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Molecular cloning and expression analysis of α2-macroglobulin in the kuruma shrimp, Marsupenaeus japonicus
Fish & Shellfish Immunology 16 (2004) 599e611 www.elsevier.com/locate/fsi
Molecular cloning and expression analysis of a2-macroglobulin in the kuruma shrimp, Marsupenaeus japonicus Achara Rattanachaia, Ikuo Hironoa, Tsuyoshi Ohiraa, Yukinori Takahashib, Takashi Aokia,) a
Laboratory of Genome Science, Graduate School of Marine Science and Technology, Tokyo University of Marine Science and Technology, Konan 4-5-7, Minato, Tokyo, Japan b Department of Applied Aquaculture, National Fisheries University, 2-7-1, Nagatahonmachi, Shimonoseki, Yamaguchi, Japan Received 12 August 2003; received in revised form 23 September 2003; accepted 29 September 2003
Abstract The cDNA encoding the kuruma shrimp, Marsupenaeus japonicus a2-macroglobulin (a2M) was obtained by screening a haemocyte cDNA library and 5# RACE PCR amplification. The full length cDNA of 4748 bp contains an open reading frame of 4518 nucleotides that translates into a 1505-amino acid putative peptide, with a 5#untranslated region (UTR) of 59 bp and a 3#UTR of 171 bp. The open reading frame encodes an N-terminal signal sequence of 17 residues and a mature protein of 1488 residues. The entire amino acid sequence is similar to the a2M sequences of arthropods (30e31% identity), mammals (26e27% identity) and fish (25e28% identity). The M. japonicus a2M sequence contains putative functional domains including a bait region, an internal thiol ester site, and a receptor-binding domain, which are present in mammalian a2Ms. In a healthy shrimp, the mRNA of a2M was mainly expressed in haemocytes. In addition, the expression level of a2M mRNA was dramatically increased by through time upon oral administration of peptidoglycan (PG), which is an immune stimulant. The highest expression of a2M mRNA was observed 7 days after feeding with PG. These results suggest that the shrimp a2M is an important molecule in immune system. Ó 2003 Elsevier Ltd. All rights reserved. Keywords: Proteinase inhibitor; a2-macroglobulin; Crustacean; Marsupenaeus japonicus; Real-time PCR; Shrimp immunity
1. Introduction Shrimp culture constitutes an important source of revenue in many developing countries. In the past decade, production has grown extensively through semi-intensive and intensive culture. However, this industry has been limited by outbreaks of infectious diseases, particularly caused by viruses and bacteria [1]. ) Corresponding author. Tel.: +81-3-5463-0556; fax: +81-3-5463-0690. E-mail address: [email protected] (T. Aoki). 1050-4648/$ - see front matter Ó 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2003.09.011
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Basic knowledge of shrimp immunity is therefore necessary to establish strategies for prophylaxis and control of diseases in shrimp aquaculture [2]. Like other invertebrates, crustaceans possess a highly efficient innate immune system consisting of both cellular and humoral mechanisms to recognise and destroy non-self materials including microbial pathogens [3]. Cellular immune responses such as cell adhesion, phagocytosis, encapsulation or nodule formation, are performed by haemocytes [4,5]. Alternatively, the humoral immune response has three major mechanisms, consisting of a clotting process, melanisation by activation of a prophenoloxidase (proPO)-activating system, and antimicrobial action [6e8]. Other components such as proteinase inhibitors are also important in arthropod immunity [9e11]. a2-macroglobulin (a2M), an inhibitor of diverse proteinases, is abundantly present in plasma of vertebrate species [12] and several invertebrate species including the chelicerate, Limulus polyphemus [13], crustaceans [14,15]and a gastropod mollusc [16]. a2M purified from some crustaceans was found to have an activity against infection of invading pathogens [17]and was necessary for activation of the proPO system [18,19]. Thus, a2M is considered to be an important element of the innate immune system in crustaceans. The a2Ms of vertebrates, especially human, have been better characterised than invertebrate a2Ms. a2M is a unique proteinase inhibitor, that does not bind the active site of the target proteinase but enfolds the target proteinase molecule in a molecular cage to block its interaction with protein substrates [20]. The interaction of a2M with proteinase is initiated by proteolytic cleavage in a motif termed the bait region. This leads to a conformational change in the a2M molecule, which is accompanied by the trapping of the proteinase, cleavage of an internal thiol ester, and exposure of the carboxy terminal receptor region that is important for the clearance of proteinase-reacted a2M from the circulation [21]. A variety of mammalian cells including hepatocytes, fibroblasts, and monocytes/macrophages, bind to the receptor of proteinasereacted a2M [22]. In arthropods, granular amebocytes, a type of blood cell, appear to participate in the clearance of the a2M-proteinase complex [23]. Recently, a2M was purified and characterised from the white shrimp (Litopenaeus vannamei) and was shown to have a broad-spectrum inhibitory effect against different proteinase types [24]. However, most reports on a2M in invertebrates have concentrated on its basic biochemical properties and functional characterisation whereas the understanding of this protein at the molecular level is limited. Only two a2M cDNAs from horseshoe crab [25]and soft tick (Genbank accession no. AF538967) have been cloned. In this study, a cDNA encoding an a2M precursor from kuruma shrimp, Marsupenaeus japonicus was cloned. This is the first isolation and characterisation of a2M cDNA in decapod crustaceans. The expression of a2M mRNA in different tissues was also examined by RTePCR and in haemocytes activated with peptidoglycan (PG) by real-time PCR.
2. Materials and methods 2.1. Preparation of the haemocyte cDNA library and screening of a2-macroglobulin (a2M) cDNA clones Haemolymph from 25 kuruma shrimps with an average weight of 21 g were collected from the ventral sinus using a sterile syringe containing precooled 10% sodium citrate as an anticoagulant. The haemocytes were collected by centrifugation at 800 g at 4 (C for 10 min. Total RNA and poly (A)+RNA of the haemocytes were prepared using Trizol (GibcoBRL, USA) and a QuickPrep micro mRNA purification kit (Amersham Biosciences, USA), respectively, according to the manufacturer’s instructions. cDNA was synthesised with the poly (A)+ RNA and ligated into the l ZipLox phagemid vector (GibcoBRL, USA) using a SuperScriptÔ Lamda system kit (GibcoBRL, USA). The ligation product was packaged using MaxPlaxÔ Lambda packaging Extract (Epicentre, USA).
