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Cloning and expression of the sorbitol dehydrogenase gene during embryonic development and temperature stress in Artemia sinica
Gene 521 (2013) 296–302
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Gene journal homepage: www.elsevier.com/locate/gene
Cloning and expression of the sorbitol dehydrogenase gene during embryonic development and temperature stress in Artemia sinica Ting Wang a, Ming Hou b, Na Zhao a, Yifei Chen a, Ying Lv a, Zengrong Li a, Rui Zhang a, Wenting Xin a, Xiangyang Zou c,⁎, Lin Hou a,⁎⁎ a b c
College of Life Sciences, Liaoning Normal University, Dalian 116081,China Affiliated Hospital, Changchun University of Traditional Chinese Medicine, Changchun 130021, China Department of Biology, Dalian Medical University, Dalian 116044, China
a r t i c l e
i n f o
Article history: Accepted 16 March 2013 Available online 2 April 2013 Keywords: Artemia sinica Sorbitol dehydrogenase Diapause termination
a b s t r a c t Sorbitol dehydrogenase (SDH) catalyzes the interconversion of polyols and ketoses, using zinc and NAD + as cofactors. SDH converts sorbitol into fructose and plays an important role in the sorbitol metabolic pathway and in the early embryonic development of many invertebrates. Sorbitol usually accumulates in diapause embryos of insects to protect the embryos from frostbite, which indicates the vital function of SDH in the diapause and diapause-termination stages of embryo development. In this study, a 1311-bp full-length cDNA of As-sdh, including a 28-bp 5′ UTR and a 59-bp 3′ UTR, was cloned from Artemia sinica. This gene encodes 348 amino-acid proteins. Bioinformatic analysis revealed that this gene is highly conserved in arthropods. The expression patterns of As-sdh were investigated during different stages of embryonic development using real-time PCR and in situ hybridization. As-sdh was expressed at relatively high levels during the 0 h embryonic stage, and transcript levels were quite high in 5- and 7-day-old embryos. In situ hybridization analysis showed that As-sdh is expressed in a widely dispersed pattern before incubation but is mainly concentrated on the body surface and the inner wall of the alimentary tract after the nauplius stage. Our results suggest that As-sdh is integral to the process of diapause and diapause termination in A. sinica. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Artemia sinica, a small crustacean widely used in commercial aquaculture, is a valuable biological resource due to its high unsaturated fatty acid and protein content (Abatzopoulos et al., 2002). A. sinica is easy to acquire, and its diapause cysts are easy to transport and preserve. These attributes, along with the relatively short life cycle and simple culturing conditions of this crustacean, make A. sinica an excellent model system for studies of genetics, development and evolution (Zheng et al., 2011). Recently, cDNAs encoding sorbitol dehydrogenase (sdh) have been cloned from rat (Karlsson et al., 1991), silkworm (Niimi et al., 1993), Bacillus (Ng et al., 1992), and yeast (Sarthy et al., 1994). Multiple sequence alignment analysis of the encoded protein sequences revealed a highly conserved structure, which indicates that this enzyme may have an important physiological function. Diapause is a specific stage of embryonic development in A. sinica. During diapause, embryo Abbreviations: ADH, alcohol dehydrogenase; As-sdh, sorbitol dehydrogenase gene of Artemia sinica; As-SDH, sorbitol dehydrogenase of Artemia sinica; DEPC, diethylpyrocarbonate; DIG, digoxigenin; NJ, neighbor-joining; ORF, open reading frame; pI, isoelectric pointand; SDH, sorbitol dehydrogenase; sdh, sorbitol dehydrogenase; UTR, untranslated region. ⁎ Corresponding author. Tel.: +86 411 86110296. ⁎⁎ Corresponding author. Tel.: +86 411 84258306. E-mail addresses: [email protected] (X. Zou), [email protected] (L. Hou). 0378-1119/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2013.03.077
development is paused at the gastrula stage to enable the organism to survive under extreme conditions (Liang et al., 1997). In the diapause stage, embryos remain dormant until they are stimulated by suitable environmental factors to resume development to the nauplius stage (Liang and MacRae, 1999). Sorbitol often accumulates in the resting eggs of most diapause-mandatory animals, such as Bombyx (Toshinobu et al., 1990), flesh flies (Michaud and Denlinger, 2007), and A. sinica, because the water absorbing ability of sorbitol makes it an excellent cryoprotectant (Wang et al., 2005). Indeed, sdh is vital to the process of diapause termination. SDH is a member of zinc-dependent alcohol dehydrogenase-like (ADH) family. Unlike other members (Jornvall et al., 1993), the holoenzyme of SDH is a tetramer (Banfield et al., 2001), which comprises four identical subunits, each with one catalytic zinc atom (Eklund et al., 1985; Karlsson et al., 1989, 1995; Maret, 1996). This coenzyme-binding motif, which consists of seven residues of the N-terminus located between two β-strands, has a classical Rossmann fold structure (Pauly et al., 2003), that allows all four subunits of the enzyme to reversibly combine with NAD(H) (Kenneth and Ruiqiong, 1992). The catalytic zinc atom in each subunit is associated with Cys, His, Glu, and a water molecule. In human SDH, these residues include Cys43, His68 and Glu150 (Maret, 1996). Although Glu155 in human SDH is not thought to be a zinc-binding site, mutation of this residue results in the loss of catalytic activity (Karlsson et al., 1995).
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The process by which SDH catalyzes the interconversion of polyols and ketoses begins when the zinc atom associates with the three amino acid residues and a water molecule; this process requires NAD + as a cofactor. Next, the oxygen atoms of C1 and C2 in the sorbitol carbon chain combine with the zinc atom, releasing Glu150 and converting the zinc to zinc 5+. In addition, C3–C5 of the carbon chain convert to a fructose conformation. The hydroxyl group of C2 loses a proton, triggering a tandem reaction after the water molecule provides a hydrogen bond for Gly155 in the oxidase and Gly150 in the SDH/NAD+ complex. The hydrogen of the hydroxyl group of C2 releases an electron by reducing NAD + to NADH. Finally, the reaction is completed when the hydroxyl group of C2 is oxidized to form keto carbonyl (Pauly et al., 2003). While the piperazine pyrimidines are the only class of in vivo SDH inhibitors (SDIs) (Geissen et al., 1992), a class of potent in vitro SDIs was identified through their interaction with catalytic zinc (Lindstad and Mckinley-Mckee, 1996). The identification of the prototypical SDI CP-166,572 by Geissen (Geissen et al., 1994) made it possible for more potent SDIs to be synthesized (Chu-Moyer et al., 2002). In addition, this discovery led to the proposal that SDH could be inhibited by directly chelating the catalytic zinc (Mylari et al., 2001). The prodrug SDI-157 increases the activity of CP-166,572 by inducing a chemical change in its pyrimidine ring (Pauly et al., 2003). CP-166,572 is noncompetitive with sorbitol, NAD + or NADH in the inhibition procedure, but competes with fructose by binding to the SDH/NADH complex in sheep (Geissen et al., 1994) and human SDH (Pauly et al., 2003). To acclimate to cold weather, insect diapause eggs often contain sorbitol, in addition to glycerol. NAD-SDH activity is not induced in overwintering eggs of Bombyx exposed to temperature of 0 °C (Toshinobu et al., 1990), and therefore sorbitol is not utilized. And the sorbitol accumulation, by overloading glucose from glycogen (Joanisse and Storey, 1995), could also be examined in the resting eggs of flesh flies (Michaud and Denlinger, 2007). Therefore, the storage of sorbitol is a direct response to extremely cold temperatures (Storey and Storey, 1983). Whether sorbitol accumulates or not based on the activity of sdh, which was not induced in the overwintering eggs. In diapause Bombyx eggs, sdh is normally expressed at a very low level. However, when the eggs are transferred from approximately 0 °C to 25 °C, NAD-SDH activity strongly increases (Toshinobu et al., 1990); this is
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also true for diapause eggs of Drosophila (Bischoff, 1978). However, it is unclear whether the same phenomena also occur in A. sinica. As the hatchability of A. sinica is vital to its value, fully understanding the mechanism underlying diapause termination in this small crustacean would greatly improve A. sinica aquiculture. The functions of SDH have been studied in many species, but little is known about the post-diapause and diapause terminations of A. sinica. In this study, we focused on the molecular characterization and expression patterns of As-sdh gene during development, and the role of As-sdh in the temperature–stress response. 2. Methods and materials 2.1. Animal preparation A. sinica cysts were collected from the salt lake of Yuncheng in Shanxi Province (China) and were hatched in filtered fresh seawater in the laboratory. The cysts were incubated for 30 min (designated 0 h), and samples of roughly 50 mg were collected at 5, 10, 20 and 40 h, and 3, 5, and 7 days (gastrula stage (0 h), diapause termination stage; embryonic stage (5 and 10 h), the embryo returns to the normal rate of development; nauplius stage (15, 20, and 40 h); metanauplius stage (3 and 5 days) alimentary system is formed which allows individuals to gain nutritions from surroundings; sub-adult (7 days) segmentation stage). Adult brine shrimp cultured at 30 °C for 48 h were employed as the control group in the low-temperature assay, and adult Artemia in the experimental group were maintained at 25 °C, 20 °C, 15 °C, 10 °C or 5 °C. 2.2. Cloning of full-length As-sdh cDNA Total RNA from each sample was extracted using the TRIzol-A + (Tiangen, Beijing, China) method, and oligo (dT) primer and MLV reverse transcriptase (Takara, Dalian, China) were used for reverse transcription. Primer Premier 5.0 was used for primer design, based on the sdh of Artemia franciscana, and the forward (5′-TGCGAAGAGGTGAGGT-3′) and reverse (5′-TGCAAGCAGGGTGTAAT-3′) primers were synthesized (Takara, Dalian, China). An As-sdh fragment of 332 bp was obtained. The full-length cDNA of As-sdh was obtained using 3′ RACE core set ver. 2.0 (Takara, Dalian, China) and the SMART RACE cDNA amplification kit
Fig. 1. Sequence analysis of the cDNA and predicted peptide sequences of As-sdh. The start codon is indicated in purple; the stop codon is indicated in yellow; the ADH_N domain is indicated in red; the ADN_zinc_N domain is indicated in green; and the polyadenylation site (AATAAA) is indicated in blue.
