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Molecular characterization and expression analysis of a complement component 3 in the sea cucumber (Apostichopus japonicus)
Fish & Shellfish Immunology 31 (2011) 540e547
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Fish & Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi
Molecular characterization and expression analysis of a complement component 3 in the sea cucumber (Apostichopus japonicus) Zunchun Zhou*, Dapeng Sun, Aifu Yang, Ying Dong, Zhong Chen, Xiaoyu Wang, Xiaoyan Guan, Bei Jiang, Bai Wang Liaoning Key Lab of Marine Fishery Molecular Biology, Liaoning Ocean and Fisheries Science Research Institute, Dalian, Liaoning 116023, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 April 2011 Received in revised form 22 June 2011 Accepted 22 June 2011 Available online 4 July 2011
The complement system has been discovered in invertebrates and vertebrates, and plays a crucial role in the innate defense against common pathogens. As a central component in the complement system, complement component 3 (C3) is an intermediary between innate and adaptive immune system. In this study, a new isoform of C3 in the sea cucumber Apostichopus japonicus, termed AjC3-2 was identified. Its open reading frame (ORF) is 5085 bp and encodes for 1695 amino acids with a putative signal peptide of 20 amino acid residues. The mature protein molecular weight of AjC3-2 was 187.72 kDa. It has a conserved thioester site and a linker R689RRR692 where AjC3-2 is splitted into b and a chain during posttranslational modification. The expression patterns of two distinct sea cucumber C3 genes, AjC3-2 and AjC3, were similar. During the different development stages from unfertilized egg to juvenile of the sea cucumber, the highest expression levels of AjC3-2 and AjC3 genes were both found in late auricularia. In the adult, the highest expression of these two genes was observed in the coelomocytes and followed by the body wall. AjC3-2 and AjC3 genes expression increased significantly at 6 h after the LPS challenge. These results indicated that these two C3 genes play a pivotal role in immune responses to the bacterial infection in sea cucumber. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Sea cucumber Apostichopus japonicus Complement Component C3 AjC3
1. Introduction The complement system, as a highly sophisticated defense system acting in the innate immunity of vertebrates, is composed of over 30 distinct humoral and cell surface proteins, and emerges as an essential link between innate and adaptive immunity [1]. In vertebrate, the complement system can be activated through one or more distinct yet overlapping pathways: the classical, alternative and lectin pathways [2]. The classical pathway is induced by antigeneantibody interactions, whereas the other two pathways function in innate immune system. The alternative pathway is initiated by the covalent binding of the complement component 3, C3, to the hydroxyl or amine groups on the surface molecules of pathogen. The lectin pathway is triggered by carbohydrate recognition through pattern-recognition receptors, such as mannosebinding lectin (MBL). Once the complement system is activated, a chain of reactions that involve proteolysis and assembly occur, and then the three activation pathways converge at formation of C3 convertases and cleavage of C3. Afterward, the lytic pathway is * Corresponding author. Tel.: þ86 411 84691884; fax: þ86 411 84671027. E-mail address: [email protected] (Z. Zhou). 1050-4648/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2011.06.023
activated, during which the membrane-attack complex (MAC) is formed. Thus it can be seen that C3 plays a central role in the three complement activation pathways [3]. Besides the function of cell lysis, C3 also has other functions in higher vertebrate. C4b2a in the classical and lectin pathways and C3bBb in the alternative pathway, known as two different forms of C3 convertase, catalyze the proteolytic cleavage of C3 into C3a and C3b. C3a is an anaphylatoxin, which induces the complement mediated inflammation. C3b, in contrast, is an opsonin, which enhance the phagocytosis of phagocytes [4]. During the past few years, the homologs of complement C3 have been identified from higher vertebrates to lower protostomes including human, fish, amphioxus, sea squirt, sea urchin, horseshoe crab, coral, and sea anemone [4e13]. Therefore, the study on the evolutionary history of C3 would contribute to further understanding the evolution of innate immune system in both invertebrates and vertebrates. Early studies indicated that both of the higher vertebrates and deuterostome invertebrates such as sea squirts and sea urchins have the C3 subfamily genes and the a2M subfamily genes, while the genomes of the model protostomes, Drosophila melanogaster and Caenorhabditis elegans [14,15], have only the a2M subfamily genes. These findings might hint that
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complement system is a unique property of deuterostomes, and the C3 subfamily derived from the a2M subfamily by gene duplication in the deuterostome after its divergence from the protostomes [16]. However, the recent discovery of C3 in the two kinds of sea anemone (Haliplanella lineate and Nematostella vectensis), horseshoe crab and clam indicated that the origin of C3 is more ancient than that was previously thought, and gene duplication and subsequent functional differentiation among C3 and noncomplement thioester-containing proteins (TEPs), such as a2M and CD109 were very ancient events predating the divergence of the cnidaria and bilateria [13,12]. Sea cucumber (Apostichopus japonicus) belongs to echinoderm and is naturally distributed in the coasts of Bohai Sea and Yellow Sea in China. It has been an economically important aquaculture species, while disease problem is one of the major hurdles for its developing aquaculture industry. As a kind of echinoderm, sea cucumber only endowed with innate immune system for fighting pathogens. We found two isoforms of C3 genes exist in sea cucumber (A. japonicus) by the expressed sequence tags (ESTs) analysis [17]. Here we report the isolation, characterization and expression analysis of one isoform of C3 genes, AjC3-2, from the sea cucumber (A. japonicus). Another isoform of C3 genes in A. japonicus, AjC3, has been cloned by other researchers. To compare the difference of expression level, tissue distribution and expression response to pathogen infection between the two C3 isoforms, the expression patterns of AjC3 gene were also done in this study. Our results will be useful not only for the research of complement immune evolution, but also for elaborating the role of C3 genes in the immune response of sea cucumber against bacterial infection. 2. Materials and methods 2.1. Cloning of the full-length cDNA of AjC3-2 gene BLAST searches were used to identify partial cDNAs for AjC3-2 gene using sea urchin Strongylocentrotus purpuratus C3 as query against the sea cucumber A. japonicus ESTs from our previous sequencing efforts [17]. One EST, which was homologous to the C3 of sea urchin (S. purpuratus) and contained a full-length 30 untranslated region (UTR), was used to clone the full-length cDNA of C3 gene in the sea cucumber. Total RNA was transcribed with the SMART PCR cDNA Synthesis Kit (Clontech), following the manufacturer instruction to obtain 50 ready cDNA. To obtain the 50 region of AjC3-2, 50 rapid amplification of cDNA ends (RACE)-PCR was performed using SMART RACE cDNA Amplification Kit from Clontech following the manufacturer instructions. The gene-specific primers were listed in Table 1. 2.2. Molecular characterization and phylogenetic analysis For sequence analysis, the AjC3-2 amino acid sequences were either identified by simple key word searches, or with BLASTP searches using sea urchin C3 amino acid sequences at NCBI (http://
