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A galectin with quadruple-domain from red abalone Haliotis rufescens involved in the immune innate response against to Vibrio anguillarum
Fish & Shellfish Immunology 40 (2014) 1e8
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Short sequence report
A galectin with quadruple-domain from red abalone Haliotis rufescens involved in the immune innate response against to Vibrio anguillarum rate* Waleska Maldonado-Aguayo, Jaime Teneb, Cristian Gallardo-Esca n, Laboratory of Biotechnology and Aquatic Genomics, Interdisciplinary Center for Sustainable Aquaculture Research (INCAR), University of Concepcio n, Chile P.O. Box 160-C, Concepcio
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 March 2014 Received in revised form 9 June 2014 Accepted 10 June 2014 Available online 18 June 2014
Galectins are proteins that recognize and bind specifically b-galactosidase residues, playing important roles in the innate immune response of vertebrates and invertebrates. The cDNA of a tandem repeat galectin from the red abalone Haliotis rufescens cDNA (HrGal) was cloned and characterized using rapid amplification of cDNA end technique. The full-length cDNA of HrGal was 2471 bp, with a 5' terminal untranslated region (UTR) of 131 bp, a 3' UTR of 672 pb, and an open reading frame (ORF) of 1668 bp encoding a polypeptide of 556 amino acid. The ORF contains four domains carbohydrate recognition (CRD) with typical conserved motifs, which are important for carbohydrate recognition, and it appear to posses neither a signal peptide nor a transmembrane domain. The deduced amino acid sequence and the multi-domain organization of HrGal were highly similar to those described for other tandem repeat galectins of invertebrate organisms. Quantitative real time PCR analyses indicated that HrGal mRNA was highly expressed in hemocytes and gills tissues. The temporal expression of HrGal mRNA in hemocytes challenged to Vibrio anguillarum was time-dependent, showing u-regulation at 32 h post challenge. The results suggest that HrGal may be involved in the immune innate response against bacterial infection. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Galectins qPCR Immune response Haliotis rufescens
1. Introduction Microbial antigens, generally called pathogen-associated molecular patterns (PAMPs), are recognized by the immune system through a series of pattern recognition receptors (PRRs) [1]. Among the seven PRR groups, galectins play an important role in innate immunity by recognizing and specifically binding glucans on the surface of potentially pathogenic microorganisms [2]. Until now, 15 galectins have been found in mammals [3] that share a conserved carbohydrate recognition domain (CRD) [4]. According to the organization of their domains, galectins have been divided into the following three subfamilies: the proto-type subfamily, which contains only one CRD; the tandem repeat subfamily, which has two homologous CRDs separated by a linker sequence; and the chimera subfamily [5]. In vertebrates, the role of galectins as modulators or effectors of the immune response has been widely studied [6], with observations revealing participation in diverse cellular processes such as proliferation and cellular adhesion [7], development and
* Corresponding author. E-mail addresses: [email protected], (C. Gallardo-Esc arate).
crisgallardo@oceanografia.udec.cl
http://dx.doi.org/10.1016/j.fsi.2014.06.013 1050-4648/© 2014 Elsevier Ltd. All rights reserved.
morphogenesis [8], inflammation [9], and phagocytosis [10], among others. However In mollusks, our knowledge of the features and biological role of galectins is still limited [11], and only a few galectins have been characterized on the functional and molecular levels. The first galectin with a quadruple CRD was identified in the oyster Crassostrea virginica and it was found to participate in the recognition and phagocytosis of the protozoan parasite Perkinsus marinus [12]. Another two galectins with quadruple CRDs were characterized from the pearl oyster Pinctada fucata (PoGal) and Argopecten irradians (AiGal), with mRNA up-regulated in the presence of a microbial challenge [12]. Similarly, in the clam Ruditapes philippinarum (McGal) [13] and freshwater snail Biomphalaria glabrata (BgGal) [11], galectins were found that participate in the immune response. So far, no tandem repeat galectin transcripts have been identified in Haliotis rufescens. The red abalone H. rufescens is a commercially important marine gastropod. The specie was introduced in Chile in 1980 and currently is positioned as one of the major exporting of resource [14]. One of the critical points in the cultivation of abalone is mainly the development of diseases caused by infectious agents such as bacteria of the genus Vibrio [15]. Therefore, understanding the mechanisms of defense red abalone, are necessary for the development of future strategies to control diseases and the development of sustainable
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aquaculture. The objective of this study was to Investigate on a transcriptomic level, the putative role played by galectins in the innate immune response of H. rufescens. The complete mRNA sequence of H. rufescens galectin (HrGal) was characterized, in addition to its expression pattern in individuals challenged by the pathogen Vibrio anguillarum. 2. Materials and methods
Table 2 BLASTx analyses for galectin from H. rufescens in accordance with NCBI curated nonredundant protein database. Query Accession no. sequence
Species
Protein Blast e value description score
HrGal
Argopecten irradians Pinctada fucata Crassostrea gigas Crassostrea virginica Takifugu rubripes
Galectin-2 Galectin Galectin-4 Galectin Galectin-4
ACS72241.1 ACO36044.1 EKC37204.1 ABG75998.1 XP_003968729.1
839 818 854 991 642
9e-177 7e-176 9e-166 2e-163 5e-46
2.1. Sample collection Adults of red abalone H. rufescens (Body length 60 ± 5 mm, weight 22.5 ± 4.0 gr) were collected from a commercial farm located in northern Chile. Then, sixty abalones were left undisturbed for 2 weeks in 200 L polyethylene tanks at 14 ± 1 C, aerated, and fed daily with fresh kelp. After the acclimation period, the specimens (n ¼ 30) were challenged with 200 mL V. anguillarum in PBS solution (3.2 108 cell/ml) by injection into the muscle. The control group (n ¼ 30) was injected with PBS. Samples (n ¼ 4) were collected at 0, 3, 6, 12, 24, 32 and 48 h post-injection for each experimental group, fixed in RNAlater® RNA Stabilization Reagent (Ambion, USA), and stored at 80 C for subsequent RNA extraction. 2.2. RNA isolation and cDNA synthesis The total RNA was extracted from 100 mg of tissue (3 technical replicates from each individual) from the muscle, gills and hemocytes, utilizing the TRizol reagent (Invitrogen®, Life Technologies, USA) according to the manufacturer's instructions. The separation phases were performed with chloroform and were precipitated with 100% isopropanol before maintenance at 20 C. Subsequently, the RNA was washed using 75% ethanol. The concentration and purity of the isolated RNA were measured in a ND-1000 spectrophotometer (NanoDrop® Technologies, Inc), and the integrity was visualized through electrophoresis on a 1.2% MOPSAgarose gel stained with 0.0001% ethidium bromide. cDNA synthesis was performed from 200 ng of RNA with the cDNA RevertAid™ H Minus First Strand cDNA Synthesis kit (Thermo scientific, Maryland, USA) according to manufacturer's instructions. 2.3. Cloning of the full-length HrGal cDNA Partial sequences of galectin were obtained from the cDNA library generated through 454 pyrosequencing of H. rufescens and identified using the bioinformatics software CLC Genomic Workbench (CLC Bio, Aarhus, Denmark) with an e value of 1E-05. The EST database is available in the Dryad Digital Repository under the access http://dx.doi.org/10.5061/dryad.dh5qs. Specific primers were designed using the Primer 3 tool included in the bioinformatics software Geneious 5.1.3 (Biomatters, New Zeland) (Table 1). The partial sequence was amplified by conventional PCR in a final reaction volume of 12.5 ml. The conditions used for amplification were as follows: 94 C for 2 min and 30 s (holding state), 35 cycles
Table 1 Sequence primers used for gene cloning and qPCR analysis. Primer
Sequence
Tm
HrGal_F HrGal_R qHrGal_F qHrGal_R qHrEF2_F qHrEF2_R
CCTACAACCAGACTATCA GAAGTTCTAGGTAACTCTG CCCTAATGTTTCCAGATTC CCTGATAGTCTGGTTGTA CTGGAAGATGAAGATGAG GAGATTCTAAATCCCAG
52 54 51
