Identification of renal cathepsin B as a human prorenin-processing enzyme

Prorenin, the inactive biosynthetic precursor of renin, is proteolytically cleaved in the renal juxtaglomerular cells to renin. The activity of renin is rate-limiting for generation of angiotensin II in the circulation. We identified a renal thiol protease which activates and accurately cleaves the 43-amino acid prosegment of human recombinant prorenin. In the current studies, 6.5 mg of this protease was purified from human renal cortex using a three-step procedure dependent upon Leu-Leu-arginyl affinity chromatography. This represented an overall 766-fold purification and resulted in three protein bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of molecular weights 30,000, 25,000, and 24,000. All three bands cross-reacted with an anti-human liver cathepsin B antibody upon immunoblot analysis; electrolution of each band and amino-terminal sequence analysis confirmed that the Mr 30,000 protein was mature cathepsin B and the Mr 25,000 and 24,000 bands were cathepsin B subunits. The pH optimum for the hydrolysis of pure human recombinant prorenin by pure renal cathepsin B was 6, and the Michaelis-Menten constant, Km, of the reaction was 1.4 x 10(-9) M. Immunostaining of human kidney using a sheep anti-human cathepsin B antibody demonstrated the presence of cathepsin B in the juxtaglomerular areas of the kidney, as well as in the renal proximal tubules. Electron microscopic immunohistochemistry using the same antibody demonstrated cathepsin B in dense secretory granules of the juxtaglomerular cells. Renin was also shown to be present in these granules. This study provides both biochemical and morphological evidence that renal cathepsin B is a human prorenin-processing enzyme.     

The molecular biology of human renin and its gene

The molecular biology of renin, prorenin, and the renin gene have been studied. A tissue-specific pattern of expression was found in rat and human tissues. In the human placenta, the transfected and endogenous renin promoters are active, and renin mRNA levels and transfected promoter activity are increased by a calcium ionophore plus cAMP. Cultured pituitary AtT-20 cells transfected with a preprorenin expression vector mimick renal renin release by converting prorenin to renin and releasing renin in response to 8Br-cAMP. Studies with mutant renin genes suggest that the body of renin directs renin to the regulated secretory pathway, and renin glycosylation affects its trafficking. Chinese hamster ovary cells were used to produce recombinant prorenin. Infused prorenin was not converted to renin in monkeys. Renin crystals were used to determine its three-dimensional structure. Renin resembles other aspartyl proteases in the active site and core, but it differs in other regions that probably explain renin's unique substrate specificity. Based on structural and mutational analysis, a model for human prorenin was built that suggests lysine -2 of the prosegment interacts with active site aspartate residues, and that the prosegment inactivation of renin is stabilized by binding of an amino terminal beta strand into a groove on renin.     

Identification of plasma inactive renin as prorenin with a site-directed antibody

A sequence-specific antibody that recognizes a portion of the prosegment of human renin precursor was raised and used to provide direct evidence that plasma inactive renin contains the prosequence of renal renin and is therefore probably prorenin rather than an inactivated form of previously active renin. The information may help not only to resolve a major controversy concerning the nature of inactive renin in human plasma but also to elucidate its exact physiological role.     

Human prorenin structure sheds light on a novel mechanism of its autoinhibition and on its non-proteolytic activation by the (pro)renin receptor

Antibodies and prorenin mutants have long been used to structurally characterize prorenin, the inactive proenzyme form of renin. They were designed on the basis of homology models built using other aspartyl protease proenzyme structures since no structure was available for prorenin. Here, we present the first X-ray structure of a prorenin. The current structure of prorenin reveals that, in this zymogene, the active site of renin is blocked by the N-terminal residues of the mature version of the renin molecule, which are, in turn, covered by an Ω-shaped prosegment. This prevents access of substrates to the active site. The departure of the prosegment on activation induces an important global conformational change in the mature renin molecule with respect to prorenin: similar to other related enzymes such as pepsin or gastricsin, the segment that constitutes the N-terminal β-strand in renin is displaced from the renin active site by about 180° straight into the position that corresponds to the N-terminal β-strand of the prorenin prosegment. This way, the renin active site will become completely exposed and capable of carrying out its catalytic functions. A unique inactivation mechanism is also revealed, which does not make use of a lysine against the catalytic aspartates, probably in order to facilitate pH-independent activation [e.g., by the (pro)renin receptor].     

