Epitope mapping of a recombinant human TSH receptor extracellular domain: Identification of a predominant epitope using animal sera

Journal of Clinical Laboratory Analysis 10:193-204 (1996)

Epitope Mapping of a Recombinant Human TSH Receptor Extracellular Domain: Identification of a Predominant

Epitope Using Animal Sera Nicholas Hunt,’ Kevan P. Willey,‘ Nicole Abend,’ Wolfgang Northemann,* and

Freimut A. Leidenberger’ ‘Institute for Hormone and Fertility Research, University of Hamburg, Hamburg, and “Department of Molecular

Biology, EL /AS Development Laboratory, Freiburg, Germany

The extracellular domain of the TSH receptor (TSHR-561, amino acids #78-389) was expressed as a hexa-histidine fusion protein in bacteria.The recombinant protein was purified to homogeneity and used to immunize porcine and ovine species. High titre antibodies were obtained from both species that recognized the recombinant protein in Western blot analysis but failed to interfere with the TSH radio receptor assay. An epitope library was constructed and screened with affinity purified ovine and porcine antisera and detected a number of positive clones. Sequence analysis revealed that all of the epitopes contained sequences derived from the carboxyl terminus of the re-

combinant immunogen. One clone defined an epitope covering 16 amino acids from the carboxyl terminus and was the common epitope found in all of the other clones. Western blot screening of a large panel of Graves’ sera with recombinant TSH receptor protein identified one patient sera that also recognized linear epitopes in theTSHR-561 protein. Experimentation demonstrated that the linear epitope recognized by this human sera was identical to the sequence recognised by the animal antisera. This sequence is unique to the TSH receptor and will be useful in further studies to analyze the TSH receptor protein. 0 1996 Wiley-Liss, Inc.

Key words: TSH receptor, recombinant protein, antibodies, Graves’ disease

INTRODUCTION

The heterodimeric glycoprotein hormones include the thy- rotrophic hormone, TSH, and the gonadotrophic hormones, FSII, LH, and hCG (1). The a subunit is common to the hormones within a species, whereas the p subunits are unique to each hormonal activity and confer receptor specificity. Spe- cific target cell endocrine activity is associated with acute activation of adenylate cyclase and a longer term trophic response.The receptors for the glycoprotein hormones are mem- bers of a larger family of G protein-coupled receptors that bind small ligands and have a characteristic domain of seven transmembrane regions ( 2 ) . The rat and porcine LH receptors were the first to be cloned by screening gonadal cDNA libraries using oligonucleotides derived from protein sequencing data (3, 4). Subsequently the dog, human, and rat TSH receptor cDNAs were cloned and their biological activity was tested in transfected cell systems (5-10). In contrast to the G protein-coupled receptors for small ligands, the glycoprotein hormone receptors have a large extracellular domain that is presumably important for binding of the hormone.

Pathological manifestations involving these hormones and their receptors are most profound in Graves’ disease, a condi-

tion of hyperthyroxemia and thyroid hyperplasia associated with low or undetectable levels of circulating TSH. The thyrotropin receptor plays a central role in the pathophysiology of Graves’ disease and is the most predominant autoantigen against which the autoantibodies are directed. The hyperstimu- lation of the thyroid in Graves’ disease has been attributed to an autoimmune reaction, resulting in the production of a spec- trum of autoantibodies, some of which interact directly with theTSH receptor and induce chronic endocrine activity (1 1). Cloning of theTSH receptor and expression of the respective protein or variants thereof in either eukaryotic (5,6,8,12,13), prokaryotic (14,15,16,17) and indeed insect cells (18,19) has increased our knowledge about this protein and to some ex- tent its interaction with the hormone. However, elucidation of the epitopes interacting with the autoantibodies still re- mains to be conclusively demonstrated and analyzed in de- tail, although a number of recent studies have prepared a foundation for our further understanding of the receptor au-

Received October 24,199.5; accepted November 21, 199.5.

Address reprint requests to Dr. N. Hunt, Institute for Hormone and Fertility Research, University of Hamburg, Grandweg 64,22529 Hamburg, Germany.

8 1996 Wiley-Liss, Inc.