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www.ncbi.nlm.nih.gov/BLAST). Protein sequences retrieved from public database were used for open reading frame (ORF) and domain searches, alignment, and phylogenetic reconstruction. ORF was predicted using Open Reading Frame Finder (http://www.ncbi. nlm.nih.gov/gorf/gorf.html). The protein domains were predicted by scanning NCBI Conserved Domains Database (http://www.ncbi. nlm.nih.gov/structure/cdd/wrpsb.cg). Translation and protein analysis were performed using ExPaSy tools (http://us.ex pasy.org/ tools). Multiple alignment of the AjC3-2 was performed using the program ClustalX version 2.0 [18]. Phylogenetic tree was constructed using the neighbor-joining method based on the deduced full-length amino acid sequences within the Molecular Evolutionary Genetics Analysis (MEGA 4.0) package [19]. Data were analyzed using Poisson correction, and gaps were removed by complete deletion. The topological stability of the tree was evaluated by 10,000 bootstrap replications. 2.3. Embryo and tissue sampling and LPS challenge Mature sea cucumbers were collected from Guanglu Island (Dalian, China) in early July. Animals were artificially spawned by sea water (20e21 C) stimulation. Developments of A. japonicus, from fertilized eggs to 1-mm long juvenile, were cultured at a temperature of 20e21 C, a salinity of 32&, a pH of 7.8, and were examined using a light microscope (OLYMPUX JM, Japan). To investigate the expression of AjC3-2 and AjC3 genes at different development stages, samples from every development stages, including unfertilized eggs, fertilized eggs, cellulous stages, blastula prior to hatching, gastrula, early auricularia, auricularia, late auricularia, doliolaria, pentactula and 1-mm long juvenile, were collected by sieving using 60 mm filter, pelleted by centrifugation (Labnet Spectrafuge, USA) and then stored in 1.5 mL microcentrifuge tubes. All samples were frozen immediately with liquid nitrogen and then stored at 80 C prior to RNA isolation. To determine tissue distribution of AjC3-2 and AjC3 genes expression in sea cucumber, the intestine, respiratory tree, coelomocytes and body wall tissues were separated from 15 healthy individuals (3 pools of 5 individuals each) (average body weight 10.2 g). These tissues from different individuals were mixed respectively, and frozen immediately with liquid nitrogen and then stored at 80 C. In order to investigate the expression patterns of AjC3-2 and AjC3 genes in responses to pathogen infection, 500 ml LPS (1 g/L) was injected into the healthy sea cucumbers (average body weight 10.2 g). Un-injection sea cucumbers were treated as the blank group (0 h). The sea cucumbers injected with 500 ml sterile sea water (SSW) were treated as the control group [20]. Coelomocytes were collected at 3 h, 6 h, 12 h, 24 h, 72 h and 96 h after injection respectively according to the methods reported by SantiagoCardona et al. [21]. For each sampling time, the coelomocytes taken from 15 different individuals (3 pools of 5 individuals each) were mixed in three 1.5 mL microcentrifuge tubes, and thrown immediately into liquid nitrogen and then stored at 80 C. 2.4. Quantitative Real-time PCR
Table 1 PCR primers used in this study. Primer
Sequence (50 30 )
Sequence Information
AjC3-2-R1: AjC3-2-R2: AjC3-2-F: AjC3-2-R: AjC3-F: AjC3-R: Cytb-F: Cytb-R:
50 -TATAGAGCCTTCGCGTTGTAT-30 50 -TGAAAC GTTCTTTCGATTCCC-30 50 -CTCTCGTGAGTTCTGGC TCAG-30 50 -GCAGCCACTGTTACCATCGCG GA-30 50 -GCGTTGTTTCGTTCAACAAGGGGA-30 50 -GCCATTCACTGGAGGTGTGGCA-30 50 -TGAGCCGCAACAGTAATC-30 50 -AAGGGAAAAGGAAGTGAAAG-30
For AjC3-2 50 -RACE For AjC3-2 Real-time PCR For AjC3 Real-time PCR For Real-time PCR
Total RNA was isolated using the UNIQ-10 Column Total RNA Isolation Kit (Sangon, Shanghai, China) according to the manufacturer’s instructions. The quality and quantity of extracted total RNA were measured using the NanoPhotometer (ImplenGmbH, Munich, Germany) and agarose gel electrophoresis. First strand cDNA synthesis was performed in a volume of 20 ml with 900 ng total RNA, 25 pmol Oligo dT Primer, 50 pmol random 6 mers primer, 1 PrimeScriptÔ buffer, 0.5 ml PrimeScriptÔ RT enzyme Mix I (PrimeScriptÔ RT reagent Kit, TaKaRa, China). Reactions were
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incubated at 37 C for 15 min, and then at 85 C for 5 s to deactivate the enzyme. Equal amounts of cDNA templates were used in Quantitative Real-time PCR (qRT-PCR). qRT-PCR was performed in triplicate using the Mx3000pÔ detection system (Applied Stratagene, USA) in 20 ml reactions containing the following components: 10 ml of 2 SYBR Green Master mix (SYBR PrimeScriptÔ RT-PCR Kit II, TaKaRa), 0.4 ml of ROX Reference Dye II, 1 ml of cDNA template, and 0.4 mM of each primer. Primers were listed in Table 1. The qRT-PCR profile was as following: One cycle of 95 C for 30 s, followed by 40 cycles of 95 C for 10 s, 56 C for 25 s and 72 C for 25 s. Melting curve analysis of amplification products was performed at the end of each PCR reaction to confirm that only one PCR product was amplified and detected. In addition, the amplicons were checked by agarose gel with a 100 bp ladder in order to confirm the correct amplicon sizes. The cytochrome b (Cytb) gene was used as the reference gene (Table 1) [22]. The relative mRNA levels of the target genes were calculated as 2DDCt [23]. Statistical calculations were performed using SPSS (version 13.0) software. Significant difference was indicated by one-way ANOVA (P < 0.05) analysis. 3. Result 3.1. Isolation of full-length cDNA of AjC3-2 gene Based on the 641 bp EST sequence with 30 -UTR obtained by cDNA libraries sequencing, the 50 -RACE reactions were performed toward 50 end of the EST. Here, the C3 sequence in this study was named AjC3-2 (GenBank accession no. HQ874435), while the other one previously submitted to GenBank by other researchers was termed AjC3 (GenBank accession no. HQ214156). The full-length cDNA sequence of AjC3-2 gene is 5748 bp barring the poly (A) tail. The sequence comprises a 26 bp 50 -terminal untranslated region (UTR), a 637 bp 30 -UTR with a polyadenylation signal AATAAA near the poly (A) tail and a 5085 bp open reading frame (ORF) encoding 1695 amino acid residues. The molecular weight of the unglycosylated mature protein of AjC3-2 is 187.72 kDa, and the estimated isoelectric point of the proteins is 5.12. 3.2. Homology analysis and characterization of AjC3-2 Sequence analysis indicated that the amino acid sequence of AjC3-2 show 43%, 35%, 35%, 31% identities with sea cucumber AjC3, sea urchin (S. purpuratus) C3, amphioxus (Branchiostoma belcheri) C3 and human C3. The result of domain prediction indicates that AjC3-2 as well as AjC3 and other C3s have not only a2M domain which exists in a2 macroglobulin, but also complement specific domains, such as complement C3/C4/C5 domain and C345C domain [12,24,25]. The protein sequence of AjC3-2 was aligned with vertebrate and invertebrate C3 sequences (Fig. 1). Like other C3s, AjC3-2 has a linker RRRR between b and a chain which is seated at 689e692 amino acids. At this site, C3 is cleaved into b and a chain by catalysis of a furin-like enzyme during posttranslational modification [26]. The molecular weight of the deduced b and a chain are 74.80 kDa and 112.31 kDa respectively. In vertebrates, C3 convertase cleaves C3 into two parts, the larger one called C3b and the smaller one called C3a. C3a comes from about 70 amino acids closed to the N-terminal of a chain, and is regarded as an anaphylatoxin together with C4a and C5a [27]. In the homologous region of AjC3-2, four out of six cysteines forming three intrachain disulfide bridges in the disulfide-linked “core” region existing in mammal anaphylatoxins. Furthermore, G714/701and F753/743 of AjC3-2 is also in accordance with mammal anaphylatoxins in which
