Conceived and designed the experiments: SMD MCG RDM. Performed the experiments: SMD SK ZC EIV ABN OG RDM. Analyzed the data: SMD SK DES OG MCG RDM. Contributed reagents/materials/analysis tools: SMD. Wrote the paper: SMD MCG RDM.
The authors have declared that no competing interests exist.
Angiotensin-converting enzyme (ACE; Kininase II; CD143) hydrolyzes small peptides such as angiotensin I, bradykinin, substance P, LH-RH and several others and thus plays a key role in blood pressure regulation and vascular remodeling. Complete absence of ACE in humans leads to renal tubular dysgenesis (RTD), a severe disorder of renal tubule development characterized by persistent fetal anuria and perinatal death.
Patient with RTD in Lisbon, Portugal, maintained by peritoneal dialysis since birth, was found to have a homozygous substitution of Arg for Glu at position 1069 in the C-terminal domain of ACE (Q1069R) resulting in absence of plasma ACE activity; both parents and a brother who are heterozygous carriers of this mutation had exactly half-normal plasma ACE activity compared to healthy individuals. We hypothesized that the Q1069R substitution impaired ACE trafficking to the cell surface and led to accumulation of catalytically inactive ACE in the cell cytoplasm. CHO cells expressing wild-type (WT) vs. Q1069R-ACE demonstrated the mutant accumulates intracellularly and also that it is significantly degraded by intracellular proteases. Q1069R-ACE retained catalytic and immunological characteristics of WT-ACE N domain whereas it had 10–20% of the nativity of the WT-ACE C domain. A combination of chemical (sodium butyrate) or pharmacological (ACE inhibitor) chaperones with proteasome inhibitors (MG 132 or bortezomib) significantly restored trafficking of Q1069R-ACE to the cell surface and increased ACE activity in the cell culture media 4-fold.
Homozygous Q1069R substitution results in an ACE trafficking and processing defect which can be rescued, at least in cell culture, by a combination of chaperones and proteasome inhibitors. Further studies are required to determine whether similar treatment of individuals with this ACE mutation would provide therapeutic benefits such as concentration of primary urine.
Angiotensin I-converting enzyme (ACE, CD143) is a Zn2+ carboxydipeptidase which plays a key role in the regulation of blood pressure and also in the development of vascular pathologies and tissue remodeling
Numerous data convincingly indicate that elevated ACE expression is a risk factor associated with several cardiovascular and renal diseases such as hypertension, cardiac hypertrophy, diabetic nephropathy, and others
Two mutations in ACE have been described which were linked to premature fetal death due to autosomal recessive renal tubular dysgenesis -RTD
Herein, we report a mechanism for the 3rd case of RTD which was observed in Lisbon, Portugal which is associated with complete absence of ACE due to a homozygous Q1069R ACE substitution. We present evidence that this novel mutation represents a “
Peripheral blood was obtained from the affected patient, her unaffected brother, and her parents by Dr. Rosario Stone (Hospital de Santa Maria, Lisboa, Portugal) after obtaining informed consent from parents. Blood samples were sent to Hôpital Necker-Enfants Malades, INSERM, U574, Université Paris Descartes, Paris, France. Experiments were done in accordance with French ethical committee recommendation. Genomic DNA was isolated by standard methods. Linkage analyses were performed using microsatellite markers based on proximity to
We used the Oligo 6.2 program (NBI) to design specific primers to amplify the coding exons and the adjacent intronic sequences of the
Heparinized plasma from patient, her parents and healthy brother, kindly provided by Dr. Rosario Stone (Hospital de Santa Maria, Lisboa, Portugal), was taken for determination of ACE activity. The study was approved by the Institutional Review Boards of the University of Illinois at Chicago and procedures followed were in accordance with institutional guidelines. ACE activity in human plasma was measured using a fluorimetric assay
cDNA encoding mutant ACE protein was provided by Dr. Tiago Outeiro (Cell and Molecular Neuroscience Unit, Instituto de Medicina Molecular, Lisboa, Portugal) and was created by mutation of the CAG codon for Gln at position 1069 to codon C
