Heterologous expression, purification, and functional characterization of recombinant ovine angiotensinogen in the methylotrophic yeast Pichia pastoris.

Angiotensinogen (AGT), a glycosylated plasma noninhibitory serpin, serves as a precursor for angiotensin peptides which regulate blood pressure and electrolyte balance. AGT is specifically cleaved by renin to produce angiotensin-I, the first product of the angiotensin-processing cascade. Ovine angiotensinogen (oAGT) is considered an effective substrate for human renin and consequently finds application in clinical renin assays. In this study, oAGT was cloned into the genome of Pichia pastoris and expressed under the control of alcohol oxidase (AOX1) promoter for high-level production. Compared to the shake flask study, the high cell density cultivation in bioreactor resulted in multifold increase in oAGT titer (420 ± 9.26 mg/L), which is its highest reported titer to date. We purified recombinant oAGT to homogeneity using two chromatography steps. The characterization studies revealed oAGT underwent a two-state transition during thermal denaturation process as assessed by differential scanning fluorimetry, and the melting temperature (Tm ) of the purified oAGT from P. pastoris was 48.3°C. Renin reactivity with recombinant oAGT from P. pastoris (0.51 nM angiotensin-I/min) was slightly lower than the renin reactivity for recombinant oAGT from Escherichia coli (0.67 nM angiotensin-I/min), possibly because of its mannosylated N-glycan content. Enhanced production of functionally active recombinant oAGT using P. pastoris expression system reported in this study envisage the effective utilization of oAGT in clinical studies related to renin in near future.

Quantitation of Total and Free Thiol Angiotensinogen as a Prognostic Marker for Preeclampsia.

Angiotensinogen mediates an important role in the pathophysiology of preeclampsia, a disorder of pregnancy characterized by hypertension and proteinuria usually after 20 weeks of gestation. Angiotensinogen is found in two distinct posttranslational forms in the plasma, an oxidized and a reduced (free thiol) form. Higher levels of the oxidized form are associated with an increased risk of preeclampsia. We have developed novel ELISA assays to quantitate the levels of total and free thiol angiotensinogen allowing for calculation of the amount of oxidized angiotensinogen species. We describe the methodology for performing these assays.

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

BACKGROUND: Angiotensinogen (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. RESULTS: When 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 K(m) values of both oANG preparations were similar; the k(cat) value of E. coli-expressed recombinant oANG was slightly higher than that of CHO-expressed oANG. CONCLUSIONS: Recombinant 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.

The His-Pro-Phe motif of angiotensinogen is a crucial determinant of the substrate specificity of renin.

The amino acid sequence His-Pro-Phe as N-terminal residues 6-8 of the natural renin substrate, angiotensinogen, is conserved among species. We investigated whether this His-Pro-Phe motif functions as the determinant of the substrate specificity of renin. Mutant angiotensinogens in which the Ile-His-Pro-Phe-His-Leu sequence at positions 5-10 of wild-type angiotensinogen was replaced by either His-Pro-Phe-His-Leu-Leu or Ala-Ile-His-Pro-Phe-His were cleaved by renin at the C-terminal side of residues 9 and 11, respectively, while wild-type angiotensinogen was cleaved at residue 10. A triple Ala substitution for the His-Pro-Phe motif of angiotensinogen prevented its cleavage by renin. In contrast, triple Ala substitution for residues 9-11, including the natural site of cleavage by renin, allowed cleavage between the two Ala residues at positions 10 and 11. Furthermore, the 33-residue C-terminal peptide of human megsin, which carries a naturally occurring His-Pro-Phe sequence, was cleaved by renin at the C-terminal side of the His-Pro-Phe-Leu-Phe sequence. These results indicate that the His-Pro-Phe motif of angiotensinogen is a crucial determinant of the substrate specificity of renin. By binding to a corresponding pocket on renin, the His-Pro-Phe motif may act as a molecular anchor to recruit the scissile peptide bond to a favorable site for catalysis.

Chromatographic behaviour of peptides on a mixed-mode stationary phase with an embedded charged group by capillary electrochromatography and high-performance liquid chromatography.

