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Original Studies |
Autoimmune Disease Unit, Cedars-Sinai Research Institute and the School of Medicine, University of California (G.D.C., J.G., Y.W., S.M.M., B.R.), Los Angeles, California 90048; Nichols Institute Diagnostics (J.S.H., D.S.), San Juan Capistrano, California 92690; Veterans Administration Medical Center and University of California (J.C.J.), San Francisco, California 94121
Address all correspondence and requests for reprints to: Basil Rapoport, M.B., Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Suite B-131, Los Angeles, California 90048.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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20–40% purity) from the culture
medium. Nanogram quantities of this material neutralized the TSH
binding inhibitory activity in all 15 Graves’ sera studied. We
generated a mouse monoclonal antibody (mAb), 3BD10, to partially
purified TSHR-289. Screening of a TSHR complementary DNA fragment
expression library localized the 3BD10 epitope to 27 amino acids at the
N-terminus of the TSHR, a cysteine-rich segment predicted to be highly
conformational. 3BD10 preferentially recognized native, as opposed to
reduced and denatured, TSHR-289, but did not interact with the TSH
holoreceptor on the cell surface. Moreover, mAb 3BD10 could extract
from culture medium TSHR-289 nonreactive with autoantibodies, but not
the lesser amount (25%) of TSHR-289 molecules capable of
neutralizing autoantibodies. Although the active form of TSHR-289 in
culture medium was stable at ambient temperature, stability was reduced
at 37 C, explaining the mixture of active and inactive molecules in
medium harvested from cell cultures. In conclusion, studies involving a TSHR ectodomain variant indicate the exquisite conformational requirements of TSHR autoantibodies. Even under "native" conditions, only a minority of molecules in highly potent TSHR-289 preparations neutralize patients’ autoantibodies. Therefore, Graves’ disease is likely to be caused by even lower concentrations of autoantibodies than previously thought. Finally, reciprocally exclusive binding to TSHR-289 by human autoantibodies and a mouse mAb with a defined epitope suggests that the extreme N-terminus of the TSHR is important for autoantibody recognition.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It is now feasible to generate large amounts of mammalian TSHR by using fermenters to propagate TSHR-expressing myeloma cells (2), by human TSHR complementary DNA (cDNA) transgenome amplification in CHO cells (3), and by vaccinia virus infection of HeLa cells (4). However, the hydrophobic, serpentine membrane-spanning region of the TSHR as well as the need to harvest cells rather than culture medium make purification of the holoreceptor difficult. Unfortunately, the TSHR ectodomain, truncated at its entry into plasma, is not secreted, but is largely retained within mammalian cells (5, 6) in a form containing immature, high mannose carbohydrate that is not recognized by patients’ autoantibodies (6). Whether autoantibody binding requires mature complex carbohydrate or whether it is incorrect folding of the truncated ectodomain that affects both autoantibody binding and normal intracellular trafficking of the ectodomain is presently unknown. It is now appreciated that correct TSHR ectodomain trafficking can be attained by attaching to its C-terminus a membrane-anchoring tail (7, 8, 9). However, another approach that we employed to achieve secretion of a highly potent, conformationally intact TSHR ectodomain was to progressively truncate the ectodomain at sites predicted to be in the vicinity of the carboxyl-terminus of the a-subunit (10).
Of the three truncated ectodomain variants that we generated, the most efficiently secreted is TSHR-261 (amino acids 22–261, after signal peptide removal) (10). Although TSHR-289 (residues 22–289) is less well secreted than TSHR-261, we have since observed that its autoantibody-neutralizing activity in cell culture medium is more stable, making TSHR-289 a better candidate for studies of TSHR autoantibodies in Graves’ disease. The present report describes our experience with this remarkable molecule. Although stable when partially purified, TSHR-289 disintegrates on purification or even on concentration of the semipurified form. However, a mouse monoclonal antibody (mAb) generated to partially purified TSHR-289 provides information of pathophysiological importance regarding the antigenic conformational requirement, epitopes, and the in vivo concentration of human autoantibodies.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The construction and expression of plasmid TSHR-289 have been described previously (10). In brief, the cDNA for TSHR residues 1–289 (1–21 representing the signal peptide) was inserted into the vector pSV2-ECE-dhfr (11). A double stranded oligonucleotide cassette coding for six histidine residues followed by two stop codons was introduced after the codon for TSHR residue 289. CHO-DG44 dhfr- cells (provided by Dr. Robert Schimke, Stanford University, Palo Alto, CA) were stably transfected with TSHR-289 cDNA (6). Transgenome amplification was achieved by progressive adaptation over approximately 1 yr to growth in methotrexate (final concentration, 10 µmol/L) (6).
