Multimerization- and glycosylation-dependent receptor binding of SARS-CoV-2 spike proteins

  1. Kim M. Bouwman
  2. Ilhan Tomris
  3. Hannah L. Turner
  4. Roosmarijn van der Woude
  5. Tatiana M. Shamorkina
  6. Gerlof P. Bosman
  7. Barry Rockx
  8. Sander Herfst
  9. Joost Snijder
  10. Bart L. Haagmans
  11. Andrew B. Ward
  12. Geert-Jan Boons
  13. Robert P. de Vries

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Abstract

Receptor binding studies on sarbecoviruses would benefit from an available toolkit of recombinant spike proteins, or domains thereof, that recapitulate receptor binding properties of native viruses. We hypothesized that trimeric Receptor Binding Domain (RBD) proteins would be suitable candidates to study receptor binding properties of SARS-CoV-1 and -2. Here we created monomeric and trimeric fluorescent RBD proteins, derived from adherent HEK293T, as well as in GnTI-/- mutant cells, to analyze the effect of complex vs high mannose glycosylation on receptor binding. The results demonstrate that trimeric, complex glycosylated proteins are superior in receptor binding compared to monomeric and immaturely glycosylated variants. Although differences in binding to commonly used cell lines were minimal between the different RBD preparations, substantial differences were observed when respiratory tissues of experimental animals were stained. The RBD trimers demonstrated distinct ACE2 expression profiles in bronchiolar ducts and confirmed the higher binding affinity of SARS-CoV-2 over SARS-CoV-1. Our results show that complex glycosylated trimeric RBD proteins are attractive to analyze sarbecovirus receptor binding and explore ACE2 expression profiles in tissues.

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  1. Version published to 10.1371/journal.ppat.1009282
  2. ScreenIT

    SciScore for 10.1101/2020.09.04.282558: (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

    Antibodies
    SentencesResources
    Next, wells were treated with goat-α-human HRP secondary antibody for 1 hour at room temperature.
    HRP
    suggested: None
    With rigorous washing steps in between the proteins were detected with mouse anti-strep-tag-antibodies (IBA) and goat anti-mouse IgG antibodies (Life Biosciences).
    anti-mouse IgG
    suggested: None
    Quantifying and plotting fluorescent image data: Image quantification was performed by measuring the intensity of gray pixels of the stained ferret and Syrian hamster lung tissue slides with ImageJ version 1.52p. For stained tissues, background correction was performed by subtracting the average signal intensity of the antibody control from the images stained with ACE2 antibody, SARS-CoV1, and SARS-CoV2 recombinant proteins.
    ACE2
    suggested: None
    SARS-CoV1
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    The adapted pCD5 expression vector with an N-terminal HA leader (MKTIIALSYIFCLVFA) peptide, ACE2, and Twin-Strep (WSHPQFEKGGGSGGGSWSHPQFEK); IBA, Germany) was purified from HEK293T cell culture supernatant.
    HEK293T
    suggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)
    Immunofluorescent cell staining: Vero-E6, A549, and MDCK cells grown on coverslips were analyzed by immunofluorescent staining.
    A549
    suggested: NCI-DTP Cat# A549, RRID:CVCL_0023)
    MDCK
    suggested: CLS Cat# 602280/p823_MDCK_(NBL-2, RRID:CVCL_0422)
    Software and Algorithms
    SentencesResources
    Micrographs were collected using Leginon [37] and then uploaded to Appion [38].
    Leginon
    suggested: (Leginon, RRID:SCR_016731)
    2D processing was undertaken using Relion.
    Relion
    suggested: (RELION, RRID:SCR_016274)
    LAS Application Suite X was used as well as ImageJ for the addition of the scale bars.
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    Plotting means ± standard deviation values were performed in GraphPad Prism v8.0.1.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)

    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

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  3. Version published to 10.1101/2020.09.04.282558v1 on bioRxiv