Twists and turns in the search for the elusive renin processing enzyme: focus on “Cathepsin B is not the processing enzyme for mouse prorenin”
2010; American Physiological Society; Volume: 298; Issue: 5 Linguagem: Inglês
10.1152/ajpregu.00188.2010
ISSN1522-1490
AutoresKenneth W. Gross, R. Ariel Gómez, Curt D. Sigmund,
Tópico(s)Receptor Mechanisms and Signaling
ResumoEditorial FocusTwists and turns in the search for the elusive renin processing enzyme: focus on "Cathepsin B is not the processing enzyme for mouse prorenin"Kenneth W. Gross, R. Ariel Gomez, and Curt D. SigmundKenneth W. GrossMolecular and Cellular Biology, Roswell Park Cancer Institute, Buffalo, New York; , R. Ariel GomezUniversity of Virginia School of Medicine, Charlottesville, Virginia; and , and Curt D. SigmundUniversity of Iowa, Iowa City, IowaPublished Online:01 May 2010https://doi.org/10.1152/ajpregu.00188.2010This is the final version - click for previous versionMoreSectionsPDF (166 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInWeChat having worked for many years on relatively basic questions germane to the venerable renin-angiotensin system, we were musing with some fascination recently on the continuing evolution of our understanding of the system. In particular, we were taking note of the considerable amount of dogma, with varying levels of verification associated with the system, as well as the almost continuous infusion of remarkable new twists and turns that seem to mark the passing of time. We were also struck by the existence of a surprising number of unresolved core issues.Research from Reudelhuber's laboratory (13a) has a direct bearing on one of these core issues, a central question that has eluded a completely satisfactory explanation for some time: namely, the identity of the prorenin processing enzyme (PPE) that generates active renin in the juxtaglomerular cell of the kidney. This is not a trivial question. For, while there may be local activation of renin at various tissue-specific sites, the experimental evidence suggests that the preponderant source of systemically circulating active renin is the kidney. As the authors correctly point out, unequivocal identification of the PPE might thus provide a new pharmaceutical target to inhibit this critical rate-limiting step of the renin-angiotensin system, thus providing a potential novel therapy for hypertension and cardiovascular disease.Their article is of significance, not for its positive identification of a novel PPE, but rather, for its rigorous exclusion of a longtime favorite candidate for the PPE, cathepsin B, at least in the specific case of mice. Satisfactory resolution of the issue has been complicated by a number of factors, including the fact that multiple enzymes appear to be able to generate "active" renin in vitro, different NH2-terminal sequences have been identified for the presumptive mature renal renin of human, mouse, and rat origin, and the in vitro cell systems in hand are, for the most part, nonoptimal or nonrepresentative of the renal site in question.It was known for some time that a number of enzymes exhibited the capability of processing prorenin to active renin in vitro, e.g., cathepsins B, D, and G, tissue kallikrein, convertases, trypsin, mouse submandibular gland prorenin converting enzyme, plasmin, pepsin, and others (2, 6, 10, 14, 17, 18). However, there were issues of proteolysis causing degradation or issues regarding the colocalization of the enzymes with renin in vivo, which lead to uncertainty about their roles. Cathepsin B has enjoyed preferential, if not quite dogmatic, status as the PPE candidate of choice for a number of reasons. It was noted early on by Taugner and Hackenthal (24) that the secretory pathway in the renal juxtaglomerular cell of rats appeared to involve granules that had the characteristics of modified lysosomes. Not surprisingly, cathepsin B, along with a number of other lysosomal enzyme candidates, was shown to exhibit cellular and organelle colocalization with prorenin.A series of straightforward studies undertaken by Hsueh and colleagues were particularly persuasive. They purified human active renin and undertook amino terminal sequencing (4). The results indicated that prorenin appeared to be converted to renin through cleavage at the carboxyl end of a Lys-Arg dibasic amino acid doublet (residues 65–66 of preprorenin). Using a recombinant human prorenin as a substrate, they then went on to purify an enzymatic activity associated with a thiol protease from human kidney that accurately processed prorenin to renin in vitro, suggesting that a cysteine protease was the authentic renal PPE (23). Subsequent studies revealed that