N‐glycosylation of the SARS‐CoV‐2 spike protein at Asn331 and Asn343 is involved in spike‐ACE2 binding, virus entry, and regulation of IL‐6

• Published in: Microbiology and immunology Vol. 68; no. 5; pp. 165 - 178
• Main Authors: Das, Tuhin, Luo, Shuhong, Tang, Hao, Fang, Jianmin, Mao, Yinging, Yen, Haw‐Han, Dash, Sabyasachi, Shajahan, Asif, Pepi, Lauren, Huang, Steven, Jones, Valerie S., Xie, Shehuo, Huang, Gordon F., Lu, Jinqiao, Anderson, Blake, Zhang, Benyue, Azadi, Parastoo, Huang, Ruo‐Pan
• Format: Journal Article
• Language: English
• Published: Australia 01.05.2024
• Subjects: ACE2; Angiotensin-Converting Enzyme 2 - metabolism; Asparagine - metabolism; COVID-19 - metabolism; COVID-19 - virology; COVID‐19; Glycosylation; HEK293 Cells; Humans; IL‐6; Interleukin-6 - metabolism; N‐glycosylation; Polysaccharides - metabolism; Protein Binding; receptor‐binding domain; SARS-CoV-2 - metabolism; SARS-CoV-2 - physiology; SARS‐CoV‐2; Spike Glycoprotein, Coronavirus - chemistry; Spike Glycoprotein, Coronavirus - genetics; Spike Glycoprotein, Coronavirus - metabolism; spike protein; Virus Internalization; ACE2; COVID‐19; SARS‐CoV‐2; receptor‐binding domain; spike protein; N‐glycosylation; IL‐6
• DOI: 10.1111/1348-0421.13121
• ISSN: 0385-5600

The coronavirus disease 2019 (COVID‐19) pandemic is an ongoing global public health crisis. The causative agent, the SARS‐CoV‐2 virus, enters host cells via molecular interactions between the viral spike protein and the host cell ACE2 surface protein. The SARS‐CoV‐2 spike protein is extensively decorated with up to 66 N‐linked glycans. Glycosylation of viral proteins is known to function in immune evasion strategies but may also function in the molecular events of viral entry into host cells. Here, we show that N‐glycosylation at Asn331 and Asn343 of SARS‐CoV‐2 spike protein is required for it to bind to ACE2 and for the entry of pseudovirus harboring the SARS‐CoV‐2 spike protein into cells. Interestingly, high‐content glycan binding screening data have shown that N‐glycosylation of Asn331 and Asn343 of the RBD is important for binding to the specific glycan molecule G4GN (Galβ−1,4 GlcNAc), which is critical for spike‐RBD‐ACE2 binding. Furthermore, IL‐6 was identified through antibody array analysis of conditioned media of the corresponding pseudovirus assay. Mutation of N‐glycosylation of Asn331 and Asn343 sites of the spike receptor‐binding domain (RBD) significantly reduced the transcriptional upregulation of pro‐inflammatory signaling molecule IL‐6. In addition, IL‐6 levels correlated with spike protein levels in COVID‐19 patients' serum. These findings establish the importance of RBD glycosylation in SARS‐CoV‐2 pathogenesis, which can be exploited for the development of novel therapeutics for COVID‐19.