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• W2094094258 abstract "Aminopeptidase A (APA; EC 3.4.11.7) is a membrane-bound zinc metalloprotease cleaving in the brain the N-terminal aspartyl residue of angiotensin II to generate angiotensin III, which exerts a tonic stimulatory effect on the central control of blood pressure in hypertensive animals. We docked the specific APA inhibitor, glutamate phosphonate, in the three-dimensional model of the mouse APA ectodomain in the presence of Ca2+. In the S1 subsite of this model, the Ca2+ atom was coordinated with Asp-213, Asp-218,y and Glu-215 and three water molecules, one of which formed a hydrogen bond with the carboxylate side chain of the inhibitor. We report here that the carboxylate side chain of glutamate phosphonate also formed a hydrogen bond with the alcohol side chain of Thr-348. Mutagenic replacement of Thr-348 with an aspartate, tyrosine, or serine residue led to a modification of the hydrolysis velocity, with no change in the affinity of the recombinant enzymes for the substrate GluNA, either in the absence or presence of Ca2+. In the absence of Ca2+, the mutations modified the substrate specificity of APA, which was nevertheless restored by the addition of Ca2+. An analysis of three-dimensional models of the corresponding Thr-348 mutants revealed that the interaction between this residue and the inhibitor was abolished or disturbed, leading to a change in the position of the inhibitor in the active site. These findings demonstrate a key role of Thr-348 in substrate specificity of APA for N-terminal acidic amino acids by insuring the optimal positioning of the substrate during catalysis. Aminopeptidase A (APA; EC 3.4.11.7) is a membrane-bound zinc metalloprotease cleaving in the brain the N-terminal aspartyl residue of angiotensin II to generate angiotensin III, which exerts a tonic stimulatory effect on the central control of blood pressure in hypertensive animals. We docked the specific APA inhibitor, glutamate phosphonate, in the three-dimensional model of the mouse APA ectodomain in the presence of Ca2+. In the S1 subsite of this model, the Ca2+ atom was coordinated with Asp-213, Asp-218,y and Glu-215 and three water molecules, one of which formed a hydrogen bond with the carboxylate side chain of the inhibitor. We report here that the carboxylate side chain of glutamate phosphonate also formed a hydrogen bond with the alcohol side chain of Thr-348. Mutagenic replacement of Thr-348 with an aspartate, tyrosine, or serine residue led to a modification of the hydrolysis velocity, with no change in the affinity of the recombinant enzymes for the substrate GluNA, either in the absence or presence of Ca2+. In the absence of Ca2+, the mutations modified the substrate specificity of APA, which was nevertheless restored by the addition of Ca2+. An analysis of three-dimensional models of the corresponding Thr-348 mutants revealed that the interaction between this residue and the inhibitor was abolished or disturbed, leading to a change in the position of the inhibitor in the active site. These findings demonstrate a key role of Thr-348 in substrate specificity of APA for N-terminal acidic amino acids by insuring the optimal positioning of the substrate during catalysis. Aminopeptidase A (APA; EC 3.4.11.7) 3The abbreviations used are: APA, aminopeptidase A; CHO, Chinese hamster ovary; GluSH, glutamate thiol; GluNA, α-l-glutamyl-β-naphtylamide; LysSH, lysine thiol; BSA, bovine serum albumin; PBS, phosphate-buffered saline; MetSH, methionine thiol. is a 160-kDa homodimeric type II membrane-bound monozinc aminopeptidase also activated by Ca2+ (1Danielsen E.M. Noren O. Sjostrom H. Ingram J. Kenny A.J. Biochem. J... 1980; 189: 591-603Google Scholar, 2Glenner G.G. Mc M.P. Folk J.E. Nature.. 