Fusing the carboxy-terminal peptide of the chorionic gonadotropin (CG) beta-subunit to the common alpha-subunit: retention of O-linked glycosylation and enhanced in vivo bioactivity of chimeric human CG.

Furuhashi, Madoka; Shikone, Toshihiko; Fares, Fuad; T, Sugahara; Hsueh, Aaron J. W.; Boime, Irving

doi:10.1210/mend.9.1.7539107

Cited by 37 publications

(41 citation statements)

References 7 publications

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“…This shows that the additional peptide portion does not interfere either with the combination of subunits or with the association of the so-formed dimer with the LH receptor. These data contrast with those of Furuhashi et al (11) who produced an hCG chimera with an additional b carboxyterminal peptide (hCGb-CTP) fused to the Cterminus of the a-subunit. This elongated a-subunit (ha-CTP) recombined efficiently with wild-type hCGbsubunit but the so-formed dimer exhibited very low binding activity to the human LH receptor expressed in the human fetal kidney 293 cell line (<5% relative to wild-type hCG).…”

Section: Discussioncontrasting

confidence: 88%

“…Thus, the +24 peptide does not exert any inhibitory activity on hCG binding to rat LH receptors when present at the C-terminus of the a-subunit. This extension is only slightly shorter than the b-CTP (28 amino acid residues) but the latter is known to bear Osaccharide chains on four of its eight serine residues (Ser 121, 127, 132, 138 ) in the natural hCG molecule (12) and was also found to be O-glycosylated when fused to the C-terminus of the ha-subunit (11). Multiple Oglycosylation in segments of proteins impedes the folding of these regions, leading to bulky extended flexible structures (13).…”

Section: Discussionmentioning

confidence: 99%

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Human chorionic gonadotropin with C-elongated alpha-subunit retains full receptor binding and partial agonist activity

Marichatou

Martinat

Guillou

et al. 2000

European Journal of Endocrinology

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Objective: To test whether extension of the C-terminus of human chorionic gonadotropin (hCG) a-subunit (ha) alters the bioactivity of the recombined ab heterodimer. Design: The stop codon of ha was mutated to produce a 24 amino acid extension. Methods:The extended ha (a +24 ) was co-expressed with hCGb in COS-7 cells and the receptor binding and in vivo bioactivity of the secreted hormone was compared with its wild-type counterpart.Results: This extension did not impair the binding of hCG to rat LH/CG receptors and provoked a sixfold reduction in its stimulatory activity of testosterone secretion in rat Leydig cells. Conclusions:The extension of a by itself does not lead to inhibition of the ab heterodimer to LH receptors but the structure of the extension appears to play an important role. It is thus possible that one-chain hCG chimeras with the b N-terminus fused to the a C-terminus might be active.

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Section: Discussioncontrasting

confidence: 88%

Section: Discussionmentioning

confidence: 99%

Human chorionic gonadotropin with C-elongated alpha-subunit retains full receptor binding and partial agonist activity

Marichatou

Martinat

Guillou

et al. 2000

European Journal of Endocrinology

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“…This may permit the appropriate interactions between the subunits. In addition, previous studies showed that ligation of the CTP to hFSH␤ (13), hTSH␤ (14), or hCG␣ (15) did not significantly affect assembly or in vitro biological activity, but was important for the in vivo potency of the chimeras.…”

Section: Thyrotropin (Tsh)mentioning

confidence: 96%

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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“…Fusion of peptides to this portion of the ␣-subunit has been shown to have varying effects on hormone activity (20,21). Therefore, when we began these studies we were concerned that the presence of a crosslink between the ␣CT and the hCG ␤-subunit might destroy heterodimer activity by a mechanism that was unrelated to the ability of this portion of the hormone to contact the LHR.…”

Section: Resultsmentioning

confidence: 99%

Only a Portion of the Small Seatbelt Loop in Human Choriogonadotropin Appears Capable of Contacting the Lutropin Receptor

Bernard

Lin

Cao

et al. 2004

Journal of Biological Chemistry

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Twenty residues of the human choriogonadotropin (hCG) ␤-subunit that are wrapped around ␣-subunit loop 2 like a "seatbelt" stabilize the heterodimer and enable the hormone to distinguish lutropin (LHR), follitropin, and thyrotropin receptors. The N-terminal portion of the seatbelt contains a small disulfide-stabilized loop needed for heterodimer assembly and is thought to mediate hCG-LHR interactions. To test the latter notion, we compared the LHR binding and signal transduction activities of hCG analogs in which the ␣-subunit C terminus (␣CT) was cross-linked to residues in the small seatbelt loop. Analogs having an intersubunit disulfide between a cysteine in place of ␣CT residue ␣Ser-92 and cysteines substituted for loop residues ␤Arg-94, ␤Arg-95, or ␤Ser-96 had high activities in LHR binding and signaling assays despite the fact that both portions of the hormone are thought to be essential for hCG activity. Use of a larger probe blocked hormone activity when the ␣CT was crosslinked to cysteines in place of residues ␤Arg-95 and ␤Asp-99, but not to cysteines in place of residues ␤Arg-94, ␤Ser-96, or ␤Thr-97. This suggested that the side chains of residues ␤Arg-95 and ␤Asp-99, which face in the same outward direction from the heterodimer, are nearer than the others to the LHR interface. The finding that residue 95 can be cross-linked to small ␣CT probes without eliminating hormone activity indicates its side chain does not participate in essential LHR contacts. We suggest that contacts between the small seatbelt loop and the LHR, if any, involve its backbone atoms and possibly the side chain of residue ␤Asp-99.The crystal structure of hCG 1 revealed that 20 residues of its ␤-subunit surround loop ␣2 like a "seatbelt" (1, 2). The seatbelt has a key role in the stability of the heterodimer; elimination of the disulfide that "latches" its C-terminal end to ␤Cys-26 in the subunit core disrupts heterodimer formation (3). Thus, glycoprotein hormones differ from most other heterodimeric proteins, which are stabilized by hydrophobic contacts or intersubunit disulfides. Whereas the evolutionary advantages of this unusual structural arrangement remain unknown, it may have facilitated the co-evolution of ligand-receptor pairs by permitting point mutations to alter the conformation of the heterodimer and thereby modulate its biological activity (4 -6).The seatbelt is divided into two regions that differ in their influence on the activities of lutropins, follitropins, and thyrotropins. Its N-terminal half contains a small disulfide-stabilized loop that has an important role in heterodimer assembly (7-9). Because a portion of this loop participates in hydrogen bonds with ␣-subunit loop 2 (1, 2, 10), mutations that affected its conformation or the stability of these hydrogen bonds would be expected to alter the positions of the subunits in the heterodimer. The seatbelt loop contains positively charged residues in mammalian lutropins and negatively charged residues in mammalian follitropins and thyrotropins. This led to the sp...

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Fusing the carboxy-terminal peptide of the chorionic gonadotropin (CG) beta-subunit to the common alpha-subunit: retention of O-linked glycosylation and enhanced in vivo bioactivity of chimeric human CG.

Cited by 37 publications

References 7 publications

Human chorionic gonadotropin with C-elongated alpha-subunit retains full receptor binding and partial agonist activity

Human chorionic gonadotropin with C-elongated alpha-subunit retains full receptor binding and partial agonist activity

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

Only a Portion of the Small Seatbelt Loop in Human Choriogonadotropin Appears Capable of Contacting the Lutropin Receptor

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