The rapid spread of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), the causative pathogen of human coronavirus disease 2019 (COVID-19), has resulted in an unprecedented pandemic and worldwide health crisis. Similar to the beta-coronaviruses SARS-CoV and Middle Eastern Respiratory Syndrome (MERS)-CoV, SARS-CoV-2 is highly transmissible and can lead to lethal pneumonia and multi-organ failure (Zhu et al., 2020). For infection and viral entry, the Spike surface protein of SARS-CoV-2 binds to angiotensin converting enzyme 2 (ACE2) on host cells (Wu et al., 2020; Zhou et al., 2020). Recombinant soluble human ACE2 (rshACE2) has been shown to bind Spike (Wang et al., 2020), can effectively neutralize SARS-CoV-2 infections (Monteil et al., 2020, Monteil et al., 2021), and the corresponding drug candidate APN01 has undergone a phase II clinical trial for the treatment of hospitalized cases of COVID-19 (ClinicalTrials.gov Identifier: NCT04335136). A first case study of its use in a patient has been reported recently (Zoufaly et al., 2020). Additionally, an aerosol formulation of APN01 has been developed and is currently undergoing Phase I clinical studies.

Multiple other therapeutic strategies attempt to target the Spike-ACE2 interaction, for example by development of neutralizing antibodies blocking the ACE2-binding site (Barnes et al., 2020) or lectins that bind to glycans on the Spike surface (Chan et al., 2021, Hoffmann et al., 2021). Using soluble ACE2 as a decoy receptor for Spike is particularly attractive, as it minimizes the risk that variants of concern may evade the treatment through mutations as has been observed for antibodies (Weisblum et al., 2020; Li et al., 2021; Yuan et al., 2021). Furthermore, protein engineering has yielded ACE2 variants with substantially improved affinities for Spike (Glasgow et al., 2020; Chan et al., 2020). Hence, soluble ACE2-based therapeutics offer considerable advantages over other therapeutic formats that aim to hamper the Spike-ACE2 interaction sterically.

Modern structural biology has been amazingly fast to respond to this pandemic. A mere 3 months after identification of SARS-CoV-2 as the etiologic agent of COVID-19, structures of the complex between ACE2 and the receptor binding domain (RBD) (Wang et al., 2020; Lan et al., 2020; Yan et al., 2020) and of the ectodomain of trimeric Spike (Walls et al., 2020; Wrapp et al., 2020) were already solved by X-ray crystallography or cryo-electron microscopy. While this provided unprecedented insight into the protein-protein interactions between Spike and ACE2, the structural impact of protein-bound glycans on the Spike-ACE2 interface could not be assessed experimentally so far due to their compositional diversity and conformational flexibility. Here, in silico modeling of the glycans offers a powerful alternative to study the effects of individual Spike and ACE2 glycans on the molecular interactions between these two proteins.

The SARS-CoV-2 Spike protein is heavily glycosylated with both complex and oligo-mannosidic type N-glycans (Zhu et al., 2020, Hoffmann et al., 2021; Watanabe et al., 2020; Zhao et al., 2020), thereby shielding a large portion of the protein surface (Casalino et al., 2020; Sikora et al., 2021; Zimmerman et al., 2021). Similarly, ACE2 is a glycoprotein with up to seven highly utilized sites of N-glycosylation (Zhao et al., 2020). Recent computational studies started to investigate protein glycosylation in the context of the interaction between Spike and ACE2 (Zhao et al., 2020; Casalino et al., 2020; Mehdipour and Hummer, 2021). Extensive all-atom molecular dynamics (MD) simulations indicated that Spike N-glycans attached to N165 and N234 could be important stabilizers of the ligand-accessible conformation of the receptor binding domain (RBD) (Casalino et al., 2020). Furthermore, it has been proposed that the N-glycan at position N343 acts as a gate facilitating RBD opening (Sztain et al., 2021). Other MD studies concluded that the glycans attached to N90 and N322 of ACE2 could be major determinants of Spike binding (Mehdipour and Hummer, 2021), while yet other simulation works postulate that glycosylation does not affect the RBD-ACE2 interaction significantly (Cong et al., 2021; Delgado et al., 2021). Genetic or pharmacological blockade of N-glycan biosynthesis at the oligomannose stage in ACE2-expressing target cells was found to dramatically reduce viral entry (Yang et al., 2020), even though several glycoforms of ACE2 were found to display comparatively moderate variation with respect to Spike binding (Allen et al., 2021). Hence, a detailed understanding on how individual glycans on both Spike and ACE2 influence their interaction and a comprehensive experimental validation of the MD findings is crucial for the rational design of novel therapeutic soluble ACE2 variants with enhanced Spike binding affinity and the capacity to block viral entry more efficiently than the native enzyme (Zhao et al., 2020). The identification of the Spike glycans essential for efficient association with ACE2 will be also critical to guide rational design of improved SARS-CoV-2 vaccines.