at 94 C for 30 s (denaturation), 60 C (T annealing) for 30 s, 72 C for 45 s, and 72 C for 5 min. The PCR product was visualized through electrophoresis on 1.2% agarose gel and posteriorly sent to Macrogen Inc. (Korea) for sequencing in the capillary sequencer ABI 3730xl (Applied Biosystems). The sequences obtained for galectin were assembled using the Geneious 5.1.3 software, and analysis with BLASTx was carried out against the non-redundant GenBank database (Table 2). Based on this sequence, new primers were designed (Table 1) for the amplification of the extreme 3' and 5' untranslated region UTR, which was performed using the FirstChoice® RLM RACE Kit (Ambion®, Life Technologies, USA) according to the manufacturer's instructions. The fragments obtained from the extreme 3' and 5' UTR were inserted in the cloning vector using the TOPO TA Cloning Kit (Invitrogen™, Life Technologies, Carlsbad, CA, USA), transformed to electrocompetent bacteria Escherichia coli JM109, and cultivated in LB/amp/IPTG/Xgal agar overnight at 37 C. Positive clones were purified to obtained plasmids using the E.Z.N.A® Plasmid DNA Mini Kit II (Omega Bio-tek, Doraville, GA, USA). The obtained plasmids were sequenced in both directions, and these sequences were then assembled using the Geneious 5.1.3 software. 2.4. Sequencing analysis The amino acid sequence of Galectin was analyzed using BLAST algorithm (http://blast.ncbi.nlm.nih.gov/Blast) and the expert Protein Analysis System (http://www.expasy.org/). The protein domains for Galectin were revealed by the simple modular architecture research tool (SMART) version 4.0 (http://smart.emblheidelberg.de/). The tertiary structure of HrGal was modeled using Modeller 9.12 [16,17] with known structures for each domain. The structures were selected by a protein blast against the Protein Data Bank (PDB). The resultant structures were visualized with The €dinger, PyMOL Molecular Graphics system (Version 1.6.0.0, Schro LLC.). ClustalW Multiple Alignment software was used to create the multiple sequence alignment. An uprooted phylogenetic three was constructed based on the sequence alignment by the neighborjoining (NJ) algorithm using the bioinformatic software Geneious 5.1.3 (Biomatters, New Zeland) using Aplysia californica as outgroup. The reliability of the branching was tested through bootstrap re-sampling (1000 pseudo-replicates). 2.5. Tissue distribution of HrGal mRNA and temporal expression pattern after bacterial challenge Quantitative expression analysis was performed using a real time thermocyler StepOnePlus (Applied Biosystems, Life Technologies, USA) to determine the tissue-specific expression of HrGal in both challenged and control individuals. For the expression analysis, a-Tubulin (a-Tub), b-Tubulin (b-Tub), Actin, and Elongation factors 1 and 2 (EF1 and EF2) were identified as housekeeping genes (HKG) in the EST database of H. rufescens. Here, EF2 was selected due to its stable value inferred through the NormFinder algorithm.
W. Maldonado-Aguayo et al. / Fish & Shellfish Immunology 40 (2014) 1e8 Table 3 Amino acid sequences used for phylogenetic analysis. Name
Organisms
Accession no.
CRD domain
AiGal2 CvGal Pf galectin Galectin-4 Galectin-4 Galectin-4 OeGal Galectin-4 MCGal GRG LEC-8 LEC-6 As_gal Galectin-8 AGL
Argopecten irradians Crassostrea virginica Pinctada fucata Canus lupus familiaris Myotis brandtii Chinchilla lanigera Ostrea edulis Haliotis discus hannai Ruditapes philippinarum Globodera rostochiensis Caenorhabditis elegans Caenorhabditis elegans Anopheles stephensi Crassostrea gigas Aphysia californica
ACS72241 ABG75998 ACO36044 ADR80620 XP_005886040 XP_005399890 ADF80416 ABN54798 ACA09732 AAB61596 BAB11964 BAA09794 AAO06842 EKC40502 XP_005103462
Multiple CRD
Tandem repeat
3
was modeled with 3i8tA and 3zxfA using multiple template function of Modeller. Each one of these alignments shows an identity over 32%. Further, the CRD adopted a typical galectin fold composed of two anti-parallel b-sheets, arranged in b-sheets sandwich motif without any a-helix. The seven conserved residues in all four CRDs maintained their positions and orientations in the binding cleft (Fig. 3). 3.3. Homology and phylogenetic analysis of HrGal
Single CRD
Out-group
PCR reactions were carried out in a total volume of 10 mL using the Maxima SYBR Green/ROX qPCR Master mix (Thermo Scientific, USA). The conditions for amplification were as follows: 95 C for 10 min (holding State), 40 cycles at 95 C for 30 s (denaturation), 60 C (T annealing) for 30 s, and 72 C for 30 s (extension). Finally, to determine the presence of nonspecific products, dimer formations, or contamination, a denaturation curve was analyzed. All samples were tested in triplicate. The comparative Ct method was used to analyze the relative expression level. The fold change was calculated as the relative folds of expression of EF2 by 2 DDCT method [18]. 2.6. Statistical analysis Genetic expression analysis was carried out with the STATISTICA 7 software (Statsoft Inc. USA). To evaluate significant differences, a ShapiroeWilk test was performed to determine data normality. Data with a non-parametric distribution were evaluated with an ANOVA test while those that had a parametric distribution were evaluated with a KruskaleWallis test. Differences were considered statistically significant at p < 0.05. 3. Results 3.1. cDNA cloning and sequence analysis of HrGal The full sequence of HrGal cDNA (GenBank accession number KJ183034) consisted of 2471 bp, with a 5' UTR of 131 bp and a 3'UTR of 672 bp containing a canonical polyadenylation signaling site and a poly (A) tail. The open reading frame (ORF) contained 1668 bp encoding for a polypeptide of 556 amino acids (Fig. 1). The sequence contained four CRDs with a theoretical isoelectric point of 4.98 and a predicted molecular mass of 68.3 kDa. SMART analysis revealed the following four dissimilarly arranged CRDs which indicated that HrGal presented a multi-domain galectin: CRD1 (Residues 43e184), CRD2 (190e131), CDR3 (325e458), and CRD4 (463e598), with the conserved amino acids characteristic involved in sugar binding activity (H-NPR and WG-ER) (Fig. 2). Analysis of homology revealed that the four CRDs shared a similarity of 33.1%, demonstrating the high site conservation characteristic of galectin. 3.2. The potential tertiary structures of HrGal The structure of the domains of HrGal was predicted by homology with structures of other galectins deposited in PDB and identified by Blastp. The structures used were 3galA, 3zxfA and 3vknA for the domains D2, D3 and D4, respectively. The D1 domain
The deduced amino acid sequence and organization of the multi-domain HrGal galectin were similar to those described for other members of the galectin superfamily The highest similarities displayed were with A. irradians (ACS72241, 67.5% identity), Pinctada fucata (ACO36044, 66.7% identity), Crassostrea gigas (EKC37204, 65.9% identity), and C. virginica (ABG75998, 65.8% identity) (Fig. 4). Based on multiple sequence alignments of HrGal with other galectins, all of the signature sequences of the galectin family were identified in every CRD of HrGal. An unrooted phylogenetic tree was constructed using the neighbor-joining method with a 1000 bootstrap test. HrGal was grouped in the same clade as galectin from Pinctada fucata, as evidenced by a 100% bootstrap, and together with galectin sequences with quadruple CRDs (Fig 5). 3.4. Tissue distribution of the HrGal Real time quantitative PCR analysis was performed to investigate the expression pattern of HrGal, mRNA with the EF2 as an internal control. The transcription expression analysis in different tissues showed that HrGal was constitutively expressed in all tested tissues including the hemocytes, gill and muscle. However, the expression level of HrGal mRNA in the unchallenged group was higher in hemocytes expressing himself 0.3 and 0.1 times more than in muscle and gills respectively (Fig. 6). 3.5. Temporal expression profile of HrGal mRNA after V. anguillarum challenge The temporal expression patterns of HrGal mRNA in hemocytes after the V. anguillarum challenge were quantified by real time PCR. At 3, 6 and 12 h post challenge, the hemocytes exhibited a low level of HrGal expression, but this was followed by significant increase to 9 fold greater than the control group at 24 h post challenge (p < 0.05). The transcripts of HrGal in hemocytes were significantly up-regulated (p < 0.05) at 32 h post-stimulation and increased to 24.5 fold higher than levels observed in the control group. Finally, the expression levels of HrGal mRNA decreased at 48 h postinjection (Fig. 7). In all sampling points, the challenged organisms had a higher expression than the control group injected with PBS solution. 4. Discussion 4.1. Characterization of HrGal mRNA The present study cloned and characterized the complete cDNA sequence for a tandem repeat galectin from H. rufescens. The sequence revealed four CRDs in tandem and connected by a peptide linker. Multiple alignments of HrGal with other galectins showed that the sequence contained crucial residues needed to bind to carbohydrates. These residues included the typical motifs H-NPR and WG-ER, which form glucan binding sites [5], thus reflecting the evolutionary conservation of galectin. As with other described galectins, such as in Pinctada fucata [12,19,20], Tegillarca granosa [21], Trachidermus fasciatus [22], Ruditapes philippinarum [13], and
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Fig. 1. Nucleotide and deduced amino acid sequences of galectin from Haliotis rufescens. The nucleotides are numbered on the left margin. The four CRD identified by SMART program are in grey boxed. The predicted N -glycosylation site is underlined. The conserved residues involved in carbohydrate binding specificity are in italic and bold. Asterisk marks the stop codon and polyadenylation signal in the 3'UTR is marked in bold. The tertiary structure prediction of each CRD domain is adjacent to the right of the sequence.
