Download Immunoblot Detection of Proteins That Contain Cysteine

Methods in Redox Signaling
Edited by Dipak Das
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Immunoblot Detection of Proteins That Contain Cysteine
Sulfinic or Sulfonic Acids with Antibodies Specific for
the Hyperoxidized Cysteine-Containing Sequence
Hyun Ae Woo and Sue Goo Rhee
Summary
The cysteine residues in the active sites of certain proteins, including peroxiredoxin (Prx), Parkinson’s disease
protein DJ-1, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), are more readily oxidized to sulfenic
acid (Cys-SOH) than are other cysteines because of their environment. The unstable sulfenic acid undergoes
further hyperoxidation to sulfinic (Cys-SO2H) and sulfonic (Cys-SO3H) acids. Hyperoxidation to sulfinic acid was
recently implicated as a reversible posttranslational modification responsible for regulation of protein function.
Studies of such a role have been hampered, however, by the technically demanding nature of assays for hyperoxidized proteins. Here we describe the production and characterization of antibodies to hyperoxidized peptides
modeled on the active sites of Prx, DJ-1, and GAPDH. The utility of these antibodies in the study of protein
function should prove similar to that of antibodies to peptides containing phosphoserine or phosphothreonine.
various isoforms of peroxiredoxin (Prx), glyceraldehyde3-phosphate dehydrogenase (GAPDH), and the Parkinson’s
disease protein DJ-1 (2).
Prxs protect cells from oxidative stress by removing hydroperoxides produced as a result of normal cellular metabolism.
Mammalian cells express six isoforms of Prx (Prx I to Prx VI),
which can be classified into three subgroups (2-Cys, atypical
2-Cys, and 1-Cys) on the basis of the number and position of
cysteine residues that participate in catalysis (3). Prx I to Prx
IV, which belong to the 2-Cys subgroup, possess two conserved cysteine residues. Prx V and Prx VI, which belong to
the atypical 2-Cys and 1-Cys subgroups, respectively, contain
only one conserved cysteine residue.
In the catalytic cycle of 2-Cys Prx enzymes, the NH2terminal conserved Cys–SH is first converted to cysteine
sulfenic acid (Cys–SOH) by a peroxide. The unstable sulfenic intermediate then reacts with the COOH-terminal conserved Cys–SH of the other subunit in the homodimer to
form a disulfide, which is subsequently reduced by a thiolcontaining reductant, such as thioredoxin, to complete the
catalytic cycle. As a result of the slow rate of its conversion to a
disulfide, the sulfenic intermediate is occasionally oxidized
further to cysteine sulfinic acid (Cys–SO2H), leading to inactivation of peroxidase activity (4). Examination of the fate of
inactivated sulfinic 2-Cys Prxs led to the unexpected finding
that sulfinic acid formation is a reversible process (5). The
enzyme responsible for the reduction of the hyperoxidized
Introduction
R
eactive oxygen species (ROS) are produced by all aerobic organisms as a by-product of aerobic metabolism. In
addition, ultraviolet and g irradiation of cells results in the
production of ROS. A substantial increase in the intracellular
concentration of ROS is generally associated with deleterious effects, including cell death by apoptosis or necrosis, in
pathological conditions such as inflammation and ischemiareperfusion. The generation of ROS also appears to be
required for many normal cellular functions, including
transduction of cell surface receptor signaling.
Thiols of cysteine residues in proteins are the most common
targets of ROS in cells. The cysteine residues in the active sites
of certain proteins are sensitive to oxidation by ROS because
their environment promotes ionization of the thiol (Cys-SH)
group, even at a neutral pH, to the thiolate anion (Cys-S ),
which is more readily oxidized to sulfenic acid. The sulfenic
acid is usually unstable and either reacts with any accessible
thiol to form a disulfide or undergoes further oxidation to
sulfinic acid (Cys-SO2H): the disulfide group is stable and resistant to further oxidation. The hyperoxidation to sulfinic acid
does not appear to be a rare event, given that 1–2% of the
cysteine residues of soluble proteins from rat liver were detected as cysteine sulfinic acid; in contrast, cysteine sulfonic
acid(Cys-SO3H)wasnotdetected(1).Hyperoxidationtosulfinic
acid has been observed with a number of proteins including
Division of Life and Pharmaceutical Sciences, Ewha Womans University, Seoul, Korea.
