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Fish & Shellfish Immunology xxx (2015) 1e5

Contents lists available at ScienceDirect

Fish & Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi

Short sequence report

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Molecular cloning, characterization, and expression analysis of two different types of lectins from the oriental river prawn, Macrobrachium nipponense Yunji Xiu a, b, Yinghui Wang a, b, Yunting Jing a, b, Yakun Qi a, Zhengfeng Ding c, Qingguo Meng a, b, **, Wen Wang a, b, * a b c

Jiangsu Key Laboratory for Biodiversity & Biotechnology, College of Life Sciences, Nanjing Normal University, 1 Wenyuan Road, Nanjing 210046, China Jiangsu Key Laboratory for Aquatic Crustacean Diseases, College of Life Sciences, Nanjing Normal University, 1 Wenyuan Road, Nanjing 210046, China Freshwater Fisheries Research Institute of Jiangsu Province, 79 Chating East Street, Nanjing 210017, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 30 November 2014 Received in revised form 15 April 2015 Accepted 18 April 2015 Available online xxx

Lectins, which are widely expressed in invertebrates, play important roles in many biological processes, including protein trafficking, cell signaling, pathogen recognition, as effector molecules, and so on (Wang and Wang, 2013). This study identified one novel M-type lectin and one L-Type lectin, designated as MnMTL1 and MnLTL1, from the oriental river prawn Macrobrachium nipponense. The full-length cDNA of MnMTL1 was 2064 bp with a 1761 bp ORF encoding a putative protein of 586 deduced amino acids. The full-length cDNA of MnLTL1 was 1744 bp with a 972 bp ORF encoding a 323-amino acid peptide. The deduced MnMTL1 protein contained a putative type II transmembrane region and a 440-aa Glycoside hydrolase family 47 (GH47) domain. One luminal carbohydrate recognition domain and a 23-aa type I transmembrane region were identified from the MnLTL1. MnMTL1 shared 78% identity with Marsupenaeus japonicus M-type lectin and MnLTL1 shared 83% similarity with M. japonicus L-type lectin. RT-PCR analysis showed that MnMTL1 and MnLTL1 were expressed in all tested tissues. Quantitative real-time PCR analysis revealed that MnMTL1 and MnLTL1 are substantially fluctuant during Aeromonas hydrophila and Aeromonas veronii infections. Based on immune responses and previous literature, we assumed that MnMTL1 and MnLTL1 might be functioned as pattern recognition receptors and play important roles in the immune response of M. nipponense. © 2015 Published by Elsevier Ltd.

Keywords: Macrobrachium nipponense L-Type lectin M-Type lectin Pattern recognition protein Immune response

1. Introduction The oriental river prawn Macrobrachium nipponense is an economically and nutritionally important crustacean. As an indigenous species, M. nipponense is widely distributed throughout

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Abbreviations: MnMTL1, M-type lectin of Macrobrachium nipponense; MnLTL1, L-type lectin of Macrobrachium nipponense; GH47, Glycoside hydrolase family 47; proPO, prophenoloxidase; PRPs, Pattern recognition proteins; PAMP, pathogen associated molecular patterns; CRD, carbohydrate recognition domain. * Corresponding author. Jiangsu Key Laboratory for Biodiversity & Biotechnology, College of Life Sciences, Nanjing Normal University, 1 Wenyuan Road, Nanjing 210046, China. Tel.: þ86 25 85891955. ** Corresponding author. Jiangsu Key Laboratory for Biodiversity & Biotechnology, College of Life Sciences, Nanjing Normal University, 1 Wenyuan Road, Nanjing 210046, China. E-mail addresses: [email protected] (Q. Meng), [email protected], [email protected] (W. Wang).

