GLYCOSIDASE DIGESTION, ELECTROPHORESIS AND CHROMATOGRAPHIC ANALYSIS OF RECOMBINANT HUMAN GRANULOCYTE-COLONY-STIMULATING FACTOR GLYCOFORMS PRODUCED IN CHINESE-HAMSTER OVARY CELLS

Recombinant human granulocyte colony stimulating factor (G-CSF) produc ed in Chinese hamster ovary cells is glycosylated. The carbohydrate co mpositional analysis indicated that G-CSF molecule contains sialic aci d, galactose and galactosamine. By isolation and characterization of t he purified glycopeptides obtained from cleavages by Staphylococcal au reus V-8 protease and cyanogen bromide, the O-linked glycosylation sit e was confirmed to be a Thr residue at position 133. Neuraminidase and O-glycanase digestion followed by sodium dodecyl sulfate polyacrylami de and isoelectric focusing gel electrophoreses distinguished two poss ible carbohydrate structures attached at Thr-133: structure A, NeuNAc- Gal-beta(1,3)-GalNAc-O-Thr; and structure B, NeuNAc-Gal-beta(1,3)-[Neu NAc]-GalNAc-O-Thr. Different glycoforms, undigested or after glycosida se digestion, can also be separated by ion-exchange or reversed-phase high-performance liquid chromatography. The approach described in this report provides a simple and valuable procedure to characterize glyco protein structures containing simple carbohydrate moieties.