Mol Biol Rep DOI 10.1007/s11033-014-3261-z
Molecular cloning and characterization of the lipopolysaccharide and b-1,3-glucan binding protein from oriental river prawn, Macrobrachium nipponense Yunji Xiu • Ting Wu • Peng Liu • Ying Huang Qian Ren • Wei Gu • Qingguo Meng • Wen Wang
Received: 30 November 2012 / Accepted: 11 February 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract The lipopolysaccharide and b-1,3-glucan binding protein (LGBP), one of the pattern recognition proteins, plays an important role in the innate immune response of invertebrates. A 1,506 bp full-length cDNA of a LGBP gene was cloned and characterized from the oriental river prawn Macrobrachium nipponense (named as MnLGBP). Analysis of the nucleotide sequence revealed that the cDNA clone has an open reading frame of 1,119 bp, encoding a protein of 372 amino acids including a 21-aa signal peptide. The calculated molecular mass of the mature protein (351 aa) was 39.9 kDa with an estimated pI of 4.63. The MnLGBP sequence contains: (1) two putative integrin-binding motifs, (2) a glucanase motif, (3) two putative N-glycosylation sites, (4) one protein kinase C phosphorylation site, and (5) a putative recognition motif for b-1,3-linkage of polysaccharides. Sequence comparison based on the deduced amino acid sequence of MnLGBP showed varied identity of 89, 76 and 74 % with those of Macrobrachium rosenbergii LGBP, Marsupenaeus japonicus b-1,3-glucan binding proteins, and Fenneropenaeus chinensis LGBP, respectively. Quantitative RT-PCR results showed that MnLGBP was expressed in nerve, intestine, muscle, gill, heart, haemocytes and at the highest level in hepatopancreas. After challenge with the pathogen, Aeromonas hydrophila and Vibrio parahaemolyticus, the
Y. Xiu T. Wu P. Liu Y. Huang Q. Ren W. Gu Q. Meng (&) W. Wang (&) 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, People’ Republic of China e-mail: [email protected]
W. Wang e-mail: [email protected]
; [email protected]
expression of MnLGBP mRNA was significantly upregulated in the hepatopancreas compared to the control group. At the same time, the mRNA level of MnproPO increased dramatically at 48 h after injection of bacteria. These data should be helpful to better understand the function of MnLGBP in the prawn immune system. Keywords Macrobrachium nipponense LGBP Pattern recognition protein Aeromonas hydrophila Vibrio parahaemolyticus Innate immunity
Introduction The oriental river prawn, Macrobrachium nipponense, a freshwater or brackish prawn, is often a commercially important species in China, Japan and Vietnam [1, 2]. Production of this species has been challenged by opportunistic pathogen infections, including Aeromonas hydrophila, which was found to be a highly pathogenic bacterium among many isolated bacteria . Beside pathogen infections, environmental stress also seems to be an important factor contributing to reduction of immunocompetence and is signalled by the appearance or the increased prevalence of disease in crustaceans. Environmental variations are often stressfull for crustacea, resulting in a reduction of immune vigour . Therefore, understanding the immune ability of M. nipponense and their defense mechanisms has become a primary concern. The innate immune system is the first line of defense of both vertebrates and invertebrates . Because they lack an adaptive immune system, invertebrates rely entirely on their innate immune response to protect against invading pathogens . A critical step in the innate immune response is to recognize an invading organism as non-self
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. The pattern recognition proteins (PRPs) play an important role in innate immunity by recognizing and binding to common epitopes on the pathogen surface such as b-1,3-glucans (BGs), lipopolysaccharides (LPSs), and peptidoglycans (PGs) [8–10]. In arthropods, the major proteins that function as PRPs are: (1) b-glucan binding or recognition protein (BGBP or BGRP), (2) LPS and b-1,3glucan binding protein (LGBP), (3) Gram-negative binding or recognition protein (GNBP or GNRP), and (4) peptidoglycan binding or recognition protein (PGBP or PGRP) [11–14]. Subsequent to recognition, these PRPs activate distinctly a series of immune responses, including encapsulation, phagocytosis, and nodule formation , clotting cascade, the synthesis of a wide array of antimicrobial peptides, and the prophenoloxidase system (proPO) [16– 19]. LGBP is one important member of the PRPs in invertebrates and displays various biological functions . In decapod crustaceans, isolation and characterization of LGBP have been reported in Indian white shrimp Fenneropenaeus indicus , Chinese mitten crab Eriocheir sinensis , Chinese shrimp Fenneropenaeus chinensis [12, 23], giant freshwater prawn Macrobrachium rosenbergii , kuruma shrimp Marsupenaeus japonicus , white shrimp Litopenaeus vannamei , freshwater crayfish Pacifastacus leninusculus  and blue shrimp Penaeus stylirostris . The aim of the present study was to present the nucleotide sequence of LGBP from the hepatopancreas of M. nipponense, and compare its sequence with other known LGBPs and BGBPs from other decapod crustaceans. The LGBP and proPO expression profiles were evaluated when challenged with A. hydrophila or V. parahaemolyticus. The important role for MnLGBP in the proPO activating system is also discussed.
