(Received for publication, August 26, 1994; and in revised form, November 21, 1994)
From the
Conventional baculovirus vectors that utilize the very late polyhedrin promoter have not proved successful for
expressing a thyrotropin (TSH) receptor capable of ligand and
Graves' disease autoantibody binding comparable to the receptor
produced in mammalian cells. Because of the clinical importance of high
level expression of this protein, we reassessed the baculovirus system
using a new transfer vector (pAcMP3) containing the late basic
protein promoter, which functions earlier than the classical polyhedrin
promoter. Maximal synthesis of the
[S]methionine-labeled TSH receptor extracellular
domain, affinity-purified using a 6-histidine tag, occurred earlier (1
day after insect cell infection) than with a vector (pVL1393)
containing the polyhedrin promoter. The pAcMP3-derived TSH receptor
extracellular domain was larger (
68 kDa) than the pVL1393-derived
protein (
63 kDa). Only the 68-kDa product was secreted, albeit in
trace amounts detectable only by precursor labeling. Enzymatic
deglycosylation reduced both 68- and 63-kDa cellular proteins to
54 kDa, indicating that the pAcMP3 vector generated a protein with
greater carbohydrate content. However, despite its greater degree of
glycosylation, most of the 68-kDa protein remained within the cell,
almost entirely in the particulate fraction. Remarkably, the trace
amounts of 68-kDa receptor protein affinity-purified from the soluble
cytosolic fraction of infected insect cells completely neutralized TSH
receptor autoantibodies in patients' sera and partly inhibited
TSH binding.
In conclusion, a baculovirus vector with a promoter active earlier than the conventional polyhedrin promoter generates a more glycosylated and functional TSH receptor extracellular domain protein, albeit at low levels. These data carry important implications for the expression by baculovirus vectors of functional, highly glycosylated proteins.
The TSH ()receptor is the primary autoantigen in
Graves' disease, one of the most common human autoimmune diseases
(reviewed in (1) ). Large amounts of conformationally intact
TSH receptor protein are a fundamental requirement for future
diagnostic and therapeutic approaches to this disease. Generating a
functional TSH receptor in stably transfected eukaryotic cells has been
straightforward (reviewed in (2) ). However, the amount of
receptor protein produced by these cells is insufficient for
purification. There has also been no difficulty in expressing large
amounts of the TSH receptor extracellular domain in prokaryotic
cells(3, 4, 5, 6) . (
)However, in this case, most of the protein is present in
inclusion bodies, and attempts at renaturation have failed to produce
protein capable of specific binding by TSH and TSH receptor
autoantibodies(3, 4) .
For the past 4 years, the
baculovirus system (7) has been viewed as a promising solution
to this dilemma. Unlike in prokaryotes, proteins expressed in insect
cells are glycosylated, albeit not identically to eukaryotic cells.
However, all attempts to generate the full-length TSH receptor in the
baculovirus system failed(8, 9) . ()Efforts
then focused on the TSH receptor extracellular domain (9, 10) because of the importance of this region in
TSH and TSH receptor autoantibody binding (reviewed in (2) ).
Again, despite much effort, the general experience using conventional
baculovirus vectors with the very late polyhedrin promoter has been
frustrating. A number of laboratories, including our own, failed to
produce functional protein (5) or have not reported their data.
Others have described the generation of receptor protein incapable of
physiological, high affinity ligand binding (9, 10) even after attempts at protein
renaturation(11) .
In this study, we have attempted to express a functional TSH receptor using a baculovirus vector with the late basic protein promoter. This promoter is active earlier than the very late polyhedrin promoter, which functions at a time when expression of the enzymes involved in post-translational protein modification is suppressed(12) . In contrast to experience with conventional baculovirus vectors, we generated a more highly glycosylated, soluble, and functional TSH receptor, although with a very low yield. These data carry important implications for the expression by baculovirus vectors of functional highly glycosylated proteins.
