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Characterization of phenoloxidase from the sea cucumber Apostichopus japonicus
Immunobiology 219 (2014) 450–456
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Characterization of phenoloxidase from the sea cucumber Apostichopus japonicus Jingwei Jiang, Zunchun Zhou ∗ , Ying Dong, Xiaoyan Guan, Bai Wang, Bei Jiang, Aifu Yang, Zhong Chen, Shan Gao, Hongjuan Sun Liaoning Key Laboratory of Marine Fishery Molecular Biology, Liaoning Ocean and Fisheries Science Research Institute, Dalian, Liaoning 116023, PR China
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Article history: Received 14 December 2013 Received in revised form 13 February 2014 Accepted 16 February 2014 Available online 25 February 2014 Keywords: Biochemical characteristics Immune-responsive characteristics Metalloenzyme Phenoloxidase Sea cucumber (Apostichopus japonicus)
a b s t r a c t Phenoloxidase (PO) is a crucial immune-related enzyme in invertebrates. In this study, three POs of the sea cucumber Apostichopus japonicus were detected in coelomic fluid using linear-gradient native-PAGE combined with catechol staining and then partially purified by gel excising. The results showed that the three POs had a color of mahogany (AjPO1), yellow (AjPO2) and purple (AjPO3) respectively with molecular weights smaller than 21 kDa in native-PAGE after staining with catechol. Enzymatic activities analysis revealed that AjPO1, AjPO2 and AjPO3 had optimal temperature of 45, 95 and 85 ◦ C and pH of 5.0, 8.0 and 8.0, respectively. Kinetic analysis showed that the Km values of AjPO1 for catechol, l-DOPA, dopamine and hydroquinone were 3.23, 0.86, 3.98 and 1.20 mmol/l, respectively, those of AjPO2 were 0.31, 0.38, 2.05 and 1.30 mmol/l, respectively, and those of AjPO3 were 5.95, 1.28, 5.81 and 0.62 mmol/l, respectively. These results suggest that the three POs are laccase-type phenoloxidase. The activities of all three A. japonicus POs were significantly promoted by Ca2+ , Mg2+ and Mn2+ , and strongly inhibited by ethylenediamine tetraacetic acid disodium (EDTA), sodium diethyldithiocarbamate (DETC) and some common antioxidants. The inhibitions by EDTA and DETC suggest that the three A. japonicus POs are copper-containing metalloenzymes. Immune-responsive analysis showed that the total PO activities in coelomocytes (TPAC) increased greatly after lipopolysaccharide (LPS) challenge and declined significantly after polyinosinic–polycytidylic acid (PolyI:C) challenge, implying that A. japonicus PO immune system, which is composed of several isoenzymes with different characteristics, is closely involved in the defense against the infection of Gram-negative bacteria and double-stranded RNA viruses. © 2014 Elsevier GmbH. All rights reserved.
Introduction Phenoloxidase (PO), a common name of proteins that can oxidize phenols directly in presence of O2 , is one of the critical components of the immune system in invertebrates. PO catalyzes phenol substrates into quinones, which are transformed into melanin following a non-enzymatic pathway (Aladaileh et al., 2007; Asokan et al., 1997; Aspán et al., 1995; Pang et al., 2010; Wu et al., 2013). The melanin and intermediate metabolites generated from the non-enzymatic reactions are involved in pathogen
Abbreviations: PO, phenoloxidase; EDTA, ethylenediamine tetraacetic acid disodium; DETC, sodium diethyldithiocarbamate; TPAC, total PO activities in coelomocytes; LPS, lipopolysaccharide; PolyI:C, polyinosinic–polycytidylic acid; PAH, PO activity in the hemolymph; PBS, phosphate buffered saline; CLS, coelomocyte lysate supernatant; l-DOPA, l-3,4-dihydroxyphenylalanine. ∗ Corresponding author. Tel.: +86 411 84691884; fax: +86 411 84691884. E-mail address: [email protected] (Z. Zhou). http://dx.doi.org/10.1016/j.imbio.2014.02.006 0171-2985/© 2014 Elsevier GmbH. All rights reserved.
extermination, phagocytosis, wound healing, and other immune processes (Cerenius and Söderhäll, 2004; Jiravanichpaisal et al., 2006; Newton et al., 2004; Zhao et al., 2007). In marine invertebrates, POs were mainly found in the hemolymph (Cerenius and Söderhäll, 2004; Jiravanichpaisal et al., 2006; Luna-Gonzalez et al., 2003), and differed notably among different species in biochemical and enzymatic characteristics, which are essential in achieving PO immune functions (Lin et al., 2011; Parrinello et al., 2003; Wright et al., 2012). PO from the scallop Argopecten irradians had optimal activities at pH of 8.0, could catalyze both o-diphenol and p-diphenol substrates, and its activity was greatly inhibited by Cu2+ , Fe2+ and Mn2+ and enhanced by Ca2+ and Mg2+ (Jiang et al., 2011). In the shrimp Penaeus chinensis and the crab Charybdis japonica (Liu et al., 2006; Wang and Fan, 2001), POs both had optimal temperature and pH of 40 ◦ C and 6.0, however, the activity of P. chinensis PO was strongly inhibited by Cu2+ and enhanced by Mg2+ , while the activity of C. japoncia PO was significantly inhibited by Zn2+ and Mg2+ but enhanced by Cu2+ . Moreover, POs from the same species also differed among each other. In the
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clam Ruditapes philippinarum (Cong et al., 2005; Jiang et al., 2012), two POs with different molecular weight were detected, one of which had high affinity to monophenol and o-diphenol substrates, while the other of which could catalyze o-diphenol and p-diphenol substrates. In addition, POs from different species also showed great difference in the immune-responsive characteristics. The stimulation of lipopolysaccharide (LPS) induced a significant increase of PO activity in the hemolymph (PAH) in the clam Chamelea gallina but a dramatic decrease in the clams R. philippinarum and Tapes decussates (Munoz et al., 2006). The infection of white spot syndrome virus resulted in a particularly higher PAH in the shrimp Fenneropenaeus indicus while a lower PAH in the shrimp Litopenaeus vannamei (Sarathi et al., 2007; Yeh et al., 2009). Understanding the biochemical, enzymatic and immuneresponsive characteristics is conducive to the exploration and application of PO immune functions. The sea cucumber Apostichopus japonicus, an important commercial species in Asian countries, is susceptible to several pathogens; however, there exist many research gaps in its PO immune system. Here, we detected and partially purified POs from A. japonicus coelomic fluid, and determined POs for the optimal temperature and pH, the kinetic parameters, the effects of divalent metal ions and inhibitors on enzymatic activities, and the temporal profile of enzymatic activities after immune stimulations. We aim to accumulate essential data on A. japonicus POs for probing deeply into their immune functions.
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fluid was loaded. Molecular weight markers ranging from 21 to 720 kDa (SERVA, Germany) were employed simultaneously. After electrophoresis in Tris–Glycine buffer (0.025 M Tris, 0.2 M Glycine, pH 8.0) at 4 ◦ C for 12 h at 20 mA, one lane of the gel was cut and stained with 1% catechol to locate the PO-containing bands. Based on the staining results, the PO-containing bands in unstained lanes were excised, homogenized in PBS and centrifuged at 4 ◦ C for 30 min at 12,000 × g. The supernatants were collected and treated with centrifugal concentrators (Millipore, USA) for condensing and desalting, and then used as PO solutions for biochemical and enzymatic analysis. PO activity assay and protein assay With dopachromes formation method (Söderhäll, 1981), PO activities were determined as following: 100 l of the sample was added to 2.0 ml of 15 mmol/l l-3,4-dihydroxyphenylalanine (l-DOPA; Sigma, USA) that was dissolved in 100 mmol/l Tris–HCl buffer (pH 7.0), then dopachromes formation in the mixture was determined spectrophotometrically at 490 nm at an interval of 3 min for 30 min, finally the PO activities were calculated based on the increase rate of A490 . An increase of 0.001 (A490 ) per min was defined as 1 unit. The protein concentrations were measured using Bradford method (Bradford, 1976), with bovine serum albumin (Sigma, USA) as protein standard.
