The structure and function of eukaryotic protein glycosylation enzymes

项目摘要 天冬酰胺(N-连接)糖基化是真核生物分泌和膜的普遍修饰 蛋白质。N-糖基化是最常见的糖基化类型，90%的糖蛋白是N-糖基化。 糖基化。N-葡聚糖最初通过多蛋白跨膜加到三联体序列N-X-S/T中 称为寡糖转移酶(OST)的复合体。大多数蛋白质的N-糖基化发生在蛋白质是 由核糖体合成并通过转运子运输。因此，OST在物理上 与核糖体和转运子相互作用并形成超复合体。蛋白质合成的机制 和新生多肽转位更好地理解得益于核糖体和 变性人。然而，我们对真核蛋白质N-糖基化的了解非常有限，因为缺乏 高分辨率OST结构。我们的目标是通过描述结构特征来弥合这一主要的知识鸿沟 以及酵母的八蛋白OST复合体的功能。最近的一项研究表明，抑制人类 OST诱导受体酪氨酸激酶驱动的肿瘤细胞衰老，提示OST可能是一个靶点 用于开发抗肿瘤药物。此外，由于正常细胞向癌细胞的转化 细胞通常伴随着N-糖链的分支和延伸，几种N-糖链已被广泛应用 作为肿瘤标志物：例如，用于检测胰腺癌的碳水化合物抗原(CA)CA19-9，以及 CA125被认为是诊断卵巢癌的金标准标记物。我们提出了一个 酵母OST复合体的全面结构和功能研究，采用冷冻-EM联合方法， X射线结晶学，基于结构的诱变和细胞生物学，以及体外活性分析。我们的工作已经完成 在肿瘤发生和癌症诊断与治疗中的重要意义。

Project Summary Asparagine (N-linked) glycosylation is a ubiquitous modification of eukaryotic secretory and membrane proteins. N-glycosylation is the most common type of glycosylation, with 90% of glycoproteins being N- glycosylated. The N-glycans are initially added to a triplet sequence N-X-S/T by a multi-protein transmembrane complex called oligosaccharyl transferase (OST). Most of the protein N-glycosylation occurs while a protein is being synthesized by the ribosome and being transported through the translocon. Hence, OST physically interacts and forms a super-complex with the ribosome and translocon. The mechanisms of protein synthesis and nascent peptide translocation are better understood thanks to available structures of ribosomes and translocons. However, our understanding of eukaryotic protein N-glycosylation is very limited due to the lack of high-resolution OST structures. Our goal is to bridge this major knowledge gap by characterizing the structure and function of the eight-protein OST complex of yeast. A recent study demonstrated that inhibiting human OST induces senescence in receptor tyrosine kinase–driven tumor cells, suggesting that OST may be a target for the development of anti-tumor agents. Furthermore, because transformation of a normal cell to a cancer cell is usually accompanied by N-glycan branching and extension, several N-glycans have been widely used as tumor markers: for example, carbohydrate antigen (CA) CA19-9 for detecting pancreatic cancer, and CA125, which is considered the gold standard marker for diagnosing ovarian cancer. We propose a comprehensive structure and function study of the yeast OST complex, with a combined approach of cryo-EM, X-ray crystallography, structure-based mutagenesis and cell biology, and in vitro activity assays. Our work has important implications in tumorigenesis and cancer diagnosis and treatment.