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Oxidative modification of neurofilament-L by the Cu,Zn-superoxide dismutase and hydrogen peroxide system
Biochimie 86 (2004) 553–559 www.elsevier.com/locate/biochi
Oxidative modification of neurofilament-L by the Cu,Zn-superoxide dismutase and hydrogen peroxide system Nam Hoon Kim a, Moon Sik Jeong a, Soo Young Choi b, Jung Hoon Kang a,* a
Department of Genetic Engineering, Cheongju University, Cheongju 360-764, Korea Department of Genetic Engineering, Hallym University, Chunchon 200-702, Korea
b
Received 28 April 2004; accepted 12 July 2004 Available online 30 July 2004
Abstract Neurofilament-L (NF-L) is a major element of neuronal cytoskeletons and known to be important for their survival in vivo. Since oxidative stress might play a critical role in the pathogenesis of neurodegenerative diseases, we investigated the role of Cu,Zn-superoxide dismutase (SOD) in the modification of NF-L. When disassembled NF-L was incubated with Cu,Zn-SOD and H2O2, the aggregation of protein was proportional to the concentration of hydrogen peroxide. Cu,Zn-SOD/H2O2-mediated modification of NF-L was significantly inhibited by radical scavenger, spin trap agents and copper chelators. Dityrosine crosslink formation was obtained in Cu,Zn-SOD/H2O2-mediated NF-L aggregates. Antioxidant molecules, carnosine and anserine significantly inhibited the aggregation of NF-L and the formation of dityrosine. This study suggests that copper-mediated NF-L modification may be closely related to oxidative reactions which play a critical role in neurodegenerative diseases. © 2004 Elsevier SAS. All rights reserved. Keywords: Neurofilament-L; Copper; Hydrogen peroxide; Modification
1. Introduction Neurofilaments give axons their structural integrity and define axonal diameter [1,2]. Neurofilaments are composed of three subunits, identified as light (NF-L), medium (NFM), and heavy (NF-H), which assemble in a 6:2:1 ratio to form long macromolecular filaments [3,4]. Consequently, NF-L is more abundant than the other two subunits in neurons. NF-L is capable of homologous assembly, whereas NF-M and NF-H are not component to assemble in the absence of NF-L [5]. Abnormal accumulation of neurofilaments in neurons is one of the pathological hallmarks of various neurodegenerative disorders. In patients of Parkinson’s disease (PD), Lewy bodies (LBs) are cytoplasmic inclusions that are present consistently and with greatest frequency in neurons of the substantia nigra and locus ceruleus. NF proteins have been identified immunohistochemically as the major protein components of LB filament [6,7]. It was reported that peroxynitrite may nitrate tyrosine residues of * Corresponding author. Tel.: +82-43-229-8562; fax: +82-43-229-8432. E-mail address: [email protected] (J. Hoon Kang). 0300-9084/$ - see front matter © 2004 Elsevier SAS. All rights reserved. doi:10.1016/j.biochi.2004.07.006
NF-L, thereby altering NF assembly and causing neurofilament accumulation in neurons [8]. Reactive oxygen species (ROS) from metabolic or adventitious reduction of O2 are associated with the etiology of human diseases [9,10]. The oxidation of cellular proteins has been described under many pathological conditions [11,12]. Cellular metabolism has been shown to generate oxygen species such as hydrogen peroxide, hydroxyl radical and superoxide anion. Transition metals such as iron and copper react with hydrogen peroxide to produce hydroxyl radical through Fenton-like reaction and can induce the oxidative modification of DNA, proteins and lipids [10]. During exposure to hydrogen peroxide, many proteins with metal binding sites will be likely susceptible to oxidative damage, and free metal ions could be released. Therefore, the reaction of copper with hydrogen peroxide may be associated with abnormal modification of neurofilament which may be involved in the pathogenesis of neurodegenerative disorders. In this context, we hypothesize that Cu,Zn-superoxide dismutase (SOD), could be a source of copper and oxidative stress that might trigger the pathological aggregation of neurofilament. The overexpression of Cu,Zn-SOD among others
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has been correlated with cell killing [13], increased lipid peroxidation [14], reduced transport of biogenic amines [15] and decomposition of brain neuron [16]. In addition to the usual superoxide dismutase activity, Cu,Zn-SOD has a free radical-generating function that utilizes its own product, H2O2 as a substrate [17,18]. It has been reported that the reaction of Cu,Zn-SOD with H2O2 induced the protein fragmentation and then free copper ions were released from the oxidatively damaged SOD [19]. Involvement of the free radical-generating function of Cu,Zn-SOD in human disease has been suggested [20–22]. Yim et al. [21,22] reported that the gain-of-function of the familial amyotrophic lateral sclerosis (FALS) mutant Cu,Zn-SODs may be partly responsible for the development of FALS symptoms due to biological damage resulting from elevated levels of ·OH. Therefore, the function of Cu,Zn-SOD as a free radical generator might be related to oxidation reactions which may play a critical role in neurodegenerative disorders. We investigated whether the Cu,Zn-SOD/H2O2 system is involved in the aggregation of NF-L. Our results revealed that the aggregation of NF-L was induced by the Cu,ZnSOD/H2O2 system via the generation of hydroxyl radical. These results suggest that the Cu,Zn-SOD/H2O2 system may be involved in the oxidative stress-induced aggregation of NF-L in PD and related disorders.