Materials and methods Experimental animals and immune challenges One-year old healthy sea cucumbers (with average body weight 10.2 ± 2.7 g) were collected from Dalian, China and kept in seawater aquaria at 15–18 ◦ C, pH 8.1–8.3 and salinity 31‰ for one week before use. Animals used in this research were obtained from commercial sea cucumber catches, therefore approval from any ethics committee or institutional review board was not necessary. The immune challenges were conducted by coelomic injection using LPS (Sigma, USA) and polyinosinic–polycytidylic acid (PolyI:C; Sigma, USA) as stimulants, which were dissolved in phosphate buffered saline (PBS, pH = 7.4) at a concentration of 1 mg/ml and 100 g/ml, respectively. Tested animals were injected with 500 l of each stimulant solution, respectively, and controls were injected with 500 l PBS instead of stimulants, then 10 animals in each group were sampled randomly at 0 h, 4 h, 12 h, 24 h, 48 h and 72 h post-injection. Cell-free coelomic fluid and coelomocyte lysate supernatant preparation With the sterilized syringes, coelomic fluid was withdrawn from the coelom and then centrifuged at 4 ◦ C for 10 min at 700 × g. The supernatant was stored at −20 ◦ C and used as cell-free coelomic fluid. The pellet was suspended in PBS, sonicated, and subsequently centrifuged at 4 ◦ C for 15 min at 12,000 × g. The resulting supernatant was stored at −20 ◦ C and used as the coelomocyte lysate supernatant (CLS). Detection and partial purification of A. japonicus POs Based on the intrinsic function of PO for oxidizing phenol substrates directly in presence of O2 , the detection of PO in the coelomic fluid of A. japonicus was performed by native-PAGE combined with the reaction to catechol, which is a common and specific substrate of invertebrate PO. In brief, the polyacrylamide gel composed of a 5% stacking gel and a 6–27% linear-gradient separating gel was prepared firstly; subsequently, the CLS or cell-free coelomic
Optimal temperature and pH of A. japonicus POs The optimal temperature was measured as following: 100 l of each PO solution was added to 2.0 ml of 15 mmol/l l-DOPA and incubated for 20 min at the range of 5–100 ◦ C with a 5 ◦ C as an interval, then PO activities were determined using dopachrome formation method. The optimal pH was measured by determination of PO activities at different pH. Briefly, 100 l of each PO solution was added to 2.0 ml of 15 mmol/l l-DOPA that was dissolved in 100 mmol/l acetate buffer (pH 3.0, 4.0 5.0, 6.0), Tris–HCl buffer (pH 7.0, 8.0, 9.0) and carbonate–bicarbonate buffer (pH 10.0, 11.0), then PO activities were measured using dopachrome formation method. Kinetic analysis To determine the kinetic parameters, 100 l of each PO solution was added to 2.0 ml of different concentrations of catechol, l-DOPA, dopamine, hydroquinone and tyrosine (Sigma, USA) that were dissolved in 100 mmol/l Tris–HCl buffer (pH 7.0), then PO activities were measured using dopachrome formation method. Finally, based on the relationships between substrate concentrations and PO activities, kinetic parameters were calculated using Lineweaver–Burk-plot method (Atkins and Nimmo, 1975). Effects of divalent metal ions on the activities of A. japonicus POs With Ca2+ , Mg2+ , Fe2+ , Zn2+ , Cu2+ , Mn2+ , Pb2+ and Cd2+ from CaCl2 , MgSO4 , FeCl2 , ZnSO4 , CuSO4 , MnCl2 , Pb(CH3 COO)2 and CdCl2 (Songon, China) as divalent metal ions, the variation profiles of PO activities were determined by incubation of 100 l of PO solution with 100 l of divalent metal ions at different concentrations dissolved in 100 mmol/l Tris–HCl buffer (pH 7.0) at 4 ◦ C for 20 min, then 1.9 ml of 15 mmol/l l-DOPA was added, and PO activities were measured using dopachrome formation method. Each PO solution was adjusted to 100 U/ml. Controls were performed by replacement of divalent metal ion solutions with 100 mmol/l Tris–HCl buffer (pH 7.0).
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Effects of inhibitors on the activities of A. japonicus POs With ethylenediaminetetraacetic acid disodium (EDTA), sodium diethyldithiocarbamate (DETC), ascorbic acid, citric acid and sodium sulfite (Songon, China) as PO inhibitors, the variation profiles of PO activities were determined by incubation of 100 l of PO solution with 100 l of 5 mmol/l PO inhibitors that were dissolved in 100 mmol/l Tris–HCl buffer (pH 7.0) at 4 ◦ C for 20 min, then 1.9 ml of 15 mmol/l l-DOPA was added, and PO activities were measured using dopachrome formation method. Each PO solution was adjusted to 100 U/ml. Controls were performed by replacement of inhibitor solutions with 100 mmol/l Tris–HCl buffer (pH 7.0). Temporal variation of total PO activities in coelomocytes (TPAC) post immune stimulations To determine the variation profile of TPAC after immune stimulations, 100 l of CLS obtained from the LPS, PolyI:C and PBS-treated sea cucumbers at different time post injection was added to 2.0 ml of 15 mmol/l l-DOPA that was dissolved in 100 mmol/l Tris–HCl buffer (pH 7.0), respectively. PO activities were measured spectrophotometrically at 490 nm. Statistical analysis
Fig. 1. Detection of POs from A. japonicus fluid. After linear-gradient native-PAGE, the gel containing lane 1 to lane 3 was stained with Coomassie brilliant blue R-250, while the gel containing lane 4 and lane 5 was stained with 1% catechol. Lane 1: marker of protein; lane 2 and lane 4: A. japonicus CLS; lane 3 and lane 5: A. japonicus cell-free coelomic fluid.
The experiments of biochemical and immune-responsive characterization were performed in triplicate, and the data from all sub-sections in Materials and Methods were expressed as the mean ± standard deviation. Statistical analysis was carried out using SPSS 11.5 (SPSS, USA). Differences in PO activities between immune stimulants-treated groups and PBS-treated groups were analyzed by one-way ANOVA. The level of significance was defined as p < 0.05.
Optimal temperature and pH of A. japonicus POs
Results
All of the substrates selected in this study except tyrosine could be catalyzed by the three A. japonicus POs. Calculated by the Lineweaver–Burk model, Km values of AjPO1 for catechol, l-DOPA, dopamine and hydroquinone were 3.23, 0.86, 3.98 and 1.20 mmol/l, respectively (Fig. 3A), those of AjPO2 were 0.31, 0.38, 2.05 and 1.30 mmol/l, respectively (Fig. 3B), and those of AjPO3 were 5.95, 1.28, 5.81 and 0.62 mmol/l, respectively (Fig. 3C).
Detection of A. japonicus POs The results of linear-gradient native-PAGE revealed that CLS or cell-free coelomic fluid had lots of protein bands, among which totally three bands with PO activity were detected using catechol staining. The three PO bands were identified by a mahogany color, a yellow color and a purple color after reacting with catechol and the corresponding POs were named as AjPO1, AjPO2 and AjPO3, respectively. However, all three A. japonicus POs had very small molecular masses which could not be measured accurately using the present molecular weight markers (Fig. 1).
