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.

中文翻译：

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轻链丝氨酸12的O-GlcNAcylation介导利妥昔单抗的产量增加了thiamet G的两倍。

O-糖基化发生在CHO细胞产生的重组蛋白中，但是这种现象尚未得到广泛研究。在这里，我们报道了利妥昔单抗是一种O-连接的N-乙酰基-葡糖酰胺基化（O-GlcNAcylated）蛋白，利妥昔单抗的产生通过O-GlcNAcase抑制剂Thiamet G的作用而增加。利妥昔单抗的产生在OGA抑制下增加了一倍，而在O-GlcNAc转移酶抑制下下降。O-GlcNAc特异性抗体和用azidO-GlcNAc进行的代谢标记证实了噻虫G导致O-GlcNAcy的增加。蛋白质质量分析显示，利妥昔单抗轻链的丝氨酸7、12和14被O-GlcNAcy酰化。轻链的S12A突变降低了利妥昔单抗的稳定性，并且在没有任何显着的mRNA水平变化的情况下，未能提高噻硫磷G的产量。细胞毒性和热稳定性试验证实，由噻虫G治疗产生的利妥昔单抗的生物学和物理特性没有差异。因此，噻虫酰胺G治疗改善了利妥昔单抗的产生而没有显着改变其功能。