Site-specific glycosylation analysis of spike proteins of SARS-CoV-2 Omicron subvariants

Multiple Omicron subvariants of SARS-CoV-2 have emerged as dominant global concerns. Although the spike (S) proteins of Omicron subvariants harbor more than 30 mutations compared to the original wild type (WT), N-glycosylation sites within these S proteins are highly conserved. Site-specific glycosylation of S proteins from Omicron subvariants, particularly in the receptor binding domain (RBD) involved in binding to neutralizing antibodies, remain largely unexplored. Here, we purified recombinant S proteins and their corresponding RBDs from two Omicron subvariants (BA.5 and XBB.1) as well as the WT, and characterized site specific glycosylation of these proteins. Our glycoproteomic analysis revealed smaller glycans with mono-fucosylation at the site N331 in the RBD region of trimeric S proteins of Omicron subvariants relative to WT, which might reduce steric constraint for antibody binding to this region. Besides, higher levels of multi-fucosylation and sialylation at the site N331 were detected in monomeric RBDs compared to corresponding trimeric S proteins, suggesting more susceptible of RBDs to modification mediated by the glycan processing enzymes. We believe that the glycosylation profiles of Omicron subvariants will facilitate our understanding of the increased infectivity and transmissibility of Omicron subvariants, and thus assist the diagnosis, prevention, and treatment of COVID-19 infection.