A novel method for determination of alpha1,6fucosyltransferase activity using a reducing oligosaccharide from egg yolk as a specific acceptor.
A new method for determination of alpha1,6fucosyltransferase activity has been described. Recently, the disialyl-biantennary undecasaccharide was prepared in high yield from egg yolk [(1996), Carbohydr Lett 2: 137-42]. By treatment of this oligosaccharide with neuraminidase and beta-galactosidase, we readily obtained an asialo-agalacto-biantennary heptasaccharide (GlcNAcbeta 1,2Manalpha1,6[GlcNAcbeta1,2Manalpha1,3]Manbeta1 ,4GlcNAcbeta1,4GlcNAc). Using this asialo-agalacto-oligosaccharide as an acceptor, fucosyltransferases from human plasma and extracts of various human hepatoma cell lines were assayed in the presence of GDP-[3H]fucose. The reaction mixture was applied to a column of GlcNAc-binding, Psathyrella velutina lectin coupled gel. All the fucosylated acceptor were bound to the column which was eluted with 50 mM GlcNAc. Structural analyses revealed that only the innermost GlcNAc residue of the acceptor was fucosylated through an alpha1,6-linkage, and the oligosaccharide prepared could be used as a specific acceptor for alpha1,6fucosyltransferase. The present method was used to screen plasma alpha1,6fucosyltransferase in several patient groups, and significantly elevated activities were found in samples from patients with liver diseases, including chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma.
Yazawa
S
S
Japan Immunresearch Laboratories, Takasaki, Japan. syazawa@po.sphere.ne.jp
Kochibe
N
N
Nishimura
T
T
Shima
C
C
Takai
I
I
Adachi
M
M
Asao
T
T
Hada
T
T
Enoki
Y
Y
Juneja
L R
LR
D000117:Acetylglucosamine / Q000031:analogs & derivatives / Q000378:metabolism; D002240:Carbohydrate Sequence; D002846:Chromatography, Affinity / Q000379:methods; D002851:Chromatography, High Pressure Liquid; D004530:Egg Yolk / Q000737:chemistry*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005647:Fucosyltransferases / Q000037:antagonists & inhibitors / Q000097:blood / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D037102:Lectins / Q000378:metabolism; D008099:Liver / Q000473:pathology; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000032:analysis
D004791:Enzyme Inhibitors; D037102:Lectins; D009844:Oligosaccharides; D005647:Fucosyltransferases; D000117:Acetylglucosamine