key: cord-0828620-ag8joijl
authors: Kiseleva, Anna A; Troisi, Elizabeth M; Hensley, Scott E; Kohli, Rahul M; Epstein, Jonathan A
title: SARS-CoV-2 spike protein binding selectively accelerates substrate-specific catalytic activity of ACE2
date: 2021-03-27
journal: J Biochem
DOI: 10.1093/jb/mvab041
sha: 67ce1b33f64d7801e904dac543255ccee717f09f
doc_id: 828620
cord_uid: ag8joijl

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus that has given rise to the devastating global pandemic. In most cases, SARS-CoV-2 infection results in the development of viral pneumonia and acute respiratory distress syndrome, known as “coronavirus disease 2019” or COVID-19. Intriguingly, besides the respiratory tract, COVID-19 affects other organs and systems of the human body. COVID-19 patients with pre-existing cardiovascular disease have a higher risk of death, and SARS-CoV-2 infection itself may cause myocardial inflammation and injury. One possible explanation of such phenomena is the fact that SARS-CoV-2 utilizes angiotensin-converting enzyme 2 (ACE2) as the receptor required for viral entry. ACE2 is expressed in the cells of many organs, including the heart. ACE2 functions as a carboxypeptidase that can cleave several endogenous substrates, including angiotensin II, thus regulating blood pressure and vascular tone. It remains largely unknown if the SARS-CoV-2 infection alters the enzymatic properties of ACE2, thereby contributing to cardiovascular complications in patients with COVID-19. Here, we demonstrate that ACE2 cleavage of des-Arg9-bradykinin substrate analog is markedly accelerated, while cleavage of angiotensin II analog is minimally affected by the binding of spike protein. These findings may have implications for a better understanding of COVID-19 pathogenesis.

The recent COVID-19 pandemic caused by the novel coronavirus SARS-CoV-2 represents a significant threat to human health. To date, more than two million people worldwide have died of COVID-19 or COVID-19-related complications. SARS-CoV-2 is both structurally and phenotypically similar to another strain of coronavirus, SARS-CoV-1, that caused an outbreak of severe acute respiratory syndrome in 2003 1 . Similarly to SARS-CoV-1, SARS-CoV-2 binds to the angiotensin-converting enzyme 2 (ACE2) receptor to gain entry to mammalian cells 2, 3 . SARS-CoV-2 attaches to ACE2 via glycosylated spike (S) protein trimers on the virion surface 4 .

Interestingly, SARS-CoV-2 spike protein has a much higher binding affinity to ACE2 than does that of SARS-CoV-1 (K D,app = 3.9x10 -11 M compared with 1.2x10 -10 M, respectively) 5 . The receptorbinding domain (RBD) of SARS-CoV-2 spike protein is recognized by the extracellular domain of ACE2 mainly through polar residues 6 . Interestingly, the virus hijacks the ACE2 proteolytic Nterminal domain, but the binding site itself is located away from the ACE2 active site 7 , suggesting that even upon binding of the virus, ACE2 is likely to maintain some proteolytic activity. The binding of spike protein to ACE2 is also conformation-dependent, suggesting that it could affect conformational states of ACE2. X-ray crystallography studies of the unbound or inhibitor-bound forms of ACE2 also reveal a striking difference in the structure of the active site that impacts a number of surrounding residues, including the ACE2 residues implicated in binding to the SARS-CoV-2 spike protein 8 .

ACE2 normally functions as a zinc-dependent carboxypeptidase 9 , and plays an important cardio-protective role as part of the renin-angiotensin-aldosterone system (RAAS) by catalyzing the cleavage of the vasoconstricting peptide angiotensin II into the vasodilating angiotensin 1-7.

ACE2 also has other known, and likely unknown, peptide-based substrates, such as apelin 13 (which has known cardioactive functions) 10, 11 , des-Arg(9)-bradykinin (an immune modulator) 12, 13 , and dynorphin A 13 , and thus may contribute to the regulation of blood pressure, vascular tone, and various immune pathways. ACE2 is expressed by pulmonary alveolar cells and cells of many other organs and tissues including vascular endothelium, heart, and kidneys 9 . The lung likely serves as the primary point of viral entry, but it remains unclear to what extent other tissues are directly infected by SARS-CoV-2. Nevertheless, there are numerous clinical reports showing that COVID-19 results in increased mortality in patients with pre-existing conditions, such as hypertension, diabetes, obesity, as well as patients with cancer and a compromised immune system 14, 15, 16, 17 . A growing number of studies highlight that COVID-19 patients develop acute myocardial injury with potentially chronic effects on the cardiovascular system. In some cases, patients without any pre-existing heart problems had signs of ongoing myocardial inflammation after apparent recovery from COVID-19 18 . However, it remains unknown if the cardiac symptoms are developed as a result of the direct infection of myocardial cells or as a secondary event associated with infection-mediated effects such as cytokine storm 19 .

