Specific Biological Activity of Equine Chorionic Gonadotropin (eCG) Glycosylation Sites in Cells Expressing Equine Luteinizing Hormone/CG (eLH/ CG) Receptor
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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 wildtype 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 receCG glycosylation sites in equidaes.
Abstract
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 wildtype 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 receCG glycosylation sites in equidaes.
참고문헌 (43)
- Apparailly F, "Role of sialic acid residues in the in vitro superactivity of human choriogonadotropin(hCG)in rat Leydig cells", Biochim Biophys Acta Mol Cell Res, 1224, pp.559~565 (1994)
- Bhaskaran RS, "The post-endocytotic fate of the gonadotropin receptors is an important determinant of the desensitization of gonadotropin responses", J Mol Endocrinol, 34, pp.447~457 (2005)
- Bishop LA, "Specific roles for the asparagine-linked carbohydrate residues of recombinant human follicle stimulating hormone in receptor binding and signal transduction", Mol Endocrinol, 8, pp.722~731 (1994)
- Boeta M, "Luteogenic and luteotropic effects of eCG during pregnancy in the mare", Anim Reprod Sci, 130, pp.57~62 (2012)
- Bousfield GR, "Demonstration of a COOH-terminal extension on equine lutropin by means of a common acid-labile bond in equine lutropin and equine chorionic gonadotropin", J Biol Chem, 260, pp.9531~9533 (1985)
- BYAMBARAGCHAA MUNKHZAYA, "Functional characterization of naturally-occurring constitutively activating", inactivating mutations in equine follicle-stimulating hormone receptor, 2022, pp.35(3), 399~409[DOI]
- Munkhzaya Byambaragchaa, "Constitutive Activating Eel Luteinizing Hormone Receptors Induce Constitutively Signal Transduction and Inactivating Mutants Impair Biological Activity", 발생과 생식, 25(3), pp.133~143 (2021), 한국발생생물학회
- Byambaragchaa M, "Site specificity of eel luteinizing hormone N-linked oligosaccharides in signal transduction", Gen Comp Endocrinol, 268, pp.50~56 (2018)
- Byambaragchaa M, "Constitutively activating mutants of equine LH", CGR constitutively induce signal transduction and inactivating mutations impair biological activity and cell-surface receptor loss in vitro, 2021, pp.22, 10723~
- Chen W, "Recombinant carbohydrate variant to human choriogonadotropin β-subunit(hCGβ)descarboxyl terminus(115-145)", J Biol Chem, 266, pp.6246~6251 (1991)
- Chopineau M, "Topography of equine chorionic gonadotropin epitopes relative to the luteinizing hormone and follicle-stimulating hormone receptor interaction sites", Mol Cell Endocrinol, 92, pp.229~239 (1993)
- Conley AJ, "Review of the reproductive endocrinology of the pregnant and parturient mare", Theriogenology, 86, pp.355~365 (2016)
- Crawford RJ, "The nucleotide sequences of baboon chorionic gonadotropin β-subunit genes have diverged from the human", Gene, 46, pp.161~169 (1986)
- Flores-Flores G, "Luteoprotective role of equine chorionic gonadotropin(eCG)during pregnancy in the mare", Reprod Domest Anim, 49, pp.420~426 (2014)
- Galet C, "The β104–109 sequence is essential for the secretion of correctly folded single-chain βα horse LH", CG and for its FSH activity, 2009, pp.203, 167~174
- Galet C, "The postendocytotic trafficking of the human lutropin receptor is mediated by a transferable motif consisting of the C-terminal cysteine and an upstream leucine", Mol Endocrinol, 18, pp.434~446 (2004)
- Galet C, "Identification of a transferable two-amino-acid motif(GT)present in the C-terminal tail of the human lutropin receptor that redirects internalized G protein-coupled receptors from a degradation to a recycling pathway", Mol Endocrinol, 17, pp.411~422 (2003)
- Hirakawa T, "The lutropin", choriogonadotropin receptor-induced phosphorylation of the extracellular signal-regulated kinases in Leydig cells is mediated by a protein kinase A-dependent activation of Ras, 2003, pp.17, 2189~2200
- Hokke CH, "Structure determination of the disialylated poly-(N-acetyllactosamine)-containing O-linked carbohydrate chains of equine chorionic gonadotropin", Glycoconj J, 11, pp.35~41 (1994)
