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Volume 272, Number 34, Issue of August 22, 1997 pp. 21312-21316
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

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

(Received for publication, May 12, 1997, and in revised form, June 10, 1997)

From the Laboratory of Molecular Endocrinology, Department of Medicine, University of Maryland School of Medicine and the Institute of Human Virology, Medical Biotechnology Center, Baltimore, Maryland 21201 and ^§ NHLBI, National Institutes of Health, Bethesda, Maryland 20892

The human thyroid-stimulating hormone (hTSH) subunits  and  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 - and -subunits using the carboxyl-terminal peptide of the hCG -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 -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.

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