From the Departments of Anesthesiology,
§ Biochemistry and Molecular Genetics,
§§ Pathology,
Pharmacology and Toxicology,
¶¶ Neurology, ¶ Center for Free Radical Biology,
Comprehensive Sickle Cell Disease Center, and
** Comprehensive Cancer Center Mass Spectrometry Shared
Facility, University of Alabama at Birmingham, Birmingham, Alabama
35233
Received for publication, August 30, 2002
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ABSTRACT |
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The intermittent vascular occlusion
occurring in sickle cell disease (SCD) leads to
ischemia-reperfusion injury and activation of inflammatory
processes including enhanced production of reactive oxygen species and
increased expression of inducible nitric-oxide synthase (NOS2).
Appreciating that impaired nitric oxide-dependent vascular
function and the concomitant formation of oxidizing and nitrating
species occur in concert with increased rates of tissue reactive oxygen
species production, liver and kidney NOS2 expression, tissue
3-nitrotyrosine (NO2Tyr) formation and apoptosis were
evaluated in human SCD tissues and a murine model of SCD. Liver and
kidney NOS2 expression and NO2Tyr immunoreactivity were
significantly increased in SCD mice and humans, but not in nondiseased
tissues. TdT-mediated nick end-label (TUNEL) staining showed
apoptotic cells in regions expressing elevated levels of NOS2 and
NO2Tyr in all SCD tissues. Gas chromatography mass
spectrometry analysis revealed increased plasma protein
NO2Tyr content and increased levels of hepatic and renal
protein NO2Tyr derivatives in SCD (21.4 ± 2.6 and
37.5 ± 7.8 ng/mg) versus wild type mice (8.2 ± 2.2 and 10 ± 1.2 ng/mg), respectively. Western blot analysis and immunoprecipitation of SCD mouse liver and kidney proteins revealed one
principal NO2Tyr-containing protein of 42 kDa, compared
with controls. Enzymatic in-gel digestion and MALDI-TOF mass
spectrometry identified this nitrated protein as actin. Electrospray
ionization and fragment analysis by tandem mass spectrometry revealed
that 3 of 15 actin tyrosine residues are nitrated
(Tyr91, Tyr198, and Tyr240)
at positions that significantly modify actin assembly. Confocal microscopy of SCD human and mouse tissues revealed that nitration led
to morphologically distinct disorganization of filamentous actin.
In aggregate, we have observed that the hemoglobin point mutation of sickle cell disease that mediates hemoglobin polymerization defects is translated, via inflammatory oxidant reactions, into defective cytoskeletal polymerization.
The intermittent vascular occlusion occurring in
SCD1 is characterized by
acute, painful crises and leads to the renal and hepatic tissue injury
and dysfunction manifested by patients with this hemoglobinopathy
(1-3). Peripheral vascular insufficiency, accompanied by periodic
restoration of blood flow, places ischemic organs at risk of additional
injury by inducing a proinflammatory state reflected by enhanced
superoxide (O Increased rates of production of reactive oxygen- and nitrogen-derived
species in tissues mediate the oxidation and nitration of lipids,
nucleotides, and susceptible protein amino acid residues. These
products also suggest an impairment of biomolecular structure and
function. For example, the protein nitration product NO2Tyr is elevated in a variety of inflammatory diseases mediated in part by
reactive inflammatory mediators, including atherosclerosis (25), acute
lung injury (26), adult respiratory distress syndrome (27), biliary
cirrhosis (28), myocardial inflammation (29), ileitis (30), rheumatoid
arthritis (31), endotoxin-induced kidney injury (32), chronic renal
allograft rejection (33), Alzheimer's disease (34), amyotrophic
lateral sclerosis (35), and sepsis (36). The development of a causal
relationship between post-translational protein nitration and impaired
tissue function is presently limited by insight into where and how the
inflammatory modification of specific protein amino acid residues
occurs in vivo and how this is linked with
biomolecule dysfunction. Herein, a combination of clinical and
knockout-transgenic mouse studies underscores the extensive occurrence
of ·NO-mediated oxidative inflammatory reactions in SCD, with
actin identified as a key target for protein nitration in kidney and liver. This identification of actin as a key tissue target for protein
nitration and its impaired polymerization properties reveals the
significance of this post-translational protein modification in the
physiopathology of SCD and related vascular inflammatory processes.
