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Division of Endocrinology and Metabolism, Department of Medicine, Mount Sinai School of Medicine, New York, New York 10029
Address all correspondence and requests for reprints to: Dr. P. N. Graves, Box 1055, Mount Sinai Medical Center, 1 Gustave L. Levy Place, New York, New York 10029.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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F'), where the majority of the
recombinant receptor was insoluble. The soluble recombinant receptor
was affinity purified and characterized. Under nonreducing SDS-PAGE
conditions, the soluble hTSHR-ecd migrated as refolded, disulfide
bond-stabilized, multimeric species, whose formation was independent of
fusion partner protein. This product was found to be biologically
active as evidenced by the inhibition of the binding of
125I-TSH to the full-length hTSHR expressed in transfected
CHO cells and was used to develop a competitive capture enzyme-linked
immunosorbent assay for mapping of hTSHR antibody epitopes. Hence,
hTSHR-ecd produced in bacteria with a thioredoxin reductase mutation
was found to be highly soluble and biologically relevant. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The ectodomain of the hTSHR contains 11 cysteine residues, some of which are likely to form intrachain disulfide bridges (3) that stabilize the native structure of the receptor. Additionally, we previously have described interchain disulfide links involved in multimerization of both recombinant and natural TSHR (4, 5). Because stable disulfide bonds cannot be formed in the cytoplasm of wild-type Escherichia coli (E. coli) strains, bacterially expressed, hTSHR-ecd is mostly insoluble (4, 6, 7, 8). Recently thioredoxin reductase (trxB) mutants of E. coli have been shown to be useful in addressing this problem (9). In the absence of the oxidized form of trxB, thioredoxin-like proteins accumulated and promoted disulfide bond formation in the cytoplasm. The mutants expressed biologically active forms of alkaline phosphatase and mouse urokinase, where secondary structure depended on disulfide bond formation (9). Therefore, we have used one of these mutants (E. coli Ad494) to express the hTSHR-ecd fused with glutathione S-transferase (GST) and have analyzed receptor folding and disulfide based interactions in vivo. We showed that the hTSHR-ecd expressed in this system was mostly soluble and formed disulfide-linked multimers with or without the fusion partner. This soluble hTSHR-ecd inhibited binding of the 125I-TSH to the hTSHR and was used in a competitive capture enzyme-linked immunosorbent assay (ELISA) for analyses of TSHR-Abs specific for the C-terminal end of the hTSHR-ecd.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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F'; Strategene, La Jolla, CA) and
trxB mutant Ad494 (9). Ampr colonies were grown
overnight in L broth (10) containing 50 µg/ml ampicillin, diluted ten
times in the same medium, and grown to a concentration of 1 absorbance
U/ml (at 600 nm). Expression of hTSHR-ecd was induced with 1
mM IPTG for 1 h (Ad494; F' expresses
constitutively), and the bacterial suspension was processed immediately
or held on ice for 1 h. The cells were pelleted, resuspended at
1/20 vol in cold 1 x PBS, and sonicated on ice (Branson Sonifier
450, Branson Ultrasonics Corporation, Danbury, CT) (Method 1).
Alternatively, cells were resuspended at 1/100 vol in 10 mM
EDTA, 20 mM Tris-HCl (pH 8.0), incubated with chicken egg
white lysozyme (2 mg/ml) for 30 min on ice, and lysed by three cycles
of freezing in a dry ice-ethanol bath and thawing in a 28 C water bath
(Method 2). The viscous suspension then was diluted twice in cold
2 x PBS and sonicated three times for 30–40 sec on ice.
