FLUORESCENT-TAGGED MANNOSYL GLYCOPEPTIDES FOR MUSCLE-EYE-BRAIN DISEASE ASSAY

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心赠款提供。子项目的主要支持 子项目的主要研究者可能是由其他来源提供的， 包括其他NIH来源。 为子项目列出的总成本可能 表示子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 几种形式的先天性肌营养不良症与从糖蛋白的中央粘蛋白样区域中不寻常的α-O-Man修饰的丝氨酸和苏氨酸残基开始的四糖聚糖组装中的错误有关。POMGnT 1酶在O-Man上添加了一个GlcNAc残基，其活性的缺陷会引起肌-眼-脑疾病，这是一种肌肉萎缩症。这种缺陷的临床测定目前依赖于苄基甘露糖作为底物，通过掺入放射性标记的GlcNAc来监测活性。这在两个方面有不利之处。首先，苄基甘露糖不是良好的底物，其次，放射性检测是不可取的。随着对来自肌营养不良蛋白聚糖序列的几种合成甘露糖基化糖肽的评价，已经鉴定出了上级底物。本项目的目的是制备相关的甘露糖基化糖肽，在N-末端掺入荧光探针，以利用其上级底物特性，并允许使用HPLC方法灵敏地检测产物和反应物，增强现有临床测定的实用性。该项目汇集了Schachter实验室的POMGnT 1及其测定的专业知识，以及CCRC的糖肽合成和体外测定。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Several forms of congenital muscular dystrophy have been related to errors in the assembly of a tetrasaccharide glycan starting from the unusual alpha O-Man modified serine and threonine residues in the central mucin-like region of the glycoprotein. The POMGnT1 enzyme adds a GlcNAc residue to the O-Man, and defects in its activity give rise to Muscle-Eye-Brain disease, a form of muscular dystrophy. A clinical assay for this defect currently relies on benzyl mannose as a substrate, with the activity monitored by incorporation of radio-labled GlcNAc. This suffers from disadvantages on two counts. First, benzyl mannose is not a good substrate, and second, detection with radioactivity is undesirable. With the evaluation of several synthetic mannosylated glycopeptides from dystroglycan sequences, superior substrates have been identified. The aim of this project is to prepare relevant mannoyslated glycopeptides, incorporating a fluorescent probe at the N-terminus, to take advantage of their superior substrate characteristics and to allow for sensitive detection of products and reactants using HPLC methods, enhancing the utility of the existing clinical assay. The project brings together expertise in POMGnT1 and its assay in the Schachter lab, and glycopeptide synthesis and in vitro assays at CCRC.