Aslan M, Ryan TM, Townes TM, Coward L, Kirk MC, Barnes S, Alexander CB, Rosenfeld SS, Freeman BA. Nitric oxide-dependent generation of reactive species in sickle cell disease. Actin tyrosine induces defective cytoskeletal polymerization

Abstract

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 (NO(2)Tyr) formation and apoptosis were evaluated in human SCD tissues and a murine model of SCD. Liver and kidney NOS2 expression and NO(2)Tyr 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 NO(2)Tyr in all SCD tissues. Gas chromatography mass spectrometry analysis revealed increased plasma protein NO(2)Tyr content and increased levels of hepatic and renal protein NO(2)Tyr 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 NO(2)Tyr-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 (Tyr(91), Tyr(198), and Tyr(240)) 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.

Full text

Nitric Oxide-dependent Generation of Reactive Species in
Sickle Cell Disease
ACTIN TYROSINE NITRATION INDUCES DEFECTIVE CYTOSKELETAL POLYMERIZATION*
Received for publication, August 30, 2002
Published, JBC Papers in Press, October 24, 2002, DOI 10.1074/jbc.M208916200
Mutay Aslan‡§
¶储
, Thomas M. Ryan§
储
, Tim M. Townes§
储
, Lori Coward**, Marion C. Kirk**,
Stephen Barnes§**‡‡, C. Bruce Alexander§§, Steven S. Rosenfeld
¶¶
, and Bruce A. Freeman‡§
¶储储储
From the Departments of ‡Anesthesiology, §Biochemistry and Molecular Genetics, §§Pathology, ‡‡Pharmacology
and Toxicology,
¶¶
Neurology,
¶
Center for Free Radical Biology,
储
Comprehensive Sickle Cell Disease Center,
and **Comprehensive Cancer Center Mass Spectrometry Shared Facility, University of Alabama at Birmingham,
Birmingham, Alabama 35233
The intermittent vascular occlusion occurring in
sickle cell disease (SCD) leads to ischemia-reperfusion
injury and activation of inflammatory processes includ-
ing enhanced production of reactive oxygen species and
increased expression of inducible nitric-oxide synthase
(NOS2). Appreciating that impaired nitric oxide-de-
pendent vascular function and the concomitant forma-
tion 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 (NO
2
Tyr) formation and apoptosis were
evaluated in human SCD tissues and a murine model of
SCD. Liver and kidney NOS2 expression and NO
2
Tyr
immunoreactivity were significantly increased in SCD
mice and humans, but not in nondiseased tissues. TdT-
mediated nick end-label (TUNEL) staining showed apo-
ptotic cells in regions expressing elevated levels of NOS2
and NO
2
Tyr in all SCD tissues. Gas chromatography
mass spectrometry analysis revealed increased plasma
protein NO
2
Tyr content and increased levels of hepatic
and renal protein NO
2
Tyr derivatives in SCD (21.4 ⴞ 2.6
and 37.5 ⴞ 7.8 ng/mg) versus wild type mice (8.2 ⴞ 2.2 and
10 ⴞ 1.2 ng/mg), respectively. Western blot analysis and
immunoprecipitation of SCD mouse liver and kidney
proteins revealed one principal NO
2
Tyr-containing pro
-
tein 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
(Tyr
91
, Tyr
198
, and Tyr
240
) at positions that significantly
modify actin assembly. Confocal microscopy of SCD hu-
man and mouse tissues revealed that nitration led to
morphologically distinct disorganization of filamentous
actin. In aggregate, we have observed that the hemoglo-
