Principles of SARS-CoV-2 glycosylation

Abstract

The structure and post-translational processing of the SARS-CoV-2 spike glycoprotein (S) is intimately associated with the function of the virus and of sterilising vaccines. The surface of the S protein is extensively modified by glycans, and their biosynthesis is driven by both the wider cellular context, and importantly, the underlining protein structure and local glycan density. Comparison of virally derived S protein with both recombinantly derived and adenovirally induced proteins, reveal hotspots of protein-directed glycosylation that drive conserved glycosylation motifs. Molecular dynamics simulations revealed that, while the S surface is extensively shielded by N-glycans, it presents regions vulnerable to neutralising antibodies. Furthermore, glycans have been shown to influence the accessibility of the receptor binding domain and the binding to the cellular receptor. The emerging picture is one of unifying, principles of S protein glycosylation and an intimate role of glycosylation in immunogen structure and efficacy.

Graphical abstract

Figure 1

Glycosylation of viral-derived and recombinant spike protein. (a) Representation of cryo-electron tomograph (Cryo-ET) slice of SARS-CoV-2 virions reproduced from Yao et al. [37]. (b) Model of representative SARS-CoV-2 virus highlighting one S protein on the envelope, has been adapted to highlight a single spike from Yao et al. [37]. (c) Fine glycan processing of viral S representing content of oligomannose-type glycans and heat plot of percentage of oligomannose-type glycans of viral-derived S protein, using reported abundances [38,39]. (d) Glycan processing of recombinant S, values of which obtained from data produced earlier [38,39]. The glycan composition is categorised in three groups based on the abundance of oligomannose-type glycan content: green (100%–80%), orange (79%–30%) and magenta (29%–0%). The heat plot represents the percentage of oligomannose-type glycans at each site from a scale of 0% (white) to 100% (green).

Figure 2

Role of glycans in stabilizing dynamics of RBD. Model representation of a) RBD ‘closed’ and b) RBD ‘open’ showing the interaction between neighbouring glycans, N165, N234 and N343. The RBD is accessible for ACE2 binding in ‘open’ state. The N343 glycan act as a “glycan gate” as it pushes the RBD from ‘down’ to the ‘up’ conformation. The N234 is modelled with Man₉GlcNAc₂ glycan represented in green, N165 and N343 are modelled with biantennary complex-type glycans [38,39]. The RBD is shown in cyan, and the remaining S protein highlighted in grey. These models are reproduced from previous studies [41,45].

Figure 3

The influence of protein structure on glycan maturation. (a) Illustration of glycan composition of recombinant trimeric S protein of which values reproduced from Eldrid et al. [7]. The N-linked glycosylation takes place at specific sequon, Asn-X-Ser/Thr (X is any amino acid except proline) whereas O-linked glycosylation is not dictated by specific sequon and occurs on serine and threonine in exposed regions. The N-linked glycosylation is presented in three categories on basis of oligomannose content as described in Figure 1, oligomannose (green), hybrid (orange) and complex-type (magenta) glycans. The O-linked glycosylation at T323 site (see magnification) on trimeric S is present at low levels (0.2%) of which values obtained from Eldrid et al. [7]. (b) The glycan composition of recombinant monomeric S1 subunit of which values reproduced from Wang et al. [27] and Brun et al. [39]. Most of the N-glycan sites on S1 subunit is highly processed represented in magenta except N657 which is unoccupied, represented in wheat color. The O-glycosylation is present on S1 subunit at sites, T323 and T678 (see magnification). (e) The glycan composition of monomeric RBD protein (cyan) which binds to main host receptor (ACE2). The N-glycan sites of RBD are highly processed and are represented in magenta, values reproduced from Allen et al. [38]. The O-glycosylation was observed on monomeric RBD protein at sites T323 and S469/T470 (see magnification) [7].

Figure 4

Antibody recognition within the breaches of S protein glycan shield. (a) Representation of trimeric S protein with glycans (blue) present on it [33]. The RBD present on the head region of the S is highlighted in cyan. The stalk region is effectively shielded with glycans. The mesh network represents the virion membrane on which S protein is embedded. (b) Illustration of antibodies binding within the glycan shield of S protein obtained by structural alignment (www.pymol.org). The 4A8 nAb (pink, PDB 7C2L) targets the NTD region of the S protein [70]. The C002 neutralising antibody (nAb) (yellow, PBD 7K8T) is binding to RBD ‘up’ conformation [73]. The S2M11 (green, PDB 7K43) nAb recognises a quaternary epitope consisting of two neighbouring RBDs and stabilises the trimeric S in closed state [72].