Results in Immunology 1 (2011) 60–69

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Cloning of crystallin from orange-spotted grouper and characterization of its activity as potential protective agent Young-Mao Chen a,b,c,1, Cham-En Kuo d,1, Chun-Mao Lin e, Pei-Shiuan Shie a,c, Tzong-Yueh Chen a,b,c,n a

Laboratory of Molecular Genetics, Institute of Biotechnology, College of Bioscience and Biotechnology, National Cheng Kung University, Tainan 70101, Taiwan Research Center of Ocean Environment and Technology, National Cheng Kung University, Tainan 70101, Taiwan c Agriculture Biotechnology Research Center, National Cheng Kung University, Tainan 70101, Taiwan d Department of Nursing, Tzu Hui Institute of Technology, Pingtung 92641, Taiwan e Graduate Institute of Medical Sciences, Taipei Medical University, Taipei 11031, Taiwan b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 9 August 2011 Received in revised form 29 August 2011 Accepted 31 August 2011 Available online 9 September 2011

Oxidative stress associated with nodavirus infection is poorly understood, especially pertaining to infection-mediated brain injury. Indirect evidence indicates that infection increases cellular abundance of reactive oxygen species (ROS) with consequent increase in cellular dityrosine production. The detection of dityrosine in nodavirus-infected grouper was demonstrated using immunohistochemical (IHC) staining. Proteomic analyses with eye tissues of healthy grouper revealed more abundant expression of crystallin protein in the eye than in various tissues, which was confirmed by real-time polymerase chain reaction and IHC analyses. Grouper crystallin belongs to a small heat shock protein family with chaperone-like function that prevents heat-induced and oxidative stress-induced protein aggregation. Recombinant crystallin induced nitric oxide (NO) production in RAW 264.7 cells after treatment. The results provide new insight into the pathogenesis of nodavirus and demonstrate an experimental rationale for antioxidant therapy research. & 2011 Elsevier B.V. All rights reserved.

Keywords: Nodavirus Crystallin Reactive oxygen species (ROS)

1. Introduction Betanodavirus is neuropathogenic and inflicts conspicuous damage that is characterized by vacuolation and degeneration of neurons throughout the central nervous system [1,2]. Piscine nodavirus, a member of the Betanodavirdae family, is the causative agent of viral nervous necrosis or fish encephalitis that produces high mortalities in hatchery-reared larvae and juveniles of marine fishes in Taiwan, Japan, Australia, and Europe [3]. This virus is unenveloped, possesses a 25–30 nm diameter icosahedral capsid, and contains a genome composed of bipartite, singlestranded, and positive-sense RNA molecules [4]. Considerable progress in the understanding of the betanodavirdae pathogenesis has been made. The activation of the host immune response and direct invasion of cells are believed to contribute to this pathogenesis [5,6], which includes induction of inflammatory cells and the host’s immune response. For example, ubiquitin conjugating enzyme 7 interacting protein, which functions in apoptosis, and interferon induced with helicase C domain protein1, which

n Corresponding author at: Laboratory of Molecular Genetics, Institute of Biotechnology, College of Bioscience and Biotechnology, National Cheng Kung University, Tainan 70101, Taiwan, ROC. Tel.: þ 886 6 2757575x65622x610; fax: þ 886 6 2766505. E-mail address: [email protected] (T.-Y. Chen). 1 Contributed equally to this work.

2211-2839/$ - see front matter & 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.rinim.2011.08.005

contributes to apoptosis and mediates type I interferon production, are differentially expressed in infected and control cells [7]. However, the mechanisms underlying betanodavirdae pathogenesis are still not completely clear. When groupers are infected by nodavirus, the virus accumulates in the brain and eyes, and also undergoes replication at these two sites [8]. These two sites are, therefore, very important in the study of virus–host interactions. In addition, fish are under a lot of stress after viral infection: therefore, the immune responses that occur at these sites suffer from the same problems described previously, which are those of antibody production by adaptive immunity, antibody–antigen binding affinity, and immunological memory. However, at the eye, the antibody titer is low. Consequently, virus-induced stress is most important in the eye. If the eye is used to study stresses produced by innate immunity, interference by antibodies can at least be excluded. Also, this allows for examination of whether a secondary antibody production response and subsequent immunological memory are present during stress responses. Therefore, the eye is a very important tissue in the study of nodavirus-induced stress. The rapid development of proteomic techniques has revolutionized the ability to study protein interactions and cellular changes on a global scale, revealing previously unknown and unanticipated associations. Interestingly, crystallins that are involved in the regulation of cellular redox status are themselves regulated, indicating that nodavirus infection may induce oxidative stress. To clarify this, the present study evaluated the generation of reactive oxygen species (ROS) in nodavirus-infected cells. In addition, in agreement with

