Zinc Superoxide Dismutase from the Cladoceran Daphnia

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Copper/zinc-superoxide dismutase from the Cladoceran Daphnia magna: Molecular cloning and expression in response to different acute environmental stressors Kai Lyu, Xuexia Zhu, Qianqian Wang, Yafen Chen, and Zhou Yang Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/es4015212 • Publication Date (Web): 01 Jul 2013 Downloaded from http://pubs.acs.org on July 8, 2013

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Environmental Science & Technology

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Copper/zinc-superoxide dismutase from the Cladoceran Daphnia magna: Molecular cloning and expression in response to different acute environmental stressors

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Kai Lyu†, Xuexia Zhu†, Qianqian Wang†, Yafen Chen‡ and Zhou Yang†*

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†Jiangsu

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Nanjing Normal University, 1 Wenyuan Road, Nanjing 210023, China

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‡State

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and Limnology, the Chinese Academy of Sciences, Nanjing, China

Key Laboratory for Biodiversity and Biotechnology, School of Biological Sciences,

Key Laboratory for Lake Science and Environment, Nanjing Institute of Geography

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Address Correspondence to

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Zhou Yang,

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Jiangsu Key Laboratory for Biodiversity and Biotechnology, School of Biological Sciences,

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Nanjing Normal University, 1 Wenyuan Road, Nanjing 210023, China

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*E-mail: [email protected].

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Tel: +86-25-85891671

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ACS Paragon Plus Environment


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ABSTRACT: The copper/zinc superoxide dismutase (Cu/Zn-SOD) is a representative

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antioxidant enzyme that is responsible for the conversion of superoxide to oxygen and

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hydrogen peroxide in aerobic organisms. Cu/Zn-SOD mRNAs have been cloned from

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many species and employed as useful biomarkers of oxidative stresses. In the present

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study, we cloned Cu/Zn-SOD cDNA from the cladoceran Daphnia magna, analyzed

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its catalytic properties, and investigated mRNA expression patterns after exposure to

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known oxidative stressors. The full-length Cu/Zn-SOD of D. magna

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(Dm-Cu/Zn-SOD) sequence consisted of 703 bp nucleotides, encoding 178 amino

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acids, showing well-conserved domains that were required for metal binding and

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several common characteristics. The deduced amino acid sequence of

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Dm-Cu/Zn-SOD showed that it shared high identity with Daphnia pulex (88%),

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Alvinella pompejana (56%) and Cristaria plicata (56%). The phylogenetic analysis

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indicated that Dm-Cu/Zn-SOD was highly homologous to D. pulex. The variation of

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Dm-Cu/Zn-SOD mRNA expression was quantified by real-time PCR, and the results

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indicated that the expression was up-regulated after 48-h exposure to copper,

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unionized ammonia and low dissolved oxygen. This study shows that the

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Dm-Cu/Zn-SOD mRNA could be successfully employed as a biomarker of oxidative

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stress, which is a common mode of toxicity for many other aquatic hazardous

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materials.

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Table of Contents Art

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INTRODUCTION

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Reactive oxygen species (ROS) are oxygen-containing molecules generated during

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normal metabolism.1 They are usually maintained at suitable levels by balanced

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production and elimination processes. However, over-elevated ROS levels lead to

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oxidative damage including lipid peroxidation, protein and DNA oxidation, and

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enzyme inactivation.2 Aquatic organisms are usually subjected to enhanced oxidative

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stress from ROS due to acute or chronic exposure to adverse factors in their

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environments.3 In response, aquatic organisms have developed defense systems

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against ROS, including the induction of antioxidant enzymes like superoxide

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dismutase (SOD) (EC 1.15.1.1).4

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The SOD family is a representative antioxidant enzyme cluster responsible for

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the conversion of superoxide to oxygen and hydrogen peroxide.5 SODs in eukaryotes

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are classified into three groups based on the metal cofactors in their active sites:

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Cu/Zn-SOD, Fe-SOD and Mn-SOD. Cu/Zn-SODs have two destinations for work.5

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One is the extracellular matrix of tissues to which they are sorted with a signal peptide;

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the other in intracellular, without a signal peptide, and includes the cytoplasm and

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nucleus.6, 7 Both extracellular and intracellular Cu/Zn-SODs have been discovered in

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crustaceans.8-10

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The micro-crustacean Daphnia magna (Daphniidae) is an important species in

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the aquatic food web as a primary consumer and a prey for secondary consumers (e.g.,

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fish). Furthermore, D. magna is widely used as a test organism in aquatic toxicology 4


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due to its association with sensitivity of aquatic pollutants (reviewed by Sarma and

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Nandini 11). In previous studies, the enzymatic activities of SOD in response to

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oxidative stress induced by ROS have been extensively employed as a biomarker for

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the evaluation of environmental stressors using D. magna.12-14 However, limited

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information has been published regarding the gene expression profiles of SOD in

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response to oxidative stress. To our knowledge, expression of the SOD gene not only

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reveals a responsive mechanism involved in antioxidation processes, but also offers

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sensitive environmental biomonitoring values in the diagnosis of environmental