With l-DOPA as a substrate, AjPO1, AjPO2 and AjPO3 showed activities at the temperature range of 5–100 ◦ C and pH range of 3.0–11.0, and had optimal activities at temperature of 45, 95 and 85 ◦ C and pH of 5.0, 8.0 and 8.0, respectively (Fig. 2). Kinetic analysis
Effects of metal ions on the activities of A. japonicus POs With l-DOPA as a substrate, the effects of divalent metal ions on the activities of A. japonicus POs were shown in Fig. 4. For all three POs, Ca2+ , Mg2+ and Mn2+ stimulated PO activity at all of the
Fig. 2. The optimal temperature and pH of A. japonicus POs. The PO activities were measured using l-DOPA (15 mmol/l) as a substrate. (A) Temperature; and (B) pH.
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Fig. 3. Kinetic parameters of A. japonicus POs. The PO activities were measured using catechol, l-DOPA, dopamine and hydroquinone as substrates and Km values were calculated based on the Lineweaver–Burk model. (A) AjPO1; (B) AjPO2; and (C) AjPO3.
determined concentrations. Fe2+ inhibited PO activity at concentration of 2.5 mmol/l and enhanced PO activity at 30.0 mmol/l, which was opposite to what Zn2+ did. Cu2+ , Pb2+ , Cd2+ enhanced PO activity at the concentration of 2.5 mmol/l.
Effects of inhibitors on the activities of A. japonicus POs With l-DOPA as a substrate, the effects of PO inhibitors on the activities of A. japonicus POs (Fig. 5) showed that, at a concentration of 5 mmol/l, EDTA, DETC, ascorbic acid, citric acid and sodium sulfite showed about 73.5%, 93.4%, 92.2%, 80.7% and 86.1% inhibitions to AjPO1, respectively, 71.9%, 96.2%, 96.2%, 86% and 84.7% inhibitions to AjPO2, respectively, and 57.4%, 100%, 100%, 59.6% and 100% inhibitions to AjPO3, respectively.
Temporal variation of TPAC post immune stimulations With l-DOPA as a substrate, the effects of challenging with LPS or PolyI:C on TPAC were shown in Fig. 6. In PBS group, the TPAC showed no significant difference among the six sampling times by comparing with each other. Comparing with PBS group, the TPAC in LPS group increased significantly at 12 and 24 h, and then returned to control level at 48 and 72 h; the TPAC in PolyI:C group decreased significantly at 4 and 12 h, then returned to the control level at 24 h, and finally dropped to less than half of control values at 48 and 72 h. Discussion Different POs have different biochemical and immuneresponsive characteristics (Lin et al., 2011; Parrinello et al., 2003;
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Fig. 4. The variation profile of A. japonicus PO activities after treatment with six metal ions. The PO activities were measured using l-DOPA (15 mmol/l) as a substrate. (A) AjPO1; (B) AjPO2; and (C) AjPO3.
Wright et al., 2012), based on which, PO functions as an important immune and physiological factor in organisms. Here, we detected and partially purified POs from the coelomic fluid of A. japonicus, and characterized POs based on the optimal temperature and pH, the kinetic parameters, the effects of divalent metal ions and inhibitors on enzymatic activities, and the temporal profile of TPAC after immune stimulations. Using native-PAGE combined with catechol staining, we detected three POs (AjPO1, AjPO2 and AjPO3) in coelomic fluid, suggesting that A. japonicus PO is composed of several isoenzymes. This phenomenon was found in many marine invertebrates such as the scallop Chlamys farreri and the clam R. philippinarum, as well as sea cucumber A. japonicus detected here (Cong et al., 2005; Jiang et al., 2012; Xing et al., 2012; Zhou et al., 2012). However, as far as molecular mass was concerned, A. japonicus POs were much smaller than those reported in R. philippinarum (563 kDa), A. irradians (555 kDa), the oyster Saccostrea glomerata (219 kDa)
and the spiny lobster Panulirus argus (300 kDa) (Aladaileh et al., 2007; Jiang et al., 2011, 2012; Perdomo-Morales et al., 2007). The functional differentiation of PO during evolution might be one of the most important factors that caused a significant interspecific difference of PO molecular mass. Specifically, besides the capacity of phenol oxidization, PO performs other physiological functions, and the multiple functions of PO which were called “moonlighting” (Sharma and Kuhad, 2008) are different among species. For example, PO in Cryptococcus neoformans is simultaneously provided with different activities of iron oxidase, endospore coat protein oxidase and polyphenol oxidase, and has a molecular mass of 66 kDa (Williamson, 1994), while PO in Scapharca kagoshimensis functions as an oxygen carrier and a polyphenol oxidase at the same time and has a molecular mass of only 28.97 kDa (Zhang et al., 2009). However, the information about multiple functions of PO is still limited, especially in marine invertebrates, and the relationship between PO molecular mass and its multiple functions
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Fig. 5. The variation profile of A. japonicus PO activities after treatment with inhibitors. The PO activities were measured using l-DOPA (15 mmol/l) as a substrate.
needs to be further studied. The optimal temperature of AjPO1 was similar to that of Penaeus setiferus PO (45 ◦ C), C. farreri PO (45 ◦ C) and C. japonica PO (40 ◦ C), while the optimal temperatures of AjPO2 and AjPO3 were much higher than that of AjPO1 and most reported marine invertebrate POs, and similar to that of thermophilic fungi POs like Scytalidium thermophilum PO (80 ◦ C) (Liu et al., 2006; Simpson et al., 1987; Sun and Li, 1999; Younes and Sayadi, 2011). The optimal pH of AjPO1 also differed greatly from that of AjPO2 and AjPO3. The temperature and pH tests on A. japonicus POs implied that AjPO1 might differ greatly from AjPO2 and AjPO3 in protein structures. In addition, all three A. japonicus POs kept high activities at low pH (3.0, 4.0 and 5.0), but activities decreased dramatically when pH was higher than 9.0, suggesting that compared with acidic environment, alkaline environment (pH > 9.0) is more harmful to the A. japonicus PO immune system. Based on the substrate specificity, invertebrate POs were classified into three types (Barret, 1987): tyrosinase type (E.C.1.14.18.1 monophenol, l-DOPA: O2 oxidoreductase), catechol oxidase type
Fig. 6. The temporal profile of A. japonicus TPAC (total PO activities in coelomocytes) after immune stimulations. The PO activities were measured using l-DOPA (15 mmol/l) as a substrate. Values were shown as means ± standard deviation (n = 3). Significant differences compared with controls were indicated with an asterisk “*” at p < 0.05.
