ACE2 binding to viral S-protein

Data supporting ACE2 as a cell entry receptor for SARS-CoV-2 as a key event (Kim Young Jun et al).

Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is initiated by virus binding to the ACE2 cell-surface receptors (Nature 579, 270–273 ; J. Virol. 94, e00127-20; Nature 588, 327–330) The SARS-CoV-2 surface spike (S) protein accommodates the entry of SARS-CoV-2 into the target cells by binding to membrane ACE2 and the subsequent internalized together with viral particles into endosomes results in a significant decrease in soluble ACE2 plasma levels. Through bioinformatics analysis, this S protein is cleaved by TMPRSS2 serine protease into the S1 and S2 subunits, which are responsible for ACE 2 receptor recognition and membrane fusion, respectively. The S1 subunit contains a receptor-binding domain (RBD) encompassing the receptor-binding motif (RBM) to bound the ACE2. The S2 contains a fusion peptide (FP), penetrate into cell membrane to enhance fusion with the viral membrane.

Binding of S protein to ACE2 receptors present in the brain (endothelial, neuronal and glial cells)  (in relation to COVID19):

The highest ACE2 expression level in the brain was found in the pons and medulla oblongata in the human brainstem, containing the medullary respiratory centers, and this may in part explain the susceptibility of many COVID-19 patients to severe respiratory distress (Lukiw et al., 2020). High ACE2 receptor expression was also found in the amygdala, cerebral cortex and in the regions involved in cardiovascular function and central regulation of blood pressure including the sub-fornical organ, nucleus of the tractus solitarius, paraventricular nucleus, and rostral ventrolateral medulla (Gowrisankar and Clark 2016; Xia and Lazartigues 2010).

The neurons and glial cells, like astrocytes and microglia also express ACE-2, thus highlighting the vulnerability of the nervous system to SARS-CoV-2 infection. Additionally, they also express transmembrane serine protease 2 (TMPRSS2) and furin, which facilitate virus entry into the host (Jakhmola et al. 2020).

Once inside the brain, the virus can infect the neural cells, astrocytes, and microglia. These cells express ACE-2, thus initiating the viral budding cycle followed by neuronal damage and inflammation (Jakhmola et al. 2020). Specifically in the brain, ACE2 is expressed in endothelium and vascular smooth muscle cells (Hamming et al., 2004), as well as in neurons and glia (Gallagher et al., 2006; Matsushita et al., 2010; Gowrisankar and Clark, 2016; Xu et al., 2017; de Morais et al., 2018) (from Murta et al., 2020).

Astrocytes are the main source of angiotensinogen and express ATR1 and MasR; neurons express ATR1, ACE2, and MasR, and microglia respond to ATR1 activation (Shi et al., 2014; de Morais et al., 2018).

Binding of S protein to ACE2 receptors present in the intestines

The highest levels of ACE2 are found at the luminal surface of the enterocytes, the differentiated epithelial cells in the small intestine, lower levels in the crypt cells and in the colon (Liang et al, 2020; Hashimoto et al., 2012, Fairweather et al. 2012; Kowalczuk et al. 2008).

In vitro methods supporting interaction between ACE2 and SARS-CoV-2 spike protein (Kim Young Jun et al)

Methodology to identify and prioritize Key Events (KEs) relevant for MIE, Several reports using surface plasmon resonance (SPR) or biolayer interferometry binding (BLI) approaches. to study the interaction between recombinant ACE2 and S proteins have determined a dissociation constant (Kd) for SARS-CoV S and SARS-CoV-2 S as follow,

