title: Human factor IX has a tetrasaccharide O-glycosidically linked to serine 61 through the fucose residue.
abstract: We have recently discovered unusual sugar chains (xylose (Xyl)-glucose (Glc) and (Xyl)2-Glc) linked to a serine residue in the epidermal growth factor (EGF)-like domains of human and bovine clotting factors VII (Ser-52), IX (Ser-53), and protein Z (Ser-53), in addition to bovine platelet glycoprotein thrombospondin. We now have evidence of another modification in the first EGF-like domain of human factor IX, which proved to be a tetrasaccharide O-fucosidically linked to Ser-61. Two large peptides containing Ser-61 (positions 44-63), named hIX-GP1 and hIX-GP2, were first isolated from the lysyl endopeptidase-digest of human factor IX, by reversed-phase high performance liquid chromatography (HPLC). Data on the component sugar analysis after pyridylamination (PA) and sialic acid analysis of the isolated peptides indicated that they contained 1 mol each of galactose (Gal), fucose (Fuc), N-acetylglucosamine (GlcNAc), and N-acetylneuraminic acid (NeuAc), in addition to Glc and Xyl. hIX-GP1 was further digested with asparaginyl endopeptidase, and two glycopeptides containing Ser-61, named N-3 (positions 59-63) and N-9 (positions 55-63), were isolated, respectively. These glycopeptides were both composed of 1 mol each of Gal, Fuc, GlcNAc, and NeuAc but did not contain Xyl and Glc. Moreover, the data on beta-elimination for N-9 and of the fast atom bombardment mass spectrometric analysis on peptide N-3 suggested the presence of a tetrasaccharide linked to Ser-61. An analysis of the PA-oligosaccharide released from hIX-GP1 by hydrazinolysis followed by pyridylamination revealed that the reducing end was PA-Fuc. All the results support the proposal that human factor IX has a novel tetrasaccharide consisting of 1 mol each of Gal, Fuc, GlcNAc, and NeuAc, which is O-glycosidically linked to Ser-61 through the Fuc residue.
authors.affiliation: Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Fukuoka, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Nishimura
authors.forename: T
authors.initials: T
authors.lastname: Takao
authors.forename: S
authors.initials: S
authors.lastname: Hase
authors.forename: Y
authors.initials: Y
authors.lastname: Shimonishi
authors.forename: S
authors.initials: S
authors.lastname: Iwanaga
mesh_terms: D000595:Amino Acid Sequence; D002241:Carbohydrates / Q000032:analysis; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D015949:Factor IXa / Q000737:chemistry*; D005643:Fucose; D006020:Glycopeptides / Q000737:chemistry / Q000302:isolation & purification; D006027:Glycosides; D006801:Humans; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry* / Q000302:isolation & purification; D012689:Sequence Homology, Nucleic Acid; D012694:Serine; D016339:Spectrometry, Mass, Fast Atom Bombardment
chemical_list: D002241:Carbohydrates; D006020:Glycopeptides; D006027:Glycosides; D009844:Oligosaccharides; D005643:Fucose; D012694:Serine; D015949:Factor IXa