Comprehensive characterization of N- and O- glycosylation of SARS-CoV-2 human receptor angiotensin converting enzyme 2

  1. Asif Shajahan
  2. Stephanie Archer-Hartmann
  3. Nitin T Supekar
  4. Anne S Gleinich
  5. Christian Heiss
  6. Parastoo Azadi

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Abstract

The emergence of the coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has created the need for development of new therapeutic strategies. Understanding the mode of viral attachment, entry and replication has become a key aspect of such interventions. The coronavirus surface features a trimeric spike (S) protein that is essential for viral attachment, entry and membrane fusion. The S protein of SARS-CoV-2 binds to human angiotensin converting enzyme 2 (hACE2) for entry. Herein, we describe glycomic and glycoproteomic analysis of hACE2 expressed in HEK293 cells. We observed high glycan occupancy (73.2 to 100%) at all seven possible N-glycosylation sites and surprisingly detected one novel O-glycosylation site. To deduce the detailed structure of glycan epitopes on hACE2 that may be involved in viral binding, we have characterized the terminal sialic acid linkages, the presence of bisecting GlcNAc and the pattern of N-glycan fucosylation. We have conducted extensive manual interpretation of each glycopeptide and glycan spectrum, in addition to using bioinformatics tools to validate the hACE2 glycosylation. Our elucidation of the site-specific glycosylation and its terminal orientations on the hACE2 receptor, along with the modeling of hACE2 glycosylation sites can aid in understanding the intriguing virus-receptor interactions and assist in the development of novel therapeutics to prevent viral entry. The relevance of studying the role of ACE2 is further increased due to some recent reports about the varying ACE2 dependent complications with regard to age, sex, race and pre-existing conditions of COVID-19 patients.

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  1. Version published to 10.1093/glycob/cwaa101
  2. Version published to 10.1101/2020.05.01.071688v2 on bioRxiv
  3. ScreenIT

    SciScore for 10.1101/2020.05.01.071688: (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

    Experimental Models: Cell Lines
    SentencesResources
    Reduction, alkylation and protease digestion of hACE2 for glycoproteomics: The purified hACE2 (20 μg) expressed on HEK293 cells in 50 mM ammonium bicarbonate solution was reduced by adding 25 mM DTT and incubating at 60 °C for 30 min.
    HEK293
    suggested: None
    Software and Algorithms
    SentencesResources
    Data analysis was performed using Byonic 2.3.5 software and manually using Thermo Fisher Xcalibur 4.2.
    Thermo Fisher Xcalibur
    suggested: (Thermo Xcalibur, RRID:SCR_014593)
    The glycomics data analysis was performed manually with the help of GlycoWorkbench software.
    GlycoWorkbench
    suggested: (GlycoWorkbench, RRID:SCR_000782)
    The relative intensities of glycans at each site were calculated by evaluating the area under the curve of each extracted glycopeptide peak (after deconvolution using Xtract module of Xcalibur 4.2 Qual Browser) on the LC-MS chromatogram.
    Xcalibur
    suggested: (Thermo Xcalibur, RRID:SCR_014593)

    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

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  4. Version published to 10.1101/2020.05.01.071688v1 on bioRxiv