From the Laboratory of Biological Chemistry,
Department of Biology, University of Konstanz, 78457 Konstanz,
Germany and the § Laboratory of Analytical Chemistry,
Department of Chemistry, University of Konstanz,
78457 Konstanz, Germany
Received for publication, August 8, 2002, and in revised form, January 30, 2003
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ABSTRACT |
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Treatment of bovine aortic microsomes containing
active prostacyclin synthase (PGI2 synthase) with
increasing concentrations of peroxynitrite (PN) up to 250 µM of PN yielded specific staining of this enzyme on
Western blots with antibodies against 3-nitrotyrosine (3-NT), whereas
above 500 µM PN staining of additional proteins was also
observed. Following treatment of aortic microsomes with 25 µM PN, PGI2 synthase was about half-maximally
nitrated and about half-inhibited. It was then isolated by gel
electrophoresis and subjected to proteolytic digestion with several
proteases. Digestion with thermolysin for 24 h provided a single
specific peptide that was isolated by high performance liquid
chromatography and identified as a tetrapeptide
Leu-Lys-Asn-Tyr(3-nitro)-COOH corresponding to positions 427-430 of
PGI2 synthase. Its structure was established by precise
mass determination using Fourier transform-ion cyclotron
resonance-nanoelectrospray mass spectrometry and Edman microsequencing
and ascertained by synthesis and mass spectrometric characterization of
the authentic Tyr-nitrated peptide. Complete digestion by Pronase to
3-nitrotyrosine was obtained only after 72 h, suggesting that the
nitrated Tyr-430 residue may be embedded in a tight fold around the
heme binding site. These results provide evidence for the specific
inhibition of PGI2 synthase by nitration at Tyr-430 that
may occur already at low levels of PN as a consequence of endothelial
co-generation of nitric oxide and superoxide.
The nitration of tyrosine residues in proteins has become a well
recognized reaction, but has been heavily disputed with regard to the
mechanisms involved and its physiological and/or pathophysiological significance (1-5). Peroxynitrite
(PN)1 generated from nitric
oxide (NO) and superoxide (O PGI2 synthase was inactivated by micromolar PN
concentrations (16, 17) but also by a continuous generation of NO
and O Beyond this physiological background no proof for the molecular basis
of enzyme inhibition has been hitherto obtained by identification of
nitrated tyrosine. Substrate analogs of prostaglandin-endoperoxide have
been recently shown to inhibit the nitration (17), which suggested a
proximity to the heme attached to the protein by the Cys-441 residue
(21-23); however, previous attempts have been unsuccessful to detect
and identify the nitrated tyrosine. In this study we present molecular
evidence for the specific nitration of bovine PGI2 synthase
at tyrosine 430 by high resolution Fourier transform-ion cyclotron
resonance (FT-ICR) mass spectrometry (24), and the presence of 3-NT
upon extended Pronase digestion. We further show an unusually slow
digestion by thermolysin, presumably because of a tight fold around the
heme, to release a tetrapeptide by an unexpected specific cleavage
adjacent to the nitrated tyrosine residue.
Materials--
Pronase from Streptomyces griseus
(lyophilized powder) was obtained from Roche Molecular Diagnostics.
Thermolysin, type X from Bacillus thermoproteolyticus rokko
was purchased from Sigma. All other chemicals were of analytical grade
or highest purity available. PN was a gift from Dr. Koppenol
(ETH Zürich, Switzerland) and was synthesized from NO and
potassium superoxide according to Kissner et al. (25).
P450BM-3 (CYP 102), a F87Y variant from Bacillus
megaterium was a kind gift from J. A. Peterson
(Southwestern Medical School, Dallas, TX) and was purified as described
(26).
A rabbit polyclonal antibody against PGI2 synthase was
produced according to Siegle et al. (27). A mouse monoclonal
antibody against 3-NT (anti-NT, clone 1A6) was obtained from Upstate
Biotechnology (Hamburg, Germany) as a stock solution of 1 mg/ml.
Secondary antibodies (goat anti-mouse IgG and goat anti-rabbit IgG)
were obtained from Pierce (stock solutions 0.8 mg/ml). The enhanced
chemiluminescence (ECL) kit and nitrocellulose transfer membranes
(Hybond C, pore size 0.5 µm) were purchased from Amersham
Biosciences. PGH2 was obtained from Cayman Chemical (Ann
Arbor, MI).
