Folding and activation of recombinant human prorenin

In vitro folding of mature renin, prorenin, and fused prorenin, all produced in denatured form in inclusion bodies in recombinant Escherichia coli, has been studied in order to evaluate the importance of prosequence in the folding of human renin. These studies have been compared with the in vivo folding and subsequent in vitro activation of recombinant human prorenin secreted by a nonbacterial expression system, namely Chinese hamster ovary (CHO) cells grown in serum-free medium. It is concluded that prosequence is essential in the folding of human renin and, therefore, the DNA coding for this sequence cannot be removed without affecting the recovery of active human renin from recombinant bacterial and nonbacterial systems.     

Extrarenal production and activation of human plasma prorenin: the evidence after venous occlusion

Venous occlusion of the left arm in consenting men was induced for 10 or 20 min to stimulate local fibrinolytic and other proteases, thereby favouring the conversion of prorenin to renin. Using the two techniques cryoactivation and tryptic activation, we found that plasma active renin increased significantly after such occlusion (10 and 20 min) while prorenin rose more convincingly and progressively from 10 to 20 min. The renin increase can be partially attributed to hemoconcentration, but in vivo production and (or) local activation of prorenin to renin cannot be excluded. The prorenin rise can apparently be attributed to local extrarenal production, and not to hemoconcentration or influx, since it was progressive and neither prorenin nor renin levels were raised at all in blood circulating outside the occluded arm. Prekallikrein and plasminogen levels were elevated in occlusion plasmas, but responsibility of these enzyme systems for any enhanced activation of prorenin was not established. The trypsin inhibitory capacity was also elevated, increasing the requirement of trypsin to achieve optimal activation of prorenin, but not changing the prorenin estimate itself. Thus, prorenin appears to be released extrarenally, within the vasculature of an occluded arm, while in vitro evidence suggests that the mechanisms for its activation were stimulated. The importance of such extrarenal production and activation of prorenin for renin production under other physiological or pathophysiological conditions remains to be determined.     

Active prorenin: Evidence for the formation of a conformational variant of recombinant human prorenin

Using highly purified recombinant human prorenin, we report the first evidence for the formation of a stable, partially active, conformational variant of the recombinant proenzyme. The enzymatically active prorenin exhibits the following characteristics: (1) the proenzyme N-terminal sequence and molecular weight are maintained; (2) the active proenzyme is capable of cleaving a novel fluorogenic peptide substrate based on the sequence of human angiotensinogen and exhibits about 30% of mature renin specific activity for the fluorogenic substrate; (3) the active proenzyme conformation binds to, and can be eluted from, a pepstatin affinity column; and (4) the activity of the active proenzyme can be inhibited by a novel peptidomimetic renin inhibitor.     

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)     

Possible activation of human prorenin by contact with the cell membrane

Semipurified human kidney prorenin was exposed in vitro to a mixture of lipids mimicking the composition of the inner leaflet of the cell membrane, in the presence of semipurified human angiotensinogen at a concentration of 1/4 Km. Prorenin was activated in a time-dependent manner over a period of 60 min. This lipid-dependent activation was completely reversed thereafter. Pre-incubation with anti-renin serum completely prevented this activated prorenin-dependent generation of angiotensin I. Our data suggest that human prorenin can be reversibly activated by contact with the cell membrane.     

Trypsin activation pathway of rotavirus infectivity.

The infectivity of rotaviruses is increased by and most probably is dependent on trypsin treatment of the virus. This proteolytic treatment specifically cleaves VP4, the protein that forms the spikes on the surface of the virions, to polypeptides VP5 and VP8. This cleavage has been reported to occur in rotavirus SA114fM at two conserved, closely spaced arginine residues located at VP4 amino acids 241 and 247. In this work, we have characterized the VP4 cleavage products of rotavirus SA114S generated by in vitro treatment of the virus with increasing concentrations of trypsin and with proteases AspN and alpha-chymotrypsin. The VP8 and VP5 polypeptides were analyzed by gel electrophoresis and by Western blotting (immunoblotting) with antibodies raised to synthetic peptides that mimic the terminal regions of VP4 generated by the trypsin cleavage. It was shown that in addition to arginine residues 241 and 247, VP4 is cleaved at arginine residue 231. These three sites were found to have different susceptibilities to trypsin, Arg-241 > Arg-231 > Arg-247, with the enhancement of infectivity correlating with cleavage at Arg-247 rather than at Arg-231 or Arg-241. Proteases AspN and alpha-chymotrypsin cleaved VP4 at Asp-242 and Tyr-246, respectively, with no significant enhancement of infectivity, although this enhancement could be achieved by further treatment of the virus with trypsin. The VP4 end products of trypsin treatment were a homogeneous VP8 polypeptide comprising VP4 amino acids 1 to 231 and a heterogeneous VP5, which is formed by two polypeptide species (present at a ratio of approximately 1:5) as a result of cleavage at either Arg-241 or Arg-247. A pathway for the trypsin activation of rotavirus infectivity is proposed.     