A better understanding of the mechanisms underlying the interaction between ACE2 and SARS-CoV-2 is of high importance. First, it will likely be crucial to developing effective strategies for COVID-19 treatment. Several studies have shown that soluble recombinant ACE2 is able to act as a molecular trap to bind SARS-CoV-2, thus preventing or limiting infection 20, 21 . Second, it will help to elucidate how SARS-CoV-2 infection affects the function of ACE2 and downstream signaling. Moreover, SARS-CoV-2 can potentially bind to a catalytically active soluble form of ACE2 (sACE2), which lacks the transmembrane domain, is present in the bloodstream, and is able to catalyze Ang II conversion 9 . Altered ACE2 function in the setting of viral infection might contribute to the regulation of proteolytic cascades that could account for some of the many unusual characteristics of COVID-19, including cardiac dysfunction, vascular disease, and thrombosis. Hence, we sought to test if SARS-CoV-2 binding to ACE2 affects its enzymatic properties. Here, we show that the binding of SARS-CoV-2 enhances ACE2 peptidase activity in vitro.

The extracellular catalytic domain of recombinant human ACE2, SARS-CoV-2 spike full- was expressed in 293F cells and the protein was purified as described previously 22 . The heatinactivated at 65°C for 30 min SARS-CoV-2 (strain 2019nCoV/USAWA1/2020) was obtained from ATCC (cat# VR1986HK, ATCC, VA). Inactivation at this temperature was intended to leave viral epitopes intact, though partial denaturation may occur 23 .

The viral genome copy (VGC) number was quantified by RT-qPCR using the calculated Ct-value determined from the standard curve. Briefly, 5 µl of the inactivated virus or 5 µl of quantitative synthetic SARS-CoV-2 RNA (cat# VR-3276SD, ATCC, VA) were reverse transcribed using SuperScript III Reverse Transcriptase (cat# 18080044, Invitrogen, CA) following the manufacturer's instructions. The resulting cDNA from the virus was diluted 1:50; cDNA from the synthetic standards were diluted 1:5, 1:10, 1:50, 1:100, and used to generate a standard curve. The RFU values for each reaction were corrected by subtracting the background. Corrected RFUs were then converted into molar concentrations by calculating a conversion factor (CF) established by the generation of a product control. Molar concentrations of the converted product for the less efficient ACE2 substrates, such as Ang I and Dynorphin A, were calculated using the CF for Ang II. The initial rates were calculated based on measurements over the first hour, at which point turnover was linear with respect to time and turnover was limited to <20% substrate conversion.

Steady-state kinetic constants were then calculated by fitting to a hyperbolic Michaelis-Menten plot. Assays were conducted for a minimum of 2 biological replicates.

Statistical analysis was performed using linear regression analysis, two-way ANOVA (Tukey test), non-linear regression analysis for Michaelis-Menten kinetics, and Exponential plateau equation to estimate the maximum of the converted substrate for each kinetic assay using GraphPad PRISM 9.0.0 software (GraphPad Software, Inc.).

Binding of SARS-CoV-2 spike protein to ACE2 accelerates its peptidase activity. As a carboxypeptidase, ACE2 cleaves many biological substrates besides angiotensin II (Ang II). In general, ACE2 hydrolyzes a bond between proline and a C-terminal hydrophobic amino acid in peptides that resembles the following sequence: Proline-X(1-3 residues)-Proline-Hydrophobic Amino Acid 13 . One well-established method to analyze the activity of peptidases in vitro is to measure the concentration of the cleaved product over time in the presence of internally quenched fluorogenic peptides 24, 25 . These peptides are usually flanked by a fluorophore and a quencher that inhibits the fluorophore in an intact state. The inhibition is liberated upon cleavage of the peptide by a protease -the peptide fragment with the fluorophore is released from the quencher, and the fluorescent signal is emitted 25 . Therefore, to test the effect of the viral spike protein binding on the peptidase activity of ACE2, we first performed experiments to evaluate the ability of ACE2 to cleave a commercially available fluorogenic pseudosubstrate MCA-YVADAPK(Dnp) (commonly used to measure ACE2 activity, and also known to be a substrate for Caspase-1). First, we measured ACE2 activity, with and without pre-incubation with SARS-CoV-2 trimeric full-length spike protein, in the presence of MCA-YVADAPK(Dnp) substrate. Under these conditions, we observed a significant increase in the ACE2 activity rate in the presence of the spike protein ( Figure 1A ). We observed a similar effect by pre-incubating ACE2 with a shorter version of recombinant spike protein that contains only the receptor binding domain (RBD, Ala319-Phe541) of SARS-CoV-2 ( Figure 1B) , suggesting that RBD spike is sufficient to accelerate ACE2 activity. The RBD domain of SARS-CoV-1 was also able to accelerate ACE2 activity, albeit to a lesser extent than that of SARS-CoV-2, consistent with the known lower binding affinity of SARS-CoV-1 ( Figure 1B) . We also quantified ACE2 steady-state kinetics in the absence and presence of SARS-CoV-1 and SARS-CoV-2 spike RBDs ( Figure 1C, D) . The addition of SARS-CoV-2 RBD results in an approximate doubling of enzyme activity (k cat ) with no change in K M . A smaller activation of k cat (~30%) was induced by SARS-CoV-1 RBD ( Figure 1C, D) . In addition, we demonstrated that SARS-CoV-2 RBD spike affects ACE2 k cat in a dose-dependent manner (Supplementary Figure 1) .

