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Department of Life Science and Division of Molecular and Life Sciences, Pohang University of Science and Technology (C.H.B., C.-B.C.), Pohang 790-784, South Korea; Center for Neurologic Disease, Harvard Institute of Medicine, Brigham and Women’s Hospital and Harvard Medical School (J.Y.P.), Boston, Massachusetts 02215; and Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine (T.A.), Kyoto 606-8507, Japan
Address all correspondence and requests for reprints to: Dr. Chi-Bom Chae, Division of Molecular and Life Sciences, POSTECH, Pohang 790-784 Korea. E-mail: cbchae{at}postech.ac.kr
Abstract
Autoantibodies against TSH receptor (TSHR) are known to be involved in the occurrence of Graves’ disease. It is obvious that mapping of epitopes of the autoantibodies found in the patients with Graves’ disease is an important step in elucidating possible mechanism of generation of the autoantibodies against TSHR as well as in developing effective diagnostic and therapeutic approaches for Graves’ disease.
In this report we have identified the peptide sequences that bind to two human monoclonal thyroid-stimulating antibodies (mTSAbs; B6B7 and 101–2) from a disulfide-constrained phage-displayed peptide library. The peptides selected by three rounds of biopanning showed half-maximal inhibitory activities for cAMP synthesis induced by mTSAbs at about 0.1 µmol/L. SPWTLGA and TQWNMQH selected for B6B7 and 101–2, respectively, show specificity for their respective antibodies. This means that different clones of mTSAbs may have different epitopes for TSHR. The IgG of the patient from whom B6B7 was derived binds with specificity to the respective immobilized peptide in an enzyme-linked immunosorbant assay format, and its cAMP generation was also inhibited by selected peptide. It may be possible that the epitopes of TSAbs identified from the phage-displayed peptide library could be used for the classification of different clones of TSAbs present in patients with Graves’ disease and for development of drugs to treat Graves’ disease.
AUTOIMMUNITY TO thyroid-specific antigens is the most common cause of thyroid diseases, including Hashimoto’s thyroiditis and Graves’ disease (1). For instance, it is generally accepted that Hashimoto’s thyroiditis is due to T cell responses directed against thyroglobulin, and that Graves’ disease is mediated by autoantibodies to TSH receptor (TSHR). An important factor contributing to the difficulty of studies on Graves’ disease is the characteristics of autoantibodies involved in Graves’ disease. There are many types of autoantibodies to TSHR, such as thyroid-stimulating antibody (TSAb), TSH binding inhibitory antibody (TBAb), thyroid-stimulating blocking antibody (TSBAb), and thyroid growth-stimulating antibody (growth Ab) (2). Among the various types of autoantibodies, TSAb is a major cause of Graves’ disease, resulting in the overproduction of thyroid hormones by binding to TSHR on thyroid membranes. TSAbs recognize discontinuous and highly conformational epitopes on TSHR, which are assembled by bringing several regions of TSHR into a close proximity (2, 3, 4). Furthermore, it is thought that different patients with Graves’ disease have different clones of TSAbs (5, 6). The complex characteristics of the TSAbs mentioned above make it difficult to understand the actions of TSAb.
Therefore, the availability of monoclonal thyroid-stimulating antibodies (mTSAbs) with the same Ig subclass and affinity as those present in sera from patients with Graves’ disease will overcome the problem of the complex heterogeneity of autoantibodies involved in Graves’ disease. In this study we used two human mTSAb clones (B6B7 and 101–2) that were previously isolated and characterized from patients with Graves’ disease (7, 8, 9). These two mTSAb clones show significant thyroid-stimulating activity, but no TSH binding inhibitory (TBII) activity.
