Human Prorenin Has “Gate and Handle” Regions for Its Non-proteolytic Activation

Abstract

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 (1-4). The prosegment has been considered to associate with mature renin to prevent interaction with angiotensinogen, its macromolecular renin substrate (5-7). Prorenin does not proteolytically self-activate like pepsinogen, and its blood circulating level is 10 times higher than that of mature renin (8, 9). Some investigators have recently proposed that prorenin is a useful marker of diabetic microvascular complications and Wilms' tumor (11-13). 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 (3, 14, 15) and has also been non-proteolytically activated under acidic pH and/or low temperature (17)(18)(19)(20)(21)(22). We recently showed that specific antibodies to the prosegment (L 1P PTDTTTFKRIFLKR 15P ) activated human prorenin non-proteolytically (23). More recently, a renin/prorenin receptor was found in several tissues with nonproteolytically activated renin as well as prorenin (24). 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 (25-27) or a hydrophobic bond (27) 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 (4), and the speed of this process was only dependent on the amino acid sequence in the prosegment with 43 amino acid residues (28). 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. EXPERIMENTAL PROCEDURESDesignation 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.

(Pro)renin receptor is associated with angiogenic activity in proliferative diabetic retinopathy

“…HRMECs were cultured following the manufacturer's instructions. To cover the handle region of the prorenin molecule, which is the binding site of (P)RR [23], we synthesised decoy peptides NH 2 -RIFLKRMPSI-COOH as a human (P)RR blocker (PRRB), and purified them by high-pressure liquid chromatography on a C-18 reversephase column. After being serum deprived, cells were pretreated with 1 μmol/l PRRB for 1 h or 10 μmol/l MAPK/ ERK kinase (MEK) inhibitor U0126 (Promega, Madison, WI, USA) for 15 min.…”

Possible roles of human (pro)renin receptor suggested by recent clinical and experimental findings

“…10 On the basis of this finding, the 'handle region' of prorenin prosegment was thought to be a candidate of the prorenin site binding (P)RR. This concept was recently confirmed by the study using the BIACORE method showing that a peptide corresponding to the 'handle region' (HRP) binds to the (P)RR and that pretreatment with HRP inhibits the prorenin binding to the (P)RR.…”

Biochemical properties of renin and prorenin binding to the (pro)renin receptor

“…Also, the data obtained from the BIAcore kinetic study showed that the association rate of prorenin to (P)RR is higher than that of mature renin (1.8Â10 7 and 2.16Â10 6 M À1 s À1 , respectively). 44,45 HRP/DECOY AND 'HINGE' REGION PEPTIDES ARE THE MOST PROBABLE CANDIDATES FOR (P)RR BLOCKER The decoy peptide mimics the R 10P IFLKRMPSI 19P region of prorenin prosegment was first indicated by Ichihara et al 30 who designed this peptide based on the 'handle' region peptide (HRP, I 11P FLKR 15P ) in prorenin prosegment reported by Suzuki et al 24 Ichihara et al 30 demonstrated complete inhibition of diabetic nephropathy by administering this peptide in the streptozotocin-induced diabetic rats. After that, many in vivo studies have investigated the effective use of the decoy containing 'handle' region peptide as (P)RR blocker and confirmed the regression of established diabetic nephropathy in heminephrectomized streptozotocin-induced diabetic rats; 46 attenuation of the development and progression of proteinuria and glomerulosclerosis and reduction of renal angiotensin levels in stroke-prone spontaneously hypertensive rats without an effect on blood pressure; 47 attenuation of cardiac fibrosis in spontaneously hypertensive rats fed on a high-salt diet.…”

“…22,23 Also, the 'gate' (T 7P FKR 10P ) and 'handle' (I 11P FLKR 15P ) regions in the prorenin prosegment were indicated in vitro to be accountable for the non-proteolytic activation of prorenin molecules through protein-protein interaction. 24 The (pro)renin receptor, 25 (P)RR, a new player in the RAS components, binds both renin and prorenin. [25][26][27][28][29] The (P)RR itself has been observed to be associated with many pathophysiology of diseases.…”