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Original Studies |
Autoimmune Disease Unit, Cedars-Sinai Research Institute and School of Medicine, University of California, Los Angeles, California 90048
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. E-mail: rapoportb{at}cshs.org
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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In summary, purification of immunologically active and stable antigen in milligram quantities provides a powerful tool for future diagnostic and therapeutic studies in Graves’ disease.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Different strategies have been used for generating substantial quantities of conformationally intact, recombinant TSHR in mammalian cells. Membrane-associated proteins have been produced in the form of the holoreceptor with its seven membrane-spanning segments (5, 6, 7, 8), as a protein with a single transmembrane region (9), or with a glycosylphosphatidylinositol-anchored domain (10, 11). Attempts to convert the TSHR ectodomain into a secreted protein by introduction of a stop codon proximal to the plasma membrane (amino acid residue 418) were unsuccessful, with intracellular retention of a protein containing immature carbohydrate (12, 13). However, more modular truncations of the TSHR ectodomain further upstream (residues 309, 289, and 261), in the vicinity of the site of intramolecular cleavage into A and B subunits (reviewed in Ref. 1), did lead to secretion of an autoantibody-reactive protein corresponding approximately to the A-subunit (14).
In principle, a secreted protein that is not encumbered by a lipid layer and that does not require release by proteolytic digestion is preferable for immunological and structural studies. Unfortunately, lability of the secreted TSHR A-subunit prevented its purification to homogeneity (15). Even more daunting, the secreted TSHR A-subunit was found to be heterogeneous, containing two distinct forms reciprocally interactive with patients’ autoantibodies and a mouse monoclonal antibody (15). In the present study, using two different mouse monoclonal antibody affinity columns in series, we report the separation and purification to near homogeneity in milligram quantities of each of these two forms of the TSHR. Moreover, using chemical chaperones we have, for the first time, been able to stabilize purified TSHR antigen in immunologically intact form. These findings will facilitate future studies on the structural properties of the TSHR ectodomain as well as on the pathogenesis of Graves’ disease.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Construction and overexpression by transgenome amplification of the plasmid for the TSHR truncated at amino acid residue 289 with six histidine residues at the C-terminus have been described previously (14, 15). TSHR-289-expressing Chinese hamster ovary (CHO) cells were cultured in Ham’s F-12 medium containing 10% FCS, antibiotics, and 2.5 mmol/L sodium butyrate. Conditioned medium was harvested every 2 days, cell debris was removed by centrifugation (400 x g for 10 min at 4 C), and the medium was stored at -80 C.
Two separate mouse monoclonal antibody (mAb) affinity columns were made by coupling to cyanogen bromide-activated Sepharose 4B beads (Amersham Pharmacia Biotech, Piscataway, NJ) mAb 3BD10 (15) (20 mL; bed volume, 1.5 mg IgG/mL) and Penta-His anti-5H mAb (QIAGEN, Chatsworth, CA; 10 mL; bed volume, 1.0 mg/mL), according to the protocol of the manufacturer. After thawing, the medium was filtered (0.22-µm pore size), and 2 L were applied (1 mL/min at 4 C) to the 3BD10 column linked in series with the anti-His column. After extensive washing of both columns (still in series) with 10 mmol/L Tris-HCl (pH 7.4) and 150 mmol/L NaCl, the columns were separated, and protein was eluted from each with 0.2 mol/L glycine, pH 2.3. Fractions (1.6 mL) were immediately neutralized with 0.4 mL 2 mol/L Tris-HCl, pH 8.0. Fractions containing the eluted protein (determined by absorption at 280 nm) were pooled; dialyzed against 10 mmol/L Tris (pH 7.4), 50 mmol/L NaCl, and 0.02% sodium azide; concentrated using a Centriprep 30 (Amicon, Beverly, MA); and stored at -80 C. Aliquots of concentrated material were subjected to 10% SDS-PAGE under reducing conditions and stained with Coomassie blue. Prestained molecular size markers (Sigma), previously calibrated against more accurate unstained markers were used to determine protein size. Samples were also analyzed after enzymatic deglycosylation with N-glycosidase F (New England Biolabs, Inc., Beverly, MA) according to the protocol of the manufacturer.
N-Terminal sequence determination
TSHR-289 protein eluted from both the 3BD10 and anti-His affinity columns was enzymatically deglycosylated, electrophoresed on 10% polyacrylamide gels (see above), electrophoretically transferred to polyvinyl difluoride membranes (Bio-Rad Laboratories, Inc., Hercules, CA), and stained with Coomassie blue. N-Terminal amino acid sequences were determined by the Protein Structure Laboratory, University of California (Davis, CA).