Culture medium (Ultra-CHO medium, Cambrex Bio-Science Walkersville, Inc, Walkersville, MD) from these cells (WT-ACE and Q1069R) was used as a source of the soluble ACEs, and cell lysates as a source of membrane-bound ACEs for biochemical and immunological characterization. To assess the effect of culture conditions and different compounds on ACE activity and localization, CHO cells stably expressing WT- and Q1069R-ACE were cultured in F12 medium containing 10% Fetal Bovine Serum (FBS). When the cells reached 70–90% confluence they were washed twice with PBS and fresh Ultra-CHO medium having no endogenous ACE activity was added. Effect of temperature (30°C versus 37°C) and inhibitors was assessed: sodium butyrate (Sigma-Aldrich, St. Louis, MO)-5 mM; ACE inhibitor enalaprilat (Sigma-Aldrich, St. Louis, MO)-1 µM; proteosome inhibitors–MG132 (Assay Designs, Ann Arbor, MI) and Bortezomib (ChemieTek, Indianapolis, IN)-both at 5 µM. After 24 hrs, the culture medium was aspirated and centrifuged (to removed detached cells) and the cells, after washing with PBS, were lysed with 50 mM Tris-HCl buffer, pH 7.5, containing 150 mM NaCl and 0.5% Triton X-100. ACE activity in the lysate and culture medium was determined using two ACE substrates as described above.
Lysates from CHO-WT-ACE cells (with ACE activity of 8 mU/ml using Hip-His-Leu) were compared to lysates from CHO-ACE–Q1069R cells normalized by equal protein loading by SDS-PAGE in 4–15% acrylamide Tris-HCl pre-cast SDS PAGE gels (Bio-Red Laboratories, Hercules, CA). After electrophoretic transfer of proteins to microporous PVDF-Plus membranes, each membrane was incubated 30 min in 10 mM Tris-HCl (pH 8.0) buffer containing 150 mM NaCl, 0.05% Tween 20, and Western Blocking solution (Sigma-Aldrich, St. Louis, MO) prior to incubation with hybridoma culture fluids (1/10 dilution in the same blocking solution) for 1 hr at room temperature. Subsequent steps were carried with secondary antibodies (ant-mouse and anti rat IgG, depending on primary antibody to ACE, conjugated with peroxidase) and peroxidase activity was developed using SuperSignal WestPico Chemiluminescense substrate (Pierce, Rockford, IL).
Total RNA was prepared from stably transfected CHO cells (CHO-WT-ACE and CHO-Q1069R-ACE) using TRIZOL reagent (Invitrogen Corp., Carlsbad, CA) and treated with DNase (Ambion., Austin, TX) to remove DNA. RNA was converted to cDNA by SuperScript® III Reverse Transcriptase (Invitrogen Corp., Carlsbad, CA) using random hexamer primers, and mRNA levels were estimated by quantitative touchdown PCR (QPCR). PCR was done using Taq DNA Polymerase (Invitrogen Corp., Carlsbad, CA) and contained SYBR Green (SybrGreen1 10,000× concentrate, diluted 1∶10,000; Molecular Probes, Eugene, OR). PCR conditions were 30 cycles of denaturation at 94°C for 10 s; annealing at 56–62°C for 15 s; and extension at 72°C for 20 s on a Corbett Rotorgene Real-Time PCR unit (Corbett, Australia). Relative mRNA concentrations were calculated from the takeoff point of reactions using manufacturer's software and normalized to α-tubulin. Melting curve analysis and agarose gel electrophoresis ensured production of single and correct-size products. The primers used were:
α-tubulin forward,
α-tubulin reverse,
ACE forward,
ACE reverse,
To assess the cell surface and intracellular localization of exogenously expressed WT- and Q1069R-ACE, CHO cells were either fixed in 0.3% paraformaldehyde on ice for 15 min, or fixed and permeabilized with 3% paraformaldehyde at 37°C for 15 min, respectively. Fluorescent secondary Ab (goat anti-mouse Alexa 488, 1∶500) labeling of anti-ACE mAb 9B9
96-well plates (Corning, Corning, NY) were coated with 50 µl affinity-purified goat anti-mouse IgG (Pierce, Rockford, IL) at 10 µg/ml and stored overnight at 4°C. After washing with PBS containing 0.05% Tween 20, the wells were incubated with different anti-ACE mAbs (2 µg/ml) directed to 16 different epitopes on the N and C domain of ACE
Diagnosis of Renal Tubular Dysgenesis (RTD) was made in the first day of life of the patient by Dr. Rosario Stone (Hospital de Santa Maria, Lisboa, Portugal) based on hypocalvaria, hypotension, and anuric renal failure. Clinical characterization of this case will be published elsewhere. Based on the results of Gribouval et al.
