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Impact of mutations on the plant-based production of recombinant SARS-CoV-2 RBD

ABSTRACT: Recombinant RBD variants from SARS-CoV-2 spike were produced by transient expression in Nicotiana benthamiana plants and purified. The purified proteins were digested with proteases and glycopeptides were analysed by MS to identify the N-glycan structures.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Nicotiana Benthamiana

TISSUE(S): Plant Cell, Leaf

SUBMITTER: Richard Strasser

LAB HEAD: Richard Strasser

PROVIDER: PXD044462 | Pride | 2024-01-26

REPOSITORIES: Pride

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			Action	DRS
	1370_1371_1372_1373.dat	Other
	1657_1658.dat	Other
	1698_1699_1701_1702.dat	Other
	BA1FUT8_1701.raw	Raw
	BA1_1372.raw	Raw

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Impact of mutations on the plant-based production of recombinant SARS-CoV-2 RBDs.

Ruocco Valentina V Vavra Ulrike U König-Beihammer Julia J Bolaños Martínez Omayra C OC Kallolimath Somanath S Maresch Daniel D Grünwald-Gruber Clemens C Strasser Richard R

Frontiers in plant science 20231006

Subunit vaccines based on recombinant viral antigens are valuable interventions to fight existing and evolving viruses and can be produced at large-scale in plant-based expression systems. The recombinant viral antigens are often derived from glycosylated envelope proteins of the virus and glycosylation plays an important role for the immunogenicity by shielding protein epitopes. The receptor-binding domain (RBD) of the SARS-CoV-2 spike is a principal target for vaccine development and has been ...[more]

PMID: 37868317

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Implications of O-glycan modifications in the hinge region of a plant-produced SARS-CoV-2-IgA antibody on functionality

Project description:In this study, we carried out host engineering to generate a therapeutic glycoprotein largely devoid of plant-endogenous O-glycans for functional characterization. We generated four variants of a potentSARS-CoV-2 neutralizing antibody, COVA2-15 IgA1. The variants that differed in the number of modified proline residues and O-glycan compositions of their hinge region were assessed regarding their physicochemical properties and functionality.

2024-02-27 | PXD050139 | Pride

An oligosaccharyltransferase from Leishmania donovani increases the N-glycan occupancy on plant-produced IgG1

Project description:The monoclonal antibodies rituximab and cetuximab were transiently expressed in leaves of Nicotiana benthamiana plants and purified by affinity chromatography using protein A. Purified antibodies were analysed by intact MS. Purified antibodies were also subjected to trypsin digestion followed by LC-ESI-MS analysis of the glycopeptide carrying the N-glycsylation site Asn297 using the LC-OTMS, Orbitrap Exploris 480 from Thermo Scientific.

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In planta deglycosylation improves the SARS-CoV-2 neutralization activity of recombinant ACE2-Fc

Project description:The ectodomain of human ACE2-Fc was transiently expressed in leaves of Nicotiana benthamiana plants and protein A and SEC purified. SEC-purified ACE2-Fc was sequentially digested with chymotrypsin and trypsin and the glycopeptides were subjected to LC-ESI-MS analysis using the LC-OTMS, Orbitrap Exploris 480 from Thermo Scientific.

2023-04-18 | PXD041320 | Pride

Sequential Glycosylations at the Multibasic Cleavage Site of SARS-CoV-2 Spike Protein Regulate Viral Activation, Assembly, and Infection

Project description:The multibasic furin cleavage site at the S1/S2 boundary of the spike protein (S protein) is a hallmark of SARS-CoV-2 and plays an crucial role in viral infection. O-glycosylation near the furin site catalyzed by host cell glycosyltransferases can theoretically hinder spike protein processing and impede viral infection, but so far such a hypothesis has not been tested with authentic viruses. The mechanism underlying furin activation also remains poorly understood. In this study, we have discovered that GalNAc-T3 and T7 jointly initiate clustered O-glycosylations in the multibasic S1/S2 boundary region. These O-glycosylations inhibit furin processing of the spike protein and surprisingly suppress the incorporation of S protein into virus-like-particles (VLPs). Mechanistic analysis revealed that the assembly of spike protein into VLPs relies on protein-protein interaction between the furin-cleaved S protein and a double aspartic motif on the membrane protein of SARS-CoV-2, suggesting a novel mechanism for furin activation of the S protein. Interestingly, a point mutation at P681, found in the alpha and delta variants of SARS-CoV-2, confers resistance to glycosylation by GalNAc-T3 and T7, thereby diminishing the inhibitory effect against furin processing. However, an additional mutation at N679 in the most recent omicron variant reverses this resistance, rendering it susceptible to glycosylation in vitro and sensitive to the expression of GalNAc-T3 and T7 in human lung cells. Together, our findings suggest a glycosylation-based defense mechanism employed by host cells against SARS-CoV-2 and unveil the intricate interplay between the host and pathogen at this critical “battlefield” as the virus initially evades and currently succumbs to host cell glycosylation..

2024-03-21 | PXD049110 | Pride

Expression data from the subclones of Lewis Lung Carcinoma (LLC) cells

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Structure of the human heparan sulfate polymerase complex EXT1-EXT2

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2022-12-09 | PXD034871 | Pride

Effect of isoflavone agonists of IRF-3 on response to RNA viruses

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2012-05-17 | E-GEOD-38016 | biostudies-arrayexpress

Chlamydophila psittaci negative ocular adnexal marginal zone lymphomas express self polyreactive B cell receptors

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