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Table 1 Predicted phosphorylation sites for As-SDH. Name
Position
Contexta
Scoreb
Ser
68 80 129 141 143 159 194 203 307 313 51 52 56 117 271 317 46
GHESSGKVI QKVTSLAVG TRYYSHPAN KLPQSMSYE PQSMSYEYG VAVYSAERA AGAASIGIT DILQSRLDF DLVSSGKVD KVDLSRFVT WDRGTTGRF DRGTTGRFT TGRFTVKDP RFCATPPVD VGRGTPEVS SRFVTHRFP SDIHYWDRG
0.574 0.680 0.709 0.536 0.625 0.985 0.547 0.827 0.559 0.673 0.850 0.985 0.964 0.695 0.987 0.773 0.950
Thr
Tyr a b
The surrounding sequences of the phosphorylation sites. The possibilities of phosphorylation sites.
(Clontech). Gene-specific primers (GSP) were designed based on the 332 bp fragment of As-sdh (GSP1: 5′-ATTGGTGAAGTTAATGCAGC-3′ for 3′ RACE and GSP2: 5′-GTGTTCCCCTTCCAACGGTTACTA-3′ for 5′ RACE). A PMD-19 T vector was used for cloning the RACE-PCR products after purification by agarose gel electrophoresis. The 3′- and 5′- termination fragments were spliced together through DNAman 6.0.3.48 (Lynnon Biosoft) to obtain the full-length cDNA of As-sdh. 2.3. Bioinformatics The open reading frame of As-sdh was found using the ORF finder program of the National Center for Biotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.gov), and a BLAST search was then performed to determine whether the ORF matched any known genes. The predicted translation product was determined using the translation tool of ExPASy (http://web.expasy.org/translate/). The molecular weight and theoretical isoelectric point of the protein were predicted using the ProtParam tool of ExPASy (http://cn.expasy.org/tools/protparam.html). SignalP 4.0 (http://www.cbs.dtu.dk/services/SignalP/) was used for signal
Fig. 2. Protein sequence analysis of As-SDH and 18 other species from GenBank. The sequences and their accession numbers are as follows: MmSDH, Mus musculus, NP_666238; BtSDH, Bos taurus, XP_585812; CgSDH, Cricetulus griseus, XP_003498072; XlSDH, Xenopus laevis, NP_001086483; SsSDH, Salmo salar NP_001086483; IpSDH, Ictalurus punctatus, NP_001187873; DrSDH, Danio rerio, NP_001165890; AsSDH, Artemia sinica, JX406418; AeSDH, Acromyrmex echinatior EGI61782; AmSDH, Apis mellifera XP_392401; DaSDH, Drosophila ananassae, XP_001953498; HsSDH, Harpegnathos saltator, EFN83387; BaSDH, Belgica antarctica, AFS17318; DmSDH, Drosophila melanogaster, NP_524311; GmSDH, Glossina morsitans, ADD19424; DvSDH, Drosophila virilis, XP_002060401; CeSDH, Caenorhabditis elegans, NP_505591, AsuSDH, Ascaris suum, ADY47014; BmSDH, Bombyx mori, ABF51482. The sequence of ADH_N domain is in the red zone, sequence of ADH_zinc_N domain is in the blue zone. The three amino acids that were marked green (Cys40, His65 and Glu147) are the zinc-binding site.
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peptide prediction. Phosphorylation site prediction was performed using TMHMM Server v. 2.0 (http://www.cbs.dtu.dk/services/TMHMM/). ProtScale (http://psort.hgc.jp/form2.html) was used to predict the subcellular localization of the protein, and SWISS-model was employed for tertiary structure prediction. Finally, a phylogenetic tree was produced with neighbor-joining (NJ) method using ClustalX 2.0 and MEGA 4.0 softwares. The Bootstrap method was used to evaluate the accuracy of the phylogenetic tree (Bootstrap value = 1000). 2.4. Comparative real-time PCR The cDNA templates were prepared from A. sinica at different stages of development, including adults, which were grown under various low temperature schemes. Two pairs of primers for As-sdh (forward 5′-CAACGCCACCAGTAGACGG-3′, reverse 5′-CCACCTCGGCTCTTTCT-3′) and β-actin (forward 5′-AGCGGTTGCCATTTATT GTT-3′, reverse 5′-G GTCGTGACTTGACGGACTATAT-3′) were used for real-time PCR analysis. Three parallel reactions were performed for each sample using SYBR Premix Ex Taq (Takara, Dalian, China). The β-actin gene of A. sinica was used to normalize the starting quantity of each sample. Thermal Cycler Dice Real Time system software (Takara, Dalian, China) was used to analyze the gene expression data, and the 2- ΔΔCt method, based on the Ct values of As-sdh and β-actin, was used to calculate the fold increase in the expression of each gene. The data obtained from real-time PCR analysis was analyzed using one-way analysis of variance (ANOVA) followed by Tukey's test with SPSS 16.0; the significance threshold was P b 0.05. 2.5. In situ hybridization The cDNA template for the probe, obtained by PCR (forward 5′-TGCGAAGAGGTGAGGT-3′, reverse 5′-TGCAAGCAGGGTGTAAT-3′) and agarose gel electrophoresis, was cloned into a pGM-T vector to add the T7 and SP6 polymerase binding sites to the gene. A DIG-labeled RNA probe was synthesized using a DIG RNA labeling kit (SP6/T7, Roche). The 0, 5 and 10 h samples were exuviated using 50% NaClO, and all samples were fully rinsed to remove surface salinity using phosphate-buffered saline (PBS) treated with diethylpyrocarbonate (DEPC). The samples were fixed in fresh 4% paraformaldehyde solution at 4 °C for 6–8 h. The samples were then cut into 10 μm-thick sections using a Radial Microtomes. Prehybridization of each sample was then performed at 37 °C for 2 h. The prehybridization buffer contained 1× Denhardt's solution, 0.5 mg/ml salmon sperm DNA, 50% deionized formamide (v/v), and 20× SSC. Hybridization was performed at 52 °C for 12–16 h by adding 10% dextran sulfate and 1 mg/ml DIG-labeled As-sdh probe to the pre-hybridization buffer. Finally, the hybridization signal was detected using a DIG Nucleic Acid Detection Kit (Roche).
