The major N-linked carbohydrate chains from human urokinase. The occurrence of 4-O-sulfated, (alpha 2-6)-sialylated or (alpha 1-3)-fucosylated N-acetylgalactosamine(beta 1-4)-N-acetylglucosamine elements.
The primary structure of the major N-linked carbohydrate chains attached to Asn302 of urinary-type plasminogen activator (urokinase) have been determined. Urokinase was completely deglycosylated with peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F from Flavobacterium meningosepticum. Released oligosaccharides were separated from the remaining protein using gel-permeation chromatography on Bio-Gel P-100, and then on Bio-Gel P-6. Fractionation of the oligosaccharides was achieved by a combination of FPLC anion-exchange chromatography on Mono Q HR 5/5 and amine-adsorption HPLC on LiChrospher 100-NH2. Analysis by 1H-NMR spectroscopy demonstrated that the collection of N-glycans comprises di-, tri-, and tri'-antennary structures. The glycans contain predominantly GalNAc beta 1-4GlcNAc beta instead of Gal beta 1-4GlcNAc beta elements. The GalNAc residue is mainly sulfated at O4, or to a lesser extent it bears N-acetylneuraminic acid at O6; alternatively the GlcNAc residue can be fucosylated at O3. The major component, which accounts for more than 30 mol/100 mol of the total oligosaccharide pool, consists of an (alpha 1-6)-fucosylated diantennary N-linked carbohydrate chain with (SO4-)-4GalNAc beta 1-4GlcNAc beta 1-2 antennae.
Bergwerff
A A
AA
Bijvoet Center, Department of Bio-Organic Chemistry, Utrecht University, The Netherlands.
Van Oostrum
J
J
Kamerling
J P
JP
Vliegenthart
J F
JF
D000117:Acetylglucosamine / Q000031:analogs & derivatives / Q000737:chemistry*; D002240:Carbohydrate Sequence; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D005643:Fucose / Q000737:chemistry; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D019158:N-Acetylneuraminic Acid; D012794:Sialic Acids / Q000737:chemistry; D013431:Sulfates / Q000737:chemistry; D014568:Urokinase-Type Plasminogen Activator / Q000737:chemistry*
D012794:Sialic Acids; D013431:Sulfates; D005643:Fucose; D014568:Urokinase-Type Plasminogen Activator; D019158:N-Acetylneuraminic Acid; D000117:Acetylglucosamine