Analysis of the Role of N-Linked Glycosylation in Cell Surface Expression, Function, and Binding Properties of SARS-CoV-2 Receptor ACE2

  1. Raymond Rowland
  2. Alberto Brandariz-Nuñez

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Abstract

Understanding the role of glycosylation in the virus-receptor interaction is important for developing approaches that disrupt infection. In this study, we showed that deglycosylation of both ACE2 and S had a minimal effect on the spike-ACE2 interaction.

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  1. Version published to 10.1128/spectrum.01199-21
  2. ScreenIT

    SciScore for 10.1101/2021.05.10.443532: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Ethicsnot detected.
    Sex as a biological variablenot detected.
    RandomizationAfter 24 h incubation, five fields were randomly selected in each well to count the number of nuclei in fused and unfused cells.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Cells, antibodies and reagents: HEK 293T cells (ATCC CRL-3216 from American Type Culture Collection, Rockville, MD) and HEK 293T Lenti-X (Clontech/Takara Bio) were cultured at 37°C in 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies) supplemented with 10% fetal bovine serum (FBS; Gibco), 2 mM L-glutamine (Life Technologies), and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin; Life Technologies).
    antibiotics
    suggested: None
    Spike protein S2 monoclonal antibody (1A9), PDI mouse antibody (Clone: RL90), Alexa 594- and Alexa 488-conjugated antibodies against mouse and rabbit IgG, respectively.
    PDI
    suggested: (Thermo Fisher Scientific Cat# MA3019A488, RRID:AB_2633336)
    rabbit IgG
    suggested: None
    rabbit monoclonal antibody, the Calnexin (C5C9) rabbit monoclonal antibody and the FLAG-tag (D6W5B)
    FLAG-tag
    suggested: None
    The eluted samples were separated by SDS-PAGE and analyzed by Western blotting using anti-FLAG M2 monoclonal antibody or SARS-CoV/SARS-CoV-2 Spike protein S2 monoclonal antibody.
    anti-FLAG
    suggested: None
    SARS-CoV/SARS-CoV-2 Spike protein S2
    suggested: (Thermo Fisher Scientific Cat# MA5-35946, RRID:AB_2866558)
    Input lysates and pulldown proteins were analyzed by western blotting with the anti-FLAG M2 monoclonal antibody (Sigma) and anti-GAPDH monoclonal antibody (Cell Signaling) as described above.
    anti-GAPDH
    suggested: None
    Digested proteins were analyzed by Western blotting with anti-M2 FLAG monoclonal antibody (Sigma).
    anti-M2 FLAG
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cells, antibodies and reagents: HEK 293T cells (ATCC CRL-3216 from American Type Culture Collection, Rockville, MD) and HEK 293T Lenti-X (Clontech/Takara Bio) were cultured at 37°C in 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies) supplemented with 10% fetal bovine serum (FBS; Gibco), 2 mM L-glutamine (Life Technologies), and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin; Life Technologies).
    HEK 293T
    suggested: ATCC Cat# CRL-3216, RRID:CVCL_0063)
    In order to transduce cells with pseudovirions, virus was added either to mock transfected 293T or ACE2-transfected 293T cell lines at 24 hr post-transfection.
    293T
    suggested: None
    Treatment with N-glycosylation processing inhibitors and glycosidases: Tunicamycin (TM) and Kifunensine (KIF) at a concentration of 1 μg/ml and 5 μg/ml, respectively, were used to inhibit N-glycosylation in transfected HEK-293T cells.
    HEK-293T
    suggested: CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063)
    Recombinant DNA
    SentencesResources
    Plasmids: The plasmids, pCDNA3.1-hACE2-FLAG and pCDNA3.1-pACE2-FLAG, which express human ACE2 (GenBank accession number NM_021804.3) and pig ACE2 (GenBank accession number NM_001123070.1) fused to a FLAG epitope, respectively, were purchased from GenScript, as well as the pCDNA3.1-TMPRSS2-FLAG expressing TMPRSS2 ( GenBank accession number NM_005656.4) fused to a FLAG epitope.
    pCDNA3.1-hACE2-FLAG
    suggested: None
    pCDNA3.1-pACE2-FLAG
    suggested: None
    pCDNA3.1-TMPRSS2-FLAG
    suggested: None
    pCDNA3.1-SARS2-Spike expressing full-lengh Spike (S) protein from SARS-CoV-2 (GenBank accession number QHD43416.1) was purchased from Addgene.
    pCDNA3.1-SARS2-Spike
    suggested: RRID:Addgene_145032)
    Cell surface biotinylation assay: Human 293T cells were transiently transfected with plasmids encoding FLAG-tagged hACE2 variants or FLAG-tagged pACE2 protein for 24h.
    pACE2
    suggested: None
    To generate these viruses, Lenti-X 293 T cells (Takara Bio) grown in 10 cm dish were transiently transfected with the following plasmids: 5 µg of pLenti-GFP (Cell Biolabs), 6 µg of psPAX2 and 0.9 µg of pCMV-VSVG (Cell Biolabs), or 0.9 µg of pCDNA3.1-SARS2-SpikeΔ19, or 0.9 µg of pCDNA3.1-SARS-Spike using the TransIT®-LT1 Transfection Reagent (Mirus) according to the manufacturer’s instructions.
    pLenti-GFP
    suggested: None
    psPAX2
    suggested: RRID:Addgene_12260)
    pCMV-VSVG
    suggested: None
    pCDNA3.1-SARS2-SpikeΔ19
    suggested: None
    pCDNA3.1-SARS-Spike
    suggested: RRID:Addgene_145031)
    Software and Algorithms
    SentencesResources
    The following antibodies were purchased from Thermo Fischer: SARS-CoV/SARS-CoV-2
    Thermo Fischer
    suggested: None
    The images were processed with NIS-elements software (Nikon).
    NIS-elements
    suggested: (NIS-Elements, RRID:SCR_014329)

    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 pages 56 and 54. 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:
    • No conflict of interest statement was detected. If there are no conflicts, we encourage authors to explicit state so.
    • No funding statement was detected.
    • No protocol registration statement was detected.

    Results from scite Reference Check: We found no unreliable references.

    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 10.1101/2021.05.10.443532v1 on bioRxiv