Assay for TSHR autoantibodies in Graves’ patients serum to recognize truncated TSHR-289
TSHR autoantibody kits were purchased from Kronus (Boise, ID). This assay measures the ability of autoantibodies to bind to TSHR in a solubilized porcine thyroid extract, thereby competing for [125I]TSH binding to the same preparation (16). We modified this TSH binding inhibition (TBI) assay to detect TSHR-289 neutralization of patients’ autoantibodies, as previously described (14). An important principle of this modified TBI assay is that only patients’ autoantibodies, but not TSH, can bind to TSHR-289 whereas both autoantibodies and TSH bind to the porcine holoreceptors. In brief, 25 µL TSHR-289 were preincubated (30 min at room temperature) with 25 µL Graves’ serum. Solubilized porcine TSHR (50 µL) was then added (20 min at room temperature), followed by 100 µL [125I]TSH (2 h at room temperature). TSHR-[125I]TSH complexes were then precipitated with polyethylene glycol. Autoantibody activity was expressed as [125I]TSH binding in the presence of Graves’ serum relative to [125I]TSH binding in the presence of serum from healthy individuals without thyroid disease (100%). Recognition of TSHR-289 by patients’ autoantibodies was evident by the ability of TSHR-289 to adsorb out TBI activity, thereby reversing the inhibition of [125I]TSH binding to the porcine holoreceptors.
In some experiments the stability of TSHR-289 recognition by patients’ autoantibodies was examined by incubating the protein at different temperatures for different periods of time in the presence of a variety of solutes; trimethylamine N-oxide (TMAO), trehalose, proline, glycerol (all from Sigma, St. Louis, MO), and Tween-20 (J. T. Baker, Phillipsburg, NJ). After these preincubations, the TSHR-289 material was tested for autoantibody functional integrity as described above.
Relative recognition by different mouse and human antibodies of the two forms of TSHR-289
Anti-His mouse mAb was radiolabeled with 125I to a specific activity of approximately 100 µCi/ug using Iodo-Gen (Pierce Chemical Co., Rockford, IL). This material was precomplexed (1 h at room temperature) to TSHR-289 eluted from either the 3BD10 or the anti-His affinity columns linked in series (0.2 µg [125I]anti-His mAb plus 0.1 µg TSHR-289). Aliquots (2 µL at the indicated dilutions) of the following mouse or human antibodies were applied to nitrocellulose filters and air-dried: mouse mAb A9 to the TSHR (provided by Dr. Paul Banga, King’s College, London, UK) (17), mouse mAb 3BD10 (15), normal mouse serum, sera from two Graves’ patients with TSHR autoantibodies, and sera from two normal individuals without thyroid disease. Filters were blocked for 1 h at room temperature with 3% milk powder in 10 mmol/L Tris (pH 7.4) and 150 mmol/L NaCl (TBS), rinsed with TBS, and then incubated (1 h at room temperature) in TBS with 1% BSA and 106 cpm/mL of the [125I]anti-His mAb plus TSHR-289 complex. After further washing with TBS buffer containing 0.05% Tween-20, radiolabeled complexes bound to antibodies on the filters were visualized by autoradiography using Kodak BioMax MS x-ray film (Eastman Kodak Co., Rochester, NY).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The TSHR truncated at amino acid residue 289 (TSHR-289) is
secreted and is potent in neutralizing TSHR autoantibodies. However,
this material could not be purified to homogeneity by nickel-chelate
chromatography using the six His residues at its C-terminus, was
heterogeneous in containing both immunologically active and inactive
molecules, and was functionally unstable in terms of autoantibody
recognition (15). Moreover, a mouse mAb (3BD10) raised to
TSHR-289 reacted specifically with the inactive, dominant component
(15). In the present study we considered the possibility
that mAb 3BD10 could be an asset rather than an ineffective reagent.
Thus, we hypothesized that preapplication of conditioned medium from
CHO cell cultures to a 3BD10 affinity column would remove inactive
TSHR-289 and that the flow-through could be applied directly to an
anti-His affinity column in series. Although both active and inactive
forms of TSHR-289 contain a His-tail, the prior removal of inactive
TSHR-289 would leave only the active form of TSHR-289 available for
capture (Fig. 1
).
