Evidence of Structural Protein Damage and Membrane Lipid Remodeling in Red Blood Cells from COVID-19 Patients

  1. Tiffany Thomas
  2. Davide Stefanoni
  3. Monika Dzieciatkowska
  4. Aaron Issaian
  5. Travis Nemkov
  6. Ryan C. Hill
  7. Richard O. Francis
  8. Krystalyn E. Hudson
  9. Paul W. Buehler
  10. James C. Zimring
  11. Eldad A. Hod
  12. Kirk C. Hansen
  13. Steven L. Spitalnik
  14. Angelo D’Alessandro

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Abstract

The SARS-CoV-2 beta coronavirus is the etiological driver of COVID-19 disease, which is primarily characterized by shortness of breath, persistent dry cough, and fever. Because they transport oxygen, red blood cells (RBCs) may play a role in the severity of hypoxemia in COVID-19 patients. The present study combines state-of-the-art metabolomics, proteomics, and lipidomics approaches to investigate the impact of COVID-19 on RBCs from 23 healthy subjects and 29 molecularly diagnosed COVID-19 patients. RBCs from COVID-19 patients had increased levels of glycolytic intermediates, accompanied by oxidation and fragmentation of ankyrin, spectrin beta, and the N-terminal cytosolic domain of band 3 (AE1). Significantly altered lipid metabolism was also observed, in particular, short- and medium-chain saturated fatty acids, acyl-carnitines, and sphingolipids. Nonetheless, there were no alterations of clinical hematological parameters, such as RBC count, hematocrit, or mean corpuscular hemoglobin concentration, with only minor increases in mean corpuscular volume. Taken together, these results suggest a significant impact of SARS-CoV-2 infection on RBC structural membrane homeostasis at the protein and lipid levels. Increases in RBC glycolytic metabolites are consistent with a theoretically improved capacity of hemoglobin to off-load oxygen as a function of allosteric modulation by high-energy phosphate compounds, perhaps to counteract COVID-19-induced hypoxia. Conversely, because the N-terminus of AE1 stabilizes deoxyhemoglobin and finely tunes oxygen off-loading and metabolic rewiring toward the hexose monophosphate shunt, RBCs from COVID-19 patients may be less capable of responding to environmental variations in hemoglobin oxygen saturation/oxidant stress when traveling from the lungs to peripheral capillaries and vice versa.

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  1. Version published to 10.1021/acs.jproteome.0c00606
  2. ScreenIT

    SciScore for 10.1101/2020.06.29.20142703: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: Blood collection and processing: This observational study was conducted according to the Declaration of Helsinki, in accordance with good clinical practice guidelines, and approved by the Columbia University Institutional Review Board.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    Pathway analyses were performed with DAVID software and Ingenuity Pathway Analysis.
    DAVID
    suggested: (DAVID, RRID:SCR_001881)
    Ingenuity Pathway Analysis
    suggested: (Ingenuity Pathway Analysis, RRID:SCR_008653)
    Graphs and statistical analyses were prepared with GraphPad Prism 8.0 (GraphPad Software, Inc, La Jolla, CA)
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Graphs and statistical analyses were prepared with GraphPad Prism 8.0, GENE E, and MetaboAnalyst 4.0.25 Spearman’s correlations and related p-values were calculated with R Studio.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    MetaboAnalyst
    suggested: (MetaboAnalyst, RRID:SCR_015539)

    Results from OddPub: Thank you for sharing your data.

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    Unfortunately, owing to logistical limitations, we could not directly measure RBC parameters directly related to gas transport physiology, a limitation that we will address in follow-up studies. It is not clear whether the alterations of the N-terminus of AE1 are driven by oxidant stress alone or by an enzymatic activity secondary to the infection (e.g., calcium-activated proteases). However, although SARS-CoV-2 encodes cleaving enzymes (e.g., papain-like proteases), comparing our proteomics data with this viral genome did not produce any positive identifications. This suggests that the virus does not penetrate RBCs or, if it does, its protein components are rapidly degraded and not resynthesized owing to the lack of organelles; alternatively, our approach may not be sensitive enough to detect trace viral proteins in the background of ∼250 million hemoglobin molecules per RBC.29 Modification and oxidation of the N-termini of AE1, ANK1, and SPTB were accompanied by altered acyl-carnitines, fatty acids (especially saturated short- and medium-chain fatty acids), and lipid metabolism (especially sphingolipids). The latter is interesting because signaling through the N-terminus of AE1 mechanistically cross-regulates with sphingolipids to promote hemoglobin oxygen off-loading in response to physiological (e.g., high-altitude) or pathological (e.g., sickle cell disease) hypoxia.15,30 This signature is consistent with impaired membrane lipid homeostasis, which is not attributable to ...

    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 10.1101/2020.06.29.20142703 on medRxiv