Fig. 1: SARS-CoV-2 spike (S) protein highly binds to N-terminal amyloid precursor protein (APP).

A and B ACE2 protein or N-terminal APP (18-612 amino acids, APP18-612), captured on the COOH chip, can bind to S protein with an affinity constant of 34.9 or 46.0 nM, respectively, as determined by localized surface plasmon resonance (LSPR) assay. C Human embryonic kidney 293T cells (HEK293T) stably overexpressing either hACE2 (HEK293T/ACE2) or hAPP (HEK293T/APP) were plated in confocal chambers at a density of 1 × 10⁵/well for 24 h. The cells were transfected with constructs encoding SARS-CoV-2 S protein for 48 h, then fixed in 4% paraformaldehyde solution and subjected to immunofluorescence staining with anti-hACE2 antibody, anti-hAPP antibody (6E10), and anti-S protein antibody. Alexa Fluor 488 goat anti-mouse immunoglobulin G (IgG) was used to detect hACE2 (green) or hAPP (green), while Alexa Fluor 555 donkey anti-rabbit IgG was used to detect S protein (red). DAPI (4’,6-diamidino-2-phenylindole) is used as a nuclear counterstain (blue) and visualized by confocal microscopy. Scale bar 20 μm. D Validation of the interaction between SARS-CoV-2 S protein (136.9 kDa) and N-terminal APP protein. SH-SY5Y cells were co-transfected with SARS-CoV-2 S protein and APP constructs for 48 h. S protein antibody or APP antibody were primarily incubated with MAg25K protein A/G agarose beads (1 h at 37 °C) to prepare the agarose beads antibody complex. The agarose beads antibody complex was then incubated with whole-cell lysates overnight at 4 °C, followed by western blotting (WB) with anti-APP antibody (6E10); An immunoprecipitation (IP) experiment in the reverse direction followed by WB with anti-S protein antibody was performed to further confirm the association of N-terminal APP protein with S protein. IP with mouse serum was used as a blank control and the lysate as the input control.