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Phosphorylation site prediction revealed ten serine, six threonine and one tyrosine targets (score > 0.5; Table 1). Multiple sequence alignment analysis of the protein sequence of As-SDH revealed a highly conserved amino acid sequence between A. sinica and other 18 species from GenBank, which was also shown by protein sequence domain analysis. The ADH_N domain (from residues 27 to 138 in A. sinica) and the N-terminus and C-terminus of ADH_zinc_N (from residues 179 to 308 in A. sinica) were highly conserved (Fig. 2). As-SDH shares 45.48–48.02% similarity with SDH from other vertebrate species (including Mus musculus, Bos taurus, Cricetulus griseus, Xenopus laevis, Salmo salar, Ictalurus punctatus and Danio rerio), 47.98–53.85% similarity with SDH from arthropods (Drosophila ananassae, Apis mellifera, Acromyrmex echinatior, Harpegnathos saltator, Belgica antarctica, Drosophila melanogaster, Glossina morsitans, Drosophila virilis and Bombyx mori), and only 47.92% similarity with SDH from nematodes (Caenorhabditis elegans, Ascaris suum). A phylogenetic tree was produced to evaluate the evolutionary relationships using the SDH amino acid sequence from the 19 species evaluated (Fig. 3). In this phylogenetic tree analysis, the species were divided into three groups, i.e., the amniote group, the arthropod group, and the nematoda group. A. sinica was assigned to the arthropod group, as expected. 3.2. In situ hybridization analysis of As-sdh To determine where As-sdh was expressed in A. sinica embryos, in situ hybridization was performed with embryos at eight different developmental stages (0, 5, 10, 15, 20 and 40 h, 3 and 5 days; Fig. 4). In embryos at the pre-nauplius stage (0, 5, and 10 h), positive As-sdh signals were detected throughout nearly the entire embryo, especially at the intercellular spaces. At the nauplius stage (15, 20 and 40 h), during which the initial differentiation of the digestive system occurs, the As-sdh hybridization signals were concentrated at the inner wall of the alimentary tract, along with the body surface. After the nauplius stage (3 and 5 days), during which the alimentary tract is fully formed, the body surface and the inner side of the digestive tract were the only regions exhibiting concentrated As-sdh hybridization
3. Results 3.1. Isolation and bioinformatic analysis of As-sdh Using 3′ and 5′ RACE, a 1131 bp full-length As-sdh cDNA, with an open reading frame of 1044 bp, was obtained (GenBank accession number JX406418). The 5′ UTR was 28 bp, and the 3′ UTR was 59 bp with the classic polyadenylation signal AATAA (Fig. 1). The ExPASy translation tool was used to obtain the predicted amino acid sequence. The predicted protein had 348 amino acid residues, with a predicted molecular mass of 37.6 kDa and the theoretical pI of 7.96. Protein sequence domain analysis revealed the presence of the highly conserved ADH_N and ADH_zinc_N domains, which are the most important domains of SDH. No signal peptide was predicted with SignalP 4.0. The subcellar localization was predicted to be as follows: 44.4% endoplasmic reticulum, 22.2% Golgi, 22.2% mitochondrial and 11.1% cytoplasmic. This protein was not predicted to be localized to the transmembrane region, indicating that the As-SDH is primarily located inside the cellular membrane.
Fig. 3. Neighbor-joining phylogenetic tree based on the amino acid sequences of As-SDH and 18 other species from GenBank using the sequence analysis tool MEGA 4.0. The sequences and their accession numbers are indicated in the legend of Fig. 3. Red dot ( ) indicates As-SDH.
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signals. In summary, As-sdh was expressed over a broad area before the nauplius stage, and then became more localized as development progressed, with expression concentrated on the body surface of the embryo and on the lining of the digestive tract.
As-sdh was dramatically upregulated at 20 °C, peaked at 15 °C, decreased markedly at 10 °C, and further decreased at 5 °C.
3.3. Real-time PCR analysis of As-sdh
In this study, we isolated the full-length cDNA of As-sdh from A. sinica. This gene encodes a putative protein containing 348 amino acid residues. Sequence analysis revealed two highly conserved domains, ADH_N and ADH_zinc_N, which are the most important domains of SDH proteins. No signal peptides were found in this protein sequence, and gene expression was primarily confined to the inner walls of membranes and the cytoplasm, indicating that the catalytic sites of this protein are located inside the membrane. Alignment of SDH protein sequences from various species indicated that SDH has a highly conserved structure, particularly in mammalian species, arthropods and nematodes. The catalytic zinc-binding motif usually consists of three residues, i.e., Cys, His, and Glu, and an activated water molecule. Multiple sequence alignment revealed that the zinc-binding residues of SDH in A. sinica are likely to be Cys40, His65 and Glu147. Although the positions of the zinc binding sites are different in different species, the residues are the same. This is also true for the NAD+/NADH binding sites, Arg, Leu, and Val (Baker et al., 1992). As-SDH lacks an extra proline found in human and rat SDH, which may be due to an
Real-time PCR analysis was used to detect the transcript levels of As-sdh during different stages of A. sinica development, from the gastrula to the metanauplius larva stage (Fig. 5). From 0 h to 10 h, the expression level decreased notably, reaching an extremely low level; at the nauplius stage (15, 20 and 40 h), the level of As-sdh expression remained quite low. At the metanauplius stage (5 days), when the polypide was almost fully developed, As-sdh expression increased dramatically, reaching a relatively high level, with the highest expression occurring at 7 days. Therefore, As-sdh expression was down-regulated during early development and up-regulated at the nauplius stage. Real-time PCR analysis was also used to determine the level of As-sdh expression in A. sinica under different temperature–stress conditions (Fig. 6). Adult brine shrimp samples were obtained after they were subjected to low-temperature treatment for 48 h. The levels of As-sdh transcript were significantly different in the shrimp subjected to 25, 20, 15, 10 and 5 °C treatment vs. the (30 °C) control group.
4. Discussion
Fig. 4. In situ hybridization analysis of As-sdh expression during different developmental stages in A. sinica. A–H: experimental group; A1–H1: control group. A: gastrula stage (0 h); B, C: embryonic stage (5 and 10 h); D, E, F: nauplius stage (15, 20, and 40 h); G, H: metanauplius stage (3 and 5 day). Arrows indicate regions with positive hybridization signals.
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Fig. 5. Real-time PCR analysis of As-sdh expression during different stages of A. sinica development. The expression level of As-sdh was set as control group. The x-axis indicates different developmental stages (0 h to 7 day). The y-axis indicates the relative expression level of As-sdh. Data are the mean ± SD of triplicate experiments. * indicates a significant difference compared with the control (P b 0.05), while ** indicates a very significant difference compared with the control (P b 0.01).
intron/exon border arising from a differently spliced mRNA (Williamson et al., 1993). This discrepancy may lead to a slight difference in the secondary structure of As-SDH (Hou et al., 2006). Leucine, rather than methionine in SDH in humans and most other species, is present in the interphase between domains and in the glutamic identification site in As-SDH. Phylogenetic tree analysis revealed that the SDH protein is highly conserved in A. sinica and other species of arthropods, especially in key domains such as the zinc-binding sites and NAD+ binding sites; differences occur in the amino acid residues at other sites. These differences indicate that A. sinica has a relatively low classification status in the arthropod group. More differences were found between the As-SDH and less closely related species such as those of the amniote group. In the human body, SDH may contribute to an increase in free NAD+/NADH levels in the cytoplasm, leading to glucose-linked oxidative stress. SDH is primarily distributed in the heart and liver (Williamson et al., 1993). Through in situ hybridization, we found that As-sdh is expressed in almost every cell of the early embryo (0, 5 and 10 h), with increasing concentrations on the body surface and inner side of the coelom at the nauplius stage (15, 20 and 40 h). After the nauplius stage (3 day to 5 day), As-sdh was mainly expressed in the inner wall of the intestine and on the body surface. This is probably because As-sdh is required to decrease the accumulation of sorbitol that occurs in the cell during diapause, so As-sdh is widely expressed throughout the early embryo. At the nauplius stage, after the digestive system is formed, As-sdh is required for glycometabolism, which mainly occurs on the inner wall of the intestine. Real-time PCR of embryos from different developmental stages showed that As-sdh was expressed at relatively high levels in 0 h embryos. At 15 h to 40 h, As-sdh was expressed at low levels. This
Fig. 6. Real-time PCR analysis of changes in As-sdh expression in response to low temperatures. The control group (red) was cultured at 30 °C. Each group was incubated at the indicated temperature for 48 h. Data are the mean ± SD of triplicate experiments. * indicates a significant difference compared with the control (P b 0.05), while ** indicates a very significant difference compared with the control (P b 0.01).