194 Hunt et al.

toantibody interactions (20,2 1). Indeed, a plausible immunogen involved in the stimulation of the autoimmune response still has to be identified, although recent studies have demonstrated that molecular mimicry may well be of importance in the initiation of an immune response (22,23). A Combination of recombinant expression technology and applied immunol- ogy will undoubtedly aid in the elucidation of this very inter- esting but difficult problem as has been recently proven successful at least in part for another autoantigen, the thyroid peroxidase molecule associated with Hashimotos thyroiditis (24-26). Increased knowledge of the molecular mechanisms involved in TSH-receptor-antibody interactions would enable an improved clinical intervention in the etiology and treat- ment of Graves' disease.

Such an understanding would thus set a basis with which one could try to understand the dysfunctioning of such a complex hormone receptor system in an overt pathology such as Graves' disease.

MATERIALS AND METHODS

Isolation of TSH Receptor cDNA Clones and Construction of a TSH Receptor Epitope cDNA Library

Poly (A') RNA was isolated from human thyroid tissue from a patient suffering from Graves' disease. The RNA was used for the construction of a cDNA library in lambda ZAP11 (Stratagene) with a primary complexity of - 1 ~ 1 0 ~ independent clones as previously described (27). For construction of theTSHR epitope library, a full-length cDNA clone of the humanTSH receptor (pTSHR6L; 4.3 kb) corresponding to both the coding and the 3' noncoding region of the receptor was used. The portion coding for the extracellular domain of theTSH receptor cDNA insert was digested out of the cloning plasmid (pTSHR.6L; XbaI- NdeI, in pBluescript; amino acids 1-389) and isolated by agarose gel electrophoresis. The isolated fragment (- 20 pg) was then fragmented by sonication with 4 pulses for 30 s at 250 W. The sonicated cDNA was size-selected into fragments of 200-500 bp by agarose gel electrophoresis, end-repaired with T4 DNA polymerase (Stratagene), and cloned into the SmaI restriction enzyme site in the polylinker of the prokaryotic expression vector pGEX-5T (28). This vector expresses the cloned gene at the C terminal of a modified 26 kDa glutathione-S-transferase containing a hexa-histidine sequence at the amino terminus. After transformation of Escherichia coli XL-1 blue (Stratagene), the resulting ampicillin-resistant colonies, representing the TSHR epitope cDNA library, were collected by washing the colonies from plates in LB medium. The library was then concentrated by centrifugation and resuspended in LB-glycerol (20%) freezing medium, titred, and stored in aliquots at -70°C.

Expression of the Extracellular Domain of the TSHR in E. coli and Generation of Antisera in Porcine and Ovine Species

A restriction fragment (Sspel-Ndel) encompassing the ex- tracellulx domain of the human TSH receptor (amino acids #78-389) was cloned into the prokaryotic expression vector pH6EX3. This fusion vector places a histidine-hexapeptide tag at the amino terminus as has been previously described (29). The resulting construct (pTSHR.561) was further veri- fied by DNA sequence analysis before being used to trans- form theE. coli strain K5254 and other related strains, GEl96 and GAG456. Bacterial transformants were grown overnight and the recombinant protein was induced as previously described (29). The recombinant hexa-histidine fusion protein TSHR.561 (343 amino acids and expected molecular weight of 38.4 kDa) was extracted from bacteria and purified over Ni" Agarose (QIAGEN) under denaturing conditions (8 M urea) according to the manufacturers recommendations. Pu- rified TSHR.561 protein was then used to immunize porcine and ovine species. Western blot analysis using the immunogen TSHR.561 protein demonstrated that both the porcine and the ovine antisera were of extremely high titre (- 150 000) when used in conjunction with the purified protein (100 ng) and Western blotting.

