Identification of 22 N-glycosites on spike glycoprotein of SARS-CoV-2 and accessible surface glycopeptide motifs: Implications for vaccination and antibody therapeutics

Abstract

Coronaviruses hijack human enzymes to assemble the sugar coat on their spike glycoproteins. The mechanisms by which human antibodies may recognize the antigenic viral peptide epitopes hidden by the sugar coat are unknown. Glycosylation by insect cells differs from the native form produced in human cells, but insect cell-derived influenza vaccines have been approved by the US Food and Drug Administration. In this study, we analyzed recombinant severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein secreted from BTI-Tn-5B1-4 insect cells, by trypsin and chymotrypsin digestion followed by mass spectrometry analysis. We acquired tandem mass spectrometry (MS/MS) spectrums for glycopeptides of all 22 predicted N-glycosylated sites. We further analyzed the surface accessibility of spike proteins according to cryogenic electron microscopy and homolog-modeled structures and available antibodies that bind to SARS-CoV-1. All 22 N-glycosylated sites of SARS-CoV-2 are modified by high-mannose N-glycans. MS/MS fragmentation clearly established the glycopeptide identities. Electron densities of glycans cover most of the spike receptor-binding domain of SARS-CoV-2, except YQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQ, similar to a region FSPDGKPCTPPALNCYWPLNDYGFYTTTGIGYQ in SARS-CoV-1. Other surface-exposed domains include those located on central helix, connecting region, heptad repeats and N-terminal domain. Because the majority of antibody paratopes bind to the peptide portion with or without sugar modification, we propose a snake-catching model for predicted paratopes: a minimal length of peptide is first clamped by a paratope and sugar modifications close to the peptide either strengthen or do not hinder the binding.

Sensing the interactions between carbohydrate-binding agents and N-linked glycans of SARS-CoV-2 spike glycoprotein using molecular docking and simulation studies

“…As the S glycoprotein of SARS-CoV-2 is having 22 N-linked glycosylation sites occupied with complex and oligomannose type of glycans (Watanabe et al, 2020;Zhou et al, 2020), these glycans pose as interesting targets for targeted and novel antiviral development. However, understanding this glycan language is lagging behind the proteins due to its complex nature and existence of various conformations.…”

“…The 3D structure of SARS-CoV-2 (PDB ID: 6WPS) deposited in Protein Data Bank (PDB) also reported having GlcNAc, a-D-Man, b-D-Man and a-L-Fucose and fucosylated Man 3 GlcNAc 2 at site N343 (Pinto et al, Preprint). In another study, (Zhou et al, 2020) analyzed the S glycoprotein secreted from the BTI-Tn-5B1-4 insect cells. Upon chymotrypsin and trypsin digestion and mass analysis, they revealed that all predicted 22 N-linked SARS-CoV-2 glycosylated sites are changed by high mannose N-glycans.…”

COVID-19 vaccine development and a potential nanomaterial path forward

“…Therefore, glycosylation of SARS-CoV-2 S vaccine candidates should be considered: DNA/RNA vaccines are produced in situ inside the patient and thus would carry native glycosylation; for recombinant subunit vaccines glycoengineering protocols may be applied. The hotspot of SARS-CoV-2, however, which is critical in binding to the ACE2 receptor, appears to be free from glycosylation 32 ; therefore, it may not be required to glycosylate corresponding peptide epitopes.…”

Cryo-EM Structures Delineate a pH-Dependent Switch that Mediates Endosomal Positioning of SARS-CoV-2 Spike Receptor-Binding Domains

“…The SARS-CoV-2 spike is a type 1 fusion machine, responsible for virus-cell entry via ACE2 receptor interactions and TMPRSS2 protease activation in the endosome 8 . It is substantially glycosylated 9,10 . It is also divergent from other beta-coronavirus pathogens known to have infected humans 2 , and it continues to diversify 7 .…”