A selenium-containing single-chain abzyme 2F3 Se-2F3-scFv that imitates glutathione peroxidase has been produced which has the capacity to remove ROS.. To evaluate the antioxidant abilit
Trang 1A selenium-containing single-chain abzyme with potent
antioxidant activity
Delin You1, Xiaojun Ren1,2, Yan Xue1, Guimin Luo1, Tongshu Yang1and Jiacong Shen2
1
Key Laboratory of Molecular Enzymology and Engineering of Ministry of Education, Jilin University, Changchun, P R China;
2
Key Laboratory for Supramolecular Structure and Materials of Ministry of Education, Jilin University, Changchun, P R China
Reactive oxygen species (ROS) are products of normal
metabolic activities and are thought to be the cause of many
diseases A selenium-containing single-chain abzyme 2F3
(Se-2F3-scFv) that imitates glutathione peroxidase has been
produced which has the capacity to remove ROS To
evaluate the antioxidant ability of Se-2F3-scFv, we
con-structed a ferrous sulfate/ascorbate (Vc/Fe2+)-induced
mito-chondrial damage model system and investigated the
capacity of Se-2F3-scFv to protect mitochondria from
oxi-dative damage Se-2F3-scFv markedly decreased mito-chondrial swelling, inhibited lipid peroxidation, and maintained the activity of cytochrome c oxidase, in com-parison with Ebselen, a well-studied glutathione peroxidase mimic, indicating that Se-2F3-scFv has potential for treating diseases mediated by ROS
Keywords: antioxidant activity; glutathione peroxidase; mitochondria; scFv; selenium
Reactive oxygen species (ROS) include free radicals such as
superoxide anion (O2•) and hydroxyl radical (•OH), as well
as nonradical intermediates such as hydrogen peroxide
(H2O2), hydroperoxide (ROOH), nitric oxide (NO) and
singlet oxygen (1O2) [1,2] All these ROS are produced from
molecular oxygen by mitochondrial electron carriers and
from enzymes during normal metabolism of oxidative
phosphorylation of aerobic mammalian cells In addition,
ROS are produced on irradiation, both ionizing and UV
irradiation
To protect themselves from oxidative injury, aerobic
cells have evolved an enzymatic and nonenzymatic defense
system The enzymatic antioxidant system is mainly
composed of glutathione peroxidase (GPX), catalase,
superoxide dismutase and thioredoxin peroxidase The
non-enzymatic antioxidant system includes vitamin E, ascorbate,
glutathione (GSH) and uric acid However, if the ROS
loading reaches a critical concentration, overwhelming the
antioxidative defense, oxidative damage to all cellular
components, such as DNA, proteins and lipids, eventually
occurs, resulting in ROS-mediated diseases [3–5]
Exam-ples of such diseases are ischemia-reperfusion injury,
inflammation, age-related diseases, neuronal apoptosis,
cancer and cataract
The individual antioxidant enzymes are located in
specific subcellular sites and reveal distinct substrate
specificity [6] Superoxide dismutase is a metalloenzyme that catalyzes the reduction of O2• to H2O2 H2O2 produced by the reduction of O2• is subsequently detoxified by catalase present in peroxisomes or by the selenoenzyme GPX located in the cytosol and mitochon-dria GPX, the most important selenium-containing peroxidase, catalyzes the reduction of a variety of hydroperoxides (ROOH and H2O2) by GSH, thereby protecting mammalian cells against oxidative damage At least five GPX isoenzymes have been identified in mammals Although their expression is ubiquitous, the levels of each isoform vary depending on the tissue type The classical cellular GPX (GPX1 or cGPX), found in the cytosol and mitochondria, reduces fatty acid hydroper-oxides and H2O2[7–9] Phospholipid hydroperoxide GPX (GPX4 or PHGPX), found in most tissues and located in both the cytosol and the membrane fraction, can directly reduce the phospholipid hydroperoxides, fatty acid hydro-peroxides, and cholesterol hydroperoxides that are produced in peroxidized membranes and oxidized lipo-proteins [10–12] Cytosolic GPX2 (or giGPX) [13,14] and extracellular GPX 3 (pGPX) [15,16] are weakly detected
in most tissues except gastrointestinal tract and kidney, respectively Recently, a new member, GPX5, expressed specifically in mouse epididymis, is interestingly selenium-independent [17] The mechanism by which cGPX cata-lyzes the reduction of hydroperoxide has been extensively investigated
Because production of selenium-containing peroxidase
is extremely difficult by traditional genetic engineering, attempts have been made to generate compounds that imitate the enzymatic action of GPX The strategies used
to generate GPX-like catalysts include chemical synthesis
of a model system and mutation of naturally occurring enzyme by chemical or protein engineering [18–20] Three different strategies have been tested for chemically synthesizing a GPX mimic: one in which the selenium atom binds directly to a heteroatom such as nitrogen
Correspondence to G Luo, Key Laboratory of Molecular
Enzymology and Engineering of Ministry of Education,
Jilin University, Changchun, P R China.