China, including rivers, lakes, reservoirs and ditches from the South to the North. In China, the farming of M. nipponense has been in stable production for 10 years up to the present, with a farming area of about 400 000 ha year1 and a farmed production of around 200 000 t year1 including monoculture and polyculture [1]. The frequency of disease outbreaks for M. nipponense has been very low in the past. However, it has become increasingly prevalent, because of genetic retrogression, deterioration of water quality and environmental stress. Recently, some bacterial diseases such as Aeromonas veronii isolated from “soft-shell syndrome” [2] and Aeromonas hydrophila isolated from “red gill disease” [3], have led to mass mortalities. Invertebrates, which lack adaptive immune systems, rely entirely on their innate immune response to protect against invading pathogens [4]. The innate defense system of invertebrates is composed of humoral and cellular immunity systems [5]. The humoral responses include the prophenoloxidase (proPO) system,

http://dx.doi.org/10.1016/j.fsi.2015.04.022 1050-4648/© 2015 Published by Elsevier Ltd.

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the clotting cascade and a wide array of antimicrobial peptides; whereas the cellular immune responses include apoptosis, encapsulation, phagocytosis, nodule formation and RNA interference critical to effective suppression of invading pathogens [6e8]. The innate immunity responses include immune recognition, signal transduction and production of effectors [9]. Pattern recognition proteins (PRPs) play important roles in recognizing pathogenassociated molecular patterns (PAMP) and triggering a number of defense responses [10,11]. Lectins, abundant in almost all living organisms [12], are identified as one of the important PRPs [13e16]. Lectins are characterized by the carbohydrate recognition domain (CRD) which could bind sugars. Different CRDs show symbolic motif and recognize specific types of sugars [17]. The Animal Lectins Homepage (http:// www.imperial.ac.uk/research/animallectins//default.html) divided animal lectins into 13 groups: C-type, F-type, I-type, M-type, Ltype, P-type, R-type, F-box lectins, chitinase-like lectins, ficolins, calnexin, galectins, and intelectins. The M-type lectins, containing a glycoside hydrolase family 47 protein structural domain, are type II transmembrane proteins. So far, only one M-type lectin has been identified from Marsupenaeus japonicus, but little information is known about it [17]. The L-type lectins, containing a leguminous lectin domain, can bind to high-mannose type oligosaccharides. In recent years, three L-type lectins have been characterized in crustaceans, including Eriocheir sinensis [18] and M. japonicus [19]. However, the lectin information remains unknown in M. nipponense. This study is the first to report on the discovery of a M-type lectin (MnMTL1) and a L-type lectin (MnLTL1) from the prawn M. nipponense. Here, the full length cDNA sequences of MnMTL1 and MnLTL1 were cloned and characterized. The deduced amino acid sequences were compared with other known lectins from other crustaceans. Different tissue distributions and expression profiles in intestine challenged with A. hydrophila and A. veronii were also analyzed. 2. Materials and methods

analyzer yielded 43289 ESTs. BLASTx analysis revealed that two ESTs were homologous to M-type lectin protein of M. japonicus (AFJ59949) and L-type lectin of M. japonicus (AFJ59950). This two ESTs were selected for further cloning of MnMTL1 and MnLTL1. 2.3. Cloning the full-length of MnMTL1 and MnLTL1 A SMARTer™ RACE cDNA Amplification Kit (Takara, Japan) was used for Rapid amplification of cDNA ends (RACE). Two pairs of gene-specific primers were designed based on the corresponding EST sequences. For the 50 -RACE, the PCR reactions were performed with MnMTL1-R1 or MnLTL1-R1 and Universal Primer A Mix (UPM). For the 30 -RACE, the PCR reactions were performed with MnMTL1-F1 or MnLTL1-F1 and UPM. PCR was conducted in accordance with the protocol of Advantage 2 PCR Kit (Clontech). The PCR procedure was conducted under the following conditions: 4 min at 94  C, 5 cycles (30 s at 94  C, 3 min at 72  C), 25 cycles (30 s at 94  C, 30 s at 60  C, 3 min at 72  C) and 5 min at 72  C. The PCR fragments were cloned into pMD19-T vector and sequenced by Springen (Nanjing) Biotechnology Company. 2.4. Sequence analysis The homology searches for nucleotide and amino acid sequence similarities were conducted with BLAST programs (http://blast. ncbi.nlm.nih.gov/Blast.cgi). The deduced amino acid sequence was analyzed with the Expert Protein Analysis System (http:// www.expasy.org/). SignalP 4.1 program was utilized to predict the presence and location of signal peptides (http://www.cbs.dtu.dk/ services/SignalP/). Domain predictions were conducted with the Simple Modular Architecture Research Tool (http://smart.emblheidelberg.de/). Multiple sequence alignments were performed using the ClustalW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/). A cladogram was constructed based on the amino sequence alignments by the neighbor-joining (NJ) algorithm embedded in the MEGA 5 program.

