O-linked sialoglycans influence cleavage of SARS-CoV-2 . spike

In a recent study published in bioRxiv* Prepress server Researchers from Spain, the United Kingdom and the United States used chemical tools to reveal the molecular details of glycan modification mediated by the processing of the coronavirus 2 (SARS-CoV-2) spike(S).

Stady: O-linked sialoglycans modulate proteolysis in SARS-CoV-2 Spike and contribute to the mutational pathway in variants of concern.. Image Credit: NIAID


SARS-CoV-2 S is a multi-domain triplet glycoprotein with a dense glycan layer. The multi-base furin cleavage site (arginine-rich peptide sequence) (FCS) cleavage site in full-length S (FL-S) enhances binding to host cell receptors after its cleavage by furin or the membrane protease serine 2 (TMPRSS2). The enhanced protein cleavage of FL-S into the S1 and S2 subunits is attributed to a polymorphism between the amino acid (AA) residues/glutamine (Gln) peptide 675 and the proline (Pro) 681 residue that precedes the FCS. Notably, this peptide region of SARS-CoV-2 S also harbors serine (Ser) and threonine (Thr) AA residues that may carry N-acetylgalactosamine (O-GalNAc) glycans. According to the authors, binding to Ser/Thr-linked O-GalNAc glycosylation affects Furin- and TRMPSS2-mediated S cleavage.

This stretch is also the most susceptible to mutations; Subsequently, in the Alpha and Delta SARS-CoV-2 variants of concern (VOCs), several mutations are harbored between Pro681 to histidine (His) and arginine (Arg), respectively. In all Omicron substrains, including BA.1, BA.2 and BA.5, mutations are present in P681H and N679K. All of these mutations have a cleavage-promoting effect. It is an analytical challenge to study this peptide extension in SARS-CoV-2 S due to its biosynthetic complexity and the absence of a peptide consensus sequence. Furthermore, there is a lack of data on glycan abundance, its attachment site(s), and its structural effect on proteolysis. Moreover, studies have hardly investigated the role of SARS-CoV-2 VOC mutations on binding to O-GalNAc glycosylation.

There is a family of 20 GalNAc transferases, GalNAc-T1 to T20 are similar with different substrates. Identification of the glycosylation binding sites of each GalNAc-T can provide insight into the biology of O-glycan. In a previous study by Ten Hagen et al., they found seven balanced enzymes that can present GalNAc in SARS-CoV-2 S.

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In this study, the researchers used a chemical engineering tactic called “bump-and-hole (BH) engineering” to study individual GalNAc-T homologous enzymes in a living cell. Furthermore, they insisted that the fragmentation-based sequencing optimization of lycopeptides could enable enhanced mass spectrometry (MS) analysis.

First, the researchers incubated the recombinant S of the wild-type (WT) SARS-CoV-2 strain with constructs of P681R (or P681H) produced in human Expi293-F cells with either WT or BH versions of GalNAc-T1 or T2. Next, they proceeded to shock these constructs with nucleotides and the sugar uridine diphosphate (UDP)-GalN6yne. They characterized the glycosylated releases by the copper(I)-azide alkene cycloaddition catalyst (CuAAC) for visualization via Streptavidin blot. This treatment introduced GalN6yne in a homologous enzyme-specific manner while conferring glycopeptides with an additional positive charge that facilitated MS analysis.

a) SARS-CoV-2 spike and carton model. Furin cleavage site and peptide alignment for COVID-19 are variables of concern and related coronaviruses. The polybasic form of SARS-CoV- is highlighted in yellow. 2. Bold and red are amino acid changes at positions 679 and 681. b) Protrusion and hole geometry allows GalNAc-T-specific labeling of glycosylation substrates using the UDPGalN6yne substrate clickable button. FCS = furuncle cleavage site; VOCs = variables of concern.

Next, the team subjected the FL-S and S1/S2 subunits to intra-gel digestion and analyzed them by tandem MS (ETD). They used high-density collision-induced dissociation (HCD) to obtain the bare peptide sequences and glycan structures and then used the I-tagged-GalN6yne diagnostic ion to catalyze the ETD fragmentation of the peptide backbone. The researchers also validated the results of the study in the laboratory. To this end, they used glycosylation of homologously expressed S proteins with soluble versions of BH-GalNAc-T1, BH-GalNAc-T2 and CuAAC with I-azide-tagged glycoprotein analysis and MS.

