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Cloning and Characterization of the Novel Thyroid and Eye Muscle Shared Protein G2s: Autoantibodies against G2s Are Closely Associated with Ophthalmopathy in Patients with Graves’ Hyperthyroidism

Department of Medicine (K.G., J.R.W., S.K.), Allegheny University of the Health Sciences, Pittsburgh, Pennsylvania 16212; Department of Medicine, Dalhousie University (A.L., M.Y.), Halifax, Canada B3H 2Y9; Endocrine Research Laboratory, Hospital de Sant Pau, Autonomous University of Barcelona (S.W.), Barcelona 08625, Spain; Department of Veterans Affairs Medical Center and Department of Biochemistry and Biophysics (B.A.C.A.), University of California, San Francisco, California; and Institute of Endocrinology, Second University of Naples (A.D.B., A.Be., A.A.S.), Naples 80131, Italy

Address all correspondence and requests for reprints to: Dr. J. R. Wall, Dalhousie University, 1278 Tower Road, Halifax, Nova Scotia, Canada B3H 2Y9. E-mail: jack.wall{at}dal.ca

	Abstract

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Serum autoantibodies against eye muscle antigens are closely linked with thyroid-associated ophthalmopathy (TAO), although their significance is unclear. The two antigens that are most often recognized are eye muscle membrane proteins with molecular masses of 55 and 64 kDa, as determined from immunoblotting with crude human or porcine eye muscle membranes. We cloned a fragment of the 55-kDa protein by screening an eye muscle expression library with affinity-purified anti-55 kDa protein antibody prepared from a TAO patient’s serum. A complementary DNA (cDNA) encoding a novel protein, which we have called G2s, was sequenced on both strands, and its size was 411 bp. The open reading frame of G2s corresponded to a 121-amino acid peptide with a size of 1.4 kb. Using the rapid amplification of 5'-cDNA ends technique we were able to clone an additional 0.3 kb of the protein. G2s did not share significant homologies with any other entered protein in computer databases and had one putative transmembrane domain. Using the 1.4 kb cDNA as probe in Northern blotting of a panel of messenger ribonucleic acids prepared from human tissues, the parent protein was shown to correspond to a large molecule of about 5.8 kb with a calculated molecular mass of approximately 220 kDa, consistent with earlier immunoblot studies performed in the absence of reducing agents. G2s was strongly expressed in eye muscle, thyroid, and other skeletal muscle and to a lesser extent in pancreas, liver, lung, and heart muscle, but not in kidney or orbital fibroblasts. We tested sera from patients with Graves’ hyperthyroidism with and without ophthalmopathy and from control patients and subjects for antibodies against a G2s fusion protein by immunoblotting and enzyme-linked immunosorbent assay. In immunoblotting, antibodies reactive with G2s were identified in 70% of patients with TAO of less than 3 yr duration, 53% with TAO of more than 3 yr duration, 36% with Graves’ hyperthyroidism without evident ophthalmopathy, 17% with Hashimoto’s thyroiditis, 3% with type 1 diabetes, 23% with nonimmunological thyroid disorders, and 16% of normal subjects. The prevalences, compared to normal values, were significant for the two groups of patients with TAO, but not for the other groups. Tests were positive in 54% of patients with active TAO, 33% with chronic ophthalmopathy, 36% with Graves’ hyperthyroidism, 54% with Hashimoto’s thyroiditis, 23% with type 1 diabetes, and in 11% of normal subjects using enzyme-linked immunosorbent assay. The antibodies predicted the development of the ocular myopathy subtype of TAO in six of seven patients and the congestive ophthalmopathy subtype in seven of eight patients, respectively, with Graves’ hyperthyroidism studied prospectively during and after antithyroid drug therapy. Antibodies reactive with G2s may be early markers of ophthalmopathy in patients with Graves’ hyperthyroidism. Because G2s is expressed in both thyroid and eye muscle, immunoreactivity against a shared epitope in the two tissues may explain the well known link between thyroid autoimmunity and ophthalmopathy.

