ARTICLE IN PRESS Developmental and Comparative Immunology ■■ (2014) ■■–■■
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Developmental and Comparative Immunology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / d c i
Short communication
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Function of a novel C-type lectin with two CRD domains from Macrobrachium rosenbergii in innate immunity Q2 Xin Huang, Ying Huang, Yan-Ru Shi, Qian Ren *, Wen Wang **
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Jiangsu Key Laboratory for Biodiversity & Biotechnology and Jiangsu Key Laboratory for Aquatic Crustacean Diseases, College of Life Sciences, Nanjing Normal University, 1 Wenyuan Road, Nanjing 210046, China
A R T I C L E
I N F O
Article history: Received 18 September 2014 Revised 19 November 2014 Accepted 20 November 2014 Available online Keywords: Macrobrachium rosenbergii C-type lectin Innate immunity Pattern recognition receptor
A B S T R A C T
C-type lectins play crucial roles in innate immunity. In the present study, a novel C-type lectin gene, designated as MrCTL, was identified from Macrobrachium rosenbergii. MrCTL contains 2 carbohydraterecognition domains (CRDs), namely MrCRD1 and MrCRD2. The MrCRD1 contains a QEP motif and MrCRD2 contains a motif of EPD. MrCTL was mainly expressed in the hepatopancreas. The expression level of MrCTL in hepatopancreas was significantly upregulated after a challenge with Vibrio parahaemolyticus or White spot syndrome virus (WSSV). The recombinant MrCTL, MrCRD1 and MrCRD2 have an ability to agglutinate both Gram-negative (V. parahaemolyticus) and Gram-positive bacteria (Staphylococcus aureus) in a calcium dependent manner. The recombinant MrCTL, MrCRD1 and MrCRD2 bind directly to all tested microorganisms. All these results suggested that MrCTL may have important roles in immune defense against invading pathogens in prawns. © 2014 Published by Elsevier Ltd.
35 1. Introduction
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Like most invertebrate animals, Macrobrachium rosenbergii does not have an adaptive immune system. When attacked by foreign microbes, the species completely relies on its innate immune system for defense (Loker et al., 2004), the first line of defense against pathogen invasion. When foreign microbes come into contact with a host, host germline-encoded pattern recognition receptors (PRRs) recognize pathogen-associated molecular patterns (PAMPs) on the surface of microorganisms, such as lipopolysaccharide (LPS), peptidoglycans (PGN), and β-1,3-glucan, and thus prevent infection at the early stage (Fearon, 1997; Fearon and Locksley, 1996; Medzhitov and Janeway, 2000; Vasta et al., 2004). Furthermore, PRR– PAMP interactions initiate a series of immune responses, such as prophenoloxidase (proPO) activated system, antimicrobial peptide (AMP) synthesis, phagocytosis, encapsulation, blood clotting and toxin or reactive oxygen species (ROS) production (Bachère et al., 2004; Cerenius and Söderhäll, 2004; Fujita et al., 2004).
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* Correspondence author. Jiangsu Key Laboratory for Biodiversity & Biotechnology, and Jiangsu Key Laboratory for Aquatic Crustacean Diseases, College of Life Sciences, Nanjing Normal University, 1 Wenyuan Road, Nanjing 210046, China. Tel.: +86 25 85891955. E-mail address: [email protected] (Q. Ren). ** Correspondence author. Jiangsu Key Laboratory for Biodiversity & Biotechnology and Jiangsu Key Laboratory for Aquatic Crustacean Diseases, College of Life Sciences, Nanjing Normal University, 1 Wenyuan Road, Nanjing 210046, China. Tel.: +86 25 85891955. E-mail address: [email protected]; [email protected] (W. Wang).
Over the years, numerous PRRs, including Gram-negative binding proteins (GNBPs), C-type lectins (CTLs), LPS-binding proteins (LBPs), peptidoglycan-binding proteins (PGBPs), β-1,3-glucan-binding proteins (GBPs), thioester-containing proteins (TEPs), fibrinogen-like domain immunolectins (FBNs) and scavenger receptors (SCRs), have been identified (Akira et al., 2006; Christophides et al., 2004; Kurata et al., 2006; Watson et al., 2005; Yu et al., 2002). Lectin was first identified in the seeds of Leguminosae in 1888 (Sharon and Lis, 2004) and they are widely distributed in nature as carbohydrate recognizing proteins (Lakhtin et al., 2011). With the ability to bind terminal sugars of glycoproteins or glycolipids, lectins are considered to be appropriate candidates for PRRs in innate immunity. Depending on their structures and functions, lectins are classified into C-, L-, P-, I-, R-, and S-type lectins (Janeway and Medzhitov, 2002), and can also be classified as mannose-, fructose-, rhamnose-, and galactose-binding lectins (Zhang et al., 2009a). C-type lectins were originally described as a group of Ca2+-dependent (Ctype) carbohydrate-binding proteins that differ from other lectins (Drickamer, 1988) and now the protein-containing domains with sequences similar to CTL domains (CTLDs/CRDs) also belong to this family. Invertebrate C-type lectins contain some specific sites unique to C-type lectins, including a carbohydrate-binding site and Ca2+binding site (Zelensky and Gready, 2005) and most C-type lectins contain one carbohydrate recognition domain (CRD), but some have two or more CRDs (East and Isacke, 2002). A CRD fold is composed of approximately 120–130 residues that form a characteristic double-loop structure. The double-loop structure is stabilized by two conserved disulfide bridges, as well as a set of conserved hydrophobic and polar interactions (Zelensky and Gready, 2005). There
http://dx.doi.org/10.1016/j.dci.2014.11.015 0145-305X/© 2014 Published by Elsevier Ltd.
