However, the protein detected in cells of mouse, human, rat and pig origin, as well as the recombinant form of AOP2, migrates in PAGE as a protein of approximately 32 kDa.. Red blood cel
Trang 1Antioxidant protein 2 prevents methemoglobin formation
in erythrocyte hemolysates
Karl M Stuhlmeier1, Janet J Kao2, Pia Wallbrandt3, Maria Lindberg3, Barbro Hammarstro¨m3,
Hans Broell1and Beverly Paigen4
1
Ludwig Boltzmann Institute for Rheumatology and Balneology, Vienna, Austria;2Maimonides Medical Center, Brooklyn, NY, USA;3Department of Molecular Biology, AstraZeneca, Umea˚, Sweden; and4The Jackson Laboratory, Bar Harbor, ME, USA
Antioxidant protein 2 (AOP2) is a member of a family of
thiol-specific antioxidants, recently renamed peroxiredoxins,
that evolved as part of an elaborate system to counteract and
control detrimental effects of oxygen radicals AOP2 is
found in endothelial cells, erythrocytes, monocytes, T and B
cells, but not in granulocytes AOP2 was found solely in the
cytoplasm and was not associated with the nuclear or
membrane fractions; neither was it detectable in plasma
Further experiments focused on the function of AOP2 in
erythrocytes where it is closely associated with the
hemo-globin complex, particularly with the heme An investigation
of the mechanism of this interaction demonstrated that the conserved cysteine-47 in AOP2 seems to play a role in AOP2-heme interactions Recombinant AOP2 prevented induced as well as noninduced methemoglobin formation in erythrocyte hemolysates, indicating its antioxidant proper-ties We conclude that AOP2 is part of a sophisticated system developed to protect and support erythrocytes in their many physiological functions
Keywords: hemoglobin; erythrocytes; reactive oxygen species; antioxidant protein 2
Evolving antioxidant defence systems to protect against O2
toxicity has been a prerequisite for an organism’s use of O2
for efficient energy production To benefit from O2 as an
energy source, multicellular organisms had to develop a
system to distribute O2 In mammals this function is carried
out by red blood cells (RBC), which utilize hemoglobin to
distribute O2to cells Not only are RBC highly specialized
O2and CO2carriers, they also serve an additional important
function, namely acting as a sink for reactive oxygen species
(ROS) [1] Erythrocytes can take up O2-radicals as well as
H2O2in plasma to protect the organism from damage by
such compounds [2–4] These tasks make erythrocytes
especially vulnerable to damage by ROS Furthermore,
carrying high concentrations of O2 and high levels of
potentially pro-oxidant heme protein inside a membrane
rich in polyunsaturated fatty acid side chains cause additional problems RBCs are therefore exposed to a constant flow of hemoglobin auto-oxidation, as approxi-mately 3% of the hemoglobin undergoes oxidation to methemoglobin (metHb) every day Moreover RBC are also exposed to repeated physical stress through deforma-tion More importantly, RBC have low metabolic activities with no ability to synthesize new proteins or lipids to replace damaged molecules [1] Due to these properties, RBC need
to be equipped with a series of enzymes that can protect cells from damage by free radicals; such enzymes include Cu-Zn-superoxide dismutase, catalase, glutathione peroxidase, metHb reductase, and glucose 6-phosphate dehydrogenase Recently, a new type of antioxidant protein has been reported to be present in RBC [5,6], the thiol-specific antioxidant proteins, which are members of a large family of more than 40 proteins found in prokaryotes as well as eukaryotes [7–10] The peroxiredoxin proteins show no significant homology with previously identified antioxidant proteins The nomenclature of these proteins is still confu-sing, as these molecules were originally described under several names e.g rehydrins, thioredoxin-dependent per-oxide reductases, but this family has been renamed as peroxiredoxins [11,12] Peroxiredoxins are grouped into 1-Cys proteins with a conserved cysteine at amino acid position 47 and 2-Cys proteins with a second conserved amino acid at position 170 (relative to yeast peroxiredoxin) They usually exist as homodimers The substrates are alkyl hydroperoxides [9], peroxynitrates [13] and hydrogen per-oxides [14], and they detoxify these substrates by oxidation
of the Cys at amino acid 47 [9,15] These proteins enzymatically detoxify hydroxyradicals using reducing equivalents from thiol-containing molecules such as thio-redoxins and glutathione As a major function of these proteins is to regulate ROS levels, they not only protect
Correspondence to K M Stuhlmeier, Ludwig Boltzmann
Institute for Rheumatology and Balneology, Kurbadstrasse 10,
PO Box 78, A-1107 Vienna, Austria.