2. Materials and methods 2.1. Expression and purification of neurofilament-L A full-length cDNA clone of mouse NF-L in a pET-3d vector, a generous gift from Dr. Beckman (University of Alabama) transfected into E. coli (BL21). Protein expression was performed as previously described [23]. Bacteria were grown in Luria broth supplemented with 1 mM isopropyl b-D-thiogalactopyranoside beginning at an OD 600 nm reading of 0.8. Incubation was at 37 °C for 3 h. Bacteria were harvested by centrifugation (4000 × g for 10 min at 4 °C), resuspended in standard buffer (50 mM MES, 170 mM NaCl, 1 mM DDT, pH 6.25). The cells were lysed with a French press at a pressure of 20,000 p.s.i. and centrifuged at 8000 × g for 15 min at 4 °C. The supernatant was incubated for 3 h at 37 °C and then was centrifuged at 100,000 × g for 20 min at 25 °C. The pellets containing the aggregated NF-L proteins were washed twice with standard buffer before they were dissolved in urea buffer (25 mM Na–phosphate, pH 7.5, 6 M urea, 1 mM EGTA and 1 mM DDT). The sample was loaded onto a DEAE-Sepharose column and the column was washed with urea buffer. The column was eluted with a linear 25– 500 mM phosphate gradient in urea buffer and 1 ml NF-L eluted between 300 and 360 mM phosphate. These fractions were pooled and either used directly or stored at –70 °C for later experiments. Protein concentration was determined by the BCA method [24].
2.2. Oxidation of protein Oxidative modification of NF-L (0.8 mg/ml) was carried out by incubation of the protein with Cu,Zn-SOD (0.25 mg/ml) and 500 µM H2O2 in 23.5 mM NaHCO3/CO2 buffer (pH 7.6) at 37 °C. After incubation of the reaction mixtures, the mixtures were then placed into Ultrafree-MC filter and centrifuged at 13,000 rpm for 1 h and then washed with Chelex 100 treated water and centrifuged for 1 h at same speed. This was repeated four times. The filtrate was dried by freeze drier and dissolved with phosphate buffer. 2.3. Analysis of modified protein After treatment with various concentrations of H2O2 for various periods of time, samples of the reaction mixtures were diluted with a 5× loading-sample buffer (62.5 mM Tris, 10% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.01% bromophenol blue) and heated in boiling water for 10 min before loading onto SDS-PAGE. An aliquot of each sample was subjected to SDS-PAGE as described by Laemmli [25], using a 12% acrylamide slab gel. The gels were stained with 0.15% Coomassie Brilliant Blue R-250. 2.4. Detection of O,O′-dityrosine The reactions for the detection of O,O′-dityrosine were carried with NF-L (0.8 mg/ml), Cu,Zn-SOD (0.25 mg/ml) and 500 µM H2O2 in a total volume of 300 µl. The samples were diluted with 2.7 ml of Chelex 100-treated water and transferred to a cuvette (3 ml). The fluorescence emission spectrum of the sample was then monitored in the 340– 500 region (excitation, 325 nm) using Spectrofluorometer SMF 25 (Bio-Tek Instruments).
3. Results We first investigated whether the structure of Cu,Zn-SOD was affected by oxidation. For this purpose, human Cu,ZnSOD was incubated with various concentrations of H2O2 under the pH 7.6 (23.5 mM NaHCO3/CO2) conditions at 37 °C for 2 h. SDS-PAGE analysis (Fig. 1A) showed that Cu,Zn-SOD was induced to be degraded by H2O2 in a concentration-dependent manner. In order to determine whether the fragmentation of Cu,Zn-SOD has any effects on NF-L aggregation, NF-L was incubated with various concentrations of H2O2 either in the presence or absence of Cu,ZnSOD. SDS-PAGE analysis showed that NF-L remained intact after incubation with Cu,Zn-SOD or H2O2 alone (Fig. 1B). In contrast, the staining intensity for the original protein was reduced and new high molecular weight material was visualized at the stacker/separator gel interface when NF-L was incubated with both Cu,Zn-SOD and H2O2. These results suggest that both Cu,Zn-SOD and H2O2 were required for the aggregation of NF-L. The aggregation of NF-L
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Fig. 1. Modification of NF-L by the Cu,Zn-SOD/H2O2 system. (A) Cu,Zn-SOD (0.25 mg/ml) was incubated with various concentrations of H2O2. (B) NF-L (0.8 mg/ml) was incubated with or without Cu,Zn-SOD and H2O2. Lane 1, NF-L control; lane 2, N-FL + Cu,Zn-SOD; lane 3, NF-L + H2O2; lane 4, NF-L + Cu,Zn-SOD + H2O2. (C) NF-L (0.8 mg/ml) was incubated with Cu,Zn-SOD and various concentrations of H2O2 in 23.5 mM NaHCO3/CO2 buffer (pH 7.6) at 37 °C for 2 h. Lane 1, NF-L control; lane 2, Cu,Zn-SOD + 0.1 mM H2O2; lane 3, Cu,Zn-SOD + 0.3 mM H2O2; lane 4, Cu,Zn-SOD + 0.5 mM H2O2; lane 5, Cu,Zn-SOD + 1 mM H2O2. The positions of molecular weight markers (kDa) are indicated on the left.
became apparent at 0.1 mM H2O2; the aggregation increased up to 1 mM H2O2 (Fig. 1C). The effect of radical scavengers on the aggregation of NF-L by the Cu,Zn-SOD/H2O2 system was studied. The aggregation of NF-L by the Cu,Zn-SOD/H2O2 system was significantly suppressed in the presence of formate and ethanol (Fig. 2, lanes 3 and 4). Spin-trapping agents have often been used to detect the presence of ·OH radical in biological system. DMPO and PBN react with ·OH radicals to form radical adducts. Cu,Zn-SOD/H2O2 -induced NF-L aggregates are believed to be free radical mediated, thus we have investigated the protective effect of the spin-trapping agents against the aggregation of NF-L. Both DMPO and PBN significantly prevented the aggregation of NF-L induced by the Cu,Zn-SOD/H2O2 system (Fig. 2, lanes 5 and 6). These results suggest that hydroxyl radical might play a critical role in the aggregation of NF-L by the Cu,Zn-SOD/H2O2 system. Because copper ion could be released from the oxidatively damage Cu,Zn-SOD by H2O2 [19], it was predicted that copper may be contributed to the Cu,Zn-SOD/H2O2–induced
Fig. 2. Effect of radical scavengers and spin trap agents on Cu,ZnSOD/H2O2 system-mediated NF-L modification. NF-L (0.8 mg/ml) was incubated with Cu,Zn-SOD (0.25 mg/ml) and 500 µM H2O2 in 23.5 mM NaHCO3/CO2 buffer (pH 7.6) at 37 °C for 2 h in the presence of radical scavengers and spin trap agents. Lane 1, NF-L control; lane 2, no addition; lane 3, 250 mM formate; lane 4, 250 mM ethanol; lane 5, 25 mM DMPO; lane 6, 25 mM PBN. The positions of molecular weight markers (kDa) are indicated on the left.
