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(Received for publication, April 4, 1997, and in revised form, April 25 1997)
From the Thyroid Molecular Biology Unit, Veterans Administration
Medical Center and the University of California,
San Francisco, California 94121
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.
Volume 272, Number 30,
Issue of July 25, 1997
pp. 18959-18965
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
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