these two amino acids are important for the folding of the polypeptide chain. It is now known that the linear sequence at the C-terminus of C3a and C4a of human contains the structural information necessary to mediate biologic responses associated with the C3a/C4a receptor and other factors [28], and the last three amino acids LXR of this linear sequence are the C3 convertase cleavage site which is generally conserved in most of vertebrate C3a or C4a [27]. Although there is not well conservation in the corresponding sites of AjC3-2, RXR sequences of AjC3-2 and AjC3 are found near the cleavage site, similar to that observed in coral, horseshoe crab and sea anemone [10,12,13]. C3b is composed of the whole b chain and a0 chain, a chain from which C3a has been removed. A typical thioester site (GCGEQ) is located in the a0 chain of AjC3-2. In human, a hydrophobic pocket protects this thioester from reacting with water or other small nucleophiles, which is formed by the space approach of F1047, M1378, Y1425 and Y1460 [24]. These residues are conserved in AjC3-2 and AjC3, except that F1047 is replaced by another aromatic residue W1098 in AjC3. H1104 and E1106 play an important role in the specific reaction between C3b and hydroxyl groups, AjC3-2 and AjC3 also have the same residues at the corresponding sites. Among all of ten pairs of Cyses that forming disulfide bonds in human C3b, eight pairs are identical in AjC3-2 and AjC3, including one uniting the b and a0 chain, one located in b chain and six located in a0 chain. More than 40 residues in N-terminal region of the a0 chain are considered to be the most important region of C3 because it encompasses the binding sites for CR1, CR2, CR3, factor B and factor H in human [28]. This corresponding region is lower homologous between the shown C3s, and the similar status can be seen in another factor H and CR2 binding sites, but properdin binding site is higher homologous. There are three factor I cleavage sites in human C3b, but none matches perfectly to AjC3-2 and AjC3. However, the sequence of R979E980 in the AjC3-2 is similar to the factor I site 1 (Fig. 1). 3.3. Phylogenetic analysis Phylogenetic analysis was conducted based on the amino acid sequences retrieved from the GenBank. Two different clades were formed (Fig. 2). One is composed of invertebrate C3 proteins including sea cucumber, sea urchin, sea anemone, and so on, and the other one is composed of vertebrate C3, C4 and C5 proteins. AjC3s formed a cluster with the SpC3 of sea urchin and this indicated that the duplication event from which AjC3-2 and AjC3 genes originated was occurred in the echinoderm lineage after the separation of the sea cucumber and sea urchin ancestor. 3.4. AjC3-2 and AjC3 genes expression profiles during development qRT-PCR analysis was used to determine the expression patterns of AjC3-2 and AjC3 mRNA at eleven developmental stages of sea cucumber A. japonicus. The result revealed that the expression patterns of these two C3 genes were highly similar, with lower expression during the early stages of development. The gene expression levels kept increasing at the three auricularia stages, and reached the peak in late auricularia, then descended down at the last three stages, doliolaria, pentactula and juvenile (Fig. 3). 3.5. AjC3-2 and AjC3 genes expression profiles in different tissues The expression levels of AjC3-2 and AjC3 genes of different tissues were also tested in intestine, respiratory tree, coelomocytes and body wall. In healthy sea cucumbers, the AjC3-2 and AjC3 genes were both expressed at a higher level in the coelomocytes, followed by body wall. Comparing with the lowest expression level
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Fig. 1. Alignment between AjC3-2 and other complement components. The abbreviations of species name and accession numbers for sequences are explained in Fig. 4. Several areas of functional significance are labeled. Boxed areas are determined according to Fig. 4 in Lambris et al. (1993) and correspond to functional sites in human C3 or C4 (Sahu and Lambris, 2001).
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Fig. 1. (continued).
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Fig. 2. Phylogenetic tree. The tree was constructed using ClustalW and MEGA (4.0). The numbers of branches are bootstrap values for 1000 replicates. The GenBank accession numbers for the sequences are as follows: AjC3-2/C3: sea cucumber (A. japonicus), HQ874435/HQ214156; SpC3: sea urchin (Strongylocentrotus purpuratus), AAC14396; HlC3-1/C3-2: sea anemone (Haliplanella lineate), AB481383/AB481384; NvC3-1/C3-2: sea anemone (Nematostella vectensis), AB450039/AB450040; BbC3: amphioxus (Branchiostoma belcheri), BAB47146; CrC3: horseshoe crab (Carcinoscorpius rotundicauda), AF517564; VdC3: clam (Venerupis decussates), FJ392025; CiC3-1/C32:sea squirt(Ciona intestinalis), NP_001027684/CAC85958; OlC3-1/C3-2/C4: medaka (Oryzias latipes), NM_001105082/NM_001105083/NM_001104697; DrC3b: zebra fish (Danio rerio), NM_131243; HsC3/C4A/C5: human, AAA85332/AAB67980.1/NM_001735; MmC3/C4b/C5: mouse (Mus musculus), NM_009778/NM_009780/M35525; LjC3:lamprey(Lethenteron japonicum), BAA00983; CcC5-1: carp (Cyprinus carpio), AB084635.
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Fig. 3. Relative mRNA levels of AjC3-2 and AjC3 genes at different development stages of A. japonicus by using qRT-PCR analysis. 1: Unfertilized egg; 2: Fertilized egg; 3: Cellulous stage; 4: Blastula; 5: Gastrula; 6: Early auricularia; 7: Auricularia; 8: Late auricularia; 9: Doliolaria; 10: Pentactula; 11: Juvenile. Each symbol and vertical bar represents the mean S.D (n ¼ 3). Different lowercase letters above the bars indicate significant differences (P < 0.05) at different stages by t-test. Expression levels at all different stages are presented relative to that in the unfertilized egg (1).
sequence RXXR, C3 convertase cleavage site L/RXR, four disulfide bridges in C3a and eight disulfide bridges in C3b (Fig. 1). The identifications of these conserved structure suggest that C3 proteins in sea cucumber possess the basic biochemical functions throughout the evolution, and SpC3 sharing a higher homology
in the intestine, the expression in the coelomocytes, body wall and respiratory tree reached about 5, 4.5 and 2 fold for AjC3-2 and 2, 3 and 2.5 fold for AjC3, respectively (Fig. 4). 3.6. AjC3-2 and AjC3 genes expression profiles after LPS challenge In the LPS challenge experiment, we measured the AjC3-2 and AjC3 genes expression levels of the coelomocytes at the different sampling times. At 3 h after injection, there was no significant expression difference between control and challenge samples. However, it is apparent that these two C3 genes were significantly induced at 6 h after injection (Fig. 5). AjC3-2 gene expression was up-regulated about 2 fold at 6 h, 4 fold at 12 h, 29 fold at 72 h and 5 fold at 96 h after injection (P < 0.05). AjC3 gene expression was upregulated about 3 fold at 6 h, 4.6 fold at 12 h and 2.5 fold at 24 h after injection (P < 0.05). 4. Discussion In the present study, we identified and characterized the fulllength cDNA of one C3 like gene, AjC3-2 of sea cucumber. The expression profiles of two isoforms of C3 genes, AjC3-2 and AjC3, in normal tissues and after LPS challenge were also analyzed. AjC3-2 shares many conserved structural characteristics with other C3 proteins, such as the thioester site GCGEQ, the b-a linker
Fig. 4. Distribution of AjC3-2 and AjC3 mRNA in the different tissues. 1: Intestine; 2: Respiratory; 3: Coelomocytes; 4: Body wall. Each symbol and vertical bar represents the mean S.D (n ¼ 3). Different lowercase letters above the bars indicate significant differences (P < 0.05) in different tissues by t-test. Expression levels in all tissues are presented relative to that in the intestine tissue (1).
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Fig. 5. Effects of LPS challenge on transcriptional levels of AjC3-2 and AjC3 genes in the coelomocytes after injection. Each symbol and vertical bar represents the mean S.D (n ¼ 3). Different lowercase letters above the bars indicate significant differences (P < 0.05) at different time points of the same group by t-test; asterisks above the bars show significant differences (P < 0.05) between two groups of the same time point by t-test.