aggregation of NF-L. We have investigated the effects of the copper chelators on the aggregation of NF-L by the Cu,ZnSOD/H2O2 system. The Cu,Zn-SOD/H2O2–induced aggregation of NF-L was significantly inhibited by copper chelators, DTPA, DDC and penicillamine (Fig. 3). These results suggest that copper ions are involved in the aggregation of NF-L by the Cu,Zn-SOD/H2O2 system. It has been reported that O,O′-dityrosine crosslink formation between dityrosine residues may play a part in the formation of oxidative covalent protein crosslink [26]. We investigated the formation of O,O′-dityrosine during the Cu,Zn-SOD/H2O2 system-mediated NF-L aggregation by measuring fluorescence emission spectrum between 340 and 500 nm with an excitation at 325 nm. The reactions were carried out with NF-L in the presence or absence of Cu,ZnSOD and H2O2. As the reactions were proceeded, the emission peak at 410 nm due to the formation of O,O′-dityrosine crosslinks was increased (Fig. 4). Oxidative protein crosslinks are produced through several means such as direct interactions between carbon-centered radical derivatives of amino acids. We investigated the effects of radical scaven-
Fig. 3. Effect of copper chelators on Cu,Zn-SOD/H2O2 system-mediated NF-L modification. NF-L (0.8 mg/ml) was incubated with Cu,Zn-SOD (0.25 mg/ml) and 500 µM H2O2 in 23.5 mM NaHCO3/CO2 buffer (pH 7.6) at 37 °C for 2 h in the presence of copper chelators. Lane 1, NF-L control; lane 2, no addition; lane 3, 5 mM DTPA; lane 4, 5 mM DDC; lane 5, 5 mM penicillamine. The positions of molecular weight markers (kDa) are indicated on the left.
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Fig. 4. Fluorescence spectra of Cu,Zn-SOD/H2O2 system-mediated NF-L modification. The fluorescence spectra of the formation of dityrosine was observed when NF-L (0.8 mg/ml) was incubated with or without Cu,ZnSOD (0.25 mg/ml) and 500 µM H2O2 at 37 °C for 2 h. (a) NF-L control, (b) NF-L + Cu,Zn-SOD, (c) NF-L + H2O2, (d) NF-L + Cu,Zn-SOD + H2O2.
gers and copper chelators on the formation of dityrosine. Radical scavengers and copper chelators inhibited the formation of dityrosine (Fig. 5). Our result suggested that the tyrosine–tyrosine crosslink formation might participate in the Cu,Zn-SOD/H2O2 system-mediated NF-L aggregation. The dipeptide carnosine (b-alanyl-L-histidine) and related dipeptides homocarnosine and anserine are found in longlived tissues in high amounts (up to 20 mM in human muscle) [27–29] and has been shown to delay aging in cultured cells [30]. Many biochemical studies have suggested that these compounds possess copper chelation and free radical scavenging function which may partly explain its apparent homeostatic function [31,32]. We investigated the effect of adding carnosine and anserine to the aggregation of NF-L induced by the Cu,Zn-SOD/H2O2 system. These compounds significantly inhibited the Cu,Zn-SOD/H2O2 systeminduced aggregation of NF-L (Fig. 6) and the formation of dityrosine (Fig. 7).
4. Discussion NF-L, a major structural protein important to the survival of motor neurons, was modified by oxidative stress. Neurofilaments are susceptible to oxidation in part because they are among the most abundant proteins in a cell. Previous studies have suggested that oxidative stress might play a critical role in the pathogenesis of PD [33–35]. Trace metals such as copper and iron, which are presented in biological system, react with hydrogen peroxide and then produce the hydroxyl radicals. This reactive oxygen might lead to crosslinking and aggregation of amyloidogenic molecules [36]. The cleavage of the metalloproteins by oxidative dam-
Fig. 5. Effect of radical scavengers and copper chelators on the formation of modified NF-L. (A) Effect of radical scavengers and spin trap agents on the formation of modified NF-L. NF-L (0.8 mg/ml) was incubated with Cu,ZnSOD (0.25 mg/ml) and 500 µM H2O2 in 23.5 mM NaHCO3/CO2 buffer (pH 7.6) at 37 °C for 2 h in the presence of radical scavengers and spin trap agents. Oxidized, NFL + Cu,Zn-SOD + H2O2 + no effector; Formate, 250 mM formate; Ethanol, 250 mM ethanol; DMPO, 25 mM DMPO; PBN, 25 mM PBN. (B) Effect of copper chelators on the formation of modified NF-L. NF-L (0.8 mg/ml) was incubated with Cu,Zn-SOD (0.25 mg/ml) and 500 µM H2O2 in 23.5 mM NaHCO3/CO2 buffer (pH 7.6) at 37 °C for 2 h in the presence of radical scavengers and spin trap agents. Oxidized, NF-L + Cu,Zn-SOD + H2O2 + no effector; DTPA, 5 mM DTPA; DDC, 5 mM DDC; Penicillamine, 5 mM penicillamine.
age may lead to increases in the levels of metal ions in some biological cells [37]. The releasing of copper ions from Cu,Zn-SOD could be induced by a free radical-generating function of the enzyme [19]. Since copper promotes NF-L self-aggregation, it is possible that aberrant accumulation of copper ion might act as a risk factor for neurodegenerative diseases. It has been reported that copper concentration was significantly increased in the cerebrospinal-fluid of PD patients [38] and the cerebrospinal-fluid copper concentration was increased 2.2-fold in the patients of Alzheimer’s disease (AD) [39]. These results suggested that iron or coppercatalyzed oxidative reaction might contribute to the pathogenesis of neurodegenerative diseases.