Please cite this article in press as: Xin Huang, Ying Huang, Yan-Ru Shi, Qian Ren, Wen Wang, Function of a novel C-type lectin with two CRD domains from Macrobrachium rosenbergii in innate immunity, Developmental and Comparative Immunology (2014), doi: 10.1016/j.dci.2014.11.015
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are four Ca2+-binding sites identified from CRDs of different species; among which, Ca2+-binding site 2 is structurally conserved and participates in the calcium-mediated carbohydrate interaction. Two characteristic motifs exist in the Ca2+-binding site 2, which are directly involved in monosaccharide binding. The first one is always Glu-Pro-Asn (EPN) or Gln-Pro-Asp (QPD), which is thought to determine binding toward mannose or galactose, respectively; the second motif is always Trp-Asn-Asp (WND) (Zelensky and Gready, 2003, 2005). Many C-type lectins have been reported in invertebrate species and they participate in various innate immune responses. For example, Penaeus monodon (PmAV) has anti-viral activity (Luo et al., 2003). Litopenaeus vannamei has a C-type lectin (LvLT) with two CRDs, which is expressed only in the hepatopancreas. It may have a role in WSSV infection (Ma et al., 2007). Fenneropenaeus chinensis (FchsL), which has a single CRD, is specifically expressed in hepatopancreas and exhibits antimicrobial activity (Sun et al., 2008a). Purified lectin from M. rosenbergii (Mrlectin) has been demonstrated to regulate the production of oxidative radicals and functions as a potential cytotoxin and microbiocide (Sierra et al., 2005). Moreover, previous published research reported that the giant freshwater prawn M. rosenbergii had a high tolerance to WSSV (Sahul Hameed et al., 2000). Vibriosis can result in high mortality rates of larval M. rosenbergii (Jayaprakash et al., 2006). In our prior published research, we have found lectins serve important immune functions in invertebrates, including the following examples: an LDLa domain-containing C-type lectin of Eriocheir sinensis was reported that could agglutinate and bind a broad range of microbes (Huang et al., 2014a). The result of this paper was in Q3 accordance with another one (Huang et al., 2014b). Ren et al. (2012) reported the immune response of four dual-CRD C-type lectins to Q4 microbial challenges in giant freshwater prawn M. rosenbergii (Ren et al., 2012), which concluded that the four dual-CRD lectins may play important roles in pathogen elimination and anti-WSSV innate immunity, among other contributions. However there is insufficient research evidence for the possible role of C-type lectin from M. rosenbergii in the innate immunity response. In the present study, we report a novel C-type lectin with 2 CRDs from the M. rosenbergii, designated as MrCTL. Its tissue distributions and expression patterns in the hepatopancreas upon challenge by Vibrio parahaemolyticus or WSSV were investigated. Recombinant MrCTL and its individual MrCRD1 and MrCRD2 proteins bind to or agglutinate bacteria. Our study provides preliminary results that may clarify the probable roles of dual-CRD lectins in the innate immunity of M. rosenbergii. 2. Materials and methods 2.1. Immune challenge of M. rosenbergii and sample collection Adult M. rosenbergii (about 15 g each) were obtained from an aquaculture market in Nanjing, Jiangsu Province, China, and cultured temporarily in freshwater tanks at room temperature (25 °C). The methods of viral inoculum preparation and quantification were according to a previous study (Wang et al., 2009b). In the experimental group, V. parahaemolyticus (approximately 3 × 107 cells per shrimp) or WSSV (approximately 3 × 107 copies per shrimp) was injected into the abdominal segment of the shrimp by using a 1 ml sterile syringe. The healthy prawns were inoculated with 50 μl of PBS as the control group. At 2, 6, 12, 24 and 48 h post injection, the hepatopancreas was collected from challenged prawns of each experimental group for RNA extraction. Hemolymph was collected from healthy prawns (untreated) by mixing an equal volume of improved anticoagulant buffer (ACD-B) (glucose, 1.47 g; citric acid, 0.48 g; trisodium citrate, 1.32 g; prepared in double distilled water and brought to 100 ml, pH 7.3) (Huang et al., 2013). The
hemolymph was centrifuged at 800 × g for 10 min (4 °C) to isolate the hemocytes. Other tissues (i.e., heart, hepatopancreas, gills, stomach, and intestine) were also collected from healthy prawns for RNA extraction. Three prawns were chosen to eliminate individual differences. 2.2. Total RNA isolation and cDNA synthesis RNAs from various tissues were extracted using an RNApure highpurity total RNA rapid extraction kit (Spin-column, BioTeke, Beijing, China) according to the manufacturer’s protocols. The first-strand cDNA of the samples was synthesized for quantitative real-time PCR (qRT-PCR) analysis by using the PrimeScript® 1st Strand cDNA Synthesis Kit (Takara, Dalian, China), with the Oligo dT Primer. The synthesized cDNA was used in the real-time polymerase chain reaction (RT-PCR) analysis. 2.3. Cloning of the full-length cDNA of MrCTL and sequence analysis Based on the expressed sequence tags (ESTs) obtained through the sequencing of the prawn hepatopancreas transcriptome, we designed the gene-specific primers (MrCTL-F: 5′-aaggaaggagac tgggtttgggctaatgac-3′, MrCTL-R: 5′-gcctccgagtgcttcgcatagtgtttta3′) to amplify the 5′ and 3′ ends of the cDNA. The 5′ and 3′ fragments of MrCTL were amplified using gene-specific primers and a Universal Primer A Mix (UPM) according to Rapid Amplification cDNA End (RACE) methods by using a Clontech SMARTer TM RACE cDNA Amplification Kit from TAKARA (Dalian, China). The full length of MrCTL cDNAs was obtained by overlapping the 5′ and 3′ fragments. The cDNA sequence and deduced amino acid sequence of MrCTL were analyzed using the BLAST algorithm (http://www.ncbi.nlm.nih Q5 .gov/blast) and the Expert Protein Analysis System (http://www .expasy.org). The signal sequence and domain organization was analyzed with the Simple Modular Architecture Research Tool (http:// smart.embl-heidelberg.de/). The ClustalW2 Multiple Alignment program (http://www.ebi.ac.uk/Tools/msa/clustalw2/) was used to create the multiple sequence alignments. Molecular Evolutionary Genetics Analysis 5.05 was used for the phylogenetic analysis of MrCTL with other C-type lectins (Kumar et al., 2008). 2.4. Real-time PCR analysis of mRNA expression The tissue distribution of MrCTL at mRNA levels in the hemocytes, heart, hepatopancreas, gills, stomach, and intestine was analyzed through RT-PCR using the primers (MrCTL-RT-F: 5′ctgccttccctctttgata-3′ and MrCTL-RT-R: 5′-cagtttgtgtgg ttggttc-3′). qRT-PCR was performed to analyze the mRNA expression level changes of MrCTL in the hepatopancreas of the prawns of M. rosenbergii after a WSSV or V. parahaemolyticus challenge at 2, 6, 12, 24 and 48 h. The qRT-PCR was performed using SYBR® Premix Ex TaqTM II (Tli RNaseH Plus) (TAKARA, Dalian, China) according to the manufacturer’s instructions. The GAPDH was amplified for internal standardization with the primers (MrGAPDH-RT-F: 5′tgccgcccagaacatcatt-3′ and MrGAPDH-RT-R: 5′-tcgtcttcggtgtagccca3′). The 2−ΔΔCt method was used to analyze the expression level of MrCTL (Livak and Schmittgen, 2001). An unpaired sample t-test was conducted, and differences were considered significant if P < 0.05. 2.5. Expression and purification of recombinant MrCTL and its individual CRD domains Primers MrCTL-ex-F (5′-tactcagaattctatgagtgcccgtccccctac-3′) and MrCTL-ex-R (5′-tactcagcggccgcttgtttcatattttgctgg-3′) were designed to amplify a cDNA fragment encoding the mature MrCTL that contained both CRDs; MrCRD1-ex-F (5′-tactcagaattctatgagt gcccgtcccccta-3′) and MrCRD1-ex-R (5′-tactcagcggccgcgtcgcagatg
Please cite this article in press as: Xin Huang, Ying Huang, Yan-Ru Shi, Qian Ren, Wen Wang, Function of a novel C-type lectin with two CRD domains from Macrobrachium rosenbergii in innate immunity, Developmental and Comparative Immunology (2014), doi: 10.1016/j.dci.2014.11.015
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actccatt-3′) primers were used to obtain the MrCRD1 domain, while MrCRD2-ex-F (5′-tactcagaattctgcccagttccgttccagatg-3′) and MrCRD2ex-R (5′-tactcagcggccgcttgttt catattttgctgg-3′) primers were used for the MrCRD2 domain. The amplified fragment was digested by EcoR I and Not I and then inserted into the pET30a(+) vector. Afterward, the recombinant plasmid was transformed to Escherichia coli BL21 (DE3) cells for IPTG-induced recombinant expression (final IPTG concentration of 0.5 mM). The temperature for protein induction was 37 °C. The rMrCTL, rMrCRD1 and rMrCRD2 were then purified through affinity chromatography by using Ni SepharoseTM 6 Fast Flow (GE Healthcare, USA) following the instructions. The purified proteins were detected by using 12.5% SDS–polyacrylamide gel electrophoresis (SDS-PAGE). The concentration of purified recombinant proteins was quantified by a BCA method (Smith et al., 1985).