The team used a panel of synthetic peptides to study the effect of S protein mutations on GalNAc-T1-mediated glycosylation. These peptides contain mutations associated with the SARS-CoV-2 VOC in the major hotspots: Gln675, Gln677, asparagine (Asn) 679 and Pro681.

Glycosylation modulates the state processing of protein S depending on the distance to the cleavage site and the composition of the glycan. Therefore, the team directly investigated whether O-GalNAc glycans on Thr678 modulated S cleavage by furin using Förster Resonance Energy Transfer (FRET)-active substrate peptides. Peptides spanning Gln residues 672 to 689 contain N-terminal 2-aminobenzoyl and C-terminal 3-nitro-Tyr as fluorescence donors and quench motifs, respectively. The increase in fluorescence intensity indicated the cleavage of S-mediated furan. First, they compared non-glycosylated substrates corresponding to WT (FRET-1) and mutant p681H (FRET-2). In addition, they exposed recombinant TMPRSS2 to FRET glycopeptide substrates FRET-1, FRET-3 and FRET-7 to FRET-9.


Computational and manual verification revealed that Thr678 carried I-tagged-modified GalN6yne in FL-S and S1 samples only in cells expressing BH-T1. Both BH-T1 and BH-T2 glycosylated threonine (Thr) 323 were not associated with any GalNAc-T-like enzyme. FRET analysis revealed that the development of O-GalNAc glycans on Thr678 of SARS-CoV-2 S glycoprotein, especially with negatively charged modifications, severely impeded the activity of furin. O-glycans in lung epithelial cells expressing GalNAc-T1 suggested that glycosylation is a physiologically important modification that restricts S-protein maturation during development.

According to the authors, disruption of O-GalNAc glycosylation binding was a major driving force behind the development of SARS-CoV-2 VOCs. Within the evolutionary pathway from SARS-CoV-2 Alpha to Delta and Omicron VOCs, the observed changes in the AA sequence preceding FCS indicated that proteolytic cleavage was progressively enhanced with increased VOC susceptibility. The similar mutation found in the more transmissible Delta VOC (P681R) has also been linked to elevated furin cleavage, suggesting an evolutionary pathway that inhibits O-glycosylation prior to further recognition of intrinsic furan.

In contrast to P681H detected in early variants such as Alpha, the N679K mutation in Omicron did not significantly affect glycosylation binding but resulted in enhanced furin cleavage of synthetic peptides. These FCS-proximal mutants acted synergistically and evolved to suppress glycosylation binding and promote furin cleavage.


The present study demonstrated that O-glycosylation is a major determinant of SARS-CoV-2 S cleavage and maturation. It may also be a remnant of SARS-CoV-2 ancestral strains lost through viral evolution.

The authors demonstrated that GalNAc-T1 primed to bind to glycosylation at Thr678 in living cells. The glycosylation binding site at Thr678 of S (not close to FCS) explained why a single GalNAc residue was insufficient to modulate furin activity and TMPRSS2 activity was only slightly affected. Conversely, articulation and navigation abolished furin activity by up to 65%. Similarly, O-glycosylation negatively affected S cleavage by TMPRSS2, with polysaccharide-containing O-glycans 1 (Galβ1-3GalNAc-) having the greatest effect. Overall, the study results emphasized the need for more advanced glycan tracking techniques to study the evolution of SARS-CoV-2.

*Important note

bioRxiv It publishes preliminary scientific reports that have not been peer-reviewed and therefore should not be considered conclusive, guide clinical practice/health-related behaviour, or be treated as established information.

Journal reference:

  • [مقال مجاني عن PMC][PubMed]Edgar Gonzalez-Rodriguez, Mia Zul-Hanlon, Janka Peniva-Todd, Andrea Marchesi, Mark Scheele, Keira Mahoney, Chloe Rostan, Annabelle Borg, Lucia de Fagno, Svend Kiar, Anthony J. Donald J. https://doi.org/10.1101/2022.09.15.508093And the