key: cord-0688631-gakayv6e
authors: Bian, Jingwei; Li, Zijian
title: Angiotensin-converting enzyme 2 (ACE2): SARS-CoV-2 receptor and RAS modulator
date: 2020-10-13
journal: Acta Pharm Sin B
DOI: 10.1016/j.apsb.2020.10.006
sha: 57761d55d8f6f8a85d3bdab2bafcf9fb127dcb7c
doc_id: 688631
cord_uid: gakayv6e

The coronavirus disease 2019 (COVID-19) outbreak is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Angiotensin-converting enzyme 2 (ACE2) was rapidly identified as the critical functional receptor for SARS-CoV-2. ACE2 is well-known as a counter-regulator of the renin-angiotensin system (RAS) and plays a key role in the cardiovascular system. Given that ACE2 functions as both a SARS-CoV-2 receptor and a RAS modulator, the treatment for COVID-19 presents a dilemma of how to limit virus entry but protect ACE2 physiological functions. Thus, an in-depth summary of the recent progress of ACE2 research and its relationship to the virus is urgently needed to provide possible solution to the dilemma. Here, we summarize the complexity and interplay between the coronavirus, ACE2 and RAS (including anti-RAS drugs). We propose five novel working modes for functional receptor for SARS-CoV-2 infection and the routes of ACE2-mediated virus entering host cells, as well as its regulatory mechanism. For the controversy of anti-RAS drugs application, we also give theoretical analysis and discussed for drug application. These will contribute to a deeper understanding of the complex mechanisms of underlying the relationship between the virus and ACE2, and provide guidance for virus intervention strategies.

The coronavirus disease 2019 outbreak is caused by a novel coronavirus, which was named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Globally, as of 16 May 2020, there have been 4,396,392 confirmed COVID-19 cases and 300,441 deaths, which has already posed a great threat to global public health security 1 . Although respiratory symptoms are predominant, multi-organ dysfunction occurs in response to SARS-CoV-2 infections, including acute cardiac and kidney injuries, arrhythmias and liver function abnormalities 2 . At present, most patients with COVID-19 have a good prognosis; however, patients with underlying disorders have greater severity and higher mortality. 25 .2% of all patients had at least one underlying disease, particularly, hypertension (14.9%) and coronary heart disease (2.5%) 3 . Therefore, integrated treatment for COVID -19 patients with underlying diseases is key to reduce mortality. SARS-CoV-2 has been shown to share the same functional receptor, angiotensin-converting enzyme 2 (ACE2), with severe acute respiratory syndrome coronavirus (SARS-CoV) 4, 5 . ACE2 is a carboxypeptidase and a negative regulator of the renin-angiotensin system (RAS) through balancing its homology angiotensin-converting enzyme (ACE). ACE mediates angiotensin (Ang) II production to activate RAS that plays a key role in cardiovascular diseases, especially hypertension 6 . Thus, ACE inhibitor (ACEI) is used widely for treatment of hypertension, which reduces Ang II levels. Since ACE2 is a homologue of ACE, disputes have arisen about whether ACEI can upregulate ACE2 and thus the risk and severity of coronavirus infection increase. It calls into question whether to continue using of ACEI in virus-infected patients with hypertension. At present, some experts suggest that COVID-19 patients with hypertension should stop using ACEI 7, 8 . On the other hand, other experts believe that not only does ACEI not increase the risk of SARS-CoV-2 infection, but ACEI could reduce lung injury and cardiovascular damage in COVID-19 patients 9 . Thus, a comprehensive was the functional receptor of SARS-CoV-2.

Excitingly, the structures of SARS-CoV-2 and ACE2 were obtained by cryo-electron microscopy (cryo-EM) quickly. Firstly, the cryo-EM structure of the SARS-CoV-2 S-protein was determined in the prefusion conformation. The predominant state of the S-protein trimer has one of the three RBDs rotated up in a receptor-accessible conformation. The biophysical and structural evidence showed that the SARS-CoV-2 S-protein bound ACE2 with higher affinity than the SARS-CoV S-protein (approximately 10-to 20-fold) 30 , which might explain why SARS-CoV-2 is more contagious than SARS-CoV. Then, the cryo-EM structure of full-length human ACE2 in complex with a neutral amino acid transporter B 0 AT1 was revealed 31 . Immediately, the crystal structure of the RBD of SARS-CoV-2 S-protein bound with ACE2 was determined 32 . This series of work strongly confirmed that ACE2 is the functional receptor of SARS-CoV-2.

