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-糖基化对生命至关重要。糖蛋白，例如钙粘蛋白，整联蛋白和层粘连蛋白是至关重要的 开发，器官发生和组织组织。 N-糖基化的缺陷是众多人 遗传疾病，包括一组未称为常染色体疾病的代谢疾病 糖基化（CDGS）的先天性疾病。 CDG出现了一系列毁灭性症状，包括 未能蓬勃发展，发育和语音延迟，视力丧失，肌畸形，癫痫发作和中风 - 喜欢情节。患有CDG的儿童遭受认知和身体残疾。尽管其中一些症状可以 可以治疗，无法治愈CDG。 我们在此R21应用中的目标是探索CDG型1N的分子基础，这是一个鲜为人知的 内质网膜蛋白RFT1中的错义突变引起的疾病。患者， 到目前为止，在北美，英国/Europe，中东和印度均被确定，通常具有独特的 除典型的严重CDG症状外，感觉性耳聋。 CDG1N没有治疗。与众不同 大多数基本分子缺陷都充分了解的CDG，例如 糖基转移酶或糖处理酶，CDG1N是一个谜，因为RFT1的功能不是 已知。 N-糖基化的途径在CDG1N细胞的关键阶段被阻塞，从而堆积 糖基化机制无法利用的脂质中间体（称为M5-DLO）。我们假设 RFT1在细胞中的关键作用是通过克服障碍来催化M5-DLO的利用 由内质网的结构/形态构成，以促进其向下游机械的交接或 通过防止其错误定位。 我们将以两个具体的目的来检验这一假设。第一个目标将重点放在RFT1在M5-DLO中的作用 利用，利用RFT1缺陷细胞（包括CDG1N患者成纤维细胞），无细胞系统和A 亚摘要内质网的新方法。我们还将测试RFT1是否是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 分子水平，第二个目标将开发RFT1的结构功能模型。在这里，我们将定义它 膜拓扑，识别关键功能残基，并启动一个程序以阐明其3维 结构。 从长远来看，我们的结果将阐明为什么RFT1缺乏会导致M5-DLO积累和 因此，CDG1N，从而为制定治疗策略和治疗剂铺平了道路 可以利用RFT1的功能来恢复糖基化并改善临床症状。