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 species other than the horse. In this study, recombinant tethered-eCG as well as its deglycosylated mutants were produced to determine if α- and β- subunits can be synthesized as a single polypeptide chain (tethered-eCG) and display biological activity. We found that tethered-eCG (T-βα) had both LH- and FSH-like activities comparable to dimeric eCG. Luteinizing hormone-like activity of tethered-eCGs deglycosylated at Asn56 (T-βα56) was decreased. In contrast, LH-like activity of eCG without O-glycosylated carboxyl-terminal peptide (CTP) (T-βcα56) was slightly decreased but still similar to T-βα. Double mutation at Asn56 and CTP (T-βcα56) caused marked decrease in the activity, indicating that both glycosylations at Asn56 and CTP are involved in LH-like activity in the tethered form. Interestingly, FSH-like activity remained in all deglycosylated eCG mutants (T-βα56, T-βcα and T-βcα56) as well as T-βα. The biological roles of oligosaccharides at Asn 56 of eCG α-subunit and O-linked peptide of β-subunit appear to be different in LH- and FSH-like activities in tethered-eCG.
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