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-converting enzyme (ACE) is a membrane-anchored ectoprotein that is proteolytically cleaved, yielding an enzymatically active soluble ACE. Two mouse monoclonal antibodies, MAbs 1B3 and 5C8, were generated to the C-terminal part of human soluble ACE. MAb 1B3 recognized the catalytically active ACE, as revealed by ELISA and precipitation assays, whereas Western blotting and immunohistochemisty on paraffin- embedded sections using MAb 5C8 detected denatured ACE. MAb 1B3 showed extensive cross-reactivity, recognizing 15 species out of the 16 tested. The binding of this MAb to ACE was greatly affected by conformational changes induced by adsorption on plastic, formalin fixation, and underglycosylation. Furthermore, MAb 1B3 binding to the mutated ACE (Pro1199Leu substitution in the juxtamembrane region, leading to a fivefold increase in serum ACE level) was markedly decreased. MAb 5C8 detected all the known expression sites of full-size ACE using formalin-fixed and paraffin-embedded human tissues. The sequential epitope for MAb 5C8 is formed by the last 11 amino acid residues of soluble ACE (Pro1193-Arg1203), whereas the conformational epitope for 1B3 is formed by a motif within these 11 amino acid residues and, in addition, by at least one stretch that includes Ala837-His839 located distal to the sequential epitope. Our findings demonstrated that MAbs 1B3 and 5C8 are very useful for the study of ACE shedding, for identification of mutations in stalk regions, and for studying alternatively spliced variants of ACE. In addition, binding of MAb 1B3 is a sensitive determinant of the integrity of soluble ACE.

and characterization, should hasten our understanding of processes at the protein level (7 ). The combination of imaging ellipsometry and protein chip technology provides a new potential biosensor system for detection and monitoring of biomolecular interaction events for the fields of proteomics, clinical laboratory testing, and biomolecular interaction research.

We demonstrated previously that the monoclonal antibody 9B9 to angiotensin-converting enzyme (ACE), which accumulates very selectively into the rat lung after systemic injection, is a powerful tool for immunotargeting of therapeutic agents or genes to the rat lung vascular bed. Bearing in mind a high research and therapeutic potential of lung targeting via ACE, we obtained a new set of rat monoclonal antibodies to different epitopes of mouse ACE in order to expand this approach to mice. Nine new monoclonal antibodies, recognizing epitopes on the N- and C-domains of catalytically active mouse ACE, were obtained and examined for their efficacy to bind ACE both in vitro and in vivo. This set of monoclonal antibodies was proved to be useful for ACE quantification (by flow cytometry and cell enzyme-linked immunosorbent assay) on the surface of different mouse ACE-expressing cells: endothelial cells, monocytes, macrophages, dendritic cells and spermatozoa. Moreover, gene delivery into mouse ACE-expressing cells using adenoviruses increased 40-fold after redirecting of these viruses to ACE (by coating these viruses with anti-ACE monoclonal antibodies). Radiolabelled (I(125)) monoclonal antibodies specifically accumulated in the mouse lung after systemic injection. Monoclonal antibodies 3G8.17, 4B10.5 and 4B10.17 demonstrated the highest level of lung uptake, 40-50% of injected dose, and high selectivity of lung uptake. Influence of monoclonal antibodies on ACE shedding was negligible, except monoclonal antibody 1D10.11. None of the tested monoclonal antibodies inhibited ACE activity in vitro. In conclusion, a new set of rat monoclonal antibodies to mouse ACE was obtained suitable to study ACE biology in mice and for ACE expression quantification on mouse cells in particular. These monoclonal antibodies also demonstrated highly efficient and selective lung accumulation and thus has the potential for targeting drugs/genes to the pulmonary vasculature in different mouse models of human lung diseases, including numerous knockout models.

Four new rat monoclonal antibodies, generated to denatured mouse somatic angiotensin-converting enzyme (ACE, CD143), detect mouse ACE with high sensitivity in Western blotting. Epitope mapping for the monoclonal antibodies--B12, 4G6 and 5C4--was also performed. Two monoclonal antibodies--B12 and 5C4--are directed to various epitopes on the N-domain--i.e., they recognized only the somatic isoform of mouse ACE. The monoclonal antibody H7 recognized an epitope on the C-domain of mouse ACE. The monoclonal antibody 4G6 was directed to a sequence on the N-domain of mouse ACE, which is homologous to a region of the C-domain and, as a result, also recognizes mouse testicular ACE (tACE) by means of Western blotting. In paraffin-embedded mouse tissues, all monoclonal antibodies detected all known expression sites of somatic ACE (sACE), e.g., the epithelial cells of the kidney proximal tubules, intestine and epididymis, and heterogeneously in endothelial cells. The monoclonal antibodies 4G6 and H7 additionally stained mouse tACE in spermatozoa and in mature spermatids. The monoclonal antibody 4G6 also demonstrated cross-reactivity with sACE from a broad spectrum of animal species, including human, rat, rabbit and bovine. However, this monoclonal antibody did not recognize the testicular isoform of ACE of these species. This set of monoclonal antibodies is useful for identifying even subtle changes in mouse ACE conformation because of denaturation. These monoclonal antibodies are also sensitive tools for the detection of mouse ACE in biological fluids and tissues by using proteomics approaches. Their high reactivity in paraffin-embedded tissues opens up opportunities to study possible changes in the pattern of ACE expression in knockout mouse models and may prove useful for correlating ACE expression in these models with human diseases.