Structures of sialylated oligosaccharides of human erythropoietin expressed in recombinant BHK-21 cells.
The native structures of the Asn-linked oligosaccharides and the O-glycans at Ser126 of human erythropoietin expressed from recombinant BHK cells have been elucidated. Enzymatically released N-glycans were studied by methylation analyses, fast-atom-bombardment mass spectrometry as well as one- and two-dimensional 1H-NMR spectrometry at 600 MHz. Many (82.7%) were found to be tetraantennary N-acetyllactosamine-type (22.8% with one, 3.6% with two and 0.4% with three N-acetyllactosamine repeats) being tetrasialylated (41%), trisialylated (29.6%) and disialylated (12.2%). A few (9.7%; 4.1% 2,4-branched, 5.6%, 2,6-branched) of the chains were triantennary (5.4% trisialyl, 4.3% disialyl) and 4.6% were of the disialyl diantennary type. Almost all of the innermost GlcNAc residues were alpha 1-6 fucosylated and NeuAc was exclusively alpha 2-3 linked to Gal beta 1-4GlcNAc-R; 60% of the protein was found to be O-glycosylated at Ser126; structures were monosialylated (70%) or disialylated (30%) forms of the Gal beta 1-3GalNAc core type. Glycosylation patterns at individual Asn-Xaa-Thr/Ser sites were determined by analytical high-pH anion-exchange chromatography with pulsed amperometric detection. Only tetraantennary chains with 0-3 N-acetyllactosamine repeats were detected at Asn38 and Asn83, while almost all of the di- and triantennary oligosaccharides were attached to Asn24. Batch analysis of different preparations of recombinant erythropoietin revealed the high reproducibility of the production procedure. Structures containing terminal GalNAc-GlcNAc were detected in small amounts in a few batches.
Nimtz
M
M
Department of Cell Biology and Genetics, GBF-Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, Federal Republic of Germany.
Martin
W
W
Wray
V
V
Klöppel
K D
KD
Augustin
J
J
Conradt
H S
HS
D000595:Amino Acid Sequence; D000818:Animals; D002240:Carbohydrate Sequence; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D006224:Cricetinae; D004921:Erythropoietin / Q000737:chemistry* / Q000235:genetics; D006031:Glycosylation; D006801:Humans; D007668:Kidney / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D015394:Molecular Structure; D019158:N-Acetylneuraminic Acid; D009844:Oligosaccharides / Q000737:chemistry*; D011134:Polysaccharides / Q000032:analysis; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D012794:Sialic Acids / Q000737:chemistry*; D016339:Spectrometry, Mass, Fast Atom Bombardment
D009844:Oligosaccharides; D011134:Polysaccharides; D011994:Recombinant Proteins; D012794:Sialic Acids; D004921:Erythropoietin; D019158:N-Acetylneuraminic Acid