A comparison of the substrate specificities of cathepsin D and pseudorenin

Cathepsin D, purified from hog spleen, releases angiotensin I from tetradecapeptide renin substrate and from protein renin substrates purified from hog and human plasma. However, the enzyme does not act on the naturally occurring renin substrate as it exists in plasma nor on purified substrate in the presence of plasma. Cathepsin D releases angiotensin I quantitatively from tetradecapeptide renin substrate and does not further degrade the angiotensin I on prolonged incubation. The pH optimum for cathepsin D prolonged incubation. The pH optimum for cathepsin D acting on tetradecapeptide renin substrate is 4.5, and there is very low activity above pH 7. These properties are very similar to those of pseudorenin, an angiotensin-forming enzyme originally isolated from human kidney, indicating that cathepsin D and pseudorenin may be identical.     

Isorenin, pseudorenin, cathepsin D and renin. A comparative enzymatic study of angiotensin-forming enzymes

1. Renin was purified 30 000-fold from rat kidneys by chromatography on DEAE-cellulose and SP-Sephadex, and by affinity chromatography on pepstatinyl-Sepharose. 2. The enzymatic properties of isorenin from rat brain, pseudorenin from hog spleen, cathepsin D from bovine spleen, and renin from rat kidneys were compared: Isorenin, pseudorenin and cathepsin D generate angiotensin from tetradecapeptide renin substrate with pH optima around 4.9, renin at 6.0. With sheep angiotensinogen as substrate, isorenin, pseudorenin and cathepsin D have similar pH profiles (pH optima at 3.9 and 5.5), in contrast to renin (pH optimum at 6.8). 3. The angiotensin-formation from tetradecapeptide by isorenin, pseudorenin and cathepsin D was inhibited by albumin, alpha-and beta-globulins. These 3 enzymes have acid protease activity at pH 3.2 with hemoglobin as the substrate. Renin is not inhibited by proteins and has no acid protease activity. 4. Renin generates angiotensin I from various angiotensinogens at least 100 000 times faster than isorenin, pseudorenin or cathepsin D, and 3000 000 times faster than isorenin when compared at pH 7.2 with rat angiotensinogen as substrate. 5. The 3 'non-renin' enzymes exhibit a high sensitivity to inhibition by pepstatin (Ki less than 5.10(-10) M), in contrast to renin (Ki approximately 6-10(-7) M), at pH 5.5. 6. It is concluded from the data that isorenin from rat brain and pseudorenin from hog spleen are closely related to, or identical with cathepsin D.     

Cathepsin D coexists with renin in the secretory granules of juxtaglomerular epithelioid cells

Mature juxtaglomerular epithelioid cell secretory granules of the rat exhibit both renin- and cathepsin D-like immunoreactivity. On the basis of the coexistence with renin at a pH which, according to previous experiments, is probably in the range of that in lysosomes, cathepsin D is suggested to be involved in the regulation of the granular renin stores available for secretion.     

Immunocytochemical localization of cathepsin B in rat anterior pituitary endocrine cells, with special reference to its co-localization with renin and prorenin in gonadotrophs

We examined by immunocytochemistry the localization of cathepsin B in endocrine cells of rat anterior pituitary lobe, using a monospecific antibody to cathepsin B. By light microscopy, granular immunodeposits for cathepsin B were detected in most endocrine cells of anterior pituitary lobe. Cells immunoreactive for luteinizing hormone (LH) were diffusely immunostained by anti-cathepsin B. By electron microscopy, immunogold particles for cathepsin B were localized in lysosomes of thyrotrophs, somatotrophs, and mammotrophs. In mammotrophs, immunogold particles for cathepsin B were also detected in crinophagic bodies. Double immunostaining co-localized immunogold particles for LH and cathepsin B in secretory granules of gonadotrophs. Immunocytochemistry was also applied to demonstrate localization of renin and prorenin in LH-producing gonadotrophs; immunogold particles for renin were co-localized with those for LH, cathepsin B, or prorenin in their secretory granules. Immunogold particles for prorenin were also co-localized with those for LH or cathepsin B in secretory granules, but prorenin-positive granules appeared less frequently than renin-positive granules. These results suggest that cathepsin B not only plays a role in the protein degradation in lysosomes of anterior pituitary endocrine cells but also participates in the activation of renin in gonadotrophs, as has been demonstrated in secretory granules of juxtaglomerular cells.     

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.     

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.     

