Journal: The Journal of Biological Chemistry

Article Title: Sphingosine prevents binding of SARS–CoV-2 spike to its cellular receptor ACE2

doi: 10.1074/jbc.RA120.015249

Figure Lengend Snippet: Sphingosine binds to ACE2 and thereby blocks the interaction of ACE2 with the viral spike protein. A , sphingosine (SPH)-coated beads or control ( Ctr ) beads were incubated with recombinant ( rec. ) Fc-ACE2 protein ( left panel ) or with lysates obtained from Vero cells ( right panel ), extensively washed, and eluted in 1× SDS sample buffer. As indicated, suspended sphingosine ( SPH ) was added (2 μ m ) to prevent binding of ACE2 to immobilized sphingosine. The samples were separated by 7.5% SDS-PAGE electrophoresis and blotted with anti-ACE2 antibodies. The Fc-ACE2 protein has a molecular mass of ∼180 kDa, and the endogenous ACE2 protein has a molecular mass of ∼120 kDa. Shown are representative results from five independent experiments. B , recombinant Fc-ACE2 protein was immobilized on protein A/G–agarose, washed, and incubated with 2 μ m sphingosine. Controls consisted of protein A/G–agarose only, incubated with 2 μ m sphingosine. The samples were washed and extracted in CHCl 3 :CH 3 OH:1N HCl (100:100:1, v/v/v), and sphingosine was quantified employing a kinase assay. Given are the means ± S.D. of the sphingosine concentrations from six independent experiments. ***, p < 0.001, ANOVA followed by post hoc Student's t tests. C , a panel of lipid-coated beads was used to test for the specificity of ACE2-binding to sphingosine-coupled beads. The samples were prepared as above. Shown is a representative result from four independent experiments. D , recombinant Fc-ACE2 protein was immobilized on protein A/G–agarose, washed, incubated with 2 μ m sphingosine or left untreated, washed again, and incubated with the recombinant receptor-binding domain of the spike protein. The samples were washed extensively, eluted, separated by 7.5% SDS-PAGE electrophoresis, blotted, and developed with anti-spike antibodies. The recombinant receptor-binding domain of the spike protein is His-tagged and has a molecular mass of ∼27 kDa. Protein A/G–agarose beads without Fc-ACE2 served as control. Shown are representative results from five independent experiments. E , a variety of other lipids were added as indicated to immobilized recombinant human Fc-ACE2. The effects of these lipids on binding of recombinant receptor-binding domain of the spike protein were determined by Western blotting. Shown is a representative result from four independent studies. F , recombinant His-tagged RBD of spike was immobilized on Ni 2+ –agarose, washed, incubated with 2 μ m sphingosine, or left untreated. Recombinant human Fc-ACE2 was added, and the samples were incubated for 60 min. Beads without the addition of RBD spike served as control. The samples were washed extensively, eluted, separated by 7.5% SDS-PAGE electrophoresis, blotted, and developed with anti-ACE2 antibodies. Shown are representative results from five independent experiments. G , a variety of other lipids were added as indicated to immobilized recombinant RBD–spike protein. The effects of these lipids on binding of recombinant human Fc-ACE2 were determined by Western blotting. Shown is a representative result from four independent studies. PC , phosphatidylcholine; PE , phosphatidylethanolamine; SM , sphingomyelin; CER , ceramide; S1P , sphingosine 1-phosphate; Clp , cardiolipin; PS , phosphatidylserine; C16-CER , C16-ceramide; LC , lactosylceramide; OGP , octylglucopyranoside.

Article Snippet: The following lipids were immobilized to agarose: sphingosine (Sphingobeads, Echelon, catalog no. S-6100), phosphatidylcholine (Echelon, catalog no. P-BOPC), phosphatidylethanolamine (Echelon, catalog no. P-BOPE), sphingomyelin (Echelon, catalog no. P-BOSM), ceramide (Echelon, catalog no. P-BCER), sphingosine 1-phosphate (Echelon, catalog no. S6110, cardiolipin (Echelon, catalog no. P-BCLP), or unloaded control beads (Echelon, catalog no. P-8000).

Techniques: Incubation, Recombinant, Binding Assay, SDS Page, Electrophoresis, Kinase Assay, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Sphingosine prevents binding of SARS–CoV-2 spike to its cellular receptor ACE2

doi: 10.1074/jbc.RA120.015249

Figure Lengend Snippet: Sphingosine binds to ACE2 and prevents binding of pp-VSV–SARS–CoV-2 spike. A , recombinant ( rec .) Fc-ACE2 protein was immobilized ( imm .) on protein A/G–agarose, washed, incubated with 1 or 2 μ m sphingosine ( SPH ) or left untreated, washed to remove unbound sphingosine, and incubated with pp-VSV–SARS–CoV-2 spike. Protein A/G–agarose without recombinant Fc-ACE2 served as control. The samples were pelleted, the supernatants containing unbound virus were collected, and Vero cells were infected with the supernatants for 24 h. The results show that pp-VSV–SARS–CoV-2 was bound to recombinant Fc-ACE2, which was prevented by sphingosine. Displayed are the means ± S.D. of the percentage of infected cells from eight independent experiments. ***, p < 0.001, ANOVA followed by post hoc Student's t tests. B , Vero cells were incubated for 30 min with each 2 μ m sphingosine, phosphatidylcholine, sphingomyelin, C16-ceramide, sphingosine 1-phosphate, lactosylceramide, or cardiolipin or were left untreated and washed, and pp-VSV–SARS–CoV-2 spike was added, and the lipids were reconstituted to the same concentration as before. The cells were incubated for 20 min. The cells were then removed from the plate, stained with FITC-coupled anti-spike antibodies, and analyzed by flow cytometry. Shown is a representative result from four independent experiments and the quantitative analysis of the fluorescence intensity. ***, p < 0.001, ANOVA followed by post hoc Student's t tests. Fluorescence is given in arbitrary units (a.u.).

Article Snippet: The following lipids were immobilized to agarose: sphingosine (Sphingobeads, Echelon, catalog no. S-6100), phosphatidylcholine (Echelon, catalog no. P-BOPC), phosphatidylethanolamine (Echelon, catalog no. P-BOPE), sphingomyelin (Echelon, catalog no. P-BOSM), ceramide (Echelon, catalog no. P-BCER), sphingosine 1-phosphate (Echelon, catalog no. S6110, cardiolipin (Echelon, catalog no. P-BCLP), or unloaded control beads (Echelon, catalog no. P-8000).

Techniques: Binding Assay, Recombinant, Incubation, Infection, Concentration Assay, Staining, Flow Cytometry, Fluorescence