Effects of elevated ammonium on glycosylation gene expression in CHO cells.
The negative effects of ammonium on recombinant protein productivity and glycosylation have been well documented, but the interaction of ammonium on glycosylation genes has not been completely elucidated. In this study, the effects of elevated ammonium on 12 glycosylation related genes in Chinese hamster ovary cells were evaluated by quantitative real time reverse transcriptase polymerase chain reaction. Numerous cytosol and endoplasmic reticulum (ER) localized genes associated with early glycosylation steps were insensitive to the ammonium condition. The initial expression of uridine diphosphate (UDP)-galactose transporter was higher for the ammonium-treated culture, while the initial expressions of cytosine monophosphate (CMP)-sialic acid transporter, beta(1,4)-galactosyltransferase, and UDP-glucose pyrophosphorylase were higher for the control culture. alpha(2,3)-sialyltransferase was observed to have lower expression level under the elevated ammonium condition compared to the control culture. This study indicates that galactosylation and sialylation inhibition is mainly due to decreased gene expression of galactosyltransferase, sialyltransferase, and CMP-sialic acid transporter and not due to sialidase. These unbalanced initial glycosylation and branching steps can explain the higher molecular heterogeneity under ammonium stress. Moreover, this study indicates that elevated ammonium has limited effects on the glycosylation genes associated with the ER and cytosol compared to the genes associated with the Golgi.
Chen
Peifeng
P
Department of Chemical and Biomolecular Engineering, Clemson University, SC 29634-0909, USA.
Harcum
Sarah W
SW
D000818:Animals; D016466:CHO Cells; D049109:Cell Proliferation / Q000187:drug effects; D006224:Cricetinae; D003412:Cricetulus; D004305:Dose-Response Relationship, Drug; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism*; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology*; D006031:Glycosylation / Q000187:drug effects*; D006056:Golgi Apparatus / Q000187:drug effects / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000378:metabolism*; D009439:Neuraminidase / Q000378:metabolism*; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*
D009097:Multienzyme Complexes; D000644:Quaternary Ammonium Compounds; D009439:Neuraminidase