SYNTHETIC PEPTIDES FOR THE STUDY OF IN VITRO KINETICS & SPECIFICITY OF POMGNT1

用于体外动力学研究的合成肽

基本信息

批准号：
8363027
负责人：
J. Michael Pierce
金额：
$ 0.34万
依托单位：
UNIVERSITY OF GEORGIA
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-06-01 至 2012-05-31
项目状态：
已结题

项目摘要

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. Alpha-Dystroglycan (¿DG) possesses a mucin-like domain with multiple serine (S) or threonine (T) residues with O-linked mannosylated (O-Man) oligosaccharides. These O-Man moieties can then be elongated by the O-mannosyl-¿1,2-N- acetylglucosaminyltransferase (POMGnT1) and by a series of glycosyltransferases. Recent reports have shown that mutations in POMGnT1 are a major cause of a form of muscular dystrophy known as muscle-eye-brain (MEB) disease. In order to gain a better understanding of the possible role of POMGnT1 in the modifications of ¿DG that lead to MEB disease, we have synthesized eight peptide sequences derived from the mucin-like domain of ¿DG, each with one or multiple O-Man sites. These peptides were designated as M1 (residues 416-420 with one O-Man at T418), M2 (residues 429-433 with two O-Man sites at S430 and T431), M3 (residues 326-331 with two O-man sites at T328 and T329), M4 (residues 411-416 with an O-Man at T414), M5 (residues 461-466 with two O-Man sites at T463 and T464), M6 (residues 480-487 with four O-Man sites at T482, T483, T484, and S485), M7 (residues 419-427 with two O-Man sites at T421 and T424), and M8 (residues 419-427 with four O-Man sites at T421, T422, T423, and T424). These peptides are being used as in vitro acceptors for recombinant POMGnT1 expressed in Human embryonic kidney (HEK-293) cells. In addition to the kinetic parameters (Km, Kcat, and Km/Kcat) of this enzyme for each substrate, MSn fragmentation experiments are being performed in the reaction products to determine whether POMGnT1 has a preference for the addition of GlcNAC to specific O-mannosylated sites, or both sites are affected by the glucosaminyltransferase. In cases where both sites are affected by POMGnT1, in order to determine whether the action of POMGnT1 is sequential, or the addition of GlcNAc to the O- mannosylated residues occurs randomly, time course enzymatic reaction experiments are conducted in which the products are analyzed by MS to determine to which mannosylated sites the GlcNac residues are being added.

该子项目是利用资源的众多研究子项目之一由 NIH/NCRR 资助的中心拨款提供该子项目的主要支持。并且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 来源的子项目可能列出的总成本。代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 α-肌营养不良聚糖 (¿DG) 拥有一个具有多个丝氨酸 (S) 或苏氨酸 (T) 残基的粘蛋白样结构域，以及 O-连接甘露糖基化 (O-Man) 寡糖，然后这些 O-Man 部分可以通过 O- 延长。甘露糖基-¿ 1,2-N-乙酰氨基葡萄糖转移酶 (POMGnT1) 和一系列糖基转移酶最近的报告表明，POMGnT1 的突变是一种称为肌眼脑 (MEB) 疾病的肌营养不良症的主要原因。更好地了解 POMGnT1 在 ¿ 修饰中的可能作用导致 MEB 疾病的 DG，我们合成了源自 ¿ 的粘蛋白样结构域的八种肽序列DG，各自具有一个或多个O-Man位点，这些肽被指定为M1（残基416-420，在T418处具有一个O-Man位点）、M2（残基429-433，在S430和T431处具有两个O-Man位点）， M3（残基 326-331，在 T328 和 T329 处有两个 O-man 位点）、M4 （残基 411-416，在 T414 处有一个 O-Man），M5（残基 461-466，在 T463 和 T464 处有两个 O-Man 位点），M6（残基 480-487，在 T482、T483、T484 处有四个 O-Man 位点），和S485），M7（残基419-427， T421 和 T424 处有两个 O-Man 位点）和 M8（T421、T422、T423 和 T424 处有四个 O-Man 位点的残基 419-427）这些肽被用作在人中表达的重组 POMGnT1 的体外受体。胚胎肾 (HEK-293) 细胞除了动力学参数（Km、Kcat 和）。对于每种底物，在反应产物中进行 MSn 片段化实验，以确定 POMGnT1 是否偏好将 GlcNAC 添加到特定的 O-甘露糖基化位点，或者两个位点都受到葡糖胺基转移酶的影响。两个位点均受 POMGnT1 影响的情况，以确定 POMGnT1 的作用是否是连续的，或向 O-甘露糖基化残基添加 GlcNAc随机发生，进行时程酶促反应实验，其中通过 MS 分析产物以确定添加 GlcNac 残基的甘露糖化位点。