Site-Specific O-Glycosylation Analysis of SARS-CoV-2 Spike Protein Produced in Insect and Human Cells

  1. Ieva Bagdonaite
  2. Andrew J. Thompson
  3. Xiaoning Wang
  4. Max Søgaard
  5. Cyrielle Fougeroux
  6. Martin Frank
  7. Jolene K. Diedrich
  8. John R. Yates
  9. Ali Salanti
  10. Sergey Y. Vakhrushev
  11. James C. Paulson
  12. Hans H. Wandall

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Abstract

Enveloped viruses hijack not only the host translation processes, but also its glycosylation machinery, and to a variable extent cover viral surface proteins with tolerogenic host-like structures. SARS-CoV-2 surface protein S presents as a trimer on the viral surface and is covered by a dense shield of N-linked glycans, and a few O-glycosites have been reported. The location of O-glycans is controlled by a large family of initiating enzymes with variable expression in cells and tissues and hence is difficult to predict. Here, we used our well-established O-glycoproteomic workflows to map the precise positions of O-linked glycosylation sites on three different entities of protein S—insect cell or human cell-produced ectodomains, or insect cell derived receptor binding domain (RBD). In total 25 O-glycosites were identified, with similar patterns in the two ectodomains of different cell origin, and a distinct pattern of the monomeric RBD. Strikingly, 16 out of 25 O-glycosites were located within three amino acids from known N-glycosites. However, O-glycosylation was primarily found on peptides that were unoccupied by N-glycans, and otherwise had low overall occupancy. This suggests possible complementary functions of O-glycans in immune shielding and negligible effects of O-glycosylation on subunit vaccine design for SARS-CoV-2.

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  1. Version published to 10.3390/v13040551
  2. ScreenIT

    SciScore for 10.1101/2021.02.03.429627: (What is this?)

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

    Table 1: Rigor

    NIH rigor criteria are not applicable to paper type.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    Protein expression and purification in HEK 293 F cells: A synthetic gene encoding SARS-CoV-2 S (sequence derived from Genbank accession: MN908947.3) was cloned into a customized DNA vector for expression in mammalian tissue culture.
    HEK 293
    suggested: None
    Pure S-encoding plasmid DNA was transfected into HEK 293F cells (approximately 106 mL−1) using PEI (polyethylimine) MAX at 5:1 w/w (final culture DNA concentration approximately 1 μg mL−1).
    HEK 293F
    suggested: RRID:CVCL_6642)
    Software and Algorithms
    SentencesResources
    For the site-specific glycopeptide identification, the corresponding HCD MS/MS and ETD MS/MS were analyzed by Proteome discoverer 2.2 software (Thermo Fisher Scientific) using Sequest HT as a searching engine.
    Proteome discoverer
    suggested: (Proteome Discoverer, RRID:SCR_014477)
    Molecular modelling: We used Conformational Analysis Tools software (www.md-simulations.de/CAT/) interfaced with STRIDE [29] to estimate the solvent accessible surface (SAS) of the amino acids.
    Conformational Analysis Tools
    suggested: None
    The O-glycans were attached to the model manually and optimized using YASARA [30].
    YASARA
    suggested: (YASARA, RRID:SCR_017591)

    Results from OddPub: Thank you for sharing your data.

    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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  3. Version published to 10.1101/2021.02.03.429627v2 on bioRxiv
  4. Version published to 10.1101/2021.02.03.429627v1 on bioRxiv