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• W2003442673 abstract "We investigated the mechanism for non-proteolytic activation of human prorenin using five kinds of antibodies. Each of the antigens, L1PPTDTTTFKRI11P, T7PFKRIFLKRMP17P, I11PFLKRMPSIRESLKER26P, M16PPSIRESLKER26P, and G27PVDMARLGPEWSQPM41P, was designed from the tertiary structure of predicted prorenin. These antibodies were labeled anti-01/06, anti-07/10, anti-11/26, anti-16/26, and anti-27/41, respectively, for their binding specificities. Inactive recombinant human prorenin (0.1 nM) bound to various concentrations of anti-01/06, anti-11/26, and anti-27/41 antibodies at 4 °C with equilibrium dissociation constants of 138, 41, and 22 nM, respectively. However, intact prorenin (0.1 nM) did not show significant binding to 200 nM anti-07/10 and anti-16/26 antibodies for 20 h. Ninety percent of prorenin (0.1 nM) was found to be non-proteolytically activated by incubation with anti-11/26 antibodies (200 nM) at 4 °C for 20 h. Prorenin was not active even under complex with either anti-01/06 or anti-27/41 antibodies. Prorenin was also reversibly activated at pH 3.3 and 4 °C for 25 h. The acid-activated prorenin bound to anti-07/10 and anti-16/26 antibodies as well as to anti-01/06, anti-11/15, and anti-27/41 antibodies at neutral pH and 4 °C in 2 h. Their dissociation constants were 13, 40, 8.6, 3.6, and 14 nM, respectively. The acid-activated prorenin was re-inactivated by incubation at pH 7.4 and 4 °C in 50 h. Anti-07/10 and anti-11/26 antibodies inhibited such re-inactivation at 25 °C by more than 90% and 50%, respectively, whereas other kinds of antibodies did not prevent the re-inactivation at 25 °C. These results indicate that prorenin has “gate” (T7PFKR10P) and “handle” (I11PFLKR15P) regions critical for its non-proteolytic activation. We investigated the mechanism for non-proteolytic activation of human prorenin using five kinds of antibodies. Each of the antigens, L1PPTDTTTFKRI11P, T7PFKRIFLKRMP17P, I11PFLKRMPSIRESLKER26P, M16PPSIRESLKER26P, and G27PVDMARLGPEWSQPM41P, was designed from the tertiary structure of predicted prorenin. These antibodies were labeled anti-01/06, anti-07/10, anti-11/26, anti-16/26, and anti-27/41, respectively, for their binding specificities. Inactive recombinant human prorenin (0.1 nM) bound to various concentrations of anti-01/06, anti-11/26, and anti-27/41 antibodies at 4 °C with equilibrium dissociation constants of 138, 41, and 22 nM, respectively. However, intact prorenin (0.1 nM) did not show significant binding to 200 nM anti-07/10 and anti-16/26 antibodies for 20 h. Ninety percent of prorenin (0.1 nM) was found to be non-proteolytically activated by incubation with anti-11/26 antibodies (200 nM) at 4 °C for 20 h. Prorenin was not active even under complex with either anti-01/06 or anti-27/41 antibodies. Prorenin was also reversibly activated at pH 3.3 and 4 °C for 25 h. The acid-activated prorenin bound to anti-07/10 and anti-16/26 antibodies as well as to anti-01/06, anti-11/15, and anti-27/41 antibodies at neutral pH and 4 °C in 2 h. Their dissociation constants were 13, 40, 8.6, 3.6, and 14 nM, respectively. The acid-activated prorenin was re-inactivated by incubation at pH 7.4 and 4 °C in 50 h. Anti-07/10 and anti-11/26 antibodies inhibited such re-inactivation at 25 °C by more than 90% and 50%, respectively, whereas other kinds of antibodies did not prevent the re-inactivation at 25 °C. These results indicate that prorenin has “gate” (T7PFKR10P) and “handle” (I11PFLKR15P) regions critical for its non-proteolytic activation. Prorenin is the inactive precursor of renin (EC 3.4.23.15), which is a key enzyme in the regulation of blood pressure and electrolyte balance. Prorenin has a prosegment with 43 residues attached to the N terminus of mature renin with 339–341 residues (1Fukamizu A. Nishi K. Cho T. Saitoh M. Nakayama K. Ohkubo H. Nakanishi S. Murakami K. J. Mol. Biol. 1988; 201: 443-450Crossref PubMed Scopus (83) Google Scholar, 2Inagami T. Hypertension. 1991; 18: 241-251Crossref PubMed Scopus (18) Google Scholar, 3Morris B.J. J. Hypertens. 