Fig. 2. Scheme of the structural domains of HrGal mRNA. Conserved amino acids that are involved in sugar binding activity (H-NPR and WG-ER) are denoted in bold. Asterisk marks the stop codon.
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Fig. 3. Diagram showing the molecular homology of HrGal tertiary structure. The seven residues of the ligand-binding site are remarked in order to show conservation at sequence and structural level.
C. gigas [23], HrGal lacked a signal peptide sequence and a hydrophobic transmembrane segment, which suggests that HrGal would be secreted through a non-classical process [24]. According to structural variations in the CRDs, the galectin superfamily is divided into three subfamilies, proto-type, chimera, or tandem repeat [5]. Interestingly, galectins with quadruple CRDs have been found only in mollusks, among which are CvGal, AiGal1, AiGal2, AiFal2, and PfGal [12,25,26]. Phylogenetic analysis revealed that HrGal was grouped in the same clade as other galectins from mollusks with quadruple CRDs, which suggests that these sequences could have a common evolutionary history. Galectin sequences containing multiple CRDs with tandem-repeats or containing only one CRD were grouped into another two clades. The three-dimensional structure prediction of HrGal revealed that the CRD motif was tightly folded and contained two anti-parallel bsheets arranged in a b-sheet sandwich structure same as the structure of galectins previously reported. These results reflect those reported in A. irradians, where the three-dimensional structure of the sequence contained four CRD domain [12]. Based in the structural and evolutional features of HrGal, we confirm that HrGal is a member of galectin subfamily. However, the three-dimensional structure of HrGal is theoretical and remains to be confirmed by xray crystallography.
4.2. mRNA expression patterns in different tissues of H. rufescens When pathogenic microorganisms break through the physical barriers of the host, this is recognized by different PRRs, which in turn trigger a series of cellular and humoral components that isolate the pathogen [27]. There are numerous studies in mammals that cite galectin as a key PRR in the activation of the immune system [28]. In order to investigate the participation that HrGal has in the immune response of H. rufescens, the expression of HrGal was analyzed in distinct tissues, and observations found that HrGal was constitutively expressed in all tissues. However, the highest expression levels were found in tissues key to the immune response in marine invertebrates, such as the hemocytes and gills [29]. Likewise, the present results agree with those found in other studies, such as that in Pinctada fucata which observed PfGal to be principally expressed in the digestive gland and hemocytes [20]. Another study in this same species found expression principally in the hemocytes [12,30,31], a result which was also found for CvGal from C. virginica [25]. Moreover, the gills represent the first barrier of defense against pathogenic microorganisms in marine invertebrates [32]. For instance, there are evidences that Lectins can be found in gill in C. virginica [33], Eriocheir sinensis [34] and Chlamys farreri [35]. This suggests that the function of HrGal could
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Fig. 4. Multiple sequence alignment among the full-length deduced amino acid sequence of HrGal and other galectin sequences with quadruple-domain, from invertebrates such as AiGal2 (ACS72241), CvGal (ABG75998) and Pf Galectin (ACO36044). The conserved amino acid residues are shaded in dark, and similar amino acids are shaded in grey. Gaps are indicated by dashes to improve the alignment Caenorhabditis elegans (BAB19964), Crassostrea gigas (EKC40502) and Asgal Anopheles stephensi (AAO06842).
Fig. 5. A phylogenetic tree of galectin family members constructed with the neighbor-joining method. Numbers at each branch indicate the support (%) in 1000 bootstrap pseudoreplication by neighbor-joining. Sequences used to generate phylogenetic analysis are shown in Table 3 and Aplysia californica (XP_005103462.1) was used as out-group.
be related to the immune response associated to haemolymph and gills tissue [36]. 4.3. mRNA expression patterns of HrGal challenged to V. anguillarum In mammals, the functions of galectins in the immune response have been well established, and these participate in amplifying the immune response [28], in activating the inflammatory response [9], and in host-pathogen interactions [7]. In contrast, little information is available for invertebrates on the processes in which galectins
have a role, especially concerning the transduction mechanisms, which these trigger. In order to elucidate the putative function of HrGal in the immune response of the red abalone, relative expression analysis was performed in hemocytes from individuals challenged by V. anguillarum. Our results showed a time-dependent expression pattern, extremely up-regulated after stimulation with V. anguillarum. Hemocytes as the primary line of immune defense against invading microbial and parasitic infection are capable of eliminating infections through phagocytic or encapsulation response [29]. Our results show that after bacterial challenge, mRNA expression in the hemocytes significantly increased, in
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Acknowledgments This study was supported by the FONDAP (15110027) project granted by CONICYT-Chile. References
Fig. 6. HrGal mRNA expression level in different tissues in individuals unchallenged. The expression levels were normalized by a-Tub. The bars are shown the standard deviation ± SD (n ¼ 3), significant differences were considered with p < 0.05.
Fig. 7. Real time PCR analysis from HrGal, gene expression in hemocytes of H. rufescens challenged with Vibrio anguillarum. Data were analyzed from three individuals. The mRNA of HrGal was measured at 0-3-6-12-24-32 and 48 h post challenge. The relative HrGal expression level as expressed by 2DDCT was determined. Elongation factor was used as an internal control. Verticals bars represented the mean ± SD. Significant differences were considered with p < 0.05.
contrast to the control group. The expression level of HrGal transcript was up-regulated at 3 h post challenge, suggesting the activation of defense against invading pathogen. The decreasing of HrGal transcripts in hemocytes after 6e12 h post challenge might be due to the translation process of HrGal in response to the bacterial infection. However, the increasing expression from 24 to 32 h evidences a recovery of the immune response. Similar results have been reported in the clam Tagillarca granosa exposed to Vibrio parahaemoliticus, where the peak of expression was recorded at 3 and 12 h post-stimulation [21], and also in the clam Ruditapes philippinarum, where a peak in expression was observed at 24 h post challenge to Vibrio alginolyticus [37]. In the same way in C. virginica, A. irradians and Tegillarca granosa, up-regulation was observed in response to pathogens [2,12,21]. The present study suggests that HrGal is associated to the molecular function of pathogens recognition. 5. Conclusion The results of the present study demonstrated that HrGal was constitutively expressed in all analyzed tissues. The highest expression of HrGal was found in hemocytes exposed to V. anguillarum, which suggests participation in innate immune response of H. rufescens.
