Engineering the human thyrotropin receptor ectodomain from a non-secreted form to a secreted, highly immunoreactive glycoprotein that neutralizes autoantibodies in Graves' patients' sera.

Abstract 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 ∼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.