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indicates that As-sdh may not be a key factor in the embryonic development process; however, this protein is crucial for the diapause termination process, as it is needed to digest the sorbitol that accumulates before diapause. The expression of As-sdh was highly upregulated after 3 day of development because the digestive system develops during this stage, and the adults could obtain the glucide from the surroundings (Hou et al., 2006). When the embryos were exposed to temperatures ranging from 25–15 °C, the level of As-sdh transcript increased dramatically with decreasing temperature. The increase in As-sdh expression mirrored the higher metabolic level required to protect against cold. However, when the temperature was below 15 °C, the expression of As-sdh was significantly reduced with decreasing temperature. Under these temperature conditions, high metabolism is not sufficient to protect the embryos from the cold. This decrease in As-sdh activity may therefore lead to the accumulation of sorbitol, along with other sugar alcohols and glycerol, to help the animal survive the cold (Banfield et al., 2001). In fact, sdh expression depends on many factors. In Drosophila, SDH enzymatic activity is at its lowest level during the larval stages, with the highest level of activity during adulthood (Bischoff, 1978). Accumulation of sorbitol decreases the level of sdh expression, which is observed in response to increased glucose concentrations in human diabetics (Carr and Markham, 1995; Nagasaka et al., 1988). Low-temperature treatment increases the expression of sdh in diapause eggs (Banfield et al., 2001; Teruyuki and Toshinobu, 1992). Before entering the diapause stage, the activity of sdh in A. franciscana is quite low (Zhijun et al., 2007). Therefore, the changes in the expression of sdh are always related to sugar levels, temperature changes or diapause. A. sinica lives in hypersaline waters, often encountering dramatic changes in temperature. As the temperature decreases, the expression of As-sdh increases, indicating that As-sdh plays an important role in the response of A. sinica to low-temperature stress. The high expression levels observed in the diapause termination stage and the metanauplius stage suggest that As-sdh is indispensable to diapause termination and the polyol pathway, but is not so important during embryo development. Acknowledgements This work was supported by grants from the National Natural Science Foundation of China (31071876, and 31272644). We thank anonymous referees for their valuable comments on an earlier version of the manuscript. References Abatzopoulos, T.J., Beardmore, J.A., Clegg, J.S., Sorgeloos, P., 2002. Artemia: Basic and Applied Biology. Kluwer Academic, Dordrecht, The Netherlands. Baker, P.J., Britton, K.L., Rice, D.W., Rob, A., Stillman, T.J., 1992. Structural consequences of sequence patterns in the fingerprint region of the nucleotide binding fold. Implications for nucleotide specificity. J. Mol. Biol. 228, 662–671. Banfield, M.J., Salvucci, M.E., Baker, E.N., Smith, C.A., 2001. Crystal structure of the NADP(H)-dependent ketose reductase from Bemisia argentifolii at 2.3 Å resolution. J. Mol. Biol. 306, 239–250. Bischoff, W.L., 1978. Ontogeny of sorbitol dehydrogenases in Drosophila melanogaster. Biochem. Genet. 5–6, 485–507. Carr, I.M., Markham, A.F., 1995. Molecular genetic analysis of the human sorbitol dehydrogenase gene. Mamm. Genome 6, 645–652. Chu-Moyer, M.Y., et al., 2002. Orally-effective, long-acting sorbitol dehydrogenase inhibitors: synthesis, structure-activity relationships, and in vivo evaluations of novel heterocycle-substituted piperazino-pyrimidines. J. Med. Chem. 45, 511–528. Eklund, R., Horjales, E., Jornvall, H., Branden, C.L., Jeffery, J., 1985. Molecular aspects of functional differences between alcohol and sorbitol dehydrogenases. Biochemistry 24, 8005–8012. Geissen, K., Utz, R., Nimmesgern, H., Lang, H.J., 1992. Substitute pyrimidin-derivate, Verfahren zu ihrer Herstellung und ihre verwendung als reagenzien. Eur. Pat. Appl. 0470, 616. Geissen, K., Utz, R., Grotsch, H., Lang, H.J., Nimmesgern, H., 1994. Sorbitol accumulating pyrimidine derivatives. Arzneimittelforschung 44, 1032–1043. Hou, L., et al., 2006. Establishment and improvement of real-time fluorescence quantitative PCR for actin gene of Artemia sinica. J. Liaoning Norm. Univ. 29, 15–19. Joanisse, D.R., Storey, K.B., 1995. Temperature acclimation and seasonal responses by enzymes in cold-hardy gall insects. Arch. Insect Biochem. Physiol. 28, 339–349.
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Jornvall, H., et al., 1993. ζ-Crystallin versus other members of the alcohol dehydrogenase super-family. Variability as a functional characteristic. Fed. Eur. Biochem. Soc. 322, 240–244. Karlsson, C., Maret, W., Auld, D.S., Hoog, J.O., Jornvall, H., 1989. Variability within mammalian sorbitol dehydrogenases. The primary structure of the human liver enzyme. Eur. J. Biochem. 186, 543–550. Karlsson, C., Jornvall, H., Hoog, J.O., 1991. Sorbitol dehydrogenase: cDNA coding for the rat enzyme. Variations within the alcohol dehydrogenase family independent of quaternary structure and metal content. Eur. J. Biochem. 198, 761–765. Karlsson, C., Jornvall, H., Hoog, J.O., 1995. Zinc binding of alcohol and sorbitol dehydrogenases. Adv. Exp. Med. Biol. 372, 397–406. Kenneth, N., Ruiqiong, Y., 1992. Sorbitol dehydrogenase from Bacillus subtilis. J. Biol. Chem. 267, 24989–24994. Liang, P., MacRae, T.H., 1999. The synthesis of a small heat shock/alpha-crystallin protein in Artemia and its relationship to stress tolerance during development. Dev. Biol. 207, 445–456. Liang, P., Amons, R., Clegg, J.S., MacRae, T.H., 1997. Molecular characterization of a small heat shock/alpha-crystallin protein in encysted Artemia embryos. J. Biol. Chem. 272, 19051–19058. Lindstad, R.I., Mckinley-Mckee, J.S., 1996. Reversible inhibition of sheep liver sorbitol dehydrogenase by thiol compounds. Eur. J. Biochem. 241, 142–148. Maret, W., 1996. Human sorbitol dehydrogenase—a secondary alcohol dehydrogenase with distinct pathophysiological roles. pH-dependent kinetic studies. Adv. Exp. Med. Biol. 414, 383–393. Michaud, M.R., Denlinger, D.L., 2007. Shifts in the carbohydrate, polyol, and amino acid pools during rapid cold-hardening and diapause-associated cold-hardening in Xesh Xies (Sarcophaga crassipalpis): a metabolomic comparison. J. Comp. Physiol. B 177, 753–763. Mylari, B.L., et al., 2001. Sorbitol dehydrogenase inhibitors (SDIs): a new potent, enantiomeric SDI, 4-[2-1R-hydroxylethyl)-pyrimidin-4-yl]-piperazine-1-sulfonic acid dimethyamide. J. Med. Chem. 44, 2695–2700.