Preparation of Bovine Aortic Microsomes--
Endothelial and
smooth muscle layers from 8 to 10 freshly received bovine aorta were
isolated by dissection at 4 °C, rapidly frozen in liquid nitrogen,
and stored at Peroxynitrite Treatment of Microsomes--
Reaction with PN was
carried out with microsomes because active enzyme is required for
nitration and further purification steps involving denaturating
detergents partially inactivate the protein. PN (10 µl) at a defined
concentration was quickly added by thorough Vortex mixing to an
ice-cold microsomal suspension (990 µl, total protein concentration 1 mg/ml in 50 mM K-phosphate buffer, pH 7.5). Controls were
treated with decomposed PN (24 h at room temperature).
Activity Test for 6-Keto-PGF1 Western Blot Analysis--
The microsomal samples were treated
for 5 min at 95 °C with Laemmli buffer and separated by 8% (v/v)
SDS-PAGE (30 mA, 1 h). The proteins were then transferred onto a
nitrocellulose membrane by a semidry blot procedure using a constant
current of 50 mA for 90 min. The blotting buffer contained 48 mM Tris, 39 mM glycine, 20% (v/v) methanol,
and 0.037% (w/v) SDS. Transfer efficiency of proteins was examined by
staining with 0.1% Ponceau S in 5% (v/v) acetic acid. After
destaining in PBS, the membrane was blocked with 5% (w/v) milk powder
in PBS, pH 7.4, for 2 h at room temperature or at 4 °C
overnight. The membrane was then incubated for 2 h with a
polyclonal antibody against PGI2 synthase (1 µg/ml PBS). After repeated washing with PBS, 0.1% Tween 20 the membrane was incubated for 45 min with a horseradish peroxidase-conjugated goat
anti-rabbit antibody at a dilution of 1:7500 for 45 min, and ECL was
used for detection of antibody binding according to the manufacturer's instructions.
Prior to staining with a second antibody the membrane was stripped by
incubation in stripping buffer (62.5 mM Tris-HCl, pH 6.7, 2% (w/v) SDS, 100 mM 2-mercaptoethanol) under gentle
shaking for 60 min at 70 °C. After washing and blocking, the
membrane was incubated with a mouse monoclonal antibody against 3-NT at a dilution of 1 µg/ml, followed by a horseradish
peroxidase-conjugated goat anti-mouse antibody at a dilution of 1:7500.
PGI2 synthase samples were always stained first with
the PGI2 synthase antibody, then stripped one or two times
before staining with the NT antibody, to ensure complete denaturation
for recognition of nitrated protein.
Isolation of Nitrated Prostacyclin Synthase--
Separation of
PGI2 synthase from microsomal membranes and solubilization
was performed by adding 1% (v/v) Triton X-100 to aortic microsomes.
The suspension was stirred for 2 h at 4 °C, then centrifuged
for 1 h at 100,000 × g to yield a clear yellow supernatant. Because SDS-PAGE is hampered by the high actin
concentration (about 80-90% of total protein) in microsomes, actin
was partially removed by precipitation with 15 mM
CaCl2 for 1 h at 4 °C and centrifugation of the
precipitate for 5 min at 10,000 × g. Because Triton
X-100 interfered with SDS-PAGE in subsequent purification steps,
detergent was removed by extracting with chloroform and Vortex mixing
for a few seconds (29). After centrifugation for 30 min at 3000 rpm,
the organic and aqueous phases were recovered. Proteins precipitated at
the interphase as a solid white layer, and remaining chloroform was
removed by evaporation. Proteins were then solubilized in
SDS-containing electrophoresis buffer (25 mM Tris, 192 mM glycine, 0.1% (w/v) SDS).
Preparative SDS-PAGE--
Proteins were treated with Laemmli
sample buffer for 5 min at 95 °C and then separated by 10% (v/v)
SDS-PAGE (100 mA, 5 h) on preparative gels (160 × 165 mm,
thickness 1.5 mm). Reverse staining by imidazole:zinc was applied for
visualization of bands (30). Gels were equilibrated for 15 min in 100 ml of 0.2 M imidazole in water with gentle shaking, and
then placed for 1 min in 100 ml of 0.3 M ZnCl2.
