Assembly and transfer of N-linked oligosaccharides

描述(申请人提供)：本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将高甘露糖低聚糖转移到粗面内质网腔内新生蛋白的天冬酰胺残留物上。在拟议的供资期间，将特别重视哺乳动物和真菌OST复合体的生化、结构和分子特征。犬OST已被分解成多种亚基组成和酶活性不同的形式。有趣的是，两个酵母OST亚基(Stt3p和Ost3p)的哺乳动物同源基因是由多个基因(STT3-A和STT3-B；N33和LAP)编码的。多组织Northern杂交表明，这些mRNAs的相对表达因组织而异。这个项目的一个目标是阐明OST亚型多样性的作用，并了解亚基组成的差异如何影响酶的活性。动力学实验表明，寡糖转移酶具有明显的催化和调节结合部位与二十二醇连接的寡糖。将开发新的分析方法和生化探针来确定Dolicol连接的寡糖供体和N-X-S/T受体在新生多肽中的结合位置。酵母遗传学方法将被用来鉴定三肽结合部位的重要氨基酸残基。该项目的第三个目标是通过对低温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复合体的低到中分辨率结构。将使用抗体和化学探针将底物结合位置以及胞液和管腔结构域映射到OST结构上。该项目的第四个目标是确定先天性糖基化障碍新亚型的分子基础。将对成纤维细胞系进行筛查，以确定是否存在二醇连接的寡糖代谢缺陷和寡糖转移酶活性缺陷。

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.