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2.6. Microbial agglutination and binding assays The pathogen bacteria V. parahaemolyticus and Staphylococcus aureus were used for agglutination assays and they were maintained in our laboratory. Gram-positive bacteria (S. aureus) and Gramnegative bacteria (V. parahaemolyticus) in mid-logarithmic phase were washed twice in sterile Tris-buffered saline (TBS: 10 mM Tris– HCl, 150 mM NaCl, pH 7.5) and resuspended in TBS at 2 × 108 cells/ ml. In the presence or absence of 10 mM CaCl2, microorganisms were incubated with the recombinant proteins (MrCTL, MrCRD1 or MrCRD2, 100 μg/ml) or BSA (100 μg/ml, as control) by adding 25 μl of bacteria to 25 μl of TBS containing the proteins. The mixture was incubated at room temperature for 1 h, and the agglutinating reactions were observed by microscopy. Gram-positive (G+) bacteria (S. aureus, Micrococcus luteus, Bacillus subtilis and Bacillus megaterium) and Gram-negative (G−) bacteria (Aeromonas hydrophila, V. parahaemolyticus, Vibrio anguillarum and E. coli) were used for the bacterial-binding assay. The overnight bacterial culture in LB was centrifuged at 6000 rpm for 5 min and then washed with TBS twice. Approximately 500 μl of recombinant rMrCTL, rMrCRD1 or rMrCRD2 (1 mg/ml) was incubated with a bacterial suspension (2 × 108 cells/ml) at room temperature for 30 min with gentle rotation. The microorganisms were washed 4 times with TBS and then eluted with 100 μl of 8 M urea solution. Afterward, the elution was analyzed by 12.5% SDS-PAGE and rMrCTL, rMrCRD1 or rMrCRD2 protein was detected by western blot using Anti-His Mouse mAb.
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3. Results and discussion 3.1. Cloning and sequence analysis of MrCTL cDNA MrCTL comprises a 52 bp 5′ untranslated region (UTR), an open reading frame (ORF) of 1002 bp encoding a 333 amino acid protein, and a 3′ UTR of 292 bp with a polyadenylated tail. The carbohydrate binding specificity of C-type lectins is mainly determined by the position of hydrogen bond donors and acceptors in the conserved motifs of Ca2+-binding site 2 in CRDs (Zelensky and Gready, 2005). The CRDs with an EPN (Glu-Pro-Asn) motif bind mannose, while the CRDs with a QPD (Gln-Pro-Asp) motif bind galactose. Compared with the residues important for stabilizing the structure, these residues seem to be more diverse in invertebrate C-type lectins (Zelensky and Gready, 2003, 2005). MrCTL consists of two carbohydrate recognition domains (CRDs). The first CRD (MrCRD1) contains a QEP motif. The second CRD (MrCRD2) contains an EPD motif (Appendix S1: Supplementary Fig. S1). The sugar-binding motifs of MrCTL are not EPN or QPD. The multiple sequence alignment showed that MrCTL has a higher similarity with the dual-CRD lectins of Palaemon modestus and M. rosenbergii (Appendix S1: Supplementary Fig. S2). BLASTX analysis revealed that MrCTL shared 71% identity to C-type lectins from P. modestus, which contained 2 CRDs. The results of BLASTX analysis is similar to the phylogenetic tree, which showed that MrCTL together with dual-CRD containing lectins such as PmCTL1, PmCTL2, Mrlectin1 and Mrlectin2, belonged to one cluster. Other dual-CRD lectins constitute another group (Appendix S1: Supplementary Fig. S3). All the results illustrated that MrCTL was a novel member of the C-type lectin superfamily in M. rosenbergii.
78 3.2. Tissue distribution and expression patterns of MrCTL Expression of MrCTL mRNA in various tissues was determined by qRT-PCR. As the counterpart of the mammalian liver and the insect fat body, the shrimp hepatopancreas is a major tissue involved in immunity (Gross et al., 2001). In the present study, the highest expression of MrCTL mRNA was detected in the hepatopancreas and it also could be detected in other tissues (heart, hemocytes, gill, stomach and intestine) with relatively low expression levels (Appendix S1: Supplementary Fig. S4). The result was consistent with studies on some other C-type lectins, such as the C-type lectin of P. monodon (PmLT) (Ma et al., 2008), the C-type lectin with two CRD
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Fig. 1. qRT-PCR analysis of MrCTL in the hepatopancreas of M. rosenbergii under V. parahaemolyticus (A) or WSSV (B) challenged at 2, 6, 12, 24 and 48 h. Three prawns were chosen to eliminate individual differences at each time point. Asterisks indicate significant differences (*P < 0.05, **P < 0.01, ***P < 0.001) compared with values of the control. Error bars represent the mean ± S.D. of three independent PCR amplifications and quantifications.
Please cite this article in press as: Xin Huang, Ying Huang, Yan-Ru Shi, Qian Ren, Wen Wang, Function of a novel C-type lectin with two CRD domains from Macrobrachium rosenbergii in innate immunity, Developmental and Comparative Immunology (2014), doi: 10.1016/j.dci.2014.11.015
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Fig. 2. Recombinant expression and purification of MrCTL, MrCRD1 and MrCRD2 and their agglutinate and binding ability toward various bacteria. i. Recombinant expression and purification of MrCTL (A), MrCRD1 (B) and MrCRD2 (C). M: protein marker; Lane 1, crude protein extracts of E. coli without induction; Lane 2, crude protein extracts of E. coli induction for 4 h by IPTG; Lane 3, recombinant protein purified by Ni SepharoseTM 6 Fast Flow. ii. Direct binding of rMrCTL, rMrCRD1 or rMrCRD2 to various bacteria. Bacteria were incubated with rMrCTL, rMrCRD1 or rMrCRD2 and then washed four times with TBS and subjected to elution with 8 M urea. Binding was confirmed by Western blot with Anti-His Mouse mAb against recombinant MrCTL, MrCRD1, MrCRD2 with His tag. iii. Agglutination of bacteria (S. aureus and V. parahaemolyticus) by rMrCTL, rMrCRD1 or rMrCRD2 with either absence or presence of calcium. BSA served as a control.