Immunofluorescence Microscopy and TUNEL
Analysis--
Paraffin-embedded kidney and liver sections were
obtained from SCD human autopsy samples and knockout-transgenic SCD
mice (37) following approval by the Institutional Review Board for Human Use and the Institutional Animal Care and Use Committee at the
University of Alabama at Birmingham. Paraffin-embedded sections were
mounted on slides, deparaffinized, and processed for
immunofluorescence. Primary antibody incubations were for 60 min at
25 °C using a rabbit polyclonal anti-NO2Tyr (Cayman, 5 µg ml Measurement of Plasma and Tissue NO2Tyr--
Blood
was collected from healthy HbA adult volunteers and homozygous HbS
patients in anticoagulated (EDTA) Vacutainers as approved by the
Institutional Review Board for Human Use at the University of
Alabama at Birmingham. Blood cells were removed by centrifugation, and
plasma was stored at Purification of Actin from Kidney and Liver--
Tissue actin
purification was performed by DNase I affinity chromatography as
described previously (39, 40). Briefly, 15 ml of Affi-Gel 10 (BioRad)
was transferred to a Buchner funnel and washed with 3 bed volumes of
cold deionized water. The gel cake was incubated with 100 mg of DNase I
(Roche Diagnostics) and dissolved in 10 ml ice-cold coupling buffer
(0.1 M Hepes, pH 7.4, 2 mM CaCl2)
for 4 h at 4 °C. The gel slurry was loaded into a column,
washed with cold deionized water, and equilibrated with buffer G (2 mM Tris-HCl, pH 7.9, 0.2 mM CaCl2,
0.2 mM ATP, and 0.2 mM dithiothreitol). Liver
and kidney were dissected, weighed, and homogenized in ice-cold buffer
G containing 10% formamide (v/v) (Sigma). Homogenates were centrifuged
(100,000 × g, 1 h, 4 °C), and supernatants
were applied to the DNase I-agarose column. The column was washed
successively with buffer G, 0.2 M NH4Cl in
buffer G containing 10% formamide (v/v) and with buffer G containing 10% formamide (v/v). Adsorbed actin was eluted with buffer G
containing 40% formamide (v/v). Pilot studies using actin treated with
0.3 mM ONOO Western Blot Analysis and Immunoprecipitation--
For Western
blotting, mouse monoclonal anti-NO2Tyr (Cayman, 2 µg
ml MALDI-TOF and Electrospray Mass Spectrometry--
In-gel protein
digests were prepared as described previously (41, 42). Briefly,
protein bands were excised from gels, destained with acetonitrile/25
mM ammonium bicarbonate (1:1, v/v), and dried. Samples were
rehydrated with 12.5 ng µl Measurement of Actin Polymerization--
Actin was purified from
rabbit hind limb, gel-filtered, and labeled with pyrenyl iodoacetamide
(pyrene-labeled actin) as described previously (43, 44). In some cases
control and pyrene-labeled actin were treated with 0.3 mM
ONOO Kidney and Liver NOS2 Expression and NO2Tyr
Formation--
There was a strong co-distribution of NOS2 and tissue
NO2Tyr immunostaining in the renal cortex of
knockout-transgenic SCD mice and humans with SCD that was not evident
in wild type (control) C57Bl/6J mice or healthy humans expressing HbA
(Fig. 1). Distribution of NOS2 expression
in SCD kidneys was in distal and proximal tubular epithelial cells and
glomeruli. The proximal and distal tubules were immunoreactive for
NO2Tyr in both SCD mouse and human but unlike mice, human
SCD kidneys were also immunoreactive for NO2Tyr in
glomeruli (Fig. 1, A and B). The expression of
NOS2 in SCD human and mouse liver was localized to hepatocytes
surrounding the central veins and co-distributed with
NO2Tyr immunoreactivity (Fig.
2, A and B).
Immunoreactive NOS2 and NO2Tyr was significantly less in control mouse and human liver (Fig. 2). Preadsorption of
anti-NO2Tyr with NO2Tyr revealed that
NO2Tyr immunostaining in kidney and liver sections was
specific (not shown). Western blot analysis of NOS2 expression and
NO2Tyr in kidney and liver homogenates revealed increased
NOS2 expression and protein NO2Tyr content in SCD mice
compared with controls (Fig. 4, A and B).
Apoptosis--
TUNEL labeling showed dark brown apoptotic cells
with pyknotic nuclei in the proximal and distal convoluted tubules and
the glomeruli of SCD human kidney (Fig.
3A) and the proximal and
distal tubules of SCD mouse kidney (Fig. 3B). TUNEL staining
in SCD human and mouse liver was localized principally to the
pericentral hepatocytes (Fig. 3, A and B).
Plasma and Tissue NO2Tyr Concentrations--
Plasma
protein NO2Tyr content was increased 2.4- and 2.8-fold over
controls in SCD humans and mice, 24.7 ± 1.7 and 37.7 ± 6.6 ng/mg protein, respectively (Table I).
There was also a marked difference in liver and kidney homogenate
protein NO2Tyr adducts in SCD mice (21.4 ± 2.6 and
37.5 ± 7.8 ng/mg protein, respectively) versus
controls (8.2 ± 2.2 and 10 ± 1.2 ng/mg protein). The
actin-enriched fraction of mouse liver and kidney showed a greater
protein NO2Tyr content in both control and SCD mice
compared with whole organ homogenates. Finally, there was a 17-24-fold
increase in actin nitration in SCD mouse liver and kidney, respectively
(Table I).
