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Molecular cloning of two C1q-like cDNAs in mandarin fish Siniperca chuatsi
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Veterinary Immunology and Immunopathology 125 (2008) 37–46 www.elsevier.com/locate/vetimm
Molecular cloning of two C1q-like cDNAs in mandarin fish Siniperca chuatsi Hai-Hua Lao a,b, Ya-Nan Sun a, Zhi-Xin Yin a,c, Jing Wang a, Chao Chen a, Shao-Ping Weng a, Wei He a, Chang-Jun Guo a, Xian-De Huang a, Xiao-Qiang Yu d,*, Jian-Guo He a,** a
State Key Laboratory for Biocontrol, School of Life Sciences, Sun Yat-sen University, 135 Xingang Road West, Guangzhou 510275, PR China b Key Laboratory of Tropical & Subtropical Fish Breeding & Cultivation of CAFS, Pearl River Fisheries Research Institute, 1 Xingyu Road, Guangzhou 510380, PR China c Institute of Genetic Engineering, Southern Medical University, Guangzhou 510515, PR China d Division of Cell Biology and Biophysics, School of Biological Sciences, University of Missouri-Kansas City, Kansas City, MO 64110, USA Received 25 October 2007; received in revised form 30 April 2008; accepted 8 May 2008
Abstract C1q, a subunit of the C1 complex, plays a key role in the recognition of immune complexes to initiate the classical complement pathway. In this study, we reported two C1q-like cDNAs from mandarin fish (Siniperca chuatsi), mC1q-like-1 (mC1qL1) and mC1q-like-2 (mC1qL2). The full-length cDNA of mC1qL1was 990 bp, containing a 71 bp 50 -untranslated region (UTR), an open reading frame (ORF) of 723 bp, and a 196 bp long 30 -UTR. mC1qL2 cDNA was 1193 bp, containing a 100 bp 50 -UTR, followed by an ORF of 756 bp and a 30 -UTR of 337 bp. mC1qL1 and mC1qL2 share 29% identity in amino acid sequence. Both mC1qL1 and mC1qL2 contained three parts: a short amino-terminal region, a collagen-like region and a carboxyl-terminal globular C1q domain. The phylogenetic analysis showed that mC1qL1 clustered with two Danio rerio hypothetical proteins and further grouped with C1q proteins, while mC1qL2 clustered with C1qA proteins from other species. In healthy mandarin fish, mC1qL1 and mC1qL2 were expressed in all tissues tested, including liver, spleen, head kidney, caudal kidney, intestine and gill. mC1qL1 was highly expressed in head kidney, while mC1qL2 was mainly expressed in spleen. The expression level of mC1qL1 and mC1qL2 in liver were not changed obviously and mC1qL2 was significantly changed ( p < 0.05) in spleen after infectious spleen and kidney necrosis virus (ISKNV) infection. Mandarin fish C1q may play a role in response to ISKNV infection. # 2008 Elsevier B.V. All rights reserved. Keywords: mC1qL1; mC1qL2; Complement; Siniperca chuats; Tissue expression; ISKNV
1. Introduction
* Corresponding author. Tel.: +1 816 235 6379; fax: +1 816 235 1503. ** Corresponding author. Tel.: +86 20 84110976; fax: +86 20 84113819. E-mail addresses: [email protected] (X.-Q. Yu), [email protected] (J.-G. He). 0165-2427/$ – see front matter # 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2008.05.004
Mandarin fish, Siniperca chuatsi (Basilewsky), is a main economic cultured fish in China. With the rapid development of mandarin fish aquaculture, diseases, especially the viral disease, have become a major constraint and the most limiting factor in the mandarin fish culture industry. ISKNV (infectious spleen and
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kidney necrosis virus), a member of the Iridoviridae family, has caused high mortalities in mandarin fish and limited largely the mandarin fish cultures in China (He et al., 1998, 2000; Deng et al., 2000). The complement system, a family of more than 35 soluble serum proteins, plays essential roles in innate and adaptive immunity in host defense (Nonaka and Smith, 2000; Boshra et al., 2006). Activation of the complement system also contributes significantly to the orchestration and development of acquired immune responses (Boshra et al., 2006). The complement system can be activated through three pathways: the antibody-dependent classical pathway, the antibodyindependent alternative pathway, and the lectin pathway (Sunyer and Lambris, 1998). The classical complement pathway (CCP) is a major defense and clearance system in the blood circulation. It is activated at the level of C1q, a subcomponent of the C1 complex, by both immune complexes that react with the C1q globular region and nonimmune substances that react with the C1q collagen-like region (Kishore and Reid, 1999; Jiang et al., 