Please cite this article in press as: Xin Huang, Ying Huang, Yan-Ru Shi, Qian Ren, Wen Wang, Function of a novel C-type lectin with two CRD domains from Macrobrachium rosenbergii in innate immunity, Developmental and Comparative Immunology (2014), doi: 10.1016/j.dci.2014.11.015
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domains from the banana shrimp Fenneropenaeus merguiensis (FmLc) (Rattanaporn and Utarabhand, 2011), the C-type lectin (FcLec5) from the Chinese white shrimp F. chinensis and the C-type lectin with two CRD domains (FcLec2) from Chinese shrimp F. chinensis (Xu et al., 2010; Zhang et al., 2009b). In total, the expression pattern proved that C-type lectins played an important role in the immune defense. Vibriosis is a major bacterial disease caused by bacteria in the genus Vibrio (Jiravanichpaisal et al., 1994). WSSV is the most serious threat to shrimp aquaculture (Escobedo-Bonilla et al., 2008), necessitating a more thorough study of the anti-Vibrio or anti-WSSV immune response in M. rosenbergii. In our study, we analyzed the temporal expression of MrCTL in prawns challenged with V. parahaemolyticus or WSSV by qRT-PCR (Fig. 1). MrCTL was downregulated at 2 h after the V. parahaemolyticus challenge, reaching the highest level at 6 h, decreasing at 12 h, and then upregulated at 24 h. The decrease of MrCTL expression at 48 h post V. parahaemolyticus infection was not significantly different from the PBS injected group (Fig. 1A). A hepatopancreas-specific lectin from Chinese white shrimp was also downregulated 2 h after the bacterial challenge, and then upregulated from 6 h to 24 h postQ6 challenge (Sun et al., 2008b). The results indicate that bacteria may achieve some protection from the host immune system attack by downregulating MrCTL originally, which may function as opsonins. The subsequent rapid upregulation of MrCTL suggests that it may be involved in the immune response against the pathogens. Lectins were the major group of pattern recognition proteins up-regulated in the hepatopancreas of L. vannamei infected with WSSV (Gross et al., 2001). L. vannamei C-type lectin (LvLT) with two CRDs, which is expressed only in the hepatopancreas, may have a role in WSSV infection (Ma et al., 2007). FcLec3, a single-CRD lectin, interacted with VP28, one of the major envelope proteins of WSSV (Wang et al., 2009a). The giant freshwater prawn M. rosenbergii had a high tolerance to WSSV (Sahul Hameed et al., 2000). In this study, expression of MrCTL in prawns injected with WSSV rapidly increased at 2 h post-injection and then downregulated from 6 h to 12 h, subsequently reaching a maximum level at 24 h post-injection and finally declined at 48 h post-injection (Fig. 1B). The observable increase of MrCTL expression at 2 and 24 h post WSSV infection suggested that MrCTL could be effectively induced by WSSV stimulation. And the remarkable up-regulation of MrCTL expression in the early stage of WSSV infection implied the importance of multirecognition characteristics of MrCTL. However, the potential mechanism is not clear presently, which requires further study. All the results indicate that the C-type lectin of M. rosenbergii may function in innate immunity against viral and bacterial challenges. 3.3. Expression and purification of rMrCTL, rMrCRD1, rMrCRD2 All the recombinant proteins (rMrCTL, rMrCRD1 and rMrCRD2) are expressed as inclusion. The predicted molecular mass of mature MrCTL from the deduced amino acid sequence was about 37.6 kDa. rMrCTL has an apparent molecular mass of 44.6 kDa with a pET30a(+) vector, while rMrCRD1 and rMrCRD2 are 28 and 27 kDa, respectively (Fig. 2.i.). The molecular mass of the pET-30a(+) vector (containing N-terminal His tag, C-terminal His tag and S tag) from recombinant protein was about 7 kDa. 3.4. Microbial agglutinating and binding activity of MrCTL and its two CRD domains Aggregation activity is the most important feature of C-type lectins. The process may be initiated by lectins binding and recognizing carbohydrate components present on the surface of microorganisms, but absent in the host. Considering binding to the surface of the foreign invaders was the first step in the immune recognition and initiation of immune response (Gross et al., 2001). In
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the microorganism binding assay, rMrCTL, rMrCRD1 and rMrCRD2 have been found to bind toward all tested microorganisms, including Gram-positive bacteria (S. aureus, M. luteus, B. subtilis and B. megaterium) and Gram-negative bacteria (A. hydrophila, V. parahaemolyticus, V. anguillarum and E. coli) (Fig. 2.ii.). The broad binding spectrum of MrCTL toward various microorganisms might suggest its important roles in the immune defense against various pathogens in prawns. In the presence of calcium, recombinant MrCTL can agglutinate all of the tested Gram-positive (S. aureus) and Gramnegative bacteria (V. parahaemolyticus) (Fig. 2.iii.). The two dualCTLD-containing proteins FcLec2 and FcLec5 also have an effect on agglutination (Xu et al., 2010; Zhang et al., 2009b). PmLec from P. monodon (Luo et al., 2006); and PjLec from Penaeus japonicus (Yang et al., 2007) can also agglutinate to the bacteria mentioned above. In summary, a new C-type lectin (MrCTL) with two CRDs from the prawn M. rosenbergii was identified in this study. MrCTL protein was mainly expressed in the hepatopancreas of prawns and its expressions are regulated by PAMPs or bacterial challenge. The recombinant MrCTLs have agglutinating, binding activities to various bacteria. All these results suggest that MrCTL functioned as a PRR involved in pattern recognition and pathogen elimination in the innate immunity of the prawn M. rosenbergii.
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Acknowledgments We appreciate Professor O. Roger Anderson (Columbia University) for editing the manuscript. The current study was supported by the National Natural Science Foundation of China (Grant Nos. 31101926, 31170120, 31272686), the Natural Science Foundation of Jiangsu Province (BK20131401), Natural Science Fund of Colleges and Universities in Jiangsu Province (13KJB240002, 14KJA240002), High level talents in Nanjing Normal University Foundation (2012104XGQ0101), and Project for Aquaculture in Jiangsu Province (No. PJ2011-65; Y2013-45; D2013-5-3; 270 D2013-5-4), and the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
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Appendix: Supplementary Material Supplementary data to this article can be found online at doi:10.1016/j.dci.2014.11.015.
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Sahul Hameed, A.S., Xavier, C.M., Anilkumar, M., 2000. Tolerance of Macrobrachium rosenbergii to white spot syndrome virus. Aquaculture 183, 207–213. Sharon, N., Lis, H., 2004. History of lectins: from hemagglutinins to biological recognition molecules. Glycobiology 14, 53R–62R. Sierra, C., Lascurain, R., Pereyra, A., Guevara, J., Martinez, G., Agundis, C., et al., 2005. Participation of serum and membrane lectins on the oxidative burst regulation in Macrobrachium rosenbergii hemocytes. Dev. Comp. Immunol. 29, 113–121. Smith, P., Krohn, R.I., Hermanson, G., Mallia, A., Gartner, F., Provenzano, M.D., et al., 1985. Measurement of protein using bicinchoninic acid. Anal. Biochem. 150, 76. Sun, J., Wang, L., Wang, B., Guo, Z., Liu, M., Jiang, K., et al., 2008a. Purification and characterization of a natural lectin from the plasma of the shrimp Fenneropenaeus chinensis. Fish Shellfish Immunol. 25, 290–297. Sun, Y.D., Fu, L.D., Jia, Y.P., Du, X.J., Wang, Q., Wang, Y.H., et al., 2008b. A hepatopancreas-specific C-type lectin from the Chinese shrimp Fenneropenaeus chinensis exhibits antimicrobial activity. Mol. Immunol. 45, 348– 361. Vasta, G.R., Ahmed, H., Odom, E.W., 2004. Structural and functional diversity of lectin repertoires in invertebrates, protochordates and ectothermic vertebrates. Curr. Opin. Struct. Biol. 14, 617–630. Wang, S., Zhao, X.F., Wang, J.X., 2009a. Molecular cloning and characterization of the translationally controlled tumor protein from Fenneropenaeus chinensis. Mol. Biol. Rep. 36, 1683–1693. Wang, X.W., Xu, W.T., Zhang, X.W., Zhao, X.F., Yu, X.Q., Wang, J.X., 2009b. A C-type lectin is involved in the innate immune response of Chinese white shrimp. Fish Shellfish Immunol. l27, 556–562. Watson, F.L., Puttmann-Holgado, R., Thomas, F., Lamar, D.L., Hughes, M., Kondo, M., et al., 2005. Extensive diversity of Ig-superfamily proteins in the immune system of insects. Science 309, 1874–1878. Xu, W.T., Wang, X.W., Zhang, X.W., Zhao, X.F., Yu, X.Q., Wang, J.X., 2010. A new C-type lectin (FcLec5) from the Chinese white shrimp Fenneropenaeus chinensis. Amino Acids 39, 1227–1239. Yang, H., Luo, T., Li, F., Li, S., Xu, X., 2007. Purification and characterisation of a calcium-independent lectin (PjLec) from the haemolymph of the shrimp Penaeus japonicus. Fish Shellfish Immunol. 22, 88–97. Yu, X., Zhu, Y., Ma, C., Fabrick, J.A., et al., 2002. Pattern recognition proteins in Manduca sexta plasma. Insect Biochem. Mol. Biol. 32, 1287–1293. Zelensky, A.N., Gready, J.E., 2003. Comparative analysis of structural properties of the C-type-lectin-like domain (CTLD). Proteins 52, 466–477. Zelensky, A.N., Gready, J.E., 2005. The C-type lectin-like domain superfamily. FEBS J. 272, 6179–6217. Zhang, Y., Qiu, L., Song, L., Zhang, H., Zhao, J., Wang, L., et al., 2009a. Cloning and characterization of a novel C-type lectin gene from shrimp Litopenaeus vannamei. Fish Shellfish Immunol. 26, 183–192. Zhang, X.W., Xu, W.T., Wang, X.W., Mu, Y., Zhao, X.F., Yu, X.Q., et al., 2009b. A novel C-type lectin with two CRD domains from Chinese shrimp Fenneropenaeus chinensis functions as a pattern recognition protein. Mol. Immunol. 46, 1626– 1637.