Fax: + 43 1 68009 9234, Tel.: + 43 1 68009 9237,
E-mail: karlms@excite.com
Abbreviations: AOP2, antioxidant protein 2; metHb, methemoglobin;
MNCs, mononuclear cells; PMNs, polymorphonuclear cells;
RBC, red blood cells; ROS, reactive oxygen species.
Note: The nomenclature of antioxidant protein 2 is currently
under-going reconsideration This protein is currently named antioxidant
protein 2 in humans and peroxiredoxin 5 in mice However,
peroxi-redoxin 5 in humans refers to a different protein (named peroxiperoxi-redoxin
6 in mouse) As the same protein is supposed to have the same name
in different species, we will use the old name of antioxidant protein 2
until this nomenclature issue is resolved by the human and mouse
nomenclature committees.
(Received 11 June 2002, revised 13 October 2002,
accepted 26 November 2002)
Trang 2macromolecules from oxidation, but also may be involved
in signal transduction as well because ROS are implicated in
physiological signaling [16,17]
We investigated the distribution and function of AOP2,
which is a 1-Cys member of the peroxiredoxin protein
family Its presence has been described for mouse, pig and
human cells Murine AOP2 was first isolated from the liver
and kidney as a cDNA corresponding to a protein variant
that differs between the C57BL/6J and DBA/2J strains of
mice [18] Subsequently, the genomic structure of AOP2 in
mice was determined [19] and the crystal structure of the
human protein determined [20] An analysis of the EST
database suggested that AOP2 may be ubiquitously
expressed [21] The encoded protein is 224 amino acids in
length with a predicted size of 25 kDa However, the protein
detected in cells of mouse, human, rat and pig origin, as well
as the recombinant form of AOP2, migrates in PAGE as a
protein of approximately 32 kDa To date, the protein
expression pattern of the native AOP2 has been reported
only for rat, which demonstrated AOP2 exclusively in the
lung [22] However, we screened organs and different cell
types in mice with an antibody raised against a unique
peptide from the AOP2 protein and found high levels of this
protein in essentially all organs (unpublished results)
Endothelial cells, erythrocytes and white blood cells are
targets as well as sources of many forms of ROS [1] We
therefore investigated the distribution and function of this
novel antioxidant protein in these cells Herein we report
our findings on AOP2 distribution and its protective effects
in RBC, as well as the mechanism of interactions
Experimental procedures
Materials
Pyrrolidine dithiocarbamate, cysteine, serine, alanine,
cross-linked hemoglobin, hemin, pig serum, Igepal CA-630, and
globin were obtained from Sigma (St Louis, MO) or Sigma
(Vienna, Austria) L-Glutamine was obtained from PAA
Labor- und ForschungsgesmbH (Vienna, Austria),
penicil-lin, streptomycin, cell culture medium and fetal bovine
serum from Gibco BRL, Life Technologies (Vienna,
Austria) Tumor necrosis factor alpha was obtained from
R & D Research (Minneapolis, MN)
Generation of AOP2 antibodies
One synthetic peptide (FPKGVFTKELPSGKKYLRYC)
corresponding to amino acids 202–220 of murine AOP2 was
generated The peptide sequence was chosen based on an
antigenicity calculation (Jamesson–Wolf, based on
hydro-philicity, surface probability, flexibility and secondary
structure predictions) and had been shown by Kang et al
[23], to be an antigen determinant An additional amino
acid, cysteine, was added to the C-terminal end of the
synthetic peptide to obtain specific coupling of the peptide
to BSA and affinity gel matrices Rabbits were immunized
with BSA-conjugated peptides and repeatedly injected to
increase the antibody response The IgG antibody fraction
was purified from the antisera obtained after IV
immuni-zations using Protein G Sepharose 4 Fast Flow (Pharmacia
LKB Biotechnology, Uppsala, Sweden) Polyclonal AOP2
