Development and Reproduction (한국발생생물학회지:발생과생식)

The Korean Society of Developmental Biology (한국발생생물학회)
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Specific Biological Activity of Equine Chorionic Gonadotropin (eCG) Glycosylation Sites in Cells Expressing Equine Luteinizing Hormone/CG (eLH/CG) Receptor

  • Received : 2021.09.22
  • Accepted : 2021.11.23
  • Published : 2021.12.31
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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 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.

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References

  1. Apparailly F, Combarnous Y (1994) Role of sialic acid residues in the in vitro superactivity of human choriogonadotropin (hCG) in rat Leydig cells. Biochim Biophys Acta Mol Cell Res 1224:559-565. https://doi.org/10.1016/0167-4889(94)90294-1
  2. Bhaskaran RS, Ascoli M (2005) The post-endocytotic fate of the gonadotropin receptors is an important determinant of the desensitization of gonadotropin responses. J Mol Endocrinol 34:447-457. https://doi.org/10.1677/jme.1.01745
  3. Bishop LA, Robertson DM, Cahir N, Schofield PR (1994) Specific roles for the asparagine-linked carbohydrate residues of recombinant human follicle stimulating hormone in receptor binding and signal transduction. Mol Endocrinol 8:722-731. https://doi.org/10.1210/mend.8.6.7935488
  4. Boeta M, Zarco L (2012) Luteogenic and luteotropic effects of eCG during pregnancy in the mare. Anim Reprod Sci 130:57-62. https://doi.org/10.1016/j.anireprosci.2012.01.001
  5. Bousfield GR, Sugino H, Ward DN (1985) 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 260:9531-9533. https://doi.org/10.1016/S0021-9258(17)39266-9
  6. Byambaragchaa M, Ahn TY, Choi SH, Kang MH, Min KS (2021a) Functional characterization of naturally-occurring constitutively activating/inactivating mutations in equine follicle-stimulating hormone receptor (eFSHR). Anim Biosci. https://doi.org/10.5713/ab.21.0246
  7. Byambaragchaa M, Choi SH, Kim DW, Min KS (2021b) Constitutive activating eel luteinizing hormone receptors induce constitutively signal transduction and inactivating mutants impair biological activity. Dev Reprod 25:133-143. https://doi.org/10.12717/DR.2021.25.3.133
  8. Byambaragchaa M, Kim DJ, Kang MH, Min KS (2018) Site specificity of eel luteinizing hormone N-linked oligosaccharides in signal transduction. Gen Comp Endocrinol 268:50-56. https://doi.org/10.1016/j.ygcen.2018.07.015
  9. Byambaragchaa M, Seong HK, Choi SH, Kim DJ, Kang MH, Min KS (2021c) Constitutively activating mutants of equine LH/CGR constitutively induce signal transduction and inactivating mutations impair biological activity and cell-surface receptor loss in vitro. Int J Mol Sci 22:10723. https://doi.org/10.3390/ijms221910723
  10. Chen W, Bahl OP (1991) Recombinant carbohydrate variant to human choriogonadotropin β-subunit (hCGβ) descarboxyl terminus (115-145). J Biol Chem 266:6246-6251. https://doi.org/10.1016/S0021-9258(18)38110-9
  11. Chopineau M, Maurel MC, Combarnous Y, Durand P (1993) Topography of equine chorionic gonadotropin epitopes relative to the luteinizing hormone and follicle-stimulating hormone receptor interaction sites. Mol Cell Endocrinol 92:229-239. https://doi.org/10.1016/0303-7207(93)90013-A
  12. Conley AJ (2016) Review of the reproductive endocrinology of the pregnant and parturient mare. Theriogenology 86:355-365. https://doi.org/10.1016/j.theriogenology.2016.04.049