Please cite this article in press as: Xin Huang, Ying Huang, Yan-Ru Shi, Qian Ren, Wen Wang, Function of a novel C-type lectin with two CRD domains from Macrobrachium rosenbergii in innate immunity, Developmental and Comparative Immunology (2014), doi: 10.1016/j.dci.2014.11.015
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Developmental and Comparative Immunology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / d c i
Short communication
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Function of a novel C-type lectin with two CRD domains from Macrobrachium rosenbergii in innate immunity Q2 Xin Huang, Ying Huang, Yan-Ru Shi, Qian Ren *, Wen Wang **
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Jiangsu Key Laboratory for Biodiversity & Biotechnology and Jiangsu Key Laboratory for Aquatic Crustacean Diseases, College of Life Sciences, Nanjing Normal University, 1 Wenyuan Road, Nanjing 210046, China
A R T I C L E
I N F O
Article history: Received 18 September 2014 Revised 19 November 2014 Accepted 20 November 2014 Available online Keywords: Macrobrachium rosenbergii C-type lectin Innate immunity Pattern recognition receptor
A B S T R A C T
C-type lectins play crucial roles in innate immunity. In the present study, a novel C-type lectin gene, designated as MrCTL, was identified from Macrobrachium rosenbergii. MrCTL contains 2 carbohydraterecognition domains (CRDs), namely MrCRD1 and MrCRD2. The MrCRD1 contains a QEP motif and MrCRD2 contains a motif of EPD. MrCTL was mainly expressed in the hepatopancreas. The expression level of MrCTL in hepatopancreas was significantly upregulated after a challenge with Vibrio parahaemolyticus or White spot syndrome virus (WSSV). The recombinant MrCTL, MrCRD1 and MrCRD2 have an ability to agglutinate both Gram-negative (V. parahaemolyticus) and Gram-positive bacteria (Staphylococcus aureus) in a calcium dependent manner. The recombinant MrCTL, MrCRD1 and MrCRD2 bind directly to all tested microorganisms. All these results suggested that MrCTL may have important roles in immune defense against invading pathogens in prawns. © 2014 Published by Elsevier Ltd.
35 1. Introduction
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Like most invertebrate animals, Macrobrachium rosenbergii does not have an adaptive immune system. When attacked by foreign microbes, the species completely relies on its innate immune system for defense (Loker et al., 2004), the first line of defense against pathogen invasion. When foreign microbes come into contact with a host, host germline-encoded pattern recognition receptors (PRRs) recognize pathogen-associated molecular patterns (PAMPs) on the surface of microorganisms, such as lipopolysaccharide (LPS), peptidoglycans (PGN), and β-1,3-glucan, and thus prevent infection at the early stage (Fearon, 1997; Fearon and Locksley, 1996; Medzhitov and Janeway, 2000; Vasta et al., 2004). Furthermore, PRR– PAMP interactions initiate a series of immune responses, such as prophenoloxidase (proPO) activated system, antimicrobial peptide (AMP) synthesis, phagocytosis, encapsulation, blood clotting and toxin or reactive oxygen species (ROS) production (Bachère et al., 2004; Cerenius and Söderhäll, 2004; Fujita et al., 2004).
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* Correspondence author. Jiangsu Key Laboratory for Biodiversity & Biotechnology, and Jiangsu Key Laboratory for Aquatic Crustacean Diseases, College of Life Sciences, Nanjing Normal University, 1 Wenyuan Road, Nanjing 210046, China. Tel.: +86 25 85891955. E-mail address: [email protected] (Q. Ren). ** Correspondence author. Jiangsu Key Laboratory for Biodiversity & Biotechnology and Jiangsu Key Laboratory for Aquatic Crustacean Diseases, College of Life Sciences, Nanjing Normal University, 1 Wenyuan Road, Nanjing 210046, China. Tel.: +86 25 85891955. E-mail address: [email protected]; [email protected] (W. Wang).
Over the years, numerous PRRs, including Gram-negative binding proteins (GNBPs), C-type lectins (CTLs), LPS-binding proteins (LBPs), peptidoglycan-binding proteins (PGBPs), β-1,3-glucan-binding proteins (GBPs), thioester-containing proteins (TEPs), fibrinogen-like domain immunolectins (FBNs) and scavenger receptors (SCRs), have been identified (Akira et al., 2006; Christophides et al., 2004; Kurata et al., 2006; Watson et al., 2005; Yu et al., 2002). Lectin was first identified in the seeds of Leguminosae in 1888 (Sharon and Lis, 2004) and they are widely distributed in nature as carbohydrate recognizing proteins (Lakhtin et al., 2011). With the ability to bind terminal sugars of glycoproteins or glycolipids, lectins are considered to be appropriate candidates for PRRs in innate immunity. Depending on their structures and functions, lectins are classified into C-, L-, P-, I-, R-, and S-type lectins (Janeway and Medzhitov, 2002), and can also be classified as mannose-, fructose-, rhamnose-, and galactose-binding lectins (Zhang et al., 2009a). C-type lectins were originally described as a group of Ca2+-dependent (Ctype) carbohydrate-binding proteins that differ from other lectins (Drickamer, 1988) and now the protein-containing domains with sequences similar to CTL domains (CTLDs/CRDs) also belong to this family. Invertebrate C-type lectins contain some specific sites unique to C-type lectins, including a carbohydrate-binding site and Ca2+binding site (Zelensky and Gready, 2005) and most C-type lectins contain one carbohydrate recognition domain (CRD), but some have two or more CRDs (East and Isacke, 2002). A CRD fold is composed of approximately 120–130 residues that form a characteristic double-loop structure. The double-loop structure is stabilized by two conserved disulfide bridges, as well as a set of conserved hydrophobic and polar interactions (Zelensky and Gready, 2005). There
http://dx.doi.org/10.1016/j.dci.2014.11.015 0145-305X/© 2014 Published by Elsevier Ltd.
