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

Abstract

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...

Extraocular muscle antibodies and the occurrence of ophthalmopathy in Graves’ disease

“…Recently, it has been shown that the appearance of FpAb in Graves' patients during methimazole therapy, accurately predicts the development of ophthalmopathy with EM impairment, while the presence of G2sAb predicts the onset of ophthalmopathy with both EM involvement and retroorbital connective inflammation (Gunji et al ., 1999(Gunji et al ., , 2000Kaspar et al ., 2002). Finally, high extraocular muscle antibody levels have been also found in patients with euthyroid Graves' ophthalmopathy (Perros & Kendall-Taylor, 1992;Amato et al ., 2000).…”

“…One of the main antigens associated with eye muscle damage is a new thyroid and eye muscle shared protein of unknown function, called G2s. It is a cell-membrane protein, with a molecular mass of 55 kDa (Gunji et al ., 2000). Moreover, another antigen has been recently identified; it is a flavoprotein (Fp) subunit of mitocondrial succinate Correspondence: Annamaria De Bellis, MD, Chair of Endocrinology, Second University of Naples, via Pansini N. 5, 80131 Naples, Italy.…”

“…Tel: +39 81 5666634; Fax: +39 81 5666628; E-mail: annamaria.debellis@unina2.it dehydrogenase, a 67 kDa protein expressed in extraocular and systemic skeletal muscle fibres, but not in the thyroid (Kubota et al ., 1998;Gunji et al ., 1999). Serum antibodies against G2s (G2sAb) and Fp (FpAb) have been frequently detected in Graves' patients with severe ophthalmopathy with prevalent clinical EM involvement (Gunji et al ., 1999(Gunji et al ., , 2000.…”

99Tcm-octreotide scintigraphy and serum eye muscle antibodies in evaluation of active thyroid-associated ophthalmopathy

“…29 The extraocular muscles and thyroid share antigens with clinical relevance in TAO, such as calsequestrin, G2s, 19 and UACA 21 antigens. In this study, we observed a close relationship between positive reactivity of serum calsequestrin antibodies (87.9%) and active TAO, which corroborates previous report.…”

“…18 G2s, the terminal 141 amino-acid fragment of the winged-helix transcription factor FOXP1, were found both in the extraocular muscles and thyroid, but not in orbital connective or adipose tissue. 19 Anti-G2s antibodies were also reported to be associated with active ophthalmopathy in the patients with Graves's hyperthyroidism, being detected in 54% of patients with active TAO compared to 33% of those with inactive disease. 20 UACA is an auto-antigen associated with panuveitis, and is expressed in eye muscle as well as human thyroid follicular cells.…”