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

Abstract

Chlamydomonas reinhardtii N-glycans carry plant typical {beta}1,2-core xylose, 1,3-fucose residues as well as plant atypical terminal {beta}1,4-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.\n\nSignificance StatementOur 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.