soluble recombinant human ace2  (Recombina)

Journal: Journal of Virology

Article Title: ACE2 acts as a novel regulator of TMPRSS2-catalyzed proteolytic activation of influenza A virus in airway cells

doi: 10.1128/jvi.00102-24

Figure Lengend Snippet: Schematic domain structures of ACE2 and TMPRSS2. ACE2 is a type I transmembrane metalloproteinase comprised of a short cytoplasmic domain and a large ectodomain that contains the collectrin domain (CD) and the carboxypeptidase domain (PD) carrying a zinc-binding motif (HEMGH). ACE2 can be cleaved by ADAM17 or TMPRSS2 enabling ectodomain shedding. TMPRSS2 is a type II transmembrane serine protease with a stem region comprising a low-density lipoprotein receptor class A domain (LDLRA), a scavenger receptor cysteine-rich domain (SRCR), and a serine protease domain containing the catalytic triad histidine (H), aspartic acid (D), and serine (S). TMPRSS2 undergoes autocatalytic activation by cleavage at R255 (indicated by an arrowhead). TM: transmembrane domain, SP: signal peptide. ADAM17: a disintegrin and metallopeptidase domain 17. Gray numbers indicate the amino acids.

Article Snippet: Recombinant soluble human ACE2 (aa 18–740) was purchased from Abcam (ab273297).

Techniques: Binding Assay, Activation Assay

Journal: Journal of Virology

Article Title: ACE2 acts as a novel regulator of TMPRSS2-catalyzed proteolytic activation of influenza A virus in airway cells

doi: 10.1128/jvi.00102-24

Figure Lengend Snippet: ACE2 increases the enzymatic activity of recombinant TMPRSS2 in a non-catalytic manner. (A and B) Kinetic measurement of TMPRSS2 activity in the absence and presence of ACE2. Recombinant human TMPRSS2 (0.12 nM) was mixed with recombinant human ACE2 (0.8 µU) prior to the addition of fluorogenic TMPRSS2 substrate Boc-Gln-Ala-Arg-AMC. Fluorescence was measured over 1 h at room temperature (RT) (A) or at 37°C (B). When indicated, ACE2 carboxypeptidase activity was inhibited by the addition of DX600 (10 µM). (C) ACE2 activity in the presence of TMPRSS2. Recombinant ACE2 (0.8 µU) was mixed with recombinant TMPRSS2 (0.12 nM) in the absence or presence of DX600 (10 µM) prior to the addition of a quenched fluorogenic ACE2 substrate. The cleavage rate of the substrate was measured over 1h. RFU: relative fluorescence units. All data are mean values ± standard deviations (SD) of two independent experiments performed in duplicate or triplicate.

Article Snippet: Recombinant soluble human ACE2 (aa 18–740) was purchased from Abcam (ab273297).

Techniques: Activity Assay, Recombinant, Fluorescence

Journal: Journal of Virology

Article Title: ACE2 acts as a novel regulator of TMPRSS2-catalyzed proteolytic activation of influenza A virus in airway cells