antibodies were further purified on Sulfolink (Pierce, Rockford, IL, USA) affinity columns to which the synthetic peptides had been coupled by their cysteine residues Affinity-purified antibodies were characterized by ELISA and Western blot analysis In immunoblotting, the AOP2 antibody recognized a polypeptide with a relative molecular mass of approximately 32 kDa in human and mouse liver homogenates N-terminal amino acid sequencing identified the 32 kDa polypeptide as AOP2 In addition, the AOP2 antibody recognized recombinant AOP2D124A and the recombinant protein encoded by the Aop2-related sequence 1 (data not shown)
Recombinant AOP2 Production and purification of recombinant AOP2 in Escherichia coli were performed mainly as described [23] Full-length Aop2 cDNA was amplified by the polymerase chain reaction (PCR) and subsequently used for cloning PCR was performed with forward primer (5¢-CGGCA TATGCCCGGAGGGTTGCTTCTC-3¢), which contains nucleotides 1–21 of the mouse Aop2 sequence, the initiation codon and a NdeI cleavage site, and reverse primer (5¢-CGCGAATTCTTATTAAGGCTGGGGTGTATAACG G-3¢), which contains nucleotides 653–671 of the mouse Aop2 sequence, two stop codons and an EcoRI cleavage site The resulting PCR products were cloned into
pGEM-T An NdeI–EcoRI fragment from pGEM-T, containing the cDNA encoding AOP2, was subcloned into pET-17b generating pETAop2D123A The nucleotide sequence of pETAop2D124A was verified by DNA sequencing
E coli cells (BL21(DE3)pLysS) were transformed with pETAop2D124A Cells were grown overnight in a small volume of liquid broth supplemented with carbencillin and chloramphenicol and thereafter transferred to new medium Production of recombinant protein was induced
by addition of isopropyl-b-D-thiogalactopyranoside Cells were harvested, disrupted and recombinant protein was purified from the soluble fraction as described [23] The purification method includes streptomycin sulfate precipi-tation, ammonium sulfate precipiprecipi-tation, hydrophobic chromatography and anion exchange chromatography The sample was applied to a Q-Sepharose column (Pharmacia LKB Biotechnology, Uppsala, Sweden), equil-ibrated with 20 mM Tris/HCl (pH 8.0), 2 mM dithio-threitol and 1 mMEDTA At this pH recombinant AOP2 was bound to the column matrix Thereafter, bound material was eluted with a linear gradient of 0–0.5M NaCl in 20 mM Tris/HCl (pH 8.0), 2 mM dithiothreitol and 1 mM EDTA
During purification recombinant AOP2 was detected by immunoblot analysis using specific polyclonal antibodies The purified protein was more than 95% pure and the yield was 50% The N-terminal amino acid sequence, purity and accurate molecular mass of recombinant AOP2 were verified by N-terminal amino acid sequencing and electro-spray mass spectroscopy (data not shown)
Preparation of cell lysate Blood from human or mouse was diluted in phosphate buffered saline (NaCl/P), and RBC separated from white
Trang 3blood cells by density gradient centrifugation on a 67% (w/v)
Percoll (Amersham Pharmacia Biotech, Uppsala, Sweden)
gradient RBC were washed three times with NaCl/Pi, and
then hemolyzed with three volumes of distilled water for
15 s Afterwards, 10· NaCl/Pi was u sed to adju st the
salinity and pH to physiological conditions Hemolysate
was used for metHb measurements, Western blots and
pre-cipitation studies following centrifugation at 20 800 g for
15 min at 4C RBC membrane extract was prepared as
follows: ghosts (pellets after hemolysis) were washed three
times with NaCl/Pi, afterwards, membranes were dissolved
in lysis buffer containing 0.32M sucrose, 3 mM CaCl2,
2 mM magnesium acetate, 0.1 mM dithiothreitol, 0.5 mM
phenylmethylsulfonyl fluoride, and 0.5% Igepal CA-630 In
some cases, RBC were lysed directly in such a buffer and the
resulting protein solution used for Western blot
experi-ments Endothelial cell cytoplasm and nuclear extract were
prepared as described [24] Polymorphonuclear cells were
