Glycobiology and Plant Extracellular Matrix

GlycoMEV UR 4358 – University of Rouen Normandy

La 21^ème édition de l'International Conference on the Cell and Molecular Biology of Chlamydomonas s'est déroulée du 24 au 29 août 2025 à Münster (Allemagne).

Quatre membres du Laboratoire GlycoMEV ont participé à ces rencontres : le Dr Elodie RIVET, le Dr Narimane MATI-BAOUCHE, et 2 doctorants Jules DELASALLE et Thomas BANCE.

Le Dr Elodie RIVET a donné une conférence sur les travaux de recherche de l'Axe 3 intitulée :

« Fine-tuning the N-glycosylation of epigenetic transgene silencing in Chlamydomonas reinhardtii. »
 

Résumé :

Microalgae are considered as attractive expression systems for the production of biologics. As photosynthetic unicellular organisms, they do not require costly and complex media for growing, and are able to secrete proteins and perform protein glycosylation.

Some biologics have been successfully produced in the green microalgae Chlamydomonas reinhardtii. However, posttranslational modifications like glycosylation of these Chlamydomonas-made biologics have poorly been investigated so far.

Therefore, we reported on the first structural investigation of glycans linked to human erythropoietin (hEPO) expressed in a wild-type C. reinhardtii strain and in mutants impaired in key Golgi glycosyltransferases.
The glycoproteomic analysis of recombinant hEPO (rhEPO) expressed in the wild-type strain demonstrated that the three N-glycosylation sites are 100% glycosylated with mature N-glycans containing four to five mannose residues and carrying core-xylose, core-fucose and O-methyl groups.
Moreover, expression in C. reinhardtii insertional mutants defective in XYLOSYLTRANSFERASES A and B and FUCOSYLTRANSFERASE resulted in drastic decreases of core-xylosylation and corefucosylation of glycans N-linked to the rhEPOs, thus demonstrating that this strategy offers perspectives for humanizing the N-glycosylation of the Chlamydomonas-made biologics.

Poster de Jules DELASALLE :

« Elucidating the role of Calnexin and Calreticulin ER chaperones in N-glycosylation pathway of proteins: Functional insights using Chlamydomonas reinhardtii and Nicotiana tabacum BY-2 cells as models. »
Jules Delasalle, Narimane Mati-Baouche, Maëlle Noël, Emmanuel Gabriel Njoku, Elodie Rivet, Carole Plasson, Patrice Lerouge, François Chaumont, Catherine Navarre, Muriel Bardor

Résumé :

N-glycosylation of proteins in eukaryotic cells is a crucial co- and post-translational modification that impacts both their physiochemical properties and biological functions.
Glycoproteins are initially synthesized in the endoplasmic reticulum (ER), where lectin chaperones like calreticulin (CRT) and calnexin (CNX) are responsible for proper folding and 3D conformation before their transit to the Golgi apparatus.

In the context of the production of biologics which are mostly glycoproteins, it is necessary to find new and efficient bioproduction systems, such as microalgae and plant cells, to offer an alternative to commonly used mammalian cells. These alternatives reduce production costs, limit cross contamination and facilitate genome editing.
However, little information is available on the folding and secretion of N-glycoproteins in microalgae and plant cells.

The objective of this study is to functionally characterize chaperones (CRT and CNX) in Chlamydomonas reinhardtii and Nicotiana tabacum BY-2 suspension cells using Knock-Out (KO) mutants or already existing mutants (from the CLiP library and Chlamydomonas Ressource Center).
For C. reinhardtii, complementary analyses were performed at different levels (molecular, physiological and glycanic) on single KO-CNX mutants as well as on single KO-CRT and double KO- CRT/CNX mutants generated by CRISPR/Cas9. We showed that deletion of cnx gene has an impact on cell physiology: stress indicators, such as high levels of starch content and a significant decrease in cell motility, were observed when compared to WT cells.
To get further insight, the same methodology is currently applied to KO BY- 2 cells generated by CRISPR/Cas9.

Poster de Narimane MATI-BAOUCHE :

« Functional characterization of the alpha glucosidase II in Chlamydomonas reinhardtii, a key glycosylhydrolase involved in the quality control of N-glycoproteins. »
Carole Plasson, Jules Delasalle, Julia Van Bockstaele, Juliette Balieu, Benjamin Bourgeois, Hélène Dauchel, Yohan Stephan, Jérémy Bellien, Elodie Rivet, Patrice Lerouge, Muriel Bardor, Narimane Mati-Baouche

Résumé :

The N-glycosylation pathway of proteins is one of the most important co- and post- translational modification of proteins in Eukaryotes.
Glycoproteins are produced firstly in the endoplasmic reticulum (ER) where molecular actors such as glucosidases I and II are responsible for the proper folding and 3D conformation of proteins before their transfer into the Golgi apparatus.

The growing interest of using C. reinhardtii as an emerging cell biofactory for the industrial production of recombinant glycoproteins require overall an in-depth understanding and analysis of protein N-glycosylation in this organism.
Despite a relatively well-investigated N-glycosylation pathway in C. reinhardtii, many glyco-enzymes involved in the N-glycosylation pathway in the ER are still not characterized.

In this work, functional characterization of the putative alpha-glucosidase II (GSII) was conducted using combinatory approaches on C. reinhardtii insertional mutant cells in order to evaluate the impact of the GSII gene deletion compared to the WT cells and to give new insights on the ER N-glycosylation pathway in this microalga. Accumulation of free oligosaccharides (fOSs) and N-glycans such as Glc₂Man₅GlcNAc₂ are observed in the GSII mutant cells.
Moreover, an over-expression of genes involved in the quality control of misfolded proteins, was observed in addition to an accumulation of reactive oxygen species (ROS), suggesting a physiological stress in the microalgae mutant cells.