A cell-based assay for rapid assessment of ACE2 catalytic function

Figures

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  Figure 1. Design for a membrane-localized ACE2 expression system. (A) Our ACE2 construct is driven by a CMV promoter followed by the first 25 residues of ACE2 containing the leader sequence that direct ACE2 to the plasma membrane. This is followed by a 3xHA tag linked to the remainder of ACE2 (20–805) and a C-terminal sfGFP. Both 3xHA and sfGFP fusions are separated from ACE2 by flexible 3xGGGGS linkers. (B) The ACE2 fusion protein is designed to be embedded in the plasma membrane where it can perform extracellular carboxypeptidase-mediated metabolism and its levels can be detected by cell staining with antibodies to HA. (C) Lysates from untransfected or 3xHA-ACE2-sfGFP-transfected HEK293 cells were analyzed by automated Jess capillary immunoassay using antibodies to HA, GFP, and two ACE2 antibodies. (D) Confocal fluorescence microscopy of HEK cells transfected with 3xHA-ACE2-sfGFP and stained with HA and the nuclear stain DAPI.

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  Figure 2. Sensitivity, specificity and time course of exogenous ACE2 carboxypeptidase activity. (A) HEK293 cells transfected with 3xHA-ACE2-sfGFP were incubated with increasing concentrations of Ang II, Ap-13 or des-Arg9-BK for 1 h followed by phenylalanine detection. (B) Untransfected or 3xHA-ACE2-sfGFP transfected HEK293 cells were incubated for 1 h with Ang II (25 µM), Ap-13 (25 µM) or des-Arg9-BK (75 µM) with or without ACE2 inhibitors DX600 or MLN4760 followed by phenylalanine detection. Unincubated substrates were used for background subtraction. (C) Untransfected or 3xHA-ACE2-sfGFP transfected HEK293 cells were incubated with Ang II (25 µM), Ap-13 (25 µM) or des-Arg9-BK (75 µM) for various time points up to 4 h followed by phenylalanine detection. (D) The free phenylalanine measurements from transfected cells in C were divided by time point-matched results from untransfected cells to determine the magnitude of ACE2 carboxypeptidase activity over background. All assays were performed in triplicate wells and are representative of three independent experiments (n = 9).

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  Figure 3. Transfection load impacts on surface receptor expression and carboxypeptidase activity. HEK293 cells were transfected in triplicate with increasing quantities of CMV-3xHA-ACE2-sfGFP plasmid (shown in ng) followed by incubation with 25 µM of Ang II for 30 min and then stained for HA by cell surface ELISA. Assay was performed in triplicate wells and is representative of three independent experiments (n = 9). RAU = relative absorbance units, RFU = relative fluorescence units.

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  Figure 4. Substrate metabolism per receptor abundance for wildtype and R273Q ACE2. HEK293 cells were transfected in triplicate with WT or R273Q ACE2, incubated with Ang II (25 µM), Ap-13 (25 µM) or des-Arg9-BK (75 µM) for 30 min followed by phenylalanine assay and then stained for HA by cell surface ELISA. Phenylalanine values were divided by ELISA values from the same well and then normalized to the WT mean of the same substrate. Assay was performed in triplicate wells and is representative of three independent experiments (n = 9). ***Shows a statistical significance of P < 0.001 as determined by a Student’s t-test.