Chromogenic and fluorogenic glycosylated and acetylglycosylated peptides as substrates for serine, thiol and aspartyl proteases.

We synthesized short chromogenic peptidyl-Arg-p-nitroanilides containing either (Galbeta)Ser or (Glcalpha,beta)Tyr at P2 or P3 sites as well as O-acetylated sugar moieties and studied their hydrolysis by bovine trypsin, papain, human tissue kallikrein and rat tonin. For comparison, the susceptibility to these enzymes of Acetyl-X-Arg-pNa and Acetyl-X-Phe-Arg-pNa series, in which X was Ala, Phe, Gln and Asn were examined. We also synthesized internally quenched fluorescent peptides with the amino acid sequence Phe8-His-Leu-Val-Ile-His-Asn14 of human angiotensinogen, in which [GlcNAcbeta]Asn was introduced before Phe8 and/or after His13 and ortho-aminobenzoic acid (Abz) and N-[2-, 4-dinitrophenyl]-ethylenediamine (EDDnp) were attached at N- and C-terminal ends as a donor/receptor fluorescent pair. These peptides were examined as substrates for human renin, human cathepsin D and porcine pepsin. The chromogenic substrates with hydrophilic sugar moiety increased their susceptibility to trypsin, tissue kallikrein and rat tonin. For papain, the effect of sugar depends on its position in the substrate, namely, at P3 it is unfavorable, in contrast to the P2 position that resulted in increasing affinity, as demonstrated by the higher inhibitory activity of Ac-(Gal3)Ser-Arg-pNa in comparison to Ac-Ser-Arg-pNa, and by the hydrolysis of Ac-(Glcalpha,beta)Tyr-Arg-pNa. On the other hand, the acetylation of sugar hydroxyl groups improved hydrolysis of the susceptible peptides to all enzymes, except tonin. The P'4 glycosylated peptide [Abz-F-H-L-V-I-H-(GIcNAcbeta)N-E-EDDnp], that corresponds to one of the natural glycosylation sites of angiotensinogen, was shown to be the only glycosylated substrate susceptible to human renin, and was hydrolysed with lower K(m) and higher k(cat) values than the same peptide without the sugar moiety. Human cathepsin D and porcine pepsin are more tolerant to substrate glycosylation, hydrolysing both the P'4 and P4 glycosylated substrates.     

Inhibition of human renin by synthetic peptides derived from its prosegment

The primary structure of human preprorenin has recently been determined from its cDNA sequence. It includes a 46-amino acid NH2-terminal prosegment. Six peptides corresponding to the entire prosegment (9-40), except for the NH2-terminal (1-8) and COOH-terminal (41-46) ends have been synthesized. These peptides were tested for their inhibitory effect on human plasma renin activity. Boc-Tyr-Thr-Thr-Phe-Lys-Arg-Ile-Phe-Leu-Lys-Arg-Met-Pro-OMe (where Boc represents t-butoxycarbonyl and OMe represents methoxy) (h Y(9-20) and its fragment Boc-Leu-Lys-Arg-Met-Pro-OMe h (16-20) were the most potent inhibitors with IC50 values of 2 X 10(-4) and 3 X 10(-4)M, respectively. Peptides located near the COOH-terminus were less inhibitory. The inhibitory capacity of h (16-20) was studied further on highly purified human renin acting on either pure human angiotensinogen or a synthetic human tetradecapeptide substrate. In both of these assays its inhibitory potency was about 10-fold greater than that found on plasma renin activity. Peptide h (16-20) was 3-6 times less potent in inhibiting human renin than its mouse counterpart m (15-19) was in inhibiting mouse renin. Kinetic studies carried out with h (16-20) showed a mixed type of inhibition. When human angiotensinogen was used as substrate, Ki and K'i values were 17.7 +/- 3.9 and 2.9 +/- 0.9 microM, respectively. These studies showed that human renin, like mouse renin and pepsin, can be inhibited by peptides derived from its prosegment. In addition, as in the case of pepsin, they suggest that the NH2-terminal part of the prosegment interacts more strongly with the active enzyme.     