We started our research by creating 3D models of the trimeric Spike in complex with human ACE2 (hACE2). The RBD of Spike exists in two distinct conformations, referred to as ‘up’ and ‘down’ (Walls et al., 2020; Wrapp et al., 2020). The ‘up’ conformation corresponds to the receptor-accessible state with the RBD of one monomer exposed. By superimposing the RBD from the RBD-hACE2 complex (Yan et al., 2020) with the single RBD in the ‘up’ conformation (monomer 3) of the trimeric Spike (Walls et al., 2020), an initial model was obtained. Of note, although cryo-EM structures with more than one RBD in the ‘up’ conformation were shown to bind to two separate hACE2 molecules (rather than one single hACE2 dimer) (Benton et al., 2020), we decided to study the effect of the glycans on the Spike hACE2 interaction using the model with a single RBD in the ‘up’ conformation. To assess the impact of all seven individual N-glycosylation sites of hACE2 on its interaction with Spike, we first elucidated the entire glycome of rshACE2 (Figure 1—figure supplement 1). This also provided information on the glycans attached to N690, a glycosylation site not covered in previous glycoproteomic studies of soluble hACE2 (Zhao et al., 2020; Allen et al., 2021). For recombinant trimeric Spike, the glyco-analysis has been reported elsewhere (Hoffmann et al., 2021; Watanabe et al., 2020; Zhao et al., 2020; Sun et al., 2021). Based on the site-specific glycosylation profiles, we added complex or oligo-mannosidic glycan trees to the respective sites of Spike and ACE2 (Supplementray File 1). We hence constructed fully glycosylated atomistic models of the trimeric Spike glycoprotein, free dimeric ACE2 and of the Spike glycoprotein in complex with dimeric hACE2 (Figure 1). As glycans are known to be particularly flexible, and these were modeled in a single low-energy conformation, we subsequently performed molecular dynamics simulations of the Spike-ACE2 complex (Video 1), and of free hACE2. This allowed us to study the conformational distribution of the glycans on the surface of the proteins and their dynamic effects on the interaction between Spike and hACE2. Inspection of the most important interacting residues on Spike and ACE2, their average distances and the electrostatic potential of the interface area identified critical contact sites (Figure 1—figure supplements 2 and 3).

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We next quantified the complete solvent-accessible surface area (SASA) of the Spike protein in complex with ACE2, both with and without glycans. The average accessible area of protein atoms for non-glycosylated and glycosylated Spike was 1395 nm² and 864 nm², respectively, indicating that glycans shield about 38% of the protein surface of Spike, a value that is comparable to what was previously found in simulations of Spike alone (Sikora et al., 2021; Grant et al., 2020). The area of protein atoms that are shielded by the individual glycans are shown in Figure 2a and Figure 2—figure supplement 1.

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Further analysis showed that glycans at N122, N165, and N343 on Spike directly interact with ACE2 or its glycans (Figures 1b, c, 2b and c). It has been reported that Spike mutants lacking the glycans at N331 and N343 display reduced infectivity, while elimination of the glycosylation motif at N234 results in increased resistance to neutralizing antibodies, without reducing infectivity of the virus (Li et al., 2020). The equilibrium between the ‘up’ and ‘down’ conformations of Spike involves various stabilizing and destabilizing effects, with possible roles for the glycans at N165, N234, N331, and N343 (Casalino et al., 2020; Sztain et al., 2021; Mori et al., 2021). Removing the glycans at N165, N234 and N343 was experimentally seen to reduce binding to ACE2 by 10%, 40%, and 56%, respectively (Casalino et al., 2020; Sztain et al., 2021). In our MD simulations, the glycan at position N343 interacts directly with ACE2 (Figure 2), while the glycan at N331 interacts with a neighboring Spike monomer (Figure 1c, Figure 2—figure supplement 2), indicating that the N331 glycosylation site only indirectly affects the interaction of Spike with ACE2. In our model, the glycan at N234 also does not interact directly with ACE2, but seems to stabilize the ‘up’ conformation. Its removal could favor the ‘down’ conformation of the RBD, possibly explaining the observed more effective shielding against neutralizing antibodies. In agreement with previous simulations (Casalino et al., 2020) the Man9 glycan at N234 of Spike partially inserts itself into the vacant space in the core of the trimer that is created when the RBD of monomer three is in the ‘up’ conformation (Figure 1—figure supplement 4). In our simulations, the free space created by the ‘up’ conformation seems slightly smaller for Spike in complex with ACE2, suggesting that binding to ACE2 has a stabilizing effect on the Spike monomer.