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). As evident by
Coomassie blue staining, a similar yield (
0.6–0.75 mg/L applied
medium) was obtained from the separated 3BD10 and anti-His columns (in
series before elution). In other preparations, the proportion of active
to inactive TSHR-289 varied from approximately 25–50%. The
electrophoretic mobility and appearance of the material eluted from
either column were indistinguishable. Remarkably, however, despite the
similarity in their physical appearance, the two preparations were very
different in their immunological properties with respect to TSHR
autoantibodies in Graves’ patient serum. Thus, TSHR-289 eluted from
the 3BD10 column did not reverse the TSH binding inhibitory activity of
TSHR autoantibodies even at the very high concentration of 1250 ng/tube
(0.2 mL total volume; Fig. 3, upper
panel). In contrast, only 5 ng TSHR-289 eluted from the anti-His
column neutralized nearly all of the TSH binding inhibitory activity in
the same patient’s serum (Fig. 3, lower panel).
Therefore, the dual affinity column strategy effectively separated
active from inactive TSHR-289.
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Determination of a structural difference between the active and
inactive forms of TSHR-289 could provide important information on the
immunological properties of TSHR autoantibodies, including their
epitopes. In particular, the immunological difference between active
and inactive TSHR-289 could be related to a change in primary amino
acid sequence (N- or C-terminal clipping) or to an alteration in
protein folding. As shown above (Fig. 2
), the two proteins were
indistinguishable on PAGE and Coomassie blue staining. However, the
large amount of complex glycan on the secreted form of TSHR-289
(14) could obscure loss of a few residues at the N- or
C-termini.
The ability to separately purify the immunologically active and
inactive forms of TSHR-289 permitted a more detailed analysis of these
properties. Enzymatic deglycosylation with endoglycosidase F reduced
the sizes of both active and inactive TSHR-289 from approximately 60 to
35 kDa, indicating similarity in their polypeptide cores and their
degree of glycosylation (Fig. 4).
However, this apparent similarity in polypeptide size does not exclude
the loss of a few amino acids occurring either within the
TSHR-289-expressing CHO cells or by proteolysis after secretion into
the culture medium. Determination of the N-terminal amino acid
sequences of purified, deglycosylated, immunologically active and
inactive TSHR-289 indicated that they were identical (MGCSSPPCE; single
amino acid code). Clipping at the C-terminus was excluded by the
immunological integrity of the six-His tail on the inactive form of
TSHR-289 as detected by the anti-His mAb (see below; Fig. 5). On this basis, an alteration in
protein folding is the most likely explanation for the immunological
difference between the two molecules.
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, upper
panel). As a control, mouse mAb A9 did not display a major
difference in recognition of the different forms of TSHR-289.
Conversely, and more importantly, Graves’ sera recognized only active,
but not inactive, TSHR-289, and recognition was markedly reduced after
stressing TSHR-289 (37 C for 4 h; Fig. 5, lower panel).
Sera from normal individuals recognized neither form of antigen. Note
that these direct binding data are qualitative and require particularly
potent sera. In contrast, the indirect TBI neutralization data (Fig. 3)
are quantitative and much more sensitive and, therefore, are reproduced
with numerous sera with varying potency. Stabilization of TSHR-289 immunological activity using chemical chaperones
Previous data indicated that unpurified TSHR-289 in culture medium
retained its immunological activity for many hours at 4 C, but this
activity was lost within a few hours at 37 C (15).
Unfortunately, this lability persisted even when active TSHR-289 was
purified to near homogeneity with removal or reduction of
contaminating, potentially modifying, enzymes or products. These data
together with evidence that the primary amino acid structure of the
inactive TSHR was intact, led us to search for agents and solutes that
might stabilize the folding of the active form of TSHR-289. After
incubation of purified, active TSHR-289 for 4 h in different
agents, the ability of TSHR-289 to be recognized by autoantibodies in
Graves’ serum was tested by reversal of the TBI activity (Table 1). Thus, a Graves’ serum chosen for
intermediate potency reduced [125I]TSH binding
to porcine holoreceptors to approximately 30% of that observed in the
presence of serum from a normal individual (maximum binding). Active
TSHR-289 maintained for 4 h at 4 C, but not at 37 C, reversed this
TBI activity. Inclusion of trehalose, Tween-20, and glycerol in various
combinations had no protective effect. In contrast, 2 mol/L proline and
2.5 mol/L TMAO, agents known to stabilize some proteins under
environmental stress (18) completely preserved the
immunological activity of TSHR-289 during the 4-h period at 37 C.
