Mouse submaxillary renin has a protease activity and converts human plasma inactive prorenin to an active form

The activation of inactive prorenin by active renin was investigated. Inactive prorenin extensively purified from human plasma was activated by active renin which had been purified from mouse submaxillary glands by multiple chromatographic steps. The apparent lack of protease activity in renin was puzzling in view of the close similarity of its active site structure with that of acid proteases. After a series of affinity chromatographic steps designed to eliminate minute contaminants, renin was found to contain a very low but finite level of a neutral protease activity which was equivalent to 1/40,000 of that of cathepsin D tested by hemoglobinolytic activity. The protease activity was considered as intrinsic to renin since it co-purified with renin persistently at a constant ratio to the renin activity, was precipitated by a monoclonal antibody specific for renin, showed a neutral pH optimum of the enzyme activity in the same pH range as that of renin, and was inhibited by pepstatin. The neutral protease activity is likely to mediate the activation of inactive prorenin.     

Nonproteolytic "activation" of prorenin by active site-directed renin inhibitors as demonstrated by renin-specific monoclonal antibody.

Incubation of human plasma prorenin (PR), the enzymatically inactive precursor of renin (EC 3.4.23.15), with a number of nonpeptide high-affinity active site-directed renin inhibitors induces a conformational change in PR, which was detected by a monoclonal antibody that reacts with active renin but not with native inactive PR. This conformational change also occurred when inactive PR was activated during exposure to low pH. Nonproteolytically acid-activated PR, and inhibitor-"activated" PR, as well as native PR, were retained on a blue Sepharose column, in contrast to proteolytically activated PR. Kinetic analysis of the activation of plasma prorenin by renin inhibitor (INH) indicated that native plasma contains an open intermediary form of prorenin, PRoi, in which the active site is exposed and which is in rapid equilibrium with the inactive closed form, PRc. PRoi reacts with inhibitor to form a reversible complex, PRoi.INH, which undergoes a conformational change resulting in a tight complex of a modified open form of prorenin, PRo, and the inhibitor, PRoi.INH-->PRo.INH. The PRoi-to-PRo conversion leads to the expression of an epitope on the renin part of the molecule that is recognized by a renin-specific monoclonal antibody. Presumably, PRo corresponds to the enzymatically active form of PR that is formed during exposure to low pH. Thus, it seems that the propeptide of PR interacts with the renin part of the molecule not only at or near the enzyme's active site but also at some distance from the active site. Interference with the first interaction by renin inhibitor leads to destabilization of the propeptide, by which the second interaction is disrupted and the enzyme assumes its active conformation. The results of this study may provide a model for substrate-mediated prorenin activation and increase the likelihood that enzymatically active prorenin is formed in vivo.     

A targeting sequence for dense secretory granules resides in the active renin protein moiety of human preprorenin

Human renin plays an important role in blood pressure homeostasis and is secreted in a regulated manner from the juxtaglomerular apparatus of the kidney in response to various physiological stimuli. Many aspects of the regulated release of renin (including accurate processing of prorenin to renin, subcellular targeting of renin to dense secretory granules, and regulated release of active renin) can be reproduced in mouse pituitary AtT-20 cells transfected with a human preprorenin expression vector. Using protein engineering, we have attempted to define the roles of various structures in prorenin that affect its production and trafficking to dense core secretory granules, resulting in its activation and regulated secretion. Replacement of the native signal peptide of human preprorenin with that of a constitutively secreted protein (immunoglobulin M) had no apparent effect on either the constitutive secretion of prorenin or the regulated secretion of active renin in transfected AtT-20 cells. Removal of the pro segment resulted in a marked reduction in total renin secretion, but did not prevent renin from entering the regulated secretory pathway. Single or combined mutations in the two glycosylation sites of human renin did not prevent its regulated secretion; however, the complete elimination of glycosylation resulted in a significant increase in the ratio of renin/prorenin secreted by the transfected cells. Thus, these results suggest that 1) at least one of the sequences that target human renin to dense secretory granules lies within the protein moiety of active renin; 2) the presence of the pro segment is important for efficient prorenin and renin production; and 3) glycosylation can quantitatively affect the proportion of active renin secreted.     

