Acidic Hydrolysis as a Mechanism for the Cleavage of the Glu298 → Asp Variant of Human Endothelial Nitric-oxide Synthase

The 894G→T polymorphism within exon 7 of the human endothelial nitric-oxide synthase (eNOS) gene codes for glutamate or aspartate, respectively, at residue 298 and has been associated with several diseases of cardiovascular origin. A recent report indicates that Asp298-eNOS (E298D) is cleaved intracellularly to 100- and 35-kDa fragments, suggesting a mechanism for reduced endothelial function. Here we have documented the precise cleavage site of the E298D variant as a unique aspartyl-prolyl (Asp298–Pro299) bond not seen in wild-type eNOS (Glu298). We show that E298D-eNOS, as isolated from cells and in vitro, is susceptible to acidic hydrolysis, and the 100-kDa fragment can be generated ex vivo by increasing temperature at low pH. Importantly, cleavage of E298D was eliminated using a sample buffer system designed to limit acidic hydrolysis of Asp–Pro bonds. These results argue against intracellular processing of E298D-eNOS and suggest that previously described fragmentation of E298D could be a product of sample preparation. We also found that eNOS turnover, NO production, and the susceptibility to cellular stress were not different in cells expressing WTversus E298D-eNOS. Finally, enzyme activities were identical for the respective recombinant enzymes. Thus, intracellular cleavage mechanisms are unlikely to account for associations between the exon 7 polymorphism and cardiovascular diseases. The 894G→T polymorphism within exon 7 of the human endothelial nitric-oxide synthase (eNOS) gene codes for glutamate or aspartate, respectively, at residue 298 and has been associated with several diseases of cardiovascular origin. A recent report indicates that Asp298-eNOS (E298D) is cleaved intracellularly to 100- and 35-kDa fragments, suggesting a mechanism for reduced endothelial function. Here we have documented the precise cleavage site of the E298D variant as a unique aspartyl-prolyl (Asp298–Pro299) bond not seen in wild-type eNOS (Glu298). We show that E298D-eNOS, as isolated from cells and in vitro, is susceptible to acidic hydrolysis, and the 100-kDa fragment can be generated ex vivo by increasing temperature at low pH. Importantly, cleavage of E298D was eliminated using a sample buffer system designed to limit acidic hydrolysis of Asp–Pro bonds. These results argue against intracellular processing of E298D-eNOS and suggest that previously described fragmentation of E298D could be a product of sample preparation. We also found that eNOS turnover, NO production, and the susceptibility to cellular stress were not different in cells expressing WTversus E298D-eNOS. Finally, enzyme activities were identical for the respective recombinant enzymes. Thus, intracellular cleavage mechanisms are unlikely to account for associations between the exon 7 polymorphism and cardiovascular diseases. nitric oxide endothelial nitric-oxide synthase wild type polymerase chain reaction polyacrylamide gel electrophoresis lithium dodecyl sulfate Endothelium-derived nitric oxide (NO)1 plays a prominent role in regulating systemic blood pressure and maintaining vascular homeostasis. NO has also been recognized as an important mediator of structural changes in the vasculature, including flow and injury evoked vascular remodeling and angiogenesis (1Rudic R.D. Shesely E.G. Maeda N. Smithies O. Segal S.S. Sessa W.C. J. Clin. Invest. 1998; 101: 731-736Crossref PubMed Scopus (706) Google Scholar, 2Murohara T. Asahara T. Silver M. Bauters C. Masuda H. Kalka C. Kearney M. Chen D. Symes J.F. Fishman M.C. Huang P.L. Isner J.M. J. Clin. Invest. 1998; 101: 2567-2578Crossref PubMed Scopus (1092) Google Scholar, 3Moroi M. Zhang L. Yasuda T. Virmani R. Gold H.K. Fishman M.C. Huang P.L. J. Clin. Invest. 1998; 101: 1225-1232Crossref PubMed Scopus (295) Google Scholar). Within endothelial cells that line the lumen of all blood vessels, endothelial nitric-oxide synthase (eNOS) catalyzes calcium-calmodulin-dependent NO synthesis through the conversion of l-arginine to l-citrulline and NO (4Forstermann U. Pollock J.S. Schmidt H.H. Heller M. Murad F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1788-1792Crossref PubMed Scopus (551) Google Scholar). Dysfunction of the endothelium, often associated with a reduction in the activity or expression of eNOS or NO bioreactivity, is a hallmark of cardiovascular diseases such as hypertension, diabetes, heart failure and atherosclerosis (5Rudic R.D. Sessa W.C. Am. J. Hum. Genet. 