ACE2-Independent Interaction of SARS-CoV-2 Spike Protein with Human Epithelial Cells Is Inhibited by Unfractionated Heparin

  1. Lynda J. Partridge
  2. Lucy Urwin
  3. Martin J. H. Nicklin
  4. David C. James
  5. Luke R. Green
  6. Peter N. Monk

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Abstract

Coronaviruses such as SARS-CoV-2, which is responsible for COVID-19, depend on virus spike protein binding to host cell receptors to cause infection. The SARS-CoV-2 spike protein binds primarily to ACE2 on target cells and is then processed by membrane proteases, including TMPRSS2, leading to viral internalisation or fusion with the plasma membrane. It has been suggested, however, that receptors other than ACE2 may be involved in virus binding. We have investigated the interactions of recombinant versions of the spike protein with human epithelial cell lines that express low/very low levels of ACE2 and TMPRSS2 in a proxy assay for interaction with host cells. A tagged form of the spike protein containing the S1 and S2 regions bound in a temperature-dependent manner to all cell lines, whereas the S1 region alone and the receptor-binding domain (RBD) interacted only weakly. Spike protein associated with cells independently of ACE2 and TMPRSS2, while RBD required the presence of high levels of ACE2 for interaction. As the spike protein has previously been shown to bind heparin, a soluble glycosaminoglycan, we tested the effects of various heparins on ACE2-independent spike protein interaction with cells. Unfractionated heparin inhibited spike protein interaction with an IC50 value of <0.05 U/mL, whereas two low-molecular-weight heparins were less effective. A mutant form of the spike protein, lacking the arginine-rich putative furin cleavage site, interacted only weakly with cells and had a lower affinity for unfractionated and low-molecular-weight heparin than the wild-type spike protein. This suggests that the furin cleavage site might also be a heparin-binding site and potentially important for interactions with host cells. The glycosaminoglycans heparan sulphate and dermatan sulphate, but not chondroitin sulphate, also inhibited the binding of spike protein, indicating that it might bind to one or both of these glycosaminoglycans on the surface of target cells.

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  1. Version published to 10.3390/cells10061419
  2. Version published to 10.1101/2020.05.21.107870v2 on bioRxiv
  3. ScreenIT

    SciScore for 10.1101/2020.05.21.107870: (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
    Fondaparinux was purchased from Merck, UK. Goat anti-human ACE2 antibody AF933 (Biotechne), goat control IgG AB-108-C (Biotechne), rabbit anti-human TMPRSS2 (MBS9215011, Gentaur), rabbit IgG control (Biolegend) were used as per manufacturers’ instructions.
    anti-human ACE2
    suggested: None
    anti-human TMPRSS2
    suggested: None
    rabbit IgG
    suggested: None
    Cells were washed once and then incubated with the appropriate fluorescently labelled secondary antibody (anti-mouse polyvalent Ig-FITC (Merck) or anti-His6 HIS.H8 DyLight 488, (Invitrogen) for 30 min at 21°C.
    anti-mouse polyvalent Ig-FITC
    suggested: None
    anti-His6
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    The A549 cell line was obtained from the European Collection of Animal Cell Cultures (ECACC) and cultured in DMEM supplemented with 10% FCS.
    A549
    suggested: None
    293TACE2 cells were kindly provided by Paul Bieniasz (The Rockefeller University, USA), cultured as described for 293T cells including selection with 5ug/ml blasticidin.
    293TACE2
    suggested: RRID:CVCL_YZ65)
    A human keratinocyte cell line, HaCaT (300493), was obtained from Cell Line Services (CLS GmbH, Germany) and routinely cultured in DMEM supplemented with 10% FCS.
    HaCaT
    suggested: CLS Cat# 300493/p800_HaCaT, RRID:CVCL_0038)
    The Caco2 cell line (ATCC® HTB-37), derived from a colorectal adenocarcinoma, was obtained from Dr. Michael Trikic (University of Sheffield, UK).
    Caco2
    suggested: ATCC Cat# HTB-37, RRID:CVCL_0025)
    Real time quantitative PCR (RT-qPCR): HaCaT, RT4, A549, 293T, 293TACE2 and Caco2 cell lines were cultured for 48hrs under standard media conditions and harvested using trypsin.
    293T
    suggested: RRID:CVCL_YZ65)
    Wild-type S1S2 (wt S1S2; Val16-Pro1213; Stratech UK) with a His6 tag at the C-terminus was expressed in baculovirus-insect cells, while S1-Fc (Val16-Arg685; Stratech UK) with a mouse IgG1 Fc region at the C-terminus was expressed in HEK293 cells.
    HEK293
    suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)

    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: 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.
    • No funding statement was detected.
    • 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.

  4. Version published to 10.1101/2020.05.21.107870v1 on bioRxiv