Reversible cryoactivation of recombinant human prorenin.

Cleavage of prorenin's prosegment causes irreversible formation of renin. In contrast, renin activity is reversibly exposed when prorenin is acidified to pH 3.3. Nonetheless, acidification of plasma results in irreversible activation of prorenin, because endogenous proteases cleave the prosegment of acid-activated prorenin. Chilling of plasma results in irreversible cryoactivation of prorenin. In this study we investigated whether cryoactivation of purified prorenin is reversible. The intrinsic renin activity of recombinant human prorenin was measured by an enzyme kinetic assay using partially purified human angiotensinogen as substrate. Results are expressed as a percent (mean +/- S.E.) of the maximal activity exposed after limited proteolysis by trypsin. The intrinsic renin activity of two pools (0.3 and 0.06 Goldblatt units/ml) was 1.5% +/- 0.3 and 1.2% +/- 0.6 at 37 degrees C. Activity increased to 19% +/- 0.3 and 26% +/- 0.5 after incubation at 0 degrees C and to 5.4% +/- 0.5 and 2.1% +/- 1.2 at room temperature. Cryoactivation did not occur in buffers containing more than 1 M NaCl. It took 8 min at 37 degrees C or 180 min at room temperature for cryoactivated prorenin to lose half of its intrinsic renin activity. It took 48 and 26 h, respectively, at 0 degree C for the two pools of prorenin at 37 degrees C to regain half of their maximum intrinsic activity at 0 degrees C. A direct immunoradiometric assay that detects active renin but not prorenin was able to detect cryoactivated prorenin. These results show that human prorenin can be reversibly cryoactivated in buffers of low ionic strength and has greater intrinsic activity at room temperature than at 37 degrees C.     

The fate of prorenin during granulopoiesis in epithelioid cells. Immunocytochemical experiments with antisera against renin and different portions of the renin prosegment

Comparative immunocytochemical experiments with antisera directed against renin and three synthetical peptides (Pro 1, Pro 2A and Pro 3) covering almost the entire span of human renin prosegment were performed on human kidney tissue. With anti-Pro 1, i.e. the antiserum which recognizes the NH2 terminus of human prorenin, no clear immunolabeling of juxtaglomerular epithelioid cell secretory granules could be obtained. It is therefore concluded that the corresponding portion of human prorenin may be cleaved off in the Golgi complex. After application of anti-Pro 3, the antiserum which recognizes the COOH terminus of the prosegment, only the juvenile secretory granules of epithelioid cells were consistently labeled, whereas, in contrast, some of the intermediate and most of the mature secretory granules were anti-Pro 3-negative. As the immunoreactivity of mature renin increased remarkably from protogranules to mature secretory granules, it is suggested that the cleavage of the COOH terminus of the prosegment, i.e. the activation of renin, takes place in juvenile and intermediate granules during condensation of the enzyme. The immunoreactivity of Pro 2A, corresponding to the middle portion of the prosegment, disappeared in a somewhat earlier stage of granulopoiesis than that of Pro 3. It is therefore concluded that the corresponding segmental cleavage, the result of which is a truncated version of intact prorenin, occurs in the protogranules of epithelioid cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

The dominant role of the prosegment of prorenin in determining the rate of activation by acid or trypsin: studies with molecular chimeras

Human prorenin activation by acid or trypsin is faster than rat prorenin by two orders of magnitude. No plausible mechanism exists to explain the difference. Two chimeric mutant prorenins were produced in CHO cells. A chimera, hPro/rRen, composed of human prorenin prosegment and rat active renin segment, was activated as fast as wild-type human prorenin at pH 3.3 and 25 degrees C or by trypsin (1 microg/ml). The other chimera, rPro/hRen, composed of rat prorenin prosegment and human active renin segment, was activated as slowly as wild-type rat prorenin at pH 3.3 and 25 degrees C or by trypsin (50 microg/ml). These results indicate that the rate of activation of prorenin is predominantly determined by the N-terminal pro-sequence. Plausible mechanisms are discussed.     