The staining solution was removed when the background became deep white
showing the transparent protein bands. The band containing
PGI2 synthase was excised with a razor blade, immersed in
2% citric acid solution 2-3 times (10 min) to remove zinc ions from
the gel matrix, and washed several times with SDS-PAGE buffer.
Recovery of PGI2 synthase was performed by electroelution
(ELUTRAP, Schleicher & Schuell, Dassel, Germany) (31). Protein bands
were cut to slices, placed in the elution chamber and covered with
SDS-PAGE buffer (25 mM Tris, 192 mM glycine,
0.1% (w/v) SDS). A voltage of 200 V corresponding to the ~50 mA
current was then applied. After an 8-h elution period proteins
accumulated inside a trap (volume 800 µl) between two membranes were
collected with a pipette. Protein solutions were finally concentrated
with an Ultrafree-4 Centrifugal filter unit (Millipore Corp.) to a 5 µM solution (0.28 mg/ml). Protein concentrations were
determined with the Bio-Rad DC assay (Bio-Rad).
Pronase Digestion of Nitrated PGI2 Synthase and HPLC
Analysis of 3-Nitrotyrosine--
Samples of 100 µl of electroeluted
PGI2 synthase were treated with different concentrations of
PN (0, 10, 50, 100, and 250 µM), heated for 10 min to
95 °C, and then mixed with 10 mM CaCl2 to
stabilize proteases. After addition of Pronase (1 mg/ml) the samples
were incubated for 24 h at 40 °C, then another 1 mg/ml Pronase
was added and incubated again for 24 h at 50 °C. Digestion was
repeated with a third and fourth portion (0.5 mg/ml) for 12 h at
50 °C. Prior to HPLC separation the samples were filtered with the
10-kDa MICROCON centrifugal filter device (Millipore Corp.) by
centrifuging for 30 min at 10,000 × g. Products were analyzed on a Jasco HPLC system consisting of a PU-980 pump, a Jasco
UV-1575 and Spectra Physics spectra focus UV-visible detector, and a
LG-980-02 low pressure mixing unit. A C18 Nucleosil 100-5 250 × 4.6 column from Macherey & Nagel (Düren, Germany) was
used with a mobile phase gradient (0-15 min, 0% (v/v) B; 15-30 min, 0-90% (v/v) B; 30-40 min, 90% (v/v) B (A: 0.1% (v/v)
trifluoroacetic acid, pH 2, B: 80% (v/v) acetonitrile with 0.08%
(v/v) trifluoroacetic acid)). The flow rate was 1 ml/min and sample
aliquots of 100 µl were injected. Tyrosine, phenylalanine,
tryptophan, and 3-NT were identified and quantified at 270 and 360 nm
by internal and external standards. The retention time of 3-NT was 12 min. As a control 3-NT was reduced with sodium dithionite to
3-aminotyrosine.
Thermolysin Digestion of Nitrated PGI2 Synthase and
HPLC Analysis of Peptides--
Because of the large amount of SDS in
the electroeluted protein solution (>1% SDS) in-gel digestion was
more suitable than digestion in solution. Protein solutions (about 0.5 nmol of PGI2 synthase isolated from treated (25 µM PN) or untreated microsomes) were incubated in Laemmli
buffer for 5 min at 95 °C and separated by SDS-PAGE on a "Novex"
8% Tris glycine gel (10 wells, Invitrogen; 30 mA, 1 h). Protein
bands were visualized by reverse staining with imidazole:zinc as
described above. Proteolytic digestion in the gel matrix was carried
out according to the procedure of Shevchenko et al. (32).
The protein bands were excised from the gel, cut to pieces and washed
with 2% citric acid, then with water to remove staining dye, gel
buffers, and SDS, and dried at room temperature in a vacuum centrifuge.