Western blotting and quantitative analysis of recombinant
hTSHR-ecd
Protein concentration was measured by the bicinchoninic acid
method (Pierce, Rockland, IL). One to ten micrograms of total protein
from soluble and insoluble fractions was dissolved in 2 x
SDS-PAGE buffer (2% SDS, 20% glycerol, 0.001% Bromphenol blue, and
0.125 M Tris-HCl pH 6.8) with or without subsequent boiling
in a presence of 2% ß-mercaptoethanol (ßME) and fractionated by
SDS-PAGE (Miniprotean II, Bio-Rad Laboratories, Richmond, CA). Proteins
were transferred onto supported nitrocellulose membranes (0.2 µm;
BA-S NC, Schleicher and Schuell, Keene, NH), and blots were developed
as described (4) using hTSHR antibodies shown in Table 1
. Bound antibodies were detected by peroxidase-labeled
antimouse or antirabbit Ig using enhanced chemiluminescence (Amersham,
Amersham, UK). The relative amount of hTSHR-ecd in different fractions
was measured using densitometric readings of autoradiographs (Molecular
Dynamics Personal Densitometer and ImageQuant V3.3 software, Sunnyvale,
CA). Scanned peaks were corrected for the total amount of protein in
each fraction calculated from the same original volume of cells. Sizes
of proteins were estimated using prestained protein molecular weight
standards (GIBCO BRL, Bethesda, MD).
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TSH binding of the affinity-purified hTSHR-ecd
Binding of TSH to the soluble purified hTSHR-ecd was tested in a
competition assay using a CHO cell system (11). As a control-binding
protein, we used similar amounts of an unrelated protein, anthrax toxin
protective antigen, which we expressed in E. coli Ad494 and
affinity purified in parallel with hTSHR-ecd. A pGEX plasmid containing
this anthrax toxin subunit was a kind gift of Dr. A. V. Teixeira,
Department of Medicine, Mount Sinai School of Medicine, New York.
Affinity-purified hTSHR-ecd or anthrax toxin was dialyzed against
binding buffer (NaCl-free HBSS containing 280 mM sucrose)
and preincubated for 1 h at room temperature with 8,000 cpm
125I-bovine TSH (bTSH) (Kronus, San Clemente, CA) in 300
µl binding buffer. The mixture was added to CHO JP09 cells stably
expressing the wild-type hTSHR (12) (gift of Dr. G. Vassart, Free
University of Bruxelles, Belgium). After 1.5 h incubation at 37 C,
the cells were washed three times with ice-cold binding buffer,
solubilized with 0.5 ml 1N NaOH, and radioactivity was measured. The
maximum amount of 125I-TSH bound to the cells in the
presence of only the binding buffer was used as a positive control, and
the amount bound in the presence of 10-6 M
unlabeled bovine TSH (Thytropar, Armour, Kankakee, IL) was used as a
control for nonspecific binding.
ELISA for epitope mapping
The 96-well plates (Immulon 2, Dynatech Laboratories, VA) were
coated overnight with A10 or A11 monoclonal antibody (mAb) (Table 1
)
diluted in carbonate-bicarbonate buffer (pH 9.6). After washing the
plate with PBS-0.05% Tween 20, nonspecific binding sites were blocked
with 1% BSA (Sigma Chemical Co., St. Louis, MO) in PBS-Tween for
1 h at 37 C. Bacterial sonicate was diluted in PBS-Tween/1
mM phenylmethylsulfonyl fluoride (PMSF) to a concentration
0.5 mg protein/ml. Sonicate (100 µl/well) was added and incubation
continued for 2 h at 37 C. After rigorous washing, captured
hTSHR-ecd was incubated with either 100 µl test serum (diluted in
PBS-Tween) or dilution buffer only. The wells were washed again and
incubated for 1 h at 37 C with reporter antibody (for example,
R397; Table 1). After washing, the reporter antibody was detected by
incubation with alkaline phosphatase-conjugated goat antirabbit IgG
(Sigma) using p-nitrophenylphosphate substrate diluted in
diethanolamine buffer (pH 9.8). The absorbance at 405 nm was measured
using an automated ELISA plate reader (Titerek Multiscan MCC/340 MK II;
Flow Laboratories, McLean, VA). The percent of competitive inhibition
of all samples was calculated after subtracting background ODs (OD of
wells containing host bacterial lysate without the hTSHR-ecd), and
inhibition by immune serum was calculated after subtracting nonspecific
inhibition using control serum. Analyses of data were performed using
the Excel 5.0 program for Windows.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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F') and
txtB mutant (Ad494) strains of E. coli is shown
in Fig. 1A. Soluble hTSHR-ecd was almost undetectable in
F' (lane 1) but was readily detected as a 74-kDa band corresponding
to the GST-fusion protein (26 kDa GST plus 48 kDa hTSHR-ecd) in the
trxB mutant strain Ad494 (lane 2). The amount of soluble
hTSHR was markedly increased by induction (lane 3) and further
increased by cooling the bacterial suspension for 1 h (lane 4).