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A partial cDNA fragment of Marsupenaeus japonicus a2M previously identified by an EST analysis (Genbank accession no. AU175379) [26] was used as a probe for screening of the cDNA library. The cDNA fragment was labelled with 32P-dCTP by using a random primer DNA labelling kit (Takara, Japan). Approximately 500,000 plaques of the haemocyte cDNA library were screened with the labelled probe. Positive plaques were isolated in secondary and tertiary screening. The recombinant plasmids were rescued from the bacteriophage clones by in vivo excision using DH10B (ZIP) (Gibco BRL, USA) according to the manufacturer’s protocol. The isolated cDNA clones were sequenced on an automated DNA sequencer LC4200 (Li-Cor, USA) using a ThermoSequenase fluorescent-labelled primer cycle sequencing kit (Amersham Biosciences, USA). 2.2. Sequence data analysis The nucleotide and the deduced amino acid sequences were analysed by using GENETYX WIN ver. 3.1 (SDC Software development, Japan). Each obtained cDNA sequence was compared with sequences deposited in DDBJ /EMBL/Genbank using the BLASTX and BLASTP programs (National Center for Biotechnology Information, NCBI; available at www.ncbi.nlm.nih.gov/BLAST) [27]. The SignalP program was used to predict the cleavage site between the signal peptide and mature a2M (available at www. cbs.dtu.dk/services/SignalP/) [28]. The possible N-linked glycosylation sites were predicted using the NetNGlyc program (available at www.cbs.dtu.dk/services/NetNGlyc/) [29]. Alignment of other known a2Ms and M. japonicus a2M was performed using the CLUSTAL X program packaged in Bioedit software [30]. 2.3. Cloning of the 5# part of kuruma shrimp a2M cDNA Two gene-specific primers, RACEA2M/1; 5#-ctgatggcttctgagctggaccgcagcg-3# and RACEA2M/2; 5#-ctcgaactcggcactccagatccaggt-3#, were designed based on the nucleotide sequence of the M. japonicus a2M cDNA fragment isolated by the screening of the haemocyte cDNA library and the first RACE PCR, respectively. 5# RACE was performed using a Smart Race cDNA amplification kit (Clontech, USA). PCR reaction was conducted with the initial denaturation step at 94 (C for 2 min, followed by 30 cycles of 94 (C for 30 s, 68 (C for 30 s and 72 (C for 2 min, and the final extension step at 72 (C for 5 min. Amplified PCR products were purified using a DNA purification kit (Toyobo, Japan) and subsequently cloned into pGEM-T Easy vector (Promega, USA). 2.4. Tissue distribution of M. japonicus a2M mRNA Total RNA from various tissues of kuruma shrimp including haemocytes, heart, hepatopancreas, gill, fore-gut, mid-gut, muscle, subcuticular epithelium and ovary were extracted as described as above. First strand cDNA synthesis from 10 mg of each total RNA was performed using an AMV Reverse Transcriptase First Strand cDNA Synthesis kit (Life Sciences, USA). Two oligonucleotide primers, A2M-F; 5#gacaacaaacttctgcagcag-3# and A2M-R; 5#-cagctcgtagtagtcgtaga-3#, were designed based on the nucleotide sequence corresponding to the nucleotide positions 4041e4061 and 4501e4520, respectively, of M. japonicus a2M cDNA. A set of b-actin primers, b-actin-F; 5#-ttcccctccatcgtgggccg-3# and b-actin-R; 5#tgtaaggtcacgtccagcca-3#, served as a control for amount and quality of each cDNA. The first strand cDNAs were used as templates and each amplification was primed by the primers. PCR reactions were conducted for 15 cycles with denaturation at 94 (C for 30 s, annealing at 55 (C for 30 s, and extension at 72 (C for 30 s. PCR products were analyzed on 1.5% agarose gel. 2.5. Northern-blot analysis Poly (A)+ RNA from haemocytes was prepared as described above. The poly (A)+RNA (1 mg) was then separated on 1% agarose gel in the presence of formaldehyde [31]and transferred to a Hybond-N+
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nitrocellulose membrane (Amersham Bioscience, USA) according to the manufacturer’s protocol. A M. japonicus a2M cDNA fragment corresponding to the nucleotide position 4041e4520 and a b-actin cDNA fragment as described above were labelled with 32P-dCTP. Hybridisation was carried out as reported previously [32]. 2.6. Detection of M. japonicus a2M mRNA in haemocyte stimulated with peptidoglycan by real-time PCR Peptidoglycan (PG) was prepared from Bifidobacterium thermophilum as reported previously [33]. Healthy kuruma shrimps with an average size of 21.8 g were acclimated for 1 week in a tank under laboratory conditions at 20G1 (C and subsequently fed with diet containing PG at 0.2 mg kgÿ1shrimp body weight per day. Haemocytes were collected before PG feeding as an initial control and at 1, 3 and 7 days post PG feeding. Total RNA from the haemocytes of each group was prepared and subsequently first strand cDNA was synthesised as described as above. The first strand cDNAs were used as templates in the following real-time PCR. To make a standard curve, serial standard DNAs of M. japonicus a2M and b-actin were prepared as described previously [34]. Primers for a2M (A2M-Frt; 5#-accatggagggtcaaggatgct-3# and A2M-Rrt; 5#aggctgaaggcatcgctcggtt-3#) and for b-actin (b-actin-Frt; 5#-tgctggactctggcgatggcgt-3# and b-actin-Rrt; 5#tgtaaggtcacgtccagcca-3#) were designed. Real-time PCR was carried out using SYBR green PCR core reagents (Perkin-Elmer, USA) according to the manufacturer’s protocol. PCR reactions were conducted with the initial denaturation step at 50 (C for 2 min and 95 (C for 10 min, and followed by 40 cycles of 95 (C for 15 s and 60 (C for 60 s using a GeneAmp5700 sequence detector (Perkin-Elmer, USA). All samples were run in triplicate. Shrimp b-actin content in a standard sample (105) was used as an internal positive control and the normalising reference for individual variation. Statistical comparisons were analysed using ANOVA (SYSTAT 8.0 software, SPPS Inc., Illinois, USA). Values were considered to be significant at P ! 0:05.