The N165Q mutant was experimentally found to be more sensitive to neutralization (Li et al., 2020). In our models, the glycan at N165 is positioned directly next to the RBD (Figure 1b) and thus could shield important antigenic sites. These data highlight the complex impact of Spike glycosylation on the intramolecular interactions of the Spike monomers and, critically, the interaction with ACE2, posing a challenge to design SARS-CoV-2 neutralizing moieties.

Since our modeling clearly confirmed that ACE2 glycosylation plays a significant role in its binding to Spike (Figure 1d and e), we also determined the area of the Spike-ACE2 interface region, by subtracting the SASA of the complex from the SASA of the individual proteins and dividing by two. The total interface area was 24.6 nm², with glycans accounting for up to 51% of the interface area, that is 12.6 nm², contributed by the four most relevant glycans at positions N53, N90, N322, and N546 of ACE2 (Figure 3). Furthermore, we scored the number of atoms of each ACE2 glycan in contact with Spike. A contact was defined as a distance of less than 0.4 nm between two atoms. This allowed us to identify the glycans at N53, N90, N322, and N546 as interacting with Spike, with the glycan at position N53 having the weakest interaction. Notably, N546 interacted with Spike for a significant amount of time only in one of the two independent simulations. See Figure 3—figure supplement 1 for an analysis of the individual MD simulations and for separate analysis of the first and second half of the simulations. Remarkably, the interactions of the glycan at N90 seem to be more pronounced in the second, as compared to the first simulation. The degree of interaction correlated with the spatial proximity between the glycans and the RBD (Figure 1e and f). Assessing the number of hydrogen bonds that formed during the simulations, the glycans at N90 and N322 appear most prominent (Figure 3b) with a maximum occurrence of 10 hydrogen bonds existing concurrently. Interestingly, the glycans at N90 and N322 interact directly with Spike protein atoms, while the glycan at N546 (red sticks in Figure 1f) interacts with the glycans at N122 and N165 of Spike (dark green and orange sticks in Figure 1b). The glycans are highly dynamic, as can be seen in the breadth of the distributions of hydrogen bond occurrences and the multimodal character of the distributions for the interface area between the glycans and Spike (Figure 3c). A single glycan at N90 is observed to form up to 30 hydrogen bonds to Spike and to be responsible for an interface area of up to 10 nm², representing almost 40% of the average total interface area between Spike and ACE2. These findings are in agreement with previously reported simulations of the complexes (Zhao et al., 2020; Mehdipour and Hummer, 2021) but raise the question if the glycans contribute favorably to the binding of the two proteins by mediating relevant interactions or unfavorably because of steric restraints and a loss of conformational freedom upon binding.

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Next, we assessed the conformational freedom of ACE2 glycans upon binding to Spike and compared their respective density maps in the simulations of free ACE2, and ACE2 in complex with Spike (Figure 4). The density map of the unbound ACE2 (Figure 4a) shows a continuous density of glycans, largely covering the interface area. Formation of the ACE2-Spike complex significantly reduces the conformational freedom of the glycans, in particular the ones at N90 and N322 (Figure 4b). We predict that the glycans at N90 and N322 hamper binding to Spike, either sterically or through an entropic penalty upon binding due to a loss of conformational freedom. These glycans have been implicated as being relevant for binding before (Zhao et al., 2020), as well as the glycan at N53 (Barros et al., 2021), but no conclusions were drawn if they contribute positively or negatively to binding. Mehdipour and Hummer predicted the glycan at N322 to contribute favorably to binding, because of the favorable interactions of this glycan with the Spike surface (Mehdipour and Hummer, 2021). We did not observe a significantly more pronounced interaction with Spike for the glycan at N322, compared to the one at N90 (Figure 3). Based on conformational considerations, we therefore rather predict a negative impact on binding for both glycans (Figure 4).

