Suppression of O-Linked Glycosylation of the SARS-CoV-2 Spike by Quaternary Structural Restraints

Abstract

Understanding the glycosylation of the envelope spike (S) protein of SARS-CoV-2 is important in defining the antigenic surface of this key viral target. However, the underlying protein architecture may significantly influence glycan occupancy and processing. There is, therefore, potential for different recombinant fragments of S protein to display divergent glycosylation. Here, we show that the receptor binding domain (RBD), when expressed as a monomer, exhibits O-linked glycosylation, which is not recapitulated in the native-like soluble trimeric protein. We unambiguously assign O-linked glycosylation by homogenizing N-linked glycosylation using the enzymatic inhibitor, kifunensine, and then analyzing the resulting structures by electron-transfer higher-energy collision dissociation (EThcD) in an Orbitrap Eclipse Tribrid instrument. In the native-like trimer, we observe a single unambiguous O-linked glycan at T323, which displays very low occupancy. In contrast, several sites of O-linked glycosylation can be identified when RBD is expressed as a monomer, with T323 being almost completely occupied. We ascribe this effect to the relaxation of steric restraints arising from quaternary protein architecture. Our analytical approach has also highlighted that fragmentation ions arising from trace levels of truncated N-linked glycans can be misassigned as proximal putative O-linked glycan structures, particularly where a paucity of diagnostic fragments were obtained. Overall, we show that in matched expression systems the quaternary protein architecture limits O-linked glycosylation of the spike protein.

Principles of SARS-CoV-2 glycosylation

“…These mannose signatures were also observed in spike protein derived from infectious virus [ 38 , 39 ]. However, when only the S1 subunit is expressed, N234 is fully processed to complex-type glycosylation [ 39 ] consistent with the relaxation of steric restrictions to processing in the monomeric form [ 7 , 27 , 39 ] ( Figure 3 B).

Figure 3 The influence of protein structure on glycan maturation.

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“…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 ].…”

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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.

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“…The receptor-binding domain (RBD) in S1 subunit interacts with the ACE2 receptor [ 4 ]. The S protein is extensively glycosylated, typically encoding 66 N-linked glycosylation sites per trimer and a lower number of O-linked glycans present at low occupancy [ 5 , 6 , 7 ]. The high density of viral glycosylation has prompted investigations into the functional role of glycans in a wide range of settings, from its impact on protein folding to its influence on immunogenicity.…”

Designed SARS‐CoV‐2 receptor binding domain variants form stable monomers

“…Other reports have also noted substantial O-glycosylation of T323 in other monomeric S-protein variants. [36,37] Interestingly, Oglycosylation of T323 is far less pronounced in the context of fulllength S-protein trimers, [37][38][39][40] indicating that this site is shielded upon trimer formation and thus not accessible to glycosylation enzymes once oligomerization has taken place. In RBD, T323 O-glycosylation, characterized as disialyl Core 1 and Core 2 structures [39] could be required to prevent oligomerization of RBD monomers by masking an otherwise aggregation-prone hydrophobic peptide stretch or by balancing the overall charge of the protein.…”