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Fig. 6. Effect of carnosine and anserine on Cu,Zn-SOD/H2O2 systemmediated NF-L modification. NF-L (0.8 mg/ml) was incubated with Cu,ZnSOD (0.25 mg/ml) and 500 µM H2O2 in 23.5 mM NaHCO3/CO2 buffer (pH 7.6) at 37 °C for 2 h in the presence of carnosine and anserine. Lane 1, NF-L control; lane 2 and 5, no addition; lane 3, 0.5 mM carnosine; lane 4, 5 mM carnosine; lane 5, 25 mM carnosine; lane 6, 0.5 mM anserine; lane 7, 5 mM anserine; lane 8, 25 mM anserine. The positions of molecular weight markers (kDa) are indicated on the left.
Fig. 7. Effect of carnosine and anserine on the formation of modified NF-L. NF-L (0.8 mg/ml) was incubated with Cu,Zn-SOD (0.25 mg/ml) and 500 µM H2O2 in 23.5 mM NaHCO3/CO2 buffer (pH 7.6) at 37 °C for 2 h in the presence of carnosine and anserine. Oxidized, NF-L + Cu,Zn-SOD + H2O2 + no effector; CA (black bar), 5 mM carnosine; CA (gray bar), 25 mM carnosine; ANS (black bar), 5 mM anserine; ANS (gray bar), 25 mM anserine.
The present study investigated the potential role of Cu,ZnSOD in aggregation of NF-L. The toxicity of Cu,Zn-SOD may be augmented by its free radical-generating function in neurodegenerative disorder. It has been shown that the released copper ions from the oxidatively damage Cu,Zn-SOD by H2O2 could enhance the Fenton-like reaction to produce hydroxyl radicals [19,39]. Since the level of free radicals was reported to be increase in PD patients [40,41], the oxidative aggregation of neurofilament has pathological significance. Our result showed that aggregation of NF-L was not induced by either Cu,Zn-SOD or H2O2 alone, but rather was significantly stimulated in the presence of both. In addition to the usual superoxide dismutation activity, Cu,Zn-SOD has a free radical-generating function that utilizes its own product, H2O2 as a substrate. Hence, it has been suggested that this
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enzyme function as a free radical generator may be responsible in part for the deleterious effects observed by enhanced activity of Cu,Zn-SOD [17]. In this study, the Cu,ZnSOD/H2O2-induced aggregation of neurofilament was inhibited by hydroxyl radical scavengers and spin-trapping agents. These results indicate that hydroxyl radicals may involve in the aggregation of neurofilament. Interestingly, many LBs were immunostained by an antibody against Cu,Zn-SOD in the locus coeruleus and substantia nigra of PD patients [42]. Therefore, Cu,Zn-SOD-positive LBs in the substantia nigra suggests that it may play a role in the aggregation of NF-L. Trace metal such as iron and copper, which are variously present in biological systems, may interact with active oxygen species, ionizing radiation or microwave radiation to damage macromolecules [43–45]. The cleavage of the metalloproteins by oxidative damage may lead to increases in the levels of metal ions in some biological cell [37]. The releasing of copper ions from Cu,Zn-SOD could be induced by a free radical-generating function of the enzyme [17]. It has been proposed that hydroxyl radicals were generated by Cu,Zn-SOD in two distinct stages. In the early stage, bound copper ion of the enzyme reacted with H2O2 and produced the radical enzymatically (the free radical-generating activity). In this stage, two distinct fates for the hydroxyl radicals were described; first, the hydroxyl radicals may exit from the active site channel, leading to the oxidative damage of other molecules within the short range of hydroxyl radical diffusion. Second, the hydroxyl radicals may react directly with Cu,Zn-SOD, irreversibly inactivating the enzyme and leading to copper ion released from the damaged enzyme. In the late stage, the Fenton-like reaction may occur nonenzymatically by the reaction of the released copper ions with H2O2. In the present study, copper specific chelators, DDC and penicillamine significantly inhibited the aggregation of NF-L. These results indicate that copper ions may involve in the Cu,Zn-SOD/H2O2–induced aggregation of NF-L. Carnosine is a dipeptide found at up to 20 mM in muscle and nerve tissues in human [27–29]. Many biochemical studies have suggested that carnosine possesses antioxidant and free radical-scavenging function which may partly explain it apparent homeostatic function [46–48]. Our results showed that carnosine and anserine could protect the aggregation of NF-L by the Cu,Zn-SOD/H2O2 system. One of the mechanisms by which antioxidants can protect their biological targets from oxidative stress is the chelation of transition metals such as copper and iron, preventing them from participating in the deleterious Fenton reaction. Carnosine and anserine have been shown to be very efficient copper chelating agents [31]. These compounds might be able to bind Cu2+ and prevent some Cu2+-dependent radical reaction [27]. Carnosine is active electrochemically as a reducing agent in cyclic voltammetric measurements, donating a hydrogen atom to the peroxyl radical [27]. It has been reported that carnosine and related compounds quench 50–95% of hydroxyl radicals produced in Fenton reaction [32]. Therefore, it can be assumed that carnosine and anserine may protect the
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aggregation of NF-L by the Cu,Zn-SOD/H2O2 system through the combination of scavenging of ·OH radicals and the chelation of copper ion. These results suggest that carnosine and related compounds may be explored as potential therapeutic agents for pathogenesis that involve the oxidative damage of protein mediated by active oxygen species. In conclusion, the present results suggest that the aggregation of NF-L was induced by the reaction of Cu,Zn-SOD with H2O2, involving ·OH generation from H2O2. The Cu,Zn-SOD/H2O2 system-mediated NF-L aggregation might therefore be involved in the pathogenesis of neurodegenerative disorders.
Acknowledgments We thank Dr. J. S. Beckman (University of Alabama) for providing cDNA of mouse neurofilament-L. This work was supported by grant a Korea Research Foundation Grant (KRF-2002-041-C00285).