Actin Nitration--
Western blot analysis of mouse kidney and
liver homogenates with anti-NO2Tyr showed one predominant
(42 kDa) immunoreactive band in SCD tissues compared with controls
(Fig. 4A). Immunoprecipitation of kidney and liver protein extracts with polyclonal
anti-NO2Tyr also revealed a NO2Tyr-containing
42-kDa protein (Fig. 4C) in SCD but not wild type mice. The
42-kDa NO2Tyr-containing protein bands for both
liver and kidney were excised from gels following electrophoresis,
digested with trypsin, and analyzed by MALDI-TOF mass spectrometry.
Mass fingerprint data sets were analyzed using a Mascot algorithm (46)
with fragment ions of m/z 976, 1132, 1153, 1198, and 1791 (Fig. 5, A and
B) matching mouse actin with a score of 84 (p < 0.05), well above the significance threshold of
71. Mouse liver and kidney actin were partially purified by DNase I
affinity chromatography (Fig. 4D), and protein nitration was
verified by immunoblotting with a polyclonal NO2Tyr
antibody (Fig. 4E). The strong co-localization of actin and
NO2Tyr immunoreactivity in the merged fluorescence images
(Fig. 6, A and B)
also further confirmed actin nitration in SCD kidney and liver. The
minor NO2Tyr-containing 53-kDa band observed in
actin-enriched SCD tissue extracts (Fig. 4E) was also in-gel
digested and analyzed by MALDI-TOF mass spectrometry. Mass fingerprint
data sets were analyzed using a Mascot algorithm (46) with ions of
m/z 1051, 1272, 1303, 1741, 2441, and 2882 (Fig,
5, C and D) identifying G-actin-associated
vitamin D-binding protein with a score of 79 (p < 0.05).
Identification of Specific Actin Tyrosine Residues Nitrated in
Vivo--
A NO2Tyr-enriched actin fraction from SCD mouse
liver and kidney homogenates was prepared by immunoprecipitation of
NO2Tyr-containing protein from the actin fraction purified
by DNase I affinity chromatography (Fig. 4F). Following
electrophoretic separation, the 42-kDa NO2Tyr-containing protein band was in-gel digested and analyzed by MALDI-TOF mass spectrometry and MS/MS. The observed mass fingerprint data sets for actin revealed nitration of three tyrosine residues in
vivo (Tyr91, Tyr198, and
Tyr240). The MALDI-TOF mass spectrum of the tryptic
fragment corresponding to residues 85-95 (Fig.
7A) showed a +45 mass unit ion
shift from m/z 1516 to 1561. The MS/MS spectrum
of the same fragment (Fig. 8A) reflected an
identical mass increase in y10, y9, y8,
y7, and y6 daughter ions, indicative of
Tyr91 nitration. The MALDI-TOF spectrum of the tryptic
fragment corresponding to residues 197-206 (Fig. 7B) showed
a shift of +45 mass units from m/z 1132 to 1177, whereas the MS/MS spectrum of the same fragment (Fig. 8B)
showed a b2 ion that shifted from m/z
221 to 266, identifying Tyr198 as the nitrated residue. The
MALDI-TOF spectrum of the tryptic fragment corresponding to residues
239-254 (Fig. 7C) showed an ion shift of +45 mass units
from m/z 1791 to 1836, whereas the MS/MS spectrum
of the same fragment (Fig. 8C) showed a b2 ion that shifted from m/z 251 to 296, revealing
Tyr240 as the site of nitration.
Actin Nitration in Vitro--
Purified rabbit muscle G-actin was
utilized to analyze the influence of tyrosine nitration on the kinetics
of actin polymerization, and hence it was essential to identify the
sites of actin tyrosine nitration ex vivo. Electrospray
ionization MS/MS analysis of proteolytic fragments from rabbit
actin treated with 0.3 mM ONOO Effect of Nitration on Actin Polymerization--
Because tyrosine
residues 198 and 240 are in the region of the "pointed" end of the
actin filament (47), critical concentration, a measure of actin
affinity for the rapidly growing "barbed" end of the filament (48),
was examined by using pyrene fluorescence emission (45). A plot of
pyrenyl fluorescence versus actin concentration is shown in
Fig. 10A. The pyrenyl
fluorescence emission of nitrated G-actin was quenched by 50% compared
with native G-actin, because of the broad absorption band of
nitrotyrosine (
Polymerization of actin is accomplished in two steps, formation of
relatively unstable nuclei followed by rapid elongation. The nucleation
event is rate-limiting and is evidenced by a lag in formation of actin
filaments during the polymerization process. The results depicted in
Fig. 10A imply that nitration stabilizes interactions
between the pointed end of G-actin and the barbed end of a growing
actin filament. This would be expected to have two effects: 1) it
should stabilize formation of actin nuclei and shorten the lag phase;
and 2) it should either accelerate the rate of filament elongation or
slow the rate of subunit dissociation, because the lower critical
concentration implies a tighter affinity of G-actin for the barbed end.