Assay for neutralization of TSHR autoantibodies in the serum of Graves’ patients
TSHR autoantibody kits were purchased from Kronus (San Clemente, CA). Our modification of this TSH binding inhibition (TBI) assay (12) to measure autoantibody neutralization has been reported previously (10). In brief, 25 µL serum from Graves’ patients were preincubated (30 min at room temperature with 25 µL conditioned medium containing TSHR-289 or with partially purified TSHR-289 (see below). Solubilized porcine TSHR (50 µL) was then added followed by radiolabeled TSH (total volume, 200 µL; 2 h at room temperature), after which TSHR-TSH complexes were precipitated with polyethylene glycol. As controls, we used serum from normal individuals and conditioned medium from CHO cells secreting a truncated form of thyroid peroxidase (11). Autoantibody activity was expressed as the percent inhibition of [125I]TSH binding relative to that of a standard serum from a normal individual without autoantibodies.
Partial purification of TSHR-289 by concanavalin A and Ni-chelate chromatography
Conditioned medium was harvested three times per week from CHO
cells expressing TSHR-289 cultured in Ham’s F-12 medium containing
10% FCS, antibiotics, and 2.5 mmol/L sodium butyrate (13). Medium (2
L) was applied to a 70-mL concanavalin A-Sepharose (Pharmacia Biotech, Piscataway, NJ) column. After washing with 10 mmol/L
Tris (pH 7.4) and 150 mmol/L NaCl, bound material was eluted with about
80 mL 0.25 mol/L 
-methylmannoside in the same buffer. The eluted
material was made up to 50 mmol/L imidazole (pH 7.2) and applied to two
5-mL His-Trap columns in series (Pharmacia Biotech).
Elution was performed with buffer containing 10 mmol/L Tris (pH 7.4),
50 mmol/L NaCl, and 100 mmol/L ethylenediamine tetraacetate. The sample
was concentrated, and the buffer was changed to 10 mmol/L Tris (pH 7.4)
and 50 mmol/L NaCl using a Centriprep 30 (Amicon, Beverly, MA). At all
stages, TSHR-289 recovery was monitored by the TBI neutralization
described above.
Mouse mAb to TSHR-289
Six BALB/c mice were immunized sc with approximately 60 µg partially purified TSHR-289 (see above) in complete Freund’s adjuvant, followed by two further injections (at 3.5 and 9 weeks) in incomplete Freund’s adjuvant. Spleen cells (3 days after the final boost) were fused with SP2/0 mouse myeloma cells using polyethylene glycol by standard techniques, and selection of resistant clones was performed in hypoxanthine-aminopterin-thymidine medium (Sigma Chemical Co., St. Louis, MO). Wells were screened by enzyme-linked immunosorbent assay (ELISA) for IgG production and for antibodies to TSHR-289. For the latter, ELISA plates were coated with partially purified TSHR-289, and detection was performed with antimouse IgG conjugated to horseradish peroxidase (Sigma Chemical Co.). The specificity of IgG-secreting, positive clones was determined by immunoblotting under native and denaturing conditions (described below). In addition, we performed flow cytometry, as previously described (14), using TSHR expressed on the surface of TSHR-10,000 cells (3) and, as second antibody, affinity-purified goat anti-mouse IgG (0.8 µL; fluorescein isothiocyanate-conjugated; Caltag, South San Francisco, CA).
Determination of TSHR-289 mAb epitopes
We screened a size-selected (200–500 bp) TSHR cDNA fragment
library in the bacteriophage vector 
-Zap (Stratagene,
La Jolla, CA; Nagayama, Y., and B. Rapoport, unpublished data) using
ascites containing three newly isolated mAb to TSHR-289 (see below).
