Structural remodeling of SARS-CoV-2 spike protein glycans reveals the regulatory roles in receptor-binding affinity

Abstract

Glycans of the SARS-CoV-2 spike protein are speculated to play functional roles in the infection processes as they extensively cover the protein surface and are highly conserved across the variants. The spike protein has been the principal target for vaccine and therapeutic development while the exact effects of its glycosylation remain elusive. Analytical reports have described the glycan heterogeneity of the spike protein. Subsequent molecular simulation studies provided a knowledge basis of the glycan functions. However, experimental data on the role of discrete glycoforms on the spike protein pathobiology remains scarce. Building an understanding of their roles in SARS-CoV-2 is important as we continue to develop effective medicines and vaccines to combat the disease. Herein, we used designed combinations of glycoengineering enzymes to simplify and control the glycosylation profile of the spike protein receptor-binding domain (RBD). Measurements of the receptor binding affinity revealed opposite regulatory effects of the RBD glycans with and without sialylation, which presents a potential strategy for modulating the spike protein behaviors through glycoengineering. Moreover, we found that the reported anti-SARS-CoV-(2) antibody, S309, neutralizes the impact of different RBD glycoforms on the receptor binding affinity. In combination with molecular dynamics simulation, this work reports the regulatory roles that glycosylation plays in the interaction between the viral spike protein and host receptor, providing new insights into the nature of SARS-CoV-2. Beyond this study, enzymatic glycan remodeling offers the opportunity to understand the fundamental role of specific glycoforms on glycoconjugates across molecular biology.

The accomplices: Heparan sulfates and N-glycans foster SARS-CoV-2 spike:ACE2 receptor binding and virus priming

“…Interactions with S C RBD residues are overall maintained with the occupancy of some interactions reduced in favour of others, including the newly gained H-bond with S C G416 and the increased interaction with S C N165 glycan (Fig.4A and S9). These results provide a mechanistic explanation for previous reports of the importance of the N17, N165 and N343 glycans of spike and the N53, N90 and N322 glycans of ACE2 for their interaction (Hsu et al, 2023; Huang et al, 2021; Li et al, 2020; Mehdipour & Hummer, 2021; Q. Yang et al, 2020), by showing that spike and ACE2 N-glycans establish stable contacts and fill the intermolecular space, promoting more favorable protein:protein interactions.…”

Inclusion of deuterated glycopeptides provides increased sequence coverage in hydrogen/deuterium exchange mass spectrometry analysis of SARS‐CoV‐2 spike glycoprotein

“…De‐glycosylation adds time and complexity to sample preparation and potentially introduces additional back‐exchange and/or artifacts during HDX‐MS analyses. In the case of SARS‐CoV‐2 spike, previous reports indicate that glycans participate in modulation of the RBD moving to “up” or “down” positions, 66 as well as in ACE2 interaction, 67,68 indicating the importance of measuring spike glycopeptide dynamics in a native state using HDX‐MS.…”

“…In the case of SARS-CoV-2 spike, previous reports indicate that glycans participate in modulation of the RBD moving to "up" or "down" positions, 66 as well as in ACE2 interaction, 67,68…”

Antigen-Heterologous Vaccination Regimen Triggers Alternate Antibody Targeting in SARS-CoV-2-DNA-Vaccinated Mice

“…Surprisingly, all three vaccinated groups targeted an epitope containing the glycan-shielded N343 with considerable intensity ( Figure 2 ). The N343 RBD glycan is, besides shielding, suspected to interact with the ACE2 backbone and stabilize ACE2-RBD binding [ 4 , 28 ]. The mAb S309 (classified as a class 3 antibody) does not bind this exact epitope; however, N343 is sandwiched between the mAb’s binding sites, and the binding of this antibody possibly compromises ACE2-RBD binding stability, thereby inhibiting infectivity [ 28 ].…”