Please cite this article in press as: Xin Huang, Ying Huang, Yan-Ru Shi, Qian Ren, Wen Wang, Function of a novel C-type lectin with two CRD domains from Macrobrachium rosenbergii in innate immunity, Developmental and Comparative Immunology (2014), doi: 10.1016/j.dci.2014.11.015
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are four Ca2+-binding sites identified from CRDs of different species; among which, Ca2+-binding site 2 is structurally conserved and participates in the calcium-mediated carbohydrate interaction. Two characteristic motifs exist in the Ca2+-binding site 2, which are directly involved in monosaccharide binding. The first one is always Glu-Pro-Asn (EPN) or Gln-Pro-Asp (QPD), which is thought to determine binding toward mannose or galactose, respectively; the second motif is always Trp-Asn-Asp (WND) (Zelensky and Gready, 2003, 2005). Many C-type lectins have been reported in invertebrate species and they participate in various innate immune responses. For example, Penaeus monodon (PmAV) has anti-viral activity (Luo et al., 2003). Litopenaeus vannamei has a C-type lectin (LvLT) with two CRDs, which is expressed only in the hepatopancreas. It may have a role in WSSV infection (Ma et al., 2007). Fenneropenaeus chinensis (FchsL), which has a single CRD, is specifically expressed in hepatopancreas and exhibits antimicrobial activity (Sun et al., 2008a). Purified lectin from M. rosenbergii (Mrlectin) has been demonstrated to regulate the production of oxidative radicals and functions as a potential cytotoxin and microbiocide (Sierra et al., 2005). Moreover, previous published research reported that the giant freshwater prawn M. rosenbergii had a high tolerance to WSSV (Sahul Hameed et al., 2000). Vibriosis can result in high mortality rates of larval M. rosenbergii (Jayaprakash et al., 2006). In our prior published research, we have found lectins serve important immune functions in invertebrates, including the following examples: an LDLa domain-containing C-type lectin of Eriocheir sinensis was reported that could agglutinate and bind a broad range of microbes (Huang et al., 2014a). The result of this paper was in Q3 accordance with another one (Huang et al., 2014b). Ren et al. (2012) reported the immune response of four dual-CRD C-type lectins to Q4 microbial challenges in giant freshwater prawn M. rosenbergii (Ren et al., 2012), which concluded that the four dual-CRD lectins may play important roles in pathogen elimination and anti-WSSV innate immunity, among other contributions. However there is insufficient research evidence for the possible role of C-type lectin from M. rosenbergii in the innate immunity response. In the present study, we report a novel C-type lectin with 2 CRDs from the M. rosenbergii, designated as MrCTL. Its tissue distributions and expression patterns in the hepatopancreas upon challenge by Vibrio parahaemolyticus or WSSV were investigated. Recombinant MrCTL and its individual MrCRD1 and MrCRD2 proteins bind to or agglutinate bacteria. Our study provides preliminary results that may clarify the probable roles of dual-CRD lectins in the innate immunity of M. rosenbergii. 2. Materials and methods 2.1. Immune challenge of M. rosenbergii and sample collection Adult M. rosenbergii (about 15 g each) were obtained from an aquaculture market in Nanjing, Jiangsu Province, China, and cultured temporarily in freshwater tanks at room temperature (25 °C). The methods of viral inoculum preparation and quantification were according to a previous study (Wang et al., 2009b). In the experimental group, V. parahaemolyticus (approximately 3 × 107 cells per shrimp) or WSSV (approximately 3 × 107 copies per shrimp) was injected into the abdominal segment of the shrimp by using a 1 ml sterile syringe. The healthy prawns were inoculated with 50 μl of PBS as the control group. At 2, 6, 12, 24 and 48 h post injection, the hepatopancreas was collected from challenged prawns of each experimental group for RNA extraction. Hemolymph was collected from healthy prawns (untreated) by mixing an equal volume of improved anticoagulant buffer (ACD-B) (glucose, 1.47 g; citric acid, 0.48 g; trisodium citrate, 1.32 g; prepared in double distilled water and brought to 100 ml, pH 7.3) (Huang et al., 2013). The
hemolymph was centrifuged at 800 × g for 10 min (4 °C) to isolate the hemocytes. Other tissues (i.e., heart, hepatopancreas, gills, stomach, and intestine) were also collected from healthy prawns for RNA extraction. Three prawns were chosen to eliminate individual differences. 2.2. Total RNA isolation and cDNA synthesis RNAs from various tissues were extracted using an RNApure highpurity total RNA rapid extraction kit (Spin-column, BioTeke, Beijing, China) according to the manufacturer’s protocols. The first-strand cDNA of the samples was synthesized for quantitative real-time PCR (qRT-PCR) analysis by using the PrimeScript® 1st Strand cDNA Synthesis Kit (Takara, Dalian, China), with the Oligo dT Primer. The synthesized cDNA was used in the real-time polymerase chain reaction (RT-PCR) analysis. 2.3. Cloning of the full-length cDNA of MrCTL and sequence analysis Based on the expressed sequence tags (ESTs) obtained through the sequencing of the prawn hepatopancreas transcriptome, we designed the gene-specific primers (MrCTL-F: 5′-aaggaaggagac tgggtttgggctaatgac-3′, MrCTL-R: 5′-gcctccgagtgcttcgcatagtgtttta3′) to amplify the 5′ and 3′ ends of the cDNA. The 5′ and 3′ fragments of MrCTL were amplified using gene-specific primers and a Universal Primer A Mix (UPM) according to Rapid Amplification cDNA End (RACE) methods by using a Clontech SMARTer TM RACE cDNA Amplification Kit from TAKARA (Dalian, China). The full length of MrCTL cDNAs was obtained by overlapping the 5′ and 3′ fragments. The cDNA sequence and deduced amino acid sequence of MrCTL were analyzed using the BLAST algorithm (http://www.ncbi.nlm.nih Q5 .gov/blast) and the Expert Protein Analysis System (http://www .expasy.org). The signal sequence and domain organization was analyzed with the Simple Modular Architecture Research Tool (http:// smart.embl-heidelberg.de/). The ClustalW2 Multiple Alignment program (http://www.ebi.ac.uk/Tools/msa/clustalw2/) was used to create the multiple sequence alignments. Molecular Evolutionary Genetics Analysis 5.05 was used for the phylogenetic analysis of MrCTL with other C-type lectins (Kumar et al., 2008). 2.4. Real-time PCR analysis of mRNA expression The tissue distribution of MrCTL at mRNA levels in the hemocytes, heart, hepatopancreas, gills, stomach, and intestine was analyzed through RT-PCR using the primers (MrCTL-RT-F: 5′ctgccttccctctttgata-3′ and MrCTL-RT-R: 5′-cagtttgtgtgg ttggttc-3′). qRT-PCR was performed to analyze the mRNA expression level changes of MrCTL in the hepatopancreas of the prawns of M. rosenbergii after a WSSV or V. parahaemolyticus challenge at 2, 6, 12, 24 and 48 h. The qRT-PCR was performed using SYBR® Premix Ex TaqTM II (Tli RNaseH Plus) (TAKARA, Dalian, China) according to the manufacturer’s instructions. The GAPDH was amplified for internal standardization with the primers (MrGAPDH-RT-F: 5′tgccgcccagaacatcatt-3′ and MrGAPDH-RT-R: 5′-tcgtcttcggtgtagccca3′). The 2−ΔΔCt method was used to analyze the expression level of MrCTL (Livak and Schmittgen, 2001). An unpaired sample t-test was conducted, and differences were considered significant if P < 0.05. 2.5. Expression and purification of recombinant MrCTL and its individual CRD domains Primers MrCTL-ex-F (5′-tactcagaattctatgagtgcccgtccccctac-3′) and MrCTL-ex-R (5′-tactcagcggccgcttgtttcatattttgctgg-3′) were designed to amplify a cDNA fragment encoding the mature MrCTL that contained both CRDs; MrCRD1-ex-F (5′-tactcagaattctatgagt gcccgtcccccta-3′) and MrCRD1-ex-R (5′-tactcagcggccgcgtcgcagatg
Please cite this article in press as: Xin Huang, Ying Huang, Yan-Ru Shi, Qian Ren, Wen Wang, Function of a novel C-type lectin with two CRD domains from Macrobrachium rosenbergii in innate immunity, Developmental and Comparative Immunology (2014), doi: 10.1016/j.dci.2014.11.015
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actccatt-3′) primers were used to obtain the MrCRD1 domain, while MrCRD2-ex-F (5′-tactcagaattctgcccagttccgttccagatg-3′) and MrCRD2ex-R (5′-tactcagcggccgcttgttt catattttgctgg-3′) primers were used for the MrCRD2 domain. The amplified fragment was digested by EcoR I and Not I and then inserted into the pET30a(+) vector. Afterward, the recombinant plasmid was transformed to Escherichia coli BL21 (DE3) cells for IPTG-induced recombinant expression (final IPTG concentration of 0.5 mM). The temperature for protein induction was 37 °C. The rMrCTL, rMrCRD1 and rMrCRD2 were then purified through affinity chromatography by using Ni SepharoseTM 6 Fast Flow (GE Healthcare, USA) following the instructions. The purified proteins were detected by using 12.5% SDS–polyacrylamide gel electrophoresis (SDS-PAGE). The concentration of purified recombinant proteins was quantified by a BCA method (Smith et al., 1985).