Human renin is correctly processed and targeted to the regulated secretory pathway in mouse pituitary AtT-20 cells

Renin is formed by intracellular processing of prorenin and catalyzes the conversion of angiotensinogen to angiotensin I, the precursor to angiotensin II. Several tissues synthesize prorenin. However, in man, the kidney is the only known source of circulating renin, raising the possibility that the processing enzyme is unique to that tissue. We have transfected a gene that directs prorenin synthesis in pituitary AtT-20 cells, which are capable of processing other prohormones. The results demonstrate that transfected AtT-20 cells can secrete inactive prorenin, accurately process prorenin to active renin, and be stimulated to release active renin in response to a secretagogue. These data imply that cellular elements capable of directing the processing of prorenin to renin and its correct subcellular compartmentalization may be present in nonrenal cell types and that critical elements of the regulated release of renin that occur in the kidney can be reconstituted in cells in culture.     

Investigation of human and rat inactive renin in plasma and kidney.

Under an initial interval of immobilization stress in rats, reciprocal changes of plasma active and inactive renin were observed, suggesting activation of circulating inactive renin. Molecular weight (MW) studies revealed that this activation might proceed via a MW shift from inactive renin with MW of 50,000 to active renin of MW 43,000. In a later interval of stress, under stimulated renin secretion, a lower MW form (38,000) of active renin was released into the circulation. This MW is close to that of active renin (39,000) found in rat kidney renin granules. In renin granules, equilibrated in fractions of 1.6 and 1.7 mol/L sucrose in discontinuous density gradient, trypsin-activatable renin activity formed 36 and 16% of total activity, respectively. In humans, under acute bicycle exercise, a lower MW form (39,000) of active renin was released into the circulation, while the content of inactive renin with MW in the range of 51,000-58,000 and at 47,000 did not substantially change. There was a slight decrease in circulating inactive renin passing through the kidney. The data suggest that, at least in rats, in vivo pathways for activation of inactive renin might exist, other than that proceeding before secretion from renin granules. Under the conditions of increased renin secretion, a lower MW form of active renin is mainly released into the circulation in both rats and humans.     

Prorenin in plasma and kidney

Circulating prorenin is an enzymatically inactive form of renin, also present in kidney, which can be activated in vitro. Its biochemical properties and physiological behavior suggest that it may be a biosynthetic precursor of active renin. However, in contrast to typical prohormones, the normal plasma concentrations of prorenin are much higher than the active hormone. The purposes and functions of prorenin are unclear. It may have no further role after its secretion into the circulation. On the other hand, it may be a transport form of renin that can enter or exit cells more easily than the active form. It is also possible that the activity of the renin-angiotensin system may be regulated by the conversion of prorenin to renin in the kidney (which may be under beta-adrenergic control) or at other possible sites. Irreversible activation of prorenin appears to be a proteolytic process. In addition, acidification causes reversible activation, perhaps through a change in molecular conformation. Such reversible activation might occur in vivo by unknown mechanisms. Future studies are needed to define the biochemical processes by which increased physiological demand for renin is translated into the production of more active enzyme.     

Immunological evidence that inactive renin is prorenin

Antibody raised to a synthetic dodecapeptide, corresponding to the C-terminal portion of the human renin pro-segment, was tested for its ability to recognize highly purified human inactive or active (mature) renins; immune complexes were detected by precipitation with protein A-Sepharose. Serial antibody dilutions caused identical binding of renal or plasma inactive renins but failed to bind active renin. In contrast, antibody to active renin recognized both active and inactive forms. Reversible acid activation of inactive renin enhanced its binding to the anti-prorenin antibody, whereas irreversible trypsin activation significantly reduced binding. Binding was abolished following prolonged exposure to trypsin or if inactive renin was acidified prior to trypsin treatment. These results indicate that inactive renin shares immunochemical determinants with prorenin; they suggest that acidification alters the conformation of the pro-segment and that trypsin can convert the molecule both to fully mature renin and to intermediate form(s).     

A new form of renin in normal human plasma: “big renin” is a mixture of inactive prorenin and the new active high molecular weight renin

A new form of active renin was separated from inactive prorenin in normal human plasma by a new affinity chromatographic method on a column of Cibacron Blue F3GA-agarose. This active renin has a molecular weight of 54,000, considerably higher than the hitherto recognized active renin of 40,000 dalton in human plasma. The molecular weight of inactive prorenin was 56,000±2,000. Active renin produced from the inactive prorenin by trypsin or pepsin digestion or by acid treatment in $\text{in vitro}$ experiments showed a molecular weight of 54,000±2,000. Active renin with a molecular weight of 40,000 was not found in 6 samples of untreated plasma of normal human subjects nor was it formed by treatment with trypsin, pepsin, or acid pH. It is concluded that a large form of active renin (54,000 dalton) exists in normal human plasma which is distinct from a smaller form and that the activatable “big renin” is a mixture of this active renin and totally inactive prorenin. This explains the absence of molecular weight change during the activation of “big renin”.     