1999; 64: 673-677Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Given the importance of eNOS to cardiovascular function, the investigation into whether mutations or polymorphisms within the eNOS gene correlate with increased risk of cardiovascular disease has become an active area of research. The human eNOS gene has 26 exon regions and covers 21 kilobase pairs on the long arm of chromosome 7 (6Marsden P.A. Heng H.H. Scherer S.W. Stewart R.J. Hall A.V. Shi X.M. Tsui L.C. Schappert K.T. J. Biol. Chem. 1993; 268: 17478-17488Abstract Full Text PDF PubMed Google Scholar). To date, there are no positive studies demonstrating that mutations in the eNOS gene are causally linked to a disease process using traditional linkage analysis (7Wang X.L. Wang J. Mol. Genet. Metab. 2000; 70: 241-251Crossref PubMed Scopus (171) Google Scholar). However, in patients with coronary spasm (8Nakayama M. Yasue H. Yoshimura M. Shimasaki Y. Ogawa H. Kugiyama K. Mizuno Y. Harada E. Nakamura S. Ito T. Saito Y. Miyamoto Y. Ogawa Y. Nakao K. Am. J. Cardiol. 2000; 86: 628-634Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar) or renal disease (9Zanchi A. Moczulski D.K. Hanna L.S. Wantman M. Warram J.H. Krolewski A.S. Kidney Int. 2000; 57: 405-413Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar), polymorphisms within the eNOS promoter have been postulated to impact levels of mRNA and protein, whereas in patients with coronary artery disease (10Hingorani A.D. Liang C.F. Fatibene J. Lyon A. Monteith S. Parsons A. Haydock S. Hopper R.V. Stephens N.G. O'Shaughnessy K.M. Brown M.J. Circulation. 1999; 100: 1515-1520Crossref PubMed Scopus (500) Google Scholar) or hypertension (11Miyamoto Y. Saito Y. Kajiyama N. Yoshimura M. Shimasaki Y. Nakayama M. Kamitani S. Harada M. Ishikawa M. Kuwahara K. Ogawa E. Hamanaka I. Takahashi N. Kaneshige T. Teraoka H. Akamizu T. Azuma N. Yoshimasa Y. Yoshimasa T. Itoh H. Masuda I. Yasue H. Nakao K. Hypertension. 1998; 32: 3-8Crossref PubMed Scopus (488) Google Scholar) polymorphisms within exon regions of eNOS may affect enzyme function. Conversely, many other studies do not show associations of the above polymorphisms with the disease (7Wang X.L. Wang J. Mol. Genet. Metab. 2000; 70: 241-251Crossref PubMed Scopus (171) Google Scholar). The exon 7 polymorphism (894G→T) that specifies either a glutamate (E) or aspartate (D) residue at position 298 in the human eNOS protein has been analyzed in several patient populations with coronary artery disease, hypertension, and cerebral vascular disease (12Hibi K. Ishigami T. Tamura K. Mizushima S. Nyui N. Fujita T. Ochiai H. Kosuge M. Watanabe Y. Yoshii Y. Kihara M. Kimura K. Ishii M. Umemura S. Hypertension. 1998; 32: 521-526Crossref PubMed Scopus (332) Google Scholar, 13Lacolley P. Gautier S. Poirier O. Pannier B. Cambien F. Benetos A. J. Hypertens. 1998; 16: 31-35Crossref PubMed Scopus (153) Google Scholar, 14MacLeod M.J. Dahiyat M.T. Cumming A. Meiklejohn D. Shaw D. St. Clair D. Neurology. 1999; 53: 418-420Crossref PubMed Google Scholar). This polymorphism is of particular interest because this conservative amino acid substitution within the oxygenase domain of eNOS may influence eNOS function. Within each disease category, there is evidence both for and against this polymorphism influencing eNOS and/or endothelial function. For example, two groups have differing results concerning the association of the exon 7 polymorphism and essential hypertension in the Japanese population (11Miyamoto Y. Saito Y. Kajiyama N. Yoshimura M. Shimasaki Y. Nakayama M. Kamitani S. Harada M. Ishikawa M. Kuwahara K. Ogawa E. Hamanaka I. Takahashi N. Kaneshige T. Teraoka H. Akamizu T. Azuma N. Yoshimasa Y. Yoshimasa T. Itoh H. Masuda I. Yasue H. Nakao K. Hypertension. 1998; 32: 3-8Crossref PubMed Scopus (488) Google Scholar, 15Kato N. Sugiyama T. Morita H. Nabika T. Kurihara H. Yamori Y. Yazaki Y. Hypertension. 