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2.6. Microbial agglutination and binding assays The pathogen bacteria V. parahaemolyticus and Staphylococcus aureus were used for agglutination assays and they were maintained in our laboratory. Gram-positive bacteria (S. aureus) and Gramnegative bacteria (V. parahaemolyticus) in mid-logarithmic phase were washed twice in sterile Tris-buffered saline (TBS: 10 mM Tris– HCl, 150 mM NaCl, pH 7.5) and resuspended in TBS at 2 × 108 cells/ ml. In the presence or absence of 10 mM CaCl2, microorganisms were incubated with the recombinant proteins (MrCTL, MrCRD1 or MrCRD2, 100 μg/ml) or BSA (100 μg/ml, as control) by adding 25 μl of bacteria to 25 μl of TBS containing the proteins. The mixture was incubated at room temperature for 1 h, and the agglutinating reactions were observed by microscopy. Gram-positive (G+) bacteria (S. aureus, Micrococcus luteus, Bacillus subtilis and Bacillus megaterium) and Gram-negative (G−) bacteria (Aeromonas hydrophila, V. parahaemolyticus, Vibrio anguillarum and E. coli) were used for the bacterial-binding assay. The overnight bacterial culture in LB was centrifuged at 6000 rpm for 5 min and then washed with TBS twice. Approximately 500 μl of recombinant rMrCTL, rMrCRD1 or rMrCRD2 (1 mg/ml) was incubated with a bacterial suspension (2 × 108 cells/ml) at room temperature for 30 min with gentle rotation. The microorganisms were washed 4 times with TBS and then eluted with 100 μl of 8 M urea solution. Afterward, the elution was analyzed by 12.5% SDS-PAGE and rMrCTL, rMrCRD1 or rMrCRD2 protein was detected by western blot using Anti-His Mouse mAb.
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3. Results and discussion 3.1. Cloning and sequence analysis of MrCTL cDNA MrCTL comprises a 52 bp 5′ untranslated region (UTR), an open reading frame (ORF) of 1002 bp encoding a 333 amino acid protein, and a 3′ UTR of 292 bp with a polyadenylated tail. The carbohydrate binding specificity of C-type lectins is mainly determined by the position of hydrogen bond donors and acceptors in the conserved motifs of Ca2+-binding site 2 in CRDs (Zelensky and Gready, 2005). The CRDs with an EPN (Glu-Pro-Asn) motif bind mannose, while the CRDs with a QPD (Gln-Pro-Asp) motif bind galactose. Compared with the residues important for stabilizing the structure, these residues seem to be more diverse in invertebrate C-type lectins (Zelensky and Gready, 2003, 2005). MrCTL consists of two carbohydrate recognition domains (CRDs). The first CRD (MrCRD1) contains a QEP motif. The second CRD (MrCRD2) contains an EPD motif (Appendix S1: Supplementary Fig. S1). The sugar-binding motifs of MrCTL are not EPN or QPD. The multiple sequence alignment showed that MrCTL has a higher similarity with the dual-CRD lectins of Palaemon modestus and M. rosenbergii (Appendix S1: Supplementary Fig. S2). BLASTX analysis revealed that MrCTL shared 71% identity to C-type lectins from P. modestus, which contained 2 CRDs. The results of BLASTX analysis is similar to the phylogenetic tree, which showed that MrCTL together with dual-CRD containing lectins such as PmCTL1, PmCTL2, Mrlectin1 and Mrlectin2, belonged to one cluster. Other dual-CRD lectins constitute another group (Appendix S1: Supplementary Fig. S3). All the results illustrated that MrCTL was a novel member of the C-type lectin superfamily in M. rosenbergii.
78 3.2. Tissue distribution and expression patterns of MrCTL Expression of MrCTL mRNA in various tissues was determined by qRT-PCR. As the counterpart of the mammalian liver and the insect fat body, the shrimp hepatopancreas is a major tissue involved in immunity (Gross et al., 2001). In the present study, the highest expression of MrCTL mRNA was detected in the hepatopancreas and it also could be detected in other tissues (heart, hemocytes, gill, stomach and intestine) with relatively low expression levels (Appendix S1: Supplementary Fig. S4). The result was consistent with studies on some other C-type lectins, such as the C-type lectin of P. monodon (PmLT) (Ma et al., 2008), the C-type lectin with two CRD
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Fig. 1. qRT-PCR analysis of MrCTL in the hepatopancreas of M. rosenbergii under V. parahaemolyticus (A) or WSSV (B) challenged at 2, 6, 12, 24 and 48 h. Three prawns were chosen to eliminate individual differences at each time point. Asterisks indicate significant differences (*P < 0.05, **P < 0.01, ***P < 0.001) compared with values of the control. Error bars represent the mean ± S.D. of three independent PCR amplifications and quantifications.