Affinity Purification of Anti-TSHR.561 Antibodies

One milligram of purified water soluble TSHR.561 protein was coupled to epoxy-activated sepharose 6B (Sigma) according to conditions recommended by the manufacturer. Two-milliliter aliquots of the porcine or ovine antisera were incubated with theTSHR.561 coupled sepharose support in a total volume of 10 ml in PBS at 4°C overnight with constant agitation. The slurry was then transferred to a disposable plastic column (Biorad) and nonadsorbed antibody was removed by repeated washing with PBS until the eluate had an < 0.0 1. The column was then washed with 5 bed volumes of 10 mM phosphate buffer pH 6.8. The immune adsorbed antibodies were then eluted with 100 mM glycine pH 2.5 and 500 p1 aliquots were collected and neutralized in 1/20 volume 1 M phosphate buffer pH 8.0. Fractions containing the eluted antibody were pooled and stored in 100 p1 aliquots at -20°C.

Screening of the Human TSHR Epitope cDNA Library With an Aff inity-Purified Anti-TSHR.561 Serum From Sheep and Pigs

TheTSHR cDNAepitope library was plated at a density of 500 clones per 150 mm dish until the colonies were -1-2 mrn in diameter. The colonies were then lifted onto nylon nitro- cellulose membranes (Hybond-N, Amersham) and placed colony side up onto prewarmed LB agar plates containing

TSH Receptor Epitope 195

for 5 h at 37°C. After induction and incubation for the respective time, 200 PI aliquots of the bacterial culture were sedimented and heat denatured at 95°C for 5 min in 20 pl SDS-loading buffer (50 mMTris-HCl pH 6.8, 100 mM DTT, 2% SDS, 0.1% bromophenol blue, 10% glycerol).The bacterial proteins were separated in 10% SDS-PAGE under reduc- ing conditions and electrophoretically transferred onto immobilon-P membranes (Millipore) using a Hoefer trans- blot apparatus at 500 mA for 1 h in transfer buffer (39 mM glycine, 48 mM Tris base, 0.037% SDS, 20% methanol, pH 8.3). The unoccupied protein binding Gtes on the filters were blocked with 5% low fat dry milk (Blotto) in TBS buffer for 1 hat 37"C.The filters were then incubated in dilutions of the various antibodies inTBS plus 5% Blotto for 16-24 hat 8°C. The diluted sera were pretreated with 0.4 mg/mlE. coli (IPTG induced pGEXST/XLl cells) cell lysate in TBS for 1 h at room temperature, prior to incubation with the filters. The

100 pg/ml ampicillin and 0.5 mM IPTG (Stratagene). The plates were incubated for 5 h at 37°C to induce the expression of the GST fusion proteins. The bacteria were lysed by exposing the filters to chloroform vapor for 20 min and fi- nally washed with TBST buffer (10 mM Tris-HC1, pH 8.0, 150 mM NaCI, 0.05% Tween-20) with gentle agitation to re- move all of the cell debris. Immuno-detection of the positive epitope clones using affinity-purified anti-TSHR antibodies was performed as described below (Western blotting). All selected clones were sequenced with a commercially available sequencing kit (Pharmacia, Freiburg) according to the chain termination method using T7 polymerase and primers encompassing the pGEX5T expression vector (28).

Western Blotting Analysis of the TSHR Fusion Proteins

For expression of the fusion proteins, overnight cultures of the positive bacterial clones were diluted 10-fold with prewarmed LB medium containing 100 pg/ml ampicillin and incubated for 90 min prior to induction with 0.5 mM IPTG

bound antibodies were visualized with alkaline phosphatase- conjugated, antiporcine/ovine IgG immunoglobulins after incubation for 1 h at room temperature in TBS-Blotto followed by color detection using NBT (nitro blue tetrazolium)

A TSHR561 fusion protein

H6 aa #78-389

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Fig. 1. A. Structure of the construct for the expression of the extraceliular domain of the humanTSH receptor. Afragrnent (SspeI-NdeI) encompassing amino acids 78-389 was cloned into the polycloning site of the prokaryotic expression vector pH6EX3. In this construct a hexa-histidine tag was constructed at the amino terminus of the mature protein for purification pur- poses. The stop codon originates from the vector sequences of the 3' cloning site. B. Expression of the recombinantTSHR-561 protein in bacterial strains

K5254, GE196, and GAG4.56. SDS PAGE and coomassie blue staining demonstrate the levels of TSHR-561 protein expression (running at 38 kDa) in the whole bacterial lysate the eluted recombinant protein after Nit+ agarose affinity chromatography. Also shown is the column flow through representing the proteins not bound to the Ni++ agarose. The purification was performed under denaturing conditions as described in Materials and Methods. M represents the molecular weight marker in kDa.