Recently, the epitope mapping of these mTSAbs was performed by the binding of iodinated monoclonal autoantibodies to the cells expressing mutant TSHR (10). Mutations of common (amino acids 58–61) as well as different (amino acids 34–37 and 52–56) segments of the N-terminal region affect the recognition of the receptor by these mTSAbs. The binding of each mTSAb to the cells expressing TSHR was mutually exclusive and was not inhibited by TSH. However, the binding sequences of the mTSAbs could not be determined in that study. If the epitopes of these mTSAb clones are revealed, the information will be helpful for understanding the pathology and mechanism of Graves’ disease and for investigation of diagnostic and therapeutic approaches to this thyroid disease.
To identify the epitopes of these two mTSAb clones (B6B7 and 101–2) whose structural or sequence information is completely unknown, we have used a phage-displayed peptide library that has strong conformational constraints in the displayed peptide sequences and so has proven useful in the identification of structural epitopes of the target molecules (11, 12). The peptide sequences identified from the phage-displayed peptide library in this study bind to the mTSAbs and IgG of the patient with specificity and inhibit cAMP synthesis induced by the mTSAbs and Graves’ IgG in CHO cells expressing human TSHR. Moreover, the peptides can be used for detection of TSAb in Graves’ patient serum that recognizes the peptide sequence.
Materials and Methods
Purification of human mTSAbs from mouse ascites
The generation of two human mTSAbs (B6B7 and 101–2) was previously reported (9). Briefly myeloma cells transfected with complementary DNA constructs of B6B7 and 101–2 were ip injected into BALB/c mice pretreated with 2,6,10,14-tetramethyl-decanoic acid (Wako Pure Chemical Industries, Ltd., Osaka, Japan). Both constructs were made for IgG1 production. Human IgGs, which were produced in ascites of mice, were purified using the protein A-agarose purification method (Pierce Chemical Co., Rockford, IL). Protein concentrations of the purified IgGs were determined by the enzyme-linked immunosorbent assay (ELISA) method described previously (7, 9).
Removal of mouse Igs from partially purified mTSAbs through Affi-Gel 10 gel immunoaffinity chromatography
After being purified by the protein A-agarose method (13), the IgG was further purified by Affi-Gel 10 gel immunoaffinity chromatography. Affi-Gel 10 gel (1 mL; Bio-Rad Laboratories, Inc., Hercules, CA) was washed with ice-cold deionized water and equilibrated with 20 mL 0.1 mol/L NaHCO3 (pH 8). For cross-linking, 250 µL antihuman IgG (Fc specific; 20 mg/mL; Sigma, St. Louis, MO; Aldrich Chemical Co., Inc., Milwaukee, WI) was incubated with the resin at 4 C overnight with shaking. To stop the reaction, 0.1 vol 1 mol/L ethanolamine-HCl (pH 8) was added to the resin, and a column was packed and equilibrated with 20 mL ice-cold 150 mmol/L Tris/50 mmol/L NaCl (pH 8). The mTSAb partially purified by the protein A purification step was loaded onto the Affi-Gel 10 gel column conjugated with antihuman IgG, and the bound antibody was eluted with 0.1 mol/L glycine-HCl (pH 2.3). The degree of purification was confirmed by ELISA method. Briefly the mTSAbs that were purified through Affi-Gel 10 gel immunoaffinity chromatography were coated (1 µg, 100 ng, and 10 ng each) onto microtiter wells and blocked with 3% BSA/PBS in duplicate. Antihuman Fc IgG conjugated with horseradish peroxidase (Sigma and Aldrich Chemical Co., Inc.) or antimouse IgG labeled with peroxidase (Amersham Pharmacia Biotech, Little Chalfont, UK) was added to the wells coated with mTSAb. After washing with PBS and Tween 20 (PBST; 0.2% Tween 20), the bound horseradish peroxidase was determined by incubation with 2,2'-azidobis-[3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt (Kirkegaard & Perry Laboratories, Gaithersburg, MD).
The purified mTSAbs were dialyzed against NaCl-free hypotonic HBSS (1.3 mmol/L CaCl2, 5 mmol/L KCl, 0.44 mmol/L KH2PO4, 0.4 mmol/L MgSO4, 0.34 mmol/L Na2HPO4, 20 mmol/L HEPES, 0.1% BSA, and 0.1% glucose, pH 7.4).
