Naturally occurring ACE2 stalk variants are differentially released from the cell

Figures

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  Fig. 1. Natural occurring point mutations in ACE2 stalk region impact ACE2 release. A) ADAM10 and ADAM17 are the main sheddases responsible for the shedding of ACE2. ACE2 is expressed in the kidneys, lungs, and heart. B) ACE2 domain structure with detailed view of the stalk region (position 720 – 740). The naturally occurring point mutations in the stalk region are shown in red. N = N-terminus, C = C-terminus, TMH = transmembrane helix. C) HEK293 cells were transiently transfected with ACE2 variants tagged with alkaline phosphatase (AP). Western blot of inputs shows cell-associated/membrane-bound ACE2 variants (mACE2). The AP tag increases the apparent molecular weight of these ACE2 fusion proteins. While ACE2 is expected to migrate at approximately 120–130 kDa, the additional AP tag (70 – 80 kDa) increases the expected apparent molecular weight of the ACE2 fusion proteins. Uncropped images of the same blot probed with the indicated antibodies are shown in Fig. S1B. D) Cells from C) were either left untreated or stimulated with 100 nm PMA for 24 h. The relative release of ACE2 was assessed by calculating the ratio of the supernatant AP activity to the total AP activity (supernatant plus…

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  Fig. 2. P734L mutation increases ACE2 and B0AT1 surface expression. A-D) HEK293 cells stably expressing wt ACE2, P734L, or GFP (control, ctr) were analysed by flow cytometry and Western blotting. Flow cytometry was used to quantify cell-surface abundance of the ACE2 variants (A), and Western blotting to assess total cellular abundance (B–C). The surface-to-total ACE2 ratio was then calculated (D). Uncropped images of the same blot probed with the indicated antibodies are shown in Fig. S3. n > 3. E) Membrane-bound ACE2 (mACE2) forms homodimers and can assemble into heterotetrameric complexes with the amino acid transporters B0AT1 or XT3. This interaction is crucial for efficient cell-surface expression of the transporters. F-G) HEK293 cells stably overexpressing wt ACE2, P734L and or GFP (control, ctr) were transiently co-transfected with HA-tagged B0AT1 and subjected to Western blotting (F) and flow cytometric analysis (G). Uncropped images of the same blot probed with the indicated antibodies are shown in Fig. S4A. Control-transfected cells stained under identical conditions served as negative controls for autofluorescence and antibody-dependent background staining and are shown in Fig.…

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  Fig. 3. Different structural regions in members of the ACE2 family determine shedding efficiency. A) Structural overview of ACE2 family members and their sensitivity to ADAM10 and ADAM17. ACE2 is an evolutional chimera from ACE and TMEM27 (collectrin). Furthermore, three ACE2 chimeras are shown. B) AP-tagged ACE2 chimeras and wt ACE2 were transiently overexpressed in HEK293 cells and incubated for 24 h. Western blotting confirmed detectable expression of ACE2 and ACE2 chimeras within a broadly comparable range. ACE2 release in the AP assay was calculated as described previously and normalized to the total ACE2-AP signal in each sample, defined as supernatant AP activity divided by the sum of supernatant and lysate AP activity. Uncropped images of the same blot probed with the indicated antibodies are shown in Fig. S5. n > 9. C) AP-tagged ACE2 chimeras and wt ACE2 were transiently overexpressed in HEK293 or in ADAM10/ADAM17 deficient HEK293 cells (dKO HEK) and incubated for 24 h either with or without marimastat (Ma). p-values) to wt: * < 0.05; ** < 0.01.

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  Fig. 4. Stalk sequence and release rates of ACE2 mutants and ACE2 chimeras. Schematic overview of ACE2 with detailed view of the stalk region (position 720 – 740). Variant point mutations and ACE2 chimeras are highlighted in red. The difference in constitutive release compared to wt ACE2 is shown. The boxes indicate the potential ADAM10/ADAM17 cleavage sites.