To generate radiolabeled TSH receptor, infected cells in
25-cm flasks were preincubated for 4 h in Ex-Cell 401
methionine-free medium (JRH Biosciences, Lenexa, KS). The medium was
then replaced with the same medium (1.5 ml) containing 100-250
µCi of [
S]methionine (>1000 Ci/mmol;
DuPont NEN). After a 4-h pulse, the medium was removed, and the cells
were rinsed once and then either lysed or a chase was performed for
16 h in Hink's medium. Intact cells were lysed directly in
1.5 ml of 8 M urea, 0.1 M sodium phosphate, 0.01 M Tris, pH 8.0, and centrifuged for 5 min in a
microcentrifuge, and the supernatant was retained.
The distribution
of the [S]methionine-labeled TSH receptor
between the particulate and cytosolic fractions of the cells was
determined by a modification of the above approach, using guanidine to
dissolve the insoluble fraction. After infection and labeling (see
above), the Sf9 cells were resuspended in 1.5 ml of 0.05 M sodium phosphate buffer containing phenylmethylsulfonyl fluoride
(100 µg/ml), leupeptin (1 µg/ml), aprotinin (1 µg/ml), and
pepstatin A (2 µg/ml) (all from Sigma). The cells were disrupted by
two freeze-thaw cycles and centrifuged (4 °C) for 60 min at 100,000
g. The supernatant was retained, and the pellet was
dissolved in 6 M guanidine HCl, 0.1 M sodium
phosphate, 0.01 M Tris, pH 8.0.
Larger scale preparations
of soluble TSH receptor protein were made in the same manner, except
that the cells were not pulsed with
[S]methionine, and we used High Five insect
cells in 175-cm
flasks. These cells were cultured in
serum-free Ex-Cell 400 medium (JRH Biosciences).
Larger scale purification of nonradioactive soluble protein involved
the addition of 0.5 ml of resin slurry to 10-20 ml of High Five
cell cytosolic proteins (see above). The resin was poured into a column
and rinsed with 20 volumes of 0.05 M phosphate buffer, pH 7.4,
followed by the same volume of 0.05 M phosphate buffer, pH
6.3. Protein was eluted in two steps with phosphate buffer, pH 5.9 and
4.5 (10 ml each), and immediately neutralized to pH 7.0. Both eluted
fractions were pooled and concentrated by Centriprep-30 (Amicon, Inc.,
Beverly, MA) to 0.7 ml.
Ten individual viral plaques were
selected for protein expression in Sf9 insect cells.
[S]Methionine-labeled proteins extracted from
intact cells with 6 M urea buffer were subjected to Ni-NTA
affinity purification. Relative to control cells infected with
baculovirus without the TSH receptor cDNA, all 10 TSHR-EX-6H clones
expressed (9/10 strongly) a labeled protein of
68 kDa (Fig. 1). Typically, in different experiments, a protein of
smaller size (
63 kDa) and variable intensity was also
affinity-purified with the Ni-NTA resin (Fig. 1). Consistent
with the relatively uniform levels of expression by most of the
individual clones, the signal with the uncloned viral stock (viral
``pool'') was similar to that of the clones. All subsequent
experiments with the pAcMP3-TSHR-EX-6H construct were performed with
viral stock amplified from clone 2.
Figure 1:
TSH receptor extracellular
domain expression in intact insect cells. Sf9 insect cells were
infected for 1 day with 10 individual viral clones isolated from a
baculovirus stock generated by homologous recombination with the
transfer vector pAcMP3-TSHR-EX-6H. This vector, with a late basic
protein promoter, contained the cDNA for the TSH receptor extracellular
domain with a 6-His tag at its carboxyl terminus (see ``Materials
and Methods''). Pool, viral stock from which the 10
individual clones were selected; Con (control), insect cells
infected with baculovirus not containing the TSH receptor cDNA. Cells
were pulsed for 4 h with [S]methionine. Urea
extracts of intact cells were subjected to Ni-NTA affinity
purification, and aliquots were electrophoresed on a SDS-polyacrylamide
gel under reducing conditions. Autoradiography was for
16
h.
The time course of radiolabeled
TSHR-EX-6H protein expression in intact Sf9 cells was determined.