doi: 10.1128/jvi.00102-24

Figure Lengend Snippet: Knockdown of ACE2 suppresses cleavage of influenza A virus HA with monobasic cleavage site in Calu-3 airway cells. (A) Knockdown of ACE2 expression in Calu-3 cells using PPMO. Calu-3 cells were treated with 25 µM of ACE2-specific PPMO or a nonsense-sequence negative-control PPMO (scramble) or remained untreated (w/o) for 24 h. Cell lysates were subjected to SDS-PAGE and western blot analysis using an ACE2-specific antibody. Actin was used as a loading control. (B) Evaluation of the effect of PPMO treatment on cell viability. Calu-3 cells were treated with 25 µM of PPMO for 24 h. Cell viability of untreated cells (w/o) was set as 100%. Results are mean values + SD of two independent experiments performed in triplicate. Treatment with 5% ethanol (EtOH) was used as a control. (C) Calu-3 cells were treated with 25 µM PPMO or remained untreated for 24 h. Cells were then inoculated with H1N1pdm at a low MOI and incubated for 72 h without further PPMO treatment. Virus titers were determined by plaque assay at indicated time points. Data shown are mean values ± SD of three independent experiments. (D) Calu-3 cells treated with 25 µM PPMO for 24 h were inoculated with H1N1pdm at a MOI of 1. Cell lysates were subjected to SDS-PAGE and western blotting using ACE2- or HA-specific antibodies at 24 h p.i. (E/G) Calu-3 cells were treated with 25 µM PPMO for 24 h, then infected with H3N2 (E) or H7N7 (G) at a low MOI and incubated in the absence of PPMO for 72 h. At indicated time points, virus titers in supernatants were determined by plaque assay. (F) Calu-3 cells treated with PPMO for 24 h were infected with H3N2 at a MOI of 1. At 24 h p.i., cell lysates were analyzed by SDS-PAGE and western blotting using antibodies against ACE2 and H3. Uninfected cells (mock) served as control. (H) Lysates of H7N7 infected cells described above were subjected to SDS-PAGE and western blot analysis with ACE2- and H7-specific antibodies at 72 h p.i. (I) Calu-3 cells were infected with H1N1pdm at a low MOI, then treated with 5 µM ACE2 inhibitor DX600 or DMSO or remained untreated and incubated for 72 h. Virus replication was determined by plaque titration at indicated time points p.i. Data are mean ± SD of three independent experiments. (J) At 72 h p.i., cell lysates were analyzed for ACE2 expression and HA cleavage by SDS-PAGE and immunoblotting. DX600 concentration: 1 and 5 µM, respectively. One representative blot from three independent experiments is shown (D, F, H, J). Actin was used as a loading control.

Article Snippet: Recombinant soluble human ACE2 (aa 18–740) was purchased from Abcam (ab273297).

Techniques: Virus, Expressing, Sequencing, Negative Control, SDS Page, Western Blot, Incubation, Plaque Assay, Infection, Titration, Concentration Assay

Journal: Journal of Virology

Article Title: ACE2 acts as a novel regulator of TMPRSS2-catalyzed proteolytic activation of influenza A virus in airway cells

doi: 10.1128/jvi.00102-24

Figure Lengend Snippet: ACE2 increases H1N1pdm activation and replication in Caco-2 cells. (A) Caco-2 cells were transfected with an ACE2-encoding plasmid or empty vector (EV). At 24 h post-transfection cells were infected with H1N1pdm at an MOI of 0.001 and incubated for 72 h. Cell lysates were analyzed by SDS-PAGE and immunoblot using antibodies against HA and ACE2, respectively. Actin was used as a loading control. Numbers indicate quantification of HA with HA0 in EV-transfected cells set as 100%. (B) Virus titers in supernatants were determined by plaque assay. Data are mean values + SD of n = 3 (24 h) and n = 2 (48 h) independent experiments.

Article Snippet: Recombinant soluble human ACE2 (aa 18–740) was purchased from Abcam (ab273297).

Techniques: Activation Assay, Transfection, Plasmid Preparation, Infection, Incubation, SDS Page, Western Blot, Virus, Plaque Assay

Journal: Journal of Virology

Article Title: ACE2 acts as a novel regulator of TMPRSS2-catalyzed proteolytic activation of influenza A virus in airway cells

doi: 10.1128/jvi.00102-24

Figure Lengend Snippet: Analysis of ACE2-TMPRSS2 interaction by co-immunoprecipitation. (A) Scheme of the domain structure of ACE2 mutants and TMPRSS2 mutants used in this study. Wildtype ACE2 and mutants were expressed with a C-terminal HA-tag, TMPRSS2 and mutants TMPRSS2(S441A) and TMPRSS2(R255Q) with a C-terminal 3x FLAG-tag. Autocatalytic activation of TMPRSS2 at R255 (arrowhead) and cleavage of ACE2 by TMPRSS2 (scissor) are indicated. TM: transmembrane domain, CD: collectrin domain, LDLRA: LDL receptor class A domain, SRCR: scavenger receptor cysteine-rich domain. (B) Co-immunoprecipitation analysis of TMPRSS2 and ACE2 mutants. HEK293 cells were transiently co-transfected with plasmids encoding wild-type ACE2 or the indicated mutants jointly with plasmids encoding TMPRSS2 or TMPRSS2 mutants or empty plasmid as control. At 48 h post-transfection, cells were lysed and subjected to HA-tag- and FLAG-tag-specific immunoprecipitation (IP), respectively, followed by western blot analysis of lysates (input) and precipitates using antibodies against the HA-tag or FLAG-tag. The results are representative of two or three independent experiments. Lanes are identified by gray numbers. ACE2-∆PD is indicated by a white arrowhead. (C) Summary of the capability of TMPRSS2 to interact with (Co-IP) and/or cleave (scissor) the indicated ACE2 mutant.