3. Results 3.1. Isolation of M. japonicus a2M cDNA By screening of the kuruma shrimp haemocyte cDNA library, 14 positive clones were obtained. Two clones (l ZIP 1 and l ZIP 2 in Fig. 1), which contained longer inserts than those of other clones, were chosen for sequencing. Both clones contained a partial nucleotide sequence without the 5# part of a2M cDNA. Therefore, 5# RACE was conducted to amplify the 5# part of a2M cDNA. The resulting PCR product with a size of 2.1 kb (pGEMrace1 in Fig. 1) encoded M. japonicus a2M. However, it lacked of the 5# end of the cDNA. Then, a second 5# RACE was performed. An additional cDNA fragment with 1.2 kb was obtained (pGEMrace2 in Fig. 1) and included the 5# end of M. japonicus a2M cDNA. The nucleotide and deduced amino acid sequences of kuruma shrimp a2M precursor cDNA are shown in Fig. 2. The full-length of a2M precursor cDNA consisted of 4748 bp comprising a 5# untranslated region (UTR) of 59 bp, an open reading frame of 4515 bp, a stop codon of 3 bp and a 3# UTR of 171 bp containing the poly A tail. The 3# UTR contained a consensus polyadenylation signal (AATAAA) 13 bp upstream from the poly A tail (Fig. 2). The open reading frame encoded a protein consisting of 1505 amino acid residues. N-terminal segment included a high proportion of hydrophobic amino acid residues and therefore the first 17 amino acid residues were predicted to be a signal peptide by the SignalP program. The mature a2M consisted of 1488 amino acids with a molecular mass of 166.2 kDa. Eight (8) potential N-linked glycosylation sites were observed at amino acid positions, 66, 124, 251, 323, 381, 892, 1046 and 1086 (Fig. 2). The amino acid sequence of M. japonicus a2M was compared with the sequences of previously known a2Ms. As shown in Table 1, M. japonicus a2M had greater similarity to the a2Ms of soft tick, Ornithodoros
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Fig. 1. Schematic diagram showing strategy for isolation of M. japonicus a2M cDNA. lZIP 1 and lZIP 2 clones were isolated by screening of a haemocytes cDNA library, and pGEMrace 1 and pGEMrace 2 clones were isolated by 5# RACE. Black box indicates the homologue a2M probe (Genbank accession no. AU175379). Black stripes and arrows indicate the position and direction of primers designed for 5# RACE, respectively.
moubata (31% identity; 55% similarity) and horseshoe crab, Limulus polyphemus (30% identity; 53% similarity) than to those of fish and mammals (25e28% identity; 49e51% similarity). 3.2. Structural features of M. japonicus a2M Multiple alignment of amino acid sequences of M. japonicus a2M and other known a2Ms using the CLUSTAL X program indicated that M. japonicus a2M contained principal domains required for the a2M mechanism (Fig. 3). The bait region of M. japonicus a2M was found to consist of 59 amino acid residues that have little identity to those of other a2Ms. A thiol ester site (GCGEQ) of M. japonicus a2M was completely identical to the thiol ester sites of others a2Ms. Cys996 and Gln999, which are essential for formation of the thiol ester bond, were especially conserved. The region surrounding the thiol ester site was also similar to the corresponding regions of all aligned a2Ms. The receptor binding domain of M. japonicus a2M located at the C-terminal region showed a high identity to the binding domains of other a2Ms, and Lys residues responsible for receptor binding were also found in this region. 3.3. Expression studies The tissue distribution of M. japonicus a2M mRNA was examined by RTePCR using specific primers. A clear band with the expected size was observed only in haemocytes and weak bands were detected in heart and ovary (Fig. 4). The PCR product was directly sequenced and found to have the same sequence as the cloned cDNA. PCR products using the b-actin primer set were observed in all tested tissues. To determine the size of the M. japonicus a2M transcript, Northern-blot hybridisation was performed using poly A+RNA from haemocytes of healthy shrimp. A single band of about 5 kb was detected as shown in Fig. 5. The size of this band was in good agreement with that of M. japonicus a2M precursor cDNA. A b-actin probe used as a positive control also specifically hybridised with a single band of about 1.5 kb. Real-time PCR was used to examine changes in expression of M. japonicus a2M mRNA in haemocytes of shrimp fed peptidoglycan (PG) for 1, 3 and 7 days. a2M expression was slightly induced after 1 day and increased dramatically after 3 and 7 days (Fig. 6). The copy numbers of a2M after PG-feeding for 1, 3 and 7 days were significantly higher compared with the 0-day unstimulated group (P ! 0:05).
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A. Rattanachai et al. / Fish & Shellfish Immunology 16 (2004) 599e611 Table 1 Amino acid comparison between kuruma shrimp a2M and other a2Ms aligned in Fig. 3 Species
Genbank accession no.
% identity
% similarity
Sorf tick, Ornithodoros moubata Horseshoe crab, Limulus polyphemus Lamprey, Lethenteron japonicum Carp, Cyprinus carpio Guinea pig, Cavia porcellus Rat, Rattus norvegicus Mouse, Mus musculus Human, Homo sapiens
AF538967 T18544 D13567 AB026128 JC5143 P06238 Q61838 P01023
31 30 28 25 26 27 27 27
55 53 51 49 51 50 49 51
4. Discussion a2-macroglobulin is a member of the a2-macroglobulin family, which includes several closely related proteinase inhibitors, and C3, C4 and C5 of the complement system [35]. The molecule, which has been isolated in all class of vertebrates and several invertebrates, is a broad-spectrum proteinase-binding protein, which leads to elimination of circulating proteinases in the plasma [23]. Therefore, a2M is thought to play an important role in innate immunity [9,11]. However, knowledge of a2M at the molecular level is very limited especially in invertebrates. Here, the cloning of a2M cDNA from kuruma shrimp, M. japonicus is described. The cloned M. japonicus a2M showed the highest sequence similarity to a2Ms from soft tick and horseshoe crab, which belong to the same phylum (Arthropoda) as kuruma shrimp (Table 1). M. japonicus a2M also showed similarity to a2Ms of mammals and fishes. However, M. japonicus a2M had a little similarity to the complement proteins C3 and C4 (data not shown). These results may be due to the difference of evolutionary pathway between a2M and complement protein from the common ancestral protein. Alignment of amino acid sequences between M. japonicus a2M and other known a2Ms indicates that M. japonicus a2M contains three functional domains found in other a2Ms. The bait region of M. japonicus a2M greatly differs in both sequence and length from the bait regions of the other a2Ms (Fig. 3). This dissimilarity in the bait regions was also found among mammalian a2Ms [36,37]. The diversity of the bait region sequences may reflect evolutionary pressure to react with proteinases produced by specific pathogens [11]. The thiol ester domain of M. japonicus a2M is completely identical with that of the other a2Ms (Fig. 3). The putative thiol ester bond is likely to be formed between Cys996 and Gln999 in the same manner as that of mammalian a2Ms [38]. Furthermore, the region around the thiol ester domain of M. japonicus a2M showed extensive similarity to the corresponding region in the other a2Ms. This suggests that the a2M molecule appeared early in evolution, and its potential for proteinase entrapment has physiological significance [39]. The receptor-binding domain located in the C-terminal region of a2M functions in the binding to a cell surface receptor for receptor-mediated endocytosis [36]. Similarly, a sequence comparison of a2Ms indicates that a region in the C-terminus of M. japonicus a2M corresponds to the receptor-binding domain of other a2Ms. The receptor-binding domain of M. japonicus a2M shows a high sequence identity with that of other a2Ms and contains conserved lysine (Lys) residues that are important for receptor Fig. 2. Nucleotide and deduced amino acid sequences of M. japonicus a2M. The nucleotide sequence is numbered from the first base at : the 5# end. The first methionine (M) is numbered as the first deduced amino acid. The arrowhead ( ) indicates the putative signal cleavage site. The predicted N-linked glycosylation sites are shown in bold italic letters. The putative bait region sequence is underlined. The cysteine and glutamine residues forming the internal thiol ester bond are circled. The polyadenylation signal (AATAAA) is shown in bold letters at the end of 3# UTR. The complete cDNA and amino acid sequences have been deposited in Genbank with the accession no. AB108542.