Screening was performed as described previously (15), with minor
modifications. BB4 cells (optical density of 1.0 in 10 mmol/L magnesium
sulfate) were infected with the bacteriophage -Zap library. After
4 h at 37 C, about 3 x 104 plaque-forming
units/150-mm diameter petri dish were overlayed with nitrocellulose
filters soaked in 10 mmol/L
isopropyl-thio-ß-D-galactopyranoside and incubated
overnight at room temperature. Filters were washed in Tris-buffered
saline (TBS) buffer (10 mmol/L Tris, pH 7.4, and 150 mmol/L NaCl)
containing 0.05% Tween, incubated for 30 min in 3% milk powder in TBS
at room temperature, rinsed, and then incubated with the antibodies
(ascites diluted 1:500) for 2.5 h at room temperature. After
washing with 10 mmol/L Tris, pH 7.4, and 150 mmol/L NaCl containing
0.05% Tween, peroxidase-conjugated affinity-purified sheep antimouse
IgG (Sigma Chemical Co.; 1:500) was applied to the filters
for 3 h at room temperature. Color was developed with 2.8 mmol/L
4-chloro-1-naphthol, 10 mmol/L imidazole, and 0.0125%
H2O2. Positive plaques were rescreened three or
four times until clonal. The nucleotide sequences of TSHR cDNA inserts
in plaque-purified clones were determined by the dideoxy-nucleotide
method (16) after rescue of pBS double stranded plasmids in XL1-blue
bacteria using the helper phage R408 according to the protocol of the
manufacturer (Stratagene).
Affinity purification of TSHR-289
Conditioned medium (2–3 days of culture, stored at -80 C) was thawed and filtered (0.22 µm pore size), and 1–2 L were applied (1.5 mL/min at room temperature) to a column with 5 mL Sepharose-linked mouse mAb 3BD10. After extensive washing with phosphate-buffered saline, pH 7.4, the protein was eluted (1.5 mL/min) with 0.2 mol/L glycine, 0.15 mol/L sodium chloride, and 0.02% sodium azide, pH 2.3. Fractions (2 mL) were immediately neutralized with 0.4 mL 2 mol/L Tris, pH 8.0. Fractions with an optical density greater than 0.1 were pooled; dialyzed against 10 mmol/L Tris (pH 7.4), 50 mmol/L NaCl, and 0.02% sodium azide; and concentrated with a Centriprep 30 (Amicon, Beverly, MA). Aliquots were applied to polyacrylamide gels and stained with Coomassie blue or were assayed for their ability to neutralize TSHR autoantibodies in patients’ sera (see above).
Immunodepletion of TSHR-289
Mouse mAb 3BD10 to the N-terminus of the molecule (see below) or Penta-His to the six histidines at the C-terminus of the molecule (Qiagen, Chatsworth, CA; 2.5 µg each) were added (16 h at 4 C) to 0.45 mL of 2-day conditioned medium from CHO cells secreting TSHR-289. Samples were then diluted to 2.0 mL in 10 mmol/L Tris-HCl (pH 7.4) and 50 mmol/L NaCl and applied to a 1-mL HiTrap protein G column (Pharmacia Biotech). After discarding the first 1.5 mL of the flow-through (diluted in column buffer), the final 0.5-mL fraction was kept to determine its ability to neutralize the BI activity of autoantibodies in Graves’ serum (see above).
Immunoblotting of TSHR-289
For immunoblotting under native conditions, TSHR-289 in 10 mmol/L Tris (pH 7.4) and 50 mmol/L NaCl was added to native sample buffer (Bio-Rad Laboratories, Inc., Hercules, CA) and applied to 10% two-dimensional well polyacrylamide gels without SDS (Bio-Rad Laboratories, Inc.). For immunoblotting under denaturing conditions and reducing conditions, TSHR-289 was added to Laemmli sample buffer (17) with 2% SDS and 0.7 mol/L (final concentration) ß-mercaptoethanol (30 min at 45 C) and applied to SDS-polyacrylamide gels (Bio-Rad). Enzymatic deglycosylation with N-glycosidase F (New England Biolabs, Beverly, MA) was performed as described previously (6). After electrophoresis, proteins were electophoretically transferred to polyvinyl- difluoride membranes (Bio-Rad Laboratories, Inc.). After blocking (45 min) in TBS containing 5.0% skim milk powder, membranes were rinsed and incubated (overnight at 4 C) in TBS-containing mouse mAb 3BD10, A9, or A10 (1:1000; the latter two were provided by Dr. Paul Banga, London, UK) (18) and 0.5% BSA. The filters were rinsed, incubated (1–2 h at room temperature) with alkaline phosphatase-conjugated goat antimouse IgG, and the signal was developed as described previously (6).