1992; 10: 209-214Crossref PubMed Scopus (29) Google Scholar, 4Suzuki F. Tanaka N. Takeuchi K. Muramoto Y. Inagami T. Murakami K. Nakamura Y. Takahashi K. Aspartic Proteinases. Plenum Publishing Co., New York1995: 267-272Google Scholar). The prosegment has been considered to associate with mature renin to prevent interaction with angiotensinogen, its macromolecular renin substrate (5Baxter J.D. James M.N. Chu W.N. Duncan K. Haidar M.A. Carilli C.T. Reudelhuber T.L. Yale J. Biol. Med. 1989; 62: 493-501PubMed Google Scholar, 6Heinrikson R.L. Hui J. Zurcher-Neely H. Poorman R.A. Am. J. Hypertens. 1989; 2: 367-380Crossref PubMed Scopus (50) Google Scholar, 7Shiratori Y. Nakagawa S. Hori H. Murakami K. Umeyama H. J. Mol. Graphics. 1990; 8 (163–167): 150Crossref Scopus (15) Google Scholar). Prorenin does not proteolytically self-activate like pepsinogen, and its blood circulating level is 10 times higher than that of mature renin (8Leckie B.J. Birnie G. Carachi R. J. Clin. Endocrinol. Metab. 1994; 79: 1742-1746PubMed Google Scholar, 9Sealey J.E. Glorioso N. Itskovitz J. Laragh J.H. Am. J. Med. 1986; 81: 1041-1046Abstract Full Text PDF PubMed Scopus (78) Google Scholar, 10Derkx F.H. Schalekamp M.A. Clin. Exp. Hypertens. 1988; A10: 1213-1225Google Scholar). Some investigators have recently proposed that prorenin is a useful marker of diabetic microvascular complications and Wilms' tumor (11Luetsher J.A. Kraemer F.B. Wilson D.M Schwartz H.C. Bryer-Ash M. N. Engl. J. Med. 1985; 312: 1412-1417Crossref PubMed Scopus (296) Google Scholar, 12Naruse M. Wasada T. Naruse K. Yoshimoto T. Omori Y. Demura H. Endocr. J. 1995; 42: 225-233Crossref PubMed Scopus (15) Google Scholar, 13Schalekamp M.A.H. Clin. Investig. 1993; 71: S3-S6Crossref PubMed Scopus (23) Google Scholar). However, much information regarding prorenin is unclear or lacking. The intrinsic activation enzyme, the activation mechanism in vivo, and its physiological role and source in the circulation remain unknown. Prorenin has reportedly been activated in vitro by endopeptidases such as trypsin and cathepsin B (3Morris B.J. J. Hypertens. 1992; 10: 209-214Crossref PubMed Scopus (29) Google Scholar, 14Hsueh W.A. Baxter J.D. Hypertension. 1991; 17: 469-477Crossref PubMed Google Scholar, 15Hsueh W.A. Do Y.S. Wang P.H. Cell Biophys. 1991; 19: 63-71Crossref PubMed Scopus (3) Google Scholar) and has also been non-proteolytically activated under acidic pH and/or low temperature (17Sealey J.E. Laragh J.H. Circ. Res. 1975; 36–37: I-10-I-16Google Scholar, 18Derkx F.H. von Gool J.M. Wenting G.J. Verhoeven R.P. Man in't V. eld A.J. Schalekamp M.A. Lancet. 1976; 1: 496-499Abstract PubMed Scopus (46) Google Scholar, 19Leckie B.J. McGhee N.K. Nature. 1980; 288: 702-705Crossref PubMed Scopus (64) Google Scholar, 20Derkx F.H.M. Margaretha P.A. Schalekamp P.A. Schalekamp A.D.H. J. Biol. Chem. 1987; 262: 2472-2477Abstract Full Text PDF PubMed Google Scholar, 21Pitarresi T.M. Rubattu S. Heinrikson R. Sealey J. J. Biol. Chem. 1992; 267: 11753-11759Abstract Full Text PDF PubMed Google Scholar, 22Reudelhuber T.L. Brechler V. Jutras I. Mercure C. Methot D. Adv. Exp. Med. Biol. 1998; 436: 229-238Crossref PubMed Scopus (10) Google Scholar). We recently showed that specific antibodies to the prosegment (L1PPTDTTTFKRIFLKR15P) activated human prorenin non-proteolytically (23Suzuki F. Hatano Y. Nakagawa T. Terazawa K. Gotoh A. Nasir U.M. Ishida Y. Nakamura Y. Biosci. Biotechnol. Biochem. 1999; 63: 550-554Crossref PubMed Scopus (8) Google Scholar). More recently, a renin/prorenin receptor was found in several tissues with nonproteolytically activated renin as well as prorenin (24Nguyen G. Delarue F. Burckle C. Bouzhir L. Giller T. Sraer J.D. J. Clin. Invest. 2002; 109: 1417-1427Crossref PubMed Scopus (1175) Google Scholar). These non-proteolytic activations have generally been thought to arise from a conformational change of the prorenin molecule in vivo. The inactivation mechanism for prorenin has been reported using recombinant prorenins mutated at single to triple residues in the prosegment that formed ionic bonds (25Derkx F.H. Deinum J. Lipovski M. Verhaar M. Fischli W. Schalekamp M.A. J. Biol. Chem. 1992; 267: 22837-22842Abstract Full Text PDF PubMed Google Scholar, 26Yamauchi T. Nagahama M. Watanabe T. Ishizuka Y. Hori H. Murakami K. J. Biochem. 