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

先天性糖基化障碍（CDG）实际上包括人类糖缀合物生物合成中的100多种不同遗传性障碍，其通常影响多种器官，并且大多数与严重的神经损伤相结合。在过去的几年里，我们已经确定了大量的患者患有各种早期糖基化缺陷。在这些缺陷中，尤其是位于ER胞质侧的甘露糖基转移酶中发生的缺陷，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，但缺乏对N-糖基化缺陷条件下的调控机制的更深入研究。我们的项目旨在研究ALG 1，ALG 2和ALG 11甘露糖基转移酶在N-糖基化途径的早期步骤之间的病理生理相关性，以及通过研究细胞和动物模型，重点关注双功能ALG 2甘露糖基转移酶，降低其功能对其他糖基化途径的影响。特别地，我们感兴趣的是发现是否通过ALG 1、ALG 2和ALG 11将来自GDP-甘露糖的甘露糖残基顺序添加到多萜醇结合的受体寡糖上，同时它们在结构上彼此缔合。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-聚糖（复杂，杂合和富含甘露糖）的组成，并可能影响O-和C-甘露糖基化以及O-岩藻糖基化。我们工作的另一个目的是进行研究以显示缩短的ALG 2转录物是否占α 1，6甘露糖基转移酶活性。在ALG 2-CDG的青鳉模型中，我们希望研究Alg 2依赖性过程，因为这些过程对胚胎发育至关重要。除了对Alg 2-Medakas进行全面分析以检测生化异常和器官异常之外，我们还对检测与野生型动物相比Alg 2缺陷型胚胎的N-聚糖组成和糖基化位点使用的变化感兴趣。

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.