Production and Functional Characterization of Recombinant Eel Follicle-Stimulating Hormone Using an hCG β-Subunit Carboxyl-Terminal Peptide and a Flexible GGSA Linker

This study aimed to develop a glycoengineered recombinant eel follicle-stimulating hormone (rec-eel FSH) with modified O-linked glycosylation and to establish a stable mammalian cell-based production platform using Chinese hamster ovary DG44 (CHO DG44) cells. To introduce additional sites potentially accessible to O-linked glycosylation, the carboxyl-terminal peptide (CTP) region of the human chorionic gonadotropin (hCG) β-subunit (amino acids 115–145) was inserted between the β- and α-subunits of wild-type single-chain eel FSH (FSH-wt). In addition, a flexible 15-amino-acid linker, [(GGSA)3GGG], was incorporated between the FSH β-subunit and the hCG β-subunit CTP region to improve the structural accessibility of the CTP sequence, generating the FSH-GGSA-hM-Strep construct. FSH-GGSA-hM-Strep was stably expressed in CHO DG44 cells and purified using Strep-Tactin XT affinity chromatography. Western blotting and silver staining revealed a heterogeneous broad band corresponding to an apparent molecular mass of approximately 54–57 kDa, consistent with extensive post-translational modification. Enzymatic deglycosylation with PNGase F, O-glycosidase, and neuraminidase markedly reduced the apparent molecular mass, indicating that N-linked and O-linked glycans and terminal sialic acid residues contribute substantially to the increased molecular mass and molecular heterogeneity of the recombinant hormone. Functional characterization confirmed that FSH-GGSA-hM-Strep exhibited biological activity and elicited a dose-dependent response through the eel FSH receptor. Receptor stimulation induced rapid, transient ERK1/2 phosphorylation, peaking at 5 min, whereas PKA inhibition with H89 markedly attenuated this early response, supporting the involvement of cAMP/PKA-dependent signaling in ERK1/2 activation. In addition, β-arrestin 1 disruption altered the temporal pattern of ERK1/2 activation, with PKA inhibition abolishing the early response and revealing delayed ERK1/2 phosphorylation, suggesting temporally distinct PKA-dependent and β-arrestin-associated signaling mechanisms. These findings demonstrate that incorporation of the flexible [(GGSA)3GGG] linker together with the hCG β-subunit CTP sequence represents an effective strategy for generating a structurally modified recombinant eel FSH with engineered glycosylation-associated molecular heterogeneity. The establishment of stable CHO DG44 producer cells provides a potential platform for the scalable production of biologically active glycoengineered eel FSH.