Fax: + 86 431 8923907, Tel.: + 86 431 8498974,
E-mail: gmluo@mail.jlu.edu.cn
Abbreviations: ROS, reactive oxygen species; GSH, glutathione; GPX,
glutathione peroxidase; TBA, thiobarbituric acid; CCO, cytochrome c
oxidase; TBARS, thiobarbituric acid reactive substances.
(Received 20 April 2003, revised 6 July 2003,
accepted 22 August 2003)
Trang 2and generates the well-known GPX mimic, 2-phenyl-1,
2-benziososelenazol-3(2H)-one (Ebselen); a second in
which the selenium atom is not directly bound to the
heteroatom (N or O), but instead is located in close
proximity to it; and the third in which cyclodextrin is
used as an enzyme model and the selenium is not directly
bound or located in close proximity to the heteroatom
Engineering of naturally occurring enzyme by chemical
or genetic means has resulted in the semisynthetic
enzyme selenosubtilisin and a mutant version of
glycer-aldehyde-3-phosphate dehydrogenase Ebselen is an
inter-esting GPX mimic and has been extensively investigated
in studies of structure–function correlation and ability to
scavenge ROS in clinical trials [21–24], but it has some
drawbacks, such as low GPX activity and water
insolubility In previous work, we produced a series of
selenium-containing catalytic antibodies [25–27] One of
them, the selenium-containing abzyme 2F3 (Se-2F3),
exhibited high catalytic activity, 4.3 times that of GPX
from rabbit liver [27] To generate a pharmacologically
useful protein and study the cause of the highly catalytic
efficiency of Se-2F3, we sequenced, cloned and expressed
the variable region genes of 2F3 as a single-chain
antibody (2F3-scFv), and then incorporated selenium
into the 2F3-scFv by chemical mutation, resulting in the
selenium-containing 2F3-scFv (Se-2F3-scFv) Se-2F3-scFv
catalyzes the reduction of H2O2at rates approaching that
of native GPX from rabbit liver [28,29] The optimal pH
and temperature for the Se-2F3-scFv-catalyzed reduction
of H2O2 were determined to be 8.27 and 47.2C,
respectively, similar to those of native GPX [29] In this
study, we constructed a biological model of ROS-induced
mitochondrial damage to study the ability of
Se-2F3-scFv to protect mitochondria from oxidative damage We
found it to be a potent antioxidant
Materials and methods
Materials
GSH was obtained from Aldrich Ebselen, glutathione
reductase (type III baker’s yeast) and NADPH (tetrasodium
salt) were obtained from Sigma Thiobarbituric acid (TBA)
was obtained from Shanghai Second Reagent Plant,
Shanghai, China Cytochrome c was obtained from Tianjin
Biochemical Plant (Tianjin, China) Hepes was from Fluka
All other chemicals were of analytical grade
Generation of Se-2F3-scFv
The expression vector pTMFscFv containing target genes
was constructed as described previously and transformed
into bacterial cells BL21 (coden plus) After isopropyl
thio-b-D-galactoside induction, the expressed amount of
2F3-scFv proteins was 25–30% of total bacterial proteins The
2F3-scFv proteins were purified and refolded into the active
form Incorporation of selenium into 2F3-scFv protein by
chemical mutation resulted in the selenoenzyme
Se-2F3-scFv The GPX activity of Se-2F3-scFv was determined by
the coupled coenzyme system One unit of activity is defined
as the amount of compound that utilizes lmol NADPHÆ
min)1at 37C [28]
Preparation of mitochondria Bovine heart mitochondria were isolated from fresh bovine heart as described previously [30] Mitochondria were suspended in 0.25Msucrose/10 mMEDTA/25 mMHepes/ NaOH buffer, pH 7.4, and maintained at 0C The concentration of the mitochondrial proteins was determined
by the method of Bradford [31] with BSA as standard
Ferrous sulfate/ascorbate (Fe2+/Vc)-induced mitochondrial damage
Mitochondria (2 mg proteinÆmL)1) suspended in peroxida-tion medium (150 mMKCl, 10 mMEDTA, 1 mM GSH,
25 mM Hepes/NaOH, pH 7.4) were subjected, in the absence and presence of Se-2F3-scFv, to oxidative stress generated by 50 lM Fe2+plus 2 mMascorbate at 37C Damage experiments were carried out without Se-2F3-scFv protein and known as the damage group; experiments carried out without Se-2F3-scFv, ascorbate, and Fe2+were known as the control group [32]
