Human Angiotensin I-converting Enzyme Research Articles

Surface Plasmon Resonance Analysis of the Binding Mechanism of Pharmacological and Peptidic Inhibitors to Human Somatic Angiotensin I-Converting Enzyme

Somatic angiotensin I-converting enzyme (ACE) possesses two catalytic domains and plays a major role in the regulation of blood pressure, thus representing a therapeutic target for the treatment of hypertension. We present a comprehensive surface plasmon resonance (SPR) study of the interaction of human somatic ACE with the pharmacological inhibitors captopril and lisinopril, the bradykinin potentiating peptide BPP-11b, and the food peptidic inhibitors from bovine αs2-casein, F(174)ALPQYLK(181) and F(174)ALPQY(179). SPR binding curves recorded with the high potency inhibitors captopril, lisinopril, and BPP-11b were evaluated both by regression analysis and by kinetic distribution analysis. The results indicated that captopril and lisinopril bound ACE with two K(D)'s differing by a factor 10-20 and >30, respectively (lowest K(D) = 0.1-0.3 nM for both inhibitors). This shows, for the first time in a direct binding assay with the two-domain enzyme, the existence of two binding modes of the pharmacological inhibitors, presumably with the two ACE domains. The BPP-11b-ACE binding curves were complex but showed a predominant interaction with K(D) in the nanomolar range. The caseinopeptides, known to inhibit ACE with an IC₅₀ of 4.3 μM, bound to ACE with K(D) = 3-4 μM. Mapping of the F(174)ALPQY(179) binding site on ACE by sequential binding studies using captopril or BPP-11b indicated that it bound to (or near) the two active sites of ACE, in agreement with the stoichiometry of 2 determined from data fitting. Our results provide a detailed characterization of ACE-inhibitor binding modes and validate SPR for predicting the inhibitory potential of new compounds.

Absence of cell surface expression of human ACE leads to perinatal death.

Renal tubular dysgenesis (RTD) is a recessive autosomal disease characterized most often by perinatal death. It is due to the inactivation of any of the major genes of the renin-angiotensin system (RAS), one of which is the angiotensin I-converting enzyme (ACE). ACE is present as a tissue-bound enzyme and circulates in plasma after its solubilization. In this report, we present the effect of different ACE mutations associated with RTD on ACE intracellular trafficking, secretion and enzymatic activity. One truncated mutant, R762X, responsible for neonatal death was found to be an enzymatically active, secreted form, not inserted in the plasma membrane. In contrast, another mutant, R1180P, was compatible with life after transient neonatal renal insufficiency. This mutant was located at the plasma membrane and rapidly secreted. These results highlight the importance of tissue-bound ACE versus circulating ACE and show that the total absence of cell surface expression of ACE is incompatible with life. In addition, two missense mutants (W594R and R828H) and two truncated mutants (Q1136X and G1145AX) were also studied. These mutants were neither inserted in the plasma membrane nor secreted. Finally, the structural implications of these ACE mutations were examined by molecular modelling, which suggested some important structural alterations such as disruption of intra-molecular non-covalent interactions (e.g. salt bridges).

The insertion/deletion polymorphism in the ACE gene and chronic obstructive pulmonary disease

An insertion/deletion (I/D) polymorphism was identified in intron 16 of the gene encoding the human angiotensin I-converting enzyme (ACE), a candidate gene for chronic obstructive pulmonary disease (COPD). We investigated the relationship between this polymorphism in the ACE gene and the risk of developing COPD. Sixty-six COPD in-patients and 40 non-smoking control individuals were recruited for this study. The distribution of ACE genotypes in these individuals was studied. The frequencies of ACE genotypes were found to be 47.0% for DD, 30.3% for ID, and 22.7% for II in the COPD group and 32.5% for DD, 47.5% for ID, and 20.0% for II in the control group. The allele frequencies were found to be 0.62% for the D allele and 0.38% for the I allele in the COPD group and 0.56% for the D allele and 0.44% for the I allele in the control group. A significant difference was found between I and D allele frequencies (P < 0.05) of the study and control groups. Our results suggest that this ACE polymorphism may be associated with the development of COPD.

