Stable Production of a Tethered Recombinant Eel Luteinizing Hormone Analog with High Potency in CHO DG44 Cells

Overview

Journal Curr Issues Mol Biol

Publisher MDPI

Specialty Molecular Biology

Date 2024 Jun 26

PMID 38921034

Authors

Munkhzaya Byambaragchaa

Sei Hyen Park

Sang-Gwon Kim

Min Gyu Shin

Shin-Kwon Kim

Sung-Pyo Hur

Myung-Hum Park

Myung-Hwa Kang

Kwan-Sik Min

Affiliations

Soon will be listed here.

Abstract

We produced a recombinant eel luteinizing hormone (rec-eel LH) analog with high potency in Chinese hamster ovary DG44 (CHO DG44) cells. The tethered eel LH mutant (LH-M), which had a linker comprising the equine chorionic gonadotropin (eLH/CG) β-subunit carboxyl-terminal peptide (CTP) region (amino acids 115 to 149), was inserted between the β-subunit and α-subunit of wild-type tethered eel LH (LH-wt). Monoclonal cells transfected with the tethered eel LH-wt and eel LH-M plasmids were isolated from five to nine clones of CHO DG44 cells, respectively. The secreted quantities abruptly increased on day 3, with peak levels of 5000-7500 ng/mL on day 9. The molecular weight of tethered rec-eel LH-wt was 32-36 kDa, while that of tethered rec-eel LH-M increased to approximately 38-44 kDa, indicating the detection of two bands. Treatment with the peptide N-glycanase F decreased the molecular weight by approximately 8 kDa. The oligosaccharides at the eCG β-subunit O-linked glycosylation sites were appropriately modified post-translation. The EC value and maximal responsiveness of eel LH-M increased by approximately 2.90- and 1.29-fold, respectively, indicating that the mutant exhibited more potent biological activity than eel LH-wt. Phosphorylated extracellular regulated kinase (pERK1/2) activation resulted in a sharp peak 5 min after agonist treatment, with a rapid decrease thereafter. These results indicate that the new tethered rec-eel LH analog had more potent activity in cAMP response than the tethered eel LH-wt in vitro. Taken together, this new eel LH analog can be produced in large quantities using a stable CHO DG44 cell system.

Citing Articles

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PMID: 39335273 PMC: 11428806. DOI: 10.3390/ani14182684.

References

Kamei H, Kawazoe I, Kaneko T, Aida K . Purification of follicle-stimulating hormone from immature Japanese eel, Anguilla japonica, and its biochemical properties and steroidogenic activities. Gen Comp Endocrinol. 2005; 143(3):257-66. DOI: 10.1016/j.ygcen.2005.03.009. View

Bhaskaran R, Ascoli M . The post-endocytotic fate of the gonadotropin receptors is an important determinant of the desensitization of gonadotropin responses. J Mol Endocrinol. 2005; 34(2):447-57. DOI: 10.1677/jme.1.01745. View

Moller T, Pedersen M, van Senten J, Seiersen S, Mathiesen J, Bouvier M . Dissecting the roles of GRK2 and GRK3 in μ-opioid receptor internalization and β-arrestin2 recruitment using CRISPR/Cas9-edited HEK293 cells. Sci Rep. 2020; 10(1):17395. PMC: 7567791. DOI: 10.1038/s41598-020-73674-0. View

Meta A, Hirashima M, Imamura T, Kawamura R, Yano K, Uehara K . High-yield preparation of recombinant human α-thrombin for therapeutic use. J Biosci Bioeng. 2015; 120(4):432-7. DOI: 10.1016/j.jbiosc.2015.02.001. View

Strasburger C, Vanuga P, Payer J, Pfeifer M, Popovic V, Bajnok L . MOD-4023, a long-acting carboxy-terminal peptide-modified human growth hormone: results of a Phase 2 study in growth hormone-deficient adults. Eur J Endocrinol. 2016; 176(3):283-294. PMC: 5292974. DOI: 10.1530/EJE-16-0748. View

Fares F, Guy R, Bar-Ilan A, Felikman Y, Fima E . Designing a long-acting human growth hormone (hGH) by fusing the carboxyl-terminal peptide of human chorionic gonadotropin beta-subunit to the coding sequence of hGH. Endocrinology. 2010; 151(9):4410-7. DOI: 10.1210/en.2009-1431. View

