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N-glycosylation in microalgae

 

Principal investigator: Muriel Bardor


Participants: Carole Burel, Patrice Lerouge, Elodie Mathieu Rivet, Marie-Christine Kiefer-Meyer

PhD students: Clément Ovide, Gaëtan Vanier, Pierre-Louis Lucas

Project:

The main objective of this project is to unravel the biosynthesis and function of N-glycans in microalgae, including the sub-cellular localisation of main steps of this biological process. This project is challenging since little is known about N-glycosylation in microalgae organisms. Moreover, this study is an incontrovertible prerequisite for any engineering of the N-glycan pathways in microalgae in order to produce in these organisms therapeutic proteins harbouring human-compatible glycans. The study will be carried out in two microalgae models: i) the diatom Phaeodactylum tricornutum and ii) the green microalgae Chlamydomonas reinhardtii. Chlamydomonas reinhardtii is a single celled green alga that swims with two flagella and is the most widely used laboratory species which means that molecular biology tools (transformation, expression vector, miRNA…) are already available for this model. In contrast, Phaeodactylum tricornutum is less studied so far. It belongs to diatom and has the property to be pleiomorphic. Indeed, P. tricornutum can exist under different morphotypes (fusiform, triradiate, and oval). These changes in cell shape can be stimulated by environmental conditions. This feature can be used to explore the physiological role of N-glycans in morphogenesis algae. Moreover, our recent findings demonstrated that these two organisms synthesise mature N-glycans via two different pathways. P. Tricornutum exhibits a functional N-acetylglucosaminyltransferase I (GnT I) and as a consequence, possess GnT I-dependent N-glycosylation pathway as observed in pluricellular organisms (Bardor et al., 2010, BRE10-01, Baïet et al., 2010 – ACL10-15). In contrast, C. Reinhardtii synthesise mature N-glycans through a GnT I-independent pathway (unpublished results) (Fig. 3a). In order to unravel these two N-glycan pathways and their physiological significance, we will first characterize the different key hydrolases and glycosyltransferases involved in the N-glycan maturation in C. reinhardtii and P. tricornutum i.e. the mannosidases, N-acetylglucosaminidases and the-fucosyltransferases (-FucTs). The core-fucosylation of glycans N-linked to secreted proteins is particularly important since it widely occurs in eukaryotes and it is a major concern in the context of the production of recombinant therapeutic proteins. Indeed, fucosylation takes place at various positions on the N-glycan core and it has already been demonstrated that the (1,3)-Fuc epitope, unlike the (1,6)-Fuc epitope, induces strong immune responses in humans (Bardor et al., 2010 - OS10-01). The specificitiy of putative FucTs will be investigated by heterologous expression in Arabidopsis (cgl, futA/B) or CHO cells (Fut8-/-; Lec1) mutants. In addition, N-glycan pathways will be investigated through the constitutive expression of heterologous GnT I or using N-glycan inhibitors affecting the N-glycan maturation steps. In C. reinhardtii, this project will also take advantage of available N-glycan mutants and miRNA-silencing strategies in the context of the ANR KBBE program. For instance, the N-glycosylation of the L23 mutant which is mutated in a putative α-mannosidase I (not published) will be investigated. This mutant will help us for a better understanding of the N-glycosylation in C. Reinhardtii but it will also be helpful to study a potential role of the N-glycan in the movement of the algae since L23 mutant lost its swimming abilities. In C. reinhardtii and P. tricornutum, a regulation of the N-glycan maturation depending on growth conditions is suspected. The expression of key N-glycosylation enzymes (GnT I, FucTs, mannosidases) will be monitored in various cultured conditions to investigate whether they are submitted to a fine regulation process due to specific physiological needs. Furthermore, in order to assess the functions of complex N-glycans in these organisms, a particular attention will be paid to the physiology of these microalgae either in GnT I-completed or miRNA-silenced lines as well as in wild-type microalgae growth in presence of N-glycan inhibitors. The putative physiological implication of glycans N-linked to Phaeodactylum tricornutum proteins in morphogenesis will also be evaluated through a comparative glycomic approach the three morphotypes. Finally, the Golgi sub-localisation of glycosyltransferases (GTs) in the two microalgae will be investigated. Both GnT I and -FucTs fused with GFP or other tags will be expressed in microalgae and localized by confocal microscopy and immunogold labelling using anti-tag antibodies and TEM taking advantage of the methodology developed in our lab for plant cells (Chevalier and Bernard et al., 2010) (Fig. 3b).

More informations on the partnership between Agilent Technology and the Glyco-MEV

 

Want to know more about microalgae, have a look to this video

https://webtv.univ-rouen.fr/videos/muriel-bardor/