Immunoprecipitation of TSHR-289
CHO cells expressing TSHR-289 were metabolically labeled with [35S]methionine/cysteine, exactly as described previously (1-h pulse and overnight chase) (3). For comparison of native vs. denatured TSHR-289 antigen, an aliquot of medium harvested after the chase was frozen at -80 C. Another aliquot underwent reduction and alkylation using dithiothreitol and iodocetamide, exactly as described previously (19), was dialyzed against 10 mmol/L Tris (pH 7.4) and 50 mmol/L NaCl, and was frozen. Aliquots (1 mL) of each were then thawed and simultaneously subjected to immunoprecipitation as previously described (3, 10). Immunoprecipitates were dissolved in Laemmli buffer with 2% SDS and 0.7 mol/L ß-mercaptoethanol and electrophoresed on 10% polyacrylamide-SDS gels. Radiolabeled proteins were visualized by autoradiography on Kodak BioMax MS x-ray film (Eastman Kodak Co., Rochester, NY).
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Results |
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Although TSHR-261 is secreted into the culture medium to a greater
extent than the less truncated TSHR ectodomain variants TSHR-289 and
TSHR-309 (10), we observed suboptimal stability of TSHR-261 in culture
medium in terms of its ability to interact with TSHR autoantibodies.
Therefore, with the goal of purifying a soluble, immunologically active
TSHR ectodomain variant, we turned to TSHR-289, which retained its
bioactivity for at least 16 h at room temperature (data not
shown). Initially, we applied the same two-step approach previously
used for TSHR-261, namely concanavalin A lectin chromatography followed
by Ni-chelate chromatography (10) (TSHR ectodomain variants were
constructed with six histidines at their carboxyl-termini). This
procedure yielded about 0.4–0.6 mg TSHR-289 glycoprotein of
approximately 20–40% purity/L conditioned medium (Fig. 1
). Shown in the same figure for
comparison is TSHR-261 partially purified by the same method. Of note,
the loss of TSHR-261 immunoactivity (see above) was not associated with
any visible change in the appearance of the glycoprotein on Coomassie
blue staining.
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). Thus, TSH binding by the
individual sera was inhibited by 52.2 ± 20.9% (±SD;
range, 23.1–80.4%) in the absence and by 10.8 ± 4.0% (range,
5.0–16.4%) in the presence of TSHR-289 (by paired t test,
P < 0.0001).
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Although TSHR-289 activity was quite stable for up to 24 h at
ambient temperature during the lectin and Ni2+-chelate
chromatography steps, this material was remarkably recalcitrant to
further purification. The major contaminating proteins following the
Ni-chelate chromatography step were of very high (>120 kDa) molecular
mass (see Fig. 1
). N-Terminal
sequencing of this material was uninformative and did not indicate that
it represented aggregated TSH-289. The wide size difference between
TSHR-289 and the high molecular mass contaminant(s) suggested that a
final gel filtration chromatographic step would be straightforward.
Application of approximately 30% pure TSHR-289 to a Sephacryl S-100
column led to the recovery of high molecular mass material in the void
volume, but only trace amounts of TSHR-289. The latter was replaced by
very low molecular fragments lacking bioactivity (data not shown).
Mono-Q ion exchange fast protein liquid chromatography similarly
resulted in the loss of all bioactivity as well as the absence of a
TSHR-289 peak (data not shown). Attempts at separation of TSHR-289 from
the high molecular mass contaminant(s) by centrifugation through a
membrane with a 100-kDa pore led to total loss of TSHR-289 protein in
terms of both bioactivity and detection by Coomassie blue. Even further
concentration of partially purified TSHR-289 to more than approximately
1 mg/mL resulted in the total loss of bioactivity and detectable
protein.
Generation of mAb to TSHR-289
Concomitantly with the attempts at TSHR-289 purification, mouse
mAb to this material were produced by immunization with TSHR-289
partially purified by lectin and Ni-chelate chromatography. Clones were
initially screened by ELISA using partially purified TSHR-289
(logistical reasons precluded screening by flow cytometry using intact
TSHR-expressing CHO cells). Approximately 50 IgG-producing clones that
recognized the TSHR-289 preparation were subsequently screened by
immunoblotting under native and denaturing conditions as well as by
flow cytometry. Three clones (3BD10, 3BE12, and 1CE1) as well as a
positive control mAb (A9) (18) interacted with partially purified
TSHR-289 on immunoblotting under both native and denatured/reduced
conditions as well as after enzymatic deglycosylation with
endoglycosidase F (Fig. 3). The remaining
clones interacted with the high molecular mass contaminating protein(s)
and were not studied further. None of these clones recognized the TSH
holoreceptor on flow cytometry, performed as previously described (14).
For example, median fluorescence with 3BD10 (3.3 U) was similar to that
with a nonspecific monoclonal antibody (2.6 U). The mAb A9 provided a
slightly higher value (6.5 U), far lower than with a potent Graves’
serum (181.0 U). 3BD10 ascites also did not inhibit
[125I]TSH binding to solubilized porcine TSHR (94% and
100% binding relative to normal mouse IgG; each value is the mean of
duplicate determinations).