196 Hunt et al.

and BCIP (5-bro,mo-4-chloro-3-indoylphosphate) as sub- strates (Promega). Alternatively, a commercially available chemiluminescence detection system (Tropix, Serva) was used to visualize the bound primary antibodies according to the manufacturer’s specifications.

RESULTS

Expression of the Extracellular Domain of the TSH Receptor in a Prokaryotic Expression System

For the expression of the extracellular domain of the human TSH receptor, a SspI-NdeI fragment encoding amino acids #78-389 was cloned in the prokaryotic expression vector pH6EX3. This construct placed a hexa-histidine peptide tag at the amino terminus of the TSH receptor protein. The

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positive cDNA clones

characterization Epitope

Fig. 3. Construction of an epitope library for the extracellular domain of the human TSH receptor. A DNA fragment encompassing the extracellular domain of the human TSH receptor was randomly fragmented by sonication and subsequently size fractionated by agarose gel electrophoresis. Fragments ranging in size from 200-500 bp were isolated and end repaired with T4 DNA polymerase before being cloned into the SmaI site of the prokaryotic expression vector pGEX5T. Resultant epitopes (I in 6 will be in the correct reading frame) are expressed as glutathione S-transferase (GST) fusion proteins. After transformation into competent XL- 1 blue bacteria, the individual colonies were pooled to form the epitope library. The library was plated and screened with the affinity purified anti-TSHR-561 antibodies according to Materials and Methods. Positive clones were further analyzed by DNA sequencing and Western blotting.

structure of this construct, designated pTSHR.561, is shown in Figure 1A.After sequence analysis of the pTSHR.561 construct it was transformed in the E. coli strains K5254, GAG456, LE392, GE196, and GAG440 and expression was induced with IPTG as described in Materials and Methods. Studies performed to analyze the kinetics of induced protein expression demonstrated that considerable quantities of the 38 kDa TSHR.561 fusion protein could be detected after 5 h postinduction as shown by SDS-PAGE and coomassie blue staining (data not shown).

Large-scale production (500 ml) of the TSHR.561 protein in the three different E. coli strains GAG456, K5254, and

Fig. 2. Affinity purification ofthe porcine antiTSHR-561 antibodies. Pu- rified recombinant TSHR.561 protein was covalently bound to a sepharose support. This solid phase bound protein was then used to batch purify anti- TSHR-561 specific antibodies by affinity purification under native conditions.The unbound nonspecific antibodies and serum proteins were collected. Specifically bound anti-TSHR-561 antibodies populations were eluted with low pH and immediately neutralized. The specificity of the different anti- bodyherum protein fractions were tested in Western blot analysis of crude protein lysates of K5254 expressing TSHR-561 protein. Approximately 50 pg of total protein was probed with either crude porcine anti-TSHR-561 antisera, the affinity column flow through or affinity purified anti-TSHR- 561 antibodies, all at a dilution of 1:10,000. Bands were visualized using an alkaline phosphatase-conjugated second antibody as described in Materials and Methods. M represents the molecular weight marker in kDa. The recombinant TSHR.561 protein was detected at - 40 kDa.

Fig. 4. SDS PAGE and Western blot analysis of epitope clones selected with anti-TSHR-561 antibodies. Clones selected after screening the library with the affinity-purified porcine antibodies were picked, and protein expression of the GST fusion protein was induced with IPTG in 5 ml cultures. After 5 h of induction, 200 pI of the bacteria were pelleted and resuspended in SDS sample buffer before being denatured and loaded onto a 10% SDS PAGE gel. Replica gels were either stained with coomassie blue or transferred to PVDF membranes for Western blot analysis. The PVDF membranes were blotted with anti-TSHR-561 porcine antibodies (affinity purified) at a dilution of 1:10,000. Lanes 1-31 represent positive clones after screening. Lanes 32 and 33 are recombinant proteins (positive controls) encompassing a large part of the extracellular domain of the TSH receptor but are distinct from TSHR-561. Lanes 34 and 35 are total protein lysates (different concentrations) from XL-1 transformed with pCEX5T and induced with IPTG. The lane M represents the molecular weight marker.