separated from mononuclear cells on a density gradient (67
and 55% Percoll, respectively), washed three times and cell
extracts prepared as described above B and T cell extracts
were obtained from whole cell lysates of human lymphoma
cells, BJAB (Santa Cruz Biotechnology, Santa Cruz, CA)
and mouse T cell lymphoma cells, CTLL-2 (Santa Cruz
Biotechnology, Santa Cruz, CA)
Cell culture
Porcine aortic endothelial cells were isolated and cultured as
previously described [24] Briefly, endothelial cells were
cultured in Dulbecco’s modified Eagle’s medium with
4.5 gÆL)1glucose and supplemented with 10% heat
inacti-vated fetal bovine serum, L-glutamine and 50 unitsÆmL)1
penicillin/streptomycin
Methemoglobin measurement
Hemolysate was placed in disposable cuvettes (polymethyl
methacrylate, 2.5 mL capacity) with a light path of 1 cm
Lysate and indicated amounts of reagents were incubated at
room temperature without shaking The metHb formed was
measured at time intervals ranging from 0–200 h A
spectrophotometer (Spectronic Genesys 5, Milton Roy,
Rochester, NY) was used in survey scan mode to measure
absorbance between 400 and 700 nm
Immunoprecipitation Cross-linked hemoglobin, hemin and globin were obtained from Sigma (Vienna, Austria) Aliquots of beads were washed three times in NaCl/Pi Red blood cell lysate, antibodies to AOP2, recombinant AOP2 protein, bovine serum albumin, or a combination of the above were incubated at 4 C on a rotating device We used either protein A/G PLUS-Agarose (Santa Cruz Biotechnology, Santa Cruz, CA) or hemoglobin (bovine) cross-linked with cyanogen bromide to beaded agarose for these precipitation studies To ensure high specificity of protein–protein interactions, after 1 h beads were collected by centrifugation and washed six times in high stringency buffer (0.1% Tween 20, 0.1% SDS, 1% Igepal CA-630, in NaCl/Pi) After the last wash solution was aspirated, 25 lL SDS sample buffer was added, heated to 95 C for 5 min, and immunoprecipitates collected for SDS/PAGE
Western blot analysis Cytosolic and nuclear extracts from endothelial cells, mononuclear cells (MNCs) and polymorphonuclear cells (PMNs) were prepared as described [25] Equal amounts of protein (immunoprecipitates, nuclear and/or cytoplasm extract) were separated by SDS/PAGE (10 or 12%), transferred to an Immobilon-P poly(vinylidene difluoride) (PVDF) membrane using a semidry transfer cell (Bio-Rad Laboratories, Hercules, CA), and probed with a rabbit polyclonal antibody no 5 against AOP2 Bands were visualized using horseradish peroxidase, conjugated donkey antirabbit IgG, and the Enhanced ChemiLuminescence assay (Amersham Life Science Inc., Arlington Heights, IL) according to the manufacturer’s instructions
Results
Expression of AOP2 Using Western blots, we determined the expression of AOP2 in various cell types High levels of AOP2 occurred in the cytoplasm of aortic endothelial cells but not in the nuclear extract (Fig 1A) Furthermore, stimulating endo-thelial cells (50 lM of pyrolidine dithiocarbamate or
5 ngÆmL)1 of tumor necrosis factor) for 10 min, 2, 8 or
Fig 1 Expression of AOP2 in various tissues Arrow indicates AOP2 (A) Western blots demonstrated that AOP2 was expressed in RBC and MNCs but not in PMNs Furthermore, in endothelial cells (EC), AOP2 is found in the cytoplasm (EC Cyto) but not in the nucleus (EC Nuc) AOP2 was expressed similarly in mouse cells (data not shown) (B) Whole cell lysate of resting B and T cells were separated by SDS/PAGE and stained with a specific anti-AOP2 Ig as described in experimental procedures section (C) RBC from two mice (A and B) were lysed and separated into membrane and cytoplasm (stroma) fractions Equal amounts of protein were separated by SDS/PAGE Membranes were exposed to films for times ranging from 30 s to 15 min The Membrane blot was exposed approximately 10 times longer than was the Stroma blot.
