Characterization of the secretable ectodomain of thyrotropin receptor produced by the recombinant baculovirus system.
Thyrotropin receptor (TSHR) is a member of the glycoprotein hormone receptor 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 mammalian cells, but most of them failed to secrete the overexpressed ectodomain. In the present study, we observed that about one-third of the ectodomain protein (sTSHR-gp), in which the signal peptide of TSHR was replaced by the baculovirus-encoded glycoprotein 67-signal peptide, was secreted into the 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 site. Carbohydrate studies using several glycosidases and lectins revealed that the secreted form of the ectodomain had biantennary carbohydrate, whereas the non-secreted form had high-mannose. Moreover, the secreted form of sTSHR-gp exhibited high-affinity ligand binding, whereas the non-secreted form did not show any significant ligand binding. Regarding the interactions of TSHR ectodomains with anti-TSHR antibodies, both the secreted and non-secreted forms of sTSHR-gp, almost completely neutralized the stimulatory and inhibitory anti-TSHR antibody activities. In conclusion, we succeeded in secreting the ectodomain of TSHR into culture medium, which was capable of binding to TSH and neutralizing anti-TSHR antibody activities.
Hattori
Y
Y
Department of Medicine and Clinical Science and Graduate School of Medicine, Kyoto University, 54 Shogoin-Kawaharacho, Sakyo-ku, Kyoto 606-8507, Japan.
Akamizu
T
T
Saijo
M
M
Kanamoto
N
N
Moriyama
K
K
Ito
N
N
Nakao
K
K
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D016367:Baculoviridae / Q000235:genetics* / Q000502:physiology; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004274:DNA, Recombinant; D006026:Glycoside Hydrolases / Q000037:antagonists & inhibitors / Q000378:metabolism; D006031:Glycosylation; D006111:Graves Disease / Q000276:immunology; D037102:Lectins / Q000378:metabolism; D009500:Neutralization Tests; D017434:Protein Structure, Tertiary; D011989:Receptors, Thyrotropin / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020539:Sequence Analysis, Protein; D018411:Spodoptera; D013972:Thyrotropin / Q000378:metabolism
D000906:Antibodies; D004274:DNA, Recombinant; D037102:Lectins; D011989:Receptors, Thyrotropin; D011994:Recombinant Proteins; D013972:Thyrotropin; D006026:Glycoside Hydrolases