  13. Crawford RJ, Tregear GW, Niall HD (1986) The nucleotide sequences of baboon chorionic gonadotropin β-subunit genes have diverged from the human. Gene 46:161-169. https://doi.org/10.1016/0378-1119(86)90400-2
  14. Flores-Flores G, Velazquez-Canton E, Boeta M, Zarco L (2014) Luteoprotective role of equine chorionic gonadotropin (eCG) during pregnancy in the mare. Reprod Domest Anim 49:420-426. https://doi.org/10.1111/rda.12290
  15. Galet C, Guillou F, Foulon-Gauze F, Combarnous Y, Chopineau M (2009) The β104-109 sequence is essential for the secretion of correctly folded single-chain βα horse LH/CG and for its FSH activity. J Endocrinol 203:167-174. https://doi.org/10.1677/JOE-09-0141
  16. Galet C, Hirakawa T, Ascoli M (2004) 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 18:434-446. https://doi.org/10.1210/me.2003-0293
  17. Galet C, Min L, Narayanan R, Kishi M, Weigel NL, Ascoli M (2003) 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 17:411-422. https://doi.org/10.1210/me.2002-0161
  18. Hirakawa T, Ascoli M (2003) 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. Mol Endocrinol 17:2189-2200. https://doi.org/10.1210/me.2003-0205
  19. Hokke CH, Roosenboom MJH, Thomas-Oates JE, Kamerling JP, Vliegenthart JFG (1994) Structure determination of the disialylated poly-(N-acetyllactosamine)-containing O-linked carbohydrate chains of equine chorionic gonadotropin. Glycoconj J 11:35-41. https://doi.org/10.1007/BF00732430
  20. Huang J, Chen F, Puett D (1993) Amino/carboxyl-terminal deletion mutants of human choriogonadotropin β. J Biol Chem 268:9311-9315. https://doi.org/10.1016/S0021-9258(18)98351-1
  21. Kim JM, Byambaragchaa M, Kang MH, Min KS (2018) The C-terminal phosphorylation sites of eel follicle-stimulating hormone receptor are important role in the signal transduction. Dev Reprod 22:143-153. https://doi.org/10.12717/DR.2018.22.2.143
  22. Kim JM, Munkhuu O, Byambaragchaa M, Lee BI, Kim SK, Kang MH, Kim DJ, Min KS (2019) Site-specific roles of N-linked oligosaccharides in recombinant eel follicle-stimulating hormone for secretion and signal transduction. Gen Comp Endocrinol 276:37-44. https://doi.org/10.1016/j.ygcen.2019.03.003
  23. Kishi M, Liu X, Hirakawa T, Reczek D, Bretscher A, Ascoli M (2001) 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 15:1624-1635. https://doi.org/10.1210/mend.15.9.0698
  24. Krishnamurthy H, Kishi H, Shi M, Galet C, Bhaskaran RS, Hirakawa T, Ascoli M (2003) Postendocytotic trafficking of the follicle-stimulating hormone (FSH)-FSH receptor complex. Mol Endocrinol 17:2162-2176. https://doi.org/10.1210/me.2003-0118
  25. Lee SY, Byambaragchaa M, Choi SH, Kang HJ, Kang MH, Min KS (2021) 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. BMC Biotechnol 21:52. https://doi.org/10.1186/s12896-021-00712-8
  26. Lee SY, Byambaragchaa M, Kim JS, Seong HK, Kang MH, Min KS (2017) Biochemical characterization of recombinant equine chorionic gonadotropin (rec-eCG), using CHO cells and PathHunter parental cells expressing equine luteinizing hormone/chorionic gonadotropin receptors (eLH/CGR). J Life Sci 27:864-872. https://doi.org/10.5352/JLS.2017.27.8.864
  27. Legardinier S, Poirier JC, Klett D, Combarnous Y, Cahoreau C (2008) Stability and biological activities of heterodimeric and single-chain equine LH/chorionic gonadotropin variants. J Mol Endocrinol 40:185-198. https://doi.org/10.1677/JME-07-0151
  28. Matzuk MM, Boime I (1988a) The role of the asparagine-linked oligosaccharides of the α subunit in the secretion and assembly of human chorionic gonadotropin. J Cell Biol 106:1049-1059. https://doi.org/10.1083/jcb.106.4.1049