Reversible cryoactivation of recombinant human prorenin.

Cleavage of prorenin's prosegment causes irreversible formation of renin. In contrast, renin activity is reversibly exposed when prorenin is acidified to pH 3.3. Nonetheless, acidification of plasma results in irreversible activation of prorenin, because endogenous proteases cleave the prosegment of acid-activated prorenin. Chilling of plasma results in irreversible cryoactivation of prorenin. In this study we investigated whether cryoactivation of purified prorenin is reversible. The intrinsic renin activity of recombinant human prorenin was measured by an enzyme kinetic assay using partially purified human angiotensinogen as substrate. Results are expressed as a percent (mean +/- S.E.) of the maximal activity exposed after limited proteolysis by trypsin. The intrinsic renin activity of two pools (0.3 and 0.06 Goldblatt units/ml) was 1.5% +/- 0.3 and 1.2% +/- 0.6 at 37 degrees C. Activity increased to 19% +/- 0.3 and 26% +/- 0.5 after incubation at 0 degrees C and to 5.4% +/- 0.5 and 2.1% +/- 1.2 at room temperature. Cryoactivation did not occur in buffers containing more than 1 M NaCl. It took 8 min at 37 degrees C or 180 min at room temperature for cryoactivated prorenin to lose half of its intrinsic renin activity. It took 48 and 26 h, respectively, at 0 degree C for the two pools of prorenin at 37 degrees C to regain half of their maximum intrinsic activity at 0 degrees C. A direct immunoradiometric assay that detects active renin but not prorenin was able to detect cryoactivated prorenin. These results show that human prorenin can be reversibly cryoactivated in buffers of low ionic strength and has greater intrinsic activity at room temperature than at 37 degrees C.     

Refolding and activation of human prorenin expressed in Escherichia coli: application of recombinant human renin for inhibitor screening

Human prorenin was expressed in Escherichia coli as a fusion protein of thioredoxin. The chimeric protein, which accumulated insoluble inclusion bodies, was solubilized in 4 M guanidine-HCl and refolded by an arginine-detergent buffer system and by systematic dialysis. The refolded fusion prorenin was activated by trypsin. The antiserum against human kidney renin specifically inhibited the recombinant human renin activity. Using the recombinant human renin, we screened its inhibitory activity in fermented soybean paste (miso) and demonstrated that miso contained renin inhibitory activity derived from soybean. The IC(50) values for soybean and steamed soybean extracts were determined to be 1.9 and 1.6 mg/ml, respectively. This is the first demonstration of renin inhibitory activity in miso and soybean.     

Trypsin induces activation and inflammatory mediator release from human eosinophils through protease-activated receptor-2.

Protease-activated receptors (PARs) are a unique class of G protein-coupled receptors, which are activated by proteolytic cleavage of the amino terminus of the receptor itself. PARs are most likely involved in various biological responses, such as hemostasis and regulation of muscle tone; however, the roles of PARs in the functions of inflammatory and immune cells are poorly understood. Because eosinophils are most likely involved in allergic inflammation and are exposed to a variety of proteases derived from allergens and other inflammatory cells, we investigated whether PARs regulate effector functions of eosinophils. Human eosinophils constitutively transcribe mRNA for PAR2 and PAR3, but not those for PAR1 and PAR4. The expression of PAR2 protein was confirmed by flow cytometry. When trypsin, an agonist for PAR2, was incubated with eosinophils, it potently induced superoxide anion production and degranulation; 5 nM trypsin induced responses that were 50-70% of those induced by 100 nM platelet-activating factor, a positive control. In contrast, thrombin, an activator for PAR1, PAR3, and PAR4, showed minimal effects. The stimulatory effect of trypsin was dependent on its serine protease activity and was blocked 59% by anti-PAR2 Ab. Furthermore, a specific tethered peptide ligand for PAR2 potently induced superoxide production and degranulation; the effects of peptide ligands for PAR1, PAR3, and PAR4 were negligible. These findings suggest that human eosinophils express functional PAR2, and serine proteases at the inflammation site may play important roles in regulating effector functions of human eosinophils. The expression and functional relevance of other PARs still need to be determined.     

Trypsin activation of inactive renin: plasma blanks and angiotensin I radioimmunoassay.

