SARS-CoV-2 spike protein predicted to form complexes with host receptor protein orthologues from a broad range of mammals.

SARS-CoV-2 has a zoonotic origin and was transmitted to humans via an undetermined intermediate host, leading to infections in humans and other mammals. To enter host cells, the viral spike protein (S-protein) binds to its receptor, ACE2, and is then processed by TMPRSS2. Whilst receptor binding contributes to the viral host range, S-protein:ACE2 complexes from other animals have not been investigated widely. To predict infection risks, we modelled S-protein:ACE2 complexes from 215 vertebrate species, calculated changes in the energy of the complex caused by mutations in each species, relative to human ACE2, and correlated these changes with COVID-19 infection data. We also analysed structural interactions to better understand the key residues contributing to affinity. We predict that mutations are more detrimental in ACE2 than TMPRSS2. Finally, we demonstrate phylogenetically that human SARS-CoV-2 strains have been isolated in animals. Our results suggest that SARS-CoV-2 can infect a broad range of mammals, but few fish, birds or reptiles. Susceptible animals could serve as reservoirs of the virus, necessitating careful ongoing animal management and surveillance.

Characterization of angiotensin I-converting enzyme from anterior gills of the mangrove crab Ucides cordatus.

Angiotensin I-converting enzyme (ACE) is a well-known metallopeptidase that is found in vertebrates, invertebrates and bacteria. We isolated from the anterior gill of the crab Ucides cordatus an isoform of ACE, here named crab-ACE, which presented catalytic properties closely resembling to those of mammalian ACE. The enzyme was purified on Sepharose-lisinopril affinity chromatography to apparent homogeneity and a band of about 72 kDa could be visualized after silver staining and Western blotting. Assays performed with fluorescence resonance energy transfer (FRET) selective ACE substrates Abz-FRK(Dnp)P-OH, Abz-SDK(Dnp)P-OH and Abz-LFK(Dnp)-OH, allowed us to verify that crab-ACE has hydrolytic profile very similar to that of the ACE C-domain. In addition, we observed that crab-ACE can hydrolyze the ACE substrates, angiotensin I and bradykinin. The enzyme was strongly inhibited by the specific ACE inhibitor lisinopril (Ki of 1.26 nM). However, in contrast to other ACE isoforms, crab-ACE presented a very particular optimum pH, being the substrate Abz-FRK(Dnp)-P-OH hydrolyzed efficiently at pH 9.5. Other interesting characteristic of crab-ACE was that the maximum hydrolytic activity was reached at around 45°C. The description of an ACE isoform in Ucides cordatus is challenging and may contribute to a better understanding of the biochemical function of this enzyme in invertebrates.

Correlation of angiotensin converting enzyme gene polymorphism with perioperative myocardial protection under extracorporeal circulation.

OBJECTIVE: To observe the expression of angiotensin converting enzyme (ACE), angiotensin II (Ang II), cardiac troponin (cTn I), creatine kinase isozymes (CK-MB) and muscle red protein(Myo) after cardiopulmonary bypass (CPB), and to investigate the association of polymorphisms in angiotensin converting enzyme genes and myocardial injury. METHODS: Sixty-three patients suffered from rheumatic mitral stenosis and scheduled for mitral valve replacement with CPB, were randomly divided into three groups according polymorphisms in angiotensin converting enzyme genes: type II, type ID, type DD (each=21). Blood samples were withdrawn from artery before operation (T1), at the beginning of CPB (T2), 30 min after CPB (T3), (T4) at the end of CPB (T5), 2 h after CPB (T6), 6 h after CPB (T7) to measure the expression of ACE, Ang II, cTn I, CK-MB, Myo. RESULTS: The level of ACE during and after CPB were significantly higher than those before CPB (P<0.05). As extension of CPB time, the expression of ACE was increased. The level of cTn I, CK-MB, Myo after CPB were significantly higher than those before CPB(P<0.05). The level of cTn I, CK-MB and Myo were highest at T7, T6 and T5 and T7, respectively. The level of ACE, Ang II cTn I in patients with DD genotype was significantly higher than the ID and II genotype (P< 0.05). Besides, the level of ACE, Ang II in patients with ID genotype was significantly higher than the II (P< 0.05). CONCLUSIONS: There is certain correlation between CPB perioperative midterm ACE and cTn I, Myo, CK-MB. ACE DD genotype is a susceptibility gene of the CPB perioperative myocardial injury.

Characterization of the first angiotensin-converting like enzyme in bacteria: Ancestor ACE is already active.