Fig. 10B shows a plot of pyrene fluorescence
versus time for the polymerization reaction using native
(closed triangles) and nitrated (open boxes)
actin. Actin nitration shortens the lag phase and accelerates filament
elongation. Data for both native and nitrated actin were fitted to a
sum of two exponential processes, representing a lag followed by a
first order increase in fluorescence. For nitrated actin, the rate
constants for the lag and rising phase were 0.0032 and 0.0038 s The repetitive episodes of tissue ischemia-reperfusion, the
pro-inflammatory state in the systemic vasculature (51, 52), and the
oxidative impairment of vascular ·NO signaling events that occur
in SCD (51) all encourage the formation of secondary oxidizing and
nitrating species and contribute to impaired vascular and organ
function. The occurrence of xanthine oxidase-derived,
O Nitration of free and protein-associated tyrosine residues to yield
NO2Tyr has been detected in multiple species, organ
systems, and cell types during both acute and chronic inflammation
(65). The existence of multiple distinct, yet redundant, pathways for tyrosine nitration underscores the potential significance of this process in inflammation and cell signaling. This post-translational protein modification is thus a marker of oxidative injury that is
frequently linked to altered protein function during inflammatory conditions (66-68). The reversible nature of protein
NO2Tyr adducts (69, 70) also implies that tyrosine
nitration may not only represent a marker of reactive nitrogen species
formation and altered protein function but may also evoke protein
conformational changes that mimic or impact on cell signaling events
such as adenylation and tyrosine phosphorylation (71).
Critical to understanding the pathogenic inflammatory reactions
occurring in SCD is the observation of NOS (2) and NO2Tyr co-distribution in humans with SCD and a mouse model of SCD (Figs. 1
and 2), where liver and kidney NO2Tyr are elevated 2.6 and
3.7-fold, respectively (Table I). Immunoprecipitation and MALDI-TOF
mass spectrometry-assisted identification of actin as the predominant nitrated protein in the liver and kidney of SCD mouse
(Figs. 4 and 5) provides critical insight into the pathogenic events to be expected from this inflammatory milieu. Particular insight in this
regard is provided by the identification of specific actin tyrosine
residues that are nitrated in vivo. Actin, one of the most
abundant proteins in eukaryotic cells, constitutes 5% or more of cell
protein (72) and serves with other cytoskeletal proteins such as
tubulin (66) as a critical target for oxidation and nitration-induced
functional impairment (73-75). As for other cytoskeletal proteins,
actin contains a high percentage of tyrosine residues, many of which
are crucial participants in protein-protein recognition motifs (76).
The introduction of an electronegative NO2 group onto a
tyrosine ring reduces the pKa of the phenolic hydroxyl to values in the range of 6.8-7.0. If such nitrotyrosine residues were involved in intersubunit interactions, they could form
ionic or hydrogen bonds with cationic residues located in the barbed
end of a growing filament. This might stabilize both actin nucleus and
filament formation, as evidenced by the effects of nitration on
polymerization kinetics and thermodynamics (Fig. 10).
Because of the cooperative nature of actin subunit assembly (77), the
functional consequences of tyrosine nitration on actin dynamics can be
profound. Relatively small proportions of modified subunits could
stabilize elongating filaments from fragmentation, as well as drive the
equilibrium toward polymerization. Although we have not examined the
effects of nitration on the severing ability of actin binding proteins
such as gelsolin, it is reasonable to assume that even modest changes
in intersubunit affinities will alter significantly the dynamics of
actin filaments in the cell cortex. This can ultimately lead to loss of
control of filament formation, with subsequent alterations in cell
motility, attachment, and intracellular transport. Interestingly, we
observed that the extent and sites of F- and G-actin tyrosine nitration
by ONOO
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
), the nitrating and oxidizing species produced by
the radical-radical reaction of O
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1) and anti-NOS2 (BD Transduction Laboratories, 16 µg ml
1). The secondary antibody was
Alexa-594-conjugated goat anti-rabbit IgG (Molecular Probes, 1:100).
Nonspecific staining was ruled out by control experiments performed by
preadsorbing anti-NO2Tyr with 10 mM
NO2Tyr (not shown). AlexaFluor-488 phalloidin (Molecular Probes, 1 unit) was used for visualizing actin. Images were acquired on
a Leitz orthoplan microscope (Leica Inc., Wetzlar, Germany) or a Leica
DMIRBE inverted epifluorescence-Nomarski microscope with Leica TCS NT
laser confocal optics. Apoptotic cells were visualized with the
terminal deoxynucleotide transferase (TdT) FragEL DNA fragmentation kit
(Oncogene) analogous to TdT-mediated nick end-labeling.
80 °C for subsequent processing and analysis.
