Human Renin Substrate Research Articles

Escherichia coli-based production of recombinant ovine angiotensinogen and its characterization as a renin substrate.

BackgroundAngiotensinogen (ANG) is a macromolecular precursor of angiotensin, which regulates blood pressure and electrolyte balance. ANG is specifically cleaved by renin, an aspartic protease, to initiate the angiotensin-processing cascade. Ovine ANG (oANG) from sheep plasma has been shown to be a better substrate for human renin, and it has been used in clinical renin assays. To expand the availability of oANG, we aimed to produce milligram levels of recombinant oANG using an Escherichia coli expression system.ResultsWhen recombinant oANG was expressed from a T7 promoter in various E. coli strains at 37 °C, it accumulated in the insoluble fraction. However, by expressing oANG at 37 °C from a tac promoter, which has weaker transcriptional activity than a T7 promoter, we significantly elevated the ratio of soluble to insoluble recombinant oANG. Using a novel culturing system and auto-induction culture medium, we purified tac-expressed recombinant oANG to homogeneity, with a yield of 4.0 mg per liter of culture. Based on size-exclusion gel filtration analysis and dynamic light scattering analysis, the resulting purified oANG is a monomer in solution. The circular dichroism spectrum of E. coli-expressed recombinant oANG was similar to that of oANG expressed in CHO cells. Differential scanning fluorimetry showed that both preparations undergo a two-state transition during thermal denaturation, and the melting temperatures of recombinant oANG expressed in E. coli and CHO cells were 49.4 ± 0.16 °C and 51.6 ± 0.19 °C, respectively. The Km values of both oANG preparations were similar; the kcat value of E. coli-expressed recombinant oANG was slightly higher than that of CHO-expressed oANG.ConclusionsRecombinant oANG expressed in E. coli functions as a human renin substrate. This study presents an E. coli-based system for the rapid production of milligram quantities of a human renin substrate, which will be useful for both fundamental and clinical studies on renin and hypertension.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0265-x) contains supplementary material, which is available to authorized users.

Corpuscles of Stannius Secrete Renin or an Isorenin That Regulates Cardiovascular Function in Freshwater North American Eels, Anguilla rostrata LeSueur

Dorsal aortic blood flow (DABF) and caudal venous blood flow (CVBF) were measured in free-swimming conscious freshwater (FW) North American eels (Anguilla rostrata) with Doppler-flow probes. DABF and CVBF increased in a dose-dependent manner following iv doses of [Asn1, Val5, Gly9]–angiotensin I (ANG I), [Asn1, Val5]–angiotensin II (ANG II), and [Val4]–angiotensin III (ANG III) ranging from 5 to 50 ng · kg bw−1. A minimum effective dose for ANG I and ANG II was 5 ng · kg bw−1; that for ANG III was 10 ng · kg bw−1. DABF and CVBF rates increased during the first 2 min and remained elevated for 20–50 min. Flow responses similar to those of ANG II in form and magnitude followed iv injections of extracts of corpuscles of Stannius (CS-EXT). Increases in DABF and CVBF following injections of ANG I, human renin substrate (hRS), and CS-EXT were all blocked by the angiotensin-converting enzyme inhibitor Captopril. Increases in DABF and CVBF which followed injections of hRS and CS-EXT were blocked completely by pepstatin A. [Sar1, Val5]–ANG II (Sarile) blocked completely the DABF and CVBF responses to ANG II and CS-EXT, but the mammalian receptor antagonists losartan (AT1) and PD123319 (AT2) only partially blocked them. These findings support strongly the hypothesis that the corpuscles of Stannius secrete renin or isorenin and that the renin–angiotensin system regulates cardiovascular function in freshwater eels and other bony fishes that possess them.

In vivo enzymatic assay reveals catalytic activity of the human renin precursor in tissues.

The aspartyl protease renin is secreted into the circulation of mammals in 2 forms: the proteolytically processed active form of the enzyme and the precursor form, prorenin. Prorenin has no detectable enzymatic activity in the circulation, but it is the exclusive form of the enzyme produced by several tissues that also produce the other components of the renin enzymatic cascade (renin-angiotensin system). To test whether prorenin might be enzymatically active in these tissues, transgenic mice expressing the human renin substrate (angiotensinogen) exclusively in the pituitary gland were mated to mice expressing either active human renin or prorenin in the same tissue. Measurement of in vivo product formation in pituitary glands of double-transgenic mice revealed that human prorenin was enzymatically active, and Western blot analysis demonstrated that this prorenin was in the precursor form with its prosegment attached. This in vivo enzymatic assay demonstrates for the first time that human prorenin can be activated within tissues by nonproteolytic means, where it could contribute to the activity of a localized renin-angiotensin system.