with AjC3-2 and AjC3 functions as an opsonin have been confirmed in another species of echinoderm, the sea urchin S. purpuratus as well [29]. In human, a chain structure of C3 contains a lot of essential structural features for interacting with specific cellular receptors, natural factors and serine proteases as shown in Fig. 1. The binding sites of CR1, CR2, C3aR, factor B and factor H or the cleavage sites of factor I with cofactor, are not conserved well in sea cucumber C3 proteins. This result suggested that sea cucumber C3 proteins may have different folding pattern, and maybe not interact with many other proteins as known in human C3. For example, CR2 is located on the cell membrane of B lymphocyte in higher vertebrate, so the CR2-like proteins and the binding sites of CR2 should not exist in the innate immune system of sea cucumber. AjC3-2 possesses the a-g chain linker RXXR which was previously found in C4s of vertebrate, but it is clearly located outside vertebrate C3/C4/C5 proteins by the phylogenetic analysis (Fig. 2). Therefore, AjC3-2 should not be the analog of C4 proteins, and this phenomenon can also be seen in other animal C3 proteins containing the homologous sequence of RXXR such as lamprey [30], amphioxus [6], horseshoe crab [10], clam [25], sea anemone [12,13] and coral [11]. In coral and sea anemone, the linker is surrounded by a similar K/R rich region. Thus, researchers presumed that this region is a characteristic for ancestor of C3, the vertebrate C3 and C5 apparently lost the entire region, whereas vertebrate C4 and some species C3 retained a part of it and used it as the a-g processing signal [11e13]. Here we described one C3 gene of sea cucumber, AjC3-2, and we also found part sequence of another AjC3 gene from the ESTs obtained by our cDNA libraries sequencing [17]. A few months ago
we found this AjC3 cDNA sequence in GenBank submitted by other researchers. So we can see two C3 genes of A. japonicus have been cloned. To date, sea anemone [12,13], ascidian [7] and numerous teleost fish [31e33] have been found to own multiple forms of C3 genes. The phenomenon was interpreted to be caused by the tandem gene duplication, and occurs frequently during evolution [4]. The studies on the trout have demonstrated that the different isoforms of C3s display different binding efficiencies to several complement activating surfaces, thereby providing a means of expanding the immune defense repertoire [34]. In this study, the expression patterns of AjC3-2 and AjC3 genes at different development stages were investigated. The expression patterns of these two C3 genes were similar. The expression difference was not significant between the unfertilized eggs and fertilized eggs. It suggested that the lower expression level in early embryos and gastrulas is maternally derived. The auricularia stages of development, with the guts were formed, the larvae begin to eat, maybe some immune related genes like C3 have higher expression is necessary to protect the larvae against microbial invasion. The researches on the mesenchyme cells of sea urchin embryos demonstrated that they have the capacities for anti bacteria and phagocytosing yeasts [35,36]. Based on these studies, mesenchyme cells are perhaps the best candidates for mediating the immune responses in the developing echinoderm embryos [37]. During embryogenesis of sea cucumber, primary mesenchyme cells first appear in the late gastrula which precedes early auricularia. Our research results revealed that the increase of C3 genes expression was corresponded with the appearance of primary mesenchyme cells. C3 genes may express highly in the mesenchyme cells of sea cucumber embryos. Coelomocytes have long been considered to be mediators of the immune response in echinoderm, so it is not surprising that AjC3-2 and AjC3 genes have intense expression in the coelomocytes like SpC3 of sea urchin [9]. However, it is interesting that AjC3-2 and AjC3 genes also expressed highly in the body wall besides the coelomocytes. The previous experiments showed that in the sea urchin injected with LPS, the appearance of SpC3 protein in coelomocytes was significantly delayed when compared to SpC3 protein in coelomic fluid [38]. This suggested that there maybe other sites where SpC3 protein was produced in sea urchins besides the coelomocytes in the coelomic fluid. In mammals, although the main biosynthesis and secretion site of C3 is the liver, several other type cells appear to produce and secrete C3, including fibroblast cells and epithelial cells [39,40]. Both these kinds of cells are involved in the developed body wall of sea cucumber, and according to the study in starfish, the coelomic epithelial cell was identified as one of the sources of new coelomocytes, which proliferates upon LPS injection [41]. Lipopolysaccharide (LPS) is the most used immune-activating substance. Lots of immune-related genes respond to the LPS challenge. Clow et al. found that the expression of Sp064 mRNA in coelomocytes was elevated after challenging the sea urchins with LPS [38], and the results of other experiments have demonstrated that SpC3 and AsC3 of the ascidian Halocynthia roretzi are the humoral opsonins in coelomic fluid [8,29]. Furthermore, it is reported that LPS can effectively increase phagocytes and phagocytic activity in the coelomocytes of the sea cucumber Holothuria glaberrima [42]. As a result, it is possible that the C3 proteins of sea cucumber could stimulate the phagocytic activity of phagocytes as an opsonin. In this study, the expression of AjC3-2 and AjC3 genes was also up-regulated clearly in the coelomocytes after LPS challenge, and the expression levels of these two genes both reached the peak at 12 h after LPS injection. AjC3-2 gene kept higher expression levels from 12 h to 96 h after LPS injection, while AjC3 expression descended down quickly. Whether they actually display
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any functional differences remains unknown. We will continue the research work related to the functional analysis of these two C3 genes in A. japonicus. Acknowledgments This work was supported by grants from National Nature Science Foundation of China (30972272) and Public Science and Technology Research Funds Projects of Ocean (200705007). References [1] Dunkelberger JR, Song WC. Complement and its role in innate and adaptive immune responses. Cell Res 2010;20:34e50. [2] Volanakis JE. Overview of the complement system. Immunol Med 1998;20: 9e32. [3] Fujita T. Evolution of the lectin-complement pathway and its role in innate immunity. Nat Rev Immunol 2002;2:346e53. [4] Boshra H, Li J, Sunyer JO. Recent advances on the complement system of teleost fish. Fish Shellfish Immunol 2006;20:239e62. [5] Bruijn MH, Fey GH. Human complement component C3: cDNA coding sequence and derived primary structure. Proc Natl Acad Sci U S A 1985;82:708e12. [6] Suzuki MM, Satoh N, Nonaka M. C6-like and C3-like molecules from the cephalochordate, amphioxus, suggest a cytolytic complement system in invertebrates. J Mol Evol 2002;54:671e9. [7] Marino R, Kimura Y, Santis RD, Lambris JD, Pinto MR. Complement in urochordates: cloning and characterization of two C3-like genes in the ascidian Ciona intestinalis. Immunogenetics 2002;53:1055e64. [8] Nonaka M, Azumi K, Ji X, Namikawa-Yamada C, Sasaki M, Saiga H, et al. Opsonic complement component C3 in the solitary ascidian, Halocynthia roretzi. J Immunol 1999;162:387e91. [9] Al-Sharif WZ, Sunyer JO, Lambris JD, Smith LC. Sea urchin coelomocytes specifically express a homologue of the complement component C3. J Immunol 1998;160:2983e97. [10] Zhu Y, Thangamani S, Ho B, Ding JL. The ancient origin of the complement system. EMBO J 2005;24:382e94. [11] Dishaw LJ, Smith SL, Bigger CH. Characterization of a C3-like cDNA in a coral: phylogenetic implications. Immunogenetics 2005;57:535e48. [12] Fujito NT, Sugimoto S, Nonaka M. Evolution of thioester-containing proteins revealed by cloning and characterization of their genes from a cnidarian sea anemone, Haliplanella lineate. Dev Comp Immunol 2010;34:775e84. [13] Kimura A, Sakaguchi E, Nonaka M. Multi-component complement system of Cnidaria: C3, Bf, and MASP genes expressed in the endodermal tissues of a sea anemone, Nematostella vectensis. Immunobiology 2009;214:165e78. [14] Adams MD, Celniker SE, Holt RA, Evans CA, Gocayne JD, Amanatides PG. The genome sequence of Drosophila melanogaster. Science 2000;287:2185e95. [15] C. elegans Sequencing Consortium. Genome sequence of the nematode C. elegans: a platform for investigating biology. Science 1998;282:2012e8. [16] Nonaka M. Evolution of the complement system. Curr Opin Immunol 2001; 13:69e73. [17] Yang AF, Zhou ZC, He CB, Hu JJ, Chen Z, Gao XG, et al. Analysis of expressed sequence tags from body wall, intestine and respiratory tree of sea cucumber (Apostichopus japonicus). Aquaculture 2009;296:193e9. [18] Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, et al. Clustal W and Clustal X version 2.0. Bioinformatics 2007;23:2947e8. [19] Tamura K, Dudley J, Nei M, Kumar S. MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 2007;24:1596e9. [20] Ramírez-Gómez F, Ortíz-Pineda PA, Rivera-Cardona G, García-Arrarás JE. LPSinduced genes in intestinal tissue of the sea cucumber Holothuria glaberrima. PLoS ONE 2009;4:61e78.
