Objectives: The main goal of the present study is to investigate the role of N-linked oligosaccharides on the structure and function of human thyrotropin (hTSH). A second aim of the present study is to design new analogs of hTSH.
Methods: Overlapping PCR technique was used to convert hTSH heterodimer to a biologically active single chain by fusing the α subunit to the carboxyl terminal end of hTSHβ subunit in the absence (hTSHβα) or presence of a ∼30 aminoacid peptide from hCGβ (CTP) as linker (hTSHβCTPα). hTSH mutants lacking the sequence site of N-linked oligosaccharides were prepared using site-directed mutagenesis. hTSH variants were expressed in CHO cells. The TSH receptor binding activities of the variants were determined by radioligand receptor assay using CHO cells stably transfected with hTSH receptor. In vitro bioactivity was tested using cultured human thyroid follicle cells and in vivo longevity and bioactivity were tested in mice animal model.
Results: The single-peptide variants of hTSH were biologic active in vitro and in vivo with a longer half-life. Variants lacking the N-linked oligosaccharides were expressed and secreted from CHO cells. Interestingly, the deglycoselated variants were significantly less potent than TSH wild type. Moreover, the deglycoselated variants blocked cAMP formation and T3 secretion stimulated by hTSH or by hTSI in the receptor level. The variants were found to bind the hTSH receptor with high affinity. In addition, deglycoselated hTSH variants had a partial activity in vivo and significantly inhibited TSH bioactivity
Conclusions: Human TSH single peptides are biologically active. Deglycosylated variants inhibit the activity of hTSH and hTSI. These variants may offer novel therapeutic strategies in the treatment of Thyroid diseases.