Glycosylated ectodomain of the human thyrotropin receptor induces antibodies capable of reacting with multiple blocking antibody epitopes

Recently, we showed that the glycosylated ectodomain of the human thyrotrop in receptor (hET-gp) reacts with autoantibodies from autoimmune thyroid dis ease (AITD) patients' sera. To better understand the effects of glycosylati on of thyrotropin receptor (TSHR) in antibody induction, we immunized rabbi ts with hET-gp protein. The rabbits developed relatively high titers of ant ibodies with highly potent TSH binding inhibitory immunoglobulin (TBII) and thyroid stimulatory blocking antibody (TSBAb) activities. Both the hET-gp and a nonglycosylated ectodomain of the human TSHR (hETSHR) protein signifi cantly reversed the TBII as,well as TSBAb activity. Based on the ability of synthetic peptides to significantly reverse the functional activity of the se rabbit antisera, me identified three discrete regions of the TSHR, repre sented by amino acids 202-221, 292-311 and 367-386, as TBII epitopes and fo ur regions represented by amino acids 352-371, 367-386, 382-401 and 392-415 as TSBAb epitopes, These data demonstrate that rabbit antibodies that bind to amino acids 367-386 mediate their TSBAb activity by inhibiting the bind ing of TSH to TSHR; whereas, antibodies to regions 352-415, excluding aa 36 7-386, exert their TSBAb activity by affecting a step subsequent to TSH bin ding. Coincident with the elevation of TBII and TSBAb activity, serum total T-4 levels declined and thus suggested that the antibodies exerted functio nal effects on thyroid in vivo. Together, these data demonstrate that glyco sylated hET-gp protein is a more potent immunogen and it can induce a broad er antibody response directed against multiple TBII and TSBAb epitopes.