ace2 activity fluorometric assay kit  (Beyotime)

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Modulation of the pharmacokinetics of soluble ACE2 decoy receptors through glycosylation

doi: 10.1016/j.omtm.2024.101301

Figure Lengend Snippet: Choice of cell line for sACE2 2 .v2.4-IgG1 production impacts pharmacokinetics and glycosylation (A and B) sACE2 2 .v2.4-IgG1 was expressed and purified from human Expi293F (gray) or nonhuman ExpiCHO-S (black) cultures that were both transiently transfected. A 10 mg/kg amount of sACE2 2 .v2.4-IgG1 was injected into the tail vein of human FcRn transgenic mice. (A) ACE2 catalytic activity and (B) protein concentrations based on ELISA were measured in plasma. Data are mean ± SEM, n = 3 mice per time point. (C) N-glycan types from glycomics analysis of sACE2 2 .v2.4-IgG1 produced in ExpiCHO-S vs. Expi293F cells. (D) Abundance of sialylated (Neu5Ac) and fucosylated N-glycan structures. (E and F) O-Glycan analysis of sACE2 2 .v2.4-IgG1 produced in (E) ExpiCHO-S and (F) Expi293F cells. After PNGaseF treatment of protein, O-glycans were released, purified, permethylated, and analyzed by MALDI-TOF-MS. O-glycan structures were assigned using Glycoworkbench software based on precursor masses and the common mammalian biosynthetic pathway. Glycan structures are indicated on the x axis by their m/z ratio. GlcNAc, blue squares; GalNAc, yellow squares; Gal, yellow circles; Neu5Ac, purple diamonds.

Article Snippet: Hydrolysis of a quenched fluorescent peptide substrate was measured on a Biotek Cytation 5 plate reader with the Fluorometric ACE2 Activity Assay Kit (BioVision) according to the manufacturer’s directions.

Techniques: Purification, Transfection, Injection, Transgenic Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Produced, Software

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Modulation of the pharmacokinetics of soluble ACE2 decoy receptors through glycosylation

doi: 10.1016/j.omtm.2024.101301

Figure Lengend Snippet: An engineered derivative of sACE2 2 .v2.4-IgG1 and its glycosylation (A) Left, the structure (PDB: 6M17 ) of dimeric ACE2 (chains “A” and “B” in dark and light green) bound to RBD (gray ribbons). Glycans are shown as orange sticks. PD, protease domain; CLD, collectrin-like dimerization domain. Center and right, residues mutated to fill cavities (blue spheres), introduce disulfides (yellow spheres), or add N-glycosylation motifs (purple spheres) are shown on a single ACE2 subunit. Lead candidate sACE2 2 .S19-IgG1 has mutations V491I, M662T, N720S. (B) N-glycan types on sACE2 2 .S19-IgG1 produced in Expi293F cells. (C) Abundance of sialylated and fucosylated N-glycan structures on sACE2 2 .S19-IgG1 produced in Expi293F cells. (D) O-Glycan structures on sACE2 2 .S19-IgG1 produced in Expi293F cells following O-glycan release and MALDI-TOF-MS analysis. (E) Occupancy of the N-glycosylation sites based on glycopeptidomics analysis of sACE2 2 .S19-IgG1 from Expi293F (green), sACE2 2 .v2.4-IgG1 from Expi293F (pale gray), and sACE2 2 .v2.4-IgG1 from ExpiCHO-S (dark gray). sACE2 2 .S19-IgG1 has added glycosylation sites at positions 660 and 718. (F) Percent of the glycoforms at each N-glycosylation site that have at least one sialic acid.

Article Snippet: Hydrolysis of a quenched fluorescent peptide substrate was measured on a Biotek Cytation 5 plate reader with the Fluorometric ACE2 Activity Assay Kit (BioVision) according to the manufacturer’s directions.

Techniques: Introduce, Produced

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Modulation of the pharmacokinetics of soluble ACE2 decoy receptors through glycosylation

doi: 10.1016/j.omtm.2024.101301

Figure Lengend Snippet: High sialylation and YTE mutations in the Fc region enhance the pharmacokinetics of sACE2 2 .S19-IgG1 (A) Intravenous administration of proteins at 10 mg/kg into human FcRn mice. Blood was collected into heparin via retroorbital route at the plotted time points. Plasma levels of the indicated proteins were measured by ELISA. (B and C) Proteins were injected s.c. in the flank of human FcRn mice at a dose of 10 mg/kg (solid lines) or 100 mg/kg (broken line). (B) ACE2 catalytic activity in plasma and (C) protein concentrations based on ELISA. (D) sACE2 2 .S19-IgG1(YTE) was treated with PNGase F or neuraminidase. Proteins (20 μg) were analyzed without further purification by SDS-PAGE under non-reducing conditions and stained with Coomassie. The calculated molecular weight (MW) of the mature polypeptide (excluding glycans) is 218 kD for the dimer. PNGase F has an MW of 36 kD. A. ureafaciens neuraminidase is a mixture of isoenzymes. (E) Proteins (10 μg) were analyzed by IEF gel electrophoresis. Glycosidase-treated proteins were analyzed without (−) and with (+) purification by gel filtration following treatment. (F) Purified proteins were injected i.v. at 10 mg/kg into human FcRN mice and concentrations in plasma were measured by ELISA for 5 days. For PK studies in this figure, data are mean ± SEM for n = 3 per group and proteins were purified from transiently transfected Expi293F.

