The missense Glu298Asp variant of the endothelial nitric oxide synthase gene is strongly associated with placental abruption

We recently identified a missense variant (Glu298Asp) that lies within exon 7 of the endothelial nitric oxide synthase (eNOS) gene, and that is associated with severe preeclampsia (proteinuric hypertension that develops as a consequence of pregnancy). Maternal hypertension is the most consistently identified factor predisposing to placental abruption. Our objective, therefore, was to analyze the association between the Glu298Asp eNOS gene variant and placental abruption. The study participants included 35 patients with histories of placental abruption and 170 control subjects. Screening for the Glu298Asp eNOS gene variant was carried out by analysis of polymerase chain reaction/restriction fragment length polymorphism. The analyses revealed that the frequency of the Glu298Asp variant (Glu298Asp homozygotes and heterozygotes) was significantly (P<0.001) higher in the placental abruption group (n=14; 40%) than in the control group (n=24; 14%). We conclude that the presence of the Glu298Asp eNOS gene variant could be a marker of increased risk of developing placental abruption.     

Functional characterization of Glu298Asp mutant human endothelial nitric oxide synthase purified from a yeast expression system.

The Glu298Asp polymorphism of human endothelial nitric oxide synthase (eNOS) has been reported to be associated with several cardiovascular diseases, including hypertension and myocardial infarction. Therefore, we investigated the effect of the Glu298Asp (E298D) mutation on the function of purified recombinant eNOS expressed in the yeast Pichia pastoris. Wild type (WT) and mutant exhibited comparable affinities for L-arginine (K(m) values 4.4+/-0.6 and 5.2+/-0.8 microM, respectively) and V(max) values (142+/-36 and 159+/-29 nmol of L-citrulline/mg min, respectively). The E298D mutation affected neither electron transfer through the reductase domain (measured as cytochrome c reduction) nor reductive O(2) activation (measured either as NADPH oxidation or as H(2)O(2) formation in the absence of L-arginine and tetrahydrobiopterin (BH4)). The mutant was activated by BH4 with an EC(50) of 0.24+/-0.04 microM, a value comparable to that obtained with WT eNOS (0.22+/-0.02 microM). Activation of the enzyme by Ca(2+) was not affected (EC(50)=0.50+/-0.04 and 0.49+/-0.02 microM for WT and E298D eNOS, respectively). Calmodulin (CaM) affinity, studied by radioligand binding using 125I-labeled CaM, revealed virtually identical K(D) (3.2+/-0.5 and 4.0+/-0.3nM) and B(max) (1.4+/-0.2 and 1.2+/-0.3 pmol/pmol subunit) values for WT and E298D eNOS, respectively. Furthermore, E298D eNOS did not differ from the WT enzyme with respect to heme and flavin content or the ability to form SDS-resistant dimers. To summarize, we obtained no evidence for altered enzyme function of the eNOS mutant that could explain endothelial dysfunction associated with the E298D polymorphism.     

A missense Glu298Asp variant in the endothelial nitric oxide synthase gene is associated with coronary spasm in the Japanese

Coronary spasm plays an important role in the pathogenesis of not only variant angina but also ischemic heart disease in general. However, the precise mechanism(s) by which coronary spasm occurs remains to be elucidated. Coronary spasm may arise from interactions between environmental and genetic factors. Endothelial derived nitric oxide (NO) has been implicated in the control of vascular tone. We have recently shown that both basal and acetylcholine (ACh)-induced NO activities are impaired in the coronary arteries of patients with coronary spasm. The purpose of this study has been to elucidate the possible variants that occur in the coding region of the endothelial nitric oxide synthase (eNOS) gene and that may be associated with coronary spasm. After initial screening in the entire 26 coding regions of the eNOS gene, we found a missense Glu298Asp variant in exon 7 in patients with coronary spasm. We subsequently performed a larger scale study involving 113 patients with coronary spasm and 100 control subjects, who were all diagnosed by intracoronary injection of ACh. The analysis revealed a significant difference in the distribution of the variant between the coronary spasm group (21.2%) and control group (9.0%; P=0.014 for dominant effect). Thus, we have found the missense Glu298Asp variant in the eNOS gene by the analysis of its entire 26 coding regions. The variant is significantly associated with coronary spasm. Received: 2 February 1998 / Accepted: 9 April 1998     