Renin, a secretory glycoprotein, acquires phosphomannosyl residues

Renin is an aspartyl protease which is highly homologous to the lysosomal aspartyl protease cathepsin D. During its biosynthesis, cathepsin D acquires phosphomannosyl residues that enable it to bind to the mannose 6-phosphate (Man-6-P) receptor and to be targeted to lysosomes. The phosphorylation of lysosomal enzymes by UDP- GlcNAc:lysosomal enzyme N-acetylglucosaminylphosphotransferase (phosphotransferase) occurs by recognition of a protein domain that is thought to be present only on lysosomal enzymes. In order to determine whether renin, being structurally similar to cathepsin D, also acquires phosphomannosyl residues, human renin was expressed from cloned DNA in Xenopus oocytes and a mouse L cell line and its biosynthesis and posttranslational modifications were characterized. In Xenopus oocytes, the majority of the renin remained intracellular and underwent a proteolytic cleavage which removed the propiece. Most of the renin synthesized by oocytes was able to bind to a Man-6-P receptor affinity column (53%, 57%, and 90%, in different experiments), indicating the presence of phosphomannosyl residues. In the L cells, the majority of the renin was secreted but 5-6% of the renin molecules contained phosphomannosyl residues as demonstrated by binding of [35S]methionine- labeled renin to the Man-6-P receptor as well as direct analysis of [2- 3H]mannose-labeled oligosaccharides. Although the level of renin phosphorylation differed greatly between the two cell types examined, these results demonstrate that renin is recognized by the phosphotransferase and suggest that renin contains at least part of the lysosomal protein recognition domain.     

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.     

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.     

Renin-like immunoreactivity in human placenta and fetal membranes

Five antibodies that stained renin in the kidney were used to investigate the presence of renin in human placenta and fetal membranes. Despite a large number of experimental approaches to enhance penetration of the immunoglobulins, only two of them showed immunostaining in placenta and fetal membranes. Staining was found in placental syncytiotrophoblast, the amnionic epithelium overlying the placenta, and in glandular epithelial cells present in the decidua adhering to the fetal membranes. It was most consistent, however, in a small infiltrating cell type dispersed through the fetoplacental layers. The two antibodies that revealed immunostaining in all preparations showed high affinity cross-reactivity with cathepsin D. Among other, less plausible, explanations, this raises the possibility that the bulk of renin found in placenta and fetal membranes is not identical to renal renin, but may be cathepsin D or a substance related to both cathepsin D and renin.     

Separation of human renal renin and pseudorenin by affinity chromatography on hemoglobin-Sepharose-2B

Human renal renin (EC 3.4.99.19) and pseudorenin were easily separated in a single step by affinity chromatography on hemoglobin-Sepharose-2B. Renin and pseudorenin were monitored by their actions on crude and partially purified hog protein renin substrates at neutral and acidic pH and on synthetic labelled polymeric renin substrate. Under the conditions employed (0.1 M sodium acetate (pH 3.5)/1 M sodium chloride at 4 degrees C) renin does not bind to the affinity adsorbent while pseudorenin is effectively bound and can be eluted only after raising the pH to 6.5. Pseudorenin-free renin prepared by this method is devoid of proteolytic activity toward hemoglobin. The chromatographic behaviour of renal pseudorenin on hemoglobin-Sepharose-2B is similar to that of cathepsin D.     

Coexistence of renin and cathepsin B in epithelioid cell secretory granules

Summary Mature juxtaglomerular epithelioid cell secretory granules of the rat exhibit both renin- and cathepsin B-like immunoreactivity. On the basis of the coexistence with renin at a pH which, according to previous experiments, is probably in the range of that in lysosomes, cathepsin B is suggested to be involved in the activation of renin prior to secretion.These studies were supported by the German Research Foundation within the SFB 90 Cardiovasculäres System     

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.     

Cathepsin D coexists with renin in the secretory granules of juxtaglomerular epithelioid cells

Summary Mature juxtaglomerular epithelioid cell secretory granules of the rat exhibit both renin-and cathepsin D-like immunoreactivity. On the basis of the coexistence with renin at a pH which, according to previous experiments, is probably in the range of that in lysosomes, cathepsin D is suggested to be involved in the regulation of the granular renin stores available for secretion.These studies were supported by the German Research Foundation within the SFB 90 Cardiovasculäres System     

High-molecular-weight renin converting enzyme from dog kidney: detection and fractionation

Renal cortical high-molecular-weight renin (Mw:60,000) of the dog is a complex of renin (low-molecular-weight renin; Mw:40,000) and a renin binding protein. We detected an enzyme-like substance that catalyzes the conversion from high- into low-molecular-weight renin. When the renal cortical extract was added to the high-molecular-weight renin and the preparation incubated at 37 degrees C for 30 min, the high-molecular-weight renin was converted into the low-molecular-weight form. No such conversion occurred in the case of renal medullary extract. This converting substance was fractionated using concanavalin A Sepharose, 70% ammonium sulfate saturation and DEAE-cellulose chromatography. The converting activity was inhibited by potassium tetrathionate, N-ethylmaleimide and 5,5'-dithiobis-(2-nitrobenzoic acid). These events suggest that this substance is an enzyme possessing sulfhydryl moieties. However, a cathepsin B inhibitor leupeptin did not affect the activity. Accordingly, the high-molecular-weight renin converting enzyme, which is sensitive to sulfhydryl oxidation, may explain the mechanism of interconversion between high- and low-molecular-weight renin involving the oxidation-reduction of tissue sulfhydryl groups.     

