SARS-CoV-2 Nucleocapsid protein is decorated with multiple N- and O-glycans
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Abstract
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease (COVID-19) started at the end of 2019 in Wuhan, China has spread rapidly and became a pandemic. Since there is no therapy available that is proven as fully protective against COVID-19, a vaccine to protect against deadly COVID-19 is urgently needed. Nucleocapsid protein (N protein), is one of the most abundant proteins in coronaviruses and is a potential target for both vaccine development and point of care diagnostics. The variable mass of N protein (45 to 60 kDa), suggests the presence of post-translational modifications (PTMs), and it is critical to clearly define these PTMs to gain the structural understanding necessary for further vaccine research. There have been several reports suggesting that the N protein is phosphorylated but lacks glycosylation. Our comprehensive glycomics and glycoproteomics experiments confirm that the N protein is highly O-glycosylated and also contains significant levels of N-glycosylation. We were able to confirm the presence of O-glycans on seven sites with substantial glycan occupancy, in addition to less abundant O-glycans on four sites. We also detected N-glycans on two out of five potential N-glycosylation sites. Moreover, we were able to confirm one phosphorylation site. Recent studies have indicated that the N protein can serve as an important diagnostic marker for coronavirus disease and a major immunogen by priming protective immune responses. Thus, detailed structural characterization of the N protein may provide useful insights for understanding the roles of glycosylation on viral pathogenesis and also in vaccine design and development.
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SciScore for 10.1101/2020.08.26.269043: (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
Experimental Models: Cell Lines Sentences Resources Protease digestion of N protein for glycoproteomics: The purified N protein (20 μg) expressed on HEK293 cells was dissolved in 50 mM ammonium bicarbonate solution and digested with trypsin and elastase separately as well as sequentially by incubating for 1 h at 37 °C. HEK293suggested: NoneSoftware and Algorithms Sentences Resources Data analysis was performed using Byonic 2.3 software and manually using Xcalibur 4.2 and GlycoWorkbench 1.1. …SciScore for 10.1101/2020.08.26.269043: (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
Experimental Models: Cell Lines Sentences Resources Protease digestion of N protein for glycoproteomics: The purified N protein (20 μg) expressed on HEK293 cells was dissolved in 50 mM ammonium bicarbonate solution and digested with trypsin and elastase separately as well as sequentially by incubating for 1 h at 37 °C. HEK293suggested: NoneSoftware and Algorithms Sentences Resources Data analysis was performed using Byonic 2.3 software and manually using Xcalibur 4.2 and GlycoWorkbench 1.1. Xcalibursuggested: (Thermo Xcalibur, RRID:SCR_014593)The N protein expressed in HEK293 cells (Cat. No. NUN-C5227) was purchased from AcroBiosystems (Newark, DE). AcroBiosystemssuggested: (ACRObiosystems, RRID:SCR_012550)Additional structural details were determined by ESI-MSn and analysis with GlycoWorkbench 1.1 software. GlycoWorkbenchsuggested: (GlycoWorkbench, RRID:SCR_000782)The LC-MS/MS spectra were also analyzed manually for the glycopeptides with the support of the Thermo Fisher Xcalibur 4.2 software, GlycoMod tool, and ProteinProspector v6.2.1. Thermo Fisher Xcalibursuggested: (Thermo Xcalibur, RRID:SCR_014593)GlycoModsuggested: (GlycoMod, RRID:SCR_001602)ProteinProspectorsuggested: (Protein Prospector, RRID:SCR_014558)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 did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
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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