Mutagenesis and gene transfer define site-specific roles of the gonadotropin oligosaccharides.
Human chorionic gonadotropin (hCG), luteinizing hormone (LH), follicle-stimulating hormone and thyroid-stimulating hormone are a family of glycoprotein hormones that share a common alpha subunit but differ in their hormone-specific beta subunits. Using site-directed mutagenesis and gene-transfer, we analyzed the role of the N-linked oligosaccharides of alpha and chorionic gonadotropin (CG)beta in the secretion, assembly, and biologic activity of hCG. Absence of carbohydrate at alpha asparagine (Asn) 52 decreased combination with CG beta but did not alter monomer secretion. Absence of the alpha Asn78 oligosaccharide increased the degradation of the alpha subunit, but the presence of CG beta stabilized this alpha mutant in an efficiently formed dimer complex. Alternatively, absence of both alpha oligosaccharides slowed both secretion and dimer formation but allowed an intermediate level of alpha secreted or dimerized compared to the single-site mutants. Analysis of the CG beta glycosylation mutants revealed that absence of the Asn30 oligosaccharide, but not Asn13, slowed secretion but not assembly, whereas absence of both oligosaccharides slowed both secretion and dimer formation. Analysis of the receptor binding of the hCG glycosylation mutants showed that absence of any or all of the hCG N-linked oligosaccharides had only a minor effect on receptor affinity of the derivatives. However, the absence of alpha Asn52, but not the alpha Asn78 or the CG beta carbohydrate units, reduced the steroidogenic effect, unmasked differences in the beta oligosaccharides, and converted the deglycosylated derivatives into antagonists.
Matzuk
M M
MM
Department of Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110.
Boime
I
I
D000818:Animals; D006063:Chorionic Gonadotropin / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D006031:Glycosylation; D006062:Gonadotropins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D009154:Mutation; D009844:Oligosaccharides / Q000235:genetics / Q000378:metabolism*; D011967:Receptors, Gonadotropin / Q000378:metabolism; D014162:Transfection
D006063:Chorionic Gonadotropin; D006062:Gonadotropins; D009844:Oligosaccharides; D011967:Receptors, Gonadotropin