Retention behaviour of biological peptides was investigated on a stationary phase bearing an embedded quaternary ammonium group in a C21 alkyl chain by both high-performance liquid chromatography (HPLC) and capillary electrochromatography (CEC). In HPLC experiments, variation of acetonitrile (ACN) content in the mobile phase showed that peptides are mainly separated by RP mechanism. The weak or negative retention factors observed as compared to C18 silica stationary phase suggested the involvement of an electrostatic repulsion phenomenon in acidic conditions. Comparison of HPLC and CEC studies indicated that (i) ion-exclusion phenomenon is more pronounced in HPLC and (ii) higher ACN percentage in mobile phase induce for some peptides an increase of retention in CEC, pointing out the existence of mechanisms of retention other than partitioning mainly involved in chromatographic process. This comparative study demonstrated the critical role of electric field on peptide retention in CEC and supports the solvatation model of hydrolytic pillow proposed by Szumski and Buszewski for CEC using mixed mode stationary phase in CEC.

Isolation and identification of proangiotensin-12, a possible component of the renin-angiotensin system.

The renin-angiotensin (RA) system plays an important role in regulating blood pressure and fluid balance. In the search for bioactive peptides with an antibody binding to the N-terminal portion of angiotensin II (Ang II), we isolated a new angiotensinogen-derived peptide from the rat small intestine. Consisting of 12 amino acids, this peptide was termed proangiotensin-12 based on its possible role of an Ang II precursor. Proangiotensin-12 constricted aortic strips and, when infused intravenously, raised blood pressure in rats, while both the vasoconstrictor and pressor response to proangiotensin-12 were abolished by captopril and by CV-11974, an Ang II type I receptor blocker. Proangiotensin-12 is abundant in a wide range of organs and tissues including the small intestine, spleen, kidneys, and liver of rats. The identification of proangiotensin-12 suggests a processing cascade of the RA system, different from the cleavage of angiotensinogen to Ang I by renin.

Quantitation of five forms of high molecular weight angiotensinogen from human placenta.

In addition to the well characterized predominant form of plasma angiotensinogen, which will be termed low molecular weight angiotensinogen (LMrA), significant quantities of a high molecular weight angiotensinogen (HMrA) occur in the human pregnant state. HMrA is the predominant form of angiotensinogen in the placenta and amniotic fluid. The ratio of HMrA/LMrA is elevated in approximately half of the women who develop pregnancy induced hypertension (PIH). This report documents a simple two step methodology for the separation and quantitation of different forms of HMrA. The two steps are gel filtration and ion exchange chromatography. The application of this methodology to amniotic fluid and plasma from normotensive pregnant women and extracts of amnion, chorion, and placental tissue has shown five distinct forms in the tissue extracts and amniotic fluid, but only three significant forms in plasma. The elution position of all forms of HMrA are highly reproducible and are the same for each tissue or fluid studied, thus lending support to the concept that the three forms seen in plasma of normotensive pregnant women are the same as three of the forms of HMrA seen in extracts of placental tissue.

Expression and purification of human angiotensinogen in Chinese hamster ovary cells.

We have produced human angiotensinogen in Chinese hamster ovary (CHO) cells. The expression products were purified to homogeneity by a single column chromatography and its 17 amino-terminal sequences were identical to those of the native protein. We demonstrated the recombinant human angiotensinogen to be a substrate for human renin.

Synthesis of angiotensinogen by renin-containing neuroblastomas.

Angiotensinogen in plasma is of hepatic origin, but many organs possess the ability to synthesize this protein because messenger RNA for angiotensinogen is widely distributed in the body. The cell types responsible for the extrahepatic synthesis of angiotensinogen remain to be identified. To examine whether renin-containing cells synthesize angiotensinogen, we have utilized a polyclonal antibody to angiotensinogen and immunoprecipitated metabolically labeled cells of two neuroblastomas known to contain renin. The results indicate that the cell line Neuro 2a synthesizes and releases a protein with a molecular mass of 57 kDa that is specifically recognized by the angiotensinogen antibody, indicating that Neuro 2a synthesizes angiotensinogen. Similarly, the cell line NB41A3 was also found to synthesize a protein specifically recognized by the antibody to angiotensinogen.

Renin substrate in human amniotic fluid.

Purified human amniotic fluid renin substrate (RS) was compared to purified plasma RS. RS in plasma and amniotic fluid were similar in molecular weight, isoelectric point and immunological properties. Immunoreactivity of radio-iodinated amniotic fluid RS was lower than that of plasma RS. Measured by direct radioimmunoassay, RS-levels were only 10-22% of those obtained with indirect assay in 22 amniotic fluid samples. This difference suggests that amniotic fluid RS is less immunoreactive than plasma RS, possibly due to biochemical alteration or complex formation. No such difference in immunoreactivity was noticed in RS of decidual and placental cytosolic fraction.

