Human factor IX has a tetrasaccharide O-glycosidically linked to serine 61 through the fucose residue

• Published in: The Journal of biological chemistry 1992-09, Vol.267 (25), p.17520-17525
• Main Authors: NISHIMURA, H, TAKAO, T, HASE, S, SHIMONISHI, Y, IWANAGA, S
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Biological and medical sciences; Blood coagulation. Blood cells; Carbohydrates - analysis; Chromatography, Gel; Chromatography, High Pressure Liquid; coagulation factor IX; Coagulation factors; Factor IXa - chemistry; Fucose; Fundamental and applied biological sciences. Psychology; Glycopeptides - chemistry; Glycopeptides - isolation & purification; Glycosides; Humans; linkage; man; Molecular and cellular biology; Molecular Sequence Data; Oligosaccharides - chemistry; Oligosaccharides - isolation & purification; properties; Sequence Homology, Nucleic Acid; Serine; Spectrometry, Mass, Fast Atom Bombardment
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/s0021-9258(19)37073-5

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.