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). In contrast, mouse mAb A10 (obtained
from Dr. P. Banga) (18) recognized both native and denatured
TSHR-289.
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), the three mAb interacted with its polypeptide component,
making epitope mapping feasible.
Screening with the three mAb of a size-selected (200–500 bp) TSHR cDNA
fragment expression library yielded numerous clones. Nucleotide
sequencing of 16 clones and cross-screening of individual clones with
the three mAb revealed that their epitopes were the same, all at the N
terminus of the TSHR. Despite characterizing this large number of
reactive TSHR polypeptide fragments, we were unable to define a linear
segment of less than 27 amino acids (residues 25–51; Fig. 5). Indeed, the sequences of 15 of the 16
3BD10-reactive clones included residue 22, the first amino acid in the
mature protein (residues 1–21 being the signal peptide).
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Preferential recognition by mAb 3BD10 of the native form of
TSHR-289 indicated the feasibility of affinity purification of this
antigen. Passage of liter quantities of conditioned medium (2–3 days)
from CHO cells secreting TSHR-289 over a 3BD10-Sepharose column readily
purified TSHR-289 (0.5 mg/L) to near homogeneity as determined by
PAGE of the freshly isolated sample (data not shown). However, this
affinity-purified material was devoid of bioactivity (ability to
neutralize TBI autoantibodies), and, as after Sephacryl S-100 gel
filtration, it was no longer detectable when reanalyzed a few days
later by PAGE.
Although the 3BD10-Sepharose affinity column effectively purified
TSHR-289 (at least in the short term), we were interested in
determining the efficiency of this procedure for extracting
immunologically active protein. For this purpose, we applied a
relatively small volume (50 mL) of conditioned medium from TSHR-289 CHO
cell cultures to the high capacity 3BD10-Sepharose column (10 mg
3BD10). The estimated concentration of TSHR-289 in conditioned medium
is less than 1 µg/mL. As anticipated given the vast excess of mAb, a
large proportion (74.5%) of TSHR-289 in the medium was extracted after
a single passage over the column (Fig. 6A). Quantitation was performed by
densitometric analysis of immunoblots of reduced and denatured TSHR-289
detected with mAb A10 that strongly reacts with denatured TSHR.
Surprisingly, however, despite the large excess capacity of the
affinity column, two additional applications of the same flow-through
did not increase the extent of TSHR-289 extraction (74.3% and 73.2%,
respectively). Moreover, even though the affinity column extracted
three quarters of the TSHR-289 molecules from the medium, it did not
extract any TSHR autoantibody-neutralizing activity from the same
medium (Fig. 6B). These data suggested the existence of two forms of
native TSHR-289, with mAb 3BD10 recognizing a dominant component
lacking biological activity.
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There existed, therefore, a perplexing situation that mAb 3BD10
preferentially recognized TSHR-289 under native conditions but was
unable to bind to biologically active TSHR-289. One possible
explanation for this phenomenon was the existence of two forms of
native TSHR-289, with mAb 3BD10 recognizing a dominant component
lacking biological activity. The basis for such a difference could be
the reduced stability of TSHR-289 in medium at tissue culture
temperature (37 C) despite its stability at ambient temperature.
Moderate TSHR-289 instability at tissue culture temperature was indeed
found. Thus, incubation of harvested conditioned medium for 7 h at
37 C reduced the ability of the material to neutralize TSHR
autoantibodies in serum (Fig. 7). No loss
of activity was seen when the medium was maintained at 21 or 28 C for
the same time period.
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Another (more disturbing) possible explanation for the inability
of mAb 3BD10 to immunopurify material with TSHR
autoantibody-neutralizing activity was that this activity in
conditioned medium was not inherent to the TSHR-289 molecule. We,
therefore, attempted to immunodeplete TBI-neutralizing activity in
conditioned medium using a mAb to the six histidine residues at the
C-terminus of TSHR-289. Such an antibody is unlikely to be influenced
by subtle conformational changes in the autoantibody-binding site.