2.1. Animal and RNA extraction

2.5. Bacterial challenge and sample preparation

The oriental river prawns, M. nipponense (2e3 g per prawn) were purchased from an aquaculture market in Nanjing, Jiangsu Province, China. They were cultured in tanks at 20  C with freshwater and an aeration system. In order to confirm the prawns were not infected, bacteria in the hemolymph was isolated by LB agar plates. Prawn health status was assessed daily during acclimation by monitoring both general activity and food intake. Total RNA from different tissues was extracted using TRIzol Reagent following the manufacturer's instructions. RNA quality was assessed by electrophoresis on 1.2% agarose gel and the RNA concentration was measured by the absorbance at 260 nm on a spectrophotometer.

During the experiment, the prawns were fed once daily with commercial feed. The prawns were randomly divided into three groups and 50 prawns injected individually with 50 mL live A. hydrophila suspension (104 cells mL1) used as first challenge group. In the second challenge group, 50 prawns were injected individually with 50 mL live A. veronii suspension (105 cells mL1). For the control group, 50 prawns were injected with 50 mL saline (0.85% NaCl) (pH ¼ 7.0). Every five individuals were randomly sampled at 0, 1, 12, 24, 36 and 48 h post challenge. Intestine tissue was collected, and all of the samples extracted at different times were stored at 80  C for subsequent total RNA extraction.

2.2. cDNA library construction and EST analysis

2.6. Expression analysis of MnMTL1 and MnLTL1 transcript

A cDNA library was constructed from the hemocytes of M. nipponense (Shanghai Hanyu Bio-Tech). The total RNA was extracted from hemocytes of healthy prawns using Trizol reagent (Invitrogen), and the mRNA was extracted with the PolyATract mRNA isolation system (Promega, USA). The cDNA library was constructed with the Creator SMART cDNA Library Construction Kit (Clontech, USA). The double strand cDNA was digested and ligated with the pDNR-LIB vector, and then transformed into competent DH5 cells. Individual colonies were randomly selected, and plasmids were extracted for sequencing from the 50 -ends. Random sequencing of the library using illumina Hiseq 2000 genome

The mRNA expressions of MnMTL1 and MnLTL1 transcripts in different tissues, including hemocytes, heart, hepatopancreas, gill, intestine, nerve and muscle of untreated prawns, were determined by quantitative real-time PCR. The temporal expressions of MnMTL1 and MnLTL1 in intestine were also determined after being challenged with A. hydrophila. The first-strand cDNA was synthesized using PrimeScript™ 1st Strand cDNA Synthesis Kit (Takara) with 1 mg of total RNA. Two pairs of gene-specific primer (MnMTL1-qRT-F, MnMTL1-qRT-R and MnLTL1-qRT-F, MnLTL1-qRT-R) (Table 1) were used to amplify MnMTL1 and MnLTL1 genes, respectively. The primers MnEF2-qF

Please cite this article in press as: Y. Xiu, et al., Molecular cloning, characterization, and expression analysis of two different types of lectins from the oriental river prawn, Macrobrachium nipponense, Fish & Shellfish Immunology (2015), http://dx.doi.org/10.1016/j.fsi.2015.04.022

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Y. Xiu et al. / Fish & Shellfish Immunology xxx (2015) 1e5 Table 1 Primers used in the current study. Name

Sequence (50 e30 )

MnMTL1-F1 MnMTL1-R1 MnLTL1-F1 MnLTL1-R1 UPM MnMTL1-qRT-F MnMTL1-qRT-R MnLTL1-qRT-F MnLTL1-qRT-R MnEF2-qF MnEF2-qR