Angiotensin-converting enzyme (ACE) is a metallopeptidase that converts angiotensin I into angiotensin II. ACE is crucial in the control of cardiovascular and renal homeostasis and fertility in mammals. In vertebrates, both transmembrane and soluble ACE, containing one or two active sites, have been characterized. So far, only soluble, single domain ACEs from invertebrates have been cloned, and these have been implicated in reproduction in insects. Furthermore, an ACE-related carboxypeptidase was recently characterized in Leishmania, a unicellular eukaryote, suggesting the existence of ACE in more distant organisms. Interestingly, in silico databank analysis revealed that bacterial DNA sequences could encode putative ACE-like proteins, strikingly similar to vertebrates' enzymes. To gain more insight into the bacterial enzymes, we cloned the putative ACE from the phytopathogenic bacterium, Xanthomonas axonopodis pv. citri, named XcACE. The 2 kb open reading frame encodes a 672-amino-acid soluble protein containing a single active site. In vitro expression and biochemical characterization revealed that XcACE is a functional 72 kDa dipeptidyl-carboxypeptidase. As in mammals, this metalloprotease hydrolyses angiotensin I into angiotensin II. XcACE is sensitive to ACE inhibitors and chloride ions concentration. Variations in the active site residues, highlighted by structural modelling, can account for the different substrate selectivity and inhibition profile compared to human ACE. XcACE characterization demonstrates that ACE is an ancestral enzyme, provoking questions about its appearance and structure/activity specialisation during the course of evolution.

Structural features of angiotensin-I converting enzyme catalytic sites: conformational studies in solution, homology models and comparison with other zinc metallopeptidases.

Angiotensin-I Converting Enzyme (ACE) is a Zinc Metallopeptidase of which the three-dimensional structure was unknown until recently, when the X-ray structure of testis isoform (C-terminal domain of somatic) was determined. ACE plays an important role in the regulation of blood pressure due to its action in the frame of the Renin-Angiotensin System. Efforts for the specific inhibition of the catalytic function of this enzyme have been made on the basis of the X-ray structures of other enzymes with analogous efficacy in the hydrolytic cleavage of peptide substrate terminal fragments. Angiotensin-I Converting Enzyme bears the sequence and topology characteristics of the well-known gluzincins, a sub-family of zincins metallopeptidases and these similarities are exploited in order to reveal common structural elements among these enzymes. 3D homology models are also built using the X-ray structure of Thermolysin as template and peptide models that represent the amino acid sequence of the ACE's two catalytic, zinc-containing sites are designed and synthesized. Conformational analysis of the zinc-free and zinc-bound peptides through high resolution 1H NMR Spectroscopy provides new insights into the solution structure of ACE catalytic centers. Structural properties of these peptides could provide valuable information towards the design and preparation of new potent ACE inhibitors.

Genetic polymorphisms in the Renin-Angiotensin system in high-altitude and low-altitude Native American populations.

Cardiovascular disease (CVD) is reportedly less common in high-altitude native populations than in lowlanders. To some extent, this is due to cultural and demographic factors; however, increased cardiovascular efficiency contributing to hypoxia adaptation may also be involved. Numerous genetic variants have been associated with cardiovascular health. If the decreased incidence of CVD in modern high-altitude populations reflects selective pressures having favoured the transmission of these alleles in their antecedents, it would be expected that these alleles would be more common in highlanders than in lowlanders. We tested this hypothesis by determining the allele frequencies of five polymorphic loci in genes encoding components of the renin-angiotensin system (RAS) that have alleles associated with hypertension and cardiovascular disease in a high-altitude native Andean population, Quechua from the Peruvian altiplano, and in a lowland Amerindian population, Maya from the Yucatan peninsula. The polymorphisms examined were 1) the insertion/deletion polymorphism in intron 16 of the angiotensin converting enzyme (ACE) gene; 2) the A/G2350 transition (ACE-8) in intron 17 of the ACE gene; 3) the A/C1166 transversion in the 3' untranslated region of the angiotensin II receptor (type 1) gene (AGTR1); 4) the G/AI9-83 transition in intron 8 of the renin gene (REN); and 5) the T/C704 (Met235Thr) transition mutation in angiotensinogen (AGT). There was no evidence for an over-representation of the RAS alleles associated with cardiovascular fitness in the high-altitude Amerindian population when compared to the lowland Amerindian population.

Presence of angiotensin-converting enzyme in follicular fluids of porcine ovaries and its possible involvement in the intrafollicular breakdown of bradykinin.

