Engineering a potential antagonist of human thyrotropin and thyroid-stimulating antibody.

Abstract

Thyrotropin (TSH) and the gonadotropins (FSH, LH, hCG) are a family of heterodimeric glycoprotein hormones composed of two noncovalently linked subunits, alpha and beta. We have recently converted the hTSH heterodimer to a biologically active single chain (hTSHbeta.CTPalpha) by fusing the common alpha-subunit to the C-terminal end of the hTSH beta-subunit in the presence of a approximately 30-amino acid peptide from hCGbeta (CTP) as a linker. The hTSHbeta.CTPalpha single chain was used to investigate the role of the N-linked oligosaccharides of alpha- and beta-subunits in the secretion and function of hTSH. Using overlapping PCR mutagenesis, two deglycosylated variants were prepared: one lacking both oligosaccharide chains on the alpha-subunit (hTSHbeta.CTPalpha(1+2)) and the other lacking the oligosaccharide chain on the beta-subunit (hTSHbeta.CTPalpha(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 hTSHbeta.CTPalpha 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 hTSHbeta.CTPalpha(1+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.

• Authors: F A Fares, F Levi, A Z Reznick, Z Kraiem
• Journal: The Journal of biological chemistry (J Biol Chem) Vol. 276 Issue 7 Pg. 4543-8 (Feb 16 2001) ISSN: 0021-9258 [Print] United States
• PMID: 11083869 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Chorionic Gonadotropin
• Chorionic Gonadotropin, beta Subunit, Human
• Immunoglobulins, Thyroid-Stimulating
• Recombinant Fusion Proteins
• hTSHbeta.CTPalpha protein, recombinant
• Triiodothyronine
• Thyrotropin
• Cyclic AMP

Topics

• Animals
• CHO Cells
• Cells, Cultured
• Chorionic Gonadotropin (metabolism, pharmacology)
• Chorionic Gonadotropin, beta Subunit, Human (genetics)
• Cricetinae
• Cyclic AMP (biosynthesis)
• Glycosylation
• Humans
• Immunoglobulins, Thyroid-Stimulating (immunology)
• Mutagenesis, Site-Directed
• Recombinant Fusion Proteins (metabolism, pharmacology)
• Thyroid Gland (drug effects, metabolism)
• Thyrotropin (antagonists & inhibitors, genetics, metabolism, pharmacology)
• Transfection
• Triiodothyronine (metabolism)