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[21] Santiago-Cardona PG, Berrios CA, Ramirez F, Garcia-Arraras JE. Lipopolysaccharides induce intestinal serum amyloid A expression in the sea cucumber Holothuria glaberrima. Dev Comp Immunol 2003;27:105e10. [22] Yang AF, Zhou ZC, Dong Y, Jiang B, Wang XY, Chen Z, et al. Expression of immune-related genes in embryos and larvae of sea cucumber Apostichopus japonicus. Fish Shellfish Immunol 2010;29:839e45. [23] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using realtime quantitative PCR and the 2DDCt method. Methods 2001;25:402e8. [24] Janssen BJ, Huizinga EG, Raaijmakers HC, Roos A, Daha MR, Nilsson-Ekdahl K, et al. Structures of complement component C3 provide insights into the function and evolution of immunity. Nature 2005;437:505e11. [25] Prado-Alvarez M, Rotllant J, Gestal C, Novoa B, Figueras A. Characterization of a C3 and a factor B-like in the carpet-shell clam, Ruditapes decussates. Fish Shellfish Immunol 2009;26:305e15. [26] Misumi Y, Oda K, Fujiwara T, Takami N, Tashiro K, Ikehara Y. Functional expression of furin demonstrating its intracellular localization and endoprotease activity for processing of proalbumin and complement pro-C3. J Biol Chem 1991;266:16954e9. [27] Hugli TE. Structure and function of the anaphylatoxins. Semin Immunopathol 1984;7:193e219. [28] Sahu A, Lambris JD. Structure and biology of complement protein C3, a connecting link between innate and acquired immunity. Immunol Rev 2001;180: 35e48. [29] Clow LA, Raftos DA, Gross PS, Smith LC. The sea urchin complement homologue, SpC3, functions as an opsonin. J Exp Biol 2004;207:2147e55. [30] Nonaka M, Takahashi M. Complete complementary DNA sequence of the third component of complement of lamprey, Implication for the evolution of thioester containing proteins. J Immunol 1992;148:3290e5. [31] Lambris JD, Lao Z, Pang J, Alsenzt J. Third component of trout complement, cDNA cloning and conservation of functional sites. J Immunol 1993;151: 6123e34. [32] Nakao M, Mutsuro J, Obo R, Fujiki K, Nonaka M, Yano T. Molecular cloning and protein analysis of divergent forms of the complement component C3 from a bony fish, the common carp (Cyprinus carpio): presence of variants lacking the catalytic histidine. Eur J Immunol 2000;30:858e66. [33] Kuroda N, Naruse K, Shima A, Nonaka M, Sasaki M. Molecular cloning and linkage analysis of complement C3 and C4 genes of the Japanese medaka fish. Immunogenetics 2000;51:117e28. [34] Sunyer JO, Zarkadis IK, Lambris JD. Complement diversity: a mechanism for generating immune diversity? Immunol Today 1998;19:519e23. [35] Service M, Wardlaw AC. Echinochrome-A as a bactericidal substance in the coelomic fluid of Echinus esculentus (L). Comp Biochem Physiol 1984;79B: 161e5. [36] Silva JR. The onset of phagocytosis and identity in the embryo of Lytechinus variegatus. Dev Comp Immunol 2000;24:733e9. [37] Shah M, Brown KM, Smith LC. The gene encoding the sea urchin complement protein, SpC3, is expressed in embryos and can be upregulated by bacteria. Dev Comp Immunol 2003;27:529e38. [38] Clow LA, Gross PS, Shih C-S, Smith LC. Expression of Sp C3, the sea urchin complement component, in response to lipopolysaccharide. Immunogenetics 2000;51:1021e33. [39] Lappin DF, Guc D, Hill A, McShane T, Whaley K. Effect of interferon-gamma on complement gene expression in different cell types. Biochem J 1992;281: 437e42. [40] Strunk RC, Fleishr JA, Katz Y, Cole FS. Developmentally regulated effects of lipopolysaccharide on bio synthesis of the third component of complement and factor B in human fibroblasts and monocytes. Immunology 1994;82: 314e20. [41] Holm K, Dupont S, Skold H, Stenius A, Thorndyke M, Hernroth B. Induced cell proliferation in putative haematopoietic tissues of the sea star, Asterias rubens (L.). J Exp Biol 2008;211:2551e8. [42] Francisco RG, Francisco AR, Lumen MC, Jose EG. Changes in holothurian coelomocyte populations following immune stimulation with different molecular patterns. Fish Shellfish Immunol 2010;29:175e85.
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Fish & Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi
Molecular characterization and expression analysis of a complement component 3 in the sea cucumber (Apostichopus japonicus) Zunchun Zhou*, Dapeng Sun, Aifu Yang, Ying Dong, Zhong Chen, Xiaoyu Wang, Xiaoyan Guan, Bei Jiang, Bai Wang Liaoning Key Lab of Marine Fishery Molecular Biology, Liaoning Ocean and Fisheries Science Research Institute, Dalian, Liaoning 116023, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 April 2011 Received in revised form 22 June 2011 Accepted 22 June 2011 Available online 4 July 2011
The complement system has been discovered in invertebrates and vertebrates, and plays a crucial role in the innate defense against common pathogens. As a central component in the complement system, complement component 3 (C3) is an intermediary between innate and adaptive immune system. In this study, a new isoform of C3 in the sea cucumber Apostichopus japonicus, termed AjC3-2 was identified. Its open reading frame (ORF) is 5085 bp and encodes for 1695 amino acids with a putative signal peptide of 20 amino acid residues. The mature protein molecular weight of AjC3-2 was 187.72 kDa. It has a conserved thioester site and a linker R689RRR692 where AjC3-2 is splitted into b and a chain during posttranslational modification. The expression patterns of two distinct sea cucumber C3 genes, AjC3-2 and AjC3, were similar. During the different development stages from unfertilized egg to juvenile of the sea cucumber, the highest expression levels of AjC3-2 and AjC3 genes were both found in late auricularia. In the adult, the highest expression of these two genes was observed in the coelomocytes and followed by the body wall. AjC3-2 and AjC3 genes expression increased significantly at 6 h after the LPS challenge. These results indicated that these two C3 genes play a pivotal role in immune responses to the bacterial infection in sea cucumber. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Sea cucumber Apostichopus japonicus Complement Component C3 AjC3
1. Introduction The complement system, as a highly sophisticated defense system acting in the innate immunity of vertebrates, is composed of over 30 distinct humoral and cell surface proteins, and emerges as an essential link between innate and adaptive immunity [1]. In vertebrate, the complement system can be activated through one or more distinct yet overlapping pathways: the classical, alternative and lectin pathways [2]. The classical pathway is induced by antigeneantibody interactions, whereas the other two pathways function in innate immune system. The alternative pathway is initiated by the covalent binding of the complement component 3, C3, to the hydroxyl or amine groups on the surface molecules of pathogen. The lectin pathway is triggered by carbohydrate recognition through pattern-recognition receptors, such as mannosebinding lectin (MBL). Once the complement system is activated, a chain of reactions that involve proteolysis and assembly occur, and then the three activation pathways converge at formation of C3 convertases and cleavage of C3. Afterward, the lytic pathway is * Corresponding author. Tel.: þ86 411 84691884; fax: þ86 411 84671027. E-mail address: [email protected] (Z. Zhou). 1050-4648/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2011.06.023
activated, during which the membrane-attack complex (MAC) is formed. Thus it can be seen that C3 plays a central role in the three complement activation pathways [3]. Besides the function of cell lysis, C3 also has other functions in higher vertebrate. C4b2a in the classical and lectin pathways and C3bBb in the alternative pathway, known as two different forms of C3 convertase, catalyze the proteolytic cleavage of C3 into C3a and C3b. C3a is an anaphylatoxin, which induces the complement mediated inflammation. C3b, in contrast, is an opsonin, which enhance the phagocytosis of phagocytes [4]. During the past few years, the homologs of complement C3 have been identified from higher vertebrates to lower protostomes including human, fish, amphioxus, sea squirt, sea urchin, horseshoe crab, coral, and sea anemone [4e13]. Therefore, the study on the evolutionary history of C3 would contribute to further understanding the evolution of innate immune system in both invertebrates and vertebrates. Early studies indicated that both of the higher vertebrates and deuterostome invertebrates such as sea squirts and sea urchins have the C3 subfamily genes and the a2M subfamily genes, while the genomes of the model protostomes, Drosophila melanogaster and Caenorhabditis elegans [14,15], have only the a2M subfamily genes. These findings might hint that
