Identification of the oligosaccharide structures of human coagulation factor X activation peptide at each glycosylation site.
Human blood coagulation factor X has two N-linked oligosaccharides at Asn39 and Asn49 residues and two O-linked oligosaccharides at Thr17 and Thr29 residues in the region of the factor X activation peptide (XAP) which is cleaved off during its activation by factor IXa. We determined the structure of oligosaccharides in the XAP region of human factor X. Four glycopeptides each containing a glycosylation site were isolated by digestion of XAP with endoproteinase Asp-N followed by reversed-phase HPLC. N-linked oligosaccharides released from the glycopeptides by glycoamidase A digestion were derivatized with 2-aminopyridine. Pyridylamino(PA)-oligosaccharides were separated by HPLC into neutral and sialyl oligosaccharides using an anion-exchange column. Structures of oligosaccharides and their contents at each glycosylation site were determined by a two-dimensional sugar mapping method. The contents of the neutral oligosaccharides at Asn39 and Asn49 residues were 32.5% and 30.0%, respectively. Six neutral and twelve monosialyl oligosaccharides isolated from both N-linked glycosylation sites showed similar elution profiles composed of bi-, tri- and tetra-antennary complex type oligosaccharides. The predominant component in neutral oligosaccharides was biantennary without a fucose residue. Two major monosialyl oligosaccharides were also biantennary without fucose and with a Neu5Ac alpha 2-->6 residue. In addition, the structures of O-linked oligosaccharides at Thr17 and Thr29 residues were suggested to be disialylated Gal beta 3GalNAc sequences by their component analyses.
Nakagawa
H
H
GlycoLab, Nakano Vinegar Co. Ltd, Aichi, Japan.
Takahashi
N
N
Fujikawa
K
K
Kawamura
Y
Y
Iino
M
M
Takeya
H
H
Ogawa
H
H
Suzuki
K
K
D001216:Asparagine / Q000737:chemistry; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid; D010450:Endopeptidases / Q000378:metabolism; D005170:Factor X / Q000737:chemistry*; D006031:Glycosylation; D006801:Humans; D008666:Metalloendopeptidases; D008969:Molecular Sequence Data; D019158:N-Acetylneuraminic Acid; D009844:Oligosaccharides / Q000032:analysis / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry; D012794:Sialic Acids / Q000737:chemistry
D009844:Oligosaccharides; D010455:Peptides; D012794:Sialic Acids; D001216:Asparagine; D005170:Factor X; D010450:Endopeptidases; D008666:Metalloendopeptidases; C058921:endoproteinase Asp-N; D019158:N-Acetylneuraminic Acid