The fate of prorenin during granulopoiesis in epithelioid cells

Summary Comparative immunocytochemical experiments with antisera directed against renin and three synthetical peptides (Pro 1, Pro 2 A and Pro 3) covering almost the entire span of human renin prosegment were performed on human kidney tissue. With anti-Pro 1, i.e. the antiserum which recognizes the NH₂ terminus of human prorenin, no clear immunolabeling of juxtaglomerular epithelioid cell secretory granules could be obtained. It is therefore concluded that the corresponding portion of human prorenin may be cleaved off in the Golgi complex.After application of anti-Pro 3, the antiserum which recognizes the COOH terminus of the prosegment, only the juvenile secretory granules of epithelioid cells were consistently labeled, whereas, in contrast, some of the intermediate and most of the mature secretory granules were anti-Pro 3-negative. As the immunoreactivity of mature renin increased remarkably from protogranules to mature secretory granules, it is suggested that the cleavage of the COOH terminus of the prosegment, i.e. the activation of renin, takes place in juvenile and intermediate granules during condensation of the enzyme.The immunoreactivity of Pro 2A, corresponding to the middle portion of the prosegment, disappeared in a some-what earlier stage of granulopoiesis than that of Pro 3. It is therefore concluded that the corresponding segmental cleavage, the result of which is a truncated version of intact prorenin, occurs in the protogranules of epithelioid cells.The data presented are consistent with the assumption that the secretion of active renin takes place by the exocytosis of mature secretory granules, while the secretion of inactive renin, which is a truncated version of intact prorenin, is mediated by the exocytosis of juvenile and intermediate granules.These studies were supported by the German Research Foundation within the Forschergruppe Niere/Heidelberg     

Expression of a deletion mutant of the prosegment of human prorenin in Chinese hamster ovary cells

Expression plasmids encoding native human preporenin and a mutant deleted in its entire prosegment were transfected into Chinese hamster ovary cells. The cells transfected with the expression plasmid of native preporenin secreted exclusively inactive prorenin, while the cells transfected with the mutant secreted the active enzyme. The secreted amount of renin from the latter cells was much lower than that of prorenin from the former ones, although these two enzymes had little difference in specific activity after trypsin activation. These results suggest that the prosegment plays an important role in the secretory process of renin, although the fully active enzyme can be formed in its absence.     

Protein modeling of human prorenin using the molecular dynamics method

To study the activation-inactivation mechanism of the renin zymogen, prorenin, a tertiary structural model of human prorenin was constructed using computer graphics and molecular dynamics calculations, based on the pepsinogen structure. This prorenin model shows that the folded prosegment polypeptide can fit into the substrate binding cleft of the renin moiety. The three positively charged residues, Arg 10, Arg 15, and Arg 20, in the prosegment make salt bridges with Asp 225, Glu 331, and Asp 60, respectively, in renin. Arg 43, which is in the processing site, forms salt bridges with the catalytic residues of Asp 81 and Asp 269. These ionic interactions between the prosegment and the renin may contribute to keeping the prorenin structure as an inactive form.     

Refolding and activation of human prorenin expressed in Escherichia coli: application of recombinant human renin for inhibitor screening

Human prorenin was expressed in Escherichia coli as a fusion protein of thioredoxin. The chimeric protein, which accumulated insoluble inclusion bodies, was solubilized in 4 M guanidine-HCl and refolded by an arginine-detergent buffer system and by systematic dialysis. The refolded fusion prorenin was activated by trypsin. The antiserum against human kidney renin specifically inhibited the recombinant human renin activity. Using the recombinant human renin, we screened its inhibitory activity in fermented soybean paste (miso) and demonstrated that miso contained renin inhibitory activity derived from soybean. The IC(50) values for soybean and steamed soybean extracts were determined to be 1.9 and 1.6 mg/ml, respectively. This is the first demonstration of renin inhibitory activity in miso and soybean.     

