Assembly and transfer of N-linked oligosaccharides

项目描述（申请人提供）：该项目的长期目标是了解寡糖转移酶（OST）的结构、功能和机制。OST将高甘露寡糖转移到粗内质网管腔中新生蛋白质的天冬酰胺残基上。在拟议的资助期内，将特别强调哺乳动物和真菌OST复合物的生化、结构和分子特征。犬OST已分解成多种形式，不同的亚基组成和酶活性。有趣的是，两个酵母OST亚基（Stt3p和Ost3p）的哺乳动物同源物由多个基因（STT3-A和STT3-B； N33和lAP）编码。多个组织Northern印迹揭示了这些mrna相对表达的组织特异性差异。该项目的目的之一是阐明OST异构体多样性的作用，并了解亚基组成的差异如何影响酶活性。动力学实验表明，低聚糖转移酶具有独特的催化和调节结合位点。将开发新的检测方法和生化探针来确定新生多肽中醇链寡糖供体和N-X-S/T受体的结合位点的位置。酵母遗传学方法将用于鉴定三肽结合位点的重要氨基酸残基。该项目的第三个目标是通过对冷冻电子显微镜获得的图像进行三维重建，获得酵母和哺乳动物OST复合物的低至中分辨率结构。抗体和化学探针将用于绘制底物结合位点和细胞质和管腔结构域到OST结构的位置。该项目的第四个目标是确定先天性糖基化疾病新亚型的分子基础。将筛选成纤维细胞系是否存在低聚糖代谢缺陷和低聚糖转移酶活性缺陷。

DESCRIPTION (provided by applicant): The long-term objective of this project is to understand the structure, function and mechanism of the oligosaccharyltransferase (OST). The OST transfers high mannose oligosaccharides onto the asparagines residues of nascent proteins in the lumen of the rough endoplasmic reticulum. During the proposed funding period, particular emphasis will be placed on a biochemical, structural and molecular characterization of the mammalian and fungal OST complexes. The canine OST has been resolved into multiple forms that differ with respect to subunit composition and enzyme activity. Interestingly, mammalian orthologues of two of the yeast OST subunits (Stt3p and Ost3p) are encoded by multiple genes (STT3-A and STT3-B; N33 and lAP). Multiple tissue Northern blots reveal tissue-specific differences in the relative expression of these mRNAs. One objective of this project is to elucidate the role of OST isoform diversity and to understand how differences in subunit composition effect enzyme activity. Kinetic experiments suggest that the oligosaccharyltransferase has distinct catalytic and regulatory binding sites for the dolichol-linked oligosaccharide. Novel assays and biochemical probes will be developed to determine the locations of the binding sites for the dolichol-linked oligosaccharide donors and the N-X-S/T acceptors in the nascent polypeptide. Yeast genetics methods will be used to identify important amino acid residues in the tripeptide-binding site. A third objective of this project will be to obtain a low to medium resolution structure of the yeast and mammalian OST complexes by 3-dimensional reconstruction of images that have been obtained by cryoelectron microscopy. Antibodies and chemical probes will be used to map the locations of substrate binding sites and cytosolic and lumenal domains onto the OST structure. The fourth objective of this project is to determine the molecular basis for novel subtypes of Congenital Disorders of Glycosylation. Fibroblast cell lines will be screened for defects in dolichol-linked oligosaccharide metabolism and deficiencies in oligosaccharyltransferase activity.