טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentBar-Shalom Rinat
SubjectBiological Effects of TSH Variants Designed by Site-directed
Mutagenesis and Gene Transfer
DepartmentDepartment of Medicine
Supervisors Professor Zaki Kraiem
Assistant Professor Fuad Fares


Abstract

Thyrotropin (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 hCG. The heterodimer was previously converted in our laboratory into a biologically active single chain protein (hTSHβ-CTP-α) by genetic fusion of the genes coding to both subunits. The C-terminal end of the β-subunit which contained  a linker peptide (CTP) from hCGβ was fused to the N-terminal end of the α subunit. Deglycosylated single-chain TSH variants were constructed: one devoid of both N-linked oligosachride chains on the α-subunit (hTSHβ-CTP-α1+2), and the other also lacking the oligosacharides on the β-subunit (hTSHβCTPα(deg)). These variants were expressed in CHO cells and secreted into the culture media, and found to block the activities of hTSH and of thyroid-stimulating immunoglobolins (TSI) in vitro. The variants also partially blocked the activity of bovine TSH in vivo with respect to T4 secretion in mice. Receptor assay studies with forskolin using CHO cells expressing the TSH receptor and experiments using forskolin, revealed that the deglycosylated TSH single-chain variants inhibit TSH activity at the receptor level.  These variants seem therefore to hold the potential of serving as antagonists to TSH in vivo, and this may offer a novel therapeutic strategy for Graves' disease and other cases of hyperthyroidism.