Screening of peptides that bind to mTSAb from phage-displayed peptide library
In this study we used Ph.D.-C7C phage displayed peptide library kit (New England Biolabs, Inc., Beverly, MA). The Ph.D.-C7C phage-displayed peptide library was composed of random disulfide-constrained heptapeptide sequences fused to the N-terminus of minor coat protein (pIII) of M13 phage. The randomized heptapeptide was flanked by a pair of cysteine residues, and under nonreducing conditions the cysteines will spontaneously form a disulfide cross-link, resulting in display of cyclized peptides. The library consisted of 3.7 x 109 sequences and was amplified once to yield approximately 50 copies of each sequence in 10 µL of the supplied phages. The biopanning process consisted of 3 rounds of affinity selection of the phages bound to the coated mTSAb purified through Affi-Gel 10 gel immunoaffinity chromatography. 101–2 or B6B7 mTSAb in PBS (2 µg/mL) was coated onto a microtiter well and blocked with 3% BSA/PBS. Ph.D.-C7C phage solution in 3% BSA/PBS was added to the well and allowed to bind for 2 h at 37 C. In the first round of biopanning, 50 µL phage library [2.5 x 1011 plaque-forming units (pfu)] were preincubated with 50 µL normal human IgG (0.1 mg/mL) in 3% BSA/PBS for preclearing before being added to the mTSAb-coated well. After washing with PBST 10 times, bound phages were eluted with 0.1 mol/L glycine/0.1% BSA (pH 2.2) and subjected to the next round of screening after being amplified. In the second round, washing stringency was elevated by increasing the Tween 20 concentration from 0.1% to 0.5%, and the well was washed 30 times. In the third round, stringency of washing steps was still increased by total of 45 washings with PBST (0.5% Tween 20). The phage fractions in all biopanning processes were titrated to determine the degree of selection.
Phage ELISA for the binding of affinity-selected phages to mTSAbs
Microtiter wells were coated with 0.1 µg purified mTSAb (B6B7 or 101–2) by incubation at 4 C overnight and then blocked with 3% BSA/PBS. Affinity-selected phages (1010 pfu in 50 µL 3% BSA/PBS) for each clone of mTSAb B6B7 and 101–2 were added to each mTSAb-coated well. After the unbound phages were removed by washing with PBST (0.5% Tween 20), the bound phages were detected by incubation with sheep anti-M13 antibodies conjugated with horseradish peroxidase (Amersham Pharmacia Biotech). The bound horseradish peroxidase (HRP) was determined by incubation with 2,2'-azidobis-[3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt as a substrate for HRP. After the reaction was stopped by the addition of an equal volume of 1% SDS, the absorbance at 405 nm was determined in an automated ELISA reader (model EL 312e, Bio-Tek Instruments, Inc., Burlington, VT).
DNA sequencing
The phage clones selected by three rounds of biopanning and confirmed for binding to mTSAb by ELISA were precipitated with polyethylene glycol after amplification, and single-strand phage DNA was extracted by the phenol extraction method. The nucleotide sequences of the epitopes of the selected phages were determined by the dideoxynucleotide chain termination reaction method (14) using the -28 gIII sequencing primer provided in the Ph.D.-C7C phage-displayed peptide library kit. Candidate peptides were synthesized by PeptidoGenic Research & Co. (Livermore, CA). The disulfide-constrained heptapeptide sequences were synthesized as a form of ACXXXXXXXCGGGS, where X positions represent the deduced heptapeptide sequences flanked by a pair of Cs (cysteines), N-terminal A (alanine), and C-terminal GGGS (glycine-glycine-glycine-serine) present in the pIII sequence of the Ph.D.-C7C phage.
