Abstract
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 factorX activationpeptide (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 Neu5Acα-2→6 residue. In addition, the structures of O-linked oligosaccharides at Thr17 and Thr29 residues were suggested to be disialylated Gal/β3GalNAc sequences by their component analyses.
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Abbreviations
- Gal:
-
d-galactose
- GlcNAc:
-
N-acetyl-d-glucosamine
- Man:
-
d-mannose
- HPLC:
-
high-performance liquid chromatography
- NDV:
-
Newcastle disease virus
- Neu5Ac:
-
5-N-acetylneuraminic acid
- ODS:
-
octadecylsilyl
- PA:
-
pyridylamino
- RVV-X:
-
Russell's viper venom factor X activator
- TBS:
-
Tris-buffered saline
- XAP:
-
factor X activation peptide.
References
DiScipo RG, Hermodson MA, Davie EW (1977)Biochemistry 16: 5253–60.
Steinberg M, Nemerson Y (1982)The activation of factor X in Hemostasis and Thrombosis (Colman RW, Hirsh J Marder VJ, Salzman EW eds) pp. 91–99. Philadelphia PA: JB Lippincott.
Fenton JW (1988)Thromb Hemostasis 14:234–40.
Jamieson GA (1988)Prog Clin Biol Res 283:137–58.
Goligorsky MS, Menton DN, Laszlo A, Lum H (1990)J Biol Chem 264:16771–75.
Iino M, Takeya H, Nishioka J, Suzuki K (1994)J Biochem (Tokyo)116:335–40.
Leytus SP, Chung DW, Kisiel W, Kurachi K, Davie EW (1984)Proc Natl Acad Sci USA 81: 3699–702.
Inoue K, Morita T (1993)Eur J Biochem 218: 153–63.
Sinha U, Wolf DL (1993)J Biol Chem 268: 3048–51.
Tomiya N, Awaya J, Kurono M, Endo S, Arata Y, Takahashi N (1988)Anal Biochem 171: 73–90.
Takahashi N, Tomiya N (1992)Handbook of Endoglycosidases and Glycoamidases (Takahashi N, Muramatsu T eds) pp. 199–332. Boca Raton FL: CRC Press.
Tsuda E, Goto M, Murakami A, Akai K, Ueda M, Kawannishi G, Takahashi N, Sasaki R, Chiba H, Ishihara H, Mori M, Tejima S, Endo S, Arata Y (1988)Biochemistry 27: 5646–54.
Takeya H, Nishida S, Miyata T, Kawada S, Saisaka Y, Morita T, Iwanaga S (1992)J Biol Chem 267: 14109–17.
Miletich JP, Broze GJ, Majerus PW (1980)Anal Biochem 105: 304–10.
Morita T, Kisiel W (1985)Biochem Biophys Res Commun 130: 841–47.
Tomiya N, Kurono M, Ishihara H, Tejima S, Endo S, Arata Y, Takahashi N (1987)Anal Biochem 163: 489–99.
Hase S, Ikenaka T, Matsushima Y (1978)Biochem Biophys Res Commun 85: 257–63.
Yamamoto S, Hase S, Fukuda S, Sano O, Ikenaka T (1989)J Biochem 105: 547–55.
Takahashi N, Wada Y, Awaya J, Kurono M, Tomiya N (1993)Anal Biochem 208: 96–109.
Jefferis R, Lund J, Mizutani H, Nakagawa H, Kawazoe Y, Arata Y, Takahashi N (1990)Biochem J 268: 529–37
Paulson JC, Weinstein J, Dorland L, van Halbeek H, Vliegenthart JFG (1982)J Biol Chem 257: 12734–38.
Hardy MR, Townsend RR, Lee YC (1988)Anal Biochem 170: 54–62.
Lee YC (1990)Anal Biochem 189: 151–62.
Hase S (1993)Glycoprotein Analysis in Biomedicine (Hounsell EF eds) pp. 69–80. Totowa NJ: Humana Press.
Mizuochi T, Yamashita K, Fujikawa K, Titani K, Kobata A (1980)J Biol Chem 255: 3526–31.
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Nakagawa, H., Takahashi, N., Fujikawa, K. et al. Identification of the oligosaccharide structures of human coagulation factor X activation peptide at each glycosylation site. Glycoconjugate J 12, 173–181 (1995). https://doi.org/10.1007/BF00731362
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DOI: https://doi.org/10.1007/BF00731362