bin point mutation of sickle cell disease that mediates
hemoglobin polymerization defects is translated, via in-
flammatory oxidant reactions, into defective cytoskel-
etal polymerization.
The intermittent vascular occlusion occurring in SCD
1
is
characterized by acute, painful crises and leads to the renal
and hepatic tissue injury and dysfunction manifested by pa-
tients 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 super-
oxide (O
2
.
) and hydrogen peroxide (H
2
O
2
) generation (4 –7).
These reactive species, derived from xanthine oxidase (8), au-
toxidation of mitochondrial respiratory chain components (9),
and activated neutrophils (10, 11), serve to impair
䡠
NO-depend-
ent vascular function and further activate tissue inflammatory
responses (12–14). Increased expression of NOS2 and a conse-
quent increase in tissue nitrite and nitrate (NO
2
⫺
⫹ NO
3
⫺
)
production occurs in cardiac, liver, and kidney ischemia-reper-
fusion (15–18). In many instances, NOS2 inhibition by arginine
analogs or ablation of NOS2 gene expression significantly lim-
its tissue ischemia-reperfusion injury (19 –21). Moreover,
NOS2 can serve as a locus for tissue O
2
.
production during
conditions of low arginine substrate availability, as observed in
SCD (22, 23). This setting favors the generation of peroxyni-
trite (ONOO
⫺
), the nitrating and oxidizing species produced by
the radical-radical reaction of O
2
.
and
䡠
NO (24).
Increased rates of production of reactive oxygen- and nitro-
gen-derived species in tissues mediate the oxidation and nitra-
tion of lipids, nucleotides, and susceptible protein amino acid
residues. These products also suggest an impairment of biomo-
lecular structure and function. For example, the protein nitra-
tion product NO
2
Tyr 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), myo-
cardial inflammation (29), ileitis (30), rheumatoid arthritis
(31), endotoxin-induced kidney injury (32), chronic renal al-
lograft rejection (33), Alzheimer’s disease (34), amyotrophic
lateral sclerosis (35), and sepsis (36). The development of a
causal relationship between post-translational protein nitra-
tion and impaired tissue function is presently limited by in-
sight into where and how the inflammatory modification of
specific protein amino acid residues occurs in vivo and how this
* This work was supported by National Institutes of Health Grants
RO1-HL64937, RO1-HL58115, and P6-HL58418. The costs of publica-
tion of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
储储
To whom correspondence should be addressed: Dept. of Anesthesi-
ology, 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.
1
The abbreviations used are: SCD, sickle cell disease; MS, mass
spectrometry; NOS2, inducible nitric-oxide synthase; MALDI-TOF, ma-
trix-assisted laser desorption ionization-time of flight; NO
3
⫺
, nitrate;
䡠
NO, nitric oxide; NO
2
⫺
, nitrite; ONOO
⫺
, peroxynitrite; O
2
.
, superoxide;
NO
2
Tyr, 3-nitrotyrosine; TUNEL, TdT-mediated nick end-label; HPLC,
high pressure liquid chromatography; MS/MS, tandem mass
spectrometry.
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 6, Issue of February 7, pp. 4194 –4204, 2003
© 2003 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
This paper is available on line at http://www.jbc.org4194

