Angiotensin-Converting Enzyme 2: SARS-CoV-2 Receptor and Regulator of the Renin-Angiotensin System: Celebrating the 20th Anniversary of the Discovery of ACE2.

Angiotensin-converting Enzyme 2–containing Small Extracellular Vesicles and Exomeres Bind the Severe Acute Respiratory Syndrome Coronavirus 2 Spike Protein

he prevalence of coronavirus disease 2019 (COVID-19) has posed a great threat to people's health worldwide, bringing a great challenges to the public healthcare systems. A recent study has confirmed that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses severe acute respiratory syndrome coronavirus (SARS-CoV) receptor angiotensin-converting enzyme 2 (ACE2) for host cell entry. 1 ACE2 expression was previously found to correlate with susceptibility to SARS-CoV infection in vitro.

Letter by Weir Regarding Article, "Angiotensin-Converting Enzyme 2: SARS-CoV-2 Receptor and Regulator of the Renin-Angiotensin System: Celebrating the 20th Anniversary of the Discovery of ACE2".

Human recombinant soluble ACE2 in severe COVID-19

Antihypertensive drugs and risk of COVID-19?

Human angiotensin-converting enzyme 2 (ACE2) is a functional receptor for SARS coronavirus (SARS-CoV). Here we identify the SARS-CoV spike (S)-protein-binding site on ACE2. We also compare S proteins of SARS-CoV isolated during the 2002–2003 SARS outbreak and during the much less severe 2003–2004 outbreak, and from palm civets, a possible source of SARS-CoV found in humans. All three S proteins bound to and utilized palm-civet ACE2 efficiently, but the latter two S proteins utilized human ACE2 markedly less efficiently than did the S protein obtained during the earlier human outbreak. The lower affinity of these S proteins could be complemented by altering specific residues within the S-protein-binding site of human ACE2 to those of civet ACE2, or by altering S-protein residues 479 and 487 to residues conserved during the 2002–2003 outbreak. Collectively, these data describe molecular interactions important to the adaptation of SARS-CoV to human cells, and provide insight into the severity of the 2002–2003 SARS epidemic.

