The structure and function of eukaryotic protein glycosylation enzymes

项目摘要 天冬酰胺（N-连接）糖基化是真核细胞分泌和膜的普遍修饰 proteins. 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-聚糖最初通过多蛋白质跨膜转运被添加到三联体序列N-X-S/T中。 寡糖基转移酶（OST）。大多数蛋白质的N-糖基化发生在蛋白质 由核糖体合成并通过易位子转运。因此，OST物理上 与核糖体和易位子相互作用并形成超复合物。蛋白质合成的机制 由于核糖体的可用结构， translocons。然而，我们对真核蛋白质N-糖基化的理解是非常有限的，由于缺乏 高分辨率OST结构。我们的目标是通过表征结构来弥合这一主要的知识差距 和酵母八蛋白OST复合物的功能。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，表明OST可能是靶点 用于开发抗肿瘤药物。此外，由于正常细胞转化为癌症 细胞内通常伴随着N-聚糖的分支和延伸，几种N-聚糖已被广泛应用 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）CA 19 -9，和 CA 125被认为是诊断卵巢癌的金标准标志物。我们提出了一个 酵母OST复合物的全面结构和功能研究，结合冷冻电镜方法， X射线晶体学、基于结构的诱变和细胞生物学以及体外活性测定。我们的工作 在肿瘤发生和癌症诊断和治疗中具有重要意义。

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.