is linked with biomolecule dysfunction. Herein, a combination
of clinical and knockout-transgenic mouse studies underscores
the extensive occurrence of
䡠
NO-mediated oxidative inflamma-
tory 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 im-
paired polymerization properties reveals the significance of
this post-translational protein modification in the physiopa-
thology of SCD and related vascular inflammatory processes.
MATERIALS AND METHODS
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 ap-
proval 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-NO
2
Tyr (Cayman, 5
␮
gml
⫺1
) and anti-NOS2 (BD Transduction
Laboratories, 16
␮
gml
⫺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 preadsorb-
ing anti-NO
2
Tyr with 10 mM NO
2
Tyr (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-No-
marski 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.
Measurement of Plasma and Tissue NO
2
Tyr—Blood was collected
from healthy HbA adult volunteers and homozygous HbS patients in
anticoagulated (EDTA) Vacutainers as approved by the Insti-
tutional Review Board for Human Use at the University of Alabama at
Birmingham. Blood cells were removed by centrifugation, and plasma
was stored at ⫺80 °C for subsequent processing and analysis. The liver
and kidneys of C57Bl/6J or knockout-transgenic SCD mice, which syn-
thesize exclusively human Hb in the murine red blood cells (37), were
dissected, weighed, and homogenized in ice-cold homogenizing buffer
(50 m
M K
2
HPO
4
,80
␮
M leupeptin, 2.1 mM Pefabloc SC, 1 mM phenyl
-
methylsulfonyl fluoride, 1
␮
gml
⫺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 NO
2
Tyr was quantified by
gas chromatography-MS as described previously (38). For use as an
internal standard, 3-[
13
C
6
]nitrotyrosine was synthesized by the addi
-
tion of 1.5 m
M ONOO
⫺
to6mM of [
13
C
6
]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 chromato-
graph 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.
Purification of Actin from Kidney and Liver—Tissue actin purifica-
tion was performed by DNase I affinity chromatography as described
previously (39, 40). Briefly, 15 ml of Affi-Gel 10 (BioRad) was trans-
ferred 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 CaCl
2
)for4hat4°C. The gel slurry was loaded
into a column, washed with cold deionized water, and equilibrated with
buffer G (2 m
M Tris-HCl, pH 7.9, 0.2 mM CaCl
2
, 0.2 mM ATP, and 0.2 mM
dithiothreitol). Liver and kidney were dissected, weighed, and homog-
enized in ice-cold buffer G containing 10% formamide (v/v) (Sigma).
Homogenates were centrifuged (100,000 ⫻ g,1h,4°C), and superna-
tants were applied to the DNase I-agarose column. The column was
washed successively with buffer G, 0.2
M NH
4
Cl 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 m
M ONOO
⫺
showed
similar chromatographic behavior of native actin.
Western Blot Analysis and Immunoprecipitation—For Western blot-
ting, mouse monoclonal anti-NO
2
Tyr (Cayman, 2
␮
gml
⫺1
), rabbit poly
-
clonal anti-NO
2
Tyr (Cayman, 2
␮
gml
⫺1
), and anti-NOS2 (BD Trans
-
duction 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 pro-
teins were visualized by chemiluminescence (ECL reagent, Amersham
Pharmacia Biotech). For immunoprecipitation, tissue homogenates
were cleared with protein A-agarose (Roche Molecular Biochemicals) for
3hat4°C. Supernatants were then incubated with rabbit polyclonal
anti-NO
2
Tyr (Cayman, 5
␮
gml
⫺1
) for 1 h followed by protein A-agarose
incubation for3hat4°C. NO
2
Tyr-containing actin was immunopre
-
cipitated from actin-enriched SCD liver and kidney extracts with a
NO
2
Tyr affinity sorbent (Cayman, 40
␮
lml
⫺1
). Immunoprecipitated
proteins were washed, separated by SDS-PAGE, and visualized by
GelCode Coomassie Blue stain reagent (Pierce).
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 m
M am-
monium bicarbonate (1:1, v/v), and dried. Samples were rehydrated
with 12.5 ng
␮
l
⫺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-hydroxyci-
nammic acid (Aldrich) (1:1, v/v), and spotted onto a gold-coated MALDI
plate. Peptide molecular ions were analyzed in the positive ion mode
FIG.1. Immunofluorescent staining of NOS2 and 3-nitroty-
rosine 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
NO
2
Tyr when compared with controls. B, sections from a knockout-
transgenic sickle cell and C57Bl/6J control mouse kidney. NOS2 stain-
ing is observed in the glomeruli and in tubular epithelial cells, whereas
NO
2
Tyr immunoreactivity is localized principally to distal and proximal
tubules. NOS2 and NO
2
Tyr staining is not evident in C57Bl/6J control
sections. Nuclei are counterstained with Hoechst in all experiments.
䡠
NO-dependent Generation of Reactive Species in SCD 4195