The liver and kidneys of C57Bl/6J or knockout-transgenic SCD mice,
which synthesize exclusively human Hb in the murine red blood
cells (37), were dissected, weighed, and homogenized in ice-cold
homogenizing buffer (50 mM K2HPO4, 80 µM leupeptin, 2.1 mM Pefabloc SC, 1 mM phenylmethylsulfonyl fluoride, 1 µg ml
1
aprotinin, pH 7.4). Homogenates were centrifuged (40,000 × g, 30 min, 4 °C), and supernatants were stored at
80 °C. Plasma and tissue protein NO2Tyr was quantified
by gas chromatography-MS as described previously (38). For use as an
internal standard, 3-[13C6]nitrotyrosine was synthesized
by the addition of 1.5 mM ONOO
to 6 mM of [13C6]tyrosine (Cambridge
Isotope Laboratories) and purified and quantified via HPLC (38).
Peroxynitrite was synthesized as described previously (24) and its
concentration determined spectrophotometrically at 302 nm
(
M = 1670 M
1
cm
1). All samples were analyzed immediately following
derivatization in the electron ionization mode (EI) with a Varian GC
3800 gas chromatograph equipped with a 30 m × 0.25 mm ID
fused silica capillary column having a DB-5 stationary phase and
interfaced with a Varian Saturn 2000 mass spectrometer.
showed similar chromatographic
behavior of native actin.
1), rabbit polyclonal anti-NO2Tyr (Cayman,
2 µg ml
1), and anti-NOS2 (BD Transduction Laboratories,
1:800 dilution) were used as primary antibodies. Horseradish
peroxidase-conjugated goat anti-mouse IgG (Pierce, 1:10000) was used as
a secondary antibody, and immunoreactive proteins were visualized by
chemiluminescence (ECL reagent, Amersham Pharmacia Biotech). For
immunoprecipitation, tissue homogenates were cleared with protein
A-agarose (Roche Molecular Biochemicals) for 3 h at 4 °C.
Supernatants were then incubated with rabbit polyclonal
anti-NO2Tyr (Cayman, 5 µg ml
1) for 1 h
followed by protein A-agarose incubation for 3 h at 4 °C.
NO2Tyr-containing actin was immunoprecipitated from
actin-enriched SCD liver and kidney extracts with a NO2Tyr
affinity sorbent (Cayman, 40 µl ml
1).
Immunoprecipitated proteins were washed, separated by SDS-PAGE, and
visualized by GelCode Coomassie Blue stain reagent (Pierce).
1 trypsin (Promega) in 25 mM ammonium bicarbonate buffer and digested overnight at
37 °C. Peptides were extracted with acetonitrile/5% formic acid
(1:1, v/v), mixed with cyano-4-hydroxycinammic acid (Aldrich) (1:1,
v/v), and spotted onto a gold-coated MALDI plate. Peptide molecular
ions were analyzed in the positive ion mode using a Voyager DePro mass
spectrometer (Applied Biosystems). The acceleration voltage was set at
20 kV, and 100 laser shots were summed. In some cases, purified rabbit
skeletal actin (Sigma, 24 µM) was modified minimally with
0.3 mM ONOO
, denatured with 6 M
guanidine hydrochloride, and reduced with 5 mM
dithiothreitol for 2 h at 37 °C. Cysteines were
alkylated with 1 mM iodoacetamide for 2 h in the dark
at 25 °C. Samples were dialyzed on 10-kDa molecular mass cut-off
Slide-A-Lyzer Cassettes (Pierce) against 100 mM ammonium
bicarbonate, pH 8, and digested with 25 µg of sequencing grade
modified trypsin (Promega). For electrospray analysis, peptide
fragments were separated by reverse-phase HPLC column (300 µm × 15 cm C18 PepMap) at a flow rate of 2 µl min
1
with a gradient from 20 to 100% acetonitrile, 0.1% formic acid over a
period of 20 min. For both rabbit- and mouse-derived actin samples,
electrospray-mass spectrometry was performed on a Q-TOF II MS
(Micromass, Manchester, UK) with automatic functional switching between
survey MS and MS/MS modes. A multiply charged peak above 6 counts
detected in the mass spectrum was selected automatically for tandem MS analysis.
, reduced with 2 mM dithiothreitol, and
dialyzed against 5 mM Tris-HCl, 0.2 mM
CaCl2, 0.2 mM ATP, pH 8.0. Control and nitrated actin (9.6 µM) were mixed with equimolar
pyrene-conjugated G-actin (1:1, v/v), and polymer formation was
monitored by pyrene actin fluorescence (45) via an automated microplate
fluorescence reader (Fluostar Galaxy, BMG Laboratory Technologies) set
at
ex = 350 nm and
em = 410 nm.
Steady-state polymerization of control and nitrated actin were assayed
by fluorescence intensity (
ex = 345 nm and
em = 407 nm) of pyrene-labeled actin in 50 mM KCl, 25 mM Hepes, 2 mM
MgCl2, 0.1 mM CaCl2, 0.2 mM ATP, and 1 mM dithiothreitol. Depolymerization kinetics were measured by mixing nitrated or native
pyrene-labeled F-actin with DNase I (with an actin:DNase ratio of 1:5
(mol/mol)) and monitoring fluorescence intensity as above.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Immunofluorescent staining of NOS2 and
3-nitrotyrosine in control and sickle cell diseased renal cortex.
