How natural alterations within the SARS-CoV-2 glycan barrier affect the dynamics of spike protein

In a recent article published on the bioRxiv* preprint server, researchers investigated how natural alterations within the glycan barrier of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) affect the dynamics of the spike (S) protein.

Background
The pandemic of SARS-CoV-2, which has caused a protracted global medical crisis, has resulted in the deaths of over 6,4 million people worldwide. The emergence of SARS-CoV-2 variants alters the efficacy of existing protection, regardless of whether it develops naturally or as a result of vaccination.

The analysis of the SARS-CoV-2 S glycoprotein structure will aid in the comprehension of the effects of antigenic surface mutations. One type of mutation affects the glycosylation attachment sites, which can influence the antigenic architecture beyond the primary attachment site.

About the research
This study aimed to comprehend potential SARS-CoV-2 immune evasion pathways involving modifications to the glycan shield.

The researchers examined the glycan barrier of the S protein of the SARS-CoV-2 variant of concern (VOC). In addition, they investigated their effect on the adhesion of angiotensin-converting enzyme 2 (ACE2). The sequences for the SARS-CoV-2 P.1 (Gamma), B.1.617.2 (Delta), and B.1.351 (Beta) variants were engineered to produce soluble recombinant native-like trimers. Using liquid chromatography-mass spectrometry, they analysed the compositional variations in the site-specific glycan shield of SARS-CoV-2 volatile organic compounds (LC-MS).

The group synthesised glycopeptides with a single asparagine (N-) glycosylation site (PNGS). They utilised chymotrypsin, alpha-lytic, and trypsin proteases for this purpose. In addition, the glycopeptide compounds were subjected to collision-induced dissociation (HCD) fragmentation at higher energies.

The authors sought to comprehend how the protein’s topology might influence the Gamma S glycoprotein’s N188 glycosylation. Conventional molecular dynamics (MD) was used to perform exhaustive sampling on two Gamma S models, one of which had Man5GlcNAc2 (Man5) at N188 and the other of which did not have glycosylation at this site. In addition, the researchers recreated the ectodomain of the P.1 S glycoprotein based on the structure obtained through cryogenic electron microscopy (cryo-EM), i.e. PDB 7SBS.

Conclusions and Discussions
By combining compositional glycan analyses with MD simulations, the study results provide a molecular understanding of how the SARS-CoV-2 Gamma variant with an additional N188 glycan site exhibits superior shielding of the S glycoprotein surface. In addition, the team discovered minimal alterations across the glycan barrier of other VOCs, which may indicate that SARS-CoV-2 has not yet fully exploited the capacity for glycan-facilitated immune evasion.

Despite having more than sixty N-glycan sites throughout the trimer, the glycan barrier density of the SARS-CoV-2 S protein was low compared to that of influenza, human immunodeficiency virus 1 (HIV-1), and Lassa virus (LASV). Consequently, a significant portion of the surface of the immunogenic protein remained accessible to antibody-mediated immune responses.

Scientists stated that SARS-CoV-2, a virus that has only recently begun to circulate among humans, has utilised simple adaptations, such as modifications to the receptor binding domain, to increase its infectivity and circumvent host immunity (RBD).

The authors noted that modifying the glycan barrier likely has a negative effect on viral infectiousness. An increase in oligomannose-type glycan aggregation in influenza H3N2 has been attributed to recurrent endemic circulation and the development of immunity in humans, as demonstrated by earlier studies. In addition, the gradual plateau of PNGS accumulation demonstrates that increasing glycan shielding impairs viral fitness.

SARS-CoV-2 lacks the RBD glycosylation site N370 compared to other coronaviruses (CoVs), which may increase its infectiousness. As infections and vaccines persist, the researchers hypothesised that SARS-CoV-2 evolution will run out of simple amino acid deletions and substitutions and begin to alter the glycan shield, with potential fitness costs.

Conclusions
In accordance with the accessibility of a few enzymes, the N188 site of SARS-CoV-2 exhibited minimal glycan maturation, as indicated by the study’s findings. MD and structural modelling revealed that N188 was positioned within a cavity in the RBD, thereby influencing the dynamics of these adhesion domains. Observations suggest that mutations in the glycosylation sites of SARS-CoV-2 affect the structural integrity of the antigenic surface.

In addition, the researchers noted that longer-circulating CoVs, such as NL63, the common cold CoV, were significantly more densely glycosylated and contained roughly twice as many glycan domains per protomer as SARS-CoV-2. In order to better comprehend how the glycans of SARS-CoV-2 S glycoproteins manipulate the host immune system when new VOCs emerge, methods similar to those described in this manuscript would be useful.

*Important notice bioRxiv publishes preliminary scientific reports that have not been peer-reviewed and, as such, should not be considered conclusive, used to guide clinical practice/health-related behaviour, or regarded as established information.

 

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