fashion. Next, we sought to determine whether the acceleration of ACE2 catalytic activity produced by spike protein binding that is observed when using the commercially available pseudosubstrate is also evident in the presence of endogenous substrates. ACE2 is known to cleave multiple physiological substrates. Besides controlling blood pressure and fluid balance as a part of the RAAS and apelin signaling 26 , ACE2 negatively regulates the plasma kallikrein-kinin system (KKS) by cleaving des-Arg9-bradykinin which is released in response to tissue injury or inflammatory stimuli 12 . Thus, we synthesized several internally quenched fluorogenic substrates that mimic known biological substrates of ACE2, including angiotensin II (Ang II) (MCA-DRVYIHPK(Dnp)), apelin 13 (MCA-QRPRLSHKGPMPK(Dnp)), and des-Arg9-bradykinin (MCA-RPPGFSPK(Dnp)). We also included two peptides that are normally less efficiently cleaved by ACE2: dynorphin A (MCA-YGGFLRRIRPKLK(Dnp)) and angiotensin I (Ang I) (MCA-DRVYIHPFK(Dnp)). We pre-incubated ACE2 with SARS-CoV-2 RBD spike, added 20 µM of each substrate, and measured the accumulation of product over time (Figure 2 ). RBD failed to activate ACE2 catalytic activity when Ang II was used as a substrate ( Figure 2B ). However, catalytic activity was increased by RBD when each of the other substrates was utilized ( Figure 2C -F). The greatest increase in the rate of ACE2 activity was observed in the presence of des-Arg9bradykinin as substrate ( Figure 2D ). Taken together, these findings suggest substrate-specific enhancement of ACE2 enzymatic activity produced by spike RBD binding.

Next, we quantified ACE2 steady-state kinetics in the presence or absence of SARS-CoV-2 spike RBD against des-Arg9-bradykinin and found there to be an approximate 2.5-fold increase in ACE2 enzymatic activity (k cat ) with no significant change in K M produced by addition of spike RBD ( Figure 2G, H) . Notably, we observed that ACE2 activity dropped off when using the highest concentration of des-Arg9-bradykinin substrate (50 µM) suggesting a possibility of substrate inhibition ( Figure 2G ). When fitting observed rates to an alternative model that includes substrate inhibition, the key conclusions did not change, and we still observed ~2.7-fold increase in k cat in the presence of spike RBD (data not shown).

In addition, we tested if two spike RBD mutants (G502D and Y505C) with a lower affinity to ACE2 5 would still affect enzymatic activity. We observed that both mutants accelerated the activity of ACE2 but significantly less than spike RBD wild type (Supplementary Figure 2) , suggesting that the increase in ACE2 activity also depends on the affinity to spike RBD.

Arg9-bradykynin. Next, we sought to determine if binding of intact SARS-CoV-2 viral particles to ACE2 would mimic the effects on ACE2 catalytic activity observed when purified spike protein or RBD was used. Therefore, we measured ACE2 activity against three substrates (des-Arg9- (Figure 3 B, C) . In the case of the des-Arg9-bradykinin substrate, ACE2 activity was significantly accelerated even upon the addition of the lowest concentration of the virus (380 VGC/µL) ( Figure 3A ).

The COVID-19 pandemic represents a significant threat to public health and there are still numerous unanswered questions regarding the mechanisms of COVID-19 pathogenesis. Our results suggest that SARS-CoV-2 binding to ACE2 can affect its catalytic activity in a substratespecific manner, suggesting that various signaling cascades normally regulated by ACE2 may be The results of our study are in general agreement with results recently published by Lu et al. 28 , although we found some significant differences. In that study, the authors also observed enhancement of ACE2 catalytic activity upon SARS-CoV-2 spike RBD binding, with some variation depending on the substrate (Caspase-1 pseudosubstrate versus a bradykinin analog) 28 .

