Site-specific O-glycosylation analysis of SARS-CoV-2 spike protein produced in insect and human cells

Abstract

Enveloped viruses hijack not only the host translation processes, but also its glycosylation machinery, and to a variable extent cover viral surface proteins with tolerogenic host-like structures. SARS-CoV-2 surface protein S presents as a trimer on the viral surface and is covered by a dense shield of N-linked glycans, and a few O-glycosites have been reported. The location of O-glycans is controlled by a large family of initiating enzymes with variable expression in cells and tissues and hence difficult to predict. Here, we used our well-established O-glycoproteomic workflows to map the precise positions of O-linked glycosylation sites on three different entities of protein S — insect cell or human cell-produced ectodomains, or insect cell derived receptor binding domain (RBD). In total 25 O-glycosites were identified, with similar patterns in the two ectodomains of different cell origin, and a distinct pattern of the monomeric RBD. Strikingly, 16 out of 25 O-glycosites were located within three amino acids from known N-glycosites. However, O-glycosylation was primarily found on peptides that were unoccupied by N-glycans, and otherwise had low overall occupancy. This suggests possible complimentary functions of O-glycans in immune shielding and negligible effects of O-glycosylation on subunit vaccine design for SARS-CoV-2.

Evolving spike-proteinN-glycosylation in SARS-CoV-2 variants

“…So, there is very high probability that N616 glycans can regulate the integrity of S1-S2 coupling in the spike-protein trimer 71 . Presence of O-glycans has been reported at N618, that may also contribute to this effect 126,127 . P681R mutation may enhance Furin cleavage by adding a basic residue to 682RRAR in Delta and D614G stabilizes the Furin-cleaved S1-protein on S2-protein to facilitate fusogenicity, thus explaining Delta's high disease severity 107,110,128,129 .…”

Immunogenicity and Neutralizing Activity Comparison of SARS-CoV-2 Spike Full-Length and Subunit Domain Proteins in Young Adult and Old-Aged Mice

“…A recent study reported that the glycosylation patterns were found to be similar on the SARS-CoV-2 spike (S) ectodomain proteins expressed in insect and human cells [34]. Du et al (2009) compared the immunogenicity and protective immunity of recombinant SARS-CoV RBD proteins expressed in mammalian cells, insect cells, and Escherichia coli [35].…”

Cancer cells and viruses share common glycoepitopes: exciting opportunities toward combined treatments

“…These host cell-derived glycoforms facilitate diverse structural and functional roles during the viral life-cycle mostly related to immune evasion since these extensive glycosylation sites usually mask peptide sequences that would be otherwise useful targets for vaccine design (38,39,59). Importantly, whether the envelope glycoproteins of most of these viruses are expressed in human cells, their expression into other vectors lead to identical glycoform patterns (60).…”