The washing step was repeated by dehydration of the gel pieces and
discarding the solution. After shrinking by vacuum centrifugation the
gel pieces were reswollen in 200 µl of digest solution containing 50 mM Tris, pH 8.0, 5 mM CaCl2, 10%
(v/v) acetonitrile, and 25 ng/µl thermolysin, and the supernatant was
removed. Proteolytic digestion was carried out for 24 h at
50 °C under gentle shaking. Peptides were extracted several times
with 0.1% trifluoroacetic acid:acetonitrile for 24 h, lyophilized
to dryness, and analyzed on the above described HPLC system. A
C18 Nucleosil 100-3 125 × 4.6 column from Macherey & Nagel was used with a mobile phase gradient (0-5 min, 0% (v/v) B;
5-50 min, 0-60% (v/v) B (A: 0.1% (v/v) trifluoroacetic acid in
water, pH 2, B: 80% (v/v) acetonitrile with 0.08% (v/v)
trifluoroacetic acid)), at a flow rate of 0.8 ml/min. Peptide fragments
were detected at 220 and 365 nm; peaks showing a strong absorption at
365 nm were collected and lyophilized for further analysis.
Sequence Determination--
Sequence determination of the
isolated peptide was achieved by Edman amino acid sequencing.
NH2-terminal Edman degradation was performed on an Procise
HT sequencing system, model 494 (PerkinElmer Life Sciences,
Weiterstadt, Germany), fitted with an online, narrow-bore HPLC-based
amino acid analyzer that utilized a 220 × 2.1-mm C18 reversed-phase column held at 55 °C in a column heater oven.
Released phenylthiohydantoin (PTH)-derivatives from each cycle were
separated under the recommended binary gradient conditions using 3.5%
tetrahydrofuran in water (buffered with sodium phosphate, pH 4.5;
solvent A) and 10% 2-propanol in acetonitrile (unbuffered; solvent B).
Prior to sequence determination, samples of peptides were applied to a
biobrene-treated glass fiber disk and allowed to dry in a stream of
argon. Reagents, operating software, and protocols were used as
described from the instrument manufacturer. Chromatographic identification of the UV signals was done by reference to the retention
times and the absorbance of a PTH standard run. PTH-derivatives display
characteristic UV spectra with an absorbance maximum at 269 nm.
Peptide Synthesis--
The nitrated tetrapeptide LKNY(nitro) was
synthesized on a semiautomated peptide synthesizer (EPS-221, Abimed)
using solid-phase peptide synthesis Fmoc chemistry methods (33) with
all chemicals of analytical grade or highest available purity. Fmoc
amino acids, NovaSyn TGR resin, PyBop, and other reagents were obtained
from Novabiochem (Laufelfingen, Switzerland). To synthesize the peptide with COOH-terminal 3-NT carboxamide the TGR resin was employed with 40 min coupling time and 5 min deprotection in 20% piperidine in
N,N-dimethylformamide. Purification of the
peptide was performed with preparative HPLC on a Grom-Sil ODS-4Me column.
Mass Spectrometry--
High resolution mass spectrometry was
performed with a 7T Bruker Daltonik (Bremen, Germany) Apex II FT-ICR
mass spectrometer equipped with an actively shielded 7.0 tesla
superconducting magnet (Magnex, Oxford, UK), an APOLLO (Bruker
Daltonik) electrospray ionization source and nano-electrospray system,
an API1600 ESI control unit, and a UNIX based Silicon Graphics
O2 work station. Details of the instrumental conditions of
ESI-FT-ICR-MS were as previously reported (34). The mass spectra were
obtained by collecting 32-124 single scans. Experimental conditions
were: full scan mode; 45-70 V capillary exit voltage; setting of
skimmer 1, 10; setting of skimmer 2, 7; RF amplitude, 500; offset 0.9; trap, 10; extract, 10; ionization pulse time, 2500 ms; ionization delay
time, 0.001 s; excitation sweep pulse 1, 2 ms; excitation sweep
attenuation, 1:2.16 dB. Acquisition of spectra was performed with the
Bruker Daltonik software XMASS and corresponding programs for mass
calculation, data calibration, and processing. Peptide samples were
dissolved in a solution of 3% acetic acid in 50% methanol:water.
MALDI-time of flight mass spectrometry was performed with a Bruker
BiFlex-DE mass spectrometer equipped with a Scout MALDI source and
video system, a nitrogen UV laser (337 nm), and a dual channel plate
detector. Sample preparation was performed with 1 µl of a freshly
prepared saturated solution of Upon treatment of isolated PGI2 synthase with PN no
nitrated tryptic peptide(s) could be initially found although
immunoprecipitation of the enzyme with a monoclonal NT antibody, as
well as conventional acid hydrolysis of the nitrated enzyme, indicated
the presence of nitrated tyrosine. In previous work with other P450
proteins a nitration with PN resulted in proteolytic peptides with
characteristic absorbance at 365 nm, from which the position of the
3-NT could be identified (13, 14).
