N-glycosites and O-glycosites on Spike glycoprotein of SARS-CoV-2
Ontology highlight
ABSTRACT:
ORGANISM(S): Severe Acute Respiratory Syndrome Coronavirus 2
SUBMITTER: 
Dapeng Zhou
PROVIDER: PXD019282 | iProX | Tue May 19 00:00:00 BST 2020
REPOSITORIES: iProX
Similar Datasets
Project description:In this work, we performed a fully descriptive analysis N- and O- linked glycosylation of SARS-COV-2 S glycoprotein. We investigated that dual-functionalized Ti-IMAC material enable the simultaneous enrichment and separation of neutral and sialyl glycopeptides of a recombinant SARS-CoV-2 S glycoprotein from HEK293, which will eliminate the signal suppression of neutral glycopeptides to sialyl glycopeptides and improve the glycoform coverage of S protein. We have profiled 19 of its 22 potential N-glycosylated sites with 398 unique glycoforms in dual-functional Ti-IMIAC approach that is 1.6-fold of that in conventional HILIC method. We also identified O-linked glycosylation site that was not found in dual-functional Ti-IMIAC approach. In addition, we have also identified mannose-6-phosphate (M6P) glycosylation, which substantially expands the current knowledge of the spike protein’s glycosylation and enables the investigation of the influence of mannose-6-Phosphate on its cell entry.
2022-01-11 | PXD026450 | Pride
Project description:The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus utilizes an extensively glycosylated spike protein that protrudes from the viral envelope to bind to angiotensin-converting enzyme-related carboxypeptidase (ACE2) as its primary receptor to mediate host-cell entry. Currently, the predominant recombinant spike protein production hosts are Chinese hamster ovary (CHO) and human embryonic kidney (HEK) cells. In this study, recombinant full-size spike protein was produced transiently in CHO and HEK cell suspensions. To further evaluate the sialic acid linkages presenting on spike glycans, a two-step amidation process, employing dimethylamine and ammonium hydroxide reactions in a solid support system, was developed to differentially modify the sialic acid linkages on glycans and glycopeptides from spike protein. We determined global and site-specific N-linked glycosylation patterns in soluble SARS-CoV-2 spike using MALDI-TOF and LC-MS/MS with electron-transfer/higher-energy collision dissociation (EThcD) fragmentation. We identified the glycan compositions at 21 and 19 out of the 22 predicted N-glycosylation sites of the SARS-CoV-2 spike proteins produced in CHO and HEK, respectively. The N-glycan site at 1158 position (N1158) and the N-glycan sites at 122 ,282 and 1158 positions (N122, N282 and N1158) were found to be unoccupied on spike secreted from CHO and HEK cells, respectively. The structural mapping of glycans of recombinant human spike proteins revealed that CHO-Spike presented more complex and higher sialylation (α2,3-linked) content while HEK-Spike displayed more high-mannose and minor content of α2,3- and α2,6-linked sialic acids. The N74 site represents the most heavily N-glycosylated site on both spike proteins while some high-mannose abundant sites (N17, N234, N343, N616, N709, N717, N801 and N1134) on HEK-Spike may differentially shield the virus compared to CHO-spike and provide a different host immune system interaction strategy. Collectively, these data underscore the importance of characterizing site-specific glycosylation on recombinant human spike protein from HEK and CHO cells in order to better understand the impact of the production host on this complex and important protein used in diagnostics and vaccines.
2022-02-17 | PXD027536 | Pride
Project description:Coronaviruses (CoVs) are enveloped pathogens causing multiple respiratory disorders in humans with varying severity. Spike protein is one of the major proteins expressed on coronavirus surface, which mediates coronavirus entry into host cells. Spike proteins are extensively glycosylated and the glycans displayed on spike proteins play a key role in host pathogenesis and immune evasion. In this study, we aim to investigate whether glycosylation patterns are conservative at certain glycosites across different coronaviruses and how different host cells impact on the glycosylation profile. We analyzed site-specific glycans of S1 subunit from SARS-CoV and MERS-CoV spike proteins using hydrophilic interaction chromatography (HILIC) and LC-MS/MS on an Orbitrap Eclipse Tribrid mass spectrometer. We also compared glycosylation of MERS-CoV spike protein derived from HEK293 and insect cells. Our results show that SARS-CoV S1 and MERS-CoV S1 N-glycosylation presents some common patterns and also reveals the similar O-glycosites locations. Consistent with published data, confirming glycan and glycan subtype in specific positions, our data support that some monoclonal antibodies recognize glycan as part of their target epitope and cross react between SARS Cov and SARS CoV2 spike. The coronavirus spike proteins are highly glycosylated. The glycosylation sites, within each virus, are conserved with few changes over time.