ATCAAGATTGGGGATGGCAGATGTTTC GGAATAGAGATTTACATCAGCAAATGGCACA TAGACAACAAGATGGCATACAAGGATTGCTTC ATTGCTGTAAGTGTCGGCAATAATGGCTAATC CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT CCCACACCGAAGCATCTCAC CTGGTAGCTCAGGAGGAGGTATAA TAAGGTCCCTCTCTCAGTCCGC AGAAATCTCTGCTCCCAAATACATC GCCACGTCTCCAGGAACCAGTAT AACGCAAGTCAGCAGTGAAACCA

and MnEF2-qR (Table 1), were used to amplify elongation factor 2 (EF2) for internal standardization. For Real Time PCR, the cycling protocol was 1 cycle of 95  C for 30 s; 24 cycles of 95  C for 5 s, 55  C for 10 s and 72  C for 15 s followed by 1 cycle of 72  C for 5 min and holding at 4  C. qRT-PCR was carried out in a total volume of 20 ml (10 ml of 2  SYBR Premix Ex Taq, 1 ml cDNA mix, 0.5 ml of each primer (10 mM), and 8 ml of sterile distilled H2O). The PCR program was 95  C for 30 s, followed by 40 cycles of 95  C for 5 s and 60  C for 30 s. All samples were run three times. All data are reported in terms of relative mRNA expression as mean ± S.E. Statistical significance was determined by one-way ANOVA and post-hoc Duncan multiple range tests. Significance was set at P < 0.05.

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Multiple alignments displayed several conserved features, e.g. one transmembrane region and GH47 domain were conserved in M-type lectins (Supplemental Fig. 3). One signal peptide, LTLD domain and one transmembrane region were also conserved in Ltype lectins (Supplemental Fig. 4). Results of similarity analyses and alignments indicate that same type lectins were highly conserved in invertebrates, which very likely confirms their important functions during evolution. 3.3. Phylogenetic analysis To analyze the relationships among MnMTL1 and MnLTL1 with other selected lectins, a phylogenetic tree was constructed using the neighbor-joining method. As shown in Supplemental Fig. 5, based on the tree topology, selected lectins could be classified into

3. Result and discussion 3.1. Molecular characterization of MnMTL1 and MnLTL1

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The cloned full-length of MnMTL1 cDNA (GenBank accession number KP123625), contained 2064 bp, with a 1761-bp ORF encoding a 586-aa protein, a 109 bp 50 UTR and a 194 bp 30 UTR. Analysis by the SMART program, a 20-aa type II transmembrane region, was identified at the N-terminal; and a 440-aa Glycoside hydrolase family 47 (GH47) domain was found at the C-terminal of MnMTL1 (Supplemental Fig. 1). A GH47 domain, conserved in all Mtype lectins [17,20], was most similar to Tribolium castaneum endoplasmic reticulum mannosyl-oligosaccharide 1,2-alpha-mannosidase (Identity ¼ 66%). Alpha-mannosidase is involved in the maturation of Asn-linked oligo-saccharides [(PUBMED:8144580)]. The enzyme hydrolyses terminal 1, 2-linked alpha-D-mannose residues contained in the oligo-mannose oligosaccharide man(9)(glcnac)(2) in a calcium-dependent manner [21]. The cloned full-length of MnLTL1 cDNA (GenBank accession number KP123626), contained 1744 bp, with a 972-bp ORF encoding a 323-aa protein, a 37 bp 50 untranslated region (UTR), and a 735 bp 30 UTR (Supplemental Fig. 2). A typical signal peptide of 15 aa residues was predicted in the N-terminal of MnLTL1. By analysis using the SMART program, a 226-aa L-type lectin domain (LTLD) and a 23-aa type I transmembrane region were identified at the C-terminal (Supplemental Fig. 2). LTLD contains a luminal carbohydrate recognition domain, which exhibits homology to leguminous lectins [22]. 3.2. Homology analysis of MnMTL1 and MnLTL1 BLAST analysis showed a significant sequence similarity between MnMTL1 or MnLTL1 with other crustacean species. For example, MnMTL1 showed 78% similarity with M. japonicus M-type lectin. MnLTL1 shared 83% similarity with M. japonicus L-type lectin containing domain protein.