cathepsin B, of both renal and liver origin, correctly corresponded with enzymatic activity and that cathepsin B hydrolyzed the 43 amino acid prosegment of prorenin without further degrading renin (25). Importantly, cathepsin B exhibited colocalization with renin in juxtaglomerular cell secretory granules. Confirmation that cathepsin B was uniquely the juxtaglomerular PPE could have been demonstrated by showing that cathepsin B inhibitors prevented prorenin processing in vivo or in cultured juxtaglomerular cells. Whether the inhibition studies were performed is unclear.Neves et al. (18) showed that cotransfection of cathepsin B and human preprorenin expression vectors into secretory granule-containing rat GH4C1 cells resulted in enhanced generation of secretable active renin relative to the preprorenin vector alone. This suggests that cathepsin B could localize to the appropriate cellular compartment to effect correct processing in an intact cell. The ratios of active renin to prorenin secreted, however, were not particularly high, and they thus carefully pointed out that, while these results were consistent with such a role for cathepsin B, they did not constitute proof that cathepsin B was the bona fide PPE.Jutras and Reudelhuber (9) used scanning mutagenesis to identify the amino acids determining site selectivity of human prorenin cleavage by human cathepsin B in vitro. Their results suggested that the basic residue, lysine, at −2 (relative to the cleavage site) was required for cleavage in vitro, consistent with predictions of cathepsin B mechanism from activity on synthetic substrates.All of the above data are consistent with cathepsin B comprising the PPE, but a number of less satisfying observations have also been reported. In particular, amino terminal sequencing of active renins from other species revealed that the NH2 terminus of active renin does not appear to be necessarily conserved. Active renin 2 enzyme, a curious nonglycosylated renin derived from an evolutionarily recent gene duplication event unique to mice, as isolated from mouse submandibular gland, exhibits an NH2 terminus homologous to human renal renin (15). In contrast, the sequence of active mouse renin 1, as isolated from the As4.1 cell line (8), a putative model for mouse renal juxtaglomerular cells derived by transgene-targeted tumorigenesis, exhibits an NH2 terminus that lies seven amino acids downstream from the site reported for human kidney renin. Interestingly, this site is homologous to that observed for rat renal renin by Kim et al (11).The primary amino acid sequences for rat, human, and mouse renin 1 and 2 are shown in Fig. 1 with the presumptive cleavage sites specified. It is interesting to note that the dibasic amino acid doublet corresponding to residues 65–66 of human preprorenin (42–43 of prorenin) is evolutionarily conserved, although there is substitution of Lys-Lys in the case of the rat for the Lys-Arg found in mouse and human, and there are potentially significant amino acid substitutions evident in the immediate vicinity of the doublet. In particular, mouse renin 1 exhibits a proline for leucine substitution, while the rat sequence bears more similarity to the mouse renin 2 sequence with a serine doublet in place of the leucine threonine of human. As pointed out in the discussion by Mercure et al, (13a), one explanation might be that the initial cleavage in all three species is performed by cathepsin B but that the mouse and rat renins are subsequently trimmed by other enzymes to generate the observed termini. Alternatively, different processing enzymes might be involved in activating human renin vs. rat and mouse renins. A lack of evolutionary conservation for the processing of an enzyme that plays such a central role in physiology as renin is unsettling, or at least intellectually unappealing, however; and the conservation of the dibasic doublet, despite the adjacent sequence variation could be taken to argue against this radical notion.Fig. 1.Primary amino acid sequences for rat, human, and mouse renin 1 and 2, with the presumptive cleavage sites specified.Download figureDownload PowerPointConsistent with the possibility that an initial cleavage at the dibasic site is followed by "nibbling" is a report by Almeida et al. (1) who used an internally quenched fluorescent peptide assay to demonstrate that the species-specific cathepsin B's could appropriately cleave peptides mimicking the respective species-specific dibasic doublet processing sites for rat and human. At present, it is unclear how the less homologous substitution of proline, in the case of mouse renin 1, would affect cleavage at this site although there are statements