1992). Fish possess complement pathways similar to those in mammals, and fish complement proteins identified thus far show highly homologies to their mammalian counterparts (Holland and Lambris, 2002). The human C1q molecule is composed of 18 polypeptide chains (six each of A-, B- and C-chains). Each of these chains has a short amino-terminal region, followed by a collagen-like region (CLR, repeating Gly-X-Y triplets, where X is often proline and Y is often hydroxylysine or hydroxyproline) and a carboxyterminal globular head region (Sellar et al., 1991). Every C1q chain also has four conserved cysteines, which can form inter- or intra-chain disulfide bridges that play an important role in formation of intact C1q molecules. C1q, the key component of the classical complement pathway in antimicrobial defense, can trigger rapid enhanced phagocytosis resulting in clearance of cellular debris, apoptotic cells and immune complexes, phagocytosis of bacteria, neutralization of retroviruses, and modulation of dendritic cells (DCs), B cells and fibroblasts (Bohlson et al., 2007; Kishore et al., 2004). Direct binding of C1q to a wide range of pathogens including viruses (Dierich et al., 1993; Kojouharova et al., 2003), parasites (Rimoldi et al., 1989), and both gram positive (Butko et al., 1999) and gram negative (Aubert et al., 1985; Kovacsovics et al., 1987) bacteria, has been reported. C1q is a versatile recognition protein and a major connecting link between classical pathway-driven innate immunity and acquired immunity (Kishore and Reid, 2000).
The complement system has been studied extensively in mammals, but considerably less known in lower vertebrates, particularly in teleost fish. The C1q just has been identified in zebrafish (Danio rerio) and Tetraodon nigroviridis because their full genomic DNA sequences are available. The full-length cDNA sequences of C1q in common carp (Cyprinus carpio) (BAD22535) and gibel carp (Carassius auratus gibelio) (AY662672) and an EST sequence (CV823415) similar to complement C1q subcomponent in killifish (Fundulus heteroclitus) can be found in the GenBank. Fish C1q has been reported in nurse shark (Ginglymostoma cirratum) (Smith, 1998) and channel catfish (Ictalurus punctatus) (Dodds and Petry, 1993), but the full-length cDNAs or genes are not available. In addition, full-length cDNA sequence containing the C1q globular domain but lacking the collagen domain has been cloned in color crucian carp (Carassius auratus color variety) (AY583317), and this C1q-like gene has unique functions in oogenesis, oocyte maturation and egg fertilization (Chen and Gui, 2004). To our knowledge, there is no relevant paper on fulllength fish C1q. Mandarin fish is an important economic fish in China. The breakouts of ISKNV do threaten the culture of mandarin fish and cause large loss every year. To understand the relationship between the complement system and ISKNV, here we report identification of two full-length C1q-like cDNAs in mandarin fish (S. chuatsi). Both mC1qL1 and mC1qL2 shared common features with mammalian C1q. The phylogenetic analysis showed that mC1qL2 were clustered with mammalian C1qAs and mC1qL1 clustered with zebrafish C1q-domain containing protein and further grouped with mammalian C1qs. mC1qL1 and mC1qL2 were expressed constitutively in all tissues tested. Both mC1qL1 and mC1qL2 were expressed at the lowest level 7 days after fish were challenged with ISKNV, while the ISKNV infected fish began to die at 7 days post-infection. The expression pattern of mC1qLs suggests that mandarin fish C1q may play a role in response to ISKNV infection. 2. Materials and methods 2.1. RNA extraction Three healthy mandarin fish, about 500 g, were obtained from fish farm in Nan Hai (Guangdong Province, China) and acclimated for 2 weeks. The fish were assured free of ISKNV infection by PCR (Deng et al., 2000). Total RNA was extracted from fresh liver