Fig. 1. (A) Tissue distribution of MnMTL1 and MnLTL1 by Real Time PCR with EF2 as a reference gene. (B) MnMTL1 and (C) MnLTL1 mRNA expression level as revealed by SYBR green quantitative RT-PCR. Each bar represents the mean ± S. D. (n ¼ 3). Hemocytes (HC), heart (HT), hepatopancreas (HP), gills (G), intestine (I), nerve (N), and muscles (M).

Please cite this article in press as: Y. Xiu, et al., Molecular cloning, characterization, and expression analysis of two different types of lectins from the oriental river prawn, Macrobrachium nipponense, Fish & Shellfish Immunology (2015), http://dx.doi.org/10.1016/j.fsi.2015.04.022

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three main clades: Group A (L-type lectin), Group B (M-type lectin), and Group C (C-type lectin). MnMTL1 was clustered with MjMTL and formed a M-type clade with endoplasmic reticulum mannosyloligosaccharide 1,2-alpha-mannosidase (MOAM). We could assume that MnMTL1 may be involved in the maturation of Asn-linked oligo-saccharides and this speculation needs further studies. MnLTL1 was clustered with PcLTL and MjLTL, and then formed a L-type clade with VIP36 (vesicular integral membrane protein of 36 kDa) and ERGIC53 (EReGolgi intermediate compartment protein of 53 kDa). It was reported that L-type lectins can be clustered into two distinct clades. One clade included all ERGIC-53 sequences and the other clade included all VIP36 genes [18]. The phylogenetic analysis demonstrated that MnLTL1 showed closest relationship with VIP36 genes. 3.4. Expression profiles in different tissues Bacteria in the hemolymph was isolated by LB agar plates and the colony results confirmed the prawns were absence of bacteria. Real Time PCR was performed on equal amounts of total RNA isolated from various tissues (Fig. 1A). The results of RT-PCR and quantitative RT-PCR analyses showed that the mRNA of MnMTL1 and MnLTL1 were expressed in all tested tissues. The highest expression of MnMTL1 was observed in intestine, with slightly less in hemocytes, and even less in other tissues (Fig. 1B). Similarly, MnLTL1 transcripts were also most abundant in intestine, with a moderate expression in the hepatopancreas and gill, and a lower expression in other tissues (Fig. 1C). The crustacean hepatopancreas and hemocytes are regarded as important tissues involved in immunity, and thus, this tissue is naturally the main source of many Ltype lectins [18,19] and C-type lectins [23]. Apart from these tissues, the intestine is also a key tissue as a source of the transcripts of shrimp C-type lectins, for example C-type lectin of Fenneropenaeus chinensis (Fchsl), C-type lectin2 of F. chinensis (FcLec2), and C-type lectin from the shrimp Litopenaeus vannamei (LvCTL1) [24e26]. These results indicate that the intestine is also a key tissue for Mtype and L-type lectins. 3.5. Expression profiles after inoculation with Aeromonas hydrophila and Aeromonas veronii Based on the tissue distribution, the time course expression of MnMTL1 and MnLTL1 in intestine was analyzed. As shown in Fig. 2A, MnMTL1 decreased significantly at 12 h (P < 0.05) post challenge with A. hydrophila, subsequently reached a minimum at 36 h (P < 0.05), and finally was slightly up-regulated at 48 h. Similar with MnMTL1, the expression of MnLTL1 decreased from 12 to 36 h after challenge and subsequently was up-regulated at 48 h (Fig. 2B). The temporal expression profiles of MnMTL1 and MnLTL1 after challenged with A. veronii are shown in Fig. 2C and D, respectively. As shown in Fig. 2C, the expression of MnMTL1 was up-regulated immediately at 1 h and 12 h. The peak of MnMTL1 mRNA transcripts was observed at 24 h post-injection (P < 0.05), which was 3.22-fold compared to the control group. For MnLTL1, the expression became down-regulated after stimulation, and at 36 h reached its minimum (P < 0.05). As time progressed, its expression level returned to a similar expression level as the control at 48 h. We note that although the expression profiles in the control group injected with saline fluctuated slightly at different time points, no significant differences were found among them. The expression patterns that appear post pathogen challenge is insufficient to draw a conclusion on the function of the lectins. However, the results provide some hints about the role of the gene. The expression of MnMTL1 and MnLTL1 is greatly affected by bacterial infections, which suggest that they may be involved in the immune