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198 Hunt et al.

aa#78 aa#389 TSHR.561

aa#159 epi#21 aa#160 epi#19

aa#234 epi#lO aa#268 - epi#l

aa#272 - epi#4 aa#311 - epi#31 aa#312 - ePi#9

aa#327 - epi#6 aa#333 - epM3

aa#338 - epM8 aa#345 - epi#2

aa#353 I epi#22 aa#353 - epi#25

The amino acid numbering refers to aa#354 - epi#15 the primary sequence of the hTSH aa#358 - epi#3 receptor where aa#l is the ATG start codon. aa#365 = epi#17 The epitopes form the carboxy terminal aa#372 epi#16 of the GST fusion protein. aa#373 I epM2

Fig. 5. Sequence analysis of the epitope clones positive after screening the epitope library and Western blot analysis of the induced protein lysate. Summary of the sequence analysis for a number of epitope clones expressing a GST fusion protein reactive with anti-TSHR-561 antibodies. The sequence array of the original immunogenTSHR-561 is shown

encompassing the amino acids 78-389. The full sequence of the respective epitopes are aligned under this sequence to demonstrate their position within the full-length protein. The amino acid number at the beginning of each bar represents the amino acid at which the epitope begins in frame with the GST protein.

GE196 (Fig. 1B) demonstrated that equivalent synthesis of the recombinant protein occurred in all three different strains and that the TSHR.561 protein accounted for - 20% of the total protein. The hexahistidine fusion protein was subsequently purified to - 95% homogeneity by one round of NI" agarose chelating chromatography under denaturing conditions (Fig. IB, eluate). Purified TSHR.561 protein was extensively dialyzed against PBS remaining in a soluble form and after lyophilization was readily soluble in aqueous solutions. This protein was then used to immunize porcine and ovine species.

Affinity Purification of the Anti-TSHR-561 Anti bodies

Initial experiments using the recombinant TSHR.561 protein and the porcine and ovine antibodies combined with Western blotting demonstrated that both antisera had very high titre of between 150-70 000. However, although the antisera were able to detect the recombinant TSHR-561 protein by Western blot analysis, neither the porcine or the ovine antisera were able to inhibit the TSH radio-receptor assay at any concentration used (1 :2-1: 15000, data not shown). Thus it appeared that the epitopes seen by these antisera (in contrast to the linear epitopes seen in the Western blot analysis) were either unable to interact with the naturally folded TSH receptor expressed in a transfected cell system, or if so did

not influence the TSHRSH receptor interaction. To define further the linear epitopes recognized by the antisera, the antibodies were affinity purified using solid phase bound TSHR.561 protein as described in Materials and Methods. Western blot analysis of crude bacterial homogenates expressing the TSHR.561 protein using sera and affinity purified porcine antibodies are shown in Figure 2. Although the nontreated sera was able to detect the TSHR.561 protein in the crude bacterial homogenate, there were some nonhomo- logous proteins detected due to cross reactivity. Indeed, the nonbound antibodies eluted in the first wash demonstrated this cross-reactivity more extensively. However, the low pH eluted antibodies (those specific for the TSHR.561 protein) demonstrated no cross reactivity even when used at low dilutions (1 500). Essentially identical results were obtained using the ovine antibodies (data not shown). Assured with the fact that the affinity-purified antibodies Contained low to nondetectable, cross-reactive activities against bacterial proteins, the antibodies were used to screen the TSH receptor epitope library.