1999; 33: 933-936Crossref PubMed Scopus (126) Google Scholar). Based upon the crystal structure of the oxygenase domain of eNOS, the substitution of a glutamate residue for an aspartate at position 298 within the protein is unlikely to alter protein conformation to an appreciable extent (16Raman C.S. Li H. Martasek P. Kral V. Masters B.S. Poulos T.L. Cell. 1998; 95: 939-950Abstract Full Text Full Text PDF PubMed Scopus (579) Google Scholar). However, a recent report demonstrated that eNOS, as isolated from patients with an 894T allele (coding for aspartate at residue 298), was cleaved intracellularly by an unknown protease, thus providing a possible mechanism to explain an impairment in eNOS function (17Tesauro M. Thompson W.C. Rogliani P. Qi L. Chaudhary P.P. Moss J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2832-2835Crossref PubMed Scopus (471) Google Scholar). Because most aspects of the proposed intracellular cleavage remain to be determined, we sought to: 1) elucidate the mechanism whereby the Glu298 → Asp variant of eNOS (E298D) is cleaved and 2) further characterize enzymatic differences between wild-type eNOS and cleaved E298D eNOS in cells andin vitro. Total RNA from single-donor human umbilical vein endothelial cells was isolated with TriZol reagent and subjected to reverse transcriptase-PCR using an oligo(dT)17 primer. The reverse transcriptase-PCR product corresponding to human eNOS was PCR-amplified using the forward primer 5′-ccccgccaaagcttggacgcacagtaccatgggcaacttgaagagcg-3′ and the reverse primer 5′-ggggctctagaggcggacctgagtcgggcagccgc-3′. The amplified product was ligated into (HindIII andXbaI) pcDNA3.1 (Invitrogen) mammalian expression vector. The wild-type human eNOS sequence was verified by DNA sequencing. The generation of E298D eNOS cDNA was performed by site-directed mutagenesis (QuikChange, Stratagene) according to the manufacturer's protocol. Complementary primers were used for mutagenesis (+ strand oligonucleotide: 5′-gcccctgctgctgcaggctccggatgatcccccagaactcttcc-3′). A 898-base pair fragment containing the mutation was subcloned into the original WT vector (FseI and NheI), and the DNA sequence was verified across the mutation site. All DNA sequencing and oligonucleotide synthesis were carried out at the W. M. Keck Biotechnology Resource Center at Yale University School of Medicine. Unless otherwise noted, COS-7 cells were cultured in high glucose Dulbecco's modified Eagle's medium containing 10% (v/v) fetal bovine serum, penicillin, streptomycin, and l-glutamine as described previously (18Garcia-Cardena G. Oh P. Liu J. Schnitzer J.E. Sessa W.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6448-6453Crossref PubMed Scopus (578) Google Scholar). Unless otherwise noted, an equal number of cells were seeded in 60-mm dishes, such that, typically, 24 h later the cells were 90% confluent and ready for transfection. Transfections were carried out using LipofectAMINE 2000 (Life Technologies) following the manufacturer's protocol. In some experiments, transfected COS cells were treated with varying conditions to evoke cell stress. To examine the effects of hypoxia, the transfected cells were placed into an incubator and equilibrated with 1% oxygen for 48 h, as described previously (19Banasiak K.J. Haddad G.G. Brain Res. 1998; 797: 295-304Crossref PubMed Scopus (137) Google Scholar). To trigger cellular apoptosis, COS cells were treated with staurosporine (1 µm) for 6 h (20Bertrand R. Solary E. O'Connor P. Kohn K.W. Pommier Y. Exp. Cell Res. 1994; 211: 314-321Crossref PubMed Scopus (471) Google Scholar). To initiate oxidative stress, transfected cells were incubated with H2O2 (5 mm) for 30 min. At the end of each incubation period, samples were prepared for electrophoresis using the NUPAGE buffer system (described below). For all experiments on lysates (except when intentionally altering pH), 48 h post-transfection COS-7 cells were lysed in modified radioimmune precipitation buffer (50 mm Tris-Cl, pH 7.4, 1% Nonidet P-40, 0.1 mm EDTA, 0.1 mm EGTA, 0.1% SDS, 0.1% deoxycholic acid, 1 mm Pefabloc, 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 2 µg/ml pepstatin). Sample preparation for SDS-PAGE was performed using either the traditional Laemmli method (21Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207538) Google Scholar) or the commercial NUPAGE system (Novex/Invitrogen) that was designed to limit in vitro degradation of protein samples. A Laemmli-style loading buffer (6× concentrated SDS/Tris-based buffer/dye at pH 6.8 (22 °C) with β-mercaptoethanol) was added to the samples, and the samples were boiled at 100 °C for 5 min. Alternatively, we followed the protocols of the NUPAGE system, where a lithium dodecyl sulfate (LDS) sample buffer (Tris/glycerol buffer, pH 8.5) was mixed with fresh dithiothreitol