1962; 194: 867Google Scholar). It specifically cleaves the N-terminal glutamyl or aspartyl residue from peptide substrates, such as angiotensin II and cholecystokinin-8, in vitro (3Nagatsu I. Nagatsu T. Yamamoto T. Glenner G.G. Mehl J.W. Biochim. Biophys. Acta.. 1970; 198: 255-270Google Scholar, 4Wilk S. Healy D. Adv. Neuroimmunol... 1993; 3: 195-207Google Scholar). APA is strongly expressed in many tissues, including the brush border of intestinal and renal epithelial cells, and in the vascular endothelium (5Lodja Z. Gossrau R. Histochemistry.. 1980; 67: 267-290Google Scholar). APA has also been detected in several brain nuclei involved in controlling body fluid homeostasis and cardiovascular function (6de Mota N. Iturrioz X. Claperon C. Bodineau L. Fassot C. Roques B.P. Palkovits M. Llorens-Cortes C. J. Neurochem... 2008; 106: 416-428Google Scholar, 7Zini S. Masdehors P. Lenkei Z. Fournie-Zaluski M.C. Roques B.P. Corvol P. Llorens-Cortes C. Neuroscience.. 1997; 78: 1187-1193Google Scholar). Studies with specific and selective APA inhibitors (8Chauvel E.N. Llorens-Cortès C. Coric P. Wilk S. Roques B. Fournié-Zaluski M.C. J. Med. Chem... 1994; 37: 2950-2956Google Scholar) have shown that APA cleaved the N-terminal aspartyl residue of brain angiotensin II to generate angiotensin III in vivo (9Zini S. Fournie-Zaluski M.C. Chauvel E. Roques B.P. Corvol P. Llorens-Cortes C. Proc. Natl. Acad. Sci. U. S. A... 1996; 93: 11968-11973Google Scholar) and that brain angiotensin III exerts a tonic stimulatory action on the control of blood pressure in hypertensive animals (10Fournie-Zaluski M.C. Fassot C. Valentin B. Djordjijevic D. Reaux-Le Goazigo A. Corvol P. Roques B.P. Llorens-Cortes C. Proc. Natl. Acad. Sci. U. S. A... 2004; 101: 7775-7780Google Scholar, 11Reaux A. Fournie-Zaluski M.C. David C. Zini S. Roques B.P. Corvol P. Llorens-Cortes C. Proc. Natl. Acad. Sci. U. S. A... 1999; 96: 13415-13420Google Scholar). Thus, the inhibition of brain APA with specific and selective inhibitors normalizes blood pressure in conscious spontaneously hypertensive rats or hypertensive deoxycorticosterone acetate salt rats (10Fournie-Zaluski M.C. Fassot C. Valentin B. Djordjijevic D. Reaux-Le Goazigo A. Corvol P. Roques B.P. Llorens-Cortes C. Proc. Natl. Acad. Sci. U. S. A... 2004; 101: 7775-7780Google Scholar, 11Reaux A. Fournie-Zaluski M.C. David C. Zini S. Roques B.P. Corvol P. Llorens-Cortes C. Proc. Natl. Acad. Sci. U. S. A... 1999; 96: 13415-13420Google Scholar-12Bodineau L. Frugiere A. Marc Y. Inguimbert N. Fassot C. Balavoine F. Roques B. Llorens-Cortes C. Hypertension.. 2008; 51: 1318-1325Google Scholar), suggesting that brain APA constitutes an interesting candidate target for the treatment of certain forms of hypertension (13Ferreira A.J. Raizada M.K. Hypertension.. 2008; 51: 1273-1274Google Scholar, 14Iturrioz X. Reaux-Le Goazigo A. Llorens-Cortes C. Aminopeptidase Inhibitor as Anti-Hypertensive Drugs.Kluwer Academic/Plenum Publishers. 2004; Google Scholar-15Reaux A. Iturrioz X. Vazeux G. Fournie-Zaluski M.C. David C. Roques B.P. Corvol P. Llorens-Cortes C. Biochem. Soc. Trans... 2000; 28: 435-440Google Scholar). This justifies the development of potent and selective APA inhibitors crossing the blood-brain barrier after oral administration for use as centrally acting antihypertensive agents. To achieve this goal, the study of the organization of the APA active site has been pursued. Using the crystal structure of leukotriene-A4 hydrolase (EC 3.3.2.6) (16Thunnissen M.M. Nordlund P. Haeggstrom J.Z. Nat. Struct. Biol... 2001; 8: 131-135Google Scholar) as a template and the functional data collected from site-directed mutagenesis studies on APA (17Iturrioz X. Rozenfeld R. Michaud A. Corvol P. Llorens-Cortes C. Biochemistry.. 