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: 10510784
• 负责人: ANANT K MENON
• 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: 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/10510784
• 关键词: Binding; Binding Proteins; Blindness; Cadherins; Categories; Cell Adhesion Molecules; Cell Surface Receptors; Cell-Free System; Cells; Chemicals; Child; Clinical; Collaborations; Congenital disorders of glycosylation; Cryoelectron Microscopy; 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; 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; 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。糖蛋白如钙粘蛋白、整联蛋白和层粘连蛋白是调节细胞凋亡的关键。 发育、器官发生和组织组织。N-糖基化缺陷是许多人类疾病的基础。 遗传性疾病包括一组异质性常染色体隐性遗传代谢疾病， 先天性糖基化障碍（CDG）。CDG表现出一系列破坏性症状，包括 发育不良、发育和言语迟缓、视力丧失、张力减退、小头畸形、癫痫发作和中风- 就像一幕幕患有CDG的儿童患有认知和身体残疾。虽然其中一些症状可能 如果没有治疗，CDG是无法治愈的。 我们在R21应用中的目标是探索CDG 1 N型的分子基础， 由内质网膜蛋白RFT 1的错义突变引起的疾病。病人， 目前在北美、英国/欧洲、中东和印度，往往呈现出独特的 感音神经性耳聋除了典型的严重CDG症状。CDG 1 N没有治疗方法。不像 大多数CDG的潜在分子缺陷是很好理解的，例如，缺陷所致 糖基转移酶或糖加工酶，CDG 1 N是一个谜，因为RFT 1的功能不是 知道的在CDG 1 N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 RFT 1在细胞中的关键作用是催化M5-DLO的利用， 由内质网结构/形态构成，以促进其向下游机器的传递，或 防止其错误定位。 我们将在两个具体目标中检验这一假设。第一个目标将集中在RFT 1在M5-DLO中的作用 利用RFT 1缺陷细胞（包括CDG 1 N患者成纤维细胞）、无细胞系统和 新的方法来细分内质网。我们还将测试RFT 1是否为M5-DLO 结合蛋白认识到结构信息对于理解RFT 1在一个特定环境下的功能至关重要， 分子水平，第二个目标将开发RFT 1的结构-功能模型。在这里，我们将定义其 膜拓扑结构，确定关键的功能残基，并启动一项计划，以阐明其三维 结构 从长远来看，我们的研究结果将阐明为什么RFT 1缺乏导致M5-DLO积累， 因此，CDG 1 N，从而为开发治疗策略和疗法铺平了道路， 可能有助于RFT 1恢复糖基化，改善临床症状。