Consistent with the time of activity of the basic protein promoter, the
major 68-kDa band, affinity-purified by Ni-NTA chromatography, was
apparent only after 1 day of infection with the pAcMP3-TSHR-EX-6H virus (Fig. 2). For comparison, insect cells were infected with virus
generated by the same TSH receptor cDNA in the transfer vector,
pVL1393, with the standard very late polyhedrin promoter. In contrast
to the earlier expression of labeled protein with the pAcMP3-TSHR-EX-6H
virus, specific protein expression was maximal on the second
post-infection day and was still evident 3 days after infection (Fig. 2). Of note, the cellular protein generated with the
polyhedrin promoter was smaller (63 kDa) than the major protein
generated under the influence of the basic protein promoter (
68
kDa) (Fig. 2).
Figure 2:
Left
panel, time course of radiolabeled TSH receptor extracellular domain
protein in insect cells. Sf9 cells were infected for up to 4 days with
recombinant baculovirus generated either with the pAcMP3 (late basic
protein promoter) or pVL1393 (very late polyhedrin promoter) transfer
vector. Both transfer vectors contained the same cDNA insert
(TSHR-EX-6H) coding for the TSH receptor extracellular region with a
6-His tag at its carboxyl-terminal end. Infected cells were pulsed for
4 h with [S]methionine. Urea extracts of intact
cells were subjected to Ni-NTA affinity purification, and aliquots were
electrophoresed on a SDS-polyacrylamide gel under reducing conditions.
Autoradiography was for
16 h. Right panel, comparison of
the sizes of affinity-purified radiolabeled protein generated in Sf9
cells infected with the pAcMP3-TSHR-EX-6H and pVL1393-TSHR-EX-6H
viruses. In this experiment, infections were for 1 and 2 days,
respectively, prior to a 4-h pulse with
[
S]methionine and extraction of intact cells
with buffer containing 6 M urea (see ``Materials and
Methods''). Autoradiography was for 16
h.
Cells infected with virus generated with pAcMP3-TSHR-EX-6H did
secrete recombinant protein into the culture medium, but only in very
small amounts (Fig. 3). Autoradiograph exposures of weeks,
rather than hours, were required for detection. A single protein of
68 kDa was present in the medium in maximal amounts after 1 day of
infection, consistent with the time course of synthesis of cellular
protein (Fig. 2). Mainly, degradation products were present in
the medium 2-4 days post-infection. In contrast, no TSH receptor
extracellular domain was detectable in the medium when insect cells
were infected with baculovirus containing the polyhedrin promoter in
the pVL1393-TSHR-EX-6H transfer vector (Fig. 3).
Figure 3:
Secretion
of the TSH receptor extracellular domain by insect cells. Sf9 cells
were infected for 1-4 days with virus that had recombined with
either the pAcMP3-TSHR-EX-6H or pVL1393-TSHR-EX-6H transfer vector.
Cells were then pulsed for 4 h with
[S]methionine. Medium was harvested after a 16-h
chase and subjected to affinity purification with Ni-NTA resin (see
``Materials and Methods''). Aliquots of affinity-purified
material were electrophoresed on a SDS-polyacrylamide gel under
reducing conditions. Autoradiography was for 3
weeks.
Figure 4:
Particulate and cytosolic distribution of
the TSH receptor extracellular domain in infected insect cells. Sf9
cells were infected with virus obtained by recombination with either
the pAcMP3-TSHR-EX-6H or pVL1393-TSHR-EX-6H transfer vector. Infections
were for 1 and 2 days, respectively. Cells were pulsed for 4 h with
[S]methionine, rinsed, and disrupted by
freeze-thaw cycles (see ``Materials and Methods''). The
100,000
g supernatants and pellets were subjected to
affinity purification with Ni-NTA resin, with elution in similar
volumes (40 µl) (see ``Materials and Methods''). Equal
aliquots (
18 µl) were electrophoresed on a SDS-polyacrylamide
gel under reducing conditions. A, affinity-purified material
from pAcMP3-TSHR-EX-6H-infected cells; B, affinity-purified
material from pVL1393-TSHR-EX-6H-infected cells. In the middlelane, the particulate fraction was diluted 20-fold.
Autoradiography in this representative experiment was for 2
days.
