Carbohydrate structure of erythropoietin expressed in Chinese hamster ovary cells by a human erythropoietin cDNA.
The proper glycosylation of erythropoietin is essential for its function in vivo. Human erythropoietins were isolated from Chinese hamster ovary cells transfected with a human erythropoietin cDNA and from human urine. Carbohydrate chains attached to these proteins were isolated and fractionated by anion-exchange high performance liquid chromatography (HPLC) and HPLC employing a Lichrosorb-NH2 column. The structures of fractionated saccharides were analyzed by fast atom bombardment-mass spectrometry and methylation analysis before and after treatment with specific exoglycosidases. Both erythropoietins were found to contain one O-linked oligosaccharide/mol of the proteins, and its major component was elucidated to be NeuNAc alpha 2----3Gal beta 1----3(NeuNAc alpha 2----6)GalNAcOH (where NeuNAc represents N-acetylneuraminic acid) in both proteins. The N-linked saccharides of recombinant erythropoietin were found to consist of biantennary (1.4% of the total saccharides), triantennary (10%), triantennary with one N-acetyllactosaminyl repeat (3.5%), tetraantennary (31.8%), and tetraantennary with one (32.1%), two (16.5%), or three (4.7%) N-acetyllactosaminyl repeats. All of these saccharides are sialylated by 2----3-linkages. Tetraantennary with or without polylactosaminyl units are mainly present as disialosyl or trisialosyl forms, and these structures exhibit the following unique features. alpha 2----3-Linked sialic acid and N-acetyllactosaminyl repeats are selectively present in the side chains attached to C-6 and C-2 of 2,6-substituted alpha-mannose and C-4 of 2,4-substituted alpha-mannose. We have also shown that the carbohydrate moiety of urinary erythropoietin is indistinguishable from recombinant erythropoietin except for a slight difference in sialylation, providing the evidence that recombinant erythropoietin is valuable for biological as well as clinical use.
D000818:Animals; D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000032:analysis*; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000032:analysis*; D004921:Erythropoietin / Q000032:analysis* / Q000235:genetics; D005260:Female; D006031:Glycosylation; D006801:Humans; D013058:Mass Spectrometry; D010053:Ovary / Q000378:metabolism; D011994:Recombinant Proteins / Q000032:analysis* / Q000235:genetics
D002241:Carbohydrates; D011994:Recombinant Proteins; D004921:Erythropoietin; D004247:DNA