Trang 424 h did not lead to changes in AOP2 levels (data not
shown) Next, we tested whether polymorphonuclear cells
(PMNs), mononuclear cells (MNCs), or erythrocytes
express AOP2 Human and mouse blood was collected,
and cells separated immediately on a Percoll gradient
AOP2 was detected in RBC and MNCs, bu t not in PMNs
(Fig 1B) Even gels loaded with maximal amou nts of PMN
whole cell extract (50 lg proteinÆlane)1) and deliberately
overexposed exhibited only a weak band of AOP2 (data not
shown), which may have resulted from RBCs
contamin-ating the PMN fraction A representative Western blot
(Fig 1B) indicates that both B cells (human BJAB cells) and
T cells (mouse lymphocyte cell line CTLL-2) contained
equal amounts of AOP2 and showed no difference in its
molecular size Similarly, AOP2 of RBC from mouse and
human origin had identical staining patterns (data not
shown)
AOP2 is located in the cytoplasm of RBC but is not
membrane bound (Fig 1C) We collected blood of A/J and
C57BL/6 strains, separated the plasma from the solid
components, fractioned the RBCs, and electrophoresed the
membrane and cytoplasm portions on a 12% gel The gels
were immunostained, and blots were exposed to films for
30 s to an hour Even when exposed for an hour, blots
revealed that AOP2 was present only in RBC cytoplasm
(Fig 1C)
AOP2 was not present in mouse, pig, or human plasma
By using SDS/PAGE (5 lL plasma per lane) and
over-exposing Western blots so that concentrations of
recom-binant AOP2 smaller than 4 ng per lane could easily be
detected (Fig 2), we determined that AOP2 was absent
from porcine, murine, and human plasma (data not shown)
To gain insight into how AOP2 interacts with itself or
other proteins, RBC stroma proteins were examined using
native PAGE Under native conditions, AOP2 migrates as a
molecule of approximately 130 kDa in contrast to the
32 kDa observed under reducing conditions (Fig 3A)
Because adding either reducing SDS or native, nonreducing
sample buffer precipitated hemoglobin, we vortexed the
sample before loading the gel to ensure uniform protein
distribution We used this precipitation of hemoglobin to
study AOP2–hemoglobin interactions When RBC stoma
protein was incubated in nonreducing native sample buffer,
and supernatant and hemoglobin pellets collected separately, electrophoresis on a native gel showed no AOP2 in the supernatant This fact suggested that AOP2 coprecipitates with the hemoglobin fraction Hemoglobin in its native state forms a 64-kDa tetramer consisting of four 16 kDa subunits; this hemoglobin complex is visible on the blots
as white areas (Fig 3A), because the large quantities of hemoglobin blocked nonspecific binding of antibodies during the immunostaining procedure
AOP2 binds to hemoglobin
As AOP2 coprecipitated with hemoglobin, we tested whether AOP2 actually binds to the hemoglobin complex Hemoglobin cross-linked to agarose beads were prepared and used as described; special care was taken to remove unbound proteins through extensive washing steps under high stringency conditions As AOP2 could be detected in liver, heart, testis, and brain (data not shown), we used whole cell extracts of RBC, liver and heart as well as recombinant AOP2 for these experiments Recombinant AOP2, as well as AOP2 isolated from liver and heart cells, binds to hemoglobin Interestingly, when red blood cell extract, which contains high levels of AOP2 (as shown in Fig 1), were used, no AOP2 bound to hemoglobin cross-linked to beads The controls, which have NaCl/Pi, dem-onstrate that purchased hemagarose beads are free of AOP2 protein Furthermore, AOP2 binding to hemagarose beads
is specific, as it could not be blocked by addition of bovine serum albumin
Next, RBC, heart and liver cell extracts were incubated
on a rolling platform for 1 h at 4 C together with the anti-AOP2 IgG and protein A/G cross-linked to agarose beads
As expected AOP2 was immunoprecipitated from the cell extracts (Fig 4B) Interestingly, large amounts of hemo-globin coprecipitated with the AOP2 from RBC extracts
Fig 2 Demonstration of sensitivity and linearity of the Western blot
system used AOP2 could be detected at 4 ng Recombinant AOP2 was
separated by SDS/PAGE under reducing conditions at increasing ng
quantities At 400 and 1000 ng, the formation of dimers can be
observed where the amount of AOP2 added exceeds the reducing
capacity of 2-mercaptoethanol.
