Title : Identification of the oligosaccharide structures of human coagulation factor X factor X activation peptide at each glycosylation site
Abstract :
Human blood coagulation factor X 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