	Introduction

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ALTHOUGH THYROID-ASSOCIATED ophthalmopathy (TAO) is generally considered to be an autoimmune disorder of the eye muscle and the surrounding orbital connective tissue and fat (1, 2, 3), the identity and nature of the principal target antigens and the significance of the corresponding serum autoantibodies are still unclear. Ophthalmopathy is closely linked with thyroid autoimmunity, and the current dogma is that this association is best explained by reactivity against a thyroid and orbital tissue shared antigen(s) (4, 5). One such candidate is the TSH receptor, which is expressed in the orbital preadipocyte (6, 7, 8). We have postulated that a shared eye muscle and thyroid antigen may also play a role in the eye muscle component of TAO (1, 4, 9) that occurs in about 30% of patients with ophthalmopathy. Serum antibodies against eye muscle antigens of 55–95 kDa are detected in approximately 70% of patients with active ophthalmopathy and eye muscle dysfunction by Western blotting (10, 11, 12), and purified antigens are recognized by a similar proportion of patients using enzyme-linked immunosorbent assay (ELISA) (13, 14). Recently, we reported that eye muscle membrane antigens of 63–67 kDa comprise three proteins: namely, the flavoprotein (Fp) subunit of the mitochondrial enzyme succinate dehydrogenase, the so-called 64-kDa protein, a nontissue-specific membrane protein called 1D, and the calcium-binding protein calsequestrin, which is localized in the sarcoplasmic reticulum of the skeletal muscle fiber. These proteins have small differences in molecular mass and band density on immunoblotting (13). Antibodies against Fp seem to be the best makers of ophthalmopathy in patients with Graves’ hyperthyroidism, and they are sensitive predictors of the development of eye muscle dysfunction in such patients after antithyroid drug treatment (14, 15).

A second candidate antigen that is associated with TAO is a 55-kDa protein of unknown structure and function. Wengrowicz et al. (16) and Chang et al. (17) found serum antibodies against a 55-kDa protein in Western blotting in over 50% of patients with TAO, confirming our own findings (18), and the antibodies were closely associated with extraocular muscle enlargement, as demonstrated by orbital computed tomography (17). Recently, we had the opportunity to study serum from an apparently normal woman with a family history of thyroiditis and colitis who developed Graves’ hyperthyroidism and ophthalmopathy 18 months later. In this patient, the serum concentration of antibodies against a 55-kDa protein decreased after she developed ophthalmopathy (19), suggesting that the antibody is an early marker of ophthalmopathy. In the present study we attempted to clone the 55-kDa protein by screening a human eye muscle expression library with affinity-purified anti-55-kDa protein antibodies prepared from a TAO patient’s serum. A 1.7-kb fragment of a positive clone, called G2s, was sequenced and partly characterized. Serum antibodies reactive with a G2s fusion protein were closely associated with both the ocular myopathy and congestive ophthalmopathy subtypes of TAO.

	Materials and Methods

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Clinical subjects

The studies concerned patients with TAO: 1) 10 men and 28 women, aged 27–61 yr (mean age, 48 yr), with active ophthalmopathy of less than 1-yr duration, of whom 13 had predominant eye muscle involvement; and 2) 5 men and 12 women, aged 30–75 yr (mean age, 51 yr), with inactive chronic ophthalmopathy of more than 3-yr duration, of whom 6 had permanent eye muscle dysfunction. The eye changes were classified according to recommendations of a committee of the International Thyroid Associations (20). In addition, patients with TAO were classified according to whether they had 1) congestive ophthalmopathy, characterized by mainly inflammatory changes including, chemosis, conjunctival injection, epiphora and periorbital swelling and no or only minor eye muscle swelling on orbital imaging; or 2) ocular myopathy, manifest as double vision, reduced eye muscle function, and marked eye muscle swelling on orbital imaging.