Human urinary kallikrein can generate angiotensin II from homologous renin substrates.

We previously proposed the "kinin-tensin system," a unique vasoregulatory system that can produce both angiotensin II and kinins. To verify whether tissue kallikrein is a part of this system in humans, we examined the ability of human urinary kallikrein (HUK) to generate angiotensin (ANG) II directly from homologous renin substrates such as purified human angiotensinogen (AOGEN) and authentic human tridecapeptide renin substrate (13 RS). HUK released ANG II not only from ANG I but also directly from both AOGEN and 13RS at an optimum pH of 7.0. The amount of generated ANG II from 7.5 nmol of each of the three substrates at pH 7.0 was as follows: ANG I, 292.7 +/- 67.2; 13 RS, 1951.7 +/- 239.6; AOGEN, 2.2 +/- 0.3 (pmol/3h, n = 3 mean +/- SE). HUK cleaved Phe-His and His-Leu bonds in 13 RS, and Tyr-Ile and Phe-His bonds in ANG I. These results suggest that HUK is a part of the "kinintensin system", i.e., HUK can not only release kinins, but can also generate ANG II mainly through ANG I conversion and from AOGEN, the latter being a minor source of ANG II. Furthermore, HUK may play a role in regulating vascular tone under certain conditions in vivo.

Site-directed double fluorescent tagging of human renin and collagenase (MMP-1) substrate peptides using the periodate oxidation of N-terminal serine. An apparently general strategy for provision of energy-transfer substrates for proteases.

Periodate in neutral aqueous solution rapidly converts N-terminal Ser or Thr to an alpha-N-glyoxylyl moiety that can serve as the locus for incorporation of a modifying group [Geoghegan, K. F., and Stroh, J. G. (1992) Bioconjugate Chem. 3, 138-146. Gaertner, H. F. et al. (1992) Bioconjugate Chem. 3, 262-268]. The usefulness of this procedure has been further illuminated in a route to "energy-transfer" substrates for endoproteases. Each such substrate is an oligopeptide cleavable by a proteinase, but modified (usually at its termini) with two chromophores that form an energy donor-acceptor pair. Production of these substrates is an exercise in double site-directed peptide modification. The new route is composed of three steps, beginning from an unprotected peptide in which a sequence recognized by the pertinent enzyme is placed between N-terminal Ser and C-terminal Lys. Lys may not occur elsewhere in the peptide. Periodate oxidation converts the N-terminal Ser to an alpha-N-glyoxylyl group, which is then allowed to form a hydrazone with the carbohydrazide derivative Lucifer Yellow CH, a hydrophilic fluor with a large Stokes shift (excitation maximum, 425 nm; emission maximum, 525 nm). Finally, the modified peptide is allowed to react with 5-carboxytetramethylrhodamine succinimidyl ester. This reaction selectively modifies the epsilon-amino group of C-terminal Lys, the only amino group remaining in the peptide. 5-Carboxytetramethylrhodamine strongly (> 90%) quenches Lucifer Yellow fluorescence by resonance energy transfer in the intact substrate, but enzyme-catalyzed cleavage eliminates the quenching. The resulting increase in fluorescence may be used to follow the hydrolytic reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Clinical Pharmacology of Enalkiren, A Novel, Dipeptide Renin Inhibitor

Enalkiren (A-64662), a potent, dipeptide renin inhibitor, mimics the transition state of the human renin substrate, angiotensinogen. Enalkiren has been shown to produce dose-related suppression of plasma renin activity (PRA) and angiotensin II when administered intravenously. Doses of enalkiren of less than 0.1 mg/kg induced little hemodynamic response in normotensive and hypertensive volunteers despite marked suppression of PRA. However, at doses of 0.3 and 1.2 mg/kg, enalkiren produced significant, dose-related decreases in systolic and diastolic blood pressure (BP) in hypertensive patients, and the BP response was enhanced by pretreatment with hydrochlorothiazide. The effects of enalkiren on PRA and BP are prolonged despite its relatively short elimination phase plasma half-life (1.6 h). Persistent pharmacologic activity without evidence of tachyphylaxis was demonstrated during 1 week of treatment in hypertensive patients. The observed dissociation between suppression of PRA and BP response and the recruitment of dose-related BP decrements, despite complete suppression of PRA, are unexplained phenomena. The results of clinical trials with enalkiren are encouraging, and suggest that renin inhibitors may be safe, useful therapeutic agents in the management of hypertension.