2001; 40: 14440-14448Google Scholar, 18Iturrioz X. Vazeux G. Celerier J. Corvol P. Llorens-Cortes C. Biochemistry.. 2000; 39: 3061-3068Google Scholar, 19Vazeux G. Iturrioz X. Corvol P. Llorens-Cortes C. Biochem. J... 1997; 327: 883-889Google Scholar, 20Vazeux G. Iturrioz X. Corvol P. Llorens-Cortes C. Biochem. J... 1998; 334: 407-413Google Scholar, 21Vazeux G. Wang J. Corvol P. Llorens-Cortes C. J. Biol. Chem... 1996; 271: 9069-9074Google Scholar-22Wang J.Y. Cooper M.D. Proc. Natl. Acad. Sci. U. S. A... 1993; 90: 1222-1226Google Scholar), we previously built a three-dimensional model of the mouse APA ectodomain from residues 79 to 559 (23Rozenfeld R. Iturrioz X. Maigret B. Llorens-Cortes C. J. Biol. Chem... 2002; 277: 29242-29252Google Scholar), including the active site of the enzyme. In this model, the zinc atom is coordinated by the two histidine residues (His-385 and His-389) of the consensus sequence HEXXH, Glu-408 and a water molecule, as previously shown (21Vazeux G. Wang J. Corvol P. Llorens-Cortes C. J. Biol. Chem... 1996; 271: 9069-9074Google Scholar, 22Wang J.Y. Cooper M.D. Proc. Natl. Acad. Sci. U. S. A... 1993; 90: 1222-1226Google Scholar). We then docked a potent and selective APA inhibitor, 4-amino-4-phosphobutyric acid (glutamate phosphonate) into the three-dimensional model of the APA active site. This inhibitor behaves as a transition state pseudoanalog, in which the replacement of the substrate scissile amide bond with a phosphonic acid group mimics the tetrahedral transition state (24Lejczak B. De Choszczak M.P. Kafarski P. J. Enzyme Inhib... 1993; 7: 97-103Google Scholar). Analysis of the APA three-dimensional model complexed with glutamate phosphonate showed that Tyr-471 interacted with the inhibitor, providing support for previous demonstrations that this residue is essential for stabilizing the transition state of catalysis in APA (19Vazeux G. Iturrioz X. Corvol P. Llorens-Cortes C. Biochem. J... 1997; 327: 883-889Google Scholar). This model also provided evidence of an interaction between the N-terminal amine of glutamate phosphonate and the Glu-352 and Glu-215 residues of APA, which are responsible for the exopeptidase specificity of this enzyme (20Vazeux G. Iturrioz X. Corvol P. Llorens-Cortes C. Biochem. J... 1998; 334: 407-413Google Scholar, 25Rozenfeld R. Iturrioz X. Okada M. Maigret B. Llorens-Cortes C. Biochemistry.. 2003; 42: 14785-14793Google Scholar). We then introduced Ca2+ into this three-dimensional model. We identified the Ca2+ ligands in the S1 subsite: Asp-213 (corresponding to Asp-221 in human APA (26Goto Y. Hattori A. Mizutani S. Tsujimoto M. J. Biol. Chem... 2007; 282: 37074-37081Google Scholar)) and Asp-218 (27Claperon C. Rozenfeld R. Iturrioz X. Inguimbert N. Okada M. Roques B. Maigret B. Llorens-Cortes C. Biochem. J... 2008; 416: 37-46Google Scholar). We also visualized another residue in the S1 subsite, Thr-348, which interacts with the side chain of the inhibitor. We characterized here the role of Thr-348 in substrate/inhibitor binding and substrate specificity of APA for N-terminal acidic amino acid residues further, through a combination of molecular modeling and site-directed mutagenesis studies. We replaced Thr-348 by a serine, tyrosine, or aspartate residue. We checked that the mutated enzymes displayed similar processing and had a similar subcellular distribution to wild type APA. We then biochemically and kinetically characterized the purified recombinant wild type and mutated enzymes and determined their sensitivity to Ca2+ and various inhibitors. We also evaluated the influence of the mutations on APA substrate specificity. The effects of the mutations on the three-dimensional model of APA were followed in parallel. Restriction endonucleases and DNA-modifying enzymes were obtained from New