A, sections from sickle cell and control (Ctl)
human kidney. Glomeruli and proximal and distal tubules display intense
immunofluorescence for NOS2 and NO2Tyr when compared with
controls. B, sections from a knockout-transgenic sickle cell
and C57Bl/6J control mouse kidney. NOS2 staining is observed in the
glomeruli and in tubular epithelial cells, whereas NO2Tyr
immunoreactivity is localized principally to distal and proximal
tubules. NOS2 and NO2Tyr staining is not evident in
C57Bl/6J control sections. Nuclei are counterstained with Hoechst in
all experiments.
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Fig. 2.
Immunofluorescent staining of NOS2 and
NO2Tyr in control and sickle cell liver.
A, sections from sickle cell and control (Ctl)
human liver. NOS2 and NO2Tyr staining is observed
predominantly in the pericentral hepatocytes of SCD liver compared with
controls. B, sections from knockout-transgenic sickle cell
and C57Bl/6J control mouse liver. Increased NOS2 and NO2Tyr
staining are localized to the pericentral hepatocytes of SCD mouse and
are not evident in controls. Nuclei are counterstained with Hoechst in
all experiments. CV, central vein.
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Fig. 3.
TUNEL staining in control and sickle cell
kidney and liver. Dark brown cells with pyknotic nuclei
indicate positive staining for apoptosis, and green to
greenish tan signifies a nonreactive cell. A,
sections from control (Ctl) and sickle cell human kidney and
liver. Apoptotic cells are seen in the tubular epithelium and glomeruli
of SCD renal cortex and in pericentral hepatocytes of SCD liver.
B, sections from a knockout-transgenic SCD and C57Bl/6J
control mouse kidney and liver. Apoptosis is prevalent in the proximal
and distal tubules of SCD kidney and in the pericentral hepatocytes of
SCD liver. CV, central vein.
3-Nitrotyrosine content in sickle cell disease
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Fig. 4.
SDS-PAGE and Western blot analysis of kidney
and liver homogenates and immunoprecipitation pellets.
A and B, kidney and liver homogenates of
knockout-transgenic SCD and C57Bl/6J control mice analyzed by
immunoblotting with mouse mAb against NO2Tyr (A)
or NOS2 (B). C, immunoprecipitation of SCD and
C57Bl/6J control (Ctl) mouse kidney (Kid) and
liver (Liv) homogenates using a polyclonal
NO2Tyr antibody. The immunoprecipitation pellet was
separated by SDS-PAGE and visualized by Coomassie Blue staining. The
nitrated protein was observed as a single 42-kDa band in SCD kidney and
liver with IgG heavy (50 kDa) and light chains (25 kDa). (D)
SDS-PAGE and Coomassie Blue staining of actin-enriched kidney and liver
extracts obtained from SCD and C57Bl/6J control mouse. E,
immunoblot analysis of actin-enriched kidney and liver extracts using a
polyclonal NO2Tyr antibody. The observed
NO2Tyr-containing proteins in SCD kidney and liver
correspond to actin (42 kDa) and actin-associated vitamin
D-binding protein (53 kDa). F,
immunoprecipitation of actin-enriched SCD liver and kidney extracts
with NO2Tyr affinity sorbent. The immunoprecipitation
pellet was separated by SDS-PAGE and visualized by Coomassie Blue
staining.
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Fig. 5.
MALDI-TOF MS identification of actin and
vitamin D-binding protein. The 42-kDa protein, immunoprecipitated
from liver and kidney homogenates of SCD mouse via a NO2Tyr
antibody, was in-gel digested and analyzed by MALDI-TOF MS. Peptide
fragments from kidney (A)- and liver (B)-matched
mouse actin with Mascot algorithm analysis. The
NO2Tyr-containing 53-kDa band observed in actin-enriched
SCD tissue extracts was in-gel digested and identified by MALDI-TOF MS.
Mass fingerprint data sets obtained from SCD liver (C) and
kidney (D) were analyzed using Mascot algorithm with
fragment ions matching mouse vitamin D-binding
protein.
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Fig. 6.
Immunohistochemical co-distribution of actin
and NO2Tyr in sickle cell kidney and liver.
A, sections from sickle cell human kidney and liver.
B, sections from knockout-transgenic SCD mouse kidney and
liver. Tissue sections were labeled for actin (green) and
NO2Tyr (red). Nuclei (blue) were
counterstained with Hoechst. To assess co-distribution of actin and
NO2Tyr, images were merged (orange).
CV, central vein.
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Fig. 7.
