• Development and Reproduction
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• v.25 no.4
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• pp.199-211
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• 2021

Equine chorionic gonadotropin (eCG), produced by the endometrial cups of the placenta after the first trimester, is a specific glycoprotein that displays dual luteinizing hormone (LH)-like and follicle-stimulating hormone (FSH)-like effects in non-equid species. However, in equidaes, eCG exhibits only LH-like activity. To identify the specific biological functions of glycosylated sites in eCG, we constructed the following site mutants of N- and O-linked glycosylation: eCGβ/αΔ56, substitution of α-subunit⁵⁶ N-linked glycosylation site; eCGβ-D/α, deletion of the O-linked glycosylation sites at the β-subunit, and eCGβ-D/αΔ56, double mutant. We produced recombinant eCG (rec-eCG) proteins in Chinese hamster ovary suspension (CHO-S) cells. We examined the biological activity of rec-eCG proteins in CHO-K1 cells expressing the eLH/CG receptor and found that signal transduction activities of deglycosylated mutants remarkably decreased. The EC₅₀ levels of eCGβ/αΔ56, eCGβ-D/α, and eCGβ-D/αΔ56 mutants decreased by 2.1-, 5.6-, and 3.4-fold, respectively, compared to that of wild-type eCG. The Rmax values of the mutants were 56%-80% those of wild-type eCG (141.9 nmol/10⁴ cells). Our results indicate that the biological activity of eCG is greatly affected by the removal of N- and O-linked glycosylation sites in cells expressing eLH/CGR. These results provide important information on rec-eCG in the regulation of specific glycosylation sites and improve our understanding of the specific biological activity of rec-eCG glycosylation sites in equidaes.

• Korean Journal of Animal Reproduction
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• v.26 no.3
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• pp.299-308
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• 2002

In horse, a single gene encodes both eCG and eLH $\beta$ subunits. The difference between eCG and eLH lies in the structure of their glycoresidues, which are both sialylated and sulfated in LH and sialylated in CG eCG consists of highly glycosyiated $\alpha$- and $\beta$-subunits and is an unique member of the gonadotropin family because it elicits response characteristics of both FSH and LH in other species than the horse. This dual activity of eCG in heterologous species is of fundamental interest to the study of gonadotropin structure-function relationships and the understanding of the molecular bases of the specific interactions of these hormones with their receptors. Thus, eCG is a dintinct molecule from the view points of its biological function and glycoresidue structures. The oligosaccharide at Asn 56 of the $\alpha$-subunit plays an indispensable role, whereas the carboxyl-terminal extension of the eCG $\beta$-subunit with its associated O-linked oligosaccharides is not improtant for, the in vitro LH-like activity of eCG. In contrast, both N- and O-linked oligosaccharides play important roles for FSH-like activity and increase FSH-like activity by removal of N- and O-linked oligosaccharides. Therefore, the dual LH- and FSH-like activities of eCG can be clearly separated by removal of either the N-linked oligosaccharide on the $\alpha$-subunit or CTP-associated O-linked oligosaccharides from its $\beta$-subunit. The glycoresidues seem to play crucial roles fer biological activities. The tethered-eCG was effciently secreted and showed similar LH-like activity to the dimeric eCG $\alpha$/ $\beta$ and native eCG. FSH-like activity of the tethered-eCG was also shown similarly in comparison with the native and wild type eCG $\alpha$/ $\beta$. Our data for the first time suggest that the tethered-eCG can be expressed efficiently and the produced product by the CHO-Kl cells is fully LH- and FSH-like activities in rat in vitro bioassay system. Our results also suggest that this molecular can imply particular models ot FSH-like activity not LH-like activity in the eCG. Taken together, these data indicate that the constructs of tethered molecule will be useful in the study of mutants that affect subunit association and/or secretion.

