The current emergence of the novel coronavirus pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) demands the development of new therapeutic strategies to prevent rapid progress of mortalities. The coronavirus spike (S) protein, which facilitates viral attachment, entry and membrane fusion is heavily glycosylated and plays a critical role in the elicitation of the host immune response. The spike protein is comprised of two protein subunits (S1 and S2), which together possess 22 potential N-glycosylation sites. Herein, we report the glycosylation mapping on spike protein subunits S1 and S2 expressed on human cells through high-resolution mass spectrometry. We have characterized the quantitative N-glycosylation profile on spike protein and interestingly, observed unexpected O-glycosylation modifications on the receptor-binding domain of spike protein subunit S1. Even though O-glycosylation has been predicted on the spike protein of SARS-CoV-2, this is the first report of experimental data for both the site of O-glycosylation and identity of the O-glycans attached on the subunit S1. Our data on the N- and O-glycosylation are strengthened by extensive manual interpretation of each glycopeptide spectra in addition to using bioinformatics tools to confirm the complexity of glycosylation in the spike protein. The elucidation of the glycan repertoire on the spike protein provides insights into the viral binding studies and more importantly, propels research toward the development of a suitable vaccine candidate.

中文翻译：

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推导新型冠状病毒 SARS-CoV-2 刺突蛋白的 N- 和 O- 糖基化谱。

当前出现的由严重急性呼吸综合征冠状病毒 2 (SARS-CoV-2) 引起的新型冠状病毒大流行需要开发新的治疗策略，以防止死亡率迅速上升。冠状病毒刺突 (S) 蛋白促进病毒附着、进入和膜融合，它被高度糖基化，在引发宿主免疫反应中发挥着关键作用。刺突蛋白由两个蛋白亚基（S1 和 S2）组成，它们共同拥有 22 个潜在的 N-糖基化位点。在此，我们通过高分辨率质谱报告了人类细胞上表达的刺突蛋白亚基 S1 和 S2 的糖基化图谱。我们对刺突蛋白的定量 N-糖基化谱进行了表征，有趣的是，在刺突蛋白亚基 S1 的受体结合域上观察到了意想不到的 O-糖基化修饰。尽管已预测 SARS-CoV-2 的刺突蛋白上有 O-糖基化，但这是首次报告 O-糖基化位点和亚基 S1 上附着的 O-聚糖的身份的实验数据。除了使用生物信息学工具确认刺突蛋白中糖基化的复杂性之外，我们还通过对每个糖肽光谱进行广泛的手动解释来加强我们关于 N- 和 O-糖基化的数据。对刺突蛋白上聚糖谱的阐明为病毒结合研究提供了见解，更重要的是，推动了研究开发合适的候选疫苗。