title: Structural study of asparagine-linked oligosaccharide moiety of taste-modifying protein, miraculin.
abstract: The structures of the N-linked oligosaccharides of miraculin, which is a taste modifying glycoprotein isolated from miracle fruits, berries of Richadella dulcifica, are reported. Asparagine-linked oligosaccharides were released from the protein by glycopeptidase (almond) digestion. The reducing ends of the oligosaccharide chains thus obtained were aminated with a fluorescent reagent, 2-aminopyridine, and the mixture of pyridylamino derivatives of the oligosaccharides was separated by high performance liquid chromatography (HPLC) on an ODS-silica column. More than five kinds of oligosaccharide fractions were separated by the one chromatographic run. The structure of each oligosaccharide thus isolated was analyzed by a combination of sequential exoglycosidase digestion and another kind of HPLC with an amidesilica column. Furthermore, high resolution proton nuclear magnetic resonance (1H NMR) measurements were carried out. It was found that 1) five oligosaccharides obtained are a series of compounds with xylose-containing common structural core, Xyl beta 1----2 (Man alpha 1----6) Man beta 1----4-GlcNAc beta 1----4 (Fuca1----3)GlcNAc, 2) a variety of oligosaccharide structures are significant for two glycosylation sites, Asn-42 and Asn-186, and 3) two new oligosaccharides, B and D, with unusual structures containing monoantennary complex-type were characterized. (formula; see text)
authors.affiliation: Department of Biochemistry, Nagoya City University College of Nursing, Japan.
authors.forename: N
authors.initials: N
authors.lastname: Takahashi
authors.forename: H
authors.initials: H
authors.lastname: Hitotsuya
authors.forename: H
authors.initials: H
authors.lastname: Hanzawa
authors.forename: Y
authors.initials: Y
authors.lastname: Arata
authors.forename: Y
authors.initials: Y
authors.lastname: Kurihara
mesh_terms: D000581:Amidohydrolases / Q000378:metabolism; D000631:Aminopyridines; D001216:Asparagine; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000032:analysis; D002851:Chromatography, High Pressure Liquid; D005456:Fluorescent Dyes; D006020:Glycopeptides / Q000302:isolation & purification / Q000378:metabolism; D006023:Glycoproteins / Q000032:analysis; D006031:Glycosylation; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000378:metabolism; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
chemical_list: D000631:Aminopyridines; D002241:Carbohydrates; D005456:Fluorescent Dyes; D006020:Glycopeptides; D006023:Glycoproteins; D009844:Oligosaccharides; C008533:miraculin protein, Synsepalum dulcificum; D001216:Asparagine; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; C032439:alpha-aminopyridine