• Development and Reproduction
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• v.22 no.1
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• pp.55-64
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• 2018

Previous studies showed that recombinant equine chorionic gonadotropin ($rec-eCG{\beta}/{\alpha}$) exhibits both follicle-stimulating hormone (FSH) and luteinizing hormone (LH)-like activities in rat LHR- and FSHR-expressing cells. In this study, we analyzed signal transduction by eelFSHR and eelLHR upon stimulation with $rec-eCG{\beta}/{\alpha}$ and native eCG. The cyclic adenosine monophosphate (cAMP) stimulation in CHO-K1 cells expressing eelLHR was determined upon exposure to different doses (0-1,450 ng/mL) of $rec-eCG{\beta}/{\alpha}$ and native eCG. The $EC_{50$ values of $rec-eCG{\beta}/{\alpha}$ and native eCG were 172.4 and 786.6 ng/mL, respectively. The activity of $rec-eCG{\beta}/{\alpha}$ was higher than that of native eCG. However, signal transduction in the CHO PathHunter Parental cells expressing eelFSHR was not enhanced by stimulation with both agonist $rec-eCG{\beta}/{\alpha}$ and native eCG. We concluded that $rec-eCG{\beta}/{\alpha}$ and native eCG were completely active in cells expressing eelLHR, similar to the activity in the mammalian cells expressing LHRs. However, $rec-eCG{\beta}/{\alpha}$ and native eCG did not invoke any signaling response in the cells expressing eelFSHR. These results suggest that eCG has a potent activity in cells expressing eelLHR. Thus, we also suggest that $rec-eCG{\beta}/{\alpha}$ can induce eel maturation by administering gonadotropic reagents (LH), such as salmon pituitary extract.

• Journal of Life Science
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• v.11 no.2
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• pp.103-107
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• 2001

The equine chorionic gonadotropin (eCG) subunits $\alpha$ and ${\beta}$ are transcribed from different genes and associate noncovalently to form the bioactive eCG heterodimer. Dimerization is rate limiting for eCG secretion, and dissociation leads to hormone inactivation. The correct conformation of the heterodimer is alto important for efficient secretion, hormone-specific post-translational modifications, receptor binding and signal transduction. To determine whether ${\alpha}$ and ${\beta}$ subunits can be synthesized as a single polypeptide chain (tethered-eCG) and also display biological activity, the tethered-eCG molecule by fusing the carboxyl terminus of the eCG ${\beta}$-subunit to the amino terminus of the af-subunit was construe-ted and transfected into chinese hamster ovary (CHO-Kl) cells. LH- and FSH-like activities were assayed in terms of testosterone production and aromatase activity in primary cultured rat Leydig cells and granulosa cells, respectively. The tethered-eCG was efficiently secreted and showed similar LH-like activity to the dimeric eCG ${\alpha}$/${\beta}$ and native eCG. FSH-like activity of the tethered-eCG was also shown similarly in comparison with the native and wild type eCG ${\alpha}$/${\beta}$. Our data for the first time suggest that the tethered-eCG can be expressed efficiently and the produced product by the CHO-K1 cells is fully LH- and FSH-like activities in rat in vitro bioassay system. Our results also suggest that this molecular can imply particular models of FSH-like activity not LH-like activity in the eCG. Taken together, these data indicate that the constructs of tethered molecule will be useful in the study of mutants that affect subunit association and/or secretion.

• Reproductive and Developmental Biology
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• v.33 no.4
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• pp.229-236
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• 2009

The glycoprotein hormone family represents a class of heterodimers, that includes the placental hormone equine chorionic gonadotropin (eCG) and the anterior pituitary hormones- follitropin (FSH), lutropin (LH), and thyrotropin (TSH). The 4 hormones are heterodimers, with a common $\alpha$-subunit and unique $\beta$-subunits. eCG is the most heavily glycosylated of the known pituitary and placental glycoprotein hormones. Recent observations using single chain glycoprotein hormone analogs in which, the $\beta$-and $\alpha$-subunits are linked, implied that heterodimeric-like quaternary configuration is not a prerequisite for receptor/signal transduction. To study the function and signal transduction of tethered rec-eCG, a single chain eCG molecule was constructed and rec-eCG protein was produced. Molecular mass of the single chain is about 45 kDa. All mice were ovulated by tethered rec-eCG treatment. The dual activity of tethered rec-eCG was determined in receptor cell lines of nonequid species; in fact, this dual activity was proven in species other than horse. Tethered rec-eCG in equids does not bind to FSH receptors, suggesting that eCG is primarily an LH-like hormone in the horse. Taken together, these data suggest that tethered rec-eCG has dual activity in nonequid species in vitro. However, it has only LH-like activity in equid species in vitro.

