The ACE2 Receptor for Coronavirus Entry Is Localized at Apical Cell-Cell Junctions of Epithelial Cells.

Florian Rouaud,Sandra Citi,Isabelle Méan

doi:10.3390/cells11040627

Abstract

Transmembrane proteins of adherens and tight junctions are known targets for viruses and bacterial toxins. The coronavirus receptor ACE2 has been localized at the apical surface of epithelial cells, but it is not clear whether ACE2 is localized at apical Cell—Cell junctions and whether it associates with junctional proteins. Here we explored the expression and localization of ACE2 and its association with transmembrane and tight junction proteins in epithelial tissues and cultured cells by data mining, immunoblotting, immunofluorescence microscopy, and co-immunoprecipitation experiments. ACE2 mRNA is abundant in epithelial tissues, where its expression correlates with the expression of the tight junction proteins cingulin and occludin. In cultured epithelial cells ACE2 mRNA is upregulated upon differentiation and ACE2 protein is widely expressed and co-immunoprecipitates with the transmembrane proteins ADAM17 and CD9. We show by immunofluorescence microscopy that ACE2 colocalizes with ADAM17 and CD9 and the tight junction protein cingulin at apical junctions of intestinal (Caco-2), mammary (Eph4) and kidney (mCCD) epithelial cells. These observations identify ACE2, ADAM17 and CD9 as new epithelial junctional transmembrane proteins and suggest that the cytokine-enhanced endocytic internalization of junction-associated protein complexes comprising ACE2 may promote coronavirus entry.

Highlights

The apical junctional complex of epithelial cells consists of tight junctions (TJ) and adherens junctions (AJ), and TJ forms a critical barrier to the entry of pathogens into the organism [1]
The strongest coexpression of ACE2, CD9 and ADAM17 mRNAs was found to occur in intestinal, kidney, breast and lung epithelial tissues (Figure 1B)
Since epithelial tissues and cells are characterized by the presence of tight junctions (TJ), we evaluated the expression of the TJ proteins cingulin (CGN) and occludin (OCLN)

Summary

Introduction

The apical junctional complex of epithelial cells consists of tight junctions (TJ) and adherens junctions (AJ), and TJ forms a critical barrier to the entry of pathogens into the organism [1]. C virus, measles virus, herpes virus and polioviruses exploit transmembrane TJ and AJ proteins, such as JAM-A, CAR, nectins and claudins to gain access to the cell interior and spread in tissues (reviewed in [2–5]). Clostridium perfringens enterotoxin and Staphylococcus aureus α-toxin bind to transmembrane junctional proteins of TJ and AJ, such as claudins [6] and ADAM10 [7], and affect epithelial junction integrity to exert their toxic effects (reviewed in [5,8,9]). It is not clear whether coronaviruses use transmembrane junctional proteins as receptors and gateways for entry into cells. Transcript, immunocytochemistry and immunofluorescence microscopy (IF) analyses show that ACE2 is expressed in epithelial cells of human airways, olfactory, kidney, intestinal tissues, and in endothelial cells [17–22]

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Conclusion