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disease’s occurrence, immunochemical staining detected protein dityrosine in atherosclerotic lesion of apo-E-deficient mice using a novel monoclonal antibody [9]. As ROS are known to alter proteins [10], the formation of dityrosine [11], which is an indicator of intracellular ROS [12], was used to determine whether nodavirus could alter proteins through ROS activity. Epidemiological studies have revealed that nodavirus infection is a complicated condition and the pathogenesis of the infection is still not fully defined [13]. Although some nodavirus markers such as coat protein, coat protein antibody, and fish immunoglobulin have been identified and used in diagnosing and monitoring the progress of disease, no single serological test on juvenile groupers can unequivocally diagnose the infection. For example, the detection of nodavirus coat protein is a hallmark for viral nervous necrosis, but the absence of detectable coat protein cannot exclude nodavirus infection. Definitive diagnosis of nodavirus infected viral nervous necrosis still relies on a combination of serological, biochemical, and histological examinations. Using proteomic profiling analysis, the present study aimed to identify biomarkers useful in the diagnosis of viral carrier states in grouper, and how the nodavirus evades host defenses. Detailed analysis of these proteins may reveal valuable information for the diagnosis of nodavirus infected viral nervous necrosis. One of the identified proteins, crystallin, whose expression was significantly higher in the eye than in other tissues [14], was confirmed by real-time polymerase chain reaction (PCR) and IHC analyses. Crystallin belongs to a small heat shock protein family with chaperone functions that prevent heat-induced and oxidative stress-induced aggregation proteins [15]. In an inflammation-activated mouse model, crystallin pretreatment reduced tumor necrosis factor-a (TNF-a) and nitric oxide (NO) production in lipopolysaccharide (LPS)-activated astrocytes [16]. This suggested the ability to prevent the inflammation-triggered neurotoxicity by crystallin. Recently, as a class of heat shock protein, crystallin exhibits protective function in LPS-induced proinflammation release and therapeutic role in neurodegenerative diseases, including Parkinson’s disease, Alzheimer’s disease and multiple sclerosis [17–19]. The role of crystallin in vitro in relation to the function of macrophage activation during nodavirus-infected grouper is not clear. In this report, the focus was on the well-characterized nodavirus-mediated neuropathogenesis of grouper, aiming to reveal any association between nodavirus infection an oxidative damage to brain area. Nodavirus infection was associated with increased production of ROS. Dityrosine, a useful marker for protein oxidation, was involved in amino acid hydroxylation of brain and eye tissue during nodavirus infection in groupers. Injury mediated by free radicals, particularly by ROS, is an important common pathway of such varied pathological processes as inflammatory damage [20] and neurodegenerative diseases [21]. These previous and present observations indicate that recombinant crystallin is capable of activation of macrophages [22], which is accompanied by production of nitric oxide (NO). A crystallin cDNA from orangespotted grouper Epinephelus coioides was cloned and its expression was characterized. Grouper crystallin possessed chaperone functions that prevented heat-induced and oxidative stressinduced aggregation proteins. Collectively, these observations indicate that crystallin has the potential to act as an antiinflammatory agent in neuroprotective processes.

2. Materials and methods 2.1. Cell culture and reagents The grouper cell line GF-1 [23] was grown at 28 1C in Leibovitz’s L-15 medium (GibcoBRL, Gaithersburg, MD, USA)

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supplemented with 5% fetal bovine serum (FBS). GF-1 grouper cells, which are susceptible to nodavirus infection and replication, were obtained from the Taiwan Bioresources Collection and Research Center. Transient transfections were performed by introducing 1–2 mg of plasmid encoding grouper crystallin into cells using Lipofectamine (Invitrogen, Carlsbad, CA, USA). After transfection, cells were grown for 24–30 h. Intracellular localization of crystallin proteins was examined using a model IX70 microscope (Olympus, Tokyo, Japan). 20 ,70 -Dichlorodihydrofluorescein diacetate (H2DCFDA; Molecular Probes, Eugene, OR, USA) was dissolved in 20% dimethylsulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, USA). An alkaline phosphatase-conjugated substrate Western blotting detection system kit was purchased from Bio-Rad (Hercules, CA, USA). Alkaline phosphatase-conjugated anti-mouse, anti-rabbit, and anti-goat IgG antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were diluted 1:5000 prior to use. 2.2. Measurement of intracellular ROS The production of intracellular ROS was measured using H2DCFDA as previously described [24]. H2DCFDA reacts with ROS to form the highly fluorescent compound dichlorofluorescein. To measure ROS GF-1 cells starved by growth in low-serum Leibovitz’s L-15 medium with 1% FBS were treated with nodavirus (104 TCID50 mL  1) for 24 h followed by 10 mM H2DCFDA for 20 min. Control cells received DMSO. Cells were collected following exposure to nodavirus or DMSO, and fluorescence was determined with excitation and emission wavelengths of 485 and 520 nm, respectively, using a microplate reader (Thermo Labsystems, Waltham, MA, USA). To determine the production of ROS during nodavirus infection, cells were first incubated with the ROS scavenger, N-acetylcysteine (NAC), for 2 h, followed by nodavirus infection. The ROS level was determined by dividing the absorbance of the infected group by the absorbance of the control group. 2.3. Preparation of dityrosine standard and isocratic reverse-phase high-pressure liquid chromatography (HPLC) Dityrosine can be formed by a horseradish peroxidase-catalyzed oxidation of tyrosine in the presence of hydrogen peroxide (H2O2). Ten milligrams of horseradish peroxidase was dispensed in 500 mL of 5 mM tyrosine prepared in 0.1 M borate buffer, pH 9.1. Then, 142 mL of 30% H2O2 was added and mixed by brief swirling. After incubation at room temperature for 60 min, 175 mL of 2-mercaptoethanol was added to the reaction mixture. The resulting solution was immediately frozen in liquid nitrogen and lyophilized. The lyophilized material was dissolved in 250 mL of distilled water and the pH was adjusted to 8.8 with a few drops of 0.01 M NaOH. The resulting solution was loaded onto a DEAE column that has been pre-equilibrated with 20 mM NaHCO3, pH 8.8, and was eluted using 200 mM borate buffer, pH 8.8. The dityrosine-containing solutions were pooled and lyophilized. The dityrosine produced in the mixture was chromatographically purified. To do this, the supernatant was loaded onto a BioGel P-2 column equilibrated with 100 mM NH4HCO3. The column was eluted with 100 mM NH4HCO3 with a flow rate 40 mL/h. The lyophilized dityrosine was dissolved in 20 mL of 100 mM formic acid and the pH was adjusted to 2.5 by adding concentrated formic acid. The column was eluted with 100 mM formic acid, and the dityrosine-containing solution was lyophilized and stored at  20 1C [10]. An isocratic reverse-phase HPLC system also was used to analyze dityrosine in conjunction with both absorbance detection systems. The 4.6  250 mm2 reverse-phase column (ODS II Spherisorb; LC-Resources, Deerfield, IL, USA) has an 11%

