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Caught in the act: precision tools to unravel and diagnose glycoprotein misfolding

陷入困境：解开和诊断糖蛋白错误折叠的精密工具

基本信息

批准号：
2597991
负责人：
金额：
--
依托单位：
Imperial College London
依托单位国家：
英国
项目类别：
Studentship
财政年份：
2021
资助国家：
英国
起止时间：
2021 至无数据
项目状态：
未结题

来源：
https://gtr.ukri.org/projects?ref=studentship-2597991
关键词：
Caught act precision tools unravel

项目摘要

Reporters of cellular glycosylation: Engineering N-acetylglucosaminyl (GlcNAc) transferases- One of the major types of protein glycosylation, Asn(N)-linked glycosylation, undergoes extensive maturation in the secretory pathway in an assembly line principle. Crucial bifurcation points in glycan maturation are generated through introduction of GlcNAc at defined positions (https://www.ncbi.nlm.nih.gov/books/NBK453020/).- Dysfunctional N-glycosylation is a major cause of rare congenital disorders of glycosylation (CDGs) that are often complex and present with severe developmental/neurological symptoms.- We have the capacity to bump-and-hole engineer the GlcNAc transferases MGAT1, MGAT2 and MGAT5 to accept bioorthogonally labeled, bumped nucleotide-sugar substrates (https://doi.org/10.1016/j.cbpa.2020.09.001) to allow identification of CDG-relevant substrates and create reporter systems for glycoprotein maturation at different stages of the secretory pathway.- Objectives:o Engineering MGAT1, MGAT2 and/or MGAT5 to accept bumped versions of the substrate UDP-GlcNAc.o Synthesis of bumped UDP-sugars using chemoenzymatic synthesis.o Cellular delivery of the substrate, click/enrichment/proteomics of model glycoproteins to benchmark the approach.o Application to profile protein trafficking by imaging (e.g. through bioorthogonal ligation with cell-permeable turn-on fluorophores).o Establishment of a toolbox to probe protein maturation/recycling and shed light on CDGs, e.g. by

细胞糖基化的报告基因：工程化N-乙酰葡糖胺（GlcNAc）转移酶-蛋白质糖基化的主要类型之一，Asn（N）-连接的糖基化，在装配线原理的分泌途径中经历广泛的成熟。通过在定义的位置引入GlcNAc产生聚糖成熟中的关键分叉点（https：//www.ncbi.nlm.nih.gov/books/NBK453020/）。功能失调的N-糖基化是罕见的先天性糖基化疾病（CDG）的主要原因，这些疾病通常很复杂，并伴有严重的发育/神经症状。我们有能力对GlcNAc转移酶MGAT 1、MGAT 2和MGAT 5进行凹凸工程改造，以接受生物正交标记的凹凸核苷酸-糖底物（https：//doi.org/10.1016/j.cbpa.2020.09.001），从而能够鉴定CDG相关底物，并在分泌途径的不同阶段创建用于糖蛋白成熟的报告系统。目的：o工程化MGAT 1、MGAT 2和/或MGAT 5以接受底物UDP-GlcNAc的碰撞形式。o使用化学酶促合成合成碰撞的UDP-糖。o底物的细胞递送，模型糖蛋白的点击/富集/蛋白质组学以基准该方法。o通过成像（例如通过与细胞可渗透的开启荧光团的生物正交连接）来分析蛋白质运输的应用。