Pseudo typed vesicular stomatitis virus expressing SARS-CoV-2 S (VSV-SARS-S2) expression system can be used efficiently infects cell lines, with Calu-3 human lung adenocarcinoma epithelial cell line, CaCo-2 human colorectal adenocarcinoma colon epithelial cell line and Vero African grey monkey kidney epithelial cell line being the most permissive (Hoffmann et al., 2020; Ou et al., 2020).  It can be measured using a wide variety of assays targeting different biological phases of infection and altered cell membrane permeability and cell organelle signaling pathway. Other assay measured alteration in the levels of permissive cell lines all express ACE2 or hACE2-expressing 293T cell (e.g. pNUO1-hACE2, pFUSE-hIgG1-Fc2), as previously demonstrated by indirect immunofluorescence (IF) or by immunoblotting are associated with ELISA(W Tai et al., nature 2020). To prioritize the identified potential KEs for selection and to select a KE to serve as a case study, further in-silico data that ACE2 binds to SARS-CoV-2 S is necessary for virus entry. The above analysis outlined can be used evidence-based assessment of molecular evidence as a MIE.

Receptor recognition is an essential determinant of molecular level in this AOP. ACE2 was reported as an entry receptor for SARS-CoV-2. The viral entry process is mediated by the envelope-embedded surface-located spike (S) glycoprotein.  Jun Lan and Walls, A.C et al (Nature 581, 215–220; Cell 180, 281–292) demonstrated a critical initial step of infection at the molecular level from the interaction of ACE2 and S protein. ACE2 has shown that receptor binding affinity to S protein is nM range. To elucidate the interaction between the SARS-CoV-2 RBD and ACE2 at a higher resolution, they also determined the structure of the SARS-CoV-2 RBD–ACE2 complex using X-ray crystallography. The expression and distribution of the ACE2 in human body may indicate the potential infection of SARS-CoV-2. Through the developed single-cell RNA sequencing (scRNA-Seq) technique and single-cell transcriptomes based on the public database, researchers analyzed the ACE2 RNA expression profile at single-cell resolution. High ACE2 expression was identified in type II alveolar cells (Zou, X. et al. Front. Med.2020)

A table on expression levels according to tissues:(Kim Young Jun et al)

SARS-CoV-2 belongs to the Coronaviridae family, which includes evolutionary related enveloped (+) strand RNA viruses of vertebrates, such as seasonal common coronaviruses, SARS-CoV and CoV-NL63, SARS-CoV (Kim Young Jun et al)

COVID19 References related to CNS:

de Morais SDB, et al. Integrative Physiological Aspects of Brain RAS in Hypertension. Curr Hypertens Rep. 2018 Feb 26; 20(2):10.

Gallagher PE, et al. Distinct roles for ANG II and ANG-(1-7) in the regulation of angiotensin-converting enzyme 2 in rat astrocytes. Am J Physiol Cell Physiol. 2006 Feb; 290(2):C420-6.

Gowrisankar YV, Clark MA. Angiotensin II regulation of angiotensin-converting enzymes in spontaneously hypertensive rat primary astrocyte cultures. J Neurochem. 2016 Jul; 138(1):74-85.

Hamming I et al. Tissue distribution of ACE2 protein, the functional receptor for SARS coronavirus. A first step in understanding SARS pathogenesis. J Pathol. 2004 Jun;203(2):631-7.

Jakhmola S, et al. SARS-CoV-2, an Underestimated Pathogen of the Nervous System. SN Compr Clin Med. 2020.

Lukiw WJ et al. SARS-CoV-2 Infectivity and Neurological Targets in the Brain. Cell Mol Neurobiol. 2020 Aug 25;1-8.

Matsushita T, et al. CSF angiotensin II and angiotensin-converting enzyme levels in anti-aquaporin-4 autoimmunity. J Neurol Sci. 2010 Aug 15; 295(1-2):41-5.

Murta et al. Severe Acute Respiratory Syndrome Coronavirus 2 Impact on the Central Nervous System: Are Astrocytes and Microglia Main Players or Merely Bystanders? ASN Neuro. 2020. PMID: 32878468

Shi A, et al. Isolation, purification and molecular mechanism of a peanut protein-derived ACE-inhibitory peptide. PLoS One. 2014; 9(10):e111188.

Xia, H. and Lazartigues, E.  Angiotensin-Converting Enzyme 2: Central Regulator for Cardiovascular Function. Curr. Hypertens. 2010  Rep. 12 (3), 170– 175