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J. Biol. Chem., Vol. 276, Issue 7, 4543-4548, February 16, 2001

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Engineering a Potential Antagonist of Human Thyrotropin and Thyroid-stimulating Antibody^*

Fuad A. Fares^§, Flonia Levi, Abraham Z. Reznick^¶, and Zaki Kraiem

From the Departments of  Biochemistry and Molecular Genetics and ^¶ Anatomy and Cell Biology and the  Endocrine Research Unit, Carmel Medical Center and the Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, 34362 Israel

Thyrotropin (TSH) and the gonadotropins (FSH, LH, hCG) are a family of heterodimeric glycoprotein hormones composed of two noncovalently linked subunits,  and . We have recently converted the hTSH heterodimer to a biologically active single chain (hTSH·CTP) by fusing the common -subunit to the C-terminal end of the hTSH -subunit in the presence of a ~30-amino acid peptide from hCG (CTP) as a linker. The hTSH·CTP single chain was used to investigate the role of the N-linked oligosaccharides of - and -subunits in the secretion and function of hTSH. Using overlapping PCR mutagenesis, two deglycosylated variants were prepared: one lacking both oligosaccharide chains on the -subunit (hTSH·CTP₁₊₂) and the other lacking the oligosaccharide chain on the -subunit (hTSH·CTP(deg)). The single chain variants were expressed in CHO cells and were secreted into the medium. hTSH variants lacking the oligosaccharide chains were less potent than hTSH·CTP wild-type with respect to cAMP formation and thyroid hormone secretion in cultured human thyroid follicles. Both deglycosylated variants competed with hTSH in a dose-dependent manner. The hTSH·CTP₁₊₂ variant blocked cAMP formation and thyroid hormone secretion stimulated by hTSH as well as by the antibody, thyroid-stimulating immunoglobulins, responsible for the most common cause of hyperthyroidism, Graves disease. Thus, this variant behaves as a potential antagonist, offering a novel therapeutic strategy in the treatment of thyrotoxicosis caused by Graves' disease and TSH-secreting pituitary adenoma.

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