OR10-2 A Modifying Autoantigen in Graves' Disease

In patients with Graves’ disease (GD), previous studies have associated SNPs in linkage disequilibrium with the TSHR gene locus with changes in expression of two TSH receptor (TSHR) variants suggesting that receptor variants may have an important role in disease etiology. Bioinformatic analyses predicts the existence of several human TSHR isoforms from alternative splicing and the most abundant is the previously described splice variant, TSHR-v1.3. In-silico modeling of TSHR v1.3 confirmed the potential binding of TSH and TSHR autoantibody to the v1.3 protein indicating its potential as an autoantigen and pseudoreceptor. To further characterize the bioactivity of v1.3 we engineered the protein by cloning the entire ORF into a mammalian expression vector. The purified protein was obtained from cell lysate by Ni-affinity chromatography. Immunoprecipitation demonstrated that both TSHR stimulating monoclonal antibody (MS1) and human recombinant TSH were able to bind TSHR v1.3 recombinant protein as predicted by the in-silico studies. TSHR-v1.3 was also able to inhibit the effect of TSH and MS-1 on cAMP generation in a dose-dependent manner. These observations confirmed the ability of purified TSHR-v1.3 protein to interact with TSH and autoantibody and modulate their activity. Using purified IgG fractions from sera of 13 patients with GD with known TSHR antibodies we also looked for TSHR-v1.3 autoantibodies. We used a peptide based ELISA against two different epitopes of the splice variant. 11 out 13 (84.6%) of the GD samples were positive for a carboxy terminal peptide (COOH) and 10 out of 13 (76.9%) were positive to a junction region peptide (JNX). A dose-dependent bocking of signal was observed in the GD samples using the specific peptide. To detemine if the truncated isoform of the receptor could serve as a potential source of antigen and modulate GD we used double transfected cells expressing both GFP tagged TSHR-v1.3 and full-length TSHR. We then induced cell stress and apoptosis using a TSHR-mAb directed to the cleavage region (Tab16) and observed a dose dependent increase in mitochondrial cell stress and increased apoptosis (viability <46%) compared to control isotype specific antibody treated or untreated cells (74/71% viability).The culture supernatant contained released V1.3 tagged GFP protein suggesting that release of a new TSHR receptor antigen by this mechanism could play an important role in modulation of the disease process. These data show that a TSHR isoform that is expressed by alternative splicing can have a duel role by acting as a potential competitor for TSHR autoantibody and TSH itself and also as a likely source of autoantigen. Therefore, expression of such altered receptor protein within tissue depots has the potential to modulate thyroid autoimmunity by serving as an alternative intracellular antigen after apoptosis of thyroid cells.