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Oxidative modification of neurofilament-L by the Cu,Zn-superoxide dismutase and hydrogen peroxide system Nam Hoon Kim a, Moon Sik Jeong a, Soo Young Choi b, Jung Hoon Kang a,* a
Department of Genetic Engineering, Cheongju University, Cheongju 360-764, Korea Department of Genetic Engineering, Hallym University, Chunchon 200-702, Korea
b
Received 28 April 2004; accepted 12 July 2004 Available online 30 July 2004
Abstract Neurofilament-L (NF-L) is a major element of neuronal cytoskeletons and known to be important for their survival in vivo. Since oxidative stress might play a critical role in the pathogenesis of neurodegenerative diseases, we investigated the role of Cu,Zn-superoxide dismutase (SOD) in the modification of NF-L. When disassembled NF-L was incubated with Cu,Zn-SOD and H2O2, the aggregation of protein was proportional to the concentration of hydrogen peroxide. Cu,Zn-SOD/H2O2-mediated modification of NF-L was significantly inhibited by radical scavenger, spin trap agents and copper chelators. Dityrosine crosslink formation was obtained in Cu,Zn-SOD/H2O2-mediated NF-L aggregates. Antioxidant molecules, carnosine and anserine significantly inhibited the aggregation of NF-L and the formation of dityrosine. This study suggests that copper-mediated NF-L modification may be closely related to oxidative reactions which play a critical role in neurodegenerative diseases. © 2004 Elsevier SAS. All rights reserved. Keywords: Neurofilament-L; Copper; Hydrogen peroxide; Modification
1. Introduction Neurofilaments give axons their structural integrity and define axonal diameter [1,2]. Neurofilaments are composed of three subunits, identified as light (NF-L), medium (NFM), and heavy (NF-H), which assemble in a 6:2:1 ratio to form long macromolecular filaments [3,4]. Consequently, NF-L is more abundant than the other two subunits in neurons. NF-L is capable of homologous assembly, whereas NF-M and NF-H are not component to assemble in the absence of NF-L [5]. Abnormal accumulation of neurofilaments in neurons is one of the pathological hallmarks of various neurodegenerative disorders. In patients of Parkinson’s disease (PD), Lewy bodies (LBs) are cytoplasmic inclusions that are present consistently and with greatest frequency in neurons of the substantia nigra and locus ceruleus. NF proteins have been identified immunohistochemically as the major protein components of LB filament [6,7]. It was reported that peroxynitrite may nitrate tyrosine residues of * Corresponding author. Tel.: +82-43-229-8562; fax: +82-43-229-8432. E-mail address: [email protected] (J. Hoon Kang). 0300-9084/$ - see front matter © 2004 Elsevier SAS. All rights reserved. doi:10.1016/j.biochi.2004.07.006
NF-L, thereby altering NF assembly and causing neurofilament accumulation in neurons [8]. Reactive oxygen species (ROS) from metabolic or adventitious reduction of O2 are associated with the etiology of human diseases [9,10]. The oxidation of cellular proteins has been described under many pathological conditions [11,12]. Cellular metabolism has been shown to generate oxygen species such as hydrogen peroxide, hydroxyl radical and superoxide anion. Transition metals such as iron and copper react with hydrogen peroxide to produce hydroxyl radical through Fenton-like reaction and can induce the oxidative modification of DNA, proteins and lipids [10]. During exposure to hydrogen peroxide, many proteins with metal binding sites will be likely susceptible to oxidative damage, and free metal ions could be released. Therefore, the reaction of copper with hydrogen peroxide may be associated with abnormal modification of neurofilament which may be involved in the pathogenesis of neurodegenerative disorders. In this context, we hypothesize that Cu,Zn-superoxide dismutase (SOD), could be a source of copper and oxidative stress that might trigger the pathological aggregation of neurofilament. The overexpression of Cu,Zn-SOD among others
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has been correlated with cell killing [13], increased lipid peroxidation [14], reduced transport of biogenic amines [15] and decomposition of brain neuron [16]. In addition to the usual superoxide dismutase activity, Cu,Zn-SOD has a free radical-generating function that utilizes its own product, H2O2 as a substrate [17,18]. It has been reported that the reaction of Cu,Zn-SOD with H2O2 induced the protein fragmentation and then free copper ions were released from the oxidatively damaged SOD [19]. Involvement of the free radical-generating function of Cu,Zn-SOD in human disease has been suggested [20–22]. Yim et al. [21,22] reported that the gain-of-function of the familial amyotrophic lateral sclerosis (FALS) mutant Cu,Zn-SODs may be partly responsible for the development of FALS symptoms due to biological damage resulting from elevated levels of ·OH. Therefore, the function of Cu,Zn-SOD as a free radical generator might be related to oxidation reactions which may play a critical role in neurodegenerative disorders. We investigated whether the Cu,Zn-SOD/H2O2 system is involved in the aggregation of NF-L. Our results revealed that the aggregation of NF-L was induced by the Cu,ZnSOD/H2O2 system via the generation of hydroxyl radical. These results suggest that the Cu,Zn-SOD/H2O2 system may be involved in the oxidative stress-induced aggregation of NF-L in PD and related disorders.
