Site-Specific Steric Control of SARS-CoV-2 Spike Glycosylation

Abstract

A central tenet in the design of vaccines is the display of native-like antigens in the elicitation of protective immunity. The abundance of N-linked glycans across the SARS-CoV-2 spike protein is a potential source of heterogeneity among the many different vaccine candidates under investigation. Here, we investigate the glycosylation of recombinant SARS-CoV-2 spike proteins from five different laboratories and compare them against S protein from infectious virus, cultured in Vero cells. We find patterns that are conserved across all samples, and this can be associated with site-specific stalling of glycan maturation that acts as a highly sensitive reporter of protein structure. Molecular dynamics simulations of a fully glycosylated spike support a model of steric restrictions that shape enzymatic processing of the glycans. These results suggest that recombinant spike-based SARS-CoV-2 immunogen glycosylation reproducibly recapitulates signatures of viral glycosylation.

Figure 1

Site-specific glycosylation of recombinant and virally derived S protein from multiple laboratories. (A) Site-specific glycan analysis of recombinant S protein expressed and purified at different locations. The bar charts represent the relative proportions of glycoforms present at each site, including the proportion of PNGSs that were not modified by an N-linked glycan. The proportions of oligomannose- and hybrid-type glycans are colored green. Processed complex-type glycans are colored pink, and the proportions of unoccupied sites are colored gray. The institution that provided the S protein for analysis is listed above each chart. The Texas/Southampton data are reproduced from Chawla et al. (unpublished data). The average compositions of recombinant S protein were calculated using all samples. Bars represent the mean ± the standard error of the mean of all recombinant samples analyzed. (B) Virally derived site-specific analysis was performed using data acquired by Yao et al. and categorized in the same manner as described above. Data for sites N657 and N1158 could not be obtained and are not represented. (C) Full length model displaying the site-specific oligomannose glycosylation of virally derived S protein. A description of how this model was generated can be found in Materials and Methods. Both protein and glycans are shown in surface representation; the former is colored gray, and the latter colored on the basis of the oligomannose content as shown in Table S1 (green for 80–100%, orange for 30–79%, and pink for 0–29%).

Figure 2

Comparison of detailed compositions between sites with differential processing states. Glycan compositions at N234, N1074, and N282. For all samples analyzed, glycans were categorized and colored according to the detected compositions. Oligomannose-type glycans (M9-M4) are colored green. Hybrid-type glycans, those containing three HexNAcs and at least five hexoses, were colored as for complex-type glycans because one arm can be processed in a similar manner. Complex-type glycans were categorized according to the number of HexNAc residues detected and the presence or absence of fucose. Core glycans represent any detected composition smaller than HexNAc₂Hex₃. For hybrid and complex-type glycans, bars are colored to represent the terminal processing present. Blue represents agalactosylated, yellow galactosylated (containing at least one galactose), and purple sialylated (containing at least one sialic acid). The proportion of unoccupied PNGSs is colored gray.

Figure 3

Comparative analysis of the glycosylation of the two PNGSs on the RBD for virally derived S protein, recombinant S protein, and the monomeric RBD. (A) Detailed site-specific glycan compositions of the two sites located in the RBD of SARS-CoV-2. Recombinant S protein data are reproduced from Chawla et al. (unpublished data), and data for the viral S protein RBD sites were obtained from ref (32). Site-specific glycan data are presented as outlined in Figure 2. (B) Site-specific compositions for N-glycan sites located in the RBD of MERS-CoV when expressed as part of a soluble recombinant S protein compared to the RBD only. Data for the MERS-CoV S protein were obtained from ref (51). Site-specific glycan data are presented as outlined in Figure 2.

Figure 4

Accessible surface area (ASA) of oligomannose-type glycans from MD simulations. (A) The ASA values were calculated for each oligomannose-type (M9) glycan for all three S protein chains. In this model, chain A is modeled in the RBD “up” conformation. The last 50 ns of the simulation was used for calculation, and error bars indicate the standard deviation along the trajectory. The probe size used was 1.5 nm. The green color bars represent the oligomannose content of respective sites, calculated as the average oligomannose-type glycan content of the recombinant S proteins analyzed in this work (Amsterdam, Harvard, Swiss, and Oxford S proteins). (B) Structure of the S protein (gray) with glycans colored on the basis of their ASA values shown in surface representation. (C) Graphs comparing glycan processing to calculated ASA values, averaged for chains A–C, for the recombinant protein average for the oligomannose-type glycan content, the proportion of glycans containing at least one sialic acid, and the proportion of glycans containing at least one fucose. The reported r values represent the Spearman’s rank correlation coefficient. (D) Oligomannose-type glycan content vs ASA for virally derived SARS-CoV-2 S protein presented as in panel C.