The increase in soluble hTSHR-ecd during storage of induced cells on
ice indicated that disulfide bond formation was more favorable under
these conditions. The low-molecular mass bands visible in lanes 3 and 4
represented products of proteolytic cleavage by host proteases during
preparation of protein extracts and varied among preparations. The
presence of some soluble hTSHR-ecd expression in the total absence of
induction (lane 2) indicated that downregulation of basal expression
was not complete.
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). The amount of hTSHR-ecd present in cells
before (lane 1) and after (lane 2) sonication showed that most
hTSHR-ecd remained in the cell pellets. This revealed that sonication
alone (Method 1) was not sufficient to break the Ad494 cells and
release hTSHR-ecd. In most experiments, therefore, the cells were
opened with lysozyme followed by freeze-thaw, and a brief sonication
was applied to decrease viscosity (Method 2). The quantitation of
densitometric readings of soluble (lane 3) and insoluble (lane 4)
fractions of sonicate relative to the amount of protein loaded (see
Materials and Methods) showed that the total amount of
soluble hTSHR-ecd was present in quantities 1.4–1.9 times higher than
the insoluble protein.
Affinity purification of recombinant hTSHR-ecd
Soluble hTSHR-ecd expressed in E. coli Ad494 was
purified on Glutathione Sepharose 4B. Binding of the 74-kDa
GST-hTSHR-ecd fusion protein and release of cleaved (48 kDa) hTSHR-ecd
from the matrix by thrombin was monitored as a function of time (Fig. 2A). Partial digestion of the fusion protein (lane 1)
occurred after 2 h (lane 2) and was nearly complete after 16
h of digestion (lane 3). Elution of hTSHR-ecd from the column also was
monitored. As shown in Fig. 2B, the amount of purified hTSHR-ecd after
one round of thrombin cleavage and elution using PBS (lane 1), was
inefficient. A second cycle of cleavage and elution with the same
buffer did not improve the recovery of the purified receptor (lanes 2
and 3). However, addition of reduced glutathione to the elution buffer
greatly enhanced the recovery of bound hTSHR-ecd protein (lanes 4–7).
This suggested that the poor recovery in PBS may have been caused by
aggregate formation by cleaved protein. The multiple small bands of
recombinant hTSHR-ecd less then 48 kDa were most likely the products of
proteolytic cleavage of hTSHR-ecd during preparation. Addition of 1
mM PMSF to the bacterial pellets before sonication was
later used to decrease this degradation, and addition of sodium
chloride (1–2 M) to PBS during elution further increased
the recovery of purified receptor (data not shown). The total amount of
purified soluble hTSHR-ecd produced varied from 10–50 µg/liter of
culture.
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). Under nonreducing conditions, the sizes of the
major species in the insoluble fraction were 74 and 215 kDa, suggesting
some formation of multimeric forms (Fig. 3A, lane 1). In
contrast, the soluble receptor formed only a 215-kDa species with no
other forms present (Fig. 3A, lane 2), indicating that the soluble
hTSHR-ecd proteins formed disulfide-linked multimers in solution with
much higher efficiency than the insoluble.
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). Thrombin
digestion removed the 26-kDa GST and generated a 48-kDa hTSHR-ecd
(lanes 3 and 4). Both digested and undigested recombinant proteins were
analyzed under denaturing (boiling of samples in the presence of 2%
ßME) and partially denaturing conditions (samples were mixed in the
same buffer and loaded directly onto the gel without boiling).