Sequencing of the 26 exons of the
Plasma ACE activity of the patient and immediate family were measured and showed that the patient had no ACE activity whereas both parents and a brother had approximately two-times less ACE compared to healthy individuals (
ACE activity in heparinized plasma (1/5 dilution in PBS) of members of the affected family, the father (F), mother (M), and brother (B) as well as in 5 healthy individuals (marked by initials) that served as controls was determined by fluorimetric assay of 40 µl diluted plasma with 200 µl each of substrates Hip-His-Leu (HHL) and Z-Phe-His-Leu(ZPHL) during 1 hour of incubation.
The localization of Gln1069 in the C-domain of human ACE which was mutated to Arg is shown using molecular surface (
There are several possibilities as to why this particular mutation leads to absence of ACE protein in the plasma and presumably the surface of endothelial cells (the main source of circulating ACE). We hypothesized that the absence of plasma ACE could be due to destabilization of mRNA, proteolytic cleavage by intracellular proteases as a result of misfolding of the mutant protein, and impaired trafficking of the mutant to the plasma membrane.
Previously, we showed that dendritic cells (DC) originating from Acute Myeloid Leukemia (AML) blasts has no cell surface ACE, in sharp contrast to DC generated from monocytes of healthy donors
Several ACE glycosylation mutants that do not have ACE catalytic activity, when transfected into CHO cells (for example a mutation in the N domain in which 4 glycosylation sites are removed, provided by Dr. E. Sturrock, University of Cape Town, South Africa) showed significant ACE catalytic activity after cultivation at lower temperature or in the presence of sodium butyrate-data not shown- personal communications). Therefore, we hypothesized that the new mutation in ACE (Q1069R substitution) caused misfolding of ACE and impaired trafficking of ACE from the endoplasmic reticulum to the cell surface, and thus accumulated inside the cells.
To decipher the mechanism by which the novel Q1069R mutation might be responsible for the absence of plasma (and perhaps tissue) ACE in the affected individuals, we performed site-directed mutagenesis of human recombinant somatic ACE, generated this mutant of somatic ACE in CHO cells, and compared the biochemical and immunological characteristics of this mutant with WT somatic ACE.
For the initial characterization of mutant ACE produced by CHO cells, we performed Western blotting of cell lysates from CHO-WT-ACE and CHO-Q1069R-ACE using several mAbs to denatured human ACE: mouse mAb 1D8, 3C5, and 2E2, which recognize sequential epitopes in the C domain
CHO cells were transiently transfected with plasmids coding for wild-type (WT) and mutant (Q1069R) ACE (4 µg of plasmid DNA per 35 mm dish). The lysates of these cells (normalized by equal protein loading of 10 µg per lane) were subjected to SDS-PAGE (4–15% gradient gel) in reducing conditions for Western blotting (
In order to determine the reason for the dramatic decrease of mutant ACE protein in transfected CHO cells, we quantified mRNA production by CHO-ACE-WT and the mutant Q1069R.
Confocal microscopy was used to determine the subcellular localization of WT- vs. Q1069R-ACE mutant expressed in CHO cells following immunostaining with anti-ACE mAb 9B9.