Divergent conclusions exist as to whether inactive renin is present in nephrectomized rat plasma. A major factor contributing to this conflict may be related to significant changes in the "plasma blank" when trypsin-treated plasma is subjected to angiotensin I (AI) radioimmunoassay (RIA). In normal, but not nephrectomized rat plasma, AI-like substances are present in direct proportion to active renin. These substances are destroyed by trypsin. However, trypsin generates additional AI-like material, in both normal and nephrectomized rat plasma. This material, which is present in proportion to the renin substrate concentration, does not appear to be tetradecapeptide (TDP). In normal plasma, however, exogenous TDP is converted to AI in proportion to the active renin concentration and AI generation from TDP is increased by activation of inactive renin. However, in nephrectomized rat plasma, no AI generation from TDP was evident either before or after trypsin treatment. The coincident tryptic generation of a substance that quenches the levels of AI detected by RIA, combined with significant changes in the levels of endogenous and trypsin generated AI-like substances, may have significant bearing on the measured levels of inactive renin.     

Glucagon-initiated human growth hormone release: a comparative study

Human growth hormone (HGH) responses in 20 healthy adults to subcutaneous glucagon, arginine infusion and tolbutamide and insulin hypoglycemia were compared. HGH rose in all four tests. HGH response to glucagon was also studied in 49 patients with suspected pituitary insufficiency, of whom 25 also later received an arginine infusion; an abnormal response to glucagon was the most frequent functional abnormality and often HGH was the only anterior pituitary hormone of which a deficiency was detectable. In seven subjects (two healthy controls and five patients with suspected hypopituitarism) there was a subnormal HGH response to arginine but a normal response to glucagon. It is concluded that glucagon is a simple and effective stimulus to HGH release, equal or superior to arginine, tolbutamide and insulin, and is an important test of anterior pituitary function.     

Recombinant human prorenin from CHO cells: Expression and purification

The gene for human preprorenin was obtained from total RNA prepared from primary human chorion cells. An expression vector was constructed containing an SV40 early promoter, a human preprorenin cDNA, bovine growth hormone poly-A addition signal, and a dihydrofolate reductase (dhfr) expression cassette. This vector was inserted into the DXB-11 Chinese hamster ovary (CHO) cell line. The recombinant protein was exported by CHO cells into the tissue culture media. At harvest the prorenin levels ranged from 1–5 mg/L. For prorenin isolation the cell culture supernatants were processed by filtration, concentration, dialysis, and batch extraction. Preparative-scale isolation of prorenin was accomplished using blue-dye chromatography and size-exclusion chromatography. The isolated prorenin yielded a single SDS-gel band with Mr 40,000. The proprotein was characterized with respect to N-terminal sequence and N-linked sugar composition. Trypsin-activated renin prepared from the proprotein was characterized with respect to N-terminal sequence andpH-activity profile. Enzyme activity was measured with a newly developed fluorogenic peptide substrate containing the P₆-P₃ sequence of human angiotensinogen.     

Expansion of human embryonic stem cells: a comparative study

Objectives: Human embryonic stem cells (hESC) are promising for tissue engineering (TE) purposes due to their unique properties. However, current standard mechanical passaging techniques limit rates of possible TE experiments, as it is difficult to obtain high enough numbers of the cells for experimentation. In this study, several dissociative solutions and application methods are tested for their applicability to, and influence on, hESC culture and expansion. Materials and methods: Expansion of two hESC lines, H1 and VUB01, subjected to different passaging techniques, was evaluated. Four dissociative solutions – TrypLE? Express, Trypsin‐EDTA, Cell Dissociation Solution and Accutase?– were combined with two application protocols. As reference conditions, manual and bead‐based passaging techniques were used. Results: Results showed that use of Cell Dissociation Solution in combination with a slow adaptation protocol, generated the best expansion profile for both cell lines. The hESC single cell lines remained pluripotent, had good expansion profiles and were capable of differentiation into representatives of all three germ layers. Reproducibility of the results was confirmed by adaptation for three other hESC lines. Conclusion: Use of Cell Dissociation Solution, combined with slow adaptation protocol, allows a fast switch from the mechanical passaging technique to a single‐cell split technique, generating stable and robust hESC cell lines, which allow for large scale expansion of hESC for TE purposes.     

beta-D-mannosidase in human polymorphonuclear leukocytes and lymphocytes: a comparative study

All lymphocytes and polymorphonuclear leukocytes (PMNL) beta-D-mannosidase activities are adsorbed on DEAE-Trisacryl column at pH 7.0. Only one form is eluted with a 0.15 M linear gradient. The two enzymes isolated from either type of cells exhibit similar properties. The chromatographic profiles of beta-D-mannosidase from leukemic lymphocytes (chronic lympho?d leukemia and hairy cells leukemia) differ from the normal ones by the presence of a more acidic minor form.