Structure and Function in alpha-Dystroglycan Glycosylation

项目摘要/摘要 基于O-甘露醇的蛋白质糖基化在高等动物中的意义已在初级研究中阐明。 依赖于α-DG(α-DG)，它确立了多糖在将细胞锚定到细胞外基质中的作用 α-DG低糖基化与一组肌营养不良有关，通常伴有 认知或其他方面的异常。寻找这些病理的分子基础揭示了新的生物合成- IC通路涉及以前未被识别的酶和其缺陷影响α-DG糖基的糖链- Ttion，增加了先天性糖基化障碍的名单。在评价O-的过程中 甘露糖化途径，人们发现该途径还参与介导某些病毒感染和 转移。α-DG上最具功能批判性的O-MAN葡聚糖类的完整结构 是在2016年通过我们和其他人的努力才成立的。附加的蛋白质O-甘露糖化途径 最近也出现了。我们的目标是进一步了解O-甘露醇的作用和调节- 基于蛋白质的糖基化以及重复双糖聚合物的制备和分布 Matriglycan，首次在α-DG M3核心三糖上鉴定。Matriglycan负责功能中的- 与ECM蛋白中层粘连蛋白G结构域的相互作用。头两个目标是解决关于 备选方案在扩展最初的O-Man站点方面的相互作用和后果。关键决定 多糖延伸中的点来自于POMGNT2在O-上添加GlcNAc时形成的连接 MAN对α-DG进行改良。目前已知，随之而来的M3葡聚糖核心只在一个蛋白质的两个位置， α-DG。我们已经确定了一些控制这些位点的蛋白质序列元件，但我们将开发一种 全面了解该问题的高度特异性，解决是否存在其他潜在的复发问题 这种多糖在蛋白质上的可感知的位置，以及这些位置是否可以被工程到其他蛋白质中进行治疗- TIC抢救表型的前景。我们将结合使用多种方法来理解POMGNT2 专一性。相反，POMGNT1可以在高尔基体中作用于插入带有 产生其他主要类型的O-Man多聚糖的不同连锁，特别是M1。的显式功能 这些多糖是未知的，但在没有M1的情况下，M3的多糖不能完全延伸，α-DG是功能的。 合规性受到了损害。我们将定义M3多糖精化在哪里被破坏，M1与什么相互作用， 并更好地理解POMGNT1函数。第三个目标涉及对母体罐头长度和 它与生化和细胞水平的功能的关系。我们还将探索Matriglycan在 非O-Man糖蛋白，不依赖于蛋白质。作为一个整体，拟议目标的完成 将在分子水平上为功能性O-甘露糖化建立规则，这些规则可以在 设计治疗方法。

PROJECT SUMMARY/ABSTRACT The significance of O-mannosyl-based protein glycosylation in higher animals was elucidated in studies primar- ily on α-dystroglycan (α-DG) that established a role for the glycans in anchoring cells to the extracellular matrix (ECM).  α-DG hypoglycosylation is associated with a subset of muscular dystrophies, often accompanied by cognitive or other abnormalities. Searching for a molecular basis of these pathologies revealed new biosynthet- ic pathways involving previously unrecognized enzymes and glycans whose defects impacted α-DG glycosyla- tion, adding to the list of congenital disorders in glycosylation. During the course of evaluating the O- mannosylation pathway, it was discovered this pathway is also involved in mediating certain viral infections and metastasis. The full structure of arguably the most functionally critical of the O-Man glycan classes on α-DG was only established in 2016 through work by us and others. Additional protein O-mannosylation pathways have also recently emerged. Our goal here is to further understand the role and regulation of O-mannosyl- based protein glycosylation as well as the elaboration and distribution of the repeating disaccharide polymer matriglycan, first identified on the α-DG M3 core trisaccharide. Matriglycan is responsible for the functional in- teractions with laminin-G domains in ECM proteins. The first two aims address outstanding questions on the interplay and consequences of the alternative options in extending the initial O-Man sites. The critical decision point in glycan extension comes in the ER from the linkage formed when POMGNT2 adds a GlcNAc on the O- Man modified α-DG. The M3 glycan core that ensues is currently known only to be at two sites on one protein, α-DG. We have already identified some protein sequence elements controlling the sites, but we will develop a comprehensive understanding of the high degree of specificity to address whether there are other potential re- ceptive sites for this glycan on proteins, and whether sites can be engineered into other proteins with therapeu- tic prospects for rescuing phenotypes. We will use a combination of approaches to understand POMGNT2 specificity. In contrast, POMGNT1 can act in the Golgi on the remaining O-Man sites inserting a GlcNAc with a different linkage generating the other major classes of O-Man glycans, particularly M1. The explicit function of these glycans is not known, but in the absence of M1, the M3 glycan is not fully extended and α-DG is func- tionality compromised. We will define where the M3 glycan elaboration is disrupted, what M1 interacts with, and better understand POMGNT1 function. The third aim addresses regulation of the matriglycan length and its relationship to function at biochemical and cellular levels. We will also explore the function of matriglycan on non-O-Man glycoproteins and independent of protein. Taken as a whole, the completion of the proposed aims will establish the rules at a molecular level for functional O-Mannosylation that can be taken advantage of in designing therapeutic approaches.