Nagasaka, Y., Fujii, S., Kaneko, T., 1988. Human erythrocyte sorbitol metabolism and the role of sorbitol dehydrogenase. Diabetologia 31, 766–770. Ng, K., Ye, R., Wu, X.C., Wong, S.L., 1992. Sorbitol dehydrogenase from Bacillus subtilis. Purification, characterization, and gene cloning. J. Biol. Chem. 267, 24989–24994. Niimi, T., Yamashita, O., Yaginuma, T., 1993. A cold-inducible Bombyx gene encoding a protein similar to mammalian sorbitol dehydrogenase. Eur. J. Biochem. 213, 1125–1131. Pauly, T.A., et al., 2003. X-ray crystallographic and kinetic studies of human sorbitol dehydrogenase. Structure 11, 1071–1085. Sarthy, A.V., Schopp, C., Idler, K.B., 1994. Cloning and sequence determination of the gene encoding sorbitol dehydrogenase from Saccharomyces cerevisiae. Gene 140, 121–126. Storey, J.M., Storey, K.B., 1983. Regulation of cryoprotectant metabolism in the overwintering galaxy larva Eurosta solidaginis: temperature control of glycerol and sorbitol levels. J. Comp. Physiol. 149, 495–502. Teruyuki, N., Toshinobu, Y., 1992. Biosynthesis of NAD-sorbitol dehydrogenase is induced by acclimation at 5 °C in diapause eggs of silkworm, Bombyx Mori. Comp. Biochem. 102 (1), 169–173. Toshinobu, Y., Michihiro, K., Okitsugu, Y., 1990. Distinct effects of different low temperatures on the induction of NAD-sorbitol dehydrogenase activity in diapause eggs of the silkworm, Bombyx mori. J. Comp. Physiol. B 160, 277–285. Wang, G., Jiang, X., Wu, L., Li, S., 2005. Di Verences in the density, sinking rate and biochemical composition of Centropages tenuiramis (Copepoda calanoida) subitaneous and diapause eggs. Mar. Ecol. Prog. Ser. 288, 165–171. Williamson, J.R., et al., 1993. Hyperglycemic pseudohypoxia and diabetic complications. Diabetes 42, 801–813. Zheng, L.P., et al., 2011. Expression pattern of a gram-negative bacteria-binding protein in early embryonic development of Artemia sinica and after bacterial challenge. Dev. Comp. Immunol. 35, 35–43. Zhijun, Q., Tsoi, Stephen C.M., MacRae, T.H., 2007. Gene expression in diapausedestined embryos of the crustacean. Artemia francisana. Mech. Dev. 124, 856–867.
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Cloning and expression of the sorbitol dehydrogenase gene during embryonic development and temperature stress in Artemia sinica Ting Wang a, Ming Hou b, Na Zhao a, Yifei Chen a, Ying Lv a, Zengrong Li a, Rui Zhang a, Wenting Xin a, Xiangyang Zou c,⁎, Lin Hou a,⁎⁎ a b c
College of Life Sciences, Liaoning Normal University, Dalian 116081,China Affiliated Hospital, Changchun University of Traditional Chinese Medicine, Changchun 130021, China Department of Biology, Dalian Medical University, Dalian 116044, China
a r t i c l e
i n f o
Article history: Accepted 16 March 2013 Available online 2 April 2013 Keywords: Artemia sinica Sorbitol dehydrogenase Diapause termination
a b s t r a c t Sorbitol dehydrogenase (SDH) catalyzes the interconversion of polyols and ketoses, using zinc and NAD + as cofactors. SDH converts sorbitol into fructose and plays an important role in the sorbitol metabolic pathway and in the early embryonic development of many invertebrates. Sorbitol usually accumulates in diapause embryos of insects to protect the embryos from frostbite, which indicates the vital function of SDH in the diapause and diapause-termination stages of embryo development. In this study, a 1311-bp full-length cDNA of As-sdh, including a 28-bp 5′ UTR and a 59-bp 3′ UTR, was cloned from Artemia sinica. This gene encodes 348 amino-acid proteins. Bioinformatic analysis revealed that this gene is highly conserved in arthropods. The expression patterns of As-sdh were investigated during different stages of embryonic development using real-time PCR and in situ hybridization. As-sdh was expressed at relatively high levels during the 0 h embryonic stage, and transcript levels were quite high in 5- and 7-day-old embryos. In situ hybridization analysis showed that As-sdh is expressed in a widely dispersed pattern before incubation but is mainly concentrated on the body surface and the inner wall of the alimentary tract after the nauplius stage. Our results suggest that As-sdh is integral to the process of diapause and diapause termination in A. sinica. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Artemia sinica, a small crustacean widely used in commercial aquaculture, is a valuable biological resource due to its high unsaturated fatty acid and protein content (Abatzopoulos et al., 2002). A. sinica is easy to acquire, and its diapause cysts are easy to transport and preserve. These attributes, along with the relatively short life cycle and simple culturing conditions of this crustacean, make A. sinica an excellent model system for studies of genetics, development and evolution (Zheng et al., 2011). Recently, cDNAs encoding sorbitol dehydrogenase (sdh) have been cloned from rat (Karlsson et al., 1991), silkworm (Niimi et al., 1993), Bacillus (Ng et al., 1992), and yeast (Sarthy et al., 1994). Multiple sequence alignment analysis of the encoded protein sequences revealed a highly conserved structure, which indicates that this enzyme may have an important physiological function. Diapause is a specific stage of embryonic development in A. sinica. During diapause, embryo Abbreviations: ADH, alcohol dehydrogenase; As-sdh, sorbitol dehydrogenase gene of Artemia sinica; As-SDH, sorbitol dehydrogenase of Artemia sinica; DEPC, diethylpyrocarbonate; DIG, digoxigenin; NJ, neighbor-joining; ORF, open reading frame; pI, isoelectric pointand; SDH, sorbitol dehydrogenase; sdh, sorbitol dehydrogenase; UTR, untranslated region. ⁎ Corresponding author. Tel.: +86 411 86110296. ⁎⁎ Corresponding author. Tel.: +86 411 84258306. E-mail addresses: [email protected] (X. Zou), [email protected] (L. Hou). 0378-1119/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2013.03.077
development is paused at the gastrula stage to enable the organism to survive under extreme conditions (Liang et al., 1997). In the diapause stage, embryos remain dormant until they are stimulated by suitable environmental factors to resume development to the nauplius stage (Liang and MacRae, 1999). Sorbitol often accumulates in the resting eggs of most diapause-mandatory animals, such as Bombyx (Toshinobu et al., 1990), flesh flies (Michaud and Denlinger, 2007), and A. sinica, because the water absorbing ability of sorbitol makes it an excellent cryoprotectant (Wang et al., 2005). Indeed, sdh is vital to the process of diapause termination. SDH is a member of zinc-dependent alcohol dehydrogenase-like (ADH) family. Unlike other members (Jornvall et al., 1993), the holoenzyme of SDH is a tetramer (Banfield et al., 2001), which comprises four identical subunits, each with one catalytic zinc atom (Eklund et al., 1985; Karlsson et al., 1989, 1995; Maret, 1996). This coenzyme-binding motif, which consists of seven residues of the N-terminus located between two β-strands, has a classical Rossmann fold structure (Pauly et al., 2003), that allows all four subunits of the enzyme to reversibly combine with NAD(H) (Kenneth and Ruiqiong, 1992). The catalytic zinc atom in each subunit is associated with Cys, His, Glu, and a water molecule. In human SDH, these residues include Cys43, His68 and Glu150 (Maret, 1996). Although Glu155 in human SDH is not thought to be a zinc-binding site, mutation of this residue results in the loss of catalytic activity (Karlsson et al., 1995).