Introduction An enzyme that metabolizes angiotensin II (Ang II) and other substrates was discovered in 2000, and was termed angiotensin converting enzyme 2 (ACE2) because of its substantial homology with ACE. Since the beginning of the coronavirus disease 2019 (COVID-19) pandemic, ACE2 has received unprecedented attention because in its membrane-bound form, it is the essential cell-entry receptor for severe acute respiratory syndrome coronavirus 2 (1). ACE2 has a single zinc metalloprotease active site and acts as a monocarboxypeptidase, cleaving a hydrophobic or basic amino acid (aa) from the C-terminus of small peptides. Within the renin angiotensin system (RAS), enzymatic actions of ACE2 are in opposition to those of its homolog ACE, such that ACE forms Ang II, whereas ACE2 fosters its degradation by forming Ang 1–7. This action is important at the tissue level, and seemingly minor in the systemic circulation, where other RAS enzymes are involved in the metabolism of Ang II (2). The ability of ACE2 to downregulate the RAS within the kidney prompted us to design shorter forms of ACE2 as novel renal therapeutics that can pass the glomerular filtration barrier (3). In this article, we discuss the different forms of enzymatically active ACE2 present in the kidney and urine. Kidney ACE2 A membrane-bound full-length (FL) form of ACE2 is abundantly expressed in the kidney, mainly at the apical surface of proximal tubules, but also in glomerular epithelial cells (podocytes), parietal cells in the Bowman's capsule, and in the renal vasculature (4). Within the renal vasculature, ACE2 is present in the tunica media, but absent or weakly expressed in endothelial cells (4). Increased albuminuria and kidney lesions were observed in aged ACE2-deficient mice, and after pharmacological ACE2 inhibition in diabetic mice, suggesting that amplifying ACE2 could have therapeutic potential for kidney disease (5). There are different forms of enzymatically active ACE2 that need to be recognized (Figure 1). The tissue form of ACE2 is bound to the cell membrane as an 805 aa–long FL protein. The main part of FL-ACE2 is present in the extracellular space (ectodomain), encompassing the 740 N-terminal aa and containing the catalytic unit, which determines enzyme activity. FL-ACE2 is anchored to the cell membrane by a short transmembrane domain (aa 741–768) and has a small intracellular C-terminal tail (aa 769–805) with cell-signaling properties (6). The ectodomain of the FL-ACE2, when detached from the plasma membrane, is referred to as soluble ACE2. The term soluble means the protein is not membrane or tissue bound, but rather is freely dissolved in plasma, interstitial fluid, and other body fluids, such as the urine and the cerebrospinal fluid. The soluble ACE2 that is shed mainly by the action of a metalloproteinase ADAM17 is catalytically active, and hydrolyzes Ang II to Ang 1–7 (7,8). The FL form of ACE2 can also circulate bound to the membrane of exosomes (9). This form, although circulating, should not be considered soluble ACE2.Figure 1.: Different forms of ACE2 with preserved enzymatic activity and hypothetical formation of small soluble fragments. Upper panel. In kidney tissue, a full-length (FL) angiotensin converting enzyme 2 (ACE2) of 805 amino acids (aa) is a plasma membrane–bound protein with a large part protruding into the extracellular space (ectodomain ACE2 with 740 aa). A small membrane domain (red) anchors the FL-ACE2 in the plasma membrane and a short C-terminal tail (green) with cell-signaling properties appears on the intracellular site of the kidney cell. The 740 aa–long ectodomain dimerizes and the total size of native FL-ACE2 is 98 kDa ×2. The ectodomain contains the catalytic unit (egg shape, brown) responsible for enzyme activity. Lower panel. In the urine, ACE2 can be found as an FL-ACE2 bound to the membrane of exosomes. The FL-ACE2 is the source of soluble ACE2 fragments, which under denaturing conditions (SDS- PAGE), would appear as large (approximately 98 kDa) and small (approximately 62 kDa) fragments. The large 98 kDa soluble ACE2 can maximally encompass the entire ectodomain (approximately 740 aa) or can be approximately 708 aa–long after cleavage by ADAM17. The intact dimerization-prone neck domain (616–726 aa) is present in the FL-ACE2 and the large 740 aa soluble ACE2 that both contain the entire ectodomain. It is not known whether in the 708 aa soluble ACE2, the neck domain is sufficiently intact to enable dimerization (blue thick line) under native conditions or not (absence of dimerization). The small 62 kDa soluble ACE2 can be proteolytically formed from longer FL or soluble ACE2 species, and does not dimerize due to the absence of the neck domain.ACE2 in the urine of patients with CKD (10) and in animal models of diabetic kidney disease is increased (11). This could suggest the possibility that this increase might be the result of more circulatory ACE2. However, when large amounts of soluble recombinant ectodomain ACE2 (aa 18–740) were infused to nondiabetic and diabetic wild-type (WT) mice (11,12) and global ACE2 knockout (KO) mice (11), the resulting marked increase in circulating (serum) ACE2 did not translate into any increase in urinary ACE2 (11,12). This was to be expected because a single chain of ectodomain of soluble ACE2 is approximately 100–110 kDa (3,11), which makes it unlikely to cross the glomerular filtration barrier. Moreover, in a native state, both the soluble and membrane-bound full-length ACE2 dimerize, doubling its monomer size (13). This makes it even more unlikely for such a large molecule to filter through an intact glomerular filtration barrier (12). Our group, moreover, has examined this question using radio-labeled ACE2 ectodomain infused to mice, and has shown there is no renal uptake, which is in contrast with shorter variants of soluble ACE2 that we have bioengineered (Wysocki et al., unpublished 2019). Therefore, increases in serum ACE2 activity as observed in diabetes or other conditions such as in patients with COVID-19, cannot account for elevated urinary ACE2 activity (if the circulating soluble ACE2 indeed comprises the entire ectodomain). Accordingly, when the glomerular filtration barrier is not severely damaged, the source of increased urinary ACE2 must be the kidney itself because the soluble ectodomain ACE2 (that includes 740 N-terminal aa), even if highly elevated in circulation, does not appear in the urine (11,12). In contrast, when the glomerular filtration barrier is highly disrupted, for example, in a mouse model of Alport syndrome, even a large molecule such as soluble ectodomain ACE2 can be recovered in the urine when the circulating levels are increased markedly (12). The next question, therefore, is what form of ACE2 is found in the urine, and where does it come from? Urine ACE2 ACE2 protein measured by ELISA and ACE2 activity is found in small amounts in mouse and human plasma, whereas urine enzymatic ACE2 activity is much higher than in plasma (approximately 5–10 times per volume unit) (11). Studies with mouse and sheep urine moreover showed that urinary ACE2 is capable of Ang II degradation (8,11). In urine of human and mice, two major species of ACE2 can be found. In mouse urine, one of the species of 100–110 kDa is likely the result of shedding from the proximal tubule apical membrane where FL-ACE2 is abundantly expressed (11) (Figure 1). The other ACE2-immunoreactive protein species detectable in mouse urine was of lower molecular size (approximately 75 kDa) (3,7,11). Both ACE2 immunoreactive bands appear enzymatically active (7,11). Of note, the FL-ACE2 and the larger soluble ACE2 (100–110 kDa) might not be easily distinguishable on gel electrophoresis because of their size proximity (11). We showed that the ectodomain of soluble ACE2 of 100–110 kDa can be catabolized by kidney and urine proteases to shorter ACE2 