Expression of biologically active fusion genes encoding the common α subunit and either the CGβ or FSHβ subunits: role of a linker sequence

T, Sugahara; Grootenhuis, Peter D. J.; Sato, Asomi; Kudo, Masataka; Ben-Menahem, David; Pixley, Mary R.; Hsuen, Aaron J.W.; Boime, Irving

doi:10.1016/s0303-7207(96)03944-5

Cited by 39 publications

(28 citation statements)

References 22 publications

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“…The similar cAMP accumulation following the activation of the FSH receptor with the different bovine SC variants is in accordance with previous studies with CTP-spaced and unspaced human tethered FSH and hCG analogs (Sugahara et al, 1996a(Sugahara et al, ,b, 1995. In these studies, similar cAMP levels were measured following stimulation of receptorexpressing cells with human SC variants that have a similar design to the bovine FSH analogs in the current study (Sugahara et al, 1996a(Sugahara et al, ,b, 1995. The apparent rank potency of the SC analogs as judged by progesterone production was FSHba P FSHbboCTPa > FSHbCTPa, implying that the unspaced design was favorable for the steroidogenic activity of FSH in vitro.…”

Section: Discussionsupporting

confidence: 91%

Modulation of the steroidogenic related activity according to the design of single-chain bovine FSH analogs

Asraf

Amsterdam

Ben-Menahem

2015

General and Comparative Endocrinology

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Section: Discussionsupporting

confidence: 91%

Modulation of the steroidogenic related activity according to the design of single-chain bovine FSH analogs

Asraf

Amsterdam

Ben-Menahem

2015

General and Comparative Endocrinology

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“…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: 99%

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

Fares¹,

Levi²,

Reznick³

et al. 2001

Journal of Biological Chemistry

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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...

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“…to receptor binding and biological activity [5][6][7][8][9]. In these studies, the C terminus of hCGβ served as a linker between the two subunits.…”

mentioning

confidence: 99%

Single chain human chorionic gonadotropin, hCGαβ: Effects of mutations in the α subunit on structure and bioactivity

Setlur

Dighe

2006

Glycoconj J

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The strategy of translationally fusing the subunits of heterodimeric proteins into single chain molecules is often used to overcome the mutagenesis-induced defects in subunit interactions. The approach of fusing the alpha and beta subunits of human Chorionic Gonadotropin (hCG) to produce a single chain hormone (phCGalphabeta) was used to investigate roles of critical residues of the alpha subunit in hormone receptor interaction and biological activity. The alpha subunit was mutated using PCR-based site-directed mutagenesis, fused to the wild type beta subunit and the fusion protein was expressed using Pichia pastoris expression system. Following partial purification, the mutant proteins were extensively characterized using immunological probes, receptor assays, and in vitro bioassays. The mutation hCGalpha P38A, which disrupts subunit interaction in the heterodimeric molecule, produced a fusion molecule exhibiting altered subunit interactions as judged by the immunological criteria, but could bind to the receptor with lower affinity and elicit biological response. Mutation of hCGalpha T54A disrupting the glycosylation at Asparagine 52, believed to be important for bioactivity, also yielded a biologically active molecule suggesting that the glycosylation at this site is not as critical for bioactivity as it is in the case of the heterodimer. The fusion protein approach was also used to generate a superagonist of hormone action. Introduction of four lysine residues in the Loop 1 of the alpha subunit led to the generation of a mutant having higher affinity for the receptor and enhanced bioactivity. Immunological characterization of single chain molecules revealed that the interactions between the subunits were not identical to those seen in the heterodimeric hormone, and the subunits appeared to retain their isolated conformations, and also retained the ability to bind to the receptors and elicit response. These data suggest the plasticity of the hormone-receptor interactions.

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Expression of biologically active fusion genes encoding the common α subunit and either the CGβ or FSHβ subunits: role of a linker sequence

Cited by 39 publications

References 22 publications

Modulation of the steroidogenic related activity according to the design of single-chain bovine FSH analogs

Modulation of the steroidogenic related activity according to the design of single-chain bovine FSH analogs

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

Single chain human chorionic gonadotropin, hCGαβ: Effects of mutations in the α subunit on structure and bioactivity

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