ER glycolipid flippases and congenital disorders of glycosylation

ER 糖脂翻转酶和先天性糖基化障碍

• 批准号: 8953730
• 负责人: Johannes Graumann
• 金额: $ 25.43万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-06-01 至 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8953730
• 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; 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; Relative (related person); Reporting; Side; Specificity; Substrate Specificity; Symptoms; Tail; Technology; Testing; Time; Tissues; Triton; Triton X100; Tritons; Vesicle; Virus Receptors; Yeasts; base; carboxypeptidase C; congenital muscular dystrophy; genetic approach; glycosylation; human disease; immunogenicity; in vivo; innovation; interest; lipid transport; mannose 6 phosphate; mannosyl(5)-N-acetyl(2)-glucose; mutant; novel; protein profiling; public health relevance; rat Piga protein; reconstitution; research study; yeast genetics

 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（申请人提供）：人类基因组编码的蛋白质中至少有一半是N-糖基化的，这是一种重要的翻译后修饰。糖基化缺陷是100多种人类遗传疾病的基础。例如，I细胞疾病是由于不能在溶酶体水解酶的N-聚糖上构建甘露糖-6-磷酸表位，导致它们从细胞分泌而不是定位于溶酶体。先天性糖基化障碍（CDG）是一种具有神经系统和其他症状的严重遗传性疾病家族，通常由蛋白质N-糖基化缺陷引起。 我们对基本糖基化途径的理解仍存在重大差距。例如，用于N-糖基化的寡糖供体的组装需要将三种不同的糖脂（Man 5GlcNAc 2-PP-多萜醇（M5-DLO）、甘露糖磷酸多萜醇（MPD）和葡萄糖磷酸多萜醇（GPD））从ER的细胞质侧翻转到内腔侧。这些糖脂具有长的烃尾和极极性的头部基团。它们代表了N-聚糖生物合成途径从细胞质向ER腔侧过渡的关键中间体。他们是如何在急诊室翻转的是一个长期的谜。虽然有令人信服的证据表明，特定的ER膜蛋白（翻转酶）是必需的，并且它们对它们转运的脂质具有精确的特异性，但翻转酶的分子身份尚不清楚。 我们在此R21应用程序中的目标是识别负责翻转M5-DLO和MPD的糖脂翻转酶。我们开发了在合成脂质囊泡中概括M5-DLO和MPD翻转的方法，该技术现在为我们将部署以实现我们的目标的策略提供了基石。我们将使用一种创新的定量蛋白质组学方法，以确定翻转酶的候选人之间的酵母ER膜蛋白。然后，我们将使用基于重组的检测来筛选候选人并评估其活性。这将提供决定性的功能证据，我们将证实 通过酵母遗传学的体内测试。有了这个策略，我们希望第一次识别翻转本身。由于我们的方法绕过了传统遗传和生物化学方法的局限性，这些方法迄今未能提供这些翻转酶的分子身份，我们相信我们有一个独特的机会来解决这个几十年的问题。我们的发现将有助于基础细胞生物学，揭示了一类新的转运蛋白，与一个毫无疑问的新的运输机制，也指出了新的遗传基因座与CDG。