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carbon loading and a particle size of 5 mm. The solvent consisted of 92% water, 8% acetonitrile, and 0.1% trifluoroacetic acid. By employing an excitation wavelength of 280 nm, the excited-state ionization characteristic of dityrosine allows fluorometric monitoring of acidic solutions [11]. The dityrosine-conjugated Keyhole Limpet Hemocyanin (KLH; 500 mL, 1 mg mL  1) was emulsified with an equal volume of complete Freund’s adjuvant to a final concentration of 0.5 mg mL  1, and the solution was then intramuscularly injected into New Zealand White rabbits. The rabbits were boosted after 4, 8, and 12 weeks with the same amount of antigen in Freund’s incomplete adjuvant until an adequate antibody generation was achieved. 2.4. Immunohistochemical analysis Immunohistochemical studies were performed as previously described [25]. Briefly, grouper at post-hatch day 40–45 were fixed in 10% formalin and embedded in paraffin following a routine procedure. Each 5mm-thick section was mounted on a polylysine-coated slide, deparaffinized in xylene, and rehydrated in descending grades (100–70%) of ethanol. Endogenous peroxidase activity was blocked by 10 min incubation at room temperature with absolute methanol containing 3% H2O2. The sections were sequentially blocked with a power block solution (BioGenex, San Ramon, CA, USA), washed with phosphate-buffered saline (PBS), and incubated with polyclonal rabbit antigrouper crystallin, dityrosine, or polyclonal rabbit anti-coat protein antibody (1:500 dilution) at 4 1C overnight. The sections were washed twice with PBS, incubated with secondary antibody (Super Sensitive Polymer-HRP IHC; BioGenex, San Ramon, CA, USA) for 30 min at room temperature. Peroxidase activity was ascertained with 3,30 -diaminobenzidine (used as chromogen) for 10 min. The sections were counterstained with Harris hematoxylin for nuclei, dehydrated, and mounted. Negative controls were performed with preimmune rabbit serum and incubation with PBS instead of anti-grouper crystallin antibodies. The sections were observed using an Axiovert 40 microscope (Carl Zeiss, Jena, Germany). The images were obtained with an SPOT RT3 camera (Diagnostic, Sterling Heights, MI, USA). 2.5. Production of polyclonal grouper crystallin antiserum for Western blotting cDNA from amino acids 1–231 for the mature crystallin of an orange-spotted grouper was cloned into a pGEM-T vector (Promega, Madison, WI, USA) and subcloned into the pET29a vector (Novagen, San Diego, CA, USA) between the EcoRI and XhoI sites to obtain pET29a-crystallin. The resulting expression vector encoded crystallin with a (His)6 and several extra amino acids at the N-terminus. This vector was transformed into the bacterial host, Escherichia coli BL21 (DE3), for expression driven by T7 polymerase. Induction by 0.5 mM isopropyl-b-thiogalactopyranoside was carried out at 37 1C for 3 h. After undergoing freezing and thawing once, cells were sonicated on ice, and the cleared lysate was obtained by centrifugation at 12,000 rpm for 15 min. The (His)6-tagged crystallin was bound to a nickel-charged HisTrap column (HisTrap HP, 5 mL bed volume; Amersham Biosciences, Piscataway, NJ, USA) pre-equilibrated with the binding buffer, and washed with the binding buffer containing 50 mM imidazole. The bound crystallin was eluted with binding buffer containing imidazole in a step-gradient manner (100–500 mM). The crystallin protein peaks eluted with 250–350 mM imidazole were combined and dialyzed against buffer A (50 mM Tris–HCl, pH 8.0, 1 mM dithiothreitol, 50 mM NaCl, 5 mM MgCl2, 10% glycerol), followed by freezing at  701C in a minimal aliquot. Protein concentrations were determined using a Bio-Rad protein

