Investigation of the N-Glycosylation of the SARS-CoV-2 S Protein Contained in VLPs Produced in Nicotiana benthamiana

Abstract

The emergence of the SARS-CoV-2 coronavirus pandemic in China in late 2019 led to the fast development of efficient therapeutics. Of the major structural proteins encoded by the SARS-CoV-2 genome, the SPIKE (S) protein has attracted considerable research interest because of the central role it plays in virus entry into host cells. Therefore, to date, most immunization strategies aim at inducing neutralizing antibodies against the surface viral S protein. The SARS-CoV-2 S protein is heavily glycosylated with 22 predicted N-glycosylation consensus sites as well as numerous mucin-type O-glycosylation sites. As a consequence, O- and N-glycosylations of this viral protein have received particular attention. Glycans N-linked to the S protein are mainly exposed at the surface and form a shield-masking specific epitope to escape the virus antigenic recognition. In this work, the N-glycosylation status of the S protein within virus-like particles (VLPs) produced in Nicotiana benthamiana (N. benthamiana) was investigated using a glycoproteomic approach. We show that 20 among the 22 predicted N-glycosylation sites are dominated by complex plant N-glycans and one carries oligomannoses. This suggests that the SARS-CoV-2 S protein produced in N. benthamiana adopts an overall 3D structure similar to that of recombinant homologues produced in mammalian cells.

A plant-produced SARS-CoV-2 spike protein elicits heterologous immunity in hamsters

“…Other studies have also reported reduced mannose processing in plants for viral glycoproteins ( Dobrica et al., 2021 ; Margolin et al., 2021a ), although the extent to which processing was arrested here remains a surprise. Interestingly the N-glycosylation pattern observed for the plant-produced spike in this study contrasts with a recent report describing the production of virus-like particles bearing the full-length protein in wild type N. benthamiana ( Balieu et al., 2022 ). In the study conducted by Balieu et al., highly processed mature N-glycans were observed, including typical plant-derived complex glycans with Lewis A epitopes, contrasting against the abundance of immature oligomannose N-glycans that we observed.…”

Performance of plant-produced RBDs as SARS-CoV-2 diagnostic reagents: a tale of two plant platforms

“…The N -glycan profiles of NB-RBD and BY2-RBD were then determined through a glycoproteomic approach as previously described in Balieu et al. (2022) .…”

“…In order to determine the respective N -glycan profiles of both RBD samples and their distribution on these two N ‐glycosylation sites, the mixtures of peptides and glycopeptides released by the endoprotease digestions were submitted to a targeted LC-ESI MS/MS analysis ( Balieu et al., 2022 ). To this end, peptides giving MS/MS spectra exhibiting N ‐glycan diagnostic fragment ions at m/z 204 ( N ‐acetylglucosamine) and 366 (Man‐GlcNAc) were selected as being glycopeptides.…”

“…Affinodetection with concanavalin A (Con A) and immunodetections with antibodies raised against core β(1,2)-xylose (anti-xylose antibodies, Agrisera) and α(1,3)-fucose (anti-fucose antibodies, Agrisera) were performed according to the literature ( Bardor, 1999 ). The N -glycan profiles of NB-RBD and BY2-RBD were determined through analysis by LC-ESI MS/MS of peptides and glycopeptides released by trypsin and Glu-C digestions of purified RBDs as previously described in Balieu et al. (2022) for glycoproteomic and glycan profiling of the SARS-CoV-2 S protein produced in N. benthamiana .…”

Microalgae as an Efficient Vehicle for the Production and Targeted Delivery of Therapeutic Glycoproteins against SARS-CoV-2 Variants

“…These plant-based vaccines showed high immunogenicity and were well-tolerated in phase I, II, and III clinical trials [95,96]. However, other reports have revealed that the plant-produced vaccine contained plant-specific N-glycan epitopes that could cause immunogenic or allergic reactions in humans [97,98]. Recently, the potential of the bacteria Escherichia coli and the yeast Pichia pastoris was evaluated for the production of SARS-CoV-2 RBD fragments [99,100].…”