2. Materials and methods 2.1. Expression and purification of neurofilament-L A full-length cDNA clone of mouse NF-L in a pET-3d vector, a generous gift from Dr. Beckman (University of Alabama) transfected into E. coli (BL21). Protein expression was performed as previously described [23]. Bacteria were grown in Luria broth supplemented with 1 mM isopropyl b-D-thiogalactopyranoside beginning at an OD 600 nm reading of 0.8. Incubation was at 37 °C for 3 h. Bacteria were harvested by centrifugation (4000 × g for 10 min at 4 °C), resuspended in standard buffer (50 mM MES, 170 mM NaCl, 1 mM DDT, pH 6.25). The cells were lysed with a French press at a pressure of 20,000 p.s.i. and centrifuged at 8000 × g for 15 min at 4 °C. The supernatant was incubated for 3 h at 37 °C and then was centrifuged at 100,000 × g for 20 min at 25 °C. The pellets containing the aggregated NF-L proteins were washed twice with standard buffer before they were dissolved in urea buffer (25 mM Na–phosphate, pH 7.5, 6 M urea, 1 mM EGTA and 1 mM DDT). The sample was loaded onto a DEAE-Sepharose column and the column was washed with urea buffer. The column was eluted with a linear 25– 500 mM phosphate gradient in urea buffer and 1 ml NF-L eluted between 300 and 360 mM phosphate. These fractions were pooled and either used directly or stored at –70 °C for later experiments. Protein concentration was determined by the BCA method [24].
2.2. Oxidation of protein Oxidative modification of NF-L (0.8 mg/ml) was carried out by incubation of the protein with Cu,Zn-SOD (0.25 mg/ml) and 500 µM H2O2 in 23.5 mM NaHCO3/CO2 buffer (pH 7.6) at 37 °C. After incubation of the reaction mixtures, the mixtures were then placed into Ultrafree-MC filter and centrifuged at 13,000 rpm for 1 h and then washed with Chelex 100 treated water and centrifuged for 1 h at same speed. This was repeated four times. The filtrate was dried by freeze drier and dissolved with phosphate buffer. 2.3. Analysis of modified protein After treatment with various concentrations of H2O2 for various periods of time, samples of the reaction mixtures were diluted with a 5× loading-sample buffer (62.5 mM Tris, 10% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.01% bromophenol blue) and heated in boiling water for 10 min before loading onto SDS-PAGE. An aliquot of each sample was subjected to SDS-PAGE as described by Laemmli [25], using a 12% acrylamide slab gel. The gels were stained with 0.15% Coomassie Brilliant Blue R-250. 2.4. Detection of O,O′-dityrosine The reactions for the detection of O,O′-dityrosine were carried with NF-L (0.8 mg/ml), Cu,Zn-SOD (0.25 mg/ml) and 500 µM H2O2 in a total volume of 300 µl. The samples were diluted with 2.7 ml of Chelex 100-treated water and transferred to a cuvette (3 ml). The fluorescence emission spectrum of the sample was then monitored in the 340– 500 region (excitation, 325 nm) using Spectrofluorometer SMF 25 (Bio-Tek Instruments).
3. Results We first investigated whether the structure of Cu,Zn-SOD was affected by oxidation. For this purpose, human Cu,ZnSOD was incubated with various concentrations of H2O2 under the pH 7.6 (23.5 mM NaHCO3/CO2) conditions at 37 °C for 2 h. SDS-PAGE analysis (Fig. 1A) showed that Cu,Zn-SOD was induced to be degraded by H2O2 in a concentration-dependent manner. In order to determine whether the fragmentation of Cu,Zn-SOD has any effects on NF-L aggregation, NF-L was incubated with various concentrations of H2O2 either in the presence or absence of Cu,ZnSOD. SDS-PAGE analysis showed that NF-L remained intact after incubation with Cu,Zn-SOD or H2O2 alone (Fig. 1B). In contrast, the staining intensity for the original protein was reduced and new high molecular weight material was visualized at the stacker/separator gel interface when NF-L was incubated with both Cu,Zn-SOD and H2O2. These results suggest that both Cu,Zn-SOD and H2O2 were required for the aggregation of NF-L. The aggregation of NF-L
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Fig. 1. Modification of NF-L by the Cu,Zn-SOD/H2O2 system. (A) Cu,Zn-SOD (0.25 mg/ml) was incubated with various concentrations of H2O2. (B) NF-L (0.8 mg/ml) was incubated with or without Cu,Zn-SOD and H2O2. Lane 1, NF-L control; lane 2, N-FL + Cu,Zn-SOD; lane 3, NF-L + H2O2; lane 4, NF-L + Cu,Zn-SOD + H2O2. (C) NF-L (0.8 mg/ml) was incubated with Cu,Zn-SOD and various concentrations of H2O2 in 23.5 mM NaHCO3/CO2 buffer (pH 7.6) at 37 °C for 2 h. Lane 1, NF-L control; lane 2, Cu,Zn-SOD + 0.1 mM H2O2; lane 3, Cu,Zn-SOD + 0.3 mM H2O2; lane 4, Cu,Zn-SOD + 0.5 mM H2O2; lane 5, Cu,Zn-SOD + 1 mM H2O2. The positions of molecular weight markers (kDa) are indicated on the left.
became apparent at 0.1 mM H2O2; the aggregation increased up to 1 mM H2O2 (Fig. 1C). The effect of radical scavengers on the aggregation of NF-L by the Cu,Zn-SOD/H2O2 system was studied. The aggregation of NF-L by the Cu,Zn-SOD/H2O2 system was significantly suppressed in the presence of formate and ethanol (Fig. 2, lanes 3 and 4). Spin-trapping agents have often been used to detect the presence of ·OH radical in biological system. DMPO and PBN react with ·OH radicals to form radical adducts. Cu,Zn-SOD/H2O2 -induced NF-L aggregates are believed to be free radical mediated, thus we have investigated the protective effect of the spin-trapping agents against the aggregation of NF-L. Both DMPO and PBN significantly prevented the aggregation of NF-L induced by the Cu,Zn-SOD/H2O2 system (Fig. 2, lanes 5 and 6). These results suggest that hydroxyl radical might play a critical role in the aggregation of NF-L by the Cu,Zn-SOD/H2O2 system. Because copper ion could be released from the oxidatively damage Cu,Zn-SOD by H2O2 [19], it was predicted that copper may be contributed to the Cu,Zn-SOD/H2O2–induced
Fig. 2. Effect of radical scavengers and spin trap agents on Cu,ZnSOD/H2O2 system-mediated NF-L modification. NF-L (0.8 mg/ml) was incubated with Cu,Zn-SOD (0.25 mg/ml) and 500 µM H2O2 in 23.5 mM NaHCO3/CO2 buffer (pH 7.6) at 37 °C for 2 h in the presence of radical scavengers and spin trap agents. Lane 1, NF-L control; lane 2, no addition; lane 3, 250 mM formate; lane 4, 250 mM ethanol; lane 5, 25 mM DMPO; lane 6, 25 mM PBN. The positions of molecular weight markers (kDa) are indicated on the left.