Article Snippet: Recombinant soluble human ACE2 (aa 18–740) was purchased from Abcam (ab273297).

Techniques: Immunoprecipitation, FLAG-tag, Activation Assay, Transfection, Plasmid Preparation, Western Blot, Co-Immunoprecipitation Assay, Mutagenesis

Journal: Journal of Virology

Article Title: ACE2 acts as a novel regulator of TMPRSS2-catalyzed proteolytic activation of influenza A virus in airway cells

doi: 10.1128/jvi.00102-24

Figure Lengend Snippet: Knockdown of ACE2 suppresses multicycle replication of MERS-CoV and cleavage of S in Calu-3 airway cells. (A) Calu-3 cells were treated with ACE2-AUG PPMO or remained untreated (w/o) for 24 h. Cells were then inoculated with MERS-CoV at a MOI of 0.001. Virus titers in supernatants were determined by TCID50 at indicated time points post-infection. The mean + SD of three independent experiments are shown. (B) Calu-3 cells treated with ACE2-AUG or scramble PPMO or without PPMO treatment were infected with MERS-CoV as described above. At 72 h p.i., cell lysates were subjected to SDS-PAGE and western blot analysis using MERS-CoV S2-specific antibodies. Uninfected cells served as control (mock).

Article Snippet: Recombinant soluble human ACE2 (aa 18–740) was purchased from Abcam (ab273297).

Techniques: Virus, Infection, SDS Page, Western Blot

Journal: Journal of Virology

Article Title: ACE2 acts as a novel regulator of TMPRSS2-catalyzed proteolytic activation of influenza A virus in airway cells

doi: 10.1128/jvi.00102-24

Figure Lengend Snippet: Model depicting how ACE2 expression may regulate the cleavage of IAV HA or MERS-CoV S by TMPRSS2. (A) Co-expression of ACE2 and TMPRSS2 in the trans-Golgi network (TGN) increases or stabilizes TMPRSS2 activity and supports efficient HA cleavage by TMPRSS2. (B) ACE2 knockdown results in reduced TMPRSS2 activity or stability in the TGN and thereby prevents cleavage of newly synthesized HA0 into HA1 + HA2. NA: neuraminidase. (C) ACE2 may regulate TMPRSS2 activity at the cell surface. Knockdown of ACE2 reduces the activity or stability of TMPRSS2 at the plasma membrane and inhibits proteolytic activation of MERS-CoV S upon entry.

Article Snippet: Recombinant soluble human ACE2 (aa 18–740) was purchased from Abcam (ab273297).

Techniques: Expressing, Activity Assay, Synthesized, Membrane, Activation Assay

Journal: iScience

Article Title: Inhalation of ACE2 as a therapeutic target on sex-bias differences in SARS-CoV-2 infection and variant of concern

doi: 10.1016/j.isci.2023.107470

Figure Lengend Snippet: SARS-CoV-2 infection in male and female k18-hace2 mice over 7 days post-infection (dpi) (A) Body weight loss in response to intranasal inoculation of SARS-CoV-2 (4 × 10 5 TCID50/50μL/mouse). ∗p < 0.05 vs. male. Data represented in median ± IQR. (B) Lung injury scoring assessed on a scale of 0–4 for each of the following criteria: 1) neutrophil numbers in the alveolar space, 2) alveolar septal thickening, 3) number of hyaline membranes, 4) alveolar hemorrhage, and 5) cellular hyperplasia. ∗p < 0.05 vs. Ctrl and male. Data represented in mean ± SEM. (C) Viral RNA levels in oropharyngeal swabs at 2, 4, and 6 dpi. ∗p < 0.05 vs. male. Data represented in median ± IQR. (D–G) Copy numbers of E-gene and RdRp-gene in multiple organ tissues. ∗p < 0.05 vs. Ctrl. Data represented in mean ± SEM. (H) Detection of SARS-CoV-2 nucleocapsid protein (red) in lung tissues. Scale bars, 50 μm (main images) and 20 μm (magnified). (I) Protein expression in lung tissue detected by western blots in vehicle control (C) and at 7 dpi. (J) Concentration of soluble RAGE protein in plasma in vehicle control and at 7 dpi. ∗p < 0.05 vs. other groups. Data represented in mean ± SEM. (K) Correlation of protein levels of ACE2 and ERα in lung tissue of male mice before and after infection at 7 dpi. (L) Estrogen receptor alpha (ERα)/androgen receptor (AR) ratio assessed from western blot analysis in relation to β-actin loading control. ∗p < 0.05 vs. male. Data represented in mean ± SEM. (M) Concentration of 17β Estradiol in lung tissue homogenates in vehicle control and at 7 dpi. ∗p < 0.05 vs. male. n = 5–7 biologically independent mice for all groups. Data represented in mean ± SEM. (see also Figure S1 ).

