SARS-CoV-2 spike proteins uptake mediated by lipid raft ganglioside GM1 in human cerebrovascular cells

  1. Conor McQuaid
  2. Alexander Solorzano
  3. Ian Dickerson
  4. Rashid Deane

Reviewed by

Read the full article See related articles

Listed in

Log in to save this article

Abstract

While there is clinical evidence of neurological manifestation in coronavirus disease-19, it’s unclear whether this is due to differential severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uptake from blood by cells of the cerebrovasculature. SARS-CoV-2 and its spike protein (SP) interact with the endothelium but the roles of extracellular peptidase domain on angiotensin converting enzyme 2 receptors (ACE2) and ACE2 independent pathways (such as glycans) are not fully elucidated. In addition, for SARS-CoV-2 to enter the brain parenchyma from blood it has to cross several cell types, including the endothelium, pericytes and vascular smooth muscle. Since SARS-CoV-2 interacts with host cells via it SP at the entry point of it life cycle, we used fluorescently labelled SP (SP-555) (wild type and mutants) to model viral behaviour, in vitro , for these cell types (endothelial, pericytes and vascular smooth muscle) to explore pathways of viral entry into brain from blood. There was differential SP uptake by these cell types. The endothelial cells had the least uptake, which may limit SP uptake into brain from blood. Uptake was mediated by ACE2, but it was dependent on SP interaction with ganglioside GM1 in the lipid raft. Mutation sites, N501Yand E484K and D614G, as seen in variants of interest, were differentially taken up by these cell types. There was greater uptake but neutralization with anti-ACE2 and anti-GM1antibodies was less effective. Our data suggested that GM1/lipid raft is an important entry point of SARS-CoV-2 into these cells since inhibition of SP uptake with both anti-ACE2 and anti-GM1 together was similar to that with only anti-GM1, and both ACE2 and GM1 are within the lipid raft region of plasma membrane. Thus, GM1 is a potential SARS-CoV-2 and therapeutic target at the cerebrovasculature.

Article activity feed

  1. ScreenIT

    SciScore for 10.1101/2022.03.20.485050: (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.
    RandomizationFive square fields with an area of 1×10−6 μm were randomly placed on each imaged.
    BlindingThe person performing the analysis were blinded to the experimental design and tracer used.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Anti-ACE2 antibody (R&D Systems, Cat# AF933) was labeled with Alexa Fluor 488 by following the manufacturer instructions (Microscale protein labeling kit; ThermoFisher Scientific).
    Anti-ACE2
    suggested: (Millipore Cat# AB9896, RRID:AB_805247)
    These include, anti ACE2 antibody (R&D Systems, Cat# AF933) used at a low (10 μg/m)l and high (60 μg/ml) concentration; anti TMPRSSE2 antibody (Invitrogen, Cat# PA5-14264) used at 13 μg/ml; anti CD147 antibody (Invitrogen, Cat# 34-5600) used at 2.5 μg/ml; anti NP-1antibody (Invitrogen, Cat# PA5-47027) used at 2 μg/ml and anti GM1 antibody (Abcam, Cat# Ab23943) used at 5 μg/ml.
    anti ACE2
    suggested: (Acris Antibodies Cat# BP2275, RRID:AB_972872)
    anti TMPRSSE2
    suggested: None
    anti CD147
    suggested: (Acris Antibodies Cat# SM2094P, RRID:AB_975010)
    anti NP-1antibody
    suggested: None
    anti GM1
    suggested: None
    The secondary antibodies, which were conjugated to Alexa Fluor 488, were donkey anti-rabbit (Thermofisher Scientific #A32790), anti-goat (Thermofisher Scientific #A32814) and anti-mouse (Thermofisher Scientific #A32766), and used at 1:200 dilution.
    anti-rabbit
    suggested: (Thermo Fisher Scientific Cat# A32790, RRID:AB_2762833)
    anti-goat
    suggested: (Thermo Fisher Scientific Cat# A32814, RRID:AB_2762838)
    anti-mouse
    suggested: (Thermo Fisher Scientific Cat# A32766, RRID:AB_2762823)
    Experimental Models: Cell Lines
    SentencesResources
    SARS-CoV-2 Spike proteins (recombinant SARS-CoV-2 Spike Protein (SP-RBD, Arg319-Phe 541; cat# RP-87678, HEK293 cell expressed and binds ACE2) was obtained from Life Technologies Corporation, Carlsbad CA, USA. Mutants SPs and its control wild type protein were obtained from RayBiotech Inc, Peachtree Corners, GA, USA.
    HEK293
    suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)
    Software and Algorithms
    SentencesResources
    All SPs were labeled separately with Alexa Fluor 555, using a kit (Microscale protein labeling kit; ThermoFisher Scientific; Waltham, MA, USA) and by following the manufacturer instructions.
    ThermoFisher Scientific
    suggested: None
    Statistic: All analysis were performed using Graphpad Prims version 9.2.0.
    Graphpad
    suggested: (GraphPad, RRID:SCR_000306)
    Outliers were identified and removed using ROUT method with Q= 10% in GraphPad Prism version 9.2.0.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)

    Results from OddPub: Thank you for sharing your data.

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    Limitations of the study: Since RBD of the SPs were used as a model of SARS-CoV-2, uptake of the actual virus might be different. However, the SP of SARS-CoV-2 is essential for viral entry into host cells. Thus, the attachment part of the viral life cycle can be explored with the SP. Also other cell types of the neurovascular unit, such as astrocytes, microglia and neurons need to be explored for SP uptake mechanisms. However, it is likely that there will be similar mechanisms since these cell types also express ACE2 and have glycans, including GM1.

    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.

    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.

  2. Version published to 10.1101/2022.03.20.485050v1 on bioRxiv