• Journal of Life Science
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• v.27 no.8
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• pp.864-872
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• 2017

Equine chorionic gonadotropin (eCG) consists of highly glycosylated ${\alpha}-$ and ${\beta}-subunits$ and is a unique member of the gonadotropin family, because it elicits the response characteristics of follicle stimulating hormone (FSH) and luteinizing hormone (LH) in species other than the horse. To directly assess the biological function of $rec-eCG{\beta}/{\alpha}$, we constructed mammalian expressing vectors of equine luteinizing hormone/chorionic gonadotropin receptors (eLH/CGR). The activity of $rec-eCG{\beta}/{\alpha}$ in vitro assayed in transient transfected CHO-K1 cells and in stably transfected PathHunter Parental cells with eLH/CGR was investigated. $rec-eCG{\beta}/{\alpha}$ was efficiently secreted in the CHO-K1 suspension cell media, and the quantity detected was about 200 mIU/ml from 1 to 7 days after transfection. In the western blot analysis, the $rec-eCG{\beta}/{\alpha}$ protein was broadly identified to be about 40~45 kDa molecular weight. The cAMP stimulation in CHO-K1 cells expressing eLH/CGR was determined to evaluate the activity of $rec-eCG{\beta}/{\alpha}$. The cAMP concentration increased in direct proportion to the concentration of the $rec-eCG{\beta}/{\alpha}$. The $EC_{50}$ value in the transient transfected CHO-K1 cells was $8.1{\pm}6.5ng$. The stable cell lines of eLH/CGR were established in the PathHunter Parental cells expressing ${\beta}-arrestin$. We found that $rec-eCG{\beta}/{\alpha}$ had full LH activity in the PathHunter Parental cells expressing eLH/CGR. The $EC_{50}$ value in transient and stable cells was $5.0{\pm}4.7ng/ml$ and $4.5{\pm}5.2ng/ml$, respectively. These results suggest that $rec-eCG{\beta}/{\alpha}$ has a biological activity in a cell expressing eLH/CGR. These stable cells expressed in PathHunter Parental cells could be useful for elucidating the functional mechanisms of deglycosylated $rec-eCG{\beta}/{\alpha}$ mutants.

• Asian-Australasian Journal of Animal Sciences
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• v.30 no.12
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• pp.1679-1683
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• 2017

Objective: An experiment was conducted to study the association between the single nucleotide polymorphisms (SNPs) in 5'-untranslated regions (5'-UTR) of equine chorionic gonadotropin (eCG) genes and the serum eCG levels. Methods: SNPs in 5'-UTR of eCG genes were screened across 10 horse breeds, including 7 Chinese indigenous breeds and 3 imported breeds using iPLEX chemistry, and the association between the serum eCG levels of 174 pregnant Da'an mares and their serum eCG levels (determined with ELISA) was analyzed. Results: Four SNPs were identified in the 5'-UTR of the $eCG{\alpha}$ gene, and one of them was unique in the indigenous breeds. There were 2 SNPs detected at the 5' end of the $eCG{\beta}$ subunit gene, and one of them was only found in the Chinese breeds. The SNP g.39948246T>C at the 5'-UTR of $eCG{\alpha}$ was associated significantly with eCG levels of 75-day pregnant mare serum (p<0.05) in Da'an mares. Prediction analysis on binding sites of transcription factors showed that the g.39948246T>C mutation causes appearance of the specific binding site of hepatocyte nuclear factor 3 forkhead homolog 2 (HFH-2), which is a transcriptional repressor belonging to the forkhead protein family of transcription factors. Conclusion: The SNP g.39948246T>C at the 5'-UTR of $eCG{\alpha}$ is associated with eCG levels of 75-day pregnant mare serum (p<0.05).

