Effect of additional N-glycosylation signal in the N-terminal region on intracellular function of the human gonadotropin alpha-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 Asn52 and Asn78. 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 Asp3 and Gln5 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 Asn52 (alpha deltaAsn1), Asn78 (alpha deltaAsn2), and both (alpha deltaAsn(1 + 2)). The new sugar chain did not affect secretion and assembly except that 1) it increased the intracellular degradation of alpha deltaAsn(1 + 2), and 2) it augmented the assembly of alpha deltaAsn1 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.
Furuhashi
Madoka
M
Department of Obstetrics and Gynecology, Japanese Red Cross Nagoya First Hospital, Nagoya 453-8511, Japan.
Suganuma
Nobuhiko
N
D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics; D016466:CHO Cells; D018997:Chorionic Gonadotropin, beta Subunit, Human / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D019281:Dimerization; D024202:Electrophoretic Mobility Shift Assay; D005973:Glutamine / Q000235:genetics; D015292:Glycoprotein Hormones, alpha Subunit / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000378:metabolism; D009154:Mutation; D020079:Terminal Repeat Sequences; D013912:Threonine / Q000235:genetics
D018997:Chorionic Gonadotropin, beta Subunit, Human; D015292:Glycoprotein Hormones, alpha Subunit; D005973:Glutamine; D013912:Threonine; D001224:Aspartic Acid; D001216:Asparagine; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase