Processing of O-linked glycosylation in the chimera consisting of alpha-subunit and carboxyl-terminal peptide of the human chorionic gonadotropin beta-subunit is affected by dimer formation with follicle-stimulating hormone beta-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. hCGbeta is unique among beta-subunits due to a carboxyl-terminal peptide (CTP) bearing four O-linked oligosaccharides. We previously reported that there were differences in O-glycosylation between two chimeras consisting of alpha-subunit and CTP, i.e. a variant with CTP at the N-terminal region (Calpha) and another analog with CTP at the C-terminus (alphaC) of the alpha-subunit. To address whether O-glycosylation is influenced by the heterodimer formation, Calpha and alphaC were expressed alone or with FSHbeta-subunit in Chinese hamster ovary cells. The O-linked glycosylation was assessed by continuous labeling with [(35)S]methionine/cysteine, immunoprecipitation with anti-alpha or anti-FSH serum, serial digestion with endoglycosidase-F and neuraminidase, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The decrease in molecular weight of dimeric chimeras digested with endoglycosidase-F was greater in Calpha than that in alphaC after treatment with neuraminidase, revealing that both chimeras have different numbers of sialic acids on O-linked carbohydrates. By treating with endoglycosidase-F, the dimeric alphaC migrated faster than its free form, whereas the mobility difference between assembled and unassembled forms of Calpha was very little. These data indicate that processing of O-glycosylation is affected by the backbone polypeptide chain(s).
Department of Obstetrics and Gynecology, Japanese Red Cross Nagoya First Hospital, Nagoya, Japan.
Madoka
M
Furuhashi
Nobuhiko
N
Suganuma
D000818:Animals; D016466:CHO Cells; D018997:Chorionic Gonadotropin, beta Subunit, Human / Q000737:chemistry* / Q000235:genetics*; D006224:Cricetinae; D003412:Cricetulus; D019281:Dimerization; D037201:Follicle Stimulating Hormone, beta Subunit / Q000737:chemistry*; D015292:Glycoprotein Hormones, alpha Subunit / Q000235:genetics*; D006031:Glycosylation; D010446:Peptide Fragments / Q000235:genetics; D011233:Precipitin Tests; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000378:metabolism*
D018997:Chorionic Gonadotropin, beta Subunit, Human; D037201:Follicle Stimulating Hormone, beta Subunit; D015292:Glycoprotein Hormones, alpha Subunit; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins