Molecular cloning and characterisation of a thioester-containing α2-macroglobulin (α2-M) from the haemocytes of mud crab Scylla serrata

Fish & Shellfish Immunology 22 (2007) 115e130 www.elsevier.com/locate/fsi

Molecular cloning and characterisation of a thioester-containing a2-macroglobulin (a2-M) from the haemocytes of mud crab Scylla serrata Baskaralingam Vaseeharan, Yong-Chin Lin, Chi-Fong Ko, Tzu-Ting Chiou, Jiann-Chu Chen* Department of Aquaculture, College of Life Sciences, National Taiwan Ocean University, Keelung 202, Taiwan Received 1 March 2006; revised 29 March 2006; accepted 29 March 2006 Available online 18 April 2006

Abstract Molecular approaches were used to clone thioester-containing a2-macroglobulin (a2-M) genes in the haemocytes of mud crab Scylla serrata. The full length sequence of a2-M was determined by RT-PCR, cloning and sequencing of overlapping PCR and rapid amplification of cDNA ends (RACE) method. Analysis of the nucleotide sequence revealed that the a2-M cDNA clone consists of 5491 bp with an open reading frame (ORF) of 4986 bp encoding a protein of 1662 amino acids with 22 residues signal sequence. The calculated molecular mass of the mature protein is 184.2 kDa with an estimated pI of 8.41. The S. serrata a2-M sequence contains putative functional domains including a GCGEQNM thioester region, a bait region, and a receptor-binding domain which are present in other invertebrate and vertebrate a2-Ms. Sequence comparison showed that a2-M deduced amino acid sequence of S. serrata has an overall similarity of 68% and 48% to that of kuruma shrimp Marsupenaeus japonicus and American horseshoe crab Limulus polyphemus, respectively. Phylogentic analysis revealed that S. serrata a2-M is closely related to other arthropod a2-M, and displays the highest similarity to M. japonicus a2-M. The a2-M was mainly expressed in haemocytes. Quantitative real-time RT-PCR analysis showed that a2-M mRNA transcript in haemocytes of S. serrata increased significantly in 24 h- and 48 h-post lipopolysaccharide (LPS) injection. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: Mud crab; Scylla serrata; a2-macroglobulin; Thioester-containing protein; Bait region; Innate immunity; Lipopolysaccharide

1. Introduction The mud crab, genus Scylla, also known as mangrove crab constitutes an important secondary crop in the traditional shrimp/fish culture ponds in the South-East Asian countries. Viral diseases such as reolike virus and white spot syndrome virus (WSSV), and vibriosis caused by Vibrio parahaemolyticus have been reported to infect crabs [1,2]. These pathogenic viruses and bacteria infected the haemocytes and epithelial cells, and are associated with mass mortalities during disease outbreaks. It is known that invertebrates lack a true adaptive immune system, and * Corresponding author. Tel./fax: þ886 2 2462 0295. E-mail address: [email protected] (J.-C. Chen). 1050-4648/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2006.03.017

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rely instead on innate responses against invading pathogens. Research on the innate immune system will provide new insights into the management and control of infectious diseases. Crustaceans possess both cellular and humoral responses to recognise and destroy non-self materials including microbial pathogens [3]. Cellular immune responses such as cell adhesion, phagocytosis, encapsulation and nodule formation are performed by haemocytes [4,5], whereas, humoral immune response has several major mechanisms including clotting process, melanisation through prophenoloxidase (proPO) activating system, and antimicrobial action [6e8]. Proteinase inhibitors like pacifastin and a2-macroglobulin (a2-M) play an important role in regulating the proPO system to avoid the deleterious effects of its active components [9e11]. Proteins of the a2-M family are abundant components of plasma of mammals and arthropods [9,12], and comprise about 3% of the total plasma protein of human [13] and with a2-M the third most abundant protein of the plasma of the American horseshoe crab Limulus polyphemus [14,15]. It functions as a protease-binding protein, and involves in the physical entrapment of target proteases within the folds of a molecule of a2-M [15,16]. This is a unique mechanism whereby the interaction between the protease and the bait region of a2-M results in the structural re-organisation of the a2-M molecule to reveal a highly reactive thioester region [15]. a2-M belongs to a superfamily of protein that possesses an internal thioester region [17]. This superfamily includes murinoglobulins, ovomacroglobulins, pregnancy zone proteins, alpha-1-inhibitor III, and complement proteins (components) C3, C4 and C5. It is known that C3, C4 and C5 arose from an ancestral a2-M before the two phyla diverged through gene duplication [18,19]. Thioester-containing proteins (TEPs) appeared early in animal evolution: members of this family have been identified in nematodes, insects, molluscs, fish, birds and mammals [20]. In invertebrates, a2-M has been purified from American horseshoe crab L. polyphemus and white shrimp Litopenaeus vannamei [21,22], and gastropod mollusc Biomphalaria glabrata [23]. a2-M has also been cloned and characterised in the American horseshoe crab L. polyphemus [24] and kuruma shrimp Marsupenaeus japonicus [25]. The aim of the present study was to present the nucleotide sequence of a2-M from the haemocytes of mud crab S. serrata, and compare its sequence with other a2-Ms, and to evaluate this a2-M expression when S. serrata was injected with lipopolysaccharide (LPS), and to examine the expression of a2-M in various tissues of S. serrata. 2. Materials and methods 2.1. Collection and maintenance of mud crab S. serrata S. serrata (200 g to 250 g) collected from a farm in Ilan, Taiwan, were acclimatised in plastic turf containing 35 & seawater for three days. They were fed daily with fish or shrimp meat at 10% of body weight. 2.2. RNA isolation from haemocyte and reverse transcription (RT) Haemolymph (10 ml) was withdrawn by inserting a syringe into the sinus at the base of right chelate leg into a 50 ml polyethylene tube containing 10 ml of precooled (4  C) anticoagulant (10% trisodium citrate) [26,27]. The diluted haemolymph was centrifuged at 500  g at 4  C for 20 min. The resulting haemocyte pellet was used for total RNA isolation. Total RNA was isolated and further purified using the guanidinium thiocyanate method [28]. First strand cDNA synthesis in RT (reverse transcription) was performed by using SuperscriptÔ III RNAse H reverse transcriptase (Invitrogen, Carlsbad, CA, USA) to transcribe poly (A)þ RNA with oligo-d (T)18 as the primers. Reaction conditions recommended by the manufacturer were followed. 2.3. Degenerate primer design and strategy of a2-M cDNA cloning Full-length a2-M cDNA of S. serrata was obtained by the procedures of reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) method. Multiple alignments and phylogenetic comparisons of a2-M amino acid sequences of S. serrata with other decapod crustaceans were performed. Degenerate primers were designed based on the highly conserved nucleotide sequence of a2-M of kuruma shrimp M. japonicus (AB108542) [25], American horseshoe crab L. polyphemus (D83196) [24] and soft tick Ornithodoros

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moubata (AF538967) [29] in the GenBank database [30] using CLUSTAL program [31]. The schematic representations of the degenerate and gene specific primers used to obtain a2-M from S. serrata are shown in Fig. 1. The primer pairs used for S. serrata a2-M cDNA clone are shown in Table 1. The degenerate primers of AMGD F and AMGD R were used to amplify the partial cDNA of a2-M and cDNA from S. serrata were used as templates for PCR (GeneAmp PCR system 2700, Applied Biosystems, Forster, CA, USA). The PCR reaction buffer was 50 mM TriseHCl buffer (pH 9) containing 50 mM KCl, 1% Triton X-100 2.5 mM MgCl2, 2.5 U Taq polymerase, 0.25 mM dNTPs, and 10 mM of each primer. The PCR reactions were performed as follows: 35 cycles of denaturation at 94  C for 1 min, annealing at 60  C for 1 min, and elongation at 72  C for 1 min, followed by a 7 min extension at 72  C and cooling to 4  C. Gene-specific primers and nested primers were designed from the previously determined DNA sequence to confirm the S. serrata a2-M partial sequence. Briefly, first fragment of partial a2-M cDNA was obtained using primer AMGGSP F and AMGGSP R, then 50 - and 30 RACE were performed to make a full-length a2-M cDNA.

α 2-macroglobulin mRNA

5

3 AAAAA

RT

Oligo d(T)18

PCR, cloning S. ser α 2-M 898 bp 5 RACE

3 -RACE AMGGSP R

AMGGSP F S. ser α 2-M5-I 1778 bp

AMGRA R1 MC α 2-M3 790 bp

AMGNRA R2

AAAAA

S. ser α 2-M5-II 1065 bp AMGRA F1

AMGRA F2 AMGRA R3 AMGNRA R4

S. ser α 2-M5-III 960 bp

AMGRA R5 AMGNRA R6 Overlapping

Scylla serrata α 2-macroglobulin 5491 bp AAAAA

Fig. 1. Sequencing strategy of Scylla serrata a2-M. Arrows indicate the direction of sequencing. Grey shaded boxes show the names of the primers and black shaded boxes indicate the name of clones. Name and the sequence of the primers are in Table 1. The lengths of the bold line correspond to the lengths of the sequences.

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Table 1 Names and sequences of the primers used in the investigation and expression of a2-M of Scylla serrata Name of the primer

Sequence 50 to 30

AMGD F AMGD R AMGGSP F AMGGSP R AMGRA R1 AMGNRA R2 AMGRA R3 AMGNRA R4 AMGRA R5 AMGNRA R6 AMGRET F AMGRET R AMGRA F1 AMGRA F2 AAP AUAP PGT1 PGT2 b actin F1 b actin R1

CTSCKCAGCWCCTGTCACARCSTACWC TCGTWAKGTGKCASCGTGTRCCTC GCCTCAGCAGTGTGTCCTCCCGCCATCCAT GTAGTAGTCGTACACCACCCACTGTGCCGG TAGGCTTTGAGGGCCAGGGTGTATGGAT ATGTCAAGAGGTGGAGCTGAAGGCTGCTCC GTTATCCAGATAGCGAAACAT TCCGGCTGTTCCAGGCTGGTGCTC GTCGTGGCAACCGGTCACGTATACC ATCAGTCGCCAGGATGTACTTGGGCGGCGT GACAGGCGATGGATGC GGGATGTAACCCGAAATGAG AGGTGGACGGCAGCAAGATCAACTTC TTCAGAGTGACGCGAACCGTGGACGTGGAG GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG GGCCACGCGTCGACTAGTAC GTTGCCGACGACGAGCCTACTTTTTTTT GTTGCCGACGACGAGCCTAC TAGGTGGTCTCGTGGATGCC GAGACCTTCAACACCCCCGC

R: A/G, W: A/T, S: G/C K: G/T.

2.4. PCR and 50 - and 30 -RACE Total RNA isolated from haemocytes was used as a starting material for both methods of 50 -RACE and 30 -RACE. For 30 RACE, 5 mg of cDNA was used for PCR reaction. The reaction buffer was 50 mM TriseHCl buffer (pH 9), containing 50 mM KCl, 1% Triton X-100 (Boehringer Mannheim, Germany), 2.5 mM MgCl2, 5 U Taq polymerase, 0.25 mM dNTPs, and 10 mM of each primer. The PCR reactions were performed with two steps as follows: In the first instance, PCR reaction was performed with 5 cycles of denaturation at 94  C for 1 min, annealing at 50  C for 1 min, then 25 cycles of denaturation at 94  C for 1 min, annealing at 55  C for 1 min, and elongation at 72  C for 1 min, followed by a 7 min extension at 72  C and cooling to 4  C. For 50 -RACE, 5 mg of total RNA was reverse-transcribed with oligo-d(T)18 primer as that described above. The 50 RACE part of the a2-M gene was obtained in three subsequent 50 -RACE reactions by using 50 -RACE system (Cat. 18374-058, Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instruction. The first-strand cDNA was tailed at the 50 -end by terminal transferase TdT and dCTP. The primer set consisted of AMGRA R1 and Abridged Anchor Primer (AAP) for the first-run PCR, and AMGNRA R2 and Abridged Universal Amplification Primer (AUAP) for the second-run PCR derived S. ser a2-M5-I fragment (Fig. 1). The PCR product (S. ser a2-M5-I) did not contain 50 UTR and signal peptide sequences. Two subsequent 50 -RACE reactions were performed as above with a new set of gene-specific primers and nested primers AMGRA R3 and AMGNRA R4 for S. ser a2-M5-II sequence and AMGRA R5 and AMGNRA R6 for S. ser a2-M5-III sequences (Fig. 1). The PCR reaction conditions were the same as that described above, except annealing temperature (58  C) of second-run PCR. For 30 -RACE, the reverse-transcribed was performed by PGT1 primer. Two successive PCRs were carried out with the primer AMGRA F1 and PGT1, and AMGRA F2 and PGT2 for first and second run, respectively (Table 1). Again, the PCR conditions were the same as that described above. 2.5. Cloning, sequencing and sequence analysis The PCR fragments were subjected to electrophoresis on 1.5% agarose gel for length differences, and amplified cDNA fragments were cloned into the pGEM-T Easy vector following the instructions provided (Promega Corporation, Madison, WI, USA). Recombinant bacteria were identified by blue/white screening and confirmed by PCR. Plasmids containing the insert were purified (Promega minipreps) and used as a template for DNA sequencing. Nucleotide sequence analysis was performed using the dideoxynucleotide chain termination method on a DNA sequencer

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(Model 373A, Applied Biosystems, Forster, CA, USA). Plasmid DNA at 1 mg was used for sequencing with a Dye Terminator Cycle Sequencing Kit (Applied Biosystems) and subjected to electrophoresis on 6% denaturing gels. Clones were sequenced with the M13 forward and reverse primers. The a2-M gene sequence was analysed and compared by using the BLASTX and BLASTP search programs (http://www.ncbi.nlm.nih.gov/BLAST/) with GenBank database search. Forward and reverse sequences were checked, and vector bases were trimmed using Chromas software (Version 2.13). Contiguous fragments were assembled using Contig Express, part of the Vector NTI software package (Version 9). Search for similarities with known genes was performed using BLAST. Translation and protein analysis were performed using the ExPASy tools (http://us.expasy.org/tools/http://us.expasy.org/tools/). 2.6. Phylogenetic analysis The multiple sequence alignment was created by using the CLUSTAL W [32], and the same software was used to analyse similarity of the aligned sequences using a neighbour joining (NJ) algorithm. A phylogenetic tree based on the deduced amino acid sequences were performed using the NJ algorithm and the reliability of the branching was tested using bootstrap re-sampling (1000 pseudo-replicates). 2.7. a2-M expression in lipopolysaccharide (LPS)-injected S. serrata For the challenge test, there were two treatments (S. serrata injected with LPS and crab injected with saline) combined with four exposure times at 0, 12, 24 and 48 h. LPS which had been dissolved in 0.85% NaCl solution to 5 mg ml1 was used as test solution. S. serrata were injected at the base of the right fifth leg with LPS solution at a rate of 100 ml per kg crab to reach a dose of 0.5 mg kg1. Crabs were injected with 200 ml saline as control. For each treatment and each exposure time, three crabs were sampled from the haemolymph. Seawater was replaced daily. Haemolymph of S. serrata in LPS challenge was taken by inserting a syringe into the base of right chelate leg. Five millilitres of haemolymph were collected into a 25 ml test tube containing 5 ml of anticoagulant [26,27]. The diluted haemolymph was centrifuged at 500  g at 4  C for 20 min. The resulting pellet was used for total RNA isolation, and used for the a2-M transcript analysis by PCR as described above. The RT-PCR was performed using gene specific primers AMGGSP F and AMGGSP R with cDNA (as a template) that had been reverse transcribed. The primers b-actin F and b-actin R were used to amplify b-actin fragment that was used as a positive control for RT-PCR. 2.7.1. Preparation of standard curve for a2-M mRNA The absolute real-time standard curve of a2-M gene was prepared according to Bustin [33]. Briefly, using the plasmid vector containing S. serrata cDNA as a template, and addition of SP6 RNA polymerase (Promerga, USA) in vitro, a2-M RNA was transcribed. After transcription, the sample was digested by the addition of RNAse-free DNAse and the RNA was quantified with a spectrophotometer. The RNA diluted in different concentrations with DEPC-water was used as RNA standard. The RNA was reverse transcribed as described above and stored at 20  C until use for quantitative real-time RT-PCR. 2.7.2. Quantification of a2-M gene expression by real-time RT-PCR The mRNA expression of S. serrata a2-M in haemocyte of saline injected and LPS-injected crabs were measured by quantitative real-time RT-PCR. The cDNA and a2-M standards were used for the assay of quantitative real-time RT-PCR. The SYBR Green I real-time RT-PCR assay was carried out in an ABI PRISMTM 7900 Sequence Detection System (Applied Biosystems). The amplifications were performed in a 96-well plate in a 25 ml reaction volume containing 12.5 ml of 2X SYBR Green Master Mix (PE Applied Biosystems), 2.5 ml (each) AMGRET F and AMGRET R primers (10 mM), 1 ml of template (1 mg and 0.5 mg cDNA of haemocyte, or different concentrations of a2-M standard) and 9 ml of DEPC-water. The thermal profile for SYBR Green real-time RT-PCR was 50  C for 2 min and 95  C for 10 min followed by 40 cycles of 95  C for 15 s and 60  C for 1 min. In a 96-well plate, each sample was conducted in duplicate. DEPC-water for the replacement of template was used as negative control. Data analysis of RT-PCR was performed with the SDS software V2.0 (Applied Biosystems, Foster City, CA). The linear relationships between Ct, threshold PCR cycle and different log concentrations of a2-M RNA standard was tested using the General Linear Model Procedure and the Regression Procedure, version 6.03, SAS (Statistical Analysis System) computer software.

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The different DCt values of a2-M were calculated to actual concentrations of a2-M mRNA in total RNA based on the a2-M standard curve. 2.8. Expression of a2-M gene in tissues Expression of mud crab S. serrata a2-M mRNA in tissues was demonstrated by RT-PCR. Heart, gill, muscle, haemocyte, hepatopancreas, antennules, eyestalk, ovary and intestine were dissected out. The tissues were blotted and weighed then homogenised in Trizol reagent using a glass homogeniser. Total RNA was extracted as described above. The extracted and purified total RNA was quantitatively determined to the same concentration, and a sample of total RNA at 5 mg was used for reverse transcription. The first strand cDNA was synthesised using a cDNA first strand synthesis Kit with superscriptÔ III RNAse H- reverse transcriptase (Invitrogen). The gene specific primer pairs AMGGSP F and AMGGSP R were used to amplify the a2-M transcript, and the primers b-actinF and b-actinR were used to amplify b-actin fragment that was used as a positive control (Table 1). The PCR reaction was the same as that described above. 2.9. Statistical analysis A multiple comparison (Duncan) test was conducted to compare the significant differences in a2-M gene expression between saline-injected crab and LPS-injected crab using SAS software (SAS Institute Inc., Cary, NC, USA). A significant level of P ¼ 0.05 was chosen. 3. Results 3.1. Isolation of S.serrata a2-M cDNA Sequencing of the degenerate PCR product revealed a high sequence similarity to other crustaceans a2-Ms, the same sequence was used to design gene specific primers to perform 50 and 30 RACE to obtain the full length cDNA sequence. The gene specific primer pairs AMGGSP F and AMGGSP R were used to amplify 898 bp fragment of partial a2-M from haemocytes. The full-length sequence of the S. serrata a2-M cDNA was completed from five overlapping PCR-amplified fragments (Fig. 1). The N-terminal portion of S. serrata a2-M and the 50 UTR were obtained by 50 -RACE PCR. The first 50 RACE product of 1778 bp (S. ser a2-M5-I) did not extend to the very 50 -end; therefore this part had to be completed by amplification of a second S. ser a2-M-II product (1065 bp) and third S. ser a2-M-III product (960 bp). The C-terminal part of S.serrata a2-M including its 3 0 UTR and polyadenylation signal was determined by cloning and sequencing of a 790 bp product obtained using 30 -RACE method. The nucleotide and deduced amino acid sequences of S.serrata a2-M precursor cDNA are shown in Fig. 2. The full-length of a2-M precursor cDNA consisted of 5491 bp comprising a 50 untranslated region (UTR) of 190 bp, an open reading frame (PRF) of 4986 bp, a stop codon of 3 bp and a 30 UTR of 312 bp. The 30 UTR contained a consensus polyadenylation signal (aataaa) 85 bp upstream from the poly A tail (Fig. 2). The open reading frame encoded a protein consisting of 1662 amino acid residues. The mature protein of a2-M consisted of 1662 amino acids with a theoretical molecular mass of 184.2 kDa. Eleven potential N-linked glycosylation sites were observed at amino acid positions, 114, 277, 287, 404, 484, 799, 925, 1090, 1130, 1366 and 1455 (Fig. 2). This sequence contains an intra-chain thioester making it a member of the a2-M super family of protein. The signal peptide is formed by the first 22 residues as predicted by the SignalP 3.0 server http://www.cbs.dtu.dk/services/SignalP/ according to Nielsen et al. [34]. The S. serrata a2-M cDNA sequence and deduced amino acid sequence has been submitted to the NCBI GenBank as accession number DQ347954. 3.2. a2-M domains of S. serrata Multiple alignments of amino acid sequences of mud crab S. serrata a2-M and other known a2-Ms using the CLUSTAL X program indicated that S. serrata a2-M contained principal domains required for the a2-M mechanism. A thioester site (GCGEQNM) of S.serrata a2-M was completely identical to other invertebrate and vertebrate a2-Ms cysteine at position 1041 is located in the highly conserved thioester domain and thus it most likely forms the reactive

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Fig. 2. Nucleotide sequence (above) and deduced amino acid sequence of the open reading frame (below) of Scylla serrata putative a2-M cDNA. Nucleotides are numbered from the first base at the 50 end. Amino acids are numbered from the initiating start codon methionine. The polyadenylation signal aataaa is shown in the italic letters with double underline. The conserved thioester sequence (GCGEQNM) is shown in bold letters with bold underline. The presumable bait region and receptor binding domain are shown in dot and solid boxes, respectively. The predicted Nlinked glycosylation sites are shown in bold italic letters. Forward and reverse gene specific primers (AMGGSP F and AMGGSP R) for the first sequence coloning are shown in bold letters, respectively. The full sequence was submitted to Genbank with accession number DQ347954.

thioester bond with glutamine residue 1044. The region surrounding the thioester site was also similar to the corresponding regions of all aligned a2-Ms (Fig. 3). Distribution of Cys residues is also well conserved throughout the entire sequence of S. serrata a2-M. The bait region (presumable positions of 681 to 785) of S. serrata a2-M showed a little identity to that of other a2-Ms. The receptor binding domain (positions at 1339 to 1523) of S. serrata a2-M

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498 1741 518 1801 538 1861 558 1921 578 1981 598 2041 618 2101 638 2161 658 2221 678 2281 698 2341 718 2401 738 2461 758 2521 778 2581 798 2641 818 2701 838 2761 858 2821 878 2881 898 2941 918 3001 938 3061 958 3121 978 3181 998 3241 1018 3301 1038 3361 1058

R G K V Q R W W N L Y S T S H T Q S A L tggcattttcagagcattagccgccgcaaagtgctgcgctgctggagcctgtatgtggat W H F Q S I S R R K V L R C W S L Y V D tgcctgccgaccatgctgccgtttaacacccagcaggcggaaaccccgcattataacagc C L P T M L P F N T Q Q A E T P H Y N S cagccgtttgtgaccggctttgtgagcattcgcattagctttccgcatgcgaccagctgc Q P F V T G F V S I R I S F P H A T S C accgtgctgctgcgctggtatacccgctatgatgtgcaggtggtgtatgatagcgtgcag T V L L R W Y T R Y D V Q V V Y D S V Q gtgatgcgcgatcgctgctggctggatgaaacctgcctgacctggagcaccagcctggaa V M R D R C W L D E T C L T W S T S L E cagccggaagaaattaccctgagcgtgcaggcgtggaccgatgcgatttgctttgtgggc Q P E E I T L S V Q A W T D A I C F V G gtggtggataaaaacgcggatctgatgaacaccgatccgcataccattaacctggataaa V V D K N A D L M N T D P H T I N L D K atgtttcgctatctggataactatattatgtatacctggctgtatagccatattgatgat M F R Y L D N Y I M Y T W L Y S H I D D attacctattgcctgcagattgtggtgcgccagcgctgcgtgcaggcgaacagcgtgccg I T Y C L Q I V V R Q R C V Q A N S V P gaaaccgaagatattctgccggcgagccgccgcggctatagcagccagagcgtggatagc E T E D I L P A S R R G Y S S Q S V D S ctgaccatgtttagcgatgcgggcctgggcggcggcagcgtgggcattgcgggcgtggcg L T M F S D A G L G G G S V G I A G V A tttgatcgcccgaaaccggcggcgccgccgcaggcgctgggcggcgtggcgattgcggaa F D R P K P A A P P Q A L G G V A I A E gatgcgctgggcccggatcgcgaacaggcgattgaacatgaaaccagcagcgaaagcagc D A L G P D R E Q A I E H E T S S E S S gcggaagaagcgccgcgcaccaactttccggaaacctggctgtggaacattgtggtgccg A E E A P R T N F P E T W L W N I V V P gaaaacggcacccgcaaagtggatcagaccctgccggataccattacccagtgggtgggc E N G T R K V D Q T L P D T I T Q W V G aaagcggtgtgcgtgcatccgcaggtgggcgtgggcctgagcgaacgcgaaagcattgcg K A V C V H P Q V G V G L S E R E S I A acctttaccagcttttttgtggatctgaccctgccgccgaccgtgaaacgcaaagaaacc T F T S F F V D L T L P P T V K R K E T ctgccggtgaaaagcgtgtttaactatcatgataaagatctgccgattaccattaccctg L P V K S V F N Y H D K D L P I T I T L gaagatagcatgctcaatttcgccgctaacgtcaacatgatggaatacctgaccgtgact E D S M L N F A A N V N M M E Y L T V T gatcagaacacccccgatagcacagggaagctcctcaggttcatgaggacgggataccaa D Q N T P D S T G K L L R F M R T G Y Q aggcaactgctgcgccgctgcaacggctcctacaccgccttggggagtgcagacgactgc R Q L L R R C N G S Y T A L G S A D D C ggctcggtggaatatgatattctggaagaaccggaactgccgggcgcgcgcggcaaacgc G S V E Y D I L E E P E L P G A R G K R agcgcgtgcattccggcgaacgataaagtggtgctgaccgtgcgcgtgaccccgggcgcg S A C I P A N D K V V L T V R V T P G A tgccgcgaaccggatagcgtgtgccgccgcggccgccatggcctgccgtatctgtggcgc C R E P D S V C R R G R H G L P Y L W R ggcgaacagaccccgccgaccgaagcagacatcgtgccggactctgagcgaggctgggtc G E Q T P P T E A D I V P D S E R G W V acggttgtcggtgacctgcttgcactctcgctccagaaccttggctcgctcatccgcctt T V V G D L L A L S L Q N L G S L I R L ccctccggttgcggtgagcagaacatgctgaactttgcgccgaacatttttctgatggat P S G C G E Q N M L N F A P N I F L M D tatctggaaaccacccgccaggcgaccccggaagcgaccgcgaaactgatgcgctataac Y L E T T R Q A T P E A T A K L M R Y N

517 1800 537 1860 557 1920 577 1980 597 2040 617 2100 637 2160 657 2220 677 2280 697 2340 717 2400 737 2460 757 2520 777 2580 797 2640 817 2700 837 2760 857 2820 877 2880 897 2940 917 3000 937 3060 957 3120 977 3180 997 3240 1017 3000 1037 3360 1057 3420 1077

Fig. 2. (continued)

located at the C-terminal region showed a high similarity to the binding domains of other a2-Ms, and Lys residues responsible for receptor binding were also found in this region (Fig. 2). The amino acid sequence of S. serrata a2-M was compared with the sequences of other a2-Ms. A search in the NCBI GenBank http://www.ncbi.nlm.nih. gov/BLAST/Blast.cgi) database, using the BLASTP program, revealed that the deduced sequence S. serrata a2-M showed high similarity with that of a2-M from other arthropods. The most similar sequences were from kuruma shrimp M. japonicus (AB108542), American horseshoe crab L. polyphemus (D83196), soft tick O. moubata

B. Vaseeharan et al. / Fish & Shellfish Immunology 22 (2007) 115e130

3421 1078 3481 1098 3541 1118 3601 1138 3661 1158 3721 1178 3781 1198 3841 1218 3901 1238 3961 1258 4021 1278 4081 1298 4141 1318 4201 1338 4261 1358 4321 1378 4381 1398 4441 1418 4501 1438 4561 1458 4621 1478 4681 1498 4741 1518 4801 1538 4861 1558 4921 1578 4981 1598 5041 1618 5101 1638 5161

accggctatcagcgccagctgctgtatcgccgcaacaacggcagctatagcgcgtttggc T G Y Q R Q L L Y R R N N G S Y S A F G aacgcggatgaaagcggcagcacctggctgaccgcgtttgtgctgaaaagctttacccag N A D E S G S T W L T A F V L K S F T Q gcgaaaaaatatattcaggtggatgaagaaaaactgaaccagacccgccgctggctgctg A K K Y I Q V D E E K L N Q T R R W L L agccatcagggcccggatggctgctttaccgcggtgggcaaagtgctgcataaaagcatg S H Q G P D G C F T A V G K V L H K S M cagggcggcgtgagcgatagcaaaaccccggcgccgctgaccccccatctctccccagct Q G G V S D S K T P A P L T P H L S P A cctgtcacagcctacgtgctaatgtcgctggtggaaggcggggagcagccttcagctcca P V T A Y V L M S L V E G G E Q P S A P cctcttgacatggccatccaatgcctcagcagtgtgtcctcccgccatccatacaccctg P L D M A I Q C L S S V S S R H P Y T L gccctcaaagcctacgccatggccctcgctggccgtcccgaggcagctgacgtcctgaca A L K A Y A M A L A G R P E A A D V L T gagctggagaatgcagccgttgtcacttcaaactccatgcactggaagctgcccgaaggc E L E N A A V V T S N S M H W K L P E G aggagcagagctgccgccgtggaggtggcggggtacgccatcctggccatgatgaccctc R S R A A A V E V A G Y A I L A M M T L aacccagagacctatgagcccaaggcgaggaaggtggtgaagtggatcaccacgaagagg N P E T Y E P K A R K V V K W I T T K R aacggccagggaggcttctattccacacaggacaccgtggtggccatgcaggcgctgaca N G Q G G F Y S T Q D T V V A M Q A L T ctgtttgagagccaccgttaccagggtcccctcaatgtagtcgcctccgtcaaggccgag L F E S H R Y Q G P L N V V A S V K A E gggctcgagcacaccttcaacgtgaatgacgacaacaagctgctgcagcagttgaagacg G L E H T F N V N D D N K L L Q Q L K T ctgcctatactaccaacacaagtgaacctcaccatgacaggcgatggatgcgccgtgctg L P I L P T Q V N L T M T G D G C A V L cagggcgtcctccgttacaacattcccaatcccgagccgagtgatgcctttgacctcacc Q G V L R Y N I P N P E P S D A F D L T gtcaacaccattacggtgccagaccgcctgtgcgccaccaaacgcatcaccgcctgcgcc V N T I T V P D R L C A T K R I T A C A tcctaccgccttcctgacggcgcctccaacatggtggtcatcgaggtggacctcatttcg S Y R L P D G A S N M V V I E V D L I S ggttacatcccggataaggatgacctcaagctcctcaccaaacaggacaagaacatcacg G Y I P D K D D L K L L T K Q D K N I T cgttacgaggtggacggcagcaagatcaacttctatataaacgagttgacggtgaaggac R Y E V D G S K I N F Y I N E L T V K D acctgtgtgaacttcagagtgacgcgaaccgtggacgtggaggacgtgaagcccggcaca T C V N F R V T R T V D V E D V K P G T gtgggtggtgtacgactactaccaggaggagttctccatctccatggaggtacacgctgc V G G V R L L P G G V L H L H G G T R C cacctaacgacgagtgccgctaacgcgcgcccgagccgcagcccgagccgcaccaccaac H L T T S A A N A R P S R S P S R T T N ccgccgaacctgcgcaacccgagcagcgtgagcccgcgcccgacccgccagagcgcgcag P P N L R N P S S V S P R P T R Q S A Q cgcctgcatgcgcgcgcgagcaaaagcaaatgcaaaagcgcgattaccgaaagcgtggcg R L H A R A S K S K C K S A I T E S V A cgccgcacctggccgagctggaaacgcagcagcccgggcaccgcgcgccgcctgctgctg R R T W P S W K R S S P G T A R R L L L gaaagcagccgcccgagcaccaccacccgccgcgcgcgactggatggaagcggcgagctt E S S R P S T T T R R A R L D G S G E L tacctggacccgcagcaccagcagccagctgagcagctgcccgagcctgaaaaccctgct Y L D P Q H Q Q P A E Q L P E P E N P A gtggacccgcagcagcccgagcccgtggcgctataccctgccgccgcgcgtgcgcgtgcg V D P Q Q P E P V A L Y P A A A R A R A cgcgcatcatgcggatagccgcggcgaaggccgccatcatagcctgcagctgcgcctgcc

1658 R

A

S

C

G

*

123

3480 1097 3540 1117 3600 1137 3660 1157 3720 1177 3780 1197 3840 1217 3900 1237 3960 1257 4020 1277 4080 1297 4140 1317 4200 1337 4260 1357 4320 1377 4380 1397 4440 1417 4500 1437 4561 1457 4621 1477 4681 1497 4740 1517 4800 1537 4861 1557 4920 1577 4981 1597 5040 1617 5100 1637 5160 1657 5220 1662

5221 ggcgaccaccaccacccagaccgcgattctgagcaaccatattaccccggtgagcattct 5280 5281 gattcataactttagccgcaaaaggcttttatgtggtggtgagcctgtttagcaaagata 5340 5341 ccgtggtgatttttgtgctgctgcagtttgtgtttctggtgcaggcgtgtttctgtttaa 5400 5401 aataaatcatgtggcgacctaagataccgtgcagtgctttagcgtggtggcgaacaaagt 5460 5461 gctgacctaaaaaaaaaaaaaaaaaaaaaaa Fig. 2. (continued)

5491

B. Vaseeharan et al. / Fish & Shellfish Immunology 22 (2007) 115e130

124 (1) 1

10

20

30

40

50

60

76

ScseA2M (1) PSGCGEQNMLNFAPNIFLMDYLETTRQATPEATAK---LMRYN-TGYQRQLLYRRNNGSYSAFGNA---DESGSTW MajaA2M (1) PSGCGEQNMVNFAPNVYMMQYLTVTEQNTPDSTGK---LLRFMRTGYQRQLLYRRSNGSYSAFGSA---DDSGSTW CycaA2M (1) PYGCGEQNMAVLSPNIYILQYLENTEQLTSAIRER---ATGFLKSGYQRQLNYKHSDGAYSTFGYG-----DGNTW MumuA2M (1) PYGCGEQNMVLFVPNIYVLNYLNETQQLTEAIKSK---AINYLISGYQRQLNYQHSDGSYSTFGNHGGGNTPGNTW RanoA2M (1) PYGCGEQNMVLFAPNIYVLDYLNETQQLTQEIKTK---AIAYLNTGYQRQLNYKHRDGSYSAFGDKPG-RNHANTW ChfaA2M (1) PYGCGEQNMASWSPNIVVLQYLTNTNQLTSKIETE---ALNYMRIGYQRQLNYRHDDGSYSAFGNS---NADGSMW LipoA2M (1) PTGCGEQNMVKFVPNIFVLDYLTATGSITDSIKEK---ALNNMRKGYARQQNYRHPDGSYSAFGNR---DKQGNLF OrmoA2M (1) PTGCGEQNMVKFTPNVYVLDYLKATGKQDADIEKK---AVENLKTGYQRQQKYRHSDGSYSAFGTN---DRQGSLF ApmeA2M (1) PKGCGEQNMILFVPNNHVIKYLDAMRINKPDLRAK---AIRNMEKGYQRELKYRFMDGSYSAFE-----EGESSIW AngaTEP-1 (1) PTGCGEQNMVKFVPNILVLDYLYATGSKEQHLIDK---ATNLLRQGYQNQMRYRQTDGSFGVWEKS-----GSSVF DrmeTEP-1 (1) PCGCGEQNMFNFVPSILALSYLKAKNRQDQEIENK---AKRYVETGYQIELNYKRNDGSFSAWGQH---DALGSTW CiinGPI-A2M (1) PSGCGEQNMLGFAPDVFVTLYLHSAGKLDAATRAK---AFKHFQTGYSNELNYKHRDGSFSAFGEG---DASGSTW HosaC3 (1) PSGCGEQNMIGMTPTVIAVHYLDETEQWEKFGLEKRQGALELIKKGYTQQLAFRQPSSAFAAFVKR-----APSTW OnmyC3-1 (1) PVGCGEQNMIYMTLPVIATHYLDNTKKWEDIGLDKRNTAIKYINIGYQRQLAYRKEDGSYAAWVSR-----QSSTW

ScseA2M MajaA2M CycaA2M MumuA2M RanoA2M ChfaA2M LipoA2M OrmoA2M ApmeA2M AngaTEP-1 DrmeTEP-1 CiinGPIA2M HosaC3 OnmyC3-1

(77) (70) (71) (69) (74) (73) (71) (71) (71) (69) (69) (71) (71) (72) (72)

77

90

100

110

120

130

140

152

LTAFVLKSFTQAKKYI-QVDEEKLNQTRRWLLSHQGP-DGCFTAVGKVLHKSMQGGVSD--SKTPAPLTPHLSPAP LTAFVLKSFVQAKEFI-YIDDSSLNRTRAWLMASELDRSGCVVPVGKVFSKGLKGGLKG--KASPVPMTAYVLIAL LTAFVLRSFGKAQKYI-FIDPQIIQSAKEWLISRRDS-DGCFIQQGRLFNNRMKGGVNDN-----VTMTAYITASL LTAFVLKAFAQAQSHI-FIEKTHITNAFNWLSMKQKE-NGCFQQSGYLLNNAMKGGVDD-----EVTLSAYITIAL LTAFVLKSFAQARKYI-FIDEVHITQALLWLSQQQKD-NGCFRSSGSLLNNAMKGGVED-----EVTLSAYITIAL LTAFVVKCFGQSRPFI-DIDNDDLVKSLNWFRRRQLE-NGCFPKIGYTHSYYLKGGISK--SSNEAAMTAFVLIAM LTAFVYRSFAQAERFI-LINKNKLNETENWILNRQRS-NGCFRKIGKLFNSALKGGISSN-DETPAPLTAYVLISL LTAFVIRSFKQAERYI-PIDEKMLQQSVNWVLNKQIPVNGCFNNVGRVLSSGLKGKVNE---SNPGPLTAYVLAAL LTAFVLKSFAQAASLI-HIDKYVLESSVSWITMNQLE-DGCFPVIGTVFHKSMKGGLQEHG--SSSALTAYILISL LTAFVATSMQTASKYMNDIDAAMVEKALDWLASKQHS-SGRFDETGKVWHKDMQGGLRN-----GVALTSYVLTAL LTAYVIRSFHQAAKYI-DIDKNVLVAGLDFLVSRQST-DGKFKELGMVIHNS--HGS-------PLALTSFVLLTF LTAFAAKCFMFARELRPTLVSASVIDQALTFLINQQNTTGTFREPGRVSHKAMQGGVDS-----PITMTAYVLITL LTAYVVKVFSLAVNLI-AIDSQVLCGAVKWLILEKQKPDGVFQEDAPVIHQEMIGGLRNN-NEKDMALTAFVLISL LTAYVVKVFAMSSTLI-SVQENVLCTAVKWLILNTQQPDGIFNEFAPVIHAEMTGNVRG--SDNDASMTAFVLIAM

Fig. 3. Multiple alignment of Scylla serrata a2-M with other thioester proteins using CLUSTAL W. The figure shows the thioester region and compares the specificity defining residues (BOLD) as described by Dodds and Day [52]. GenBank accession numbers for the sequences used are listed in Table 3. Black shaded sequence indicates positions where all the sequences share the same amino acid residue, gray shaded sequence indicates conserved amino acid substitutions; Light gray shaded sequences indicates semi-conserved amino acid substitutions.

(AF538967), sea scallop Chlamys farreri (AY395573), honey bee Apis mellifera (XM_392454) and human Homo sapiens (AB209614.1) (Table 2). Comparing a full-length multiple alignment of the a2-M sequence with members of the a2-M (Table 3), a phylogeny tree was constructed based on the neighbour-joining method (Fig. 4). The phylogeny tree is composed of three main distinct branches, comprising the group of a2-Ms, complement proteins, and insect TEPs, respectively. Mud crab S. serrata shows an overall sequence similarity with kuruma shrimp M. japonicus a2-M, and they apparently form a monophyletic group. 3.3. Expression analysis of S. serrata a2-M mRNA The tissue-specific expression of a2-M was investigated by RT-PCR analysis using total RNA isolated from heart, gill, muscle, haemocyte, hepatopancreas, antennules, eyestalk, ovary and intestine of S. serrata. The primer set used for quantitative PCR (AMGGSP F and AMGGSP R) was designed to the putative a2-M sequence to yield a 898 bp product within the conserved domain (Fig. 5A). All reactions using b-actin as the positive control were detected in all Table 2 Similarity comparison of mud crab Scylla serrata a2-M with other species Species

a2-M

GenBank accession no.

% identity/similarity

Kuruma shrimp Marsupenaeus japonicus Horseshoe crab Limulus polyphemus Soft tick Ornithodoros moubata Sea scallop Chlamys farreri Honey bee Apis mellifera Human Homo sapiens Mud crab Scylla serrata

a2-macroglobulin homolog a2-macroglobulin a2-macroglobulin a2-macroglobulin a2-macroglobulin a2-macroglobulin a2-macroglobulin

AB108542 D83196 AF538967 AY395573 XM_392454 AB209614.1 DQ347954

60/68 30/48 30/47 30/47 34/53 34/52 Present study

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125

Table 3 Species, protein and GenBank accession number of a2-M sequences used to construct phylogenetic tree Abbreviation

Species and protein

GenBank accession number

AngaTEP-1 ApmeA2M BrbeC3 Cael A2M ChfaA2M Ciin GPI-A2M CiinC3-1 CiinC3-2 CycaA2M CycaC3-H1 CycaC3-H2 CycaC3-Q1 CycaC3-Q2 CycaC3-S CycaC4-1 CycaC4-2 DrmeTEP-1 DrmeTEP-2 DrmeTEP-3 DrmeTEP-4 EutaA2M GagaC3 HosaC3 HosaC5 HosaCD109 A2M HosaGPI-A2M LipoA2M MajaA2M MumuA2M MumuC3 MumuC4 MumuGPI-A2M NanaC3 OnmyC3-1 OnmyC3-3 OnmyC3-4 OrlaC3-1 OrlaC3-2 OrlaC4 OrmoA2M PaolC3 RanoA2M RanoC3 ScseA2M StpuC3 XelaC4

Anopheles gambiae thioester protein-1 Apis mellifera a2-macroglobulin Branchiostoma belcheri C3 Caenorhabditis elegans a2-macroglobulin Chlamys farreri a2-macroglobulin Ciona intestinalis GPI-a2-macroglobulin Ciona intestinalis C3-1 Ciona intestinalis C3-2 Cyprinus carpio a2-macroglobulin Cyprinus carpio C3-H1 Cyprinus carpio C3-H2 Cyprinus carpio C3-Q1 Cyprinus carpio C3-Q2 Cyprinus carpio C3-S Cyprinus carpio C4-1 Cyprinus carpio C4-2 Drosophila melanogaster thioester protein-1 Drosophila melanogaster thioester protein-2 Drosophila melanogaster thioester protein-3 Drosophila melanogaster thioester protein-4 Euphaedusa tau a2-macroglobulin Gallus gallus C3 Homo sapiens C3 Homo sapiens C5 Homo sapines CD109 a2-macroglobulin Homo sapiens GPI-a2-macroglobulin Limulus polyphemus a2-macroglobulin Marsupenaeus japonicus a2-macroglobulin Mus musculus a2-macroglobulin Mus musculus C3 Mus musculus C4 Mus musculus GPI-a2-macroglobulin Naja naja C3 Oncorhynchus mykiss C3-1 Oncorhynchus mykiss C3-3 Oncorhynchus mykiss C3-4 Oryzias latipes C3-1 Oryzias latipes C3-2 Oryzias latipes C4 Ornithodoros moubata a2-macroglobulin Paralichthys olivaceus C3 Rattus norvegicus a2-macroglobulin Rattus norvegicus C3 Scylla serrata a2-macroglobulin Strongylocentrotus purpuratus C3 Xenopus laevis C4

AF291654 XM_392454 AB050668 CAB05007 AY395573 AJ431688 AJ320542 AJ320543 AB026128 AB016210 AB016212 AB016214 AB016215 AB016213 AB037278 AB037279 AJ269538 AJ269539 AJ269540 AJ269541 AB206788 U16848 AY513239 M57729 NM_133493 AF410459 D83196 AB108542 M93264 K02782 AF049850 AY083458 L02365 L24433 AF271079 AF271080 AB025575 AB025576 AB025577 AF538967 AB021653 J02635 X52477 DQ347954 AF025526 D78003

tissues (Fig. 5B). A predominant a2-M transcript expression with the expected size was observed only in haemocytes but not in other tissues tested (Fig. 5A). 3.4. a2-M expression of S. serrata injected with LPS Real-time PCR was used to determine the changes in expression of S. serrata a2-M mRNA in haemocytes of mud crab injected with LPS for 12 h, 24 h and 48 h. The a2-M expression slightly induced after 12 h, and increased significantly after 24 h and 48 h LPS injection (Fig. 6). The copy numbers of a2-M transcript in 24 h- and 48 h-post LPS injection were significantly higher (P < 0.05), as compared with the saline injected S. serrata.

B. Vaseeharan et al. / Fish & Shellfish Immunology 22 (2007) 115e130

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Fig. 4. Phylogenetic tree produced using the neighbour joining method. GenBank accession numbers used to construct phylogenetic tree are given in Table 3.

4. Discussion In the present study, the full-length sequence of thioester containing a2-M, a macromolecular proteinase inhibitor of the a2-M family was cloned from the mud crab S. serrata. S. serrata a2-M deduced amino acid sequences showed the highest percentage similarity to a2-M of M. japonicus (68%) and L. polyphemus (48%), which belong to the same class of Crustacea and Merostomata in the phylum Arthropoda, respectively. The a2-M deduced amino acid of

A

M

1

2

3

4

5

6

7

8

9

1000 bp

2-M

500 bp

B

M

1

2

3

4

5

6

7

8

9

1000 bp actin

500 bp Fig. 5. Expression of mud crab Scylla serrata a2-M in various tissues (A). b-actin was used as control in all tissues (B). M indicates the molecular weight marker. Lane 1: Haemocytes, Lane 2: Heart, Lane 3: Gill, Lane 4: Muscle, Lane 5: Hepatopancreas, Lane 6: Antennules, Lane 7: Eyestalk, Lane 8: Ovary, Lane 9: Intestine.

B. Vaseeharan et al. / Fish & Shellfish Immunology 22 (2007) 115e130 9.00E+08

Saline

LPS

127

a

8.00E+08

Copy number

7.00E+08 6.00E+08 5.00E+08 4.00E+08 a

3.00E+08 2.00E+08 1.00E+08

a

a

a

a

b

b

0.00E+00 0

12

24

48

Time elapsed (h) Fig. 6. Expression analysis of a2-M in the haemocytes of Scylla serrata injected with LPS and saline (control). a2-M mRNA levels were analysed by Real time PCR and standardised according to the respective b-actin mRNA levels. Bars the same exposure time with different letters are significantly different (P < 0.05) between treatments.

S. serrata (1662 aa) is longer than that of M. japonicus (1505 aa). This difference may be considered due to evolutionary pressure. The sequence alignment of a2-M revealed that the deduced amino acid of S. serrata showed similarity to a2-Ms of other invertebrates and vertebrates species. NCBI database blast search revealed that the deduced amino acid sequence of S. Serrata a2-M also showed the presence of C3 complement proteins features as compared to that of horseshoe crab L. polyphemus [24]. This fact also indicated that it may be due to the variation in the evolutionary pathway between a2-M and complement protein from the common ancestral protein. The a2-M sequence of mud crab S. serrata contains 11 potential N-glycosylation sites. The glycosylation level of a2-M is possibly higher than that of kuruma shrimp M. japonicus, horseshoe crab L.polyphemus, and human H. sapiens a2-M, which possess only 8, 7, and 8 glycosylation sites, respectively [25,35,36]. Recently 13 N-glycosylation sites were reported in soft tick O. moubata a2-M sequence [29]. Alignment of amino acid sequences revealed that S. serrata a2-M contains three functional domains which were also observed in other a2-Ms. The thioester site of S. serrata a2-M is completely identical with that of the other a2-Ms (Fig. 3). The putative thioester bond is likely to be formed between Cys1041 and Gln1044 as that of mammalian a2-Ms [37]. Moreover, the region around the thioester site of S. serrata a2-M showed extensive similarity to the corresponding region in the other a2-Ms. This suggests that the a2-M molecule appeared early in evolution, and its potential for proteinase entrapment has physiological significance [38]. In a2-Ms, proteolytic cleavage of the bait region activates the unique intrachain b-cysteinyl-g-glutamyl thioester, which then covalently binds lysine side chains on the attacking proteinase [39,40]. The thioester of some forms of a2-M from arthropods forms intra g-glutamyl-e-lysine bonds with the second chain of a2-M, rather than with the protease molecule [41]. The thioester can be cleaved by small primary amines, and in most a2-Ms this results in a conformational change similar to that initiated by bait region cleavage [42]. The bait region of mud crab S. serrata a2-M greatly differs in both sequence and length from that of other a2-Ms. Arthropod a2-M showed pleiotropy of the bait region in soft tick O. moubata and horseshoe crab L. polyphemus [29,36]. This pleiotropy of bait region was also found in mammalian a2-Ms and Mud crab S. serrata a2-M [12,17,43,44]. The diversity of the bait region sequences may reflect evolutionary pressure to react with proteinases produced by specific pathogens [45]. The receptor-binding domain located in the C-terminal region of a2-M functions in the binding to a cell surface receptor for receptormediated endocytosis [20]. Similarly, a sequence comparison of a2-Ms indicates that a region in the C-terminus of S. serrata a2-M corresponds to the receptor-binding domain of other a2-Ms. The receptor-binding domain of S. serrata a2-M shows a similarity with that of other a2-Ms, and contains conserved lysine (Lys) residues that are important for receptor binding [45,46]. In mammalian a2-M, four lysine residues (Lys1361, Lys1370, Lys1374 and Lys1425; numbering for human a2-M) have been reported to contribute significantly to receptor-binding [47]. In S. serrata, a2-M showed three conserved lysines (Lys1350, Lys1356 and Lys1447), and these play a similar role. The carboxyl end of the S. serrata a2-M does not contain KPTVK motif which is required for a2-M receptor binding (Fig. 2), as observed in mammalian a2-M sequences, but this is common to other more ancient a2-M

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sequences, such as horseshoe crab L. polyphemus (D83196) [24], European lobster Homarus gammarus (AJ697860), Ascidian Ciona intestinalis (AJ431688) [48] and kuruma shrimp M. japonicus (AB108542) [25]. Phylogenetic analysis revealed that S. serrata a2-M unequivocally belongs to the group of a2-M proteinase inhibitors, and is distinct from complement proteins and insect TEPs. The similarity of S. serrata a2-M full-length sequence exists in kuruma shrimp M. japonicus, soft tick O. mobulata and horseshoe crab L. polyphemus a2-M (Fig. 4). Multiple alignments indicate that the a2-M of S. serrata with that of the other crustacean form a monophyletic group. In mud crab S. serrata, a2-M transcript appears to be secreted by haemocytes. a2-M mRNA was also expressed in haemocytes of American horseshoe crab L. polyphemus [24], and kuruma shrimp M. japonicus [25]. The corresponding organ in mammals, the liver, is the site of synthesis of a2-M in mammals [49]. Ascidian Ciona intestinalis a2-M was expressed in circulating blood cells, hepatopancreas and gut; sites often associated with host defence [48]. The expression of a2-M gene was not detected in the hepatopancreas of kuruma shrimp M. japonicus and horseshoe crab L. polyphemus, and was not detected in mud crab S. serrata in the present study either. In crayfish, Pacifastacus leniusculus, a2-M was found to be synthesised in haemocytes but not in hepatopancreas [50]. These results suggest that, haemocyte is the main site for the synthesis of a2-M in invertebrates. The fact that mud crab S. serrata a2-M in haemocytes coupled with its sequence characteristics indicates that the encoded protein probably assists in host defence by entrapping and inhibiting proteases from micro-organisms a2--M expression in haemocytes of kuruma shrimp M. japonicus was significantly induced by peptidoglucan [25]. In the present study, mud crab S. serrata a2-M expression in haemocytes increased significantly after 48 h LPS injection. Similarly, Gunnarsson et al. [51] reported that incubation of peripheral blood mononuclear cell populations with lipopolysaccharide induced a2-M mRNA expression. In conclusion, a 5491 bp a2-M cDNA cloned from the haemocytes of S. serrata indicates that a2-M is synthesised in haemocytes. It encodes 1662 amino acids (a mature protein of 1662 amino acids) with 22 residues of signal peptide. The calculated molecular mass of the S. serrata a2-M mature proteins is 184.2 kDa with an estimated pI of 8.14. 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