[1] Medzhitov R, Janeway Jr C. Innate immune recognition: mechanisms and pathways. Immunol Rev 2000;173:89e97. [2] Tasumi S, Vasta GR. A galectin of unique domain organization from hemocytes of the eastern oyster (Crassostrea virginica) is a receptor for the protistan parasite Perkinsus marinus. J Immunol 2007;179:3086e98. [3] Arata Y, Hirabayashi J, Kasai KI. Sugar binding properties of the two lectin domains of the tandem repeat-type galectin LEC-1 (N32) of Caenorhabditis elegans : detailed analysis by an improved frontal affinity chromatography method. J Biol Chem 2001;276:3068e77. [4] Barondes SH, Cooper DN, Gitt MA, Leffler H. Galectins. Structure and function of a large family of animal lectins. J Biol Chem 1994;269:20807e10. [5] Hirabayashi J, Hashidate T, Arata Y, Nishi N, Nakamura T, Hirashima M, et al. Oligosaccharide specificity of galectins: a search by frontal affinity chromatography. Biochim Biophys Acta 2002;1572:232e54. [6] Kasai KI, Hirabayashi J. Galectins: a family of animal lectins that decipher glycocodes. J Biochem 1996;119:1e8. [7] Colin Hughes R. Galectins as modulators of cell adhesion. Biochimie 2001;83: 667e76. [8] Vasta GR, Ahmed H, Du S, Henrikson D. Galectins in teleost fish: zebrafish (Danio rerio) as a model species to address their biological roles in development and innate immunity. Glycoconj J 2004;21:503e21. [9] Liu FT, Yang RY, Hsu DK. Galectins in acute and chronic inflammation. Ann N Y Acad Sci 2012;1253:80e91. [10] Rabinovich GA, Gruppi A. Galectins as immunoregulators during infectious processes: from microbial invasion to the resolution of the disease. Parasite Immunol 2005;27:103e14. [11] Yoshino TP, Dinguirard N, Kunert J, Hokke CH. Molecular and functional characterization of a tandem-repeat galectin from the freshwater snail Biomphalaria glabrata, intermediate host of the human blood fluke Schistosoma mansoni. Gene 2008;411:46e58. [12] Song X, Zhang H, Wang L, Zhao J, Mu C, Song L, et al. A galectin with quadruple-domain from bay scallop Argopecten irradians is involved in innate immune response. Dev Comp Immunol 2011;35:592e602. [13] Kim JY, Kim YM, Cho SK, Choi KS, Cho M. Noble tandem-repeat galectin of Manila clam Ruditapes philippinarum is induced upon infection with the protozoan parasite Perkinsus olseni. Dev Comp Immunol 2008;32:1131e41. ~ a: Editorial Mundi[14] Bautista C. Moluscos, Tecnología de Cultivo. Madrid Espan Prensa; 1989. pp. 54e5. y 143-4 pp. n en [15] C aceres J. Enfermedades de los abalones En: Seminario: El cultivo del abalo n actual y perspectivas. Puerto Montt, Chile: Fundacio n Chile; 2000. Chile, situacio [16] Eswar N, Webb B, Marti-Renom MA, Madhusudhan MS, Eramian D, Shen MY, et al. Comparative protein structure modeling using modeller. Current Protocols in Bioinformatics: John Wiley & Sons, Inc; 2002. [17] Sali A, Blundell TL. Comparative protein modelling by satisfaction of spatial restraints. J Mol Biol 1993;234:779e815. [18] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using realtime quantitative PCR and the 2DDCT method. Methods 2001;25:402e8. [19] Jin XK, Li WW, He L, Lu W, Chen LL, Wang Y, et al. Molecular cloning, characterization and expression analysis of two apoptosis genes, caspase and nm23, involved in the antibacterial response in Chinese mitten crab, Eriocheir sinensis. Fish Shellfish Immunol 2011;30:263e72. [20] Zhang D, Hu Y, Guo H, Cui S, Su T, Jiang S. cDNA cloning and mRNA expression of a tandem-repeat galectin (PoGal2) from the pearl oyster, Pinctada fucata. Genet Mol Res 2011;10:1963e74. [21] Bao Y, Shen H, Zhou H, Dong Y, Lin Z. A tandem-repeat galectin from blood clam Tegillarca granosa and its induced mRNA expression response against bacterial challenge. Genes Genom 2013;35:733e40. [22] Yang H, Yu S, Chai Y, Liu Y, Zhang Q, Ding X, et al. A galectin from roughskin sculpin, Trachidermus fasciatus: molecular cloning and characterization. Fish Shellfish Immunol 2013;35:815e24. [23] Yamaura K, Takahashi KG, Suzuki T. Identification and tissue expression analysis of C-type lectin and galectin in the Pacific oyster, Crassostrea gigas. Comp Biochem Physiol B Biochem Mol Biol 2008;149:168e75. [24] Wada J, Kanwar YS. Identification and characterization of galectin-9, a novel b-galactoside-binding mammalian lectin. J Biol Chem 1997;272:6078e86. [25] Kurz S, Jin C, Hykollari A, Gregorich D, Giomarelli B, Vasta GR, et al. Haemocytes and plasma of the eastern oyster (Crassostrea virginica) display a diverse repertoire of sulphated and blood group A-modified N-glycans. J Biol Chem 2013;288: 24410e28. http://dx.doi.org/10.1074/jbc.M113.478933. [26] Song X, Zhang H, Zhao J, Wang L, Qiu L, Mu C, et al. An immune responsive multidomain galectin from bay scallop Argopectens irradians. Fish Shellfish Immunol 2010;28:326e32. [27] Rowley A, Powell A. Invertebrate immune systems- specific, quasi-specific, or nonspecific? J Immunol 2007;179:7209e14. [28] Pace K, Baum L. Insect galectins: roles in immunity and development. Glycoconj J 2004;19:607e14.
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[29] Anderson RS. Hemocyte-derived reactive oxygen intermediate production in four bivalve mollusks. Dev Comp Immunol 1994;18:89e96. [30] Zhang D, Jiang S, Hu Y, Cui S, Guo H, Wu K, et al. A multidomain galectin involved in innate immune response of pearl oyster Pinctada fucata. Dev Comp Immunol 2011;35:1e6. [31] Li WW, He L, Jin XK, Jiang H, Chen LL, Wang Y, et al. Molecular cloning, characterization and expression analysis of cathepsin A gene in Chinese mitten crab, Eriocheir sinensis. Peptides 2011;32:518e25. re E, Destoumieux[32] Schmitt P, Rosa RD, Duperthuy M, De Lorgeril J, Bache n D. The antimicrobial defense of the Pacific oyster, Crassostrea gigas. Garzo How diversity may compensate for scarcity in the regulation of resident/ pathogenic microflora. Front Microbiol 2012;3:1e28. [33] Espinosa EP, Perrigault M, Ward JE, Shumway SE, Allam B. Lectins associated with the feeding organs of the oyster Crassostrea virginica can mediate particle selection. Biol Bull 2009;217:130e41.
[34] Jin XK, Li WW, Cheng L, Li S, Guo XN, Yu AQ, et al. Two novel short C-type lectin from Chinese mitten crab, Eriocheir sinensis, are induced in response to LPS challenged. Fish Shellfish Immunol 2012;33:1149e58. [35] Wang H, Song L, Li C, Zhao J, Zhang H, Ni D, et al. Cloning and characterization of a novel C-type lectin from zhikong scallop Chlamys farreri. Mol Immunol 2007;44:722e31. [36] Yu Y, Yuan S, Yu Y, Huang H, Feng K, Pan M, et al. Molecular and biochemical characterization of galectin from amphioxus: primitive galectin of chordates participated in the infection processes. Glycobiology 2007;17:774e83. [37] Moreira R, Balseiro P, Romero A, Dios S, Posada D, Novoa B, et al. Gene expression analysis of clams Ruditapes philippinarum and Ruditapes decussatus following bacterial infection yields molecular insights into pathogen resistance and immunity. Dev Comp Immunol 2012;36:140e9.