Figure 5:
Enzymatic deglycosylation of the TSH
receptor extracellular domain. Sf9 insect cells were infected with
virus obtained by recombination with either the pAcMP3-TSHR-EX-6H or
pVL1393-TSHR-EX-6H transfer vector. Infections were for 1 and 2 days,
respectively. Cells were pulsed for 4 h with
[S]methionine, proteins were extracted from the
intact cells with buffer containing 6 M urea, and specific
proteins were purified with Ni-NTA resin (see ``Materials and
Methods''). Aliquots were treated with endoglycosidase F (Endo
F) for 16 h (see ``Materials and Methods''), followed by
electrophoresis on a SDS-polyacrylamide gel under reducing conditions.
Autoradiography was for 4 days. Con,
control.
We
therefore tested for the presence of TSH receptor functional activity
in affinity-purified soluble protein from the 100,000 g supernatant of infected insect cells. Despite the fact that the
yield of unlabeled TSH receptor extracellular domain was too low to
quantitate, TSH binding activity was clearly observed in a standard
radiolabeled TSH binding assay. Thus, the column eluate inhibited in a
dose-dependent manner
I-TSH binding to the wild-type TSH
receptor expressed on the surface of CHO cells (Fig. 6). A
20-fold dilution of the eluate reduced TSH binding by
50%. Control
affinity column buffer was without effect. Much less bioactivity was
observed in the affinity column eluate following application of cytosol
from insect cells infected with pVL1393-derived virus. This low level
of activity serves as further control for the specificity of the
inhibition observed with the pAcMP3-derived material (Fig. 6).
In separate experiments, direct binding of
I-TSH to the
column eluate could be demonstrated in a dose-dependent manner using
polyethylene glycol to precipitate hormone-receptor
complexes(21) . The control eluate was without effect. However,
the high background (
50%) with this assay, at least in our hands,
precludes us from reporting these data. As examples, however, we
observed 20 and 14% binding of
I-TSH with 1:7 and 1:10
dilutions of the receptor extract, respectively.
Figure 6:
Competition by the soluble TSH receptor
extracellular domain for I-TSH binding to the wild-type
receptor on intact cells. Soluble receptor protein was obtained by
affinity purification on Ni-NTA of the 100,000
g supernatant of
3
10
High Five cells
infected for 1 day with virus derived from the pAcMP3-TSHR-EX-6H
transfer vector. The eluate was concentrated to 0.7 ml. Fifty µl of
this eluate or dilutions of this eluate in the same buffer were
preincubated with
I-TSH (total volume of 0.5 ml) for 1 h
prior to addition of the ligand to CHO cells stably expressing the
wild-type TSH receptor on their surface (see ``Materials and
Methods''). Con (control), TSH binding buffer (see
``Materials and Methods'') alone. Specific
I-TSH binding (see ``Materials and Methods'')
is expressed as a percent of maximum binding in the absence of the
soluble TSH receptor extracellular domain. Each bar indicates
the mean and range of determinations on duplicate dishes of cells. The
results shown are representative of three separate experiments using
different TSH receptor extracellular domain
preparations.
The pAcMP3-derived
soluble TSH receptor extracellular domain protein was also tested for
its functional activity by it ability to interact with TSH receptor
autoantibodies in the sera of patients with Graves' disease.
These autoantibodies, which recognize the TSH-binding site on the
native receptor, are present in too low a concentration to be detected
by direct binding. However, they can be assayed by their ability to
inhibit I-TSH binding to the immobilized TSH receptor,
for example on the surface of cultured cells (TSH binding inhibition
assay)(19) .
We therefore performed a modified three-stage
assay. TSH receptor autoantibodies were first preincubated with the
affinity-purified protein TSH receptor extracellular domain. The
mixture was then added to CHO cell monolayers expressing the wild-type
TSH receptor on their surface. After removal of the mixture and rinsing
the cells, residual unoccupied wild-type TSH receptors were detected
with I-TSH. TSH receptor protein completely reversed the
ability of autoantibodies to occupy TSH-binding sites on the cultured
cells (Fig. 7). This was the case with IgG preparations from
three different patients, each with different degrees of TSH binding
inhibitory activity. Boiling abolished the functional activity of the
TSH receptor extracellular domain protein present in the affinity
column eluate (data not shown).
