Multiple xylosyltransferases heterogeneously xylosylate proteinN‐linked glycans inChlamydomonas reinhardtii

Abstract

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.

Novel Insights Into N-Glycan Fucosylation and Core Xylosylation in C. reinhardtii

“…When XylT1B was knocked out in addition to XylT1A and Man1A, N-glycans lost one Pent as revealed by mass spectrometric analyses. Immunoblotting results confirmed that b1,2-xylose was lost by knocking down XylT1B in the double mutant instead of a b1,4-xylose, which is attached to an outer mannose residue in C. reinhardtii (Mathieu-Rivet et al, 2013;Lucas et al, 2019). Therefore, it can be concluded that XylT1B is a core XylT.…”

“…The position of the remaining Pent could not be resolved due to a lack of specific antibodies. However, newest data suggest that the additional Pent is a xylose attached to the first or second Hex of the a1,3branch (Lucas et al, 2019).…”

“…There are several candidates for further XylT encoded in C. reinhardtii (e.g. Cre10.g458950, Cre13.g588750 and Cre08.g361250, proposed in Lucas et al, 2019) which can be targeted in further studies to obtain strains producing N-glycans completely devoid of xylose or fucose.…”

Perspectives for Glyco-Engineering of Recombinant Biopharmaceuticals from Microalgae

“…Another interesting result is the possible presence of a plant-like N-linked β(1,2)-xylosyltransferase (XYLT). C. reinhardtii is known to have xylose residues linked to the core GlcNAc and the antennas, and a previous study has demonstrated the presence of two xylosyltransferases, named XTA and XTB [129]. The absence of HPAT1 and HPAT3 in this microalga was unexpected, given that this microalga was shown to produce O-arabinose structures [124].…”

N- and O-Glycosylation Pathways in the Microalgae Polyphyletic Group

“…In microalgae, current knowledge regarding the N-glycosylation processes is available essentially in species belonging to the Chlorophytes (Table 1). Among these, the Chlorophyceae Chlamydomonas reinhardtii and the Trebouxiophycea Chlorella vulgaris have been the most investigated microalgae so far (Mathieu-Rivet et al, 2013;Vanier et al, 2017;Lucas et al, 2018Lucas et al, , 2020Schulze et al, 2018;Mócsai et al, 2019Mócsai et al, , 2020aOltmanns et al, 2020). Concerning microalgae with secondary plastids, the structural data available regarding N-glycans have been obtained from the diatom Phaeodactylum tricornutum (Baïet et al, 2011;Vanier et al, 2015;Lucas et al, 2018) and from the Euglenoid Euglena gracilis (De La Canal and Parodi, 1985;O'Neill et al, 2017).…”