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

Abstract

<i>Chlamydomonas reinhardtii (C. reinhardtii) N-</i>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. <i>N-</i>Glycans found in a XylT1A/Man1A double mutant carried core xylose residues, suggesting the existence of a second core xylosyltransferase in <i>C. reinhardtii</i>. To further elucidate enzymes important for <i>N</i>-glycosylation, novel single knockdown mutants of candidate genes involved in the <i>N</i>-glycosylation pathway were characterized. In addition, double, triple, and quadruple mutants affecting already known <i>N</i>-glycosylation pathway genes were generated. By characterizing <i>N</i>-glycan compositions of intact <i>N</i>-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 <i>N-</i>glycans with strongly diminished core modifications. Thus, the mutant strains generated will pave the way for further investigations on how single <i>N-</i>glycan core epitopes modulate protein function in <i>C. reinhardtii</i>.