606 A. Rattanachai et al. / Fish & Shellfish Immunology 16 (2004) 599e611 Fig. 3. Alignment of a2M amino acid sequences between M. japonicus and other known species. The entire amino acid sequences of M. japonicus a2M (A2MK.SHRIMP, Genbank accession no. AB108542) was aligned with those of O. moubata a2M (A2MSOFT.TICK, Genbank accession no. AF538967), Limulus polyphemus a2M (A2MH.CRAB, Genbank accession no. T18544), Lethenteron japonicum a2M (A2MLAMPREY, Genbank accession no. D13567), Cyprinus carpio a2M (A2MCARP, Genbank accession no. AB02618), Mus musculus a2M (A2MMOUSE, Genbank accession no. Q61838), Rattus norvegicus a2M (A2MRAT, Genbank accession no. P06238), Cavia porcellus a2M (A2MPIG, Genbank accession no. JC5143) and Homo sapiens a2M (A2MHUMAN, Genbank accession no. P01023) using the CLUSTAL X program. Conservation of amino acid identity is shown with an asterisk ‘)’ whereas ‘:’ and ‘.’ indicate high and low levels of amino acid similarity, respectively. The signal peptide sequences are shown in italic letters at the N-terminal part.
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Fig. 3 (continued).
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Fig. 3 (continued ).
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Fig. 4. Tissue distribution of M. japonicus a2M mRNA. Lane 1, haemocytes; lane 2, heart; lane 3, hepatopancreas; lane 4, gill; lane 5, fore-gut; lane 6, mid-gut; lane 7, muscle; lane 8, subcuticular epithelium; lane 9, ovary. M indicates molecular weight marker (100 bp ladder).
binding [40,41]. Four Lys are strongly conserved in the aligned a2Ms (1361, 1370, 1374, 1425; human a2M numbering) (Fig. 3). The first three lysines are conserved in M. japonicus a2M but the last one is replaced by valine (Val). The replacement of the last lysine was also found in horseshoe crab a2M and this may contribute to its reduced affinity for mammalian cell surface receptors [25]. Expression studies revealed constitutive expression of a2M in kuruma shrimp haemocytes. Expression appeared to be low in other tissues, but this may have been due to contamination with haemocytes. a2M mRNA was also expressed in haemocytes of horseshoe crab [25]. Expression of a2M gene was not detected in the hepatopancreas of either kuruma shrimp or horseshoe crab. The corresponding organ in mammals, the liver, is the site of synthesis of a2M in mammals [42]. In the crayfish, Pacifastacus leniusculus, a2M is found to be synthesised in haemocytes but not in the hepatopancreas [43]. These data suggest that haemocytes are the main site of a2M synthesis in invertebrates. a2M expression in haemocytes of kuruma shrimp was significantly induced by administration of PG, which is well known as an immunostimulant. Lipopolysaccharide (LPS) was also found to induce the expression of a2M [44]. These results strongly suggest that M. japonicus a2M functions in the immune system.
Fig. 5. Northern-blot hybridisation of M. japonicus a2M using haemocyte mRNA.
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Fig. 6. Expression pattern of M. japonicus a2M in haemocytes from M. japonicus administered with PG. Shrimp were collected at 0, 1, 3 and 7 days post PG feeding. M. japonicus mRNA levels were determined by real-time PCR and standardised according to the respective b-actin mRNA levels. Data are presented as mean G SE in triplicates. Points with asterisks indicate values significantly different from that day 0 (P ! 0:05).