Purification and partial characterization of canine angiotensinogen.

A procedure is described to isolate angiotensinogen (renin substrate) from canine plasma. The isolation procedure resulted in an 800-fold purification with a rate of recovery of approximately 12%. The purified protein has a specific activity of 24 micrograms of angiotensin I/mg protein. The amino terminal amino acid sequence of canine angiotensinogen was found to be identical to that of the horse but to differ from that of human and rat angiotensinogens. Canine angiotensinogen was heterogeneous with respect to molecular weight and isoelectric point. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of pure angiotensinogen revealed two closely spaced bands with apparent molecular weights of 58,000 and 56,000. Chromatofocusing showed four isoforms: Peaks of pure angiotensinogen eluted at pH levels of 4.32, 4.23, 4.15, and 4.04. Isoelectric focusing confirmed the presence of four isoforms. Thus, the purification procedure identified two molecular weight forms and four isoforms of canine angiotensinogen. Isolation of the four isoforms will allow their characterization and the study of their physiological significance.

The rapid purification and partial characterization of human serum angiotensinogen.

Human angiotensinogen has been purified 390-fold from serum by a rapid high-yielding procedure that involved chromatography on Blue Sepharose, phenyl-Sepharose, hydroxyapatite and immobilized 5-hydroxytryptamine (5-HT). Angiotensinogen was specifically bound to immobilized 5-HT, which effected a partial resolution into multiple forms, which were also evident when analysed by SDS/polyacrylamide-gel electrophoresis (Mr 59,400, 60,600, 62,600 and 63,800). This heterogeneity was confirmed by resolution into six main bands on isoelectric focusing, ranging from pI 4.40 to 4.82. N-terminal analysis, digestion with human renal renin and deglycosylation studies implied that the preparation comprised several forms of angiotensinogen, varying in their degree of glycosylation. The presence of sialic acid was shown to be a major factor in determining the heterogeneity.

Purification of multiple forms of plasma angiotensinogen: molecular weight and charge heterogeneity.

A method of purifying angiotensinogen (C.A. 11002-13-14) from dog plasma using a monoclonal antibody is presented. More than 98% of applied angiotensinogen was sequestered by the 2-C: G7F7 anti-angiotensinogen monoclonal antibody in solid-phase adsorption assays. Employing immunoaffinity column chromatography, sufficient angiotensinogen was purified for subsequent investigation of angiotensinogen molecular heterogeneity. SDS-polyacrylamide gel electrophoretic analysis resolved two molecular weight forms, an alpha-form at Mr 59,000 and a beta-form at Mr 57,000. These alpha- and beta-plasma angiotensinogen forms were separated by anion-exchange HPLC; an additional gamma-form was also identified. Homogeneous dog angiotensinogen, recovered by reverse-phase HPLC, was partially sequenced. The dog angiotensinogen N-terminal sequence was shown to be more similar to rat than to human angiotensinogen. Since neuraminidase reduced the molecular weight of alpha-angiotensinogen by 4000 and beta-angiotensinogen by 2000, these two forms of the prohormone in dog plasma contain two and one carbohydrate side chain, respectively.

Active and inactive renin in the cat.

Pentobarbitone-anesthetized cats underwent peritoneal dialysis and had blood samples removed and kidneys deep frozen at sacrifice. Inactive renin is easily measurable in cat plasma and peritoneal dialysate fluid. Only small amounts are found after acid activation at pH 4.0, but large amounts after trypsin 2 mg/ml at 4 degrees C for 10 minutes. Mean active renin in pentobarbitone-anesthetized cats was 1.8 +/- 0.4 pmoles AI/ml/hr, while inactive renin was 2.3 +/- 0.5 pmoles AI/ml/hr. The increased angiotensin I producing activity after trypsin in peritoneal dialysate was most active at pH 7.0 (plasma and kidney active renin 7.25 and 7.85), and had an apparent molecular weight of 39-40,000. (Plasma active renin had an apparent MW of 33,500 and kidney active renin 36,000. Plasma inactive renin had an apparent MW of 35,500) Cat plasma after cibacron-blue affinity chromatography showed mainly active renin in the breakthrough buffer (30% of total renin eluted), and renin which is almost entirely inactive in the bound peak (70% of total renin eluted). Active renin from plasma and kidney, and activated inactive renin from concentrated peritoneal fluid, showed exactly similar inhibition by the renin inhibitor H77 (IC50 0.3 microM). Cat plasma angiotensinogen had an apparent MW of 53,000.