Matzuk M, Hsueh A, Lapolt P, Tsafriri A, Keene J, Boime I . The biological role of the carboxyl-terminal extension of human chorionic gonadotropin [corrected] beta-subunit. Endocrinology. 1990; 126(1):376-83. DOI: 10.1210/endo-126-1-376. View

Fares F, Yamabe S, Ben-Menahem D, Pixley M, Hsueh A, Boime I . Conversion of thyrotropin heterodimer to a biologically active single-chain. Endocrinology. 1998; 139(5):2459-64. DOI: 10.1210/endo.139.5.6021. View

Ren X, Reiter E, Ahn S, Kim J, Chen W, Lefkowitz R . Different G protein-coupled receptor kinases govern G protein and beta-arrestin-mediated signaling of V2 vasopressin receptor. Proc Natl Acad Sci U S A. 2005; 102(5):1448-53. PMC: 547876. DOI: 10.1073/pnas.0409534102. View

10.

Tao Y, Abell A, Liu X, Nakamura K, Segaloff D . Constitutive activation of G protein-coupled receptors as a result of selective substitution of a conserved leucine residue in transmembrane helix III. Mol Endocrinol. 2000; 14(8):1272-82. DOI: 10.1210/mend.14.8.0503. View

11.

Joshi L, Murata Y, Wondisford F, Szkudlinski M, Desai R, Weintraub B . Recombinant thyrotropin containing a beta-subunit chimera with the human chorionic gonadotropin-beta carboxy-terminus is biologically active, with a prolonged plasma half-life: role of carbohydrate in bioactivity and metabolic clearance. Endocrinology. 1995; 136(9):3839-48. DOI: 10.1210/endo.136.9.7544273. View

12.

Jazayeri S, Amiri-Yekta A, Gourabi H, Abd Emami B, Halfinezhad Z, Abolghasemi S . Comparative Assessment on the Expression Level of Recombinant Human Follicle-Stimulating Hormone (FSH) in Serum-Containing Versus Protein-Free Culture Media. Mol Biotechnol. 2017; 59(11-12):490-498. DOI: 10.1007/s12033-017-0037-4. View

13.

Furuhashi M, Shikone T, Fares F, Sugahara T, Hsueh A, Boime I . 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. Mol Endocrinol. 1995; 9(1):54-63. DOI: 10.1210/mend.9.1.7539107. View

14.

Banerjee A, Mahale S . Extracellular loop 3 substitutions K589N and A590S in FSH receptor increase FSH-induced receptor internalization and along with S588T substitution exhibit impaired ERK1/2 phosphorylation. Arch Biochem Biophys. 2018; 659:57-65. DOI: 10.1016/j.abb.2018.09.011. View

15.

Shenoy S, Drake M, Nelson C, Houtz D, Xiao K, Madabushi S . beta-arrestin-dependent, G protein-independent ERK1/2 activation by the beta2 adrenergic receptor. J Biol Chem. 2005; 281(2):1261-73. DOI: 10.1074/jbc.M506576200. View

16.

Hershkovitz O, Bar-Ilan A, Guy R, Felikman Y, Moschcovich L, Hwa V . In Vitro and in Vivo Characterization of MOD-4023, a Long-Acting Carboxy-Terminal Peptide (CTP)-Modified Human Growth Hormone. Mol Pharm. 2015; 13(2):631-9. DOI: 10.1021/acs.molpharmaceut.5b00868. View

17.

Boeta M, Zarco L . Luteogenic and luteotropic effects of eCG during pregnancy in the mare. Anim Reprod Sci. 2012; 130(1-2):57-62. DOI: 10.1016/j.anireprosci.2012.01.001. View

18.

Kara E, Crepieux P, Gauthier C, Martinat N, Piketty V, Guillou F . A phosphorylation cluster of five serine and threonine residues in the C-terminus of the follicle-stimulating hormone receptor is important for desensitization but not for beta-arrestin-mediated ERK activation. Mol Endocrinol. 2006; 20(11):3014-26. DOI: 10.1210/me.2006-0098. View

19.

Min K, Hattori N, Aikawa J, Shiota K, Ogawa T . 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. 1996; 43(5):585-93. DOI: 10.1507/endocrj.43.585. View

20.

Reiter E, Lefkowitz R . GRKs and beta-arrestins: roles in receptor silencing, trafficking and signaling. Trends Endocrinol Metab. 2006; 17(4):159-65. DOI: 10.1016/j.tem.2006.03.008. View