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Short sequence report
A galectin with quadruple-domain from red abalone Haliotis rufescens involved in the immune innate response against to Vibrio anguillarum rate* Waleska Maldonado-Aguayo, Jaime Teneb, Cristian Gallardo-Esca n, Laboratory of Biotechnology and Aquatic Genomics, Interdisciplinary Center for Sustainable Aquaculture Research (INCAR), University of Concepcio n, Chile P.O. Box 160-C, Concepcio
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 March 2014 Received in revised form 9 June 2014 Accepted 10 June 2014 Available online 18 June 2014
Galectins are proteins that recognize and bind specifically b-galactosidase residues, playing important roles in the innate immune response of vertebrates and invertebrates. The cDNA of a tandem repeat galectin from the red abalone Haliotis rufescens cDNA (HrGal) was cloned and characterized using rapid amplification of cDNA end technique. The full-length cDNA of HrGal was 2471 bp, with a 5' terminal untranslated region (UTR) of 131 bp, a 3' UTR of 672 pb, and an open reading frame (ORF) of 1668 bp encoding a polypeptide of 556 amino acid. The ORF contains four domains carbohydrate recognition (CRD) with typical conserved motifs, which are important for carbohydrate recognition, and it appear to posses neither a signal peptide nor a transmembrane domain. The deduced amino acid sequence and the multi-domain organization of HrGal were highly similar to those described for other tandem repeat galectins of invertebrate organisms. Quantitative real time PCR analyses indicated that HrGal mRNA was highly expressed in hemocytes and gills tissues. The temporal expression of HrGal mRNA in hemocytes challenged to Vibrio anguillarum was time-dependent, showing u-regulation at 32 h post challenge. The results suggest that HrGal may be involved in the immune innate response against bacterial infection. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Galectins qPCR Immune response Haliotis rufescens
1. Introduction Microbial antigens, generally called pathogen-associated molecular patterns (PAMPs), are recognized by the immune system through a series of pattern recognition receptors (PRRs) [1]. Among the seven PRR groups, galectins play an important role in innate immunity by recognizing and specifically binding glucans on the surface of potentially pathogenic microorganisms [2]. Until now, 15 galectins have been found in mammals [3] that share a conserved carbohydrate recognition domain (CRD) [4]. According to the organization of their domains, galectins have been divided into the following three subfamilies: the proto-type subfamily, which contains only one CRD; the tandem repeat subfamily, which has two homologous CRDs separated by a linker sequence; and the chimera subfamily [5]. In vertebrates, the role of galectins as modulators or effectors of the immune response has been widely studied [6], with observations revealing participation in diverse cellular processes such as proliferation and cellular adhesion [7], development and
* Corresponding author. E-mail addresses: [email protected], (C. Gallardo-Esc arate).
crisgallardo@oceanografia.udec.cl
http://dx.doi.org/10.1016/j.fsi.2014.06.013 1050-4648/© 2014 Elsevier Ltd. All rights reserved.
morphogenesis [8], inflammation [9], and phagocytosis [10], among others. However In mollusks, our knowledge of the features and biological role of galectins is still limited [11], and only a few galectins have been characterized on the functional and molecular levels. The first galectin with a quadruple CRD was identified in the oyster Crassostrea virginica and it was found to participate in the recognition and phagocytosis of the protozoan parasite Perkinsus marinus [12]. Another two galectins with quadruple CRDs were characterized from the pearl oyster Pinctada fucata (PoGal) and Argopecten irradians (AiGal), with mRNA up-regulated in the presence of a microbial challenge [12]. Similarly, in the clam Ruditapes philippinarum (McGal) [13] and freshwater snail Biomphalaria glabrata (BgGal) [11], galectins were found that participate in the immune response. So far, no tandem repeat galectin transcripts have been identified in Haliotis rufescens. The red abalone H. rufescens is a commercially important marine gastropod. The specie was introduced in Chile in 1980 and currently is positioned as one of the major exporting of resource [14]. One of the critical points in the cultivation of abalone is mainly the development of diseases caused by infectious agents such as bacteria of the genus Vibrio [15]. Therefore, understanding the mechanisms of defense red abalone, are necessary for the development of future strategies to control diseases and the development of sustainable
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aquaculture. The objective of this study was to Investigate on a transcriptomic level, the putative role played by galectins in the innate immune response of H. rufescens. The complete mRNA sequence of H. rufescens galectin (HrGal) was characterized, in addition to its expression pattern in individuals challenged by the pathogen Vibrio anguillarum. 2. Materials and methods
Table 2 BLASTx analyses for galectin from H. rufescens in accordance with NCBI curated nonredundant protein database. Query Accession no. sequence
Species
Protein Blast e value description score
HrGal
Argopecten irradians Pinctada fucata Crassostrea gigas Crassostrea virginica Takifugu rubripes
Galectin-2 Galectin Galectin-4 Galectin Galectin-4
ACS72241.1 ACO36044.1 EKC37204.1 ABG75998.1 XP_003968729.1
839 818 854 991 642
9e-177 7e-176 9e-166 2e-163 5e-46
2.1. Sample collection Adults of red abalone H. rufescens (Body length 60 ± 5 mm, weight 22.5 ± 4.0 gr) were collected from a commercial farm located in northern Chile. Then, sixty abalones were left undisturbed for 2 weeks in 200 L polyethylene tanks at 14 ± 1 C, aerated, and fed daily with fresh kelp. After the acclimation period, the specimens (n ¼ 30) were challenged with 200 mL V. anguillarum in PBS solution (3.2 108 cell/ml) by injection into the muscle. The control group (n ¼ 30) was injected with PBS. Samples (n ¼ 4) were collected at 0, 3, 6, 12, 24, 32 and 48 h post-injection for each experimental group, fixed in RNAlater® RNA Stabilization Reagent (Ambion, USA), and stored at 80 C for subsequent RNA extraction. 2.2. RNA isolation and cDNA synthesis The total RNA was extracted from 100 mg of tissue (3 technical replicates from each individual) from the muscle, gills and hemocytes, utilizing the TRizol reagent (Invitrogen®, Life Technologies, USA) according to the manufacturer's instructions. The separation phases were performed with chloroform and were precipitated with 100% isopropanol before maintenance at 20 C. Subsequently, the RNA was washed using 75% ethanol. The concentration and purity of the isolated RNA were measured in a ND-1000 spectrophotometer (NanoDrop® Technologies, Inc), and the integrity was visualized through electrophoresis on a 1.2% MOPSAgarose gel stained with 0.0001% ethidium bromide. cDNA synthesis was performed from 200 ng of RNA with the cDNA RevertAid™ H Minus First Strand cDNA Synthesis kit (Thermo scientific, Maryland, USA) according to manufacturer's instructions. 2.3. Cloning of the full-length HrGal cDNA Partial sequences of galectin were obtained from the cDNA library generated through 454 pyrosequencing of H. rufescens and identified using the bioinformatics software CLC Genomic Workbench (CLC Bio, Aarhus, Denmark) with an e value of 1E-05. The EST database is available in the Dryad Digital Repository under the access http://dx.doi.org/10.5061/dryad.dh5qs. Specific primers were designed using the Primer 3 tool included in the bioinformatics software Geneious 5.1.3 (Biomatters, New Zeland) (Table 1). The partial sequence was amplified by conventional PCR in a final reaction volume of 12.5 ml. The conditions used for amplification were as follows: 94 C for 2 min and 30 s (holding state), 35 cycles
Table 1 Sequence primers used for gene cloning and qPCR analysis. Primer
Sequence
Tm
HrGal_F HrGal_R qHrGal_F qHrGal_R qHrEF2_F qHrEF2_R
CCTACAACCAGACTATCA GAAGTTCTAGGTAACTCTG CCCTAATGTTTCCAGATTC CCTGATAGTCTGGTTGTA CTGGAAGATGAAGATGAG GAGATTCTAAATCCCAG
52 54 51