Because the postulated mechanism (35, 36) suggested that only active
PGI2 synthase can be nitrated, bovine aortic microsomes were first nitrated with increasing concentrations of PN and then the
enzyme was isolated by gel electrophoresis (Fig.
1). Western blot analyses shown in Fig. 1
provided identical, specific bands at approximately 52 kDa up to a
concentration of 500 µM PN that were stained by a
polyclonal antibody against PGI2 synthase, and a monoclonal
antibody against 3-NT, whereas higher concentrations than 500 µM caused unspecific additional staining of other
proteins. The control also stained weakly, which probably was because
of the presence of some atherosclerotic plaques in bovine arteries (37).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
70 °C. Frozen strips were homogenized at 0-4 °C
in a Waring blender in 100 mM K-phosphate buffer, pH 7.5, containing 1 mM EDTA, 0.1 mM dithiothreitol,
0.1 mM butylated hydroxytoluene, and 44 mg/liter
phenylmethylsulfonyl fluoride. The microsomal fraction was obtained by
centrifugation as described (28) to a final volume of 75-100 ml, with
a protein concentration of 10-20 mg/ml. The homogenization buffer
contained 50 mM K2HPO4, pH 7.5, without additional protease inhibitors.
--
The activity
for PGI2 formation of PN-treated microsomes was tested by
incubation of 100 µl of microsomal suspension (1 mg of protein/ml) in
100 mM potassium phosphate buffer, pH 7.4, with PGH2 (1 µg) for 1 min at 20 °C. To avoid
cross-reactivities with PGH2 degradation products the
incubation mixture was stopped with 20 µl of 1 M citric
acid and extracted two times with 300 µl of ethyl acetate and
separated by TLC (Silica Gel 60, Merck, Darmstadt, Germany); solvent:
ethyl acetate:2,2,4-trimethylpentane:acetic acid:water, 10:50:20:100).
6-Keto-PGF1
was identified by an iodine-stained
reference (RF value about 0.18). The area of
6-keto-PGF1
was excised, extracted with ethyl acetate, and evaporated to complete dryness. After addition of 100 µl of PBS
three dilutions of 1:100, 1:1000, and 1:10000 were prepared and tested
by EIA (Assay Designs Inc., BioTrend, Köln, Germany) according to
the manufacturer's protocol.
-cyano-4-hydroxycinnamic acid in
acetonitrile, 0.1% trifluoroacetic acid (2:1), which was mixed with
0.5 µl of the peptide solution (34). Spectra were recorded at an
accelerating voltage of 25 kV and were averaged over 40 single laser shots.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Western blot of aortic microsomes treated
with increasing concentrations of PN. A,
immunodetection of PGI2 synthase (52 kDa band) was
performed with a polyclonal antibody against PGI2 synthase.
B, immunodetection of 3-NT-positive proteins was achieved
with a monoclonal antibody against 3-NT from the same blot. Each lane
of the preceding 8% Tris glycine gel contained 20 µg of
protein.
A clear concentration dependence of PN on the extent of nitration was
found up to 250 µM, which was at variance with the high affinity seen with the isolated enzyme (16, 17), but may be explained
by competitive targets for PN in the microsomal fraction. Indeed, when
aortic microsomes were added to P450 BM-3 (Mr
116,000) as a model protein for PGI2 synthase its
Tyr-nitration was sharply decreased (Fig.
2). Because the concentration of
PGI2 synthase on the gel is very low its nitration hardly
shows. If microsomes were treated with 5,5'-dithiobis(2-nitrobenzoic
acid) to block SH groups their inhibitory effect is much less (data not
shown), indicating that in microsomes protein thiols compete for PN and therefore higher PN concentrations are required as with the isolated enzyme.
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Previous studies with other P450 proteins and with model proteins (13,
14, 38) had indicated that multiple Tyr nitrations may occur;
therefore, in this study a PN concentration of 25 µM was
selected that appeared most suitable to yield selective modification of
a single Tyr residue. The inhibition of 6-keto-PGF1
formation was about 50 ± 15% compared with the inhibition at 250 µM, thus matching the NT staining intensities at 25 versus 250 µM. Treatment and isolation of
bovine aortic microsomes under these conditions (see "Experimental
Procedures") provided ~20 µg of PGI2 synthase isolated on SDS-PAGE from the 52-kDa band (Fig.