Human placental chorionic renin: production, purification and characterization

Native human renin, produced from the culture of human chorionic trophoblasts, has been purified to homogeneity on a milligram scale using a five-step purification scheme. The chorion cells secrete 50-200 milliGoldblatt Units of trypsin-activatable prorenin per ml into the medium. The pro-enzyme is partially purified by ammonium sulfate fractionation and chromatographies on QAE-Sephadex and cibracon blue-agarose. Following conversion of prorenin to the active enzyme by porcine trypsin, the renin is purified to homogeneity by affinity chromatography and gel filtration. Chorionic prorenin has a molecular weight of 43,000; the active enzyme 40,000. Both proteins exist as a single polypeptide chain as determined by SDS-polyacrylamide gel electrophoresis under reducing conditions. The average specific activity of six different preparations was found to be 1072 Goldblatt Units/mg. The amino acid composition and N-terminal sequence of the active enzyme has been determined and is identical to the human kidney enzyme. Microheterogeneity of chorionic renin was demonstrated by isoelectrofocusing analysis. The physical characterization of chorionic renin is compared with that reported for the human kidney enzyme.     

High-affinity prorenin binding to cardiac man-6-P/IGF-II receptors precedes proteolytic activation to renin

Mannose-6-phosphate (man-6-P)/insulin-like growth factor-II (man-6-P/IgF-II) receptors are involved in the activation of recombinant human prorenin by cardiomyocytes. To investigate the kinetics of this process, the nature of activation, the existence of other prorenin receptors, and binding of native prorenin, neonatal rat cardiomyocytes were incubated with recombinant, renal, or amniotic fluid prorenin with or without man-6-P. Intact and activated prorenin were measured in cell lysates with prosegment- and renin-specific antibodies, respectively. The dissociation constant (K(d)) and maximum number of binding sites (B(max)) for prorenin binding to man-6-P/IGF-II receptors were 0.6 +/- 0.1 nM and 3,840 +/- 510 receptors/myocyte, respectively. The capacity for prorenin internalization was greater than 10 times B(max). Levels of internalized intact prorenin decreased rapidly (half-life = 5 +/- 3 min) indicating proteolytic prosegment removal. Prorenin subdivision into man-6-P-free and man-6-P-containing fractions revealed that only the latter was bound. Cells also bound and activated renal but not amniotic fluid prorenin. We concluded that cardiomyocytes display high-affinity binding of renal but not extrarenal prorenin exclusively via man-6-P/IGF-II receptors. Binding precedes internalization and proteolytic activation to renin thereby supporting the concept of cardiac angiotensin formation by renal prorenin.     

Pure human inactive renin. Evidence that native inactive renin is prorenin

To clarify contradicting observations on the identity of inactive renin and prorenin, inactive renin was completely purified from native human chorion laeve and the culture medium of human chorion cells. A 720,000-fold purification with 14% recovery was achieved from chorion laeve in 6 steps, including immunoaffinity chromatography on a monoclonal antibody to human renin coupled to Protein A-Sepharose CL-4B. A 3,100-fold purification with 40% recovery was achieved from chorion culture medium in 4 steps, including immunoaffinity chromatography. Inactive renin purified from the two different sources migrated as a single protein band with the same molecular weight of 47,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and consisted of multiple components that could be resolved by isoelectric focusing. Both had the same pI values which shifted downward upon activation by trypsin; however, relative peak heights were different between the two preparations. The purified inactive renin from chorion laeve was completely inactive and did not bind to pepstatin-aminohexyl-Sepharose; however, that from chorion culture medium was partially active and completely bound to the pepstatin gel, indicating that each molecule is partially activated. Trypsin-activated inactive renins from both sources were identical with human renal renin in terms of pH optimum and Km. Specific activities of trypsin-activated inactive renin from chorion laeve and chorion culture medium were 529 Goldblatt units/mg of protein and 449 Goldblatt units/mg of protein, respectively. Amino acid sequence analysis of both of the purified inactive renin preparations demonstrated a leucine residue at the amino terminus. The sequence of 11 additional amino acids was identical in both and agreed with that predicted from the base sequence of the renin gene. These findings indicate that preprorenin is converted to prorenin following removal of a 23-amino acid signal peptide and that the native inactive renin, whose amino acid sequence commences with Leu-Pro-Thr..., is prorenin.     