England Biolabs Inc. and were used according to the manufacturer's instructions. The Expand high fidelity Taq polymerase PCR system was purchased from Roche Applied Science. The liposomal transfection reagent, Lipofectamine 2000, the pcDNA 3.1-His vector, and the monoclonal anti-Xpress antibody were purchased from Invitrogen. The monoclonal anti-α-tubulin antibody and the horseradish peroxidase-conjugated goat anti-mouse antibody was purchased from Sigma-Aldrich. Immobilized cobalt affinity columns (Talon) were obtained from Clontech (Heidelberg, Germany). The synthetic substrate, α-l-glutamyl-β-naphthylamide (GluNA), was purchased from Bachem (Bunderdorf, Switzerland). Modeling of the Mutated APA + Ca2+ System—The protein + ligand + Zn2+ + Ca2+ system was investigated as previously described (27Claperon C. Rozenfeld R. Iturrioz X. Inguimbert N. Okada M. Roques B. Maigret B. Llorens-Cortes C. Biochem. J... 2008; 416: 37-46Google Scholar); we checked the stability of the system by carrying out several rounds of energy minimization followed by short molecular dynamics runs, using a molecular mechanics program and simulation protocol similar to those described in a previous study (23Rozenfeld R. Iturrioz X. Maigret B. Llorens-Cortes C. J. Biol. Chem... 2002; 277: 29242-29252Google Scholar). The refined whole model of APA + glutamate phosphonate + Zn2+ + Ca2+ + water obtained in this way was used for all of the subsequent calculations. The mutants were obtained by changing only the side chains of the amino acid residue 348 and subjecting the corresponding structures to a new cycle of refinement. Cloning and Site-directed Mutagenesis—The mouse cDNA encoding APA was used as a template for the generation of mutants by PCR-based site-directed mutagenesis, as previously described (28Herlitze S. Koenen M. Gene (Amst.).. 1990; 91: 143-147Google Scholar). Two overlapping regions of the cDNA were amplified separately, using two flanking oligonucleotides: A (5′-TTAATACGACTCACTATAGGGA-3′; corresponding to nucleotides 862–883) as a forward primer and B (5′-GAATCCTAAGATAGAGGCCCGGAG-3′; corresponding to nucleotides 3215–3238) as a reverse primer, together with two overlapping oligonucleotides corresponding to nucleotides 2045–2065 and containing the mutated residue at the appropriate position (C1D1 for Ala-348, C2D2 for Asp-348, C3D3 for Ser-348, and C4D4 for Tyr-348). The forward primers were: C1, 5′-GATTTTGGCGCCGGCGCCATG-3′; C2, 5′-GATTTTGGCGACGGCGCCATG-3′; C3, 5′-GATTTTGGCTCAGGCGCCATG-3′; and C4, 5′-GATTTTGGCTACGGCGCCATG-3′. The reverse primers were: D1, 5′-CATGGCGCCGGCGCCAAAATC-3′; D2, 5′-CATGGCGCCGTCGCCAAAATC-3′; D3, 5′-CATGGCGCCTGAGCCAAAATC-3′; and D4, 5′-CATGGCGCCGTAGCCAAAATC-3′. The underlined bases encode the new amino acid residue and replace the naturally occurring codon at position 348. Nucleotide numbering is as for the mouse APA sequence (29Wu Q. Lahti J.M. Air G.M. Burrows P.D. Cooper M.D. Proc. Natl. Acad. Sci. U. S. A... 1990; 87: 993-997Google Scholar) deposited in the GenBank™/EBI data base (accession number M29961). The products of the first two rounds of amplification (A-D1–4 and B-C1–4) were used as the template for a second PCR with the two flanking oligonucleotides, A and B. For all PCRs, high fidelity Taq polymerase (Roche Applied Science) (1 unit) was used (25 cycles: 94 °C for 30 s, 54 °C for 45 s, and 72 °C for 2 min). The final 2376-bp PCR product was digested with HindIII and EcoRV, and the resulting 1505-bp HindIII-EcoRV fragment containing the mutation was used to replace the corresponding nonmutated region (HindIII-EcoRV) of the full-length APA cDNA. The presence of the mutation and the absence of nonspecific mutations were confirmed by automated sequencing on an Applied Biosystems 377 DNA sequencer with dye deoxyterminator