at 94 C for 30 s (denaturation), 60 C (T annealing) for 30 s, 72 C for 45 s, and 72 C for 5 min. The PCR product was visualized through electrophoresis on 1.2% agarose gel and posteriorly sent to Macrogen Inc. (Korea) for sequencing in the capillary sequencer ABI 3730xl (Applied Biosystems). The sequences obtained for galectin were assembled using the Geneious 5.1.3 software, and analysis with BLASTx was carried out against the non-redundant GenBank database (Table 2). Based on this sequence, new primers were designed (Table 1) for the amplification of the extreme 3' and 5' untranslated region UTR, which was performed using the FirstChoice® RLM RACE Kit (Ambion®, Life Technologies, USA) according to the manufacturer's instructions. The fragments obtained from the extreme 3' and 5' UTR were inserted in the cloning vector using the TOPO TA Cloning Kit (Invitrogen™, Life Technologies, Carlsbad, CA, USA), transformed to electrocompetent bacteria Escherichia coli JM109, and cultivated in LB/amp/IPTG/Xgal agar overnight at 37 C. Positive clones were purified to obtained plasmids using the E.Z.N.A® Plasmid DNA Mini Kit II (Omega Bio-tek, Doraville, GA, USA). The obtained plasmids were sequenced in both directions, and these sequences were then assembled using the Geneious 5.1.3 software. 2.4. Sequencing analysis The amino acid sequence of Galectin was analyzed using BLAST algorithm (http://blast.ncbi.nlm.nih.gov/Blast) and the expert Protein Analysis System (http://www.expasy.org/). The protein domains for Galectin were revealed by the simple modular architecture research tool (SMART) version 4.0 (http://smart.emblheidelberg.de/). The tertiary structure of HrGal was modeled using Modeller 9.12 [16,17] with known structures for each domain. The structures were selected by a protein blast against the Protein Data Bank (PDB). The resultant structures were visualized with The €dinger, PyMOL Molecular Graphics system (Version 1.6.0.0, Schro LLC.). ClustalW Multiple Alignment software was used to create the multiple sequence alignment. An uprooted phylogenetic three was constructed based on the sequence alignment by the neighborjoining (NJ) algorithm using the bioinformatic software Geneious 5.1.3 (Biomatters, New Zeland) using Aplysia californica as outgroup. The reliability of the branching was tested through bootstrap re-sampling (1000 pseudo-replicates). 2.5. Tissue distribution of HrGal mRNA and temporal expression pattern after bacterial challenge Quantitative expression analysis was performed using a real time thermocyler StepOnePlus (Applied Biosystems, Life Technologies, USA) to determine the tissue-specific expression of HrGal in both challenged and control individuals. For the expression analysis, a-Tubulin (a-Tub), b-Tubulin (b-Tub), Actin, and Elongation factors 1 and 2 (EF1 and EF2) were identified as housekeeping genes (HKG) in the EST database of H. rufescens. Here, EF2 was selected due to its stable value inferred through the NormFinder algorithm.
W. Maldonado-Aguayo et al. / Fish & Shellfish Immunology 40 (2014) 1e8 Table 3 Amino acid sequences used for phylogenetic analysis. Name
Organisms
Accession no.
CRD domain
AiGal2 CvGal Pf galectin Galectin-4 Galectin-4 Galectin-4 OeGal Galectin-4 MCGal GRG LEC-8 LEC-6 As_gal Galectin-8 AGL
Argopecten irradians Crassostrea virginica Pinctada fucata Canus lupus familiaris Myotis brandtii Chinchilla lanigera Ostrea edulis Haliotis discus hannai Ruditapes philippinarum Globodera rostochiensis Caenorhabditis elegans Caenorhabditis elegans Anopheles stephensi Crassostrea gigas Aphysia californica
ACS72241 ABG75998 ACO36044 ADR80620 XP_005886040 XP_005399890 ADF80416 ABN54798 ACA09732 AAB61596 BAB11964 BAA09794 AAO06842 EKC40502 XP_005103462
Multiple CRD
Tandem repeat
3
was modeled with 3i8tA and 3zxfA using multiple template function of Modeller. Each one of these alignments shows an identity over 32%. Further, the CRD adopted a typical galectin fold composed of two anti-parallel b-sheets, arranged in b-sheets sandwich motif without any a-helix. The seven conserved residues in all four CRDs maintained their positions and orientations in the binding cleft (Fig. 3). 3.3. Homology and phylogenetic analysis of HrGal
Single CRD
Out-group
PCR reactions were carried out in a total volume of 10 mL using the Maxima SYBR Green/ROX qPCR Master mix (Thermo Scientific, USA). The conditions for amplification were as follows: 95 C for 10 min (holding State), 40 cycles at 95 C for 30 s (denaturation), 60 C (T annealing) for 30 s, and 72 C for 30 s (extension). Finally, to determine the presence of nonspecific products, dimer formations, or contamination, a denaturation curve was analyzed. All samples were tested in triplicate. The comparative Ct method was used to analyze the relative expression level. The fold change was calculated as the relative folds of expression of EF2 by 2 DDCT method [18]. 2.6. Statistical analysis Genetic expression analysis was carried out with the STATISTICA 7 software (Statsoft Inc. USA). To evaluate significant differences, a ShapiroeWilk test was performed to determine data normality. Data with a non-parametric distribution were evaluated with an ANOVA test while those that had a parametric distribution were evaluated with a KruskaleWallis test. Differences were considered statistically significant at p < 0.05. 3. Results 3.1. cDNA cloning and sequence analysis of HrGal The full sequence of HrGal cDNA (GenBank accession number KJ183034) consisted of 2471 bp, with a 5' UTR of 131 bp and a 3'UTR of 672 bp containing a canonical polyadenylation signaling site and a poly (A) tail. The open reading frame (ORF) contained 1668 bp encoding for a polypeptide of 556 amino acids (Fig. 1). The sequence contained four CRDs with a theoretical isoelectric point of 4.98 and a predicted molecular mass of 68.3 kDa. SMART analysis revealed the following four dissimilarly arranged CRDs which indicated that HrGal presented a multi-domain galectin: CRD1 (Residues 43e184), CRD2 (190e131), CDR3 (325e458), and CRD4 (463e598), with the conserved amino acids characteristic involved in sugar binding activity (H-NPR and WG-ER) (Fig. 2). Analysis of homology revealed that the four CRDs shared a similarity of 33.1%, demonstrating the high site conservation characteristic of galectin. 3.2. The potential tertiary structures of HrGal The structure of the domains of HrGal was predicted by homology with structures of other galectins deposited in PDB and identified by Blastp. The structures used were 3galA, 3zxfA and 3vknA for the domains D2, D3 and D4, respectively. The D1 domain
The deduced amino acid sequence and organization of the multi-domain HrGal galectin were similar to those described for other members of the galectin superfamily The highest similarities displayed were with A. irradians (ACS72241, 67.5% identity), Pinctada fucata (ACO36044, 66.7% identity), Crassostrea gigas (EKC37204, 65.9% identity), and C. virginica (ABG75998, 65.8% identity) (Fig. 4). Based on multiple sequence alignments of HrGal with other galectins, all of the signature sequences of the galectin family were identified in every CRD of HrGal. An unrooted phylogenetic tree was constructed using the neighbor-joining method with a 1000 bootstrap test. HrGal was grouped in the same clade as galectin from Pinctada fucata, as evidenced by a 100% bootstrap, and together with galectin sequences with quadruple CRDs (Fig 5). 3.4. Tissue distribution of the HrGal Real time quantitative PCR analysis was performed to investigate the expression pattern of HrGal, mRNA with the EF2 as an internal control. The transcription expression analysis in different tissues showed that HrGal was constitutively expressed in all tested tissues including the hemocytes, gill and muscle. However, the expression level of HrGal mRNA in the unchallenged group was higher in hemocytes expressing himself 0.3 and 0.1 times more than in muscle and gills respectively (Fig. 6). 3.5. Temporal expression profile of HrGal mRNA after V. anguillarum challenge The temporal expression patterns of HrGal mRNA in hemocytes after the V. anguillarum challenge were quantified by real time PCR. At 3, 6 and 12 h post challenge, the hemocytes exhibited a low level of HrGal expression, but this was followed by significant increase to 9 fold greater than the control group at 24 h post challenge (p < 0.05). The transcripts of HrGal in hemocytes were significantly up-regulated (p < 0.05) at 32 h post-stimulation and increased to 24.5 fold higher than levels observed in the control group. Finally, the expression levels of HrGal mRNA decreased at 48 h postinjection (Fig. 7). In all sampling points, the challenged organisms had a higher expression than the control group injected with PBS solution. 4. Discussion 4.1. Characterization of HrGal mRNA The present study cloned and characterized the complete cDNA sequence for a tandem repeat galectin from H. rufescens. The sequence revealed four CRDs in tandem and connected by a peptide linker. Multiple alignments of HrGal with other galectins showed that the sequence contained crucial residues needed to bind to carbohydrates. These residues included the typical motifs H-NPR and WG-ER, which form glucan binding sites [5], thus reflecting the evolutionary conservation of galectin. As with other described galectins, such as in Pinctada fucata [12,19,20], Tegillarca granosa [21], Trachidermus fasciatus [22], Ruditapes philippinarum [13], and
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Fig. 1. Nucleotide and deduced amino acid sequences of galectin from Haliotis rufescens. The nucleotides are numbered on the left margin. The four CRD identified by SMART program are in grey boxed. The predicted N -glycosylation site is underlined. The conserved residues involved in carbohydrate binding specificity are in italic and bold. Asterisk marks the stop codon and polyadenylation signal in the 3'UTR is marked in bold. The tertiary structure prediction of each CRD domain is adjacent to the right of the sequence.