in previous papers (12) suggesting three enzymes that have been found to be capable of activating human renin, cathepsin B, and the convertases PC1 and PC5, are incapable of activating mouse renin 1 and rat renin in the cell transfection assays employed. As a point of information, the convertases PC1 (2) and PC5 (14) have been shown to be capable of activating human renin and mouse renin 2, but it appears unclear that PC1 and PC5 exhibit appropriate cell specificity to fulfill the role of the renal PPE in humans. They have been proposed to potentially perform this role at other sites, e.g., adrenal, in humans. Again, however, there appears to be a disturbing lack of evolutionary conservation.In vivo release of renin from juxtaglomerular cells is thought to be mediated via two pathways (20). Processed active renin is secreted from dense modified lysosomal storage granules in response to physiological cues, while the inactive zymogen is constitutively released via clear vesicle fusion with the plasma membrane. Support that the secretory granules are, in fact, secretory lysosomes has been provided by studying the influence on granule morphology by genetic mutations such as the Beige mutation, which causes a lysosome secretory defect (7). In contrast, many of the cellular assays that have been described above have been performed in cultured cell lines that are characterized as having regulated secretory pathways characteristic of other tissue sites, e.g., rat pituitary GH4C1 cells (18) and mouse pituitary AtT20 cells (13). Renin release from juxtaglomerular cells is a highly regulated process (21). Renin release is stimulated by activation of the cAMP pathway and inhibited by elevation of intracellular Ca levels. It is unclear whether the cellular models that have been employed to study prorenin processing are genuinely representative of the processing/secretory pathway of juxtaglomerular cells. As pointed out by Jutras and Reudelhuber (9) in their cellular cotransfection studies of cathepsin B and human prorenin in AtT20 cells, it appears that cathepsin B is correctly sorted to lysosomes through modified mannose residues and does not appear to be cosecreted with renin from the regulated pathway. These observations raise the question of how cathepsin B could be mediating the processing if the two proteins are not coresident within the cells. It has been proposed that in the overexpression systems employed there may be sufficient activation of cathepsin B in early stages of the secretory process, where the proteins are coresident in the trans-Golgi and immature granules to achieve processing of prorenin before they segregate to their respective compartments.In contrast, there is a large body of evidence suggesting colocalization of renin and lysosomal enzymes in renal juxtaglomerular cells (24). Renin gene ablation studies in vivo have provided some interesting insights, suggesting that the trafficking and maturation processes studied in the in vitro cell culture systems may not be mirroring the intact kidney. The Mullins laboratory (22) differentially knocked out the Ren1d and Ren2d loci in a strain that harbors both loci. Ren1d gives rise to the glycosylated renin 1, whereas Ren2d gives rise to the atypical nonglycosylated renin 2. The Ren2 knockout resulted in no observable histomorphological alterations to juxtaglomerular cell morphology as monitored by electron microscopy (22), while ablating expression of Ren1d led to a change in immunostaining from a punctuate, abundant granular pattern to diffuse, weak cytoplasmic staining. Electron microscopy revealed a complete absence of dense granule formation in the Ren1d-deficient mice (3). The results suggest that the signals required for sorting to the regulated pathway of juxtaglomerular cells in vivo are restricted to the renin 1 protein and further suggest that renin 1 and renin 2 are secreted by distinct pathways in vivo. Parallel studies by Gomez's laboratory in Ren1d-Ren2d mice (19) and Fukamizu's laboratory in strains of mice harboring only the single Ren1c locus (26) exhibited a similar phenotype. In an elegant set of studies, Mullins et al. (16) went on to demonstrate that a bacterial artificial chromosome (BAC) harboring wild-type Ren1d and Ren2d could completely rescue granulation and other aspects of the phenotype in otherwise renin-deficient mice. On the contrary, a BAC harboring a Ren1d locus into which a Lac reporter cassette had been inserted, disrupting Ren transcription, was incapable of rescuing the granulation phenotype. Importantly, reporter expression clearly indicated expression in bona fide juxtaglomerular cells. These studies also demonstrated that overexpression