MALDI-TOF MS of identification nitrated actin
fragments. NO2Tyr-enriched actin fractions, obtained
from actin-enriched SCD liver and kidney extracts via
NO2Tyr antibody immunoprecipitation, were in-gel digested
and analyzed by MALDI-TOF MS. A, MS spectrum of the nitrated
tryptic fragment 85IWHHTFYNELR95
[M+H]+ (m/z 1561). B, MS
spectrum of the nitrated tryptic fragment
197GYSFTTTAER206 [M+H]+
(m/z 1177). C, MS spectrum of the
nitrated tryptic fragment
239SYELPDGQVITIGNER254 [M+H]+
(m/z 1836).
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Fig. 8.
MS/MS identification and
representation of in vivo nitrated actin
residues. A, MS/MS spectrum of the tryptic fragment
85IWHHTFYNELR95 [M+2H]2+
(m/z 781). B, MS/MS spectrum of the
tryptic fragment 197GYSFTTTAER206
[M+2H]2+ (m/z 589). C,
MS/MS spectrum of the tryptic fragment
239SYELPDGQVITIGNER254 [M+2H]2+
(m/z 918). D, ribbon representation of
actin (Ref. 47; PDB Id: 1J6Z) produced using Rasmol version 2.6. Actin subdomains are
represented in green (subdomain 1), gray
(subdomain 2), magenta (subdomain 3), and silver
(subdomain 4). The regions contributing to longitudinal actin contacts
in domains II, III, and IV are depicted in blue, and the
bound nucleotide is shown as yellow sticks. Nitrated
tyrosine residues are shown as red sticks and are labeled
with single-letter codes.
revealed
nitration of four residues (Tyr53, Tyr198,
Tyr240, and Tyr362), with nitration of
Tyr362 not consistently observed in some experiments. MS/MS
spectra obtained by collision-induced dissociation of
[M+2H]2+-nitrated tryptic fragments resulted in dominant
fragmentation at the amide bonds yielding type b or
y ions (Fig 9).
Again, fragment ions containing the NO2 group were shifted
by +45 mass units. These ions are designated by circles and
numbered according to their position along the sequence
(Fig. 9). The MS/MS spectrum of the tryptic fragment
corresponding to residues 51-61 (parent ion, m/z
622.2) showed a y9 ion that shifted from
m/z 996.4 to 1041.5 and a b3 ion that
shifted from m/z 366.1 to 411.1, thus identifying
Tyr53 in the amino acid sequence DSYVGDEAQSK as the site of
nitration (Fig. 9A). The MS/MS spectrum of the tryptic
fragment corresponding to residues 197-206 (parent ion
m/z 588.7) showed a b2 ion that shifted from m/z 221 to 266, identifying
Tyr198 in the amino acid sequence GYSFVTTAER as the
nitrated residue (Fig. 9B). The MS/MS spectrum of the
tryptic fragment corresponding to residues 239-254 (parent ion
m/z 918.4) showed a b2 ion that shifted from m/z 251 to 296, revealing
Tyr240 in the amino acid sequence SYELPDGQVITIGNER as the
site of nitration (Fig. 9C). The MS/MS spectrum of the
tryptic fragment corresponding to residues 360-372 (parent ion
m/z 773.8) showed a y11 ion that shifted from m/z 1243 to 1288, exposing
Tyr362 in the amino acid sequence QEYDEAGPSIVHR as the
nitrated residue (Fig. 9D).
View larger version (22K):
[in a new window]
Fig. 9.
Tandem 32
MS/MS identification and representation of
in vitro nitrated actin residues. A,
MS/MS spectrum of the tryptic fragment
51DSYVGDEAQSK61 [M+2H]2+
(m/z 622.2). B, MS/MS spectrum of the tryptic fragment
197GYSFVTTAER206 [M+2H]2+
(m/z 588.7). C, MS/MS spectrum of the
tryptic fragment 239SYELPDGQVITIGNER254
[M+2H]2+ (m/z 918.4). D,
MS/MS spectrum of the tryptic fragment
360QEYDEAGPSIVHR372 [M+2H]2+
(m/z 773.8). E, ribbon representation
of actin (Ref. 47; PDB Id: 1J6Z) produced using Rasmol version 2.6. Actin subdomains are represented in green (subdomain 1),
gray (subdomain 2), magenta (subdomain 3), and
silver (subdomain 4). The regions contributing to
longitudinal actin contacts in domains II, III, and IV are depicted in
blue, and bound nucleotides are shown as
yellow sticks. Nitrated tyrosine residues are shown as
red sticks and are labeled with single-letter codes.
430 = 4400 M
1cm
1 (49)). Even in the
presence of this quenching, a significant effect of nitration on
critical concentration was observed. For native actin (open
circles), the extrapolated critical concentration is 89 ± 16 nM, similar to previous measurements at this ionic strength
in 50 mM KCl (50). By contrast, the curve for nitrated actin extrapolated to the origin, implying a critical
concentration < 10 nM.
View larger version (17K):
[in a new window]
Fig. 10.
