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

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

Bioprocess and biosystems engineering (2020-01-26)
Hye-Yeon Kim, Minseong Park, Choeun Kang, Woon Heo, Sei Mee Yoon, Jinu Lee, Joo Young Kim
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Ciprofloxacin, ≥98% (HPLC)
BRAND® 96-well microplate, U-bottom, round bottom, non-sterile
Sigma-Aldrich
OSMI-1, ≥98% (HPLC)