From the Department of Pharmacology, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, Connecticut 06536
Received for publication, April 24, 2001
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ABSTRACT |
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The 894G 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 (1-3). 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
(4). 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 (5). 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 (6). 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
(7). However, in patients with coronary spasm (8) or renal disease (9),
polymorphisms within the eNOS promoter have been postulated to impact
levels of mRNA and protein, whereas in patients with coronary
artery disease (10) or hypertension (11) 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 (7).
The exon 7 polymorphism (894G 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 (16). 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 (17). Because most aspects of the proposed
intracellular cleavage remain to be determined, we sought to: 1)
elucidate the mechanism whereby the Glu298 Generation of Wild-type (WT) Human eNOS and E298D eNOS
cDNA--
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 and
XbaI) 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.
Cell Culture and Reagents--
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 (18).
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 (19). To trigger cellular apoptosis, COS cells were treated
with staurosporine (1 µM) for 6 h (20). 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).
Western Blot of Total Cell Lysates--
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 (21)
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 Isolation and Sequencing of 100-kDa Fragment--
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 (22). 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).
Generation of Purified Human eNOS from Escherichia coli--
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. coli together with a vector coding
for the chaperone proteins groES and groEL. Bacterial culture,
induction, and eNOS purification were performed as previously described
(23).
Preparation of Cell Lysates at Acidic pH--
Phosphate-citrate
buffer solutions were mixed to pH 7.0, 5.0, and 3.0. Transfected cells
were harvested using the above pH lysis solutions (buffers plus 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). Lysates were rotated for 10 h
at 37 °C. Lysates were adjusted to pH 7.4 using sodium hydroxide and
prepared for SDS-PAGE using NUPAGE sample preparation.
NO Release Assay--
Chemiluminescent measurement of nitrite in
media was performed as described previously (25). Basal levels of NO
release were acquired by sampling media from cells just prior to
harvesting (48 h post-transfection).
Biochemical Assays on Purified eNOS--
Hemoglobin capture
assays and cytochrome c reduction assays were all
performed as described previously (22).
Pulse-Chase Experiments--
Twelve hours post-transfection,
confluent COS-7 cells from 100 mm cell culture plates were trypsinized
and divided equally into 60-mm cell plates. Cells were grown to 70%
confluency in complete medium. Medium was changed to
methionine/cystine-free Dulbecco's modified Eagle's medium (Life
Technologies, Inc.) for 20 min prior to labeling.
35S-labeled methionine/cysteine (Amersham Pharmacia
Biotech) was added to methionine/cystine-deficient medium, and
cells were incubated with the 150 µCi/ml 35S medium (2 ml) for 1 h. Cells were rinsed quickly and incubated in complete
Dulbecco's modified Eagle's medium before harvesting at various time
points. Lysates were immunoprecipitated using anti-eNOS antibody
(Transduction Laboratories) and protein A-Sepharose. Eluted proteins
were separated by SDS-PAGE. Gels were dried for 3 h and then
exposed to film at The allele frequency of the 894G polymorphism is greater
than the 894T variant in populations studied to date (e.g.
67.5% in Australians and 92.2% in Japanese (27)). For this reason we
have termed eNOS with glutamate at 298 as wild type and eNOS with aspartate at 298 as E298D eNOS. Recently it was shown that E298D
eNOS may undergo enhanced intracellular cleavage (17). To investigate
the mechanism of this cleavage, we transiently expressed both
polymorphic versions of eNOS in COS-7 cells that do not express eNOS
endogenously. As seen in Fig.
1A, a unique 100-kDa cleavage
fragment (see arrow) was present in cell lysates containing
the E298D eNOS, as previously reported (17). As the antibody used was
raised against an epitope derived from the extreme carboxyl terminus of
human eNOS, it was apparent that the cleavage took place within the
amino-terminal domain of the protein. To determine the precise location
of the cleaved bond, we isolated the 100-kDa fragment and sequenced the
amino terminus by Edman degradation. The sequenced portion of the
fragment corresponds precisely to residues Pro299 through
Val310 of eNOS, showing that E298D eNOS was cleaved on the
carboxyl side of the aspartate at position 298, as depicted in Fig.
1B.