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
␮
M)
was modified minimally with 0.3 mM ONOO
⫺
, denatured with 6 M
guanidine hydrochloride, and reduced with 5 mM dithiothreitol for 2 h
FIG.2. Immunofluorescent staining of NOS2 and NO
2
Tyr in
control and sickle cell liver. A, sections from sickle cell and control
(Ctl) human liver. NOS2 and NO
2
Tyr staining is observed predomi
-
nantly in the pericentral hepatocytes of SCD liver compared with con-
trols. B, sections from knockout-transgenic sickle cell and C57Bl/6J
control mouse liver. Increased NOS2 and NO
2
Tyr 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.
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.
䡠
NO-dependent Generation of Reactive Species in SCD4196

at 37 °C. Cysteines were alkylated with 1 mM iodoacetamide for2hin
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
␮
g of sequencing grade
modified trypsin (Promega). For electrospray analysis, peptide frag-
ments were separated by reverse-phase HPLC column (300
␮
m ⫻ 15
cm C18 PepMap) at a flow rate of 2
␮
l 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 sur-
vey MS and MS/MS modes. A multiply charged peak above 6 counts
detected in the mass spectrum was selected automatically for tandem
MS analysis.
Measurement of Actin Polymerization—Actin was purified from rab-
bit 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 m
M ONOO
⫺
,
reduced with 2 m
M dithiothreitol, and dialyzed against 5 mM Tris-HCl,
0.2 mM CaCl
2
, 0.2 mM ATP, pH 8.0. Control and nitrated actin (9.6
␮
M)
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 m
M KCl, 25 mM Hepes, 2 mM MgCl
2
, 0.1 mM CaCl
2
,
0.2 m
M 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
Kidney and Liver NOS2 Expression and NO
2
Tyr Forma
-
tion—There was a strong co-distribution of NOS2 and tissue
NO
2
Tyr immunostaining in the renal cortex of knockout-trans
-
genic SCD mice and humans with SCD that was not evident in
wild type (control) C57Bl/6J mice or healthy humans express-
ing 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 immunoreac-
tive for NO
2
Tyr in both SCD mouse and human but unlike
mice, human SCD kidneys were also immunoreactive for
NO
2
Tyr 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 NO
2
Tyr
immunoreactivity (Fig. 2, A and B). Immunoreactive NOS2 and
NO
2
Tyr was significantly less in control mouse and human
liver (Fig. 2). Preadsorption of anti-NO
2
Tyr with NO
2
Tyr re
-
vealed that NO
2
Tyr immunostaining in kidney and liver sec
-
tions was specific (not shown). Western blot analysis of NOS2
expression and NO
2
Tyr in kidney and liver homogenates re
-
vealed increased NOS2 expression and protein NO
2
Tyr 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 NO
2
Tyr Concentrations—Plasma protein
NO
2
Tyr 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 differ-
ence in liver and kidney homogenate protein NO
2
Tyr adducts
in SCD mice (21.4 ⫾ 2.6 and 37.5 ⫾ 7.8 ng/mg protein, respec-
tively) versus controls (8.2 ⫾ 2.2 and 10 ⫾ 1.2 ng/mg protein).
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 NO
2
Tyr (A) or NOS2 (B). C, immunoprecipitation of SCD and
C57Bl/6J control (Ctl) mouse kidney (Kid) and liver (Liv) homogenates
using a polyclonal NO
2
Tyr antibody. The immunoprecipitation pellet
was separated by SDS-PAGE and visualized by Coomassie Blue stain-
ing. 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, im-
munoblot analysis of actin-enriched kidney and liver extracts using a
polyclonal NO
2
Tyr antibody. The observed NO
2
Tyr-containing proteins
in SCD kidney and liver correspond to actin (42 kDa) and actin-associ-
ated vitamin
D-binding protein (53 kDa). F, immunoprecipitation of
actin-enriched SCD liver and kidney extracts with NO
2
Tyr affinity
sorbent. The immunoprecipitation pellet was separated by SDS-PAGE
and visualized by Coomassie Blue staining.
T
ABLE I
3-Nitrotyrosine content in sickle cell disease
Measurement
3-Nitrotyrosine
Control SCD
Human
Plasma (ng/mg protein) 10.1 ⫾ 3.2 (3)
a
24.7 ⫾ 1.7
b
(4)
Mouse tissue
Plasma (ng/mg protein) 13.1 ⫾ 2.2 (3) 37.7 ⫾ 6.6
b
(4)
Liver (ng/mg protein) 8.2 ⫾ 2.2 (3) 21.4 ⫾ 2.6
b
(5)
Kidney (ng/mg protein) 10.0 ⫾ 1.2 (3) 37.5 ⫾ 7.8
b
(4)
Mouse actin-enriched fraction
Liver (
␮
g/mg protein) 0.13 ⫾ 0.03 (2) 0.34 ⫾ 0.06 (3)
Kidney (
␮
g/mg protein) 0.17 ⫾ 0.03 (2) 0.92 ⫾ 0.08
b
(3)
a
n for each measurement is in parentheses.
b
p ⬍0.05 from control.
䡠
NO-dependent Generation of Reactive Species in SCD 4197

