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

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH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 中心，不一定是研究者的机构。 α-肌营养不良聚糖（DG）具有粘蛋白样结构域，具有多个丝氨酸（S）或苏氨酸（T）残基， 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-甘露糖基延长。 乙酰葡糖胺基转移酶（POMGnT 1）和一系列糖基转移酶。 最近的报告显示， POMGnT 1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。 在 为了更好地了解POMGnT 1在导致MEB疾病的DG修饰中的可能作用， 我们已经合成了八个来自DG粘蛋白样结构域的肽序列，每个都有一个或多个O-Man 网站. 将这些肽命名为M1（残基416-420在T418处具有一个O-Man）、M2（残基429-433在T418处具有两个O-Man）、M3（残基429-433在T418处具有一个O-Man）和M4（残基429-433在T418处具有两个O-Man）。 S430和T431处的O-Man位点）、M3（残基326-331，T328和T329处有两个O-Man位点）、M4（残基411-416 在T414处具有O-Man）、M5（在T463和T464处具有两个O-Man位点的残基461-466）、M6（在T463和T464处具有四个O-Man位点的残基480 - 487）、M6（在T463和T464处具有两个O-Man位点的残基461-466）、M6（在T463和T464处具有四个O-Man位点的残基480-487）、M6（在T463和T464处具有四个O-Man位点的残基480-487）、M6（在T463和T464处具有四个O-Man位点的残基480-487）和M6（在T463和T464处具有四个O-Man位点的残基480-487）。 T482、T483、T484和S485处的O-Man位点）、M7（残基419-427，T421和T424处有两个O-Man位点）和M8 （残基419-427在T421、T422、T423和T424处具有四个O-Man位点）。 这些肽被用作体外 在人胚肾（HEK-293）细胞中表达的重组POMGnT 1的受体。 除了动力学 对于每种底物，该酶的参数（Km、Kcat和Km/Kcat），MSn片段化实验正在进行。 在反应产物中进行，以确定POMGnT 1是否偏好将GlcNAC添加到特定的 O-甘露糖基化位点或两个位点均受葡糖胺基转移酶影响。 如果两个站点都受到 为了确定POMGnT 1的作用是否是顺序的，或者将GlcNAc添加到O-葡萄糖链中， 甘露糖基化残基随机出现，进行时程酶促反应实验，其中产物 通过MS分析以确定GlcNac残基被添加到哪些甘露糖基化位点。

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.