fragments that retain ACE2 enzyme activity (3). The cleavage likely happens from the C-terminal end, which contains a fairly large structural portion (from aa 616 upwards) that has no enzymatic function and can be removed without loss of enzyme activity (3). Part of this structural entity is the so called neck domain (616–726), which is largely responsible for the dimerization of soluble ectodomain (13). A recent study by Nelson et al. (14) reports that ACE2 can be filtered in the urine. On the basis of current views of protein passage across the glomerular filtration, however, only a short form of ACE2 (less than about 75 kDa) could be filtered. In the study of Nelson et al., kidney cross-transplantation between ACE2 deficient and WT mice was carried out to dissect the contribution of ACE2 originating from the kidney versus extrarenal sites to urine ACE2. In an elegant design, four different models were generated: (1) "kidney ACE2KO," with ACE2 deletion confined to the kidney (a kidney transplanted from ACE2KO into WT mouse), (2) "systemic ACE2KO," where ACE2 was deleted from extrarenal tissues (ACE2KO mouse with a kidney transplanted from WT), (3) "total ACE2KO" (ACE2KO mouse with a kidney transplanted from ACE2KO), and (4) "global WT" (WT mouse with a kidney transplanted from WT). When all four types of mice were challenged with a pressor dose of Ang II, increased albuminuria (and cardiac hypertrophy indicative of higher BP) was observed in the total ACE2 KO compared with the other groups. In urine of WT mice, two ACE2 species were detected (sizes of approximately 62 kDa and 98 kDa), which was consistent with previous studies (3,7,11). Intriguingly, in the urine from kidney ACE2KO mice only a shorter form of soluble ACE2 (approximately 62 kDa) was detected and the authors suggest that in response to a pressor dose of Ang II, the 62 kDa urinary ACE2 observed in the "kidney ACE2KO" is being shed from cells outside of the kidney, enters the circulation, and crosses the glomerular filtration barrier, ultimately being excreted in the urine (14). Consistent with this, Ang II has been shown to induce ADAM17 (the protease that can cleave membrane-bound ACE2, forming soluble ACE2) in cardiac tissue with concomitantly increased plasma ACE2 levels (15). Although the cross-transplantation model is useful, it may also permit confounding influence from the interfaces between donor ACE2KO kidney, and the host WT tissues as a source of ACE2 proteins originating from more distal urogenital tissues. Therefore, one might argue that some of the soluble 62 kDa ACE2 observed in the urine from "kidney ACE2KO" might, at least in part, originate from the genitourinary tissues of the host WT animals, such as testicle epithelia, which are very rich in ACE2. To substantiate the notion of soluble ACE2 passage through the glomerular filtration barrier, Nelson et al. (14) performed micropuncture studies in WT mice, which revealed the presence of ACE2 protein in the filtration fluid collected from the Bowman's capsule. This would have been indeed a convincing piece of evidence if the fluid had been collected from the Bowman's space of the "kidney ACE2KO" mice. However, because WT mice were used, this does not provide a definite answer to filterability of the soluble ACE2. Of note, peptide alignment showed that the ACE2 recovered in the Bowman's capsule in the micropuncture experiment and that examined from the blood must be of a much larger size than 62 kDa (14). The peptide alignment suggested a size above the putative ADAM17 cleavage site (between the aa Arg708 and Ser709 for human ACE2 [16] and Met706 for mouse ACE2 [7]). The length of such soluble ACE2 protein that extends beyond ADAM17 cleavage site would be consistent with the approximately 98 kDa soluble ACE2 protein. Because parietal epithelial cells of the Bowman's capsule abundantly express ACE2 on the apical surface, shedding of ACE2 and not filtered ACE2 might have accounted for the observed ACE2 protein. In addition, ACE2 is expressed, albeit much less abundantly, in the podocytes (4). The soluble ACE2, in the Bowman's space therefore, might have been generated through shedding from parietal and visceral (podocytes) epithelial cells and not necessarily originate from filtered plasma. Therefore, if indeed such a large ACE2 (approximately 98 kDa) is filtered from the blood (which we think is only possible with substantial disruption of glomerular filtration barrier), one must infer that this larger form is subsequently converted to the smaller 62 kDa ACE2 within the lumen of the nephron by the action of tubular proteases. The formation of this smaller 62 kDa ACE2 fragment could be the result of proteolytic cleavage of the 98 kDa ACE2 species (3) (Figure 1). Indeed, in a previous study when the recombinant ectodomain soluble ACE2 18–740 was exposed to ACE2KO mouse kidney lysates and urine, the 100–110 kDa ectodomain ACE2 was digested to smaller enzymatically active ACE2 fragments of 75 and 60–70 kDa (3). One also cannot exclude a possibility that ADAM17 has more than one cleavage site within mouse ACE2 (7), and could also be involved in this process. This seems plausible because ADAM17 expression has been found to be augmented by Ang II in cultured proximal tubule cells and associated with increased release of ACE2 protein in the culture media as two bands of 90 kDa and 70 kDa (7). We developed shorter forms of recombinant soluble ACE2 that, unlike the large ectodomain soluble ACE2 (aa 18–740), can indeed be filtered into the urine as demonstrated by injecting them intravenously to global ACE2KO mice (3). The intravenously injected recombinant short soluble ACE2 protein was not only recovered in the final urine in an enzymatically active form, but in addition, was found in the kidney, indicating there is a tubular reabsorption of ACE2 that had been filtered into the urine (3). In summary, the urine contains enzymatically active ACE2 in at least three forms: (1) FL-ACE2 present in exosomes originating in the kidney most likely the proximal tubule, (2) soluble ACE2 with a molecular size of about 98 kDa, which is also likely of renal origin, and (3) smaller fragments that may be the result of action of kidney proteases, or possibly originate from the circulation and be filtered (Figure 1). The main form of circulating soluble ACE2, to our knowledge, has not been completely identified, because western blot analysis is not sensitive to detect the low levels in plasma. Recombinant soluble ACE2 proteins of human or murine origin with 740 aa are too large to pass the glomerular filtration barrier under normal conditions (11). To overcome this limitation, we bioengineered shorter forms of ACE2 that pass the glomerular filtration barrier under normal conditions and that are taken up by the kidney parenchyma and capable to foster the formation of Ang 1–7 from Ang II within the kidney (3). The therapeutic potential of soluble ACE2 for the kidney and beyond clearly deserves more attention than it has had during the two decades since its discovery. Disclosures D. Batlle is a founder of Angiotensin Therapeutics Inc.; and reports receiving a grant and consulting fees from AstraZeneca, and consulting fees from Advicenne, all unrelated to this work. D. Batlle and J. Wysocki are coinventors of patents entitled "Active Low Molecular Weight Variants of Angiotensin Converting Enzyme 2," "Active Low Molecular Weight Variants of Angiotensin Converting Enzyme 2 (ACE2) for the treatment of diseases and conditions of the eye," and "Soluble ACE2 Variants and Uses Therefor", which includes the use of prevention and treatment of COVID-19. J. Wysocki reports having a scientific advisory capacity for Angiotensin Therapeutics Inc. Funding This work was supported by a gift from the Joseph and Bessie Feinberg Foundation and National Institutes of Health grant 1R21 AI166940-01 (to D. Batlle).

