Engineering the human thyrotropin receptor ectodomain from a non-secreted form to a secreted, highly immunoreactive glycoprotein that neutralizes autoantibodies in Graves' patients' sera.
Previous attempts to generate autoantibody-reactive, secreted thyrotropin receptor (TSHR) ectodomain in mammalian cells have failed because of retention within the cell of material with immature carbohydrate. We have overcome this difficulty by performing progressive carboxyl-terminal truncations of the human TSHR ectodomain (418 amino acid residues including signal peptide). Three ectodomain variants (TSHR-261, TSHR-289, and TSHR-309) were truncated at residues 261, 289, and 309, respectively. Unlike the full ectodomain, ectodomain variants were secreted with an efficiency inversely proportional to their size. Secreted ectodomain variants contained approximately 20 kDa of complex carbohydrate. TSHR-261 was chosen for further study because it was secreted very efficiently and neutralized autoantibodies in Graves' patients' sera. This ectodomain variant was partially purified using sequential lectin and nickel-chelate chromatography, permitting the first direct visualization and quantitation of the mammalian TSHR. Most important, very small (nanogram) quantities of this material neutralized 70-100% of TSHR autoantibody activity in all 18 Graves' sera studied. In summary, carboxyl-terminal truncation of the human TSHR ectodomain generates a secreted protein with complex carbohydrate that neutralizes autoantibodies in Graves' patients' sera. Antigenically active TSHR will be valuable for future studies on the diagnosis, pathogenesis, and immunotherapy of Graves' disease.
Chazenbalk
G D
GD
Thyroid Molecular Biology Unit, Veterans Administration Medical Center and the University of California, San Francisco, California 94121, USA.
Jaume
J C
JC
McLachlan
S M
SM
Rapoport
B
B
D000327:Adsorption; D000818:Animals; D001323:Autoantibodies / Q000276:immunology*; D016466:CHO Cells; D006224:Cricetinae; D003470:Culture Media; D006023:Glycoproteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D006111:Graves Disease / Q000276:immunology* / Q000503:physiopathology; D006801:Humans; D037102:Lectins / Q000378:metabolism; D008958:Models, Molecular; D009500:Neutralization Tests; D010446:Peptide Fragments / Q000276:immunology; D015202:Protein Engineering; D011989:Receptors, Thyrotropin / Q000235:genetics / Q000276:immunology*
D001323:Autoantibodies; D003470:Culture Media; D006023:Glycoproteins; D037102:Lectins; D010446:Peptide Fragments; D011989:Receptors, Thyrotropin