These studies also concerned patients with Graves’ hyperthyroidism without evident ophthalmopathy: 6 men and 27 women, aged 25–75 yr (mean age, 50 yr), including 19 patients who were studied prospectively for up to 18 months, 15 of whom developed predominant ocular myopathy (8 patients) or mainly congestive ophthalmopathy (7 patients). The diagnosis was made from the presence of hyperthyroidism, diffuse thyroid enlargement, and detectable serum antibodies against the TSH receptor. These patients did not undergo orbital computed tomography scan, although ultrasonography was performed at baseline and after each 6 months of follow-up on all patients studied prospectively.

We also studied patients with Hashimoto’s thyroiditis without evident ophthalmopathy: 5 men and 25 women, aged 25–69 yr (mean age, 44 yr), in whom the diagnosis was made from the usual clinical signs, including a firm nodular goiter and significant (>1:400) titers of antithyroid peroxidase antibodies. Six of the patients were hypothyroid, and 25 were euthyroid.

Patients with type 1 diabetes mellitus were studied: 18 men and 12 women, aged 14–68 yr (mean age, 29 yr), in whom the diagnosis was made from the usual clinical and biochemical criteria, including abnormal fasting blood glucose level and detectable antibodies against GAD65 in the ELISA and against islet cell antigen in immunofluorescence. None of the patients had evidence of thyroid autoimmunity or ophthalmopathy.

We also studied patients, 8 men and 22 women, aged 22–75 yr (mean age, 54 yr), with nonimmunological thyroid disorders, namely, multinodular goiter (14 patients), single adenoma (10 patients), or thyroid cancer (6 patients).

The normal subjects included 12 men and 19 women, aged 22–54 yr (mean age, 40 yr), with no personal or family history of thyroid or other autoimmunity, ophthalmopathy, eyelid lag, or goiter.

The study was approved by the human ethics committee of Allegheny General Hospital, and written informed consent was obtained from all subjects.

Preparation of eye muscle membranes

Fresh-frozen pig eye muscle was thawed in phosphate-buffered saline (PBS) containing a cocktail of protease inhibitors [aprotinin, phenylmethylsulfonylfluoride, benzamidine, epsilon-aminocapronic acid, orthopenanthroline (PBS/PI; Boehringer Mannheim], rinsed of blood, separated from adipose tissue, and homogenized. The homogenate was centrifuged at 600 x g for 15 min to remove cell debris and nuclei, and the supernatant from this step was centrifuged at 100,000 x g for 60 min. The resulting pellet was resuspended in PBS/PI and recentrifuged at 100,000 x g for 60 min. Finally, the pellet was resuspended in PBS/PI, and the protein concentration was determined. This fraction was termed pig eye muscle membrane (PEMM).

SDS-PAGE and Western blotting

SDS-PAGE was performed according to the standard method of Laemmli (21). Briefly, two kinds of gels were used: an 8% separating gel/4% stacking gel in a minigel apparatus (Bio-Rad Laboratories, Inc., Richmond, CA) for eye muscle membranes and a 16.5% separating gel/10% spacer gel/4% stacking gel for G2s fusion protein. In each experiment, molecular mass standards (Bio-Rad Laboratories, Inc.) were included. The gels were run at 110 V for 90 min in running buffer (25 mmol/L Tris, 192 mmol/L glycine, and 0.2% SDS for 8% gel) or at 150 V for 120 min in 0.1 mol/L Tris, 0.1 mol/L Tricine, and 0.1 mol/L SDS, as cathode buffer and in 0.2 mol/L Tris, pH 8.9, as anode buffer, respectively, then transferred to Immobilon-P polyvinylidene difluoride (PVDF; Millipore Corp., Bedford, MA) paper at 100 V for 60 min in transfer buffer (25 mmol/L Tris, 192 mmol/L glycine, and 20% methanol, pH 8.3). The strips containing purified protein or molecular mass standards were stained with Coomassie blue. Filter strips containing protein were blocked with 10% polyvinylpyrolidone in Tris-buffered saline (50 mmol/L Tris and 150 mmol/L NaCl, pH 7.4; TBS), then incubated with diluted patient or control (normal) serum, or affinity-purified anti-55 kDa PEMM protein antibody (see below) at room temperature for 2 h. Each strip was washed with Tween-TBS (TBS-0.05% Tween-20) and incubated with alkaline phosphatase-conjugated IgG diluted with TBS at 37 C for 2 h. Strips were again washed with Tween-TBS and developed with 5-bromo-4-chloro-3-indolylphosphate-toluidine and p-nitro blue tetrazolium chloride (Bio-Rad Laboratories, Inc.) for 20 s. Finally, strips were soaked in distilled water for 5 min.