chemistry. Cell Culture, Establishment of Stable CHO-K1 Cell Lines Producing Wild type and Mutated His-APAs, and Purification of Recombinant His-APAs—CHO-K1 (American Type Culture Collection, Manassas, VA) cells were maintained in Ham's F-12 medium supplemented with 7% fetal calf serum, 0.5 mm glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin (all from Invitrogen). The cells were transfected with 1 μg of plasmid containing the polyhistidine-tagged wild type or a polyhistidine-tagged mutated APA cDNA, using Lipofectamine 2000 (Invitrogen), and stable cell lines producing the polyhistidine-tagged wild type and mutated His-APAs were established as previously described (18Iturrioz X. Vazeux G. Celerier J. Corvol P. Llorens-Cortes C. Biochemistry.. 2000; 39: 3061-3068Google Scholar). Stably transfected CHO cells were harvested, and a crude membrane preparation was obtained, as previously described (18Iturrioz X. Vazeux G. Celerier J. Corvol P. Llorens-Cortes C. Biochemistry.. 2000; 39: 3061-3068Google Scholar). Wild type and mutated His-APAs were purified from the solubilized crude membrane preparation by metal affinity chromatography with a metal chelate resin column (Talon Co2+), as previously described (18Iturrioz X. Vazeux G. Celerier J. Corvol P. Llorens-Cortes C. Biochemistry.. 2000; 39: 3061-3068Google Scholar). The purity of the final preparation was assessed by SDS-PAGE in 8% polyacrylamide gels, as described by Laemmli (30Laemmli U.K. Nature.. 1970; 227: 680-685Google Scholar). The proteins were stained with Coomassie Brilliant Blue R-250. We routinely obtain a purified protein >80% pure with an overall yield of about 14% and a purification factor of 10. Protein concentrations were determined with the BCA protein assay kit (Pierce), using bovine serum albumin (BSA) as the standard. Western Blotting—Purified wild type and mutated His-APAs were resolved by 8% SDS-PAGE, and the proteins were transferred to a polyvinylidene difluoride membrane in 25 mm Tris/glycine buffer, pH 8.3, supplemented with 20% (V/V) methanol. His-tagged recombinant proteins were detected with the anti-Xpress antibody (dilution 1:5000), and loading control was done with the anti-α-tubulin antibody (dilution 1: 4000). The resulting immune complex was then detected with the ECL Plus Western blotting detection system (GE Healthcare). Immunofluorescence Analysis of Stably Transfected CHO Cells—CHO cells producing wild type and mutated His-APAs were dispensed (25,000 cells) onto 14-mm-diameter coverslips. The cells were cultured for 24 h in Ham's F-12 medium in a humidified atmosphere of 5% CO2, 95% air. They were then fixed and permeabilized by incubation for 5 min in 100% ice-cold methanol. The cells were rinsed three times in 0.1 m PBS, pH 7.4, and then saturated by incubation with 5% BSA for 30 min at room temperature. They were then incubated with a 1:1000 dilution of rabbit polyclonal anti-(rat APA) serum (31Song L. Ye M. Troyanovskaya M. Wilk E. Wilk S. Healy D.P. Am. J. Physiol... 1994; 267: F546-F557Google Scholar) in 0.5% BSA in PBS for 2 h at room temperature. The coverslips were washed three times with cold PBS and then incubated with a 1:1000 dilution of cyanin 3-conjugated polyclonal anti-rabbit antibody in 0.5% BSA in PBS for 2 h at room temperature. The coverslips were washed three times with PBS and once with water and mounted in Mowiol. The cells were examined with a Leica TCS SP II (Leica Microsystems, Heidelberg, Germany) confocal laser scanning microscope equipped with an argon/krypton laser and configured with a Leica DM IRBE inverted microscope. Cyanin 3 fluorescence was detected after 100% excitation at 568 nm. The images (1024 × 1024 pixels) were obtained with a 63× magnification oil-immersion objective. Each image corresponded