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

Wysocki, Jan; Schulze, Arndt; Batlle, Daniel

To confirm the consistency of the time course degradation studies just described, three different ACE2KO urines and three different kidney lysates were then incubated with the native 100-110 kD mrACE2 at 37 • C. The six hour timepoint was chosen for the robustness of the bands found in urine and kidney lysates. Consistent with data shown in Figure 1 , the 100-110 kD native mrACE2 protein was converted in ACE2KO urines and kidney lysates into a band of an apparent molecular size of 75 kD ( Figure 2 ). This 75 kD ACE2 immunoreactive band appeared to exhibit high specific enzyme activity, similar to that of the original 100-110 kD rACE2 band that has been used for spiking ( Figure 2 ). This suggests that kidney and urine contain proteases capable of digesting native ACE2 into an enzymatically active truncate of~75 kD. The native mrACE2 that was used for spiking contains a C-terminal 10-His tag. Re-probing of the membranes with anti-His antibody revealed a band of the size of the original native mrACE2 at 100-110 kDa but failed to detect the lower band at~75 kD ( Figure 3 ). While N-terminal cleavage of native ACE2 cannot be disproved, this finding clearly shows that proteolysis by mouse urine and kidneys takes place at the C-terminal site. Figure 2 was re-probed using an anti-His antibody. Western blot image shows disappearance of the 100-110 kD mrACE2 band and no appearance of a smaller 75 kD ACE2 immunoreactive band in urine and kidney lysates from ACE2KO mice, suggesting the proteolysis of native mrACE2 from its c-terminal end; urines and kidney lysates from three independent degradation experiments.