The carboxypeptidase Angiotensin Converting Enzyme 2 (ACE2) is a crucial player of the Renin-Angiotensin-System (RAS) as it balances the ratio between vasoconstrictive and fibrotic Angiotensin II and vasodilative and anti-fibrotic Angiotensin 1-7. This balance is affected by ACE2 either by direct conversion of Angiotensin II to Angiotensin 1-7, or by cleavage of Angiotensin I to Angiotensin 1-9, which is further converted to Ang 1-7 by Angiotensin Converting Enzyme (ACE) or Neutral Endopeptidase (NEP). An increased activity of ACE2 leads to a shift of the RAS towards an Angiotensin 1-7 dominated pattern, which was shown to be protective in various models of cardiovascular and fibrotic diseases. We performed a comparative pharmacologic characterization of recombinant murine and human ACE2 using natural peptide substrates for the evaluation of the substrate specific ACE2 activity in whole blood. Furthermore, we investigated the impact of ACE2 activators and inhibitors on murine and human ACE2 in whole blood using an LC-MS/MS based approach allowing the simultaneous quantification of 10 different angiotensin peptides (RAS-Fingerprinting). The investigation of inhibitors and activators of ACE2 using our experimental setting revealed the presence of species-specific enzymatic features, which was indicated by differences between the human and murine enzyme regarding the susceptibility to pharmacologic manipulation and the specificity for different endogenous peptide substrates. Our studies demonstrate that sequence diversity observed between recombinant human and murine ACE2 significantly affects substrate specificity and pharmacologic enzyme properties. The utilization of Angiotensin I by ACE2 generates Angiotensin 1-7 via Angiotensin 1-9 and represents a pathway of alternative RAS activation, which might have been systematically underestimated in previous studies. The strongly reduced affinity of murine ACE2 for Angiotensin I results in a lack of production of Angiotensin 1-7 via this pathway, which might be of particular importance during anti-hypertensive treatments with ACE inhibitors, where the formation of Angiotensin II is prevented while increased levels of Angiotensin I are observed in circulation.