Notably, the structure of full-length human ACE2 in complex with B 0 AT1 revealed a novel mode that showed SARS-CoV-2 bound to ACE2 in a homodimer manner (Fig. 1B) . In this mode, B 0 AT1 might be a key factor in determining the formation of an ACE2 homodimer. Thus, these results suggest that SARS-CoV-2 binds with ACE2 by two working modes: i) SARS-CoV-2 binds directly with an ACE2 monomer without the B 0 AT1 protein (Fig. 1A) and ii) SARS-CoV-2 binds with ACE2 homodimer when with the existing of B 0 AT1 protein (Fig. 1B) . However, the binding affinity and biological function of these two different modes are still unclear.

ACE2 is widely expressed across a variety of organs and its expression is higher in many organs (such as heart, kidney, etc.) than that in lung. However, the lung is the major organ infected by SARS-CoV-2 33 . In addition, although ACE2 knockout mice showed a significant decrease in SARS-CoV infection, it did not completely prevent virus infection from occurring 34 . Those data suggested that there could be other receptors involved in a viral invasion. More recently, CD147, a transmembrane glycoprotein that belongs to the immunoglobulin superfamily, was identified as a receptor for SARS-CoV-2 17, 35 . Interestingly, previous studies have shown that CD147 played a key role in SARS-CoV invasion into host cells, while CD147 antagonistic peptides have an inhibitory effect on SARS-CoV 36 . These results further suggested that CD147 might be a novel receptor for SARS-CoV-2. Another possible receptor is CD209L (L-SIGN), a type transmembrane glycoprotein in the C-type lectin family 37, 38 , which has been identified as the receptor of SARS-CoV from in vitro studies 25 . Thus, given that SARS-CoV-2 is similar to SARS-CoV, CD209L could be another potential receptor for SARS-CoV-2. Therefore, besides ACE2, there are several other potential receptors for SARS-CoV-2. SARS-CoV-2 might invade cells through an alternative J o u r n a l P r e -p r o o f receptor mode (Fig. 1C ) or a co-receptors mode (Fig. 1D) .

Interestingly, viruses are very tricky, as they can also infect host cells in a detour or bait-and-switch strategy. For example, SARS-CoV can first attach to the surface of dendritic cells (DC) through the DC-SIGN receptor, which is a DC-specific intercellular adhesion molecule-3-grabbing non-integrin, a homologue of CD209L. Then the virus is presented to host cells by the DC cells and binds to ACE2 of host cell, which eventually leads to infection 26 . Similarly, SARS-CoV-2 might also use this subterfuge to infect a host cell. Since the DC-SIGN receptor plays only a transmitting role, it could be called the "transmissive receptor" (Fig. 1E ).

In summary, we propose five distinctive working modes of SARS-CoV-2 receptors: monomer receptor, homodimer receptor, alternative receptors, co-receptors and transmissive receptor. The five modes are distinct but also interconnected. 

ACE2 was discovered as a homologue of ACE in 2000 39, 40 . It plays a physiological and pathological role in cardiovascular, renal, intestinal and respiratory systems [41] [42] [43] [44] . ACE2 is a type I transmembrane protein, which consists of 805 amino acids with an extracellular N-terminal domain and a short to be internalized into cytoplasm upon virus binding 47, 48 . In addition to the membrane-bound form, the soluble form of ACE2 can be detected in the plasma and urine through its extracellular domain shedding 49, 50 . A disintegrin and metallopeptidase domain 17 (ADAM17) and type II transmembrane serine proteases (TMPRSS2) are considered as the proteases for ACE2 proteolytic cleavage 51-53 .

Identifying the distribution of ACE2 in organs has major implications for understanding the pathogenesis and treatment options for SARS-CoV-2. ACE2 was initially only detected in the heart, kidneys and testes 39, 41, 54 . Then, ACE2 mRNA expression was revealed in virtually all organs (72 human tissues) by real-time PCR 55 . In 2004, Hamming et al. 56 investigated the localization of the ACE2 protein in human organs by immunohistochemistry and ACE2 protein expression was found in various organs, including oral and nasal mucosa, nasopharynx, lung, stomach, small intestine, colon, skin, lymph nodes, thymus, bone marrow, spleen, liver, kidney, and brain. More recently, single-cell RNA-seq datasets revealed that the abundance of ACE2 expression from high to low is in the ileum, heart, kidney, bladder, respiratory tract, lung, esophagus, stomach and liver 57 . Here, we present a map of ACE2 distribution and expression in major organs based on the previous results and the BioGPS website (http://biogps.org) (Fig. 3 , left panel).