assay kit with a bovine serum albumin (BSA) standard. To obtain anti-grouper crystallin rabbit polyclonal antibody, the (His)6tagged crystallin were used to immunize two New Zealand White rabbits with a primary injection emulsified in Freund’s incomplete adjuvant at 1 mg/mL, and 1 mL was injected subcutaneously into two rabbits. The rabbits were boosted after 4, 8, and 12 weeks with the same amount of antigen in the adjuvant. The crystallin antibody was obtained after clotting overnight at 41C followed by centrifugation at 1200 rpm [26]. 2.6. Two-dimensional (2-D) gel electrophoresis Healthy grouper eyes were crushed in liquid nitrogen and homogenized with 10% trichloroacetic acid and 0.07% b-mercaptoethanol in cold acetone. After centrifugation, each pellet was washed twice with cold acetone. Supernatants were discarded and the pellets were vacuum-dried to a protein powder. The powder was solubilized in 1 mL of lysis buffer [9.5 M urea, 2% (w/ v) CHAPS, 0.8% (w/v) Pharmalyte pH 3–10, and 1% (w/v) dithiothreitol]. For isoelectric focusing, 50 mL of each sample was mixed with 300 mL of a rehydration solution [8 M urea, 2% (w/v) CHAPS, 0.8% (w/v), 15 mM dithiothreitol, and 0.5% (v/v) (Immobilized pH Gradient (IPG)-buffer pH 3–10)] to produce a final protein amount of 150 mg per sample. Isoelectric focusing was performed using immobilized pH gradient strips. The IPG strips (13 cm, pH 3–10 NL) were rehydrated overnight at 50 V and focused for 3 h at 8000 V at 20 1C under mineral oil. Strips were then incubated for 10 min in equilibration buffer I [6 M urea, 30% (w/v) glycerol, 2% (w/v) sodium dodecyl sulfate (SDS), 1% (w/v) dithiothreitol in 0.05 M Tris–HCl buffer pH 8.8] following by incubation in equilibration buffer II [6 M urea, 30% (w/v) glycerol, 2% (w/v) SDS, and 4% (w/v) iodacetamide in 0.05 M Tris/HCl buffer pH 8.8]. After the equilibration steps the strips were transferred to a 22 cm  22 cm 10% SDS–polyacrylamide gel electrophoresis (SDS–PAGE) system. Electrophoresis in the second dimension was performed at 150 V and 150 mA at 20 1C for approximately 18 h [27]. 2.7. Cloning and expression analysis of grouper crystallin Total RNA was isolated from post-hatch day 40–45 orangespotted grouper, Epinephelus coioides, following the single-step acid guanidinium thiocyanate-phenol-chlorofrom extraction method [28]. Extracted cellular total RNA (5 mg) as template was incubated at 42 1C for 60 min in 20 mL of 1X reaction buffer containing 2 U Moloney murine leukemia virus (M-MLV) reverse transcriptase (Promega), 0.25 mM dNTP and 4 mM oligo(dT)15 primer, and 0.4 U RNase (Boehringer Mannheim Biochemicals, Mannheim, Germany). Primers used for PCR amplification were osgshsp-F TACCTgTTTgAgTgCggTgACT and osgshsp-R gATACgACgCATggACTggA; they were designed from four peptide sequences obtained by mass spectrometry of protein (Supplementary Fig. 2A, boxed region) and matched those of crystallin. Putative crystallin sequences were examined for homogeneity by published crystallin sequences with Blast (http://www.ncbi.nlm.nih.gov/BLAST). RACE-PCR was carried out as previously described [29]. Based on the verified sequence of the 979 bps fragment of crystallin cDNA, two primers crystallin-5RACE, osgshsp-a387 gCggTAggAgTTgCTCC, osgshsp-a516 CCAgAgggTgggCACATC, and crystallin-3RACE, osgshsp-s300 TCgCTgggATACCTggAgC and osgshsps600, gATCTgCCTgTATgAgCTCTCCg were PCR-amplified and cloned for the 50 and 30 ends, respectively. Two adapter primers for both ends were provided in the Marathon cDNA Amplication Kit (Clontech, Mountain View, CA, USA). The (RACE)-PCR thermal cycle profile was as follows: 94 1C for 1 min; 30 s at 94 1C, 4 min at 72 1C, and 10 min at 72 1C for 30 cycles; followed by an extension at 72 1C for 10 min. The amplified fragment was

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verified with subcloning into a pCR II vector for sequencing. To minimize polymerization errors during PCR, a proofreading polymerase was employed in PCR reactions. Real time reverse transcription (RT)-PCR was used to quantify the expression of mRNA for crystallin with expression of actin as reference. The primers osgshsp-F TACCTgTTTgAgTgCggTgACT and osgshsp-R gATACgACgCATggACTggA were used to amplify a 130 bp fragment of grouper crystallin. A standard curve was constructed by serially diluting a linearized plasmid containing the open reading frame of grouper crystallin. Grouper beta actin primers were applied to normalize the starting quantity of RNA. Typical profile times used were initial step, 95 1C for 15 min, followed by a second step at 94 1C for 15 s, 60 1C for 30 s and 72 1C for 30 s for 40 cycles with melting curve analysis. The level of target mRNA was normalized to the level of actin and compared to control (healthy grouper) and the values were calculated by the 2  DDCT method. To elucidate the evolutionary history of crystallin, novel crystallin sequences were identified in zebrafish and eight mammalian species, and used to explore the phylogenetic relationships of the crystallin gene family. A neighbor-joining phylogenetic tree was produced by the MEGA3.1 program [30] on the basis of a ClustalW alignment of the nucleotide sequences of the openreading frames along with all known complete sequences of crystallin cDNA with the complete deletion of gaps for 1000 bootstrap replications. Numbers indicated bootstrap confidence values through 1000 replications; only bootstrap values over 70% are exhibited. 2.8. Determination of nitric oxide (NO) production

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coverslip was then removed from the plate, mounted on a glass slide, and observed with an Olympus IX70 fluorescence microscope.