Two-step prorenin-renin conversion. Isolation of an intermediary form of activated prorenin

Prorenin is an inactive form of the aspartic protease renin. Like pepsinogen, it is activated at low pH. The kinetics of acid activation of prorenin were studied in human amniotic fluid and plasma and in preparations of purified prorenin isolated from amniotic fluid and plasma. Conversion of prorenin (pR) into active renin (R) appeared to be a two-step process involving the generation of an intermediary form of activated prorenin (pRa). The pR----pRa step is an acid-induced reversible change in the conformation of the molecule, and the pRa----R step is proteolytic. pRa----R conversion occurred in amniotic fluid at low pH by the action of an endogenous aspartic protease. In plasma pRa----R conversion occurs after restoration of pH to neutral and is caused by the serine protease plasma kallikrein. pRa----R conversion did not occur in purified preparations of prorenin. Thus, in contrast to pepsinogen, the acid-induced reversible conformational change is not followed by autocatalysis. pRa of amniotic fluid and plasma could be separated from R by affinity chromatography on Cibacron blue F3GA-agarose, and R but not pRa was detected by an immunoassay using monoclonal antibodies reacting with R and not with pR. The first-order rate constant for pR----pRa conversion depends on the protonation of a polar group (or groups) with pK approximately 3.4, the rate constant being proportional to the fraction of pR molecules that have this group protonated. This is analogous to the reversible acid-induced conformational change of pepsinogen that occurs before its proteolytic conversion into pepsin. kcat/Km for pRa----R conversion by plasmin and plasma kallikrein at pH 7.4 and 37 degrees C was 7.8 X 10(6) and 5.2 X 10(6) M-1 min-1, respectively, which was about 50-70 times greater than for pR----R conversion. The susceptibility of pRa to proteolytic attack is high enough for the intrinsic factor XII-kallikrein pathway to cause rapid pRa----R conversion at 37 degrees C even in whole blood with its abundance of serine protease inhibitors. Formation of pRa may occur in vivo in an acidic cellular compartment, such as exo- or endocytotic vesicles.     

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].     

Conversion between renin and high-molecular-weight renin in the dog.

Two forms of renin, one of mol.wt. 43,000 and the other 60,000, were found in the dog kidney. Conversion between the two forms of renin was reversible at neutral pH. Though the molecular weight of renin in kidney-cortex homogenate was 43,000, it was completely converted into high-molecular-weight renin in the presence of substances that react with thiol groups. On the contrary, stored renin in the granules was the form of normal size (mol. wt. 43,000) regardless of the absence or presence of such substances. The present experiments indicated that renin is stored in the granules as the form of normal size and might be converted into high-molecular-weight renin when it is released from the granules and attached to some substance in the soluble fraction of renal-cortical tissue.     

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.     

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.     

Studies on the isolation and properties of renin granules from the rat kidney cortex.

The present study was undertaken to isolate and investigate some physicochemical properties of renin granules from the rat kidney cortex. Two preparations of subcellular organelles were used: a primary-granule fraction, which allowed the properties of lysosomes to be compared simultaneously with those of renin granules, and a semi-purified preparation of the latter. The specific activity of renin in the primary-granule preparations was about 4-fold higher than in the original homogenate; that of the semi-purified renin-granule preparation was about 18-fold higher than in the homogenate, and consisted mainly of electron-dense granules but some mitochondria were also observed. Renin and acid phosphatase release from the primary-granule preparation was increased by lowering osmolality, by a low-molecular-weight solute (glucose) and by Triton X-100 or digitonin. Enzyme release was decreased by lowering the incubation temperature (4 degrees C) or the presence of CaCl2. Renin release from the partially purified granule preparation was not affected by cyclic AMP, cyclic GMP and ATP.     

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.     

Purification and characterization of human truncated prorenin.

Posttranslational processing of enzymatically inactive prorenin to an active form participates in the control of the activity of a key system involved in blood pressure regulation, growth, and other important functions. The issue is complicated because renin can be produced by a number of tissues throughout the body, in addition to the kidney, but the mechanism by which they process prorenin to renin is unknown and difficult to determine because of the small amounts of renin present. In the juxtaglomerular cell of the kidney, a 43 amino acid prosegment is cleaved from the amino terminus of prorenin to generate renin of molecular weight 44,000 [Do, Y. S., Shinagawa, T., Tam, H., Inagami, T., & Hsueh, W. A. (1987) J. Biol. Chem. 262, 1037-1043]. Using human uterine lining or a recombinant human prorenin system, we employed the same approach as that used in kidney, ammonium sulfate precipitation at pH 3.1 followed by pepstatin and H-77 affinity chromatography or gel filtration, to purify to homogeneity a 45,500-MW totally active renin. The specific activity of the active truncated prorenin was 850 Goldblatt units (GU)/mg of protein for chorion-decidua renin and 946 GU/mg of protein for recombinant renin, both similar to that reported for pure human renal renin. Both forms of renin cross-reacted with an antibody generated against 44,00-MW pure human renal renin and with an antibody generated against a peptide identical to the carboxy-terminal one-third of the prosegment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Low molecular weight renin as a storage form in renin granules of the dog