Effects of nitration on actin polymerization
thermodynamics and kinetics. A, critical concentration
plot of native pyrene-labeled actin (open circles) compared
with nitrated pyrene-labeled actin (closed triangles, 2 mol
of nitrotyrosine/mol of G-actin monomer). Although the plot of
fluorescence versus total actin concentration shows the
inflection typical of native actin, defining a critical concentration
of 89 ± 16 nM, the plot for nitrated actin
extrapolates to <10 nM. B, kinetics of
polymerization for native (closed triangles)
versus nitrated (open boxes) actin (2 mol of
nitrotyrosine/mol of actin). Polymerization data were fitted to a sum
of two exponentials to describe a lag phase followed by a first order
polymerization step. For nitrated actin, the rate constants for the lag
and rising phase were 0.0032 and 0.0038 s 1, respectively,
whereas for native actin the corresponding rates were 0.000763 and
0.000564 s
1. C, kinetics of depolymerization
of native (closed triangles) versus nitrated
(open boxes) actin filaments. Polymerized actin at a
concentration of 4 µM was depolymerized by the addition
of 20 µM DNase I. The rate of depolymerization was
monitored by loss of pyrene fluorescence and followed a first order
process for both preparations. The rate constants were 0.00029 and
0.00055 s
1 for nitrated and native actin,
respectively.
1, respectively, 4-7-fold faster than the corresponding
values for native actin. The higher affinity of nitrated G-actin for the actin filament is also consistent with the kinetics of subunit dissociation. The addition of a 5-fold molar excess of DNase I leads to
complete depolymerization of the actin filament, with the rate for this
process ~2-fold slower for nitrated actin (0.00029 s
1,
open boxes) compared with native actin (0.000545 s
1, closed triangles, Fig.
10C).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
occurs in SCD. A
major target of tissue ONOO
reactivity is with carbon
dioxide (CO2) to yield the secondary nitrating species,
nitrosoperoxocarbonate (ONOOCO2) (56). Tissue hypercapnia
is often a consequence of impaired vascular function and is observed in
SCD (57), thus creating a setting for enhanced ONOO
-mediated nitration reactions and the amplification
of NOS2 expression that occurs during hypercapnia (58). Additionally,
neutrophil myeloperoxidase and other heme proteins abundantly present
in SCD (59) can oxidize NO
were similar (not shown), suggesting that minimal
or no steric hindrance exists for the readily diffusible species that
mediate nitration of either monomeric or polymerized actin. Confocal
microscopy images of tissue actin distribution and morphology strongly
affirm this influence of tyrosine nitration on actin polymerization
properties, by reflecting a disorganized actin assembly in regions of
both mouse and human SCD kidney where nitrotyrosine-containing
actin was localized (Fig. 11).
View larger version (140K):
[in a new window]
Fig. 11.
Immunofluorescent actin filament staining in
control and sickle cell kidney. F-actin prevalent in the brush
border of control (Ctrl) human and mouse kidney tubules is
disorganized and aggregated in regions indicated by arrows
in SCD human and mouse kidney. The nuclei were counterstained with the
blue fluorescent DNA stain Hoechst, and image acquisition was performed
using laser confocal microscopy. CV, central vein.
A dynamic network of cytoskeletal actin is required for cell function by compartmentalizing metabolic pathways (78), promoting intracellular motility (79), and maintaining a dynamic cytoskeleton (80). The organization of actin filaments is also necessary for a direct physical link between the extracellular matrix and the cytoskeleton (81). Importantly, multiple stimuli for actin filament depolymerization will induce apoptosis (62, 82, 83). The ability of actin tyrosine nitration to alter actin polymerization (75) thus also links actin nitration with the enhanced apoptosis observed in regions of NO2Tyr immunoreactivity in the liver and kidney of SCD mouse and human (Fig. 3).
In summary, we have observed that an oxidative inflammatory milieu
exists in the vasculature, kidney, and liver of SCD patients, with
·NO-mediated nitration reactions catalyzing the
post-translational modification and functional impairment of a key cell
cytoskeletal protein, actin. In addition to adversely affecting
vascular function, the selective nitration of liver and kidney actin
tyrosine residues can also lead to the apoptotic cell death and loss of
organ function observed in SCD.
![]() |
ACKNOWLEDGEMENTS |
---|
We appreciate the insights and assistance provided by Drs. Phil Allen, Denyse Thornley-Brown, Elizabeth Lowenthal, Phil Chumley, and Scott Sweeney.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants RO1-HL64937, RO1-HL58115, and P6-HL58418.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: Dept. of
Anesthesiology, Biomedical Research Bldg. II, 901 19th Street So.,
University of Alabama at Birmingham, Birmingham, AL 35233. Tel.:
205-934-4234; Fax: 205-934-7437; E-mail:
Bruce.Freeman@ccc.uab.edu.
Published, JBC Papers in Press, October 24, 2002, DOI 10.1074/jbc.M208916200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
SCD, sickle cell
disease;
MS, mass spectrometry;
NOS2, inducible nitric-oxide synthase;
MALDI-TOF, matrix-assisted laser desorption ionization-time of flight;
NO, peroxynitrite;
O
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