Conformational Changes of Blood ACE in Chronic Uremia

BackgroundThe pattern of binding of monoclonal antibodies (mAbs) to 16 epitopes on human angiotensin I-converting enzyme (ACE) comprise a conformational ACE fingerprint and is a sensitive marker of subtle protein conformational changes.HypothesisToxic substances in the blood of patients with uremia due to End Stage Renal Disease (ESRD) can induce local conformational changes in the ACE protein globule and alter the efficacy of ACE inhibitors.Methodology/Principal FindingsThe recognition of ACE by 16 mAbs to the epitopes on the N and C domains of ACE was estimated using an immune-capture enzymatic plate precipitation assay. The precipitation pattern of blood ACE by a set of mAbs was substantially influenced by the presence of ACE inhibitors with the most dramatic local conformational change noted in the N-domain region recognized by mAb 1G12. The “short” ACE inhibitor enalaprilat (tripeptide analog) and “long” inhibitor teprotide (nonapeptide) produced strikingly different mAb 1G12 binding with enalaprilat strongly increasing mAb 1G12 binding and teprotide decreasing binding. Reduction in S-S bonds via glutathione and dithiothreitol treatment increased 1G12 binding to blood ACE in a manner comparable to enalaprilat. Some patients with uremia due to ESRD exhibited significantly increased mAb 1G12 binding to blood ACE and increased ACE activity towards angiotensin I accompanied by reduced ACE inhibition by inhibitory mAbs and ACE inhibitors.Conclusions/SignificanceThe estimation of relative mAb 1G12 binding to blood ACE detects a subpopulation of ESRD patients with conformationally changed ACE, which activity is less suppressible by ACE inhibitors. This parameter may potentially serve as a biomarker for those patients who may need higher concentrations of ACE inhibitors upon anti-hypertensive therapy.

Molecular recognition and regulation of human angiotensin-I converting enzyme (ACE) activity by natural inhibitory peptides

Angiotensin-I converting enzyme (ACE), a two-domain dipeptidylcarboxypeptidase, is a key regulator of blood pressure as a result of its critical role in the renin-angiotensin-aldosterone and kallikrein-kinin systems. Hence it is an important drug target in the treatment of cardiovascular diseases. ACE is primarily known for its ability to cleave angiotensin I (Ang I) to the vasoactive octapeptide angiotensin II (Ang II), but is also able to cleave a number of other substrates including the vasodilator bradykinin and N-acetyl-Ser-Asp-Lys-Pro (Ac-SDKP), a physiological modulator of hematopoiesis. For the first time we provide a detailed biochemical and structural basis for the domain selectivity of the natural peptide inhibitors of ACE, bradykinin potentiating peptide b and Ang II. Moreover, Ang II showed selective competitive inhibition of the carboxy-terminal domain of human somatic ACE providing evidence for a regulatory role in the human renin-angiotensin system (RAS).

Structural characterization of angiotensin I‐converting enzyme in complex with a selenium analogue of captopril

Human somatic angiotensin I-converting enzyme (ACE), a zinc-dependent dipeptidyl carboxypeptidase, is central to the regulation of the renin–angiotensin aldosterone system. It is a well-known target for combating hypertension and related cardiovascular diseases. In a recent study by Bhuyan and Mugesh [Org. Biomol. Chem. (2011) 9, 1356–1365], it was shown that the selenium analogues of captopril (a well-known clinical inhibitor of ACE) not only inhibit ACE, but also protect against peroxynitrite-mediated nitration of peptides and proteins. Here, we report the crystal structures of human testis ACE (tACE) and a homologue of ACE, known as AnCE, from Drosophila melanogaster in complex with the most promising selenium analogue of captopril (SeCap) determined at 2.4 and 2.35 Å resolution, respectively. The inhibitor binds at the active site of tACE and AnCE in an analogous fashion to that observed for captopril and provide the first examples of a protein–selenolate interaction. These new structures of tACE–SeCap and AnCE–SeCap inhibitor complexes presented here provide important information for further exploration of zinc coordinating selenium-based ACE inhibitor pharmacophores with significant antioxidant activity.DatabaseStructural data for the two SeCap complexes with ACE and AnCE have been deposited with the RCSB Protein Data Bank under the codes 2YDM and 3ZQZ, respectively.

