSHR produced in E. coli is unglycosylated (28, 29), and TSHR produced in the baculovirus system is only partially glycosylated, containing mainly high mannose-type sugar residues (30, 31, 32) and neither of the receptors bound TSH with high affinity (29, 30, 31, 32, 33). Furthermore, [35S]TSHR produced in vitro in the transcription/translation reaction system is unglycosylated and does not bind TSH (34, 35). In addition, the importance of TSHR’s glycosylation for TSH binding has been indicated also in studies using TSHR preparations containing mutated asparagine sites (36) or TSHR fragments expressed in High Five insect cells (37).
Consequently, current studies indicate that formation of the TSH-binding site on the TSHR is dependent on complex posttranslational modifications of the peptide chain in which carbohydrate residues play an important role. In particular, acquisition of complex-type sugar residues by the TSHR appears to be an important requirement for the formation of the TSH-binding site.
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Acknowledgments |
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Footnotes |
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Received September 3, 1998.
Revised January 28, 1999.
Accepted March 8, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-2,3 to penultimate galactose
residues. J Biol Chem. 263:4576–4585.This article has been cited by other articles:
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  R Nunez Miguel, J Sanders, D Y Chirgadze, T L Blundell, J Furmaniak, and B Rees Smith FSH and TSH binding to their respective receptors: similarities, differences and implication for glycoprotein hormone specificity J. Mol. Endocrinol., September 1, 2008; 41(3): 145 - 164. [Abstract] [Full Text] [PDF]
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 A. Bell, A. Gagnon, L. Grunder, S. J. Parikh, T. J. Smith, and A. Sorisky Functional TSH receptor in human abdominal preadipocytes and orbital fibroblasts Am J Physiol Cell Physiol, August 1, 2000; 279(2): C335 - C340. [Abstract] [Full Text] [PDF]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
, Material from DSL affinity chromatography. W2–W9,
Unretarded (wash) fractions. E4–E12, Fractions eluted with sugar. %
TSH bound = mean of % 125I-labeled TSH bound to
duplicate 50-µL aliquots of each column fraction (diluted 1:8) minus
% 125I-labeled TSH bound in the presence of 50 µL buffer
only (nonspecific binding). See text for full experimental details.
Results shown are typical of three separate experiments (see also Table 2