and added to samples. The LDS samples were then heated to 70 °C for 10 min. All cell lysates were separated by electrophoresis on 7.5% polyacrylamide gels and transferred to nitrocellulose membranes. WT and the E298D eNOS were detected using a monoclonal antibody (Transduction Laboratories,N30020) directed at the carboxyl terminus of human eNOS, an epitope conserved in WT and E298D eNOS. Ten plates of COS-7 cells (100-m plates) were transfected with WT or E298D eNOS cDNA. Cells were lysed with modified radioimmune precipitation buffer, and eNOS was isolated using 2′-5′ ADP-Sepharose (Amersham Pharmacia Biotech) as described previously (22McCabe T.J. Fulton D. Roman L.J. Sessa W.C. J. Biol. Chem. 2000; 275: 6123-6128Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar). Bound proteins were eluted by adding one volume of Laemmli-style sample buffer to the Sepharose bed and boiling for 5 min in a 100 °C block heater. Eluted proteins were subjected to a 7.5% SDS-PAGE and then transferred to a polyvinylidene difluoride membrane. Proteins were visualized by staining the membrane with a Coomassie Brilliant Blue stain (Sigma), and the unique 100-kDa band generated from the E298D mutant was sequenced by Edman degradation (W. M. Keck Biotechnology Resource Center at Yale University School of Medicine). WT and E298D cDNAs were subcloned into the bacterial expression vector pCW (NdeI and XbaI). pCW-WT or pCW-E298D were transformed into BL-21 E. with a vector for the proteins and and eNOS were performed as previously described P. Liu Liu J. Roman L.J. S.S. Sessa W.C. Masters B.S. Res. 1996; PubMed Scopus Google Scholar). buffer were mixed to pH and cells were using the above pH 1% Nonidet P-40, 0.1 mm EDTA, 0.1 mm EGTA, 0.1% SDS, 1 mm Pefabloc, 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 2 µg/ml pepstatin). were for 10 h at were to pH using and prepared for SDS-PAGE using NUPAGE sample preparation. of in was performed as described previously D. T.J. J. Y. K. A. Sessa W.C. Nature. 1999; PubMed Scopus Google Scholar). levels of NO were by from cells to h and reduction were all performed as described previously (22McCabe T.J. Fulton D. Roman L.J. Sessa W.C. J. Biol. Chem. 2000; 275: 6123-6128Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar). confluent COS-7 cells from 100 mm cell plates were and into 60-mm cell Cells were to in was to Dulbecco's modified Eagle's medium (Life for min to (Amersham Pharmacia Biotech) was added to and cells were incubated with the medium for 1 Cells were and incubated in Dulbecco's modified Eagle's medium at were using antibody (Transduction and protein Eluted proteins were separated by were for h and then to at °C for at was by J. G. Sessa W.C. PubMed Scopus Google Scholar). The allele of the polymorphism is the 894T variant in populations to in and in Japanese H. Wang X.L. J. Mol. 1999; PubMed Scopus Google For this we have eNOS with glutamate at 298 as wild type and eNOS with aspartate at 298 as E298D eNOS. was that E298D eNOS may intracellular cleavage (17Tesauro M. Thompson W.C. Rogliani P. Qi L. Chaudhary P.P. Moss J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2832-2835Crossref PubMed Scopus (471) Google Scholar). To the mechanism of this we both of eNOS in COS-7 cells that do not eNOS seen in a unique 100-kDa cleavage fragment was in cell lysates containing the E298D eNOS, as previously (17Tesauro M. Thompson W.C. Rogliani P. Qi L. Chaudhary P.P. Moss J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2832-2835Crossref PubMed Scopus (471) Google Scholar). the antibody used was against an epitope from the carboxyl terminus of human eNOS, was that the cleavage within the domain of the To the precise of the cleaved we isolated the 100-kDa fragment and sequenced the amino terminus by Edman The sequenced of the fragment to through of eNOS, that E298D eNOS was cleaved on the carboxyl of the aspartate at position as in B. Because the cleavage site at the Asp–Pro we for that may at this site and found However, has been recognized for many that are susceptible to acid hydrolysis D. M. Res. 1970; PubMed Scopus Google Scholar). In one of using the method of Laemmli when samples for SDS-PAGE is that proteins are boiled in an acidic a of conditions that can protein degradation J. F. PubMed Scopus Google Scholar). the loading used in SDS-PAGE are pH the become acidic when example, a mm buffer with pH 6.8 at °C to pH at 100 