3, lane D). The protein band
was excised and subjected to proteolytic digestion using trypsin and
mass spectrometric proteome analysis by MALDI-TOF as well as high
resolution FT-ICR (39) (data not shown), which yielded unequivocal
peptide fragment identification of the PGI2 synthase
sequence. However, no NT-containing peptides or other modified peptide
sites were detected by these mass spectrometric data (see Table
I). With low abundance, ATP synthase (56 kDa) and peptide ions because of additional (unidentified)
contaminating proteins in very low amounts were found by protein
sequence data base analyses (SwissProt data base; data not shown). The
contaminating proteins were estimated to account for maximally
20-30% of the protein band (see Fig. 3).
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The isolated protein was subjected to Pronase digestion under
conditions that should lead to quantitative release of the 3-NT residue
for HPLC analysis. Initial digestion for 12 h provided positive
Western blot staining with an NT antibody but did not yield detectable
3-NT by HPLC, although nitrated BSA as a reference protein was
completely digested under these conditions (Fig.
4A). However, prolonged
digestion for 72 h provided the complete liberation and HPLC
detection of 3-NT after the lag phase of about 12 h, suggesting a
decreased accessibility for degradation in the microenvironment of the
nitration site (Fig. 4A). A quantitative estimation of 3-NT
(Fig. 4B) yielded ~1.5 µM 3-NT at a PN
concentration of 25 µM, with the assumption of a pure
protein band of PGI2 synthase. Correcting for 20-30% of
contaminating proteins (ATP-synthase fragments were found) and a
nitration of about 50% one could estimate a nitrotyrosine
concentration of less than 2 µM with 25 µM
PN-treated microsomes. This value would agree with the result in Fig.
4B, which also indicates that at higher PN concentrations
(>100 µM) secondary Tyr nitrations may occur. Therefore,
at 25 µM PN only the specific nitration site could be
expected.
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The isolated PGI2 synthase was subjected to proteolytic
digestion with trypsin under a variety of conditions, followed by mass
spectrometric peptide mapping using both MALDI-TOF-MS and MALDI-FT-ICR-MS (Table I). These studies provided the detailed characterization of the primary structure of the protein; however, HPLC
analysis of the digestion mixture did not result in any peptide with an
absorption at 365 nm indicative for nitrated tyrosine. Experiments with
other proteases (Asp-N, Lys-C, or pepsin) had also shown not to be
successful. Based on the experience with Pronase digestion, thermolysin
was then selected as a protease under comparatively extensive digestion
conditions (24 h, 50 °C). At these conditions a distinct, abundant
peak was found in the PN-treated protein by HPLC at 365 nm with a
retention time of approximately 31 min (Fig.
5C). Typical peptide patterns
were obtained at 220 nm, suggesting that a large portion of the protein had been digested (Fig. 5, B and D). A small peak
was also observed in the untreated control enzyme, confirming a small
basal nitration of PGI2 synthase (Fig. 5A).
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The HPLC-isolated peptide was analyzed by ESI-FT-ICR-MS (Fig.
6A) and Edman sequencing,
which resulted in the unequivocal structure determination and
identification of the nitration site. The ESI-FT-ICR mass spectrum
yielded a single major protonated molecular ion at
m/z 582.29073, corresponding to the monoisotopic composition of the tetrapeptide, LKNY(nitro)-COOH (PGI2
synthase (427-430)); in addition a less abundant (M + Na)+
was obtained. Several less abundant ions were also found indicating some contamination of the HPLC peak, but did not interfere with the
precise mass determination of the tetrapeptide. The specificity of the
FT-ICR-MS analysis was ascertained by comparison with all possible
thermolysin fragments and their tyrosine-containing products, none of
which could account for the MS data of the nitrated peptide (Table I).
Additional proof for the nitration at Tyr-430 came from Edman
microsequencing, which yielded the sequence LKN-Y(nitro), using 3-NT as
a standard and the FT-ICR mass spectrum of the synthetic tetrapeptide
in the carboxamide form (Fig. 6B).