to a cross-section of the cell. Enzyme Assay—Enzyme assays were carried out for wild type and mutated His-APA in 50 mm Tris-HCl buffer, pH 7.4, with or without 4 mm CaCl2, by monitoring the rate of hydrolysis of GluNA, as previously described (8Chauvel E.N. Llorens-Cortès C. Coric P. Wilk S. Roques B. Fournié-Zaluski M.C. J. Med. Chem... 1994; 37: 2950-2956Google Scholar). The sensitivity to Ca2+ of purified wild type and mutated His-APAs was determined by incubating 0.5 mm GluNA with 0 or 4 mm CaCl2 in a final volume of 100 ml of 50 mm Tris-HCl buffer, pH 7.4. All of the assays were performed in black 96-well plates (solid black 96-well flat-bottomed nontreated plates with no lid; Corning Costar). All of the enzymatic studies were performed such that substrate hydrolysis rates were maintained below 10% (initial rate conditions). The progress curves were monitored by following the increase in fluorescence at 460 nm (lex = 330 nm), induced by the release of the β-naphthylamine fluorogenic part of the substrate by APA. Fluorescence signals were monitored by counting photons with a spectrophotometer (Fusion™ universal microplate analyzer; Packard) equipped with a temperature control device and a plate shaker. The kinetic parameters (Kmand kcat) were determined from Michaelis-Menten plots, using Enzfitter software (Biosoft™), with a final concentration of substrate (GluNA) from 0.005 to 2 mm. For evaluation of the substrate specificity of APA in the absence or presence of Ca2+, the rate of hydrolysis of two additional substrates, LeuNA and LysNA, was determined under the same experimental conditions as described for GluNA. The sensitivity of wild type and mutated His-APAs to inhibition by glutamate thiol (GluSH), lysine thiol (LysSH), and methionine thiol (MetSH) was determined by establishing dose-dependent inhibition curves for a final GluNA concentration of 0.5 mm, in the presence of 0 or 4 mm CaCl2. Because these compounds are linear competitive inhibitors, their Ki were calculated from the formula Ki = IC50/(1+ [GluNA]/Km). Statistical Analysis—Statistical comparisons were carried out with Student's unpaired t test. Differences were considered significant if p was less than 0.05. Modeling of APA Mutants in the Absence of Ca2+ and in the Presence of the APA Inhibitor Glutamate Phosphonate—The three-dimensional model of wild type APA complexed with glutamate phosphonate obtained in the absence of Ca2+ showed the presence of a hydrophilic pocket containing two aspartate residues (Asp-213 and Asp-218) and a threonine residue (Thr-348), corresponding to the S1 subsite (Fig. 1A) (27Claperon C. Rozenfeld R. Iturrioz X. Inguimbert N. Okada M. Roques B. Maigret B. Llorens-Cortes C. Biochem. J... 2008; 416: 37-46Google Scholar). The acidic side chain of the inhibitor is pointing toward this pocket and the Cα amine of Thr-348 is engaged in one hydrogen bond with the acidic side chain of Asp-213. Moreover, the Thr-348 alcohol side chain is engaged in two balanced hydrogen bonds, one with the carboxylate side chain of Asp-213 and the other with the carboxylate side chain of glutamate phosphonate. In the absence of Ca2+, this interaction is skewed toward the carboxylate side chain of Asp-213. We investigated the role of Thr-348, by replacing this residue with a serine, aspartate, or tyrosine residue and constructing the corresponding three-dimensional active site models. The three-dimensional active site model of the Ser-348 mutated APA (Fig. 2) is similar to that of wild type APA, as described above, except that the Ser-348 hydroxyl group is hydrogen-bonded only with the carboxylate side chain of glutamate phosphonate. In the three-dimensional active site model of the Asp-348 mutated APA (Fig. 2), only the hydrogen bond between the Asp-348 backbone