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Prxs was identified in yeast (6) and mammals (7) and was
named sulfiredoxin (Srx). Reduction by Srx is specific to 2-Cys
Prxs; the sulfinic forms of Prx V and Prx VI are thus not reduced by Srx (8). Moreover, Srx acts on neither the sulfinic
form of GAPDH nor DJ-1 (8). This specificity is due to the fact
that Srx physically associates with the 2-Cys Prxs but not
with other sulfinic proteins.
In addition to peroxidase activity, 2-Cys Prxs function as
molecular chaperones. While the hyperoxidation of the activesite cysteine results in inactivation of peroxides activity, the
chaperone function is enhanced by the hyperoxidation (9).
DJ-1 also has chaperone activity, and only the sulfinic form of
DJ-1 has been shown to have significant antiaggregation
properties against a-synuclein (10). Hyperoxidation induces
the translocation of DJ-1 from the cytosol to the outer mitochondrial membrane and protects against mitochondrial
damage (2). Accordingly, hyperoxidation of critical cysteine
residue to sulfinic acid has recently drawn wide attention as a
novel posttranslational modification responsible for regulation of protein function. Studies of such a role have been
hampered, however, by the technically demanding nature
of assays for sulfinylated proteins. Proteins that contain hyperoxidized cysteine residues (Cys–SO2H or Cys–SO3H)
are detected as the more acidic satellite spots of the spots
corresponding to the reduced form of the protein on twodimensional (2D) polyacrylamide gels. However, given that
such an acidic shift is also caused by protein phosphorylation,
as is the case with Prx I (11) and GAPDH (12), mass spectral
analysis of the acidic forms of proteins is required to ascertain
the presence of hyperoxidized Cys residues. Even with mass
spectral analysis, quantitation of hyperoxidized proteins has
been possible only by the application of complex procedures.
We here describe the production of antibodies to sulfonylated peptides modeled on the active sites of Prx isoforms,
DJ-1, and GAPDH. Immunoblot analysis with the respective
antibodies detected hyperoxidized Prxs, DJ-1, or GAPDH in
H2O2-treated cells with high sensitivity and specificity. The
utility of these antibodies in the study of protein function
should prove similar to that of antibodies to peptides containing phosphoserine or phosphothreonine.
Materials and Methods
Preparation of recombinant human Prx I, V, and VI was
previously described (13–15). Rabbit antisera specific for each
Prx isoform were obtained from Young In Frontier (Seoul,
Korea). Rabbit muscle GAPDH proteins and mouse monoclonal antibodies to GAPDH were obtained from Sigma
(St. Louis, MO) and Millipore (Billerica, MA), respectively.
Aprotinin, leupeptin, and 4-(2-aminoethyl)-benzenesulfonyl
fluoride (AEBSF) were obtained from ICN Biomedicals (Costa
Mesa, CA). Mouse monoclonal antibodies to DJ-1 were kindly
provided by Dr. Benoit Giasson (University of Pennsylvania
School of Medicine).
Preparation of sulfonylated peptides
Five peptides, DFTFVCPTEI, AFTPGCSKTH, DFTPVCTTEL, KIISNASCTTN, and GLIAAICAGPTA, which correspond to the active site of 2-Cys Prxs (Prx I to IV), Prx V, Prx
VI, GAPDH, and DJ-1, respectively, were oxidized by dissolving 5 mg of each in 50 ml of performic acid (freshly prepared by mixing formic acid and hydrogen peroxide, 9:1
WOO AND RHEE
(v=v), and incubating the mixture for 1 h at 258C. The peptides
were then dried for 15 min under vacuum without heating,
and the resulting residue was dissolved in 500 ml of water to
give a concentration of 2 mg=ml. Portions (10 mg) of the oxidized peptides were then analyzed by high-performance liquid chromatography on a Vydac C18 column that had been
equilibrated with 0.1% trifluoroacetic acid in water; elution
was performed over 60 min with a linear gradient of 0 to 100%
acetonitrile in 0.1% trifluoroacetic acid. Major peaks (>95%
for each peptide) were collected and subjected to analysis with
a Voyager-STR matrix-assisted laser desorption ionization–
time of flight (MALDI-TOF) instrument to confirm their sulfonic oxidation state.