Article Snippet: Hydrolysis of a quenched fluorescent peptide substrate was measured on a Biotek Cytation 5 plate reader with the Fluorometric ACE2 Activity Assay Kit (BioVision) according to the manufacturer’s directions.

Techniques: Enzyme-linked Immunosorbent Assay, Injection, Activity Assay, Purification, SDS Page, Staining, Molecular Weight, Nucleic Acid Electrophoresis, Filtration, Transfection

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Modulation of the pharmacokinetics of soluble ACE2 decoy receptors through glycosylation

doi: 10.1016/j.omtm.2024.101301

Figure Lengend Snippet: The v2.4 and S19 mutations in ACE2 are predicted to have low immunogenicity (A) Computational analysis of calculated affinity of peptides to HLA-II allotypes. The wild-type sACE2 2 -IgG1 sequence (top row) is scanned for peptides predicted to be displayed on a common set of 14 HLA-II allotypes. In this analysis, the minimum threshold for a peptide to be considered an antigen is predicted affinity for four HLA-II allotypes (Nhits ≥4) from a set of 14 common alleles. For sACE2 2 -IgG1 derivatives, the sequence is grayed out except for the regions where mutations are introduced to highlight whether a mutation is within a predicted epitope and/or changes the probability of presentation. Peptides that were analyzed experimentally by yeast display are indicated below with dark red bars. (B) Peptides were displayed on yeast and binding to HLA-II following an HLA-DM-dependent peptide loading reaction was measured by flow cytometry. The correlation plot shows the agreement between two independent replicates measuring mean fluorescence units (MFU) for bound HLA-II. Polyserine negative control reactions are blue, positive control peptide/HLA-II pairs are orange, and ACE2 peptides are gray. (C) Yeast display measurements of HLA-II binding to ACE2 peptides is plotted from no signal (dark blue) to high binding signal (orange).

Article Snippet: Hydrolysis of a quenched fluorescent peptide substrate was measured on a Biotek Cytation 5 plate reader with the Fluorometric ACE2 Activity Assay Kit (BioVision) according to the manufacturer’s directions.

Techniques: Sequencing, Mutagenesis, Binding Assay, Flow Cytometry, Fluorescence, Negative Control, Positive Control

Journal: Signal Transduction and Targeted Therapy

Article Title: FGF7 enhances the expression of ACE2 in human islet organoids aggravating SARS-CoV-2 infection

doi: 10.1038/s41392-024-01790-8

Figure Lengend Snippet: Dynamic expression of ACE2 during the differentiation of human islet organoids from hESCs. a Schematic diagram outlining the differentiation protocol. The cells from the ES to S5 stages were subjected to 2D culture and the cells in S6 and S7 were suspended and 3D aggregated at the first day of S6. ES embryonic stem cells, DE definitive endoderm, PGT primitive gut tube, PF posterior foregut, PE pancreatic endocrine, EP endocrine progenitor. b Dynamic changes in ACE2 mRNA expression relative to ES from S1 to S7 relative to that in the ES stage. The dashed line indicates ACE2 mRNA expression in ES cells, which was set as 1. c Western blot analysis of ACE2 protein levels relative to that of β-actin from S1 to S7. d Quantification of the ACE2 protein level relative to that of β-actin from S1 to S7. The dashed line represents the intensity of β-actin, which was set to 1. e Immunofluorescence (IF) staining of differentiated cells from each stage for ACE2 (green) and stage-specific markers (red or white, S1: SOX17; S2: FOXA2; S3: PDX1; S4: PDX1 and NKX6.1; S5: PDX1 and NKX6.1). The white arrowheads represent the colocalization of ACE2 with stage-specific markers. The image at the bottom provides an enlarged perspective of the enclosed region within the dashed box. f Cryosections of islet organoids at S6D5 and S7D14 were subjected to IF staining for ACE2 and endocrine markers (GCG, INS, and SST). The image in the top-right corner of the picture represents an enlarged view of the region enclosed within the dashed box. g Quantification of ( f ) showing the percentage (%) of ACE2 + cells among GCG + , INS + , and SST + cells in islet organoids. h IF staining of ACE2 in purified α (GCG + ), purified β (INS + ), and δ (SST + ) cells from S7D14 islet organoids. The image on the right-hand side represents an enlarged view of the region enclosed within the dashed box. i Quantification of ( h ) revealing the percentages of ACE2 + cells among GCG + cells, INS + cells and SST + cells. j Western blot image and quantification of ACE2 protein levels relative to β-actin in purified α cells, purified β cells and α − β − cells (the remaining cells after sequentially sorting α and β cells) from S7D14 islet organoids. Images are from one representative experiment from 3 to 5 independent experiments. For all statistical plots, the data are presented as mean ± SD, the distinct dots are represented as the individual values of 3 to 5 replicates with 3 repeats in each experiment. p values were calculated by one-way ANOVA and Tukey’s multiple comparison test. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: The enzymatic activity of ACE2 in islet organoids was measured using an ACE2 activity assay kit (Fluorometric) (Abcam, ab273297), which leverages the ability of active ACE2 to cleave a synthetic MCA-based peptide substrate, resulting in the release of a free fluorophore.