The follicular fluid of porcine ovaries contains a metalloenzyme capable of hydrolyzing the synthetic substrate, benzyloxycarbonyl-Val-Lys-Met-MCA. This enzyme was purified by ammonium sulfate fractionation followed by column chromatography on DEAE-cellulose, CM-cellulose, Zn(2+)-chelating Cellulofine, and Diol-300 gel-filtration columns. The molecular weight of the purified enzyme was estimated to be 170,000 by SDS-PAGE and 400,000 by gel-filtration analysis, suggesting that the native enzyme is a dimer of the 170-kDa subunit polypeptide. The enzyme activity was drastically enhanced by the presence of chloride ion, and strongly inhibited by captopril and bradykinin potentiator B. A 9-residue peptide containing a processing site of human amyloid precursor protein was degraded by its dipeptidyl carboxypeptidase activity. Furthermore, the purified protein was recognized by specific antibody raised against human angiotensin-converting enzyme. The enzyme rapidly degraded bradykinin in vitro. These results indicate that benzyloxycarbonyl-Val-Lys-Met-MCA-hydrolyzing enzyme is a porcine angiotensin-converting enzyme, and that the enzyme may play a role in bradykinin turnover within the follicles of porcine ovaries.

A DNA polymorphism at the alpha2-macroglobulin gene is associated with the severity of rheumatoid arthritis.

OBJECTIVE: To determine if DNA polymorphisms at the alpha2-macroglobulin (alpha2m) and angiotensin converting enzyme (ACE) genes were associated with rheumatoid arthritis (RA). METHODS: A total of 160 patients (71 with early active severe RA, 89 with non-severe RA) were genotyped (polymerase chain reaction) for the alpha2m (5 bp deletion/insertion) and ACE (I/D) polymorphisms. We also genotyped 500 healthy controls from the same Caucasian population (Asturias, Northern Spain). RESULTS: Carriers of the alpha2m deletion allele were at a significantly higher frequency among patients with an early active severe form of the disease, compared to patients with non-severe RA (p = 0.037). The frequency of the alpha2m deletion allele was significantly higher in patients with severe compared to nonsevere RA (p = 0.017). In addition, the frequency of the deletion allele was significantly higher among patients with 5 or more episodes of acute exacerbation of disease activity per year (n = 39) compared to those with none (n = 46) (p = 0.002). Gene and genotype frequencies for the ACE-I/D polymorphism did not differ between those with early active severe and non-severe RA. CONCLUSION: The genetic variation at alpha2m is associated with the severity of RA. Carriers of the alpha2m deletion allele would have increased risk of developing an early active severe form of the disease. Our data suggest that alpha2m could be a valuable target in the treatment of RA.

Purification and characterization of a dipeptidyl carboxypeptidase from the polychaete Neanthes virens resembling angiotensin I converting enzyme.

Dipeptidyl carboxypeptidase (DCP) is well known as a mammalian angiotensin I converting enzyme (ACE) which plays an important role in blood pressure homeostasis. DCP was purified from the whole body of a polychaete, Neanthes virens. The purified enzyme was homogeneous by SDS-PAGE, with a molecular mass of 71 kDa by SDS-PAGE and 69 kDa by gel filtration, indicating that it is monomeric. The isoelectric point was 4.5 and optimum pH for the activity was 8.0. It showed a specific activity of 466.8 U/mg, which is the highest of known DCPs. The enzyme hydrolyzed angiotensin I to angiotensin II and sequentially released Phe-Arg and Ser-Pro from the C-terminus bradykinin, but does not cleave imido-bonds. Activity was completely inhibited by 1 mM EDTA and 5 mM o-phenanthroline, but it was not affected by serine and aspartic protease inhibitors. The original activity of EDTA-inactivated DCP was restored by addition of cobalt, manganese or low concentrations of zinc. The Km and Vmax values of the enzyme for Bz-Gly-His-Leu were 0.56 mM and 600 mumol/min per mg, respectively. The Ki values for specific mammalian ACE inhibitors, such as captopril and lisinopril, were 1.38 and 2.07 nM, respectively. In conclusion, we have shown the existence of a DCP from the polychaete, N. virens, with similar properties to those of mammalian ACE.

Angiotensin-converting enzyme (ACE) alleles in the Quechua, a high altitude South American native population.

Recently it was reported that an allelic variant of the gene encoding angiotensin-converting enzyme (ACE) was significantly over-represented in a cohort of elite British mountaineers. It was proposed that this may be evidence for a specific genetic factor influencing the human capacity for physical performance. The implication that this allele could enhance performance at high altitude prompted us to determine its frequency in Quechua speaking natives living at altitudes greater than 3000m on the Andean Altiplano in South America. We found that the frequency of the putative performance allele in the Quechuas, although significantly higher than in Caucasians, was not different from lowland Native American populations. This observation suggests that, although the higher frequency of the 'performance allele' may have facilitated the migration of the ancestral Quechua to the highlands, the ACE insertion allele has not been subsequently selected for in this high altitude population.