The actin-enriched fraction of mouse liver and kidney showed a
greater protein NO
2
Tyr 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-NO
2
Tyr 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-NO
2
Tyr also revealed a
NO
2
Tyr-containing 42-kDa protein (Fig. 4C) in SCD but not
wild type mice. The 42-kDa NO
2
Tyr-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 NO
2
Tyr antibody (Fig. 4E). The strong co-
localization of actin and NO
2
Tyr immunoreactivity in the
merged fluorescence images (Fig. 6, A and B) also further
confirmed actin nitration in SCD kidney and liver. The minor
NO
2
Tyr-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
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 NO
2
Tyr antibody,
was in-gel digested and analyzed by MALDI-TOF MS. Peptide frag-
ments from kidney (A)- and liver (B)-matched mouse actin with Mascot
algorithm analysis. The NO
2
Tyr-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 frag-
ment ions matching mouse vitamin
D-binding protein.
FIG.6. Immunohistochemical co-distribution of actin and
NO
2
Tyr 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 NO
2
Tyr (red). Nuclei (blue) were counterstained with Hoechst. To
assess co-distribution of actin and NO
2
Tyr, images were merged (or
-
ange). CV, central vein.
䡠
NO-dependent Generation of Reactive Species in SCD4198

ions of m/z 1051, 1272, 1303, 1741, 2441, and 2882 (Fig, 5, C
and D) identifying G-actin-associated vitamin
D-binding pro-
tein with a score of 79 (p ⬍ 0.05).
Identification of Specific Actin Tyrosine Residues Nitrated in
Vivo—ANO
2
Tyr-enriched actin fraction from SCD mouse liver
and kidney homogenates was prepared by immunoprecipita-
tion of NO
2
Tyr-containing protein from the actin fraction pu
-
rified by DNase I affinity chromatography (Fig. 4F). Following
electrophoretic separation, the 42-kDa NO
2
Tyr-containing pro
-
tein 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 (Tyr
91
, Tyr
198
, and Tyr
240
). 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 Tyr
91
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 frag-
ment (Fig. 8B) showed a b2 ion that shifted from m/z 221 to 266,
identifying Tyr
198
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 Tyr
240
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. Electro-
spray ionization MS/MS analysis of proteolytic fragments from
rabbit actin treated with 0.3 m
M ONOO
⫺
revealed nitration of
four residues (Tyr
53
, Tyr
198
, Tyr
240
, and Tyr
362
), with nitration
of Tyr
362
not consistently observed in some experiments.
MS/MS spectra obtained by collision-induced dissociation of
[M⫹2H]
2⫹
-nitrated tryptic fragments resulted in dominant
FIG.7.MALDI-TOF MS of identification nitrated actin fragments. NO
2
Tyr-enriched actin fractions, obtained from actin-enriched SCD
liver and kidney extracts via NO
2
Tyr antibody immunoprecipitation, were in-gel digested and analyzed by MALDI-TOF MS. A, MS spectrum of
the nitrated tryptic fragment
85
IWHHTFYNELR
95
[M⫹H]
⫹
(m/z 1561). B, MS spectrum of the nitrated tryptic fragment
197
GYSFTTTAER
206
[M⫹H]
⫹
(m/z 1177). C, MS spectrum of the nitrated tryptic fragment
239
SYELPDGQVITIGNER
254
[M⫹H]
⫹
(m/z 1836).
䡠
NO-dependent Generation of Reactive Species in SCD 4199

