Structure-guided glyco-engineering of ACE2 for improved potency as soluble SARS-CoV-2 decoy receptor

  1. Tümay Capraz
  2. Nikolaus F Kienzl
  3. Elisabeth Laurent
  4. Jan W Perthold
  5. Esther Föderl-Höbenreich
  6. Clemens Grünwald-Gruber
  7. Daniel Maresch
  8. Vanessa Monteil
  9. Janine Niederhöfer
  10. Gerald Wirnsberger
  11. Ali Mirazimi
  12. Kurt Zatloukal
  13. Lukas Mach
  14. Josef M Penninger
  15. Chris Oostenbrink
  16. Johannes Stadlmann

  • Curated by eLife

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    Evaluation Summary:

    This manuscript is of broad interest in light of the COVID-19 pandemic. Characterization of how glycosylation affects interactions between the viral Spike protein and ACE2 receptor can inform efforts to inhibit the SARS-CoV-2. The molecular modeling and functional analysis need to be improved.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

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Abstract

Infection and viral entry of SARS-CoV-2 crucially depends on the binding of its Spike protein to angiotensin converting enzyme 2 (ACE2) presented on host cells. Glycosylation of both proteins is critical for this interaction. Recombinant soluble human ACE2 can neutralize SARS-CoV-2 and is currently undergoing clinical tests for the treatment of COVID-19. We used 3D structural models and molecular dynamics simulations to define the ACE2 N-glycans that critically influence Spike-ACE2 complex formation. Engineering of ACE2 N-glycosylation by site-directed mutagenesis or glycosidase treatment resulted in enhanced binding affinities and improved virus neutralization without notable deleterious effects on the structural stability and catalytic activity of the protein. Importantly, simultaneous removal of all accessible N-glycans from recombinant soluble human ACE2 yields a superior SARS-CoV-2 decoy receptor with promise as effective treatment for COVID-19 patients.

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  1. Version published to 10.7554/elife.73641 on eLife
  2. eLife

    Evaluation Summary:

    This manuscript is of broad interest in light of the COVID-19 pandemic. Characterization of how glycosylation affects interactions between the viral Spike protein and ACE2 receptor can inform efforts to inhibit the SARS-CoV-2. The molecular modeling and functional analysis need to be improved.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

  3. eLife

    Reviewer #1 (Public Review):

    Capraz et al extend previous studies of ACE2 protein as an antiviral agent against SARS-CoV-2, which acts by binding to spike. Their work indicates that ACE2 with engineered decreased glycosylation has increased binding (as measured by BLI) and antiviral activity against SARS-CoV-2, without decreasing enzymatic activity of ACE2. Glycosylation of ACE2 is decreased using point mutations and enzymatically.

  4. eLife

    Reviewer #2 (Public Review):

    Summary: The authors provide a succinct review of Spike and ACE2 glycosylation and its apparent importance in modulating the interaction of these two proteins. Of particular translational value is the idea that understanding the configurations and effects of ACE2 glycans could inform efforts to develop soluble ACE2 variants as SARS-CoV-2 therapeutics. The authors make a good case for studies to understand the functional roles of glycosylation in this context. For their analysis, the authors begin with 3D modeling of the Spike trimer in complex with one molecule of ACE2. The interaction is mediated by a single RBD (receptor binding domain) in Spike that is modeled in the "up" conformation previously shown to be competent for receptor binding. They then generated a fully glycosylated model and then performed molecular dynamics simulation. Post-simulation structural analysis focused primarily on identifying effects of the glycans on the Spike:ACE2 interaction. Two types of apparent effects were concluded: (1) direct interactions between glycans and proteins, and (2) indirect effects. Finally, the authors present functional data on glycan effects on protein stability, binding, and inhibitory activity of soluble ACE2.

    Critique: Data and analysis presented in this manuscript generally support the general idea that glycans can have direct and/or modulatory effects on the interaction between the SARS-CoV-2 Spike protein and its receptor ACE2. The molecular dynamics methodology and functional/biophysical experiments themselves appear to have been performed expertly. However, there are deficiencies in the both the experimental design and data analysis, at least some of which can be addressed by more detailed and clearer description of the what was done and how the data were analyzed.

  5. ScreenIT

    SciScore for 10.1101/2021.08.31.458325: (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.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    Recombinant expression of all other proteins was performed by transient transfection of HEK293-6E cells, licensed from National Research Council (NRC) of Canada, as previously described.
    HEK293-6E
    suggested: RRID:CVCL_HF20)
    A German 2019-nCoV isolate (Ref-SKU: 026V-03883, Charité, Berlin, Germany) was propagated in Vero E6 cells.
    Vero E6
    suggested: RRID:CVCL_XD71)
    Recombinant DNA
    SentencesResources
    pCAGGS vector constructs containing either the sequence of the SARS-CoV-2 RBD (residues R319-F541) or the complete luminal domain of Spike, modified in terms of removal of the polybasic furin cleavage site and introduction of two stabilizing point mutations (K986P and V987P), were kindly provided by Florian Krammer
    pCAGGS
    suggested: RRID:Addgene_18926)
    62,63 Plasmid constructs pcDNA3-sACE2(WT)-Fc(IgG1) and pcDNA3-sACE2-T92Q-Fc(IgG1) were obtained from Addgene (United States).
    pcDNA3-sACE2
    suggested: None
    pcDNA3-sACE2-T92Q-Fc(IgG1
    suggested: RRID:Addgene_145170)
    Software and Algorithms
    SentencesResources
    Protein bands were visualized with the Gel Doc XR+ Imager (Bio-Rad Laboratories).
    Bio-Rad Laboratories
    suggested: (Bio-Rad Laboratories, RRID:SCR_008426)
    Fitting was done with Origin 7.0 for DSC software using the non-2-state transition model.
    Origin
    suggested: (Origin, RRID:SCR_014212)
    Statistical analyses were conducted using GraphPad Prism 8.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    5 Molecular models and simulation trajectories are available through the BioExcel COVID-19 Molecular Structure and Therapeutics Hub (https://covid.bioexcel.eu/simulations/).
    BioExcel
    suggested: None

    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: We found the following clinical trial numbers in your paper:

    IdentifierStatusTitle
    NCT04335136CompletedRecombinant Human Angiotensin-converting Enzyme 2 (rhACE2) a…

    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.
    • 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.

    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.

  6. Version published to 10.1101/2021.08.31.458325v1 on bioRxiv