Screening of the TSHR Epitope Library The cDNA fragment coding for the extracellular domain

of the TSH receptor was used to construct an epitope library in the pGEX-ST expression vector in the E . roli strain XL-1 as described in Materials and Methods and depicted in Figure 3. The original unamplified library contained - 10,000 indi-


1 2 3 4 5 7 11 12 13 17

34 32 31 30 29 28 27 26 25 24 23 22 21

Fig. 6. Western blot analysis to demonstrate monospecificity of the porcine anti-TSHR-561 serum. Protein lysates identical to those used in Figure 4 were separated on 10% SDS-Page gels and the proteins were transferred to PVDF membranes. Before addition of the porcine anti-TSHR-56 I antibodies (affiiity purified) to the filters, they were incubated for 1 h with an excess of epi#12 fusion protein (500 pgiml) as in Materials and Methods. The final

vidual clones of which theoretically 16% could give rise to a GST fusion protein in the correct reading frame. The library was titred and 5,000 clones were plated at a density of 500 clones/l5 cm plate. The bacteria were induced and screened as described. From the primary screening, 30 positive clones were identified on duplicate filters by immuno detection with the affinity-purified porcine antibodies. These clones together with control clones were then further analyzed by SDS-PAGE and subsequent Western blotting of IPTG-induced, small scale cultures. The results of this analysis are shown in Figure 4. As can be seen from the stained gels, some of the GST fusion proteins were expressed in considerable quantities, whereas some TSH receptor epitope clones were only detected after Western blotting. The clones that were positive after Western blotting were then further analyzed by DNA sequence analysis using primers upstream and downstream of the epitope cloning sites. The results of the sequencing are shown in Fig- ure 5. The analysis demonstrated that the affinity-purified antibodies recognized linear epitopes, all of which contained sequences localized at the carboxyl end of the recombinant protein construct, with the smallest epitope being 16 amino

dilution of the anti-TSHF-561 antibodies was 1:10,000. Lanes 1-31 represent positive clones after screening. Lanes 32 and 33 are recombinant proteins encompassing a large part of the extracellular domain of theTSH receptor but are distinct from TSHR-561. Lanes 34 md 35 are total protein lysates (different concentrations) from XL-1 transformed with pCEX5T and induced with IPTG. M represents the molecular weight marker in kDa.

acids long and corresponding to the last 16 amino acids of the TSHR.561 protein (amino acids #373-389). That this sequence was present in all of the clones obtained thus sug- gested that a common linear epitope recognized by the anti-561 antibodies was delineated by the amino acids #373-389. To further analyze this, the Westem blotting experiment described above was repeated, except that the affinity-purified anti-561 antibodies were preadsorbed with purified recombinant epitope clone #12 GST fusion protein (1 mg) as described in Materials and Methods. The results of this analysis are shown in Figure 6. All of the detectable immuno reactivity of the anti-561 antibodies was removed by preincubation of the recombinant fusion proteins with the recombinant clone #12 GST fusion protein. This thus demonstrated that the amino acids #373-389 constituted the predominant linear epitope recognized by the affinity purified anti-561 antibodies. Thus it appeared that we had generated a polyclonal mono-specific antisera to the carboxyl terminal 16 amino acids of the recombinant TSHR.561 protein. Screening with a battery of the epitope clones covering all of the recombinant TSHR- 561 protein with the ovine sera (not affinity purified at a dilu-

200 Hunt et al.

3 4 6 10 11 12 13 15 17 16 18 21 24 25 27 31 33 34

Fig. 7. Western blot analysis epitope clones spanning the entire TSHR- 561 protein with a Graves’ patient serum. Induced bacterial protein lysates of respective epitope clones were separated on 10% SDS PAGE gels and either stained with coomassie blue or transferred to PVDF membranes. The membrane was incubated with a Graves’ patient serum showing marked reactivity to TSHR-561 protein at a dilution of 1,500. The blot was developed

tion of 1:5,000) demonstrated that the same linear epitope (epitope clone #12, corresponding to amino acid sequence 373-389) was also the predominant linear epitope detectable with this sera (data not shown).

Screening of Graves’ Sera for lmmuno Reactivity to TSHR-561 Protein Linear Epitopes

During the course of this work, we used the recombi- nantTSHR.561 protein to screen a large number of Graves’ sera using Western blotting to determine whether we could identify linear determinants in the extracellular domain of the TSH receptor detectable by patient antibodies. Screening of a large panel of Graves’ sera (>200) revealed one patient’s sera that reacted strongly (at a titre of 1500) with the recombinant TSHR.561 protein (100 ng). This serum sample was used to screen the panel of linear epitopes defined during the course of the preceding study with the porcine and ovine anti-561 antibodies. Interest- ingly, the same linear determinant was recognized by this patient’s sera as compared to the pig and ovine antisera.