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stressors.15, 16 In this study, we tested three wide-spread environmental stressors

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having different modes of oxidative response, copper (Cu), unionized ammonia (NH3)

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and low dissolved oxygen (DO).17

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High levels of Cu occur in the aquatic environment due to irregular human

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activities and accumulate at various levels in aquatic food chains.18 Toxic effects of

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excessive Cu have been studied in many aquatic species, such as algae, invertebrates

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and fish.17 As a ubiquitous heavy metal pollutant, attention needs to be paid to the

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biological toxicity of Cu in aquatic ecosystem. In addition, the incidence of

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cyanobacterial blooms has increased on a global scale in recent decades due to the

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combination of eutrophication and climate change.19 Die-off of the blooms released

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high levels of nitrogenous pollutants (e.g. ammonia) which can cause toxic effects on

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aquatic organisms, particularly to animals.20 More often, low DO frequently occurs in

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poorly mixed waters or polluted waters and usually is associated with increased 5



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ammonia in eutrophic lakes.21 The aim of this study was to identify the molecular characterization of

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Cu/Zn-SOD from D. magna (Dm-Cu/Zn-SOD) and evaluate the mRNA expression

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patterns after the exposure to known environmental stressors: Cu, NH3 and low DO.

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This will allow us to understand the oxidative response mechanisms and test the

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applicability of Dm-Cu/Zn-SOD as biomarkers for oxidative stress, to be used in

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ecosystem risk assessments. We also examined the total SOD enzyme activity of D.

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magna exposed to the three stressors mentioned above.

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MATERIALS AND METHODS

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Maintenance of D. magna Culture. We used a D. magna clone that has been

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maintained in our laboratory for over ten years.22 We identified this species based on

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its morphological characteristics and sequencing its mitochondrial DNA CO1. D.

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magna were cultured in 200-mL beakers at 25 ºC and fed Scenedesmus obliquus (5 ×

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104 cells mL-1) daily, under fluorescent light at 40 µmol photons m-2 s-1, with a

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light-dark period of 12:12 h. M4 medium 23 was used as recommended by OECD 24

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and was gently aerated with clear air for 24 h before used and renewed twice weekly.

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Treatment with Cu, NH3 and low DO. Separate 48-h exposure (Cu, NH3 and

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low DO) experiments were carried out using D. magna juveniles (4-day-old). The

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concentrations of Cu, NH3-N and DO were prepared based on the results from

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previous acute and chronic toxicity tests.25-27 Cu exposure levels were 0, 5.0 and 20.0

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µg L-1, and NH3-N exposure levels were 0, 0.3, 0.5 and 1.0 mg L-1. Cu test solutions 6


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were prepared by dissolving copper chloride (CuCl2). The pH was stable at 7.2 in

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Daphnia experimental cultures and was measured daily by using a multi-parameter

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meter. DO levels of 2.0, 4.0, 8.0 mg L-1 were prepared using compressed nitrogen gas

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and compressed air for the battery of DO levels, as described previously.28 DO in each

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beaker was regularly checked using a dissolved oxygen meter (Bante A820, Bante

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Instrument, China) and adjusted if necessary. All experiments were conducted under

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the conditions as same as D. magna maintenance.

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Three replicates of 20 juvenile D. magna were used in all exposure conditions.

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Live D. magna were collected for gene expression assay and total SOD enzyme

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activity. For quantification of Dm-Cu/Zn-SOD mRNA expression, a pair of

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gene-specific primers (S-Q-F, S-Q-R) was used in qPCR (Supporting Information (SI)

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Table S1). The qPCR amplification was carried out in EU Thin-wall 8-tube strip in a

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20 µL reaction volume, which contained 10ml SYBR Premix Ex Taq (2×) (TaKaRa,

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Japan), Then the 106 bp product was amplified with S-Q-F and S-Q-R, and was

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sequenced to verify the PCR specificity.29 Furthermore, the qPCR procedure was

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consisted of 95 ºC for 2 min, followed by 95 ºC for 5 s, 56 ºC for 20 s and 72 ºC for

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10 s per cycle for 40 cycles. We also used actin (GenBank accession number:

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AJ292554.1) as an internal control and amplified it with a primer set actin-F and

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actin-R (SI Table S1). Expression levels of the SOD genes were according to the 2-∆∆T

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method.30

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Total RNA Extraction and cDNA Synthesis. We collected the treated D. magna 7



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individuals and homogenized them using a pestle. Total RNA was then extracted

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using the Trizol technique following the manufacturer’s instructions (Takara, Japan).

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The total RNA concentrations were determined by absorbance at OD260 and RNA

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integrity was checked by electrophoresis. Total RNA was reverse-transcribed to

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cDNA with oligo-dT primers and a cDNA Synthesis Kit (Takara, Japan), according to

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the manufacturer’s instructions.

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Next, we used S-F1 and S-R1 (SI Table S1) as the PCR primers to amplify SOD

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coding sequences. The primers set was designed against the conserved regions of the

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corresponding genes in congeneric Daphnia pulex (GenBank accession number:

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ACJG01001954.1), the deep-sea worm Alvinella pompejana (EU178106.1), and the

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freshwater mussel Cristaria plicata (FJ194441.1). PCR program and vector

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connection were detailedly described in SI.