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 WT
versus 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.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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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
(12-14). 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 (11, 15).
Asp variant
of eNOS (E298D) is cleaved and 2) further characterize enzymatic
differences between wild-type eNOS and cleaved E298D eNOS in cells and
in vitro.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-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.
80 °C for 12 h. Band intensity at 135 kDa
was assayed by densitometry (26).
RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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View larger version (38K):
[in a new window]
Fig. 1.
Glutamate to aspartate
substitution at residue 298 creates a labile Asp-Pro bond in human
eNOS. A, COS-7 cells were transiently transfected with
vectors coding for the WT or Glu298 Asp variant
(E298D) of eNOS. Lysates were Western blotted using
a carboxyl-terminal eNOS monoclonal antibody. An
arrow indicates a unique 100-kDa band in the E298D lysate.
In B, Edman sequencing of the purified 100-kDa fragment
confirms cleavage at the Asp298-Pro299
bond.
Because the cleavage site occurred at the Asp-Pro bond, we searched
for proteases that may cleave at this site and found none. However, it
has been recognized for many years that Asp-Pro bonds are particularly
susceptible to acid hydrolysis (28). In fact, one criticism of using
the method of Laemmli when preparing samples for SDS-PAGE is that
proteins are boiled in an acidic solution, a combination of conditions
that can cause significant protein degradation (29). Although the
Tris-based loading buffers commonly used in SDS-PAGE are near neutral
pH (6.8), the solutions become significantly more acidic when heated.
(For example, a 50 mM Tris buffer with pH 6.8 at 22 °C
will drop to pH 5.3 at 100 °C.) Therefore, before investigating an
in vivo mechanism of eNOS fragmentation, we investigated
whether the cleavage phenomenon could be an artifact of our sample
preparation method. As shown in Fig.
2A, when we used Laemmli-style
buffers and increased the boiling time during sample preparation, we
saw increased generation of the 100-kDa fragment of interest in E298D
samples (arrow; compare 100-kDa fragment in
lanes 3-8). All other degradation bands were found in both WT and E298D eNOS. Importantly, when we used a commercially available sample buffer containing LDS formulated to maintain a more
basic pH throughout sample preparation, we observed absolutely no
fragmentation of E298D (compare lanes 1 and
2 with lanes 3-8). This suggests that the unique
100-kDa fragment generated in E298D eNOS may arise from acid hydrolysis
of the Asp-Pro bond. Next, we surmised that if the cleavage of E298D
eNOS occurs intracellularly, this should be reflected in a change in
protein half-life relative to WT. To examine this hypothesis
directly, we performed pulse-chase studies to determine the
biosynthetic half-life of WT versus E298D eNOS. As shown in
Fig. 2B, the half-lives of the two proteins were virtually
indistinguishable and close to the reported values (26). From these
experiments, we conclude that sample preparation alone could account
for cleavage of E298D eNOS and that the turnover of the two proteins
was not different.
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Having established acid hydrolysis as a likely mechanism of cleavage of
E298D eNOS, we investigated whether acidic conditions alone, without
boiling, could account for fragmentation of the enzyme. To separate the
heating of samples from the accompanying acidification, we switched to
cell lysis solutions buffered by citrate-phosphate mixtures. The
citrate-phosphate lysis solutions were much more resistant to
acidification upon heating than Tris-based buffers (e.g. a
pH 7.0 solution of citrate-phosphate buffer at 22 °C remained
at pH 7.0 when temperature was raised to 100 °C, whereas Tris
buffers commonly dropped 1-2 pH units upon heating to 100 °C).
Experiments in total cell lysates were performed to test whether
fragmentation of E298D could occur upon chronic exposure to acidic
conditions. Cells were transfected with appropriate cDNAs and
lysates processed and further incubated in various pH solutions while
rotating, at 37 °C for 10 h. As seen in Fig.