Materials and methods Animals and RNA isolation The oriental river prawns, M. nipponense (2–3 g in weight, 4–5 cm in length) obtained from a market in Nanjing, China, were cultivated at 20 °C in tanks with recirculating water. The prawns were acclimated for 10 days before processing. Total RNA was extracted from the hepatopancreas using TRIzol Reagent (Takara) following the manufacturer’s protocol. RNA quality was assessed by electrophoresis on 1.2 % agarose gel, and the total RNA concentration was determined by measuring absorbance at 260 nm using a spectrophotometer (NanoDrop 2000c ThermoScientific).
cDNA synthesis and gene cloning A total of 5 lg RNA was reversely transcribed using the Takara PrimerScriptTM RT reagent Kit (Perfect Real Time) (RR037) according to the manufacturer’s instruction. Degenerate primers, MnLGBP-F1 and MnLGBP-R1, designed based on the highly conserved nucleotides of LGBP using the CLUSTAL program, were used in the reverse-transcriptase polymerase chain reaction (RT-PCR). Amplification primers for MnLGBP are shown in Table 1. Rapid amplification of cDNA ends (RACE) of MnLGBP About 5 lg of Total RNA was reverse-transcribed with MnLGBP-R2 primer to generate a 50 -RACE template. MnLGBP-R2 primer was designed based on the sequence amplified by the degenerate primers. For the 50 -RACE, part of the MnLGBP gene was obtained using a 50 -RACE System (Invitrogen, USA) according to the manufacturer’s instruction. The primer set consisted of MnLGBP-R3 with Abridged Anchor Primer (AAP) for the first-run PCR, and MnLGBP-R4 with the Abridged Universal Amplification Primer (AUAP) for the second-run PCR. For the 30 -RACE, the RT-PCR was performed using the Oligo-d(T)18 ACP primer with MnLGBP-F2, and the nested PCR was performed using MnLGBP-F3 with 30 -RACE primer. The amplified products were cloned into pMD19-T vector and sequenced by Springen (Nanjing) Biotechnology Company. Nucleotide and amino acid sequences analysis The homology search for the nucleotide and protein sequences was performed using the BLAST algorithm at NCBI (http://www.ncbi.nlm.nih.gov/). The deduced amino acid sequence was analyzed with the Expert Protein Analysis System (http://www.expasy.org/). The signal peptide and motif were predicted by the SignalP 3.0 program (http://www.cbs.dtu.dk/services/SignalP/) and Motif scan program (http://hits.isb-sib.ch/cgi-bin/ PFSCAN), respectively. The full-length MnLGBP sequence was compared with LGBPs, BGBPs, GNBPs and b-1,3-glucanase (GNs) from other organisms. Amino acid sequences for various species were retrieved from the NCBI and analyzed using the ClustalW Multiple Alignment program (http://www.ebi.ac. uk/clustalw/). A cladogram was constructed based on the amino sequences alignment by the neighborjoining (NJ) algorithm embedded in the MEGA 4 program. The reliability of the branching was tested by bootstrap resampling (1,000 pseudoreplicates).