The renin-angiotensin system in the perinatal period in rats

Plasma concentrations of renin, angiotensinogen, kininogen, total protein, and renal renin concentration were measured in rats before spontaneous birth, immediately after vaginal delivery, during the subsequent 48 h, as well as at the ages of 10, 20 and 80 days. Preterm rats had a plasma renin concentration about 15 times higher than adults, which increased further in 1 h-old vaginally-delivered rats. Thereafter renin fell to very low levels within 2 h, rose again during the first day and remained at 4 times the adults level until day 10. Renal renin content and concentration increased over the whole observation period, except for a slight fall of renin concentration in the first 3 h after birth. In pre- and full-terms rats, angiotensinogen concentration was only 20% that of adults, reaching even lower values immediately after delivery, due to excessive consumption by renin. Thereafter, angiotensinogen increased more than 10 fold within 48 h. Kininogen concentration in plasma was higher than in adults and stable up to the 10th postnatal day. We conclude that vaginal delivery is a strong stimulus for renin release, the resulting high concentration of renin being responsible both for the increased turnover of angiotensinogen and the subsequent inhibition of renin release. The cause and biological significance of the dramatic increase of angiotensinogen during the first 48 h of life remains obscure.     

Comparative enzymatic studies of human renin acting on pure natural or synthetic substrates

Some of the essential structural requirements for the enzymatic reaction of pure human renin acting on pure human and rat angiotensinogen and on their synthetic tetradecapeptide substrates were investigated. The five carboxy terminal amino acids of synthetic tetradecapeptides played a significant role in substrate recognition and/or hydrolysis by human renin. Kinetic constants Km, Kcat and kcat/Km of the various human renin assays were different according to the substrate used. The presence of either an asparagine or a threonine residue in the S'4 renin subsite did not affect significantly the kinetic constant values. A tyrosine residue, rather than a histidine residue, in the S'3 renin subsite gave the best synthetic substrate studied. When tyrosine residue was present in the S'2 renin subsite an important decrease in kcat was observed. Human angiotensinogen was hydrolysed by human renin with lower Km and kcat values than those measured with human and porcine synthetic substrates, suggesting that the 3-dimensional structure of human angiotensinogen plays a key role in the hydrolysis. This finding was supported by assays performed with rat angiotensinogen, which was cleared by human renin with the same kcat value as rat tetradecapeptide, but with a 49-fold lower Km. Between human and rat angiotensinogen a kcat/Km value of only 2-fold higher has been found in the renin assay using human substrate.     

Assignment by in situ hybridization of the angiotensinogen gene to chromosome band 1q4, the same region as the human renin gene

Summary A 1.8kb human cDNA probe for angiotensinogen (renin substrate) was used to determine the chromosomal location of the angiotensinogen gene by in situ hybridization. The results show that human chromosome region 1q4 contains the angiotensinogen gene. The human renin gene has also recently been assigned to the same band of chromosome 1. Thus, the angiotensinogen and renin genes are located in the same region of chromosome 1.     

Changes in activity of the renin-angiotensin system of the rat by induction of acute inflammation

Angiotensinogen is the precursor of biologically active peptide angiotensin II and its hepatic synthesis is increased by the induction of acute inflammation. Studies were carried out to know whether the rise in plasma angiotensinogen is actually involved in the activity of the renin-angiotensin system during acute inflammation. The plasma level of angiotensinogen in rats was increased to 2.5 times the normal level 16 h after the induction of acute inflammation by administration of lipopolysaccharide (LPS). The plasma renin concentration (PRC) was decreased to about 40% of the normal level concomitantly with a reduction of plasma renin activity (PRA) at 4 h after LPS administration. In contrast, 16 h after LPS injection, when plasma angiotensinogen showed a high level and PRC had recovered to the normal range, PRA was increased to 1.7 times the normal level. These results indicate that acute inflammation induced by LPS causes a biphasic change in the generation of angiotensin I, i.e., an early decrease depending upon the reduction of PRC and later increase depending upon elevation of the angiotensinogen concentration.     

Expression and characterization of recombinant human angiotensinogen in a heterologous eukaryotic cell line

Transfection of Chinese hamster ovary cells with an expression plasmid containing a full length human angiotensinogen cDNA has provided cell lines that secrete recombinant angiotensinogen in large quantities. This angiotensinogen is immunologically identical to plasma angiotensinogen and can be cleaved by human kidney renin (EC 3.4.23.15.). The peptide liberated by renin cleavage is immunologically identical to standard angiotensin I and shows a retention time on isocratic reversed-phase high-pressure liquid chromatography identical to that of standard angiotensin I. The heterogeneity of recombinant angiotensinogen on sodium dodecyl sulfate-polyacrylamide gel electrophoresis differs from that of plasma angiotensinogen. Treatment with endoglycosidases demonstrated that this difference is restricted to that of N-glycans and that N-glycans correspond to the quasi-totality of the carbohydrate content of both recombinant and plasma angiotensinogens. The development of a system capable of expressing human angiotensinogen cDNA in mammalian cells and the ability to obtain the corresponding angiotensinogen in large quantities will allow new studies on structure-function relationships of this protein.     

