Site specific N- and O-glycosylation mapping of the spike proteins of SARS-CoV-2 variants of concern

Abstract

The glycosylation on the spike (S) protein of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19, modulates the viral infection by altering conformational dynamics, receptor interaction and host immune responses. Several variants of concern (VOCs) of SARS-CoV-2 have evolved during the pandemic, and crucial mutations on the S protein of the virus have led to increased transmissibility and immune escape. In this study, we compare the site-specific glycosylation and overall glycomic profiles of the wild type Wuhan-Hu-1 strain (WT) S protein and five VOCs of SARS-CoV-2: Alpha, Beta, Gamma, Delta and Omicron. Interestingly, both N- and O-glycosylation sites on the S protein are highly conserved among the spike mutant variants, particularly at the sites on the receptor-binding domain (RBD). The conservation of glycosylation sites is noteworthy, as over 2 million SARS-CoV-2 S protein sequences have been reported with various amino acid mutations. Our detailed profiling of the glycosylation at each of the individual sites of the S protein across the variants revealed intriguing possible association of glycosylation pattern on the variants and their previously reported infectivity. While the sites are conserved, we observed changes in the N- and O-glycosylation profile across the variants. The newly emerged variants, which showed higher resistance to neutralizing antibodies and vaccines, displayed a decrease in the overall abundance of complex-type glycans with both fucosylation and sialylation and an increase in the oligomannose-type glycans across the sites. Among the variants, the glycosylation sites with significant changes in glycan profile were observed at both the N-terminal domain and RBD of S protein, with Omicron showing the highest deviation. The increase in oligomannose-type happens sequentially from Alpha through Delta. Interestingly, Omicron does not contain more oligomannose-type glycans compared to Delta but does contain more compared to the WT and other VOCs. O-glycosylation at the RBD showed lower occupancy in the VOCs in comparison to the WT. Our study on the sites and pattern of glycosylation on the SARS-CoV-2 S proteins across the VOCs may help to understand how the virus evolved to trick the host immune system. Our study also highlights how the SARS-CoV-2 virus has conserved both N- and O- glycosylation sites on the S protein of the most successful variants even after undergoing extensive mutations, suggesting a correlation between infectivity/ transmissibility and glycosylation.

Figure 1

The mutations across the variants of concerns (VOCs) of SARS-CoV-2. Mutations shown represent the exact protein sequences used for this analysis, based on the sequence information provided by R&D systems. The mutations from the Wuhan-Hu-1 strain (WT) happened predominantly in the NTD and RBD domains and mutations increased significantly in the case of Omicron variant. NTD, N-terminal Domain; RBD, Receptor Binding Domain; SD1, subdomain 1; SD2, subdomain 2; FP1, Fusion Peptide 1; FP2, Fusion Peptide 2; HR1, Heptad repeat 1; HR2, Heptad repeat 2; S1, Subunit 1; S2, Subunit 2.

Figure 2

Most abundant glycan structure on each site of the WT and VOCs. Glycosylation sites are conserved in all the variants except for the presence of an additional site N20 in Gamma variant and loss of N17 in Delta variant. Most abundant glycoforms are similar across sites (examples being N61, N331, N1134, N1173 and N1194).

Figure 3

Site specific N- and O- glycans on SARS-CoV-2 spike protein across the WT and VOCs. Each pie chart denotes glycosylation sites of the S protein for each variant and shows the distribution of N- and O-glycosylation types at each site of the WT and VOCs.

Figure 4

Relative abundances of four most intense glycoforms at each site of WT and VOCs.

Figure 5

Confirmation of the presence of N-glycosylation at N20 site and absence of N-glycosylation at N17 site on the spike protein of the Gamma variant by stable ¹⁸O isotope labelling technique. HCD MS/MS spectrum of the peptide VNFTNR showing b and y fragment ion series, which confirms the ¹⁸O label is only at N20 (MS1, y2, y3, y4, y5 with 2.98 Da mass) thereby confirming the presence of N-glycosylation. Inset: in silico predicted b and y ion masses of peptide VNFTNR without deamidation or ¹⁸O label (left) and with ¹⁸O label (right).There was no deamidation or ¹⁸O label noted on N17.

Figure 6

PCA analysis and heat map distribution of the N-linked glycans on sites at RBD, (A) N331 and (B) N343 showed distinct differences in the glycosylation on the VOCs in comparison to the WT, particularly the Omicron variant.

Figure 7

Distribution of (A) N- and (B) O- glycans among the VOCs determined by glycomics (released glycans). All variants showed differences in the type of N-glycans with respect to WT. (C). Relative abundance comparison of most abundant ten glycoforms of WT and VOCs. Omicron showed most significant overall N-glycan differences with 43% high mannose structures. Error bars represent standard deviation from duplicate analysis.