Figure 7:
TSH receptor extracellular domain protein
prevents occupancy by TSH receptor autoantibodies of the wild-type TSH
receptor on the surface of cultured CHO cells. The assay for detection
of TSH receptor autoantibodies in the sera of patients with
Graves' disease (TSH binding inhibition assay) was modified (see
``Materials and Methods''). Fifty µl of TSH receptor
extracellular domain protein in the affinity column eluate or dilutions
of this eluate (described in the legend to Fig. 6) were
preincubated (1 h at room temperature) with 1.5 mg/ml Graves' IgG
(total volume of 0.5 ml). The mixture was then added to CHO cells (8 h
at 4 °C) expressing the wild-type TSH receptor on their surface.
After removing the mixture and rinsing the cells, residual unoccupied
wild-type TSH receptors were detected with I-TSH (see
``Materials and Methods''). Each panel depicts data with IgG
preparations of different potency from three different Graves'
patients. The whitebar (Con (control))
indicates maximal
I-TSH binding in the absence of IgG or
TSH receptor extracellular domain protein. In the absence of TSH
receptor extracellular domain protein (eluate buffer alone),
I-TSH binding to the cells is inhibited to varying
degrees by the different IgG samples. This effect is progressively
reversed by increasing amounts of eluate containing TSH receptor
extracellular domain protein. Each bar indicates the mean and
range of determinations on duplicate dishes of cells. The results shown
are representative of three separate experiments using different TSH
receptor extracellular domain preparations.
The present ``roadblock'' to fundamental progress
in understanding the pathogenesis of Graves' disease remains the
unavailability of purified TSH receptor protein in a form satisfactory
for TSH and TSH receptor autoantibody binding. Despite many attempts
over 2 decades, there are no convincing data that functional TSH
receptor protein has ever been purified from thyroid tissue.
Purification has been hampered by the small number of TSH receptors in
thyroid cells (5
10
receptors/cell)(1) . Furthermore, ligand affinity
purification of the receptor is extremely difficult because of the
narrow differential between the affinity of TSH for receptor
(
10
M) and for unrelated proteins, or
even plastic (
10
M) (reviewed in (2) ). The most encouraging study involves the use of a murine
monoclonal antibody to affinity purify the TSH holoreceptor from human
thyroid tissue(5) . However, no data have appeared to indicate
that the purified receptor can be recognized by TSH or autoantibodies,
suggesting that the protein may not be conformationally intact.
The availability of recombinant human TSH receptor has not altered the reputation of this receptor as a difficult protein to investigate. Even though a functional TSH receptor can be expressed in mammalian cells (unlike in prokaryotic cells) (see above), purification of this material has not been achieved. Previous experience with the baculovirus system has not been satisfactory. Furthermore, there are no reports that the first TSH receptor monoclonal antibodies of unequivocal specificity (5, 9) have been used successfully for affinity purification of a functional recombinant protein of any source. These antibodies were generated by immunization with TSH receptor protein of prokaryotic origin and may not recognize the native protein very well.
The negative experience with the TSH
receptor in the baculovirus system contrasts with many other
membrane-associated receptors. For example, high level expression of
functional protein has been attained for the
-adrenergic receptor(22, 23) ,
muscarinic acetylcholine receptor(24) , insulin
receptor(25, 26) , and epidermal growth factor
receptor(27) . The full-length follicle-stimulating hormone
receptor has been expressed in the baculovirus system, although at low
levels(28) . In trying to understand why the TSH receptor has
been particularly difficult to study, an important consideration is its
very high degree of glycosylation. Remarkably, the
45-kDa
polypeptide backbone of the TSH receptor extracellular domain contains
15-20 kDa of carbohydrate moieties(5, 29) .
Even the much larger insulin receptor is only glycosylated to the
extent of 5-7 kDa(26) . It must be recognized, however,
that the importance of TSH receptor glycosylation in ligand binding is
an unanswered question (reviewed in (2) ), especially in light
of some(30, 31) , but not all(32) , data
suggesting that glycosylation is unimportant for lutropin binding to
its receptor. A distinction must also be made between the role of
glycosylation in receptor function and receptor expression (as in a
baculovirus system).
