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的一个 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。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 α-肌营养不良聚糖（DG）具有粘蛋白样结构域，该结构域具有多个丝氨酸（S）或苏氨酸（T）残基和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-乙酰葡糖胺转移酶（POMGnT 1）和一系列糖基转移酶延长。最近的报告表明，POMGnT 1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POMGnT 1在导致MEB疾病的<$DG修饰中的可能作用，我们合成了8个来自<$DG粘蛋白样结构域的肽序列，每个肽序列具有一个或多个O-Man位点。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S430和T431处具有两个O-Man位点），M3（残基326-331，在T328和T329处具有两个O-man位点），M4（残基411-416在T414处具有O-Man），M5（残基461-466，在T463和T464处具有两个O-Man位点），M6（残基480-487，在T482、T483、T484和S485处具有四个O-Man位点），M7（在T421和T424处具有两个O-Man位点的残基419-427）和M8（在T421、T422、T423和T424处具有四个O-Man位点的残基419-427）。这些肽被用作在人胚肾（HEK-293）细胞中表达的重组POMGnT 1的体外受体。除了动力学参数（Km，Kcat，和Km/Kcat）的这种酶的每种底物，MSn片段化实验正在进行的反应产物，以确定是否POMGnT 1有一个偏好的GlcNAC添加到特定的O-甘露糖基化的网站，或两个网站的葡糖胺转移酶的影响。在两个位点都受POMGnT 1影响的情况下，为了确定POMGnT 1的作用是顺序的，还是GlcNAc向O-甘露糖基化残基的添加随机发生，进行时程酶促反应实验，其中通过MS分析产物以确定GlcNAc残基被添加到哪个甘露糖基化位点。

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.