Measurement of cAMP
The function of TSAbs was assessed by determining the amount of cAMP produced in response to TSAbs using CHO cells expressing human TSHR (CHO-TSHR cells) (15, 16). The CHO-TSHR cells were seeded at 2 x 105 cells/well in 24-well plates for 24 h before the cAMP assay. After the culture medium was removed, cells were preincubated with NaCl-free hypotonic HBSS for 30 min at 37 C. mTSAb (50 µg/mL) or Graves’ IgG (2 mg/mL) was added to the CHO-TSHR cells in 200 µL NaCl-free hypotonic HBSS containing 0.5 mmol/L 3-isobutyl-1-methylxanthine for 3 h at 37 C. Then the intracellular cAMP was extracted with ice-cold absolute ethanol, and the amount of cAMP was determined by a commercial RIA kit (17) (Amersham Pharmacia Biotech). For investigation of the inhibitory activity of peptides on the synthesis of cAMP induced by TSAbs, IgGs were preincubated with various concentrations of peptides for 30 min at room temperature before being added to the culture dish containing CHO-TSHR cells. The CHO-TSHR cells were maintained under 5% CO2 at 37 C and cultured in Ham’s F-12 mixture medium supplemented with 10% FBS and 0.5 mg/mL Geneticin (G418, Life Technologies, Inc., Gaithersburg, MD).
Immobilization of the peptides via covalent coupling in an ELISA format
For immobilization of the peptides, two peptides were conjugated to the amino surface of CovaLink NH2 wells (Nalge Nunc International, Milwaukee, WI) by primary amino groups grafted on the surface. Water-soluble 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) (Pierce Chemical Co.) was used to activate the carboxylic acid group of peptides in the presence of N-hydroxysuccinimide (NHS; Pierce Chemical Co.). Each peptide (10 nmol/well) in 50 µL 0.1 mol/L 2-[N-morpholino] ethanesulfonic acid and 0.9 mol/L NaCl (pH 4.8) was added to CovaLink NH2 wells. Freshly prepared solutions of NHS (0.17 mg/mL, 1 µL/well) followed by EDC (0.15 mg/mL, 1 µL/well) were added to each well and incubated for 30 min at room temperature with gentle agitation. The wells were washed with distilled water and PBST (0.1% Tween 20) three times each and were treated with 50 µL mTSAb or Graves’ IgG for 2 h at 37 C. The bound IgG was detected using goat antihuman IgG Fc conjugated with HRP.
To investigate the specific interaction between Graves’ IgG and peptide, competitive ELISA was performed in CovaLink NH2 wells coupled with peptide as described above. Before addition of Graves’ IgG, it was preincubated with soluble peptide for 1 h at room temperature.
Results
Selection of phages bound to mTSAbs
Two human mTSAbs, B6B7 and 101–2, were partially purified from
mouse ascites by a protein A purification method. The mTSAb
fractionated by the protein A method was further purified through
Affi-Gel 10 gel cross-linked with antihuman IgG for a biopanning
procedure. An ELISA showed that the purified mTSAb preparation was free
of mouse Igs (data not shown). To select the phage clones that bind to
mTSAbs, the Ph.D.-C7C phage-displayed peptide library was incubated
with the mTSAbs (B6B7 or 101–2) coated on a microtiter well. For each
round of biopanning, phages were titrated for pfu in the inputs and
outputs to determine the degree of selection (Table 1
). The result was compared with that of
the well coated with 3% BSA/PBS blocking buffer only (blank). The
fraction of total number of phages bound to mTSAb was increased from
3.7 x 10-6 ()
and 3.9 x 10-6
(B6B7) in the first round to 1.1 x
10-3 () and 1.3
x 10-4 (B6B7) in the
third round, almost 3500- and 300-fold increases for 101–2 and B6B7,
respectively, but the yields of the phages binding to the blank well
increased by about 10-fold for both 101–2 and B6B7. Therefore, it
appears that the proportion of the phages that bound to mTSAbs with
high specificity and affinity was gradually increased at each round of
panning.