and the Asp-213 side chain is maintained, because the carboxylate side chain of Asp-348 now interacts with a water molecule. Greater disturbance of the binding pocket is observed in the three-dimensional model of Tyr-348 mutated APA (Fig. 2), because of the presence of an aromatic group involved in a pi-anion interaction with the carboxyl end of the inhibitor (32Schottel B.L. Chifotides H.T. Dunbar K.R. Chem. Soc. Rev... 2008; 37: 68-83Google Scholar). In this case, the position of the inhibitor differs from that in the wild type. Modeling of APA in the Presence of Ca2+ and in the Presence of the APA Inhibitor Glutamate Phosphonate—In the three-dimensional model of wild type or Ser-348 mutated APAs in the presence of Ca2+ and glutamate phosphonate (Figs. 1B and 2), the alcohol side chain of Thr- or Ser-348 in the S1 subsite establishes a hydrogen bond with the carboxylate side chain of glutamate phosphonate. Moreover, the nitrogen of the Cα amine moiety of residue 348 interacts with Asp-213 (27Claperon C. Rozenfeld R. Iturrioz X. Inguimbert N. Okada M. Roques B. Maigret B. Llorens-Cortes C. Biochem. J... 2008; 416: 37-46Google Scholar). The Ca2+ atom is thus connected to glutamate phosphonate through a water molecule. In the three-dimensional model of Asp-348 mutated APA (Fig. 2), the glutamate phosphonate-water-Ca2+ link is maintained, but the carboxylate side chain of Asp-348 displaces the water molecule away from the Ca2+ coordination shell. As in the three-dimensional model of the Tyr-348 mutated APA obtained in the absence of Ca2+, in the presence of Ca2+ (Fig. 2), the phenol ring of Tyr-348 strongly modifies the binding pocket and, consequently, the position of the inhibitor; Tyr-348 establishes a hydrogen bond with the carboxylate side chain of Glu-215, whereas a new hydrogen bond is created between the Gly-349 backbone and glutamate phosphonate, which is now held away from the Ca2+ atom through a network of two water molecules. Site-directed Mutagenesis, Expression, and Purification of Recombinant Wild type and Mutant His-APAs—We investigated the role of Thr-348 in substrate/inhibitor binding and substrate specificity by replacing this residue with an alanine, serine, aspartate, or tyrosine residue by site-directed mutagenesis. Wild type and mutated His-APAs were then stably expressed, and the recombinant enzymes were purified by metal affinity chromatography, as previously described (18Iturrioz X. Vazeux G. Celerier J. Corvol P. Llorens-Cortes C. Biochemistry.. 2000; 39: 3061-3068Google Scholar). We first checked that the mutations did not affect the production and processing of the recombinant proteins. For this purpose, we analyzed by Western blot wild type and mutated His-APAs transitory transfected in CHO cells. We showed that all recombinant proteins, except the Ala-348 mutant, displayed a major mature 160-kDa form and a minor immature 140-kDa form. The Ala-348 mutant displayed a truncated form of 40-kDa (Fig. 3A), probably reflecting the cleavage of this misfolded mutated protein. In an additional set of experiments (data not shown) performed on CHO cells stably expressing the Ala-348 mutant, we also detected the presence of a truncated form of 40-kDa, but only when cells were treated with proteasome inhibitor, MG132 (16 μm, cell treatment for 16 h), suggesting that the mutation induces the cleavage of a nonmature unglycosylated form of APA rapidly degraded by the proteasome machinery. Moreover, we investigated the subcellular distribution of wild type and mutant His-APAs in stably transfected CHO cells by immunofluorescence staining with a rabbit polyclonal anti-rat APA antibody and a cyanin-3-conjugated anti-rabbit secondary antibody. Confocal microscopy analysis of CHO cells stably expressing wild type and mutated