aggregation of NF-L. We have investigated the effects of the copper chelators on the aggregation of NF-L by the Cu,ZnSOD/H2O2 system. The Cu,Zn-SOD/H2O2–induced aggregation of NF-L was significantly inhibited by copper chelators, DTPA, DDC and penicillamine (Fig. 3). These results suggest that copper ions are involved in the aggregation of NF-L by the Cu,Zn-SOD/H2O2 system. It has been reported that O,O′-dityrosine crosslink formation between dityrosine residues may play a part in the formation of oxidative covalent protein crosslink [26]. We investigated the formation of O,O′-dityrosine during the Cu,Zn-SOD/H2O2 system-mediated NF-L aggregation by measuring fluorescence emission spectrum between 340 and 500 nm with an excitation at 325 nm. The reactions were carried out with NF-L in the presence or absence of Cu,ZnSOD and H2O2. As the reactions were proceeded, the emission peak at 410 nm due to the formation of O,O′-dityrosine crosslinks was increased (Fig. 4). Oxidative protein crosslinks are produced through several means such as direct interactions between carbon-centered radical derivatives of amino acids. We investigated the effects of radical scaven-
Fig. 3. Effect of copper chelators on Cu,Zn-SOD/H2O2 system-mediated NF-L modification. NF-L (0.8 mg/ml) was incubated with Cu,Zn-SOD (0.25 mg/ml) and 500 µM H2O2 in 23.5 mM NaHCO3/CO2 buffer (pH 7.6) at 37 °C for 2 h in the presence of copper chelators. Lane 1, NF-L control; lane 2, no addition; lane 3, 5 mM DTPA; lane 4, 5 mM DDC; lane 5, 5 mM penicillamine. The positions of molecular weight markers (kDa) are indicated on the left.
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Fig. 4. Fluorescence spectra of Cu,Zn-SOD/H2O2 system-mediated NF-L modification. The fluorescence spectra of the formation of dityrosine was observed when NF-L (0.8 mg/ml) was incubated with or without Cu,ZnSOD (0.25 mg/ml) and 500 µM H2O2 at 37 °C for 2 h. (a) NF-L control, (b) NF-L + Cu,Zn-SOD, (c) NF-L + H2O2, (d) NF-L + Cu,Zn-SOD + H2O2.
gers and copper chelators on the formation of dityrosine. Radical scavengers and copper chelators inhibited the formation of dityrosine (Fig. 5). Our result suggested that the tyrosine–tyrosine crosslink formation might participate in the Cu,Zn-SOD/H2O2 system-mediated NF-L aggregation. The dipeptide carnosine (b-alanyl-L-histidine) and related dipeptides homocarnosine and anserine are found in longlived tissues in high amounts (up to 20 mM in human muscle) [27–29] and has been shown to delay aging in cultured cells [30]. Many biochemical studies have suggested that these compounds possess copper chelation and free radical scavenging function which may partly explain its apparent homeostatic function [31,32]. We investigated the effect of adding carnosine and anserine to the aggregation of NF-L induced by the Cu,Zn-SOD/H2O2 system. These compounds significantly inhibited the Cu,Zn-SOD/H2O2 systeminduced aggregation of NF-L (Fig. 6) and the formation of dityrosine (Fig. 7).
4. Discussion NF-L, a major structural protein important to the survival of motor neurons, was modified by oxidative stress. Neurofilaments are susceptible to oxidation in part because they are among the most abundant proteins in a cell. Previous studies have suggested that oxidative stress might play a critical role in the pathogenesis of PD [33–35]. Trace metals such as copper and iron, which are presented in biological system, react with hydrogen peroxide and then produce the hydroxyl radicals. This reactive oxygen might lead to crosslinking and aggregation of amyloidogenic molecules [36]. The cleavage of the metalloproteins by oxidative dam-
Fig. 5. Effect of radical scavengers and copper chelators on the formation of modified NF-L. (A) Effect of radical scavengers and spin trap agents on the formation of modified NF-L. NF-L (0.8 mg/ml) was incubated with Cu,ZnSOD (0.25 mg/ml) and 500 µM H2O2 in 23.5 mM NaHCO3/CO2 buffer (pH 7.6) at 37 °C for 2 h in the presence of radical scavengers and spin trap agents. Oxidized, NFL + Cu,Zn-SOD + H2O2 + no effector; Formate, 250 mM formate; Ethanol, 250 mM ethanol; DMPO, 25 mM DMPO; PBN, 25 mM PBN. (B) Effect of copper chelators on the formation of modified NF-L. NF-L (0.8 mg/ml) was incubated with Cu,Zn-SOD (0.25 mg/ml) and 500 µM H2O2 in 23.5 mM NaHCO3/CO2 buffer (pH 7.6) at 37 °C for 2 h in the presence of radical scavengers and spin trap agents. Oxidized, NF-L + Cu,Zn-SOD + H2O2 + no effector; DTPA, 5 mM DTPA; DDC, 5 mM DDC; Penicillamine, 5 mM penicillamine.
age may lead to increases in the levels of metal ions in some biological cells [37]. The releasing of copper ions from Cu,Zn-SOD could be induced by a free radical-generating function of the enzyme [19]. Since copper promotes NF-L self-aggregation, it is possible that aberrant accumulation of copper ion might act as a risk factor for neurodegenerative diseases. It has been reported that copper concentration was significantly increased in the cerebrospinal-fluid of PD patients [38] and the cerebrospinal-fluid copper concentration was increased 2.2-fold in the patients of Alzheimer’s disease (AD) [39]. These results suggested that iron or coppercatalyzed oxidative reaction might contribute to the pathogenesis of neurodegenerative diseases.