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The process by which SDH catalyzes the interconversion of polyols and ketoses begins when the zinc atom associates with the three amino acid residues and a water molecule; this process requires NAD + as a cofactor. Next, the oxygen atoms of C1 and C2 in the sorbitol carbon chain combine with the zinc atom, releasing Glu150 and converting the zinc to zinc 5+. In addition, C3–C5 of the carbon chain convert to a fructose conformation. The hydroxyl group of C2 loses a proton, triggering a tandem reaction after the water molecule provides a hydrogen bond for Gly155 in the oxidase and Gly150 in the SDH/NAD+ complex. The hydrogen of the hydroxyl group of C2 releases an electron by reducing NAD + to NADH. Finally, the reaction is completed when the hydroxyl group of C2 is oxidized to form keto carbonyl (Pauly et al., 2003). While the piperazine pyrimidines are the only class of in vivo SDH inhibitors (SDIs) (Geissen et al., 1992), a class of potent in vitro SDIs was identified through their interaction with catalytic zinc (Lindstad and Mckinley-Mckee, 1996). The identification of the prototypical SDI CP-166,572 by Geissen (Geissen et al., 1994) made it possible for more potent SDIs to be synthesized (Chu-Moyer et al., 2002). In addition, this discovery led to the proposal that SDH could be inhibited by directly chelating the catalytic zinc (Mylari et al., 2001). The prodrug SDI-157 increases the activity of CP-166,572 by inducing a chemical change in its pyrimidine ring (Pauly et al., 2003). CP-166,572 is noncompetitive with sorbitol, NAD + or NADH in the inhibition procedure, but competes with fructose by binding to the SDH/NADH complex in sheep (Geissen et al., 1994) and human SDH (Pauly et al., 2003). To acclimate to cold weather, insect diapause eggs often contain sorbitol, in addition to glycerol. NAD-SDH activity is not induced in overwintering eggs of Bombyx exposed to temperature of 0 °C (Toshinobu et al., 1990), and therefore sorbitol is not utilized. And the sorbitol accumulation, by overloading glucose from glycogen (Joanisse and Storey, 1995), could also be examined in the resting eggs of flesh flies (Michaud and Denlinger, 2007). Therefore, the storage of sorbitol is a direct response to extremely cold temperatures (Storey and Storey, 1983). Whether sorbitol accumulates or not based on the activity of sdh, which was not induced in the overwintering eggs. In diapause Bombyx eggs, sdh is normally expressed at a very low level. However, when the eggs are transferred from approximately 0 °C to 25 °C, NAD-SDH activity strongly increases (Toshinobu et al., 1990); this is
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also true for diapause eggs of Drosophila (Bischoff, 1978). However, it is unclear whether the same phenomena also occur in A. sinica. As the hatchability of A. sinica is vital to its value, fully understanding the mechanism underlying diapause termination in this small crustacean would greatly improve A. sinica aquiculture. The functions of SDH have been studied in many species, but little is known about the post-diapause and diapause terminations of A. sinica. In this study, we focused on the molecular characterization and expression patterns of As-sdh gene during development, and the role of As-sdh in the temperature–stress response. 2. Methods and materials 2.1. Animal preparation A. sinica cysts were collected from the salt lake of Yuncheng in Shanxi Province (China) and were hatched in filtered fresh seawater in the laboratory. The cysts were incubated for 30 min (designated 0 h), and samples of roughly 50 mg were collected at 5, 10, 20 and 40 h, and 3, 5, and 7 days (gastrula stage (0 h), diapause termination stage; embryonic stage (5 and 10 h), the embryo returns to the normal rate of development; nauplius stage (15, 20, and 40 h); metanauplius stage (3 and 5 days) alimentary system is formed which allows individuals to gain nutritions from surroundings; sub-adult (7 days) segmentation stage). Adult brine shrimp cultured at 30 °C for 48 h were employed as the control group in the low-temperature assay, and adult Artemia in the experimental group were maintained at 25 °C, 20 °C, 15 °C, 10 °C or 5 °C. 2.2. Cloning of full-length As-sdh cDNA Total RNA from each sample was extracted using the TRIzol-A + (Tiangen, Beijing, China) method, and oligo (dT) primer and MLV reverse transcriptase (Takara, Dalian, China) were used for reverse transcription. Primer Premier 5.0 was used for primer design, based on the sdh of Artemia franciscana, and the forward (5′-TGCGAAGAGGTGAGGT-3′) and reverse (5′-TGCAAGCAGGGTGTAAT-3′) primers were synthesized (Takara, Dalian, China). An As-sdh fragment of 332 bp was obtained. The full-length cDNA of As-sdh was obtained using 3′ RACE core set ver. 2.0 (Takara, Dalian, China) and the SMART RACE cDNA amplification kit
Fig. 1. Sequence analysis of the cDNA and predicted peptide sequences of As-sdh. The start codon is indicated in purple; the stop codon is indicated in yellow; the ADH_N domain is indicated in red; the ADN_zinc_N domain is indicated in green; and the polyadenylation site (AATAAA) is indicated in blue.
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Table 1 Predicted phosphorylation sites for As-SDH. Name
Position
Contexta
Scoreb
Ser
68 80 129 141 143 159 194 203 307 313 51 52 56 117 271 317 46
GHESSGKVI QKVTSLAVG TRYYSHPAN KLPQSMSYE PQSMSYEYG VAVYSAERA AGAASIGIT DILQSRLDF DLVSSGKVD KVDLSRFVT WDRGTTGRF DRGTTGRFT TGRFTVKDP RFCATPPVD VGRGTPEVS SRFVTHRFP SDIHYWDRG
0.574 0.680 0.709 0.536 0.625 0.985 0.547 0.827 0.559 0.673 0.850 0.985 0.964 0.695 0.987 0.773 0.950
Thr
Tyr a b
The surrounding sequences of the phosphorylation sites. The possibilities of phosphorylation sites.
(Clontech). Gene-specific primers (GSP) were designed based on the 332 bp fragment of As-sdh (GSP1: 5′-ATTGGTGAAGTTAATGCAGC-3′ for 3′ RACE and GSP2: 5′-GTGTTCCCCTTCCAACGGTTACTA-3′ for 5′ RACE). A PMD-19 T vector was used for cloning the RACE-PCR products after purification by agarose gel electrophoresis. The 3′- and 5′- termination fragments were spliced together through DNAman 6.0.3.48 (Lynnon Biosoft) to obtain the full-length cDNA of As-sdh. 2.3. Bioinformatics The open reading frame of As-sdh was found using the ORF finder program of the National Center for Biotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.gov), and a BLAST search was then performed to determine whether the ORF matched any known genes. The predicted translation product was determined using the translation tool of ExPASy (http://web.expasy.org/translate/). The molecular weight and theoretical isoelectric point of the protein were predicted using the ProtParam tool of ExPASy (http://cn.expasy.org/tools/protparam.html). SignalP 4.0 (http://www.cbs.dtu.dk/services/SignalP/) was used for signal
Fig. 2. Protein sequence analysis of As-SDH and 18 other species from GenBank. The sequences and their accession numbers are as follows: MmSDH, Mus musculus, NP_666238; BtSDH, Bos taurus, XP_585812; CgSDH, Cricetulus griseus, XP_003498072; XlSDH, Xenopus laevis, NP_001086483; SsSDH, Salmo salar NP_001086483; IpSDH, Ictalurus punctatus, NP_001187873; DrSDH, Danio rerio, NP_001165890; AsSDH, Artemia sinica, JX406418; AeSDH, Acromyrmex echinatior EGI61782; AmSDH, Apis mellifera XP_392401; DaSDH, Drosophila ananassae, XP_001953498; HsSDH, Harpegnathos saltator, EFN83387; BaSDH, Belgica antarctica, AFS17318; DmSDH, Drosophila melanogaster, NP_524311; GmSDH, Glossina morsitans, ADD19424; DvSDH, Drosophila virilis, XP_002060401; CeSDH, Caenorhabditis elegans, NP_505591, AsuSDH, Ascaris suum, ADY47014; BmSDH, Bombyx mori, ABF51482. The sequence of ADH_N domain is in the red zone, sequence of ADH_zinc_N domain is in the blue zone. The three amino acids that were marked green (Cys40, His65 and Glu147) are the zinc-binding site.