Glu298Asp polymorphism of the endothelial nitric oxide synthase gene in Turkish patients with ischemic stroke

The low plasma nitric oxide concentrations and reduced vascular reactivity are considered major proatherogenic mechanisms in cardiovascular diseases. The present study aimed to assess the allelic frequency and the genotypic distribution of the Glu298Asp gene polymorphism at exon 7 of endothelial nitric oxide synthase (eNOS) gene in Turkish ischemic stroke patients compared to appropriate healthy controls, and to correlate the genetic findings with stroke subtypes. The study population included 146 (75 males, 71 females) patients with ischemic stroke which were categorized according to the Trial of ORG 10172 in Acute Stroke Treatment (TOAST) and 133 (34 males, 99 females) healthy subjects. The eNOS polymorphism was identified with a PCR followed by RFLP with the restriction enzyme BanII. Genotypes were defined as GG, GT, and TT according to the presence of the G and T alleles. In this case-control study, we did not find any significant difference in either the genotypic distribution or allelic frequency of Glu298Asp gene polymorphism between the patients and the controls. In addition, there was also no significant difference for the genotype distribution and the allelic frequency among the stroke subtypes. The results suggested the lack of the association between the Glu298Asp gene polymorphism and ischemic stroke or subtypes of ischemic stroke in the Turkish population.     

Relationship between hemorheology and Glu(298)Asp polymorphism of endothelial nitric oxide synthase gene in patients with coronary artery disease

This study aimed to investigate the relationship between endothelial nitric oxide synthase Glu(298)Asp gene polymorphism and hemorheological parameters. Red blood cell (RBC) deformability, aggregation were measured using an ectacytometry, whole blood, plasma viscosities were determined by a viscometer. Restriction fragment length polymorphism was used to detect polymorphism. Plasma nitrite, nitrate concentrations were determined by Griess method. The genotype distribution of the control group was as follows: 50 (67.5%) GG, 21 (28.4%) GT, 3 (4.1%) TT. A 48 (57.8%) of the patients with CAD had GG, 28 (33.7%) GT, 7 (8.5%) of them TT genotype. RBC aggregation index of CAD patients with G allele was higher and t½ lower compared to controls carrying the same allele. The amplitude of RBC aggregation of healthy subjects with T allele, who are under increased cardiovascular risk was lower compared to control subjects with G allele. The results of this study indicate that, alterations in RBC aggregation seem to be a consequence of CAD, more than being a preexisting cause. Additionally, some compensatory mechanisms by causing decrements in RBC aggregation, may help regulation of circulation in healthy individuals with high cardiovascular risk.     

NO Level and Endothelial NO Synthase Gene Polymorphism （Glu298Asp） in the Patients with Coronary Artery Disease from the Turkish Population

A healthy endothelium plays a core role in cardiovascu-lar control [1]. In the endothelial cell, nitric oxide (NO) issynthesized by the endothelial nitric oxide synthase (eNOS)encoded by a 26-exon gene (NOS 3) located on chromo-some 7 [2]. Besides its regulatory functions on vasomotortone and blood flow, endothelial NO is known to inhibitthe platelet activation and modulate migration and growthof the vascular smooth muscle [3]. Indirect evidence sug-gests that alterations of the NO pathwa…     

Association of the Glu298Asp polymorphism in the endothelial nitric oxide synthase gene with essential hypertension resistant to conventional therapy

