Searchable abstracts of presentations at key conferences in endocrinology
Endocrine Abstracts (2008) 16 P689

ECE2008 Poster Presentations Thyroid (146 abstracts)

Structure-function studies of thyrotropin using site-directed mutagenesis and gene transfer: development of new agonists and antagonists

Fuad Fares 1 , Naiel Azzam 1 , Rinat Bar-Shalom 1 & Zaki Kraiem 2

1University of Haifa, Haifa, Israel; 2Technion-Israel Institute of Technology, Haifa, Israel.

Thyrotropin (TSH) and the gonadotropins (FSH, LH, hCG) are a family of heterodimeric glycoprotein hormones composed of two noncovalently linked subunits, α and β. The hTSH heterodimer was converted to a biologically active single-peptide chain (hTSHβCTPα), by fusing the common α subunit to the carboxyl terminal end of hTSHβ subunits in the presence of a ~30 aminoacid peptide from hCGβ (CTP) as a linker. Ligation of the CTP to the carboxyl-end of hFSH resulted in increasing the biological activity and longivity in vivo. In addition, the single chain constructs are more active and have longer half-lives compared to dimeric constructs.

The hTSHβCTPα, was used to investigate the role of the N-linked oligosaccharides of α and β subunits on secretion and function of hTSH. Two deglycosylated variants were prepared: one lacks both oligosaccharide chains on α subunit (hTSHβCTPα1+2), and the other lacks also the oligosaccharide chain on β subunit of the single chain (hTSHβCTPα(deg)). The single-peptide chain variants were expressed in CHO cells and they are secreted into the medium. Absence of the N-linked oligosaccharides on α or β subunits does not affect the secretion of the variants. These results may indicate that the signal for the secretion exists in the single peptide chain and is independent of the oligosaccharides. hTSH variants lack of the oligosaccharide chains is less potent than hTSHβCTPα on cAMP accumulation and T3 secretion in human cultured thyroid follicles. Moreover, both deglycosylated variants compete with normal hTSH and human thyroid stimulating immunoglobulin (hTSI) in a dose dependent manner and decreased significantly the hTSH and hTSI-stimulated levels of cAMP and T3 secretion. Thus, this variant, behaves as potential antagonist, who may offer a novel therapeutic strategy in the treatment of Grave’s disease, the most common form of hyperthyroidism.

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