Confocal microscopy was used to determine the localization of WT- vs. Q1069R-ACE mutant expressed in CHO cells following immunostaining with anti-ACE mAb 9B9 and Alexa 488 goat anti-mouse secondary Ab. A-C: WT-ACE expressing cells; D-F: Q1069R-ACE expressing cells. A,B and D,E. Immunostaining of WT and Q1069R ACE in 0.3% paraformaldehye for 15 min at 4C (fixed only). B and E. Treatment with the chemical chaperone sodium butyrate and proteosome inhibitor MG132 for 24 hrs followed by immunostaining with 9B9 mAb. C and F. CHO cells fixed and permeabilized with 3% paraformaldehyde for 15 min at 37C and labeled as above show WT-ACE on the plasma membrane and to a lesser extent in the cytosol and Q1069R-ACE in perinuclear areas.
During the last 20 years, numerous ACE mutations have been generated (more than 40) and among them, several where identified which had no enzymatic activity but measurable mRNA expression or ACE protein production; these were shown to be due to mutations of potential glycosylation sites. Thus several multiple and single glycosylation mutants were characterized by the arrest of mutant protein in the endoplasmic reticulum and by rapid intracellular degradation
Kinetic analysis of soluble mutant ACE as well as membrane bound mutant in comparison with WT ACE using two widely used ACE substrates showed a very unexpected result. ACE activity of the lysates of CHO cells expressing mutant human ACE determined with Hip-His-Leu was very low and consisted of about 2.6% of the WT ACE activity in CHO cells (
ACE activity of the membrane-bound form of ACE in lysates of CHO cells expressing WT and mutant ACE was determined by fluorimetric assay of 40 µl aliquotes of cells with 200 µl each of substrates Hip-His-Leu (HHL) and Z-Phe-His-Leu (ZPHL) during 1 hr incubation.
ACE activity in cell lysates of CHO-Q1069R-ACE cells determined using the substrate Z-Phe-His-Leu was significantly greater (14.1%) than observed with Hip-His-Leu (2.6%), although still much less than WT ACE (
The two domains of ACE hydrolyze the same range of natural and synthetic substrates, but with rather different efficiencies
Data from
We (and others) hypothesized that several amino acid residues might determine a significant substrate specificity of the N- and C-domains. Based on analysis of the rate of hydrolysis of different substrates and the response to different inhibitors and effectors (sulfate ions, dinitrofluorobenzene) by ACE from different species, we hypothesized that the amino acid residues Ser858, Met907, Lys1005, Glu1029, Val1104 in the C-domain might determine preferential hydrolysis of HHL by the C-domain and low efficiency of HHL hydrolysis by the N-domain, giving rise to a high ZPHL/HHL ratio for the truncated N-domain [Danilov, unpublished observation]. Recently, analysis of the 3D structure of the C-domain with novel C-domain selective inhibitors revealed several amino acid residues that may contribute to the domain specificity of ACE inhibition
A closer look to the structure of the active center of the C-domain ruled out the possibility that the Q1069R substitution is responsible for the dramatic increase in ZPHL/HHL ratio of mutant ACE.
The localization of amino acid residue Arg1069 relative to the active center of the human ACE C-domain is shown in the ribbon representation of the substrate-bound crystal structure of the C domain fragment in which the 36 amino acid residues unique to tACE have been deleted
We developed a set of mAbs that recognized 16 different conformational epitopes on the surface of the N- and C-domains of human ACE useful for epitope mapping. Eight mAbs recognize epitopes on the catalytically active N-domain
We thus immunoprecipitated mutant membrane-bound ACE from lysates of CHO cells expressing WT vs. Q1069R ACE (as well as soluble ACE from culture fluids from these cells) to fingerprint the conformation of the mutant. The ability of each mAb to immunoprecipitate ACE is influenced by the local ACE conformation which of course can be influenced by changes of amino acid sequence induced by genetic mutations or by local, selective denaturation. As clearly apparent from
Membrane-bound WT and mutant ACE lysates were normalized to achieve 5 mU/ml ACE activity with Z-Phe-His-Leu as substrate and incubated in microtiter plate wells covered with 16 mAbs to human ACE
Increased fragility of the C-domain in comparison with the N-domain was demonstrated first by studying thermal stability of the separated N- and C-domains
Selective inactivation of the active center in the C-domain and partial denaturation of this domain was confirmed by analysis of mutant Q1069R ACE inhibition by ACE inhibitor enalaprilat and by anti-N-domain catalytic mAbs 3A5 and i2H5 (
Lysates form CHO cells expressing mutant and WT membrane-bound ACE were incubated with ACE inhibitor enalaprilat (10−9–10−8 M) (
We also assessed ZPHL/HHL hydrolysis by mutant ACE bound to antibodies.
