©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Expression of the Extracellular Domain of the Thyrotropin Receptor in the Baculovirus System Using a Promoter Active Earlier than the Polyhedrin Promoter
IMPLICATIONS FOR THE EXPRESSION OF FUNCTIONAL HIGHLY GLYCOSYLATED PROTEINS (*)

(Received for publication, August 26, 1994; and in revised form, November 21, 1994)

Gregorio D. Chazenbalk (§) Basil Rapoport

From the Thyroid Molecular Biology Unit, Veterans Administration Medical Center and the University of California, San Francisco, California 94121

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

The TSH (^1)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) . (^2)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) . (^3)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.


MATERIALS AND METHODS

Truncation of the 5`-Untranslated Region of the TSH Receptor cDNA

False ATG initiation codons in frame with a stop codon in the 5`-untranslated region of the TSH receptor were removed by generating a 0.23-kilobase fragment of the TSH receptor by the polymerase chain reaction (13) using the pBS-hTSHR-mod cDNA (14) as template. The upstream primer (with SalI and EcoRI restriction sites) was 5`TAAGTCGACGAATTCCCCGAGTCCCGTGGAAAATGAGG, and the downstream primer (with an SnaBI site) was 5`-ATCTATAGATACGTAGATTCTGGA. The polymerase chain reaction was performed using Pfu DNA polymerase (Stratagene, La Jolla, CA). The 0.23-kilobase fragment was restricted with SalI and SnaBI and used to replace the same fragment in the full-length TSH receptor cDNA in pSV2-NEO-ECE-TSHR(14) . The nucleotide sequence was confirmed by the dideoxynucleotide termination method(15) .

Baculovirus Expression Vector Construction

A cassette formed by two oligonucleotides (5`-CTAGTGGGAGGTGGACATCACCATCACCATCACTGATAATCTA and 5`-GATCTAGATTATCAGTGATGGTGATGGTGATGTCCACCTCCCA) was used to introduce 3 glycine and 6 histidine residues followed by two stop codons at the carboxyl terminus of the 420-amino acid residue TSH receptor extracellular domain (including the signal peptide). This cassette was inserted into the SpeI and BglII sites in the modified human TSH receptor cDNA in pBluescript (pBS-hTSHR-mod)(14) . The 1.6-kilobase MluI-XbaI fragment including the 6-His cassette was excised and used to replace the corresponding fragment of the modified pSV2-NEO-ECE-TSHR (see above). From this plasmid, the 1.3-kilobase TSH receptor extracellular domain cDNA with the 6-His tag was released with EcoRI and BglII and subcloned into the same sites in the baculovirus transfer vectors pAcMP3 and pVL1393 (Pharmingen, San Diego, CA) to generate the plasmids pAcMP3-TSHR-EX-6H and pVL1393-TSHR-EX-6H, respectively.

Expression of TSH Receptor Extracellular Domain (TSHR-EX-6H) Protein

Purified pAcMP3-TSHR-EX-6H or pVL1393-TSHR-EX-6H plasmid (2 µg) was cotransfected with 0.5 µg of BaculoGold viral DNA (Pharmingen) according to the protocol of the manufacturer. Monolayers of Sf9 insect cells were cultured at 27 °C in Hink's medium with 10% heat-inactivated fetal calf serum. After 5 days of incubation, the virus in the supernatant was titered, and individual plaques were isolated and amplified 2-4 times using Sf9 cell suspensions (Pharmingen)(16) . TSHR-EX-6H protein was generated by infecting Sf9 or High Five insect cells (Pharmingen) in monolayer for different periods of time as indicated in the text for individual experiments. Infections were performed with viral stocks of 0.5-2 times 10^8 plaque-forming units/ml at multiplicities of infection of 10-20 plaque-forming units/cell.

To generate radiolabeled TSH receptor, infected cells in 25-cm^2 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 times 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^2 flasks. These cells were cultured in serum-free Ex-Cell 400 medium (JRH Biosciences).

Nickel Chelate Column Purification of the TSH Receptor Protein

Affinity purification of [S]methionine-labeled TSHR-EX-6H from (i) urea-solubilized intact cells, (ii) culture medium following chase (secreted protein), (iii) the cytosolic fraction of disrupted cells, and (iv) the guanidine-solubilized particulate fraction of disrupted cells was performed by adding 50 µl of a 50% slurry of Ni-NTA resin (QIAGEN Inc., Chatsworth, CA) to 1.5 ml of these solutions. After mixing for 60 min at room temperature, the resin was washed three times with 1 ml of buffer B (8 M urea, 0.1 M sodium phosphate, 0.01 M Tris, pH 6.3), and protein was released with 40 µl of buffer B containing 0.1 M EDTA. Aliquots (20 µl) in Laemmli buffer (17) containing 0.7 M beta-mercaptoethanol were electrophoresed on SDS-polyacrylamide gels, followed by autoradiography (XAR-5 film, Eastman Kodak Co.).