Indeed, almost all TBI-neutralizing activity was removed by addition to
the medium of the antihistidine mAb followed by passage over a protein
G column (Fig. 8). Consistent with the
previous data, mAb 3BD10 was largely ineffective.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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On the other hand, our data provide valuable new insight into this remarkable molecule that will contribute to the long term goal of understanding its interaction with TSH and disease-causing autoantibodies. It is apparent that two forms of native TSHR-289 exist, only one of these being recognized by TSHR autoantibodies. In a reciprocal manner, mouse mAb 3BD10 only recognizes TSHR-289 that does not interact with autoantibodies and vice versa. In our view, the most reasonable explanation for this phenomenon is that 3BD10 has a conformational epitope that is cryptic on the "super" native molecule but is revealed by subtle unfolding, imperceptible other than by loss of autoantibody binding. Further denaturation of TSHR-289 (reduction and alkylation) leads to loss of the 3BD10 epitope.
These observations carry a number of important implications. First, the data help to resolve the still controversial concept that TSHR autoantibodies recognize exquisitely conformational epitopes. The discontinuous nature of these epitopes was revealed by studies using chimeric TSH-LH/CG receptor molecules (25). On the other hand, studies too numerous to describe have used TSHR-based synthetic peptides and prokaryotic fragments to report a myriad of linear epitopes apparently recognized by patients’ autoantibodies (reviewed in Ref. 1). Parenthetically, the evidence most commonly cited for the conformational nature of TSHR autoantibody epitopes is the inability of Graves’ patients’ sera to detect TSHR cDNA fragments expressed in a prokaryotic library (26). However, this conclusion depends on recognition by Graves’ sera of the TSH holoreceptor in such libraries, a finding that does not occur (26) (Nagayama, Y., and B. Rapoport, unpublished data).
A second intriguing conclusion from our study is that TSHR autoantibody
concentrations in patients’ sera are even lower than previously
estimated, a phenomenon of pathophysiological relevance. TSHR
autoantibodies in the majority of Graves’ patients cannot be detected
by indirect immunofluorescence (27) or flow cytometry (14) on
TSHR-expressing mammalian cells (thyroidal or nonthyroidal). In
contrast, thyroid peroxidase (TPO) autoantibodies in autoimmune
Hashimoto’s thyroiditis, with titers typically 50-fold higher, are
easy to detect by the same approach (14). The availability of partially
purified ectodomain variant TSHR-261 permitted quantitative
neutralization studies of TSHR autoantibodies in patients’ sera (10).
By this means, relatively few sera were estimated to have TSHR
autoantibodies in the microgram per mL range (10), whereas TPO
autoantibody levels can attain 1 mg/mL (28). The present study with
TSHR-289 indicates that only approximately 25% of the antigen
molecules in conditioned medium contain the TSH binding-neutralizing
activity. Therefore, TSHR autoantibody levels in patients’ sera are
clearly in the nanogram per mL range. The extremely low level of TSHR
autoantibody in serum is consistent with the hypothesis (29) that these
antibodies arise at a very early stage of the autoimmune process.
Support for this concept is provided by restricted 
or light
chain usage (30, 31, 32) and relative restriction to the IgG1 subclass (33)
of TSHR autoantibodies.
The epitope for mouse mAb 3BD10 as well as those for the other two mAb raised against TSHR-289 are also of interest. Previously, using the same cDNA fragment library approach for mouse mAb that bind to denatured TPO, we could readily narrow the cognate region to 15 amino acids (15). Likewise, a mAb (A9) that recognizes the TSHR after reduction has an epitope of only 14 amino acids (18). In contrast, despite analyzing 16 clones with TSHR fragments recognized by 3BD10, the linear epitope of 3BD10 could not be narrowed to less than 27 residues (amino acids 25–51). Because a typical antibody makes contact with 15–22 amino acid residues (34), the large size of the 3BD10 epitope is consistent with our data demonstrating its conformational nature and suggests that it may even be discontinuous. Support for this concept is that 15 of the 16 clones analyzed contained the cluster of 4 cysteine residues (Cys24, Cys29, Cys31, and Cys41) at the extreme N-terminus of the ectodomain (after signal peptide deletion). Modeling of the structurally rigid, leucine-rich repeats in the TSHR ectodomain (35) suggests that these four cysteines are spatially distant from the other 7 cysteines in the 397-amino acid residue ectodomain. Disulfide bonds are therefore likely to occur between the four N-terminal cysteines. Such bonding in a relatively small segment could, in turn, create a highly structured, possibly discontinuous, epitope. It is possible that folding variability in this region could result in the reciprocally exclusive recognition of mAb 3BD10 and Graves’ TSHR autoantibodies. Indeed, chimeric receptor and other mutagenesis studies have implicated residues Ser25-Glu30 (36) and Thr40 (37) as being a part of the TSHR autoantibody-binding site. Mutation of Cys41 also eliminates TSH binding (38), although whether this receptor trafficks to the cell surface is unknown.