After baculovirus infection of insect cells, activation of the very late polyhedrin promoter is associated with a general suppression of expression of other cellular proteins, including those responsible for post-translational glycosylation. Obviously, this effect may be detrimental to a very highly glycosylated protein such as the TSH receptor. The use of recently developed vectors with promoters active at an earlier stage after infection could overcome this problem(12) . However, we are unaware of data confirming this advantage for a heavily glycosylated receptor protein. We therefore used a baculovirus transfer vector (pAcMP3) with a basic protein promoter to determine whether or not the TSH receptor extracellular domain protein was more highly glycosylated, and perhaps functional, in comparison with the same protein generated under the influence of the polyhedrin promoter. This was indeed the case.
The TSH receptor
extracellular domain was 68 kDa in size when synthesized under the
influence of the basic protein promoter. In contrast, the same cDNA
driven by the conventional polyhedrin promoter was 63 kDa in size,
as described previously by Huang et al.(9) . The
50-kDa protein reported by Seetharamaiah et al.(10) ,
encoded by cDNA lacking a signal peptide, is likely to be very poorly
glycosylated. Sizes of 68, 63, and 50 kDa obviously represent major
differences for a protein with a polypeptide backbone of 45 kDa.
Enzymatic deglycosylation with endoglycosidase F reduced both 68-
and 63-kDa proteins to the same size (54 kDa), indicating that the
size difference reflects their N-linked carbohydrate content.
The enzymatically treated receptor is still
9 kDa larger than the
predicted size of the polypeptide backbone. Possible contributing
factors to this difference could include (i) a retained signal peptide
(see below), (ii) contribution of the glycine spacer and 6-His tag, and
(iii) O-linked glycosylation. Huang et al.(9) reported a slightly smaller (49-kDa) deglycosylated
TSH receptor extracellular domain. However, after subtraction of the
contribution of the glycine/histidine tag in our construct, this value
and ours are within the range of experimental variation.
An important observation in this study was the functional activity of the more highly glycosylated, 68-kDa TSH receptor extracellular domain generated by pAcMP3-infected insect cells. Although produced in very small amounts and only detected by precursor radiolabeling, the affinity-purified material completely neutralized TSH receptor autoantibodies in patients' sera. Inhibition of TSH binding was only partial. The latter observation may indicate the importance of the transmembrane segment of the TSH receptor in ligand binding(33) , although there are data contrary to this concept(34) . Consistent with our findings, the 63-kDa protein of Huang et al.(9) generated using a vector with a polyhedrin promoter appeared to have less functional activity. Thus, much larger quantities of material than used in our study only partially neutralized TSH receptor autoantibody binding and did not appear to interact with TSH. More information is required to evaluate the nature of the relatively low affinity TSH binding to the 50-kDa TSH receptor extracellular domain also generated under the influence of the polyhedrin promoter(11) . This 50-kDa protein is initially insoluble and requires renaturation.
It must be emphasized that, although more highly glycosylated and functional than the 63-kDa TSH receptor extracellular domain protein, only a trace amount of the 68-kDa protein generated under the influence of the basic promoter was secreted. Lack of secretion is unlikely to be attributable to incomplete glycosylation because the retained material was the same size as the secreted protein. On the other hand, the different glycosylation pattern of insect cells and mammalian cells may be a factor. Another possible explanation for lack of secretion is non-cleavage of the signal peptide, as suggested by the larger than expected size of the polypeptide backbone (see above). However, the illuminating and sobering experience of Jarvis et al.(35) indicates that even insect-derived signal peptides cannot always enhance the expression or secretion of proteins that are not normally generated by insect cells. Many other potential factors can be implicated (discussed in (35) ).
In conclusion, a baculovirus vector with a promoter active at a relatively early stage after infection expresses a more highly glycosylated and functional extracellular domain of the human TSH receptor than that generated with the conventional polyhedrin promoter. However, even though glycosylated to a greater extent, the TSH receptor extracellular domain was expressed at low levels, and most was retained within the cell. Our data and those of Jarvis et al.(35) suggest that factors other than the degree of glycosylation and signal peptides play an important role in the high level expression of some secreted proteins. A recombinant soluble TSH receptor preparation would be of great value in a direct assay for TSH receptor autoantibodies. Unfortunately, the very small amount of functional receptor generated in the baculovirus system does not, at least at present, make this goal feasible.