Engineering a potential antagonist of human thyrotropin and thyroid-stimulating antibody.
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.
Fares
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Department of Biochemistry, Carmel Medical Center and the Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, 34362 Israel. fares@actcom.co.il
Levi
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Reznick
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Kraiem
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D000818:Animals; D016466:CHO Cells; D002478:Cells, Cultured; D006063:Chorionic Gonadotropin / Q000378:metabolism / Q000494:pharmacology*; D018997:Chorionic Gonadotropin, beta Subunit, Human / Q000235:genetics; D006224:Cricetinae; D000242:Cyclic AMP / Q000096:biosynthesis; D006031:Glycosylation; D006801:Humans; D018828:Immunoglobulins, Thyroid-Stimulating / Q000276:immunology*; D016297:Mutagenesis, Site-Directed; D011993:Recombinant Fusion Proteins / Q000378:metabolism / Q000494:pharmacology; D013961:Thyroid Gland / Q000187:drug effects* / Q000378:metabolism; D013972:Thyrotropin / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D014162:Transfection; D014284:Triiodothyronine / Q000378:metabolism
D006063:Chorionic Gonadotropin; D018997:Chorionic Gonadotropin, beta Subunit, Human; D018828:Immunoglobulins, Thyroid-Stimulating; D011993:Recombinant Fusion Proteins; C429013:hTSHbeta.CTPalpha protein, recombinant; D014284:Triiodothyronine; D013972:Thyrotropin; D000242:Cyclic AMP