Elsevier

Journal of Biotechnology

Volume 282, 20 September 2018, Pages 101-110
Journal of Biotechnology

The contributions of individual galactosyltransferases to protein specific N-glycan processing in Chinese Hamster Ovary cells

https://doi.org/10.1016/j.jbiotec.2018.07.015Get rights and content
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Highlights

  • Study of individual galactosyltransferases involved in N-glycosylation in triple and quadruple CHO-K1 deletion cell lines.

  • Contributions of B4GALT2 and B4GALT3 are protein-dependent.

  • B4GALT1 alone is not sufficient for full galactosylation of EPO-Fc.

  • Link between impaired galactosylation and occurance of less processed glycan structures with terminal mannose.

Abstract

Galactosylation as part of N-glycan processing is conducted by a set of beta-1,4-galactosyltransferases (B4GALTs), with B4GALT1 as the dominant isoenzyme for this reaction. Nevertheless, the exact contributions of this key-player as well as of the other isoenzymes involved in N-glycosylation, B4GALT2, B4GALT3 and B4GALT4, have not been studied in-depth.

To increase the understanding of the protein- and site-specific activities of individual galactosyltransferases in Chinese Hamster Ovary cells, a panel of triple deletion cell lines was generated that expressed only one isoform of B4GALT each. Two model proteins were selected for this study to cover a large spectrum of possible N-glycan structures: erythropoietin and deamine-oxidase. They were expressed as Fc-fusion constructs (EPO-Fc and Fc-DAO) and their N-glycan processing status was analyzed by site-specific mass spectrometry. The sole activity of B4GALT1 resulted in a decrease of 15–21 % of fully galactosylated structures for erythropoietin, emphasizing the involvement of other isoenzymes. Interestingly, the contributions of B4GALT2 and B4GALT3 differed for the two model proteins. Unexpectedly, removal of galactosyltransferases influenced the overall process of N-glycan maturation, with the result of a higher occurrence of poorly processed oligosaccharides.

In the context of high productivity cell lines, which can push N-glycan maturation towards incomplete galactosylation, galactosyltransferases are potential targets to ensure stable product quality. In view of our results, specifically engineered “designer” cell lines may be required for different proteins.

Abbreviations

CHO cells
Chinese Hamster Ovary cells
CRISPR
clustered regularly interspaced short palindromic repeat
sgRNA
single guide RNA
ST3GAL
alpha-2,3-sialyltransferase
ST6GAL
alpha-2,6-sialyltransferase
EPO
erythropoietin
Fc
Fragment crystallizable
DAO
diamine-oxidase
B4GALT
beta-1,4-galactosyltransferase
MGAT
N-acetylglucosaminyltransferase
MS
mass spectrometry
VCD
viable cell density

Keywords

CHO cells
N-glycosylation
beta-1,4-galactosyltransferases
Mass spectrometry

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