3. Results 3.1. ROS production induced by nodavirus infection To assess the effect of nodavirus-infected grouper on ROS production, the ROS-sensitive fluorescent probe DCFDA was used. Nodavirus-infected cells increased ROS production by 2.4-fold, comparing to noninfected cells, whereas ROS production was blocked by the antioxidant compound N-acetylcysteine (NAC; Fig. 1A). Because nodavirus infection leads to an inflammatory response and an increase in the basal oxidative stress, the next experiment determined the formation of dityrosine, serving as a marker of oxidatively modified proteins, in cells exposed to oxygen-free radicals. Dityrosine was prepared by incubation of tyrosine with horseradish peroxidase in the presence of H2O2. Dityrosine could be distinguished by the intense 420 nm fluorescence, measurable upon ultraviolet spectroscopy over a wavelength range of 254–365 nm, as shown in Supplementary Fig. 1A. Tyrosine and dityrosine were analyzed by reverse-phase HPLC with an ultraviolet detection wavelength of 280 nm. A typical chromatogram is shown in Supplementary Fig. 1B. Tyrosine and dityrosine eluted at 5.2 and 6.8 min, respectively. Crystallin, which is the structural constituent of the lens of the eye, is significantly affected by oxidative stress via Fenton-type

RAW 264.7 cells, a murine macrophage cell line, were obtained from American Type Culture Collection (ATCC) and cultured at 37 1C in Dulbecco’s Modified Essential Medium supplemented with 10% fetal bovine serum (FBS), 100 units mL  1 penicillin, and 100 mg mL  1 streptomycin in a 5% CO2 incubator as recommended by ATCC. Briefly, RAW 264.7 cells grown on a 75 cm2 culture dish were seeded in 96 well plates at a density of 2  105 cells/well. Adhered cells were then incubated for 24 h with or without 1 mg mL  1 of E. coli lipopolysaccharide (LPS), in the absence or presence of 10 mM recombinant crystallin. The activated continuously with E. coli lipopolysaccharide (LPS; 1 mg mL  1) for 24 h as the positive control. Nitrite in culture supernatants was measured by adding 100 mL of Griess reagent (1% sulfanilamide and 0.1% N-[1-naphthyl]-ethylenediamine dihydrochloride in 5% phosphoric acid) to 100 mL samples of the medium for 10 min at room temperature. The optical density at 570 nm was measured using a Multiskan RC photometric microplate reader (Labsystems, Helsinki, Finland). A NO standard curve was made with sodium nitrite. The detection limit of the assay was 0.5 mM. 2.9. Immunofluorescence microscopy Immunofluorescence microscopy to observe the formation of punctated crystallin foci was conducted essentially as described previously [31]. Briefly, cells were seeded into wells of a six-well culture plate containing a glass coverslip in each well. After treatment, cells were fixed in 4% paraformaldehyde for 15 min, washed with PBS, and permeabilized in 0.2% Triton X-100. After blockage with blocking serum for 1 h, samples were incubated with a rabbit polyclonal anti-crystallin antibody overnight at 4 1C, followed by Alexa-Fluor 594-conjugated goat anti-rabbit secondary antibody at room temperature for 1 h. To stain the nuclei, 40 ,60 -diamidino-2-phenylindole dihydrochloride was added to the cells, which were then incubated for another 15 min. Each

Fig. 1. Nodavirus induces generation of intracellular ROS in GF-1 cells. (A) ROS level was measured with H2DCFDA. Relative ROS level indicates the fluorescence level per total protein unit and is normalized to control cells. The control cells served as the calibrator cells (ROS level ¼ 1). (B) Dityrosine structure was probed with anti-dityrosine antibody as observed in recombinant crystallin protein and not detected in BSA by Western blot analysis (panel B, left) and Coomassie staining (panel B, right).

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reactions, by virtue of being enriched in aromatic amino acids; the reaction generates the dityrosine structure (Fig. 1B). In this manner, ROS generation in nodavirus-infected cells was examined to determine oxidative stress. As well, free radical species were effectively reduced by the NAC antioxidant. These observations suggested that nodavirus infection may induce oxidative stress in cells.

oxidative stress that occurs during nodavirus infection. Dityrosine was considered to be a marker for organisms exposed to oxidative stress, such as occur from systemic bacterial infections [32] and lens cataracts [33].

3.2. Dityrosine accumulation was significantly higher in those cases of nodavirus-infected grouper

Molecular characterization and expression of grouper crystallins were shown in Supplementary Fig. 2. These results prompted an experiment to determine the normal biological function of crystallin. To answer this, the E. coli expression system was exploited to express recombinant crystallin protein as antigen, which was then injected into rabbits to obtain anti-crystallin antibodies. Western blotting was used to determine whether there were any differences in crystallin protein expression between nodavirus-infected and healthy grouper eye. b-actin was used as an internal control. Crystallins expression did not differ significantly in Supplementary Fig. 2. Next, stimulating macrophages with recombinant crystallins caused a gradual nitrite elevation in cell supernatants. Nitrite monitoring was performed using the Griess reaction as isolated measurements at 24 h after the stimulation by recombinant crystallins. The same experiments using cells with lipopolysaccharide (LPS) stimulation were performed as positive controls (Fig. 4A). In order to evaluate the effect of cystallin on activated macrophage cells, crystallins were added to RAW 264.7 macrophage cells 1 h before LPS stimulation. As shown in Fig. 4A, the NO production that stimulates macrophages with recombinant crystallins was lower when compared to positive controls (cells with LPS stimulation). However, we also evaluated recombinant crystallins to determine the elevated NO production levels. Fig. 4B showed that the NO production levels were increased 3.25-fold in the presence of 4 mM and 12 mM recombinant crystallins. Take together, incubation with crystallins with LPS activation give rise to releasing of NO; then, pretreatment with crystallin for 1 h reduced LPS-induced production of NO by about 18%.