Please cite this article in press as: Xin Huang, Ying Huang, Yan-Ru Shi, Qian Ren, Wen Wang, Function of a novel C-type lectin with two CRD domains from Macrobrachium rosenbergii in innate immunity, Developmental and Comparative Immunology (2014), doi: 10.1016/j.dci.2014.11.015
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Fig. 2. Recombinant expression and purification of MrCTL, MrCRD1 and MrCRD2 and their agglutinate and binding ability toward various bacteria. i. Recombinant expression and purification of MrCTL (A), MrCRD1 (B) and MrCRD2 (C). M: protein marker; Lane 1, crude protein extracts of E. coli without induction; Lane 2, crude protein extracts of E. coli induction for 4 h by IPTG; Lane 3, recombinant protein purified by Ni SepharoseTM 6 Fast Flow. ii. Direct binding of rMrCTL, rMrCRD1 or rMrCRD2 to various bacteria. Bacteria were incubated with rMrCTL, rMrCRD1 or rMrCRD2 and then washed four times with TBS and subjected to elution with 8 M urea. Binding was confirmed by Western blot with Anti-His Mouse mAb against recombinant MrCTL, MrCRD1, MrCRD2 with His tag. iii. Agglutination of bacteria (S. aureus and V. parahaemolyticus) by rMrCTL, rMrCRD1 or rMrCRD2 with either absence or presence of calcium. BSA served as a control.
Please cite this article in press as: Xin Huang, Ying Huang, Yan-Ru Shi, Qian Ren, Wen Wang, Function of a novel C-type lectin with two CRD domains from Macrobrachium rosenbergii in innate immunity, Developmental and Comparative Immunology (2014), doi: 10.1016/j.dci.2014.11.015
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domains from the banana shrimp Fenneropenaeus merguiensis (FmLc) (Rattanaporn and Utarabhand, 2011), the C-type lectin (FcLec5) from the Chinese white shrimp F. chinensis and the C-type lectin with two CRD domains (FcLec2) from Chinese shrimp F. chinensis (Xu et al., 2010; Zhang et al., 2009b). In total, the expression pattern proved that C-type lectins played an important role in the immune defense. Vibriosis is a major bacterial disease caused by bacteria in the genus Vibrio (Jiravanichpaisal et al., 1994). WSSV is the most serious threat to shrimp aquaculture (Escobedo-Bonilla et al., 2008), necessitating a more thorough study of the anti-Vibrio or anti-WSSV immune response in M. rosenbergii. In our study, we analyzed the temporal expression of MrCTL in prawns challenged with V. parahaemolyticus or WSSV by qRT-PCR (Fig. 1). MrCTL was downregulated at 2 h after the V. parahaemolyticus challenge, reaching the highest level at 6 h, decreasing at 12 h, and then upregulated at 24 h. The decrease of MrCTL expression at 48 h post V. parahaemolyticus infection was not significantly different from the PBS injected group (Fig. 1A). A hepatopancreas-specific lectin from Chinese white shrimp was also downregulated 2 h after the bacterial challenge, and then upregulated from 6 h to 24 h postQ6 challenge (Sun et al., 2008b). The results indicate that bacteria may achieve some protection from the host immune system attack by downregulating MrCTL originally, which may function as opsonins. The subsequent rapid upregulation of MrCTL suggests that it may be involved in the immune response against the pathogens. Lectins were the major group of pattern recognition proteins up-regulated in the hepatopancreas of L. vannamei infected with WSSV (Gross et al., 2001). L. vannamei C-type lectin (LvLT) with two CRDs, which is expressed only in the hepatopancreas, may have a role in WSSV infection (Ma et al., 2007). FcLec3, a single-CRD lectin, interacted with VP28, one of the major envelope proteins of WSSV (Wang et al., 2009a). The giant freshwater prawn M. rosenbergii had a high tolerance to WSSV (Sahul Hameed et al., 2000). In this study, expression of MrCTL in prawns injected with WSSV rapidly increased at 2 h post-injection and then downregulated from 6 h to 12 h, subsequently reaching a maximum level at 24 h post-injection and finally declined at 48 h post-injection (Fig. 1B). The observable increase of MrCTL expression at 2 and 24 h post WSSV infection suggested that MrCTL could be effectively induced by WSSV stimulation. And the remarkable up-regulation of MrCTL expression in the early stage of WSSV infection implied the importance of multirecognition characteristics of MrCTL. However, the potential mechanism is not clear presently, which requires further study. All the results indicate that the C-type lectin of M. rosenbergii may function in innate immunity against viral and bacterial challenges. 3.3. Expression and purification of rMrCTL, rMrCRD1, rMrCRD2 All the recombinant proteins (rMrCTL, rMrCRD1 and rMrCRD2) are expressed as inclusion. The predicted molecular mass of mature MrCTL from the deduced amino acid sequence was about 37.6 kDa. rMrCTL has an apparent molecular mass of 44.6 kDa with a pET30a(+) vector, while rMrCRD1 and rMrCRD2 are 28 and 27 kDa, respectively (Fig. 2.i.). The molecular mass of the pET-30a(+) vector (containing N-terminal His tag, C-terminal His tag and S tag) from recombinant protein was about 7 kDa. 3.4. Microbial agglutinating and binding activity of MrCTL and its two CRD domains Aggregation activity is the most important feature of C-type lectins. The process may be initiated by lectins binding and recognizing carbohydrate components present on the surface of microorganisms, but absent in the host. Considering binding to the surface of the foreign invaders was the first step in the immune recognition and initiation of immune response (Gross et al., 2001). In
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the microorganism binding assay, rMrCTL, rMrCRD1 and rMrCRD2 have been found to bind toward all tested microorganisms, including Gram-positive bacteria (S. aureus, M. luteus, B. subtilis and B. megaterium) and Gram-negative bacteria (A. hydrophila, V. parahaemolyticus, V. anguillarum and E. coli) (Fig. 2.ii.). The broad binding spectrum of MrCTL toward various microorganisms might suggest its important roles in the immune defense against various pathogens in prawns. In the presence of calcium, recombinant MrCTL can agglutinate all of the tested Gram-positive (S. aureus) and Gramnegative bacteria (V. parahaemolyticus) (Fig. 2.iii.). The two dualCTLD-containing proteins FcLec2 and FcLec5 also have an effect on agglutination (Xu et al., 2010; Zhang et al., 2009b). PmLec from P. monodon (Luo et al., 2006); and PjLec from Penaeus japonicus (Yang et al., 2007) can also agglutinate to the bacteria mentioned above. In summary, a new C-type lectin (MrCTL) with two CRDs from the prawn M. rosenbergii was identified in this study. MrCTL protein was mainly expressed in the hepatopancreas of prawns and its expressions are regulated by PAMPs or bacterial challenge. The recombinant MrCTLs have agglutinating, binding activities to various bacteria. All these results suggest that MrCTL functioned as a PRR involved in pattern recognition and pathogen elimination in the innate immunity of the prawn M. rosenbergii.
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Acknowledgments We appreciate Professor O. Roger Anderson (Columbia University) for editing the manuscript. The current study was supported by the National Natural Science Foundation of China (Grant Nos. 31101926, 31170120, 31272686), the Natural Science Foundation of Jiangsu Province (BK20131401), Natural Science Fund of Colleges and Universities in Jiangsu Province (13KJB240002, 14KJA240002), High level talents in Nanjing Normal University Foundation (2012104XGQ0101), and Project for Aquaculture in Jiangsu Province (No. PJ2011-65; Y2013-45; D2013-5-3; 270 D2013-5-4), and the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
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Appendix: Supplementary Material Supplementary data to this article can be found online at doi:10.1016/j.dci.2014.11.015.
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Please cite this article in press as: Xin Huang, Ying Huang, Yan-Ru Shi, Qian Ren, Wen Wang, Function of a novel C-type lectin with two CRD domains from Macrobrachium rosenbergii in innate immunity, Developmental and Comparative Immunology (2014), doi: 10.1016/j.dci.2014.11.015
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