Suppression of O-linked glycosylation of the SARS-CoV-2 spike by quaternary structural restraints
Suppression of O-linked glycosylation of the SARS-CoV-2 spike by quaternary structural restraints
Understanding the glycosylation of the envelope spike (S) protein of SARS-CoV-2 is important in defining the antigenic surface of this key viral target. However, the underlying protein architecture may significantly influence glycan occupancy and processing. There is, therefore, potential for different recombinant fragments of S protein to display divergent glycosylation. Here, we show that the receptor binding domain (RBD), when expressed as a monomer, exhibits O-linked glycosylation, which is not recapitulated in the native-like soluble trimeric protein. We unambiguously assign O-linked glycosylation by homogenizing N-linked glycosylation using the enzymatic inhibitor, kifunensine, and then analyzing the resulting structures by electron-transfer higher-energy collision dissociation (EThcD) in an Orbitrap Eclipse Tribrid instrument. In the native-like trimer, we observe a single unambiguous O-linked glycan at T323, which displays very low occupancy. In contrast, several sites of O-linked glycosylation can be identified when RBD is expressed as a monomer, with T323 being almost completely occupied. We ascribe this effect to the relaxation of steric restraints arising from quaternary protein architecture. Our analytical approach has also highlighted that fragmentation ions arising from trace levels of truncated N-linked glycans can be misassigned as proximal putative O-linked glycan structures, particularly where a paucity of diagnostic fragments were obtained. Overall, we show that in matched expression systems the quaternary protein architecture limits O-linked glycosylation of the spike protein.
COVID-19, Glycosylation, Humans, Polysaccharides, Protein Binding, SARS-CoV-2, Spike Glycoprotein, Coronavirus/genetics
14392-14400
Eldrid, Charles F S
caf78c85-1eae-4700-814a-01b5f0635a0b
Allen, Joel D
c89d5569-7659-4835-b535-c9586e956b3a
Newby, Maddy L
750f2bf7-92fe-45ee-afdc-95d06dbaff18
Crispin, Max
cd980957-0943-4b89-b2b2-710f01f33bc9
2 November 2021
Eldrid, Charles F S
caf78c85-1eae-4700-814a-01b5f0635a0b
Allen, Joel D
c89d5569-7659-4835-b535-c9586e956b3a
Newby, Maddy L
750f2bf7-92fe-45ee-afdc-95d06dbaff18
Crispin, Max
cd980957-0943-4b89-b2b2-710f01f33bc9
Eldrid, Charles F S, Allen, Joel D, Newby, Maddy L and Crispin, Max
(2021)
Suppression of O-linked glycosylation of the SARS-CoV-2 spike by quaternary structural restraints.
Analytical Chemistry, 93 (43), .
(doi:10.1021/acs.analchem.1c01772).
Abstract
Understanding the glycosylation of the envelope spike (S) protein of SARS-CoV-2 is important in defining the antigenic surface of this key viral target. However, the underlying protein architecture may significantly influence glycan occupancy and processing. There is, therefore, potential for different recombinant fragments of S protein to display divergent glycosylation. Here, we show that the receptor binding domain (RBD), when expressed as a monomer, exhibits O-linked glycosylation, which is not recapitulated in the native-like soluble trimeric protein. We unambiguously assign O-linked glycosylation by homogenizing N-linked glycosylation using the enzymatic inhibitor, kifunensine, and then analyzing the resulting structures by electron-transfer higher-energy collision dissociation (EThcD) in an Orbitrap Eclipse Tribrid instrument. In the native-like trimer, we observe a single unambiguous O-linked glycan at T323, which displays very low occupancy. In contrast, several sites of O-linked glycosylation can be identified when RBD is expressed as a monomer, with T323 being almost completely occupied. We ascribe this effect to the relaxation of steric restraints arising from quaternary protein architecture. Our analytical approach has also highlighted that fragmentation ions arising from trace levels of truncated N-linked glycans can be misassigned as proximal putative O-linked glycan structures, particularly where a paucity of diagnostic fragments were obtained. Overall, we show that in matched expression systems the quaternary protein architecture limits O-linked glycosylation of the spike protein.
Text
acs.analchem.1c01772
- Version of Record
More information
e-pub ahead of print date: 20 October 2021
Published date: 2 November 2021
Keywords:
COVID-19, Glycosylation, Humans, Polysaccharides, Protein Binding, SARS-CoV-2, Spike Glycoprotein, Coronavirus/genetics
Identifiers
Local EPrints ID: 452212
URI: http://eprints.soton.ac.uk/id/eprint/452212
ISSN: 0003-2700
PURE UUID: b43748fb-9445-4379-8f4f-d259c703d3a9
Catalogue record
Date deposited: 30 Nov 2021 17:31
Last modified: 17 Mar 2024 04:09
Export record
Altmetrics
Download statistics
Downloads from ePrints over the past year. Other digital versions may also be available to download e.g. from the publisher's website.
View more statistics