During nodavirus infection, one of the mechanisms to alleviate cellular oxidative stress associated cytotoxicity may involve reaction with tyrosine amino acid to form dityrosine. The potent initial step in dityrosine formation is the creation of a tyrosine radical, which promotes the intermolecular cross-linking of two monomeric tyrosine containing proteins to generate dityrosine as a stable end product. Evidence of in vivo generation can be demonstrated by the presence of dityrosine in cells from nodavirusinfected tissues. Thus, the detection of dityrosine can serve as an indicator of ROS. More exuberant expression of dityrosine was found in nodavirus-infected injured eye and brain tissues (Fig. 2A and B). Dityrosine staining was observed mainly in the eye, with a diffuse distribution pattern throughout the retina. Notably, the expression pattern of crystallin was similar in both healthy and nodavirus-infected grouper tissues (Fig. 2C and D). Serial biopsy sections obtained from nodavirus-infected grouper confirmed that all dityrosine-labeling retina coexpressed crystallin. In contrast, histological and immunohistochemical examinations of eye and brain tissue from nodavirus-infected grouper revealed immunoreactivity to coat protein in eye and brain tissues (Fig. 2E and F). A higher magnification view clearly revealed brain vacuolization (Fig. 2G and H). To summarize, no significant difference in the retina staining score of crystallin between healthy and nodavirusinfected grouper was found, but dityrosine formation was significantly different compared with that obtained in eye tissue with non-viral healthy grouper. In all cases studied the majority of dityrosine-expressing cellular region, which were distributed throughout the retina of nodavirus infected groupers, were located in close proximity to nodavirus antigen-expressing eye tissue. ROSmediated dityrosine formation of crystallin protein was markedly induced during nodavirus infection, and appeared to be associated with the histological severity of viral nervous necrosis (VNN). 3.3. Proteomic analysis of crystallin expression characteristic of grouper lenses Two-dimensional electrophoresis (2-DE) and mass spectrometry (MS) were used to demonstrate and analyze the expression of crystallins in grouper lenses. Abundant protein spots detected at the 20–31 kDa range (pH 6–9) appeared likely to represent crystallins. Peptide mass fingerprinting of 24 of these spots demonstrated that 12 were crystallins (Table 1). The proteome patterns of revealed great similarity between grouper and zebrafish crystallin expression. Grouper crystallins contained many phosphorylated and dimerized isoforms (Fig. 3). It could not be established from these experiments whether cellular stress interfered with the modification of aromatic amino acids in crystallin. This implicated the formation of dityrosine in crystallins as a biomarker of nodavirus infection. While the formation of dityrosine could lead to alteration in conformation, ligand binding, and biological activity, this specific modification of amino acids in crystallins might reduce ROS production and protect antioxidant enzymes, even under condition of nodavirus infection, resulting in oxidative stress. Dityrosine cross-linked crystallin could be susceptible to proteolysis in response to insults, including

3.4. Recombinant crystallins induce nitric oxide (NO) production by mimicking macrophages activation

3.5. Influence of nodavirus infection on crystallin expression and chaperone-like activity Naturally nodavirus-infected groupers were divided into groups of 10 and crystalline expression was compared by RT-PCR with 10 groups of experimentally nodavirus infected groupers. No significant differences were evident. Crystallin is present in the eye proteome and is abundantly expressed in the eye, but nodavirus infection does not result in different expression. However, 2-D proteomic analyses indicated three groups of crystallin. The most likely explanation is that crystallin undergoes post-translational modification, dimerization, and oligomerization. It is not known whether nodavirus infection in groupers affects these processes, thereby interfering with the biological functions of crystallins. Crystallin is regulated by temperature [34] and undergoes folding under normal physiological conditions, which support a chaperone-like activity. Therefore, an experiment was done where grouper cells were infected with the nodavirus at 28 1C and 32 1C. No temperature-related differences in expression mRNA and protein were evident. However, after viral infection, immunohistochemical staining showed that crystallin proteins clustered within cells forming puncture spots (Fig. 5). With increased temperature and viral infection, the probability of intracellular puncture formation also increased. The results are consistent with the view that crystallin is a stress-induced protein. Using human crystallins as an example, under normal conditions, crystallin assembles into high order forms [34]. However, under stress situations, grouper crystallin assembles into puncture forms, which helps to unfold abnormal proteins back into normal proteins, performing chaperone-like functions.

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Fig. 2. The expression pattern of dityrosine, crystalline, and nodavirus coat protein in nodavirus-infected grouper compared with healthy grouper. Histological sections of brain and eye tissues from grouper with pathological changes that could be attributed to nodavirus, immunohistochemistry for dityrosine (A, B), crystallin (C, D), nodavirus coat protein (E, F), and staining with H&E (G, H).

3.6. Analysis of polyglutamine (polyQ) aggregation and its prevention by recombinant crystallin in vitro PolyQ proteins fused with green fluorescent protein (GFP) were used to monitor the aggregation of misfolded proteins [35], therefore,

we evaluated firstly whether recombinant polyQ-GFP reproduces key features, accumulated in inclusion body-like aggregation in vitro. GFP fluorescence was observed under the control of E. coli expression system. In marked contrast, when different length polyglutamine fused to GFP were expressed, distinct fluorescent images were

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Table 1 Protein identification isolated from 2-D gels. Spot no.

Protein name

Acc. No.