of renin 2 could not compensate for the loss of renin 1 and indicate that an active Ren1 locus is essential and sufficient for normal morphology of the juxtaglomerular apparatus. They note that the salient difference between renin 1 and renin 2 lies in the three N-linked glycosylation sites that characterize the renin 1d and renin 1c sequences in contrast to the renin 2 sequence, and that human and rat renin also exhibit two of the three potential glycosylation sites. It is hypothesized that these may be the signal for trafficking and generation of dense granules, presumably via a modified lysosome pathway (5).The present study by Mercure et al. (13a) demonstrates by the most rigorous state-of-the-art means that cathepsin B is not supported to be the relevant PPE in vivo for generation of renin in the mouse. In view of the disparities observed between human and mouse renin 2 vs. rat and mouse renin 1 for in vitro processing by cathepsin B, it might be of interest to assess the effect of the cathepsin B knockout on a Ren1d knockout line where Ren2 is the only contributing locus, or a complete mouse Ren knockout harboring a human Ren transgene (or humanized renin-angiotensin system). In the end, it is ironic that it has taken so long to perform this critical test; in some respects it would appear to confirm that a negative result needs to be performed in a sexier context in order to see the light of day in the literature. We can thank the Reudlhuber lab for definitively reopening the search for the elusive PPE by reinforcing what is known and not known and opening our thoughts to other explanations and possibilities.DISCLOSURESNo conflicts of interest, financial, or otherwise, are declared by the author(s).REFERENCES1. Almeida PC , Oliveira V , Chagas JR , Meldal M , Juliano MA , Juliano L. Hydrolysis by cathepsin B of fluorescent peptides derived from human prorenin. Hypertension 35: 1278–1283, 2000.Crossref | PubMed | ISI | Google Scholar2. Benjannet S , Reudelhuber T , Mercure C , Rondeau N , Chretien M , Seidah NG. Proprotein conversion is determined by a multiplicity of factors including convertase processing, substrate specificity, and intracellular environment. Cell type-specific processing of human prorenin by the convertase PC1. J Biol Chem 267: 11417–11423, 1992.Crossref | PubMed | ISI | Google Scholar3. Clark AF , Sharp MG , Morley SD , Fleming S , Peters J , Mullins JJ. Renin-1 is essential for normal renal juxtaglomerular cell granulation and macula densa morphology. J Biol Chem 272: 18185–18190, 1997.Crossref | PubMed | ISI | Google Scholar4. Do YS , Shinagawa T , Tam H , Inagami T , Hsueh WA. Characterization of pure human renal renin. Evidence for a subunit structure. J Biol Chem 262: 1037–1043, 1987.Crossref | PubMed | ISI | Google Scholar5. Faust PL , Chirgwin JM , Kornfeld S. Renin, a secretory glycoprotein, acquires phosphomannosyl residues. J Cell Biol 105: 1947–1955, 1987.Crossref | PubMed | ISI | Google Scholar6. Hsueh WA , Baxter JD. Human prorenin. Hypertension 17: 469–477, 1991.Crossref | PubMed | ISI | Google Scholar7. Jensen BL , Rasch R , Nyengaard JR , Skott O. Giant renin secretory granules in beige mouse renal afferent arterioles. Cell Tissue Res 288: 399–406, 1997.Crossref | PubMed | ISI | Google Scholar8. Jones CA , Petrovic N , Novak EK , Swank RT , Sigmund CD , Gross KW. Biosynthesis of renin in mouse kidney tumor As4.1 cells. Eur J Biochem 243: 181–190, 1997.Crossref | PubMed | Google Scholar9. Jutras I , Reudelhuber TL. Prorenin processing by cathepsin B in vitro and in transfected cells. FEBS Lett 443: 48–52, 1999.Crossref | PubMed | ISI | Google Scholar10. Kikkawa Y , Yamanaka N , Tada J , Kanamori N , Tsumura K , Hosoi K. Prorenin processing and restricted endoproteolysis by mouse tissue kallikrein family enzymes (mK1, mK9, mK13, and mK22). Biochim Biophys Acta 1382: 55–64, 1998.Crossref | PubMed | ISI | Google Scholar11. Kim S , Hosoi M , Kikuchi N , Yamamoto K. Amino-terminal amino acid sequence and heterogeneity in glycosylation of rat renal renin. J Biol Chem 266: 7044–7050, 1991.Crossref | PubMed | ISI | Google Scholar12. Laframboise M , Reudelhuber TL , Jutras I , Brechler V , Seidah NG , Day R , Gross KW , Deschepper CF. Prorenin activation and prohormone convertases in the mouse As4.1 cell line. Kidney Int 51: 104–109, 1997.Crossref | PubMed | ISI | Google Scholar13. Landenheim RG , Seidah N , Lutfalla G , Rougeon F. Stalle and transient expression of mouse submaxillary gland renin cDNA in AtT20 cells: proteolytic processing and secretory pathways. FEBS Lett 245: 70–74, 1989.Crossref | PubMed | ISI | Google Scholar13a. Mercure C , Lacombe MJ , Khazaie K , Reudelhuber TL. Cathepsin