Measurement of lipid peroxidation Lipid peroxidation in the Vc/Fe2+-induced mitochondrial damage system was analyzed by the TBA assay In this assay, TBA reacts with malonaldehyde and/or other carbonyl by-products of free-radical-mediated lipid per-oxidation to give 2 : 1 (mol/mol) colored conjugates [33] Before and during incubation with the different concentra-tions of Se-scFv-2F3, a 1.0 mL aliquot was taken and vortex-mixed with 1 mL 75% (w/v) trichloroacetic acid and
1 mL 0.5% (w/v) TBA in water The assay mixtures were heated for 40 min at 80C After cooling and centrifuga-tion, A532of the supernatants was recorded These readings (corrected for blanks) were converted into thiobarbituric acid reactive substance (TBARS) values, using an absorp-tion coefficient obtained for authentic malonaldehyde, 1.56· 105
M )1Æcm)1
Assay of mitochondrial swelling Swelling of mitochondria was assayed as described by Hunter et al [34] Changes in light scattering are correlated with mitochondrial swelling Mitochondrial swelling was measured as the decrease in turbidity of the reaction mixture
at 520 nm The decrease in absorbance indicates an increase
in mitochondrial swelling and a decrease in mitochondrial integrity
Assay of cytochromec oxidase (CCO) activity
An aliquot of incubation mixture from the Damage group
or Control group was taken at different time intervals and centrifuged (10 000 g, 4C, 2 min).The pellet was washed with 10 mMpotassium phosphate buffer, pH 7.4, contain-ing 125 mMKCl, 1 mMMgCl2, and 5 mMglutamate Then
it was suspended in a small amount of 100 mMpotassium phosphate buffer, pH 7.0, and an aliquot was taken for assay of CCO activity [35] The CCO activity was measured
in 2 mL of the reaction system, in which the cytochrome c concentration was 15 lM The absorbance was decreased
Trang 3with oxidation of cytochrome c in the sample cell, into
which 5 lL 10 mM K3Fe(CN)6 was added to oxidize
cytochrome c thoroughly when the reaction was completed
The absorbance intensity at this time was recorded as A1
The plot of ln(At) A1) vs time was made The absolute
value of the line slope, Kapp, was the apparent rate constant
of cytochrome c oxidation and was used to express CCO
activity
Results
The GPX activity of Se-2F3-scFv
We successfully cloned the variable regions of antibody 2F3
genes and expressed them as inclusion body proteins [28]
After refolding of inactive 2F3-scFv protein, the catalytic
residue Sec was incorporated into the binding site by
chemical modification to produce the selenium-containing
abzyme Se-2F3-scFv Se-2F3-scFv catalyzed the reduction
of H2O2 by GSH as listed in Table 1 The activity was
2840 ± 113.6 UÆlmol)1, which is 49.1% of that of rabbit
liver GPX This is a relatively high figure, although it is only
11.7% of that of the intact monoclonal catalytic antibody
Se-2F3 This activity is 2870 times that of the well-studied
GPX mimic Ebselen (PZ51) These results are similar to
previous reports [28,29]
Inhibition of lipid peroxidation by Se-2F3-scFv
The polyunsaturated fatty acid in mitochondrial membrane
is readily attacked by ROS, especially•OH produced by the
Fenton reaction, producing TBARS TBARS therefore was
used to measure the extent of lipid peroxidation TBA reacts
with malonaldehyde and/or other carbonyl by-products of
free-radical-mediated lipid peroxidation to give 2 : 1 (mol/
mol) colored conjugates [33], which have an A532value
Bovine heart mitochondria exposed to (Fe2+ plus
ascorbate)-induced oxidative stress are peroxidized in a
time-dependent manner as indicated by the formation of
TBARS from membrane lipids Over 50 min, the amount of
TBARS accumulated in the damage group was between
2.40 ± 0.02 and 3.14 ± 0.03 nmol per mg protein and for
the control group it was between 2.03 ± 0.02 and 2.32 ±
0.02 nmol per mg protein The increased TBARS in the
damage group was 2.2-fold higher than that in the control
group