1990; 107: 27-31Crossref PubMed Scopus (25) Google Scholar, 27Mercure C. Thibault G. Lussier-Cacan S. Davignon J. Schiffrin E.L. Reudelhuber T.L. J. Biol. Chem. 1995; 270: 16355-16359Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) or a hydrophobic bond (27Mercure C. Thibault G. Lussier-Cacan S. Davignon J. Schiffrin E.L. Reudelhuber T.L. J. Biol. Chem. 1995; 270: 16355-16359Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) between the prosegment and mature renin. Advanced research on the role of the prosegment in the non-proteolytic activation of prorenin may provide clues to solving those problems. Recently, we found that the acid activation rate of rat prorenin was less than one-fifth of that of human prorenin (4Suzuki F. Tanaka N. Takeuchi K. Muramoto Y. Inagami T. Murakami K. Nakamura Y. Takahashi K. Aspartic Proteinases. Plenum Publishing Co., New York1995: 267-272Google Scholar), and the speed of this process was only dependent on the amino acid sequence in the prosegment with 43 amino acid residues (28Suzuki F. Nakagawa T. Kakidachi H. Murakami K. Inagami T. Nakamura Y. Biochem. Biophys. Res. Commun. 2000; 267: 577-580Crossref PubMed Scopus (26) Google Scholar). These results led to our working hypothesis that there was an essential region in the N-terminal side of the prosegment for non-proteolytic activation of prorenin. In this study, we propose a hypothesis that there are two key regions, “gate” and “handle,” in prorenin non-proteolytic activation using several kinds of prorenin-specific antibodies. Designation of Antigen Peptides—Antigen peptides in several regions of the prorenin prosegment were designed on the basis of the primary structure of the prosegment and the stereo structure of human prorenin predicted by the homology modeling method, as shown in Fig. 1. Preparations of Several Kinds of Anti-prorenin Prosegment Antibodies—Each of the anti-prosegment antisera was raised against a peptide, L1PPTDTTTFKRI11P-C, T7PFKRIFLKRMP17P-C, I11PFLKRMPSIRESLKER26P-C, M16PPSIRESLKER26P-C, or G27PVDMARLGPEWSQPM41P-C, conjugated with keyhole limpet hemocyanin in rabbits. Each of the peptides was also used for the titer test of the antiserum using a commercial kit (Vectastatin ABC-AP rabbit IgG kit, Vector Laboratories, Inc.) and the ligand of affinity column for purification of each of the antibodies. High titer antisera were obtained 6 weeks after the first immunization. Each of the affinity gels was prepared by conjugation of Biogel 102 (amine coupled gel; Bio-Rad) through a cysteine residue of antigen peptide L1PPTDTTTFKRI11P-C, T7PFKRIFLKRMP17P-C, I11P-FLKRMPSIRESLKER26P-C, M16PPSIRESLKER26P-C, or G27PVDMARLGPEWSQPM41P-C as a ligand. With these affinity columns, each of the antibodies was purified and stored at –80 °C until use. The concentration of each of the purified antibodies was calculated using an extinction coefficient of 1.35 at 1 mg/ml IgG and 280 nm (29Little J.R. Donahue H. Methods of Immunology and Immunochemistry. Academic Press, New York1967Google Scholar). Epitope Mapping of Each Purified Antibodies by a Set of Affinity Gels with Their Antigen Peptide as a Ligand—Each of the affinity gels (50 μl) was incubated with the 32 nm of the antibodies (25 μl) and protein A-horseradish peroxidase (25 μl; 1:30,000 dilution; Bio-Rad) for 2 h at 4 °C under gentle shaking. After centrifugation, the pellet was washed with 450 μl of phosphate-buffered saline three times and incubated with 100 μl of 55 mm 3,3′,5,5′-tetramethylbenzidine and 50 μl of 0.01% H2O2 for 10 min at 37 °C. The reaction was stopped by adding 100 μl of 2 m H2SO4. The absorbance of each of the final solutions was measured at 450 nm. The cross-reactivity of five kinds of antibodies to five antigen peptides was examined by comparison of their binding ability to these affinity gels under these assay conditions, as shown in Fig. 2. These antibodies were labeled anti-01/06, anti-07/10, anti-11/26, anti-16/26, and anti-27/41, respectively, by their binding specificities. Determination of Dissociation Constant of Anti-prorenin Antibodies· Prorenin Complexes—To separate prorenin bound to each of the antibodies from the unbound prorenin fraction, 50 μl of protein A-Sepharose CL-4B gel (Amersham Biosciences) was suspended for 1 min at 4 °Cin50 μl of the reaction mixture of prorenin (0.1 nm) at various concentrations (2, 6, 20, 60, and 200 nm as IgG) of each of the antibodies (anti-01/06, anti-07/10, anti-11/26, anti-16/26, and anti-27/41) for 20 h (for inactive prorenin) or 2 h (for acid-activated prorenin), after which the suspension was centrifuged. The renin activity in the supernatant containing unbound prorenin was measured after trypsin treatment. The percentage of prorenin bound to the IgG was defined in the following equation: B(%)={[Ab]/([Ab]+KD)}×100(Eq. 1) where B (%) equals the percentage of prorenin bound to IgG in the total amount of prorenin, [Ab] is the concentration (nm) of IgG, and KD is the dissociation constant of prorenin-IgG complex. Kinetic Analysis of Prorenin Activation by Anti-11/26 Antibodies— Prorenin was reported to make two inactive forms at neutral pH by Derkx et al. (25Derkx F.H. Deinum J. Lipovski M. Verhaar M. Fischli W. Schalekamp M.A. J. Biol. Chem. 1992; 267: 22837-22842Abstract Full Text PDF PubMed Google Scholar). One was a closed form, whereas the other was an intermediately open form inactive. In this study, we also assumed that anti-11/26 antibodies bound not to the closed form (Prenc), but to the intermediately open form inactive (Prenoi), and that prorenin was completely activated under the complex is shown in the following reaction sequence. Ka and Kb are defined as follows: Kb=[Prenoi]·[Ab]/[Prenoi·Ab](Eq. 2) Ka=[Prenc]/[Prenoi](Eq. 3) [Prenc], [Prenoi], [Ab], and [Prenoi·Ab] are the concentrations of Prenc, Prenoi, antibodies, and Prenoi·antibody complex. The renin activity obtained in this study is equivalent to [Prenoi·Ab]. The anticipated reaction leads to the following equation. −d[Pren]/dt=[Prenoi·Ab]·kmax(Eq. 4) [Pren] is defined as follows: [Pren]=[Prenc]+[Prenoi]+[Prenoi·Ab](Eq. 5) On the basis of Eqs. 2, 3, and 5, [Prenoi·Ab] is expressed as follows. [Prenoi·Ab]=[Ab]/Kb1+1/Ka+[Ab]/Kb·[Pren](Eq. 6) The solution of Eq. 4 is [PR]t=[Pren]oe−Akmaxt(Eq. 7) in which A=[Ab]/Kb1+1/Ka+[Ab]/Kb (Eq. 8) and [PR]t and [Pren]o are the concentrations of Pren at t = t and t = 0, respectively. From Eqs. 7 and 8, it follows that kobs=[Ab]/Kb1+1/Ka+[Ab]/Kb·kmax(Eq. 9) or 1kobs=Kb(1+1/Ka)kmax·1[Ab]+1kmax (Eq. 10) in which kobs is the observed first-order rate constant for Preno· Ab complex. By plotting 1/kobs on the y axis against 1/[Ab] on the x axis, a straight line is obtained intersecting the x axis at –1/[Kb(1 + 1/Ka)] and the y axis at 1/kmax. Assuming the KD obtained by the other method in this study (Eq. 1) is the same as Kb, Ka was calculated from this plot. Non-proteolytic Activation of Prorenin by Specific Antibodies—The prorenin preparation was stored at –80 °C before use and then incubated at 37 °C for 1 h to avoid cryo-activation of the prorenin. By this treatment, the inactive level of prorenin in each preparation was synchronized at less than 2% of its total potential activity obtained by the trypsin treatment. The prorenin preparation (0.1 nm prorenin) was incubated with 200 nm of each antibody (anti-01/06, anti-07/10, anti-11/26, anti-16/26, and anti-27/41), and then the renin activity in each aliquot was determined under the standard assay conditions. Non-proteolytic Activation of Prorenin at Acidic pH—The prorenin preparation was acidified for 25 h at pH 3.3 and 4 °C in the presence of 5 mm EDTA and 1 mg/ml bovine serum albumin treated thermally, after which the renin activity was determined by standard assay conditions. Prorenin was activated by 80% through this treatment and was used as the acid-activated prorenin in this study. Blunting Re-inactivation of Acid-activated Prorenin by Antibodies— The 0.1 nm acid-activated prorenin, which had an 80% trypsin-activated activity level, was incubated at pH 7.4 and 25 °C for 2, 25, and 50 h with 200 nm of each of the antibodies (anti-01/06, anti-07/10, anti-11/26, anti-16/26, and anti-27/41), and then the renin activity was measured in each aliquot under standard assay conditions. Proteolytic Activation of Prorenin by Trypsin—Next, 100 μl of the prorenin preparation was incubated with 5 μl of 1 mg/ml trypsin (Sigma) for 2 min at 25 °C. The trypsin action was stopped by adding 10 μl of 2 mg/ml soybean trypsin inhibitor (type I-S; Sigma). By this treatment, 0.1 nm human prorenin was activated to a plateau level. Determination of Prorenin Concentration—A conditioned medium of Chinese hamster ovary cells harboring human renin cDNA was used as a recombinant human prorenin source (30Poorman R.A. Palermo D.P. Post L.E. Murakami K. Kinner J.H. Smith C.W. Reardon I. Heinrikson R.L. Proteins. 