Substrate-dependent angiotensin II formation in the peripheral circulation

An alternative angiotensin II-forming system distinct from the vascular renin-angiotensin system was demonstrated using a rat hindlimb perfusion system and synthetic substrates. This pathway was resistant to captopril and aprotinin, but was highly sensitive to chymostatin. Moreover, angiotensin II formation was substrate-dependent, i.e. angiotensin II formation from tridecapeptide human renin substrate in the presence of captopril was more than twice that that from an equimolar amount of angiotensin I. Both pathways may play a role in regulating the peripheral circulation.

Clonidine-Displacing Substance is Present in Peripheral Tissues of the Rat

Activation of semipurified human kidney prorenin was found to occur in vitro in presence of a mixture of lipids that mimics the composition of the inner human cell membrane. The lipid-dependent activation was indeed only partial (38 +/- 4%) when compared to that obtained by trypsin in liquid phase (100 micrograms/mL) used as a control of maximal activation (100%) under our experimental conditions (semipurified human kidney prorenin in presence of semipurified human plasma renin substrate at a concentration of 1400 ng/mL, at pH 7.2). The phenomenon was time-dependent up to 60 min whereas the angiotensin I generated after 120 min was virtually the same as that generated after 60 min thus indicating a possible reversible activation of human prorenin. We speculate that prorenin may be reversibly activated by contact with the lipidic portion of the cell membrane either inside or outside the cells thus allowing a limited angiotensin II-generating cascade at a local site initiated by prorenin independently from the presence of active renin.

Immunological comparison of renin substrate with erythropoietin.

Immunological similarities between human renin substrate and erythropoietin were studied. Purified renin substrate was coupled to erythrocytes, and haemagglutination was demonstrated both with antisera against renin substrate and erythropoietin. Extensive purification of renin substrate was performed on fast protein liquid chromatography (FPLC), and fractions were collected and assayed for renin substrate and erythropoietin. Following FPLC, the major protein peak corresponded to renin substrate, whereas erythropoietin immunoactivity dissociated from this peak. Iodinated renin substrate and erythropoietin-containing fractions showed specific binding to both renin substrate and erythropoietin antisera. The results suggest that human erythropoietin or erythropoietin-like material may co-purify with renin substrate, and that preparative procedures like FPLC should be included to obtain pure angiotensinogen preparations. While renin substrate and erythropoietin, judging from the present results and cloned DNA sequences, appear completely unrelated, the possible relationship between renin substrate and other erythropoietic factors remains unsettled.

Erythropoietin and Renin Substrate in Cerebellar Haemangioblastoma

We examined eight cerebellar haemangioblastoma tumours from eight patients, aged 16-63 years, 5 females and 3 males. Preoperative haemoglobin values exceeded 180 g/l in four patients, and 150 g/l in four. All high Hb values were normalized upon surgical removal of the tumours. All tumours contained scattered cells which stained positively with antisera against pure human urinary erythropoietin and plasma renin substrate. We conclude that cerebellar haemangioblastomas produce immunoreactive erythropoietin, which shares common antigenic determinants with renin substrate.

Biochemical properties of renin in human pituitary tissue

The biochemical properties of renin, extracted from human pituitary specimens obtained at autopsy, were studied using a specific antirenin antibody raised against human kidney renin. The following results were obtained. The molecular weight of pituitary renin was estimated to be about 37,000 daltons by gel filtration through Sephadex G-100. The optimum pH of pituitary renin was between 6.0 approximately 7.0, while that of a renin-like substance which did not react with the antirenin antibody had an acidic pH of 4.0, with a pH comparable to that of the cathepsin D-like enzyme in the pituitary tissue. The presence of two different isoelectric-point species of pituitary renin was revealed by isoelectric focusing, one with a point of pH 4.47 and the other with that of pH 5.77. The Km value of pituitary renin was 37.9 microM for synthetic human renin substrate. Affinity chromatography of the pituitary renin on a Concanavalin-Sepharose column showed that most (87.4%) of the pituitary renin did not contain glycoprotein residues. Treatment with either trypsin or glandular kallikrein increased the renin activity, indicating the presence of an inactive form of renin in the pituitary tissue. From these findings, it is concluded that specific renin exists in human pituitary tissue. It seems likely that the pituitary renin is of local origin rather than contamination of the circulating enzyme.