Techniques: Expressing, Western Blot, Immunofluorescence, Staining, Purification, Comparison

Journal: Signal Transduction and Targeted Therapy

Article Title: FGF7 enhances the expression of ACE2 in human islet organoids aggravating SARS-CoV-2 infection

doi: 10.1038/s41392-024-01790-8

Figure Lengend Snippet: FGF7 regulates ACE2 expression during the early developmental stages of hESC-derived pancreatic endocrine progenitors via the FGF7-FGFR2/1 pathway. a Scheme of the dosages of FGF7 and FGFR inhibitors (FGFRis, including FGFR1 and FGFR2 inhibitors) administered to endocrine progenitors in group 1 (G1, control, without FGF7 and FGFRi), group 2 (G2, 50 ng/ml FGF7) and group 3 (G3, 50 ng/ml FGF7 + 5 μM FGFRis). The red arrow represents the sample collection time at S5D3. b ACE2 mRNA expression in endocrine progenitors from the G1, G2 and G3 groups. c Western blot analysis of ACE2 protein levels in the G1, G2 and G3 groups. d Co-localization of ACE2 with the stage-specific marker PDX1 after FGF7-treatment in S5. Green, ACE2; red, PDX1; blue, DAPI. e Quantification of ( d ) showing the ratio of ACE2 + cells relative to total nuclei in the G1 and G2 groups. f Quantification of ( d ) showing the ratio of PDX1 + and ACE2 + double-positive cells (PDX1 + ACE2 + ) relative to ACE2 + cells in G1 and G2. g Heatmap illustrating the proximity of G1 to G2. Significant ACE2-related genes are labeled on the right. h Volcano plots showing selected genes in the comparison of G1 and G2. Significant ACE2-related genes with p < 0.05 and log2 (fold change) >1 are highlighted in red, while log2 (fold change) < −1 are highlighted in blue. i Heatmap illustrating the proximity of G2 to G3. Significant ACE2-related genes are labeled on the right. j Volcano plots showing selected genes in the comparison of G2 and G3. Significant ACE2-related genes with p < 0.05 and log2 (fold change) >1 are shown in red while those with log2 (fold change) < −1 are shown in blue. k Selection of significantly enriched KEGG gene sets in G1 versus G2. l Selection of significantly enriched KEGG gene sets in G2 versus G3. Images are from one representative experiment from 3 to 5 independent experiments. For all statistical plots, the data are presented as mean ± SD, the distinct dots are represented as the individual values of 3 to 5 replicates. p values were calculated using an unpaired Student’s t test ( e , f ) or one-way ANOVA and Tukey’s multiple comparison test ( b , c ). * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: The enzymatic activity of ACE2 in islet organoids was measured using an ACE2 activity assay kit (Fluorometric) (Abcam, ab273297), which leverages the ability of active ACE2 to cleave a synthetic MCA-based peptide substrate, resulting in the release of a free fluorophore.

Techniques: Expressing, Derivative Assay, Western Blot, Marker, Labeling, Comparison, Selection

