In a recent preprint study posted to Research Square* and under consideration at the journal Human Genomics, researchers investigated the connecting link between viral genetic variability and clinical heterogeneity of coronavirus disease 2019 (COVID-19) by analyzing the effect of several missense mutations on the angiotensin-converting enzyme (ACE2) receptor coding sequence.
Study: Characterization of ACE2 naturally occurring missense variants: impact on subcellular localization and trafficking. Image Credit: Kateryna Kon/Shutterstock
Although essential for the physiological functioning of the renal renin-angiotensin system (RAS), the ACE2 receptor facilitates transmembrane invasion of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into cells of the human body by the attachment of the spike (S) glycoprotein of the virus onto its surface.
This news article was a review of a preliminary scientific report that had not undergone peer-review at the time of publication. Since its initial publication, the scientific report has now been peer reviewed and accepted for publication in a Scientific Journal. Links to the preliminary and peer-reviewed reports are available in the Sources section at the bottom of this article. View Sources
The researchers hypothesized that ACE2 genomic variations could bring about diversifications in the clinical presentation and differing inter-individual severity of COVID-19. This implies that ACE2, the primary mediator of viral entry, could be a potential target of therapeutic strategies against SARS-CoV-2 infections.
ACE2 is a type I metalloproteinase receptor protein produced in the endoplasmic reticulum (ER) and transferred to the plasma membrane and cellular surface after undergoing folding and post-translational alterations in the Golgi apparatus. This trafficking is regulated by the endoplasmic reticulum-associated degradation (ERAD) system.
Previous studies have suggested that genomic variations could affect the binding affinity of viral S proteins to the surface of ACE2 receptors. However, data on the effect of missense mutations on the subcellular folding, localization, targeting, and trafficking of ACE2 are sparse.
About the study
The researchers generated 28 ACE2 receptor missense mutant variants in several cell lines in the present study. These cells were then subjected to immunofluorescence analysis for the estimation of protein expression and western blot testing for the quantification of proteins.
The proteins were overexpressed for a day, after which a protein synthesis inhibitor was added to assess the half-life, turnover, and stability of ACE2. The potential effect of genomic missense mutant variants on intracellular trafficking and localization was evaluated using confocal microscopy (CFM) and confirmed by their N-glycosylation results. Data obtained from the Ensembl database was subjected to a combination of bioinformatic tools and experimental analysis.
Results and discussion
The present study results have indicated that from a total of 28 mutant variants generated, 25 variants, for example, the R710H variant located especially at the neck region of protein dimers, decreased protein molecular structure stability. Thirteen mutant variants caused functional alterations, and only three variants (R768W, G173S, and G575V) were destructive. The highest molecular instability was brought about by the N33D, F592L, I21T, V801G, and D785N variants.
Ten substitutions at specific positions in the amino acid sequence, such as the substitution of serine with proline, isoleucine with threonine, and asparagine to aspartic acid, along with the E668K missense mutation led to disruptions in protein conformation. Protein modeling analysis results indicated that the majority (13/28) of variants were situated in the peptidase region of the ACE2 structure, a prospective site for binding of the viral spike proteins.
The presence of the S19P missense variant along with the generation of amino acid residues such as K26, D38, and N33 increased viral glycoprotein-ACE2 receptor binding. The presence of S692P and R708W variants contributed to improper protein folding, while the R768W variant exerted a profound impact on the ACE2 structure and the G173 variant affected the ACE2 catalytic activity. The expressed proteins had a half-life of 12 hours. ACE2 was reported to possess seven N-glycosylation sites.
Conclusion
The present study concluded that the generation of missense mutant variants in the endogenous ACE2 receptor protein did not affect its intracellular trafficking, targeting, and localization. The loss of proper intracellular trafficking and localization mechanisms of ACE2 receptors would otherwise lead to loss of their protein function.
Since ACE2 serves important physiological and biological functions as an enzyme in the human body, the opinion of the authors based on the present research resonates with the hypothesis that functional loss in the ACE2 receptor protein is intolerable.
Studies evaluating the role of patient demographic factors such as gender and age, lifestyle factors as well as potential confounding variables such as the presence of underlying medical disorders such as obesity, diabetes, hypertension, and cardiovascular disease should be carried out as these could affect the host immune response and contribute to the varied presentation and severity of COVID-19 among different individuals.
This news article was a review of a preliminary scientific report that had not undergone peer-review at the time of publication. Since its initial publication, the scientific report has now been peer reviewed and accepted for publication in a Scientific Journal. Links to the preliminary and peer-reviewed reports are available in the Sources section at the bottom of this article. View Sources
Article Revisions
- May 12 2023 - The preprint preliminary research paper that this article was based upon was accepted for publication in a peer-reviewed Scientific Journal. This article was edited accordingly to include a link to the final peer-reviewed paper, now shown in the sources section.
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