Investigation of the core α1,3-fucosyltransferase activity in Nicotiana tabacum BY-2 GnTI-KO suspension cells

Details

Supervisors

Chaumont, François ; Navarre, Catherine

Faculty

Faculté des bioingénieurs

Degree label

Master [120] : bioingénieur en chimie et bioindustries, à finalité spécialisée

Abstract

Core α1,3-fucosyltransferases are glycosyltransferase enzymes that catalyse L-fucose transfer to core N-acetylglucosamine residues of oligosaccharides by a 1,3 glycosidic bond. In plants, these enzymes play a role in the synthesis of complex glycans in the N-glycosylation pathway of membrane and secreted proteins. Notably, they have a substrate specificity to hybrid and complex glycans already processed by the β1,2-N-acetylglucosaminyltransferase I (GnTI). Nicotiana tabacum BY-2 suspension cells are a plant expression platform developed for molecular farming. This technology aims to propose an alternative to the production of pharmaceutical proteins in mammalian cells thanks to their lower cost, rapid growth, and easy transformation. Another advantage of plant expression platforms is the presence of an N- glycosylation pathway similar to that of mammalian cells with the synthesis of biantennary complex structures. However, the complex glycans generated are heterogenous and different from human glycans, causing variability and possible immune responses. A solution to this problem is the glycoengineering of BY-2 cells by suppression or insertion of specific glycosyltransferases. In this context, the GnTI-KO BY-2 cell line was previously generated in our laboratory. This deletion was expected to suppress all complex glycans on secreted proteins but a little residual fucose signal was detected in Western blot. Generation of GnTI/FucT-KO BY-2 cell lines was necessary to suppress this signal. The aim of this study was to understand which α1,3-FucT isoform of N. tabacum was responsible for the fucose signal in GnTI-KO cells and what glycan is generated, allowing to better understand the substrate specificity of core α1,3-FucT in plants. The α1,3-FucTB and α1,3-FucTC expression cassettes were constructed by Golden Gate Modular Cloning assembly method and transformed into GnTI/FucT-KO BY-2 cells. Screening of the generated cell lines showed that the α1,3-FucTC isoform is capable to fucosylate glycans in absence of GnTI, probably due to relaxed substrate specificity. To identify the fucosylated glycans synthesised, a pool of secreted proteins will be sent to the GlycoMEV Lab, Rouen.