FIG.8. MS/MS identification and representation of in vivo nitrated actin residues. A, MS/MS spectrum of the tryptic fragment
85
IWHHTFYNELR
95
[M⫹2H]
2⫹
(m/z 781). B, MS/MS spectrum of the tryptic fragment
197
GYSFTTTAER
206
[M⫹2H]
2⫹
(m/z 589). C, MS/MS
spectrum of the tryptic fragment
239
SYELPDGQVITIGNER
254
[M⫹2H]
2⫹
(m/z 918). D, ribbon representation of actin (Ref. 47; PDB Id: 1J6Z)
䡠
NO-dependent Generation of Reactive Species in SCD4200

fragmentation at the amide bonds yielding type b or y ions (Fig
9). Again, fragment ions containing the NO
2
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 Tyr
53
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 Tyr
198
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 Tyr
240
in the amino acid
sequence SYELPDGQVITIGNER as the site of nitration (Fig.
9C). The MS/MS spectrum of the tryptic fragment correspond-
ing to residues 360–372 (parent ion m/z 773.8) showed a y11
ion that shifted from m/z 1243 to 1288, exposing Tyr
362
in the
amino acid sequence QEYDEAGPSIVHR as the nitrated resi-
due (Fig. 9D).
Effect of Nitration on Actin Polymerization—Because tyro-
sine 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 fila-
ment (48), was examined by using pyrene fluorescence emis-
sion (45). A plot of pyrenyl fluorescence versus actin concentra-
tion 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
(
⑀
430
⫽ 4400 M
⫺1
cm
⫺1
(49)). Even in the presence of this
quenching, a significant effect of nitration on critical concen-
tration was observed. For native actin (open circles), the ex-
trapolated critical concentration is 89 ⫾ 16 n
M, similar to
previous measurements at this ionic strength in 50 m
M KCl
(50). By contrast, the curve for nitrated actin extrapolated to
the origin, implying a critical concentration ⬍ 10 n
M.
Polymerization of actin is accomplished in two steps, forma-
tion 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 proc-
ess. The results depicted in Fig. 10A imply that nitration sta-
bilizes 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 accel-
erate 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 accel-
erates filament elongation. Data for both native and nitrated
actin were fitted to a sum of two exponential processes, repre-
senting 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
⫺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 con-
sistent with the kinetics of subunit dissociation. The addition of
a 5-fold molar excess of DNase I leads to complete depolymer-
ization 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
The repetitive episodes of tissue ischemia-reperfusion, the
pro-inflammatory state in the systemic vasculature (51, 52),
and the oxidative impairment of vascular
䡠
NO signaling events
that occur in SCD (51) all encourage the formation of secondary
oxidizing and nitrating species and contribute to impaired vas-
cular and organ function. The occurrence of xanthine oxidase-
derived, O
2
.
-dependent inhibition of
䡠
NO-mediated vascular re-
laxation in SCD vessels (53) and the elevated expression of
NOS2 (54, 55) in the kidney and liver of SCD mice and human
(Figs. 1 and 2) also reinforces the concept that elevated rates of
production of the oxidizing and nitrating species ONOO
⫺
oc
-
curs in SCD. A major target of tissue ONOO
⫺
reactivity is with
carbon dioxide (CO
2
) to yield the secondary nitrating species,
nitrosoperoxocarbonate (ONOOCO
2
) (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). Addi-
tionally, neutrophil myeloperoxidase and other heme proteins
abundantly present in SCD (59) can oxidize NO
2
⫺
(60, 61), an
䡠
NO metabolite elevated in SCD (23), to the nitrating species
nitrogen dioxide (
䡠
NO
2
). Myeloperoxidase-catalyzed protein ni
-
tration frequently displays spatial and temporal colocalization
with tyrosine nitration and has been observed to occur in the
vessel wall (63). Finally, the acidotic conditions present in
poorly perfused tissue compartments may promote protonation
of NO
2
⫺
, conferring a chemistry that can also result in nitrous
acid (HNO
2
)-mediated tyrosine nitration (64). The observation
of significant increases in plasma and tissue protein NO
2
Tyr
derivatives in an animal model and clinical SCD reinforces that
one or more of these oxidative inflammatory pathways are
operative and are mediating pathogenic tissue responses fol-
lowing the post-translational nitration of structurally and func-
tionally important target molecules.
Nitration of free and protein-associated tyrosine residues to
yield NO
2
Tyr has been detected in multiple species, organ
systems, and cell types during both acute and chronic inflam-
mation (65). The existence of multiple distinct, yet redundant,
pathways for tyrosine nitration underscores the potential sig-
nificance 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 revers-
ible nature of protein NO
2
Tyr 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 tyro-
sine phosphorylation (71).
Critical to understanding the pathogenic inflammatory reac-
tions occurring in SCD is the observation of NOS (2) and
NO
2
Tyr co-distribution in humans with SCD and a mouse
model of SCD (Figs. 1 and 2), where liver and kidney NO
2
Tyr
are elevated 2.6 and 3.7-fold, respectively (Table I). Immuno-
precipitation and MALDI-TOF mass spectrometry-assisted
identification of actin as the predominant nitrated protein in
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.
䡠
NO-dependent Generation of Reactive Species in SCD 4201

