Expression of Biologically Active Fusion Genes Encoding the Common α Subunit and the Follicle-stimulating Hormone β Subunit

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

doi:10.1074/jbc.271.18.10445

Cited by 73 publications

(49 citation statements)

References 38 publications

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“…In addition, it was reported that CTP and associated O-linked oligosaccharides in hCG are not important for receptor binding or in vitro signal transduction but are critical for in vivo biological response (23). Moreover, it has been shown that the secretion of the hFSH single chain increased in the presence of CTP as a linker between the subunits (8). The fact that deglycosylated variants of hTSH are secreted efficiently from CHO and ldlD cells (ldlD cells have a reversible defect in synthesizing oligosaccharide chains, data not shown) indicates that Nlinked and O-linked oligosaccharides are not vital for the secretion of the hTSH single chain.…”

Section: Discussionmentioning

confidence: 99%

“…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%

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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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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Thyrotropin (Tsh)mentioning

confidence: 95%

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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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“…In glycoprotein hormones, tethered-hCG and hFSH molecules with the C-terminus of the complete β-subunit conjoined to the N-terminus of the α-subunit, were not only efficiently secreted but displayed an increased biological activity both in vitro and in vivo [28][29][30][31].…”

mentioning

confidence: 99%

Biological Activities of Tethered Equine Chorionic Gonadotropin (eCG) and Its Deglycosylated Mutants

Min

Hiyama

Seong³

et al. 2004

J. Reprod. Dev.

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Abstract. Equine chorionic gonadotropin (eCG), which consists of highly glycosylated α-and β-subunits, is a unique member of the gonadotropin family because it elicits response characteristics of both follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in species other than the horse. In this study, recombinant tethered-eCG as well as its deglycosylated mutants were produced to determine if α-and β-subunits can be synthesized as a single polypeptide chain (tethered-eCG) and display biological activity. We found that tethered-eCG (T-βα) had both LH-and FSH-like activities comparable to dimeric eCG. Luteinizing hormone-like activity of tethered-eCGs deglycosylated at Asn 56 (T-βα56) was decreased. In contrast, LH-like activity of eCG without O-glycosylated carboxylterminal peptide (CTP) (T-βcα) was slightly decreased but still similar to T-βα. Double mutation at Asn 56 and CTP (T-βcα56) caused marked decrease in the activity, indicating that both glycosylations at Asn 56 and CTP are involved in LH-like activity in the tethered form. Interestingly, FSH-like activity remained in all deglycosylated eCG mutants (T-βα56, T-βcα and T-βcα56) as well as T-βα. The biological roles of oligosaccharides at Asn 56 of eCG α-subunit and O-linked peptide of β-subunit appear to be different in LH-and FSH-like activities in tethered-eCG. Key words: Equine chorionic gonadotropin, Luteinizing hormone, Follicular stimulating hormone, Glycosylation, Tethered hormone (J. Reprod. Dev. 50: [297][298][299][300][301][302][303][304] 2004) quine chorionic gonadotropin (eCG) is a member of the glycoprotein hormone family which includes luteinizing hormone (LH), follicles t i m u l a t i n g h o r m o n e ( F S H ) a n d t h y r o i dstimulating hormone (TSH). This hormone family is characterized by a heterodimeric structure composed of a common α-subunit noncovalently linked to a hormone-specific β-subunit [1]. The β-subunits of eCG and eLH, translated from the same gene, have identical primary structures [2,3]. The difference between eCG and eLH lies in the structure of their carbohydrates, which are both sialylated and sulfated in LH and sialylated in CG [4,5]. Equine chorionic gonadotropin shows both LH-and FSH-like activities in many species but not the horse [6][7][8][9][10][11]. The LH-and FSH-like activities of eCG in vitro observed in heterologous species is of fundamental interest to the study of structurefunction relationships of gonadotropins and their receptors.We previously prepared recombinant eCG in CHO-K1 cells, and found that these recombinant