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(E.C.1.10.3.1 diphenol: O2 oxidoreductase) and laccase type (E.C.1.10.3.2; p-diphenol: O2 oxidoreductase. The oxidizing reactions of all three A. japonicus POs with catechol (o-diphenol), l-DOPA (o-diphenol), dopamine (o-diphenol) and hydroquinone except tyrosine (monophenol) imply that these enzymes are laccase-type POs, similar to POs in Crassostrea gigas, Crassostrea virginica, C. farreri, A. irradians, and R. philippinarum (Jiang et al., 2011, 2012; Jordan and Deaton, 2005; Luna-Acosta et al., 2011; Xing et al., 2012). In addition, the Km values indicated that AjPO1, AjPO2 and AjPO3 had the highest affinity to l-DOPA, catechol and hydroquinone respectively, which suggested that the active sites of three A. japonicus POs might be different. All of the eight metal ions tested in this study showed no regular trends during the experiments, indicating that their effects on PO activities were dose-independent. The activities of all three A. japonicus POs were strongly increased in the presence of Ca2+ , Mg2+ and Mn2+ , which suggested that the three metal ions played positive roles during the catalysis of A. japonicus POs and may be employed as potential immunopotentiators at appropriate concentrations. Actually, Ca2+ and Mg2+ are always involved in enzymes activation, and their activation of PO has been reported in several invertebrates including the moth Galleria mellonella, the pyralid Ostrinia furnacalis, the cockroach Blaberus craniifer, the silkworm Bombyx mori, the bay scallop A. irradians, etc. (Ashida et al., 1983; Dunphy, 1991; Feng et al., 2008; Jiang et al., 2011; Leonard et al., 1985). In addition, Fe2+ and Zn2+ had opposite effects at a high concentration (30 mmol/l) and a low concentration (2.5 mmol/l) in all three A. japonicus POs, indicating that Fe2+ and Zn2+ may play opposite roles during the catalysis of A. japonicus POs. Overall, the effects of eight metal ions on enzymatic activities had some difference among AjPO1, AjPO2 and AjPO3. The mechanism of metal ions affecting PO activity is still not clear. Some studies speculated that metal ions modulate PO activity by means of activating electrophile and nucleophile or changing the secondary structure of certain peptides of PO (Feng et al., 2008; Zibaee et al., 2010), however, the irregular variation trends of PO activities after treatment with divalent metal ions cannot be explained clearly by these hypothetical mechanisms, which drive us to investigate the detailed mechanism in further studies. The common antioxidants including ascorbic acid, citric acid and sodium sulfite each showed strong inhibition to the activities of three A. japonicus POs. Besides that, the divalent cation chelator – EDTA and the specific copper chelator – DETC also inhibited the activities of three A. japonicus POs greatly, implying that AjPO1, AjPO2 and AjPO3 obtained in this study were all copper-containing metalloenzymes, similar to the POs in other marine invertebrates like A. irradians, R. philippinarum, S. glomerata, C. japonica and ascidians Styela plicata, Phallusia mammillata and Ciona intestinalis (Aladaileh et al., 2007; Cong et al., 2005; Jiang et al., 2011; Liu et al., 2006; Parrinello et al., 2003). The temporal profile of A. japonicus TPAC indicated that A. japonicus PO immune system, composed of several isoenzymes with different characteristics, was very sensitive to the immune stimulations. The significant increase of TPAC at 12 h and 24 h post injection of LPS, which represent Gram-negative bacteria, is similar to results in C. gallina and C. gigas (Hellio et al., 2007; Munoz et al., 2006), suggesting that A. japonicus PO immune system is efficient in the response against Gram-negative bacteria challenge. However, the significant decline of TPAC after injection of PolyI:C, which represent double-stranded RNA viruses, revealed that A. japonicus PO immune system might suffer a depression from the infection of double-stranded RNA viruses. In the present study, we reported the detection, partial purification, biochemical characterization and immune-responsive analysis of POs from A. japonicus. The isolated PO isoenzymes showed clear difference in biochemical characteristics, and as a
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whole, were closely involved in the response against immune challenge. However, the difference among A. japonicus PO isoenzymes in immune and physiological process is not clear and further study on gene cloning and monoclonal antibody production to investigate the functional differentiation of A. japonicus POs is expected. Conflict of interest statement None declared. Acknowledgements This work was supported by grants from National Nature Science Foundation of China (31272687), State 863 High-Technology R&D Project of China (2012AA10A412), Science and Technology Project of Liaoning Province (2011203005), and Doctoral Startup Foundation of Liaoning Province (20111072). The funders had no role in study design, data analysis, decision to publish, or preparation of the manuscript. References Aladaileh, S., Rodney, P., Nair, S.V., Raftos, D.A., 2007. Characterization of phenoloxidase activity in Sydney rock oysters (Saccostrea glomerata). Comp. Biochem. Physiol. B 148, 470–480. Ashida, M., Ishizaki, Y., Iwahana, H., 1983. Activation of pro-phenoloxidase by bacterial cell walls or beta-1,3-glucans in plasma of the silkworm, Bombyx mori. Biochem. Biophys. Res. Commun. 113, 562–568. Asokan, R., Arumugam, M., Mullainadhan, P., 1997. Activation of prophenoloxidase in the plasma and haemocytes of the marine mussel, Perna viridis Linnaeus. Dev. Comp. Immunol. 52, 1–12. Aspán, A., Huang, T.S., Cerenius, L., Söderhäll, K., 1995. cDNA cloning of prophenoloxidase from the freshwater crayfish Pacifastacus leniusculus and its activation. Proc. Natl. Acad. Sci. U. S. A. 92, 939–943. Atkins, G.L., Nimmo, I.A., 1975. A comparison of seven methods for fitting the Michaelis–Menten equation. Biochem. J. 149, 775–777. Barret, F.M., 1987. Phenoloxidases from larval cuticle of the sheep blowfly, Lucilia cuprina: characterization, developmental changes, and inhibition by antiphenoloxidase antibodies. Arch. Insect Biochem. Physiol. 5, 99–118. Bradford, M., 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of dye binding. Anal. Biochem. 72, 248–254. Cerenius, L., Söderhäll, K., 2004. The prophenoloxidase-activating system in invertebrates. Immunol. Rev. 198, 116–126. Cong, R.S., Sun, W.J., Liu, G.X., Fan, T.J., Meng, X.H., Yang, L.L., Zhu, L.Y., 2005. Purification and characterization of phenoloxidase from clam Ruditapes philippinarum. Fish Shellfish Immunol. 18, 61–70. Dunphy, G.B., 1991. Phenoloxidase activity in the serum of two species of insects, the gypsy moth, Lymantria dispar (Lymantriidae) and the greater wax moth, Galleria mellonella (Pyralidae). Comp. Biochem. Physiol. B 98, 535–538. Feng, C.J., Song, Q.S., Lü, W.J., Lu, J.F., 2008. Purification and characterization of hemolymph prophenoloxidase from Ostrinia furnacalis (Lepidoptera: Pyralidae) larvae. Comp. Biochem. Physiol. B 151, 139–146. Hellio, C., Bado-Nilles, A., Gagnaire, B., Renault, T., Thomas-Guyon, H., 2007. Demonstration of a true phenoloxidase activity and activation of a ProPO cascade in Pacific oyster, Crassostrea gigas (Thunberg) in vitro. Fish Shellfish Immunol. 22, 433–440. Jiang, J.W., Xing, J., Sheng, X.J., Zhan, W.B., 2011. Characterization of phenoloxidase from the bay scallop Argopecten irradians. J. Shellfish Res. 30, 273–277. Jiang, J.W., Xing, J., Zhan, W.B., 2012. Purification and characterization of laccase-type phenoloxidase from the clam Ruditapes philippinarum. Oceanol. Limnol. Sin. 43, 294–298. Jiravanichpaisal, P., Lee, B.L., Söderhäll, K., 2006. Cell-mediated immunity in arthropods: hematopoiesis, coagulation, melanization and opsonization. Immunobiology 211, 213–236. Jordan, P.J., Deaton, L.E., 2005. Characterization of phenoloxidase from Crassostrea virginica hemocytes and the effect of Perkinsus marinus on phenoloxidase activity in the hemolymph of Crassostrea virginica and Geukensia demissa. J. Shellfish Res. 24, 477–482.