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Since only the glycans at N90 and N322 directly interact with the protein atoms of the Spike proteins, while the glycan on N546 forms hydrogen bonds with glycans present on Spike, we set out to confirm the negative influence of N90 and N322 glycosylation on the interactions with Spike experimentally. First, we ablated N-glycosylation at N90 and N322 individually using the ACE2-Fc fusion constructs ACE2-T92Q-Fc (Chan et al., 2020) and ACE2-N322Q-Fc. Note that (Chan et al., 2020) indeed suggests that removal of the glycan at N90 through a mutation of T92 leads to enhanced interaction with Spike. The same data set, however, suggests that removal of the glycan at N322 through a mutation of T324 most likely leads to reduced affinity to Spike. However, T324 is itself part of the interface with Spike (Figure 1—figure supplement 5), and any mutation of this residue could easily disrupt ACE2 – Spike binding directly, rather than through its effect on the N322 glycosite. We therefore decided to mutate N322 into glutamine to prevent glycosylation at this position.

The wild-type and mutant ACE2-Fc constructs were expressed in HEK293-6E cells and purified from the culture supernatants by protein A affinity chromatography to apparent homogeneity (Figure 5—figure supplement 1). Analysis by size-exclusion chromatography combined with detection by multi-angle light scattering (SEC-MALS) demonstrated that all purified proteins were dimers of the expected native molecular mass (Figure 5—figure supplement 2). The impact of the introduced mutations on the overall fold of ACE2-Fc was tested with differential scanning calorimetry (DSC), a sensitive biophysical method for the assessment of the thermal stability of proteins. Three thermal transitions could be discriminated. The first midpoint of transition (T_m1) is due to the unfolding of ACE2, whereas the second and third midpoints of transitions (T_m2 and T_m3) reflect the thermal denaturation of the C_H2 and C_H3 domains of the Fc part of the fusion proteins (Lobner et al., 2017). The T_m1 midpoint transition temperatures of the ACE2-Fc glycomutants (53.3°C–54.0°C) were slightly higher than for the wild-type protein (52.2 °C), while T_m2 and T_m3 remained unchanged (Figure 5). This indicates that removal of the N90 and N322 glycans does not compromise the structural integrity of ACE2.

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The Spike-binding properties of the purified ACE2-Fc variants were characterized by biolayer interferometry (BLI). For this, ACE2-wt-Fc, ACE2-T92Q-Fc, and ACE2-N322Q-Fc were biotinylated, immobilized on streptavidin biosensor tips and dipped into serial dilutions of trimeric Spike. Since we did not observe appreciable dissociation of ACE2-Fc/trimeric Spike complexes in our analyses (Figure 6—figure supplement 1), we evaluated the association rates (k_obs; Figure 6a). To determine equilibrium affinity constants (K_D), we analyzed the interactions between the immobilized ACE2-Fc constructs and monomeric RBD (Figure 6b, Figure 6—figure supplement 2). The BLI data are in good agreement with our computational models, confirming that the removal of protein N-glycosylation at either N90 or N322 results in up to twofold higher binding affinities, when compared to ACE2-wt-Fc (ACE2-wt-Fc: K_D = 16.2 ± 0.7 nM; ACE2-T92Q-Fc: K_D = 8.0 ± 0.7 nM; ACE2-N322Q-Fc: K_D = 11.4 ± 0.3 nM; Figure 6b; Figure 6—figure supplement 2). Thus, structure-guided glyco-engineering at N90 and N322 results in ACE2 forms with increased affinity for SARS-CoV-2 Spike binding.

