Structures of the sugar chains of recombinant macrophage colony-stimulating factor produced in Chinese hamster ovary cells.
The structures of the N- and O-linked sugar chains of recombinant human macrophage colony-stimulating factor (rhM-CSF) from Chinese hamster ovary (CHO) cells were studied. rhM-CSF is a homodimeric glycoprotein. Sugar composition analysis revealed that rhM-CSF contained 4.1 mol N-acetylgalactosamine, 10.3 mol N-acetylglucosamine, 5.0 mol mannose, 10.0 mol galactose, 1.4 mol fucose, and 11.8 mol sialic acid per mol of the monomer. The N- and O-linked sugar chains liberated by hydrazinolysis were N-acetylated, and the reducing-end sugar residues were tagged with 2-aminopyridine. The pyridylamino (PA-) sugar chains thus obtained were purified by HPLC. The structures of the PA-sugar chains were analyzed by a combination of reversed-phase and size-fractionation HPLC, and exoglycosidase digestions, from which the structures of the rhM-CSF sugar chains were estimated to be as follows: monosialo biantennary sugar chain (9 mol%), monosialo fucosylbiantennary sugar chain (10 mol%), disialo biantennary sugar chain (30 mol%), disialo fucosylbiantennary sugar chain (28 mol%), disialo triantennary sugar chain (7 mol%), trisialo triantennary sugar chain (11 mol%), and trisialo fucosyltriantennary sugar chain (5 mol%) for the N-linked sugar chains, and asialo (27 mol%), monosialo (51 mol%), and disialo (22 mol%) Galbeta1-3GalNAc for the O-linked sugar chains. Sialic acid residues were linked to the N-linked sugar chains through an alpha2-3 linkage.
Ushida
Y
Y
Department of Chemistry, Graduate School of Science, Osaka University.
Natsuka
S
S
Shimokawa
Y
Y
Takatsu
Z
Z
Shimamura
S
S
Hase
S
S
D000818:Animals; D016466:CHO Cells / Q000378:metabolism*; D002236:Carbohydrate Conformation; D050260:Carbohydrate Metabolism; D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000032:analysis* / Q000737:chemistry* / Q000302:isolation & purification; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006224:Cricetinae; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D016173:Macrophage Colony-Stimulating Factor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D019158:N-Acetylneuraminic Acid / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001616:beta-Galactosidase / Q000378:metabolism
D002241:Carbohydrates; D006023:Glycoproteins; D011994:Recombinant Proteins; D016173:Macrophage Colony-Stimulating Factor; D001616:beta-Galactosidase; D019158:N-Acetylneuraminic Acid