Mol Biol Rep Table 1 Primers used in the present study Name
Sequence(50 –30 )
30 -RACE primer MnLGBP-QF
Tissue expression of MnLGBP Tissue distribution of MnLGBP mRNA in nerve, intestine, muscle, gill, heart, haemocytes and hepatopancreas were demonstrated by quantitative real-time PCR analysis. Total RNA was extracted as described above, 5 lg of total RNA was used to synthesis the first strand cDNA. For quantification of the MnLGBP expression, a pair of gene-specific primers (MnLGBP-QF, MnLGBP-QR) were used, and the primers b-actin F and b-actin R  were used to amplify b-actin as an internal control. The difference in gene expression was calculated by the 2-DDCT method . Statistical analysis was performed using SPSS software (Ver11.0). Data are presented as the mean ± standard error (n = 3). Statistical significance was determined by oneway ANOVA and post hoc Duncan multiple range tests. Expression pattern of MnLGBP and MnproPO in hepatopancreas in response to A. hydrophila or V. parahaemolyticus stimulation For each experimental group, prawns were inoculated individually with 50 ll of bacterial suspension (104 cells/ ml). At the same time, prawns inoculated with 50 ll saline (0.85 % NaCl, pH 7.0) were used as the control group. M. nipponense were sampled at 0, 1, 12, 24, 36 and 48 h postinjection. For each treatment and each exposure time, the hepatopancreas of five prawns was sampled and total RNA was extracted using TRIzol Reagent. The extracted RNA was determined quantitatively, and 5 lg of total RNA from
the hepatopancreas was used for reverse transcription. Gene expression of MnLGBP and MnproPO was determined by quantitative real-time RT-PCR. The quantitative real-time PCR method was the same as that described above.
Results cDNA cloning and sequence characterization of the MnLGBP A 729 bp partial cDNA fragment was obtained through the degenerate primers, MnLGBP-F1 and MnLGBP-R1. The full-length MnLGBP cDNA was comprised of 1,506 bp, containing 116 bp in the 50 -untranslated region (UTR), 1,119 bp in the open reading frame (ORF) and 271 bp in 30 -UTR with a poly (A) tail of 23 bp. The ORF encodes a polypeptide of 372 amino acids, including a 21 amino acid signal peptide. The conserved domain of MnLGBP has been identified in Fig. 1. The calculated molecular mass of the mature protein (351 amino acids) was 39.9 kDa with an estimated pI of 4.63. The cDNA sequence and deduced amino acid sequence of MnLGBP were submitted to NCBI as GenBank accession number JX171291. Multiple alignment of MnLGBP with LGBPs and BGBP from different crustaceans showed conservation of motifs, including two putative integrin binding motifs (RGD 110–112, 161–163). Four amino acid residues (Trp 181, Glu 186, Ile 187, Asp 188) involved in the cleavage of
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Fig. 1 Nucleotide sequence of the cDNA encoding MnLGBP and its deduced amino acid sequence. The nucleotide sequence is numbered from the 50 end, and the amino acid code is presented below the corresponding codon. The sequence of the signal peptide is underlined. Potential N-glycosylation sites are red shaded. Amino acids
shaded in yellow color represent a potential recognition motif for b1,3-linkage of polysaccharides. Two RGD putative cell adhesive sites (integrin-binding motifs) are boxed. The amino acids composing the functional motif of glucanase are shown in red letters. A dotted line marks a potential kinase C phosphorylation site. (Color figure omline)
b-1,3- or b-1,4-glucosidic linkages of bacterial glucanase were conserved. Two potential N-glycosylation sites (NRS 70–72, NTS 324–326) and one protein kinase C phosphorylation site (SAR 137–139) were found in the MnLGBP molecule (Fig. 2).
A phylogenetic tree was constructed by the NJ method based on the amino acid sequences of selected LGBPs, BGBPs, GNBPs and GNs. The first group was comprised of LGBPs and BGBPs; the second group was comprised of GNBPs and GNs. MnLGBP grouped with M. rosenbergii LGBP, which belonged to the first group (Fig. 3).