His-APA constructs showed that APA was located at the plasma membrane, except for the Ala-348 mutant APA, which remained trapped in an intracellular network possibly corresponding to the endoplasmic reticulum (Fig. 3B). We therefore did not carry out enzymatic characterization of this mutant. Finally, Western blot analysis of purified wild type and mutated His-APAs stably expressed in CHO cells showed that all proteins displayed a mature 160-kDa form (Fig. 3C). Enzymatic Activity of Purified Wild type and Mutated His-APAs—Because APA activity is enhanced by Ca2+, we evaluated the enzymatic activity of purified wild type and mutated APAs in the presence or absence of 4 mm Ca2+, using GluNA as a substrate (Table 1). In the absence of Ca2+, the replacement of Thr-348 by a serine residue led to a significant increase in substrate hydrolysis, by a factor of 1.5 compared with wild type His-APA. By contrast, the level of enzymatic activity detected for the Asp-348 and Tyr-348 mutants was significantly lower, corresponding to 40 and 13% of wild type His-APA activity levels, respectively. In the presence of 4 mm Ca2+, the enzymatic activity of Ser-348 remained significantly (2 times) higher than that of the wild type His-APA, whereas those of Asp-348 and Tyr-348 APA were significantly lower, corresponding to 11 and 50% of wild type His-APA activity, respectively. The presence of 4 mm Ca2+ enhanced the enzymatic activity of wild type, Ser-348, Asp-348, or Tyr-348 His-APA by factors of 8, 10, 2, and 30, respectively (Table 1).Table 1Enzymatic activity of purified recombinant wild type and mutated His-APAs Shown are the enzymatic activity (μmol of GluNA hydrolyzed/min/mg of protein) means ± the standard error from three to six separate experiments with duplicate determinations. Recombinant His-APAs No Ca2+ 4 mm Ca2+ Factor of stimulation Wild type 1.98 ± 0.12 14.67 ± 0.42dp < 0.05 8 Ser-348 3.05 ± 0.15ap < 0.05 (corresponding mutated His-APA vs. corresponding wild-type APA) 31.54 ± 1.33ap < 0.05 (corresponding mutated His-APA vs. corresponding wild-type APA),cNot significant when compared to the corresponding recombinant enzyme activity in the absence of Ca2+ 10 Asp-348 0.8 ± 0.05ap < 0.05 (corresponding mutated His-APA vs. corresponding wild-type APA) 1.6 ± 0.14ap < 0.05 (corresponding mutated His-APA vs. corresponding wild-type APA),dp < 0.05 2 Tyr-348 0.25 ± 0.007bp < 0.01 (corresponding mutated His-APA vs. corresponding wild-type APA) 7.4 ± 0.13ap < 0.05 (corresponding mutated His-APA vs. corresponding wild-type APA),dp < 0.05 30a p < 0.05 (corresponding mutated His-APA vs. corresponding wild-type APA)b p < 0.01 (corresponding mutated His-APA vs. corresponding wild-type APA)c Not significant when compared to the corresponding recombinant enzyme activity in the absence of Ca2+d p < 0.05 Open table in a new tab Kinetic Parameters for Purified Recombinant Wild type and Mutated His-APAs—The enzymatic activities of purified recombinant His-APAs were analyzed by determining the catalytic constants (Km and kcat) in the absence or presence of 4 mm Ca2+, using GluNA as a substrate. The results are summarized in Table 2. In the absence of Ca2+, the replacement of Thr-348 with a serine residue doubled hydrolysis efficiency. By contrast, the replacement of Thr-348 with an aspartate or a tyrosine residue led to decreases in kcat/Km by factors" @default.
• W2094094258 created "2016-06-24" @default.
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• W2094094258 date "2009-04-01" @default.
• W2094094258 modified "2023-09-30" @default.
• W2094094258 title "Identification of Threonine 348 as a Residue Involved in Aminopeptidase A Substrate Specificity" @default.
• W2094094258 cites W1858848970 @default.
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