2025-04-07 | PXD062150 | Pride
Project description:mRNA vaccination of individuals with prior SARS-CoV-2 infection provides superior protection against breakthrough infections with variants of concern compared to vaccination in the absence of prior infection. However, the immune mechanisms by which this ‘hybrid immunity’ is generated and maintained are unknown. While genetic variation in spike glycoprotein effectively subverts neutralizing antibodies, spike-specific T cells are generally maintained against SARS-CoV-2 variants. Thus, we comprehensively profiled T cell responses against the S1 and S2 domains of spike glycoprotein in a cohort of SARS-CoV-2-naive or convalescent individuals who received two-dose mRNA vaccine series and were matched by age, sex, and vaccine type. Using flow cytometry, we observed that the overall functional breadth of CD4 T cells as well as polyfunctional Th1 responses were similar between the two groups. However, polyfunctional cytotoxic CD4 T cell responses against both S1 and S2 domains trended higher among convalescent subjects. Multi-modal single-cell RNA sequencing revealed diverse functional programs in spike-specific CD4 and CD8 T cells in both groups. However, convalescent individuals displayed enhanced cytotoxic and antiviral CD8 T cell responses to both S1 and S2 in the absence of cytokine production. Taken together, our data suggest that cytotoxic CD4 and CD8 T cells targeting spike glycoprotein may partially account for hybrid immunity and protection against breakthrough infections with SARS-CoV-2.
2023-03-01 | GSE223236 | GEO
Project description:The spike protein of SARS-CoV-2, the virus responsible for the global pandemic of COVID-19, is an abundant, heavily glycosylated surface protein that plays a key role in receptor binding and host cell fusion, and is the focus of all current vaccine development efforts. Variants of concern are now circulating worldwide that exhibit mutations in the spike protein. Protein sequence and glycosylation variations of the spike may affect viral fitness, antigenicity, and immune evasion. Global surveillance of the virus currently involves genome sequencing, but tracking emerging variants should include quantitative measurement of changes in site-specific glycosylation as well. In this work, we used data-dependent acquisition (DDA) and data-independent acquisition (DIA) mass spectrometry to quantitatively characterize the five N-linked glycosylation sites of the glycoprotein standard alpha-1-acid glycoprotein (AGP), as well as the 22 sites of SARS-CoV-2 spike protein. We found that DIA compared favorably to DDA in sensitivity, resulting in more assignments of low abundance glycopeptides. However, the reproducibility across replicates of DIA-identified glycopeptides was lower than that of DDA, possibly due to the difficulty of reliably assigning low abundance glycopeptides confidently. The differences in the data acquired between the two methods suggest that DIA out-performs DDA in terms of glycoprotein coverage but that overall performance is a balance of sensitivity, selectivity, and statistical confidence in glycoproteomics. We assert that these analytical and bioinformatics methods for assigning and quantifying glycoforms would benefit the process of tracking viral variants as well as for vaccine development.
2022-09-12 | PXD027279 | Pride
Project description:The spike protein of SARS-CoV-2, the virus responsible for the global pandemic of COVID-19, is an abundant, heavily glycosylated surface protein that plays a key role in receptor binding and host cell fusion, and is the focus of all current vaccine development efforts. Variants of concern are now circulating worldwide that exhibit mutations in the spike protein. Protein sequence and glycosylation variations of the spike may affect viral fitness, antigenicity, and immune evasion. Global surveillance of the virus currently involves genome sequencing, but tracking emerging variants should include quantitative measurement of changes in site-specific glycosylation as well. In this work, we used data-dependent acquisition (DDA) and data-independent acquisition (DIA) mass spectrometry to quantitatively characterize the five N-linked glycosylation sites of the glycoprotein standard alpha-1-acid glycoprotein (AGP), as well as the 22 sites of SARS-CoV-2 spike protein. We found that DIA compared favorably to DDA in sensitivity, resulting in more assignments of low abundance glycopeptides. However, the reproducibility across replicates of DIA-identified glycopeptides was lower than that of DDA, possibly due to the difficulty of reliably assigning low abundance glycopeptides confidently. The differences in the data acquired between the two methods suggest that DIA out-performs DDA in terms of glycoprotein coverage but that overall performance is a balance of sensitivity, selectivity, and statistical confidence in glycoproteomics. We assert that these analytical and bioinformatics methods for assigning and quantifying glycoforms would benefit the process of tracking viral variants as well as for vaccine development.
