期刊
ANALYTICAL CHEMISTRY
卷 93, 期 43, 页码 14392-14400出版社
AMER CHEMICAL SOC
DOI: 10.1021/acs.analchem.1c01772
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资金
- International AIDS Vaccine Initiative (IAVI) [INV-008352/OPP1153692]
- IAVI Neutralizing Antibody Center through the Collaboration for AIDS Vaccine Discovery [OPP1196345/INV-008813]
- Bill and Melinda Gates Foundation
- Bright Future Trust
- University of Southampton Coronavirus Response Fund
Understanding the glycosylation of the SARS-CoV-2 envelope spike protein is crucial in determining the antigenic surface of this viral target. Different recombinant fragments of the S protein may exhibit varying glycosylation patterns. The quaternary protein architecture in matched expression systems has been shown to limit O-linked glycosylation of the spike protein.
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. Her; 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.
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