Homology and phylogenetic analysis Tissues expressions of MnLGBP The deduced amino acid sequence of MnLGBP is homologous to that of M. rosenbergii LGBP (identities = 89 %), M. japonicus BGBP (identities = 76 %), F. chinensis LGBP (identities = 74 %), and L. stylirostris LGBP (identities = 74 %).
Quantitative real-time RT-PCR analyses were employed to quantify the MnLGBP mRNA expressions in nerve, intestine, muscle, gill, heart, haemocytes and hepatopancreas. b-Actin was the endogenous positive reference for
Mol Biol Rep Fig. 2 Multiple alignment of MnLGBP amino acid sequence with other crustacean homologues: F. chinensis LGBP (AAX63902), L. stylirostris LGBP (AAM73871), M. japonicus BGBP (BAD36807), M. rosenbergii LGBP (ACT33045). Identical (*) and similar (. or :) amino acid residues were indicated. Gaps (-) were introduced to maximize the alignment. The RGD (Arg-Gly-Asp) putative cell adhesive sites are marked as black color boxes. One kinase C phosphorylation site (SAR) and two putative N-linked glycosylation sites are marked with red- and green- color boxes, respectively. The yellowcolored box shows the glucanase motif. (Color figure online)
Mol Biol Rep Fig. 3 NJ phylogenetic tree of LGBPs, BGBPs, GNBPs, and GNs. Amino acid sequences are from different organisms. The respective accession numbers were retrieved from NCBI GenBank
1.2 1.0 .8 .6 .4 *
t ar he
e cl us m
in st te in
Relative expression of MnLGBP
Fig. 4 Expression level of the MnLGBP mRNA in different tissues. Quantitative real-time RT-PCR was carried out with RNA samples from nerve, intestine, muscle, gill, heart, haemocytes and hepatopancreas of the M. nipponense. Significant differences from hepatopancreas are indicated with an asterisk at p \ 0.05
all tissues. As shown in Fig. 4, the MnLGBP was detected in all of those tissue samples. A predominant MnLGBP transcript was found in the hepatopancreas, but with reduced expression in haemocytes. Expression of MnLGBP post A. hydrophila or V. parahaemolyticus challenge To study the function of MnLGBP in the immune response of the prawn, the expression of MnLGBP in hepatopancreas after A. hydrophila challenge was measured through quantitative real-time RT-PCR. The temporal expression
profiles of the MnLGBP gene in the control and bacteriachallenged samples are shown in Fig. 5a. At 1 h postinjection of bacteria, the expression of MnLGBP was upregulated immediately, followed by a step-by-step recovery at 12 and 24 h. The peak of MnLGBP mRNA transcripts was observed at 36 h post-injection, which was 5.86-fold greater compared to that in the control group. As time progressed, the expression level of the MnLGBP became down-regulated, and at 48 h it returned to a similar expression level as the control. Expression profiles of MnLGBP in hepatopancrease after injection of V. parahaemolyticus are shown in Fig. 5b. MnLGBP gene expression was most easily induced when challenged with V. parahaemolyticus. MnLGBP expression level showed a significant change after challenge with V. parahaemolyticus when compared to the A. hydrophila injection. The level of MnLGBP transcripts sharply increased from 12 h (2.71-fold) to 36 h (7.01-fold) and then sharply decreased at 48 h. Expression of MnproPO post A. hydrophila or V. parahaemolyticus challenge In order to elucidate that MnLGBP functions as a PRP in the prawn proPO activating system, the transcript expression profiles of MnproPO in response to A. hydrophila or V. parahaemolyticus challenges were also examined. As shown in Fig. 6a, the first peak of MnproPO mRNA transcripts was observed at 12 h post injection with A. hydrophila, which was 2.57-fold greater compared to that in the control group. Thereafter, MnproPO expression gradually
Saline injected A. hydrophila injected
8 6 4
The relative expression of proPO
A The relative expression of MnLGBP
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Saline injected A. hydrophila injected
Time post challenge
Saline injected V. parahaemolyticus
Time post challenge 25 Saline injected V. parahaemolyticus injected
The relative expression of proPO
The relative expression of MnLGBP
Time post challenge 0
Fig. 5 Temporal expression profile of the MnLGBP transcript in hepatopancreas after A. hydrophila (a) or V. parahaemolyticus (b) challenge. The hepatopancreas tissues collected from prawns injected with bacteria or those injected with saline were compared with respect to MnLGBP mRNA expression (relative to b-actin) using Students t tests. Significant differences from samples at 0 h post injection are indicated with an asterisk at p \ 0.05
decreased during 12–36 h post-injection, and reached the same level as that of the control. The expression level reached a peak at 48 h, which was 5.67 times higher than that of the control. The level of MnproPO transcripts upon V. parahaemolyticus challenge showed no significant changes during the initial 12 h and then quickly increased from 12 to 24 h, becoming 7.81 times higher than that of the control. And the peak level also appeared at 48 h after stimulation, which was 17.2 times higher than that of the control.