Fig 3 AOP2 binds to and coprecipitates with hemoglobin (A) Under nonreducing conditions, AOP2 migrates as a molecule complex of approximately 130 kDa Hemoglobin (Hb) tetramers can be observed
at 64 kDa as a light unstained band These samples were RBC lysates separated on a 10% PAGE gel (B) Under native conditions, AOP2 precipitated with hemoglobin This Western blot was prepared after RBC lysate in native sample bu ffer was warmed to precipitate hemo-globin After centrifuging the lysate and the precipitated hemoglobin,
10 and 20 lL aliquots of supernatant and the washed and resuspended hemoglobin pellet were electrophoresed by native-PAGE on a 10% gel.
Trang 5(Fig 4B), supporting the hypothesis that AOP2 binds
tightly to hemoglobin complexes Hemoglobin is visible
as the unstained (lighter) areas in Western blots and as
reddish-colored dots on PVDF membranes following
protein transfer Hemoglobin monomers (16 kDa), dimers
(32 kDa) and tetramers (64 kDa) are readily recognizable
on the blots because the large amounts of hemoglobin could
not be reduced completely by the 2-mercaptoethanol
present in sample buffer These experiments indicated that
AOP2 is tightly bound to the hemoglobin complex The
failure to precipitate any AOP2 with hemagarose (Fig 4A)
may indicate that all AOP2 in RBC is tightly bound to
hemoglobin so that no free AOP2 protein is available to
react with added hemagarose
To determine if AOP2 binds to the globin or the heme
portion of hemoglobin, we incubated hemin, globulin and
hemoglobin cross-linked to agarose beads with red blood
cell lysate for 1 h at 4 C Beads were collected by
centrifugation and extensively washed with high stringency
buffer Western blots revealed that AOP2 in red blood cell
lysate bound only to hemin but not to globin (Fig 4C) It is
somewhat puzzling that AOP2 in red blood cell lysate
bound to heme alone, but not to heme in hemoglobin This
might result if AOP2 has a higher affinity for isolated heme
so that heme on agarose beads can successfully compete for
AOP2 already bound to the endogenous hemoglobin in
RBC lysate
Cysteine 47 is essential for AOP2-heme binding
The binding of AOP2 and heme led us to investigate the
mechanism by which AOP2 binds to heme All
peroxire-doxin proteins have a conserved cysteine residue
corres-ponding to Cys47 in yeast peroxiredoxin [26] To determine
if the sulfur group of this cysteine bound to the iron in heme, heme cross-linked to agarose beads were preincubated for
30 min at 4 C with 25 mM cysteine, 25 mM alanine,
25 mMserine, or NaCl/Pi(as control) Subsequently, equal amounts of recombinant AOP2 were added and the mixture incubated for an additional hour at 4 C on a rotary platform The supernatants were aspirated after a final wash and the pellets resuspended in 40 lL of reducing sample buffer Aliquots of 20 lL were loaded per lane and separated by PAGE Quantitation of the immunoblot reveals that hemagarose beads preincubated with cysteine inhibited the subsequent binding of AOP2 by 86%, while preincubation with alanine or serine had no effect (Fig 5), indicating that AOP2 does bind to heme by its conserved cysteine
AOP2 prevents induced and spontaneous methemoglobin formation
ROS oxidize hemoglobin to methemoglobin (metHb), which is unable to deliver oxygen to tissues MetHb can form either spontaneously or be induced to form by many substances, including ascorbic acid at high doses [1,27] To determine if the tight binding of AOP2 to hemoglobin could prevent MetHb from forming spontaneously, we compared the amount of MetHb in fresh hemoglobin to that formed after 72 h in three solutions: untreated hemoglobin, hemo-globin treated with 4 lgÆmL)1 AOP2, and hemoglobin treated with 5 mM ascorbic acid After comparing the absorbance of these three solutions to that of MetHb (characteristic peak at 620–640 nm) [28] and unoxidized fresh Hb (576 nm), we concluded that treatment with AOP2 prevented the oxidation of Hb to metHb (Fig 6)
