N-Glycoproteomic Characterization of Mannosidase and Xylosyltransferase Mutant Strains of Chlamydomonas reinhardtii

摘要

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 beta 1,2-N-acetylglucosaminyltransferase I. Here we have performed comparative N-glycoproteomic analyses of insertional mutants of mannosidase 1A (IMMan1A) and xylosyltransferase 1A (IMXylT1A). The disruption of man1A affected methylation of Man and the addition of terminal Xyl. The absence of XylT1A led to shorter Nglycans compared to the wild type. The use of a IM(Man1A)xIM(XylT1A) double mutant revealed that the absence of Man1A suppressed the IMXylT1A phenotype, indicating that the increased N-glycan trimming is regulated by core beta 1,2-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.