Antibody production
Sulfonylated peptides (2 mg) were individually coupled to
10 mg of keyhole limpet hemocyanin (Pierce Biotechnology,
Rockford, IL) by incubation overnight at room temperature in
the presence of 7 mM glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.0). The peptide-hemocyanin conjugates
were mixed with incomplete Freund’s adjuvant for the initial
injection and with complete Freund’s adjuvant for booster
injections. After the initial injection with 1 mg of peptide,
rabbits were subjected to two booster injections, each of 500 mg
of peptide, administered (at multiple subcutaneous sites) at
4-week intervals. Antisera (20–60 ml) were collected 1 week
after the second booster injection, and the immunoglobulin G
fraction was precipitated with 50% (w=v) ammonium sulfate.
Antibodies that recognized the corresponding nonoxidized
peptide were removed by treating the immunoglobulin G
fraction with the thiol peptide coupled to Affigel-15 affinity
gel (Bio-Rad Laboratories, Hercules, CA).
Preparation of oxidized proteins
Recombinant human Prx I (250 mg) was incubated in a
250 ml reaction mixture containing 20 mg recombinant human
Trx1, 1 mM EDTA, 10 mM DTT, 1 mM H2O2, and 50 mM TrisHCl (pH 7.5). The oxidation reaction was initiated by the
addition of H2O2 and continued for 30 min at 308C. Addition
of 5 ml of 50 mM H2O2 to the reaction mixture and incubation
for 30 min were repeated three times. A similar procedure was
followed for the preparation of the sulfinic form of Prx V with
the exception that the concentration of H2O2 was increased to
5 mM for Prx V. Recombinant human Prx VI (250 mg) or rabbit
muscle GAPDH (250 mg) was incubated for 10 min at 308C in a
250 ml reaction mixture containing 4 mM H2O2 and 50 mM
Tris-HCl (pH 7.5). The residual DTT and H2O2 were removed
by ultrafiltration. The sulfinic state of the oxidized proteins
was verified by MALDI-TOF mass spectrometry.
Cell culture, treatment with H2O2,
and preparation of cell extracts
HeLa (human cervical cancer) cells were maintained in
Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen,
Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) (Invitrogen) and penicillin-streptomycin. Raw264.7
(mouse macrophage) cells were maintained in DMEM supplemented with 10% FBS. A549 (human lung epithelial type II)
cells were maintained in Ham’s F-12 nutrient mixture medium (Invitrogen) supplemented with 10% FBS. Cells (5106
cells=100 mm plate) were washed with Hank’s balanced salt
METHODS IN REDOX SIGNALING
solution (HBSS) twice and exposed to the indicated amount of
H2O2 in 10 ml of prewarmed HBSS for 10 min. Then cells were
rinsed twice with cold HBSS and lysed in 1 ml of lysis buffer
(20 mM HEPES, pH 7.4, 1 mM EDTA, 100 mM NaCl, 1% Triton
X-100, 10% glycerol, and protease inhibitor [5 g=ml leupeptin,
5 g=ml aprotinin, and 1 mM AEBSF]).
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separated proteins were transferred electrophoretically to a
nitrocellulose membrane, which was then incubated with the
antibodies to the sulfonylated peptides. Immune complexes
were detected with horseradish peroxidase–conjugated secondary antibodies and enhanced chemiluminescence reagents
(GE Healthcare). The membranes were then stripped and reprobed with antibodies to the corresponding whole proteins.
