O-GlcNAcylation of light chain serine 12 mediates rituximab production doubled by thiamet G

Abstract

O-Glycosylation occurs in recombinant proteins produced by CHO cells, but this phenomenon has not been studied extensively. Here, we report that rituximab is an O-linked N-acetyl-glucosaminylated (O-GlcNAcylated) protein and the production of rituximab is increased by thiamet G, an inhibitor of O-GlcNAcase. The production of rituximab doubled with OGA inhibition and decreased with O-GlcNAc transferase inhibition. O-GlcNAc-specific antibody and metabolic labelling with azidO-GlcNAc confirmed the increased O-GlcNAcylation with thiamet G. Protein mass analysis revealed that serine 7, 12, and 14 of the rituximab light chain were O-GlcNAcylated. S12A mutation of the light chain decreased rituximab stability and failed to increase the production with thiamet G without any significant changes of mRNA level. Cytotoxicity and thermal stability assays confirmed that there were no differences in the biological and physical properties of rituximab produced by thiamet G treatment. Therefore, thiamet G treatment improves the production of rituximab without significantly altering its function.

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Abbreviations

OGT:

N-Acetylglucosamine transferase

OGA:

O-Linked N-acetylglucosaminidase

ADCC:

Antibody-dependent cell cytotoxicity

CDC:

Complement-dependent cytotoxicity

PTMs:

Post-translational modifications

GlcNAc:

N-Acetylglucosamine

GalNAc:

N-Acetylgalactosamine

CHO-K1:

Chinese Hamster Ovary cell line

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Acknowledgements

This work was supported by grants from the National Research Foundation of Korea, Project No. NFR-2018R1A2B4010319 and a faculty research grant from Yonsei University College of Medicine (6-2018-0070) to J. Y Kim.

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Kim, HY., Park, M., Kang, C. et al. O-GlcNAcylation of light chain serine 12 mediates rituximab production doubled by thiamet G. Bioprocess Biosyst Eng 43, 863–875 (2020). https://doi.org/10.1007/s00449-020-02282-z

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Keywords

  • Rituximab
  • O-GlcNAc
  • Production yield
  • Thiamet G
  • ADCC
  • CDC
  • Thermal stability