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

hCG, LH, FSH, and TSH are a family of heterodimeric glycoprotein hormones that contain a common alpha-subunit, but differ in their hormone-specific beta-subunits. The alpha-subunit has two N-glycosylation sites at Asn(52) and Asn(78). To obtain more information on the relationship between the structure and function of the alpha-subunit, we introduced a novel N-glycosylation site in the N-terminal region by mutating Asp(3) and Gln(5) into Asn and Thr, respectively. Glycosylation mutants were expressed alone or with hCGbeta-subunit in Chinese hamster ovary cells. New N-linked oligosaccharides were efficiently added to the wild-type and mutant alpha-subunits lacking N-glycan at Asn(52) (alphaDeltaAsn1), Asn(78) (alphaDeltaAsn2), and both (alphaDeltaAsn(1 + 2)). The new sugar chain did not affect secretion and assembly except that 1) it increased the intracellular degradation of alphaDeltaAsn(1 + 2), and 2) it augmented the assembly of alphaDeltaAsn1 with hCGbeta-subunit. Amino acid changes generated the attachment of O-glycosylation in free alpha-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 alpha-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 alpha-subunit.