Podocyte-specific overexpression of human angiotensin-converting enzyme 2 attenuates diabetic nephropathy in mice

The severe acute respiratory syndrome coronavirus (SARS-CoV) from palm civets has twice evolved the capacity to infect humans by gaining binding affinity for human receptor angiotensin-converting enzyme 2 (ACE2). Numerous mutations have been identified in the receptor-binding domain (RBD) of different SARS-CoV strains isolated from humans or civets. Why these mutations were naturally selected or how SARS-CoV evolved to adapt to different host receptors has been poorly understood, presenting evolutionary and epidemic conundrums. In this study, we investigated the impact of these mutations on receptor recognition, an important determinant of SARS-CoV infection and pathogenesis. Using a combination of biochemical, functional, and crystallographic approaches, we elucidated the molecular and structural mechanisms of each of these naturally selected RBD mutations. These mutations either strengthen favorable interactions or reduce unfavorable interactions with two virus-binding hot spots on ACE2, and by doing so, they enhance viral interactions with either human (hACE2) or civet (cACE2) ACE2. Therefore, these mutations were viral adaptations to either hACE2 or cACE2. To corroborate the above analysis, we designed and characterized two optimized RBDs. The human-optimized RBD contains all of the hACE2-adapted residues (Phe-442, Phe-472, Asn-479, Asp-480, and Thr-487) and possesses exceptionally high affinity for hACE2 but relative low affinity for cACE2. The civet-optimized RBD contains all of the cACE2-adapted residues (Tyr-442, Pro-472, Arg-479, Gly-480, and Thr-487) and possesses exceptionally high affinity for cACE2 and also substantial affinity for hACE2. These results not only illustrate the detailed mechanisms of host receptor adaptation by SARS-CoV but also provide a molecular and structural basis for tracking future SARS-CoV evolution in animals.

Response by Gheblawi et al to Letter Regarding Article, "Angiotensin-Converting Enzyme 2: SARS-CoV-2 Receptor and Regulator of the Renin-Angiotensin System: Celebrating the 20th Anniversary of the Discovery of ACE2".

