O-glycosylation in BY-2 cells: The use of a hyper-glycosylated Ser-Pro motif fused to a reporter protein to investigate the role of P4H in proline hydroxylation

Details

Supervisors

Chaumont, François ; Bailly, Nicolas J.

Faculty

Faculté des sciences

Degree label

Master [120] en biochimie et biologie moléculaire et cellulaire, à finalité approfondie

Abstract

The production of biopharmaceuticals proteins by plants has made substantial progress, particularly with the FDA’s approval of taliglucerase alfa produced by carrot cell lines (Daucus carota L.) to treat Gaucher’s disease. The most recent FDA approval of such proteins concerns ELFABRIO, developed in BY-2 cell suspension cultures to treat Fabry disease. Plant cell cultures offer several advantages over animal cell cultures. In particular, the presence of post-translational modifications typical of higher eukaryotes makes them an excellent platform for the production of human proteins. Nevertheless, the low yields of these cultures represent a challenge for plant biotechnology research. Post-translational modifications are extremely important for the function and structure of proteins, and are therefore being studied in depth. These include glycosylation, comprising N- and O-glycosylation. N-glycosylation has been extensively studied and is well known, but more remains to be discovered about O-glycosylation. In plants, O-glycosylation involves two steps: hydroxylation of proline residues by prolyl-4-hydroxylases, followed by the addition of glycans to the newly formed hydroxyproline residues. In order to study O-glycosylation, tags with Ser-Pro repeat motifs (representing either Pro contiguous or non-contiguous) were fused to GFP and sent into the secretory pathway by using different signal peptides. These tags were composed of motifs recognized for arabinogalactan or extensin O-glycosylation and were O-glycosylated while passing through the secretory pathway. The glycosylation profile of the recombinant proteins produced in Nicotiana tabacum BY-2 and Nicotiana benthamiana leaves was determined by western blot. A high molecular mass shift was observed in the presence of the tags. Finally, to study the action of the different P4H families, chemical inhibition was conducted and genetic constructs were produced for CRISPR-Cas9 silencing. Chemical was quantified by western blot but the results did not confirm the arrest of O-glycosylated GFP accumulation in the protein extracts.