Comparative structural study of N-linked oligosaccharides of urinary and recombinant erythropoietins.
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)
Tsuda
E
E
Research Institute of Life Science, Snow Brand Milk Products Company Ltd., Tochigi, Japan.
Goto
M
M
Murakami
A
A
Akai
K
K
Ueda
M
M
Kawanishi
G
G
Takahashi
N
N
Sasaki
R
R
Chiba
H
H
Ishihara
H
H
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
D006023:Glycoproteins; D009844:Oligosaccharides; D011994:Recombinant Proteins; D004921:Erythropoietin