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Designing new analogs; agonists and antagonists, for the glycoprotein hormones using site directed mutagenesis and gene transfer” Fuad Fares Department of Molecular Genetics, Carmel Medical Center and the Bruce Rappaport, Faculty of Medicine, Technion, Haifa, Israel. Email: fares@clalit.org.il
Thyrotropin (TSH) and the gonadotropins (FSH, LH, hCG) are a family of heterodimeric glycoprotein hormones composed of two noncovalently linked subunits, a and b. The hTSH heterodimer was converted to a biologically active single-peptide chain, by fusing the common a subunit to the carboxyl-terminal end of hTSHb subunit in the absence (hTSHba) or presence of a ~30 aminoacid peptide from hCGb (CTP) as a linker (hTSHbCTPa). Ligation of the CTP to the carboxyl-end of hFSH, hCGa subunit and to hTSH resulted in increasing the biological activity and longivity in vivo. In the present study, the hTSHbCTPa, was used to investigate the role of the N-linked oligosaccharides of a and b subunits on secretion and function of hTSH. Two deglycosylated variants were prepared: one lacks both oligosaccharide chains on a subunit (hTSHbCTPa1+2), and the other lacks also the oligosaccharide chain on b subunit of the single chain (hTSHbCTPa(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 a or b subunits and the O-linked oligosaccharides on the CTP, does not affect the secretion of the variants. These results indicate that the signal for the secretion exists in the single peptide chain is independent of the oligosaccharides. hTSH variants lack of the oligosaccharide chains is less potent than hTSHbCTPa on cAMP accumulation and T3 secretion in human cultured thyroid follicles. Moreover, both deglycosylated variants compete with normal hTSH and hTSI in a dose dependent manner in in vitro and in in vivo systems. 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. Our studies indicated that super agonists with long half-life in vivo can be prepared by addition of O-linked oligosaccharides to the protein. On the other hand, antagonists can be prepared by deletion of N-linked oligosaccharide.
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Presented at the
International Congress of Nanotechnology, November 7-10, 2004
San Francisco, USA
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