Fig. 2. Scheme of the structural domains of HrGal mRNA. Conserved amino acids that are involved in sugar binding activity (H-NPR and WG-ER) are denoted in bold. Asterisk marks the stop codon.
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Fig. 3. Diagram showing the molecular homology of HrGal tertiary structure. The seven residues of the ligand-binding site are remarked in order to show conservation at sequence and structural level.
C. gigas [23], HrGal lacked a signal peptide sequence and a hydrophobic transmembrane segment, which suggests that HrGal would be secreted through a non-classical process [24]. According to structural variations in the CRDs, the galectin superfamily is divided into three subfamilies, proto-type, chimera, or tandem repeat [5]. Interestingly, galectins with quadruple CRDs have been found only in mollusks, among which are CvGal, AiGal1, AiGal2, AiFal2, and PfGal [12,25,26]. Phylogenetic analysis revealed that HrGal was grouped in the same clade as other galectins from mollusks with quadruple CRDs, which suggests that these sequences could have a common evolutionary history. Galectin sequences containing multiple CRDs with tandem-repeats or containing only one CRD were grouped into another two clades. The three-dimensional structure prediction of HrGal revealed that the CRD motif was tightly folded and contained two anti-parallel bsheets arranged in a b-sheet sandwich structure same as the structure of galectins previously reported. These results reflect those reported in A. irradians, where the three-dimensional structure of the sequence contained four CRD domain [12]. Based in the structural and evolutional features of HrGal, we confirm that HrGal is a member of galectin subfamily. However, the three-dimensional structure of HrGal is theoretical and remains to be confirmed by xray crystallography.
4.2. mRNA expression patterns in different tissues of H. rufescens When pathogenic microorganisms break through the physical barriers of the host, this is recognized by different PRRs, which in turn trigger a series of cellular and humoral components that isolate the pathogen [27]. There are numerous studies in mammals that cite galectin as a key PRR in the activation of the immune system [28]. In order to investigate the participation that HrGal has in the immune response of H. rufescens, the expression of HrGal was analyzed in distinct tissues, and observations found that HrGal was constitutively expressed in all tissues. However, the highest expression levels were found in tissues key to the immune response in marine invertebrates, such as the hemocytes and gills [29]. Likewise, the present results agree with those found in other studies, such as that in Pinctada fucata which observed PfGal to be principally expressed in the digestive gland and hemocytes [20]. Another study in this same species found expression principally in the hemocytes [12,30,31], a result which was also found for CvGal from C. virginica [25]. Moreover, the gills represent the first barrier of defense against pathogenic microorganisms in marine invertebrates [32]. For instance, there are evidences that Lectins can be found in gill in C. virginica [33], Eriocheir sinensis [34] and Chlamys farreri [35]. This suggests that the function of HrGal could
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Fig. 4. Multiple sequence alignment among the full-length deduced amino acid sequence of HrGal and other galectin sequences with quadruple-domain, from invertebrates such as AiGal2 (ACS72241), CvGal (ABG75998) and Pf Galectin (ACO36044). The conserved amino acid residues are shaded in dark, and similar amino acids are shaded in grey. Gaps are indicated by dashes to improve the alignment Caenorhabditis elegans (BAB19964), Crassostrea gigas (EKC40502) and Asgal Anopheles stephensi (AAO06842).
Fig. 5. A phylogenetic tree of galectin family members constructed with the neighbor-joining method. Numbers at each branch indicate the support (%) in 1000 bootstrap pseudoreplication by neighbor-joining. Sequences used to generate phylogenetic analysis are shown in Table 3 and Aplysia californica (XP_005103462.1) was used as out-group.
be related to the immune response associated to haemolymph and gills tissue [36]. 4.3. mRNA expression patterns of HrGal challenged to V. anguillarum In mammals, the functions of galectins in the immune response have been well established, and these participate in amplifying the immune response [28], in activating the inflammatory response [9], and in host-pathogen interactions [7]. In contrast, little information is available for invertebrates on the processes in which galectins
have a role, especially concerning the transduction mechanisms, which these trigger. In order to elucidate the putative function of HrGal in the immune response of the red abalone, relative expression analysis was performed in hemocytes from individuals challenged by V. anguillarum. Our results showed a time-dependent expression pattern, extremely up-regulated after stimulation with V. anguillarum. Hemocytes as the primary line of immune defense against invading microbial and parasitic infection are capable of eliminating infections through phagocytic or encapsulation response [29]. Our results show that after bacterial challenge, mRNA expression in the hemocytes significantly increased, in
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Acknowledgments This study was supported by the FONDAP (15110027) project granted by CONICYT-Chile. References
Fig. 6. HrGal mRNA expression level in different tissues in individuals unchallenged. The expression levels were normalized by a-Tub. The bars are shown the standard deviation ± SD (n ¼ 3), significant differences were considered with p < 0.05.
Fig. 7. Real time PCR analysis from HrGal, gene expression in hemocytes of H. rufescens challenged with Vibrio anguillarum. Data were analyzed from three individuals. The mRNA of HrGal was measured at 0-3-6-12-24-32 and 48 h post challenge. The relative HrGal expression level as expressed by 2DDCT was determined. Elongation factor was used as an internal control. Verticals bars represented the mean ± SD. Significant differences were considered with p < 0.05.
contrast to the control group. The expression level of HrGal transcript was up-regulated at 3 h post challenge, suggesting the activation of defense against invading pathogen. The decreasing of HrGal transcripts in hemocytes after 6e12 h post challenge might be due to the translation process of HrGal in response to the bacterial infection. However, the increasing expression from 24 to 32 h evidences a recovery of the immune response. Similar results have been reported in the clam Tagillarca granosa exposed to Vibrio parahaemoliticus, where the peak of expression was recorded at 3 and 12 h post-stimulation [21], and also in the clam Ruditapes philippinarum, where a peak in expression was observed at 24 h post challenge to Vibrio alginolyticus [37]. In the same way in C. virginica, A. irradians and Tegillarca granosa, up-regulation was observed in response to pathogens [2,12,21]. The present study suggests that HrGal is associated to the molecular function of pathogens recognition. 5. Conclusion The results of the present study demonstrated that HrGal was constitutively expressed in all analyzed tissues. The highest expression of HrGal was found in hemocytes exposed to V. anguillarum, which suggests participation in innate immune response of H. rufescens.