Article Snippet: Male K18-hACE2 mice aged 8–10 weeks received soluble human recombinant ACE2 (rACE2, Apeiron Biologics, Vienna, Austria) either by intravenous injection (0.4 mg/kg) or nebulization (DD 12 mg/kg).

Techniques: Infection, Expressing, Western Blot, Control, Concentration Assay, Clinical Proteomics

Journal: iScience

Article Title: Inhalation of ACE2 as a therapeutic target on sex-bias differences in SARS-CoV-2 infection and variant of concern

doi: 10.1016/j.isci.2023.107470

Figure Lengend Snippet: Inhalation of rACE2 attenuates SARS-CoV-2 infection, replication, and lung injury and recovers expression of endogenous ACE2 and ERα in K18-hACE2 male mice at 7 dpi (A) Study scheme. Mice were intranasally inoculated with SARS-CoV-2 (4 × 10 5 TCID50/50μL/mouse) and 48 h later received daily nebulization of rACE2 (12 mg/kg) or vehicle control solution for 5 days. (B and C) Copy numbers of viral envelope (E) gene by RT-qPCR and titers (TCID50) in lung tissue in the SARS-CoV-2 infection alone and rACE2-treated groups. ∗p < 0.05 vs. SARS-CoV-2. Data represented in mean ± SEM. (D) Gene expression of IL-6 in lung tissue in the infection alone and rACE2-treated groups. ∗p < 0.05 vs. SARS-CoV-2. Data represented in mean ± SEM. (E) Concentration of soluble RAGE protein in plasma in infection alone and rACE2-treated mice. ∗p < 0.05 vs. SARS-CoV-2. Data represented in mean ± SEM. (F) Detection of SARS-CoV-2 nucleocapsid protein (red) in lung tissues. Scale bars, 50 μm (main images) and 20 μm (magnified). (G) Representative lung histology by H&E staining. Arrows indicate neutrophil infiltration in the alveolar space. Lung injury scores from 6 animals per group are also shown. ∗p < 0.05 vs. naive control (C), ♰p < 0.05 vs. SARS-CoV-2 alone & (C). Data represented in mean ± SEM. (H and I) Protein expression in lung tissue detected by western blots in naive control (C), SARS-CoV-2 (CoV2) alone, and rACE2-treated groups. ∗p < 0.05 vs. C & rACE2-treated groups. Data represented in mean ± SEM. (J and K). Fold changes in the concentrations of 17β Estradiol and ERα/AR ratio assessed from western blot analysis in relation to β-actin loading control in lung tissue in naive control (C), SARS-CoV-2 (CoV2) alone, and rACE2-treated groups. ∗p < 0.05 vs. C and SARS-CoV-2, respectively. n = 5–7 biologically independent mice for all groups. Data represented in mean ± SEM. (see also Figure S3 ).

Article Snippet: Male K18-hACE2 mice aged 8–10 weeks received soluble human recombinant ACE2 (rACE2, Apeiron Biologics, Vienna, Austria) either by intravenous injection (0.4 mg/kg) or nebulization (DD 12 mg/kg).

Techniques: Infection, Expressing, Control, Quantitative RT-PCR, Gene Expression, Concentration Assay, Clinical Proteomics, Staining, Western Blot

Journal: iScience

Article Title: Inhalation of ACE2 as a therapeutic target on sex-bias differences in SARS-CoV-2 infection and variant of concern

doi: 10.1016/j.isci.2023.107470

Figure Lengend Snippet: Nebulization of rACE2 partially restores the ERα-associated signaling proteomic profiles in lung tissue at 7 dpi Proteomic analysis revealed that treatment with rACE2 resulted in an increase in 31 ERα-associated proteins and a decrease in 10 proteins. Of note, 16 of these proteins (highlighted in purple) were also found in female mice without rACE2 treatment at 7 dpi.