Acknowledgements This research was supported in part by a Grant-in-Aid for Scientific Research (S) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
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Molecular cloning, characterization, and expression of TNF cDNA and gene from Japanese flounder Paralichthys olivaceus. J Immunol 2000;165:4423e7. [33] Itami T, Asano M, Tokushige K, Kubono K, Nakagawa A, Takeno N, et al. Enhancement of disease resistance of kuruma shrimp, Penaeus japonicus, after oral administration of peptidoglycan derived from Bifidobacterium thermophilus. Aquaculture 1998;164:277e88. [34] Park C, Kurobe T, Hirono I, Aoki T. Cloning and characterization of cDNAs for two distinct tumor necrosis factor receptor superfamily genes from Japanese flounder Paralichthys olivaceus. Dev Comp Immunol 2003;27:365e75. [35] Armstrong PB, Quigley JP. a2-macroglobulin: an evolutionarily conserved arm of the innate immune system. Dev Comp Immunol 1999;23:375e90. [36] Sottrup-Jensen L. a-macroglobulins: structure, shape and mechanism of proteinase complex formation. J Biol Chem 1989;264:11539e42. [37] Sottrup-Jensen L, Sand O, Kristensen L, Fey GH. The alpha-macroglobulin bait region sequence diversity and localization of cleavage site for proteinases in five mammalian alpha-macroglobulins. J Biol Chem 1989;264:15781e9. [38] Sottrup-Jensen L, Hansen HF, Mortensen SB, Petersen TE, Magnusson S. Sequence location of the reactive thiol ester in human a2-macroglobulin. FEBS Lett 1981;123:145e8. [39] Hall M, Soderhall K, Sottrup-Jensen L. Amino acid sequence around the thiolester of a2-macroglobulin from plasma of the crayfish, Pacifastacus leniusculus. FEBS Lett 1989;254:111e4. [40] Van Leuven F, Marynen P, Cassiman J-J, Van Den Berghe H. The epitopes of two complex-specific monoclonal antibodies, related to the receptor recognition site, map to the COOH-terminal end of human a2-macroglobulin. J Biol Chem 1986;261:6933e7. [41] Huang W, Dolmer K, Liao X, Guttins PG. Localization of basic residues required for receptor binding to the single alpha-helix of the receptor binding domain of human alpha 2-macroglobulin. Protein Sci 1998;7:2602e12. [42] Andus T, Gross V, Tran-Thi T-A, Schreiber G, Nagashima M, Heinrich PC. The biosynthesis of acute-phase proteins in primary cultures of rat hepatocytes. Eur J Biochem 1983;133:561e71. [43] Liang Z, Lindblad P, Beauvais A, Johansson MW, Latge J-P, Hall M, et al. Crayfish a-macroglobulin and 76 kDa protein; their biosynthesis and subcellular localization of the 76 kDa protein. J Insect Physiol 1992;38:987e95. [44] Gunnarsson M, Frangsmyr L, Stigbrand T, Jensen PEH. Stimulation of peripheral blood mononuclear cells with lipopolysaccharide induces expression of the plasma protein a2-macroglobulin. Protein Expr Purif 2003;27:238e43.
Molecular cloning and expression analysis of a2-macroglobulin in the kuruma shrimp, Marsupenaeus japonicus Achara Rattanachaia, Ikuo Hironoa, Tsuyoshi Ohiraa, Yukinori Takahashib, Takashi Aokia,) a
Laboratory of Genome Science, Graduate School of Marine Science and Technology, Tokyo University of Marine Science and Technology, Konan 4-5-7, Minato, Tokyo, Japan b Department of Applied Aquaculture, National Fisheries University, 2-7-1, Nagatahonmachi, Shimonoseki, Yamaguchi, Japan Received 12 August 2003; received in revised form 23 September 2003; accepted 29 September 2003
Abstract The cDNA encoding the kuruma shrimp, Marsupenaeus japonicus a2-macroglobulin (a2M) was obtained by screening a haemocyte cDNA library and 5# RACE PCR amplification. The full length cDNA of 4748 bp contains an open reading frame of 4518 nucleotides that translates into a 1505-amino acid putative peptide, with a 5#untranslated region (UTR) of 59 bp and a 3#UTR of 171 bp. The open reading frame encodes an N-terminal signal sequence of 17 residues and a mature protein of 1488 residues. The entire amino acid sequence is similar to the a2M sequences of arthropods (30e31% identity), mammals (26e27% identity) and fish (25e28% identity). The M. japonicus a2M sequence contains putative functional domains including a bait region, an internal thiol ester site, and a receptor-binding domain, which are present in mammalian a2Ms. In a healthy shrimp, the mRNA of a2M was mainly expressed in haemocytes. In addition, the expression level of a2M mRNA was dramatically increased by through time upon oral administration of peptidoglycan (PG), which is an immune stimulant. The highest expression of a2M mRNA was observed 7 days after feeding with PG. These results suggest that the shrimp a2M is an important molecule in immune system. Ó 2003 Elsevier Ltd. All rights reserved. Keywords: Proteinase inhibitor; a2-macroglobulin; Crustacean; Marsupenaeus japonicus; Real-time PCR; Shrimp immunity
1. Introduction Shrimp culture constitutes an important source of revenue in many developing countries. In the past decade, production has grown extensively through semi-intensive and intensive culture. However, this industry has been limited by outbreaks of infectious diseases, particularly caused by viruses and bacteria [1]. ) Corresponding author. Tel.: +81-3-5463-0556; fax: +81-3-5463-0690. E-mail address: [email protected] (T. Aoki). 1050-4648/$ - see front matter Ó 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2003.09.011
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Basic knowledge of shrimp immunity is therefore necessary to establish strategies for prophylaxis and control of diseases in shrimp aquaculture [2]. Like other invertebrates, crustaceans possess a highly efficient innate immune system consisting of both cellular and humoral mechanisms to recognise and destroy non-self materials including microbial pathogens [3]. Cellular immune responses such as cell adhesion, phagocytosis, encapsulation or nodule formation, are performed by haemocytes [4,5]. Alternatively, the humoral immune response has three major mechanisms, consisting of a clotting process, melanisation by activation of a prophenoloxidase (proPO)-activating system, and antimicrobial action [6e8]. Other components such as proteinase inhibitors are also important in arthropod immunity [9e11]. a2-macroglobulin (a2M), an inhibitor of diverse proteinases, is abundantly present in plasma of vertebrate species [12] and several invertebrate species including the chelicerate, Limulus polyphemus [13], crustaceans [14,15]and a gastropod mollusc [16]. a2M purified from some crustaceans was found to have an activity against infection of invading pathogens [17]and was necessary for activation of the proPO system [18,19]. Thus, a2M is considered to be an important element of the innate immune system in crustaceans. The a2Ms of vertebrates, especially human, have been better characterised than invertebrate a2Ms. a2M is a unique proteinase inhibitor, that does not bind the active site of the target proteinase but enfolds the target proteinase molecule in a molecular cage to block its interaction with protein substrates [20]. The interaction of a2M with proteinase is initiated by proteolytic cleavage in a motif termed the bait region. This leads to a conformational change in the a2M molecule, which is accompanied by the trapping of the proteinase, cleavage of an internal thiol ester, and exposure of the carboxy terminal receptor region that is important for the clearance of proteinase-reacted a2M from the circulation [21]. A variety of mammalian cells including hepatocytes, fibroblasts, and monocytes/macrophages, bind to the receptor of proteinasereacted a2M [22]. In arthropods, granular amebocytes, a type of blood cell, appear to participate in the clearance of the a2M-proteinase complex [23]. Recently, a2M was purified and characterised from the white shrimp (Litopenaeus vannamei) and was shown to have a broad-spectrum inhibitory effect against different proteinase types [24]. However, most reports on a2M in invertebrates have concentrated on its basic biochemical properties and functional characterisation whereas the understanding of this protein at the molecular level is limited. Only two a2M cDNAs from horseshoe crab [25]and soft tick (Genbank accession no. AF538967) have been cloned. In this study, a cDNA encoding an a2M precursor from kuruma shrimp, Marsupenaeus japonicus was cloned. This is the first isolation and characterisation of a2M cDNA in decapod crustaceans. The expression of a2M mRNA in different tissues was also examined by RTePCR and in haemocytes activated with peptidoglycan (PG) by real-time PCR.