vivo mechanism of eNOS we whether the cleavage could be an of sample preparation in when we used Laemmli-style and increased the boiling sample we increased generation of the 100-kDa fragment of interest in E298D samples 100-kDa fragment All other degradation were found in both WT and E298D eNOS. Importantly, when we used a sample buffer containing LDS to a pH sample we no fragmentation of E298D 1 with This that the unique 100-kDa fragment generated in E298D eNOS may from acid hydrolysis of the we that the cleavage of E298D eNOS this be in a in protein to To examine this we performed studies to the of WT E298D eNOS. in 2 the of the two proteins were and to the J. G. Sessa W.C. PubMed Scopus Google Scholar). experiments, we that sample preparation could account for cleavage of E298D eNOS and that the of the two proteins was not acid hydrolysis as a mechanism of cleavage of E298D eNOS, we whether acidic conditions could account for fragmentation of the To the of samples from the we to cell by The were to upon a pH of buffer at °C at pH when temperature was to 100 whereas pH upon to 100 in cell lysates were performed to whether fragmentation of E298D could upon to acidic Cells were transfected with cDNAs and lysates and further incubated in pH at °C for 10 seen in fragmentation of both E298D and WT eNOS at pH The fragmentation of E298D in pH a unique 100-kDa band of the a prominent band that is also seen in the WT eNOS, pH and At pH the prominent band seen at pH is not a a unique band in the the pH at pH the of the The unique 100-kDa band in E298D is in with the fragment of interest seen in 1 and These experiments evidence that upon to pH h at pH E298D be the enzyme to levels °C not cleavage 2 1 and where the samples are heated to 70 thus suggesting that both a in pH and temperature were for the However, to whether to cellular stress could evoke the cleavage of eNOS, transfected COS cells were to a of and samples were in LDS seen in of cells to h, 1% H2O2 to evoke oxidative stress or a of (20Bertrand R. Solary E. O'Connor P. Kohn K.W. Pommier Y. Exp. Cell Res. 1994; 211: 314-321Crossref PubMed Scopus (471) Google Scholar), not in the of the 100-kDa eNOS fragment the above suggest that the cleavage of the E298D eNOS to the 100-kDa may be generated in vitro. However, we out the that in the E298D eNOS may have and activities different from wild-type eNOS that may account for disease risk in of the 894T Thus, COS cells were transfected with WT and E298D eNOS cDNA and the of NO a was using in and the of and the levels of eNOS protein expression in experiments were not different from cells expressing the two eNOS. we both WT and E298D eNOS cDNAs into a bacterial expression and and the recombinant seen in both proteins were on Coomassie analysis of the activity of recombinant WT or E298D eNOS using of NO as an to the of NO or reduction as an to the of the eNOS domain to to an that proteins were 5 To that cleavage of E298D is a product of sample preparation cleaved we prepared recombinant for using NUPAGE or Laemmli-style to samples. 5 that recombinant E298D is cleaved in a to that from cell lysates prepared activity and cleavage of WT and E298D eNOS Coomassie gel of recombinant WT or E298D eNOS, from E. of activities of WT and E298D eNOS as by and In recombinant WT or E298D was prepared for analysis using either LDS or Laemmli sample The results in are the of Here we show that the documented E298D polymorphism in eNOS not to influence the or activity of the enzyme isolated from cells or the enzymatic activity of the recombinant of samples in Laemmli buffer for SDS-PAGE a 100-kDa fragment cleaved at the Asp–Pro suggesting that the of the 100-kDa fragment in human from of the E298D polymorphism is to because of sample preparation. However, we out the that this fragment can be generated in an in vivo by an unknown In we generated a 100-kDa protein corresponding to the fragment from the cleavage of E298D 1 and 2) and as a recombinant protein in E. analysis of this protein the domain that was an active on this cleavage can in a cellular is possible for this protein to oxygen to can to endothelial Alternatively, because eNOS is subjected to levels of including and may be by the In a linkage between the polymorphism E298D in eNOS and the of cardiovascular disease be on the cleavage or function of the We Haddad for the of

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