Endothelial nitric oxide synthase (eNOS) produces nitric oxide (NO) which, after diffusing into vascular smooth muscle cells, activates guanylate cyclase leading to vasodilatation. A polymorphism (894G to T) in exon 7 of the eNOS gene causes the conversion of Glu to Asp in position 298. The recently described crystal structure of the heme domain of eNOS protein shows that Glu298 is fully solvent accessible and distant from regions integral to enzyme function. Searching for phenotypic expression of eNOS gene variants, we genotyped a group of patients with essential hypertension (H, n = 119) for the Glu298Asp polymorphism and compared them with age- and sex-matched healthy normals (N, n = 85). To specify phenotypic expression further, the hypertensive patients were subdivided into one group that responded well to regular antihypertensive therapy (CH, n = 45) and one group that was resistant to the therapy (RH, n = 74). Patients with BP higher than 140/90 mmHg when on adequate lifestyle modification and triple-combination therapy (including diuretics) were considered resistant. In RH and H groups, a significantly higher frequency of T alleles (P = 0.022 and P = 0.046, respectively) was found compared to normotonics (N). In well-controlled hypertonics, the same tendency was found, but did not reach statistical significance. The Glu298Asp polymorphism may contribute to the complex pathogenesis of essential hypertension and may be a factor in the resistance of these patients to conventional antihypertensive therapy. The presence of this allele may thus be predictive of the patients' therapeutic response.     

Neuronal nitric-oxide synthase mutant (Ser-1412 --> Asp) demonstrates surprising connections between heme reduction, NO complex formation, and catalysis

Rat neuronal NO synthase (nNOS) contains an Akt-dependent phosphorylation motif in its reductase domain. We mutated a target residue in that site (Ser-1412 to Asp) to mimic phosphorylation and then characterized the mutant using conventional and stopped-flow spectroscopies. Compared with wild-type, S1412D nNOS catalyzed faster cytochrome c and ferricyanide reduction but displayed slower steady-state NO synthesis with greater uncoupling of NADPH oxidation. Paradoxically, the mutant had faster heme reduction, faster heme-NO complex formation, and greater heme-NO complex accumulation at steady state. To understand how these behaviors related to flavin and heme reduction rates, we utilized three soybean calmodulins (CaMs) that supported a range of slower flavin and heme reduction rates in mutant and wild-type nNOS. Reductase activity and two catalytic parameters (speed and amount of heme-NO complex formation) related directly to the speed of flavin and heme reduction. In contrast, steady-state NO synthesis increased, reached a plateau, and then fell at the highest rate of heme reduction that was obtained with S1412D nNOS + CaM. Substituting with soybean CaM slowed heme reduction and increased steady-state NO synthesis by the mutant. We conclude the following. 1) The S1412D mutation speeds electron transfer out of the reductase domain. 2) Faster heme reduction speeds intrinsic NO synthesis but diminishes NO release in the steady state. 3) Heme reduction displays an optimum regarding NO release during steady state. The unique behavior of S1412D nNOS reveals the importance of heme reduction rate in controlling steady-state activity and suggests that nNOS already has a near-optimal rate of heme reduction.     

Kinetics of CO and NO ligation with the Cys(331)-->Ala mutant of neuronal nitric-oxide synthase

Nitric-oxide synthases (NOS) catalyze the conversion of l-arginine to NO, which then stimulates many physiological processes. In the active form, each NOS is a dimer; each strand has both a heme-binding oxygenase domain and a reductase domain. In neuronal NOS (nNOS), there is a conserved cysteine motif (CX(4)C) that participates in a ZnS(4) center, which stabilizes the dimer interface and/or the flavoprotein-heme domain interface. Previously, the Cys(331) --> Ala mutant was produced, and it proved to be inactive in catalysis and to have structural defects that disrupt the binding of l-Arg and tetrahydrobiopterin (BH(4)). Because binding l-Arg and BH(4) to wild type nNOS profoundly affects CO binding with little effect on NO binding, ligand binding to the mutant was characterized as follows. 1) The mutant initially has behavior different from native protein but reminiscent of isolated heme domain subchains. 2) Adding l-Arg and BH(4) has little effect immediately but substantial effect after extended incubation. 3) Incubation for 12 h restores behavior similar but not quite identical to that of wild type nNOS. Such incubation was shown previously to restore most but not all catalytic activity. These kinetic studies substantiate the hypothesis that zinc content is related to a structural rather than a catalytic role in maintaining active nNOS.     