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complement system is a unique property of deuterostomes, and the C3 subfamily derived from the a2M subfamily by gene duplication in the deuterostome after its divergence from the protostomes [16]. However, the recent discovery of C3 in the two kinds of sea anemone (Haliplanella lineate and Nematostella vectensis), horseshoe crab and clam indicated that the origin of C3 is more ancient than that was previously thought, and gene duplication and subsequent functional differentiation among C3 and noncomplement thioester-containing proteins (TEPs), such as a2M and CD109 were very ancient events predating the divergence of the cnidaria and bilateria [13,12]. Sea cucumber (Apostichopus japonicus) belongs to echinoderm and is naturally distributed in the coasts of Bohai Sea and Yellow Sea in China. It has been an economically important aquaculture species, while disease problem is one of the major hurdles for its developing aquaculture industry. As a kind of echinoderm, sea cucumber only endowed with innate immune system for fighting pathogens. We found two isoforms of C3 genes exist in sea cucumber (A. japonicus) by the expressed sequence tags (ESTs) analysis [17]. Here we report the isolation, characterization and expression analysis of one isoform of C3 genes, AjC3-2, from the sea cucumber (A. japonicus). Another isoform of C3 genes in A. japonicus, AjC3, has been cloned by other researchers. To compare the difference of expression level, tissue distribution and expression response to pathogen infection between the two C3 isoforms, the expression patterns of AjC3 gene were also done in this study. Our results will be useful not only for the research of complement immune evolution, but also for elaborating the role of C3 genes in the immune response of sea cucumber against bacterial infection. 2. Materials and methods 2.1. Cloning of the full-length cDNA of AjC3-2 gene BLAST searches were used to identify partial cDNAs for AjC3-2 gene using sea urchin Strongylocentrotus purpuratus C3 as query against the sea cucumber A. japonicus ESTs from our previous sequencing efforts [17]. One EST, which was homologous to the C3 of sea urchin (S. purpuratus) and contained a full-length 30 untranslated region (UTR), was used to clone the full-length cDNA of C3 gene in the sea cucumber. Total RNA was transcribed with the SMART PCR cDNA Synthesis Kit (Clontech), following the manufacturer instruction to obtain 50 ready cDNA. To obtain the 50 region of AjC3-2, 50 rapid amplification of cDNA ends (RACE)-PCR was performed using SMART RACE cDNA Amplification Kit from Clontech following the manufacturer instructions. The gene-specific primers were listed in Table 1. 2.2. Molecular characterization and phylogenetic analysis For sequence analysis, the AjC3-2 amino acid sequences were either identified by simple key word searches, or with BLASTP searches using sea urchin C3 amino acid sequences at NCBI (http://
541
www.ncbi.nlm.nih.gov/BLAST). Protein sequences retrieved from public database were used for open reading frame (ORF) and domain searches, alignment, and phylogenetic reconstruction. ORF was predicted using Open Reading Frame Finder (http://www.ncbi. nlm.nih.gov/gorf/gorf.html). The protein domains were predicted by scanning NCBI Conserved Domains Database (http://www.ncbi. nlm.nih.gov/structure/cdd/wrpsb.cg). Translation and protein analysis were performed using ExPaSy tools (http://us.ex pasy.org/ tools). Multiple alignment of the AjC3-2 was performed using the program ClustalX version 2.0 [18]. Phylogenetic tree was constructed using the neighbor-joining method based on the deduced full-length amino acid sequences within the Molecular Evolutionary Genetics Analysis (MEGA 4.0) package [19]. Data were analyzed using Poisson correction, and gaps were removed by complete deletion. The topological stability of the tree was evaluated by 10,000 bootstrap replications. 2.3. Embryo and tissue sampling and LPS challenge Mature sea cucumbers were collected from Guanglu Island (Dalian, China) in early July. Animals were artificially spawned by sea water (20e21 C) stimulation. Developments of A. japonicus, from fertilized eggs to 1-mm long juvenile, were cultured at a temperature of 20e21 C, a salinity of 32&, a pH of 7.8, and were examined using a light microscope (OLYMPUX JM, Japan). To investigate the expression of AjC3-2 and AjC3 genes at different development stages, samples from every development stages, including unfertilized eggs, fertilized eggs, cellulous stages, blastula prior to hatching, gastrula, early auricularia, auricularia, late auricularia, doliolaria, pentactula and 1-mm long juvenile, were collected by sieving using 60 mm filter, pelleted by centrifugation (Labnet Spectrafuge, USA) and then stored in 1.5 mL microcentrifuge tubes. All samples were frozen immediately with liquid nitrogen and then stored at 80 C prior to RNA isolation. To determine tissue distribution of AjC3-2 and AjC3 genes expression in sea cucumber, the intestine, respiratory tree, coelomocytes and body wall tissues were separated from 15 healthy individuals (3 pools of 5 individuals each) (average body weight 10.2 g). These tissues from different individuals were mixed respectively, and frozen immediately with liquid nitrogen and then stored at 80 C. In order to investigate the expression patterns of AjC3-2 and AjC3 genes in responses to pathogen infection, 500 ml LPS (1 g/L) was injected into the healthy sea cucumbers (average body weight 10.2 g). Un-injection sea cucumbers were treated as the blank group (0 h). The sea cucumbers injected with 500 ml sterile sea water (SSW) were treated as the control group [20]. Coelomocytes were collected at 3 h, 6 h, 12 h, 24 h, 72 h and 96 h after injection respectively according to the methods reported by SantiagoCardona et al. [21]. For each sampling time, the coelomocytes taken from 15 different individuals (3 pools of 5 individuals each) were mixed in three 1.5 mL microcentrifuge tubes, and thrown immediately into liquid nitrogen and then stored at 80 C. 2.4. Quantitative Real-time PCR
Table 1 PCR primers used in this study. Primer
Sequence (50 30 )
Sequence Information
AjC3-2-R1: AjC3-2-R2: AjC3-2-F: AjC3-2-R: AjC3-F: AjC3-R: Cytb-F: Cytb-R:
50 -TATAGAGCCTTCGCGTTGTAT-30 50 -TGAAAC GTTCTTTCGATTCCC-30 50 -CTCTCGTGAGTTCTGGC TCAG-30 50 -GCAGCCACTGTTACCATCGCG GA-30 50 -GCGTTGTTTCGTTCAACAAGGGGA-30 50 -GCCATTCACTGGAGGTGTGGCA-30 50 -TGAGCCGCAACAGTAATC-30 50 -AAGGGAAAAGGAAGTGAAAG-30
For AjC3-2 50 -RACE For AjC3-2 Real-time PCR For AjC3 Real-time PCR For Real-time PCR
Total RNA was isolated using the UNIQ-10 Column Total RNA Isolation Kit (Sangon, Shanghai, China) according to the manufacturer’s instructions. The quality and quantity of extracted total RNA were measured using the NanoPhotometer (ImplenGmbH, Munich, Germany) and agarose gel electrophoresis. First strand cDNA synthesis was performed in a volume of 20 ml with 900 ng total RNA, 25 pmol Oligo dT Primer, 50 pmol random 6 mers primer, 1 PrimeScriptÔ buffer, 0.5 ml PrimeScriptÔ RT enzyme Mix I (PrimeScriptÔ RT reagent Kit, TaKaRa, China). Reactions were