B is not the processing enzyme for mouse prorenin. Am J Physiol Regul Integr Comp Physiol (February 17, 2010). doi:10.1152/ajpregu.00830.2009.Google Scholar14. Mercure C , Jutras I , Day R , Seidah NG , Reudelhuber TL. Prohormone convertase PC5 is a candidate processing enzyme for prorenin in the human adrenal cortex. Hypertension 28: 840–846, 1996.Crossref | PubMed | ISI | Google Scholar15. Misono KS , Chang JJ , Inagami T. Amino acid sequence of mouse submaxillary gland renin. Proc Natl Acad Sci USA 79: 4858–4862, 1982.Crossref | PubMed | ISI | Google Scholar16. Mullins LJ , Payne CM , Kotelevtseva N , Brooker G , Fleming S , Harris S , Mullins JJ. Granulation rescue and developmental marking of juxtaglomerular cells using "piggy-BAC" recombination of the mouse ren locus. J Biol Chem 275: 40378–40384, 2000.Crossref | PubMed | ISI | Google Scholar17. Nakayama K , Kim WS , Nakagawa T , Nagahama M , Murakami K. Substrate specificity of prorenin converting enzyme of mouse submandibular gland. Analysis using site-directed mutagenesis. J Biol Chem 265: 21027–21031, 1990.Crossref | PubMed | ISI | Google Scholar18. Neves FA , Duncan KG , Baxter JD. Cathepsin B is a prorenin processing enzyme. Hypertension 27: 514–517, 1996.Crossref | PubMed | ISI | Google Scholar19. Pentz ES , Lopez ML , Kim HS , Carretero O , Smithies O , Gomez RA. Ren1d and Ren2 cooperate to preserve homeostasis: evidence from mice expressing GFP in place of Ren1d. Physiol Genomics 6: 45–55, 2001.Link | ISI | Google Scholar20. Pratt RE , Carleton JE , Richie JP , Heusser C , Dzau VJ. Human renin biosynthesis and secretion in normal and ischemic kidneys. Proc Natl Acad Sci USA 84: 7837–7840, 1987.Crossref | PubMed | ISI | Google Scholar21. Schweda F , Friis U , Wagner C , Skott O , Kurtz A. Renin release. Physiology (Bethesda) 22: 310–319, 2007.Link | ISI | Google Scholar22. Sharp MG , Fettes D , Brooker G , Clark AF , Peters J , Fleming S , Mullins JJ. Targeted inactivation of the Ren-2 gene in mice. Hypertension 28: 1126–1131, 1996.Crossref | PubMed | ISI | Google Scholar23. Shinagawa T , Do YS , Baxter JD , Carilli C , Schilling J , Hsueh WA. Identification of an enzyme in human kidney that correctly processes prorenin. Proc Natl Acad Sci USA 87: 1927–1931, 1990.Crossref | PubMed | ISI | Google Scholar24. Taugner R , Hackenthal E. The Juxtaglomerular Apparatus. Berlin: Springer-Verlag, 1989.Crossref | Google Scholar25. Wang PH , Do YS , Macaulay L , Shinagawa T , Anderson PW , Baxter JD , Hsueh WA. Identification of renal cathepsin B as a human prorenin-processing enzyme. J Biol Chem 266: 12633–12638, 1991.Crossref | PubMed | ISI | Google Scholar26. Yanai K , Saito T , Kakinuma Y , Kon Y , Hirota K , Taniguchi-Yanai K , Nishijo N , Shigematsu Y , Horiguchi H , Kasuya Y , Sugiyama F , Yagami K , Murakami K , Fukamizu A. Renin-dependent cardiovascular functions and renin-independent blood-brain barrier functions revealed by renin-deficient mice. J Biol Chem 275: 5–8, 2000.Crossref | PubMed | ISI | Google ScholarAUTHOR NOTESAddress for reprint requests and other correspondence: K. W. Gross, Molecular and Cellular Biology, Elm and Carlton St., Buffalo, NY 14263 (e-mail: kenneth.gross@roswellpark.org). Download PDF Previous Back to Top Next FiguresReferencesRelatedInformation Cited ByBehind every smile there's teeth: Cathepsin B's function in health and disease with a kidney viewBiochimica et Biophysica Acta (BBA) - Molecular Cell Research, Vol. 1869, No. 4Transgenic Mice Overexpressing Human Alpha-1 Antitrypsin Exhibit Low Blood Pressure and Altered Epithelial Transport Mechanisms in the Inactive and Active Cycles22 September 2021 | Frontiers in Physiology, Vol. 12Protein Kinase G Is Involved in Acute but Not in Long-Term Regulation of Renin Secretion18 July 2019 | Frontiers in Pharmacology, Vol. 10Classical Renin‐Angiotensin System in Kidney Physiology7 August 2014Parallel regulation of renin and lysosomal integral membrane protein 2 in renin-producing cells: further evidence for a lysosomal nature of renin secretory vesicles11 December 2012 | Pflügers Archiv - European Journal of Physiology, Vol. 465, No. 6Regulation of renin secretion by renal juxtaglomerular cells26 June 2012 | Pflügers Archiv - European Journal of Physiology, Vol. 465, No. 1Pharmacological and genetic evidence that cathepsin B is not the physiological activator of rodent proreninBiological Chemistry, Vol. 391, No. 12 More from this issue > Volume 298Issue 5May 2010Pages R1209-R1211 Copyright & PermissionsCopyright © 2010 the American Physiological Societyhttps://doi.org/10.1152/ajpregu.00188.2010PubMed20237305History Published online 1 May 2010 Published in print 1 May 2010 Metrics
Referência(s)