Rat submaxillary gland serine protease, tonin. Structure solution and refinement at 1.8 A resolution

Tonin is a mammalian serine protease that is capable of generating the vasoconstrictive agent, angiotensin II, directly from its precursor protein, angiotensinogen, a process that normally requires two enzymes, renin and angiotensin-converting enzyme. The X-ray crystallographic structure determination and refinement of tonin at 1.8 A resolution and the analysis of the resulting model are reported. The initial phases were obtained by the method of molecular replacement using as the search model the structure of bovine trypsin. The refined model of tonin consists of 227 amino acid residues out of the 235 in the complete molecule, 149 water molecules, and one zinc ion. The R-factor (R = sigma Fo - Fc/sigma Fo) is 0.196 for the 14,997 measured data between 8 and 1.8 A resolution with I greater than or equal to sigma (I). It is estimated that the overall root-mean-square error in the coordinates is about 0.3 A. The structure of tonin that has been determined is not in its active conformation, but one that has been perturbed by the binding of Zn2+ in the active site. Zn2+ was included in the buffer to aid the crystallization. Nevertheless, the structure of tonin that is described is for the most part similar to its native form as indicated by the close tertiary structural homology with kallikrein. The differences in the structures of the two enzymes are concentrated in several loop regions; these structural differences are probably responsible for the differences in their reactivities and specificities.     

Tonin as activator of renin

Tonin, a new serine protease, found in high concentration in rat submaxillary glands, leads to a significant activation of human amniotic fluid renin. The optimum pH on renin activation by tonin was found at pH 6.0. The reaction was time dependent and the initial rate of angiotensin I generation was constant up to 2 h. The two amniontic fluid samples studied showed an increase in renin activity after incubation with tonin to about five times the control level (268 to 1240 pmol x h-1 x mL-1 and 1490 to 7480 pmol x h-1 x mL-1).     

Measurement of inactive renin in normal, nephrectomized, and adrenalectomized rats.

In a new method for measurement of inactive rat plasma renin, the trypsin generated angiotensin I immunoreactive material, which was HPLC characterized as similar to tetradecapeptide renin substrate, is removed by a cation exchange resin before the renin incubation step. The method also corrects for trypsin destruction of endogenous angiotensinogen by the addition of exogenous angiotensinogen. When measured with this method inactive renin in rat plasma decreased after nephrectomy and increased after adrenalectomy. This is in accordance with findings in humans. A sexual dimorphism of prorenin (inactive renin) in rat plasma, similar to that reported in humans and mice, was demonstrated. Thus, inactive renin in the rat is no exception among species, and the rat might be a suitable animal model for further studies dealing with the physiology of prorenin in plasma and tissues.     

Correlation of 24-hour fluctuations in renin granules of juxtaglomerular cells and in renin and angiotensinogen in blood plasma of the rat

Summary Subcellular structures of juxtaglomerular (JG) cells in the rat kidney were morphometrically examined at six evenly spaced times over 24 h. Plasma renin activities and angiotensinogen concentrations were also measured at these times. The cell volumes were larger at 20.00 h and 04.00 h than at 00.00 h, whereas the nuclear volumes peaked at 20.00 h and 08.00 h, decreasing at 00.00 h and 16.00 h. The volume and surface densities of renin granules and their individual volumes and surface areas peaked at 16.00 h and 00.00 h, decreasing at 20.00 h and 08.00 h, whereas their numerical densities peaked at 20.00 h, decreasing at 12.00 h. The surface densities of the rough endoplasmic reticulum (rER) peaked at 20.00 h, decreasing at other times, except at 08.00 h, when rER volume and surface density were relatively high. The plasma renin activity was maximal at 20.00 h, whereas it was minimal at 08.00 h. The variation in plasma angiotensinogen concentrations was inversely correlated with that in plasma renin activities. These results suggest that JG cells actively synthesize and release renin during the dark period, especially at 20.00 h, whereas during the light period they gradually synthesize renin and produce the granules, most of which may be stored in the cells during this period.     

