Engineering O-glycosylation in BY-2 cells
(2025)
Files
ThesisPrint.pdf
Open Access - Adobe PDF
- 38.12 MB
Details
- Authors
- Supervisors
- Abstract
- Glycosylation has significant impacts on therapeutic proteins and is described as a critical quality attribute by the industry. Glycoengineering is a powerful strategy for producing safer and improved therapeutics. In this context, Nicotiana tabacum BY-2 cell suspensions are a particularly attractive recombinant expression system as they display a simple N-glycosylation profile that can be easily engineered to yield a homogeneous and humanised profile. However, the O-glycosylation capacity of BY-2 cells is different from that of human cells. Indeed, mammalian GalNAc-O-glycosylation is absent in plants, while plant O-glycosylation consists of the hydroxylation of prolines by P4Hs and their glycosylation with arabinose chains or arabinogalactan polysaccharides. These residues, which are absent in humans and potentially immunogenic, have been observed on several plant-produced therapeutics. To improve the BY-2 cell platform developed in our laboratory, we engineered BY-2 cell line with humanised N-glycosylation to produce GalNAc-O-glycans. W expressed the enzymes responsible for initiating glycosylation and generating the core 1 structure, GalNAcT2 and C1GalT1, respectively, and showed that this cell line can produce the immunoglobulin A1 with humanised O-glycosylation of the hinge region. However, it was also modified with hydroxyprolines and pentoses. We have identified 21 putative P4H genes in N. tabacum and have started a CRISPR/Cas9 KO screen in reporter cell lines based on the fusion of Ser-Pro glycomodules with GFP to enable a simple phenotypic characterisation by western blotting. This screening has not yet identified P4Hs required for O-glycosylation of the modules. This work paved the way for the humanisation of O-glycosylation profile in BY-2 cells and a better understanding of the different eukaryotic O-glycosylation pathways.
- Affiliations
Citations
Bailly, N. J. (2025). Engineering O-glycosylation in BY-2 cells.