Direct radioimmunoassay for human renin substrate and its measurement in plasma from essential hypertensive patients, diabetic patients and pregnant women.

We homogeneously purified human renin substrate from outdated bank plasma and raised antibody against it. The antibody did not cross-react with angiotensin I, angiotensin II or synthetic tetradecapeptide at concentrations of up to 160 nmol, nor did it cross-react with up to 0.2 ml of plasma from rat, rabbit or sheep. It did, however, completely cross-react with human des-angiotensin I renin substrate. A direct radioimmunoassay for human renin substrate was developed using the antibody, and the minimum detectable value of renin substrate found to be 70 pg of protein. Plasma renin substrate concentrations of normal subjects, essential hypertensive patients, diabetic patients and pregnant women were measured by direct and indirect assays. Hypertensive patients had similar concentrations of plasma renin substrate to normal subjects, whereas diabetic patients had significantly lower plasma renin substrate concentrations (p less than 0.01). The direct assay always gave a higher value than the indirect assay, and the ratios of the two assays (direct assay/indirect assay) were similar in normal subjects, hypertensive patients, diabetic patients and pregnant women.

New inhibitors of human renin tested in vitro and in vivo in the anaesthetized baboon.

A new inhibitor of human renin (H. 189) is described. It is a decapeptide analogue of human renin substrate with the amino acid, statine, substituted for leucine in the scissile bond. Its inhibitory potency as shown by IC50 is 1.0 X 10(-8) M with human plasma renin and 1.5 X 10(-8) M with baboon plasma renin. It is less effective with dog and rat renin, but its inhibitory potency with human renin is similar to that of another inhibitor of ours (H. 142) having a reduced isostere in the scissile bond. H. 189 has some inhibitory effect on cathepsin D (IC50 6.5 X 10(-5) M) but H. 142 has no discernible effect. Pepstatin, on the other hand, was highly effective against cathepsin D (IC50 1.2 X 10(-8) M). H. 142 and H. 189 were infused intravenously at 10 mg/kg/h in four anaesthetized salt-deplete baboons (Papio hamadryas). The activity of renin in plasma decreased markedly as did the circulating concentration of its products, angiotensin I and angiotensin II.

Immunochemical differences between angiotensin I-forming enzymes in man.

1. Human plasma, amniotic fluid and acidified amniotic fluid were incubated at pH 5.5 with the same concentrations of human plasma renin substrate and rat plasma renin substrate. They produced three to eight times more angiotensin I with human than with rat renin substrate. By contrast, human brain extracts generated 20 times more angiotensin I when incubated with rat plasma renin substrate than with human plasma renin substrate. 2. Serial dilutions of anti-(human renin) antibody inhibited, in a dose-dependent manner, the production of angiotension I when plasma, amniotic fluid and brain extracts were incubated with human plasma renin substrate. They also inhibited the production of angiotensin I when plasma and amniotic fluid were incubated with rat plasma renin substrate. They were ineffective on the angiotensin I generation by human brain extracts acting on rat plasma renin substrate. 3. Affinity chromatography on an haemoglobin-Sepharose gel separated the fraction of brain extract acting on human renin substrate and inhibited by anti-(human renin) antiserum; this was not retained on the gel at pH 3.3. Part of the angiotensin I-forming activity detected by rat renin substrate hydrolysis was not retained on the gel and part was eluted at pH 8.5. These angiotensin I-forming activities did not hydrolyse human renin substrate, and were not neutralized by anti-(human renin) antibody. 4. These results demonstrate that a renin, immunochemically identical with renal, plasma amd amniotic fluid renin, is present in the human brain. Other angiotensin I-forming activity, acting on an heterologous substrate at a more acidic pH, is also present in human brain.

Kinetic studies of the human renin and human substrate reaction

The hydrolysis of human renin substrate by human renin was studied simultaneously in sera of women receiving or not receiving oral contraceptives. The kinetic parameters K m and V m of the Michaelis-Menten equation and the relevant standard errors were estimated by Wilkinson's method for the statistical analysis of kinetic data. Significant differences in the kinetic constants between these two groups were not observed. These studies indicate that the higher velocity in sera from women receiving estrogen is related to the substrate concentration rather than modifying factors or multiple forms of renin substrate.

Human renal renin. Complete purification and characterization.