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Next, we tested the virus neutralization properties of ACE2-wt-Fc, ACE2-T92Q-Fc, and ACE2-N322Q-Fc. For this, we infected Vero E6 cells with 60 plaque-forming units (PFU; multiplicity of infection (MOI): 0.002) of SARS-CoV-2 in the presence of 10–50 µg/mL ACE2-wt-Fc, ACE2-T92Q-Fc, or ACE2-N322Q-Fc. The extent of SARS-CoV-2 infection and replication was quantified by RT-qPCR detection of viral RNA present in the culture supernatants. Untreated SARS-CoV-2 infected cells released up to 10 times more viral RNA than ACE2-wt-Fc-treated cells. Importantly, co-incubation of cells with SARS-CoV-2 and ACE2-T92Q-Fc resulted in significant further reduction of the viral load when compared to ACE2-wt-Fc. Enhanced SARS-CoV-2 neutralization was also observed for ACE2-N322Q-Fc. However, this mutant was less effective in promoting virus neutralization than ACE2-T92Q-Fc (Figure 7 and Figure 7—figure supplement 1). Similar results were obtained when SARS-CoV-2 neutralization assays were performed with much larger amounts of inoculated virus (MOI: 20) and concomitantly increased ACE2-Fc concentrations (Figure 7—figure supplement 2). Hence, in line with our structural glycan interaction map, the removal of either of the N-glycans attached to N90 and N322 gives rise to ACE2 decoy receptors with improved SARS-CoV-2 neutralization properties.

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To investigate a potential additive effect of simultaneous elimination of N-glycosylation at N90 and N322, we generated a double mutant ACE2-T92Q-N322Q-Fc construct. We also digested ACE2-wt-Fc with peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F (PNGase F) to remove all accessible N-glycans (deglyco-ACE2-wt-Fc) and neuraminidase to release terminal sialic acid residues (desialo-ACE2-wt-Fc). Purity and homogeneity of these additional ACE2-Fc variants was ascertained by SDS-PAGE and SEC-MALS (Figure 5—figure supplements 1 and 2). The absence of N-glycans attached to N90 and/or N322 in ACE2-T92Q-N322Q-Fc and the respective single mutants was demonstrated by LC-ESI-MS (Figure 7—figure supplement 3). Quantitative release of sialic acids and complete removal of N-glycans from all ACE2-wt-Fc N-glycosylation sites with the exception of N546 was also confirmed (Figure 7—figure supplements 4 and 5). The glycans at N546 of ACE2-wt-Fc exhibited partial resistance (40%) to PNGase F treatment (Figure 7—figure supplement 4). Combined introduction of the mutations T92Q and N322Q as well as enzymatic desialylation did not reduce the thermal stability of ACE2-Fc as assessed by DSC, while close-to-complete removal of N-glycans by PNGase F led to a slightly decreased midpoint transition temperature of the ACE2 domain (Figure 5). Studies of the interaction between ACE2-T92Q-N322Q-Fc and deglyco-ACE2-wt-Fc with RBD by BLI analysis yielded K_D values similar to those determined for the single mutant ACE2-T92Q-Fc (ACE2-T92Q-N322Q-Fc: K_D = 8.2 ± 0.2 nM; deglyco-ACE2-wt-Fc: K_D = 7.6 ± 0.3 nM). The affinity of desialo-ACE2-wt-Fc for RBD (K_D = 11.3 ± 0.4 nM) was also higher than that of native ACE2-wt-Fc (Figure 6b). The increased affinities of these ACE2-Fc variants for Spike correlate with their potencies to neutralize SARS-CoV-2, with deglyco-ACE2-wt-Fc followed by ACE2-T92Q-N322Q-Fc displaying the highest neutralization potencies (Figure 7 and Figure 7—figure supplement 2). The effect of desialo-ACE2-wt-Fc on SARS-CoV-2 infections of Vero E6 cells was less pronounced and comparable to that of the single mutant ACE2-N322Q-Fc (Figure 7), in good agreement with the almost identical RBD-binding affinities of these two ACE2-Fc variants (Figure 6b). Taken together, these data identify critical glycans at position N90 and N322 of ACE2 that structurally and functionally interfere with Spike-ACE2 binding; ablation of these glycans via site-directed mutagenesis or enzymatic deglycosylation generated ACE2 variants with improved Spike-binding properties and increased neutralization strength.