Partially denaturing conditions were chosen because they allowed us to
have a mixture of both forms of proteins (reduced and nonreduced). The
band running above the 210-kDa marker under partially reduced
conditions with the GST-fusion protein (lane 1) was resolved into a
major 74-kDa band after complete denaturation (lane 2). The hTSHR-ecd
without the GST partner formed 180-kDa aggregates under partially
denaturing conditions (lane 3), which were resolved into 48-kDa
proteins during denaturation (lane 4). This suggested that the purified
soluble recombinant hTSHR-ecd protein formed stable structures under
nondenaturing conditions. The apparent mass of these structures,
estimated from migration rates, was compatible with four monomers held
together by disulfide bridges. The GST sequence did not contribute to
this process.
Binding of TSH to hTSHR-ecd
To determine whether the soluble hTSHR-ecd prepared from
E. coli Ad494 cells was biologically active, we tested its
ability to compete for binding of radioiodinated TSH to functional
hTSHR expressed on CHO JP09 cells (12). Affinity-purified hTSHR-ecd
(without the fusion partner) reduced binding of 125I-TSH to
the hTSHR expressed in CHO cells in a dose-dependent manner, reaching
approximately 40% at 150 µl/well of eluate containing approximately
10 µg/ml hTSHR-ecd (Fig. 4). The same amount of
control anthrax toxin protein reduced binding by approximately 12%.
Binding data analyzed by Fourier-derived affinity spectrum analysis
(FASA, 13) using the MacFASA program (Biomath Explorations, Port
Washington, NY) showed that the affinity of binding of labeled TSH to
the purified hTSHR-ecd was 4.2 x 10-8 M.
This affinity was about 100 times less than achieved using porcine
thyroid membranes as a natural source of full length TSHR (14).
Nevertheless, these data reflected increased biological activity of the
soluble hTSHR-ecd compared with the insoluble hTSHR-ecd, where no
inhibition of labeled TSH binding was observed using similar
concentrations of the receptor (6).
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). Using the capture antibody A11 approximately 100 ng ecd was bound
when bacterial sonicate, containing 50 µg of total protein, was added
per well; whereas 50 ng was captured using A10 (Fig. 5A). The potential competitive inhibition of the rabbit
antibodies binding to the captured hTSHR-ecd was tested initially
using, as a competitor, mAb A7 specific for aminoacids 402–415 of
hTSHR ecd (Table 1). As shown in Fig. 5B, detection of the captured
hTSHR-ecd by R397 was inhibited by more than 50% by preincubation with
mAb A7 (sample 1) but by less than 5% by preincubation with control
mAb (sample 2), demonstrating the specificity of detection of
R397-binding sites (aa 397–415). We then analyzed serum from seven
CBA/J mice immunized with hTSHR-ecd (15) for the presence of antibodies
to the C-terminal residues of the receptor ecd. Incubation of the
captured receptor with immune serum reduced specific R397 binding by
more than 30% (Fig. 5B, sample 3), compared with no specific
inhibition by pooled serum from seven mice immunized with control
insect cell extract (15). These results showed that the immunized mice
had developed an immune response to the same C-terminal residues of the
hTSHR-ecd.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We showed that the truncated receptor expressed in E. coli
Ad494 was mostly soluble. This was in contrast to the expression of the
truncated receptor in other E. coli strains (DH5 and
F’), where most of the recombinant protein was insoluble (4, 6).
This difference in solubility of recombinant hTSHR-ecd in different
E. coli strains reflected the fact that proper folding of
the hTSHR-ecd depended on S-S bond formation, which cannot be achieved
in the cytoplasm of commonly used E. coli strains (9).
However, it was possible to stabilize the structure by allowing
disulfide bond formation in a bacterial strain containing mutations in
the txt gene (9). The soluble hTSHR-ecd without the GST fusion protein
was affinity purified on a glutathione Sepharose column using thrombin
digestion of the fusion partner and elution under mild conditions.
However, the total amount of hTSHR-ecd produced in E.
coli Ad494 was less than reported for the expression of insoluble
hTSHR-ecd (9) secondary to proteolytic degradation of the hTSHR-ecd and
low efficiency of its elution from the affinity column.
