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Angiotensin I-converting enzyme (ACE), a key enzyme in cardiovascular pathophysiology, consists of two homologous domains (N and C), each bearing a Zn-dependent active site. We modeled the 3D-structure of the ACE N-domain using known structures of the C-domain of human ACE and the ACE homologue, ACE2, as templates. Two monoclonal antibodies (mAb), 3A5 and i2H5, developed against the human N-domain of ACE, demonstrated anticatalytic activity. N-domain modeling and mutagenesis of 21 amino acid residues allowed us to define the epitopes for these mAbs. Their epitopes partially overlap: amino acid residues K407, E403, Y521, E522, G523, P524, D529 are present in both epitopes. Mutation of 4 amino acid residues within the 3A5 epitope, N203E, R550A, D558L, and K557Q, increased the apparent binding of mAb 3A5 with the mutated N-domain 3-fold in plate precipitation assay, but abolished the inhibitory potency of this mAb. Moreover, mutation D558L dramatically decreased 3A5-induced ACE shedding from the surface of CHO cells expressing human somatic ACE. The inhibition of N-domain activity by mAbs 3A5 and i2H5 obeys similar kinetics. Both mAbs can bind to the free enzyme and enzyme-substrate complex, forming E.mAb and E.S.mAb complexes, respectively; however, only complex E.S can form a product. Kinetic analysis indicates that both mAbs bind better with the ACE N-domain in the presence of a substrate, which, in turn, implies that binding of a substrate causes conformational adjustments in the N-domain structure. Independent experiments with ELISA demonstrated better binding of mAbs 3A5 and i2H5 in the presence of the inhibitor lisinopril as well. This effect can be attributed to better binding of both mAbs with the "closed" conformation of ACE, therefore, disturbing the hinge-bending movement of the enzyme, which is necessary for catalysis.

Angiotensin I-converting enzyme (ACE), a key enzyme in cardiovascular pathophysiology, consists of 2 homologous domains, each bearing a Zn-dependent active site. The ratio of the rates of hydrolysis of 2 synthetic substrates, Z-Phe-His-Leu (ZPHL) and Hip-His-Leu (HHL), is characteristic for each type of ACE: somatic 2-domain 1, N-domain 4.5, and C-domain 0.7 (Williams et al, 1996). We hypothesized that inactivation or selective inhibition of the C-domain within the somatic ACE should increase the ratio from 1 toward higher values, whereas inactivation or inhibition of the N-domain should decrease the ratio to lower values. Temperatures in the 40-60 degrees C range cause preferential inactivation of the C-domain in somatic ACE and an increase in the ZPHL/HHL ratio. Determination of the ZPHL/HHL ratio in freshly 100-fold concentrated urine ruled out the existence of the N-domain fragment in human urine, which appears only as a result of long storage. Inhibition of ACE by common inhibitors also increases the ZPHL/HHL ratio for 2-domain ACE, thus providing a sensitive method for the detection of ACE inhibitors in biological fluids. Therefore, simultaneous measurements of ACE activity with 2 substrates (ZPHL and HHL) and calculation of their ratio allow us to monitor the status of the ACE molecule and detect ACE inhibitors (endogenous and exogenous) in human blood and other biological fluids. This method should find wide application in monitoring clinical trials with ACE inhibitors and in the development of the approach for personalized medicine by these effective drugs.

