Tian Yuan, Parsons Lisa M, Jankowska Ewa, Cipollo John F
Food and Drug Administration, Center for Biologics Evaluation and Research, Division of Bacterial, Parasitic and Allergenic Products, Silver Spring, MD, United States.
Front Chem. 2021 Nov 19;9:767448. doi: 10.3389/fchem.2021.767448. eCollection 2021.
The SARS-CoV-2 spike protein is heavily glycosylated, having 22 predicted N-glycosylation sites per monomer. It is also O-glycosylated, although the number of O-glycosites is less defined. Recent studies show that spike protein glycans play critical roles in viral entry and infection. The spike monomer has two subdomains, S1 and S2, and a receptor-binding domain (RBD) within the S1 domain. In this study, we have characterized the site-specific glycosylation patterns of the HEK293 recombinant spike RBD and S1 domains as well as the intact spike derived from the whole virus produced in Vero cells. The Vero cell-derived spike from the WA1 strain and a D614G variant was analyzed. All spike proteins, S1, and RBDs were analyzed using hydrophilic interaction chromatography (HILIC) and LC-MS/MS on an Orbitrap Eclipse Tribrid mass spectrometer. N-glycans identified in HEK293-derived S1 were structurally diverse. Those found in the HEK293-derived RBD were highly similar to those in HEK293 S1 where N-glycosites were shared. Comparison of the whole cell-derived WA1 and D614G spike proteins revealed that N-glycosites local to the mutation site appeared to be more readily detected, hinting that these sites are more exposed to glycosylation machinery. Moreover, recombinant HEK293-derived S1 was occupied almost completely with complex glycan, while both WA1 and D614G derived from the Vero E6 cell whole virus were predominantly high-mannose glycans. This stands in stark contrast to glycosylation patterns seen in both CHO- and HEK cell-derived recombinant S1, S2, and the whole spike previously reported. Concerning O-glycosylation, our analyses revealed that HEK293 recombinant proteins possessed a range of O-glycosites with compositions consistent with Core type 1 and 2 glycans. The O-glycosites shared between the S1 and RBD constructs, sites T323 and T523, were occupied by a similar range of Core 1 and 2 type O-glycans. Overall, this study reveals that the sample nature and cell substrate used for production of these proteins can have a dramatic impact on the glycosylation profile. SARS-CoV-2 spike glycans are associated with host ACE2 receptor interaction efficiency. Therefore, understanding such differences will serve to better understand these host-pathogen interactions and inform the choice of cell substrates to suite downstream investigations.
严重急性呼吸综合征冠状病毒2（SARS-CoV-2）刺突蛋白高度糖基化，每个单体有22个预测的N-糖基化位点。它也存在O-糖基化，尽管O-糖基化位点的数量不太明确。最近的研究表明，刺突蛋白聚糖在病毒进入和感染中起关键作用。刺突单体有两个亚结构域，S1和S2，以及S1结构域内的一个受体结合结构域（RBD）。在本研究中，我们表征了人胚肾293（HEK293）重组刺突RBD和S1结构域以及从Vero细胞中产生的完整病毒衍生的完整刺突的位点特异性糖基化模式。分析了来自WA1毒株和D614G变体的Vero细胞衍生刺突。所有刺突蛋白、S1和RBD均使用亲水相互作用色谱（HILIC）和在Orbitrap Eclipse Tribrid质谱仪上的液相色谱-串联质谱（LC-MS/MS）进行分析。在HEK293衍生的S1中鉴定出的N-聚糖结构多样。在HEK293衍生的RBD中发现的N-聚糖与HEK293 S1中共享N-糖基化位点的那些高度相似。对全细胞衍生的WA1和D614G刺突蛋白的比较表明，突变位点处的N-糖基化位点似乎更容易被检测到，这表明这些位点更容易暴露于糖基化机制。此外，HEK293重组衍生的S1几乎完全被复合聚糖占据，而来自Vero E6细胞全病毒的WA1和D614G主要是高甘露糖聚糖。这与先前报道的在CHO细胞和HEK细胞衍生的重组S1、S2和完整刺突中看到的糖基化模式形成鲜明对比。关于O-糖基化，我们的分析表明，HEK293重组蛋白具有一系列O-糖基化位点，其组成与核心1型和2型聚糖一致。S1和RBD构建体之间共享的O-糖基化位点，即T323和T523位点，被一系列相似的核心1型和2型O-聚糖占据。总体而言，本研究表明，用于生产这些蛋白质的样品性质和细胞底物可对糖基化谱产生显著影响。SARS-CoV-2刺突聚糖与宿主血管紧张素转换酶2（ACE2）受体相互作用效率相关。因此，了解这些差异将有助于更好地理解这些宿主-病原体相互作用，并为选择适合下游研究的细胞底物提供参考。