Given that ACE2 is the functional receptor of SARS-CoV-2, its expression is associated with the organic vulnerability to SARS-CoV-2. Indeed, the tissue expression of ACE2 shows some correlation with the sites of SARS-CoV-2 infection. For example, ACE2 protein is abundantly expressed in the lung and heart which are the vulnerable organs for SARS-CoV-2 33 . However, the level of ACE2 expression is not completely related with the vulnerability to SARS-CoV-2. Here, we present a comparative map of ACE2 expression and vulnerability to SARS-CoV-2 in different organs (Fig. 3 ).

Based on the comparative map, the ACE2 expression is the highest in ileum, but the ileum is not the most vulnerable organ, suggesting other complicated mechanisms might be involve in virus infection.

The possible mechanisms for inconsistency between ACE2 expression and virus vulnerability include: i) There might be other unknown receptor-mediated virus infection; ii) ACE2 alone is not enough to mediate virus infection, which needs another co-receptor's assistance (such as angiotensin II type 2 receptor (AT2R), a potential receptor for SARS-CoV-2 58 ); iii) The function of the ACE2 receptor is regulated by some protein as yet unidentified. For example, B 0 AT1, a neutral amino acid transporter also expressed in the ileum 59 , can bind ACE2 and promotes ACE2 to form a homodimer.

The formation of ACE2 homodimer hides the cleavage-sites for ACE2 ectodomain shedding, which J o u r n a l P r e -p r o o f reduces ACE2 endocytosis and further decreases virus infection in the ileum at least 31 ; and/or iv) Some proteases, notably TMPRSS2, are also involved in the virus infection. TMPRSS2 can promote the membrane fusion between virus and host cell upon ACE2 engagement and SARS-CoV-2 S-protein activation 60 .

In summary, the inconsistency between ACE2 expression and virus vulnerability reflects the complex mechanisms involved in virus infection and also further supports the proposed working modes shown in Fig. 1 .

Understanding how ACE2-mediated SARS-CoV-2 entering cell will provide valuable information for virus pathogenesis and drug target. After SARS-CoV-2 binds to ACE2, there are two routes that SARS-CoV-2 enters host cell: endocytosis and membrane fusion.

Coronaviruses, such as SARS-CoV and MERS-CoV, have been shown to enter host cells via receptor-mediated endocytosis 48, 61 . Recently, using SARS-CoV-2 S-protein pseudovirus system, it was found that, after binding with ACE2, SARS-CoV-2 was also endocytosed into cell (Fig. 4A) 62 .

Receptor-mediated endocytosis, including both clathrin-dependent pathways and caveolae-dependent pathways, is the most important mechanism for virus internalization 48, 63 .

Clathrin-dependent endocytosis is considered the primary endocytic route for the coronavirus. The infection of both SARS-CoV and MERS-CoV have been shown to occur by clathrin-dependent endocytosis 48, 61 . As an alternative to clathrin, caveolae-dependent endocytosis has been also described as the endocytic route for some viruses, such as simian virus 40 (SV40) 63 . However, the caveolae-dependent endocytosis does not seem to involve ACE2 endocytosis after SARS-CoV infection. Additionally, lipid raft-dependent endocytosis was found in SARS-CoV as a novel pathway, which is both clathrin-and caveolae-independent, may constitute a specialized high capacity endocytic pathway for lipids and fluids 47, 64 . Therefore, it is reasonable to assume that SARS-CoV-2 binds to ACE2 and might be able to either initiate clathrin-dependent and/or non-caveolae lipid raft-dependent endocytosis to enter host cell (Fig. 4A ), but these need to be further verified by biological experiments.

In addition to ACE2-mediated virus endocytosis, TMPRSS2-mediated direct membrane fusion is another important way for SARS-CoV-2 entry. Unlike the route that ACE2 binds with SARS-CoV-2 J o u r n a l P r e -p r o o f to mediated virus endocytosis together, during the process of TMPRSS2-mediated membranes fusion, ACE2 plays a role in arresting and fixing the SARS-CoV-2 at the surface. After the viruses arrested and fixed, TMPRSS2 induces direct membrane fusion between virus and host cell 60 (Fig. 4B) .

Notably, the route of TMPRSS2-mediated membrane fusion is also crucial for SARS-CoV and MERS-CoV entry 65 .