Two-dimensional (2D) gel electrophoresis
To remove nonprotein impurities such as salts and nucleic
acids, trichloroacetic acid was added to the cell lysates (250 mg
of protein) to a final concentration of 10%, and the proteins
were allowed to precipitate on ice for 30 min. The precipitated
proteins were washed twice with ice-cold acetone and then
resuspended in 250 ml of Rehydration buffer (GE Healthcare,
Uppsala, Sweden) containing 3 ml of DeStreak Solution (GE
Healthcare). After removal of insoluble material, each sample
was applied to 13 cm Immobiline DryStrips (pH 3 to 10,
nonlinear; GE Healthcare). Isoelectric focusing was performed with an IPGPhor unit (GE Healthcare), after which
the focused proteins were subjected to reduction and alkylation on the strips as recommended by the manufacturer. The
second-dimension SDS-PAGE was conducted on 12% gels,
then subjected to immunoblot analysis.
Results
Cell lysates were fractionated by SDS-PAGE on a 14%
gel or by 2D gel electrophoresis as described above. The
To explore the possibility of immunological detection of
hyperoxidized Cys-containing proteins, we prepared rabbit
antibodies to five sulfonylated peptides based on the active
site sequences of 2-Cys Prx (DFTFVCPTEI), Prx V (AFTPGC
SKTH), Prx VI (DFTPVCTTEL), GAPDH (KIISNASCTTN),
and DJ-1(GLIAAICAGPTA). Because the active-site sequence
(DFTFVCPTEI) is the same for 2-Cys Prxs (Prx I to IV) and
because the sizes of Prx I and Prx II are identical, the sulfinic
forms of Prx I and Prx II cannot be differentiated by immunoblot
of a one-dimensional gel. Sulfinic Prx III and Prx IV, however,
can be distinguished because their sizes are different from that of
Prx I= II and from each other. The Prx peptide sequence is
common to four mammalian isoforms of the protein (Prx I to IV).
To assess the specificity of the antibodies generated in response
to the sulfonylated Prx peptide, we combined oxidized (sulfinylated) and nonoxidized forms of Prx I in various ratios and
then subjected equal amounts of these mixtures to immunoblot
analysis with the antibodies (Fig. 4–1A). The intensity of the Prx I
FIG. 4–1. Characterization of antibodies to sulfonylated
2-Cys Prx peptide. A, Nonoxidized Prx I (Re. Prx I) and Prx I
hyperoxidized on Cys-51 (Ox. Prx I) were mixed in the indicated ratios to yield samples each containing a total of
50 ng of Prx I. The samples were subjected to immunoblot
analysis with antibodies to the sulfonylated Prx peptide
(a-Prx-SO3) and with antibodies to Prx I (a-Prx I). B, HeLa,
A549, or Raw264.7 cells were incubated for 30 min in the
absence or presence of 1 mM H2O2, after which cell lysates
(25 mg of protein) were subjected to immunoblot analysis
with a-Prx-SO3 and a-Prx I. Prx I and Prx II, which contain
the same number of amino acid residues, comigrate.
FIG. 4–2. Characterization of antibodies to sulfonylated
GAPDH peptide. A, Nonoxidized GAPDH (Re. GAPDH) and
GAPDH hyperoxidized on Cys-149 (Ox. GAPDH) were mixed
in the indicated ratios to yield samples each containing a total
of 100 ng of GAPDH. The samples were subjected to immunoblot analysis with antibodies to the sulfonylated GAPDH
peptide (a-GAPDH-SO3) and with antibodies to GAPDH (aGAPDH). B, HeLa, A549, or Raw264.7 cells were incubated for
30 min in the absence or presence of 1 mM H2O2, after which
cell lysates (25 mg of protein) were subjected to immunoblot
analysis with a-GAPDH-SO3 and a-GAPDH.
Immunoblot analysis
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FIG. 4–3. Characterization of antibodies to sulfonylated
Prx VI peptide. A, Nonoxidized Prx VI (Re. Prx VI) and Prx
VI hyperoxidized on Cys-47 (Ox. Prx VI) were mixed in the
indicated ratios to yield samples each containing a total of
100 ng of Prx VI. The samples were subjected to immunoblot
analysis with antibodies to the sulfonylated Prx VI peptide
(a-Prx VI-SO3) and with antibodies to Prx VI (a-Prx VI). B,
HeLa or A549 cells were incubated for 30 min in the absence
or presence of 1 mM H2O2, after which cell lysates (25 mg of
protein) were subjected to immunoblot analysis with a-Prx
VI-SO3 and a-Prx VI.
