The University of Southampton
University of Southampton Institutional Repository
Warning ePrints Soton is experiencing an issue with some file downloads not being available. We are working hard to fix this. Please bear with us.

MassIVE MSV000087897

MassIVE MSV000087897
MassIVE MSV000087897
O-linked Glycosylation of the SARS-CoV-2 Spike is Suppressed by Quaternary Structural Restraints,Understanding the glycosylation of 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 structures, particularly where a paucity of diagnostic fragments were obtained. Overall, we and show that in matched expression systems quaternary protein architecture limits O-linked glycosylation of the spike protein.,
University of Southampton
Crispin, Max
cd980957-0943-4b89-b2b2-710f01f33bc9
Crispin, Max
cd980957-0943-4b89-b2b2-710f01f33bc9

(2021) MassIVE MSV000087897. University of Southampton doi:10.25345/c5425b [Dataset]

Record type: Dataset

Abstract

O-linked Glycosylation of the SARS-CoV-2 Spike is Suppressed by Quaternary Structural Restraints,Understanding the glycosylation of 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 structures, particularly where a paucity of diagnostic fragments were obtained. Overall, we and show that in matched expression systems quaternary protein architecture limits O-linked glycosylation of the spike protein.,

This record has no associated files available for download.

More information

Published date: 1 January 2021

Identifiers

Local EPrints ID: 451643
URI: http://eprints.soton.ac.uk/id/eprint/451643
PURE UUID: 6a834bd3-c350-47bd-a480-107ccc15773b
ORCID for Max Crispin: ORCID iD orcid.org/0000-0002-1072-2694

Catalogue record

Date deposited: 18 Oct 2021 16:30
Last modified: 19 Oct 2021 01:52

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

Atom RSS 1.0 RSS 2.0

Contact ePrints Soton: eprints@soton.ac.uk

ePrints Soton supports OAI 2.0 with a base URL of http://eprints.soton.ac.uk/cgi/oai2

This repository has been built using EPrints software, developed at the University of Southampton, but available to everyone to use.

We use cookies to ensure that we give you the best experience on our website. If you continue without changing your settings, we will assume that you are happy to receive cookies on the University of Southampton website.

×