A novel ACE2 isoform is expressed in human respiratory epithelia and is upregulated in response to interferons and RNA respiratory virus infection

  1. Cornelia Blume
  2. Claire L. Jackson
  3. Cosma Mirella Spalluto
  4. Jelmer Legebeke
  5. Liliya Nazlamova
  6. Franco Conforti
  7. Jeanne-Marie Perotin
  8. Martin Frank
  9. John Butler
  10. Max Crispin
  11. Janice Coles
  12. James Thompson
  13. Robert A. Ridley
  14. Lareb S. N. Dean
  15. Matthew Loxham
  16. Stephanie Reikine
  17. Adnan Azim
  18. Kamran Tariq
  19. David A. Johnston
  20. Paul J. Skipp
  21. Ratko Djukanovic
  22. Diana Baralle
  23. Christopher J. McCormick
  24. Donna E. Davies
  25. Jane S. Lucas
  26. Gabrielle Wheway
  27. Vito Mennella

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Abstract

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    SciScore for 10.1101/2020.07.31.230870: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: Airway samples for the study were collected following approval by South-Central Hampshire A, Research Ethics Committee, UK (reference numbers: 07/Q1702/109, 13/SC/0182 and 14/WM/1226) and all participants gave their informed consent.
    Consent: Airway samples for the study were collected following approval by South-Central Hampshire A, Research Ethics Committee, UK (reference numbers: 07/Q1702/109, 13/SC/0182 and 14/WM/1226) and all participants gave their informed consent.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Briefly, ice cold methanol fixed and 4% dried milk blocked cilia pellets and deciliated cell membranes (excised from Transwell inserts) were labelled for 1 hour at room temperature with a mouse anti-alpha-tubulin antibody (T9026, Sigma) diluted 1:500 in PBST.
    anti-alpha-tubulin
    suggested: None
    Following three PBST washes a secondary goat antimouse Alexa488 antibody (Molecular Probes) was incubated for 30 minutes at room temperature before PBST washes.
    antimouse
    suggested: (C. Birchmeier, Max Delbruck Center for Molecular Medicine; Berlin; Germany Cat# Guinea pig anti-mouse Tlx3 polyclonal antibody, RRID:AB_2532145)
    HRP-conjugated anti-β-actin antibody (Sigma) was used as a loading control.
    anti-β-actin
    suggested: None
    Membranes were cut from the inserts and epithelial cells were stained with anti-ACE2 antibodies (ab15348 (Abcam), AF933 (R&D systems) and #NBP2-67692 (Novus)), anti-alpha tubulin (T9026 Sigma), and appropriate fluorescently labelled secondary antibodies (Alexa-488 labelled anti-mouse (Invitrogen), Alexa649 labelled anti-goat (Abcam), DyLight 647 labelled anti-rabbit (Biolegend)).
    anti-ACE2
    suggested: (Abcam Cat# ab15348, RRID:AB_301861)
    #NBP2-67692
    suggested: None
    anti-alpha tubulin
    suggested: None
    Alexa-488 labelled anti-mouse ( Invitrogen)
    suggested: None
    anti-goat ( Abcam)
    suggested: None
    anti-rabbit
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Vero E6 (ECACC Vero C1008) cells were cultured in DMEM medium supplemented with 10% FBS and penicillin/streptomycin (Gibco) at 37°C with 5% CO2.
    Vero E6
    suggested: RRID:CVCL_XD71)
    NCI-H441 [H441] (ATCC HTB-174) cells were cultured in RPMI 1640 medium (Gibco) supplemented with 10% FCS, sodium pyruvate, L-glutamine and penicillin/streptomycin at 37°C with 5% CO2 and passaged every 3-4 days.
    NCI-H441
    suggested: ATCC Cat# HTB-174, RRID:CVCL_1561)
    Cells were washed with modified HBSS with calcium and magnesium (HyClone) before detaching with 0.2% trypsin EDTA. hTERT RPE-1 (ATCC CRL-4000) were cultured in DMEM/F12 medium (Gibco) supplemented with 10% FCS at 37°C with 5% CO2 and passaged every 4-6 days. 293 [HEK-293] (ATCC® CRL-1573™) were cultured in DMEM high glucose supplemented with 10% FCS at 5% CO2 and passaged every 4-6 days at a ratio of 1:8.
    293
    suggested: None
    Long-range RT-PCR: Phusion High-Fidelity PCR Master Mix with HF Buffer (NEB) was used to amplify the novel and annotated transcript using custom primers (IDT) from cDNA produced from nasal brushings and BCi-NS1.1 cells.
    BCi-NS1.1
    suggested: RRID:CVCL_T029)
    Antibodies: Human rhinovirus (RV) 16 propagation and titration: Human rhinovirus (HRV16; ATCC VR-283™, Teddington, UK) was amplified using H1 HeLa cells as previously described76,77.
    HeLa
    suggested: None
    Software and Algorithms
    SentencesResources
    Library quality was assessed using a broad range DNA chip on the Agilent Bioanalyser 2100.
    Agilent Bioanalyser
    suggested: None
    Quality of sequence was assessed using FastQC v0.11.5 (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/).
    FastQC
    suggested: (FastQC, RRID:SCR_014583)
    Alignment to reference genome and quality control: Paired FASTQ files were aligned to GRCh38 human genome reference using GENCODE v33 gene annotations and STAR v2.6.0a splice aware aligner31, using ENCODE recommend options (3.2.2 in the STAR manual (https://github.com/alexdobin/STAR/blob/master/doc/STARmanual.pdf).
    GENCODE
    suggested: (GENCODE, RRID:SCR_014966)
    STAR
    suggested: (STAR, RRID:SCR_015899)
    Alignment files were assessed for saturation of known splice junctions using RSeqQC v3.0.171
    RSeqQC
    suggested: None
    Alignment to reference transcriptome and transcript level abundance estimates: SALMON tool v1.3.073 was used to perform transcript abundance estimates from raw FASTQ files using selective alignment with a decoy-aware transcriptome assembled using Scallop tool.
    SALMON
    suggested: (Salmon, RRID:SCR_017036)
    Samples were size separated against Hyperladder 1kb (BioLine)
    BioLine
    suggested: None
    Electropherograms were visualised using 4Peaks.
    4Peaks
    suggested: (4Peaks, RRID:SCR_000015)
    ImageJ was used for densitometry.
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    Structural analysis: PyMOL Molecular Graphics System, Version 2.0 (Schrödinger, LLC) or UCSF Chimera78 was used to model the novel protein isoform based on full-length isoform 6M18.
    PyMOL
    suggested: (PyMOL, RRID:SCR_000305)
    Molecular dynamics simulations in explicit solvent were performed using YASARA with GPU acceleration80 on an Intel i9-9940X CPU (using 28 Threads) and GeForce RTX 2080 Ti.
    YASARA
    suggested: (YASARA, RRID:SCR_017591)
    Statistics: Statistical analyses were performed in GraphPad Prism v7.02 (
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    GraphPad Software Inc.
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    All sequence files to be deposited on Sequence Read Archive.
    Sequence Read Archive
    suggested: (DDBJ Sequence Read Archive, RRID:SCR_001370)

    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 page 49. 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:
    • 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.

  2. Version published to 10.1038/s41588-020-00759-x
  3. Version published to 10.1101/2020.07.31.230870v1 on bioRxiv