SARS-CoV-2 Nucleocapsid protein is decorated with multiple N- and O-glycans

  1. Nitin T. Supekar
  2. Asif Shajahan
  3. Anne S. Gleinich
  4. Daniel Rouhani
  5. Christian Heiss
  6. Parastoo Azadi

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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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  1. ScreenIT

    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 Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    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.
    HEK293
    suggested: None
    Software and Algorithms
    SentencesResources
    Data analysis was performed using Byonic 2.3 software and manually using Xcalibur 4.2 and GlycoWorkbench 1.1.
    Xcalibur
    suggested: (Thermo Xcalibur, RRID:SCR_014593)
    The N protein expressed in HEK293 cells (Cat. No. NUN-C5227) was purchased from AcroBiosystems (Newark, DE).
    AcroBiosystems
    suggested: (ACRObiosystems, RRID:SCR_012550)
    Additional structural details were determined by ESI-MSn and analysis with GlycoWorkbench 1.1 software.
    GlycoWorkbench
    suggested: (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 Xcalibur
    suggested: (Thermo Xcalibur, RRID:SCR_014593)
    GlycoMod
    suggested: (GlycoMod, RRID:SCR_001602)
    ProteinProspector
    suggested: (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.

    About SciScore

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  2. Version published to 10.1101/2020.08.26.269043v1 on bioRxiv