The results presented above uncover the critical importance of N-glycans located at the ACE2-Spike interface for the infection of host cells by SARS-CoV-2. This prompted us to test the feasibility of removing all N-glycans from clinical-grade rshACE2, which has undergone placebo-controlled phase II clinical testing in 178 COVID-19 patients (ClinicalTrials.gov Identifier: NCT04335136), and to test for its SARS-CoV-2 neutralization properties. To this end, we generated enzymatically deglycosylated clinical-grade rshACE2 (deglyco-rshACE2) using PNGase F. The quantitative release of all N-glycans, with the exceptions of those attached to the N432 and N546 glycosites, was confirmed by LC-ESI-MS/MS (Figure 7—figure supplement 4), and the integrity and homogeneity of dimeric deglyco-rshACE2 was demonstrated by SEC-MALS (Figure 5—figure supplement 2). Paralleling our observations with deglyco-ACE2-wt-Fc, we found the binding affinity of deglyco-rshACE2 to RBD (K_D = 5.1 ± 0.5 nM) to be two times higher than for native rshACE2 (K_D = 10.5 ± 0.4 nM; Figure 6b). Furthermore, deglyco-rshACE2 displayed improved SARS-CoV-2 neutralization properties in Vero E6 cell infection assays. At a final concentration of 200 µg/mL deglyco-rshACE2, we observed a significant reduction in SARS-CoV-2 replication when compared to treatment with the native form of the protein (Figure 8).

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Besides serving as a soluble decoy receptor to prevent SARS-CoV-2 infection of ACE2-expressing host cells, rshACE2 also regulates blood pressure and protects multiple organs such as the heart, kidney and lung as well as blood vessels via enzymatic degradation of angiotensin II (Vickers et al., 2002). In contrast to other recently described ACE2 mutants displaying improved Spike binding concomitant with inadvertently or intentionally impaired enzymatic activity (Glasgow et al., 2020; Chan et al., 2020), the catalytic activities of ACE2-T92Q-Fc, ACE2-N322Q-Fc and ACE2-T92Q-N322Q-Fc were found to be only modestly reduced as compared to ACE2-wt-Fc (ACE2-T92Q-Fc: 65% ± 11%; ACE2-N322Q-Fc: 69% ± 7%; ACE2-T92Q-N322Q-Fc: 79% ± 11%; Figure 9).

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Interestingly, deglyco-ACE2-wt-Fc (149% ± 1 %) and desialo-ACE2-wt-Fc (160% ± 2 %) exhibited higher enzymatic activities than native ACE2-wt-Fc (Figure 10 and Figure 10—figure supplement 1). A similar observation was made for deglyco-rshACE2, although the enhancing effects of enzymatic deglycosylation on catalytic efficiency were less pronounced (113% ± 2% as compared to native rshACE2; Figure 10). These results show that enzymatic removal of N-glycans from ACE2-Fc and clinical-grade rshACE2 results in increased Spike binding and enhanced SARS-CoV-2 neutralization while preserving its potentially critical enzymatic activity.

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Our data demonstrate that structure-guided glycoengineering is a powerful means to develop ACE2 variants with improved SARS-CoV-2 neutralization properties without compromising the structural stability and catalytic activity of the enzyme. Our in silico models of the Spike-ACE2 complex combined with simulations of its spatial and temporal dynamics rationalized previously published data and led to predictions that were confirmed by in vitro binding studies and cell-based SARS-CoV-2 neutralization assays. However, it requires further explanation how the moderately enhanced affinity of ACE2 glycovariants for monomeric RBD observed in biolayer interferometry experiments can relate to a far more pronounced increase of their virus-neutralization potency in Vero E6 cells. First, while the binding affinity is represented by an equilibrium constant, which is the ACE2 concentration at which 50% is in a bound state, the inhibitory strength in cell-based assays is measured at three distinct concentrations, at which one protein may show little inhibition (the relative concentration in the assay is below the K_d) while another variant shows strong inhibition (the relative concentration in the assay is above the K_d). Second, a cooperative effect may be expected for the association of trimeric Spike molecules present in the viral envelope with ACE2 dimers. In this supramolecular setting, a subtle increase in the affinity of ACE2 for RBD can lead to a dynamic equilibrium of binding and unbinding events with up to six potential interactions, leading to an overall much stronger avidity effect. The dynamic equilibrium of the RBDs between ‘down’ and ‘up’ conformations can further steepen the dose-response curves (Monod et al., 1965). Third, a slight advantage of the soluble ACE2 decoy receptor over endogenous native ACE2 may be sufficient to tip the balance between SARS-CoV-2 attachment and shedding of viral particles from the host cell surface. The inhibitory strength is not a function of the number of Spike proteins that are bound, but of the probability that a high enough fraction of Spike proteins is bound, potentiating a small increase in affinity (Magnus, 2013). Fourth, the SARS-CoV-2 neutralization assays shown in Figure 7 were performed at a low multiplicity of infection. This can lead to complete neutralization of all virus particles in the inoculum (Monteil et al., 2020) as becomes evident from the individual data points of our experiments (Figure 7—figure supplement 1). Hence, the data are presented as medians and not as arithmetic means, which potentially accentuates the numeric differences between individual samples. Finally, it is possible that the N-glycan moiety of ACE2 also modulates other aspects of viral entry besides promoting the docking of Spike to the cell surface (Yang et al., 2020).