2. Materials and methods 2.1. Preparation of the haemocyte cDNA library and screening of a2-macroglobulin (a2M) cDNA clones Haemolymph from 25 kuruma shrimps with an average weight of 21 g were collected from the ventral sinus using a sterile syringe containing precooled 10% sodium citrate as an anticoagulant. The haemocytes were collected by centrifugation at 800 g at 4 (C for 10 min. Total RNA and poly (A)+RNA of the haemocytes were prepared using Trizol (GibcoBRL, USA) and a QuickPrep micro mRNA purification kit (Amersham Biosciences, USA), respectively, according to the manufacturer’s instructions. cDNA was synthesised with the poly (A)+ RNA and ligated into the l ZipLox phagemid vector (GibcoBRL, USA) using a SuperScriptÔ Lamda system kit (GibcoBRL, USA). The ligation product was packaged using MaxPlaxÔ Lambda packaging Extract (Epicentre, USA).
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A partial cDNA fragment of Marsupenaeus japonicus a2M previously identified by an EST analysis (Genbank accession no. AU175379) [26] was used as a probe for screening of the cDNA library. The cDNA fragment was labelled with 32P-dCTP by using a random primer DNA labelling kit (Takara, Japan). Approximately 500,000 plaques of the haemocyte cDNA library were screened with the labelled probe. Positive plaques were isolated in secondary and tertiary screening. The recombinant plasmids were rescued from the bacteriophage clones by in vivo excision using DH10B (ZIP) (Gibco BRL, USA) according to the manufacturer’s protocol. The isolated cDNA clones were sequenced on an automated DNA sequencer LC4200 (Li-Cor, USA) using a ThermoSequenase fluorescent-labelled primer cycle sequencing kit (Amersham Biosciences, USA). 2.2. Sequence data analysis The nucleotide and the deduced amino acid sequences were analysed by using GENETYX WIN ver. 3.1 (SDC Software development, Japan). Each obtained cDNA sequence was compared with sequences deposited in DDBJ /EMBL/Genbank using the BLASTX and BLASTP programs (National Center for Biotechnology Information, NCBI; available at www.ncbi.nlm.nih.gov/BLAST) [27]. The SignalP program was used to predict the cleavage site between the signal peptide and mature a2M (available at www. cbs.dtu.dk/services/SignalP/) [28]. The possible N-linked glycosylation sites were predicted using the NetNGlyc program (available at www.cbs.dtu.dk/services/NetNGlyc/) [29]. Alignment of other known a2Ms and M. japonicus a2M was performed using the CLUSTAL X program packaged in Bioedit software [30]. 2.3. Cloning of the 5# part of kuruma shrimp a2M cDNA Two gene-specific primers, RACEA2M/1; 5#-ctgatggcttctgagctggaccgcagcg-3# and RACEA2M/2; 5#-ctcgaactcggcactccagatccaggt-3#, were designed based on the nucleotide sequence of the M. japonicus a2M cDNA fragment isolated by the screening of the haemocyte cDNA library and the first RACE PCR, respectively. 5# RACE was performed using a Smart Race cDNA amplification kit (Clontech, USA). PCR reaction was conducted with the initial denaturation step at 94 (C for 2 min, followed by 30 cycles of 94 (C for 30 s, 68 (C for 30 s and 72 (C for 2 min, and the final extension step at 72 (C for 5 min. Amplified PCR products were purified using a DNA purification kit (Toyobo, Japan) and subsequently cloned into pGEM-T Easy vector (Promega, USA). 2.4. Tissue distribution of M. japonicus a2M mRNA Total RNA from various tissues of kuruma shrimp including haemocytes, heart, hepatopancreas, gill, fore-gut, mid-gut, muscle, subcuticular epithelium and ovary were extracted as described as above. First strand cDNA synthesis from 10 mg of each total RNA was performed using an AMV Reverse Transcriptase First Strand cDNA Synthesis kit (Life Sciences, USA). Two oligonucleotide primers, A2M-F; 5#gacaacaaacttctgcagcag-3# and A2M-R; 5#-cagctcgtagtagtcgtaga-3#, were designed based on the nucleotide sequence corresponding to the nucleotide positions 4041e4061 and 4501e4520, respectively, of M. japonicus a2M cDNA. A set of b-actin primers, b-actin-F; 5#-ttcccctccatcgtgggccg-3# and b-actin-R; 5#tgtaaggtcacgtccagcca-3#, served as a control for amount and quality of each cDNA. The first strand cDNAs were used as templates and each amplification was primed by the primers. PCR reactions were conducted for 15 cycles with denaturation at 94 (C for 30 s, annealing at 55 (C for 30 s, and extension at 72 (C for 30 s. PCR products were analyzed on 1.5% agarose gel. 2.5. Northern-blot analysis Poly (A)+ RNA from haemocytes was prepared as described above. The poly (A)+RNA (1 mg) was then separated on 1% agarose gel in the presence of formaldehyde [31]and transferred to a Hybond-N+
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nitrocellulose membrane (Amersham Bioscience, USA) according to the manufacturer’s protocol. A M. japonicus a2M cDNA fragment corresponding to the nucleotide position 4041e4520 and a b-actin cDNA fragment as described above were labelled with 32P-dCTP. Hybridisation was carried out as reported previously [32]. 2.6. Detection of M. japonicus a2M mRNA in haemocyte stimulated with peptidoglycan by real-time PCR Peptidoglycan (PG) was prepared from Bifidobacterium thermophilum as reported previously [33]. Healthy kuruma shrimps with an average size of 21.8 g were acclimated for 1 week in a tank under laboratory conditions at 20G1 (C and subsequently fed with diet containing PG at 0.2 mg kgÿ1shrimp body weight per day. Haemocytes were collected before PG feeding as an initial control and at 1, 3 and 7 days post PG feeding. Total RNA from the haemocytes of each group was prepared and subsequently first strand cDNA was synthesised as described as above. The first strand cDNAs were used as templates in the following real-time PCR. To make a standard curve, serial standard DNAs of M. japonicus a2M and b-actin were prepared as described previously [34]. Primers for a2M (A2M-Frt; 5#-accatggagggtcaaggatgct-3# and A2M-Rrt; 5#aggctgaaggcatcgctcggtt-3#) and for b-actin (b-actin-Frt; 5#-tgctggactctggcgatggcgt-3# and b-actin-Rrt; 5#tgtaaggtcacgtccagcca-3#) were designed. Real-time PCR was carried out using SYBR green PCR core reagents (Perkin-Elmer, USA) according to the manufacturer’s protocol. PCR reactions were conducted with the initial denaturation step at 50 (C for 2 min and 95 (C for 10 min, and followed by 40 cycles of 95 (C for 15 s and 60 (C for 60 s using a GeneAmp5700 sequence detector (Perkin-Elmer, USA). All samples were run in triplicate. Shrimp b-actin content in a standard sample (105) was used as an internal positive control and the normalising reference for individual variation. Statistical comparisons were analysed using ANOVA (SYSTAT 8.0 software, SPPS Inc., Illinois, USA). Values were considered to be significant at P ! 0:05.