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.

Enzymatic Deglycosylation of TSHR-EX-6H Protein

Following Ni-NTA affinity purification, radiolabeled samples (10 µl in buffer B with 0.1 M EDTA) were added to 0.25 ml of 0.05 M sodium phosphate buffer, pH 7.4, containing 25 mM EDTA, 1% SDS, 1% beta-mercaptoethanol, and 0.5% Nonidet P-40. Samples were incubated for 16 h at 37 °C with 2 units of endoglycosidase F (Boehringer Mannheim) and then subjected to SDS-polyacrylamide gel electrophoresis, followed by autoradiography.

Effect of TSHR-EX-6H Protein on I-TSH Binding

Radiolabeled TSH binding to the wild-type human TSH receptor stably expressed on the surface of Chinese hamster ovary cells in monolayer culture was performed as described previously (18) with the following modifications. Dilutions of affinity-purified TSHR-EX-6H in 0.05 M sodium phosphate buffer, pH 7.4, were prepared (see ``Results''). Fifty µl of each dilution was preincubated for 1 h at room temperature with 10,000 cpm I-TSH in 450 µl of TSH binding buffer(18) . Aliquots (230 µl) were then added to the CHO cells for 8 h at 4 °C, and the cells were then processed as described previously(18) . Specific TSH binding was calculated by subtraction of nonspecific binding in the presence of 10M TSH (2-5% of total counts).

TSH Receptor Autoantibody Neutralization by TSHR-EX-6H Protein

A standard TSH binding inhibition assay (19, 20) was used, with the addition of a preincubation step to test for autoantibody neutralization. IgG was prepared from the sera of patients containing TSH receptor autoantibodies by ammonium sulfate precipitation; dialyzed against 10 mM phosphate buffer, pH 7.4; and diluted to 1.5 mg/ml in TSH binding buffer (see above). Dilutions of affinity-purified TSHR-EX-6H protein (50 µl each) were added to 450 µl of IgG in TSH binding buffer and preincubated for 1 h at room temperature. The mixture was then added to the CHO TSH receptor cells for 8 h at 4 °C. The IgG/TSHR-EX-6H mixture was removed, the cells were rinsed once with 1 ml of ice-cold TSH binding buffer, and 10,000 cpm I-TSH in 0.25 ml of TSH binding buffer was added for 12-16 h at 4 °C. Specifically bound TSH was then determined as described above.


RESULTS

TSH Receptor Extracellular Domain Expression in Intact Insect Cells

We approached the problem of high level expression of a functional TSH receptor extracellular domain by using a new baculovirus transfer vector (pAcMP3) with a late basic protein promoter active at an earlier stage than the conventional very late polyhedrin promoter. After removing the 5`-untranslated region, we inserted the cDNA for the TSH receptor extracellular domain into the pAcMP3 transfer vector and generated by homologous recombination the baculovirus pAcMP3-TSHR-EX-6H. This cDNA (TSHR-EX-6H) encodes the first 420 amino acid residues of the TSH receptor followed by a 3-Gly spacer and a 6-His tag at its 3`-end.

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.



Secretion of the TSH Receptor Extracellular Domain by Insect Cells

An extracellular protein that has traversed the secretory pathway is most likely to be correctly processed and folded. It was therefore important to determine whether the extracellular domain of the TSH receptor could be secreted by infected insect cells. For this purpose, Sf9 cells infected for 1-4 days were pulsed for 4 h with [S]methionine. Medium harvested after a 16-h chase was subjected to affinity purification with Ni-NTA resin.

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.



Particulate and Cytosolic Distribution of the TSH Receptor Extracellular Domain

The very low level of secretion of TSH receptor extracellular domain protein by infected insect cells suggested that most of this protein was retained within the cells. Regardless of whether the pAcMP3- or pVL1393-derived virus was used, most of the affinity-purified, [S]methionine-labeled TSH receptor protein was in the particulate fraction of the infected insect cells (Fig. 4). This material could not be solubilized with detergent (1% Triton X-100; data not shown). As seen with urea extracts of whole cells (Fig. 2), the major labeled protein obtained with the pAcMP3-derived virus (68 kDa) was larger than that obtained with the pVL1393-derived virus (63 kDa). Coomassie Blue staining of these proteins revealed a similar pattern (data not shown).


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 times 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.