Renin activity determination using human plasma as a substrate

A method for human renin activity determination using human plasma as the substrate is described. Angiotensin I is generated by incubating renin with human plasma, which is available along with the commercial radioimmunoassay kit for angiotensin I. The method obviates the need to isolate and purify the substrate, human angiotensinogen, from human plasma. In addition, the assay is highly renin specific, sensitive, and convenient to use for the routine determination of active human renin during its isolation and purification from tissue extracts or from genetically engineered bacterial and nonbacterial expression systems.     

Physicochemical characteristics of human high molecular weight angiotensinogen

A high molecular weight angiotensinogen (Mr. 332,000 daltons) was prepared from plasma of pregnant women by gel filtration on Sephacryl S-300. The molecular weight was reduced to 81,000 by treatment with dithiothreitol (DTT), but not by treatment with SDS. DTT-treated high molecular weight (HMW) angiotensinogen was very similar to low molecular weight (LMW) angiotensinogen with respect to molecular weight, pH profile for angiotensin formation by human kidney renin, thermostability, Km value and isoelectric point. The antibody against LMW-angiotensinogen completely cross-reacted with HMW-angiotensinogen. These results suggest that HMW-angiotensinogen is probably a complex of LMW-angiotensinogen and other protein(s) which might be bound by disulfide bond.     

Identification of the active site of human renin with use of new fluorogenic peptides

Fluorogenic peptide substrates were synthesized in which amino acid residues corresponded to the C-terminal and the N-terminal sides of the site of human angiotensinogen cleaved by renin. Compared with the synthetic substrates of renin previously reported, these fluorogenic substrates had practical advantages in that their digestion products could be rapidly separated and sensitively detected by high-pressure liquid chromatography with a fluorescence detector. The recombinant human renin and human plasma split Leu-Val, which cleavage site is similar to that in human angiotensinogen. The kinetic parameters of the reaction of renin using these substrates were calculated. There seemed to be at least eight subsites in the active site of recombinant human renin, to judge from the enzyme-substrate binding characteristics. The two histidine residues (S5 and S'3) in the octapeptide His-Pro-Phe-His-Leu-Val-Ile-His were important in the enzyme action.     

Comparison of angiotensinogen and tetradecapeptide as substrates for human renin. Substrate dependence of the mode of inhibition of renin by a statine-containing hexapeptide

The kinetic properties of two different substrates for human renin, a synthetic tetradecapeptide and the natural substrate human angiotensinogen, have been compared. While the Vmax was similar for the two substrates, the Km values differed by a factor of 10, i.e., 11.7 +/- 0.7 microM (tetradecapeptide) and 1.0 +/- 0.1 microM (angiotensinogen). The mode of inhibition of renin by a statine (Sta)-containing hexapeptide, BW897C, that is a close structural analog of residues 8-13 of human angiotensinogen (Phe-His-Sta-Val-Ile-His-OMe), was determined for the two substrates. Competitive inhibition was observed when tetradecapeptide was the substrate (Ki = 2.0 +/- 0.2 microM), but a more complex mixed inhibition mode (Ki = 1.7 +/- 0.1 microM, Ki' = 3.0 +/- 0.23 microM) was found with angiotensinogen as substrate. This mixed inhibition probably results from the formation of an enzyme-inhibitor-substrate or enzyme-inhibitor-product complex and reflects the more extensive interactions that the protein angiotensinogen, as opposed to the small tetradecapeptide substrate, can make with renin. We conclude that the mixed inhibition observed when angiotensinogen is used as renin substrate could be important in the clinical application of renin inhibitors because it is less readily reversed by increased concentrations of substrate than is simple competitive inhibition.     

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.     

Characterization of a human angiotensinogen cleaved in its reactive center loop by a proteolytic activity from Chinese hamster ovary cells

Angiotensinogen, the renin (E.C. 3.4.23.15) substrate, belongs to the serpins superfamily and has been classified as a noninhibitory serpin. Using mass spectroscopy, angiotensinogen purified from Chinese hamster ovary cell supernatant shows a broad spectrum. The absence of protease inhibitors throughout the purification leads to an angiotensinogen cleaved within the reactive center loop. This cleavage does not affect the Ang I generation because kinetic parameters are similar to the values of the full-length angiotensinogen. Although cleavage is complete, the cleaved angiotensinogen migrates after deglycosylation on SDS-polyacrylamide gel electrophoresis as a doublet differing by 4 kDa. To test whether the circulating angiotensinogen is cleaved in the reactive center loop, it was purified from a pool of human plasma and was shown to be uncleaved. Its migration was obviously slower than of cleaved angiotensinogen but also consisted of two bands pointing to a so far unexplained residual heterogeneity. We then compared the heat-induced polymerization of full-length- and reactive center loop-cleaved angiotensinogens. Both monomers were able to aggregate, revealing a particular behavior of angiotensinogen distinct from that of reactive center loop-cleaved serpins. Lacking the three-dimensional structure of angiotensinogen, we propose and discuss a structural model of the serpin fold within the renin substrate.     

