N- and O-Glycosylation of the SARS-CoV-2 Spike Protein

Šanda, Miloslav; Morrison, Lindsay; Goldman, Radoslav

doi:10.1021/acs.analchem.0c03173

Cited by 185 publications

(268 citation statements)

References 22 publications

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“…Regardless, low occupancy of O-glycosites close to N-glycosites fits well with published data on S N-glycosylation. The 22 SARS-CoV-2 S N-glycosites are reported to be more than 95 % occupied in recent glycoproteomic analyses of SARS-CoV-2 S [13][14][15][16][17]. Based on our data, it is unlikely, that both N-and O-glycans exist in close vicinity to each other, as we found such O-glycosites almost exclusively on peptides with unoccupied N-X-S/T sequons.…”

Section: Discussionsupporting

confidence: 54%

“…We report 25 unique O-glycosites, most of which were identified unambiguously. This includes the previously identified T323 and T678 adjacent to S1/S2 cleavage site [14][15][16][17].…”

Section: Discussionmentioning

confidence: 99%

“…A number of different protein formulations and expression systems have been used to deduce structure and post-translational modifications (PTMs) of SARS-CoV-2 spike protein (S), and some of those have been explored as candidates in vaccine research. So far, 22 highly occupied N-linked glycosylation sites have been identified on protein S, as well as a variable number of O-linked glycosylation sites at low stoichiometries [13][14][15][16][17][18].…”

Section: Introductionmentioning

confidence: 99%

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

Bagdonaite

Thompson

Wang

et al. 2021

Viruses

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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 is 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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Section: Discussionsupporting

confidence: 54%

“…We report 25 unique O-glycosites, most of which were identified unambiguously. This includes the previously identified T323 and T678 adjacent to S1/S2 cleavage site [14][15][16][17].…”

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Bagdonaite

Thompson

Wang

et al. 2021

Viruses

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“…that the spike protein's LacdiNAc structural motifs and polyLacNAc structures (Sanda et al, 2021). The spike glycoprotein (S1) interacts with host cell epithelial angiotensin-converting enzyme 2 (ACE-2) receptors (ACE2).…”

Section: Mechanism Of Sars-cov-2 Pathogenesismentioning

confidence: 99%

SARS-CoV-2: Pathogenesis, Molecular Targets and Experimental Models

Kanimozhi¹,

Pradhapsingh

Pawar

et al. 2021

Front. Pharmacol.

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The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a recent pandemic outbreak threatening human beings worldwide. This novel coronavirus disease-19 (COVID-19) infection causes severe morbidity and mortality and rapidly spreading across the countries. Therefore, there is an urgent need for basic fundamental research to understand the pathogenesis and druggable molecular targets of SARS-CoV-2. Recent sequencing data of the viral genome and X-ray crystallographic data of the viral proteins illustrate potential molecular targets that need to be investigated for structure-based drug design. Further, the SARS-CoV-2 viral pathogen isolated from clinical samples needs to be cultivated and titrated. All of these scenarios demand suitable laboratory experimental models. The experimental models should mimic the viral life cycle as it happens in the human lung epithelial cells. Recently, researchers employing primary human lung epithelial cells, intestinal epithelial cells, experimental cell lines like Vero cells, CaCo-2 cells, HEK-293, H1299, Calu-3 for understanding viral titer values. The human iPSC-derived lung organoids, small intestinal organoids, and blood vessel organoids increase interest among researchers to understand SARS-CoV-2 biology and treatment outcome. The SARS-CoV-2 enters the human lung epithelial cells using viral Spike (S1) protein and human angiotensin-converting enzyme 2 (ACE-2) receptor. The laboratory mouse show poor ACE-2 expression and thereby inefficient SARS-CoV-2 infection. Therefore, there was an urgent need to develop transgenic hACE-2 mouse models to understand antiviral agents’ therapeutic outcomes. This review highlighted the viral pathogenesis, potential druggable molecular targets, and suitable experimental models for basic fundamental research.

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“…for example, deletions of the N-glycosites on N331 and N343 could drastically reduce the viral infectivity (Li et al, 2020b). The O-glycosylation of S protein was also previously characterized using mass spectrometry (MS) analysis (Sanda et al, 2021;Shajahan et al, 2020;Watanabe et al, 2020;Zhang et al, 2020;Zhao et al, 2020a).…”

Section: Introductionmentioning

confidence: 99%

Altered O-glycosylation Level of SARS-CoV-2 Spike Protein by Host O-glycosyltransferase Strengthens Its Trimeric Structure

Tian

et al. 2021

Preprint

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SummaryThe trimeric spike protein (S) mediates host-cell entry and membrane fusion of SARS-CoV-2. S protein is highly glycosylated, whereas its O-glycosylation is still poorly understood. Herein, we site-specifically examine the O-glycosylation of S protein through a mass spectrometric approach with HCD-triggered-ETD model. We identify 15 high-confidence O-glycosites and at least 10 distinct O-glycan structures on S protein. Peptide microarray assays prove that human ppGalNAc-T6 actively participates in O-glycosylation of S protein. Importantly, the upregulation of ppGalNAc-T6 expression can profoundly enhance the O-glycosylation level by generating new O-glycosites and increasing both O-glycan heterogeneity and intensities. Further molecular dynamics simulations reveal that the O-glycosylation on the protomer-interface regions, which are mainly modified by ppGalNAc-T6, can potentially stabilize the trimeric S protein structure. Our work provides deep molecular insights of how viral infection harnesses the host O-glycosyltransferases to dynamically regulate the O-glycosylation level of the viral envelope protein responsible for membrane fusion.

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N- and O-Glycosylation of the SARS-CoV-2 Spike Protein

Cited by 185 publications

References 22 publications

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

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

SARS-CoV-2: Pathogenesis, Molecular Targets and Experimental Models

Altered O-glycosylation Level of SARS-CoV-2 Spike Protein by Host O-glycosyltransferase Strengthens Its Trimeric Structure

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