Mass spectrometry analysis of newly emerging coronavirus HCoV-19 spike S protein and human ACE2 reveals camouflaging glycans and unique post-translational modifications

  1. Zeyu Sun
  2. Keyi Ren
  3. Xing Zhang
  4. Jinghua Chen
  5. Zhengyi Jiang
  6. Jing Jiang
  7. Feiyang Ji
  8. Xiaoxi Ouyang
  9. Lanjuan Li

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Abstract

The pneumonia-causing COVID-19 pandemia has prompt worldwide efforts to understand its biological and clinical traits of newly identified HCoV-19 virus. In this study, post-translational modification (PTM) of recombinant HCoV-19 S and hACE2 were characterized by LC-MSMS. We revealed that both proteins were highly decorated with specific proportions of N-glycan subtypes. Out of 21 possible glycosites in HCoV-19 S protein, 20 were confirmed completely occupied by N-glycans, with oligomannose glycans being the most abundant type. All 7 possible glycosylation sites in hACE2 were completely occupied mainly by complex type N-glycans. However, we showed that glycosylation did not directly contribute to the binding affinity between SARS-CoV spike protein and hACE2. Additionally, we also identified multiple sites methylated in both proteins, and multiple prolines in hACE2 were converted to hydroxylproline. Refined structural models were built by adding N-glycan and PTMs to recently published cryo-EM structure of the HCoV-19 S and hACE2 generated with glycosylation sites in the vicinity of binding surface. The PTM and glycan maps of both HCoV-19 S and hACE2 provide additional structural details to study mechanisms underlying host attachment, immune response mediated by S protein and hACE2, as well as knowledge to develop remedies and vaccines desperately needed nowadays.

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  1. ScreenIT

    SciScore for 10.1101/2020.04.29.068098: (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
    Expression and purification of HCoV-19 spike ectodomain and hACE2: HCoV-19 S gene (virus isolate: Wuhan Hu-1; GenBank number QHD43416.1) was synthesized (Genscript) with codons optimized for insect cell expression.
    HCoV-19
    suggested: None
    The extracellular domain of hACE2 (Gln18-Ser740, NP_068576.1) with C-terminal Fc tag was expressed in HEK 293 cells were purified by protein A sepharose beads (GE Healthcare).
    HEK 293
    suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)
    Software and Algorithms
    SentencesResources
    The MS and MS/MS spectra were recorded using the Xcalibur software 2.3 (Thermo Scientific, USA)
    Xcalibur
    suggested: (Thermo Xcalibur, RRID:SCR_014593)
    Bioinformatics: The acquired MS raw files from deglycosylated peptides were searched by MaxQuant (version 1.6.10.43) against the human ACE2 sequence from UniProtKB and HCoV-19 S sequence (YP_009724390.1_3) from NCBI.
    MaxQuant
    suggested: (MaxQuant, RRID:SCR_014485)
    UniProtKB
    suggested: (UniProtKB, RRID:SCR_004426)
    The modified residues within each model were generated with Coot (Emsley et al., 2010).
    Coot
    suggested: (Coot, RRID:SCR_014222)
    All structural figures were performed by PyMOL (Schrödinger Inc. U.S.A.).
    PyMOL
    suggested: (PyMOL, RRID:SCR_000305)

    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.

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  2. Version published to 10.1101/2020.04.29.068098 on bioRxiv