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INTRODUCTION

By 2024, COVID-19 has become endemic, with new variants contributing to its continued spread. The Spike protein forms trimers that bind to the ACE2 receptor on host cells, with the S1 subunit being a primary target for vaccines and antiviral treatments.

METHODS

Herein, we performed an in-depth analysis of the N-glycosylation of the recombinant Spike S1 protein (S1 protein) across the wild-type (WT) virus and its 5 variants, including Alpha, Beta, Gamma, Delta, and Lambda, by integrating ultrahigh-performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry (UHPLC-Q-TOF MS) and unique TiO₂-PGC chip-based LC/MS techniques.

RESULTS

A total of 332 glycan structures arising from 180 compositions on the S1 and RBD regions were identified, revealing remarkable glycosylation diversity of the S1 protein. Complex glycan was shown to be the dominant structure across variants. Neutral N-glycans are mainly di-antennary with two fucosyl groups, while the majority of acidic N-glycans were multi-antennary with mono-fucosyl residues. In addition, sialic acid linkages of the N-glycans were extensively studied by utilizing ¹³C-labeled standards and specific enzymes for the first time, showing the existence of both α-2,3 and α-2,6 linkages across WT and five variants. It should be noted that the Lambda variant shows more complex α-2,3 and α-2,6-linked glycans in the RBD region, which may potentially enhance its glycan shield effect. Acetylated glycans, which were identified on S protein for the first time, were found to be fully fucosylated on the S1 region and sialylated on the RBD region across all variants. UHPLC-TOF MS analysis revealed unoccupied N-glycosylation sites in S1-Gamma (N657), S1-Delta (N61), and S1-Lambda (N17, N61, N657), with N17 and N61 showing low glycan occupancy (0%-3.4%), suggesting these sites may lack glycan shield protection.

DISCUSSION

This study provides a comprehensive N-glycosylation profile of the S1 protein across different variants, offering an essential structural basis for future vaccine development and research on viral functions.

INTRODUCTION

METHODS

RESULTS

DISCUSSION

引言

到2024年，新冠病毒已成为地方性流行病，新变种导致其持续传播。刺突蛋白形成三聚体，与宿主细胞上的血管紧张素转换酶2（ACE2）受体结合，S1亚基是疫苗和抗病毒治疗的主要靶点。

方法

在此，我们通过整合超高效液相色谱与四极杆飞行时间质谱（UHPLC-Q-TOF MS）以及基于独特的二氧化钛-多孔石墨化碳芯片的液相色谱/质谱技术，对野生型（WT）病毒及其5种变体（包括阿尔法、贝塔、伽马、德尔塔和拉姆达）的重组刺突S1蛋白（S1蛋白）的N-糖基化进行了深入分析。

结果

在S1和受体结合域（RBD）区域共鉴定出180种组成产生的332种聚糖结构，揭示了S1蛋白显著的糖基化多样性。复合聚糖是各变体中的主要结构。中性N-聚糖主要是带有两个岩藻糖基的双天线型，而大多数酸性N-聚糖是带有单岩藻糖基残基的多天线型。此外，首次利用¹³C标记标准品和特定酶对N-聚糖的唾液酸连接进行了广泛研究，结果表明WT和五种变体中均存在α-2,3和α-2,6连接。值得注意的是，拉姆达变体在RBD区域显示出更复杂的α-2,3和α-2,6连接聚糖，这可能会增强其聚糖屏蔽效应。首次在刺突蛋白上鉴定出的乙酰化聚糖在所有变体的S1区域完全被岩藻糖基化，在RBD区域被唾液酸化。UHPLC-TOF MS分析揭示了S1-伽马（N657）、S1-德尔塔（N61）和S1-拉姆达（N17、N61、N657）中未被占据的N-糖基化位点，其中N17和N61的聚糖占据率较低（0%-3.4%），表明这些位点可能缺乏聚糖屏蔽保护。