Structure and Function in alpha-Dystroglycan Glycosylation

描述（由申请人提供）：蛋白质α-肌营养不良蛋白上的O-甘露糖基聚糖修饰的畸变与功能丧失和肌营养不良形式相关，这些形式也表现出显著的神经学后果。这是第一个在动物界直接鉴定出O-甘露糖基化和相关聚糖的蛋白质，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的应用和动物模型系统的操作。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该过程方面存在局限性，体内蛋白质糖基化的内在微观异质性也使其复杂化。需要化学生物学方法来充分阐明这些聚糖结构、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限定的糖肽作为定量酶促加工的中间体，从而允许详细表征酶活性并解码其底物要求，再加上质谱分析聚糖结构。这些信息对于充分揭示这些聚糖的结构/功能关系、疾病诊断和合理设计治疗策略至关重要。O-甘露糖基化及其生物合成途径与其他蛋白质的更广泛相关性正变得越来越明显，最近有几篇报道描述了其他靶点，包括其在钙粘蛋白上的流行。在这个建议中，我们将集中在复杂的O-甘露糖基聚糖结构的关键与细胞外基质的相互作用。第一个目标是确定调节最近鉴定的酶POMGNT 2的作用的因素。这在一个关键步骤起作用，选择O-Man残基用于确定核心聚糖，介导与细胞外基质相互作用的层粘连蛋白结合结构附着于该核心聚糖。第二个目标是确定连接核心三糖聚糖和远端层粘连蛋白结合寡糖的未知结构，从而了解组装它的活性。第三个目标是利用我们的糖肽合成方法制备特定结构的抗体，提供有希望用于诊断的试剂，这将促进对 发现这种糖基化形式的糖蛋白的范围更广。

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.