Chlamydomonas reinhardtii Mannosidase 1A and Xylosyltransferase1A N-glycosylation LC/MS-MS

We have identified insertional mutants of mannosidase 1A and xylosyltransferase 1A in Chlamydomonas reinhardtii. Mass spectrometric analyses of intact N-glycopeptides revealed that disruption of the genes altered the N-glycan composition of the alga (in single as well as double mutant). In contrast to wildtype, methylated hexoses and terminal xylose were nearly absent in N-glycopeptides from the Man1A mutant strain whereas the XylT1A mutant showed a lack of core xylose and excessively trimmed N-glycans. The double mutant did not show this excessive trimming, thus Man1A-dependent trimming can be concluded in C. reinhardtii. Furthermore, these data indicate that methylation of hexoses is tightly interlinked with Man1A function, pointing to an enzymatic cascade in the N-glycosylation pathway.

我们在莱茵衣藻（Chlamydomonas reinhardtii）中鉴定得到了甘露糖苷酶1A（mannosidase 1A）与木糖基转移酶1A（xylosyltransferase 1A）的插入突变体。通过对完整N-糖肽的质谱分析发现，上述基因的功能失活会改变该藻类的N-糖链组成，单突变体与双突变体均呈现该变化。与野生型菌株相比，Man1A突变株的N-糖肽中几乎完全缺失甲基化己糖与末端木糖；而XylT1A突变株则存在核心木糖缺失以及N-糖链过度修剪的表型。双突变体未出现该过度修剪现象，由此可推断莱茵衣藻中存在依赖于Man1A的糖链修剪过程。此外，本数据集的分析结果显示，己糖的甲基化与Man1A的功能紧密关联，提示N-糖基化通路中存在酶促级联反应。