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after incubation with an alkaline phosphatase secondary antibody and subsequent chemiluminescent detection. Lanes 3-31 represent positive clones spanning the entire length of the TSHR.561 protein. Lanes 34 and 35 are total protein lysates (different concentrations) from XL-1 transformed with pGEXST and induced with IPTG. M represents the molecular weight marker in kDa.

The Western blot analysis is shown in Figure 7. Again, the smallest linear epitope encompassed the carboxyl end of the TSHR.561 protein and was restricted to amino acids #373-389 as demonstrated by the preadsorption of the patient sera with purified clone #12 GST fusion protein. The summary of the results obtained with the porcine, ovine, and patient sera are presented in Figure 8.

DISCUSSION

The understanding of the TSH/TSH receptor interaction is of utmost importance if we are to ascertain the protein sequences important for this interaction and thus sequences recognized by the circulating auto-antibodies found in Graves’ disease. To this aim we started a study to produce polyclonal antisera to a recombinant protein corresponding to the extracellular domain of the human TSH receptor. A construct encompassing amino acids #78-389 (where theATG corresponds to amino acid #1) with an amino terminal hexa-histidine tag was expressed in a prokaryotic expression system. This construct in constrast to many other TSHR extracellular domain


a#78 ITSHR561+ + +

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+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

Fig. 8. Combined results for the epitope mapping using porcine, ovine, and one positive Graves’ serum. Summary of the sequence analysis for a number of epitope clones expressing a GST fusion protein reactive with porcine ovine and Graves’patient anti-TSHR-561 antibodies. The sequence array of the original immunogen TSHR-561 is shown encompassing the

amino acids 78-389.The full sequence of the respective epitopes are aligned under this sequence to demonstrate their position within the full-length protein. The amino acid number at the beginning of each bar represents the amino acid at which the epitope begins in frame with the GST protein. Re- activity of each serum sample is shown in the righthand columns.

proteins (Northemann et al., unpub. data) was not refractory to high levels of expression and indeed in the corresponding bacterial strain (K5254, GE196, and GAG456) and repre- sented - 20-30% of the total protein content after 5 h of induction. However, the high levels of expression resulted in aggregation of the protein and subsequent inclusion body for- mation. The recombinant TSHR-561 protein was purified to - 95% homogeneity (as detected by SDS-PAGE and coomassie staining) by one round of affinity chromatography over a Ni++Agarose column under denaturing conditions (6 M guanidine-hydrochloride/8M urea). However, after ex- tensive dialysis against PBS and subsequent lyophilization, the purified recombinant TSHR-56 1 protein was readily soluble in aqueous solutions, although the folded structure of this recombinant protein is surely not comparable to the native TSHR extracellular domain. Indeed this recombinant protein does not interact with a TSH radio-ligand directly nor is it able to interfere with the TSH radio receptor assay in a transfected cell system (27) (data not shown). Attempts to express the whole of the TSH receptor excluding the leader sequence. to the last arginine reside, 418 of the extracellular domain in both prokaryotic and insect expression systems have met with a number of difficulties with respect to yield, solubil-

ity, and functionality (1 8,19). However, when one considers that this portion of the TSHR contains 11 cysteine residues, it is not surprising that the recombinant protein aggregates and becomes insoluble, possibly through dimer- ization as has recently been demonstrated (20 and Hunt et al., unpub. data).

The recombinant TSHR-561 protein was, however, immunogenic after immunization o f porcine and ovine species, although these animals did not display any overt thyroid dysfunction during the course of the immunization scheme. The antiera had a high titre in Western blot toward the immunogen, thus demonstrating that at least some of the antibodies were directed against linear epitopes and not just conformational epitopes. T h s was indeed substantiated by the epitope mapping using the affinity purified antisera. We expected that the affinity-purified antisera as well as being depleted of any nonspecific antibodies would also contain antibodies directed against any conformational epitopes, as both the immunogen and the protein used for the antibody purification were indeed soluble and cross-linked to the epoxy resin under mild conditions.