The snoring child, implications for adulthood

The brain renin-angiotensin system plays a role in both cardiovascular homeostasis and neurosecretory functions. Since the mechanisms of angiotensin (Ang) II formation in the human brain have not been clarified, the aims of the present study were to determine the presence of human chymase and angiotensin I-converting enzyme (ACE) in human and non-human brains. In the human brain, the total Ang II-forming activity was significantly higher in the pineal and pituitary glands than those in other regions. In other species (rat, bovine and porcine), the level of chymase as well as total Ang II-forming activities in pineal glands were significantly lower than those in human glands. High levels of chymase-like immunoreactivity (ir) were found in the arteriolar endothelial cells, adventitial mesenchymal cells and in parenchymal cells of the human pineal and pituitary glands while ACE-ir was mostly observed in the endothelial cells and occasionally found in parenchymal cells. Our study provides the first evidence that human chymase exists in the pineal and pituitary glands. The remarkable regional and species differences in mechanisms of Ang II formation suggest a specific role of chymase or ACE in the human brain.

Novel mechanism of inhibition of human angiotensin-I-converting enzyme (ACE) by a highly specific phosphinic tripeptide

Human ACE (angiotensin-I-converting enzyme) has long been regarded as an excellent target for the treatment of hypertension and related cardiovascular diseases. Highly potent inhibitors have been developed and are extensively used in the clinic. To develop inhibitors with higher therapeutic efficacy and reduced side effects, recent efforts have been directed towards the discovery of compounds able to simultaneously block more than one zinc metallopeptidase (apart from ACE) involved in blood pressure regulation in humans, such as neprilysin and ECE-1 (endothelin-converting enzyme-1). In the present paper, we show the first structures of testis ACE [C-ACE, which is identical with the C-domain of somatic ACE and the dominant domain responsible for blood pressure regulation, at 1.97Å (1 Å=0.1 nm)] and the N-domain of somatic ACE (N-ACE, at 2.15Å) in complex with a highly potent and selective dual ACE/ECE-1 inhibitor. The structural determinants revealed unique features of the binding of two molecules of the dual inhibitor in the active site of C-ACE. In both structures, the first molecule is positioned in the obligatory binding site and has a bulky bicyclic P1′ residue with the unusual R configuration which, surprisingly, is accommodated by the large S2′ pocket. In the C-ACE complex, the isoxazole phenyl group of the second molecule makes strong pi–pi stacking interactions with the amino benzoyl group of the first molecule locking them in a ‘hand-shake’ conformation. These features, for the first time, highlight the unusual architecture and flexibility of the active site of C-ACE, which could be further utilized for structure-based design of new C-ACE or vasopeptidase inhibitors.

