The first intimation of the current COVID-19 crisis came with the news of severe pneumonic illness caused by an unknown agent in a Chinese city called Wuhan, issued on the last day of 2019. Following this, researchers found the cause to be a novel coronavirus, closely related to the earlier SARS virus, now called severe acute respiratory syndrome coronavirus 2.
Efforts are underway to develop an effective vaccine or treatment to prevent the further spread of this global infection, which has already taken well over 543,000 lives among the approximately 11.8 million reported cases. The detailed structure of the viral particle is vital in generating an effective vaccine that will elicit high levels of specific neutralizing antibodies.
Spike Protein Analysis
The virus takes its name from the numerous spikes on its surface, composed of glycoprotein. The transmembrane spike (S) protein engages the angiotensin-converting enzyme (ACE) 2 on the host cell surface to trigger the process of viral entry. The ACE2 receptor is also a glycoprotein, and the sugar residues, therefore, play a key role in the interaction of these proteins.
This news article was a review of a preliminary scientific report that had not undergone peer-review at the time of publication. Since its initial publication, the scientific report has now been peer reviewed and accepted for publication in a Scientific Journal. Links to the preliminary and peer-reviewed reports are available in the Sources section at the bottom of this article. View Sources
The spike is present as a trimer of three S proteins, or protomers, each having its S1 and S2 subunits. The S1 binds to the ACE2 receptor, while the S2 is responsible for the fusion of the virus with the host cell membrane to initiate viral entry.
While both SARS-CoV and SARS-CoV-2 share the ACE2 receptor, their affinity varies considerably, with the latter showing 10-fold to 20-fold higher affinity. This again, may be traced to glycosylation patterns.
Heavy S Protein Glycosylation
The SARS-CoV-2 carries 22 or more N-glycan residues, with 3 predicted O-glycosylations. However, these last have not yet been proved to exist. The most recent data show complex, multimolecular short mannose chains at 20/22 N-glycosylation sites. Researchers have also reported one O-glycopeptide at a site separate from the furin cleavage site at the S1/S2 interface.
N-Glycan Structural Motifs
A new study by scientists at Georgetown University and Waters Corporation published on the preprint server bioRxiv* analyzed the sugars at the various sites, emphasizing repeated structures at the O and N glycans identified so far, on a recombinant full-length S protein expressed in a laboratory cell line. The researchers identified 17 N-glycans of the spike protein, containing mannose, hybrid, and complex sugar residues.
While most of the amino acids forming the attachment site sequence (sequon) for the N-linked sugar are fully occupied, they found two that were not glycosylated, and another that was occupied by high-mannose sugars. The rest of the sequons are attached to complex glycans, with fucose residues at the core of 15 sequons.
The study also found structural motifs on all the occupied sequons, namely, a LacdiNAc motif attached asymmetrically within an N-glycan with two sialic acid residues. This is different from the LacNAc residues because of the presence of the m/z 366/407 ions.
The researchers also saw 6 N-glycans that had polyLacNAc structures, showing numerous fucose residues in the core and on the outer arms of the glycoproteins. They were able to confirm the presence of fucose sugars at the core of 15 sequons and outer arm fucosyl residues on the outer arms of the N-glycopeptides on 7 sequons.
Differences Explained
These findings are different from earlier studies, which could be because of the difference in the way the laboratory cell line used in the current experiments or the variation in the analytical method. For one, this study used a modified full-length protein without using convertases to cleave it into fragments. The method of study can potentially cause differences in the results of the analysis.
However, the researchers say, the more probable explanation is that the optimization of the workflows in terms of energy use produced a higher resolution of structural analysis. This helped to establish the structural motifs clearly, an important feature often linked to specific biological roles in the living organism. However, all the linkages and the isobaric structures related to the glycopeptide could not be assigned with complete confidence.
Overall, the scientists were able to show the presence of polyLacNAc motifs carried on 6 glycopeptides. All the sequons carrying complex glycans carried LacdiNAc to some extent. On residues N165 and N1098, more than 50% of the glycoforms were composed of these structural motifs. This could be important for vaccine development since the HEK293 cell line used in this study is commonly used to study the function of the S glycoprotein and to produce vaccine candidates.
O-Glycopeptide Analysis
The study also identified the O-glycans reported by earlier research, and another 8 glycoproteins occupied by core-1 and core-2 structures. Unlike the N-glycans, the site occupancy is variable, from less than 1% to 57%, and for three of them, it is very low.
The polybasic furin cleavage site, a novel feature of the SARS-CoV-2, located at the interface of S1 and S2, has a T678 residue nearby with core-1 and core-2 structures, and 13% occupancy. This is biologically important because the O-glycans occurring before the convertase cleavage sites of a protein are involved in regulating protein cleavage, and could even affect the rate of activation of the S protein in this virus.
Using further site identification tools, they found 9 O-glycoproteins occupied by O-glycans. The T678 residue is the primary occupied site, with the z6 carrying a glycan while the z4 does not, while the peptide has both core-1 and core-2 residues. They established more structural details of various O-glycoproteins. The functional importance of these remains to be established.
Future Directions
Overall, the study will stimulate further research on the effect of glycosylation on the function of the spike protein in this virus.
The study concludes, “We resolved, for the first time, LacdiNAc and polyLacNAc structural motifs associated with the N-glycopeptides and we identified novel O-glycopeptides including a glycopeptide near the furin cleavage site of the spike glycoprotein.”
This news article was a review of a preliminary scientific report that had not undergone peer-review at the time of publication. Since its initial publication, the scientific report has now been peer reviewed and accepted for publication in a Scientific Journal. Links to the preliminary and peer-reviewed reports are available in the Sources section at the bottom of this article. View Sources
Article Revisions
- Mar 25 2023 - The preprint preliminary research paper that this article was based upon was accepted for publication in a peer-reviewed Scientific Journal. This article was edited accordingly to include a link to the final peer-reviewed paper, now shown in the sources section.
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