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Fig. 6. Effect of carnosine and anserine on Cu,Zn-SOD/H2O2 systemmediated NF-L modification. NF-L (0.8 mg/ml) was incubated with Cu,ZnSOD (0.25 mg/ml) and 500 µM H2O2 in 23.5 mM NaHCO3/CO2 buffer (pH 7.6) at 37 °C for 2 h in the presence of carnosine and anserine. Lane 1, NF-L control; lane 2 and 5, no addition; lane 3, 0.5 mM carnosine; lane 4, 5 mM carnosine; lane 5, 25 mM carnosine; lane 6, 0.5 mM anserine; lane 7, 5 mM anserine; lane 8, 25 mM anserine. The positions of molecular weight markers (kDa) are indicated on the left.
Fig. 7. Effect of carnosine and anserine on the formation of modified NF-L. NF-L (0.8 mg/ml) was incubated with Cu,Zn-SOD (0.25 mg/ml) and 500 µM H2O2 in 23.5 mM NaHCO3/CO2 buffer (pH 7.6) at 37 °C for 2 h in the presence of carnosine and anserine. Oxidized, NF-L + Cu,Zn-SOD + H2O2 + no effector; CA (black bar), 5 mM carnosine; CA (gray bar), 25 mM carnosine; ANS (black bar), 5 mM anserine; ANS (gray bar), 25 mM anserine.
The present study investigated the potential role of Cu,ZnSOD in aggregation of NF-L. The toxicity of Cu,Zn-SOD may be augmented by its free radical-generating function in neurodegenerative disorder. It has been shown that the released copper ions from the oxidatively damage Cu,Zn-SOD by H2O2 could enhance the Fenton-like reaction to produce hydroxyl radicals [19,39]. Since the level of free radicals was reported to be increase in PD patients [40,41], the oxidative aggregation of neurofilament has pathological significance. Our result showed that aggregation of NF-L was not induced by either Cu,Zn-SOD or H2O2 alone, but rather was significantly stimulated in the presence of both. In addition to the usual superoxide dismutation activity, Cu,Zn-SOD has a free radical-generating function that utilizes its own product, H2O2 as a substrate. Hence, it has been suggested that this
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enzyme function as a free radical generator may be responsible in part for the deleterious effects observed by enhanced activity of Cu,Zn-SOD [17]. In this study, the Cu,ZnSOD/H2O2-induced aggregation of neurofilament was inhibited by hydroxyl radical scavengers and spin-trapping agents. These results indicate that hydroxyl radicals may involve in the aggregation of neurofilament. Interestingly, many LBs were immunostained by an antibody against Cu,Zn-SOD in the locus coeruleus and substantia nigra of PD patients [42]. Therefore, Cu,Zn-SOD-positive LBs in the substantia nigra suggests that it may play a role in the aggregation of NF-L. Trace metal such as iron and copper, which are variously present in biological systems, may interact with active oxygen species, ionizing radiation or microwave radiation to damage macromolecules [43–45]. The cleavage of the metalloproteins by oxidative damage may lead to increases in the levels of metal ions in some biological cell [37]. The releasing of copper ions from Cu,Zn-SOD could be induced by a free radical-generating function of the enzyme [17]. It has been proposed that hydroxyl radicals were generated by Cu,Zn-SOD in two distinct stages. In the early stage, bound copper ion of the enzyme reacted with H2O2 and produced the radical enzymatically (the free radical-generating activity). In this stage, two distinct fates for the hydroxyl radicals were described; first, the hydroxyl radicals may exit from the active site channel, leading to the oxidative damage of other molecules within the short range of hydroxyl radical diffusion. Second, the hydroxyl radicals may react directly with Cu,Zn-SOD, irreversibly inactivating the enzyme and leading to copper ion released from the damaged enzyme. In the late stage, the Fenton-like reaction may occur nonenzymatically by the reaction of the released copper ions with H2O2. In the present study, copper specific chelators, DDC and penicillamine significantly inhibited the aggregation of NF-L. These results indicate that copper ions may involve in the Cu,Zn-SOD/H2O2–induced aggregation of NF-L. Carnosine is a dipeptide found at up to 20 mM in muscle and nerve tissues in human [27–29]. Many biochemical studies have suggested that carnosine possesses antioxidant and free radical-scavenging function which may partly explain it apparent homeostatic function [46–48]. Our results showed that carnosine and anserine could protect the aggregation of NF-L by the Cu,Zn-SOD/H2O2 system. One of the mechanisms by which antioxidants can protect their biological targets from oxidative stress is the chelation of transition metals such as copper and iron, preventing them from participating in the deleterious Fenton reaction. Carnosine and anserine have been shown to be very efficient copper chelating agents [31]. These compounds might be able to bind Cu2+ and prevent some Cu2+-dependent radical reaction [27]. Carnosine is active electrochemically as a reducing agent in cyclic voltammetric measurements, donating a hydrogen atom to the peroxyl radical [27]. It has been reported that carnosine and related compounds quench 50–95% of hydroxyl radicals produced in Fenton reaction [32]. Therefore, it can be assumed that carnosine and anserine may protect the
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aggregation of NF-L by the Cu,Zn-SOD/H2O2 system through the combination of scavenging of ·OH radicals and the chelation of copper ion. These results suggest that carnosine and related compounds may be explored as potential therapeutic agents for pathogenesis that involve the oxidative damage of protein mediated by active oxygen species. In conclusion, the present results suggest that the aggregation of NF-L was induced by the reaction of Cu,Zn-SOD with H2O2, involving ·OH generation from H2O2. The Cu,Zn-SOD/H2O2 system-mediated NF-L aggregation might therefore be involved in the pathogenesis of neurodegenerative disorders.
Acknowledgments We thank Dr. J. S. Beckman (University of Alabama) for providing cDNA of mouse neurofilament-L. This work was supported by grant a Korea Research Foundation Grant (KRF-2002-041-C00285).
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