Preparation of affinity-purified antibody (APAb) against the 55-kDa PEMM protein (APAb-55kDa)

PEMM was subjected to SDS-PAGE using a minigel apparatus. After electrophoresis, gel proteins were transferred to a PVDF membrane as described above. The membrane was stained with Ponseau S for 10 min at room temperature, and the molecular mass standards and a vertical strip, respectively, were cut from each side of the membrane. Molecular mass standards were stained with Coomassie and subjected to Western blotting as a guide to identify the 55-kDa protein on the second membrane. The 55-kDa protein was cut from this membrane, destained with TBS, blocked with 10% PVP in TBS for 2 h at 37 C and then incubated at 4 C for 16 h with a TAO patient serum strongly reactive against a 55-kDa PEMM in Western blotting. After this strip was soaked in 0.1 mol/L KCl for 2 min, antibodies were eluted with 1 mL 0.2 mol/L glycine-HCl (pH 3.0) containing 1 mg/mL BSA and neutralized with 0.2 mL 1 mol/L Tris, followed by dialysis against TBS. Reactivity of APAb-55 kDa with eye muscle membrane is shown in Fig. 1.

View larger version (103K): [in this window] [in a new window]   Figure 1. Western blot of porcine eye muscle membrane with an affinity-purified Ab against the 55-kDa PEMM protein (APAb-55 kDa). Lane 1, TBS; lane 2, APAb-55 kDa; lane 3, anti-55kDa-protein positive serum from patient with TAO. The arrow shows the reactive band corresponding to a molecular mass of 55 kDa. Serum dilution was 1:10. MW, Molecular mass standard.

Immunoscreening of a 11 human eye muscle complementary DNA (cDNA) library with APAb-55kDa

Five hundred thousand plaques from a gt11 human eye muscle cDNA library (donated by Dr. Marian Ludgate, Cardiff, UK) were transferred to 10 mmol/L IPTG-saturated nitrocellulose membranes. After the membranes were washed with Tween-TBS and blocked with 10% PVP in TBS for 1 h at 37 C, they were screened with APAb-55kDa, diluted 1:100. Membranes were then incubated with secondary antibody, and the reactions were developed as described for Western blotting. After three rounds of screening, positive clones were isolated.

Subcloning, sequencing, and production of recombinant protein of positive clones from a human cDNA eye muscle library

A 1.4-kb insert of a positive clone, called G2s, was digested with EcoRI and ligated into pBluescript (Stratagene, La Jolla, CA). The nucleotide sequence of the insert was determined on both strands using the standard Sanger’s dideoxy chain termination method (22). Based on sequencing data, a pair of specific primers to this positive clone was designed. The insert of the positive clone was amplified with these primers and subcloned into a pFlag-ATS Escherichia coli expression vector (Sigma-Aldrich) in XOL-II blue. After induction of recombinant G2s fusion protein with 100 mmol/L isopropylthiogalactoside, cells in which recombinant G2s fusion protein was induced were destroyed by osmotic shock. The supernatant of the cell lysate was applied to a column containing M2, an eight-amino acids peptide marker, and the G2s fusion protein was isolated in a pure form for use as an antigen in Western blotting and ELISA.