The emergence of new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mutants and breakthrough infections despite available coronavirus disease 2019 (COVID-19) vaccines calls for antiviral therapeutics. The application of soluble angiotensin converting enzyme 2 (ACE2) as a SARS-CoV-2 decoy that reduces cell bound ACE2-mediated virus entry is limited by a short plasma half-life. This work presents a recombinant human albumin ACE2 genetic fusion (rHA-ACE2) to increase the plasma half-life by an FcRn-driven cellular recycling mechanism, investigated using a wild type (WT) albumin sequence and sequence engineered with null FcRn binding (NB). Binding of rHA-ACE2 fusions to SARS-CoV-2 spike protein subdomain 1 (S1) was demonstrated (WT-ACE2 KD = 32.8 nM and NB-ACE2 KD = 31.7 nM) using Bio-Layer Interferometry and dose-dependent in vitro inhibition of host cell infection of pseudotyped viruses displaying surface SARS-CoV-2 spike (S) protein. FcRn-mediated in vitro recycling was translated to a five times greater plasma half-life of WT-ACE2 (t½ β = 13.5 h) than soluble ACE2 (t½ β = 2.8 h) in humanised FcRn/albumin double transgenic mice. The rHA-ACE2-based SARS-CoV-2 decoy system exhibiting FcRn-driven circulatory half-life extension introduced in this work offers the potential to expand and improve the anti-COVID-19 anti-viral drug armoury. Statement of significanceThe COVID-19 pandemic has highlighted the need for rapid development of efficient antiviral therapeutics to combat SARS-CoV-2 and new mutants to lower morbidity and mortality in severe cases, and for people that are unable to receive a vaccine. Here we report a therapeutic albumin ACE2 fusion protein (rHA-ACE2), that can bind SARS-CoV-2 S protein decorated virus-like particles to inhibit viral infection, and exhibits extended in vivo half-life compared to ACE2 alone. Employing ACE2 as a binding decoy for the virus is expected to efficiently inhibit all SARS-CoV-2 mutants as they all rely on binding with endogenous ACE2 for viral cell entry and, therefore, rHA-ACE2 constitutes a versatile addition to the therapeutic arsenal for combatting COVID-19.

Angiotensin converting enzyme 2 (ACE2), a transmembrane glycoprotein, is an important part of the renin-angiotensin system (RAS). In the COVID-19 epidemic, it was found to be the receptor of severe acute respiratory syndrome coronavirus 2 (SARS-COV-2). ACE2 maintains homeostasis by inhibiting the Ang II-AT1R axis and activating the Ang I (1-7)-MasR axis, protecting against lung, heart and kidney injury. In addition, ACE2 helps transport amino acids across the membrane. ACE2 sheds from the membrane, producing soluble ACE2 (sACE2). Previous studies have pointed out that sACE2 plays a role in the pathology of the disease, but the underlying mechanism is not yet clear. Recent studies have confirmed that sACE2 can also act as the receptor of SARS-COV-2, mediating viral entry into the cell and then spreading to the infective area. Elevated concentrations of sACE2 are more related to disease. Recombinant human ACE2, an exogenous soluble ACE2, can be used to supplement endogenous ACE2. It may represent a potent COVID-19 treatment in the future. However, the specific administration concentration needs to be further investigated.

Coronavirus disease 2019 (COVID-19) is caused by a novel strain of coronavirus, designated as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It has caused a global pandemic rapidly s...

SummaryThe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike N-terminal domain (NTD) remains poorly characterized despite enrichment of mutations in this region across variants of concern (VOCs). Here, we examine the contribution of the NTD to infection and cell-cell fusion by constructing chimeric spikes bearing B.1.617 lineage (Delta and Kappa variants) NTDs and generating spike pseudotyped lentivirus. We find that the Delta NTD on a Kappa or wild-type (WT) background increases S1/S2 cleavage efficiency and virus entry, specifically in lung cells and airway organoids, through use of TMPRSS2. Delta exhibits increased cell-cell fusogenicity that could be conferred to WT and Kappa spikes by Delta NTD transfer. However, chimeras of Omicron BA.1 and BA.2 spikes with a Delta NTD do not show more efficient TMPRSS2 use or fusogenicity. We conclude that the NTD allosterically modulates S1/S2 cleavage and spike-mediated functions in a spike context-dependent manner, and allosteric interactions may be lost when combining regions from more distantly related VOCs.