• Development and Reproduction
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• v.21 no.2
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• pp.111-120
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• 2017

Equine chorionic gonadotropin (eCG) is a unique molecule that elicits the response characteristics of both follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in other species. Previous studies from this laboratory had demonstrated that recombinant eCG (rec-eCG) from Chinese hamster ovary (CHO-K1) cells exhibited both FSH- and LH-like activity in rat granulosa and Leydig cells. In this study, we analyzed receptor internalization through rec-eCGs, wild type eCG ($eCG{\beta}/{\alpha}$) and mutant eCG ($eCG{\beta}/{\alpha}{\Delta}56$) with an N-linked oligosaccharide at $Asn^{56}$ of the ${\alpha}-subunit$. Both the rec-eCGs were obtained from CHO-K1 cells. The agonist activation of receptors was analyzed by measuring stimulation time and concentrations of rec-eCGs. Internalization values in the stably selected rat follicle-stimulating hormone receptor (rFSHR) and rat luteinizing/chorionic gonadotropin receptor (rLH/CGR) were highest at 50 min after stimulation with 10 ng of $rec-eCG{\beta}/{\alpha}$. The dose-dependent response was highest when 10 ng of $rec-eCG{\beta}/{\alpha}$ was used. The deglycosylated $eCG{\beta}/{\alpha}{\Delta}56$ mutant did not enhance the agonist-stimulated internalization. We concluded that the state of activation of rFSHR and rLH/CGR could be modulated through agonist-stimulated internalization. Our results suggested that the eLH/CGRs are mostly internalized within 60 min by agonist-stimulation by rec-eCG. We also suggested that the lack of responsiveness of the deglycosylated $eCG{\beta}/{\alpha}{\Delta}56$ was likely because the site of glycosylation played a pivotal role in agonist-stimulated internalization in cells expressing rFSHR and rLH/CGR.

• Reproductive and Developmental Biology
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• v.34 no.1
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• pp.33-40
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• 2010

Equine chorionic gonadotropin (eCG) is a heavily glycosylated glycoprotein composed of non-covalently linked $\alpha$- and $\beta$-subunits. To study the function and signal transduction of tethered recombinant-eCG (rec-eCG), a single chain eCG molecule was constructed, and the rec-eCG protein was prepared. In this study, we constructed 5 mutants (${\Delta}1$, ${\Delta}2$, ${\Delta}3$, ${\Delta}4$, and ${\Delta}5$) of rec-eCG using data about known glycoprotein hormones to analyze the role of specific follicle stimulating homone (FSH)-like activity. Three amino acids of certain specific sites were replaced with alanine. The expression vectors were transfected into CHO cells and subjected to G418 selection for 2~3 weeks. The media were collected and the quantity of secreted tethered rec-eCGs was quantified by ELISA. The LH- and FSH-like activities were assayed in terms of cAMP production by rat LH/CG and rat FSH receptors. Then, the metabolic clearance rate analyzed by the injection of rec-eCG (5 IU) into the tail vein was analyzed. The mutant eCGs (${\Delta}l$, ${\Delta}4$, and ${\Delta}5$) were transcripted, but not translated into proteins. Rec-eCG A2 was secreted in much lower amounts than the wild type. Only the rec-eCG ${\Delta}3$ ($\beta$-subunit: $Gln^{94}-Ile^{95}-Lys^{96}{\rightarrow}Ala^{94}-Ala^{95}-Ala^{96}$) was efficiently secreted. Although activity is low, its LH-like activity was similar to that of tethered $eCG{\beta\alpha}$. However, the FSH-like activity of rec-$eCG{\beta\alpha\Delta}3$ was completely flat. The result of the analysis of the metabolic clearance rate shoed the persistence of the mutant in the blood until 4 hours after the injection. After then, it almost disappeared at 8 hours. Taken together, these data suggest that 94~96 amino acid sequences in eCG $\beta$-subunit appear to be of utmost importance for signal transduction of the FSH receptor.

• Proceedings of the KSAR Conference
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• 2004.06a
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• pp.221-221
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• 2004

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 other species than the horse. To determine whether α and β subunits can be synthesized as a single polypeptide chain (tethered-eCG) and also display biological activity, the tethered-eCG molecule was constructed and transfected into Chinese hamster ovary (CHO-K1) cells. (omitted)