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

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 几种形式的先天性肌营养不良与四糖糖的错误组装有关，这些错误始于糖蛋白中央粘蛋白样区的不寻常的αO-Man修饰丝氨酸和苏氨酸残基。POMGNT1酶将GlcNAc残基添加到O-Man中，其活性缺陷会导致肌肉-眼-脑疾病，这是肌肉营养不良的一种形式。目前针对这一缺陷的临床检测依赖于以苄基甘露糖为底物，通过掺入放射性标记的GlcNAc来监测其活性。这在两个方面受到了不利影响。首先，苄基甘露糖不是一个好的底物，其次，用放射性进行检测是不可取的。通过对几种从营养不良多聚糖序列合成的甘露糖化糖肽的评价，已经确定了更好的底物。本项目的目的是制备相关的甘露糖糖肽，在N端加入荧光探针，以利用它们优越的底物特性，并允许使用高效液相方法灵敏地检测产物和反应物，提高现有临床检测的实用性。该项目汇集了沙赫特实验室的POMGNT1及其检测，以及CCRC的糖肽合成和体外检测方面的专业知识。

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.