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Fungal cell wall -1,3-glucans induce clotting and phenoloxidase attachment to foreign surfaces of crayfish haemocyte lysate. Dev. Comp. Immunol. 5, 565–573. Sun, H.S., Li, G.Y., 1999. Phenoloxidase and myeloperoxidase activity in the haemocytes and serum of Chlamys farreri. J. Fish. Sci. China 6, 9–13. Wang, X.F., Fan, T.J., 2001. Some biochemical specificity studies of phenoloxidase from Penaeus chinensis. Mar. Sci. 27, 71–75. Williamson, P.R., 1994. Biochemical and molecular characterization of the diphenol oxidase of Cryptococcus neoformans: identification as a laccase. J. Bacteriol. 176, 656–664. Wright, J., Clark, W.M., Cain, J.A., Patterson, A., Coates, C.J., Nairn, J., 2012. Effects of known phenoloxidase inhibitors on hemocyanin-derived phenoloxidase from Limulus polyphemus. Comp. Biochem. Physiol. B 163, 303–308. Wu, C.L., Charoensapsri, W., Nakamura, S., Tassanakajon, A., Söderhäll, I., Söderhäll, K., 2013. An MBL-like protein may interfere with the activation of the proPOsystem, an important innate immune reaction in invertebrates. Immunobiology 218, 159–168. Xing, J., Jiang, J.W., Zhan, W.B., 2012. Phenoloxidase in the scallop Chlamys farreri: purification and antibacterial activity of its reaction products generated in vitro. Fish Shellfish Immunol. 32, 89–93. Yeh, S.P., Chen, Y.N., Hsieh, S.L., Cheng, W., Liu, C.H., 2009. Immune response of white shrimp, Litopenaeus vannamei, after a concurrent infection with white spot syndrome virus and infectious hypodermal and hematopoietic necrosis virus. Fish Shellfish Immunol. 26, 582–588. Younes, S.B., Sayadi, S., 2011. Purification and characterization of a novel trimeric and thermotolerant laccase produced from the ascomycete Scytalidium thermophilum strain. J. Mol. Catal. B 73, 35–42. Zhang, Y.N., Fan, T.J., Xu, B., Jing, Z., 2009. Research advances in animal respiratory proteins. J. Shandong Univ. (Nat. Sci.) 44, 1–7. 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Characterization of phenoloxidase from the sea cucumber Apostichopus japonicus Jingwei Jiang, Zunchun Zhou ∗ , Ying Dong, Xiaoyan Guan, Bai Wang, Bei Jiang, Aifu Yang, Zhong Chen, Shan Gao, Hongjuan Sun Liaoning Key Laboratory of Marine Fishery Molecular Biology, Liaoning Ocean and Fisheries Science Research Institute, Dalian, Liaoning 116023, PR China
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Article history: Received 14 December 2013 Received in revised form 13 February 2014 Accepted 16 February 2014 Available online 25 February 2014 Keywords: Biochemical characteristics Immune-responsive characteristics Metalloenzyme Phenoloxidase Sea cucumber (Apostichopus japonicus)
a b s t r a c t Phenoloxidase (PO) is a crucial immune-related enzyme in invertebrates. In this study, three POs of the sea cucumber Apostichopus japonicus were detected in coelomic fluid using linear-gradient native-PAGE combined with catechol staining and then partially purified by gel excising. The results showed that the three POs had a color of mahogany (AjPO1), yellow (AjPO2) and purple (AjPO3) respectively with molecular weights smaller than 21 kDa in native-PAGE after staining with catechol. Enzymatic activities analysis revealed that AjPO1, AjPO2 and AjPO3 had optimal temperature of 45, 95 and 85 ◦ C and pH of 5.0, 8.0 and 8.0, respectively. Kinetic analysis showed that the Km values of AjPO1 for catechol, l-DOPA, dopamine and hydroquinone were 3.23, 0.86, 3.98 and 1.20 mmol/l, respectively, those of AjPO2 were 0.31, 0.38, 2.05 and 1.30 mmol/l, respectively, and those of AjPO3 were 5.95, 1.28, 5.81 and 0.62 mmol/l, respectively. These results suggest that the three POs are laccase-type phenoloxidase. The activities of all three A. japonicus POs were significantly promoted by Ca2+ , Mg2+ and Mn2+ , and strongly inhibited by ethylenediamine tetraacetic acid disodium (EDTA), sodium diethyldithiocarbamate (DETC) and some common antioxidants. The inhibitions by EDTA and DETC suggest that the three A. japonicus POs are copper-containing metalloenzymes. Immune-responsive analysis showed that the total PO activities in coelomocytes (TPAC) increased greatly after lipopolysaccharide (LPS) challenge and declined significantly after polyinosinic–polycytidylic acid (PolyI:C) challenge, implying that A. japonicus PO immune system, which is composed of several isoenzymes with different characteristics, is closely involved in the defense against the infection of Gram-negative bacteria and double-stranded RNA viruses. © 2014 Elsevier GmbH. All rights reserved.
Introduction Phenoloxidase (PO), a common name of proteins that can oxidize phenols directly in presence of O2 , is one of the critical components of the immune system in invertebrates. PO catalyzes phenol substrates into quinones, which are transformed into melanin following a non-enzymatic pathway (Aladaileh et al., 2007; Asokan et al., 1997; Aspán et al., 1995; Pang et al., 2010; Wu et al., 2013). The melanin and intermediate metabolites generated from the non-enzymatic reactions are involved in pathogen
Abbreviations: PO, phenoloxidase; EDTA, ethylenediamine tetraacetic acid disodium; DETC, sodium diethyldithiocarbamate; TPAC, total PO activities in coelomocytes; LPS, lipopolysaccharide; PolyI:C, polyinosinic–polycytidylic acid; PAH, PO activity in the hemolymph; PBS, phosphate buffered saline; CLS, coelomocyte lysate supernatant; l-DOPA, l-3,4-dihydroxyphenylalanine. ∗ Corresponding author. Tel.: +86 411 84691884; fax: +86 411 84691884. E-mail address: [email protected] (Z. Zhou). http://dx.doi.org/10.1016/j.imbio.2014.02.006 0171-2985/© 2014 Elsevier GmbH. All rights reserved.
extermination, phagocytosis, wound healing, and other immune processes (Cerenius and Söderhäll, 2004; Jiravanichpaisal et al., 2006; Newton et al., 2004; Zhao et al., 2007). In marine invertebrates, POs were mainly found in the hemolymph (Cerenius and Söderhäll, 2004; Jiravanichpaisal et al., 2006; Luna-Gonzalez et al., 2003), and differed notably among different species in biochemical and enzymatic characteristics, which are essential in achieving PO immune functions (Lin et al., 2011; Parrinello et al., 2003; Wright et al., 2012). PO from the scallop Argopecten irradians had optimal activities at pH of 8.0, could catalyze both o-diphenol and p-diphenol substrates, and its activity was greatly inhibited by Cu2+ , Fe2+ and Mn2+ and enhanced by Ca2+ and Mg2+ (Jiang et al., 2011). In the shrimp Penaeus chinensis and the crab Charybdis japonica (Liu et al., 2006; Wang and Fan, 2001), POs both had optimal temperature and pH of 40 ◦ C and 6.0, however, the activity of P. chinensis PO was strongly inhibited by Cu2+ and enhanced by Mg2+ , while the activity of C. japoncia PO was significantly inhibited by Zn2+ and Mg2+ but enhanced by Cu2+ . Moreover, POs from the same species also differed among each other. In the
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clam Ruditapes philippinarum (Cong et al., 2005; Jiang et al., 2012), two POs with different molecular weight were detected, one of which had high affinity to monophenol and o-diphenol substrates, while the other of which could catalyze o-diphenol and p-diphenol substrates. In addition, POs from different species also showed great difference in the immune-responsive characteristics. The stimulation of lipopolysaccharide (LPS) induced a significant increase of PO activity in the hemolymph (PAH) in the clam Chamelea gallina but a dramatic decrease in the clams R. philippinarum and Tapes decussates (Munoz et al., 2006). The infection of white spot syndrome virus resulted in a particularly higher PAH in the shrimp Fenneropenaeus indicus while a lower PAH in the shrimp Litopenaeus vannamei (Sarathi et al., 2007; Yeh et al., 2009). Understanding the biochemical, enzymatic and immuneresponsive characteristics is conducive to the exploration and application of PO immune functions. The sea cucumber Apostichopus japonicus, an important commercial species in Asian countries, is susceptible to several pathogens; however, there exist many research gaps in its PO immune system. Here, we detected and partially purified POs from A. japonicus coelomic fluid, and determined POs for the optimal temperature and pH, the kinetic parameters, the effects of divalent metal ions and inhibitors on enzymatic activities, and the temporal profile of enzymatic activities after immune stimulations. We aim to accumulate essential data on A. japonicus POs for probing deeply into their immune functions.
