PDF(Pubmed)
Abstract：
Ebola virus glycoprotein (EBOV GP) is one of the most heavily O-glycosylated viral glycoproteins, yet we still lack a fundamental understanding of the structure of its large O-glycosylated mucin-like domain and to what degree the host O-glycosylation capacity influences EBOV replication. Using tandem mass spectrometry, we identified 47 O-glycosites on EBOV GP and found similar glycosylation signatures on virus-like particle- and cell lysate-derived GP. Furthermore, we performed quantitative differential O-glycoproteomics on proteins produced in wild-type HEK293 cells and cell lines ablated for the three key initiators of O-linked glycosylation, GalNAc-T1, -T2, and -T3. The data show that 12 out of the 47 O-glycosylated sites were regulated, predominantly by GalNAc-T1. Using the glycoengineered cell lines for authentic EBOV propagation, we demonstrate the importance of O-linked glycan initiation and elongation for the production of viral particles and the titers of progeny virus. The mapped O-glycan positions and structures allowed to generate molecular dynamics simulations probing the largely unknown spatial arrangements of the mucin-like domain. The data highlight targeting GALNT1 or C1GALT1C1 as a possible way to modulate O-glycan density on EBOV GP for novel vaccine designs and tailored intervention approaches.IMPORTANCEEbola virus glycoprotein acquires its extensive glycan shield in the host cell, where it is decorated with N-linked glycans and mucin-type O-linked glycans. The latter is initiated by a family of polypeptide GalNAc-transferases that have different preferences for optimal peptide substrates resulting in a spectrum of both very selective and redundant substrates for each isoform. In this work, we map the exact locations of O-glycans on Ebola virus glycoprotein and identify subsets of sites preferentially initiated by one of the three key isoforms of GalNAc-Ts, demonstrating that each enzyme contributes to the glycan shield integrity. We further show that altering host O-glycosylation capacity has detrimental effects on Ebola virus replication, with both isoform-specific initiation and elongation playing a role. The combined structural and functional data highlight glycoengineered cell lines as useful tools for investigating molecular mechanisms imposed by specific glycans and for steering the immune responses in future vaccine designs.
摘要：
埃博拉病毒糖蛋白(EBOVGP)是最严重的O-糖基化病毒糖蛋白之一,然而，我们仍然缺乏对其大型O-糖基化粘蛋白样结构域的结构以及宿主O-糖基化能力在多大程度上影响EBOV复制的基本了解。使用串联质谱,我们在EBOVGP上鉴定了47个O-糖蛋白位点,并在病毒样颗粒和细胞裂解物衍生的GP上发现了相似的糖基化特征.此外，我们对野生型HEK293细胞和细胞系中产生的蛋白质进行了定量差异O-糖蛋白质组学，GalNAc-T1、-T2和-T3。数据显示，47个O-糖基化位点中有12个受到调节，主要由GalNAc-T1。使用糖工程细胞系进行真正的EBOV繁殖，我们证明了O-连接的聚糖起始和延伸对于病毒颗粒的产生和子代病毒滴度的重要性。映射的O-聚糖位置和结构允许产生分子动力学模拟，探测粘蛋白样结构域的大部分未知的空间排列。数据突出了靶向GALNT1或C1GALT1C1作为调节EBOVGP上O-聚糖密度的可能方法，用于新型疫苗设计和定制的干预方法。重要埃博拉病毒糖蛋白在宿主细胞中获得其广泛的聚糖屏蔽，用N-连接的聚糖和粘蛋白型O-连接的聚糖装饰。后者由多肽GalNAc-转移酶家族起始,所述多肽GalNAc-转移酶家族对最佳肽底物具有不同的偏好,导致每种同种型的非常选择性和冗余的底物谱。在这项工作中,我们绘制了埃博拉病毒糖蛋白上O-聚糖的确切位置，并确定了由GalNAc-Ts的三种关键亚型之一优先启动的位点子集，证明每种酶都有助于聚糖屏蔽的完整性。我们进一步表明，改变宿主O-糖基化能力对埃博拉病毒的复制有不利影响，同工型特异性起始和伸长都起作用。组合的结构和功能数据突出了糖工程化细胞系作为用于研究由特定聚糖施加的分子机制和用于指导未来疫苗设计中的免疫应答的有用工具。