Conformational Fingerprinting of the Angiotensin I-Converting Enzyme (ACE). 1. Application in Sarcoidosis

Fine epitope mapping of monoclonal antibodies (mAbs) to 16 epitopes on human angiotensin I-converting enzyme (ACE) revealed that the epitopes of all mAbs contained putative glycosylation sites. ACE glycosylation is both cell- and tissue-specific and, therefore, the local conformation of ACE produced by different cells could be also unique. The pattern of ACE binding by a set of mAbs to 16 epitopes of human ACE - "conformational fingerprint of ACE" - is the most sensitive marker of ACE conformation and could be cell- and tissue-specific. The recognition of ACEs by mAbs to ACE was estimated using an immune-capture enzymatic plate precipitation assay. Precipitation patterns of soluble recombinant ACE released from Chinese hamster ovary (CHO)-ACE cells was influenced by conditions that alter ACE glycosylation. This pattern was also strongly cell type specific. Patients with sarcoidosis exhibited conformational fingerprints of tissue ACE (lungs and lymph nodes), as well as blood ACE, which were distinct from controls. Conformational fingerprinting of ACE may detect ACE originated from the cells other than endothelial cells in the blood and when combined with elevated blood ACE levels in patients with sarcoidosis may potentially reflect extrapulmonary sarcoidosis involvement (bone marrow, spleen, liver). If proven true, this would serve as a biomarker of enormous potential clinical significance.

ACE activity is modulated by the enzyme α-galactosidase A

Fabry disease is a multisystem X-linked disorder resulting from α-galactosidase A (α-GalA) gene mutations leading to the accumulation of globotriaosylceramide mainly in endothelium compromising heart, kidney, and brain. In Fabry patients, progressive renal failure is frequently treated with angiotensin I-converting enzyme (ACE) inhibitors. We were interested in the possible interactions between ACE inhibitors therapy and the only causative therapy for Fabry disease, the enzyme replacement therapy (ERT) using recombinant human α-GalA (rhα-GalA). Our results suggest that ACE activity was significantly inhibited in plasma of Fabry patients and the blood pressure level decreased just after ERT (at the end of the rhα-GalA infusion). Interestingly, 2 weeks later, ACE activity was significantly upregulated and the plasma levels of angiotensin II increased in the patients treated with rhα-GalA following the elevations of ACE activity. The same inhibitory effect on ACE activity was also observed in rats after rhα-GalA infusion. Furthermore, ACE activity in CHO cells transfected with the human ACE was inhibited dose and time-dependently by rhα-GalA. In vitro, the incubation of plasma from healthy volunteers with rhα-GalA significantly reduced ACE activity. Finally, rhα-GalA also inhibited ACE activity and released galactose residues from purified rabbit lung ACE dose-dependently. In summary, our results suggest that rhα-GalA interacts with ACE and inhibits its activity, possibly by removing the galactose residues from the enzyme. This modulation might have profound impact on the clinical outcome of Fabry patients treated with rhα-GalA.

High-Resolution Crystal Structures of Drosophila melanogaster Angiotensin-Converting Enzyme in Complex with Novel Inhibitors and Antihypertensive Drugs

Angiotensin I-converting enzyme (ACE), one of the central components of the renin–angiotensin system, is a key therapeutic target for the treatment of hypertension and cardiovascular disorders. Human somatic ACE (sACE) has two homologous domains (N and C). The N- and C-domain catalytic sites have different activities toward various substrates. Moreover, some of the undesirable side effects of the currently available and widely used ACE inhibitors may arise from their targeting both domains leading to defects in other pathways. In addition, structural studies have shown that although both these domains have much in common at the inhibitor binding site, there are significant differences and these are greater at the peptide binding sites than regions distal to the active site. As a model system, we have used an ACE homologue from Drosophila melanogaster (AnCE, a single domain protein with ACE activity) to study ACE inhibitor binding. In an extensive study, we present high-resolution structures for native AnCE and in complex with six known antihypertensive drugs, a novel C-domain sACE specific inhibitor, lisW-S, and two sACE domain-specific phosphinic peptidyl inhibitors, RXPA380 and RXP407 (i.e., nine structures). These structures show detailed binding features of the inhibitors and highlight subtle changes in the orientation of side chains at different binding pockets in the active site in comparison with the active site of N- and C-domains of sACE. This study provides information about the structure–activity relationships that could be utilized for designing new inhibitors with improved domain selectivity for sACE.