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incubated at 37 C for 15 min, and then at 85 C for 5 s to deactivate the enzyme. Equal amounts of cDNA templates were used in Quantitative Real-time PCR (qRT-PCR). qRT-PCR was performed in triplicate using the Mx3000pÔ detection system (Applied Stratagene, USA) in 20 ml reactions containing the following components: 10 ml of 2 SYBR Green Master mix (SYBR PrimeScriptÔ RT-PCR Kit II, TaKaRa), 0.4 ml of ROX Reference Dye II, 1 ml of cDNA template, and 0.4 mM of each primer. Primers were listed in Table 1. The qRT-PCR profile was as following: One cycle of 95 C for 30 s, followed by 40 cycles of 95 C for 10 s, 56 C for 25 s and 72 C for 25 s. Melting curve analysis of amplification products was performed at the end of each PCR reaction to confirm that only one PCR product was amplified and detected. In addition, the amplicons were checked by agarose gel with a 100 bp ladder in order to confirm the correct amplicon sizes. The cytochrome b (Cytb) gene was used as the reference gene (Table 1) [22]. The relative mRNA levels of the target genes were calculated as 2DDCt [23]. Statistical calculations were performed using SPSS (version 13.0) software. Significant difference was indicated by one-way ANOVA (P < 0.05) analysis. 3. Result 3.1. Isolation of full-length cDNA of AjC3-2 gene Based on the 641 bp EST sequence with 30 -UTR obtained by cDNA libraries sequencing, the 50 -RACE reactions were performed toward 50 end of the EST. Here, the C3 sequence in this study was named AjC3-2 (GenBank accession no. HQ874435), while the other one previously submitted to GenBank by other researchers was termed AjC3 (GenBank accession no. HQ214156). The full-length cDNA sequence of AjC3-2 gene is 5748 bp barring the poly (A) tail. The sequence comprises a 26 bp 50 -terminal untranslated region (UTR), a 637 bp 30 -UTR with a polyadenylation signal AATAAA near the poly (A) tail and a 5085 bp open reading frame (ORF) encoding 1695 amino acid residues. The molecular weight of the unglycosylated mature protein of AjC3-2 is 187.72 kDa, and the estimated isoelectric point of the proteins is 5.12. 3.2. Homology analysis and characterization of AjC3-2 Sequence analysis indicated that the amino acid sequence of AjC3-2 show 43%, 35%, 35%, 31% identities with sea cucumber AjC3, sea urchin (S. purpuratus) C3, amphioxus (Branchiostoma belcheri) C3 and human C3. The result of domain prediction indicates that AjC3-2 as well as AjC3 and other C3s have not only a2M domain which exists in a2 macroglobulin, but also complement specific domains, such as complement C3/C4/C5 domain and C345C domain [12,24,25]. The protein sequence of AjC3-2 was aligned with vertebrate and invertebrate C3 sequences (Fig. 1). Like other C3s, AjC3-2 has a linker RRRR between b and a chain which is seated at 689e692 amino acids. At this site, C3 is cleaved into b and a chain by catalysis of a furin-like enzyme during posttranslational modification [26]. The molecular weight of the deduced b and a chain are 74.80 kDa and 112.31 kDa respectively. In vertebrates, C3 convertase cleaves C3 into two parts, the larger one called C3b and the smaller one called C3a. C3a comes from about 70 amino acids closed to the N-terminal of a chain, and is regarded as an anaphylatoxin together with C4a and C5a [27]. In the homologous region of AjC3-2, four out of six cysteines forming three intrachain disulfide bridges in the disulfide-linked “core” region existing in mammal anaphylatoxins. Furthermore, G714/701and F753/743 of AjC3-2 is also in accordance with mammal anaphylatoxins in which
these two amino acids are important for the folding of the polypeptide chain. It is now known that the linear sequence at the C-terminus of C3a and C4a of human contains the structural information necessary to mediate biologic responses associated with the C3a/C4a receptor and other factors [28], and the last three amino acids LXR of this linear sequence are the C3 convertase cleavage site which is generally conserved in most of vertebrate C3a or C4a [27]. Although there is not well conservation in the corresponding sites of AjC3-2, RXR sequences of AjC3-2 and AjC3 are found near the cleavage site, similar to that observed in coral, horseshoe crab and sea anemone [10,12,13]. C3b is composed of the whole b chain and a0 chain, a chain from which C3a has been removed. A typical thioester site (GCGEQ) is located in the a0 chain of AjC3-2. In human, a hydrophobic pocket protects this thioester from reacting with water or other small nucleophiles, which is formed by the space approach of F1047, M1378, Y1425 and Y1460 [24]. These residues are conserved in AjC3-2 and AjC3, except that F1047 is replaced by another aromatic residue W1098 in AjC3. H1104 and E1106 play an important role in the specific reaction between C3b and hydroxyl groups, AjC3-2 and AjC3 also have the same residues at the corresponding sites. Among all of ten pairs of Cyses that forming disulfide bonds in human C3b, eight pairs are identical in AjC3-2 and AjC3, including one uniting the b and a0 chain, one located in b chain and six located in a0 chain. More than 40 residues in N-terminal region of the a0 chain are considered to be the most important region of C3 because it encompasses the binding sites for CR1, CR2, CR3, factor B and factor H in human [28]. This corresponding region is lower homologous between the shown C3s, and the similar status can be seen in another factor H and CR2 binding sites, but properdin binding site is higher homologous. There are three factor I cleavage sites in human C3b, but none matches perfectly to AjC3-2 and AjC3. However, the sequence of R979E980 in the AjC3-2 is similar to the factor I site 1 (Fig. 1). 3.3. Phylogenetic analysis Phylogenetic analysis was conducted based on the amino acid sequences retrieved from the GenBank. Two different clades were formed (Fig. 2). One is composed of invertebrate C3 proteins including sea cucumber, sea urchin, sea anemone, and so on, and the other one is composed of vertebrate C3, C4 and C5 proteins. AjC3s formed a cluster with the SpC3 of sea urchin and this indicated that the duplication event from which AjC3-2 and AjC3 genes originated was occurred in the echinoderm lineage after the separation of the sea cucumber and sea urchin ancestor. 3.4. AjC3-2 and AjC3 genes expression profiles during development qRT-PCR analysis was used to determine the expression patterns of AjC3-2 and AjC3 mRNA at eleven developmental stages of sea cucumber A. japonicus. The result revealed that the expression patterns of these two C3 genes were highly similar, with lower expression during the early stages of development. The gene expression levels kept increasing at the three auricularia stages, and reached the peak in late auricularia, then descended down at the last three stages, doliolaria, pentactula and juvenile (Fig. 3). 3.5. AjC3-2 and AjC3 genes expression profiles in different tissues The expression levels of AjC3-2 and AjC3 genes of different tissues were also tested in intestine, respiratory tree, coelomocytes and body wall. In healthy sea cucumbers, the AjC3-2 and AjC3 genes were both expressed at a higher level in the coelomocytes, followed by body wall. Comparing with the lowest expression level
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Fig. 1. Alignment between AjC3-2 and other complement components. The abbreviations of species name and accession numbers for sequences are explained in Fig. 4. Several areas of functional significance are labeled. Boxed areas are determined according to Fig. 4 in Lambris et al. (1993) and correspond to functional sites in human C3 or C4 (Sahu and Lambris, 2001).
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Fig. 1. (continued).
Z. Zhou et al. / Fish & Shellfish Immunology 31 (2011) 540e547
Fig. 2. Phylogenetic tree. The tree was constructed using ClustalW and MEGA (4.0). The numbers of branches are bootstrap values for 1000 replicates. The GenBank accession numbers for the sequences are as follows: AjC3-2/C3: sea cucumber (A. japonicus), HQ874435/HQ214156; SpC3: sea urchin (Strongylocentrotus purpuratus), AAC14396; HlC3-1/C3-2: sea anemone (Haliplanella lineate), AB481383/AB481384; NvC3-1/C3-2: sea anemone (Nematostella vectensis), AB450039/AB450040; BbC3: amphioxus (Branchiostoma belcheri), BAB47146; CrC3: horseshoe crab (Carcinoscorpius rotundicauda), AF517564; VdC3: clam (Venerupis decussates), FJ392025; CiC3-1/C32:sea squirt(Ciona intestinalis), NP_001027684/CAC85958; OlC3-1/C3-2/C4: medaka (Oryzias latipes), NM_001105082/NM_001105083/NM_001104697; DrC3b: zebra fish (Danio rerio), NM_131243; HsC3/C4A/C5: human, AAA85332/AAB67980.1/NM_001735; MmC3/C4b/C5: mouse (Mus musculus), NM_009778/NM_009780/M35525; LjC3:lamprey(Lethenteron japonicum), BAA00983; CcC5-1: carp (Cyprinus carpio), AB084635.