1986; 1: 139-145Crossref PubMed Scopus (95) Google Scholar). This cultured medium was dialyzed against 0.1 m phosphate, pH 7.0, containing 10 mm EDTA and 1 mm diisopropylfluorophosphate. This dialysate was stored in 1 mg/ml bovine serum albumin at –80 °C as the prorenin preparation. The prorenin concentration was measured at 80 ng/ml by an enzyme-linked immunosorbent assay for human renin, as described previously after the trypsin treatment. Determination of Renin Activity—Prorenin activated by the antibodies, either by the acidic or trypsin treatment, was incubated with 0.8 μm recombinant sheep angiotensinogen in the presence of 10 mm diisopropylfluorophosphate and 10 mm EDTA at pH 6.5 and 37 °C for 20 min, and the Ang 1The abbreviation used is: Ang, angiotensin. I generated was assayed by Ang I enzyme-linked immunosorbent assay. The renin activity was expressed as the amount of Ang I generated for 1 h of incubation with 1 ml of the original prorenin preparation. Antibodies Binding to Inactive and Acid-activated Prorenin—Inactive prorenin (0.1 nM) bound to 2, 6, 20, 60, and 200 nM anti-01/06, anti-11/26, and anti-27/41 antibodies at 4 °C (Fig. 3). The equilibrium dissociation constants (KD) for these complexes were 138, 41, and 22 nM, respectively. The inactive prorenin insignificantly associated with anti-07/10 and anti-16/26 antibodies under the present study conditions (Fig. 3). The inactive recombinant prorenin was activated to have an 80% trypsin-activated activity level by incubation at pH 3.3 and 4 °C for 25 h. The acid-activated prorenin (0.1 nM) bound to 2, 6, 20, 60, and 200 nM anti-01/06, anti-07/10, anti-11/26, anti-16/26, and anti-27/41 antibodies, respectively (Fig. 4). The equilibrium dissociation constants (KD) for their complexes were 8.6, 13, 3.6, 40, and 14 nM, respectively. Non-proteolytic Activation of Prorenin by Specific Antibodies—Inactive prorenin (0.1 nM) was clearly activated by incubation with 2, 6, 20, 60, and 200 nM anti-11/26 antibodies at 4 °C for 20 h (Fig. 5). More than 90% of prorenin was activated by 200 nM of the antibodies. Prorenin was slightly activated up to the 20% level by the anti-01/06 antibodies. There was insignificant activation by the anti-27/41 antibodies, although inactive and acid-activated prorenins could easily bind to them. Inactive prorenin (0.1 nM) was incubated with anti-11/26 antibodies at 4 °C for 0, 1, 2, 3.5, 5, 8, 16, and 24 h to observe the time dependence for making a complex and activation of prorenin with 200 nM anti-11/26 antibodies (Fig. 6). Both levels increased at the same rate and reached 50% and 90% in 4 and 15 h, respectively. Inactive prorenin (0.1 nM) was incubated with anti-11/26 antibodies at 4 °C for 0, 1, 2, 3.5, 5, 8, 16, and 24 h to observe the time dependence and kinetics for activation of prorenin with 6, 20, 60, 200, and 600 nM anti-11/26 antibodies (Fig. 7A). The initial rate of the activation increased with the dose of the antibodies. From the x intercept and y intercept, kmax and Kb(1 + 1/Ka) were calculated at 0.25 h–1 and 60 nM, respectively (Fig. 7B). Blunting Re-inactivation of Acid-activated Prorenin by Specific Antibodies—More than 90% of the acid-activated prorenin was re-inactivated by incubation at pH 7.4 and 4 °C within 24 h (Fig. 8). To examine whether antibodies prepared in this study block such re-inactivation, renin activity was measured in the acid-activated prorenin preparation after incubation with anti-01/06, anti-07/10, anti-11/26, anti-16/26, and anti-27/41 antibodies for 2, 25, and 50 h. The anti-01/06, anti-16/26, and anti-27/41 antibodies did not prevent the re-inactivation, as shown in Fig. 8. The anti-11/26 antibodies inhibited the reinactivation by 50% in 50 h. The anti-07/10 antibodies sustained an activity level of more than 90%, even after 50 h of incubation. Prorenin has been considered to be activated in vivo proteolytically and/or non-proteolytically in many published reports (3Morris B.J. J. Hypertens. 1992; 10: 209-214Crossref PubMed Scopus (29) Google Scholar, 14Hsueh W.A. Baxter J.D. Hypertension. 