Identification of the first Oomycete annexin as a (1-->3)-beta-D-glucan synthase activator

(1-->3)-beta-D-Glucans are major components of the cell walls of Oomycetes and as such they play an essential role in the morphogenesis and growth of these microorganisms. Despite the biological importance of (1-->3)-beta-D-glucans, their mechanisms of biosynthesis are poorly understood. Previous studies on (1-->3)-beta-D-glucan synthases from Saprolegnia monoica have shown that three protein bands of an apparent molecular weight of 34, 48 and 50 kDa co-purify with enzyme activity. However, none of the corresponding proteins have been identified. Here we have identified, purified, sequenced and characterized a protein from the 34 kDa band and clearly shown that it has all the biochemical properties of proteins from the annexin family. In addition, we have unequivocally demonstrated that the purified protein is an activator of (1-->3)-beta-D-glucan synthase. This represents a new type of function for proteins belonging to the annexin family. Two other proteins from the 48 and 50 kDa bands were identified as ATP synthase subunits, which most likely arise from contaminations by mitochondria during membrane preparation. The results, which are discussed in relation with the possible regulation mechanisms of (1-->3)-beta-D-glucan synthases, represent a first step towards a better understanding of cell wall polysaccharide biosynthesis in Oomycetes.     

Characterization of recombinant human endothelial nitric-oxide synthase purified from the yeast Pichia pastoris

Human endothelial nitric-oxide synthase (eNOS) was expressed in the methylotrophic yeast Pichia pastoris, making use of the highly inducible alcohol oxidase promoter. The recombinant protein constituted approximately 3% of total protein and was largely soluble (>75%). About 1 mg of purified eNOS was obtained from 100-ml yeast cell cultures by affinity chromatography of crude cell supernatants. The purified enzyme had a V(max) of 192 +/- 18 nmol of L-citrulline x mg(-1) x min(-1), had a K(m) for L-arginine of 3.9 +/- 0.2 microM, and showed an absolute requirement for tetrahydrobiopterin (H(4)biopterin). NADPH oxidase activity was 136 +/- 9 and 342 +/- 24 nmol x mg(-1) x min(-1) in the absence and presence of 0.1 mM L-arginine, respectively, and not affected by H(4)biopterin. The protein contained 0.56 +/- 0.06 equivalents of FAD and 0.79 +/- 0.08 equivalents of FMN. On-line gel filtration/inductively coupled plasma mass spectrometry analysis confirmed that both iron (0.80 +/- 0.09 mol/subunit) and zinc (0.43 +/- 0.03 mol/subunit) were bound to the enzyme. Graphite furnace-atomic absorption spectroscopy yielded a value for bound iron of 0.84 +/- 0.04 mol/subunit. The absorbance of the enzyme at 398 nm implied a heme content of 0.85 +/- 0.09 mol/subunit, and the high pressure liquid chromatography heme assay gave an estimate of 0.71 +/- 0.02 mol heme/subunit. Gel permeation chromatography yielded one single peak with a Stokes radius of 6.62 +/- 0.7 nm, indicating that the native protein is dimeric. Upon low temperature gel electrophoresis the untreated protein appeared mainly as a monomer (88 +/- 3%), but pretreatment with H(4)biopterin and L-arginine led to a pronounced shift toward dimers (77 +/- 4%). Thus, in contrast to bovine eNOS (List, B. M., Kl?sch, B., V?lker, C., Gorren, A. C. F., Sessa, W. C., Werner, E. R., Kukovetz, W. R., Schmidt, K., and Mayer, B. (1997) Biochem. J. 323, 159-165; Rodriguez-Crespo, I., Gerber, N. C., and Ortiz de Montellano, P. R. (1996) J. Biol. Chem. 271, 11462-11467), the human eNOS appears to be markedly stabilized by H(4)biopterin.     

