Naturally occurring ACE2 stalk variants are differentially released from the cell

Abstract Angiotensin-converting enzyme 2 (ACE2) is a key regulator of the renin-angiotensin-aldosterone system (RAAS). It also acts as a receptor for SARS-CoV-2 and stabilises the B0AT1 amino acid transporter at the cell surface. Therefore, surface expression of ACE2 is crucial for these physiological processes. ACE2 is released as a soluble, catalytically active form, partly through ectodomain shedding. This process mainly involves the sheddases ADAM10 and ADAM17, but the exact regulatory mechanisms remain unclear. We assessed 11 naturally occurring single-point mutations in the ACE2 stalk region. Most variants showed significantly reduced release compared to wild-type (WT) ACE2; however, the single point mutations P734L and G726R significantly increased their release. ACE2_P734L also exhibits higher surface expression, directly increasing the surface levels of B0AT1. Despite B0AT1 and ACE2 forming a tight tetrameric complex, this did not affect ACE2 shedding. This suggests that complex formation does not restrict sheddase access. Overall, these data identify the ACE2 stalk region as a major determinant of shedding efficiency. Naturally occurring variants in this region can substantially affect the release of soluble ACE2, potentially contributing to interindividual differences that are relevant for pathophysiological processes. Competing Interest Statement The authors have declared no competing interest. Footnotes Funding: To S.D.: START grant (#691903-06/19) of the Medical Faculty of the RWTH Aachen University. To A.L.: Grant from the Bundesministerium für Bildung und Forschung (01KI20207). Data Availability Statement The datasets and materials generated during the current study are available from the corresponding author upon request. Declaration of interest The authors declare no competing interests. Abbreviations - ACE2 - angiotensin-converting enzyme 2 - AP - alkaline phosphatase - BSA - bovine serum albumin - DMSO - dimethyl sulfoxide - FCS - fetal calve serum - GI - GI254023X - GW - GW280264X - HEK - human embryonic kidney cells - mACE2 - membrane-bound ACE2 - PBS - phosphate buffered saline - PCR - polymerase chain reaction - PNP - p-nitrophenol - PNPP - p-nitrophenyl phosphate - sACE2 - soluble ACE2 (ectodomain) - TAPI-1 - Tumor necrosis factor α protease inhibitor TBS tris buffered saline - wt - wildtype