Membrane-bound WT (
We confirmed that this chaperone-like effect of these mAbs to ACE was also retained when mutant ACE was in solution as in the situation described in
Membrane-bound (
A chaperone-like effect of the mAbs and the partial or complete renaturation of
There are now hundreds if not a thousands of examples of transport-defective mutations to proteins. Mutations resulting in the changes in protein sequences often result in the production of misfolded and disease-causing proteins that are transcribed and translated at normal levels but are unable to reach their functional destination in cells (for review see Sanders and Nagy,
In the past decade, extraordinary efforts have been made to understand how abnormal folding relates to certain pathologies and to design therapeutic interventions that prevent or correct the structural abnormality of the disease-causing misfolded proteins. In this regard, rescue of misfolded “trafficking-defective” proteins by chemical and pharmacological chaperones and by inhibitors of intracellular degradation is emerging as one of the most promising therapeutic strategies for such disorders
The clarification of the molecular mechanism of this particular ACE mutation has not only scientific, but also clinical value. If the treatment of CHO cells expressing mutant ACE with a combination of chemical and pharmacological chaperones for ACE and proteasome inhibitors can effectively restore ACE trafficking to the cell surface
In order to improve the trafficking of mutant ACE to the cell surface, we cultivated CHO cells expressing mutant and WT ACE in various conditions known to increase trafficking of proteins to the cell surface and prevent intracellular degradation.
ACE activity of membrane-bound form of WT and mutant ACE after culturing cells 24 hrs in different conditions listed below as described in
We should note that a combination of similar treatments, for example low temperature and sodium butyrate, did not show an additive effect (data not shown), whereas the combination of low temperature with the proteasome inhibitor bortezomib had a profound additive effect (
Based on these results (
The complete absence of ACE during fetal development caused structural malformations of the renal tubular system of the index patient. Thus, it is possible that restoration of impaired ACE trafficking to the surface of ACE expressing cells after treatment with chemical or pharmacological chaperones and the generation of AII by renal proximal tubule epithelial cells may not improve clinical symptoms in this patient. However, there are at least two arguments based on mouse models that did not develop anuria but had urine concentrating defects that support the hypothesis and goal of improving clinical symptoms in this patient.
First, the ACE.2 strain of mice developed by the K. Bernstein lab
The second argument comes from studies demonstrating that administration of exogenous angiotensin II caused a restoration of tubuloglomerular feedback (TGF) responsiveness to 71% of control in heterozygous and to 62% of control in homozygous ACE null mice. These data support previous conclusions that angiotensin II is an essential component in the signal transmission pathway that links the
Thus, we identified the molecular mechanism by which a novel mutation of ACE in which codon CAG coding for Gln at position 1069 is substituted in both alleles by codon C
We characterized the molecular basis of defective ACE processing due to this mutation using a cellular CHO expression model in which mutant ACE (Q1069R) activity, immunoreactivity, and trafficking could be defined. Using a novel conformational fingerprinting approach for ACE, we demonstrated that mutant ACE was selectively denatured. The C-domain of somatic ACE was practically non-functional and only the N-domain active center was able to cleave ACE substrates. Moreover, using this model we were able to prevent, to some extent, intracellular degradation of misfolded mutant ACE, and, to rescue, to some degree, trafficking of mutant ACE to the cell surface.
Therefore, this study provides important insights into the mechanism by which the novel ACE mutation (Q1069R) results in dysfunctional membrane ACE expression due to a trafficking defect as well as severe renal tubular dysgenesis. Moreover, we demonstrated an
The authors thank Dr. Edward Sturrock (University of Cape Town, South Africa) for help with analysis of the ACE 3-dimensional structure and discussion of the results. We also wish to thank I. Naperova (Moscow State University, Moscow, Russia) for technical assistance.