Functional significance of cytosolic endothelial nitric-oxide synthase (eNOS): regulation of hyperpermeability

Endothelial NOS (eNOS)-derived NO is a key factor in regulating microvascular permeability. We demonstrated previously that eNOS translocation from the plasma membrane to the cytosol is required for hyperpermeability. Herein, we tested the hypothesis that eNOS activation in the cytosol is necessary for agonist-induced hyperpermeability. To study the fundamental properties of endothelial cell monolayer permeability, we generated ECV-304 cells that stably express cDNA constructs targeting eNOS to the cytosol or plasma membrane. eNOS-transfected ECV-304 cells recapitulate the eNOS translocation and permeability properties of postcapillary venular endothelial cells (Sánchez, F. A., Rana, R., Kim, D. D., Iwahashi, T., Zheng, R., Lal, B. K., Gordon, D. M., Meininger, C. J., and Durán, W. N. (2009) Proc. Natl. Acad. Sci. U.S.A. 106, 6849-6853). We used platelet-activating factor (PAF) as a proinflammatory agonist. PAF activated eNOS by increasing phosphorylation of Ser-1177 and inducing dephosphorylation of Thr-495, increasing NO production, and elevating permeability to FITC-dextran 70 in monolayers of cells expressing wild-type and cytosolic eNOS. PAF failed to increase permeability to FITC-dextran 70 in monolayers of cells transfected with eNOS targeted to the plasma membrane. Interestingly, this occurred despite eNOS Ser-1177 phosphorylation and production of comparable amounts of NO. Our results demonstrate that the presence of eNOS in the cytosol is necessary for PAF-induced hyperpermeability. Our data provide new insights into the dynamics of eNOS and eNOS-derived NO in the process of inflammation.     

Crystal structures of zinc-free and -bound heme domain of human inducible nitric-oxide synthase. Implications for dimer stability and comparison with endothelial nitric-oxide synthase.

The crystal structures of the heme domain of human inducible nitric-oxide synthase (NOS-2) in zinc-free and -bound states have been solved. In the zinc-free structure, two symmetry-related cysteine residues form a disulfide bond. In the zinc-bound state, these same two cysteine residues form part of a zinc-tetrathiolate (ZnS(4)) center indistinguishable from that observed in the endothelial isoform (NOS-3). As in NOS-3, ZnS(4) plays a key role in stabilizing intersubunit contacts and in maintaining the integrity of the cofactor (tetrahydrobiopterin) binding site of NOS-2. A comparison of NOS-2 and NOS-3 structures illustrates the conservation of quaternary structure, tertiary topology, and substrate and cofactor binding sites, in addition to providing insights on isoform-specific inhibitor design. The structural comparison also reveals that pterin binding does not preferentially stabilize the dimer interface of NOS-2 over NOS-3.     

The Asp298 allele of endothelial nitric oxide synthase is a risk factor for myocardial infarction among patients with type 2 diabetes mellitus

Background

To establish an efficient prophylaxis of coronary artery disease reliable risk stratification is crucial, especially in the high risk population of patients suffering from diabetes mellitus. This prospective study determined the predictive value of coronary calcifications for future cardiovascular events in asymptomatic patients with diabetes mellitus.

Methods

We included 716 patients suffering from diabetes mellitus (430 men, 286 women, age 55.2 ± 15.2 years) in this study. On study entry all patients were asymptomatic and had no history of coronary artery disease. In addition, all patients showed no signs of coronary artery disease in ECG, stress ECG or echocardiography. Coronary calcifications were determined with the Imatron C 150 XP electron beam computed tomograph. For quantification of coronary calcifications we calculated the Agatston score. After a mean observation period of 8.1 ± 1.1 years patients were contacted and the event rate of cardiac death (CD) and myocardial infarction (MI) was determined.

