The Role of ACE2 in SARS-CoV-2

The global spread of the novel coronavirus has sparked international interest in understanding the cellular mechanisms of host infiltration. SARS-Cov-2 (formerly 2019-nCoV) has been identified as the causative viral agent of the disease COVID-19.  

Recent efforts to understand SARS-CoV-2 have yielded crucial knowledge in the fight against coronaviruses. Angiotensin-converting enzyme 2 (ACE2) has been implicated as the cellular receptor which facilitates uptake of the virus into the host cell. Thanks to the tireless efforts of researchers around the world, further understanding of the structural and functional features of the SARS-CoV-2 virus has been made publically available.

As the number of cases continues to rise, it is of critical importance that diagnostic kits be made available to researchers. Detection kits for SARS-CoV-2 and assay kits for ACE2 activity and inhibition are available. Assay Genie has recently released a SARS-CoV-2 Detection Kit which utilises one-step RT-qPCR for coronavirus detection. Similarly, multiple ACE2 assays to measure the activity of this key enzyme are available below.

Details about the role of ACE2 in SARS-Cov-2/COVID-19 infection— as well as relevant assays and kits for detection— are detailed here.

SARS-CoV-2 shares many features with other coronaviruses. It is part of the subgenus Sarbecovirus (Beta-CoV lineage B) which was responsible for a similar disease outbreak in the early 2000s. Unique and relevant structural features are outlined below.

> The four base structural proteins found in CoV viruses are: spike (S), envelope (E), membrane (M), and nucleocapsid (N).

> The transmembrane spike (S) glycoprotein is a homotrimeric protrusion from the surface of the virus. This is a key target for therapeutic intervention and for immune antibodies.

> S is heavily glycosylated with N-linked glycans. These glycans are necessary for proper folding. (Rossen et al., 1998)

> S is functionally divided into two subunits, named S1 and S2.

    > S1 subunit: binds to the host cell receptor and contains receptor binding domain (RBD).

    > S2 subunit: fuses the viral and cellular membranes

> A furin cleavage site at the boundary of the two functional S subunits of SARS-CoV-2 was recently discovered. It is cleaved during biosynthesis, making it a novel feature unique to the CoV-2 strain.

> SARS-CoV-2 is a positive-sense, single-stranded RNA virus, approximately 30,000 bp long.

Fig 1: Schematic labelled diagram of SARS-CoV particle. Nucleocapsid protein (N), membrane glycoprotein (M), Spike protein (S). (Finlay et al., 2004)

> Entry to the host cell is facilitated by ACE2 and S1/S2 interaction.

> S is a class 1 viral fusion protein that allows host attachment and enables the viral and cellular membranes to fuse. (Bosch et al., 2003)

> The distal S1 subunit comprises the receptor-binding domains, and helps stabilise the prefusion state of the membrane-bound S2 subunit that contains the machinery necessary for host fusion.

> In all coronaviruses, S is further cleaved by host proteases at the S2 site immediately upstream of the fusion peptide. This cleavage activates the protein for membrane fusion via permanent and extensive conformational changes.

> Coronavirus entry into vulnerable cells requires receptor-binding and proteolytic processing of the S protein to facilitate fusion of the virus to the cell.

> SARS-CoV-2 has a higher affinity to human ACE2 than the original SARS virus strain.

Angiotensin converting enzyme II (ACE2) is part of the renin-angiotensin system (RAS) that controls the regulation of blood pressure by cleaving the C-terminal dipeptide of Angiotensin II to convert it into Angiotensin 1-7. ACE2 is a receptor of human coronaviruses, such as SARS-CoV-2.

> The angiotensin-converting enzyme 2 is a zinc metalloprotease and carboxypeptidase that is similar in structure to human ACE-1.

> Localisation: Alveolar epithelial cells and enterocytes in the small intestine are target cells of SARS-CoV-2 infection. ACE2 is expressed and active in various cell types including alveolar epithelial cells, surface enterocytes of the small intestine, and heart and kidney endothelial cells.