The proteolytic shedding of a transmembrane protein like ACE2 can result in release of the soluble extracellular domain (ectodomain) from the membrane and a fragment that remains bound to the membrane. The shedding is an important regulatory mechanism to control the function and distribution of membrane proteins, which can terminate the function of a full-length membrane protein or release the biologically active ectodomain to activate the membrane protein. In addition, the shedding contributes to membrane protein endocytosis 66, 67 . ACE2 shedding can increase SARS-CoV entry [51] [52] [53] , but the exact molecular mechanism responsible for increased virus entry is unclear at present. Some researchers have suggested that ACE2 shedding promoted ACE2-mediated virus internalization and increased virus uptake into target cells 53 . Here, we summarize two distinct modes for ACE2 shedding: ADAM17-dependent shedding and TMPRSS2-dependent shedding (Fig.   5 ). ADAM17, a disintegrin and metalloproteinase, has an important established role in the regulation of ACE2 shedding 68 . ADAM17 functions in the shedding of ACE2 via arginine and lysine residues within ACE2 amino acids 652 to 659 53 (Fig. 5) . SARS-CoV S-protein binding facilitates ADAM17-dependent ACE2 shedding and has been shown to induce viral entry into the cell 52 . On the other hand, there is also another research has suggested that only type II transmembrane serine proteases TMPPSS2, but not ADAM-17, promotes SARS-CoV entry via ACE2 shedding 53 . Different from ADAM17, TMPPSS2 requires arginine and lysine residues within ACE2 amino acids 697 to 716 for ACE2 cleavage 53 . Notably, the neutral amino acid transporter B 0 AT1 might inhibit TMPRSS2-dependent ACE2 shedding (Fig. 5B) since B 0 AT1 binds ACE2 to form a protein complex.

The structure of the complex of ACE2/B 0 AT1 reveals that the TMPRSS2 cleavage sites (residues of 697-716) are hidden in the dimeric interface of ACE2 31 . It indicates that B 0 AT1 may block the access of TMPRSS2 to the cleavage sites on ACE2 which would result in decrease of ACE2

shedding.

In addition, the proprotein convertase furin has been also identified as an important factor for regulation of ACE2-mediated virus entering host cells. Furin mainly preactivates SARS-CoV-2

S-protein during viral packaging, and enhances virus entry into target cells 69, 70 . However, recent study showed that furin reduced SARS-CoV-2 entry into Vero cells 61 shows that the increase of ADAM-17-dependent ACE2 shedding is associated with myocardial hypertrophy and fibrosis 73 . In addition, elevated soluble ACE2 activity is associated with severer of myocardial dysfunction and is an independent predictor of adverse clinical events 73 . However, the question whether soluble ACE2, produced by ACE2 shedding, can bind with SARS-CoV-2 S-protein to clear away virus is confusing, which will be the future direction for research on soluble ACE2.

RAS plays a critical role in maintaining blood pressure homeostasis, as well as fluid and salt balance.

Therefore, RAS is intimately connected the pathophysiology of heart and kidney diseases 74, 75 .

Production of angiotensins from angiotensinogen requires the participation and coordination of many J o u r n a l P r e -p r o o f on Ang II, because the affinity of ACE2 with Ang II is 400 times higher than that with Ang I 80 . Ang (1) (2) (3) (4) (5) (6) (7) binds to the Mas receptor (MasR), which is a seven transmembrane G-protein-coupled receptor, and forms an ACE2-Ang (1-7)-MasR axis to mediate vasodilation and decreases blood pressure 81, 82 .

In summary, current knowledge of the RAS has been transformed from a linear hormonal system to a complex counter-regulatory system. The counter-regulatory RAS axis, ACE2-Ang (1-7)-MasR axis, opposes the effect of the ACE-Ang II-AT1R axis and has been show to reverse organ damage in renal or cardiovascular diseases 78 (Fig. 6) .

Insert Fig. 6 

led to significant down-regulation of ACE2 expression 34 . In post-mortem autopsy heart tissues from 20 patients who succumbed to SARS-CoV, seven heart samples had detectable viral SARS-CoV genome. These patients were also characterized by reduced myocardial ACE2 expression 83 , which further confirmed that ACE2 expression was decrease after virus infection.

Given that ACE2 functions as a counter-regulator of RAS, the decrease of ACE2 expression leads to the weakened ACE2-Ang (1-7)-MasR axis, mainly manifested as the increase of Ang II and decrease of vasodilator Ang (1-7) level. At present, a cohort study of 12 COVID-19 patients have partly confirmed this view. It was found that the level of Ang II in the plasma sample from SARS-CoV-2 infected patients was significantly higher than that from uninfected individuals 84 

Since ACE2 has been identified as the functional receptor of SARS-CoV-2, some disputes have At present, it is not clear whether ACEI and ARB increase ACE2 expression at the protein level.