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fluid was loaded. Molecular weight markers ranging from 21 to 720 kDa (SERVA, Germany) were employed simultaneously. After electrophoresis in Tris–Glycine buffer (0.025 M Tris, 0.2 M Glycine, pH 8.0) at 4 ◦ C for 12 h at 20 mA, one lane of the gel was cut and stained with 1% catechol to locate the PO-containing bands. Based on the staining results, the PO-containing bands in unstained lanes were excised, homogenized in PBS and centrifuged at 4 ◦ C for 30 min at 12,000 × g. The supernatants were collected and treated with centrifugal concentrators (Millipore, USA) for condensing and desalting, and then used as PO solutions for biochemical and enzymatic analysis. PO activity assay and protein assay With dopachromes formation method (Söderhäll, 1981), PO activities were determined as following: 100 l of the sample was added to 2.0 ml of 15 mmol/l l-3,4-dihydroxyphenylalanine (l-DOPA; Sigma, USA) that was dissolved in 100 mmol/l Tris–HCl buffer (pH 7.0), then dopachromes formation in the mixture was determined spectrophotometrically at 490 nm at an interval of 3 min for 30 min, finally the PO activities were calculated based on the increase rate of A490 . An increase of 0.001 (A490 ) per min was defined as 1 unit. The protein concentrations were measured using Bradford method (Bradford, 1976), with bovine serum albumin (Sigma, USA) as protein standard.
Materials and methods Experimental animals and immune challenges One-year old healthy sea cucumbers (with average body weight 10.2 ± 2.7 g) were collected from Dalian, China and kept in seawater aquaria at 15–18 ◦ C, pH 8.1–8.3 and salinity 31‰ for one week before use. Animals used in this research were obtained from commercial sea cucumber catches, therefore approval from any ethics committee or institutional review board was not necessary. The immune challenges were conducted by coelomic injection using LPS (Sigma, USA) and polyinosinic–polycytidylic acid (PolyI:C; Sigma, USA) as stimulants, which were dissolved in phosphate buffered saline (PBS, pH = 7.4) at a concentration of 1 mg/ml and 100 g/ml, respectively. Tested animals were injected with 500 l of each stimulant solution, respectively, and controls were injected with 500 l PBS instead of stimulants, then 10 animals in each group were sampled randomly at 0 h, 4 h, 12 h, 24 h, 48 h and 72 h post-injection. Cell-free coelomic fluid and coelomocyte lysate supernatant preparation With the sterilized syringes, coelomic fluid was withdrawn from the coelom and then centrifuged at 4 ◦ C for 10 min at 700 × g. The supernatant was stored at −20 ◦ C and used as cell-free coelomic fluid. The pellet was suspended in PBS, sonicated, and subsequently centrifuged at 4 ◦ C for 15 min at 12,000 × g. The resulting supernatant was stored at −20 ◦ C and used as the coelomocyte lysate supernatant (CLS). Detection and partial purification of A. japonicus POs Based on the intrinsic function of PO for oxidizing phenol substrates directly in presence of O2 , the detection of PO in the coelomic fluid of A. japonicus was performed by native-PAGE combined with the reaction to catechol, which is a common and specific substrate of invertebrate PO. In brief, the polyacrylamide gel composed of a 5% stacking gel and a 6–27% linear-gradient separating gel was prepared firstly; subsequently, the CLS or cell-free coelomic
Optimal temperature and pH of A. japonicus POs The optimal temperature was measured as following: 100 l of each PO solution was added to 2.0 ml of 15 mmol/l l-DOPA and incubated for 20 min at the range of 5–100 ◦ C with a 5 ◦ C as an interval, then PO activities were determined using dopachrome formation method. The optimal pH was measured by determination of PO activities at different pH. Briefly, 100 l of each PO solution was added to 2.0 ml of 15 mmol/l l-DOPA that was dissolved in 100 mmol/l acetate buffer (pH 3.0, 4.0 5.0, 6.0), Tris–HCl buffer (pH 7.0, 8.0, 9.0) and carbonate–bicarbonate buffer (pH 10.0, 11.0), then PO activities were measured using dopachrome formation method. Kinetic analysis To determine the kinetic parameters, 100 l of each PO solution was added to 2.0 ml of different concentrations of catechol, l-DOPA, dopamine, hydroquinone and tyrosine (Sigma, USA) that were dissolved in 100 mmol/l Tris–HCl buffer (pH 7.0), then PO activities were measured using dopachrome formation method. Finally, based on the relationships between substrate concentrations and PO activities, kinetic parameters were calculated using Lineweaver–Burk-plot method (Atkins and Nimmo, 1975). Effects of divalent metal ions on the activities of A. japonicus POs With Ca2+ , Mg2+ , Fe2+ , Zn2+ , Cu2+ , Mn2+ , Pb2+ and Cd2+ from CaCl2 , MgSO4 , FeCl2 , ZnSO4 , CuSO4 , MnCl2 , Pb(CH3 COO)2 and CdCl2 (Songon, China) as divalent metal ions, the variation profiles of PO activities were determined by incubation of 100 l of PO solution with 100 l of divalent metal ions at different concentrations dissolved in 100 mmol/l Tris–HCl buffer (pH 7.0) at 4 ◦ C for 20 min, then 1.9 ml of 15 mmol/l l-DOPA was added, and PO activities were measured using dopachrome formation method. Each PO solution was adjusted to 100 U/ml. Controls were performed by replacement of divalent metal ion solutions with 100 mmol/l Tris–HCl buffer (pH 7.0).
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Effects of inhibitors on the activities of A. japonicus POs With ethylenediaminetetraacetic acid disodium (EDTA), sodium diethyldithiocarbamate (DETC), ascorbic acid, citric acid and sodium sulfite (Songon, China) as PO inhibitors, the variation profiles of PO activities were determined by incubation of 100 l of PO solution with 100 l of 5 mmol/l PO inhibitors that were dissolved in 100 mmol/l Tris–HCl buffer (pH 7.0) at 4 ◦ C for 20 min, then 1.9 ml of 15 mmol/l l-DOPA was added, and PO activities were measured using dopachrome formation method. Each PO solution was adjusted to 100 U/ml. Controls were performed by replacement of inhibitor solutions with 100 mmol/l Tris–HCl buffer (pH 7.0). Temporal variation of total PO activities in coelomocytes (TPAC) post immune stimulations To determine the variation profile of TPAC after immune stimulations, 100 l of CLS obtained from the LPS, PolyI:C and PBS-treated sea cucumbers at different time post injection was added to 2.0 ml of 15 mmol/l l-DOPA that was dissolved in 100 mmol/l Tris–HCl buffer (pH 7.0), respectively. PO activities were measured spectrophotometrically at 490 nm. Statistical analysis
Fig. 1. Detection of POs from A. japonicus fluid. After linear-gradient native-PAGE, the gel containing lane 1 to lane 3 was stained with Coomassie brilliant blue R-250, while the gel containing lane 4 and lane 5 was stained with 1% catechol. Lane 1: marker of protein; lane 2 and lane 4: A. japonicus CLS; lane 3 and lane 5: A. japonicus cell-free coelomic fluid.
The experiments of biochemical and immune-responsive characterization were performed in triplicate, and the data from all sub-sections in Materials and Methods were expressed as the mean ± standard deviation. Statistical analysis was carried out using SPSS 11.5 (SPSS, USA). Differences in PO activities between immune stimulants-treated groups and PBS-treated groups were analyzed by one-way ANOVA. The level of significance was defined as p < 0.05.
Optimal temperature and pH of A. japonicus POs
Results
All of the substrates selected in this study except tyrosine could be catalyzed by the three A. japonicus POs. Calculated by the Lineweaver–Burk model, Km values of AjPO1 for catechol, l-DOPA, dopamine and hydroquinone were 3.23, 0.86, 3.98 and 1.20 mmol/l, respectively (Fig. 3A), those of AjPO2 were 0.31, 0.38, 2.05 and 1.30 mmol/l, respectively (Fig. 3B), and those of AjPO3 were 5.95, 1.28, 5.81 and 0.62 mmol/l, respectively (Fig. 3C).