In conclusion, studies involving a TSHR ectodomain variant truncated at residue 289 indicate the exquisite conformational requirements of TSHR autoantibodies. Even under native conditions, only a minority of molecules in highly potent TSHR-289 preparations neutralize patients’ autoantibodies. Therefore, Graves’ disease is caused by even lower concentrations of autoantibodies than previously thought. Finally, reciprocally exclusive binding to TSHR-289 by human autoantibodies and a mouse mAb with a defined epitope provides strong complementary evidence to the results of mutagenesis studies that the extreme N-terminus of the TSHR is important for autoantibody recognition.
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Acknowledgments |
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Footnotes |
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Received August 12, 1998.
Revised October 7, 1998.
Accepted November 5, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-light
chains: evidence for the "forbidden clone" theory. J Clin
Endocrinol Metab. 62:342–347./ immunoglobulin distribution in Graves’
thyroid-stimulating antibodies. Simulataneous analysis of C gene
polymorphisms. J Clin Invest. 82:1306–1312.[CrossRef]
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S. M. McLachlan, H. A. Aliesky, P. N. Pichurin, C.-R. Chen, R. W. Williams, and B. Rapoport Shared and Unique Susceptibility Genes in a Mouse Model of Graves' Disease Determined in BXH and CXB Recombinant Inbred Mice Endocrinology, April 1, 2008; 149(4): 2001 - 2009. [Abstract] [Full Text] [PDF]
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S. M. McLachlan, Y. Nagayama, P. N. Pichurin, Y. Mizutori, C.-R. Chen, A. Misharin, H. A. Aliesky, and B. Rapoport The Link between Graves' Disease and Hashimoto's Thyroiditis: A Role for Regulatory T Cells Endocrinology, December 1, 2007; 148(12): 5724 - 5733. [Abstract] [Full Text] [PDF]
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 S.-C. Ho, S.-S. Goh, I. H C Kee, P. K H Chow, C.-P. Yeo, and D. H C Khoo Effects of genetic immunization of Swiss outbred mice with human thyroid stimulating hormone receptor cDNA plasmids harboring gain-of-function mutations J. Mol. Endocrinol., February 1, 2007; 38(2): 277 - 288. [Abstract] [Full Text] [PDF]
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H. A. Aliesky, P. N. Pichurin, C.-R. Chen, R. W. Williams, B. Rapoport, and S. M. McLachlan Probing the Genetic Basis for Thyrotropin Receptor Antibodies and Hyperthyroidism in Immunized CXB Recombinant Inbred Mice Endocrinology, June 1, 2006; 147(6): 2789 - 2800. [Abstract] [Full Text] [PDF]
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 P. N. Pichurin, C.-R. Chen, G. D. Chazenbalk, H. Aliesky, N. Pham, B. Rapoport, and S. M. McLachlan Targeted Expression of the Human Thyrotropin Receptor A-Subunit to the Mouse Thyroid: Insight into Overcoming the Lack of Response to A-Subunit Adenovirus Immunization J. Immunol., January 1, 2006; 176(1): 668 - 676. [Abstract] [Full Text] [PDF]
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 S. M. McLachlan, Y. Nagayama, and B. Rapoport Insight into Graves' Hyperthyroidism from Animal Models Endocr. Rev., October 1, 2005; 26(6): 800 - 832. [Abstract] [Full Text] [PDF]
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S. Costagliola, M. Bonomi, N. G. Morgenthaler, J. Van Durme, V. Panneels, S. Refetoff, and G. Vassart Delineation of the Discontinuous-Conformational Epitope of a Monoclonal Antibody Displaying Full in Vitro and in Vivo Thyrotropin Activity Mol. Endocrinol., December 1, 2004; 18(12): 3020 - 3034. [Abstract] [Full Text] [PDF]
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 L. J. Dawson, H. E. Allison, J. Stanbury, D. Fitzgerald, and P. M. Smith Putative anti-muscarinic antibodies cannot be detected in patients with primary Sjogren's syndrome using conventional immunological approaches Rheumatology, December 1, 2004; 43(12): 1488 - 1495. [Abstract] [Full Text] [PDF]