Mistyping frequency of the angiotensin-converting enzyme gene polymorphism and an improved method for its avoidance.

A DD genotype of the angiotensin I-converting enzyme gene has been implicated in various diseases. However, genotype frequencies differ between previous reports, and data on the association of DD genotype with disease are sometimes conflicting. Although elimination of mistyping is of crucial importance, assessment of the accuracy of currently adopted typing methods has rarely been performed. Mistyping of the DD genotype is reported to occur by a conventional method with insertion/ deletion (I/D) flanking primers using polymerase chain reaction (PCR). We investigated whether currently adopted genotyping methods by PCR are reliable or not. We genotyped 248 patients by conventional PCR methods with I/D flanking primers with or without dimethyl sulfoxide (DMSO), and confirmed the DD genotype with insertion-specific primers with or without DMSO. Mistyping occurred frequently, not only in both methods without DMSO but also in a modified method with I/D flanking primers with inclusion of DMSO. Typing by these methods proved to lead to erroneous results more frequently than had been previously thought. To reduce mistyping frequency, initial PCR genotyping with I/D flanking primers with an inclusion of DMSO, followed by confirmation of the DD genotype by insertion-specific primers with DMSO, is recommended.

The angiotensin I-converting enzyme (ACE) locus is strongly associated with age and duration of diabetes in patients with type I diabetes.

We have investigated the frequency of the angiotensin I-converting enzyme (ACE) insertion/ deletion (I/D) polymorphism in 249 patients with type I diabetes and 162 normal healthy controls. There was no significant difference in the frequency of ACE genotypes between those patients with diabetic nephropathy (n = 72) (nephropaths) compared to those with no proteinuria after 20 years duration of diabetes (n = 86) (normoalbuminurics). There was, however, a significant difference in the frequency of ACE genotypes between the short-duration and long-term normoalbuminuric group (chi = 11.5, p = 0.001). Analysis of the ACE genotypes with respect to age and duration of diabetes showed that homozygosity for the insertion (I/I) genotype was significantly decreased with longer duration of diabetes (r2 = 92.7%, p < 0.009). No association was found with age in the normal controls. In conclusion, these results suggest that the ACE locus may be associated with longevity and survival in patients with type I diabetes rather than diabetic nephropathy or microvascular disease per se.

The Acer gene of Drosophila codes for an angiotensin-converting enzyme homologue.

Mammalian angiotensin-converting enzyme (ACE) exists as two forms, somatic (sACE), controlling blood pressure via angiotensin II, and testicular (tACE), whose function is unknown. The former has two highly homologous N- and C-terminal Zn2+ metallopeptidase active sites, whereas the latter only has one, which is identical to the C-terminal domain of sACE. We have sequenced 2452 bases of a 3.1-kb mRNA whose predicted translation product shows 40% identity with mammalian testicular ACE, and 48% identity with an already identified Drosophila homologue of ACE (Ance). We have termed this gene Acer (Angiotensin converting enzyme-related). Acer mRNA is found in the developing dorsal vessel (heart) during embryogenesis. Phylogenetic analysis indicates that duplication of an ancestral ACE gene occurred in the lineage leading to the arthropods, independently of the duplication which gave rise to the two domain somatic ACE of mammals.

Serum angiotensin-I-converting enzyme activity in women with cardiological syndrome X: relation to blood pressure and lipid and carbohydrate metabolic risk markers for coronary heart disease.

Polymorphism of the angiotensin-I-converting enzyme (ACE) gene is associated with variations in serum ACE activity and the incidence of cardiovascular disease. Whether a similar association exists with cardiological syndrome X (chest pain, positive ECG exercise test, and normal angiography) is unclear. As ACE activity affects vascular tone, it could be involved in the pathogenesis of this syndrome. We measured serum ACE activity in 18 postmenopausal women with syndrome X and in 18 healthy controls matched for age, adiposity, and menopausal status. The relationship of ACE activity to coronary heart disease risk factors was also examined within these groups. Serum ACE activity did not differ significantly between women with syndrome X and controls. Neither blood pressure, serum total cholesterol, triglyceride, nor high density lipoprotein subfraction 2 cholesterol was associated with ACE activity in either patient group. However, serum high density lipoprotein cholesterol, high density lipoprotein subfraction 3 cholesterol, and apolipoprotein-A-I and -A-II were inversely associated with ACE activity in women with syndrome X, but not in controls. Insulin sensitivity, fasting plasma glucose, insulin, and C-peptide as well as their postglucose challenge concentration profiles were not significantly associated with ACE activity in either group. We conclude that serum ACE activity and, consequently, ACE polymorphism are not associated with syndrome X and are largely independent of metabolic risk factors in these women.