> Structure: ACE2 is a type I integral membrane protein, meaning that the N-terminus domain of the protein points towards the extracellular domain of the cell leading to a more negatively net charge on the cell.

> Amino Acid Sequence: The protein is made up of 805 amino acids. The type I integral protein contains a 17 AA N-terminal sequence and a 22 AA hydrophobic transmembrane sequence close to the C-terminus. In addition, the protein has a cytoplasmic domain consisting of 43 amino acids. This domain contains potential phosphorylation sites.

> In addition, the protein contains a zinc binding domain which is involved in signal transduction pathways in alveolar epithelial cells and enterocytes when activated.

> It has been noted that the first SARS-CoV elicited polyclonal antibody responses which potently neutralised SARS-CoV-2, indicating that there is high structural similarity between the two strains. The produced antibodies against SARS-CoV-2 targeted the S2 subunit, including the fusion peptide region.

> Neutralisation of this subunit prevents viral entry into target cells.

> ACE2 is an entry receptor for SARS-CoV-2.

> The receptor binding domain on S1, located at residues 318–510, recognises and binds to the LYS341 residue of ACE2 with high affinity.

> ACE2 is involved in the regulation of cardiovascular and renal function and also regulates blood pressure.

> Loss of ACE2 can be detrimental, as it leads to the impaired function of the heart and progression of cardiac, renal, and vascular pathologies.

> The binding of the RBD to ACE2 mediates the recruitment of a host protease to cleave the spike protein. This leads to the production of a spike fusion peptide which mediates viral entry into the target cell.

> ACE2 plays an important role in viral entry by facilitating infection. Onset of COVID-19 leads to alveolar cell damage and death which contributes to the progression of the disease.

Assay Genie has recently launched a COVID-19 Rapid POC CE-IVD Test. It is a 10 minute test which qualitatively detects severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from patient samples. Using a rapid COVID-19 test provides an opportunity to identify more individuals who are unaware they have been infected.

The COVID-19 Rapid POC CE-IVD Test is a qualitative membrane-based immunoassay for the detection of IgG and IgM patient-generated antibodies to the SARS-CoV-2 virus, using whole blood, serum or plasma specimens.

This kit utilises the ability of an active ACE2 to cleave a synthetic MCA based peptide substrate to release a free fluorophore. The released MCA can be easily quantified using a fluorescence microplate reader. Also provided is an ACE2 specific inhibitor that can differentiate the ACE2 activity from other proteolytic activity. The kit can detect as low as 0.4 mU. The assay kit is simple and can be used in a high-throughput format.

The ACE2 Activity Assay Kit contains ACE2 assay buffer, ACE2 dilution buffer, ACE2 lysis buffer, ACE2 positive control, ACE2 substrate, ACE2 inhibitor (22 mM), and MCA-Standard (1 mM). It is intended for research use only.  

This kit can be used for screening, characterizing, or studying ACE2 inhibitors and activators. Screening for small molecule and peptide inhibitors might also help in finding treatment for coronavirus-mediated infections, such as COVID-19.

Assay Genie's ACE2 Inhibitor Screening Kit can be used to screen for potent inhibitors of ACE2 activity. It utilizes the ability of an active ACE2 to cleave a synthetic MCA based peptide substrate to release a free fluorophore. The released MCA can be easily quantified using a fluorescence microplate reader. In the presence of an ACE2 specific inhibitor, the enzyme loses its peptidase activity which results in the decrease of fluorescence intensity. This assay kit is user-friendly and can identify and characterize ACE2 inhibitors in a high-throughput format.

The kit contains ACE2 Assay Buffer, ACE2 Dilution Buffer, ACE2 Enzyme, ACE2 Substrate, and ACE2 Inhibitor (0.5 mM).

Fig 3. Names associated with SARS-Cov-2