Effect of additional N-glycosylation signal in the N-terminal region on intracellular function of the human gonadotropin α-subunit

Abstract

hCG, LH, FSH, and TSH are a family of heterodimeric glycoprotein hormones that contain a common α-subunit, but differ in their hormone-specific β-subunits. The α-subunit has two N-glycosylation sites at Asn52 and Asn78. To obtain more information on the relationship between the structure and function of the α-subunit, we introduced a novel N-glycosylation site in the N-terminal region by mutating Asp3 and Gln5 into Asn and Thr, respectively. Glycosylation mutants were expressed alone or with hCGβ-subunit in Chinese hamster ovary cells. New N-linked oligosaccharides were efficiently added to the wild-type and mutant α-subunits lacking N-glycan at Asn52 (αΔAsn1), Asn78 (αΔAsn2), and both (αΔAsn(1 + 2)). The new sugar chain did not affect secretion and assembly except that 1) it increased the intracellular degradation of αΔAsn(1 + 2), and 2) it augmented the assembly of αΔAsn1 with hCGβ-subunit. Amino acid changes generated the attachment of O-glycosylation in free α-subunit but not in assembled form. These data indicate that the newly introduced N-glycosylation consensus sequence is functional, and that the N-terminal region of the α-subunit is flexible and can be modified without affecting the intracellular function. Furthermore, amino acid sequences in the N-terminus are involved in the O-glycosylation in free α-subunit.