title: Comparative structural study of N-linked oligosaccharides of urinary and recombinant erythropoietins.
abstract: The structures of the N-linked oligosaccharides of the urinary erythropoietin (u-EPO) purified from urine of aplastic anemic patients were analyzed and compared with those for recombinant erythropoietin (r-EPO) prepared with baby hamster kidney (BHK) cells. Asparagine-linked neutral oligosaccharides were released from each EPO protein by N-oligosaccharide 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 8 and 13 kinds of oligosaccharide fractions for u-EPO and r-EPO (BHK), respectively, were completely separated by the one-step HPLC procedure. The structure of each oligosaccharide thus isolated was analyzed by a combination of sequential exoglycosidase digestion and another kind of HPLC with an amide-silica column. Furthermore, high-resolution proton nuclear magnetic resonance (1H NMR) spectroscopy and methylation analyses were carried out in the case of r-EPO (BHK).(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Research Institute of Life Science, Snow Brand Milk Products Company Ltd., Tochigi, Japan.
authors.forename: E
authors.initials: E
authors.lastname: Tsuda
authors.forename: M
authors.initials: M
authors.lastname: Goto
authors.forename: A
authors.initials: A
authors.lastname: Murakami
authors.forename: K
authors.initials: K
authors.lastname: Akai
authors.forename: M
authors.initials: M
authors.lastname: Ueda
authors.forename: G
authors.initials: G
authors.lastname: Kawanishi
authors.forename: N
authors.initials: N
authors.lastname: Takahashi
authors.forename: R
authors.initials: R
authors.lastname: Sasaki
authors.forename: H
authors.initials: H
authors.lastname: Chiba
authors.forename: H
authors.initials: H
authors.lastname: Ishihara
mesh_terms: D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid; D004921:Erythropoietin / Q000032:analysis* / Q000652:urine; D006023:Glycoproteins / Q000032:analysis*; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000032:analysis*; D011994:Recombinant Proteins / Q000032:analysis
chemical_list: D006023:Glycoproteins; D009844:Oligosaccharides; D011994:Recombinant Proteins; D004921:Erythropoietin