That the predominantly detectable linear epitope recognized by both the porcine and ovine antibodies were identical and

202 Hunt et al.

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Fig. 9. Amino acid alignment of the human TSH, LH, and FSH receptor sequences to demonstrate the location of the common epitope sequence. The protein sequences for the humanTSH, LH, and FSH receptor sequences were aligned according to the MultAlign program. The epitope defined in the experimentation is defined by the striped bar and encompasses the

amino acid sequence PQEETLQAFDSHYDYT. This alignment demon- strates the uniqueness of the epitope and also the shared homology of the three receptor protein sequences, as demonstrated by the shaded boxes, which represent 100% identity.

corresponded to the free carboxyl terminus of the recombinant protein were somewhat surprising. However, when one considers that in the native receptor a further 300 amino acids are normally to be found juxtaposed to this sequence, then one could speculate that the free carboxy terminus in this construct is extremely free being unable to form its own natural

conformation. Such a sequence would in a globular soluble protein thus represent a particularly immunogenic determinant, a supposition substantiated in both the immunized species described here. However, this sequence in the native-transfected, full-length TSH receptor is either nonaccessible or not influenced by the porcine and ovine an-


dearly missed. We extend appreciation to those responsible for the stimulating research environment provided within the Institute for Hormone and Fertility Research. This study was supported by the Bundesminister fur Forschung und Technologie, Bonn, Germany, as part of a larger concerted project “Fertilitatsstorungen” (01 KY 9103).

tibodies. Although recent studies using these antibodies demonstrate that in immunohistochemical analysib Fab fragments compared to whole antibodies are able to detect TSHR protein in human thyroid sections (Hunt, unpub. data).

Using the recombinant TSHR-561 protein, we screened over 200 Graves’ sera for immunoreactivity in Western blot analysis. We were somewhat surprised to detect only one positive serum considering the ease with which one can detect interfering autoantibodies in either endogenous or transfected receptor preparations, although one could argue that the majority of these interfering entities interact with conformational epitopes of either theTSHR polypeptide or possible complex as has been postulated on a number of occasions and more recently demonstrated with recombinant extracellular domain of the TSHR expressed in E . coli (20,21). Even more surprising was the fact that the linear epitopes recognised by this patient serum were identical to the epitopes delineated by the experimental sera, which had been shown not to influence the TSH/TSHR interaction in an in vitro assay system. Se- quence analysis of this epitope, amino acids #373-389 demonstrated that this sequence as compared to the related LH/ hCG and PSH receptors is unique to the TSH receptor and is found within the 50 amino acid insertion characteristic to the TSH receptor (Fig. 9).

Unfortunately the limited amounts of this patient’s sera will make further analysis of this antibody difficult. Al- though the sera had a high TRAK titre and was indeed able to inhibit TSHDSHR interaction in a transfected assay system, it will be difficult to demonstrate and inter- pret the proportions of antibodies directed against the various immunogenic determinants. It thus appears from this study that a great majority of the anti-TSHR autoantibodies are indeed directed toward conformational epitopes of the TSHR complex. Indeed, if the free cysteine found in the human TSHR extracellular domain is able to form an intermolecular disulfide bridge resulting in either a homo- or even possibly a heterodimer, this could explain the difficulties encountered while trying to delin- eate the epitopes seen by autoantibodies (20,21). This could also explain the apparent difficulties encountered while attempting to produce large quantities of functional extracellular domain of theTSH receptor protein in a number of recombinant expression systems. However, a number of studies are at present underway to determine whether such complexes are indeed the functional conformations seen by the large percentage of Graves’ autoantibodies. Such studies will be of great relevance and may well result in the production of recombinant epitopes for use in sensitive ELISA type diagnostics specific for Graves’ disease.

ACKNOWLEDGMENTS

This work is dedicated to Dr. Wolfgang Northemann, who died during the final stages of this work. His presence will be

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Epitope mapping of a recombinant human TSH receptor extracellular domain: Identification of a predominant epitope using animal sera