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

Abstract

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.

Genetic analysis of ACE2 peptidase domain in SARS-CoV-2-positive and SARS-CoV-2-negative individuals from Pakistan

“…Consistently, this variant has been previously classified as a benign variant using various in silico analysis tools including SIFT, MUTTASTER, PROVEAN, PolyPhen-2, CADD, and REVEL [24,31,32]. Other studies, based on structural modeling and molecular docking simulations suggested that the p.Lys26Arg increased binding affinity of SARS-CoV-2 spike protein to the ACE2 receptor and showed increased SARS-CoV-2 infection as compared to the wildtype ACE2 [9,14,24,26,33,34].…”

Genetic analysis of ACE2 peptidase domain in SARS-CoV-2-positive and SARS-CoV-2-negative individuals from Pakistan

“…In the current study, p.Lys26Arg was predicted as a benign variant using VarCards in silico analysis tool. Consistently, this variant has been previously classi ed as a benign variant using various in silico analysis tools including SIFT, MUTTASTER, PROVEAN, PolyPhen-2, CADD, and REVEL [24,32,33]. Other studies, based on structural modeling and molecular docking simulations suggested that the p.Lys26Arg increased binding a nity of SARS-CoV-2 spike protein to the ACE2 receptor and showed increased SARS-CoV-2 infection as compared to the wildtype ACE2 [9,14,24,26,34,35].…”

Unveiling the pathogenic mechanisms of NPR2 missense variants: insights into the genotype-associated severity in acromesomelic dysplasia and short stature

“…HEK293T and HeLa cells were cultured in Dulbecco's Modified Eagle Medium (Gibco) supplemented with 10% fetal bovine serum (Gibco), and antibiotic-antimycotic (Gibco) at 37 °C and 5% CO 2 as recently described (Badawi et al, 2022). Cells were grown in 6-well and 24-well tissue culture plates.…”