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Rapid Amplification of cDNA Ends (RACE). The D. magna Cu/Zn-SOD

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partial cDNA sequence was extended using 5’ and 3’ RACE (SMART™ cDNA kit,

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Life Technologies). More details of RACE process were found in SI.

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Sequence Analysis of Cu/Zn-SOD. The cDNA sequence and deduced amino

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acid sequence of Cu/Zn-SOD were analyzed using the BLAST algorithm

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(http://www.ncbi.nlm.nih.gov/blast). Translation and protein analyses were performed

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using ExPASy tools (http://us.expasy.org/tools/). The ClustalW Multiple Alignment

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program (http://www.ebi.ac.uk/clustalw/) was used to create the multiple sequence

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alignment. The predicted molecular weight was calculated using the online tool 8


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(http://expasy.org/cgi-bin/pi_tool). To establish the tertiary structure of

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Dm-Cu/Zn-SOD, a 1.86 Å crystal structure of Homo Cu/Zn-SOD dimer from PDB

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database (ID, 1n19B) was selected as template by Swiss-Model

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(http://swissmodel.expasy.org/). An unrooted phylogenetic tree was constructed based

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on the amino sequences alignment by the neighbor-joining (NJ) algorithm embedded

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in MEGA 5 program. The reliability of the branching was tested by bootstrap

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resampling (1000 pseudoreplicates).

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Measurement of Total SOD Enzymatic Activity. To determine the responses in

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SOD enzymatic activity of D. magna, 15 individuals were sampled from each beaker,

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pooled in a microcentrifuge tube and immediately frozen at −70 °C until further

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biochemical analysis (for more details see SI).

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Statistical Analysis. All biochemical data were expressed as means ±1 SE.

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Significant differences were evaluated by one-way analysis of variance (ANOVA)

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followed by Duncan multiple range test (α=0.05). All tests were conducted using

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SigmaPlot (V 11.0).

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RESULTS

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Sequence of Dm-Cu/Zn-SOD cDNA. The full-length Dm-Cu/Zn-SOD cDNA

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consisted of 703 bp, including 46 bp in the 5’-untranslated region, an open reading

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frame (ORF) of 537, and 120 bp in the 3’-untranslated region with a poly A tail

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(Figure 1). The ORF of Dm-Cu/Zn-SOD cDNA was composed of 178 amino acids

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(aa), including a 21-aa signal peptide predicted by SignalP software (ExPASy) in the 9



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N-terminus. The calculated molecular mass of Dm-Cu/Zn-SOD protein was 18.30

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kDa with an estimated pI of 6.11. The Dm-Cu/Zn-SOD cDNA sequence and its

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deduced amino acid sequence were submitted to the NCBI GenBank under the

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accession number JX456347.

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Dm-Cu/Zn-SOD Motifs. Multiple alignment of amino acid sequences revealed

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that Dm-Cu/Zn-SOD contained two Cu and Zn signatures from 66 to 75

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(GFHVHQFGDV), four copper binding sites (His68, 70, 86, 145), four Zn binding

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sites (His86, 94, 105, and Asp 108), two cysteines (73 and 163) that form a disulfide

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bond (Figure 1). Comparison of the deduced amino acid sequence of Dm-Cu/Zn-SOD

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with those of from D. pulex, A. pompejana and C. plicata manifested all of these

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Cu/Zn-SODs and had the common features of four copper binding sites, four zinc

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sites and two Cu and Zn signatures (Figure 2).

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Phylogenetic Analysis of Dm-Cu/Zn-SOD. ClustalW analysis revealed that the

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deduced amino acid sequence of Dm-Cu/Zn-SOD shared high identity with D. pulex

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(88%), A. pompejana (56%) and C. plicata (56%). The phylogenetic analysis

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indicated that Dm-Cu/Zn-SOD was highly homologous to that of D. pulex, and the

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two SODs were somewhat close to the Insecta: Anopheles gambiae (XP 001651859.1)

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and Leptopilina boulardi (AET83769.1). However, Dm-Cu/Zn-SOD was only

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remotely related to the Cu/Zn-SOD of the Chordata and Mollusca (Figure 3).

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The SWISS-MODEL prediction algorithm established the potential tertiary

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structure of the Cu/Zn-SODs from D. magna, D. pulex and A. pompejana based on the 10


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template of Homo Cu/Zn-SOD. Result showed that D. magna, D. pulex and A.

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pompejana shared different identities with Homo: 51.299 %, 52.597 % and 61.438 %,

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indicating that the three-dimensional structure of Dm-Cu/Zn-SOD was more similar

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with D. pulex than A. pompejana (Figure 4).

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Changes in Dm-Cu/Zn-SOD mRNA Expression and Total SOD Enzymatic

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Activity after Harmful Exposure. After 48-h exposures to Cu, Dm-Cu/Zn-SOD

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transcript levels was significantly elevated due to increased Cu concentrations (Figure

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5A; P

Zinc Superoxide Dismutase from the Cladoceran Daphnia

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