Constraints on the transport and glycosylation of recombinant IFN-gamma in Chinese hamster ovary and insect cells.
In this study we compare intracellular transport and processing of a recombinant glycoprotein in mammalian and insect cells. Detailed analysis of the N-glycosylation of recombinant human IFN-gamma by matrix-assisted laser-desorption mass spectrometry showed that the protein secreted by Chinese hamster ovary and baculovirus-infected insect Sf9 cells was associated with complex sialylated or truncated tri-mannosyl core glycans, respectively. However, the intracellular proteins were predominantly associated with high-mannose type oligosaccharides (Man-6 to Man-9) in both cases, indicating that endoplasmic reticulum to cis-Golgi transport is a predominant rate-limiting step in both expression systems. In CHO cells, although there was a minor intracellular subpopulation of sialylated IFN-gamma glycoforms identical to the secreted product (therefore associated with late-Golgi compartments or secretory vesicles), no other intermediates were evident. Therefore, anterograde transport processes in the Golgi stack do not limit secretion. In Sf9 insect cells, there was no direct evidence of post-ER glycan-processing events other than core fucosylation and de-mannosylation, both of which were glycosylation site-specific. To investigate the influence of nucleotide-sugar availability on cell-specific glycosylation, the cellular content of nucleotide-sugar substrates in both mammalian and insect cells was quantitatively determined by anion-exchange HPLC. In both host cell types, UDP-hexose and UDP-N-acetylhexosamine were in greater abundance relative to other substrates. However, unlike CHO cells, sialyltransferase activity and CMP-NeuAc substrate were not present in uninfected or baculovirus-infected Sf9 cells. Similar data were obtained for other insect cell hosts, Sf21 and Ea4. We conclude that although the limitations on intracellular transport and secretion of recombinant proteins in mammalian and insect cells are similar, N-glycan processing in Sf insect cells is limited, and that genetic modification of N-glycan processing in these insect cell lines will be constrained by substrate availability to terminal galactosylation.
Hooker
A D
AD
Research School of Biosciences, University of Kent, Canterbury CT2 7NJ, United Kingdom.
Green
N H
NH
Baines
A J
AJ
Bull
A T
AT
Jenkins
N
N
Strange
P G
PG
James
D C
DC
D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D006031:Glycosylation; D006801:Humans; D007371:Interferon-gamma / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D011134:Polysaccharides / Q000032:analysis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D018411:Spodoptera / Q000166:cytology / Q000378:metabolism
D011134:Polysaccharides; D011994:Recombinant Proteins; D007371:Interferon-gamma