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DISCUSSION |
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In this study we present the definite, molecular identification of
the nitration of PGI2 synthase at tyrosine 430 as an
unusual post-translational modification that occurs with the so far
highest reported affinity for PN. Unequivocal identification of this
nitrated peptide, as well as previously studied Tyr-nitrated peptides, was obtained by high resolution electrospray-FT-ICR-MS, in combination with microsequence analysis of 3-NT; in contrast, MALDI-MS of nitrated
peptides yields extensive fragmentation by cleavage of the nitro group
with elimination of NO and O, hence the resulting ions may obscure the
assignment of nitration sites in complex proteolytic peptide mixtures
(40). Although the specificity of the NT antibodies has been found
sufficiently high to exclude cross-reactivity with other oxidatively
modified amino acids, it was essential to define the tyrosine residue
responsible for the inhibition of enzyme activity. The proteolytic
degradation of the Tyr-nitrated domain was hampered by the high
stability in the microenvironment at the nitration site, whereas the
previously used acid hydrolysis is ambiguous in its specificity because
traces of nitrite may yield artifacts and false-positive results. Using prolonged Pronase digestion we could establish in this study a PN
concentration-dependent increase of the 3-NT formation
providing qualitative and quantitative evidence for the nitration
reaction. By treatment of aortic microsomes with only 25 µM PN a specific nitration at Tyr-430 was established
that was important in view of the possibility that at higher
concentrations a heme-catalyzed formation of a ferryl complex and the
·NO2 radical may lead to secondary modification of
other Tyr residues in PGI2 synthase. Under the
conditions employed for the nitration of PGI2 synthase by
PN only about half of the enzyme was nitrated as judged from the
staining intensity in the Western blots and about half of the
PGH2 conversion to 6-keto-PGF1 was lost.
From previous experiments with aortic microsomes (16) we also know that the untreated enzyme contains a basal level of about 5% nitration (100% activity) and that with 150-250 µM PN about 85% inhibition is reached. Higher PN concentrations did not result in more inhibition but from Fig. 4B a higher 3-NT formation is apparent that probably reflects Tyr residues other than Tyr-430. If only 25 µM PN at a given concentration of microsomal protein are used it is likely that only Tyr-430 is being nitrated. Indeed no other nitrated peptides were found. A direct correlation of the 50% inhibition of activity under these conditions with 50% of enzyme being nitrated is difficult to obtain, because the gel region of 52 kDa isolated contained not only PGI2 synthase, as judged from other peptides (found e.g. from ATP synthase). But assuming about 70% purity the agreement is satisfactory (1.75 µM PGI2 synthase for 1.5 µM 3-NT).
Our results provide definite proof for Tyr-nitration by PN that has
been questioned repeatedly (9-12). The specificity of this
post-translational modification may be provided by the heme catalysis
involved that allows the exclusive nitration of Tyr residues closely
located to the heme (17). The low levels of PN originating from the
reaction of NO and O
The most puzzling finding was the low accessibility of Tyr-430 that hampered the identification of its nitration for some time. The previously observed poor yield of sequence analysis of tryptic peptides (approximately up to 50% at best per experiment of the expected peptide yield) may be explained by the high resistance of the core protein to protease digestion. Accordingly, we postulate that Tyr-430 must be close to the heme because substrate analogs block the nitration; furthermore, because Cys-441 binds heme at its fifth coordination site the domain, Tyr-430 to Cys-441 is expected to form a loop around the heme and this structure may be embedded in a tightly folded conformation. This structure would explain the high resistance to proteases, and is probably also the reason for the incomplete transfer of the enzyme to blotting membranes and the difficulties in obtaining reproducible Western blots, caused by refolding on the blotting membrane. Also the incomplete incorporation of the heme into the protein in expression systems may be associated with a tight secondary structure at the active site.2 Obviously mutation of the Tyr-430 residue should give further clues to the importance of this amino acid. Such experiments are ongoing but hampered by the difficulty of incorporating the heme. However, first data show that a substitution of Tyr-430 by phenylalanine does not block the activity suggesting that the nitro group may only block sterically access to the active site.3 This would be in agreement with the observation that nitration could never block the activity by more than ~80% (17).