1991; 17: 469-477Crossref PubMed Google Scholar, 15Hsueh W.A. Do Y.S. Wang P.H. Cell Biophys. 1991; 19: 63-71Crossref PubMed Scopus (3) Google Scholar, 17Sealey J.E. Laragh J.H. Circ. Res. 1975; 36–37: I-10-I-16Google Scholar, 18Derkx F.H. von Gool J.M. Wenting G.J. Verhoeven R.P. Man in't V. eld A.J. Schalekamp M.A. Lancet. 1976; 1: 496-499Abstract PubMed Scopus (46) Google Scholar, 19Leckie B.J. McGhee N.K. Nature. 1980; 288: 702-705Crossref PubMed Scopus (64) Google Scholar, 20Derkx F.H.M. Margaretha P.A. Schalekamp P.A. Schalekamp A.D.H. J. Biol. Chem. 1987; 262: 2472-2477Abstract Full Text PDF PubMed Google Scholar, 21Pitarresi T.M. Rubattu S. Heinrikson R. Sealey J. J. Biol. Chem. 1992; 267: 11753-11759Abstract Full Text PDF PubMed Google Scholar, 22Reudelhuber T.L. Brechler V. Jutras I. Mercure C. Methot D. Adv. Exp. Med. Biol. 1998; 436: 229-238Crossref PubMed Scopus (10) Google Scholar, 23Suzuki F. Hatano Y. Nakagawa T. Terazawa K. Gotoh A. Nasir U.M. Ishida Y. Nakamura Y. Biosci. Biotechnol. Biochem. 1999; 63: 550-554Crossref PubMed Scopus (8) Google Scholar, 24Nguyen G. Delarue F. Burckle C. Bouzhir L. Giller T. Sraer J.D. J. Clin. Invest. 2002; 109: 1417-1427Crossref PubMed Scopus (1175) Google Scholar, 25Derkx F.H. Deinum J. Lipovski M. Verhaar M. Fischli W. Schalekamp M.A. J. Biol. Chem. 1992; 267: 22837-22842Abstract Full Text PDF PubMed Google Scholar, 26Yamauchi T. Nagahama M. Watanabe T. Ishizuka Y. Hori H. Murakami K. J. Biochem. 1990; 107: 27-31Crossref PubMed Scopus (25) Google Scholar, 27Mercure C. Thibault G. Lussier-Cacan S. Davignon J. Schiffrin E.L. Reudelhuber T.L. J. Biol. Chem. 1995; 270: 16355-16359Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 28Suzuki F. Nakagawa T. Kakidachi H. Murakami K. Inagami T. Nakamura Y. Biochem. Biophys. Res. Commun. 2000; 267: 577-580Crossref PubMed Scopus (26) Google Scholar). In this study, we demonstrated in vitro the “gate” and “handle” regions in the prorenin molecule crucial for its non-proteolytic activation by a protein-protein interaction and proposed a possible mechanism for its non-proteolytic activation in vivo. Inactive prorenin could not bind to anti-07/10 and anti-16/26 antibodies but associated with anti-01/06, anti-11/26, and anti-27/41 antibodies (Fig. 3). However, acid-activated prorenin made complexes with all of them (Fig. 4). The affinity of prorenin to anti-01/06 and anti-11/26 antibodies significantly increased more than 10 times after the acidification, but no significant change was observed in its affinity to anti-27/41 antibodies (Figs. 3 and 4). It is well known that the acidification of prorenin locally changes its conformation to open an active site (17Sealey J.E. Laragh J.H. Circ. Res. 1975; 36–37: I-10-I-16Google Scholar, 18Derkx F.H. von Gool J.M. Wenting G.J. Verhoeven R.P. Man in't V. eld A.J. Schalekamp M.A. Lancet. 1976; 1: 496-499Abstract PubMed Scopus (46) Google Scholar, 19Leckie B.J. McGhee N.K. Nature. 1980; 288: 702-705Crossref PubMed Scopus (64) Google Scholar, 20Derkx F.H.M. Margaretha P.A. Schalekamp P.A. Schalekamp A.D.H. J. Biol. Chem. 1987; 262: 2472-2477Abstract Full Text PDF PubMed Google Scholar, 21Pitarresi T.M. Rubattu S. Heinrikson R. Sealey J. J. Biol. Chem. 1992; 267: 11753-11759Abstract Full Text PDF PubMed Google Scholar, 22Reudelhuber T.L. Brechler V. Jutras I. Mercure C. Methot D. Adv. Exp. Med. Biol. 1998; 436: 229-238Crossref PubMed Scopus (10) Google Scholar). We previously proposed that the acid activation rate depended only on the amino acid sequence of prorenin prosegment (28Suzuki F. Nakagawa T. Kakidachi H. Murakami K. Inagami T. Nakamura Y. Biochem. Biophys. Res. Commun. 2000; 267: 577-580Crossref PubMed Scopus (26) Google Scholar) and that inactive human prorenin was activated by anti-01/15 antibodies (pf#1 antibodies) (23Suzuki F. Hatano Y. Nakagawa T. Terazawa K. Gotoh A. Nasir U.M. Ishida Y. Nakamura Y. Biosci. Biotechnol. Biochem. 