Journal: Signal Transduction and Targeted Therapy

Article Title: FGF7 enhances the expression of ACE2 in human islet organoids aggravating SARS-CoV-2 infection

doi: 10.1038/s41392-024-01790-8

Figure Lengend Snippet: FGF7 regulates ACE2 expression and activity during the late stages of development in mature islet organoids, which affects β-cell function. a Scheme illustrating the dosage and duration of FGF7 and FGFRi treatment in Group 1 (G1, control), Group 4 (G4, treated with 50 ng/ml FGF7), and Group 5 (G5, treated with 50 ng/ml FGF7 + 5 μM FGFRi) in the late mature islet stage (S7). The red arrow represents the sample collection time at S7D14. b ACE2 mRNA expression in mature islet organoids from G1, G4 and G5. c Western blot analysis of ACE2 protein levels in G1, G4 and G5. d IF staining of ACE2 (green) and nuclei (blue) in S7D14 islet organoids from G1, G4 and G5. e Quantification of ( d ) to calculate the number of ACE2 + cells in islet organoids divided by the numbers of nuclei. f Co-staining of ACE2 (green) and GCG (red) in purified α cells from G1, G4 and G5. g Co-staining of ACE2 (green) and INS (red) in purified β cells from G1, G4 and G5. h Measurement of the fluorescence intensity (FI) of ( f ) and ( g ) for ACE2 + cells in purified α and β cells (ACE2 + /GCG + or ACE2 + /INS + double-positive cells) from G1, G4 and G5. i Western blot images of ACE2, β-actin and GCG in purified α cells from G1, G4 and G5. j Western blot images of ACE2, β-actin and INS in purified β cells from G1, G4 and G5. k Quantification of ( i ) and ( j ) normalized to G1. l ACE2 enzymatic activity curves of islet organoids from G1, G4 and G5 subjected to a kinetics model for 130 min. m ACE2 enzymatic activity (mU) per mg protein relative to G1. n Insulin secretion of islet organoids in the static GSIS assay. Images are from one representative experiment from 3 to 4 independent experiments. The data are represented as the individual values of 3 to 4 replicates, with 3 replicates in each experiment. p values were calculated by one-way ANOVA and Tukey’s multiple comparison test. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: The enzymatic activity of ACE2 in islet organoids was measured using an ACE2 activity assay kit (Fluorometric) (Abcam, ab273297), which leverages the ability of active ACE2 to cleave a synthetic MCA-based peptide substrate, resulting in the release of a free fluorophore.

Techniques: Expressing, Activity Assay, Cell Function Assay, Western Blot, Staining, Purification, Fluorescence, Comparison

Journal: Signal Transduction and Targeted Therapy

Article Title: FGF7 enhances the expression of ACE2 in human islet organoids aggravating SARS-CoV-2 infection

doi: 10.1038/s41392-024-01790-8

Figure Lengend Snippet: FGF7 exacerbates SARS-CoV-2 infection in islet organoids, thus impairing the insulin secretion. a Schematic representation of the methodology used to infect islet organoids from G1, G4 and G5 with SARS-CoV-2: viral replication was tested in the organoids and the virus was released into the supernatant at 72 h post-infection (hpi). Sample collection time at S7D14. b qPCR analysis of SARS-CoV-2 replication in the supernatants of G1, G4 and G5 cells. The values are normalized to those of G1 after 3 independent inoculations. c qPCR analysis of intracellular viral RNA in islet organoids from G1, G4 and G5. d ACE2 mRNA expression in G1, G4 and G5 at 72 hpi. The values are normalized to G1. e Western blot analysis of ACE2 and β-actin in islet organoids at 72 hpi in G1, G4 and G5. f The ACE2 enzymatic activity curves of G1, G4 and G5 from 72 hpi islet organoids subjected to a kinetics model for 130 min. g ACE2 enzymatic activity (mU) per mg of protein from G1, G4 and G5 islet organoids relative to G1 at 72 hpi. h The co-localization of SARS-CoV-2 (CoV-2, green), ACE2 (white), and INS (red) in G1, G4 and G5 from 72 hpi islet organoids. i Quantification of ( h ) showing the percentage of ACE2 + cells relative to total cells according to the number of nuclei. j Quantification of ( h ) showing the percentage of SARS-CoV-2-infected cells ratio in 72 hpi islet organoids from G1, G4 and G5. k Tricolor IF co-staining of CoV-2 pseudo-virus spike (pseudo-V, green), ACE2 (red) and INS (purple) in purified β cells from G1, G4 and G5. The image in the top-right corner of the picture represents an enlarged view of the region enclosed within the dashed box. l Quantification of ( k ) revealing the fluorescence intensity of pseudo-V in purified β cells from G1, G4 and G5. m Insulin secretion of islet organoids at 72 hpi in static GSIS assay. Images are from one representative experiment from 3 to 4 independent experiment. The data are presented as the individual values of 3 to 4 replicates with 3 repeats in each experiment. p values were calculated by one-way ANOVA and Tukey’s multiple comparison test, * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: The enzymatic activity of ACE2 in islet organoids was measured using an ACE2 activity assay kit (Fluorometric) (Abcam, ab273297), which leverages the ability of active ACE2 to cleave a synthetic MCA-based peptide substrate, resulting in the release of a free fluorophore.

Techniques: Infection, Virus, Expressing, Western Blot, Activity Assay, Staining, Purification, Fluorescence, Comparison