Structure and Function in alpha-Dystroglycan Glycosylation

α-肌营养不良聚糖糖基化的结构和功能

• 批准号: 9906935
• 负责人: DAVID H LIVE
• 金额: $ 32.08万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9906935
• 关键词: Actins; Address; Affect; Amino Acid Sequence; Animals; Antibodies; Arenavirus; Biochemical; Biological Assay; Brain; Cardiac; Cell Line; Cells; Cognitive; Complex; Congenital Disorders; Consensus; Cytoskeleton; Defect; Disaccharides; Disseminated Malignant Neoplasm; Dystrophin; Elements; Engineering; Enzymes; Estrogen receptor positive; Extracellular Matrix; Extracellular Matrix Proteins; Funding; Gatekeeping; Generations; Glycopeptides; Glycoproteins; Goals; Golgi Apparatus; Human; Impairment; Individual; Infection; Laminin; Length; Life; Ligand Binding; Limb-Girdle Muscular Dystrophies; Longevity; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Methods; Molecular; Muscle Development; Muscle eye brain disease; Muscle function; Muscular Dystrophies; Mutation; Myoblasts; Neoplasm Metastasis; Neurologic; Null Lymphocytes; Pathology; Pathway interactions; Patients; Phenotype; Play; Polymers; Polysaccharides; Protein Glycosylation; Proteins; Regulation; Role; Severity of illness; Site; Specificity; Structure; System; Testing; Therapeutic; Therapeutic Intervention; Trisaccharides; Ursidae Family; Vesicular stomatitis Indiana virus; Viral; Virus Diseases; Walker-Warburg syndrome; Work; X-Ray Crystallography; alpha Dystroglycan; axon guidance; base; causal variant; cognitive function; congenital muscular dystrophy; design; dystroglycanopathy; enzyme pathway; glycosylation; glycosyltransferase; improved; man; novel; overexpression; protein complex; receptor; relating to nervous system; three dimensional structure; tool

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.

项目总结/摘要 在高等动物中，基于O-甘露糖基的蛋白质糖基化的意义在研究中得到阐明， 研究α-肌营养不良聚糖（α-DG），该聚糖在将细胞锚定到细胞外基质中发挥作用 α-DG低糖基化与肌营养不良症的一个子集相关，通常伴有 认知或其他异常。寻找这些病理学的分子基础揭示了新的生物合成， ic途径涉及以前未识别的酶和聚糖，其缺陷影响α-DG糖基化， 在糖基化的先天性疾病的列表中添加。在评估O- 甘露糖基化途径，发现该途径也参与介导某些病毒感染， 转移可以说是α-DG上功能最关键的O-Man聚糖类的完整结构 是在2016年通过我们和其他人的工作才成立的。其他蛋白质O-甘露糖基化途径 最近也出现了。我们的目标是进一步了解O-甘露糖基的作用和调节， 基于蛋白质糖基化以及重复二糖聚合物的加工和分布 基质聚糖，首先在α-DG M3核心三糖上鉴定。基质聚糖负责功能性的- 与ECM蛋白中的层粘连蛋白-G结构域的相互作用。前两个目标解决的是 在扩展最初的O-Man站点时，各种备选方案的相互作用和后果。关键的决定 聚糖延伸的点来自于当POMGNT 2在O-末端上添加GlcNAc时形成的连接， 人改造的α-DG。目前已知的M3聚糖核心仅位于一种蛋白质上的两个位点， 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，但我们将开发一种新的蛋白质序列元件。 全面了解的高度特异性，以解决是否有其他潜在的再- 蛋白质上这种聚糖的受体位点，以及这些位点是否可以通过治疗工程化到其他蛋白质中， 拯救表型的前景。我们将结合使用各种方法来理解POMGNT 2 的特异性相比之下，POMGNT 1可以在高尔基体中作用于剩余的O-Man位点上，插入具有 不同的连接产生其他主要类别的O-Man聚糖，特别是M1。的显函数 这些聚糖是未知的，但在没有M1的情况下，M3聚糖没有完全延伸，α-DG是功能性的。 安全性受损我们将定义M3聚糖的加工在哪里被破坏，M1与什么相互作用， 更好地理解POMGNT 1功能。第三个目的是调节基质聚糖长度， 它与生物化学和细胞水平功能的关系。我们还将探讨基质聚糖在 非O-Man糖蛋白，不依赖于蛋白质。总体而言，完成拟议目标 将在分子水平上建立功能性O-甘露糖基化的规则， 设计治疗方法。