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.

Summary Nowadays, little information is available regarding the N‐glycosylation pathway in the green microalga Chlamydomonas reinhardtii. Recent investigation demonstrated that C. reinhardtii synthesizes linear oligomannosides. Maturation of these oligomannosides results in N‐glycans that are partially methylated and carry one or two xylose residues. One xylose residue was demonstrated to be a core β(1,2)‐xylose. Recently, N‐glycoproteomic analysis performed on glycoproteins secreted by C. reinhardtii demonstrated that the xylosyltransferase A (XTA) was responsible for the addition of the core β(1,2)‐xylose. Furthermore, another xylosyltransferase candidate named XTB was suggested to be involved in the xylosylation in C. reinhardtii. In the present study, we focus especially on the characterization of the structures of the xylosylated N‐glycans from C. reinhardtii taking advantage of insertional mutants of XTA and XTB, and of the XTA/XTB double‐mutant. The combination of mass spectrometry approaches allowed us to identify the major N‐glycan structures bearing one or two xylose residues. They confirm that XTA is responsible for the addition of the core β(1,2)‐xylose, whereas XTB is involved in the addition of the xylose residue onto the linear branch of the N‐glycan as well as in the partial addition of the core β(1,2)‐xylose suggesting that this transferase exhibits a low substrate specificity. Analysis of the double‐mutant suggests that an additional xylosyltransferase is involved in the xylosylation process in C. reinhardtii. Additional putative candidates have been identified in the C. reinhardtii genome. Altogether, these results pave the way for a better understanding of the C. reinhardtii N‐glycosylation pathway.