The molecular weight of renin extracted from isolated renin granules of the dog was estimated by gel filtration, using tetradecapeptide as substrate, and was approximately 43,000 daltons. Neither big renin nor big big renin was demonstrable. On the other hand, crude extract of kidney cortex showed angiotensin I generating enzymes other than 43,000 dalton form of renin, whose molecular weight were over 100,000 and around 70,000 daltons. They seemed nonspecific proteases, since they hydrolyzed tetradecapeptide but not plasma angiotensinogen. Therefore renin is stored in the renin granules as a low molecular weight form.     

Activation of urinary inactive kallikrein by an extract from the rat kidney cortex

Activation of purified urinary inactive kallikrein by an extract from the rat kidney cortex was investigated. The extract produced a dose-dependent activation of the inactive kallikrein and the optimum pH for this activation was 5.0. Marked depression of the activation was observed when the extract was pre-incubated with E-64, p-CMB and iodoacetate, but not with DFP, PMSF or pepstatin A. The molecular weight of the inactive kallikrein (Mr 44,000) was reduced to 38,000 by treatment with the extract, this molecular weight value being identical with that of urinary active kallikrein. These results indicate that the rat kidney cortex contains a protease catalyzing conversion of urinary inactive kallikrein into its active form, and that the protease has properties compatible with those of a thiol protease, but not of trypsin which has been used as a tool for the activation of urinary inactive kallikrein. The thiol protease is probably one of regulators of the kallikrein-kinin system in the kidney.     

Site-directed mutagenesis of human prorenin. Substitution of three arginine residues in the propeptide with glutamine residues yields active prorenin

Human prorenin is an inactive zymogen comprising 43 amino acid residues at the amino terminus of human renin. The aim of this work was to determine why prorenin is inactive at neutral pH. Eighteen different mutant prorenins, in which positively charged residues in the propeptide were substituted with either glutamine (Gln) or lysine (Lys) residues by site-directed mutagenesis, were expressed in COS-7 cells and characterized. By replacing each of the three arginine (Arg) residues (Arg10P, Arg15P, and Arg20P) with Gln residues, partially active prorenins were produced, which exhibited significant but not full renin activity without trypsin activation. The effect of double or triple amino acid substitutions on the appearance of active prorenin was cumulative, the activity reaching about 80% in a mutant in which all the three Arg residues were replaced by Gln residues. In contrast, mutant prorenins with Lys residues substituted for the Arg residues were inactive. These results clearly indicate that the positive charges of the three Arg residues are essential for maintenance of the human prorenin in an inactive form.     

Renin processing studied by immunogold localization of prorenin and renin in granular juxtaglomerular cells in mice treated with enalapril

Summary Immunogold techniques were used to investigate renin processing within granular juxtaglomerular cells following short-term (6 h and 1 day) and long-term (4 weeks) enalapril treatment in female BALB/c mice. In control animals, renin protein labelling was localized to all types of granules (proto-, polymorphous, intermediate and mature) and to transport vesicles, whilst prorenin labelling was found in all these sites except mature granules, confirming that active renin is localized to mature granules only. Following short-term enalapril treatment, the exocytosis of renin protein from mature granules was increased. Long-term enalapril treatment resulted in increased numbers of transport vesicles and all types of granules, consistent with increased synthesis and storage of renin. More large intermediate granules contained discrete regions labelled for prorenin. Renin protein was exocytosed from individual and multiple granules, whilst prorenin was exocytosed from protoand intermediate granules. It is concluded that under normal conditions prorenin is secreted constitutively by bulk flow from transport vesicles. On the other hand, active renin is secreted regulatively from mature granules. In conditions of intense stimulation (angiotensin-converting enzyme inhibition treatment), increased synthesis of prorenin leads to enhanced secretion of prorenin by both constitutive and regulative pathways. Under these conditions, the conversion of prorenin to active renin is increased, with increased secretion of active renin occurring in a regulative manner. Furthermore, the localization of prorenin to one discrete region of large intermediate granules leads us to conclude, that cleavage of the prosegment of renin occurs with the transition of intermediate to mature granules.