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“…The three single chain human choriogonadotropins presented in this work have distinct features that differ from the published single chain glycoprotein hormones. The spacer moiety in our single chains does not consist of (parts of) the C-terminal peptide of the subunit [3,4,6, 71 but of Ser-Gly repeats. Since mutants 1 and 2 resemble heterodimeric lutropin in terms of receptor binding and to a lesser extent bioactivity, it can be concluded that the composition of the spacer only has minor effects on the structure and function of the single chain human choriogonadotropins.…”

Section: Discussionmentioning

confidence: 99%

Evaluation of Subunit Truncation and the Nature of the Spacer for Single Chain Human Gonadotropins

Heikoop¹,

Vaan²,

Boogaart³

et al. 1997

European Journal of Biochemistry

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Three single chain gonadotropins were designed based on the three-dimensional-structure of human choriogonadotropin and structure/activity relationships of the glycoprotein hormones. In each single chain, the C-terminal end of the human choriogonadotropin /? subunit is connected via Ser-Gly repeats to the N-terminal end of the a subunit. In addition, two of the single chains have truncated subunits. The three mutants were expressed in CHO cells. In vitro binding of two of the three mutants to the human lutropinl choriogonadotropin receptor was found to be comparable to wild-type lutropin. In contrast, both the receptor binding and the in vitro bioactivity of the mutant with truncated (I and /? subunits in which the p:26-110 disulphide bond cannot be formed, are lowered relative to wild-type lutropin. The fact that this mutant still displays biological activity shows that the seat-belt arrangement proposed by Isaacs and coworkers [Lapthorn, A. J., Harris, D. C., Littlejohn, A., Lustbader, J. W., Canfield, R. E., Machin, K. Keywords: gonadotropin ; single chain ; structure-based design; spacer; truncation.The glycoprotein hormones 11 1 form a family of structurally related proteins. Members include choriogonadotropin, follitropin, lutropin and thyrotropin. Follitropin, lutropin and thyrotropin are present in most vertebrate species and are synthesized and secreted by the pituitary. Choriogonadotropin has so far been found only in primates, including humans, and in horses, and is synthesized by placental tissue. The glycoprotein hormones are heterodimers composed of two dissimilar subunits, named 0: and /?, which are associated by non-covalent bonds.Within a species, the a subunit is the same for each member of the gonadotropin family. The , / 3 subunits are different for each member, i.e. choriogonadotropin, follitropin, thyrotropin and lutropin, and confer receptor-binding specificity on the hormones.The association of the (x and the p subunit is a very important step in the biosynthesis of the glycoprotein hormones, since only the intact dimers are biologically active. The correct assembly of the heterodimer is essential for efficient secretion, receptor binding, and signal transduction of the hormone. Free a and / 3 subunits are biologically inactive 121. Numerous mutation studies have shown that the dimerization is a highly specific process and can easily be prohibited by single point mutations. Structure/function analysis of the glycoprotein hormones is often hampered by unwanted secondary effects on the assembly of the subunits.Therefore, we and others [3 -71 have pursued approaches to determine whether the active conformation of the glycoprotein hormones can also be formed when the two subunits are synthesized in tandem on a single polypeptide chain. It has been dem- onstrated that covalently linked u and /j subunits are efficiently secreted and able to fold in a conformation that binds the lutropin/choriogonadotropin receptor with high affinity and stimulates adenylate cyclase. Thus, it can be concluded th...

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Expression of Biologically Active Fusion Genes Encoding the Common α Subunit and the Follicle-stimulating Hormone β Subunit

Cited by 73 publications

References 38 publications

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

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

Biological Activities of Tethered Equine Chorionic Gonadotropin (eCG) and Its Deglycosylated Mutants

Evaluation of Subunit Truncation and the Nature of the Spacer for Single Chain Human Gonadotropins

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