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peptide prediction. Phosphorylation site prediction was performed using TMHMM Server v. 2.0 (http://www.cbs.dtu.dk/services/TMHMM/). ProtScale (http://psort.hgc.jp/form2.html) was used to predict the subcellular localization of the protein, and SWISS-model was employed for tertiary structure prediction. Finally, a phylogenetic tree was produced with neighbor-joining (NJ) method using ClustalX 2.0 and MEGA 4.0 softwares. The Bootstrap method was used to evaluate the accuracy of the phylogenetic tree (Bootstrap value = 1000). 2.4. Comparative real-time PCR The cDNA templates were prepared from A. sinica at different stages of development, including adults, which were grown under various low temperature schemes. Two pairs of primers for As-sdh (forward 5′-CAACGCCACCAGTAGACGG-3′, reverse 5′-CCACCTCGGCTCTTTCT-3′) and β-actin (forward 5′-AGCGGTTGCCATTTATT GTT-3′, reverse 5′-G GTCGTGACTTGACGGACTATAT-3′) were used for real-time PCR analysis. Three parallel reactions were performed for each sample using SYBR Premix Ex Taq (Takara, Dalian, China). The β-actin gene of A. sinica was used to normalize the starting quantity of each sample. Thermal Cycler Dice Real Time system software (Takara, Dalian, China) was used to analyze the gene expression data, and the 2- ΔΔCt method, based on the Ct values of As-sdh and β-actin, was used to calculate the fold increase in the expression of each gene. The data obtained from real-time PCR analysis was analyzed using one-way analysis of variance (ANOVA) followed by Tukey's test with SPSS 16.0; the significance threshold was P b 0.05. 2.5. In situ hybridization The cDNA template for the probe, obtained by PCR (forward 5′-TGCGAAGAGGTGAGGT-3′, reverse 5′-TGCAAGCAGGGTGTAAT-3′) and agarose gel electrophoresis, was cloned into a pGM-T vector to add the T7 and SP6 polymerase binding sites to the gene. A DIG-labeled RNA probe was synthesized using a DIG RNA labeling kit (SP6/T7, Roche). The 0, 5 and 10 h samples were exuviated using 50% NaClO, and all samples were fully rinsed to remove surface salinity using phosphate-buffered saline (PBS) treated with diethylpyrocarbonate (DEPC). The samples were fixed in fresh 4% paraformaldehyde solution at 4 °C for 6–8 h. The samples were then cut into 10 μm-thick sections using a Radial Microtomes. Prehybridization of each sample was then performed at 37 °C for 2 h. The prehybridization buffer contained 1× Denhardt's solution, 0.5 mg/ml salmon sperm DNA, 50% deionized formamide (v/v), and 20× SSC. Hybridization was performed at 52 °C for 12–16 h by adding 10% dextran sulfate and 1 mg/ml DIG-labeled As-sdh probe to the pre-hybridization buffer. Finally, the hybridization signal was detected using a DIG Nucleic Acid Detection Kit (Roche).
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Phosphorylation site prediction revealed ten serine, six threonine and one tyrosine targets (score > 0.5; Table 1). Multiple sequence alignment analysis of the protein sequence of As-SDH revealed a highly conserved amino acid sequence between A. sinica and other 18 species from GenBank, which was also shown by protein sequence domain analysis. The ADH_N domain (from residues 27 to 138 in A. sinica) and the N-terminus and C-terminus of ADH_zinc_N (from residues 179 to 308 in A. sinica) were highly conserved (Fig. 2). As-SDH shares 45.48–48.02% similarity with SDH from other vertebrate species (including Mus musculus, Bos taurus, Cricetulus griseus, Xenopus laevis, Salmo salar, Ictalurus punctatus and Danio rerio), 47.98–53.85% similarity with SDH from arthropods (Drosophila ananassae, Apis mellifera, Acromyrmex echinatior, Harpegnathos saltator, Belgica antarctica, Drosophila melanogaster, Glossina morsitans, Drosophila virilis and Bombyx mori), and only 47.92% similarity with SDH from nematodes (Caenorhabditis elegans, Ascaris suum). A phylogenetic tree was produced to evaluate the evolutionary relationships using the SDH amino acid sequence from the 19 species evaluated (Fig. 3). In this phylogenetic tree analysis, the species were divided into three groups, i.e., the amniote group, the arthropod group, and the nematoda group. A. sinica was assigned to the arthropod group, as expected. 3.2. In situ hybridization analysis of As-sdh To determine where As-sdh was expressed in A. sinica embryos, in situ hybridization was performed with embryos at eight different developmental stages (0, 5, 10, 15, 20 and 40 h, 3 and 5 days; Fig. 4). In embryos at the pre-nauplius stage (0, 5, and 10 h), positive As-sdh signals were detected throughout nearly the entire embryo, especially at the intercellular spaces. At the nauplius stage (15, 20 and 40 h), during which the initial differentiation of the digestive system occurs, the As-sdh hybridization signals were concentrated at the inner wall of the alimentary tract, along with the body surface. After the nauplius stage (3 and 5 days), during which the alimentary tract is fully formed, the body surface and the inner side of the digestive tract were the only regions exhibiting concentrated As-sdh hybridization
3. Results 3.1. Isolation and bioinformatic analysis of As-sdh Using 3′ and 5′ RACE, a 1131 bp full-length As-sdh cDNA, with an open reading frame of 1044 bp, was obtained (GenBank accession number JX406418). The 5′ UTR was 28 bp, and the 3′ UTR was 59 bp with the classic polyadenylation signal AATAA (Fig. 1). The ExPASy translation tool was used to obtain the predicted amino acid sequence. The predicted protein had 348 amino acid residues, with a predicted molecular mass of 37.6 kDa and the theoretical pI of 7.96. Protein sequence domain analysis revealed the presence of the highly conserved ADH_N and ADH_zinc_N domains, which are the most important domains of SDH. No signal peptide was predicted with SignalP 4.0. The subcellar localization was predicted to be as follows: 44.4% endoplasmic reticulum, 22.2% Golgi, 22.2% mitochondrial and 11.1% cytoplasmic. This protein was not predicted to be localized to the transmembrane region, indicating that the As-SDH is primarily located inside the cellular membrane.
Fig. 3. Neighbor-joining phylogenetic tree based on the amino acid sequences of As-SDH and 18 other species from GenBank using the sequence analysis tool MEGA 4.0. The sequences and their accession numbers are indicated in the legend of Fig. 3. Red dot ( ) indicates As-SDH.
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signals. In summary, As-sdh was expressed over a broad area before the nauplius stage, and then became more localized as development progressed, with expression concentrated on the body surface of the embryo and on the lining of the digestive tract.
As-sdh was dramatically upregulated at 20 °C, peaked at 15 °C, decreased markedly at 10 °C, and further decreased at 5 °C.
3.3. Real-time PCR analysis of As-sdh
In this study, we isolated the full-length cDNA of As-sdh from A. sinica. This gene encodes a putative protein containing 348 amino acid residues. Sequence analysis revealed two highly conserved domains, ADH_N and ADH_zinc_N, which are the most important domains of SDH proteins. No signal peptides were found in this protein sequence, and gene expression was primarily confined to the inner walls of membranes and the cytoplasm, indicating that the catalytic sites of this protein are located inside the membrane. Alignment of SDH protein sequences from various species indicated that SDH has a highly conserved structure, particularly in mammalian species, arthropods and nematodes. The catalytic zinc-binding motif usually consists of three residues, i.e., Cys, His, and Glu, and an activated water molecule. Multiple sequence alignment revealed that the zinc-binding residues of SDH in A. sinica are likely to be Cys40, His65 and Glu147. Although the positions of the zinc binding sites are different in different species, the residues are the same. This is also true for the NAD+/NADH binding sites, Arg, Leu, and Val (Baker et al., 1992). As-SDH lacks an extra proline found in human and rat SDH, which may be due to an
Real-time PCR analysis was used to detect the transcript levels of As-sdh during different stages of A. sinica development, from the gastrula to the metanauplius larva stage (Fig. 5). From 0 h to 10 h, the expression level decreased notably, reaching an extremely low level; at the nauplius stage (15, 20 and 40 h), the level of As-sdh expression remained quite low. At the metanauplius stage (5 days), when the polypide was almost fully developed, As-sdh expression increased dramatically, reaching a relatively high level, with the highest expression occurring at 7 days. Therefore, As-sdh expression was down-regulated during early development and up-regulated at the nauplius stage. Real-time PCR analysis was also used to determine the level of As-sdh expression in A. sinica under different temperature–stress conditions (Fig. 6). Adult brine shrimp samples were obtained after they were subjected to low-temperature treatment for 48 h. The levels of As-sdh transcript were significantly different in the shrimp subjected to 25, 20, 15, 10 and 5 °C treatment vs. the (30 °C) control group.
