Complex formation, dolichol (re)cycling and pathological mechanisms in the initial steps of the N-glycosylation pathway

先天性糖基化紊乱(CDG)实际上包括100多种不同的遗传性糖共轭生物合成障碍，一般累及多种器官，大多合并严重的神经功能障碍。在过去的几年里，我们发现了大量患有各种早期糖基化缺陷的患者。在这些缺陷中，尤其是位于内质网胞浆侧的甘露糖转移酶的缺陷会导致极其严重的临床表型。虽然全面的生化分析已经证明了突变蛋白的功能障碍，但对N-糖基化缺陷条件下的调控机制缺乏更深入的研究。我们的项目旨在通过研究细胞和动物模型来研究ALG1、ALG2和ALG11甘露糖基转移酶在N-糖基化途径早期阶段的病理生理学相关性，以及它们的功能降低对其他糖基化途径的影响。特别是，我们感兴趣的是，ALG1、ALG2和ALG11是否在结构上相互关联的情况下，将GDP-甘露糖中的甘露糖残基顺序加成到二元醇结合的低聚糖上。此外，我们将确定这些甘露糖基转移酶的故障如何改变三种类型的N-多糖(复合型、杂合型和富含甘露糖的)的组成，并潜在地影响O-和C-甘露糖化以及O-岩藻糖化。我们工作的另一个目标是进行研究，以表明缩短的ALG2转录本是否占α1，6甘露糖基转移酶的活性。在ALG2-CDG的Medaka模型中，我们希望研究阿尔法2依赖的过程，因为这些过程对胚胎发育至关重要。除了对阿尔法2-Medakas进行全面分析以检测生化异常和器官异常外，我们还有兴趣检测与野生型动物相比，阿尔法2基因缺陷胚胎的N-糖链组成和糖基化位点使用的变化。

Congenital Disorders of Glycosylation (CDG) actually comprise more than 100 different inherited disorders in glycoconjugate biosynthesis in man which in general affect a variety of organs and are mostly combined with severe neurological impairment. Over the last few years we have identified a large number of patients suffering from a variety of early glycosylation deficiencies. Among these defects especially those occurring in mannosyltransferases localized at the cytosolic side of the ER lead to extremely severe clinical phenotypes. Although comprehensive biochemical analysis has demonstrated the dysfunction of the mutated protein responsible, more in-depth studies on regulatory mechanisms under N-glycosylation deficient conditions are lacking. Our project aims to investigate the pathophysiologic correlation between the ALG1, ALG2 and ALG11 mannosyltransferases in the early steps of the N-glycosylation pathway and the impact of their reduced functionality on other glycosylation pathways by investigating cellular and animal models with a focus on the bifunctional ALG2 mannosyltransferase. In particular we are interested to find out whether the sequential addition of mannose residues from GDP-Mannose onto a dolichol-bound acceptor oligosaccharide by ALG1, ALG2 and ALG11 is carried out while they are structurally associated with each other. Besides, we will determine how malfunctions of these mannosyltransferases change the composition of the three types of N-glycans (complex, hybrid and mannose-rich) and potentially influence O- and C-mannosylation as well as O-fucosylation. An additional aim of our work will be to perform studies to show whether shortened ALG2 transcripts account for alpha1,6 mannosyltransferase activity. In a Medaka model for ALG2-CDG we want to study Alg2-dependent processes as these are crucial for embryonic development. In addition to comprehensive analyses of the Alg2-Medakas to detect biochemical irregularities and organ abnormalities, we are also interested in detecting changes in the N-glycan composition and glycosylation site usage of Alg2-deficient embryos in comparison to wildtype animals.