Abstract：
N-linked glycosylation is an essential and highly conserved co- and post-translational protein modification in all domains of life. In humans, genetic defects in N-linked glycosylation pathways result in metabolic diseases collectively called Congenital Disorders of Glycosylation. In this modification reaction, a mannose rich oligosaccharide is transferred from a lipid-linked donor substrate to a specific asparagine side-chain within the -N-X-T/S- sequence (where X ≠ Proline) of the nascent protein. Oligosaccharyltransferase (OST), a multi-subunit membrane embedded enzyme catalyzes this glycosylation reaction in eukaryotes. In yeast, Ost4 is the smallest of nine subunits and bridges the interaction of the catalytic subunit, Stt3, with Ost3 (or its homolog, Ost6). Mutations of any C-terminal hydrophobic residues in Ost4 to a charged residue destabilizes the enzyme and negatively impacts its function. Specifically, the V23D mutation results in a temperature-sensitive phenotype in yeast. Here, we report the reconstitution of both purified recombinant Ost4 and Ost4V23D each in a POPC/POPE lipid bilayer and their resonance assignments using heteronuclear 2D and 3D solid-state NMR with magic-angle spinning. The chemical shifts of Ost4 changed significantly upon the V23D mutation, suggesting a dramatic change in its chemical environment.
摘要：
N-连接的糖基化是生命所有域中必需的且高度保守的翻译后蛋白质修饰。在人类中,N-连接糖基化途径中的遗传缺陷导致代谢疾病统称为先天性糖基化障碍。在这种改性反应中，富含甘露糖的寡糖从脂质连接的供体底物转移到新生蛋白的-N-X-T/S-序列(其中X#脯氨酸)内的特定天冬酰胺侧链。寡糖糖基转移酶(OST),一种多亚基膜包埋酶在真核生物中催化这种糖基化反应。在酵母中，Ost4是最小的九个亚基和桥梁的催化亚基的相互作用，stt3，与Ost3（或其同系物，Ost6).Ost4中的任何C末端疏水残基突变为带电残基会使酶不稳定并负面影响其功能。具体来说,V23D突变导致酵母的温度敏感表型.这里,我们报告了纯化的重组Ost4和Ost4V23D在POPC/POPE脂质双层中的重建以及使用异核2D和3D固态NMR和魔角旋转的共振分配。在V23D突变后，Ost4的化学位移发生了显著变化，表明其化学环境发生了戏剧性的变化。
