Identification of 22 N-glycosites on Spike Glycoprotein of SARS-CoV-2 and Accessible Surface Glycopeptide Motifs: Implications on Vaccination and Antibody Therapeutics

Abstract

Corona viruses hijack human enzymes to assembly sugar coat on Spike glycoproteins. The mechanism that human antibodies may uncover the antigenic viral peptide epitopes hidden by sugar coat are unknown. In this study, we analyzed recombinant SARS-CoV-2 Spike protein secreted from BTI-Tn-5B1-4 cells, by trypsin and chymotrypsin digestion followed by mass spectrometry analysis. We acquired MS/MS spectrums for glycopeptides of all 22 predicted N-glycosylated sites. We further analyzed the surface accessibility of Spike proteins according to Cryo-EM and homolog-modeled structures, and available antibodies that bind to SARS-CoV-1. The results showed that all 22 N-glycosylated sites of SARS-CoV-2 are modified by high-mannose type of 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 included those located on Central Helix, between amino acids 967 and 1016 of SARS-CoV-1, and 985 to 1034 of SARS-CoV-2 Spike protein. As the majority of antibody paratopes bind to peptide portion with or without sugar modification, we propose a snake-catcher model that a minimal length of peptide is first clamped by a paratope, and the binding is either strengthened by sugars close to peptide, or not interfered by sugar modification.

Epitope-based chimeric peptide vaccine design against S, M and E proteins of SARS-CoV-2 etiologic agent of global pandemic COVID-19: an in silico approach

“…Moreover, some antibodies such as mAb 8ANC195 have evolved to recognize peptide epitope with no dependence on glycan binding (Kong et al, 2015). However, there is no data available for antibodies specific to spike glycoproteins of SARS-CoV-2, whether their recognition is interfered by the glycosylation of spike or may either be strengthened by sugars close to the peptide epitope, or not interfered by sugar modification (Zhou et al, 2020b). Furthermore, most of the epitopes of the CoV-RMEN harbor no glycoside apart from the NTD region (Watanabe et al, 2020).…”

Techniques assisting peptide vaccine and peptidomimetic design. Sidechain exposure in the SARS-CoV-2 spike glycoprotein

“…With the rise of COV-19, a great deal of diverse work has also been done that relates to the immunogenicity of the spike protein and subsequences including glycosylated subsequences in it (e.g. Refs [ 30 , 31 , 32 ]), including some novel methods of assessing accessibility as a basis of design (e.g. Ref [ 33 ]).…”

“…Despite the comments in Section 1.1 regarding the relative lack of detailed general analysis of the proteins produced by the SARS-COV-2 genome in the understandable rush for vaccines and antiviral agents, there have been many other studies of the accessible surface including the glycosylation of the SARS-C-V-2 glycoprotein (e.g. refs [ 30 , 31 , 32 ]) in order to facilitate development of such weapons against the virus. As one may expect, SARS-CoV-2 has also stimulated some unusual approaches of this general kind [ 33 ].…”

The broad-spectrum antiviral recommendations for drug discovery against COVID-19

“…It is reported that several other critical structures of spike molecules, including heptad repeat 1 and 2 (HR1 and HR2) domains, Central Helix (CH) are involved in the fusion of infected and host cells (Yuan et al 2017;Xia et al 2019). Most recently the Zhou and his research group checked the ASA profiling of RBD of a novel coronavirus (COVID-19) and founded a vulnerable region YQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQ named as "Achilles heel" (see Glossary), which counted helpful during the binding of S protein and ACE-2 receptor (see Figure 2) (Zhou, Qi, et al 2020).…”