Our results also shed some light on the reactivity of PN in biological
systems that has been a matter of debate in recent literature. Because
of its short lifetime of less than 1 s under physiological
conditions one cannot define an affinity for PN and a given target
because the effective concentration for PN varies with other potential
targets present and their concentration. Hence, using isolated
PGI2 synthase it was easy to obtain nitration and
inhibition of activity at submicromolar levels of PN (16, 17), however,
with a complex mixture of macromolecules present in microsomes
substantially higher PN concentrations are required. This is evident
from our data that show in microsomes no Tyr-nitration other than in
PGI2 synthase up to about 250 µM. By
eliminating reactive thiol groups by 5,5'-dithiobis(2-nitrobenzoic
acid) the sensitivity of PGI2 synthase for nitration was
increased. It is known that PN is an efficient oxidant for zinc finger
proteins (41) and in the presence of metal ions further potential
targets may compete for reaction with PN. Despite this straightforward explanation, the observation that micromolar PN can nitrate
PGI2 synthase in whole endothelial (17, 42) or mesangial
cells (42) is not readily understood. A possible clue to this
phenomenon may be the recent observation that PGI2 synthase
is mainly located to the caveolae at the outer cell membrane (20).
Thus, added PN may find PGI2 synthase as a target before
the complex and antioxidative environment of the cell will compete. The
fact that endothelial NO synthase is found in the same compartment
suggests that the generation of NO and its reaction with O
Recently we could demonstrate the nitration of PGI2 synthase by endotoxin exposure of aortic rings and found nitration, loss of activity, and tissue contraction correlated (43). Inhibition of NO synthesis and the presence of superoxide dismutase could prevent all three effects.
Another still open question is the reversibility of the nitration. The
presence of a denitratase has been postulated but could not yet be
established (44, 45). From own
observations4 there may be
indeed a recovery of PGI2 synthase activity that is faster
than new protein synthesis. If this would turn out as a process
involving denitration a new redox-regulatory mechanism would be
established. But even without this reversibility the superoxide-mediated trapping of NO with a concomitant down-regulation of PGI2 synthase, followed by thromboxane
A2/PGH2 receptor activation, represents
a key event in our understanding of endothelial activation (5).
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Markus Kohlmann and Eugen Damoc for expert assistance with the proteome analysis and proteolytic digestion studies. We are also grateful to Prof. Dr. F. Lottspeich (MPI Martinsried, Germany), Prof. Dr. W. D. Lehmann (DKFZ Heidelberg, Germany), A. von Kriegsheim (University of Glasgow, UK), and Dr. C. Schöneich (Department of Pharmacological Chemistry, University of Kansas) for preliminary experiments. The collaboration of Dr. T. Tanabe (Osaka, Japan) is highly appreciated.
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FOOTNOTES |
---|
* This work was supported by grants from the Deutsche Forschungsgemeinschaft, Forschergruppe "Endotheliale Gewebszerstörung-Mechanismen der Autodestruktion" (to V. U.), and "High Resolution Biopolymer Mass Spectrometry" (to M. P.).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.
¶ To whom correspondence should be addressed: Fakultät für Biologie, Universität Konstanz, Fach X910, Sonnenbühl, 78457 Konstanz, Germany. Tel.: 49-7531-88-2287; Fax: 49-7531-88-4084; E-mail: volker.ullrich@uni-konstanz.de.
Published, JBC Papers in Press, January 31, 2003, DOI 10.1074/jbc.M208080200
2 M. Wada and T. Tanabe, personal communication.
3 M. Wada, T. Tanabe, and V. Ullrich, unpublished results.
4 P. Schmidt, M. Bachschmid, and V. Ullrich, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are:
PN, peroxynitrite
(oxoperoxonitrate (1));
PGI2, prostacyclin;
PGI2 synthase, prostacyclin synthase;
NT, 3-nitrotyrosine;
PGH2, prostaglandin endoperoxide;
6-keto-PGF1
, 6-keto-prostaglandin F1
;
EIA, enzyme immunoassay;
NO, nitric oxide;
PBS, phosphate-buffered
saline;
BSA, bovine serum albumin;
P450BM-3, bacterial
monooxygenase-3 from Bacillus megaterium (CYP 102);
FT-ICR, Fourier transform ion cyclotron resonance;
HPLC, high performance
liquid chromatography;
PTH, phenylthiohydantoin;
MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight;
Fmoc, N-(9-fluorenyl)methoxycarbonyl.
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