A selenium-containing single-chain abzyme with potent antioxidant activity Delin You 1 , Xiaojun Ren 1,2 , Yan Xue 1 , Guimin Luo 1 , Tongshu Yang 1 and Jiacong Shen 2 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/Fe 2+ )-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 (O 2 –• ) and hydroxyl radical ( • OH), as well as nonradical intermediates such as hydrogen peroxide (H 2 O 2 ), hydroperoxide (ROOH), nitric oxide (NO) and singlet oxygen ( 1 O 2 ) [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 O 2 –• to H 2 O 2 .H 2 O 2 produced by the reduction of O 2 –• 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 H 2 O 2 ) 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 H 2 O 2 [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) Eur. J. Biochem. 270, 4326–4331 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03825.x and 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 H 2 O 2 at rates approaching that of native GPX from rabbit liver [28,29]. The optimal pH and temperature for the Se-2F3-scFv-catalyzed reduction of H 2 O 2 were determined to be 8.27 and 47.2 °C, 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 )1 at 37 °C [28]. Preparation of mitochondria Bovine heart mitochondria were isolated from fresh bovine heart as described previously [30]. Mitochondria were suspended in 0.25 M sucrose/10 m M EDTA/25 m M Hepes/ NaOH buffer, pH 7.4, and maintained at 0 °C. The concentration of the mitochondrial proteins was determined by the method of Bradford [31] with BSA as standard. Ferrous sulfate/ascorbate (Fe 2+ /Vc)-induced mitochondrial damage Mitochondria (2 mg proteinÆmL )1 ) suspended in Ôperoxida- tion mediumÕ (150 m M KCl, 10 m M EDTA, 1 m M GSH, 25 m M Hepes/NaOH, pH 7.4) were subjected, in the absence and presence of Se-2F3-scFv, to oxidative stress generated by 50 l M Fe 2+ plus 2 m M ascorbate at 37 °C. 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 Fe 2+ were known as the control group [32]. Measurement of lipid peroxidation LipidperoxidationintheVc/Fe 2+ -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 80 °C. After cooling and centrifuga- tion, A 532 of 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 · 10 5 M )1 Æcm )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 cytochrome c 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,4°C, 2 min).The pellet was washed with 10 m M potassium phosphate buffer, pH 7.4, contain- ing 125 m M KCl, 1 m M MgCl 2 ,and5m M glutamate. Then it was suspended in a small amount of 100 m M potassium 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 l M . The absorbance was decreased Ó FEBS 2003 Se-containing abzyme with potent antioxidant activity (Eur. J. Biochem. 270) 4327 with oxidation of cytochrome c in the sample cell, into which 5 lL10m M K 3 Fe(CN) 6 was added to oxidize cytochrome c thoroughly when the reaction was completed. The absorbance intensity at this time was recorded as A 1 . The plot of ln(A t ) A 1 ) vs. time was made. The absolute value of the line slope, K app , was the apparent rate constant of cytochrome c oxidationandwasusedtoexpressCCO 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 H 2 O 2 by GSH as listed in Table 1. The activity was 2840 ± 113.6 UÆlmol )1 ,whichis% 49.1%ofthatofrabbit 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 A 532 value. Bovine heart mitochondria exposed to (Fe 2+ 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 Fe 2+ /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 l M , 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 l M , 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 A 520 reflects an increase in mitochondrial swelling and a decrease in mitochondrial integrity. The A 520 for the control group remained basically constant, whereas that for the damage group decreased considerably with time, indicating that the Fe 2+ /Vc-induced damage resulted in extensive mitochondrial swelling. The reason for the swelling is that H 2 O 2 produced by Fe 2+ /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 .Dataare 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 Ebselen 0.99 3.61 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 )/ Fe 2+ (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. 4328 D. You et al.(Eur. J. Biochem. 270) Ó FEBS 2003 lipid peroxidation and destroys the structure of the mem- brane. When different concentrations (0.46, 1.39, and 3.95 l M ) 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 l M (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 Fe 2+ /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 l M , over 60 min, 90.2 ± 2.0% of CCO activity was retained; for 8.00 l M Ebselen 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 H 2 O 2 and t-BuOOH [41,42]. Another approach is to use a ROS-producing system such as Fe 2+ /Vc or XO/HX [43,44]. The reactions for Fe 2+ /Vc-induced mitochondrial damage are proposed to be as follows: Ascorbic acid þ 2Fe 3þ ! dehydroascorbic acid þ 2Fe 2þ þ 2H þ ð1Þ Fe 2þ þ H 2 O 2 ! Fe 3þ þ OH À þ  OH ð2Þ  OH þ LH ! H 2 O þ L  ð3Þ L  þ O 2 ! LOO  ð4Þ LOO  þ LH ! LOOH þ L  ð5Þ 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)8.00l M Ebselen; (,) damage. Bovine heart mitochondria were incubated for 50mintoascorbate(2m M )/Fe 2+ (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). Ó FEBS 2003 Se-containing abzyme with potent antioxidant activity (Eur. J. Biochem. 270) 4329 LOOH þ Fe 2þ ! LOO  þ Fe 3þ ð6Þ where L represents lipid compounds. Unsaturated lipids appear to be prominent targets. Lipid peroxida- tion is triggered by hydrogen abstraction 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 H 2 O 2 and LOOH (Eqns 2 and 6), therefore scavenging of H 2 O 2 and LOOH would be an alternat- ive approach to protecting cells from oxidative 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). ROOH þ 2GSH ÀÀÀÀ! GPX ROH þ GSSG þ H 2 O 2 ð7Þ NADPH þ H þ þ GSSG ÀÀÀÀÀÀÀÀÀÀÀÀÀÀ! Glutathione reductase NADP þ þ2GSH ð8Þ Se-2F3-scFv exhibited high GPX activity, efficiently catalyzed the reduction of hydroperoxides by GSH, and blocked lipid peroxidation. In the Fe 2+ /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. 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Acknowledgements We are grateful to the Major State Basic. peroxidation LipidperoxidationintheVc/Fe 2+ -induced mitochondrial damage system was analyzed by the TBA assay. In this assay, TBA reacts with malonaldehyde and/or