Angiotensin I-converting enzyme (ACE; CD143, EC 3.4.15.1) is a type-1 integral membrane protein that can also be released into extracellular fluids (such as plasma, and seminal and cerebrospinal fluids) as a soluble enzyme following cleavage mediated by an unidentified protease(s), referred to as ACE secretase, in a process known as "shedding". The effects of monoclonal antibodies (mAbs) to eight different epitopes on the N-terminal domain of ACE on shedding was investigated using Chinese hamster ovary cells (CHO cells) expressing an ACE transgene and using human umbilical vein endothelial cells. Antibody-induced shedding of ACE was strongly epitope-specific: most of the antibodies increased the shedding by 20-40%, mAbs 9B9 and 3A5 increased the shedding by 270 and 410% respectively, whereas binding of mAb 3G8 decreased ACE shedding by 36%. The ACE released following mAb treatment lacked a hydrophobic transmembrane domain anchor. The antibody-induced shedding was completely inhibited at 4 degrees C and by zinc chelation using 1,10-phenanthroline, suggesting involvement of a metalloprotease in this process. A hydroxamate-based metalloprotease inhibitor (batimastat, BB-94) was 15 times more efficacious in inhibiting mAb-induced ACE shedding than basal (constitutive) ACE release. Treatment of CHO-ACE cells with BB-94 more effectively prevented elevation in antibody-dependent (but not basal) ACE release induced by 3,4-dichloroisocoumarin and iodoacetamide. These data suggest that different secretases might be responsible for ACE release under basal compared with antibody-induced shedding. Further experiments with more than 40 protease inhibitors suggest that calpains, furin and the proteasome may participate in this process.

In a biomembrane modeling system, reverse micelles, somatic ACE forms dimers via carbohydrate-mediated interaction, providing evidence for the existence of a carbohydrate-recognizing domain on the ACE molecule. We localized this putative region on the N-domain of ACE using monoclonal antibodies (mAbs) to seven different epitopes of ACE. Two mAbs, 9B9 and 3G8, directed to distinct, but overlapping, epitopes of the N-domain of ACE shielded the CRD. Only "simple" ACE-antibody complexes were found in the system. Five mAbs allowed the formation of "double" antibody-ACE-ACE-antibody complexes via carbohydrate-mediated interactions. The results were confirmed using the ACE N- and C-domains. Testicular ACE was unable to form carbohydrate-mediated ACE dimers in the reverse micelles, while the N-domain of ACE, obtained by limited proteolysis of the parent full-length ACE, retained the ability to form dimers. Furthermore, mAb 3G8, which blocked ACE dimerization in micelles, significantly inhibited ACE shedding from the surface of ACE-expressing cells. Galactose prevented ACE dimerization in reverse micelles and also affected antibody-induced ACE shedding in an epitope-dependent manner. Restricted glycosylation of somatic ACE, obtained by the treatment of CHO-ACE cells with the glucosidase inhibitor N-butyldeoxynojirimycin, significantly increased the rate of basal ACE shedding and altered antibody-induced ACE shedding. A chemical cross-linking approach was used to show that ACE is present (at least in part) as noncovalently linked dimers on the surface of CHO-ACE cells. These results suggest a possible link between putative ACE dimerization on the cell surface and the proteolytic cleavage (shedding) of ACE.

ACE chimeric proteins and N domain monoclonal antibodies (mAbs) were used to determine the influence of the N domain, and particular regions thereof, on the rate of ACE ectodomain shedding. Somatic ACE (having both N and C domains) was shed at a rate of 20%/24 h. Deletion of the C domain of somatic ACE generated an N domain construct (ACEDeltaC) which demonstrated the lowest rate of shedding (12%). However, deletion of the N domain of somatic ACE (ACEDeltaN) dramatically increased shedding (212%). Testicular ACE (tACE) having 36 amino acid residues (heavily O-glycosylated) at the N-terminus of the C domain shows a 4-fold decrease in the rate of shedding (49%) compared to that of ACEDeltaN. When the N-terminal region of the C domain was replaced with the corresponding homologous 141 amino acids of the N domain (N-delACE) the rate of shedding of the ACEDeltaN was only slightly decreased (174%), but shedding was still 3.5-fold more efficient than wild-type testicular ACE. Monoclonal antibodies specific for distinct, but overlapping, N-domain epitopes altered the rate of ACE shedding. The mAb 3G8 decreased the rate of shedding by 30%, whereas mAbs 9B9 and 3A5 stimulated ACE shedding 2- to 4-fold. Epitope mapping of these mAbs in conjunction with a homology model of ACE N domain structure, localized a region in the N-domain that may play a role in determining the relatively low rate of shedding of somatic ACE from the cell surface.