We then determined whether AOP2 could prevent ascorbic acid-induced metHb from forming and whether such an effect would be dose-dependent We prepared two samples of fresh hemoglobin combined with 5 mMascorbic acid and containing either 2 lg or 7 lg of recombinant AOP2 per ml MetHb formation was measured in these two samples after they had incubated for 48 and 120 h at 25 C MetHb was calculated as a ratio of absorbance at 575 and
626 nm according to published methods [28], with the slight modification of using the second peak of the hemoglobin spectrum instead the first at around 546 nm As Table 1 shows, AOP2 did indeed prevent ascorbic acid-induced metHb from forming, and it did so in a dose-dependent manner
Fig 4 AOP2 binds to hemoglobin (A) Recombinant AOP2, and
cytoplasm extracts from heart, liver, and RBC were incubated with
hemoglobin bound to agarose beads High stringency washing
con-ditions were used to remove unbound proteins Aliquots of beads were
added to reducing SDS sample buffer, and the solutions loaded on a
10% gel Recombinant AOP2, as well as AOP2 in heart and liver cells
bound to hemoglobin, while the AOP2 present in RBC did not BSA
was added to some samples to block nonspecific binding sites on
hemoglobin agarose beads (B) Lysates of liver, heart, or RBC were
incubated with anti-AOP2 Ig and protein A/G cross linked to agarose
beads The anti-AOP2 Ig not only precipitates AOP2 in red blood cell
lysate but also pulls down hemoglobin (indicated as lighter area in the
Western blot) bound to AOP2 This is a further indicator of the tight
interactions of AOP2 and hemoglobin in RBC (C) Red blood cell
lysate was incubated for 1 h at 4 C on a rotating platform with
agarose beads cross linked to hemin, globin, or hemoglobin AOP2
bound to the heme molecule but not to globin or hemoglobin.
Fig 5 Cysteine is essential for AOP2 heme binding Hemin, cross-linked to agarose beads, was used to study the involvement of cysteine
in AOP2–heme interactions AOP2 was incubated for 1 h at 4 C with hemin-beads preincubated with NaCl/P i or (25 m M ) cysteine, serine or alanine for 30 min at 4 C Unbound AOP2 was removed by exces-sive washing Cysteine blocks the binding of AOP2 to hemin while serine and alanine had no effect.
Trang 6We report on the distribution and function of AOP2 in
the four cell types tested: granulocytes, lymphocytes,
endothelial cells, and RBCs All these cells are either
exposed to or can release ROS and would therefore
appear to benefit from mechanisms to protect themselves
from ROS RBCs are especially vulnerable because they
have a high iron content, are exposed to high oxygen
pressure [29], and cannot repair proteins or other
molecules damaged by ROS We demonstrated that
AOP2 is a cytoplasmic protein widely present in
lympho-cytes, endothelial cells, and RBC, but not in granulocytes
It was not present in plasma or in RBC membranes or
nucleus
Aop2 belongs to a multigene family, the members of
which each have multiple bands detectable by Southern
blot [21,30,31] It is unknown if these proteins function
similarly in different cell types Our data indicate that
AOP2, in a dose-dependent manner, significantly protects
RBCs from ROS by binding to hemoglobin, thus
preventing both its induced and spontaneous oxidation
to metHb Another member of the peroxiredoxin protein
family, the 25-kDa protector protein [32], has also been
shown to suppress metHb formation and membrane lipid
peroxidation
The alignment of the peroxiredoxin family members reveals that many of them have two highly conserved cysteine residues corresponding to the Cys47 and Cys170
in yeast peroxiredoxin The N-terminal cysteine is con-served in all family members and the C-terminal is conserved in all except six members AOP2 is among the family members with only one conserved cysteine The eight other amino acids in the cysteine region of mamma-lian peroxiredoxin are 100% conserved, indicating an essential role in peroxiredoxin function Furthermore, there
is an additional motif of six conserved amino acids out of
11, as well as a region with six out of 15 conserved amino acids [21] Both Cys47 and Cys170 were shown to be necessary to maintain peroxiredoxin polymers, but only the Cys47 region seems to be essential for antioxidant activity [33]
