Post-Translation Processing of Alpha-Dystroglycan

描述(申请人提供)：拟议研究的目标是了解1-DG(1-DG)的翻译后处理，1-DG是Dystrophin-糖蛋白复合体的关键成分，将肌肉细胞锚定在细胞外基质上。高度保守的中央粘蛋白样区1-DG糖基化缺陷，大量糖基化的S和T残基是几种形式的遗传性肌营养不良的原因。悬挂的葡聚糖通过N-乙酰氨基葡萄糖(GalNAc)或更不寻常的甘露糖(Man)残基与蛋白质相连，这些残基的位置现在刚刚被绘制出来。O-Man四糖的组装问题与疾病之间的关系已经确定。我们最近发现的证据表明GalNAc残基在结构上是重要的。研究中将整合合成、生化和结构方法，以了解这一复杂过程中的各个步骤，提供酶转化的定量描述，并促进对1-DG参与的关键相互作用的深入了解。对这一过程步骤的理解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1-DG的初始O-Man修改之后的两个步骤上。本研究的两个主要目的是：1)分析POMGNT1的底物谱，POMGNT1参与O-Man糖链组装的关键步骤，其缺陷与肌肉-眼-脑疾病相关，以更好地了解如何挽救缺陷并建立更好的检测缺陷酶的方法；2)阐明多肽GalNAc转移酶在启动1-DG上O-GalNAc位点中的活性及其与POMGNT1糖基化的关系。与公共卫生相关：几种形式的肌营养不良症与碳水化合物与糖蛋白1-营养不良糖链的附着异常有关，这些异常是由于执行导致碳水化合物特定安装在其上的一系列复杂步骤的酶的缺陷引起的。这项拟议的研究将使用一种综合的方法，结合从1-肌营养不良多糖合成糖肽、糖基转移酶对糖肽修饰的生化分析以及反应物和产物的结构分析，在分子水平上了解这一重要糖蛋白的翻译后修饰。所获得的信息将有助于了解几种形式的肌营养不良的病理机制，改进临床检测，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.