Tian Yuan, Cipollo John F
Food and Drug Administration, Center for Biologics Evaluation and Research, Division of Product Quality Assessment V, Parasitic and Allergenic Products, 10903 New Hampshire Avenue, Silver Spring, Maryland 20993, United States.
Food and Drug Administration, Center for Drug Evaluation and Research, Division of Product Quality Assessment, Parasitic and Allergenic Products, 10903 New Hampshire Avenue, Silver Spring, Maryland 20993, United States.
J Proteome Res. 2025 May 2;24(5):2256-2265. doi: 10.1021/acs.jproteome.4c00716. Epub 2025 Apr 7.
SARS-CoV-1 and MERS-CoV were the infective agents of the 2002 and 2012 coronavirus outbreaks, respectively. Here, we report a comparative liquid chromatography/mass spectrometry (LC/MS) Orbitrap and glycosylation glycoproteomics study of the recombinant S1 spike derived from these two viruses. The former was produced in HEK293 cells and the latter in both HEK293 and insect cells. Both proteins were highly glycosylated, with SARS-CoV-1 S1 having 13 and MERS-CoV S1 having 12 glycosites. Nearly all were occupied at 85% or more. Between 2 and 113 unique glycan compositions were detected at each glycosite across the three proteins. Complex -glycans dominated in HEK293 cell-derived spike S1 proteins. While glycosylation differs between HEK293 and insect cells, the extent of glycan processing at glycosites was similar for the two MERS-CoV S1 forms. The HEK293-derived SARS-CoV-1 S1 glycans were more highly sialylated and fucosylated compared to MERS S1, while the latter had more high-mannose glycosides, particularly in the -terminus and near the RBD. Seven and 8 -glycosites were identified in SARS-CoV-1 S1 and MERS-CoV S1, respectively. Mapping of predicted antigenic and glycosylation sites reveals colocalization consistent with a role for glycosylation in immune system avoidance. Glycosylation patterns of these S1 proteins differ from those of other SARS-CoV-1 and MERS-CoV spike reported forms such as recombinant trimeric and virus-propagated forms, which has implications for virus research, including vaccine development, as glycosylation plays a role in spike function and epitope structure.
严重急性呼吸综合征冠状病毒1型（SARS-CoV-1）和中东呼吸综合征冠状病毒（MERS-CoV）分别是2002年和2012年冠状病毒爆发的致病因子。在此，我们报告了一项针对源自这两种病毒的重组S1刺突蛋白的液相色谱/质谱联用（LC/MS）Orbitrap和糖基化糖蛋白组学的比较研究。前者在人胚肾293（HEK293）细胞中产生，后者在HEK293细胞和昆虫细胞中均有产生。两种蛋白都高度糖基化，SARS-CoV-1 S1有13个糖基化位点，MERS-CoV S1有12个糖基化位点。几乎所有位点的占据率都达到85%或更高。在这三种蛋白的每个糖基化位点检测到2至113种独特的聚糖组成。复杂聚糖在HEK293细胞来源的刺突S1蛋白中占主导。虽然HEK293细胞和昆虫细胞中的糖基化有所不同，但两种MERS-CoV S1形式在糖基化位点的聚糖加工程度相似。与MERS S1相比，HEK293来源的SARS-CoV-1 S1聚糖的唾液酸化和岩藻糖基化程度更高，而后者有更多的高甘露糖糖苷，特别是在N端和受体结合域（RBD）附近。在SARS-CoV-1 S1和MERS-CoV S1中分别鉴定出7个和8个N-糖基化位点。预测的抗原位点和糖基化位点的映射显示共定位，这与糖基化在逃避免疫系统中的作用一致。这些S1蛋白的糖基化模式与其他报道的SARS-CoV-1和MERS-CoV刺突形式（如重组三聚体和病毒传播形式）不同，这对病毒研究（包括疫苗开发）有影响，因为糖基化在刺突功能和表位结构中起作用。
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