Discussion PRPs play an important role in activating the innate immune defense [10, 30–32]. LGBP is one of the important invertebrate PRPs, and has been isolated from several crustaceans: Indian white shrimp F. indicus , Chinese Shrimp F. chinensis , Chinese mitten crab E. sinensis
Time post challenge
Fig. 6 MnproPO mRNA expression profiles post A. hydrophila (a) or V. parahaemolyticus (b) injection. Samples stimulated by saline were adopted as a control. Values are shown as mean ± S.E. Significant differences from the blank are indicated with an asterisk at p \ 0.05
, and giant freshwater prawn M. rosenbergii . However, less research has been reported for M. nipponense. In the present study, we cloned and characterized the LGBP of M. nipponense. Alignment of amino acid sequences of MnLGBP with other known crustacean LGBPs and BGBP indicated that MnLGBP contained several functional domains. Two integrin binding sites (RGD) were found in the MnLGBP. The RGD motif was the putative cell adhesion site through which the PRPs can bind to the haemocyte membrane and induce a series of immune reactions . In the prediction of the 3D structure of kuruma shrimp M. japonicus LGBP, two RGDs extend to the surface of the structure and probably provide the binding site for integrin . The RGD motif is also present in the peroxinectin molecule where the adhesive function is likely to be mediated by the integrin-binding motif, KGD (Lys-Gly-Asp) or RGD motifs . Therefore, it is possible that the LGBP could bind to the integrin through the RGD site and induce a
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series of immune reactions, such as the activation of the proPO system. Further investigations of the complex relationships among MnLGBP and integrin are required to better understand the mechanisms of LGBP. Another conserved domain was detected in MnLGBP, namely the glucanase catalytic site with WEID, which binds the LPS and b-1,3-glucans. It validated the hypothesis that the invertebrate LGBP genes might have evolved from bacterial glucanase and that the glucanase activity may have been lost during evolution; but the glucan-binding properties were retained, and therefore can play a role in the innate immune response . The constructed phylogenetic tree was composed of two main distinct branches with strong bootstrap values, thus suggesting that the LGBPs, GNBPs, GNs and GNBPs share a common ancestor. Sequence data suggest that MnLGBP shared high similarity with other members of LGBPs and BGBPs that all contain glucanase domains. We thus speculate that MnLGBP, such as LGBPs, BGBPs and GNBPs genes of other species, also evolved from a b-1,3glucanase. The distribution of LGBP in different tissues has been investigated in a variety of organisms, but inconsistent results were reported. For example, the LGBP mRNA transcript expression was higher in haemocytes when compared to hepatopancreas in the freshwater crayfish P. leninusculus  and the black tiger shrimp P. monodon . Meanwhile, LGBP transcript expression was also detected in the non-hemocytic cells and hepatopancreas of white shrimp L. vannamei  and in penaeid shrimp P. stylirostris . In the present study, the MnLGBP mRNA was expressed in all detected tissues; especially in the hepatopancreas, and appeared lower in haemocytes. The hepatopancreas is considered to be another key organ in the crustacean immune responses ; and the expression of immune-related genes, including LGBP, have been detected in this organ in different animals [37–39]. Thus, we speculate that the MnLGBP is mainly synthesised in the hepatopancreas and the others in haemocytes. In addition, it is consistent with the conclusion that hepatopancreas and haemocytes were major sites in our experimental preparations for the synthesis of immune defense molecules that are involved in eliminating the pathogens [26, 31, 38, 40, 41]. To further understand the possible biological function of the MnLGBP, its temporal expression pattern was quantified at different time points after bacteria stimulation in the hepatopancreas tissue. A. hydrophila is a ubiquitous Gramnegative bacteria in aquatic ecosystems , and was found to be a highly pathogenic bacterium to M. nipponense  and E. sinensis . The Gram-negative bacterium Vibrio parahaemolyticus was used for the study because Vibrios have been reported to be fish and invertebrate