Complete purification of human renin from noncancerous, autopsied kidneys is reported. A 480,000-fold purification was achieved to yield renin with a specific activity of 950 Goldblatt units/mg. This preparation satisfied multiple criteria of purity as tested by polyacrylamide gel electrophoresis, isoelectric focusing, specific activity, analytical ultracentrifugation, and immunodouble diffusion. The molecular weight of the pure enzyme determined by sedimentation equilibrium is 40,000. The apparent molecular weight estimated by gel filtration is 41,000. The enzyme has an isoelectric point of pH 5.7. Human renin shows an affinity for concanavalin A, suggesting the presence of carbohydrates. These properties and the amino acid composition of human renin are different from those of renin obtained from other mammalian species. Human renin antibodies prepared with the pure enzyme preparation showed negligible cross-reactivity with renin from other mammalian species. The activity with homologous human renin substrate has a pH optimum of 6, whereas with substrates from other mammalian species the optima were in higher or lower pH ranges.

Isolation and characterization of human renin substrate.

Human renin substrate (angiotensinogen) was purified from outdated bank plasma. Purification procedures included ammonium sulfate precipitation, DEAE-cellulose column chromatography, concanavalin A-Sepharose column chromatography, Hydroxylapatite column chromatography preparative isoelectric focusing and Ultrogel AcA 44 gel filtration. The final recovery was 10% and the specific angiotensin I content of 10.5 micrograms/mg of protein was obtained. Polyacrylamide gel and SDS-polyacrylamide gel electrophoresis and analytical ultracentrifugal analyses showed the homogeneity of the purified renin substrate. The molecular weight of 60900 was determined by sedimentation equilibrium studies. Human renin substrate was a glycoprotein containing 13% carbohydrate. Cystine could not be detected on amino acid analysis. The purified renin substrate showed the isoelectric point heterogeneity (pI, 4.6 and 4.9).

Multiple forms of human plasma renin substrate.

The objective of this investigation was to determine whether heterogeneity of plasma renin substrate could be observed in states of steroid excess and various forms of hypertensive disease. In states of stimulated renin substrate production by estrogens or glucocorticoids, multiple forms of renin substrate were apparent when stimulation was excessive. Stimulation of substrate production caused by uremia associated with hypertension showed similar results. None, or only trace quantities of the additional forms of renin substrate were evident in subjects with normal or suppressed levels of plasma renin substrate. The additional forms of renin substrate could be distinguished from the normal form on the basis of cross-reactivity with a specific antiserum to the normal form, electrophoretic mobility, and kinetic rate constants. Differences in rate constants of the various forms of plasma renin substrate may account for the altered rate of the renin reaction associated with several states of hypertension. In plasma of patients with renovascular hypertension, significant quantities of a protein which cross-reacted with the antiserum but could not generate angiotensin I were observed.

In vitro and in vivo inhibition of renin by fatty acids.

Circulating neutral lipids inhibit the in vitro renin reaction. To identify the inhibitor(s), free fatty acids were added to human renin and homologous substrate. Capric, lauric, palmitoleic, linoleic, and arachidonic acids each inhibited the rate of angiotensin I production in vitro (P less than 0.01). Inhibition by polysaturated fatty acids (linoleic and arachidonic) was less (P less than 0.01) after catalytic hydrogenation of the double bonds. To evaluate an in vivo effect of renin inhibition intra-arterial blood pressure responses to infusions of renin and angiotensin II (5.0 microgram) were measured in anephric rats (n = 6) before and after infusion of linoleic acid (10 mg iv). Mean increase of blood pressure to angiotensin II before (75 mmHg +/- 9) and after (90 +/- 12) linoleic acid did not differ (P greater than 0.05). However, the pressor response to renin after linoleic acid (18 +/- 3) was less (P less than 0.00)) than that before (102 +/- 13). In summary, several fatty acids inhibit the in vitro renin reaction, and in part inhibition is dependent on unsaturation. Linoleic acid also inhibits the in vivo pressor response to renin. These results suggest that fatty acids may modify the measurement of plasma renin activity and may also affect angiotensin production in vivo.

Purification of human renin substrate

Renin substrate, angiotensinogen, has been purified from human plasma by methods which permit the processing of large amounts of outdated bank blood. The purified protein is homogeneous by disc gel electrophoresis at pH 9.5. The specific activity of 18 nmol/mg corresponds to a molecular weight of 56,000, while a higher value, 90,000, is found by gel filtration. Chromatography of partially purified renin substrate on DEAE-cellulose in a descending pH gradient shows evidence for the existence of multiple forms. However, some of these forms appear to be lost after chromatography on hydroxylapatite.