Catalytic properties of recombinant dipeptidyl carboxypeptidase from Escherichia coli: a comparative study with angiotensin I-converting enzyme

Dipeptidyl carboxypeptidase from Escherichia coli (EcDcp) is a zinc metallopeptidase with catalytic properties closely resembling those of angiotensin I-converting enzyme (ACE). However, EcDcp and ACE are classified in different enzyme families (M3 and M2, respectively) due to differences in their primary sequences. We cloned and expressed EcDcp and studied in detail the enzyme's S(3) to S(1)' substrate specificity using positional-scanning synthetic combinatorial (PS-SC) libraries of fluorescence resonance energy transfer (FRET) peptides. These peptides contain ortho-aminobenzoic acid (Abz) and 2,4-dinitrophenyl (Dnp) as donor/acceptor pair. In addition, using FRET substrates developed for ACE [Abz-FRK(Dnp)P-OH, Abz-SDK(Dnp)P-OH and Abz-LFK(Dnp)-OH] as well as natural ACE substrates (angiotensin I, bradykinin, and Ac-SDKP-OH), we show that EcDcp has catalytic properties very similar to human testis ACE. EcDcp inhibition studies were performed with the ACE inhibitors captopril (K(i)=3 nM) and lisinopril (K(i)=4.4 microM) and with two C-domain-selective ACE inhibitors, 5-S-5-benzamido-4-oxo-6-phenylhexanoyl-L-tryptophan (kAW; K(i)=22.0 microM) and lisinopril-Trp (K(i)=0.8 nM). Molecular modeling was used to provide the basis for the differences found in the inhibitors potency. The phylogenetic relationship of EcDcp and related enzymes belonging to the M3 and M2 families was also investigated and the results corroborate the distinct origins of EcDcp and ACE.

Simulated Interactions between Angiotensin-Converting Enzyme and Substrate Gonadotropin-Releasing Hormone: Novel Insights into Domain Selectivity

Human angiotensin-I converting enzyme (ACE) is a central component of the renin-angiotensin system and a major target for cardiovascular therapies. The somatic form of the enzyme (sACE) comprises two homologous metallopeptidase domains (N and C), each bearing a zinc active site with similar but distinct substrate and inhibitor specificities. On the basis of the recently determined crystal structures of both ACE domains, we have studied their complexes with gonadotropin-releasing hormone (GnRH), which is cleaved releasing both the protected NH2- and COOH-terminal tripeptides. This is the first molecular modeling study of an ACE-peptide substrate complex that examines the structural basis of ACE's endopeptidase activity and offers novel insights into subsites that are distant from the obligatory binding site and were not identified in the crystal structures. Our data indicate that a bridging interaction between Arg500 of the N-domain and Arg8 of GnRH that involves a buried chloride ion may account for its role in the specificity of the N-domain for endoproteolytic cleavage of the substrate at the NH2-terminus in vitro. In support of this, the protected NH2-terminal dipeptide of GnRH exhibits stronger interactions than the protected COOH-terminal dipeptide with the N-domain of ACE. Further comparison of the models of ACE-substrate complexes promotes our understanding of how the two domains differ in their function and specificity and provides an extension of the pharmacophore model used for structure-based drug design up to the S7 subsite of the enzyme.

Monoclonal Antibodies 1G12 and 6A12 to the N-Domain of Human Angiotensin-Converting Enzyme: Fine Epitope Mapping and Antibody-Based Detection of ACE Inhibitors in Human Blood

