title: Identification and structural analysis of the tetrasaccharide NeuAc alpha(2-->6)Gal beta(1-->4)GlcNAc beta(1-->3)Fuc alpha 1-->O-linked to serine 61 of human factor IX.
abstract: O-Linked fucose has been found attached to Thr/Ser residues within the sequence Cys-X-X-Gly-Gly-Thr/Ser-Cys in the N-terminal EGF domains of several coagulation/fibrinolytic proteins. Carbohydrate composition and mass spectrometric analyses of tryptic and thermolytic peptides containing the corresponding site (Ser-61) in the first EGF domain of human factor IX indicated the presence of a tetrasaccharide containing one residue each of sialic acid, galactose, N-acetylglucosamine, and fucose. The Ser-61 tetrasaccharide was not susceptible to alpha-fucosidase digestion. Fragments generated during mass spectrometric analysis indicated that fucose was the attachment sugar residue. The involvement of fucose in the carbohydrate-peptide linkage was confirmed by two-dimensional 1H NMR spectroscopic analysis of the glycopeptide containing factor IX residues 57-65. The complete structure of the tetrasaccharide was obtained by methylation analysis and two-dimensional 1H TOCSY and ROESY experiments as NeuAc alpha(2-->6)Gal beta(1-->4)GlcNAc beta(1-->3)Fuc alpha 1-->O-Ser61.
authors.affiliation: Department of Medicinal and Analytical Chemistry, Genentech, Inc., South San Francisco, California 94080.
authors.forename: R J
authors.initials: RJ
authors.lastname: Harris
authors.forename: H
authors.initials: H
authors.lastname: van Halbeek
authors.forename: J
authors.initials: J
authors.lastname: Glushka
authors.forename: L J
authors.initials: LJ
authors.lastname: Basa
authors.forename: V T
authors.initials: VT
authors.lastname: Ling
authors.forename: K J
authors.initials: KJ
authors.lastname: Smith
authors.forename: M W
authors.initials: MW
authors.lastname: Spellman
mesh_terms: D000595:Amino Acid Sequence; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid; D005164:Factor IX / Q000737:chemistry*; D005643:Fucose / Q000032:analysis; D006020:Glycopeptides / Q000302:isolation & purification; D006026:Glycoside Hydrolases; D006801:Humans; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry* / Q000302:isolation & purification; D010446:Peptide Fragments / Q000302:isolation & purification; D012694:Serine; D013912:Threonine
chemical_list: D006020:Glycopeptides; D009844:Oligosaccharides; D010446:Peptide Fragments; D005643:Fucose; D013912:Threonine; D012694:Serine; D005164:Factor IX; D006026:Glycoside Hydrolases