[1] Medzhitov R, Janeway Jr C. Innate immune recognition: mechanisms and pathways. Immunol Rev 2000;173:89e97. [2] Tasumi S, Vasta GR. A galectin of unique domain organization from hemocytes of the eastern oyster (Crassostrea virginica) is a receptor for the protistan parasite Perkinsus marinus. J Immunol 2007;179:3086e98. [3] Arata Y, Hirabayashi J, Kasai KI. Sugar binding properties of the two lectin domains of the tandem repeat-type galectin LEC-1 (N32) of Caenorhabditis elegans : detailed analysis by an improved frontal affinity chromatography method. J Biol Chem 2001;276:3068e77. [4] Barondes SH, Cooper DN, Gitt MA, Leffler H. Galectins. Structure and function of a large family of animal lectins. J Biol Chem 1994;269:20807e10. [5] Hirabayashi J, Hashidate T, Arata Y, Nishi N, Nakamura T, Hirashima M, et al. Oligosaccharide specificity of galectins: a search by frontal affinity chromatography. Biochim Biophys Acta 2002;1572:232e54. [6] Kasai KI, Hirabayashi J. Galectins: a family of animal lectins that decipher glycocodes. J Biochem 1996;119:1e8. [7] Colin Hughes R. Galectins as modulators of cell adhesion. Biochimie 2001;83: 667e76. [8] Vasta GR, Ahmed H, Du S, Henrikson D. Galectins in teleost fish: zebrafish (Danio rerio) as a model species to address their biological roles in development and innate immunity. Glycoconj J 2004;21:503e21. [9] Liu FT, Yang RY, Hsu DK. Galectins in acute and chronic inflammation. Ann N Y Acad Sci 2012;1253:80e91. [10] Rabinovich GA, Gruppi A. Galectins as immunoregulators during infectious processes: from microbial invasion to the resolution of the disease. Parasite Immunol 2005;27:103e14. [11] Yoshino TP, Dinguirard N, Kunert J, Hokke CH. Molecular and functional characterization of a tandem-repeat galectin from the freshwater snail Biomphalaria glabrata, intermediate host of the human blood fluke Schistosoma mansoni. Gene 2008;411:46e58. [12] Song X, Zhang H, Wang L, Zhao J, Mu C, Song L, et al. A galectin with quadruple-domain from bay scallop Argopecten irradians is involved in innate immune response. Dev Comp Immunol 2011;35:592e602. [13] Kim JY, Kim YM, Cho SK, Choi KS, Cho M. Noble tandem-repeat galectin of Manila clam Ruditapes philippinarum is induced upon infection with the protozoan parasite Perkinsus olseni. Dev Comp Immunol 2008;32:1131e41. ~ a: Editorial Mundi[14] Bautista C. Moluscos, Tecnología de Cultivo. Madrid Espan Prensa; 1989. pp. 54e5. y 143-4 pp. n en [15] C aceres J. Enfermedades de los abalones En: Seminario: El cultivo del abalo n actual y perspectivas. Puerto Montt, Chile: Fundacio n Chile; 2000. Chile, situacio [16] Eswar N, Webb B, Marti-Renom MA, Madhusudhan MS, Eramian D, Shen MY, et al. Comparative protein structure modeling using modeller. Current Protocols in Bioinformatics: John Wiley & Sons, Inc; 2002. [17] Sali A, Blundell TL. Comparative protein modelling by satisfaction of spatial restraints. J Mol Biol 1993;234:779e815. [18] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using realtime quantitative PCR and the 2DDCT method. Methods 2001;25:402e8. [19] Jin XK, Li WW, He L, Lu W, Chen LL, Wang Y, et al. Molecular cloning, characterization and expression analysis of two apoptosis genes, caspase and nm23, involved in the antibacterial response in Chinese mitten crab, Eriocheir sinensis. Fish Shellfish Immunol 2011;30:263e72. [20] Zhang D, Hu Y, Guo H, Cui S, Su T, Jiang S. cDNA cloning and mRNA expression of a tandem-repeat galectin (PoGal2) from the pearl oyster, Pinctada fucata. Genet Mol Res 2011;10:1963e74. [21] Bao Y, Shen H, Zhou H, Dong Y, Lin Z. A tandem-repeat galectin from blood clam Tegillarca granosa and its induced mRNA expression response against bacterial challenge. Genes Genom 2013;35:733e40. [22] Yang H, Yu S, Chai Y, Liu Y, Zhang Q, Ding X, et al. A galectin from roughskin sculpin, Trachidermus fasciatus: molecular cloning and characterization. Fish Shellfish Immunol 2013;35:815e24. [23] Yamaura K, Takahashi KG, Suzuki T. Identification and tissue expression analysis of C-type lectin and galectin in the Pacific oyster, Crassostrea gigas. Comp Biochem Physiol B Biochem Mol Biol 2008;149:168e75. [24] Wada J, Kanwar YS. Identification and characterization of galectin-9, a novel b-galactoside-binding mammalian lectin. J Biol Chem 1997;272:6078e86. [25] Kurz S, Jin C, Hykollari A, Gregorich D, Giomarelli B, Vasta GR, et al. Haemocytes and plasma of the eastern oyster (Crassostrea virginica) display a diverse repertoire of sulphated and blood group A-modified N-glycans. J Biol Chem 2013;288: 24410e28. http://dx.doi.org/10.1074/jbc.M113.478933. [26] Song X, Zhang H, Zhao J, Wang L, Qiu L, Mu C, et al. An immune responsive multidomain galectin from bay scallop Argopectens irradians. Fish Shellfish Immunol 2010;28:326e32. [27] Rowley A, Powell A. Invertebrate immune systems- specific, quasi-specific, or nonspecific? J Immunol 2007;179:7209e14. [28] Pace K, Baum L. Insect galectins: roles in immunity and development. Glycoconj J 2004;19:607e14.
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W. Maldonado-Aguayo et al. / Fish & Shellfish Immunology 40 (2014) 1e8
[29] Anderson RS. Hemocyte-derived reactive oxygen intermediate production in four bivalve mollusks. Dev Comp Immunol 1994;18:89e96. [30] Zhang D, Jiang S, Hu Y, Cui S, Guo H, Wu K, et al. A multidomain galectin involved in innate immune response of pearl oyster Pinctada fucata. Dev Comp Immunol 2011;35:1e6. [31] Li WW, He L, Jin XK, Jiang H, Chen LL, Wang Y, et al. Molecular cloning, characterization and expression analysis of cathepsin A gene in Chinese mitten crab, Eriocheir sinensis. Peptides 2011;32:518e25. re E, Destoumieux[32] Schmitt P, Rosa RD, Duperthuy M, De Lorgeril J, Bache n D. The antimicrobial defense of the Pacific oyster, Crassostrea gigas. Garzo How diversity may compensate for scarcity in the regulation of resident/ pathogenic microflora. Front Microbiol 2012;3:1e28. [33] Espinosa EP, Perrigault M, Ward JE, Shumway SE, Allam B. Lectins associated with the feeding organs of the oyster Crassostrea virginica can mediate particle selection. Biol Bull 2009;217:130e41.
[34] Jin XK, Li WW, Cheng L, Li S, Guo XN, Yu AQ, et al. Two novel short C-type lectin from Chinese mitten crab, Eriocheir sinensis, are induced in response to LPS challenged. Fish Shellfish Immunol 2012;33:1149e58. [35] Wang H, Song L, Li C, Zhao J, Zhang H, Ni D, et al. Cloning and characterization of a novel C-type lectin from zhikong scallop Chlamys farreri. Mol Immunol 2007;44:722e31. [36] Yu Y, Yuan S, Yu Y, Huang H, Feng K, Pan M, et al. Molecular and biochemical characterization of galectin from amphioxus: primitive galectin of chordates participated in the infection processes. Glycobiology 2007;17:774e83. [37] Moreira R, Balseiro P, Romero A, Dios S, Posada D, Novoa B, et al. Gene expression analysis of clams Ruditapes philippinarum and Ruditapes decussatus following bacterial infection yields molecular insights into pathogen resistance and immunity. Dev Comp Immunol 2012;36:140e9.