Observations on the renal processing and sorting of prorenin.

Human prorenin is the biosynthetic precursor of renin. In general, prorenin is enzymatically inactive until it is converted to renin. The kidney is the major source of renin in the circulation, and is also an important source of circulating prorenin. The mechanisms of prorenin sorting and processing to renin in the juxtaglomerular cell may be a determinant of renal renin production. Therefore, our studies have focused on renal enzymes involved in "limited proteolysis" of prorenin to renin and on the morphology of prorenin sorting in the human juxtaglomerular cell.     

Amino-terminal amino acid sequence and heterogeneity in glycosylation of rat renal renin

We isolated 7.4 mg of pure renin from 2 kg of rat kidneys using affinity chromatography on pepstatin-aminohexyl-Sepharose and an octapeptide renin inhibitor, H-77-Sepharose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that renin consists of two polypeptide chains linked by a disulfide bond, one of Mr = 36,000 (heavy chain) and the other of Mr = 3,000 (light chain). The amino-terminal 10-amino acid sequences of the heavy and the light chains were identical to the sequences beginning at Ser72 and Asp355, respectively, of the amino acid sequence of preprorenin deduced from the renin cDNA sequence. Amino acid sequencing of the carboxyl-terminal peptide of the heavy chain, generated by digestion with lysyl endopeptidase, showed that the carboxyl-terminal residue of the heavy chain is Phe. Thus, the propeptide of prorenin is cleaved after Thr71, followed by removal of two amino acids, Arg353 and Asn354, the result being formation of the heavy and light chains. Thus, the site of cleavage of rat prorenin is after a nonbasic amino acid, in contrast to the cleavage of the propeptide after a pair of basic amino acids in mouse submaxillary renin, human renal renin, and many secretory proteins. Treatment of renin with neuraminidase or glycopeptidase F had no apparent effect on the charge heterogeneity of renin. Glycosylation probably does not contribute to charge heterogeneity.     

Analysis of inactive renin by renin profragment monoclonal antibodies

Two peptides were synthesized, corresponding to the sequences (-19 to -7) and (-26 to -17) of the prorenin prosegment. Monoclonal antibodies were raised to these sequences and used to characterize human plasma inactive renin. Only anti (-19 to -7) reacted with inactive renin, as measured by direct assay or affinity chromatography. The data were used to evaluate two possible inactive renin stuctures: plasma inactive renin is a truncated prorenin lacking the prosegment N-terminal portion; its spatial conformation masks the N-terminal extremity, preventing interaction of this region with specific antibodies.     

In vitro biosynthesis of human renin and identification of plasma inactive renin as an activation intermediate

The biosynthesis and post-translational modifications, including proteolytic processing and core glycosylation, of the human renin precursor have been studied in vitro in a cell-free system. For this purpose, highly enriched renin mRNA was isolated from a renin-producing juxtaglomerular cell tumor and translated in rabbit reticulocyte lysate containing [35S]methionine in the presence or absence of dog pancreas microsomal membranes. Fluorographic analysis of the radioactive translation products, immunoprecipitated and then resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, revealed that the primary translation product, preprorenin (Mr = 45,000), is initially processed to glycosylated prorenin (Mr = 47,000) during or shortly after its sequestration into the lumen of the microsomal membranes. The vectorial translocation across the membrane was confirmed by the observation that the proform was resistant to digestion with trypsin while preprorenin was sensitive. Radiosequencing and the use of prorenin-specific antibodies established the cleavage points of the pre- and profragment and showed that the in vitro precursor of human renin contains a 23-residue signal peptide and a 43-residue prosegment. The post-translational modification which, despite the removal of signal peptide, resulted in an increase in apparent Mr, reflects the glycosylation as examined using Xenopus oocytes microinjected with renin mRNA in the presence of tunicamycin, an inhibitor of protein glycosylation. Four anti-peptide antibodies which specifically recognize the NH2 terminus (Pro 1), two middle parts (Pro 2A and Pro 2B), and COOH terminus (Pro 3) of the prosegment, respectively, have been raised and used to characterize plasma prorenin. Renin precursors (pre- and prorenin) synthesized in vitro or in the kidney reacted with these antibodies (anti-Pro 1, anti-Pro 2A, anti-Pro 2B, and anti-Pro 3). However, quite unexpectedly, human plasma prorenin was recognized only by anti-Pro 3, indicating that plasma prorenin is a truncated version of intact prorenin, which lacks a large portion of the NH2 terminus of the prosegment and may represent an activation intermediate. This somewhat surprising result may lead to a better understanding of the exact roles and activation mechanisms of plasma prorenin existing in a relatively large amount.     