pathogens [44–47]. In addition, it has been reported that in shimp, LGBPs show strong binding activity to Gram-negative bacteria compared to Gram-positive bacteria and yeast [10, 12, 22]. Thus, we chose A. hydrophila and V. parahaemolyticus as pathogens to infect the prawns in an attempt to investigate the MnLGBP expression pattern within the pathological infection strategy used in our research. In previous studies, most of the LGBPs were upregulated once stimulated by pathogens and then decreased to the initial level step-by-step [21, 22, 26]. In the present study, the expression of MnLGBP was up-regulated immediately upon the injection of A. hydrophila or V. parahaemolyticus; and the remarkable up-regulation of MnLGBP was observed at 36 and 24 h, respectively after stimulation. The up-regulation of MnLGBP expression at 24 and 36 h might indicate a protection mechanism against bacteria through active proliferation of hepatopancreatic cells. This result would suggest that MnLGBP plays an important signaling role in shrimp infection during the early stage. The prawns apparently attempted to clear up the infection with an up-regulation of MnLGBP expression. It has been reported that binding of LPS or b-1,3-glucans to LGBP triggers a serine proteinase cascade, eventually leading to the cleavage of the inactive proPO to the active PO that functions to produce the melanin and toxic reactive intermediates against invading pathogens . To further elucidate the role of prawn LGBPs in the proPO activating system, the responses of MnproPO expression to the Gram-negative bacteria A. hydrophila and V. parahaemolyticus were also investigated. In the present study, the proPO transcript of M. nipponense increased at 12 h post-A. hydrophila injection, and continued to maintain higher levels at 48 h when compared to the control group. A significant increase in the expression of the MnproPO gene was found after 24 h incubation with V. parahaemolyticus. In this study, the highest two expression levels of MnproPO were found at 48 h post challenge. MnLGBP, which was expressed dramatically at 36 h, acts as a functional PRP and played a role in the recognition of microbes. It may also play important roles in inducing expression of proPO gene. To our knowledge, this is the first report to analyze the LGBP gene and its expression pattern in M. nipponense. The MnLGBP mRNA was widely found in nerve, intestine, muscle, gill, heart, haemocytes and hepatopancreas. The transcription of MnLGBP was upregulated when prawns received an injection with A. hydrophila or V. parahaemolyticus. Concurrent with changes of MnLGBP, the expression level of MnproPO mRNA also showed significant changes. In conclusion, MnLGBP is believed to play an important role in the immune responses against infection in M. nipponense.
Mol Biol Rep Acknowledgments We thank Professor O. Roger Anderson for editing the manuscript. This work was supported by Grants from the National Natural Sciences Foundation of China (NSFC Nos. 31170120; 31101926; 31272686), Project for aquaculture in Jiangsu Province (No. PJ011-65), a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), Jiangsu Agriculture Science and Technology Innovation Fund (JASTIF), CX(12)3066, and the Cultivation Plan for Excellent Doctorial Dissertations of Nanjing Normal University (No. CXLX13_383), and Incubation Program for a New Type Academic Ph.D Student of Nanjing Normal University.
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