Site specificity of eel luteinizing hormone N-linked oligosaccharides in signal transduction

Eel luteinizing hormone (eelLH) is composed of a common α-subunit and hormone specific β-subunit, both of which contain asparagine-linked carbohydrate residues, located at positions 56 and 79 on the α-subunit and position 10 on the β-subunit. The specific roles of the individual carbohydrate chains are poorly defined in eel. Thus, we characterized the biologically active single chains by fusing the α-subunit to the carboxyl terminal region of the eelLH β-subunit. Site-directed mutagenesis of the three N-linked glycosylation sites was performed to examine the function of individual glycosylation sites in secretion and signal transduction. The absence of the Asn⁷⁹N-linked sugar chain slightly reduced secretion in Chinese hamster ovary (CHO) cells. The expression of eelLHβ/α (wild-type) in CHO suspension cells was increased by approximately 2-fold higher than that of attached CHO cells. By western blotting analysis, the molecular weight of wild-type was found to be 32 kDa. Mutants (β/α△56, β/α△79, and β△10/α) of the oligosaccharide chain at a single site showed molecular weights that were reduced by approximately 10%. However, the double mutant (β/α△56.79) molecular weight was decreased by more than 20% compared to the wild-type. Enzymatic digestion of oligosaccharides using PNGaseF treatment showed that the molecular weights of all mutants, including wild-type, were reduced to 25 kDa. The results obtained in the absence of carbohydrates at Asn⁵⁶ of the α-subunit and at Asn¹⁰ of the β-subunit revealed their roles in signal transduction through the eelLH receptor. The EC₅₀ value of the cAMP response at Asn⁷⁹ of the α-subunit was increased by 5-fold, whereas the maximum response was dramatically reduced to 17.8% of wild-type levels. Specifically, removal of the carbohydrates at double mutant (β/α△56.79) is approximately 85% to wild-type levels in biopotency. These results revealed the site-specific roles of eelLH carbohydrate residues. Our data suggest that N-linked oligosaccharide chains play a pivotal role in biological activity through the eelLH receptor.