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

Abstract

Chlamydomonas reinhardtii (C. reinhardtii) N-glycans carry plant typical b1,2-core xylose, a1,3-fucose residues, as well as plant atypical terminal b1,4-xylose and methylated mannoses. In a recent study, XylT1A was shown to act as core xylosyltransferase, whereby its action was of importance for an inhibition of excessive Man1A dependent trimming. N-Glycans found in a XylT1A/Man1A double mutant carried core xylose residues, suggesting the existence of a second core xylosyltransferase in C. reinhardtii. To further elucidate enzymes important for N-glycosylation, novel single knockdown mutants of candidate genes involved in the N-glycosylation pathway were characterized. In addition, double, triple, and quadruple mutants affecting already known N-glycosylation pathway genes were generated. By characterizing N-glycan compositions of intact Nglycopeptides from these mutant strains by mass spectrometry, a candidate gene encoding for a second putative core xylosyltransferase (XylT1B) was identified. Additionally, the role of a putative fucosyltransferase was revealed. Mutant strains with knockdown of both xylosyltransferases and the fucosyltransferase resulted in the formation of N-glycans with strongly diminished core modifications. Thus, the mutant strains generated will pave the way for further investigations on how single N-glycan core epitopes modulate protein function in C. reinhardtii.

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).…”

“…Thus, glycoproteins harboring the non-canonical Man 5 GlcNAc 2 are submitted in the Golgi apparatus to the action of glycosyltransferases responsible for the addition of decorations. Mass spectrometry analyses carried out on C. reinhardtii secreted and membrane-bound proteins have shown that mature N-glycans are partially O-methylated Man 3 GlcNAc 2 to Man 5 GlcNAc 2 substituted by one or two Xyl residues (Mathieu-Rivet et al, 2013), and for a minor part by one fucose residue (Oltmanns et al, 2020). Recently, Schulze et al (2018) and Lucas et al (2020) have showed that the first Xyl is linked in β(1,2) to the β-Man via the action of the xylosyltransferase A (XylTA) similarly to the plant xylosylation process.…”

“…However, although it is clearly established that these two XylT play a major role in the N-glycan xylosylation processing, the remaining presence of structures containing Xyl residues in a double knockdown mutant XylTA × XylTB has suggested that other uncharacterized enzymes could also contribute to the N-glycan xylosylation in C. reinhardtii. The fucosylation mechanism remains uncleared as the analysis of an insertional mutant in which the candidate gene encoding for a putative FucT was disrupted, did not affect N-glycans harboring Fuc residues (Oltmanns et al, 2020). In addition, a bioinformatic analysis Mócsai et al, 2020a,b Analysis of N-glycans in a strain collection and commercial products derived from C. vulgaris and C. sorokiniana highlighted for the first time a huge heterogeneity of N-glycan structures and the presence of arabinose residues in N-glycans that has never been reported before.…”

Decision letter: Altered N-glycan composition impacts flagella-mediated adhesion in Chlamydomonas reinhardtii

“…In order to assign glycopeptides, samples were measured employing In-Source collision induced dissociation (IS-CID) as described previously followed by analysis of data with Ursgal and SugarPy (Kremer et al, 2016;Oltmanns et al, 2020;Schulze et al, 2020).…”

“…In land plants, Golgi maturation leads to N-glycans modified with β1,2-core xylose and α1,3-core fucose (Strasser, 2016). In Chlamydomonas reinhardtii, a unicellular biflagellate green alga, N-glycans can be decorated with core xylose and -fucose (Lucas et al, 2020;Oltmanns et al, 2020;Schulze et al, 2018). Additionally, 6O-methylation of mannose and addition of a terminally linked β1,4-xylose were reported (Mathieu-Rivet et al, 2013).…”