2022-02-12 | PXD027425 | Pride
Project description:This experiment aims to profile polyclonal antibody binding profiles in serum from vaccinated animals relative to antibody function in a virus neutralization assay. Rabbits received three vaccinations with a DNA vaccine encoding the spike protein of the SARS-CoV-2 index strain. Serum samples were selected based on a three-tier (low, intermediate, and high) capacity to cross-neutralize SARS-CoV-2 strains with known neutralization resistance. Following normalization of total anti-spike IgG levels, serum of each animal (n=3) were evaluated for antibody binding to 10mer cyclic constrained peptides spanning the entire spike protein and regions with known SARS-CoV-2 variant of concern spike mutations.
2023-08-31 | E-MTAB-13040 | biostudies-arrayexpress
Project description:The SARS-CoV-2 spike protein mediates attachment of the virus to host cell receptor and fusion between the virus and cell membrane. The S1 subunit of the spike glycoprotein (S1 protein) contains the angiotensin converting enzyme 2 (ACE2) receptor binding domain. The SARS-CoV-2 variants of concern contain mutations in the S1 subunit. The spike protein is the primary target of neutralizing antibodies generated following infection and constitutes the viral component of mRNA based COVID-19 vaccines. Therefore, in this work we assessed the effect of exposure (24 hours) of 10 nM SARS-CoV-2 recombinant S1 protein on physiologically relevant human bronchial (bro) and alveolar (alv) lung mucosa models cultured at air-liquid interface (ALI) (n=6/experimental group). Corresponding sham exposed samples served as control. The bro-ALI model was developed using primary bronchial epithelial cells and the alv-ALI model using representative type II pneumocytes. Exposure of S1 protein induced the surface expression of ACE2, toll like receptor (TLR) 2, and TLR4 in both bro-ALI and alv-ALI models. Transcript expression analysis identified 117 (bro-ALI) and 97 (alv-ALI) differentially regulated genes (p< 0.01). Pathway analysis revealed enriched terms as specific as COVID-19, interferon (IFN) signaling, influenza, Corona virus, anti-viral response in the bro-ALI. Secreted levels of Interleukin (IL) 4 and IL12 were significantly (p<0.05; non-parametric) increased whereas IL6 decreased in the bro-ALI. In case of alv-ALI, enriched terms involving p53, APRIL, tight junction, integrin kinase, IL1 signaling were identified. These terms are associated with lung fibrosis. Further, significantly (p<0.05; non-parametric) increased levels of secreted pro-inflammatory cytokines IFNγ, IL1ꞵ, IL2, IL4, IL6, IL8, IL10, IL12, IL13, and tumor necrosis factor alpha were detected in alv-ALI. Altered levels of these cytokines are also associated with lung fibrotic response. Taken together, we observed a typical anti-viral response in the bronchial model whereas a pro-fibrotic response in the alveolar model. Therefore the bro-ALI and alv-ALI models may serve as an easy and robust platform for assessing the pathogenicity of variants of concern at different lung regions.
2021-12-13 | GSE185657 | GEO
Project description:A central tenet in the design of vaccines is the display of native-like antigens in the elicitation of protective immunity. The abundance of N-linked glycans across the SARS-CoV-2 spike protein is a potential source of heterogeneity between the many different vaccine candidates under investigation. Here, we investigate the glycosylation of recombinant SARS-CoV-2 spike proteins from five different laboratories and compare them against infectious virus S protein. We find patterns which are conserved across all samples and this can be associated with site-specific stalling of glycan maturation which act as a highly sensitive reporter of protein structure. Molecular dynamics (MD) simulations of a fully glycosylated spike support s a model of steric restrictions that shape enzymatic processing of the glycans. These results suggest that recombinant spike-based SARS-CoV-2 immunogen glycosylation reproducibly recapitulates signatures of viral glycosylation.
https://doi.org/10.1101/2021.03.08.433764
This folder contains the RAW MS files used in the glycopeptide analysis for recombinant SARS proteins from a range of different labs outlined in Figure 1 and 2 and additionally the analysis performed on monomeric RBD
2021-04-27 | MSV000087308 | MassIVE
Project description:We here identified that the trimeric spike protein of SARS-CoV-2 could bind to TLR4 directly and robustly activate downstream signaling in monocytes and neutrophils. Moreover, specific TLR4 or NFKB inhibitor, or knockout of MyD88 could significantly block IL-1B induction by spike protein. We thus reveal that spike protein of SARS-CoV-2 functions as a potent stimulus causing TLR4 activation and sepsis related abnormal responses.
2021-02-25 | GSE167480 | GEO