Naturally occurring ACE2 stalk variants are differentially released from the cell

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  Figure 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). D) Cells from C) were either left untreated or stimulated with 100 nm PMA for 24 hours. The relative release of ACE2 was assessed by calculating the ratio of the supernatant AP activity to the total AP activity (supernatant plus lysate). n > 3. E) HEK293 cells that transiently express the ACE2 variants ACE2_G726R or ACE2_P734L (tagged with AP) were either left untreated, or stimulated with 100 nM PMA, and/or treated with either 10 μM TAPI or 10 μM GI, for 24 hours. n > 3 F) HEK293 cells stably expressing myc-tagged ACE2 or the myc-tagged ACE2 variant ACE2_P734L were either…

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  Figure 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). 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). n = 4. H) AP-based shedding assay showing relative ACE2 release in the presence or absence of B0AT1 and upon inhibition with GW or GI for 24 h. n = 3. p-values) to wt: * < 0.05; ** < 0.01.

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  Figure 3:. Different structural regions in members of the ACE2 family determiner 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 hours. Release of soluble proteins was quantified as before via AP-activity measuring. Western blotting shows equal expression. 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 hours either with or without marimastat (Ma). p-values) to wt: * < 0.05; ** < 0.01.

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  Figure 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.