title: Human thyroid-stimulating hormone (hTSH) subunit gene fusion produces hTSH with increased stability and serum half-life and compensates for mutagenesis-induced defects in subunit association.
abstract: The human thyroid-stimulating hormone (hTSH) subunits alpha and beta are transcribed from different genes and associate noncovalently to form the bioactive hTSH heterodimer. Dimerization is rate-limiting for hTSH secretion, and dissociation leads to hormone inactivation. Previous studies on human chorionic gonadotropin (hCG) and human follicle-stimulating hormone had shown that it was possible by subunit gene fusion to produce a bioactive, single chain hormone. However, neither the stability nor the clearance from the circulation of such fused glycoprotein hormones has been studied. We show here that genetic fusion of the hTSH alpha- and beta-subunits using the carboxyl-terminal peptide of the hCG beta-subunit as a linker created unimolecular hTSH whose receptor binding and bioactivity were comparable to native hTSH. Interestingly, the fused hTSH had higher thermostability and a longer plasma half-life than either native or dimeric hTSH containing the hCG beta-subunit-carboxyl-terminal peptide, suggesting that dimer dissociation may contribute to glycoprotein hormone inactivation in vivo. In addition, we show for the first time that synthesis of hTSH as a single polypeptide chain could overcome certain mutagenesis-induced defects in hTSH secretion, therefore enabling functional studies of such mutants. Thus, in addition to prolongation of plasma half-life, genetic fusion of hTSH subunits should be particularly relevant for the engineering of novel analogs where desirable features are offset by decreased dimer formation or stability. Such methods provide a general approach to expand the spectrum of novel recombinant glycoprotein hormones available for in vitro and in vivo study.
authors.affiliation: Department of Medicine, University of Maryland School of Medicine and the Institute of Human Virology, Medical Biotechnology Center, Baltimore, Maryland 21201, USA. grossman@umbi.umd.edu
authors.forename: M
authors.initials: M
authors.lastname: Grossmann
authors.forename: R
authors.initials: R
authors.lastname: Wong
authors.forename: M W
authors.initials: MW
authors.lastname: Szkudlinski
authors.forename: B D
authors.initials: BD
authors.lastname: Weintraub
mesh_terms: D000818:Animals; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D019281:Dimerization; D006801:Humans; D008657:Metabolic Clearance Rate; D016297:Mutagenesis, Site-Directed; D051381:Rats; D011989:Receptors, Thyrotropin / Q000378:metabolism*; D013552:Swine; D013972:Thyrotropin / Q000737:chemistry* / Q000378:metabolism / Q000493:pharmacokinetics
chemical_list: D011989:Receptors, Thyrotropin; D013972:Thyrotropin