Heterologous expression of the N-acetylglucosaminyltransferase I dictates a reinvestigation of the N-glycosylation pathway in Chlamydomonas reinhardtii

Gaëtan Vanier (1) , Pierre-Louis Lucas (1) , Corinne Loutelier-Bourhis (2) , Jessica Vanier (1) , Carole Plasson (1) , Marie-Laure Walet-Balieu (3) , Philippe Chan Tchi-Song (3) , Isabelle Remy-Jouet (4) , Vincent Richard (4) , Sophie Bernard (1) , Azeddine Driouich (1) , Carlos Afonso (2) , Patrice Lerouge (1) , Elodie Mathieu-Rivet (1) , Muriel Bardor (1)

1 Glyco-MEV - Laboratoire de Glycobiologie et Matrice Extracellulaire Végétale
2 COBRA - Chimie Organique et Bioorganique : Réactivité et Analyse
3 Plate-forme de Protéomique PISSARO
4 UNIROUEN - Université de Rouen Normandie

Eukaryotic N-glycosylation pathways are dependent of N-acetylglucosaminyltransferase I (GnTI), a key glycosyltransferase opening the door to the formation of complex-type N-glycans by transferring a N-acetylglucosamine residue onto the Man5GlcNAc2 intermediate. In contrast, glycans N-linked to Chlamydomonas reinhardtii proteins arise from a GnTI-independent Golgi processing of oligomannosides giving rise to Man5GlcNAc2 substituted eventually with one or two xylose(s). Here, complementation of C. reinhardtii with heterologous GnTI was investigated by expression of GnTI cDNAs originated from Arabidopsis and the diatom Phaeodactylum tricornutum. No modification of the N-glycans was observed in the GnTI transformed cells. Consequently, the structure of the Man5GlcNAc2 synthesized by C. reinhardtii was reinvestigated. Mass spectrometry analyses combined with enzyme sequencing showed that C. reinhardtii proteins carry linear Man5GlcNAc2 instead of the branched structure usually found in eukaryotes. Moreover, characterization of the lipid-linked oligosaccharide precursor demonstrated that C. reinhardtii exhibit a Glc3Man5GlcNAc2 dolichol pyrophosphate precursor. We propose that this precursor is then trimmed into a linear Man5GlcNAc2 that is not substrate for GnTI. Furthermore, cells expressing GnTI exhibited an altered phenotype with large vacuoles, increase of ROS production and accumulation of starch granules, suggesting the activation of stress responses likely due to the perturbation of the Golgi apparatus.

Domaines: Polymères; Amélioration des plantes; Biotechnologies; Gamétogenèse; Organisation et fonctions cellulaires [q-bio.SC]; Interactions cellulaires [q-bio.CB]; Biochimie [q-bio.BM]; Biologie moléculaire; Génomique, Transcriptomique et Protéomique [q-bio.GN]; Réseaux moléculaires [q-bio.MN]; Phytopathologie et phytopharmacie

Gaëtan Vanier, Pierre-Louis Lucas, Corinne Loutelier-Bourhis, Jessica Vanier, Carole Plasson, et al.. Heterologous expression of the N-acetylglucosaminyltransferase I dictates a reinvestigation of the N-glycosylation pathway in Chlamydomonas reinhardtii. Scientific Reports, 2017, 7, pp.10156-10156. ⟨10.1038/s41598-017-10698-z⟩. ⟨hal-01777653⟩