1999; 63: 550-554Crossref PubMed Scopus (8) Google Scholar). These results suggested that the antibodies that remarkably decreased the KD after the acidification of prorenin should be considered the candidates for prorenin activators. In fact, anti-11/26 antibodies was found to activate prorenin significantly in a dose- and time-dependent manner (Figs. 5, 6, 7), whereas anti-01/06 and anti-27/41 antibodies did not contribute to such activation, even when bound to it (Figs. 3 and 5). The regions T7PFKR10P and M16PPSIRESLKER26P were therefore closely associated in some regions with renin and/or prorenin molecule, so as to block the prorenin activation. The region I11P FLKR15P, which bound not to anti-16/26 but to anti-11/26 antibody, must be the key region for a non-proteolytic activation of prorenin by a protein-protein interaction. The acid treatment is thought to stimulate the protonation of amino acid residues containing carboxylate, e.g. Glu or Asp, so that the quick dissociation of the ionic bond should induce local conformational changes with slow dissociation of the hydrophobic bond at a low temperature in the prorenin molecule (17Sealey J.E. Laragh J.H. Circ. Res. 1975; 36–37: I-10-I-16Google Scholar, 18Derkx F.H. von Gool J.M. Wenting G.J. Verhoeven R.P. Man in't V. eld A.J. Schalekamp M.A. Lancet. 1976; 1: 496-499Abstract PubMed Scopus (46) Google Scholar, 19Leckie B.J. McGhee N.K. Nature. 1980; 288: 702-705Crossref PubMed Scopus (64) Google Scholar, 20Derkx F.H.M. Margaretha P.A. Schalekamp P.A. Schalekamp A.D.H. J. Biol. Chem. 1987; 262: 2472-2477Abstract Full Text PDF PubMed Google Scholar, 21Pitarresi T.M. Rubattu S. Heinrikson R. Sealey J. J. Biol. Chem. 1992; 267: 11753-11759Abstract Full Text PDF PubMed Google Scholar, 22Reudelhuber T.L. Brechler V. Jutras I. Mercure C. Methot D. Adv. Exp. Med. Biol. 1998; 436: 229-238Crossref PubMed Scopus (10) Google Scholar). Acidified prorenin was observed to reach maximum binding levels with each of the antibodies faster than inactive prorenin, as shown in Figs. 3 and 4. Binding of prorenin to the anti-11/26 antibodies reached an equilibrium level in 25 h (Fig. 6), although this process was achieved within 2 h for acidified prorenin. The KD for the complex of prorenin and anti-11/26 antibodies was calculated to be 40 nM by the equilibrium method. This value should be identical to the Kb in the kinetics study (Fig. 7). From the x intercept in that figure, the Ka value was estimated to be 50 pM. This Ka level is 1000 times lower than that reported by Derkx et al. (25Derkx F.H. Deinum J. Lipovski M. Verhaar M. Fischli W. Schalekamp M.A. J. Biol. Chem. 1992; 267: 22837-22842Abstract Full Text PDF PubMed Google Scholar). Therefore, our present data suggest that prorenin has intermediately inactive forms at neutral pH. Because the anti-16/26 antibodies did not bind to inactive prorenin (Figs. 3 and 6), the region R16PMPSIRESLKER26P was thought not to interact with a prorenin activator protein such as anti-16/26 antibodies in vivo. In this study, we clearly showed that the rate-limiting step for prorenin activation was the binding of the I11PFLKR15P region to the antibodies by the protein-protein interaction. Acid activation of prorenin is thought to be reversibly and inversely dependent on temperature. The cryo-activation of prorenin should therefore be suppressed at 25 °C. As shown in Fig. 8, almost all of the acid-activated prorenin was re-inactivated in 24 h at pH 7.4. Anti-01/06, anti-16/26, and anti-27/41 antibodies had no effect on this re-inactivation of acid-activated prorenin. However, we found that the re-inactivation was inhibited by the anti-07/10 antibodies for 50 h (Fig. 8). The anti-11/26 antibodies suppressed 50% of the re-inactivation for 24 h (Fig. 8). This finding confirms that prorenin was irreversibly activated by anti-11/26 antibodies (Fig. 5). These results show that the region from 7P to 10P is essential for the inactivation of prorenin. The region from 11P to 15P probably associates with an “opener protein” such as anti-11/26 antibodies and/or prorenin receptor at physiological temperatures. Prorenin is well known to be irreversibly activated by several enzymes such as trypsin, cathepsin B, and plasmin (3Morris B.J. J. Hypertens. 1992; 10: 209-214Crossref PubMed Scopus (29) Google Scholar, 14Hsueh W.A. Baxter J.D. Hypertension. 1991; 17: 469-477Crossref PubMed Google Scholar, 15Hsueh W.A. Do Y.S. Wang P.H. Cell Biophys. 