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: 8767819
• 负责人: DAVID H LIVE
• 金额: $ 28.31万
• 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 2018-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8767819
• 关键词: Address; Anabolism; Animal Model; Animals; Antibodies; Binding; Biochemical; Biochemistry; Biological Assay; Biological Models; Biological Process; Biology; Brain; Cadherins; Carbohydrates; Cell Surface Proteins; Cell surface; Cells; Chemical Structure; Chemicals; Complex; Coupled; Defect; Detection; Diagnosis; Disease; Distal; Dystroglycan; Elements; Enzymes; Extracellular Matrix; Future; Generations; Glycopeptides; Glycoproteins; Goals; Health; In Vitro; Investigation; Laminin; Lead; Link; Mass Spectrum Analysis; Mediating; Modification; Molecular; Molecular Structure; Monoclonal Antibodies; Mus; Muscle; Muscular Dystrophies; Mutation; Neurologic; Oligosaccharides; Paper; Pathology; Pathway interactions; Phosphorylation; Play; Polysaccharides; Post-Translational Protein Processing; Prevalence; Process; Protein Glycosylation; Proteins; Reagent; Regulation; Reporting; Research; Role; Route; Science; Site; Specificity; Structure; Structure-Activity Relationship; Syndrome; Therapeutic; Therapeutic Studies; Tissues; Trisaccharides; alpha Dystroglycan; base; carbohydrate structure; chemical synthesis; congenital muscular dystrophy; design; disease diagnosis; dystroglycanopathy; enzyme activity; enzyme substrate; genetic analysis; genetic manipulation; glycosylation; glycosyltransferase; in vivo; insight; loss of function; man; mannose 6 phosphate; member; mutant; novel; phosphodiester; practical application; research study; therapy design; tool

DESCRIPTION (provided by applicant): Aberrations in O-mannosyl glycan modifications on the protein alpha- dystroglyan, have been associated with loss of function and forms of muscular dystrophy those also manifest significant neurological consequences. This was the first protein on which O-mannosylation and associated glycans were directly identified in the animal kingdom, and has led to the emerging understanding of several new glycosylation biosynthetic pathways. Until now, investigations have relied heavily on the application of genetic analysis and manipulation of animal model systems. The strategy has identified a number of players, but it has limitations in fully elaborating the process at a molecular level, also complicated by the intrinsic microheterogeneity of protein glycosylation in vivo. A chemical biology approach is needed to fully elucidate these glycan structures, the regulation of their synthesis, and the molecular basis of disease. This will entail the use of chemical synthesis to access defined glycopeptides as intermediates in quantifying enzymatic processing, thereby allowing detailed characterization of enzyme activities and decoding of their substrate requirements, coupled with mass spectrometry to analyze glycan structures. This information is critical in fully revealing the structure/function relationships of these glycans, in disease diagnosis, and in rational design of therapeutic strategies. The broader relevance of O- mannosylation and its biosynthetic pathway to other proteins are becoming more apparent, with several recent reports describing additional targets, including its prevalence on cadherins. In this proposal we will focus on the complex O-mannosyl glycan structure critical to the interactions with the extracellular matrix. The first aim will determine the factors that regulate he action of the recently identified enzyme POMGNT2. This functions at a key step, selecting O-Man residues for commitment to elaborate the core glycan to which the laminin binding structure, that mediates interaction with the extracellular matrix, is attached. The second aim wil identify the as yet unknown structure that links this core trisaccharide glycan with the distal laminin binding oligosaccharide, and thus gain insight into the activities that assemble it. The third aim will leverage our glycopeptide synthetic approach to make antibodies to specific structures, providing reagents that offer promise in diagnosis and which will facilitate studies of the broader range of glycoproteins on which this form of glycosylation is found.

描述(由申请人提供)：O-甘露醇多聚糖在蛋白质α-dystroglan上的修饰异常与功能丧失和肌肉营养不良的形式有关，这些也表现出显著的神经后果。这是在动物界中第一个直接鉴定出O-甘露糖化和相关多糖的蛋白质，并导致了对几种新的糖基化生物合成途径的理解。到目前为止，研究在很大程度上依赖于遗传分析和动物模型系统操纵的应用。该策略已经确定了一些参与者，但它在分子水平上充分阐述这一过程方面存在局限性，而且由于体内蛋白质糖基化的内在微观异质性而变得复杂。需要一种化学生物学方法来充分阐明这些多糖结构、其合成的调节以及疾病的分子基础。这将需要使用化学合成来获得确定的糖肽作为量化酶处理的中间体，从而允许详细地表征酶的活性和解码其底物要求，并结合质谱学来分析糖的结构。这些信息对于充分揭示这些多糖的结构/功能关系、疾病诊断和合理设计治疗策略是至关重要的。O-甘露糖化及其生物合成途径与其他蛋白质的更广泛的相关性正变得更加明显，最近的几篇报道描述了更多的靶点，包括它在钙粘附素上的流行。在这项提案中，我们将重点介绍复杂的O-甘露糖多糖结构，该结构对与细胞外基质的相互作用至关重要。第一个目标将确定调节最近发现的POMGNT2酶的作用的因素。这在一个关键步骤发挥作用，选择O-Man残基进行承诺，以详细阐述核心多糖，层粘连蛋白结合结构与细胞外基质相互作用的中介作用。第二个目标是确定将这个核心三糖与远端层粘连蛋白结合的寡糖连接的未知结构，从而深入了解组装它的活动。第三个目标将利用我们的糖肽合成方法来制造针对特定结构的抗体，提供有望在诊断中提供希望的试剂，并将促进对在其上发现这种形式的糖基化的更广泛糖蛋白的研究。