Abstract Chlamydomonas reinhardtii N -glycans carry plant typical β1,2-core xylose, α1,3-fucose residues as well as plant atypical terminal β1,4-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 N -glycopeptides 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 . Significance Statement Our data provide novel insights into the function of XylT1B and FucT in C. reinhardtii as N -glycan core modifying enzymes. In the course of our study, different mutants were created by genetic crosses showing either varying or a lack of N -glycan core modification, enabling comparative analyses in relation to single N -glycan core epitope and overall protein function in C. reinhardtii .

Novel insights into N-glycan fucosylation and core xylosylation in C. reinhardtii