For example, perindopril (ACEI) was able to increase hepatic ACE2 expression at the protein level under conditions of liver fibrosis 89 . However, another ACEI drug ramipril decreased ACE2 protein expression after myocardial infarction 90 . For ARB drugs, olmesartan up-regulated ACE2 protein expression in the carotid arteries after balloon injury. But there were no changes in ACE2 protein expression in uninjured carotid arteries in olmesartan-treated rats 91 . Therefore, from an ACE2 

ACE2 functions as both a SARS-CoV-2 receptor and RAS modulator, which presents the dilemma of J o u r n a l P r e -p r o o f how to limit virus entry while protecting its physiological function. Therefore, it is of great significance to understand fully the function and mechanism of ACE2 and the relationships among virus, ACE2 and RAS. Although ACE2 has been identified as a SARS-CoV-2 receptor, there might be other receptors or co-receptors for this virus that are yet to be discovered. In this review, we propose five working modes of functional receptors for SARS-CoV-2: monomer receptor, homodimer receptor, alternative receptors, co-receptors and transmissive receptor. We summarize the routes of ACE2 receptor-mediated virus entering host cells (ACE2-mediated virus endocytosis via clathrin-dependent pathways and non-caveolar lipid raft dependent pathways, and TMPRSS2-mediated membrane fusion upon ACE2 engagement) and its regulatory mechanism. In addition, a comparative map of ACE2 expression and vulnerability to SARS-CoV-2 in different organs was described. Moreover, the complex relationship among coronavirus, ACE2 and RAS (including anti-RAS drugs) is also summarized and discussed. These will contribute to a deeper understanding of the complex mechanisms and intervention strategies for virus infection and target organ damage. These raise further important implications for therapeutic targets for SARS-CoV-2.

Effective therapies against SARS-CoV-2 are urgently required due to the severity of the outbreak.

Based on the above theoretical summary, five steps are important anti-viral targets, including 1) the binding between coronavirus and ACE2; 2) virus entry mediated by ACE2; 3) virus replication; 4) virus assembly, and 5) virus exit (Fig. 7) .

One of important strategies to control viral infections is to block the initial binding of virus to its In addition, ACE2 interference is another way to block the binding between the SARS-CoV-2 S-protein and ACE2. For example, chloroquine can impact terminal glycosylation of ACE2, thereby preventing it from binding to the S-protein and inhibiting the SARS-CoV-2 infection [103] [104] [105] .

Furthermore, interfering with other receptors (such as CD147) proposed in Fig. 1 also can be considered as the therapeutic targets 17 . For example, meplazumab, an anti-CD147 humanized antibody, significantly inhibited the viruses from invading host cells 35 .

J o u r n a l P r e -p r o o f ②). ACE2 receptor-mediated endocytosis is a complex process regulated by a variety of proteins, which are potential target for interference. For example, AP2-associated protein kinase 1 (AAK1) is a well-known positive regulator of receptor endocytosis. An inhibitor of AAK1 (baricitinib) is predicted to reduce the ability of the SARS-CoV-2 entry by inhibiting ACE2 receptor-mediated endocytosis 106 . In addition, camostat mesylate, a TMPRSS2 inhibitor, has been shown to inhibit SARS-CoV-2 entry into cells 60 .

Of course, in addition to the steps related to ACE2 (the binding between coronavirus and ACE2, and virus entry mediated by ACE2), virus replication has been the hotspot for antiviral drug research.

Some drugs have entered clinical practice (Fig. 7 ③) . 

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HCoV Year Disease Functional receptor Ref. SARS-CoV-2 2019 Corona virus disease ACE2

Middle-East respiratory syndrome DPP4 18,19 HKU1 2005 Acute respiratory tract infection 9-O-Ac-Sia 20,21 NL63 2004 Acute lower respiratory tract infection in children ACE2

Severe acute respiratory syndrome ACE2, CD209L, DC-SIGN 24-26 OC43 1967 Common cold 9-O-Ac-Sia

ACE2, angiotensin-converting enzyme 2; DDP4, dipeptidyl peptidase 4; CD209L (also called L-SIGN), liver/lymph node-specific intercellular adhesion molecule-3-grabbing integrin

dendritic cell-specific intercellular adhesion molecule-3-grabbing integrin

The authors acknowledge funding support from the National Natural Science Foundation of China (91939301, 81820108031, 91539123, and 81471893) and Beijing Municipal Natural Science Foundation (7172235, China).

All authors researched data for the article and discussed its content. Jingwei Bian wrote the manuscript. Zijian Li designed, reviewed and edited this manuscript before submission.

On behalf of all authors, the corresponding author states that there is no conflict of interest.