Characterization of the secretable ectodomain of thyrotropin receptor produced by the recombinant baculovirus system

Thyrotropin receptor (TSHR) is a member of the glycoprotein hormone recepto r family and an autoantigen of Graves' disease. Various attempts have been made to obtain a large amount of soluble ectodomain of TSHR in insect or ma mmalian cells, but most of them failed to secrete the overexpressed ectodom ain. In the present study, we observed that about one-third of the ectodoma in protein (sTSHR-gp), in which the signal peptide of TSHR was replaced by the baculovirus-encoded glycoprotein 67-signal peptide, was secreted into t he culture medium and the remainder stayed within cells in the recombinant baculovirus system. Microsequencing the N-terminal of the purified protein confirmed that the baculovirus signal peptide was cleaved at the expected s ite. Carbohydrate studies using several glycosidases and lectins revealed t hat the secreted form of the ectodomain had biantennary carbohydrate, where as the non-secreted form had high-mannose. Moreover, the secreted form of s TSHR-gp exhibited high-affinity ligand binding, whereas the non-secreted fo rm did not show any significant ligand binding. Regarding the interactions of TSHR ectodomains with anti-TSHR antibodies, both the secreted and non-se creted forms of sTSHR-gp, almost completely neutralized the stimulatory and inhibitory anti-TSHR antibody activities. In conclusion, we succeeded in s ecreting the ectodomain of TSHR into culture medium, which was capable of b inding to TSH and neutralizing anti-TSHR antibody activities. (C) 2001 Else vier Science Ireland Ltd. All rights reserved.