Results

During the observation period 40 patients suffered from MI, 36 patients died from acute CD. The initial Agatston score in patients that suffered from MI or died from CD (475 ± 208) was significantly higher compared to those without cardiac events (236 ± 199, p < 0.01). An Agatston score above 400 was associated with a significantly higher annualised event rate for cardiovascular events (5.6% versus 0.7%, p < 0.01). No cardiac events were observed in patients with exclusion of coronary calcifications. Compared to the Framingham risk score and the UKPDS score the Agatston score showed a significantly higher diagnostic accuracy in the prediction of MI with an area under the ROC curve of 0.77 versus 0.68, and 0.71, respectively, p < 0.01.

Conclusion

By determination of coronary calcifications patients at risk for future MI and CD could be identified within an asymptomatic high risk group of patients suffering from diabetes mellitus. On the other hand future events could be excluded in patients without coronary calcifications.     

Monitoring the pH dependence of IR carboxylic acid signals upon Q(B)- formation in the Glu-L212 --> Asp/Asp-L213 --> Glu swap mutant reaction center from Rhodobacter sphaeroides

In the photosynthetic reaction center (RC) from the purple bacterium Rhodobacter sphaeroides, proton-coupled electron-transfer reactions occur at the secondary quinone (QB) site. Involved in the proton uptake steps are carboxylic acids, which have characteristic infrared vibrations in the 1770-1700 cm-1 spectral range that are sensitive to 1H/2H isotopic exchange. With respect to the native RC, a novel protonation pattern for carboxylic acids upon QB photoreduction has been identified in the Glu-L212 --> Asp/Asp-L213 --> Glu mutant RC using light-induced FTIR difference spectroscopy (Nabedryk, E., Breton, J., Okamura, M. Y., and Paddock, M. L. (2004) Biochemistry 43, 7236-7243). These carboxylic acids are structurally close and have been implicated in proton transfer to reduced QB. In this work, we extend previous studies by measuring the pH dependence of the QB-/QB FTIR difference spectra of the mutant in 1H2O and 2H2O. Large pH dependent changes were observed in the 1770-1700 cm-1 spectral range between pH 8 and pH 4. The IR fingerprints of the protonating carboxylic acids upon QB- formation were obtained from the calculated double-difference spectra 1H2O minus 2H2O. These IR fingerprints are specific for each pH, indicative of the contribution of different titrating groups. In particular, the 1752 cm-1 signal indicates that Glu-L213 protonates upon QB- formation at pH >or= 5, whereas the 1746 cm-1 signal indicates protonation of Asp-L212 even at pH 4. An unidentified carboxylic acid absorbing at approximately 1765 cm-1 could be the proton donor between pH 8 and 5. The observation that in the swap mutant there are several uniquely behaving carboxylic acids shows that electrostatic interactions occurring between them are sufficiently modified from the native RC to reveal their IR signatures.     

Physicochemical characterisation of the two active site mutants Trp(52)-->Phe and Asp(55)-->Val of glucoamylase from Aspergillus niger

Glucoamylase 1 (GA1) from Aspergillus niger is a multidomain starch hydrolysing enzyme that consists of a catalytic domain and a starch-binding domain connected by an O-glycosylated linker. The fungus also produces a truncated form without the starch-binding domain (GA2). The active site mutant Trp(52)-->Phe of both forms and the Asp(55)-->Val mutant of the GA1 form have been prepared and physicochemically characterised and compared to recombinant wild-type enzymes. The characterisation included substrate hydrolysis, inhibitor binding, denaturant stability, and thermal stability, and the consequences for the active site of glucoamylase are discussed. The circular dichroic (CD) spectra of the mutants were very similar to the wild-type enzymes, indicating that they have similar tertiary structures. The D55V GA1 mutant showed slower kinetics of hydrolysis of maltose and maltoheptaose with delta delta G(double dagger) congruent with 22 kJ mol(-1), whereas the binding of the strong inhibitor acarbose was greatly diminished by delta delta G degrees congruent with 52 kJ mol(-1). Both W52F mutant forms have almost the same stability as the wild-type enzyme, whereas the D55V GA1 mutant showed slight destabilisation both towards denaturant and heat (DSC). The difference between the CD unfolding curves recorded by near- and far-UV indicated that D55V GA1 unfolds through a molten globule intermediate.