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Japanese eel Luteinizing Hormone (LH) : The Role of ASN–LINKED Oligosaccharides For Hormonal Activity
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- URLhttps://db.koreascholar.com/Article/Detail/318112
The glycoprotein hormone family consists of luteinizing hormone (LH), follicle stimulating hormone (FSH) and thyroid stimulating hormone, which are secreted by the pituitary gland in all mammalian species, and choriogonadotropin (CG), which is secreted by the placenta in primates and equids. The hormones are composed of a common α subunit and a hormone specific β subunit which are non-covalently associated. Recent advances in biotechnology, particularly in the production of recombinant proteins, have provided opportunities to produce sufficient quantities of recombinant fish GTHs using various expression hosts. Japanese eel Anguilla japonica is one of the most important fish species being aquacultured in Japan but is hampered from the fact that this species does not reproduce in captivity. Artificial induction of gonadal maturation has been successful by administration of pituitary extracts or human chorionic gonadotropin, but the understanding the regulatory mechanism of gonadal development moderated by follicle stimulating hormone (FSH) and luteinizing hormone (LH) remains elusive due to lack of suitable amounts of eel gonadotropins (GTHs). In the present study, we produced tethered rec-eel LH and deglycosylated mutants (56, 79 and 56-79 of α subunit; 10 of β-subunit) of Asn-linked oligosaccharides in CHO suspension cells.
Luteinizing hormone acts through binding to its specific receptor. Binding of ligand to the receptor activates the adenosine 3',5'-cyclic monophosphate (cAMP) pathway (McFarland et al., 1989; Ji and Ji, 1991a; Rose, 1998) and the inositol 1 phosphate (IP1) secondary messenger systems. After stimulation of eelLH/CG receptor transfected CHO cells with rec-LH wild type (wt) and mutant hormones as a ligand, production of cAMP and IP-1 were evaluated (Cisbio). cAMP IC-50 values by rec-eelLH wt; αΔ56; αΔ79; αΔ56_79 and βΔ10 were 606.2; 374.9; 100.3; 14.2 and 210.9 ng/ml, respectively. IP-1 IC-50 values by rec-eelLH wt; αΔ56; αΔ79; αΔ56.79 and βΔ10 were 28.3; 16.04; 4.3; 2.1 and 3.6ng/ml, respectively too. As seen in both of the second messenger production, general stimulatory pattern is analogous. cAMP and IP-1 stimulation by wild type and αΔ56, as well as αΔ79 and βΔ10 were approximate, but the stimulating effect of double mutant (αΔ56_ 79) was drastically higher. According to the data, deglycosylated eelLH may bind to the receptor with high affinity and cAMP production is gradually increased.
Furthermore, receptor activation by tethered rec-eel mutant ligands (FreeStyle CHO-MAX Expression System) will be evaluated with β arrestin recruitment and GPCR internalization for N-linked oligosaccharides’ biological role in activation of eelLH/CGR.