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The phages obtained from the third round of biopanning were
amplified and analyzed. Seventy-two phage clones were selected for each
mTSAb, and the phages were tested for binding to mTSAb by ELISA. Some
phage clones show higher binding to mTSAbs than others (Fig. 1). The DNA from the 30 and 15 candidate
phage clones for mTSAb 101–2 and B6B7, respectively, were sequenced,
and their deduced peptide sequences are shown in Table 2. For the phages bound to 101–2 mTSAb,
26 phage clones of 30 encoded the same peptide sequence, TQWNMQH. Among
15 selected clones that bound to B6B7 mTSAb, 8 clones encoded
SPWTLGA, and 5 clones SPWSIGA (Table 2). The peptides that showed
the highest affinity and frequency for the two mTSAb were synthesized
as a cyclized form, including some flanking sequences:
ACSPWTLGACGGGS for B6B7 mTSAb and
ACTQWNMQHCGGGS for 101–2. The cyclic peptides will be
referred to as SPWTLGA and TQWNMQH.
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Both mTSAbs induced the cAMP synthesis in CHO-TSHR cells by about
200% of the basal level at 25 µg/mL as previously reported
(9). To investigate whether the selected peptide sequences
inhibit the cAMP synthesis induced by each mTSAb, we determined the
amount of cAMP produced in response to mTSAb in the presence or absence
of the peptides. The peptides were preincubated for 30 min at room
temperature with 50 µg/mL mTSAb before being added to CHO-TSHR cells.
After treatment of the cells with mTSAb alone or mTSAb plus the
selected peptides, the level of cAMP synthesized in CHO-TSHR cells was
determined as described in Materials and Methods. The
peptides SPWTLGA and TQWNMQH inhibited the cAMP synthesis induced by
the respective mTSAb B6B7 and 101–2 in a dose-dependent manner (Fig. 2). The two peptides showed half-maximal
inhibitory activities at about 0.1 µmol/L. The peptides did not
inhibit the cAMP synthesis induced by TSH (data not shown). Also, the
selected peptides did not show the cross-inhibitory activities for the
two mTSAbs at a significantly high concentration (30 µmol/L). The
peptide sequence of less prevalent phage clones showed lower inhibitory
activity (data not shown).
|
). The
reason for the incomplete inhibition of the stimulatory activity of
total patient’s IgG is most likely due to the presence of more than
one clone of TSAb in patient’s serum, as others have suggested.
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To test the possibility that different types of TSAb can be
classified by the peptides that recognize TSAbs, we investigated
whether the patient’s autoantibodies bind to immobilized peptide. At
first, the binding of TSAbs to immobilized peptide was assessed in an
ELISA format. We found that monoclonal antibody did not bind to the
peptides immobilized to plastic wells, perhaps due to the shortness of
the peptides. Therefore, CovaLink NH2 plate was
used, which offers a surface for direct covalent coupling of protein to
the primary amino groups grafted on the surface. The -COOH group of
peptides that was activated in the presence of water-soluble
carbodiimide (EDC) and NHS was cross-linked covalently to the
NH2 group of the CovaLink
NH2 plate. mTSAb B6B7 and 101–2 showed
significant binding activity for the respective peptides (Fig. 4). 101–2 showed higher binding activity
for the peptide than B6B7. We also demonstrated specific binding of
total IgG of the patient’s serum from which the B6B7 clone was derived
to immobilized SPWTLGA. The binding was reduced in the presence of
free SPWTLGA, but not by TQWNMQH (Fig. 5). The patient’s serum from which mTSAb
101–2 was derived was exhausted by the time of this study, and we
could not conduct a similar study.