- Huang J, "Amino", carboxyl-terminal deletion mutants of human choriogonadotropin β, 1993, pp.268, 9311~9315
- Jeong-Min Kim, "The C-terminal Phosphorylation Sites of eel Follicle-Stimulating Hormone Receptor are Important Role in the Signal Transduction", 발생과 생식, 22(2), pp.143~153 (2018), 한국발생생물학회
- Kim JM, "Site-specific roles of N-linked oligosaccharides in recombinant eel follicle-stimulating hormone for secretion and signal transduction", Gen Comp Endocrinol, 276, pp.37~44 (2019)
- Kishi M, "Identification of two distinct structural motifs that, when added to the C-terminal tail of the rat LH receptor, redirect the internalized hormone-receptor complex from a degradation to a recycling pathway", Mol Endocrinol, 15, pp.1624~1635 (2001)
- Krishnamurthy H, "Postendocytotic trafficking of the follicle-stimulating hormone(FSH)-FSH receptor complex", Mol Endocrinol, 17, pp.2162~2176 (2003)
- Lee SY, "Roles of N-linked and O-linked glycosylation sites in the activity of equine chorionic gonadotropin in cells expressing rat luteinizing hormone", chorionic gonadotropin receptor and follicle-stimulating hormone receptor, 2021, pp.21, 52~
- 이소연, "Biochemical Characterization of Recombinant Equine Chorionic Gonadotropin (rec-eCG), Using CHO Cells and PathHunter Parental Cells Expressing Equine Luteinizing Hormone", Chorionic Gonadotropin Receptors (eLH, 생명과학회지, pp.2017, 27(8)[DOI]
- Legardinier S, "Stability and biological activities of heterodimeric and single-chain equine LH", chorionic gonadotropin variants, 2008, pp.40, 185~198
- Matzuk MM, "The role of the asparagine-linked oligosaccharides of the α subunit in the secretion and assembly of human chorionic gonadotropin", J Cell Biol, 106, pp.1049~1059 (1988)
- Matzuk MM, "Site-specific mutagenesis defines the intracellular role of the asparagine-linked oligosaccharides of chorionic gonadotrophin β subunit", J Biol Chem, 263, pp.17106~17111 (1988)
- Matzuk MM, "The biological role of the carboxyl-terminal extension of human chorionic gonadotroin β-subunit", Endocrinology, 126, pp.376~383 (1990)
- Matzuk MM, "Site specificity of the chorionic gonadotropin N-linked oligosaccharides in signal transduction", J Biol Chem, 264, pp.2409~2414 (1989)
- Min KS, "Site-directed mutagenesis of recombinant equine chorionic gonadotropin", luteinizing hormone : Differential role of oligosaccharides in luteinizing hormone-and follicle-stimulating hormone-like activities, 1996, pp.43, 585~593
- Min KS, "Biological activities of tethered equine chorionic gonadotropin(eCG)and its deglycosylated mutants", J Reprod Dev, 50, pp.297~304 (2004)
- Min KS, "Characterization of tethered equine chorionic gonadotropin and its deglycosylated mutants by ovulation stimulation in mice", BMC Biotechnol, 19, pp.60~ (2019)
- Min KS, "Comparative gene expression profiling of mouse ovaries upon stimulation with natural equine chorionic gonadotropin(N-eCG)and tethered recombinant-eCG(R-eCG)", BMC Biotechnol, 20, pp.59~ (2020)
- Murphy BD, "Equine chorionic gonadotropin", Endocr Rev, 12, pp.27~44 (1991)
- 박종주, "Internalization of Rat FSH and LH", CG Receptors by rec-eCG in CHO-K1 Cells, 2017, pp.21(2), 111~120[DOI]
- Saneyoshi T, "Equine folliclestimulating hormone : Molecular cloning of β subunit and biological role of the asparaginelinked oligosaccharide at asparagine56 of α subunit", Biol Reprod, 65, pp.1686~1690 (2001)
- Sherman GB, "A single gene encodes the β-subunits of equine luteinizing hormone and chorionic gonadotropin", Mol Endocrinol, 6, pp.951~959 (1992)
- Sugahara T, "Expression of biologically active fusion genes encoding the common α subunit and either the CGβ or FSHβ subunits : Role of a linker sequence", Mol Cell Endocrinol, 125, pp.71~77 (1996)
- Sugino H, "Structural studies on equine glycoprotein hormones : Amino acid sequence of equine chorionic gonadotropin β-subunit", J Biol Chem, 262, pp.8603~8609 (1987)
- Talmadge K, "Evolution of the genes for the β subunits of human chorionic gonadotropin and luteinizing hormone", Nature, 307, pp.37~40 (1984)
- Valove FM, "Receptor binding and signal transduction are dissociable functions requiring different sites on follicle-stimulating hormone", Endocrinology, 135, pp.2657~2661 (1994)