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. 2021 Feb;160(3):958-961.e3.
doi: 10.1053/j.gastro.2020.09.042. Epub 2020 Oct 3.

Angiotensin-converting Enzyme 2-containing Small Extracellular Vesicles and Exomeres Bind the Severe Acute Respiratory Syndrome Coronavirus 2 Spike Protein

Qin Zhang  1 , Dennis K Jeppesen  1 , James N Higginbotham  1 , Jeffrey L Franklin  1 , James E Crowe Jr  2 , Robert J Coffey  3
Affiliations

Angiotensin-converting Enzyme 2-containing Small Extracellular Vesicles and Exomeres Bind the Severe Acute Respiratory Syndrome Coronavirus 2 Spike Protein

Qin Zhang et al. Gastroenterology. 2021 Feb.
No abstract available

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Figures

Figure 1
Figure 1
sEVs and exomeres containing ACE2 can bind SARS-CoV-2 through the virus spike (S) protein. (A) Immunoblot analysis of cells and sEVs purified by high-resolution density gradients. (B) LIM1215 (left) and DiFi (right) samples were fractionated followed by immunoblotting. Arrow, full-length ACE2; ∗Ectodomain glycoform 1 of ACE2; ∗∗Ectodomain glycoform 2 of ACE2; ACE2 (ecto), ectodomain-specific monoclonal antibody; ACE2 (intra), intracellular-specific monoclonal antibody; I, glycosylated DPP4; II, nonglycosylated DPP4; anti-CD63 and anti-HSPA13 are used as exosomal and exomere markers, respectively; NV, nonvesicular; Exom, exomere. (C) Fluorescence-activated vesicle sorting analysis for ACE2 expression in sEV pellets (P) and exomeres from DiFi cells using an ectodomain-specific polyclonal antibody directly conjugated to PE. (D) Immunoblot analysis of inducible nitric oxide synthase (iNOS) and ACE2 expression in LIM1215 cells treated with indicated doses of interferon gamma (INF-γ) and TNF-α. (E) Sample inputs from DiFi cells (left) and agarose-conjugated Sambucus nigra agglutinin (SNA) lectin pull-downs (right) were analyzed by immunoblotting. β1-Integrin and epidermal growth factor receptor (EGFR) are known to be α2,6-sialylated and were used as positive controls. (F) Binding of human recombinant soluble ACE2 to the RBD of SARS-CoV-2 S protein. IP, immunoprecipitation; IB, immunoblotting. (G) Binding of DiFi exomeres with RBD of SARS-CoV-2 S protein. (H) Binding of DiFi cells, sEV-P, and exomeres with the S1 subunit of SARS-CoV-2 S protein. (I) Fluorescence-activated vesicle sorting analysis for binding of DiFi sEV-P and exomere to a mouse Fc-tagged RBD of SARS-CoV-2 S protein. (J) Schematic illustration of (i) SARS-CoV-2 viral infection of ACE2-positive cells and (ii) inhibition of infection due to hypothetical viral decoy binding by ACE2-positive sEVs and exomeres. Similarly, DPP4-positive sEVs and exomeres may act as decoys for MERS-CoV.
Supplementary Figure 1
Supplementary Figure 1
(A) Immunoblot analysis of colorectal cancer cells with indicated antibodies. (B) Immunoblot analysis of DKO-1 cells, sEV, and nonvesicular (NV) samples with indicated antibodies. (C) Fluorescence-activated vesicle sorting analysis for ACE2 expression in DiFi sEV-P using an ectodomain-specific monoclonal ACE2 primary antibody followed by an Alexa-647 secondary antibody. ß-actin serves as a loading control.
Supplementary Figure 2
Supplementary Figure 2
(A) Immunoblot analysis of inducible nitric oxide synthase (iNOS) and ACE2 expression in cells cultured under standard conditions for EV and exomere isolation. (B) Immunoblot analysis of iNOS and ACE2 expression in cells treated with indicated concentrations of interferon gamma (INF-γ) and TNF-α. (C) Immunoblot analysis for ACE2 from LS174T cells, sEV-Ps, and Exom. (D) Fluorescence-activated vesicle sorting analysis of LS174T, sEV-Ps, and exomeres binding to a mouse Fc-tagged RBD of SARS-CoV-2 spike protein S1. Actin serves as a loading control.
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