App. Mt (kDa)/pI

Mowse score (Queries matched)

Sequence coverage (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

Mitochondrial ATP-synthase beta subunit-like protein Enolase 1 (alpha) Enolase 1 (alpha) Crystallin, beta A4 protein Keratin 5 Keratin 15 Actin, alpha1, skeletal muscle Glyceraldehyde-3-phosphate dehydrogenase Aldolase a, fructose-bisphosphate, b Crystallin B1 protein isoform 10 Crystallin B1 protein isoform 10 Crystallin B1 protein Crystallin, beta B1 Crystallin, beta B1 Crystallin, gamma M2c Crystallin, beta B1 Crystallin, beta B2 Crystallin, beta A4 Crystallin, gamma N2 Crystallin, alpha A Nucleoside-diphosphate kinase Peripheral myelin protein 2 Fatty acid binding protein 11a Beta globin

NP_001019600 AAH59511 AAH59511 NP_001018135 NP_571231 NP_998688 NP_5711666 NP_998259 NP_998380 XP_001923189 XP_001923189 CAC84899 XP_700835 XP_688538 NP_001007784 XP_700835 AAY18969 NP_001018135 NP_001003428 AAH83177 CAI11562 NP_001004682 NP_001004682 AAB05405

65.2/5.46 47.0/6.16 47.4/6.25 23.0/6.25 58.5/5.34 49.1/5.13 41.9/5.23 36.1/6.55 39.5/8.48 26.8/6.44 26.8/6.44 26.8/6.44 25.6/6.88 25.9/6.53 21.4/8.3 25.6/6.88 23.7/6.65 23.0/6.25 21.7/5.86 19.7/5.84 17.2/5.43 15.1/7.63 15.1/7.63 11.7/7.93

725(37) 254(6) 141(2) 72(1) 53(1) 244(12) 457(21) 124(3) 180(6) 571(22) 497(23) 211(4) 368(15) 238(20) 127(7) 459(16) 101(2) 151(11) 46(6) 132(2) 62(2) 182(4) 94(2) 93(5)

34 14 7 5 2 7 28 8 13 44 31 24 47 20 26 51 12 34 16 12 11 11 13 18

(r-crystallin) and heat shock protein 90 (r-HSP90) reduced r-Q9GFP aggregation in vitro. In contrast, both r-crystallin and r-HSP90 did not alter aggregated r-Q9GFP to form soluble r-Q9GFP (Fig. 6D). In this study, grouper crystallin processes chaperone-like properties, including the ability to prevent the aggregation of misfolded proteins.

4. Discussion

Fig. 3. 2-DE profiles of total grouper lens protein. Separation was performed on 11 cm IPG strips with a nonlinear pH 3–10 gradient. Gels were stained using Coomassie Blue and spots were identified by Matrix-Assisted Laser Desorption/ Ionization Time of Flight Mass Spectrometer (MALDI-TOF MS). Mass standards are indicated on the left in kDa. Spots labeled with numbers detected at the 20–31 kDa range (pH 6–9) appeared likely to represent crystallins. Large scale of cellular proteins separated in 2-D gels. By means of the liquid chromatographytandem mass spectrometry set-up, some peptide sequences could be deduced (Table 1), which identified the protein as crystallin on the basis of homology with a protein from zebrafish.

observed in expressing proteins. Recombinant GFP (r-GFP) and recombinant Q5GFP (r-Q5GFP) were diffusely distributed, whereas the recombinant Q9GFP (r-Q9GFP) accumulated partially in inclusion body-like aggregation (Fig. 6A). Analysis of total extracts proteins were subjected to high speed centrifugation, r-GFP remained exclusively in the soluble supernatant fraction, whereas a significant portion of both r-Q15GFP and r-Q21GFP were found in the insoluble pellet fraction (Fig. 6B). Next, we evaluated whether grouper crystallin reduced the amount of aggregates of r-Q9GFP. As shown in Fig. 6C, fluorescence microscopy revealed that both recombinant crystallin

With a proteomics approach, a proteome map has been constructed for grouper eye tissue. However, the effects of nodavirus infection on host cell protein expression have not been investigated. Twenty-four proteins were presently identified; all were shown to be involved in cellular metabolism, mobility, or stress response. Among them, several proteins related to cell redox regulation were of particular interest. Crystallin, which is rich in aromatic amino acids, is a lens structural protein that participates in cellular antioxidation reactions against ROS, including superoxide anion, H2O2, and hydroxyl radicals. Interestingly, two other proteins identified in this study, dehydrogenase and mitochondrial ATP-synthase, are also involved in the cellular redox milieu. The present study sought to examine ROS generation in nodavirus-infected cells in an effort to substantiate the connection. As shown in Fig. 1, infection with nodavirus increased ROS levels in grouper cells. Our proteomic approach resurrects the notion that ROS has an important role in the pathogenesis of nodavirus, and the results fit well with the severe deleterious effects on cells elicited by ROS. One of the deleterious consequences of ROS generation is abnormal protein formation. There is no mention in the literature that nodavirus is able to induce abnormal protein formation, but nodavirus may contribute to apoptosis [36]. On the one hand, apoptosis from ROS stimulated by nodavirus infection is quite plausible; since nodavirus leads to accumulation of genetic aberrations in cell [37], let alone inducing apoptosis the profound abnormal protein formation in infected cells was hard to imagine. The present results strongly correlate nodavirus-induced ROS with severe abnormal protein formation, and, during treatment with NAC, occurred with a minimal decrease in cell viability. In particular,

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Fig. 4. Recombinant crystallins were suggested to induce NO production in RAW 264.7 macrophage cells. (A) Nitrite accumulation in the supernatant collected from RAW 264.7 macrophage cell culture after stimulation by recombinant crystallins (8 mM) for the given 24 h. The same experiments were performed in the presence of LPS (5 ng mL  1) as a positive control. Each point of the curve was constructed from three individual experiments. a, Po 0.05, LPS vs. LPS þcrystallin; b, Po 0.01, LPS þcrystallin vs. crystallin (n¼ 3). (B) NO production in medium containing different concentration of recombinant crystallins. RAW 264.7 macrophage cells were cultured in medium supplemented with different concentration of recombinant crystallins and incubated for 24 h. NO production was determined by Griess reaction.

