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.

Keywords: COVID; N-glycans; Nicotiana benthamiana; SPIKE; biologics; biopharmaceuticals; mass spectrometry; plant molecular farming; vaccine.

Figure 1

MALDI-TOF mass spectrum profile of [M+Na]⁺ molecular ions of permethylated N-glycans released from the SARS-CoV-2 S protein produced in N. benthamiana. Structures of main oligosaccharides are depicted. Each N-glycan has been drawn according to the international nomenclature [22]. Blue square: GlcNAc; green circle: Man; yellow circle: Gal; red triangle: Fuc and yellow star: Xyl. Linkages of monosaccharides to the core GlcNAc₂Man₃GlcNAc₂ and linkage of the Le^a terminal antennae are indicated. See legend of Figure 2 for nomenclature of plant N-linked glycans.

Figure 2

Oligomannoses, hybrid and complex glycans N-linked to the SARS-CoV-2 S protein produced in plants. Complex N-glycans are named according to the terminal glycan residues attached to Man₃GlcNAc₂ N-glycan sequence (i.e., M₂XF stands for Man₃(Xyl)(Fuc)GlcNAc₂). Glycan isomers differing by their attachment of GlcNAc (Gn) or Le^a epitopes on the two α-mannose arms of the core are represented with a bracket (GnMXF, Le^aMXF and Le^aGNXF). Each N-glycan has been drawn according to the international nomenclature [22]. Linkages of monosaccharides to the core GlcNAc₂Man₃GlcNAc₂ and of the Le^a antennae are indicated.

Figure 3

MS/MS spectra of (a) dicharged ion at m/z 1327.09 assigned to the peptide D₂₂₈-R₂₃₇ N-linked to Man-7, (b) dicharged ion at m/z 1041.44 assigned to the peptide V₃₄₁-R₃₄₆ N-linked to GnMXF and (c) dicharged ion at m/z 1296.54 assigned to the peptide V₃₄₁-R₃₄₆ N-linked to Le^aGnXF. Structures of fragment ions are depicted. Each N-glycan has been drawn according to the international nomenclature [22]. Blue square: GlcNAc; green circle: Man; red triangle: Fuc; yellow circle: Gal and yellow star: Xyl.

Figure 3

MS/MS spectra of (a) dicharged ion at m/z 1327.09 assigned to the peptide D₂₂₈-R₂₃₇ N-linked to Man-7, (b) dicharged ion at m/z 1041.44 assigned to the peptide V₃₄₁-R₃₄₆ N-linked to GnMXF and (c) dicharged ion at m/z 1296.54 assigned to the peptide V₃₄₁-R₃₄₆ N-linked to Le^aGnXF. Structures of fragment ions are depicted. Each N-glycan has been drawn according to the international nomenclature [22]. Blue square: GlcNAc; green circle: Man; red triangle: Fuc; yellow circle: Gal and yellow star: Xyl.

Figure 4

Distribution of main N-glycans on the 22 N-glycosylation consensus sites of the SARS-CoV-2 S protein produced in plants. Distribution of all N-glycans per glycosite are reported in Table S1. Blue square: GlcNAc; green circle: Man; red triangle: Fuc; yellow circle: Gal and yellow star: Xyl.