Remarkably, both 2 mol/L proline and 2.5 mol/L TMAO stabilized the
folding of the active form of TSHR-289 for up to 29 h at 37 C
(Fig. 6). TMAO did not convert inactive
TSHR-289 into its active form, nor could we restore activity by
refolding after denaturation in guanidine or by treatment with protein
disulfide isomerase (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The inactive form of TSHR-289 is not simply a denatured molecule. Thus, denaturation of inactive TSHR-289 leads to loss of mAb 3BD10 recognition (15). The 3BD10 epitope on inactive TSHR-289 is, therefore, conformational. This epitope has been mapped to a 27-amino acid, cysteine-rich segment at the extreme N-terminus of the TSHR ectodomain (residues 25–51; numbering includes the signal peptide) (15). Twenty-seven residues is larger than a typical antibody footprint (19). The inability of the mAb to interact with smaller polypeptide fragments (15) and the high cysteine content of the epitope suggest that besides being conformational, the epitope may also be discontinuous. From the perspective of the present study the most remarkable aspect of the 3BD10 epitope is that it overlaps with one component (amino acid residues 25–30) of the discontinuous epitope(s) for TSHR autoantibodies (3, 20). Reciprocal binding by 3BD10 and autoantibodies to conformational determinants involving the same amino acid residues in the TSHR raises the possibility of a prion-like shift between folded states of a native molecule. Prions are proteins synthesized by the same gene, containing identical amino acid sequences. A change in folding and conformation converts the normal, benign form into an infective pathogenic form (reviewed in Ref. 21). This analogy is not meant to imply that the TSHR can become infective, simply that it can convert from one native form to another.
An important observation in the present study was the ability to use
cellular osmolytes, or chemical chaperones (reviewed in Ref.
18), to stabilize a labile molecule. Obviously, producing
milligram quantities of purified TSHR antigen for future structural and
immunological studies is of limited value if the material is unstable.
Previously, we had been unable to stabilize the TSHR in terms of its
autoantibody binding properties with a wide variety of proteolytic
inhibitors. Among the commonly available molecular chaperones, we find
that proline and TMAO, but not trehalose or glycerol, are effective. Of
interest, to pursue the analogy with prions, chemical chaperones
prevent the conversion of cellular prion protein to the pathogenic
isoform, possibly by stabilizing the 
-helical conformation of the
former (22).
Besides providing the means to generate milligram quantities of purified, soluble, autoantibody-reactive TSHR antigen, the present study provides additional insight into TSHR structure and immunological properties. First, the direct sequence of purified TSHR-289 establishes unequivocally the signal peptide cleavage site. Second, the influence of a minor folding change on antibody recognition reinforces why previous studies using other forms of recombinant antigen, in particular peptides, were not very fruitful. Third, the potency of only a few nanograms of purified, active TSHR-289 in neutralizing TSHR autoantibodies in Graves’ sera emphasizes that the concentration of the latter is even lower than considered previously when a heterogeneous preparation of TSHR-289 was available (15). These quantitative neutralization data are consistent with previous immunohistochemical (23) and flow cytometry (24) data, indicating that in thyroid autoimmunity, TSHR autoantibody concentrations are 2–3 orders of magnitude lower than those of thyroid peroxidase autoantibodies (typically present in the same patient), an observation of possible pathogenetic significance (reviewed in Ref. 1).
Finally, an important future goal is to determine the three-dimensional structure of the TSHR ectodomain. Our studies indicate subtle folding heterogeneity in the TSHR-289 molecule that would be incompatible with this objective. Whether similar heterogeneity exists in TSHR preparations from other laboratories is unknown. However, our ability to separate different folded forms of the TSHR, generate large quantities of these molecules and maintain their stability, as well as expressing a smaller modular portion of the molecule rather than the entire ectodomain, improves the chances of obtaining crystals. Nevertheless, major hurdles remain, including the very high TSHR-289 glycan content. In addition, it is unknown how molar concentrations of the molecular chaperones necessary for the stabilization of TSHR-289 will affect crystal formation. However, even if crystallization of this very difficult molecule is never achieved, the generation of milligram quantities of purified, immunologically active, and stable antigen represents a major advance in the field and provides a powerful tool for future diagnostic and therapeutic studies.
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Acknowledgments |
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Footnotes |
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Received September 12, 2000.
Revised November 14, 2000.
Accepted November 29, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The hatched area between 40% and 100% depicts
the range of TBI activities in the presence of varying concentrations
of TSHR-289 (up to 1250 ng/tube; volume, 0.2 mL). Only the material
eluted from the anti-His mAb column (lower panel)
neutralized TSHR autoantibody binding and was, therefore,
immunologically active.
