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Site-specific glycan analysis of the SARS-CoV-2 spike.

ABSTRACT: The emergence of the betacoronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), represents a considerable threat to global human health. Vaccine development is focused on the principal target of the humoral immune response, the spike (S) glycoprotein, which mediates cell entry and membrane fusion. The SARS-CoV-2 S gene encodes 22 N-linked glycan sequons per protomer, which likely play a role in protein folding and immune evasion. Here, using a site-specific mass spectrometric approach, we reveal the glycan structures on a recombinant SARS-CoV-2 S immunogen. This analysis enables mapping of the glycan-processing states across the trimeric viral spike. We show how SARS-CoV-2 S glycans differ from typical host glycan processing, which may have implications in viral pathobiology and vaccine design.

SUBMITTER: Watanabe Y

PROVIDER: S-EPMC7199903 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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Site-specific glycan analysis of the SARS-CoV-2 spike.

Watanabe Yasunori Y Allen Joel D JD Wrapp Daniel D McLellan Jason S JS Crispin Max M

Science (New York, N.Y.) 20200504 6501

The emergence of the betacoronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), represents a considerable threat to global human health. Vaccine development is focused on the principal target of the humoral immune response, the spike (S) glycoprotein, which mediates cell entry and membrane fusion. The SARS-CoV-2 S gene encodes 22 N-linked glycan sequons per protomer, which likely play a role in protein folding and im ...[more]

PMID: 32366695

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Site-specific analysis of the SARS-CoV-2 glycan shield.

Project description:The emergence of the betacoronavirus, SARS-CoV-2 that causes COVID-19, represents a significant threat to global human health. Vaccine development is focused on the principal target of the humoral immune response, the spike (S) glycoprotein, that mediates cell entry and membrane fusion. SARS-CoV-2 S gene encodes 22 N-linked glycan sequons per protomer, which likely play a role in immune evasion and occluding immunogenic protein epitopes. Here, using a site-specific mass spectrometric approach, we reveal the glycan structures on a recombinant SARS-CoV-2 S immunogen. This analysis enables mapping of the glycan-processing states across the trimeric viral spike. We show how SARS-CoV-2 S glycans differ from typical host glycan processing, which may have implications in viral pathobiology and vaccine design.

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Site-specific N-glycosylation Characterization of Recombinant SARS-CoV-2 Spike Proteins.

Project description:The glycoprotein spike (S) on the surface of severe acute respiratory syndrome coronavirus (SARS-CoV-2) is a determinant for viral invasion and host immune response. Herein, we characterized the site-specific N-glycosylation of S protein at the level of intact glycopeptides. All 22 potential N-glycosites were identified in the S-protein protomer and were found to be preserved among the 753 SARS-CoV-2 genome sequences. The glycosites exhibited glycoform heterogeneity as expected for a human cell-expressed protein subunit. We identified masses that correspond to 157 N-glycans, primarily of the complex type. In contrast, the insect cell-expressed S protein contained 38 N-glycans, completely of the high-mannose type. Our results revealed that the glycan types were highly determined by the differential processing of N-glycans among human and insect cells, regardless of the glycosites' location. Moreover, the N-glycan compositions were conserved among different sizes of subunits. Our study indicates that the S protein N-glycosylation occurs regularly at each site, albeit the occupied N-glycans were diverse and heterogenous. This N-glycosylation landscape and the differential N-glycan patterns among distinct host cells are expected to shed light on the infection mechanism and present a positive view for the development of vaccines and targeted drugs.

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Analysis of the SARS-CoV-2 spike protein glycan shield: implications for immune recognition.

Project description:Here we have generated 3D structures of glycoforms of the spike (S) glycoprotein from SARS-CoV-2, based on reported 3D structures and glycomics data for the protein produced in HEK293 cells. We also analyze structures for glycoforms representing those present in the nascent glycoproteins (prior to enzymatic modifications in the Golgi), as well as those that are commonly observed on antigens present in other viruses. These models were subjected to molecular dynamics (MD) simulation to determine the extent to which glycan microheterogeneity impacts the antigenicity of the S glycoprotein. Lastly, we have identified peptides in the S glycoprotein that are likely to be presented in human leukocyte antigen (HLA) complexes, and discuss the role of S protein glycosylation in potentially modulating the adaptive immune response to the SARS-CoV-2 virus or to a related vaccine. The 3D structures show that the protein surface is extensively shielded from antibody recognition by glycans, with the exception of the ACE2 receptor binding domain, and also that the degree of shielding is largely insensitive to the specific glycoform. Despite the relatively modest contribution of the glycans to the total molecular weight (17% for the HEK293 glycoform) the level of surface shielding is disproportionately high at 42%.

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Site-specific O-glycosylation analysis of SARS-CoV-2 spike protein produced in insect and human cells.

Project description:Enveloped viruses hijack not only the host translation processes, but also its glycosylation machinery, and to a variable extent cover viral surface proteins with tolerogenic host-like structures. SARS-CoV-2 surface protein S presents as a trimer on the viral surface and is covered by a dense shield of N-linked glycans, and a few O-glycosites have been reported. The location of O-glycans is controlled by a large family of initiating enzymes with variable expression in cells and tissues and hence difficult to predict. Here, we used our well-established O-glycoproteomic workflows to map the precise positions of O-linked glycosylation sites on three different entities of protein S - insect cell or human cell-produced ectodomains, or insect cell derived receptor binding domain (RBD). In total 25 O-glycosites were identified, with similar patterns in the two ectodomains of different cell origin, and a distinct pattern of the monomeric RBD. Strikingly, 16 out of 25 O-glycosites were located within three amino acids from known N-glycosites. However, O-glycosylation was primarily found on peptides that were unoccupied by N-glycans, and otherwise had low overall occupancy. This suggests possible complementary functions of O-glycans in immune shielding and negligible effects of O-glycosylation on subunit vaccine design for SARS-CoV-2.

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Site-Specific O-Glycosylation Analysis of SARS-CoV-2 Spike Protein Produced in Insect and Human Cells.

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