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

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

doi:10.1038/s41598-017-10698-z

Abstract

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.

Highlights

N-linked glycosylation is an extensive eukaryotic post-translational modification of secreted proteins consisting of the covalent attachment of an oligosaccharide onto asparagine residues belonging to the consensus sequence Asn-X-Ser/Thr/Cys, where X represents any amino acid except proline[1,2,3,4]
We characterized N-glycan structures linked to endogenous proteins in C. reinhardtii and showed they were mostly oligomannosides and for 30% novel mature structures containing xylose residues and methylated mannoses on Man4-5GlcNAc215
These structures resulted from a Golgi GnTI-independent processing of oligomannosides as the bioinformatics analysis of the genome has revealed that it lacks GnTI

Summary

Introduction

N-linked glycosylation is an extensive eukaryotic post-translational modification of secreted proteins consisting of the covalent attachment of an oligosaccharide onto asparagine residues belonging to the consensus sequence Asn-X-Ser/Thr/Cys, where X represents any amino acid except proline[1,2,3,4]. The Man5GlcNAc2-PP-Dol is flipped into the lumen of the ER5, 6 where its extension occurs by the addition of several Man and glucose (Glc) residues into a complete oligosaccharide precursor Glc3M an9GlcNAc2-PP-Dol[7] This LLO is thereafter transferred by the oligosaccharyltransferase (OST) complex onto the asparagine of the N-glycosylation consensus sequence of the nascent polypeptides[8]. N-acetylglucosaminyltransferase I (GnTI), α-mannosidase II and N-acetylglucosaminyltransferase II (GnTII) give rise to the canonical GlcNAc2Man3GlcNAc2 core common to mammals, insects and land plants[11, 12] This core undergoes further maturation into organism-specific complex-type N-glycans that are involved in several physiological functions like cell-cell interaction, intracellular communication and signaling[13, 14]. Our findings demonstrate that the expression of GnTI induces stress responses in C. reinhardtii and reveal a truncated ER N-glycosylation pathway in C. reinhardtii

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Results

Conclusion