Ribonucleic acid (RNA) isolation and Northern blotting

Total RNA was prepared by the acid guanidinium thiocyanate-phenol-chloroform method (23) from human neck skeletal muscle, eye muscle, thyroid, and orbital fibroblasts. Briefly, 100 mg tissues were powdered in liquid nitrogen and homogenized in 1 mL denatured solution [4 mol/L guanidinium thiocyanate, 25 mmol/L sodium citrate (pH 7), 0.5% sarcosyl, and 0.1 mol/L 2-mercaptoethanol]. Subsequently, 0.1 mL 2 mol/L sodium acetate, pH 4, and 1 mL water-saturated phenol-chloroform (1:1) were carefully mixed with the homogenates, and the solutions were cooled for 15 min. After centrifugation at 1000 x g for 20 min, the supernatants were extracted with 1 mL water-saturated phenol-chloroform. RNA pellets were rinsed with 80% ethanol, dried, and resuspended in deionized formamide for Northern blotting or water treated with diethylpyrocarbonate for preparation of messenger RNA from total RNA with the Oligotex-dT kit (QIAGEN, Chatsworth, CA). Two milligrams of messenger RNA or 0.5–5 mg total RNA were applied to each well, electrophoresed in 0.9% agarose gel, then transferred to nylon membrane. Human calsequestrin cDNA was used as control cDNA. Northern blot and hybridization were performed as previously described (24). Complete, even, transfer of RNA from the agarose gel to the blots was verified by monitoring of the ethidium bromide staining patterns. Finally, filters were exposed to Kodak XAR-5 film (Eastman Kodak Co., Rochester, NY).

ELISA

A standard ELISA, as described in previous publications from this laboratory (25, 26), was used to measure anti-G2s antibodies. In preliminary checkerboard studies, the assay was optimized with respect to antigen concentration (0.125 µg/mL) and serum dilution (1:50). A positive test was defined as an optical density (OD) more than the mean + 2 SD for a panel of 10 age- and sex-matched normal subjects. The interassay variation was 8%, and the intraassay variation was 7%.

	Results

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An eye muscle expression library was screened with APAb-55 kDa. We obtained a positive clone of 411 bp, which we called G2s. The open reading frame of G2s corresponded to a 121-amino acid peptide. The DNA sequence of the 1.4-kb fragment of G2s is shown in Fig. 2A. Extensive searches of protein databases failed to reveal significant homologies with any other entered protein. Despite using a variety of techniques, including rapid amplification of 5'- and 3'-cDNA ends, PCR-based methods, and rescreening of the eye muscle expression library with the original G2s cDNA, we were only able to obtain and sequence another 0.3 kb of the G2s gene. The reasons for this are unclear, although the occurrence of stop codons in amplified G2s products raised the possibility that G2s is an abnormal variant of some other protein. From database analysis G2s is predicted to have one transmembrane domain (Fig. 2B); its predicted orientation across the plasma membrane is shown in Fig. 2C.

View larger version (18K): [in this window] [in a new window]   Figure 2. Hydrophobicity and predicted topology of the 1.4 kb G2s fragment. A, Predicted amino acid sequences of G2s. B, Hydropathy plot. A putative transmembrane domain (amino acids 24–45) is indicated by the horizontal bar. C, Schematic representation of G2s. N and C, NH2- and COOH-termini, respectively.

View larger version (41K): [in this window] [in a new window]   Figure 3. Tissue distribution of G2s. Northern blot of human tissues probed with a 400-bp fragment of G2s. A: Lane 1, Pancreas; lane 2, kidney; lane 3, skeletal muscle; lane 4, liver; lane 5, lung; lane 6, placenta; lane 7, brain; lane 8, heart (each 0.5 mg polyadenylated RNA/lane). B: Lane 1, 5 mg/lane of eye muscle RNA; lanes 2–5, 0.5, 1.0, 2.5, and 5.0 mg/lane, respectively, of skeletal muscle RNA. C: Lane 1, Fibroblasts from retroorbital tissues; lane 2, eye muscle; lane 3, thyroid (each 0.5 mg polyadenylated RNA/lane). The arrow indicates a 5.87-kb transcript.

View larger version (73K): [in this window] [in a new window]   Figure 4. SDS-PAGE of G2s fusion protein and Western blotting with sera from patients with TAO, Graves’ disease, Hashimoto’s thyroiditis, nonimmunologic thyroid disorders, and type 1 diabetes and from normal subjects. A: Lane 1, G2s fusion protein stained with Coomassie blue; lane 2, TBS; lane 3, anti-M2 monoclonal antibody (M2 is an eight-amino acid peptide used as a marker in this protein expression system); lanes 4–11, TAO; lanes 12–17, Graves’ hyperthyroidism; lanes 18–23, normal subjects. B: Lanes 1 and 2, As in A; lanes 3–9, Hashimoto’s thyroiditis; lanes 10–16, nonimmunologic thyroid disorders; lanes 17–23, type 1 diabetes. Serum dilution was 1/40. MW, Molecular mass standard.