Characterization of pure human renal renin. Evidence for a subunit structure

Renin was completely purified from human kidney cortex employing a rapid three-step procedure which included homogenization and ammonium sulfate precipitation, aminohexyl-pepstatin affinity chromatography, and affinity chromatography using a synthetic octapeptide renin inhibitor (H-77) with a reduced peptide bond (-CH2-NH- instead of -CO-NH-) between Leu5-Leu6, Three kg of cortex dissected from 10 kg of human cadaver kidney yielded 1.7 +/- 0.5 mg of protein (mean +/- S.E. for five procedures) with a specific activity of 1094 +/- 166 Goldblatt units/mg of protein and an overall recovery of 52 +/- 2%. Both gel filtration high performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed a molecular weight of 44,000, although Mr = 22,000 and 18,000 bands were also identified by SDS-PAGE. The pH optima with sheep angiotensinogen were 5.5 and 7.8 and the Km was 0.31 microM. With pure human substrate the pH optimum was 6.0 and the Km was 1.15 microM. Enzyme activity was inhibited by two different anti-human renal renin antibodies. Amino-terminal sequencing demonstrated a leucine residue at the 1-position. Sequencing of 15 additional amino acids agreed with that predicted from the gene sequence and indicated that prorenin is converted to renin following cleavage at the carboxyl end of two basic residues, Lys-2 Arg-1. As with SDS-PAGE analysis, high performance liquid chromatography in the presence of 6 M urea demonstrated Mr = 44,000, 22,000, and 18,000 bands. Immunoblot studies revealed that all of these bands cross-reacted with antihuman renin antibody. Amino-terminal sequencing indicated the Mm = 22,000 band is the amino terminus and the Mr = 18,000 band the carboxyl terminus of Mr = 44,000 renin. In the aqueous phase, these subunits bound to H-77 suggesting that they represent components of the active enzyme complex. Unlike mouse renin, there was no evidence of disulfide bonds. These results raise the question of whether human renin circulates as a subunit aggregation as well as a single chain protein. This may serve as a possible mechanism to regulate renin activity in plasma and tissues.     

Prorenins activation by an enzyme from rat plasma (PreR-Co)

The aim of the present research was to explore the capacity of PreR-Co to process prorenin purified from kidney and corpora lutea (CL) and to study its action on extrarenal tissues. The PreR-Co was obtained from plasma as a single electrophoretic band by (NH4)2SO4 precipitation, gel filtration, anti-rat albumin immunoaffinity, and ion-exchange chromatography. Prorenin free of renin was obtained after (NH4)2SO4 precipitation, gel filtration, and ion-exchange chromatography by a passage through an affinity gel of H-77 Sepharose. SDS-PAGE of supernatant and of acidic elution from gel, exhibited a single band of 43 kDa and 35 kDa, respectively; both recognized by the specific anti rat renin antibody. The isolated renin was not attacked by PreR-Co; on the contrary prorenin was completely activated. The product of PreR-Co-activated prorenin showed an analogous MW to that of renin and was recognized by the specific antibody. In addition to processing kidney prorenin, PreR-Co was able to cleave inactive renin from ovary, CL, uterus and adrenal gland homogenates. However, the amount of active renin generated from these tissues was lower than those produced by trypsin activation. PreR-Co is a good candidate for the role of the enzyme involved in tissues prorenin activation.