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. ACE inhibitors are among the most prescribed drugs in the treatment of hypertension and cardiac failure. Fine epitope mapping of two monoclonal antibodies (mAb), 1G12 and 6A12, against the N-domain of human ACE, was developed using the N-domain 3D-structure and 21 single and double N-domain mutants. The binding of both mAbs to their epitopes on the N-domain of ACE is significantly diminished by the presence of the C-domain in the two-domain somatic tissue ACE and further diminished by the presence of sialic acid residues on the surface of blood ACE. The binding of these mAbs to blood ACE, however, increased dramatically (5-10-fold) in the presence of ACE inhibitors or EDTA, whereas the effect of these compounds on the binding of the mAbs to somatic tissue ACE was less pronounced and even less for truncated N-domain. This implies that the binding of ACE inhibitors or removal of Zn2+ from ACE active centers causes conformational adjustments in the mutual arrangement of N- and C-domains in the two-domain ACE molecule. As a result, the regions of the epitopes for mAb 1G12 and 6A12 on the N-domain, shielded in somatic ACE by the C-domain globule and additionally shielded in blood ACE by sialic acid residues in the oligosaccharide chains localized on Asn289 and Asn416, become unmasked. Therefore, we demonstrated a possibility to employ these mAbs (1G12 or 6A12) for detection and quantification of the presence of ACE inhibitors in human blood. This method should find wide application in monitoring clinical trials with ACE inhibitors as well as in the development of the approach for personalized medicine by these effective drugs.

Human ACE and bradykinin B 2 receptors form a complex at the plasma membrane

To investigate how angiotensin I-converting enzyme (ACE) inhibitors enhance the actions of bradykinin (BK) on B2 receptors independent of blocking BK inactivation, we expressed human somatic ACE and B2 receptors in CHO cells. Bradykinin and its ACE-resistant analog were the receptor agonists. B2 fused with green fluorescent protein (GFP) and ACE were coprecipitated with antisera to GFP or ACE shown in Western blots. Immunohistochemistry of fixed cells localized ACE by red color and B2-GFP by green. Yellow on plasma membranes of coexpressing cells also indicated enzyme-receptor complex formation. Using ACE-fused cyan fluorescent protein donor and B2-fused yellow fluorescent protein (YFP) acceptor, we registered fluorescence resonance energy transfer (FRET) by the enhanced fluorescence of donor on acceptor photobleaching, establishing close (within 10 nm) positions of B2 receptors and ACE. Bradykinin stimulation cointernalized ACE and B2 receptors. We expressed ACE fused to N terminus of B2 receptors, anchoring only receptors to plasma membranes. Here, in contrast to cells, where both ACE and B2 receptors are separately anchored, ACE inhibitors neither enhance activation of chimeric B2 nor resensitize desensitized B2 receptors. Heterodimer formation between ACE and B2 receptors can be a mechanism for ACE inhibitors to augment kinin activity at cellular level.

Inhibitory Antibodies to Human Angiotensin-Converting Enzyme: Fine Epitope Mapping and Mechanism of Action

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.

Crystal Structure of the N Domain of Human Somatic Angiotensin I-converting Enzyme Provides a Structural Basis for Domain-specific Inhibitor Design

Human somatic angiotensin I-converting enzyme (sACE) is a key regulator of blood pressure and an important drug target for combating cardiovascular and renal disease. sACE comprises two homologous metallopeptidase domains, N and C, joined by an inter-domain linker. Both domains are capable of cleaving the two hemoregulatory peptides angiotensin I and bradykinin, but differ in their affinities for a range of other substrates and inhibitors. Previously we determined the structure of testis ACE (C domain); here we present the crystal structure of the N domain of sACE (both in the presence and absence of the antihypertensive drug lisinopril) in order to aid the understanding of how these two domains differ in specificity and function. In addition, the structure of most of the inter-domain linker allows us to propose relative domain positions for sACE that may contribute to the domain cooperativity. The structure now provides a platform for the design of "domain-specific" second-generation ACE inhibitors.