Detection of A. japonicus POs The results of linear-gradient native-PAGE revealed that CLS or cell-free coelomic fluid had lots of protein bands, among which totally three bands with PO activity were detected using catechol staining. The three PO bands were identified by a mahogany color, a yellow color and a purple color after reacting with catechol and the corresponding POs were named as AjPO1, AjPO2 and AjPO3, respectively. However, all three A. japonicus POs had very small molecular masses which could not be measured accurately using the present molecular weight markers (Fig. 1).
With l-DOPA as a substrate, AjPO1, AjPO2 and AjPO3 showed activities at the temperature range of 5–100 ◦ C and pH range of 3.0–11.0, and had optimal activities at temperature of 45, 95 and 85 ◦ C and pH of 5.0, 8.0 and 8.0, respectively (Fig. 2). Kinetic analysis
Effects of metal ions on the activities of A. japonicus POs With l-DOPA as a substrate, the effects of divalent metal ions on the activities of A. japonicus POs were shown in Fig. 4. For all three POs, Ca2+ , Mg2+ and Mn2+ stimulated PO activity at all of the
Fig. 2. The optimal temperature and pH of A. japonicus POs. The PO activities were measured using l-DOPA (15 mmol/l) as a substrate. (A) Temperature; and (B) pH.
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Fig. 3. Kinetic parameters of A. japonicus POs. The PO activities were measured using catechol, l-DOPA, dopamine and hydroquinone as substrates and Km values were calculated based on the Lineweaver–Burk model. (A) AjPO1; (B) AjPO2; and (C) AjPO3.
determined concentrations. Fe2+ inhibited PO activity at concentration of 2.5 mmol/l and enhanced PO activity at 30.0 mmol/l, which was opposite to what Zn2+ did. Cu2+ , Pb2+ , Cd2+ enhanced PO activity at the concentration of 2.5 mmol/l.
Effects of inhibitors on the activities of A. japonicus POs With l-DOPA as a substrate, the effects of PO inhibitors on the activities of A. japonicus POs (Fig. 5) showed that, at a concentration of 5 mmol/l, EDTA, DETC, ascorbic acid, citric acid and sodium sulfite showed about 73.5%, 93.4%, 92.2%, 80.7% and 86.1% inhibitions to AjPO1, respectively, 71.9%, 96.2%, 96.2%, 86% and 84.7% inhibitions to AjPO2, respectively, and 57.4%, 100%, 100%, 59.6% and 100% inhibitions to AjPO3, respectively.
Temporal variation of TPAC post immune stimulations With l-DOPA as a substrate, the effects of challenging with LPS or PolyI:C on TPAC were shown in Fig. 6. In PBS group, the TPAC showed no significant difference among the six sampling times by comparing with each other. Comparing with PBS group, the TPAC in LPS group increased significantly at 12 and 24 h, and then returned to control level at 48 and 72 h; the TPAC in PolyI:C group decreased significantly at 4 and 12 h, then returned to the control level at 24 h, and finally dropped to less than half of control values at 48 and 72 h. Discussion Different POs have different biochemical and immuneresponsive characteristics (Lin et al., 2011; Parrinello et al., 2003;
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Fig. 4. The variation profile of A. japonicus PO activities after treatment with six metal ions. The PO activities were measured using l-DOPA (15 mmol/l) as a substrate. (A) AjPO1; (B) AjPO2; and (C) AjPO3.
Wright et al., 2012), based on which, PO functions as an important immune and physiological factor in organisms. Here, we detected and partially purified POs from the coelomic fluid of A. japonicus, and characterized POs based on the optimal temperature and pH, the kinetic parameters, the effects of divalent metal ions and inhibitors on enzymatic activities, and the temporal profile of TPAC after immune stimulations. Using native-PAGE combined with catechol staining, we detected three POs (AjPO1, AjPO2 and AjPO3) in coelomic fluid, suggesting that A. japonicus PO is composed of several isoenzymes. This phenomenon was found in many marine invertebrates such as the scallop Chlamys farreri and the clam R. philippinarum, as well as sea cucumber A. japonicus detected here (Cong et al., 2005; Jiang et al., 2012; Xing et al., 2012; Zhou et al., 2012). However, as far as molecular mass was concerned, A. japonicus POs were much smaller than those reported in R. philippinarum (563 kDa), A. irradians (555 kDa), the oyster Saccostrea glomerata (219 kDa)
and the spiny lobster Panulirus argus (300 kDa) (Aladaileh et al., 2007; Jiang et al., 2011, 2012; Perdomo-Morales et al., 2007). The functional differentiation of PO during evolution might be one of the most important factors that caused a significant interspecific difference of PO molecular mass. Specifically, besides the capacity of phenol oxidization, PO performs other physiological functions, and the multiple functions of PO which were called “moonlighting” (Sharma and Kuhad, 2008) are different among species. For example, PO in Cryptococcus neoformans is simultaneously provided with different activities of iron oxidase, endospore coat protein oxidase and polyphenol oxidase, and has a molecular mass of 66 kDa (Williamson, 1994), while PO in Scapharca kagoshimensis functions as an oxygen carrier and a polyphenol oxidase at the same time and has a molecular mass of only 28.97 kDa (Zhang et al., 2009). However, the information about multiple functions of PO is still limited, especially in marine invertebrates, and the relationship between PO molecular mass and its multiple functions
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Fig. 5. The variation profile of A. japonicus PO activities after treatment with inhibitors. The PO activities were measured using l-DOPA (15 mmol/l) as a substrate.
needs to be further studied. The optimal temperature of AjPO1 was similar to that of Penaeus setiferus PO (45 ◦ C), C. farreri PO (45 ◦ C) and C. japonica PO (40 ◦ C), while the optimal temperatures of AjPO2 and AjPO3 were much higher than that of AjPO1 and most reported marine invertebrate POs, and similar to that of thermophilic fungi POs like Scytalidium thermophilum PO (80 ◦ C) (Liu et al., 2006; Simpson et al., 1987; Sun and Li, 1999; Younes and Sayadi, 2011). The optimal pH of AjPO1 also differed greatly from that of AjPO2 and AjPO3. The temperature and pH tests on A. japonicus POs implied that AjPO1 might differ greatly from AjPO2 and AjPO3 in protein structures. In addition, all three A. japonicus POs kept high activities at low pH (3.0, 4.0 and 5.0), but activities decreased dramatically when pH was higher than 9.0, suggesting that compared with acidic environment, alkaline environment (pH > 9.0) is more harmful to the A. japonicus PO immune system. Based on the substrate specificity, invertebrate POs were classified into three types (Barret, 1987): tyrosinase type (E.C.1.14.18.1 monophenol, l-DOPA: O2 oxidoreductase), catechol oxidase type
Fig. 6. The temporal profile of A. japonicus TPAC (total PO activities in coelomocytes) after immune stimulations. The PO activities were measured using l-DOPA (15 mmol/l) as a substrate. Values were shown as means ± standard deviation (n = 3). Significant differences compared with controls were indicated with an asterisk “*” at p < 0.05.
