N-Glycoproteomic Characterization of Mannosidase and Xylosyltransferase Mutant Strains of Chlamydomonasreinhardtii.

ABSTRACT

At present, only little is known about the enzymatic machinery required for N-glycosylation in Chlamydomonas reinhardtii, leading to the formation of N-glycans harboring Xyl and methylated Man. This machinery possesses new enzymatic features, as C. reinhardtii N-glycans are independent of ß1,2-N-acetylglucosaminyltransferase I. Here we have performed comparative N-glycoproteomic analyses of insertional mutants of mannosidase 1A (IM Man1A ) and xylosyltransferase 1A (IM XylT1A ). The disruption of man1A affected methylation of Man and the addition of terminal Xyl. The absence of XylT1A led to shorter N-glycans compared to the wild type. The use of a IM Man1A xIM XylT1A double mutant revealed that the absence of Man1A suppressed the IM XylT1A phenotype, indicating that the increased N-glycan trimming is regulated by core ß1,2-Xyl and is dependent on Man1A activity. These data point toward an enzymatic cascade in the N-glycosylation pathway of C. reinhardtii with interlinked roles of Man1A and XylT1A. The results described herein represent the first step toward a functional characterization of the enzymatic N-glycosylation machinery in C. reinhardtii.