3A, fragmentation of both
E298D and WT eNOS occurs markedly at pH 3.0. The fragmentation pattern
of E298D in pH 3.0 includes a unique 100-kDa band (to the
right of the asterisk) just below a larger, more
prominent band that is also seen in the WT eNOS, pH 3.0 lane (lanes 3 and 6). At pH 5.0, the larger,
prominent band seen at pH 3.0 is not present. With a darker exposure, a
unique band present in the E298D, the pH 3.0 lane, becomes apparent at
pH 5.0 (to the left of the asterisk). The unique
100-kDa band in E298D is consistent in size with the fragment of
interest seen in Figs. 1 and 2. These experiments provide evidence that
only upon chronic exposure to nonphysiologic pH ranges (10 h at less
than pH 5.0) will E298D be partially cleaved. Heating the enzyme to
levels beyond 37 °C without substantial acidification does not cause significant cleavage (i.e. see Fig. 2A,
lanes 1 and 2, where the samples are
heated to 70 °C), thus suggesting that both a change in pH and
temperature (acidic hydrolysis) were responsible for the cleavage.
However, to test whether various manipulations to promote cellular
stress could evoke the cleavage of eNOS, transfected COS cells were
exposed to a variety of stimuli and samples were processed in LDS
buffer. As seen in Fig. 3B exposure of cells to hypoxia (48 h, 1% ambient oxygen), H2O2 to evoke oxidative stress or staurosporine, a known inducer of apoptosis (20), did not
result in the formation of the 100-kDa eNOS fragment under these
extreme conditions.
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Collectively, the above data suggest that the cleavage of the E298D
eNOS to the 100-kDa species may be artifactually generated in
vitro. However, we cannot rule out the possibility that in the
uncleaved state, E298D eNOS may have properties and activities different from wild-type eNOS that may account for higher cardiac disease risk in carriers of the 894T allele. Thus, COS cells
were transfected with WT and E298D eNOS cDNA and the amount of
NO produced over a 16-h period was quantified using NO-specific
chemiluminescence. As shown in Fig. 4,
A and B, the production of
NO
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Here we show that the extensively documented E298D polymorphism in eNOS
does not appear to influence the stability, half-life, or biologic
activity of the enzyme isolated from cells or the enzymatic activity of
the recombinant protein. Most importantly, isolation of samples in
standard Laemmli buffer for SDS-PAGE will generate a 100-kDa fragment
cleaved precisely at the Asp-Pro bond, suggesting that the presence of
the 100-kDa fragment in human tissue from carriers of the E298D
polymorphism is likely to arise because of sample preparation. However,
we cannot rule out the possibility that this fragment can be generated
in an in vivo context by an unknown proteolytic mechanism.
In preliminary studies, we generated a 100-kDa protein corresponding to
the fragment derived from the cleavage of E298D (Figs. 1 and 2) and
expressed it as a recombinant protein in E. coli. Functional
analysis of this protein missing the heme domain showed that it was an
active reductase (based on cytochrome c reduction). If this
cleavage can occur in a cellular context, it is possible for this
protein to reduce oxygen to superoxide, which can contribute to
endothelial dysfunction. Alternatively, because eNOS is subjected to
various levels of regulation including protein-protein interactions,
fatty acylation, and phosphorylation, these control steps may be
influenced by the polymorphism. In conclusion, a causative linkage
between the common polymorphism E298D in eNOS and the incidence of
cardiovascular disease cannot be explained based on the cleavage or
impaired function of the enzyme.
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ACKNOWLEDGEMENT |
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We thank Dr. Gabriel Haddad for the use of hypoxic incubators.
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FOOTNOTES |
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* This work was supported by grants from the National Institutes of Health (RO1 HL57665, RO1 HL61371, and RO1 HL64793 to W. C. S.; T32HL10183 to D. F.) and a grant-in-aid from the American Heart Association (national grant to W. C. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
A Howard Hughes Medical Institute Medical Student Research
Training Fellow.
§ In receipt of a fellowship from the Canadian Institutes of Health Research.
¶ An Established Investigator of the American Heart Association. To whom correspondence should be addressed. Tel.: 203-737-2291; Fax: 203-737-2290; E-mail: william.sessa@yale.edu.
Published, JBC Papers in Press, April 30, 2001, DOI 10.1074/jbc.M103647200
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ABBREVIATIONS |
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The abbreviations used are: NO, nitric oxide; eNOS, endothelial nitric-oxide synthase; WT, wild type; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; LDS, lithium dodecyl sulfate.
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