Enzymatic Deglycosylation of the TSH Receptor Extracellular Domain

We examined whether or not the different sizes of the TSH receptor extracellular domain generated in insect cells under the influence of the two different promoters (Fig. 2) could be explained by different degrees of glycosylation. This was indeed the case. Radiolabeled TSH receptor was affinity-purified with Ni-NTA resin and subjected to enzymatic deglycosylation with endoglycosidase F (see ``Material and Methods''). The proteins generated by both the pAcMP3- and pVL1393-derived viruses were reduced to the same size, 54 kDa (Fig. 5).


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.



Functional Activity of the TSH Receptor Extracellular Domain Generated in Insect Cells

Although high levels of TSH receptor protein can be generated in insect cells under the influence of the very late polyhedrin promoter, this material is nonfunctional, or poorly functional, in terms of ligand and autoantibody binding (see above). Because of the greater extent of glycosylation of TSH receptor extracellular protein generated with the pAcMP3-derived virus, we tested the functional activity of this material. To generate larger amounts of unlabeled TSH receptor protein, we used High Five insect cells, which were more efficient in generating the protein than Sf9 cells (data not shown). Despite this greater efficiency, the yield with the pAcMP3 vector remained small. Typically, guanidine solubilization of 3 times 10^7 intact High Five cells yielded 20 µg of affinity-purified TSH receptor protein. Furthermore, the inability to remove urea from the eluting buffer without loss of solubility precluded testing the protein in a functional assay.

We therefore tested for the presence of TSH receptor functional activity in affinity-purified soluble protein from the 100,000 times 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 times g supernatant of 3 times 10^7 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.




DISCUSSION

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 times 10^3 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 (10M) and for unrelated proteins, or even plastic (10M) (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 beta(2)-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.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant DK19289. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Veterans Affairs Medical Center, Thyroid Molecular Biology Unit (111T), 4150 Clement St., San Francisco, CA 94121. Tel.: 415-476-5984; Fax: 415-752-6745.

(^1)
The abbreviations used are: TSH, thyrotropin; Ni-NTA, nickel-nitrilotriacetic acid; CHO, Chinese hamster ovary.

(^2)
G. D. Chazenbalk and B. Rapoport, unpublished data.

(^3)
B. Rapoport, unpublished data.


ACKNOWLEDGEMENTS

We thank Pui Seto for technical assistance as well as Drs. Sandra McLachlan and Sebastiano Filetti for critical review of the manuscript. We also thank the National Hormone and Distribution Program, NIDDK, the Center for Population Research of NICHD, the Agricultural Research Service of the U. S. Department of Agriculture, and the University of Maryland School of Medicine for kindly providing the highly purified bovine TSH for radioiodination.


