ER glycolipid flippases and congenital disorders of glycosylation

ER 糖脂翻转酶和先天性糖基化障碍

基本信息

批准号：
9069623
负责人：
Johannes Graumann
金额：
$ 21.19万
依托单位：
WEILL MEDICAL COLL OF CORNELL UNIV
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-06-01 至 2018-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9069623
关键词：
Affect Anabolism Antibodies Asparagine Binding Biochemical Biochemical Genetics Biological Assay Bypass Carrier Proteins Cell Adhesion Molecules Cell Surface Receptors Cell secretion Cell surface Cells Cellular biology Characteristics Congenital Disorders Defect Development Disease Dolichol Dolichol Phosphates Endoplasmic Reticulum Epitopes Eukaryota Family Fractionation G-Protein-Coupled Receptors Genetic Genetic screening method Glucosephosphates Glycolipids Glycoproteins Goals HIV Envelope Protein gp120 Head Health Hereditary Disease Hormones Human Human Genetics Human Genome Hydrocarbons Hydrolase I-Cell Disease Individual Inherited Laboratories Lipids Lysosomes Mannose Membrane Membrane Proteins Methods Microsomes Molecular Neurologic Oligosaccharides Pathway interactions Pharmaceutical Preparations Phenotype Polysaccharides Post-Translational Protein Processing Protein Glycosylation Proteins Proteomics Publications Reporting Side Specificity Substrate Specificity Symptoms Tail Technology Testing Time Tissues Triton Triton X100 Vesicle Virus Receptors Yeasts base carboxypeptidase C conditional mutant congenital muscular dystrophy genetic approach glycosylation human disease immunogenicity in vivo innovation interest lipid transport mannose 6 phosphate mannosyl(5)-N-acetyl(2)-glucose novel protein profiling rat Piga protein reconstitution research study yeast genetics

项目摘要

DESCRIPTION (provided by applicant): At least half the proteins encoded by the human genome are N-glycosylated, an essential post-translational modification. Defects in glycosylation underlie more than 100 human genetic disorders. For example, I-cell disease is caused by the inability to construct mannose-6-phosphate epitopes on N-glycans of lysosomal hydrolases, resulting in their secretion from cells rather than localization to lysosomes. Congenital Disorders of Glycosylation (CDGs), a family of severe inherited diseases with neurological and other symptoms, frequently result from defects in protein N-glycosylation. Major gaps remain in our understanding of basic glycosylation pathways. For example, assembly of the oligosaccharide donor for N-glycosylation requires flipping of three different glycolipids (Man5GlcNAc2-PP- dolichol (M5-DLO), mannose phosphate dolichol (MPD) and glucose phosphate dolichol (GPD)) from the cytoplasmic to the luminal side of the ER. These glycolipids have long hydrocarbon tails and very polar head groups. They represent key intermediates in the transition of the N-glycan biosynthetic pathway from the cytoplasmic to the luminal side of the ER. How they are flipped across the ER is a long-standing mystery. While there is compelling evidence that specific ER membrane proteins (flippases) are required, and that they have exquisite specificity for the lipids that they transport, the molecular identity of he flippases is not known. Our goal in this R21 application is to identify the glycolipid flippases responsible for flipping M5-DLO and MPD. We developed methods that recapitulate M5-DLO and MPD flipping in synthetic lipid vesicles and this technology now provides the cornerstone of the strategy that we will deploy to achieve our goal. We will use an innovative quantitative proteomics approach to identify flippase candidates from amongst yeast ER membrane proteins. We will then use our reconstitution-based assays to screen the candidates and evaluate their activity. This will provide conclusive evidence of function that we will corroborate by in vivo tests using yeast genetics. With this strategy we expect to identify, for the first time the flippases themselves. As our approach bypasses the limitations of traditional genetic and biochemical approaches that have thus far failed to provide the molecular identity of these flippases, we believe that we have a unique opportunity to solve this decades-old problem. Our discovery will contribute to basic cell biology by revealing a new class of transport proteins, associated with an undoubtedly novel transport mechanism, and also point to new genetic loci that are associated with CDGs.

描述（由申请人提供）：人类基因组编码的蛋白质至少有一半是 N-糖基化的，这是一种重要的翻译后修饰。糖基化缺陷是 100 多种人类遗传性疾病的基础。无法在溶酶体水解酶的 N-聚糖上构建 6-磷酸甘露糖表位，导致它们从细胞中分泌而不是定位到先天性糖基化障碍 (CDG) 是一类具有神经系统和其他症状的严重遗传性疾病，通常是由蛋白质 N-糖基化缺陷引起的。我们对基本糖基化途径（例如寡糖的组装）的理解仍然存在重大差距。 N-糖基化供体需要翻转三种不同的糖脂（Man5GlcNAc2-PP-dolichol (M5-DLO)、磷酸甘露糖糖脂（MPD）和磷酸葡萄糖糖脂（GPD））从细胞质到内质网腔侧，这些糖脂具有长烃尾和极性极强的头部基团，它们是 N-聚糖生物合成途径转变的关键中间体。尽管有令人信服的证据表明特定的 ER 膜蛋白，但它们如何在 ER 上翻转一直是个谜。（翻转酶）是必需的，并且它们对其运输的脂质具有精确的特异性，但翻转酶的分子身份尚不清楚。我们在该 R21 应用中的目标是鉴定负责翻转 M5-DLO 和 MPD 的糖脂翻转酶。我们开发了在合成脂质囊泡中重现 M5-DLO 和 MPD 翻转的方法，这项技术现在为我们为实现我们的目标而部署的策略提供了基石。一种创新的定量蛋白质组学方法，用于从酵母 ER 膜蛋白中识别翻转酶候选者，然后我们将使用基于重组的测定来筛选候选者并评估其活性，这将提供我们将证实的功能的决定性证据。通过使用酵母遗传学的体内测试，我们希望首次识别翻转酶本身，因为我们的方法绕过了迄今为止未能提供这些翻转酶分子身份的传统遗传和生化方法的局限性，我们相信，我们有一个独特的机会来解决这个几十年前的问题，我们的发现将通过揭示一类与无疑新颖的转运机制相关的新型转运蛋白，为基础细胞生物学做出贡献，并指出新的基因位点。与 CDG 相关。