The relevance of the structure of hTSHR-ecd, expressed in bacteria as soluble protein, to the native receptor conformation was addressed by analyzing formation of multimers in vivo. The ecd of the hTSHR contains 11 cysteine residues, some of which are likely to be involved in dimerization/multimerization of subunits. Recently, we detected the multimerization of the natural TSHR and receptor subunits in detergent-solubilized porcine thyroid membranes (5). We also have previously demonstrated that multimers formed in vitro from recombinant hTSHR-ecd proteins purified from E. coli, and insect cells (4, 16) were recognized by autoantibodies from patients with Graves’ disease (16). In this report, we demonstrated that the soluble hTSHR-ecd formed such multimers in vivo. To exclude the possible involvement of the GST fusion partner in multimerization, soluble hTSHR was analyzed with and without the GST portion of the molecule. SDS-PAGE analysis of the soluble fraction showed that the soluble hTSHR-ecd formed multimers under nonreducing conditions, and their formation was independent of the presence of the GST fusion partner. This was in agreement with the formation of hTSHR-ecd multimers using in vitro refolding of urea-solubilized recombinant receptor, expressed as insoluble aggregates in bacteria (4) and insect cells (16). The relevance of such multimeric organization of recombinant hTSHR-ecd and the natural TSHR was supported by the fact that lutropin-choriogonadotropin and FSH receptors, which have a high degree of similarity with the TSHR, have been shown also to form tetramers (17, 18, 19, 20).
The function of the expressed hTSHR-ecd was analyzed by TSH binding-assay. Our results showed that 30 pM of the soluble, affinity-purified, recombinant receptor inhibited TSH binding to hTSHR expressed on CHO cells by approximately 40%. Previous studies with the same amount of hTSHR-ecd, produced in E. coli as insoluble protein, and refolded in vitro have shown no inhibition of TSH binding (6). These data indicated that solubility of recombinant hTSHR-ecd was an important step in producing biologically active protein and that, perhaps, previous solubilization under harsh denaturing conditions (8 M urea) and refolding in vitro did not allow the formation of the same structure as was formed in the present studies in the cytoplasm of the bacteria. At the same time, inhibition of TSH binding by soluble recombinant hTSHR-ecd was much less efficient than inhibition by the TSHR present in porcine thyroid membranes, as reported in the literature (14), indicating that other factors (for example, the presence of the transmembrane domain and the proper posttranslational modification of the hTSHR) are even more important for the high-efficiency binding of the TSH to its receptor.
We used the soluble prokaryotic hTSHR-ecd to develop a competitive capture ELISA assay for detection of epitope-specific hTSHR-Abs. The hTSHR-ecd was captured on a plastic surface by antibody generated against the N-terminal end of the receptor, and the amount of hTSHR-Ab specific for the C-terminal end (aa 397–415) was detected by inhibition of detecting rabbit antibody (R397) binding. To test the system, we used mAb A7 specific for aa 402–415 of the hTSHR. This mAb blocked binding of rabbit antibody by 50%, compared with only 5% blocking by unrelated antibody. We also analyzed sera from mice immunized with hTSHR-ecd. The 30% of specific inhibition by this serum suggested the presence of hTSHR-Ab to aa 397–415 in the immunized mice. This region of the receptor previously was shown to be important in hormone binding because the same rabbit antibody inhibited binding of TSH to porcine TSHR by up to 33% (5). However, mice which developed hTSHR(397–415)-Ab remained euthyroid (15), suggesting that the interaction with mouse TSHR was insufficient to significantly inhibit thyroid function or that such function was compensated for by other mechanisms. By changing the detecting antibody, it should be possible to measure the entire epitope repertoire recognized by the mouse sera. This technique will give us the opportunity to analyze sera for the presence of antibodies to a variety of epitopes on the unglycosylated hTSHR-ecd, thus providing their epitope characterization.
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Acknowledgments |
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Footnotes |
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Received July 1, 1996.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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