A potent radiolabeled human renin inhibitor, [3H]SR42128: enzymatic, kinetic, and binding studies to renin and other aspartic proteases

The in vitro binding of [3H]SR42128 (Iva-Phe-Nle-Sta-Ala-Sta-Arg), a potent inhibitor of human renin activity, to purified human renin and a number of other aspartic proteases was examined. SR42128 was found to be a competitive inhibitor of human renin, with a Ki of 0.35 nM at pH 5.7 and 2.0 nM at pH 7.4; it was thus more effective at pH 5.7 than at pH 7.4. Scatchard analysis of the interaction binding of [3H]SR42128 to human renin indicated that binding was reversible and saturable at both pH 5.7 and pH 7.4. There was a single class of binding sites, and the KD was 0.9 nM at pH 5.7 and 1 nM at pH 7.4. The association rate was 10 times more rapid at pH 5.7 than at pH 7.4, but there was no difference between the rates of dissociation of the enzyme-inhibitor complex at the two pHs. The effect of pH on the binding of [3H]SR42128 to human renin, cathepsin D, pepsin, and gastricsin was also examined over the pH range 3-8. All the aspartic proteases had a high affinity for the inhibitor at low pH. However, at pH 7.4, [3H]SR42128 was bound only to human renin and to none of the other aspartic proteases. Competitive binding studies with [3H]SR42128 and a number of other inhibitors on human renin or cathepsin D were used to examine the relationships between structure and activity in these systems.(ABSTRACT TRUNCATED AT 250 WORDS)     

The selectivity of statine-based inhibitors against various human aspartic proteinases.

The interactions of five human enzymes (renin, pepsin, gastricsin, cathepsin D and cathepsin E) and the aspartic proteinase from Endothia parasitica with several series of synthetic inhibitors were examined. All of the inhibitors contained the dipeptide analogue statine or its phenylalanine or cyclohexylalanine homologues in the P1-P1' positions. The residues occupying the peripheral sub-sites (P4 to P3') were varied systematically and inhibitory constants were determined for the interactions with each of the proteinases. Inhibitors were elucidated that specifically inhibited human renin and did not affect any of the other human enzymes or the fungal proteinase. With suitable selection of residues to occupy individual sub-sites, effective inhibitors of specific human aspartic proteinases may now be designed.     

Pepsin-mediated processing of synthetic precursor-like sequence yields neurotensin-like peptide.

A 15 amino acid synthetic peptide, which spanned the dibasic cleavage site C-terminal to neurotensin (NT), in its 170-residue canine precursor, was synthesized by solid-phase methods. Using this substrate in combination with a radioimmunoassay specific for the C-terminal region of NT, a simple assay was developed to monitor protease-mediated cleavage of the Leu8-Lys9 bond in the substrate. Hog pepsin and the related enzymes, rhizopus pepsin, bovine cathepsin D, and mouse renin, were found to be effective in this assay, pepsin cleaving only this bond to liberate the NT-like sequence. The pH dependence of the reaction indicated that pepsin, cathepsin D, and renin exhibited significant activity at pH's characteristic for secretory vesicles (pH 5.5-6.5). In addition, pepsin and cathepsin D were shown to process the native precursor at pH's as high as 5.5. These results, although not proof, are consistent with the idea that endoproteases with pepsin-like specificity may be involved in the processing of the NT precursor in neural/endocrine cells.     

Multiple forms of immunoreactive renin in human pituitary tissue

Immunoreactive renin was demonstrated in pituitary tissues of postmortem human subjects with different diseases. The specific immunoreactive renin activity comprised the majority of the tissue renin-like activity (mean, 83%), indicating the absence of nonspecific actions of proteases such as cathepsin D. We used three pituitary specimens with high levels of the specific renin activity for further biochemical characterization of the enzyme. Small differences were found in the molecular mass (45 K, 42 K and 37 K), binding to concanavalin A-Sepharose, and isoelectric points (pI) (4.72, 4.78, 4.86, 5.06, 5.28 and 5.44). These results seem to be interpreted as evidence for the presence of specific renin in the human pituitary with microheterogeneity.     

Subcellular Localization in Rat Brain of Angiotensin I-Generating Endopeptidase Activity Distinct from Cathepsin D

Abstract: The generation of angiotensin I from the artificial renin substrate tetradecapeptide by proteolytic enzymes in rat brain tissue was studied. The involvement of endopeptidase activity in the enzymatical cleavage of the renin substrate was inferred from the simultaneous accumulation of both angiotensin I and the complementary tetrapeptide Leu-Val-Tyr-Ser on incubation of tetradecapeptide with rat brain tissue. This endopeptidase activity was active over a pH range of 3.5–7.5. In contrast, cathepsin D released angiotensin I from tetradecapeptide only at acidic pH. The angiotensin I accumulation on incubation of tetradecapeptide with brain endopeptidase activity was only partly inhibited in the presence of an excess of the carboxyl protease inhibitor N -acetyl pepstatin. Further, the brain endopeptidase activity displayed a subcellular localization different from that of acid protease activity. It is concluded that angiotensin I can be generated in the brain by soluble endopeptidases, which are distinct from cathepsin D.