The extracellular domain of the glycosylated human thyrotropin receptor (ET-gp) contains epitopes that can adsorb pathogenic antibodies from sera of patients with Graves' disease (GD). In an attempt to define the regions within the ETSHR with which autoantibodies interact, we expressed extracellular domains of eight thyrotropin receptor/chorionic gonadotropin receptor (TSHR/LH-CGR) chimeric proteins in insect cells. The levels of expression were high and chimeric proteins were glycosylated. Chimeric proteins designated as EMc2+4 and EMc2+3+4, in which amino acids (aa) 90-165 and 261-370, and aa 90-370, respectively, of TSHR were replaced with corresponding aa of LH-CGR, partially reversed the thyrotropin binding inhibitory immunoglobulin (TBII) activity of experimental anti-TSHR antisera (anti-ET-gp). The other six chimeras almost completely reversed the TBn activity of these anti-ET-GP antisera. Next, we tested the ability of these chimeric proteins to reverse the TBII activity of GD patients' sera. Similar to our earlier study, ET-gp protein reversed the TBII activity of all eight GD patients' sera tested. Chimera EMc2, in which aa 90-165 of TSHR has been replaced with corresponding aa of LH-CGR, and EMc2+4 partially reversed the TBII activity of only three of the eight GD patients' sera. However, the other six chimeric proteins failed to neutralize the TBII activity of any of GD patients' sera. These data showed the following: (1) There is considerable heterogeneity amongst autoantibodies in GD patients' sera, (2) The TBII activity of some, but not others, is dependent on aa 90-165 and 261-370, and (3) Most Graves' sera, with TBII activity, failed to react with chimeric proteins in which either N-terminal or C-terminal regions of the extra cellular domain of the TSHR were replaced with corresponding regions of LH- CGR. These results suggest that the TBII activity of GD patients' sera is dependent on conformational epitopes and replacement of certain regions of TSHR with homologous regions of LH-CGR results in sufficient alteration in the conformation of the protein leading to loss of reactivity.
ASJC Scopus subject areas
- Endocrinology, Diabetes and Metabolism