title: Isolation and structure determination of the intact sialylated N-linked carbohydrate chains of recombinant human follitropin expressed in Chinese hamster ovary cells.
abstract: Biologically active recombinant human follitropin has been expressed in Chinese hamster ovary cells. The carbohydrate chains of the recombinant glycoprotein hormone were enzymatically released by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F. The oligosaccharides were separated from the N-deglycosylated protein by gel-permeation chromatography on Bio-Gel P-100, and fractionated by a combination of FPLC on Mono Q and HPLC on Lichrosorb-NH2. The structures of the carbohydrate chains were determined by 500- or 600-MHz 1H-NMR spectroscopy. The following types of carbohydrates occur: monosialylated diantennary (10%), disialylated diantennary (43%), disialylated tri-antennary (5%), trisialylated tri-antennary (13%), trisialylated tri'-antennary (8%), and tetrasialylated tetraantennary (12%) N-acetyllactosamine type of carbohydrate chains, all bearing exclusively alpha 2-3-linked N-acetylneuraminic acid (Neu5Ac). Previously, for pituitary follitropin mono-, di-, tri-, tri'-, and tetra-antennary oligosaccharides containing alpha 2-3- as well as alpha 2-6-linked Neu5Ac residues were reported. The bisecting GlcNAc residues present in native follitropin were not detected in the recombinant glycoprotein. Of the oligosaccharides 29% have an alpha 1-6-linked Fuc residue at the asparagine-bound GlcNAc, whereas this amount is about 50% in pituitary follitropin. In some of the tri-, tri'- and tetra-antennary oligosaccharide fractions small amounts (less than 5%) of compounds were detected having one or more additional N-acetyllactosamine units.
authors.affiliation: Bijvoet Center, Department of Bio-Organic Chemistry, Utrecht University, The Netherlands.
authors.forename: K
authors.initials: K
authors.lastname: Hård
authors.forename: A
authors.initials: A
authors.lastname: Mekking
authors.forename: J B
authors.initials: JB
authors.lastname: Damm
authors.forename: J P
authors.initials: JP
authors.lastname: Kamerling
authors.forename: W
authors.initials: W
authors.lastname: de Boer
authors.forename: R A
authors.initials: RA
authors.lastname: Wijnands
authors.forename: J F
authors.initials: JF
authors.lastname: Vliegenthart
mesh_terms: D000818:Animals; D002240:Carbohydrate Sequence; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D005640:Follicle Stimulating Hormone / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D008969:Molecular Sequence Data; D009005:Monosaccharides / Q000032:analysis; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012794:Sialic Acids / Q000032:analysis
chemical_list: D009005:Monosaccharides; D011994:Recombinant Proteins; D012794:Sialic Acids; D005640:Follicle Stimulating Hormone