Figure 1 shows that Se-2F3-scFv effectively protects membrane lipids from Fe2+/Vc-induced oxidative damage The inhibition of lipid peroxidation by Se-2F3-scFv proteins was strongly dependent on the concentration of Se-2F3-scFv The amount of TBARS produced decreased with an increase in Se-2F3-scFv concentration When the concen-tration of Se-2F3-scFv protein was 8.35 lM, the TBARS content was 56 ± 1.7% of the damage group, indicating that TBARS production was inhibited by 44% The antioxidant activity of Ebselen was also determined in this experiment When the concentration of Ebselen was 8.00 lM, the TBARS content was only 89.4 ± 2% of the damage group, indicating that TBARS production was inhibited by 10.6% Therefore, Se-2F3-scFv was more protective than Ebselen This is in agreement with their GPX activities
Effect of Se-2F3-scFv on swelling of the damaged mitochondria
Swelling and shrinking of mitochondria is a normal physiological phenomenon during respiration However, abnormal swelling will disrupt the mitochondrial membrane resulting in cell death Mitochondrial swelling therefore characterizes its integrity It can be correlated with changes
in light scattering A decrease in A520reflects an increase in mitochondrial swelling and a decrease in mitochondrial integrity
The A520for the control group remained basically constant, whereas that for the damage group decreased considerably with time, indicating that the Fe2+/Vc-induced damage resulted in extensive mitochondrial swelling The reason for the swelling is that H2O2produced by Fe2+/Vc is converted into•OH by the Fenton reaction, which initiates
Table 1 GPX activity of Se-2F3-scFv, Se-2F3, Ebselen and native GPX
from rabbit liver GPX activity was assayed by the coupled coenzyme
system Reactions were carried out in 50 m M potassium phosphate
buffer, pH 7.0, at 37 C, 1 m M GSH, 0.5 m M H 2 O 2 Data are
means ± SD (n ¼ three separate experiments).
Species
GPX activity UÆlmol)1 UÆmg)1 Se-2F3-scFv 2840.0 ± 113.6 94.7 ± 3.8
Se-2F3 24 300 ± 729 162.0 ± 4.9
Native GPX 5780 85.7
Fig 1 Effect of Se-2F3-scFv on the production of TBARS Bovine heart mitochondria were incubated for 50 min with ascorbate (2 m M )/
Fe2+(50 l M ) in the presence of various concentrations of Se-2F3-scFv
at 37 C The extent of lipid peroxidation was measured as accumu-lation of TBARS as described in Materials and methods Data are expressed as mean ± SD for five independent preparations.
Trang 4lipid peroxidation and destroys the structure of the
mem-brane When different concentrations (0.46, 1.39, and
3.95 lM) of Se-2F3-scFv protein were added, the
mito-chondrial swelling was apparently inhibited compared with
the damage group, and this was dependent on Se-2F3-scFv
concentration Furthermore, the protection afforded by
Se-2F3-scFv was much greater than that by Ebselen at
8.00 lM(Fig 2)
Protection of CCO activity in damaged mitochondria
CCO is one of the key redox enzymes in the
electron-transport chain of mitochondria and is also the marker
enzyme of mitochondria The integrity of the mitochondrial
membrane is important for enzyme activity Mitochondria
exposed to Fe2+/Vc-induced oxidative stress are
peroxi-dized, producing TBARS The integrity of the mitochondria
therefore is destroyed, resulting in a decrease in CCO
activity Over 60 min, CCO activity in the damage group
decreased from 0.356 ± 0.012 to 0.208 ± 0.010 U per mg
protein, i.e by 41.6% Figure 3 shows that CCO
protection increased with increasing Se-2F3-scFv
concen-tration When the Se-2F3-scFv concentration was 4.15 lM,
over 60 min, 90.2 ± 2.0% of CCO activity was retained;
for 8.00 lMEbselen only 71 ± 2.5% of CCO activity was
retained
Discussion
The involvement of ROS in a wide variety of diseases and
the ageing process is now widely accepted [36,37] Natural
antioxidants have been shown to play an important part in
the protection of mitochondria from damage by scavenging
ROS GPX catalyzes the reduction of a variety of
hydro-peroxides, and therefore protects the cell from oxidative damage Ebselen is an interesting small GPX mimic and has been widely studied as an antioxidant [21–24], but it has some drawbacks, such as low GPX activity and water solubility Se-2F3-scFv overcomes these shortcomings and shows much better protection of mitochondria
Mitochondria are a major source of ROS production in the cell and are particularly susceptible to oxidative stress [36,38] In addition, highly energized mitochondria are also dangerous for the cell, as the reduced state of respiratory chain electron carriers supports the formation of superoxide
