Structure and Function in alpha-Dystroglycan Glycosylation

α-肌营养不良聚糖糖基化的结构和功能

基本信息

批准号：
8898155
负责人：
DAVID H LIVE
金额：
$ 28.47万
依托单位：
UNIVERSITY OF GEORGIA
依托单位国家：
美国
项目类别：
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/8898155
关键词：
Address Anabolism Animal Model Animals Antibodies Binding 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 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 biochemical tools 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

项目摘要

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-甘露糖基聚糖修饰的畸变对蛋白质α-多糖的修饰与功能的丧失和肌肉营养不良的丧失有关，这些肌营养不良也表现出显着的神经系统后果。这是第一个在动物界直接鉴定出O-甘露糖基化和相关的聚糖的蛋白质，并导致对几种新的糖基化生物合成途径的新兴理解。到目前为止，研究严重依赖于遗传分析和动物模型系统操纵的应用。该策略已经确定了许多参与者，但是它在分子水平完全阐述过程方面存在局限性，这也因体内蛋白质糖基化的内在微分差而变得复杂。需要一种化学生物学方法来充分阐明这些聚糖结构，调节其合成以及疾病的分子基础。这将需要使用化学合成来访问定义的糖肽作为中间体来量化酶促加工，从而允许详细的酶活性表征并解码其底物需求，并加上质谱法以分析聚糖结构。这些信息对于充分揭示这些聚糖，疾病诊断以及治疗策略的理性设计的结构/功能关系至关重要。 O-甘露糖基化及其与其他蛋白质的生物合成途径的更广泛相关性变得越来越明显，最近有几份报告描述了其他靶标，包括其对钙粘蛋白的流行。在此提案中，我们将重点关注与细胞外基质相互作用至关重要的复杂的O-甘露糖基聚糖结构。第一个目标将确定调节最近确定的酶Pomgnt2作用的因素。该功能在关键步骤中，选择O-MAN残基以详细说明层粘连蛋白结合结构（介导与细胞外基质的相互作用）的核心聚糖。第二个目的将确定将这种核心三糖聚糖与远端层粘连蛋白结合寡糖联系起来的尚未知道的结构，从而深入了解共同的活动。第三个目标将利用我们的糖肽合成方法来制造特定结构的抗体，提供提供诊断前景的试剂，并有助于研究发现这种形式的糖基化形式的糖蛋白更广泛。