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)     

Development and fate of the secretory granules of juxtaglomerular epithelioid cells

Summary The development and fate of the secretory granules in murine, rat and human juxtaglomerular epithelioid cells were examined using ultrastructural and immunocytochemical methods. The formation of mature renin granules occurs by fusion of rhomboid protogranules followed by coalescence of their paracrystalline contents, and by the fusion of roundish juvenile granules having an amorphous internum. Protogranules with paracrystalline contents are prominent in animals with stimulated renin synthesis, indicating an overcharge in processing and/or packaging of the secretory product, renin, under these conditions. Various similarities between lysosomes/multivesicular bodies (MVBs) and juvenile renin granules have been observed. With the exception of small MVBs, no renin-negative organelles that could be regarded as lysosomes were found in epithelioid cells of mice and rats. Therefore, we suggest that renin granules are modified lysosomes. Immunocytochemical findings indicate that juvenile secretory granules of epithelioid cells represent the converting and activating compartment for prorenin. Endocytosed foreign tracers such as HRP or cationized ferritin are preferentially internalized by juvenile renin granules, which hence appear to be outstanding by their fusogeneity. Consequently, juvenile granules are probably responsible for the secretion of prorenin, and mature granules for that of active renin.These studies were supported by the German Research Foundation within the Forschergruppe Niere/Heidelberg     

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.     

Immunocytochemical localization of prorenin, renin, and cathepsins B, H, and L in juxtaglomerular cells of rat kidney

To examine the correlation of localization of prorenin, renin, and cathepsins B, H, and L, immunocytochemistry was applied to rat renal tissue, using a sequence-specific anti-body (anti-prorenin) that recognizes the COOH terminus of the rat renin prosegment. In serial semi-thin sections, immunodeposits for prorenin, renin, and cathepsins B, H, and L were localized in the same juxtaglomerular (JG) cells. Immunodeposits for renin were detected throughout the cytoplasm of the cells, whereas those for prorenin were detected in the perinuclear region. Immunoreactivity for cathepsin B was stronger than that for cathepsins H and L. By electron microscopy, prorenin was localized in small (immature) granules but not in large mature granules, whereas renin was localized mainly in mature granules. In serial thin sections, prorenin, renin, and cathepsin B were colocalized in the same immature granules containing heterogeneously dense material (intermediate granules). By double immunostaining, co-localization of renin with cathepsins B, H, or L was demonstrated in mature granules. The results suggest the possibility that processing of prorenin to renin occurs in immature granules of rat JG cells, and cathepsin B detected in JG cells may be a major candidate for the maturation of renin.     

Renin activation in juvenile secretory granules?

Summary In immunocytochemical experiments on human kidney tissue with an antiserum directed against the prosegment of renin, only juvenile granules were clearly labeled. As the concentration of renin increases from protogranules to more mature granules, while the concentration of its prosegment decreases to subthreshold levels, it is assumed that the cleavage of the prosegment, i.e. the activation of renin takes place in juvenile granules parallel to the condensation of the enzyme.These studies were supported by the German Research Foundation within the SFB 90 Cardiovasculäres System     

Renin activation in juvenile secretory granules? Immunocytochemical experiments with an antiserum directed against the prosegment of human renin

In immunocytochemical experiments on human kidney tissue with an antiserum directed against the prosegment of renin, only juvenile granules were clearly labeled. As the concentration of renin increases from protogranules to more mature granules, while the concentration of its prosegment decreases to subthreshold levels, it is assumed that the cleavage of the prosegment, i.e. the activation of renin, takes place in juvenile granules parallel to the condensation of the enzyme.     

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.     

Are the renin-containing granules of juxtaglomerular epithelioid cells modified lysosomes?