It has been reported that the sialylation status of ACE2 affects its interactions with SARS-CoV-2 Spike (Allen et al., 2021). We have found that enzymatic desialylation of ACE2 results in a reproducible increase of its affinity to RBD without detectable structural penalties. Importantly, desialylated ACE2 is more efficient in neutralizing SARS-CoV-2 than its native counterpart. Molecular simulations suggest that the terminal sialic acids of the N-glycans attached to ACE2 residues N90 and N322 mask parts of the Spike-ACE2 interface and thus could interfere with Spike binding through steric clashes and/or electrostatic effects (Figure 4—figure supplement 1). This provides a structural rationale how sialic acids present on ACE2 might dampen interactions with Spike during SARS-CoV-2 attachment to host cells (Chu et al., 2021).

We want to point out that all virus-neutralization experiments presented in this paper were performed using Vero E6 cells. Although this cell line is very popular for SARS-CoV-2 propagation and infectivity studies, it will be important to repeat our neutralization assays with a cell line derived from human lung (e.g. Calu-3) (Johnson et al., 2021). Furthermore, treatment with deglycosylated ACE2 could have per se a negative impact on cell viability and thus affect virus replication in the treated cultures. We will assess the potential cellular toxicity of deglycosylated ACE2 in future studies. However, we have shown previously that native ACE2 does not display any cytotoxic effects even when used at very high concentrations (Monteil et al., 2020).

In line with other reports (Chan et al., 2020; Chu et al., 2021), our results indicate that the elimination of the N-glycans attached to N90 is largely responsible for the improved Spike-binding properties of enzymatically deglycosylated ACE2. As proposed (Glasgow et al., 2020; Chan et al., 2020) and corroborated by our mutational analysis, substitution of ACE2 residues N90 or T92 could indeed provide an alternative approach for the development of ACE2 variants with improved SARS-CoV-2 sequestering properties. Our data indicate that ablation of N90 glycosylation could be combined with mutations of N322 and possibly other ACE2 N-glycosylation sites to achieve an even higher SARS-CoV-2 neutralizing potency. However, expression of an ACE2 variant lacking all potential N-glycosylation sites in ACE2-negative host cells led to reduced rather than enhanced susceptibility of the cells to SARS-CoV-2 as compared to transduction with wild-type ACE2 (Chu et al., 2021). This was attributed to the much lower cellular content of the mutant protein relative to the native enzyme, thus demonstrating that the importance of N-glycosylation for proper folding of glycoproteins during their biosynthesis (Xu and Ng, 2015) also applies to ACE2. Given the inferior expression yields of glycan-free ACE2 and the potential of unwanted immunological side effects when non-natural mutations are introduced into a therapeutic glycoprotein, we believe that the clinical potential of enzymatically deglycosylated rshACE2 is superior to that of any of our ACE2 glycomutants. In our opinion, treatment of clinical-grade rshACE2 with deglycosylation enzymes such as PNGase F followed by a final polishing step represents a straightforward, Good Manufacturing Practice (GMP)-compliant and industrially feasible alternative to generate a potent therapeutic drug for the treatment of SARS-CoV-2 infected persons and patients.

