Binding of the SARS-CoV-2 Spike Protein to the Asialoglycoprotein Receptor on Human Primary Hepatocytes and Immortalized Hepatocyte-Like Cells by Confocal Analysis

Abstract

Background: The SARS-CoV-2 virus may have direct or indirect effects on other human organs beyond the respiratory system and including the liver, via binding of the spike protein. This study investigated the potential direct interactions with the liver by comparing the binding of SARS-CoV-2 spike proteins to human AT2-like cells, primary human hepatocytes and immortalized hepatocyte-like hybrid cells. Receptors with binding specificity for SARS-CoV-2 spike protein on AT2 cells and hepatocytes were identified.

Methods: The specific binding of biotinylated spike and spike 1 proteins to undifferentiated human E12 MLPC (E12), E12 differentiated alveolar type 2 (AT2) cells, primary human hepatocytes (PHH) and E12 human hepatocyte-like hybrid cells (HLC) was studied by confocal microscopy. We investigated the expression of ACE-2, binding of biotinylated spike protein, biotinylated spike 1 and inhibition of binding by unlabeled spike protein, two neutralizing antibodies and an antibody directed against the hepatocyte asialoglycoprotein receptor 1 (ASGr1).

Results: E12 MLPC did not express ACE-2 and did not bind either of spike or spike 1 proteins. AT2-like cells expressed ACE-2 and bound both spike and spike 1. Both PHH and HLC did not express ACE-2 and did not bind spike 1 protein. However, both PHH and HLC actively bound the spike protein. Biotinylated spike protein binding was inhibited by unlabeled spike but not spike 1 protein on PHH and HLC. Two commercial neutralizing antibodies blocked the binding of the spike to PHH and HLC but only one blocked binding to AT2. An antibody to the hepatocyte ASGr1 blocked the binding of the spike protein to PHH and HLC.

Conclusion: The absence of ACE-2 receptors and inhibition of spike binding by an antibody to the ASGr1 on both PHH and HLC suggested that the spike protein interacts with the ASGr1. The differential antibody blocking of spike binding to AT2, PHH and HLC indicated that neutralizing activity of SARS-CoV-2 binding might involve additional mechanisms beyond RBD binding to ACE-2.

Keywords: AT2; E12 MLPC; SARS-CoV-2; asialoglycoprotein receptor; human hepatocytes; spike proteins.

Figure 1

Biotinylated spike and spike 1 protein binding to E12 differentiated AT2-like cells and inhibition by unlabeled spike protein and neutralizing antibodies. Bound biotinylated spike proteins were visualized by sequential labeling with streptavidin-alexa 594. Cells positive for binding are shown by red fluorescence. Blue nuclei were visualized by counterstaining with DAPI. (A) Binding of biotinylated spike protein (containing RBD). (B) Inhibition of biotinylated spike protein binding by co-incubation with a 5 molar excess of unlabeled spike protein (RBD). (C) Binding of spike 1 protein. (D) Inhibition of biotinylated spike protein binding by preincubation with a neutralizing antibody from ACROBiosystems. (E) Lack of binding inhibition by neutralizing antibody from Novatein Bio. (F) Inhibition of biotinylated spike 1 binding by unlabeled spike (RBD) protein.

Figure 2

Expression of ACE-2, ASGr1 and serum albumin. Undifferentiated E12 MLPC data are shown in (A–D). E12 HLC fusion cell results are presented in (E–H). Primary human hepatocytes (PHH) are shown in (I–L). Cells were incubated with unlabeled primary antibody and sequentially stained with secondary antibody labeled with alexa-594. Positive binding is shown by red fluorescence. Blue nuclei were visualized by DAPI counterstaining. Figures (A, E and I) were stained with isotype control antibodies. Figures (B, F and J) were stained with antibody specific for ACE-2. Figures (C, G and K) were stained with antibody specific for the asialoglycoprotein receptor 1 (ASGr1). Figures (D, H and L) were stained with antibody specific for serum albumin.

Figure 3

Undifferentiated E12 MLPC confocal microscopy is shown in (A–D). E12 HLC fusion cell data are presented in (E-H). Primary human hepatocytes (PHH) are shown in (I–L). Positive binding is indicated by red fluorescence. Blue nuclei were visualized with DAPI counterstaining. Figures (A, E and I) were labeled with Sav-594. Figures (B, F and J) were labeled with biotinylated spike protein followed by sequential staining with streptavidin-alexa 594. Figures (C, G and K) were labeled with biotinylated spike 1 protein followed by sequential staining with streptavidin-alexa 594. Figures (D, H and L) biotinylated spike protein binding was blocked by a 5 molar excess of unlabeled spike protein (RBD) followed by sequential staining with streptavidin-alexa 594.

Figure 4

Inhibition of biotinylated spike binding by neutralizing antibodies to spike 1, spike and ASGr1. E12 HLC fusion cell data are shown in (A–D). Primary human hepatocytes (PHH) are shown in (E–H). Positive binding of biotinylated spike proteins is shown by red fluorescence. Blue nuclei are visualized by counterstaining with DAPI. Figures (A and E) confocals show the inability of a 5 molar excess of unlabeled spike 1 protein to block the binding of biotinylated spike protein. Figures (B and F) show inhibition of binding of biotinylated spike protein by neutralizing antibody from ACROBiosystems. Figures (C and G) show binding inhibition of biotinylated spike protein by neutralizing antibody from Novatein Bio. Figures (D and H) demonstrate inhibition of any detectable binding of biotinylated spike protein by antibody specific for the hepatocyte membrane ASGr1.