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

Grossmann, Mathis; Wong, Rosemary; Szkudlinski, Mariusz W.; Weintraub, Bruce D.

doi:10.1074/jbc.272.34.21312

Cited by 55 publications

(28 citation statements)

References 34 publications

(45 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…To bypass the problem of dimerization of deglycosylated subunits, the subunits ␣ and ␤ were genetically fused in a single chain hormone. Single chains of hCG (7), hFSH (8), and hTSH (11,12) retained a biologically active conformation similar to that of the heterodimer (11,12). Therefore, fusion of ␣-and ␤-subunits in a single chain bypasses the assembly of the subunits, which is a rate-limiting step for hormone secretion and bioactivity.…”

Section: Discussionmentioning

confidence: 99%

“…To overcome these limitations, the genes encoding the common ␣-subunit and either the hCG ␤-, FSH ␤-, or TSH ␤-subunits have been genetically fused. The resulting polypeptide chains were efficiently secreted and were biologically active (7)(8)(9)(10)(11)(12). These studies presumed that addition of the human CG␤ C-terminal peptide (CTP) as a linker sequence between the subunits would be required for flexibility, hydrophilicity, stability, and successful expression of the single chain forms.…”

Section: Thyrotropin (Tsh)mentioning

confidence: 95%

“…Therefore, fusion of ␣-and ␤-subunits in a single chain bypasses the assembly of the subunits, which is a rate-limiting step for hormone secretion and bioactivity. It is apparent that despite the single chain structure, correct conformation of the subunits occurs, and the single chains have a normal biological activity (11,12).…”

Section: Discussionmentioning

confidence: 99%

See 2 more Smart Citations

Engineering a Potential Antagonist of Human Thyrotropin and Thyroid-stimulating Antibody

Fares¹,

Levi²,

Reznick³

et al. 2001

Journal of Biological Chemistry

View full text Add to dashboard Cite

Thyrotropin (TSH) and the gonadotropins (FSH, LH, hCG) are a family of heterodimeric glycoprotein hormones composed of two noncovalently linked subunits, ␣ and ␤. We have recently converted the hTSH heterodimer to a biologically active single chain (hTSH␤⅐CTP␣) by fusing the common ␣-subunit to the C-terminal end of the hTSH ␤-subunit in the presence of a ϳ30-amino acid peptide from hCG␤ (CTP) as a linker. The hTSH␤⅐CTP␣ single chain was used to investigate the role of the Nlinked oligosaccharides of ␣-and ␤-subunits in the secretion and function of hTSH. Using overlapping PCR mutagenesis, two deglycosylated variants were prepared: one lacking both oligosaccharide chains on the ␣-subunit (hTSH␤⅐CTP␣ 1؉2 ) and the other lacking the oligosaccharide chain on the ␤-subunit (hTSH␤⅐CTP␣(deg)). The single chain variants were expressed in CHO cells and were secreted into the medium. hTSH variants lacking the oligosaccharide chains were less potent than hTSH␤⅐CTP␣ wild-type with respect to cAMP formation and thyroid hormone secretion in cultured human thyroid follicles. Both deglycosylated variants competed with hTSH in a dose-dependent manner. The hTSH␤⅐CTP␣ 1؉2 variant blocked cAMP formation and thyroid hormone secretion stimulated by hTSH as well as by the antibody, thyroidstimulating immunoglobulins, responsible for the most common cause of hyperthyroidism, Graves disease. Thus, this variant behaves as a potential antagonist, offering a novel therapeutic strategy in the treatment of thyrotoxicosis caused by Graves' disease and TSH-secreting pituitary adenoma. Thyrotropin (TSH)1 is a member of the glycoprotein hormone family, which includes lutropin (LH), follitropin (FSH), and human chorionic gonadotropin (hCG). These are heterodimers composed of two noncovalent-linked subunits, a common ␣-subunit and a hormone-specific ␤-subunit (1, 2). Assembly of glycoprotein subunits is vital to the function of these hormones. The ␣-and ␤-subunits contain one (TSH␤ and LH␤) or two (␣, FSH␤, and hCG␤) asparagine-linked (N-linked) oligosaccharides (1, 2). These residues have been shown to play a role in determining the biological activity of glycoprotein hormones, including the maintenance of intracellular stability, assembly, secretion, signal transduction, and modulation of plasma halflife (1, 3). Deglycosylation of glycoprotein hormones have been utilized using chemical or enzymatic treatments, but these however cannot discriminate between individual sites, are nonspecific, and provide only completely deglycosylated hormones.Site-directed mutagenesis has become an important tool for studying the structure and function of glycoprotein hormones. However, mutations in either ␣-or ␤-subunits can alter the folding and ultimately inhibit subunit assembly and secretion of the hormone (4 -6). To overcome these limitations, the genes encoding the common ␣-subunit and either the hCG ␤-, FSH ␤-, or TSH ␤-subunits have been genetically fused. The resulting polypeptide chains were efficiently secreted and were biologically active (7-12). The...