Article Snippet: Male K18-hACE2 mice aged 8–10 weeks received soluble human recombinant ACE2 (rACE2, Apeiron Biologics, Vienna, Austria) either by intravenous injection (0.4 mg/kg) or nebulization (DD 12 mg/kg).

Techniques:

Journal: iScience

Article Title: Inhalation of ACE2 as a therapeutic target on sex-bias differences in SARS-CoV-2 infection and variant of concern

doi: 10.1016/j.isci.2023.107470

Figure Lengend Snippet: Treatment with rACE2 attenuates Delta variant replication and viral load and decreases cytopathic effects in human lung organoids (A) Human lung organoids expressing pro-surfactant protein C (SPC) and epithelial cell adhesion molecule (EpCAM). Scale bars, 20 μm (main images). (B) Representative images of human lung organoids in naive control (Ctrl, upper panel), Delta variant-challenged (middle panel), and treated with a pre-mixture of rACE2 (400 μg/mL) and Delta variant (1.8 × 10 7 TCID50/mL) for 30 min (lower panel). The expression of ACE2 (red) and viral nucleocapsid protein (green) were detected at 4 h post-infection. Scale bars, 5 μm. (see also Figure S4 ). (C) Levels of viral envelope (E) gene RNA were assessed by qRT-PCR after treatment with various concentrations of rACE2 in human lung organoids at 3 dpi. Supernatants of the Delta variant-infected lung organoids were collected and used to infect Vero E6 cells for TCID50 assay. ∗p < 0.05 vs. Mock; ♰ p < 0.05 vs. Delta variant alone. Data represented in mean ± SEM. (D) Representative images of cytopathic effects of human lung organoids in Delta variant infection and rACE2-treated groups at 3 dpi. Scale bars, 1000 μm (main images) and 400 μm (magnified). Data presented are representatives of three independent experiments.

Article Snippet: Male K18-hACE2 mice aged 8–10 weeks received soluble human recombinant ACE2 (rACE2, Apeiron Biologics, Vienna, Austria) either by intravenous injection (0.4 mg/kg) or nebulization (DD 12 mg/kg).

Techniques: Variant Assay, Expressing, Control, Infection, Quantitative RT-PCR, TCID50 Assay

Journal: iScience

Article Title: Inhalation of ACE2 as a therapeutic target on sex-bias differences in SARS-CoV-2 infection and variant of concern

doi: 10.1016/j.isci.2023.107470

Figure Lengend Snippet: Proposed mechanisms underlying biological sex differences in outcomes of SARS-CoV-2 and Delta variant infections and the therapeutic potential of inhaled ACE2 (A) Under physiological conditions, ACE2 catalyzes the conversion of Ang II to Ang1-7 for organ protection. The ACE2 gene is located at X chromosome p22.2, where genes are known to escape X-inactivation, contributing to phenotypic differences of ACE2 between biological sexes. Membrane expression of ACE2 can be upregulated by estrogens through estrogen receptor α (ERα), which exerts anti-inflammatory properties and further enhances organ protection. In contrast, androgen receptor (AR) can cleave ACE2 and mediate pro-inflammation and tissue injury. . (B) During SARS-CoV-2 infection, the virus binds to and decreases host cell surface ACE2. For a given infection, the reduction of endogenous ACE2 expression is associated with a dramatic decrease in ERα expression, resulting in loss of organ protection in males. In contrast, expression of endogenous ACE2 was sustained in females, potentially due to the extra X-linked ACE2 and well-maintained ERα expression, leading to compensatory mechanisms and persistent organ protection. (C) Inhalation of rACE2 can restore the balance of endogenous ACE2 and ERα by blocking or attenuating SARS-CoV-2 from binding to membrane ACE2 in males.

Article Snippet: Male K18-hACE2 mice aged 8–10 weeks received soluble human recombinant ACE2 (rACE2, Apeiron Biologics, Vienna, Austria) either by intravenous injection (0.4 mg/kg) or nebulization (DD 12 mg/kg).