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Author details

1. Tümay Capraz

   Institute for Molecular Modeling and Simulation, University of Natural Resources and Life Sciences (BOKU), Vienna, Austria

   Contribution

   Formal analysis, Investigation, Visualization, Writing – original draft

   Contributed equally with

   Nikolaus F Kienzl and Elisabeth Laurent

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-2547-067X

2. Nikolaus F Kienzl

   Institute of Plant Biotechnology and Cell Biology, Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences (BOKU), Vienna, Austria

   Contribution

   Formal analysis, Investigation, Visualization

   Contributed equally with

   Tümay Capraz and Elisabeth Laurent

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-8057-3930

3. Elisabeth Laurent

   Institute of Molecular Biotechnology, Department of Biotechnology and Core Facility Biomolecular & Cellular Analysis, University of Natural Resources and Life Sciences (BOKU), Vienna, Austria

   Contribution

   Formal analysis, Investigation, Visualization, Writing – original draft

   Contributed equally with

   Tümay Capraz and Nikolaus F Kienzl

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-5234-5524

4. Jan W Perthold

   Institute for Molecular Modeling and Simulation, University of Natural Resources and Life Sciences (BOKU), Vienna, Austria

   Contribution

   Investigation, Supervision, Writing – original draft

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-8575-0278

5. Esther Föderl-Höbenreich

   Diagnostic and Research Institute of Pathology, Medical University of Graz, Graz, Austria

   Contribution

   Formal analysis, Investigation, Visualization

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-2066-1036

6. Clemens Grünwald-Gruber

   Institute of Biochemistry, Department of Chemistry, University of Natural Resources and Life Sciences, Vienna, Austria

   Contribution

   Investigation

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-6097-8348

7. Daniel Maresch

   Institute of Biochemistry, Department of Chemistry, University of Natural Resources and Life Sciences, Vienna, Austria

   Contribution

   Investigation

   Competing interests

   No competing interests declared

8. Vanessa Monteil

   Karolinska Institute, Department of Laboratory Medicine, Stockholm, Sweden

   Contribution

   Investigation

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-2652-5695

9. Janine Niederhöfer

   Apeiron Biologics, Vienna, Austria

   Contribution

   Investigation

   Competing interests

   employee of Apeiron Biologics. Apeiron holds a patent on the use of ACE2 for the treatment of lung, heart, or kidney injury and applied for a patent to treat COVID-19 with rshACE2

10. Gerald Wirnsberger

    Apeiron Biologics, Vienna, Austria

    Contribution

    Investigation, Resources, Supervision

    Competing interests

    employee of Apeiron Biologics. Apeiron holds a patent on the use of ACE2 for the treatment of lung, heart, or kidney injury and applied for a patent to treat COVID-19 with rshACE2

    "This ORCID iD identifies the author of this article:" 0000-0001-7035-7038

11. Ali Mirazimi

    1. Karolinska Institute, Department of Laboratory Medicine, Stockholm, Sweden
    2. National Veterinary Institute, Uppsala, Sweden

    Contribution

    Supervision

    Competing interests

    No competing interests declared

12. Kurt Zatloukal

    Diagnostic and Research Institute of Pathology, Medical University of Graz, Graz, Austria

    Contribution

    Supervision

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0001-5299-7218

13. Lukas Mach

    Institute of Plant Biotechnology and Cell Biology, Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences (BOKU), Vienna, Austria

    Contribution

    Formal analysis, Investigation, Supervision, Writing – original draft, Writing – review and editing

    For correspondence

    lukas.mach@boku.ac.at

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0001-9013-5408

14. Josef M Penninger

    1. IMBA - Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Dr. Bohr, Vienna, Austria
    2. Department of Medical Genetics, Life Sciences Institute, University of British Columbia, Vancouver, Canada

    Contribution

    Conceptualization, Writing – review and editing

    For correspondence

    josef.penninger@ubc.ca

    Competing interests

    declares a conflict of interest as a founder, supervisory board member, and shareholder of Apeiron Biologics. Apeiron holds a patent on the use of ACE2 for the treatment of lung, heart, or kidney injury and applied for a patent to treat COVID-19 with rshACE2

    "This ORCID iD identifies the author of this article:" 0000-0002-8194-3777

15. Chris Oostenbrink

    Institute for Molecular Modeling and Simulation, University of Natural Resources and Life Sciences (BOKU), Vienna, Austria

    Contribution

    Conceptualization, Supervision, Writing – original draft, Writing – review and editing

    For correspondence

    chris.oostenbrink@boku.ac.at

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-4232-2556

16. Johannes Stadlmann

    1. Institute of Biochemistry, Department of Chemistry, University of Natural Resources and Life Sciences, Vienna, Austria
    2. IMBA - Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Dr. Bohr, Vienna, Austria

    Contribution

    Conceptualization, Formal analysis, Investigation, Supervision, Writing – original draft, Writing – review and editing

    For correspondence

    j.stadlmann@boku.ac.at

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0001-5693-6690

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