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 oligosacchride 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). It has been found that (1) significantly different distributions of oligosaccharide components exist in u-EPO preparations isolated from different individuals, whereas the distribution of oligosaccharide components is quite similar for different r-EPO (BHK) preparations, (2) in r-EPO (BHK) a tetraantennary oligosaccharide with a fucose residue is predominant and various higher molecular weight tetraantennary oligosaccharides with one to three Gal(.beta.1 .fwdarw. 4)GlcNAc repeating units exist, (3) all of the eight kinds of oligosaccharides of u-EPO also exist in r-EPO (BHK), although the distribution of the oligosaccharides is different for the two proteins, and (4) oligosacchrides with unusual structures containing the Gal(.beta.1 .fwdarw. 3)GlcNAc group exist in both u- and r-EPO (BHK) (fraction G), and in r-EPO (BHK) (fraction K).