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: 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
• 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
• 来源: https://reporter.nih.gov/project-details/8363027
• 关键词: Affect; Biochemical Reaction; Cells; Embryo; Enzymes; Funding; Grant; Human; In Vitro; Kidney; Kinetics; Lead; Link; Modification; Mucins; Muscle eye brain disease; Muscular Dystrophies; Mutation; N-Acetylglucosaminyltransferases; National Center for Research Resources; Oligosaccharides; Peptides; Principal Investigator; Reaction; Recombinants; Reporting; Research; Research Infrastructure; Resources; Role; Series; Serine; Site; Source; Specificity; Technology; Threonine; Time; United States National Institutes of Health; alpha Dystroglycan; cost; glycosyltransferase; man; 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. 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.

该副本是利用资源的众多研究子项目之一 由NIH/NCRR资助的中心赠款提供。对该子弹的主要支持 而且，副投影的主要研究员可能是其他来源提供的 包括其他NIH来源。列出的总费用可能 代表subproject使用的中心基础架构的估计量， NCRR赠款不直接向子弹或副本人员提供的直接资金。 Alpha-dystroglycan（DG）具有带有多种丝氨酸或苏氨酸（T）残基的粘蛋白样结构域，具有O连接的甘露糖基化（O-Man）寡糖。然后，这些O-Man部分可以通过O-甘露糖基 - 1,2-N-乙酰氨基氨基氨基链酰酯转移酶（POMGNT1）和一系列的糖基转移酶拉长。最近的报道表明，POMGNT1中的突变是一种肌肉营养不良形式的主要原因，称为肌肉 - 脑脑（MEB）疾病。为了更好地了解pomgnt1在导致MEB疾病的DG修饰中的可能作用，我们合成了源自从一个或多个O-Man位点的粘蛋白样结构域得出的八个胡椒序列。这些宠物被指定为M1（残基416-420，一个O-MAN，在T418处），M2（残基429-433，有两个O-Man站点在S430和T431和T431），M3（残留326-331（残留326-331），带有两个O-MAN站点，在T328和T328和T328和T329），M41411-41 AN ORE 45 ANO ORE） （残留461-466，有两个O-Man站点在T463和T464），M6（残基480-487，有四个O-Man站点在T482，T483，T483，T484和S485和S485和S485），M7，M7（残基419-427）（残留419-427）与T421和T421和T421和T424的两个O-424）； T421，T422，T423和T424的O-Man站点。这些肽被用作在人类胚胎肾（HEK-293）细胞中表达的重组POMGNT1的体外受体。 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 glucosamiyltransferase.在两个站点都受Pomgnt1影响的情况下，为了确定Pomgnt1的作用是顺序的，或者在O-甘露诺诺糖基化残差中添加GlcNAC是随机发生的，时间过程酶促反应实验是通过MS进行分析的，以确定glcnAc的STOES glcnAc VERES添加了MS的分析。