N-glycosylation of recombinant human interferon-gamma produced in different animal expression systems.
Recombinant human interferon-gamma (IFN-gamma) was expressed in Chinese hamster ovary cells, baculovirus-infected Sf9 insect cells and the mammary gland of transgenic mice. The N-linked carbohydrate populations associated with both Asn25 and Asn97 glycosylation sites were characterized by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) in combination with exoglycosidase array sequencing. A site-specific analysis of dual (2N) and single (1N) site-occupancy variants of IFN-gamma derived from Chinese hamster ovary cells showed that N-glycans were predominantly of the complex bi- and triantennary type. Although Asn25-linked glycans were substituted with a core fucose residue, Asn97 N-glycans were predominantly non-fucosylated, and truncated complex and high-mannose oligosaccharide chains were also evident. Transgenic mouse derived IFN-gamma exhibited considerable site-specific variation in N-glycan structures. Asn25-linked carbohydrates were of the complex, core fucosylated type, Asn97-linked carbohydrates were mainly of the oligomannose type, with smaller proportions of hybrid and complex N-glycans. Carbohydrates associated with both glycosylation sites of IFN-gamma from Sf9 insect cells were mainly tri-mannosyl core structures, with fucosylation confined to the Asn25 site. These data demonstrate the profound influence of host cell type and protein structure on the N-glycosylation of recombinant proteins.
James
D C
DC
Research School of Biosciences, University of Kent, Canterbury, U.K. d.c.james@ukc.ac.uk
Freedman
R B
RB
Hoare
M
M
Ogonah
O W
OW
Rooney
B C
BC
Larionov
O A
OA
Dobrovolsky
V N
VN
Lagutin
O V
OV
Jenkins
N
N
D000818:Animals; D016367:Baculoviridae; D016466:CHO Cells; D002236:Carbohydrate Conformation; D002460:Cell Line; D006224:Cricetinae; D005260:Female; D006020:Glycopeptides / Q000032:analysis*; D006031:Glycosylation; D006801:Humans; D007313:Insecta / Q000166:cytology / Q000821:virology; D007371:Interferon-gamma / Q000378:metabolism*; D008321:Mammary Glands, Animal / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D009844:Oligosaccharides / Q000032:analysis*; D011994:Recombinant Proteins; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D006020:Glycopeptides; D009844:Oligosaccharides; D011994:Recombinant Proteins; D007371:Interferon-gamma