Post-Translation Processing of Alpha-Dystroglycan

描述（由申请人提供）：拟议研究的目的是了解1-肌营养不良蛋白聚糖（1-DG）的翻译后加工，1-DG是一种糖蛋白，是肌营养不良蛋白-糖蛋白复合物的关键组分，将肌细胞锚定到细胞外基质。1-DG在其高度保守的中央粘蛋白样区域的糖基化缺陷，具有许多糖基化的S和T残基，是几种形式的遗传性肌营养不良症的原因。悬垂聚糖通过N-乙酰葡糖胺（GalNAc）或更不寻常的甘露糖（Man）残基与蛋白质连接，后者的位点目前正在绘制中。已经建立了O-Man四糖组装中的问题与疾病之间的关系。我们最近发现的证据表明，GalNAc残基是重要的结构。合成，生物化学和结构方法将被整合到研究中，以了解这个复杂过程中的步骤，提供酶促转化的定量描述，并促进对1-DG参与的关键相互作用的深入了解。了解这一过程中的步骤将为合理设计治疗方法以纠正缺陷提供基础，包括基因治疗和上调其他可能补充缺陷的酶。重点将放在两个步骤后，最初的O-Man修改1-DG。两个主要目的是1）分析POMGnT 1的底物谱，POMGnT 1是一种参与O-Man聚糖组装中关键步骤的酶，其缺陷与肌-眼-脑疾病相关，以更好地理解如何挽救缺陷并开发用于检测缺陷酶的更好测定法，2）阐明多肽GalNAc转移酶在1-DG上起始O-GalNAc位点的活性及其与POMGnT 1糖基化的关系。 公共卫生相关性：几种形式的肌营养不良症与糖蛋白1-肌营养不良聚糖上碳水化合物附着的异常有关，这些异常是由酶的缺陷引起的，这些酶执行导致碳水化合物在其上特异性安装的一系列复杂步骤。拟议的研究将使用一种综合方法，结合从1-肌营养不良聚糖化学合成糖肽，通过糖基转移酶对其修饰进行生物化学分析，以及反应物和产物的结构分析，以在分子水平上了解这种重要糖蛋白的翻译后修饰。所得信息将有助于了解几种形式的肌营养不良症的病理机制，改善临床检测，并合理设计治疗干预措施。

DESCRIPTION (provided by applicant): The objective of the proposed research is to understand the post-translational processing of 1- dystroglycan (1-DG), a glycoprotein that is a key component of the dystrophin-glycoprotein complex, anchoring muscle cells to the extracellular matrix. Defective glycosylation of 1-DG in its highly conserved central mucin-like region, with numerous glycosylated S and T residues is the cause of several forms of hereditary muscular dystrophy. Pendant glycans are linked to the protein by either N-acetylglucosamine (GalNAc) or the more unusual mannose (Man) residue whose sites are now just being mapped. Relationships between problems in assembly of the O-Man tetra-saccharide and disease have been established. We have recently found evidence that the GalNAc residues are important structurally. Synthetic, biochemical and structural methods will be integrated in the research to develop an understanding of the steps in this complex process, providing a quantitative description of enzymatic transformations and also facilitating insights into key interactions in which 1-DG participates. An understanding of the steps in this process will provide basis for rational design of therapies to correct the defects, including gene therapy and up-regulation of other enzymes that may complement the defective ones. The focus will be on two steps following the initial O-Man modifications of 1-DG. The two major aims are 1) to analyze the substrate profile of POMGnT1, an enzyme involved in a key step in the O-Man glycan assembly whose defects are associated with muscle-eye-brain disease, to better understand how rescue the defect and to develop a better assay for detection of defective enzyme, and 2) to elucidate the activity of polypeptide GalNAc transferases in initiating O-GalNAc sites on 1-DG and the relationship of these to POMGnT1 glycosylation. PUBLIC HEALTH RELEVANCE: Several forms of muscular dystrophy are associated with aberrations in the attachment of carbohydrates to the glycoprotein 1-dystroglycan arising from defects in the enzymes that carry out the complex series of steps leading to the specific installation of carbohydrates on it. The proposed research would use an integrated approach combining chemical synthesis of glycopeptides from1- dystroglycan, biochemical analysis of their modification by glycosyltransferase enzymes, and structural analysis of the reactants and products, to develop an understanding at a molecular level of the post-translational modification of this important glycoprotein. The information derived will contribute to understanding the pathological mechanisms of several forms of muscular dystrophies, improving clinical assay, and rational design of therapeutic interventions.