The associations of ACE polymorphisms with physical, physiological and skill parameters in adolescents

Genetic variation in the human Angiotensin I-Converting Enzyme (ACE) gene has been associated with many heritable traits, including physical performance. Herein we report the results of a study of several physical, physiological and skill parameters and lifestyle in 1,027 teenage Greeks. We show that there is a strong association (P < 0.001) between the ACE I/D (insertion/deletion) polymorphism and both handgrip strength and vertical jump in females, homozygotes for the I-allele exhibiting higher performance-related phenotype scores, accounting for up to 4.5% of the phenotypic variance. The association is best explained by a model in which the D-allele is dominant, with the mean phenotypic value in the I/D heterozygotes being close to that of the mean of the DD homozygotes. The association acts across the phenotype distribution in a classical polygenic manner. Other polymorphisms that define major ACE haplotypes in European populations (rs4424958, rs4311) show weaker associations with these performance-related phenotypes than does I/D. Similarly, diplotypes defined by these polymorphisms do not explain significantly larger amounts of the variance than I/D alone. As ACE I/D is the polymorphism most strongly associated with circulating ACE activity in European populations, we propose that the functional allelic differences that influence ACE activity also mediate the associations with the performance-related phenotypes studied here.

Angiotensin I-converting enzyme inhibitor peptides derived from the endostatin-containing NC1 fragment of human collagen XVIII

Extracellular matrix and soluble plasma proteins generate peptides that regulate biological activities such as cell growth, differentiation and migration. Bradykinin, a peptide released from kininogen by kallikreins, stimulates vasodilatation and endothelial cell proliferation. Various classes of substances can potentiate these biological actions of bradykinin. Among them, the best studied are bradykinin potentiating peptides (BPPs) derived from snake venom, which can also strongly inhibit angiotensin I-converting enzyme (ACE) activity. We identified and synthesized sequences resembling BPPs in the vicinity of potential proteolytic cleavage sites in the collagen XVIII molecule, close to endostatin. These peptides were screened as inhibitors of human recombinant wild-type ACE containing two intact functional domains; two full-length ACE mutants containing only a functional C- or N-domain catalytic site; and human testicular ACE, a natural form of the enzyme that only contains the C-domain. The BPP-like peptides inhibited ACE in the micromolar range and interacted preferentially with the C-domain. The proteolytic activity involved in the release of BPP-like peptides was studied in human serum and human umbilical-vein endothelial cells. The presence of enzymes able to release these peptides in blood led us to speculate on a physiological mechanism for the control of ACE activities.

Angiotensin-converting Enzyme 2 (ACE2), But Not ACE, Is Preferentially Localized to the Apical Surface of Polarized Kidney Cells

Angiotensin-converting enzyme-2 (ACE2) is a homologue of angiotensin-I converting enzyme (ACE), the central enzyme of the renin-angiotensin system (RAS). ACE2 is abundant in human kidney and heart and has been implicated in renal and cardiac function through its ability to hydrolyze Angiotensin II. Although ACE2 and ACE are both type I integral membrane proteins and share 61% protein sequence similarity, they display distinct modes of enzyme action and tissue distribution. This study characterized ACE2 at the plasma membrane of non-polarized Chinese hamster ovary (CHO) cells and polarized Madin-Darby canine kidney (MDCKII) epithelial cells and compared its cellular localization to its related enzyme, ACE, using indirect immunofluorescence, cell-surface biotinylation, Western analysis, and enzyme activity assays. This study shows ACE2 and ACE are both cell-surface proteins distributed evenly to detergent-soluble regions of the plasma membrane in CHO cells. However, in polarized MDCKII cells under steady-state conditions the two enzymes are differentially expressed. ACE2 is localized predominantly to the apical surface ( approximately 92%) where it is proteolytically cleaved within its ectodomain to release a soluble form. Comparatively, ACE is present on both the apical ( approximately 55%) and basolateral membranes ( approximately 45%) where it is also secreted but differentially; the ectodomain cleavage of ACE is 2.5-fold greater from the apical surface than the basolateral surface. These studies suggest that both ACE2 and ACE are ectoenzymes that have distinct localization and secretion patterns that determine their role on the cell surface in kidney epithelium and in urine.