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) produced in Chinese hamster ovary cells is glycosylated. The carbohydrate compositional analysis indicated that G-CSF molecule contains sialic acid, galactose and galactosamine. By isolation and characterization of the purified glycopeptides obtained from cleavages by Staphylococcal aureus V-8 protease and cyanogen bromide, the O-linked glycosylation site was confirmed to be a Thr residue at position 133. Neuraminidase and O-glycanase digestion followed by sodium dodecyl sulfate polyacrylamide and isoelectric focusing gel electrophoreses distinguished two possible carbohydrate structures attached at Thr-133: structure A, NeuNAc-Gal-beta(1,3)-GalNAc-O-Thr; and structure B, NeuNAc-Gal-beta(1,3)-[NeuNAc]-GalNAc-O-Thr. Different glycoforms, undigested or after glycosidase 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 glycoprotein structures containing simple carbohydrate moieties.
Clogston
C L
CL
Amgen Inc., Amgen Center, Thousand Oaks, CA 91320.
Hu
S
S
Boone
T C
TC
Lu
H S
HS
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000032:analysis; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D006026:Glycoside Hydrolases / Q000378:metabolism*; D016179:Granulocyte Colony-Stimulating Factor / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry
D002241:Carbohydrates; D011994:Recombinant Proteins; D016179:Granulocyte Colony-Stimulating Factor; D006026:Glycoside Hydrolases