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(E.C.1.10.3.1 diphenol: O2 oxidoreductase) and laccase type (E.C.1.10.3.2; p-diphenol: O2 oxidoreductase. The oxidizing reactions of all three A. japonicus POs with catechol (o-diphenol), l-DOPA (o-diphenol), dopamine (o-diphenol) and hydroquinone except tyrosine (monophenol) imply that these enzymes are laccase-type POs, similar to POs in Crassostrea gigas, Crassostrea virginica, C. farreri, A. irradians, and R. philippinarum (Jiang et al., 2011, 2012; Jordan and Deaton, 2005; Luna-Acosta et al., 2011; Xing et al., 2012). In addition, the Km values indicated that AjPO1, AjPO2 and AjPO3 had the highest affinity to l-DOPA, catechol and hydroquinone respectively, which suggested that the active sites of three A. japonicus POs might be different. All of the eight metal ions tested in this study showed no regular trends during the experiments, indicating that their effects on PO activities were dose-independent. The activities of all three A. japonicus POs were strongly increased in the presence of Ca2+ , Mg2+ and Mn2+ , which suggested that the three metal ions played positive roles during the catalysis of A. japonicus POs and may be employed as potential immunopotentiators at appropriate concentrations. Actually, Ca2+ and Mg2+ are always involved in enzymes activation, and their activation of PO has been reported in several invertebrates including the moth Galleria mellonella, the pyralid Ostrinia furnacalis, the cockroach Blaberus craniifer, the silkworm Bombyx mori, the bay scallop A. irradians, etc. (Ashida et al., 1983; Dunphy, 1991; Feng et al., 2008; Jiang et al., 2011; Leonard et al., 1985). In addition, Fe2+ and Zn2+ had opposite effects at a high concentration (30 mmol/l) and a low concentration (2.5 mmol/l) in all three A. japonicus POs, indicating that Fe2+ and Zn2+ may play opposite roles during the catalysis of A. japonicus POs. Overall, the effects of eight metal ions on enzymatic activities had some difference among AjPO1, AjPO2 and AjPO3. The mechanism of metal ions affecting PO activity is still not clear. Some studies speculated that metal ions modulate PO activity by means of activating electrophile and nucleophile or changing the secondary structure of certain peptides of PO (Feng et al., 2008; Zibaee et al., 2010), however, the irregular variation trends of PO activities after treatment with divalent metal ions cannot be explained clearly by these hypothetical mechanisms, which drive us to investigate the detailed mechanism in further studies. The common antioxidants including ascorbic acid, citric acid and sodium sulfite each showed strong inhibition to the activities of three A. japonicus POs. Besides that, the divalent cation chelator – EDTA and the specific copper chelator – DETC also inhibited the activities of three A. japonicus POs greatly, implying that AjPO1, AjPO2 and AjPO3 obtained in this study were all copper-containing metalloenzymes, similar to the POs in other marine invertebrates like A. irradians, R. philippinarum, S. glomerata, C. japonica and ascidians Styela plicata, Phallusia mammillata and Ciona intestinalis (Aladaileh et al., 2007; Cong et al., 2005; Jiang et al., 2011; Liu et al., 2006; Parrinello et al., 2003). The temporal profile of A. japonicus TPAC indicated that A. japonicus PO immune system, composed of several isoenzymes with different characteristics, was very sensitive to the immune stimulations. The significant increase of TPAC at 12 h and 24 h post injection of LPS, which represent Gram-negative bacteria, is similar to results in C. gallina and C. gigas (Hellio et al., 2007; Munoz et al., 2006), suggesting that A. japonicus PO immune system is efficient in the response against Gram-negative bacteria challenge. However, the significant decline of TPAC after injection of PolyI:C, which represent double-stranded RNA viruses, revealed that A. japonicus PO immune system might suffer a depression from the infection of double-stranded RNA viruses. In the present study, we reported the detection, partial purification, biochemical characterization and immune-responsive analysis of POs from A. japonicus. The isolated PO isoenzymes showed clear difference in biochemical characteristics, and as a
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whole, were closely involved in the response against immune challenge. However, the difference among A. japonicus PO isoenzymes in immune and physiological process is not clear and further study on gene cloning and monoclonal antibody production to investigate the functional differentiation of A. japonicus POs is expected. Conflict of interest statement None declared. Acknowledgements This work was supported by grants from National Nature Science Foundation of China (31272687), State 863 High-Technology R&D Project of China (2012AA10A412), Science and Technology Project of Liaoning Province (2011203005), and Doctoral Startup Foundation of Liaoning Province (20111072). The funders had no role in study design, data analysis, decision to publish, or preparation of the manuscript. References Aladaileh, S., Rodney, P., Nair, S.V., Raftos, D.A., 2007. Characterization of phenoloxidase activity in Sydney rock oysters (Saccostrea glomerata). Comp. Biochem. Physiol. B 148, 470–480. Ashida, M., Ishizaki, Y., Iwahana, H., 1983. Activation of pro-phenoloxidase by bacterial cell walls or beta-1,3-glucans in plasma of the silkworm, Bombyx mori. Biochem. Biophys. Res. Commun. 113, 562–568. Asokan, R., Arumugam, M., Mullainadhan, P., 1997. Activation of prophenoloxidase in the plasma and haemocytes of the marine mussel, Perna viridis Linnaeus. Dev. Comp. Immunol. 52, 1–12. Aspán, A., Huang, T.S., Cerenius, L., Söderhäll, K., 1995. cDNA cloning of prophenoloxidase from the freshwater crayfish Pacifastacus leniusculus and its activation. Proc. Natl. Acad. Sci. U. S. A. 92, 939–943. Atkins, G.L., Nimmo, I.A., 1975. A comparison of seven methods for fitting the Michaelis–Menten equation. Biochem. J. 149, 775–777. Barret, F.M., 1987. Phenoloxidases from larval cuticle of the sheep blowfly, Lucilia cuprina: characterization, developmental changes, and inhibition by antiphenoloxidase antibodies. Arch. Insect Biochem. Physiol. 5, 99–118. Bradford, M., 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of dye binding. Anal. Biochem. 72, 248–254. Cerenius, L., Söderhäll, K., 2004. The prophenoloxidase-activating system in invertebrates. Immunol. Rev. 198, 116–126. Cong, R.S., Sun, W.J., Liu, G.X., Fan, T.J., Meng, X.H., Yang, L.L., Zhu, L.Y., 2005. Purification and characterization of phenoloxidase from clam Ruditapes philippinarum. Fish Shellfish Immunol. 18, 61–70. Dunphy, G.B., 1991. Phenoloxidase activity in the serum of two species of insects, the gypsy moth, Lymantria dispar (Lymantriidae) and the greater wax moth, Galleria mellonella (Pyralidae). Comp. Biochem. Physiol. B 98, 535–538. Feng, C.J., Song, Q.S., Lü, W.J., Lu, J.F., 2008. Purification and characterization of hemolymph prophenoloxidase from Ostrinia furnacalis (Lepidoptera: Pyralidae) larvae. Comp. Biochem. Physiol. B 151, 139–146. Hellio, C., Bado-Nilles, A., Gagnaire, B., Renault, T., Thomas-Guyon, H., 2007. Demonstration of a true phenoloxidase activity and activation of a ProPO cascade in Pacific oyster, Crassostrea gigas (Thunberg) in vitro. Fish Shellfish Immunol. 22, 433–440. Jiang, J.W., Xing, J., Sheng, X.J., Zhan, W.B., 2011. Characterization of phenoloxidase from the bay scallop Argopecten irradians. J. Shellfish Res. 30, 273–277. Jiang, J.W., Xing, J., Zhan, W.B., 2012. Purification and characterization of laccase-type phenoloxidase from the clam Ruditapes philippinarum. Oceanol. Limnol. Sin. 43, 294–298. Jiravanichpaisal, P., Lee, B.L., Söderhäll, K., 2006. Cell-mediated immunity in arthropods: hematopoiesis, coagulation, melanization and opsonization. Immunobiology 211, 213–236. Jordan, P.J., Deaton, L.E., 2005. Characterization of phenoloxidase from Crassostrea virginica hemocytes and the effect of Perkinsus marinus on phenoloxidase activity in the hemolymph of Crassostrea virginica and Geukensia demissa. J. Shellfish Res. 24, 477–482.
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