	Discussion

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The significance of anti-G2s antibodies in patients with Hashimoto’s thyroiditis who do not have ophthalmopathy is not known, but may reflect the propensity for the production of multiple autoantibodies in this disease. Antibodies against a G2s fusion protein predicted the development of both congestive ophthalmopathy and ocular myopathy in patients with Graves’ hyperthyroidism studied prospectively after antithyroid drug therapy, whereas anti-Fp antibodies were detected only in those patients who developed the ocular myopathy subtype of TAO (15). Several other autoantibodies against eye muscle antigens have been identified in patients with TAO, including those targeting the Fp subunit of mitochondrial succinate dehydrogenase, the so-called 64-kDa protein, other mitochondrial enzymes and their flavine adenine dinucleotide cofactor (28), 1D (29, 30), and calsequestrin (31). G2s is potentially an important and interesting antigen in TAO because it is strongly expressed in both thyroid and eye muscle, but not in orbital connective tissue, and thus is a good candidate for an eye muscle and thyroid shared antigen, which we have postulated to be the mechanism for the link between ophthalmopathy and thyroid autoimmunity. The fact that G2s is also expressed in some other tissues that are not the sites of autoimmune attack in Graves’ disease is not explained, but may reflect the unique characteristics of the eye muscle fiber. Others have postulated that the TSH receptor is the orbital tissue and thyroid shared antigen in TAO (6, 7, 8), although this would not explain the immune reaction against eye muscle. The relationship between G2s and the other eye muscle antigens that we have identified is unknown. Although antibodies against the flavine adenine dinucleotide cofactor, which is used by several mitochondrial enzymes, and G2s are usually detected in the same sera (De Bellis, A., J. R. Wall et al., unpublished data), this may reflect a common pathogenetic mechanism. On the other hand, all of these antibodies may be secondary to autoimmune attack against another, as yet unidentified, cell membrane antigen. Our working hypothesis for TAO is as follows; recognition of a TSH receptor protein in the orbital preadipocytes by antibodies may be the initial event leading to homing of lymphocytes into the orbital tissue (32). In the course of thyroid inflammation, antibodies and T cells reactive against G2s expressed in thyroid membrane cross-react with the protein in the eye muscle fiber, leading to eye muscle damage and dysfunction. Only those patients with anti-G2s antibodies develop ocular myopathy. Antibodies against Fp, which are produced in the context of eye muscle fiber damage, are sensitive markers of immune-mediated fiber necrosis in patients with Graves’ hyperthyroidism (15). Antibodies against type XIII collagen have recently been identified as a new marker for the congestive ophthalmopathy subtype (33). We propose that orbital connective tissue inflammation and ocular myopathy are separate components of TAO, which may occur together or separately. The relationships between the various antibodies and the clinical findings in TAO are being assessed in an experimental model for TAO produced by genetic immunization of susceptible mice with cDNAs for the candidate proteins. In addition, we are examining the relationship between the various orbital tissue antibodies in patients with Graves’ hyperthyroidism treated with radioactive iodine or antithyroid drugs in a prospective clinical study. These studies should give insight into the nature and significance of the various orbital antibodies. Finally, we are attempting to clone full-length G2s as the basis for determining its complete structure, its localization in the thyroid and eye muscle cell membranes, and its antibody- and, possibly, T cell-reactive sites.

Received June 30, 1999.

Revised November 22, 1999.

Accepted January 6, 2000.

	References

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1. Kiljanski J, Nebes V, Wall JR. 1995 The ocular muscle the target of the immune system in endocrine ophthalmopathy. Proc Int Arch Allergy Immunol. 106:204–212.
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