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Topic III: Towards deciphering the N-glycosylation pathways and their regulation in microalgae
Principal investigator: M. Bardor, Professor.


Participants: A. Driouich, Professor; P. Lerouge, Professor; E. Mathieu-Rivet, Associate Professor; B. Gügi, Associate Professor; A. Mareck, Associate Professor; L. Menu-Bouaouiche, Associate Professor; M-C. Kiefer-Meyer, Research Engineer; C. Burel, Technician; C. Plasson, Technician. Post-doctoral fellow from GRR IRIB (to hire).

The current project aims to position the Glyco-MEV lab as a forerunner considering the lack of knowledge regarding biosynthesis and structures of glycans N-linked to proteins as well as their physiological significance in microalgae. It will also contribute to expand/enhance the knowledge in the field of “phycoglycobiology”, specifically with regards to protein post-translational modifications in microalgae.

Functional characterization of the N-glycan biosynthetic pathways in microalgae

We propose to further investigate the N-glycosylation pathways of microalgae, especially in P. tricornutum and C. reinhardtii, by functionally characterizing the Golgi actors (glycosyltransferases, glycosidases and sugar transporters) using in vivo complementation of mutants. For example, genes encoding putative α-mannosidase I, α-mannosidase II, GDP-Fucose transporter, N-acetylhexosaminidases and fucosyltransferases from P. tricornutum will be expressed in CHO, human embryonic kidney (HEK) or Arabidopsis thaliana mutants. The structural analysis of the N-glycans in the respective transformants will be performed for the evaluation of the complementation efficiency. If no complementation is observed in these in vivo assays, the corresponding flag and/or His-tagged enzymes will be purified by affinity chromatography. Then, their activity will be assayed in vitro using different acceptors and substrates.
N-glycosylation inhibitors or fluoronylated sugars (see transverse project 2) will also be used to interfere with specific steps of the N-glycosylation and N-glycan processing in the ER or in the Golgi apparatus. After treatments, N-glycan profiles of proteins will be determined by structural analyses. The effect of the treatment on the physiology and phenotype of the microalgae cells will also be investigated. In parallel, glyco-engineering through heterologous expression of glycoenzymes in the diatom model will be investigated. Indeed, It would be interesting to evaluate the physiological impact of the remodeling of the N-glycosylation pathway in P. tricornutum by over-expressing heterologous glycoenzymes or actors of the N-glycosylation pathway, especially the ones which has been demonstrated to be up and down regulated in the RNA-Seq project (please refer to report session of Topic III p 17). Altogether, this will help deciphering the protein N-glycosylation pathways in C. reinhardtii and in the three morphotypes of P. tricornutum as well unraveling the N-glycans biological function in microalgae.

Cartography of the glycosyltransferases within the Golgi apparatus of microalgae

We will work on the functional mapping of the glyco-enzymes within the Golgi stacks of the microalgae C. reinhardtii and P. tricornutum, as to determine whether these enzymes are localized within specific Golgi cisternae. Localization will be investigated in different cisternal subtypes: from the early (cis-Golgi) to trans-Golgi cisternae and Trans Golgi Network as it has been already done for mammals and land plants (Schoberer & Strasser, 2011; Berger & Hesford, 1985). This compartmentation of enzymes within Golgi stacks is belived to control the step-by-step maturation of glycans N-linked to proteins along the secretory pathway. This task will be performed by high pressure freezing/freeze substitution technique with transmission electron microscopy (TEM). Indeed, during the last years, the Glyco-MEV has gained a significant expertise in the localization of plant glycosyltransferases involved in the biosynthesis of N-glycoproteins and cell wall polysaccharides within Golgi cisternae (Chevalier et al., 2010; Driouich et al., 2012). The functional organization of the Golgi apparatus will be studied via expression of differently tagged glycosyltransferases and glycosidases. The transformants will be analyzed with confocal microscopes in order to assess the cellular localization and dynamics of the tagged protein. Then, the precise Golgi sub-cellular localization of the specific enzymes will be investigated using immunocytochemistry and TEM. This work has already been initiated and allowed the observation of a Golgi apparatus from C. reinhardtii (Figure 15A) and the Golgi subcellular localization of a GnT I-V5 tagged in P. tricornutum (Figure 15B). We started with the GnT I as we already proved its in vivo functionality within the Golgi apparatus of CHO cells (Baiet et al., 2011, please refer to report p 16). As soon as the other activities, e.g. alpha-mannosidase II, xylosyltransferases and fucosyltransferases, are confirmed, their localization in both microalgae models will be investigated after fusion with other tags and co-expression with the GnT I. This will help determining whether these glyco-enzymes are specifically localized within Golgi cisternae and associated vesicles according to their sequential involvement in the N-glycosylation pathway, as previously described for mammals and land plants (Schoberer & Strasser, 2011; Berger & Hesford, 1985).

Evolution of the N-glycosylation pathways in microalgae

As described in the report of Topic III p 17, our results suggest that evolutionary adaptation of N-glycan processing in the diatom P. tricornutum and the chlorophyceae C. reinhardtii has given rise to two distinct pathways: a GnT I-dependent pathway in the diatom and a GnT I-independent pathway in the chlorophyceae. Therefore, in the new contract, we will focus on the understanding of the evolutionary relationships among microalgae with regards to the protein N-glycosylation pathway. In this respect, we will use multidisciplinary approaches such as in silico analyses of the available genomes and transcriptomes, molecular phylogenetic studies and structural analysis of the N-glycans structures attached to the endogeneous proteins of new models chosen in different phyla. As a start, specific focus will be made on the understanding of the putative loss of the GnT I in the chlorophyte clade as compared to the land plants, charophytes, red algae and glaucophytes. To address this question, we will also extend our biochemical models and start analysing new chlorophytes such as different strains of Ostreococcus in the framework of a new collaboration with Dr F. Y. Bouget (Laboratoire LOMIC, Banyuls-sur mer, UPMC). The green algae Volvox will also be studied as it is a useful model for investigating the evolution of multicellularity and the regulation of cell differentiation. This model will be of particular interest as the apparition of complex-type N-glycans is thought to be linked to the apparition of multicellularity in eukaryotes. Other chlorophytes such as the Trebouxiophycea, Coccomyxa subellipsoidea or Chlorella variabilis will also be including in our studies. By investigating those new models, we hope to understand why and when certain microalgae have lost the GnT I expression and activity.

In parallel, we decided to complement microalgae of the chlorophyceae clade exhibiting a GnT I-independent pathway, with a heterologous GnT I in order to investigate the impact of such restauration on the glycosylation and physiology of the algae. Therefore, C. reinhardtii has already been complemented with a codon optimized version of the GnT I gene from A. thaliana. GnT I expressing C. reinhardtii cells exhibits a stressed phenotype with accumulation of starch (Figure 16). The relationship between GnT I expression, N-glycan maturation and phenotype are currently under investigation.


References:  Berger & Hesford (1985) Proc. Natl. Acad. Sci. USA, 82, 4736–4739; Schoberer & Strasser (2011) Mol Plant 4, 220–228.

 

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