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

细胞糖基化记者：工程N-乙酰氨基葡萄糖(GlcNAc)转移酶是蛋白质糖基化的主要类型之一，ASN(N)连接的糖基化在流水线原理的分泌途径中经历了广泛的成熟。糖链成熟的关键分叉点是通过在确定的位置引入(https://www.ncbi.nlm.nih.gov/books/NBK453020/).-产生的。功能不全的N-糖基化是罕见的先天性糖基化障碍(CDG)的主要原因，这些疾病通常是复杂的，并出现严重的发育/神经症状。-我们有能力对GlcNAc转移酶MGAT1、MGAT2和MGAT5进行改造，以接受生物正交标记，突变的核苷酸-糖底物(https://doi.org/10.1016/j.cbpa.2020.09.001))，以鉴定CDG相关的底物，并为分泌途径的不同阶段的糖蛋白成熟创建报告系统。-目的：o改造MGAT1、MGAT2和/或MGAT5以接受底物UDP-GlcNAc.o利用化学酶合成合成凹凸型UDP-糖。o底物的细胞递送，点击/浓缩/蛋白质组学的模型糖蛋白，以基准方法。o应用于通过成像来描述蛋白质的运输(例如，通过与细胞可渗透的打开的荧光团的生物正交连接)。o建立一个工具箱来探测蛋白质的成熟/再循环，并阐明CDG，例如通过

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