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

• 批准号: 8170749
• 负责人: J. Michael Pierce
• 金额: $ 0.26万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8170749
• 关键词: Affect; Biochemical Reaction; Cells; Computer Retrieval of Information on Scientific Projects Database; Embryo; Enzymes; Funding; Grant; Human; In Vitro; Institution; Kidney; Kinetics; Lead; Link; Modification; Mucins; Muscle eye brain disease; Muscular Dystrophies; Mutation; N-Acetylglucosaminyltransferases; Oligosaccharides; Peptides; Reaction; Recombinants; Reporting; Research; Research Personnel; Resources; Role; Series; Serine; Site; Source; Specificity; Threonine; Time; United States National Institutes of Health; alpha Dystroglycan; glycosyltransferase; preference; protein O-mannose beta-1,2-N-acetylglucosaminyltransferase; protein aminoacid sequence; research study; synthetic peptide

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.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 α-肌营养不良聚糖 (¿DG) 拥有一个粘蛋白样结构域，其中包含多个丝氨酸 (S) 或苏氨酸 (T) 残基以及 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-甘露糖基-1,2-N-乙酰葡糖胺基转移酶 (POMGnT1) 和一系列糖基转移酶来延长。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。为了更好地了解 POMGnT1 在导致 MEB 疾病的 ¿DG 修饰中可能发挥的作用，我们合成了源自 ¿DG 粘蛋白样结构域的 8 个肽序列，每个肽序列均具有一个或多个 O-Man 位点。这些肽被指定为M1（残基416-420，在T418处有一个O-Man位点），M2（残基429-433，在S430和T431处有两个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S430和T431处有两个O-man位点）。 O-Man (T414), 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 和 S485 处有四个 O-Man 位点）、M7（残基 419-427，在 T421 和 T424 处有两个 O-Man 位点）和 M8（残基419-427 带四 O-Man 站点位于 T421、T422、T423 和 T424）。这些肽被用作人胚肾 (HEK-293) 细胞中表达的重组 POMGnT1 的体外受体。除了该酶对于每种底物的动力学参数（Km、Kcat 和 Km/Kcat）之外，还在反应产物中进行 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。在两个位点都受到 POMGnT1 影响的情况下，为了确定 POMGnT1 的作用是否是连续的，或者 GlcNAc 添加到 O-甘露糖基化残基上是随机发生的，进行时程酶促反应实验，其中通过 MS 分析产物以确定 GlcNac 残基添加到哪个甘露糖基化位点。