However, they reported a significant decrease in K M upon spike binding which we did not reproduce under our assay conditions. Also, Lu et al. did not directly measure catalytic activity using an Ang II substrate analog, an important distinction as we did not observe significant enhancement with this substrate. Notably, the authors used a bradykinin analog substrate (Mca-RPPGFSAFK-Dnp)) that does not fully replicate the sequence of the "classic" bradykinin peptide (RPPGFSPFR, Sequence ID: AAH60039.1). It is also known that ACE2 has no catalytic activity against bradykinin 13 . Additionally, the rates of catalysis (k cat ) that the authors reported with the pseudosubstrate are lower than what we observed, likely attributable to different assay conditions or enzyme and substrate preparations. Importantly, however, our studies support the general observation that spike RBD binding to ACE2 can lead to higher rates of catalysis, and our work extends these findings by emphasizing the significant differences in this effect dependent upon substrate and the affinity to SARS-CoV-2 spike. Furthermore, our results newly demonstrate that the effect is not confined to isolated RBD, but that intact SARS-CoV-2 virus can also enhance ACE2 activity.

The relevance of our in vitro studies to COVID-19 pathogenesis will require further studies including the assessment of both cell surface and soluble ACE2 activity and various cleavage products in infected patients. Prior work has suggested that SARS-CoV-1 infection results in downregulation of cell surface ACE2, likely due to internalization and/or shedding of bound receptor 29, 30 . However, similar down-regulation upon SARS-CoV-2 binding has not been reported to date in experimental models up to several days after infection 31, 32 

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MCA-DRVYIHPK(Dnp)) substrate (C)

Supplementary Figure 1. Binding of the SARS-CoV-2 spike RBD protein enhances ACE2 activity in a dose-dependent manner. (A) Michaelis-Menten plot showing the effect of two different concentrations (1 µg and 10 µg) of SARS-CoV-2 spike RBD on catalytic activity of ACE2

k obs , observed rate constant. (B) Catalytic rate (k cat ) and K M of ACE2 in the absence or presence of SARS-CoV-2 RBD spike protein

Biding of the SARS-CoV-2 spike RBD mutant (G502Dand Y505C) proteins accelerates ACE2 activity less than spike RBD wild type. (A)-(B) Kinetic curves showing the effect of two different concentrations (1 µg (A) and 10 µg (B)) of SARS-CoV-2 spike RBD mutants (G502D and Y505C) and RBD wild type in the presence of des-Arg9-bradykinin mimic (MCA-RPPGFSPK(Dnp)) substrate

Kinetic curves showing the effect of full-length SARS-CoV-2 spike on ACE2 activity. (B) Kinetic curves showing the effect of SARS-CoV-1 and SARS-CoV-2 RBD spike on ACE2 activity. (C) Michaelis-Menten plot showing effect of SARS-CoV-1 and SARS-CoV-2 RBD on catalytic activity of ACE2. kobs, observed rate constant. (D) Catalytic rate (kcat) and KM of ACE2 in the absence or presence of SARS-CoV-1 and SARS-CoV-2 RBD spike protein (mean (95% confidence intervals)). Pseudosubstrate concentration in (A) and (B) is 20 µM

Kinetic curves showing the effect of SARS-CoV-2 spike RBD binding on ACE2 activity in the presence of (A) pseudosubstrate MCA-YVADAPK

B) angiotensin II mimic (MCA-DRVYIHPK(Dnp))

D) des-Arg9-bradykinin mimic (MCA-RPPGFSPK(Dnp)) (E) angiotensin I mimic

F) dynorphin A mimic (MCA-YGGFLRRIRPKLK(Dnp)) substrates; (G)

Michaelis-Menten plot showing effect of SARS-CoV-2 RBD on catalytic activity of ACE2 in the presence of des-Arg9-bradykinin mimic substrate. (H) Catalytic rate (kcat) and KM of ACE2 in the absence or presence of SARS-CoV-2 RBD spike protein and des-Arg9-bradykinin mimic substrate (mean (95% confidence intervals)). Substrate concentration in (A)-(F) is 20 µM

Binding of the heat-inactivated SARS-CoV-2 spike protein accelerates ACE2 catalytic activity. (A-C) Kinetic curves showing the effect of different concentrations of heat-inactivated SARS-CoV-2 on ACE2 activity in the presence of des-Arg9-bradykinin mimic (MCA-RPPGFSPK(Dnp)) substrate (A)

Dnp) substrate (B); and angiotensin II mimic (MCA-DRVYIHPK(Dnp)) substrate (C)

The authors declare no conflict of interest.