REFERENCES

  1. Rees Smith, B., McLachlan, S. M., and Furmaniak, J. (1988) Endocr. Rev. 9, 106-121 [Abstract]
  2. Nagayama, Y., and Rapoport, B. (1992) Mol. Endocrinol. 6, 145-156 [Abstract]
  3. Takai, O., Desai, R. K., Seetharamaiah, G. S., Jones, C. A., Allaway, G. P., Akamizu, T., Kohn, L. D., and Prabhakar, B. S. (1991) Biochem. Biophys. Res. Commun. 179, 319-326 [Medline] [Order article via Infotrieve]
  4. Huang, G. C., Collison, K. S., McGregor, A. M., and Banga, J. P. (1992) J. Mol. Endocrinol. 8, 137-144 [Abstract]
  5. Loosfelt, H., Pichon, C., Jolivet, A., Misrahi, M., Caillou, B., Jamous, M., Vannier, B., and Milgrom, E. (1992) Proc. Natl. Acad. Sci. U. S. A. 895, 3765-3769
  6. Harfst, E., Johnstone, A. P., and Nussey, S. S. (1992) J. Mol. Endocrinol. 9, 227-236 [Abstract]
  7. Summers, M. D., and Smith, G. E. (1987) A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures , Texas A & M University, College Station, TX
  8. Harfst, E., Johnstone, A. P., Gout, I., Taylor, A. H., Waterfield, M. D., and Nussey, S. S. (1992) Mol. Cell. Endocrinol. 83, 117-123 [CrossRef][Medline] [Order article via Infotrieve]
  9. Huang, G. C., Page, M. J., Nicholson, L. B., Collison, K. S., McGregor, A. M., and Banga, J. P. (1993) J. Mol. Endocrinol. 10, 127-142 [Abstract]
  10. Seetharamaiah, G. S., Desai, R. K., Dallas, J. S., Tahara, K., Kohn, L. D., and Prabhakar, B. S. (1993) Autoimmunity 14, 315-320 [Medline] [Order article via Infotrieve]
  11. Seetharamaiah, G. S., Kurosky, A., Desai, R. K., Dallas, J. S., and Prabhakar, B. S. (1994) Endocrinology 134, 549-554 [Abstract]
  12. Gruenwald, S., and Heitz, J. (1993) Baculovirus Expression Vector System: Procedures and Methods Manual , Pharmingen, San Diego, CA
  13. Saiki, R. K., Gelfand, D. N., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T., Mullis, K. B., and Erlich, H. A. (1988) Science 239, 487-491 [Medline] [Order article via Infotrieve]
  14. Nagayama, Y., Wadsworth, H. L., Chazenbalk, G. D., Russo, D., Seto, P., and Rapoport, B. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 902-905 [Abstract]
  15. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 5463-5467 [Abstract]
  16. O'Reilly, D. R., Miller, L. K., and Luckow, V. A. (1992) Baculovirus Expression Vectors: A Laboratory Manual , W. H. Freeman & Co., New York
  17. Laemmli, U. K. (1970) Nature 227, 680-685 [Medline] [Order article via Infotrieve]
  18. Chazenbalk, G. D., Nagayama, Y., Russo, D., Wadsworth, H. L., and Rapoport, B. (1990) J. Biol. Chem. 265, 20970-20975 [Abstract/Free Full Text]
  19. Filetti, S., Foti, D., Costante, G., and Rapoport, B. (1991) J. Clin. Endocrinol. & Metab. 72, 1096-1101 [Abstract]
  20. Nagayama, Y., Wadsworth, H. L., Russo, D., Chazenbalk, G. D., and Rapoport, B. (1991) J. Clin. Invest. 88, 336-340 [Medline] [Order article via Infotrieve]
  21. Shewring, G. A., and Rees Smith, B. (1982) Clin. Endocrinol. 17, 409-417 [Medline] [Order article via Infotrieve]
  22. Reilander, H., Boege, F., Vasudevan, S., Maul, G., Hekman, M., Dees, C., Hampe, W., Helmreich, E. J. M., and Michel, H. (1991) FEBS Lett. 2, 441-444 [CrossRef]
  23. Parker, E. M., Kameyama, K., Higashijima, T., and Ross, E. M. (1991) J. Biol. Chem. 266, 519-527 [Abstract/Free Full Text]
  24. Vasudevan, S., Reilander, H., Maul, G., and Michel, H. (1991) FEBS Lett. 283, 52-56 [CrossRef][Medline] [Order article via Infotrieve]
  25. Sissom, J., and Ellis, L. (1994) Biochem. J. 261, 119-126
  26. Paul, J. I., Tavare, J., Denton, R. M., and Steiner, D. F. (1990) J. Biol. Chem. 265, 13074-13083 [Abstract/Free Full Text]
  27. Greenfield, C., Hiles, I., Waterfield, M. D., Federwisch, M., Wollmer, A., Blundell, T. L., and McDonald, N. (1989) EMBO J. 8, 4115-4123 [Abstract]
  28. Christophe, S., Robert, P., Maugain, S., Bellet, D., Koman, A., and Bidart, J.-M. (1993) Biochem. Biophys. Res. Commun. 196, 402-408 [CrossRef][Medline] [Order article via Infotrieve]
  29. Harfst, E., Ross, M. S., Nussey, S. S., and Johnstone, A. P. (1994) Mol. Cell. Endocrinol. 102, 77-84 [CrossRef][Medline] [Order article via Infotrieve]
  30. Liu, X., Davis, D., and Segaloff, D. L. (1993) J. Biol. Chem. 268, 1513-1516 [Abstract/Free Full Text]
  31. Petäjä-Repo, U. E., Merz, W. E., and Rajaniemi, H. J. (1993) Biochem. J. 292, 839-844 [Medline] [Order article via Infotrieve]
  32. Zhang, R., Tsai-Morris, C. H., Kitamura, M., Buczko, E., and Dufau, M. L. (1991) Biochem. Biophys. Res. Commun. 181, 804-808 [Medline] [Order article via Infotrieve]
  33. Harfst, E., Johnstone, A. P., and Nussey, S. S. (1992) Lancet 339, 193-194
  34. Shi, Y., Zou, M., Parhar, R. S., and Farid, N. R. (1993) Thyroid 3, 129-133 [Medline] [Order article via Infotrieve]
  35. Jarvis, D. L., Summers, M. D., Garcia, A., Jr., and Bohlmeyer, D. A. (1993) J. Biol. Chem. 268, 16754-16762 [Abstract/Free Full Text]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.