Fig. 5. Chaperone-like activity of crystallin as observed by fluorescence microscope after nodavirus infection and thermo-challenge. Subcellular distribution of cellular crystallin in nodavirus-infected and thermo challenge grouper. Grouper cells were fixed at indicated times. The samples were stained with antibodies against crystallin. Samples were then reacted with anti-rabbit IgG-conjugated Texas-Red 594.

the proteomic results indicate that ROS are important factors in nodavirus pathogenesis. Crystallin is an aromatic amino acid-rich lens structural protein that protects many enzymes from inactivation and heat-induced aggregation, and reduces intracellular ROS levels [38]. ROS will attack crystallin easily, thus forming dityrosine; many abnormal proteins will then be created at the same time, inducing activated macrophages to release NO, mediating leukocyte trafficking. Crystallin directly interacts with the cell death machinery to suppress apoptosis by inhibiting caspase-3 activation and restraining the

mitochondrial translocation of proapoptotic Bcl-2 family by various agents including tumor necrosis factor [39], ultraviolet radiation [40] and H2O2 [40]. Crystallin is commonly expressed outside the lens, in particular within retinal and hippocampal neurons [41]. The crystallin functional role in these neurons might constitute a new group of factors that promote axon outgrowth [41]. Consistent with its growth of axons in retinal explants function, crystallin is secreted by cultured retinas during axonal regeneration and is involved in the elongation process of retinal ganglion cells axons [42]. In addition, crystallin may mimic the effects of lens injury [43],

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Fig. 6. Grouper crystallin inhibits polyQ-mediated aggregation. (A) Fluorescent microscopic images of expressing various recombinant polyQ(n)GFP (n¼ 0, 5, 9, 15, 21). (B) PolyQ length-dependent fused to GFP formation of aggregates. Equal protein amounts of cell lysates were analyzed after incubation in SDS/DTT at 95 1C. S and P, supernatant and pellet fractions after centrifugation of total cell lysate (12,000 rpm, 20 min), respectively. (C) Effects of expressing of recombinant crystallin and HSP90 on polyQ aggregation in vitro. (D) Both r-crystallin and r-HSP90 were not able to change the aggregated polyQ-GFP properties in vitro.

and its expression correlates with poor clinical outcomes in basallike breast cancer [44]. Dityrosine may become a useful marker for the evaluation of oxidative stress in vivo. These products that originate from tyrosine of crystallin may serve as oxidative stress biomarkers. Immunochemical approaches have been used to detect modified tyrosines and dityrosine. Antibodies to dityrosine have been prepared and are widely used for immunohistochemical staining, enzyme-linked immunosorbent assay, and Western blot analyses [45]. Dityrosine has been immunochemically identified in the lipofuscin of pyramidal neurons in aged human brain [45] and in atherosclerotic lesions in Apo-E-deficient mice [9]. Positive staining of dityrosine has been reported in models of Parkinson and Alzheimer diseases [46,47]. These immunochemical approaches can visually show the localization of dityrosine, which can thus become a universal protein oxidation marker because it can be generated by various ROS, such as peroxynitrite, metal-catalyzed oxidation, and UV irradiation [48]. The precise role of grouper crystallin during nodavirus infection remains to be elucidated. In humans, the ability of virus to generate ROS from phagocytes is an influential stress-related event. It is possible that generation of ROS exerts an antiviral effect on cells, but it is damaging to the host cells, which may give rise to abundant denatured protein. In view of nodavirus infection and then ROS production, aggregation of misfolded proteins in the host cell may lead to release of expressed crystallin. The increased level of grouper crystallin participates in macrophage activation when NO is released under physiologically-relevant stress conditions, consistent with microglial activation by alpha-crystallin resulting in an increased production of iNOS and TNF-a [22]. In addition, Wu et al., reported alpha-crystallin downregulated NO release and TNF-a production in activated microglia in vitro [49]. In agreement with alpha-crystallin and LPS may have different effects on specific pathways that compete with each other in activating the microglia [50]. Our data demonstrate that grouper crystallin downregulates the production of NO in treated LPS with

grouper cells. Crystallin can reduce the release of inflammationinduced cytokines, and it has been suggested that crystallin has the potential to act as an anti-inflammatory agent in the neuroprotective process [16]. In human neurodegenerative diseases such as Parkinson’s and Alzheimer’s disease, protein misfolding and inclusion formation play important roles [51]. Small heat shock (sHSP), HSP27, had chaperone activity capable of assisting the proper folding of misfolded proteins [17] and suggested that sHSPs inhibit amyloid-b (Ab) protein aggregation and cerebrovascular Ab protein toxicity [18]. At present, grouper crystallin exhibits chaperone-like properties, including the ability to increase cellular tolerance on stress condition and to prevent the aggregation of misfolded proteins.

Acknowledgements We thank Dr. Brian D Hoyle for editing the manuscript. This research was supported by the National Science Council (NSC972313-B-006-001-MY3, NSC98-2811-B-006-003, NSC99-2811-B-006002, NSC100-2811-B-006-005, NSC100-2313-B-006-002-MY3) and the Landmark Project (B0127) of National Cheng Kung University, the plan of University Advancement, Ministry of Education, Taiwan.

Appendix A. Supplementary materials Supplementary data associated with this article can be found in the online version at doi:10.1016/j.rinim.2011.08.005.

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