Fig. 2. Relative expressions of MnMTL1 and MnLTL1 in intestine at different time points under A. hydrophila and A. veronii challenge analyzed by quantitative RT-PCR. Graphs A and B represent expression profiles of MnMTL1 and MnLTL1 under A. hydrophila challenge, respectively. Graphs C and D represent expression profiles of MnMTL1 and MnLTL1 under A. veronii challenge, respectively. Asterisks indicate there was a significant difference between control prawns and bacteria challenged prawns.

Please cite this article in press as: Y. Xiu, et al., Molecular cloning, characterization, and expression analysis of two different types of lectins from the oriental river prawn, Macrobrachium nipponense, Fish & Shellfish Immunology (2015), http://dx.doi.org/10.1016/j.fsi.2015.04.022

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response against the pathogens. Similar results have been reported in E. sinensis, where expression of two different L-type lectins were significantly induced by different bacteria, binding experiments and clearance test suggested that this two lectins functioned as pattern recognition receptors in the immune system of E. sinensis [18]. Recently, it was suggested that L-type lectin from M. japonicus could promoted hemocyte phagocytosis and function as an opsonin involved in the antibacterial immune responses in shrimp [19]. In conclusion, we cloned and functionally characterized the MnMTL1 and MnLTL1 genes for the first time. Our results indicate that these two genes possibly participated in the antibacterial immunity of M. nipponense. Our current study should provide some valuable information that may yield insights into the molecular mechanism of the innate immune system, and the evolution of invertebrate M-type and L-type lectins. Acknowledgments We thank Professor O. Roger Anderson for editing the manuscript. We are grateful to Yongjie Liu for the presentation of bacterium. This work was supported by grants from the National Natural Sciences Foundation of China (NSFC No. 31170120, 31200139 and 31272686), the Natural Science Foundation of Jiangsu Province (Grant No. BK2011786), Jiangsu Agriculture Science and Technology Innovation Fund (No. CX(12)3066), Project for aquaculture in Jiangsu Province (Y2013-45) and A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). National Natural Sciences Foundation of China (NSFC No. 31402332), Natural Sciences Foundation of Jiangsu Province (BK20131453). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.fsi.2015.04.022. References [1] H.T. Fu, S.F. Jiang, Y.W. Xiong, Current status and prospects of farming the giant river prawn (Macrobrachium rosenbergii) and the oriental river prawn (Macrobrachium nipponense) in China, Aquac. Res. 43 (2012) 993e998. [2] X.Y. Pan, J.Y. Shen, J.Y. Li, B.X. He, W.L. Yin, G.J. Hao, et al., Identification and biological characteristics of the pathogen causing Macrobrachium nipponense soft-shell syndrome, Microbiol. China 36 (2009) 1571e1576. [3] J. Shen, D. Qian, W. Liu, W. Yin, Z. Shen, Z. Cao, et al., Studies on the pathogens of bacterial diseases of Macrobrachium nipponense, J. Zhejiang Ocean Univ. 3 (2000) 222e224. [4] J.A. Hoffmann, F.C. Kafatos, C.A. Janeway, R.A. Ezekowitz, Phylogenetic perspectives in innate immunity, Science 284 (1999) 1313e1318.

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Please cite this article in press as: Y. Xiu, et al., Molecular cloning, characterization, and expression analysis of two different types of lectins from the oriental river prawn, Macrobrachium nipponense, Fish & Shellfish Immunology (2015), http://dx.doi.org/10.1016/j.fsi.2015.04.022

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Molecular cloning, characterization, and expression analysis of two different types of lectins from the oriental river prawn, Macrobrachium nipponense.

Lectins, which are widely expressed in invertebrates, play important roles in many biological processes, including protein trafficking, cell signaling...
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