4. Discussion
Fig. 4. In situ hybridization analysis of As-sdh expression during different developmental stages in A. sinica. A–H: experimental group; A1–H1: control group. A: gastrula stage (0 h); B, C: embryonic stage (5 and 10 h); D, E, F: nauplius stage (15, 20, and 40 h); G, H: metanauplius stage (3 and 5 day). Arrows indicate regions with positive hybridization signals.
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Fig. 5. Real-time PCR analysis of As-sdh expression during different stages of A. sinica development. The expression level of As-sdh was set as control group. The x-axis indicates different developmental stages (0 h to 7 day). The y-axis indicates the relative expression level of As-sdh. Data are the mean ± SD of triplicate experiments. * indicates a significant difference compared with the control (P b 0.05), while ** indicates a very significant difference compared with the control (P b 0.01).
intron/exon border arising from a differently spliced mRNA (Williamson et al., 1993). This discrepancy may lead to a slight difference in the secondary structure of As-SDH (Hou et al., 2006). Leucine, rather than methionine in SDH in humans and most other species, is present in the interphase between domains and in the glutamic identification site in As-SDH. Phylogenetic tree analysis revealed that the SDH protein is highly conserved in A. sinica and other species of arthropods, especially in key domains such as the zinc-binding sites and NAD+ binding sites; differences occur in the amino acid residues at other sites. These differences indicate that A. sinica has a relatively low classification status in the arthropod group. More differences were found between the As-SDH and less closely related species such as those of the amniote group. In the human body, SDH may contribute to an increase in free NAD+/NADH levels in the cytoplasm, leading to glucose-linked oxidative stress. SDH is primarily distributed in the heart and liver (Williamson et al., 1993). Through in situ hybridization, we found that As-sdh is expressed in almost every cell of the early embryo (0, 5 and 10 h), with increasing concentrations on the body surface and inner side of the coelom at the nauplius stage (15, 20 and 40 h). After the nauplius stage (3 day to 5 day), As-sdh was mainly expressed in the inner wall of the intestine and on the body surface. This is probably because As-sdh is required to decrease the accumulation of sorbitol that occurs in the cell during diapause, so As-sdh is widely expressed throughout the early embryo. At the nauplius stage, after the digestive system is formed, As-sdh is required for glycometabolism, which mainly occurs on the inner wall of the intestine. Real-time PCR of embryos from different developmental stages showed that As-sdh was expressed at relatively high levels in 0 h embryos. At 15 h to 40 h, As-sdh was expressed at low levels. This
Fig. 6. Real-time PCR analysis of changes in As-sdh expression in response to low temperatures. The control group (red) was cultured at 30 °C. Each group was incubated at the indicated temperature for 48 h. Data are the mean ± SD of triplicate experiments. * indicates a significant difference compared with the control (P b 0.05), while ** indicates a very significant difference compared with the control (P b 0.01).
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indicates that As-sdh may not be a key factor in the embryonic development process; however, this protein is crucial for the diapause termination process, as it is needed to digest the sorbitol that accumulates before diapause. The expression of As-sdh was highly upregulated after 3 day of development because the digestive system develops during this stage, and the adults could obtain the glucide from the surroundings (Hou et al., 2006). When the embryos were exposed to temperatures ranging from 25–15 °C, the level of As-sdh transcript increased dramatically with decreasing temperature. The increase in As-sdh expression mirrored the higher metabolic level required to protect against cold. However, when the temperature was below 15 °C, the expression of As-sdh was significantly reduced with decreasing temperature. Under these temperature conditions, high metabolism is not sufficient to protect the embryos from the cold. This decrease in As-sdh activity may therefore lead to the accumulation of sorbitol, along with other sugar alcohols and glycerol, to help the animal survive the cold (Banfield et al., 2001). In fact, sdh expression depends on many factors. In Drosophila, SDH enzymatic activity is at its lowest level during the larval stages, with the highest level of activity during adulthood (Bischoff, 1978). Accumulation of sorbitol decreases the level of sdh expression, which is observed in response to increased glucose concentrations in human diabetics (Carr and Markham, 1995; Nagasaka et al., 1988). Low-temperature treatment increases the expression of sdh in diapause eggs (Banfield et al., 2001; Teruyuki and Toshinobu, 1992). Before entering the diapause stage, the activity of sdh in A. franciscana is quite low (Zhijun et al., 2007). Therefore, the changes in the expression of sdh are always related to sugar levels, temperature changes or diapause. A. sinica lives in hypersaline waters, often encountering dramatic changes in temperature. As the temperature decreases, the expression of As-sdh increases, indicating that As-sdh plays an important role in the response of A. sinica to low-temperature stress. The high expression levels observed in the diapause termination stage and the metanauplius stage suggest that As-sdh is indispensable to diapause termination and the polyol pathway, but is not so important during embryo development. Acknowledgements This work was supported by grants from the National Natural Science Foundation of China (31071876, and 31272644). We thank anonymous referees for their valuable comments on an earlier version of the manuscript. References Abatzopoulos, T.J., Beardmore, J.A., Clegg, J.S., Sorgeloos, P., 2002. Artemia: Basic and Applied Biology. Kluwer Academic, Dordrecht, The Netherlands. Baker, P.J., Britton, K.L., Rice, D.W., Rob, A., Stillman, T.J., 1992. Structural consequences of sequence patterns in the fingerprint region of the nucleotide binding fold. Implications for nucleotide specificity. J. Mol. Biol. 228, 662–671. Banfield, M.J., Salvucci, M.E., Baker, E.N., Smith, C.A., 2001. Crystal structure of the NADP(H)-dependent ketose reductase from Bemisia argentifolii at 2.3 Å resolution. J. Mol. Biol. 306, 239–250. Bischoff, W.L., 1978. Ontogeny of sorbitol dehydrogenases in Drosophila melanogaster. Biochem. Genet. 5–6, 485–507. Carr, I.M., Markham, A.F., 1995. Molecular genetic analysis of the human sorbitol dehydrogenase gene. Mamm. Genome 6, 645–652. Chu-Moyer, M.Y., et al., 2002. Orally-effective, long-acting sorbitol dehydrogenase inhibitors: synthesis, structure-activity relationships, and in vivo evaluations of novel heterocycle-substituted piperazino-pyrimidines. J. Med. Chem. 45, 511–528. Eklund, R., Horjales, E., Jornvall, H., Branden, C.L., Jeffery, J., 1985. Molecular aspects of functional differences between alcohol and sorbitol dehydrogenases. Biochemistry 24, 8005–8012. Geissen, K., Utz, R., Nimmesgern, H., Lang, H.J., 1992. Substitute pyrimidin-derivate, Verfahren zu ihrer Herstellung und ihre verwendung als reagenzien. Eur. Pat. Appl. 0470, 616. Geissen, K., Utz, R., Grotsch, H., Lang, H.J., Nimmesgern, H., 1994. Sorbitol accumulating pyrimidine derivatives. Arzneimittelforschung 44, 1032–1043. Hou, L., et al., 2006. Establishment and improvement of real-time fluorescence quantitative PCR for actin gene of Artemia sinica. J. Liaoning Norm. Univ. 29, 15–19. Joanisse, D.R., Storey, K.B., 1995. Temperature acclimation and seasonal responses by enzymes in cold-hardy gall insects. Arch. Insect Biochem. Physiol. 28, 339–349.
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