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

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 α-营养不良聚糖(α-DG)具有粘蛋白样结构域，含有多个丝氨酸(S)或苏氨酸(T)残基，以及O-连接甘露糖苷(O-Man)寡糖。这些O-Man部分可以被O-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延长。最近的报道表明，POMGNT1的突变是一种被称为肌肉-眼-脑(MEB)疾病的肌肉营养不良的主要原因。为了更好地了解POMGNT1在导致MEB病的DG修饰中的可能作用，我们合成了8个源自DG的粘蛋白样结构域的肽序列，每个序列都有一个或多个O-Man位点。这些多肽被命名为M1(残基416-420，在T418有一个O-Man)，M2(残基429-433，在S430和T431有两个O-Man位)，M3(残基326-331，在T328和T329有两个O-Man位)，M4(残基411-416，在T414有一个O-Man)，M5(残基461-466，在T463和T464有两个O-Man位)，M6(残基480-487，在T482、T483、T484和S485有四个O-Man位)，M7(残基419-427，在T421和T424有两个O-Man位)，和M8(残基419-427，在T421、T422、T423和T424有四个O-Man位点)。这些多肽被用作在人胚胎肾脏(HEK-293)细胞中表达的重组POMGNT1的体外受体。除了该酶对每种底物的动力学参数(Km、Kcat和Km/Kcat)外，还在反应产物中进行MSN碎片实验，以确定POMGNT1是否倾向于将GlcNAc添加到特定的O-甘露糖基位置，或者这两个位置都受到氨基葡萄糖转移酶的影响。在两个位点都受到POMGNT1影响的情况下，为了确定POMGNT1的作用是连续的，还是将GlcNAc添加到O-甘露糖基上是随机发生的，进行了时程酶反应实验，其中产物被MS分析以确定GlcNAc残基被添加到哪些甘露糖基上。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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.