O-Linked oligosaccharide chains were identified in the activation peptide (AP) of human blood coagulation factor IX. The peptide obtained from human factor IX was separated into three molecular species (APα, AP β, and ??γ) by reversed-phase high-performance liquid chromatography. Amino acid analysis showed that APα, but not AP β and ??γ, contained galactosamine in addition to glucosamine, thereby suggesting the presence of an O-linked sugar chain(s) in the molecule of APa. A nonapeptide (APα-D4, residues 157–165) and an undecapeptide (APα-D5, 166–176) derived from APa contained Thr-159 and Thr-169, neither of which could be identified using a gas-phase protein sequencer. All other serine and threonine residues present in APα were identified by peptide sequencing. Component sugar and sialic acid analyses of APα-D4 and APα-D5 revealed that they contained 1 mol each of TV-acetyl-D-galactosamine (GalNAc), D-galactose (Gal), and sialic acid. Fast atom bombardment tandem mass spectrometric analysis of APα-D4 suggested the existence of Gal-GalNAc-Thr, NeuNAc-(Gal-)GalNAc-Thr, and NeuNAc-Gal-GalNAc-Thr structures. On the basis of amino acid analysis after the isolation of APα, it accounted for approximately 35% of the total activation peptide obtained. From these results, it was concluded that a part of the activation peptide of human factor IX in circulating blood has tri- and tetrasaccharides O-glycosidically linked to the threonine residues at 159 and 169.
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