Novel Variants of Angiotensin Converting Enzyme-2 of Shorter Molecular Size to Target the Kidney Renin Angiotensin System

Wysocki, Jan; Schulze, Arndt; Batlle, Daniel

We and others have previously identified two main ACE2-immunoreactive bands in mouse urine [12, [18] [19] [20] [21] . One band at about 110 kD corresponded to the molecular weight of native ACE2 and was likely a product of shedding, likely mediated by the metalloprotease ADAM17 [20, 30, 31] from the kidney apical tubular membrane, where ACE2 is abundantly expressed [12, 31] . These observations prompted us to investigate if these shorter forms of about 75 kD are enzymatically active and, if so, to design shorter ones and test them for potential activity in vitro and in vivo. Here, we demonstrate that the natural ACE2 of 100-110 kDa can be degraded in the urine and kidney to shorter ACE2 fragments which retain ACE2 enzyme activity. That the 75 kD ACE2 band is a result of a proteolytic cleavage of the 110 kDa ACE2 was determined by demonstrating that a recombinantly generated native recombinant, ACE2 110 kDa, can be digested by kidney and urine proteases to a shorter band of similar size to the 75 kDa ACE2 fragment that was naturally observed in the urine (Figures 1 and 2 ). In these experiments, we were also able to ascertain that the C-terminal end is the site, which is removed from native rACE2 without a loss in enzyme activity (Figure 3 ). This finding is consistent with crystallographic and homology studies for human ACE2 that indicated that on the C-terminal end there is a fairly large structural portion that has non-enzymatic function [11, 32] .