TSH is a heterodimeric glycoprotein hormone synthesized in the pituitary and composed of a specific β-subunit and a common α-subunit shared with FSH, LH, and human chorionic gonadotropin. The heterodimer was previously converted into a biologically active single chain protein by genetic fusion of the genes coding to both subunits in the presence of the carboxy-terminal sequence of human (h) chorionic gonadotropin-β subunit as a linker [hTSHβ-carboxyl-terminal peptide (CTP)-α]. N-linked carbohydrate-free single-chain TSH variants were constructed by site-directed mutagenesis and overlapping PCR: one devoid of both N-linked oligosaccharide chains on the α-subunit (hTSHβ-CTP-αdeg) and the other lacking also the oligosaccharides on the β-subunit (hTSHβdeg-CTP- αdeg). These variants were expressed in Chinese hamster ovary cells and secreted into the culture media. We have previously reported that the variants block the activities of hTSH and thyroid-stimulating immunoglobulins in cultured human thyroid follicles. In the present study, binding affinity of hTSH variants to hTSH receptor and the localization of the antagonistic effect were examined. Moreover, the effect of these variants on TSH activity was tested in vivo. The results of the present study indicate that the hTSH variants bind to the hTSH receptor with high affinity. Experiments using forskolin also indicated that the N-linked carbohydrate-free TSH single-chain variants inhibit TSH activity at the receptor-binding site and not at a postreceptor level. Moreover, the variants significantly inhibited (about 50%) TSH activity with respect to thyroid hormone secretion in vivo in mice. These variants may offer a novel therapeutic strategy in treating hyperthyroidism.
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