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Our goal in this study was identification of the peptides that bind and inhibit the function of two human mTSAbs (B6B7 and 101–2) using a phage-displayed peptide library. There is much supporting evidence that TSAbs recognize discontinuous and highly conformational epitopes on TSHR (18, 19). Therefore, the peptide library may be suited for discovery of the peptides that bind to TSAb. The phage-displayed peptide library is a powerful tool for the development of pharmacologically active peptide agonists or antagonists (20). In this study we used a commercially available Ph.D.-C7C phage-displayed peptide library that consisted of disulfide-constrained heptapeptide library. There are reports that high affinity ligands can be more easily identified if a disulfide-constrained library instead of a linear peptide library is used (21).
The two mTSAb clones (B6B7 and 101–2) were previously isolated from two different patients with Graves’ disease and were found to illicit a significant increase in cAMP-synthesizing activity in CHO-TSHR cells (9). Disulfide-constrained heptapeptides that bind to either of two mTSAb clones were selected from the peptide library. Most of the phages that bound to each mTSAb clone had consensus motives in their epitope sequences: SPWTLGA for mTSAb B6B7 and TQWNMQH for mTSAb 101–2. Among the consensus sequences defined above, the N-terminal-SP-motif found in SPWTLGA and the N-terminal -TQ- motif found in TQWNMQH were observed in the extracellular domain of human TSHR: -SP- (amino acids 26 and 27) and-TQ-(amino acids 54 and 55) motifs in the N-terminal region of the receptor. In a previous study it was reported that the N-terminal region (especially residues 22–61) is a site of major TSAb epitopes in the TSHR (22). Also, there was a report that cAMP generation induced by B6B7 was significantly lowered in the cells expressing the receptors mutated in amino acid residues 34–37 and 58–61. The residues 52–56 and 58–61 of the receptor contributed to the stimulatory effect of 101–2 (10). Whether the peptides found in this study have any relationship to the key TSAb-binding regions of TSHR found in the previous studies remains to be seen. However, there is a report that the participation of two or three amino acid residues in different regions of a protein contributes to the conformational epitope (23).
We have previously identified pentapeptides that bind to mTSAb B6B7 and 101–2 from linear peptide libraries linked to resin. A similar sequence (for example RWLLP) bound to both mTSAbs and had a rather high inhibitory concentration (30 mmol/L) for synthesis of cAMP induced by the mTSAbs. The peptide is either too short, or cyclic peptides are preferred for binding to TSAb with specificity.
In this report we were able to demonstrate binding of mTSAb as well as the total IgG of the patient from which the mTSAb was derived to the identified peptides. The binding of patient’s IgG to the peptide was specific. However, the peptide identified in this study does not completely inhibit the stimulatory activity of patient’s serum from which the monoclonal antibody was derived. This is most likely due to presence of more than one clone of TSAb in patient’s serum (6, 19). We also investigated the prevalence of the clones of TSAb studied in this report of Grave’s patients. A preliminary study indicated that one in seven patients had the TSAb 101–2 clone (data not shown). More detailed study will be needed.
The results described in this report suggest that once the epitopes or peptide sequences that are recognized by different clones of TSAbs are determined, the peptide sequence would be helpful for determination of specific TSAb clones in the serum of Graves’ patient as well as for investigation of other aspects of Graves’ disease, such as pathology and epitope spreading (24, 25). The peptides could also be used in the development of drugs for the treatment of Graves’ disease.
Footnotes
1 This work was supported by the Biotechnology 2000 Program of the
Ministry of Science and Technology, Korea. 
Received November 20, 2000.
Revised March 2, 2001.
Accepted March 13, 2001.
References
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  C. H. Na, M. H. Lee, B. Y. Cho, and C.-B. Chae A Method for Identification of the Peptides That Bind to a Clone of Thyroid-Stimulating Antibodies in the Serum of Graves' Disease Patients J. Clin. Endocrinol. Metab., April 1, 2003; 88(4): 1570 - 1576. [Abstract] [Full Text] [PDF]
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, DNA-sequenced phage clones
(15 and 30 for B6B7 and 101–2, respectively).
, 10 nmol; 
, 1 nmol). Each
point represents the mean of duplicate determinations
along with the SD.