High Salt Intake Damages the Heart through Activation of Cardiac (Pro) Renin Receptors Even at an Early Stage of Hypertension

Objective

It has not yet been fully elucidated whether cardiac tissue levels of prorenin, renin and (P)RR are activated in hypertension with a high salt intake. We hypothesized that a high salt intake activates the cardiac tissue renin angiotensin system and prorenin-(pro)renin receptor system, and damages the heart at an early stage of hypertension.

Methods

Wistar Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) received regular (normal-salt diet, 0.9%) and high-salt (8.9%) chow for 6 weeks from 6 to 12 weeks of age. The systolic blood pressure, plasma renin activity (PRA) and plasma angiotensin II concentration were measured, and the protein expressions of prorenin, (pro)renin receptor, angiotensinogen, angiotensin II AT1 receptor, ERK1/2, TGF-β, p38MAPK and HSP27 in the myocardium were investigated. The cardiac function was assessed by echocardiography, and histological analysis of the myocardium was performed.

Results

The high-salt diet significantly increased the systolic blood pressure, and significantly reduced the PRA and plasma angiotensin II concentration both in the WKYs and SHRs. Cardiac expressions of prorenin, renin, (P)RR, angiotensinogen, angiotensin II AT1 receptor, phosphorylated (p)-ERK1/2, p-p38MAPK, TGF-β and p-HSP27 were significantly increased by the high salt diet both in the WKYs and SHRs. The high-salt diet significantly increased the interventricular septum thickness and cardiomyocyte size, and accelerated cardiac interstitial and perivascular fibrosis both in the WKYs and SHRs. On the other hand, dilatation of left ventricular end-diastolic dimension and impairment of left ventricular fractional shortening was shown only in salt loaded SHRs.

Conclusion

The high-salt diet markedly accelerated cardiac damage through the stimulation of cardiac (P)RR and angiotensin II AT1 receptor by increasing tissue prorenin, renin and angiotensinogen and the activation of ERK1/2, TGF-β, p38MAPK and HSP27 under higher blood pressure.     

Role of the pituitary in the effects of tonin on adrenal secretion of the rat

Chronically catheterized conscious rats were infused intravenously with tonin at 2.4 and 12 micrograms x kg-1 x min-1 for 2 h. Plasma aldosterone concentration (PAC) at the end of the experiment was 11.2 +/- 2.4 ng% in controls, 8.5 +/- 2.8 ng% in rats infused with tonin at the lower rate, and 26.2 +/- 3.6 ng% (p less than 0.01 vs. controls) in rats infused at the higher rate. Plasma corticosterone (PC) was significantly higher (p less than 0.05) in the group infused at the high rate while plasma renin activity (PRA) was significantly reduced in this group of rats. Plasma angiotensin II (AII) concentration was similar in all three groups. PAC was elevated after tonin infusion in the presence of AII blockade. PAC in conscious sodium-depleted rats infused with tonin was not significantly changed, but PRA was significantly reduced (p less than 0.01). In chronically hypophysectomized rats, PAC remained unchanged by tonin infusion. The failure of tonin to stimulate aldosterone in hypophysectomized animals indicates a role of a pituitary hormone (probably ACTH) in the effect of tonin on adrenal secretion.     

N-Terminal amino acid sequence of rat tonin: homology with serine proteases.

In this paper, we present the amino-terminal sequence of rat tonin, an endopeptidase responsible for the conversion of angiotensinogen, the tetradecapeptide renin substrate, or angiotensin I to angiotensin II. It is shown that isoleucine and proline occupy the amino- and carboxy-terminal residues respectively. The N-terminal sequence analysis permitted the identification of 34 out of the first 40 residues of the single polypeptide chain composed of 272 amino acids. These results showed an extensive homology with the sequence of many serine proteases of the trypsin-chymotrypsin family. This information, coupled with the slow inhibition of tonin by diisopropylfluorophosphate, classified this enzyme as a selective endopeptidase of the active serine protease family.