Techniques: Variant Assay, Membrane, Expressing, Infection, Virus, Blocking Assay, Binding Assay

Journal: iScience

Article Title: Inhalation of ACE2 as a therapeutic target on sex-bias differences in SARS-CoV-2 infection and variant of concern

doi: 10.1016/j.isci.2023.107470

Figure Lengend Snippet:

Article Snippet: Male K18-hACE2 mice aged 8–10 weeks received soluble human recombinant ACE2 (rACE2, Apeiron Biologics, Vienna, Austria) either by intravenous injection (0.4 mg/kg) or nebulization (DD 12 mg/kg).

Techniques: Virus, Isolation, Recombinant, Western Blot, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Software

Journal: Nature Communications

Article Title: Force-tuned avidity of spike variant-ACE2 interactions viewed on the single-molecule level

doi: 10.1038/s41467-022-35641-3

Figure Lengend Snippet: a Spike trimer in complex with one or two ACE2 dimers (indicated by red arrows). b AFM image (left) and simulation using MDS modeling (right) of Spike trimer bound to one ACE2. c Schematic illustration of single-molecule force spectroscopy experiments, in which dissociation forces between the Spike trimer (or RBD) on AFM tips and membrane-anchored ACE2 expressed on VeroE6 cells were measured. d Exemplary force–distance curves from the experiments. The Spike trimer (green lines) exhibits up to three interactions per force curve. Multiple bonds either break sequentially ((2) and (3)) or simultaneously ((II) and (III)). Monomeric RBD shows only one interaction per force curve (red line). e Experimental probability density functions (PDF) at a fixed pulling velocity. Force maxima for the Spike trimer (green curve) correspond to the simultaneous dissociation of one (I) two (II) or three (III) bonds. Monomeric RBD (red curve) shows only one force maximum, reflecting the dissociation of a single bond. f Dynamic force spectroscopy from experiments at different pulling speeds. Dependence of dissociation forces on force loading rate. Data of the first peaks were fitted with the Bell–Evans model (thin green line), which assumes that a sharp single energy barrier is crossed for dissociation and yields the kinetic off rate, k off , and the dissociation path length, x B (Table ). Using the fitting parameter of the Bell–Evans theory, the orange line and blue line were calculated using the Markov binding model for uncorrelated failure of two and three bonds, respectively. The thick magenta line is the Bell–Evans fit of raw force data for RBD. g Dependence of binding activity on preset contact (interaction) time between the AFM tip and cellular surfaces for a Spike trimer (green circles) and for a single RBD (pink circles). Binding activity ratios for two (orange diamonds) and three (blue triangles) bonds of Spike trimer are also shown. Kinetic on-rates are retrieved from exponential fittings using pseudo-first-order kinetics of a bimolecular reaction (solid lines). Source data are provided as a Source Data file.

Article Snippet: Clinical-grade soluble recombinant human ACE2 (amino acids 1–740) was produced by Polymun Scientific (contract manufacturer) from CHO cells according to Good Manufacturing Practice guidelines and formulated as a physiologic aqueous solution.

Techniques: Force Spectroscopy, Membrane, Binding Assay, Activity Assay

Journal: Nature Communications

Article Title: Force-tuned avidity of spike variant-ACE2 interactions viewed on the single-molecule level

doi: 10.1038/s41467-022-35641-3

Figure Lengend Snippet: a Simulations of one, two, and three dimeric ACE2 receptors (left to right; shades of violet, green, blue; intracellular domain not shown) bound to a single Spike trimer (shades of red). The nanodisc membrane is shown in gray. Glycans on Spike and ACE2 are shown as gold sticks. b Force-extension ( d ) curves (top; 10-ns window average in solid red, raw data in pink) and fraction Q of Spike:ACE2 contacts (bottom). Vertical lines, yellow arrow, and star indicate dissociation events, the partial unfolding of the ACE2 linker domain, and the intermediate state with ~50% of the contacts, respectively. c Snapshots from the Spike:ACE2 simulation at a force loading rate of 0.0166 N/s (orange arrows, other symbols as in b left) until detachment (right). d Zoom-in on the Spike:ACE2 interface of ( c ). Key residues are indicated in red (RBD) and violet (ACE2, including its N322 glycan involved in RBD binding). The asterisk indicates the intermediate state of ( b ) and ( c ). Source data are provided as a Source Data file.