Because cysteine is often present to help bind heme groups to substrates, we investigated whether it was essential for binding the heme iron to AOP2 We found that heme preincubated with cysteine did not bind to AOP2, whereas heme preincubated with several other unrelated amino acids freely bound to AOP2 This result contrasted somewhat to those of others showing that yeast peroxiredoxin with mutant Cys47 cannot prevent the inactivation of glutamine synthetase induced by dithio-threitol/Fe3+/O2, indicating that Cys47 has an antioxidant rather than a binding function [33] Nevertheless, the same study found that Cys47 is essential to maintain peroxire-doxin dimers, indicating that it does have a binding function This was supported by our findings that recom-binant AOP2, not disassociated by a reducing agent, separated into large complexes of dimers and tetramers (data not shown) Because cysteine is almost always present to help bind heme groups to substrates, we investigated whether it was essential for binding the heme iron to AOP2 Through immunoprecipitation studies, we found that heme preincubated with cysteine did not bind
to AOP2, whereas heme preincubated with several other unrelated amino acids freely bound to AOP2 Another member of the peroxiredoxin family also uses cysteine for binding to heme [34]
We addressed the functional importance of AOP2 in erythrocytes and found that, as can the well-known superoxide dismutase and catalase antioxidants, it was able to prevent hemoglobin oxidation (both induced and spontaneous) Hemoglobin is oxidized only at its iron atoms, which are sheltered in its hydrophobic pocket Our
Table 1 AOP2 prevents spontaneous as well as induced methemoglobin formation The indicated reagents were added to aliquots of a freshly prepared hemoglobin solution After 48 and 120 hours respectively at 25 C, absorbency readings were recorded and the 570/626 nm ratio calculated AOP2 was added at a molar ratio of 1 : 1060 (AOP2 (2 lgÆml)1) + ascorbic acid (5 m M )) and 1 : 303 (AOP2 (7 lgÆml)1) + ascorbic acid (5 m M )) respectively.
Reagent added to Hb solution Ratio 570/626 nm (48 h) Ratio 570/626 nm (120 h)
AOP2 (2 lgÆmL)1) + ascorbic acid (5 m M ) 6.6 6.2
AOP2 (7 lgÆmL)1) + ascorbic acid (5 m M ) 7.5 8.3
Fig 6 Methemoglobin formation is prevented by AOP2 AOP2
pre-vented metHb from forming spontaneously over a 72-h period MetHb
(peak absorbance around 622 nm) is nearly absent from fresh
hemo-globin Hemoglobin (peak absorbance at 576 nm) is partially oxidized
over 72 h and oxidized more by ascorbic acid AOP2 prevents Hb from
being spontaneously oxidized to metHb.
Trang 7immunoprecipitation studies indicated that AOP2 bound
with high affinity to the heme complex, supporting the
hypothesis that it protects or restores hemoglobin to its
active form This hypothesis has yet to be confirmed
Because metHb is so undesirable, organisms need a
redundant system to reduce its formation RBCs, lacking
the ability to replace damaged molecules, depend more
than do other cells on efficient detoxifying systems: a lack
of protective enzymes in RBCs would result in a surge of
oxidation Abnormal metHb formation has not been
found in human acatalasemics [1], a further indicator
that other molecules such as AOP2 might compensate
for the missing catalase in these patients AOP2 may well
be a major antioxidant in RBCs, and its role in other cells
is being investigated in our laboratories Heme is the
prosthetic group of several proteins and enzymes
(myo-globin, cytochrome c, cytochrome P450,
ubiquinol-cyto-chrome c reductase, cytoubiquinol-cyto-chrome c oxidase, tryptophan
pyrrolase, and NO synthase), and AOP2 may protect them
also This would explain its nearly ubiquitous presence and
provide further evidence for its importance as a major
protective protein
Acknowledgements
The authors thank Ray Lambert for his editing skills This work
was supported in part by a grant from AstraZeneca, by the
Austrian Ministry of Education, Science and Culture, Austrian
Ministry of Social Security and Generations GZ.236.065/6-VI/B/10/01
(GZ:236.065/7-VIII/A/6/00), and the City of Vienna.
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