Purification and characterization of ovine angiotensinogen.

The two major forms of ovine angiotensinogen (renin substrate) have been purified to homogeneity from plasma and a third form has been partially purified. The purification procedure involved ammonium sulphate fractionation, gel filtration, ion-exchange chromatography and chromatofocusing. The pure proteins have apparent relative molecular masses of 56 000 as determined by sodium dodecyl sulphate gel electrophoresis. The amino acid compositions of the two major forms appear to be the same; however, they do have different carbohydrate compositions. Antibodies raised against the a form showed complete cross-reactivity with the b and c forms. Both major forms have the same amino-terminal sequence, which includes that of ovine angiotensin I: Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu. Thus ovine angiotensin is the Ile5 form and not the Val5 form as had previously been suggested.

Purification of rat angiotensinogen.

A method of purifying rat angiotensinogen in three chromatography steps with a yield 3-4 times better than previous methods is described. Using chromatofocusing media for two steps and DEAE-affigel blue for the third step it was possible to separate angiotensinogen into three major peaks with pI of 5.25 (peak B), 4.80 (peak C) and 4.50 (peak D). Peaks B and C were completely purified with recoveries of 12% and 17% and specific activities of 21.8 and 20.0 micrograms AI/mg protein respectively. Analytical SDS-PAGE showed a 53,000 dalton band in both peaks with additional 51,000 and 57,000 dalton bands in peak C.

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.

Identity of angiotensinogen precursors of rat brain and liver.

Angiotensin modifies a number of functions of the central nervous system (CNS) including blood pressure regulation, thirst, and the secretion of the antidiuretic and adrenocorticotropic hormones. Whereas peripheral administration of angiotensin produces these effects, accumulating evidence implicates a brain angiotensin system which operates independently of the circulating renin-angiotensin system (reviewed in refs 1-3). All components of the renin-angiotensin system have been identified in the brain, but there is considerable controversy over whether these components are synthesized in the brain or derived from plasma and the extent to which these components interact to generate angiotensin in the brain in vivo. Whereas many different enzymes are able to cleave angiotensinogen (renin substrate) to release angiotensin I (AI) or angiotensin II (AII), angiotensinogen is the only known precursor of AI and AII. Definitive evidence for an independent brain angiotensin system requires the demonstration that the brain synthesizes an angiotensin precursor. Here we report the identification of angiotensinogen mRNA in rat brain by cell-free translation and show that the brain angiotensinogen precursors are identical to those previously identified for liver, the source of plasma angiotensinogen.

Granulocyte-angiotensin system. Identification of angiotensinogen as the plasma protein substrate of leukocyte cathepsin G.

Cathepsin G, a human lysosomal neutral protease, converts angiotensin I to angiotensin II and cleaves angiotensin II from a plasma protein substrate. Experiments were designed that identified and characterized cathepsin G substrate as human angiotensinogen. A total of 2, 5, and 10 micrograms of purified substrate, incubated with 2 microL of partially purified human renin (2 Goldblatt units/mg) for 60 min at 37 degrees C, generated 2, 9, and 22 pmol of angiotensin I. Cathepsin G substrate and renin substrate activities copurified during Affi-Gel Blue affinity chromatography, hydroxylapatite chromatography, phenyl-Sepharose chromatography, and S-200 gel filtration. Disc gel electrophoresis of 10 micrograms of purified protein gave a single band containing both activities. The amino-terminal sequence contained the covalent structure of angiotensin I and was Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Val-Ile-His-X-Glu-Ser-Thr-Cys-Gl u-. Reduced and unreduced angiotensinogens were subjected to sodium dodecyl sulfate gel electrophoresis, and each gel showed two bands of Mr 65 000 and 62 000. The isoelectric point of the Mr 65 000 form was pH 4.5-4.3 and the Mr 62 000 form was pH 4.9. Functional, structural, and physiochemical evidence demonstrates that the substrate of cathepsin G is angiotensinogen. Thus, human neutrophils may utilize angiotensin I or angiotensinogen as substrate for angiotensin II generation. The granulocyte-angiotensin system does not require renin or converting enzyme and may function as a mobile effector pathway which modulates tissue blood flow and/or vascular permeability.