Article Snippet: Clinical-grade soluble recombinant human ACE2 (amino acids 1–740) was produced by Polymun Scientific (contract manufacturer) from CHO cells according to Good Manufacturing Practice guidelines and formulated as a physiologic aqueous solution.

Techniques: Membrane, Glycoproteomics, Binding Assay

Journal: Nature Communications

Article Title: Force-tuned avidity of spike variant-ACE2 interactions viewed on the single-molecule level

doi: 10.1038/s41467-022-35641-3

Figure Lengend Snippet: a First row: Single Spike trimer N234Q mutant imaged using high-speed AFM. No details of RBDs or stalk are visible and only small conformational changes were observed. Second row: Single Spike trimer N234Q imaged in buffer solution containing free ACE2. One RBD in the open conformation (indicated by a white arrowhead) is bound to ACE2 (red arrowhead). b Left panels: AFM images of a single Spike mutant N234Q without ACE2 (upper image) and complexed with ACE2 (lower image). Right panels: simulations of a closed Spike trimer without ACE2 (upper image) and complexed with ACE2 (lower image). c Spike trimer N234Q shows only one force maximum, reflecting the dissociation of a single bond. d Dependence of dissociation forces on the force loading rate at different pulling speeds. Solid line is the Bell–Evans fit of dissociation force data (red stars) for Spike trimer N234Q. e Dependence of binding activity on contact time between AFM tip and cellular surfaces for the Spike trimer N234Q (red stars). Solid line represents fittings using pseudo-first-order kinetics of a bimolecular reaction. Source data are provided as a Source Data file.

Article Snippet: Clinical-grade soluble recombinant human ACE2 (amino acids 1–740) was produced by Polymun Scientific (contract manufacturer) from CHO cells according to Good Manufacturing Practice guidelines and formulated as a physiologic aqueous solution.

Techniques: Mutagenesis, Binding Assay, Activity Assay

Journal: Nature Communications

Article Title: Force-tuned avidity of spike variant-ACE2 interactions viewed on the single-molecule level

doi: 10.1038/s41467-022-35641-3

Figure Lengend Snippet: a , d Experimental PDFs from measured dissociation forces between Spike and ACE2. Force maxima for the Spike trimer Delta ( a ) and Omicron (B.1.1.529) ( d ) variants correspond to the simultaneous dissociation of one (I), two (II), or three (III) bonds. b , e Dependence of dissociation forces on the force loading rate. Data of the first peak ( b , green triangles for Delta variant and e olive circles for Omicron (B.1.1.529)) were fitted with the Bell–Evans theory ( b , green line for Delta variant and e , olive line for Omicron (B.1.1.529)). The Markov binding model for two and three bonds are computed and shown as orange and cyan lines for the Delta Spike ( b ) and wine and blue lines for Omicron (B.1.1.529) Spike ( e ), respectively. c , f Dependence of binding activity on contact time between AFM tip and cellular surfaces for the Delta Spike ( c olive triangle) and the Omicron (B.1.1.529) Spike ( f green circles). Ratio of force curves containing two ( c , orange triangles for Delta and f , yellow circles for Omicron (B.1.1.529) Spikes) and three ( c blue triangles for Delta and f cyan circles for Omicron (B.1.1.529) Spikes) bond breakages are shown and fitted with pseudo-first-order kinetics ( c for Delta and f for Omicron (B.1.1.529) Spike; orange lines for two bonds and blue lines for three bonds). g Lifetime of the RBD:ACE2 complex under force for the Wuhan reference strain (WT), the indicated mutants, and variants. h Lifetime of the Spike:ACE2 complex at increasing external force for WT and the indicated variants. Dotted or solid lines represent one RBD or full trimeric Spike. Arrows (gray for WT, blue for Delta, brown for Omicron) indicate lifetime increase arising from multivalent Spike binding. i Ratio of bound lifetimes for RBD triple bonds compared to individual RBD bonds, termed as avidity gain factor τ multi / τ 1Spike . The lifetimes of WT and the indicated variant Spikes were calculated according to the kinetic model of Williams except for N234Q, where only a single RBD binds. Source data are provided as a Source Data file.

Article Snippet: Clinical-grade soluble recombinant human ACE2 (amino acids 1–740) was produced by Polymun Scientific (contract manufacturer) from CHO cells according to Good Manufacturing Practice guidelines and formulated as a physiologic aqueous solution.

Techniques: Variant Assay, Binding Assay, Activity Assay