  29. Matzuk MM, Boime I (1988b) Site-specific mutagenesis defines the intracellular role of the asparagine-linked oligosaccharides of chorionic gonadotrophin β subunit. J Biol Chem 263:17106-17111. https://doi.org/10.1016/S0021-9258(18)37504-5
  30. Matzuk MM, Hsueh AJW, Lapolt P, Tsafriri A, Keene JL, Boime I (1990) The biological role of the carboxyl-terminal extension of human chorionic gonadotroin β-subunit. Endocrinology 126:376-383. https://doi.org/10.1210/endo-126-1-376
  31. Matzuk MM, Keene JL, Boime I (1989) Site specificity of the chorionic gonadotropin N-linked oligosaccharides in signal transduction. J Biol Chem 264:2409-2414. https://doi.org/10.1016/S0021-9258(19)81628-9
  32. Min KS, Hattori N, Aikawa JI, Shiota K, Ogawa T (1996) Site-directed mutagenesis of recombinant equine chorionic gonadotropin/luteinizing hormone: Differential role of oligosaccharides in luteinizing hormone- and follicle-stimulating hormone-like activities. Endocr J 43:585-593. https://doi.org/10.1507/endocrj.43.585
  33. Min KS, Hiyama T, Seong HH, Hattori N, Tanaka S, Shiota K (2004) Biological activities of tethered equine chorionic gonadotropin (eCG) and its deglycosylated mutants. J Reprod Dev 50:297-304. https://doi.org/10.1262/jrd.50.297
  34. Min KS, Park JJ, Byambaragchaa M, Kang MH (2019) Characterization of tethered equine chorionic gonadotropin and its deglycosylated mutants by ovulation stimulation in mice. BMC Biotechnol 19:60. https://doi.org/10.1186/s12896-019-0550-6
  35. Min KS, Park JJ, Lee SY, Byambaragchaa M, Kang MH (2020) Comparative gene expression profiling of mouse ovaries upon stimulation with natural equine chorionic gonadotropin (N-eCG) and tethered recombinant-eCG (R-eCG). BMC Biotechnol 20:59. https://doi.org/10.1186/s12896-020-00653-8
  36. Murphy BD, Martinuk SD (1991) Equine chorionic gonadotropin. Endocr Rev 12:27-44. https://doi.org/10.1210/edrv-12-1-27
  37. Park JJ, Seong HK, Kim JS, Munkhzaya B, Kang MH, Min KS (2017) Internalization of rat FSH and LH/CG receptors by rec-eCG in CHO-K1 cells. Dev Reprod 21:111-120. https://doi.org/10.12717/DR.2017.21.2.111
  38. Saneyoshi T, Min KS, Ma XJ, Nambo Y, Hiyama T, Tanaka S, Shiota K (2001) Equine follicle-stimulating hormone: Molecular cloning of β subunit and biological role of the asparaginelinked oligosaccharide at asparagine56 of α subunit. Biol Reprod 65:1686-1690. https://doi.org/10.1095/biolreprod65.6.1686
  39. Sherman GB, Wolfe MW, Farmerie TA, Clay CM, Threadgill DS, Sharp DC, Nilson JH (1992) A single gene encodes the β-subunits of equine luteinizing hormone and chorionic gonadotropin. Mol Endocrinol 6:951-959. https://doi.org/10.1210/me.6.6.951
  40. Sugahara T, Grootenhuis PDJ, Sato A, Kudo M, Ben-Menahem D, Pixley MR, Hsuen AJW, Boime I (1996) 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 125:71-77. https://doi.org/10.1016/S0303-7207(96)03944-5
  41. Sugino H, Bousfield GR, Moore Jr. WT, Ward DN (1987) Structural studies on equine glycoprotein hormones: Amino acid sequence of equine chorionic gonadotropin β-subunit. J Biol Chem 262:8603-8609. https://doi.org/10.1016/S0021-9258(18)47456-X
  42. Talmadge K, Vamvakopoulos NC, Fiddes JC (1984) Evolution of the genes for the β subunits of human chorionic gonadotropin and luteinizing hormone. Nature 307:37-40. https://doi.org/10.1038/307037a0
  43. Valove FM, Finch C, Anasti JN, Froehlich J, Flack MR (1994) Receptor binding and signal transduction are dissociable functions requiring different sites on follicle-stimulating hormone. Endocrinology 135:2657-2661. https://doi.org/10.1210/en.135.6.2657