Fig. 2: Recombinant Spike protein of SARS-CoV-2 downregulates ACE2 expression.

a Constructs of recombinant Spike proteins; Spike-6P, S1-Fc, and RBD-Fc. b Binding of S1-Fc, RBD-Fc or Spike-6P to human ACE2 and hamster ACE2 in pull-down assays. S1-Fc, RBD-Fc, and Spike-6P but not control-Fc or vehicle pulled down human ACE2 and hamster ACE2 from lysates of Caco2 human colon cells and hamster lungs, respectively. Total lysates are shown as controls. * unfolded or short form of ACE2 or non-specific band. Western Blot of S1-Fc, RBD-Fc or Spike-6P are shown in Supplementary Fig. 1d–g. c, d Decreased cell-surface expression of ACE2 after binding to Spike RBD-Fc protein at 37 °C compared to 4 °C in Vero E6 cells. ACE2 surface expression was detected at 3 h of incubation with RBD-Fc using Fc-specific antibody to directly detect surface-bound RBD-Fc and to avoid masking of the ACE2 epitope. Representative FACS histograms (c) and quantification of mean fluorescence intensity (d) are shown including a background control with an isotype-matched antibody (n = 3 per group). FSC/SSC plot was gated for live and healthy Vero E6 cells. One-way ANOVA with Sidak’s multiple comparisons test. Numbers above square brackets show P values. e Experimental protocol of acid and Spike protein (Spike-6P, S1-Fc or RBD-Fc)-induced lung injury in hamsters. S1-Fc, RBD-Fc, control-Fc (11 nmol/kg for each) or Spike-6P (3.7 nmol/kg) was intraperitoneally injected, and acid was intratracheally instilled (0.1 N HCl, 100 μl per body i.t.) under anesthesia. f Immunohistochemistry of hamster lungs to detect S1-Fc, RBD-Fc or control-Fc protein using a human Fc–specific antibody. Injected S1-Fc or RBD-Fc but not control-Fc localizes to bronchial epithelial cells, inflammatory cells and alveolar pneumocytes. Bars indicate 100 μm. g–j ACE2 protein expression in hamster lungs. Representative Western blot (g, i) and quantification of ACE2 protein abundance (h, j) are shown. * unfolded or short form of ACE2 or non-specific band. All values are means ± SEM. n = 5 hamsters per group and two-way ANOVA with Sidak’s multiple comparisons test (h) or n = 4 hamsters per group and two-tailed unpaired t-test (j). Numbers above square brackets show significant or nearly significant P values. Independent experiments were performed one time (b–d) or two times (f–j), and consistent results were obtained.