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Fig. 3. Relative mRNA levels of AjC3-2 and AjC3 genes at different development stages of A. japonicus by using qRT-PCR analysis. 1: Unfertilized egg; 2: Fertilized egg; 3: Cellulous stage; 4: Blastula; 5: Gastrula; 6: Early auricularia; 7: Auricularia; 8: Late auricularia; 9: Doliolaria; 10: Pentactula; 11: Juvenile. Each symbol and vertical bar represents the mean S.D (n ¼ 3). Different lowercase letters above the bars indicate significant differences (P < 0.05) at different stages by t-test. Expression levels at all different stages are presented relative to that in the unfertilized egg (1).
sequence RXXR, C3 convertase cleavage site L/RXR, four disulfide bridges in C3a and eight disulfide bridges in C3b (Fig. 1). The identifications of these conserved structure suggest that C3 proteins in sea cucumber possess the basic biochemical functions throughout the evolution, and SpC3 sharing a higher homology
in the intestine, the expression in the coelomocytes, body wall and respiratory tree reached about 5, 4.5 and 2 fold for AjC3-2 and 2, 3 and 2.5 fold for AjC3, respectively (Fig. 4). 3.6. AjC3-2 and AjC3 genes expression profiles after LPS challenge In the LPS challenge experiment, we measured the AjC3-2 and AjC3 genes expression levels of the coelomocytes at the different sampling times. At 3 h after injection, there was no significant expression difference between control and challenge samples. However, it is apparent that these two C3 genes were significantly induced at 6 h after injection (Fig. 5). AjC3-2 gene expression was up-regulated about 2 fold at 6 h, 4 fold at 12 h, 29 fold at 72 h and 5 fold at 96 h after injection (P < 0.05). AjC3 gene expression was upregulated about 3 fold at 6 h, 4.6 fold at 12 h and 2.5 fold at 24 h after injection (P < 0.05). 4. Discussion In the present study, we identified and characterized the fulllength cDNA of one C3 like gene, AjC3-2 of sea cucumber. The expression profiles of two isoforms of C3 genes, AjC3-2 and AjC3, in normal tissues and after LPS challenge were also analyzed. AjC3-2 shares many conserved structural characteristics with other C3 proteins, such as the thioester site GCGEQ, the b-a linker
Fig. 4. Distribution of AjC3-2 and AjC3 mRNA in the different tissues. 1: Intestine; 2: Respiratory; 3: Coelomocytes; 4: Body wall. Each symbol and vertical bar represents the mean S.D (n ¼ 3). Different lowercase letters above the bars indicate significant differences (P < 0.05) in different tissues by t-test. Expression levels in all tissues are presented relative to that in the intestine tissue (1).
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Fig. 5. Effects of LPS challenge on transcriptional levels of AjC3-2 and AjC3 genes in the coelomocytes after injection. Each symbol and vertical bar represents the mean S.D (n ¼ 3). Different lowercase letters above the bars indicate significant differences (P < 0.05) at different time points of the same group by t-test; asterisks above the bars show significant differences (P < 0.05) between two groups of the same time point by t-test.
with AjC3-2 and AjC3 functions as an opsonin have been confirmed in another species of echinoderm, the sea urchin S. purpuratus as well [29]. In human, a chain structure of C3 contains a lot of essential structural features for interacting with specific cellular receptors, natural factors and serine proteases as shown in Fig. 1. The binding sites of CR1, CR2, C3aR, factor B and factor H or the cleavage sites of factor I with cofactor, are not conserved well in sea cucumber C3 proteins. This result suggested that sea cucumber C3 proteins may have different folding pattern, and maybe not interact with many other proteins as known in human C3. For example, CR2 is located on the cell membrane of B lymphocyte in higher vertebrate, so the CR2-like proteins and the binding sites of CR2 should not exist in the innate immune system of sea cucumber. AjC3-2 possesses the a-g chain linker RXXR which was previously found in C4s of vertebrate, but it is clearly located outside vertebrate C3/C4/C5 proteins by the phylogenetic analysis (Fig. 2). Therefore, AjC3-2 should not be the analog of C4 proteins, and this phenomenon can also be seen in other animal C3 proteins containing the homologous sequence of RXXR such as lamprey [30], amphioxus [6], horseshoe crab [10], clam [25], sea anemone [12,13] and coral [11]. In coral and sea anemone, the linker is surrounded by a similar K/R rich region. Thus, researchers presumed that this region is a characteristic for ancestor of C3, the vertebrate C3 and C5 apparently lost the entire region, whereas vertebrate C4 and some species C3 retained a part of it and used it as the a-g processing signal [11e13]. Here we described one C3 gene of sea cucumber, AjC3-2, and we also found part sequence of another AjC3 gene from the ESTs obtained by our cDNA libraries sequencing [17]. A few months ago
we found this AjC3 cDNA sequence in GenBank submitted by other researchers. So we can see two C3 genes of A. japonicus have been cloned. To date, sea anemone [12,13], ascidian [7] and numerous teleost fish [31e33] have been found to own multiple forms of C3 genes. The phenomenon was interpreted to be caused by the tandem gene duplication, and occurs frequently during evolution [4]. The studies on the trout have demonstrated that the different isoforms of C3s display different binding efficiencies to several complement activating surfaces, thereby providing a means of expanding the immune defense repertoire [34]. In this study, the expression patterns of AjC3-2 and AjC3 genes at different development stages were investigated. The expression patterns of these two C3 genes were similar. The expression difference was not significant between the unfertilized eggs and fertilized eggs. It suggested that the lower expression level in early embryos and gastrulas is maternally derived. The auricularia stages of development, with the guts were formed, the larvae begin to eat, maybe some immune related genes like C3 have higher expression is necessary to protect the larvae against microbial invasion. The researches on the mesenchyme cells of sea urchin embryos demonstrated that they have the capacities for anti bacteria and phagocytosing yeasts [35,36]. Based on these studies, mesenchyme cells are perhaps the best candidates for mediating the immune responses in the developing echinoderm embryos [37]. During embryogenesis of sea cucumber, primary mesenchyme cells first appear in the late gastrula which precedes early auricularia. Our research results revealed that the increase of C3 genes expression was corresponded with the appearance of primary mesenchyme cells. C3 genes may express highly in the mesenchyme cells of sea cucumber embryos. Coelomocytes have long been considered to be mediators of the immune response in echinoderm, so it is not surprising that AjC3-2 and AjC3 genes have intense expression in the coelomocytes like SpC3 of sea urchin [9]. However, it is interesting that AjC3-2 and AjC3 genes also expressed highly in the body wall besides the coelomocytes. The previous experiments showed that in the sea urchin injected with LPS, the appearance of SpC3 protein in coelomocytes was significantly delayed when compared to SpC3 protein in coelomic fluid [38]. This suggested that there maybe other sites where SpC3 protein was produced in sea urchins besides the coelomocytes in the coelomic fluid. In mammals, although the main biosynthesis and secretion site of C3 is the liver, several other type cells appear to produce and secrete C3, including fibroblast cells and epithelial cells [39,40]. Both these kinds of cells are involved in the developed body wall of sea cucumber, and according to the study in starfish, the coelomic epithelial cell was identified as one of the sources of new coelomocytes, which proliferates upon LPS injection [41]. Lipopolysaccharide (LPS) is the most used immune-activating substance. Lots of immune-related genes respond to the LPS challenge. Clow et al. found that the expression of Sp064 mRNA in coelomocytes was elevated after challenging the sea urchins with LPS [38], and the results of other experiments have demonstrated that SpC3 and AsC3 of the ascidian Halocynthia roretzi are the humoral opsonins in coelomic fluid [8,29]. Furthermore, it is reported that LPS can effectively increase phagocytes and phagocytic activity in the coelomocytes of the sea cucumber Holothuria glaberrima [42]. As a result, it is possible that the C3 proteins of sea cucumber could stimulate the phagocytic activity of phagocytes as an opsonin. In this study, the expression of AjC3-2 and AjC3 genes was also up-regulated clearly in the coelomocytes after LPS challenge, and the expression levels of these two genes both reached the peak at 12 h after LPS injection. AjC3-2 gene kept higher expression levels from 12 h to 96 h after LPS injection, while AjC3 expression descended down quickly. Whether they actually display
Z. Zhou et al. / Fish & Shellfish Immunology 31 (2011) 540e547
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