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Thyrotropin (Tsh)mentioning

confidence: 95%

See 1 more Smart Citation

Engineering a Potential Antagonist of Human Thyrotropin and Thyroid-stimulating Antibody

Fares¹,

Levi²,

Reznick³

et al. 2001

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…Such results were consistent with ours. However, Grossmann et al (1997) showed that the fusion enzyme was more stable against thermal denaturation than the individual enzymes.…”

Section: Discussionmentioning

confidence: 99%

In-fusion expression and characterization of β-xylanase and β-1,3-1,4-glucanase in Pichia pastoris

Qiao

Cao

2012

Biologia

View full text Add to dashboard Cite

Two chimeric genes, XynA-Bs-Glu-1 and XynA-Bs-Glu-2, encoding Aspergillus sulphureus β-xylanase (XynA, 26 kDa) and Bacillus subtilis β-1,3-1,4-glucanase (Bs-Glu, 30 kDa), were constructed via in-fusion by different linkers and expressed successfully in Pichia pastoris. The fusion protein (50 kDa) exhibited both β-xylanase and β-1,3-1,4-glucanase activities. Compared with parental enzymes, the moiety activities were decreased in fermentation supernatants. Parental XynA and Bs-Glu were superior to corresponding moieties in each fusion enzymes because of lower Km and higher kcat. Despite some variations, common optima were generally 50• C and pH 3.4 for the XynA moiety and parent, and 40• C and pH 6.4 for the Bs-Glu counterparts. Thus, the fusion enzyme XynA-Bs-Glu-1 and XynA-Bs-Glu-2 were bifunctional.

show abstract

“…The b-a configuration was widely used to produce recombinant single-chain glycoprotein hormones of many species (Narayan et al 1995, Boime & BenMenahem 1999, Dirnberger et al 2001, Fidler et al 2003, Min et al 2004, Jablonka-Shariff et al 2007, sometimes with heterologous CTP from hCG inserted between fused a-and b-subunits to enhance the secretion of these single-chain protein variants (Sugahara et al 1996, Garcia-Campayo et al 1997, Grossmann et al 1997, Fares et al 1998. These earlier studies showed that the CTP played the role of a flexible hydrophilic spacer allowing genetically fused a-and b-subunits to adopt a functional conformation and that secretion in the presence of CTP was enhanced due to its specific O-glycans.…”

Section: Discussionmentioning

confidence: 99%

Stability and biological activities of heterodimeric and single-chain equine LH/chorionic gonadotropin variants

Legardinier¹,

Poirier²,

Klett³

et al. 2008

Journal of Molecular Endocrinology

View full text Add to dashboard Cite

Recombinant equine LH/chorionic gonadotropin (eLH/CG) was expressed in the baculovirus-Sf9 insect cell system either as a single-chain with the C-terminus of the b-subunit fused to the N-terminus of the a-subunit or as non-covalently linked heterodimers with or without a polyhistidine tag at various locations. All these non-covalently linked eLH/CG variants were secreted as stable heterodimers in the medium of infected Sf9 cells. To assess the influence of the presence and the position of polyhistidine tag on LH bioactivity, we expressed four non-covalently linked tagged heterodimeric eLH/CG variants that were secreted in threefold higher quantities than the single chain. Among them, only two exhibited full in vitro LH bioactivity, relative to untagged heterodimers, namely the one His-tagged at the N-terminus of a-subunit and the other at the C-terminus of the b-subunit both of which are amenable to nickel-affinity purification. Furthermore, single-chain eLH/CG was found to be N-and O-glycosylated but nevertheless less active in in vitro LH bioassays than natural eCG and heterodimeric recombinant eLH/CG. The thermal stability of natural and recombinant hormones was assessed by the initial rates of dissociation from 20 to 90 8C. Heterodimeric eLH/CG from Sf9 cells was found to be as stable as pituitary eLH and serum eCG (T 1/2 , 74-77 8C). Although Sf9 cells only elaborated short immature-type carbohydrate side chains on glycoproteins, recombinant eLH/CG produced in these cells exhibited stabilities similar to that of pituitary eLH. In conclusion, recombinant heterodimeric eLH/CG exhibits the same thermal stability as natural pituitary LH and its advantages over the single-chain eLH/CG include higher secretion, higher in vitro bioactivity, and reduced potential risk of immunogenicity.

show abstract

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

Cited by 55 publications

References 34 publications

Engineering a Potential Antagonist of Human Thyrotropin and Thyroid-stimulating Antibody

Engineering a Potential Antagonist of Human Thyrotropin and Thyroid-stimulating Antibody

In-fusion expression and characterization of β-xylanase and β-1,3-1,4-glucanase in Pichia pastoris

Stability and biological activities of heterodimeric and single-chain equine LH/chorionic gonadotropin variants

Contact Info

Product

Resources

About