Characterization of ACE2 naturally occurring missense variants: impact on subcellular localization and trafficking

Background: Human angiotensin-converting enzyme 2 (ACE2), a type I transmembrane receptor physiologically acting as a carboxypeptidase enzyme within the renin-angiotensin system (RAS), is a critical mediator of infection by several severe acute respiratory syndrome (SARS) corona viruses. For instance, it has been demonstrated that ACE2 is the primary receptor for the SARS-CoV-2 entry to many human cells through binding to the viral spike S protein. Consequently, genetic variability in ACE2 gene has been suggested to contribute to the variable clinical manifestations in COVID-19. Many of those genetic variations result in missense variants within the amino acid sequence of ACE2. The potential effects of those variations on binding to the spike protein have been speculated and, in some cases, demonstrated experimentally. However, their effects on ACE2 protein folding, trafficking and subcellular targeting have not been established. Results: In this study we aimed to examine the potential effects of 28 missense variants (V801G, D785N, R768W, I753T, L731F, L731I, I727V, N720D, R710H, R708W, S692P, E668K, V658I, N638S, A627V, F592L, G575V, A501T, I468V, M383I, G173S, N159S, N149S, D38E, N33D, K26R, I21T, and S19P) distributed across the ACE2 receptor domains on its subcellular trafficking and targeting through combinatorial approach involving in silico analysis and experimental subcellular localization analysis. Our data show that none of the studied missense variants (including 3 variants predicted to be deleterious R768W, G575V, and G173S) has a significant effect on ACE2 intracellular trafficking and subcellular targeting to the plasma membrane. Conclusion: Although the selected missense variants display no significant change in ACE2 trafficking and subcellular localization, this does not rule out their effect on viral susceptibility and severity. Further studies are required to investigate the effect of ACE2 variants on its expression, binding, and internalization which might explain the variable clinical manifestations associated with the infection.