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F. Latrofa, G. D. Chazenbalk, P. Pichurin, C.-R. Chen, S. M. McLachlan, and B. Rapoport Affinity-Enrichment of Thyrotropin Receptor Autoantibodies from Graves' Patients and Normal Individuals Provides Insight into Their Properties and Possible Origin from Natural Antibodies J. Clin. Endocrinol. Metab., September 1, 2004; 89(9): 4734 - 4745. [Abstract] [Full Text] [PDF]
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G. D. Chazenbalk, F. Latrofa, S. M. McLachlan, and B. Rapoport Thyroid Stimulation Does Not Require Antibodies with Identical Epitopes But Does Involve Recognition of a Critical Conformation at the N Terminus of the Thyrotropin Receptor A-Subunit J. Clin. Endocrinol. Metab., April 1, 2004; 89(4): 1788 - 1793. [Abstract] [Full Text] [PDF]
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P. N. Pichurin, O. Pichurina, R. C. Marians, C.-R. Chen, Terry. F. Davies, B. Rapoport, and S. M. McLachlan Thyrotropin Receptor Knockout Mice: Studies on Immunological Tolerance to a Major Thyroid Autoantigen Endocrinology, March 1, 2004; 145(3): 1294 - 1301. [Abstract] [Full Text] [PDF]
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C.-R. Chen, P. Pichurin, G. D. Chazenbalk, H. Aliesky, Y. Nagayama, S. M. McLachlan, and B. Rapoport Low-Dose Immunization with Adenovirus Expressing the Thyroid-Stimulating Hormone Receptor A-Subunit Deviates the Antibody Response toward That of Autoantibodies in Human Graves' Disease Endocrinology, January 1, 2004; 145(1): 228 - 233. [Abstract] [Full Text] [PDF]
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L. Schwarz-Lauer, P. N. Pichurin, C.-R. Chen, Y. Nagayama, C. Paras, J. C. Morris, B. Rapoport, and S. M. McLachlan The Cysteine-Rich Amino Terminus of the Thyrotropin Receptor Is the Immunodominant Linear Antibody Epitope in Mice Immunized Using Naked Deoxyribonucleic Acid or Adenovirus Vectors Endocrinology, May 1, 2003; 144(5): 1718 - 1725. [Abstract] [Full Text] [PDF]
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P. Pichurin, O. Pichurina, G. D. Chazenbalk, C. Paras, C.-R. Chen, B. Rapoport, and S. M. McLachlan Immune Deviation Away from Th1 in Interferon-{gamma} Knockout Mice Does Not Enhance TSH Receptor Antibody Production after Naked DNA Vaccination Endocrinology, April 1, 2002; 143(4): 1182 - 1189. [Abstract] [Full Text] [PDF]
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J. G. Cundiff, S. Kaithamana, G. S. Seetharamaiah, J. R. Baker Jr., and B. S. Prabhakar Studies Using Recombinant Fragments of Human TSH Receptor Reveal Apparent Diversity in the Binding Specificities of Antibodies That Block TSH Binding to Its Receptor or Stimulate Thyroid Hormone Production J. Clin. Endocrinol. Metab., September 1, 2001; 86(9): 4254 - 4260. [Abstract] [Full Text] [PDF]
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P. Pichurin, X.-M. Yan, L. Farilla, J. Guo, G. D. Chazenbalk, B. Rapoport, and S. M. McLachlan Naked TSH Receptor DNA Vaccination: A TH1 T Cell Response in Which Interferon-{gamma} Production, Rather than Antibody, Dominates the Immune Response in Mice Endocrinology, August 1, 2001; 142(8): 3530 - 3536. [Abstract] [Full Text] [PDF]
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G. D. Chazenbalk, S. M. McLachlan, P. Pichurin, X.-M. Yan, and B. Rapoport A Prion-Like Shift between Two Conformational Forms of a Recombinant Thyrotropin Receptor A-Subunit Module: Purification and Stabilization Using Chemical Chaperones of the Form Reactive with Graves' Autoantibodies J. Clin. Endocrinol. Metab., March 1, 2001; 86(3): 1287 - 1293. [Abstract] [Full Text]
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 A. P. Weetman Graves' Disease N. Engl. J. Med., October 26, 2000; 343(17): 1236 - 1248. [Full Text] [PDF]
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 C.-R. Chen, K. Tanaka, G. D. Chazenbalk, S. M. McLachlan, and B. Rapoport A Full Biological Response to Autoantibodies in Graves' Disease Requires a Disulfide-bonded Loop in the Thyrotropin Receptor N Terminus Homologous to a Laminin Epidermal Growth Factor-like Domain J. Biol. Chem., April 27, 2001; 276(18): 14767 - 14772. [Abstract] [Full Text] [PDF]
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