1991; 19: 63-71Crossref PubMed Scopus (3) Google Scholar, 25Derkx F.H. Deinum J. Lipovski M. Verhaar M. Fischli W. Schalekamp M.A. J. Biol. Chem. 1992; 267: 22837-22842Abstract Full Text PDF PubMed Google Scholar). This activation is thought to digest the prosegment region of prorenin to release it from the renin region. These enzymes cleave the peptide bond on the carboxyl side of the basic amino acid residues. Human prorenin has Lys14P and Arg15P at the key region for non-proteolytic activation, as described above. This region must also be the target for such activation enzymes. The initial proteolysis on the carboxyl sides of Lys9P and Arg10P is probably an activation trigger of prorenin, as well as the essential region for the prorenin receptor. A prorenin/renin receptor mRNA was recently detected at high levels in the heart, brain, and placenta and at lower levels in the kidney and liver (24Nguyen G. Delarue F. Burckle C. Bouzhir L. Giller T. Sraer J.D. J. Clin. Invest. 2002; 109: 1417-1427Crossref PubMed Scopus (1175) Google Scholar). By confocal microscopy, the receptor is seen to be localized in the mesangium of the glomeruli and in the subendothelium of the coronary and kidney arteries, associated with smooth muscle cells and co-localized with renin. It activated prorenin by non-proteolytical binding (24Nguyen G. Delarue F. Burckle C. Bouzhir L. Giller T. Sraer J.D. J. Clin. Invest. 2002; 109: 1417-1427Crossref PubMed Scopus (1175) Google Scholar). This finding is important for the study of prorenin and the local prorenin/renin angiotensin system. If our findings in this study could be commonly applied to non-proteolytic activation of prorenin through receptor binding, then region 7PTFKR10P would probably be dissociated from the renin molecule through the association of region I11PFLKR15P with the receptor. The amino acid sequence of the N-terminal region in prorenin has attracted the attention of several investigators to design human renin inhibitors. Mercure et al. (27Mercure C. Thibault G. Lussier-Cacan S. Davignon J. Schiffrin E.L. Reudelhuber T.L. J. Biol. Chem. 1995; 270: 16355-16359Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) reported that R10PIFLKRMPSIR20P maintained the enzymatically inactive state of prorenin. Cumin et al. (31Cumin F. Evin G. Fehrentz J.A. Seyer R. Castro B. Menard J. Corvol P. J. Biol. Chem. 1985; 260: 9154-91547Abstract Full Text PDF PubMed Google Scholar) reported that L13PKRMP17P inhibited renin activity with the 10 and 1 micromole order of Ki and K′i, respectively. Heinrikson et al. (32Heinrikson R.L. Hui J. Zurcher-Neely H. Poorman R.A. Am. J. Hypertens. 1989; 2: 367-380Crossref PubMed Scopus (51) Google Scholar) suggested that region R10PIFLK14P was essential for the reversible refolding of the prosegment that leads to prorenin inactivation. The data from the present study confirm their findings in some respects. We have shown here that regions M16PPSIRESLKER26P and T7PFKR10P were buried in the main body of the prorenin molecule. To our knowledge, this is the first study to indicate that region T7PFKR10P is crucial for refolding and the maintenance of the inactive state of prorenin (Fig. 8). Region I11PFLKR15P is another important region identified in this study. The Ki and K′i values for peptide L13PKRMP17P to renin (31Cumin F. Evin G. Fehrentz J.A. Seyer R. Castro B. Menard J. Corvol P. J. Biol. Chem. 1985; 260: 9154-91547Abstract Full Text PDF PubMed Google Scholar) are 10 times higher than the KD of 41 nM for the anti-11/16 antibodies to prorenin (Fig. 3). In addition, the region localizes at a space far removed from the active site in the cleft in the predicted tertiary model of prorenin (Fig. 1). Moreover, this region probably protrudes outside the main body of prorenin molecule. The re-inactivation of acid-activated prorenin was significantly inhibited by anti-11/26 and anti-07/10 antibodies (Fig. 8). Thus, region I11PFLKR15P should be another important region for prorenin refolding. In this study, we proposed that two regions, T7PFKR10P and I11PFLKR15P, referred to here as the “gate” and “handle,” respectively, are crucial for non-proteolytic activation of prorenin. The present findings thus provide important clues for further study of a novel renin-angiotensin system." @default.
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• W2003442673 title "Human Prorenin Has “Gate and Handle” Regions for Its Non-proteolytic Activation" @default.
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