Molecular basis of congenital disorder of glycosylation type 1N

1N型先天性糖基化障碍的分子基础

• 批准号: 10700974
• 负责人: ANANT K MENON
• 金额: $ 21.19万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-08 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10700974
• 关键词: Binding; Binding Proteins; Blindness; Cadherins; Cell Adhesion Molecules; Cell Surface Receptors; Cell-Free System; Cells; Chemicals; Child; Clinical; Collaborations; Congenital disorders of glycosylation; Cryoelectron Microscopy; Cytoplasm; Defect; Development; Developmental Delay Disorders; Disease; Dolichol; Endoplasmic Reticulum; Enzymes; Epitopes; Europe; Extracellular Matrix Proteins; Face; Failure to Thrive; Family; Fibroblasts; Genetic Diseases; Glycoproteins; Goals; Human Genetics; India; Integrins; Laminin; Lateral; Life; Link; Lipids; Location; Maps; Membrane; Membrane Biology; Membrane Proteins; Metabolic Diseases; Microcephaly; Microsomes; Middle East; Missense Mutation; Modeling; Molecular; Morphology; Muscle hypotonia; National Institute of Child Health and Human Development; Nature; North America; Oligosaccharides; Organogenesis; Pathway interactions; Patients; Permeability; Phenotype; Polysaccharides; Property; Proteins; Role; Seizures; Sensorineural Hearing Loss; Side; Site; Speech Delay; Structure; Symptoms; Testing; Therapeutic; Tissues; United States National Institutes of Health; Vesicle; X-Ray Crystallography; autosome; cognitive disability; experience; experimental study; glycosylation; glycosyltransferase; improved; interest; isoprenoid; novel strategies; physically handicapped; prevent; programs; seal; stroke-like episode; sugar; therapy development; three dimensional structure; treatment strategy

N-glycosylation is essential for life. Glycoproteins such as cadherins, integrins and laminins are key to development, organogenesis, and tissue organization. Defects in N-glycosylation underlie numerous human genetic disorders including a heterogeneous group of autosomal-recessive, metabolic diseases termed Congenital Disorders of Glycosylation (CDGs). CDGs present with a range of devastating symptoms, including failure to thrive, developmental and speech delays, vision loss, hypotonia, microcephaly, seizures, and stroke- like episodes. Children with CDGs suffer cognitive and physical disabilities. While some of these symptoms can be treated, there is no cure for CDGs. Our goal in this R21 application is to explore the molecular basis of CDG type 1N, a poorly understood disease caused by missense mutations in the endoplasmic reticulum membrane protein RFT1. Patients, identified thus far in North America, U.K./Europe, the Middle East, and India, often present with a unique sensorineural deafness in addition to typical severe CDG symptoms. There is no therapy for CDG1N. Unlike most CDGs for which the underlying molecular defect is well understood, e.g., deficiency of a glycosyltransferase or sugar-processing enzyme, CDG1N is an enigma because the function of RFT1 is not known. The pathway of N-glycosylation is blocked at a critical stage in CDG1N cells, resulting in the build-up of a lipid intermediate (termed M5-DLO) that cannot be utilized by the glycosylation machinery. We hypothesize that the critical role of RFT1 in cells is to catalyze utilization of M5-DLO either by overcoming an impediment posed by endoplasmic reticulum structure/morphology to facilitate its handoff to downstream machinery, or by preventing its mis-localization. We will test this hypothesis in two specific aims. The first aim will focus on the role of RFT1 in M5-DLO utilization, making use of RFT1-deficient cells (including CDG1N patient fibroblasts), cell-free systems, and a novel approach to sub-fractionating the endoplasmic reticulum. We will also test whether RFT1 is an M5-DLO binding protein. Recognizing that structural information is critical to understand the function of RFT1 at a molecular level, the second aim will develop a structure-function model of RFT1. Here we will define its membrane topology, identify key functional residues, and initiate a program to elucidate its 3-dimensional structure. In the long term, our results will illuminate why RFT1 deficiency results in M5-DLO accumulation and consequently CDG1N, thereby paving the way for the development of treatment strategies and therapeutics that could subserve RFT1's function to restore glycosylation and improve clinical symptoms.

N-糖基化是生命所必需的。钙粘附素、整合素和层粘连蛋白等糖蛋白是 发育、器官发生和组织结构。N-糖基化缺陷是许多人类 遗传性疾病包括一组异质性常染色体隐性遗传代谢性疾病，称为 先天性糖基化紊乱(CDGs)。CDG出现了一系列毁灭性的症状，包括 发育迟缓、发育迟缓、视力丧失、低眼压、小头畸形、癫痫发作和中风-- 就像小插曲。患有CDG的儿童患有认知和身体残疾。虽然这些症状中的一些可能 如果接受治疗，慢性萎缩性胃炎无法治愈。 我们在R21应用中的目标是探索CDG 1N的分子基础，CDG 1N是一种知之甚少的 由内质网膜蛋白RFT1错义突变引起的疾病。病人们， 到目前为止在北美、英国/欧洲、中东和印度都发现了一种独特的 除了典型的严重的CDG症状外，还有感觉神经性耳聋。CDG1N目前还没有治疗方法。不像 大多数CDG的潜在分子缺陷被很好地理解，例如 糖基转移酶或糖加工酶CDG1N是一个谜，因为RFT1的功能不是 为人所知。在CDG1N细胞中，N-糖基化途径在关键阶段被阻断，导致 一种不能被糖基化机制利用的脂质中间体(称为M5-DLO)。我们假设 RFT1在细胞中的关键作用是催化利用M5-DLO，无论是通过克服障碍 由内质网结构/形态构成，以便于其移交给下游机械，或 通过防止其错误本地化。 我们将在两个具体目标上检验这一假设。第一个目标将集中在RFT1在M5-DLO中的作用 利用RFT1缺陷细胞(包括CDG1N患者成纤维细胞)、无细胞系统和 内质网细分的新方法。我们还将测试RFT1是否为M5-DLO 结合蛋白。认识到结构信息对于理解RFT1的功能至关重要 在分子水平上，第二个目标是建立RFT1的结构-功能模型。在这里，我们将定义其 膜拓扑结构，确定关键功能残基，并启动一个程序来阐明其三维结构 结构。 从长远来看，我们的结果将解释为什么RFT1缺陷会导致M5-DLO积聚和 因此，CDG1N，从而为制定治疗战略和治疗方法铺平了道路 可替代RFT1‘S功能，恢复糖基化，改善临床症状。