by one-electron transfer reactions [39] Moreover, oxidative stress seems to differentially damage the components of the oxidative phosphorylation machinery Generally, oxidative stress decreases the activity of the components of oxidative phosphorylation and promotes the permeability transition
of mitochondria [40], resulting in loss of functional integrity Therefore protection of mitochondria from oxidative dam-age may be important in the prevention or treatment of ROS-related diseases Naturally occurring oxidative dam-age can be mimicked by exposing cells or organelles in vitro
to redox-active xenobiotics such as H2O2 and t-BuOOH [41,42] Another approach is to use a ROS-producing system such as Fe2+/Vc or XO/HX [43,44] The reactions for
Fe2+/Vc-induced mitochondrial damage are proposed to be
as follows:
Ascorbic acidþ 2Fe3þ! dehydroascorbic acid
Fe2þþ H2O2! Fe3þþ OHþ OH ð2Þ
Fig 2 Effect of Se-2F3-scFv on the swelling of mitochondria (h)
Control; (s) damage + 0.46 l M Se-F3-scFv; (n) damage + 1.39 l M
Se-2F3-scFv; (e) damage + 2.78 l M Se-2F3-scFv; (q) 8.00 l M
Ebselen; (,) damage Bovine heart mitochondria were incubated for
50 min to ascorbate (2 m M )/Fe2+(50 l M ) in the presence of various
concentrations of Se-2F3-scFv at 37 C The extent of the swelling
was measured as described in Materials and methods Data are
means ± SD (n ¼ three separate experiments).
Fig 3 Effect of Se-2F3-scFv on CCO activity of mitochondria Bovine heart mitochondria were incubated for 60 min to ascorbate (2 m M )/
Fe 2+ (50 l M ) in the presence of various concentrations of Se-2F3-scFv
at 37 C CCO activity was measured as described in Materials and methods Data are means ± SD (n ¼ three separate experiments).
Trang 5LOOHþ Fe2þ! LOO þ Fe3þ ð6Þ
where L represents lipid compounds Unsaturated
lipids appear to be prominent targets Lipid
peroxida-tion is triggered by hydrogen abstracperoxida-tion from an
unsaturated lipid (Eqn 3) Subsequent chain
propaga-tion steps (Eqns 4 and 5) generate lipid hydroperoxides
(LOOHs), with accompanying disruption of membrane
structure and function Lipid hydroperoxides could
also produce free radicals (LOO•) (Eqn 6) to continue
subsequent chain propagation (Eqn 5) As discussed
above,•OH and LOO• are the active reagents, which
initiate lipid peroxidation There is a great deal of
evidence that free-radical traps protect cells from
oxidative damage [45].•OH and LOO• are produced
from H2O2 and LOOH (Eqns 2 and 6), therefore
scavenging of H2O2and LOOH would be an
alternat-ive approach to protecting cells from oxidatalternat-ive damage
In many mitochondria, catalase is lacking [46] Thus,
GPXs, including cGPX and PHGPX, play an
import-ant role in scavenging hydroperoxides GPX mimics
with high activity can efficiently scavenge
hydroper-oxides, block lipid peroxidation, and protect
mito-chondria from oxidative damage (Eqn 7) In the living
organism, oxidized GSH (GSSG) produced in the first
step (Eqn 7) would be reduced to be GSH by GSH
reductase (Eqn 8)
ROOHþ 2GSH GPX!ROHþ GSSG þ H2O2 ð7Þ
NADPHþ Hþþ GSSG Glutathione reductase!NADPþ
Se-2F3-scFv exhibited high GPX activity, efficiently
catalyzed the reduction of hydroperoxides by GSH, and
blocked lipid peroxidation In the Fe2+/Vc-induced
mitochondrial damage model system, Se-2F3-scFv
decreased the maximal level of TBARS accumulation
and dose-dependently inhibited lipid peroxidation
Increasing concentrations of Se-2F3-scFv prevented
TBARS accumulation and mitochondrial swelling and
preserved CCO activity In all these experiments,
Se-2F3-scFv was better than Ebselen at protecting
mitochondria against oxidative injury This is in
agree-ment with their GPX activity
In summary, our results demonstrate that Se-2F3-scFv
exhibits high GPX activity and has excellent antioxidant
activity in the model of Fe2+/Vc-induced mitochondria
damage Se-2F3-scFv may therefore have potential for
curing ROS-related diseases, such as chronic inflammation,
cardiovascular disease, cancer and cataract
Acknowledgements
We are grateful to the Major State Basic Research Development
Program (Grant no G2000078102) and the High Technology Research
Development Plan (2001 AA 213 513) for financial support.
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