3. Results 3.1. Isolation of M. japonicus a2M cDNA By screening of the kuruma shrimp haemocyte cDNA library, 14 positive clones were obtained. Two clones (l ZIP 1 and l ZIP 2 in Fig. 1), which contained longer inserts than those of other clones, were chosen for sequencing. Both clones contained a partial nucleotide sequence without the 5# part of a2M cDNA. Therefore, 5# RACE was conducted to amplify the 5# part of a2M cDNA. The resulting PCR product with a size of 2.1 kb (pGEMrace1 in Fig. 1) encoded M. japonicus a2M. However, it lacked of the 5# end of the cDNA. Then, a second 5# RACE was performed. An additional cDNA fragment with 1.2 kb was obtained (pGEMrace2 in Fig. 1) and included the 5# end of M. japonicus a2M cDNA. The nucleotide and deduced amino acid sequences of kuruma shrimp a2M precursor cDNA are shown in Fig. 2. The full-length of a2M precursor cDNA consisted of 4748 bp comprising a 5# untranslated region (UTR) of 59 bp, an open reading frame of 4515 bp, a stop codon of 3 bp and a 3# UTR of 171 bp containing the poly A tail. The 3# UTR contained a consensus polyadenylation signal (AATAAA) 13 bp upstream from the poly A tail (Fig. 2). The open reading frame encoded a protein consisting of 1505 amino acid residues. N-terminal segment included a high proportion of hydrophobic amino acid residues and therefore the first 17 amino acid residues were predicted to be a signal peptide by the SignalP program. The mature a2M consisted of 1488 amino acids with a molecular mass of 166.2 kDa. Eight (8) potential N-linked glycosylation sites were observed at amino acid positions, 66, 124, 251, 323, 381, 892, 1046 and 1086 (Fig. 2). The amino acid sequence of M. japonicus a2M was compared with the sequences of previously known a2Ms. As shown in Table 1, M. japonicus a2M had greater similarity to the a2Ms of soft tick, Ornithodoros
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Fig. 1. Schematic diagram showing strategy for isolation of M. japonicus a2M cDNA. lZIP 1 and lZIP 2 clones were isolated by screening of a haemocytes cDNA library, and pGEMrace 1 and pGEMrace 2 clones were isolated by 5# RACE. Black box indicates the homologue a2M probe (Genbank accession no. AU175379). Black stripes and arrows indicate the position and direction of primers designed for 5# RACE, respectively.
moubata (31% identity; 55% similarity) and horseshoe crab, Limulus polyphemus (30% identity; 53% similarity) than to those of fish and mammals (25e28% identity; 49e51% similarity). 3.2. Structural features of M. japonicus a2M Multiple alignment of amino acid sequences of M. japonicus a2M and other known a2Ms using the CLUSTAL X program indicated that M. japonicus a2M contained principal domains required for the a2M mechanism (Fig. 3). The bait region of M. japonicus a2M was found to consist of 59 amino acid residues that have little identity to those of other a2Ms. A thiol ester site (GCGEQ) of M. japonicus a2M was completely identical to the thiol ester sites of others a2Ms. Cys996 and Gln999, which are essential for formation of the thiol ester bond, were especially conserved. The region surrounding the thiol ester site was also similar to the corresponding regions of all aligned a2Ms. The receptor binding domain of M. japonicus a2M located at the C-terminal region showed a high identity to the binding domains of other a2Ms, and Lys residues responsible for receptor binding were also found in this region. 3.3. Expression studies The tissue distribution of M. japonicus a2M mRNA was examined by RTePCR using specific primers. A clear band with the expected size was observed only in haemocytes and weak bands were detected in heart and ovary (Fig. 4). The PCR product was directly sequenced and found to have the same sequence as the cloned cDNA. PCR products using the b-actin primer set were observed in all tested tissues. To determine the size of the M. japonicus a2M transcript, Northern-blot hybridisation was performed using poly A+RNA from haemocytes of healthy shrimp. A single band of about 5 kb was detected as shown in Fig. 5. The size of this band was in good agreement with that of M. japonicus a2M precursor cDNA. A b-actin probe used as a positive control also specifically hybridised with a single band of about 1.5 kb. Real-time PCR was used to examine changes in expression of M. japonicus a2M mRNA in haemocytes of shrimp fed peptidoglycan (PG) for 1, 3 and 7 days. a2M expression was slightly induced after 1 day and increased dramatically after 3 and 7 days (Fig. 6). The copy numbers of a2M after PG-feeding for 1, 3 and 7 days were significantly higher compared with the 0-day unstimulated group (P ! 0:05).
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A. Rattanachai et al. / Fish & Shellfish Immunology 16 (2004) 599e611 Table 1 Amino acid comparison between kuruma shrimp a2M and other a2Ms aligned in Fig. 3 Species
Genbank accession no.