Summary Mature secretory granules of epithelioid cells — the so-called renin granules — exhibit certain properties, which in this particular combination are expressed only by lysosomes: Renin granules have autophagic capabilities; they react to the application of lipidosis-inducing, lysosomotropic substances by the gradual accumulation of polar lipids; all secretory granules of epithelioid cells contain acid phosphatase until maturity; and exogenous tracers reach renin granules without labeling the Golgi complex. Several functional implications can therefore be considered. Hydrolytic enzymes, constitutive elements of the granule matrix, might either cleave inactive prorenin to yield active renin within the granules or, by unspecific hydrolysis of renin, participate in the regulation of the overall quantity of secretory product. Autophagic phenomena, the involvement of renin granules in the traffic of exogenous tracers, and the build-up of polar lipids following experimental interference with lipid catabolism indicate a large turnover of membrane material in renin granules. They also suggest that cytoplasmic and extracellular fluid gains access to the granule content and may thus be involved there in the regulation of biochemical reactions by changing the intragranular milieu or via signal molecules.In addition to the lysosome-like properties of epithelioid cell secretory granules, the secretory product, renin, as a carboxyl protease, is structurally related to other acidic proteases. In the case of cathepsin D, even functional similarities exist.These studies were supported by the German Research Foundation within the SFB 90 Cardiovasculäres System.     

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.     

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.     

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].     

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.     

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)     

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.     

Efficient Production of Recombinant Human (Pro)renin Utilizing a Decahistidine Tag Attached at the C-Terminus

Human prorenin attached by a decahistidine tag at the C-terminus was produced in Chinese hamster ovary cells. The tagged protein secreted into the culture medium was in the inactive prorenin form, and was activated to mature renin by proteolytic removal of its prosegment by trypsin in the same manner as native prorenin. The tagged (pro)renin was efficiently purified by metal-chelate affinity chromatography. The enzymatic properties of mature renin carrying the tag were similar to native renin. These results indicate that the introduction of a decahistidine tag at the C-terminus does not interfere with either the correct folding of prorenin or the catalytic activity of mature renin.     

Renin and cathepsin B in human pituitary lactotroph cells. An ultrastructural study

Renin, prorenin and cathepsin B were localized in human lactotrophs using immunoelectron microscopic techniques. Renin and prorenin were found in numerous cytoplasmic granules. Cathepsin B, a lysosomal enzyme known to be able to activate prorenin into renin, was also present in cytoplasmic granules of lactotrophs. The co-localization of renin and prolactin in the same secretory granules was demonstrated by double immunolabelling. Renin and cathepsin B were co-localized in some granules by the same technique. These results suggest a local activation of renin in the secretory granules of lactotrophs and support the hypothesis of a possible autocrine action of the renin-angiotensin system on prolactin release.     

Efficient production of recombinant human (pro)renin utilizing a decahistidine tag attached at the C-terminus

Human prorenin attached by a decahistidine tag at the C-terminus was produced in Chinese hamster ovary cells. The tagged protein secreted into the culture medium was in the inactive prorenin form, and was activated to mature renin by proteolytic removal of its prosegment by trypsin in the same manner as native prorenin. The tagged (pro)renin was efficiently purified by metal-chelate affinity chromatography. The enzymatic properties of mature renin carrying the tag were similar to native renin. These results indicate that the introduction of a decahistidine tag at the C-terminus does not interfere with either the correct folding of prorenin or the catalytic activity of mature renin.     

Renin gene expression, biosynthesis, and cellular pathways of secretion

The molecular biology of the human renin gene is reviewed. This 12.5 kb gene contains 10 exons and 9 introns. In its 5' flanking region, major control elements are present. These include promoters and enhancers as well as regulatory elements. The combined action of these elements would result in tissue specific expression and regulation of the gene. In addition to the control at the gene expression level, renin is also regulated at the posttranslational and secretory levels. The translational product of renin mRNA is preprorenin, which is cotranslationally cleaved to prorenin, an inactive precursor of renin. The majority of new synthesized human prorenin is constitutively secreted. However, prorenin is also processed intracellularly to the mature single chain active renin which is stored in secretory granules. Active renin is released by a regulated mechanism which can be stimulated by cAMP and other secretagogues. Studies are under way to examine the responses of renin gene expression, biosynthesis and secretion to various physiological conditions.     

Renin and cathepsin B in human pituitary lactotroph cells

Summary Renin, prorenin and cathepsin B were localized in human lactotrophs using immunoelectron microscopic techniques. Renin and prorenin were found in numerous cytoplasmic granules. Cathepsin B, a lysosomal enzyme known to be able to activate prorenin into renin, was also present in cytoplasmic granules of lactotrophs. The co-localization of renin and prolactin in the same secretory granules was demonstrated by double immunolabelling. Renin and cathepsin B were co-localized in some granules by the same technique. These results suggest a local activation of renin in the secretory granules of lactotrophs and support the hypothesis of a possible autocrine action of the renin-angiotensin system on prolactin release.     

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.