trum of the tryptic fragment
197
GYSFVTTAER
206
[M⫹2H]
2⫹
(m/z
588.7). C, MS/MS spectrum of the tryptic fragment
239
SYELP
-
DGQVITIGNER
254
[M⫹2H]
2⫹
(m/z 918.4). D, MS/MS spectrum of the
tryptic fragment
360
QEYDEAGPSIVHR
372
[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 (subdo-
main 1), gray (subdomain 2), magenta (subdomain 3), and silver (sub-
domain 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.
FIG.9.Tandem
32
MS/MS identification and representation of
in vitro nitrated actin residues. A, MS/MS spectrum of the tryptic
fragment
51
DSYVGDEAQSK
61
[M⫹2H]
2⫹
(m/z 622.2). B, MS/MS spec-
FIG. 10. Effects of nitration on actin polymerization thermo-
dynamics and kinetics. A, critical concentration plot of native py-
rene-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 n
M, 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. Polymer-
ized actin at a concentration of 4
␮
M was depolymerized by the addition
of 20
␮
M DNase I. The rate of depolymerization was monitored by loss
of pyrene fluorescence and followed a first order process for both prep-
arations. The rate constants were 0.00029 and 0.00055 s
⫺1
for nitrated
and native actin, respectively.
䡠
NO-dependent Generation of Reactive Species in SCD4202

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 pro-
teins 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 func-
tional 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 mo-
tifs (76). The introduction of an electronegative NO
2
group onto a
tyrosine ring reduces the pK
a
of the phenolic hydroxyl to values
in the range of 6.8 –7.0. If such nitrotyrosine residues were in-
volved in intersubunit interactions, they could form ionic or hy-
drogen 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 mod-
ified subunits could stabilize elongating filaments from frag-
mentation, as well as drive the equilibrium toward polymeri-
zation. Although we have not examined the effects of nitration
on the severing ability of actin binding proteins such as gelso-
lin, 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 alter-
ations in cell motility, attachment, and intracellular transport.
Interestingly, we observed that the extent and sites of F- and
G-actin tyrosine nitration by ONOO
⫺
were similar (not shown),
suggesting that minimal or no steric hindrance exists for the
readily diffusible species that mediate nitration of either mo-
nomeric or polymerized actin. Confocal microscopy images of
tissue actin distribution and morphology strongly affirm this
influence of tyrosine nitration on actin polymerization proper-
ties, by reflecting a disorganized actin assembly in regions of
both mouse and human SCD kidney where nitrotyrosine-con-
taining actin was localized (Fig. 11).
A dynamic network of cytoskeletal actin is required for cell
function by compartmentalizing metabolic pathways (78), pro-
moting 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,
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.
䡠
NO-dependent Generation of Reactive Species in SCD 4203

83). The ability of actin tyrosine nitration to alter actin polymer-
ization (75) thus also links actin nitration with the enhanced
apoptosis observed in regions of NO
2
Tyr immunoreactivity in the
liver and kidney of SCD mouse and human (Fig. 3).
In summary, we have observed that an oxidative inflamma-
tory milieu exists in the vasculature, kidney, and liver of SCD
patients, with
䡠
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.
Acknowledgments—We appreciate the insights and assistance pro-
vided by Drs. Phil Allen, Denyse Thornley-Brown, Elizabeth
Lowenthal, Phil Chumley, and Scott Sweeney.
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NO-dependent Generation of Reactive Species in SCD4204