% identity
% similarity
Sorf tick, Ornithodoros moubata Horseshoe crab, Limulus polyphemus Lamprey, Lethenteron japonicum Carp, Cyprinus carpio Guinea pig, Cavia porcellus Rat, Rattus norvegicus Mouse, Mus musculus Human, Homo sapiens
AF538967 T18544 D13567 AB026128 JC5143 P06238 Q61838 P01023
31 30 28 25 26 27 27 27
55 53 51 49 51 50 49 51
4. Discussion a2-macroglobulin is a member of the a2-macroglobulin family, which includes several closely related proteinase inhibitors, and C3, C4 and C5 of the complement system [35]. The molecule, which has been isolated in all class of vertebrates and several invertebrates, is a broad-spectrum proteinase-binding protein, which leads to elimination of circulating proteinases in the plasma [23]. Therefore, a2M is thought to play an important role in innate immunity [9,11]. However, knowledge of a2M at the molecular level is very limited especially in invertebrates. Here, the cloning of a2M cDNA from kuruma shrimp, M. japonicus is described. The cloned M. japonicus a2M showed the highest sequence similarity to a2Ms from soft tick and horseshoe crab, which belong to the same phylum (Arthropoda) as kuruma shrimp (Table 1). M. japonicus a2M also showed similarity to a2Ms of mammals and fishes. However, M. japonicus a2M had a little similarity to the complement proteins C3 and C4 (data not shown). These results may be due to the difference of evolutionary pathway between a2M and complement protein from the common ancestral protein. Alignment of amino acid sequences between M. japonicus a2M and other known a2Ms indicates that M. japonicus a2M contains three functional domains found in other a2Ms. The bait region of M. japonicus a2M greatly differs in both sequence and length from the bait regions of the other a2Ms (Fig. 3). This dissimilarity in the bait regions was also found among mammalian a2Ms [36,37]. The diversity of the bait region sequences may reflect evolutionary pressure to react with proteinases produced by specific pathogens [11]. The thiol ester domain of M. japonicus a2M is completely identical with that of the other a2Ms (Fig. 3). The putative thiol ester bond is likely to be formed between Cys996 and Gln999 in the same manner as that of mammalian a2Ms [38]. Furthermore, the region around the thiol ester domain of M. japonicus a2M showed extensive similarity to the corresponding region in the other a2Ms. This suggests that the a2M molecule appeared early in evolution, and its potential for proteinase entrapment has physiological significance [39]. The receptor-binding domain located in the C-terminal region of a2M functions in the binding to a cell surface receptor for receptor-mediated endocytosis [36]. Similarly, a sequence comparison of a2Ms indicates that a region in the C-terminus of M. japonicus a2M corresponds to the receptor-binding domain of other a2Ms. The receptor-binding domain of M. japonicus a2M shows a high sequence identity with that of other a2Ms and contains conserved lysine (Lys) residues that are important for receptor Fig. 2. Nucleotide and deduced amino acid sequences of M. japonicus a2M. The nucleotide sequence is numbered from the first base at : the 5# end. The first methionine (M) is numbered as the first deduced amino acid. The arrowhead ( ) indicates the putative signal cleavage site. The predicted N-linked glycosylation sites are shown in bold italic letters. The putative bait region sequence is underlined. The cysteine and glutamine residues forming the internal thiol ester bond are circled. The polyadenylation signal (AATAAA) is shown in bold letters at the end of 3# UTR. The complete cDNA and amino acid sequences have been deposited in Genbank with the accession no. AB108542.
606 A. Rattanachai et al. / Fish & Shellfish Immunology 16 (2004) 599e611 Fig. 3. Alignment of a2M amino acid sequences between M. japonicus and other known species. The entire amino acid sequences of M. japonicus a2M (A2MK.SHRIMP, Genbank accession no. AB108542) was aligned with those of O. moubata a2M (A2MSOFT.TICK, Genbank accession no. AF538967), Limulus polyphemus a2M (A2MH.CRAB, Genbank accession no. T18544), Lethenteron japonicum a2M (A2MLAMPREY, Genbank accession no. D13567), Cyprinus carpio a2M (A2MCARP, Genbank accession no. AB02618), Mus musculus a2M (A2MMOUSE, Genbank accession no. Q61838), Rattus norvegicus a2M (A2MRAT, Genbank accession no. P06238), Cavia porcellus a2M (A2MPIG, Genbank accession no. JC5143) and Homo sapiens a2M (A2MHUMAN, Genbank accession no. P01023) using the CLUSTAL X program. Conservation of amino acid identity is shown with an asterisk ‘)’ whereas ‘:’ and ‘.’ indicate high and low levels of amino acid similarity, respectively. The signal peptide sequences are shown in italic letters at the N-terminal part.
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Fig. 3 (continued).
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Fig. 3 (continued ).
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Fig. 4. Tissue distribution of M. japonicus a2M mRNA. Lane 1, haemocytes; lane 2, heart; lane 3, hepatopancreas; lane 4, gill; lane 5, fore-gut; lane 6, mid-gut; lane 7, muscle; lane 8, subcuticular epithelium; lane 9, ovary. M indicates molecular weight marker (100 bp ladder).
binding [40,41]. Four Lys are strongly conserved in the aligned a2Ms (1361, 1370, 1374, 1425; human a2M numbering) (Fig. 3). The first three lysines are conserved in M. japonicus a2M but the last one is replaced by valine (Val). The replacement of the last lysine was also found in horseshoe crab a2M and this may contribute to its reduced affinity for mammalian cell surface receptors [25]. Expression studies revealed constitutive expression of a2M in kuruma shrimp haemocytes. Expression appeared to be low in other tissues, but this may have been due to contamination with haemocytes. a2M mRNA was also expressed in haemocytes of horseshoe crab [25]. Expression of a2M gene was not detected in the hepatopancreas of either kuruma shrimp or horseshoe crab. The corresponding organ in mammals, the liver, is the site of synthesis of a2M in mammals [42]. In the crayfish, Pacifastacus leniusculus, a2M is found to be synthesised in haemocytes but not in the hepatopancreas [43]. These data suggest that haemocytes are the main site of a2M synthesis in invertebrates. a2M expression in haemocytes of kuruma shrimp was significantly induced by administration of PG, which is well known as an immunostimulant. Lipopolysaccharide (LPS) was also found to induce the expression of a2M [44]. These results strongly suggest that M. japonicus a2M functions in the immune system.
Fig. 5. Northern-blot hybridisation of M. japonicus a2M using haemocyte mRNA.
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Fig. 6. Expression pattern of M. japonicus a2M in haemocytes from M. japonicus administered with PG. Shrimp were collected at 0, 1, 3 and 7 days post PG feeding. M. japonicus mRNA levels were determined by real-time PCR and standardised according to the respective b-actin mRNA levels. Data are presented as mean G SE in triplicates. Points with asterisks indicate values significantly different from that day 0 (P ! 0:05).
Acknowledgements This research was supported in part by a Grant-in-Aid for Scientific Research (S) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
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