band detected by the antibodies increased as the mole fraction of
oxidized Prx I increased, suggesting that the antibodies are
specific for oxidized Prx. The specificity of the antibody preparations was also apparent from immunoblot analysis of lysates
derived from HeLa, A549, or Raw264.7 cells that had been
treated or not with H2O2 (Fig. 4–1B). For all three cell types,
the antibodies to the sulfonylated Prx peptides detected Prx
isoforms (one band for Prx I and Prx II, another for Prx III) only
in the H2O2-treated cells—not in the nontreated cells.
Similar experiments with a mixture of oxidized and nonoxidized GAPDH revealed the specificity of the antibodies
WOO AND RHEE
FIG. 4–5. Characterization of antibodies to sulfonylated
Prx V and DJ-1 peptides. A, Nonoxidized (Re) and hyperoxidized on Cys-48 (Ox) Prx V recombinant proteins and
50 mg of cell extracts from HeLa cells, incubated for 30 min in
the absence or presence of 1 mM H2O2, were subjected to
immunoblot analysis with a-Prx V-SO3 and a-Prx V. B, HeLa
cells were incubated for 30 min in the absence or presence
of 1 mM H2O2, after which cell lysates (25 mg of protein)
were subjected to immunoblot analysis with a-DJ-1-SO3 and
a-DJ-1.
prepared with the sulfonylated GAPDH peptide for oxidized
GAPDH (Fig. 4–2A). The antibodies to the sulfonylated
GAPDH peptides also detected GAPDH only in the H2O2treated cells—not in the nontreated cells (Fig. 4–2B).
The antibodies prepared with the sulfonylated Prx VI
peptide showed similar specificity for oxidized Prx VI both in
recombinant proteins and cell lysates (Fig. 4–3A and B). Proteins that contain hyperoxidized cysteine residues (Cys-SO2H
or Cys-SO3H) are detected at a more acidic position than are
the corresponding reduced proteins on 2D gels because of the
presence of the negatively charged sulfinic group. By incubation with H2O2 in cells, Prx I, II, III, and VI were partially
hyperoxidized and were separated as acidic, hyperoxidized
spots and basic, normal spots by 2D gels. The antibodies
specific to the hyperoxidized 2-Cys Prx or Prx VI recognized
the acidic spots but not the normal spots (Fig. 4–4).
The anti–Prx V-SO2 antibodies could recognize the hyperoxidized recombinant Prx V proteins specifically; however, the hyperoxidized Prx V band in cell lysate was
barely detectable (Fig. 4–5A). Unlike typical 2-Cys Prxs, Prx V
formed intramolecular disulfide bonds (15) and was highly
resistant to the sulfinylation by H2O2 (unpublished data).
DJ-1 is a ubiquitously expressed protein of the DJ-1=ThiJ=PfpI
superfamily. DJ-1 functions as an atypical peroxiredoxinlike
peroxidase. Furthermore, oxidative conditions induce the
formation of sulfinic acid of Cys-106 of DJ-1 (16). The antibodies to the sulfonylated Cys-106 peptides of DJ-1 could
recognize the oxidized DJ-1 from the H2O2-treated cells
(Fig. 4–5).
Acknowledgments
FIG. 4–4. Antibodies to sulfonylated 2-Cys Prx or Prx VI
peptides only recognized the sulfinylated Prxs on 2D gels.
HeLa cells were incubated with 50 mM H2O2 (for Prx I, II) or
150 mM H2O2 (for Prx III, VI) for 10 min. Cell lysates (250 mg)
were then separated by 2D-PAGE, and the resulting gels
were subjected to immunoblot analysis with antibodies to
sulfonylated 2-Cys Prx or Prx VI peptides. The membranes
were then stripped and reprobed with antibodies to the
corresponding whole proteins.
This study was supported by grants from the Korean Science and Engineering Foundation (National Honor Scientist
Program grant 2006-05106 and Bio R7D program grant
M10642040001-07N4204-00110) to SGR.
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