Altered O-glycosylation Level of SARS-CoV-2 Spike Protein by Host O-glycosyltransferase Strengthens Its Trimeric Structure
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Abstract
The 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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SciScore for 10.1101/2021.04.06.438614: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources , acetonitrile (ACN), ammonium bicarbonate (NH4HCO3), anti-FALG antibody, anti-FLAG M2 affinity gel, and neuraminidase were purchased from Sigma-Aldrich (St. Louis, MO, USA). anti-FALGsuggested: Noneanti-FLAGsuggested: Noneneuraminidasesuggested: NoneExperimental Models: Cell Lines Sentences Resources The SARS-CoV-2 S protein subunit 1 (His tag) expressed by HEK-293 cells were purchased from Sanyoubio (Shanghai, HEK-293suggested: CLS Cat# 300192/p777_HEK293, …SciScore for 10.1101/2021.04.06.438614: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources , acetonitrile (ACN), ammonium bicarbonate (NH4HCO3), anti-FALG antibody, anti-FLAG M2 affinity gel, and neuraminidase were purchased from Sigma-Aldrich (St. Louis, MO, USA). anti-FALGsuggested: Noneanti-FLAGsuggested: Noneneuraminidasesuggested: NoneExperimental Models: Cell Lines Sentences Resources The SARS-CoV-2 S protein subunit 1 (His tag) expressed by HEK-293 cells were purchased from Sanyoubio (Shanghai, HEK-293suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)The glycosites and glycoforms of S protein purified from HEK-293T cells were confirmed according to the mass spectrometry results of commercial S1 using a ‘match between runs’ analysis. HEK-293Tsuggested: CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063)Software and Algorithms Sentences Resources Mass spectrometric data analysis: The LC-MS/MS raw data were analyzed manually for each glycopeptide using Xcalibur 3.0.63 (Thermo Fisher Scientific). Xcalibursuggested: (Thermo Xcalibur, RRID:SCR_014593)All the MD simulations were performed using the Amber14 package(Case et al., 2014). Amber14suggested: (Assisted Model Building with Energy Refinement (AMBER, RRID:SCR_014230)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).
Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on page 29. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
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