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of mandarin fish using SV Total RNA isolation Kit (Promega, USA). The quality and concentration were determined by agarose gel electrophoresis and spectrophotometer (Amersham, Sweden). 2.2. Cloning of C1q cDNA sequences from mandarin fish liver Degenerate oligonucleotide primers, C1qL1-R1 (50 -GTGATAACTGAAGAAAATAGACGCC-30 ) for mC1qL1 50 rapid amplification of cDNA ends (RACE), C1qL2-F1 (50 -TCAGGTGGCGTGGTCTTACAGC-30 ) and C1qL2-R1 (50 -ACAGACACATGCTGACCTTGGC-30 ) for the 30 and 50 RACE of mC1qL2, were designed based on two different EST sequences obtained previously from mandarin fish in our lab (AY909459, and another EST sequence has not been submitted in GenBank Database). The 30 and 50 end cDNA sequences of mandarin fish mC1qL1 and mC1qL2 were obtained by RACE using total RNA from mandarin fish liver as template. cDNA templates for the 50 and 30 RACE were synthesized using the BD SMARTTM RACE cDNA amplification kit (Clontech, USA) following the manufacture’s instruction. All the amplified fragments were cloned into pMD19-T vector (TaKaRa, Japan) and sequenced. The nucleotide sequences were determined from both strands by the dideoxy-chain termination method using a model 3730 sequencer. Then two fulllength cDNAs were obtained by aligning the overlapped fragments and the EST sequences. 2.3. Sequence analysis and alignment Homology searches and identity/similarity assessment were performed by BLAST at web servers of National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/blast) (Altschul et al., 1990). The signal peptide cleavage site was predicted with signalP 3.0 service (http://www.cbs.dtu.dk/ services/SignalP/) (Nielsen et al., 1997). SMART (http://smart.embl-heidelberg.de/) (Letunic et al., 2004) was used for protein domain determination and characterization. The predicted amino acid sequences of mC1qLs were multiple aligned with Fig. 1. Nucleotide and deduced amino acid sequences of mC1qL1 (A) and mC1qL2 (B) from Siniperca chuatsi. Amino acid sequence is shown with one-letter codes above the nucleotide sequence. The initiation codon (ATG) and the stop codon (TAG) are highlighted in bold. The predicted signal peptide is underlined (—). The Kozak sequence is marked by undee line ( ). The globular C1q domain is shown in gray. The two potential N-linked glycosylation sites in
mC1qL1 are shown in dot underlined ( ). The repeating triplet Glycin-X-Y is double underlined ( ). The conserved cysteine residues are boxed, a free one is in italic. The GE(K/Q/R)GEP motif in collagen-like region is shown in gray and double underlined ( ). The RGD sequence in collagen-like region of mC1qL2 is boxed. The residues for Ca2+ binding are undee lined ( ). The putative polyadenylation signals were shown in dotted underlined ( ).
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Fig. 2. Multiple alignment of mC1qLs with other C1qs. The C1q and C1q-like (C1qL) proteins from different organisms were aligned using ClusterX V1.83. Identical or highly conserved residues are shaded in black, while similar residues are shaded in grey. Sequences for the alignment are obtained from GenBank: cC1qA (AAI05346), cC1qB (AAI12491), cC1qC (AAI14894), ccC1qL (AAS93680), gcC1qL (AAT76300), chC1q (NP_996874), cpC1qL (BAD22535), dC1qA (XP_535367), dC1qB (XP_544507), dC1qC (XP_544508), fC1qC (XP_417653), fC1qB (XP_425756), hC1qA (AAH71986), hC1qB (AAH08983), hC1qC (AAP97191), hC1qL2 (NP_872334), lC1qL (BAD22833), mkC1qA (XP_001101933), mkC1qB (XP_001110783), mkC1qC (XP_001102112), msC1qA (NP_031598), msC1qB (AAH67001), msC1qC (NP_031600), msC1qL1 (NP_035925), pC1qA (AAR20892), pC1qB (AAR20888), pC1qC (AAR20893), PtC1qA (XP_001166821), PtC1qC (XP_513188), rC1qA (AAH86605), rC1qB (CAA50440), rC1qC (AAH86409), rbC1qB (AAC61578), TnC1qD (CAF95737), zC1qD (NP_001003482), zC1qD-1 (NP_001018363), and zC1qD-2 (XP_697055). Abbreviations are A, alpha; B, beta; C, gamma; D, domain; L, like;
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C1qs from other organisms using the Clustal X V1.83 (Thompson et al., 1997). Phylogenetic tree was constructed based on the full-length amino acid sequences of C1q and C1q-related proteins using the neighbor-joining (NJ) algorithm (Saitou and Nei, 1987) within MEGA V3.1 (Kumar et al., 2004). The branch points of phylogenetic tree were validated by 1000 bootstrap replications. 2.4. Expression pattern of mC1qL1 and mC1qL2 by RT-PCR and Northern blotting analyses To determine the tissue expression profiles of mandarin fish mC1qL1 and mC1qL2, semi-quantitative RT-PCR and Northern blotting were performed using equal amounts of total RNA from various tissues of healthy mandarin fish, including liver, spleen, head kidney, caudal kidney, intestine and gill. The total RNAs were extracted using TRIzol reagent (Invitrogen, USA) and quantified. The first strand cDNAs were synthesized using RTase (promega, USA) and oligo-dT and random primer (9 bp) (Sangon, China) as primers. The PCR was carried out using C1qL1-F2 (50 -AACGGCATAGTCGGTCCCAAGG-30 ) and C1qL1-R1 for mC1qL1, C1qL2F4 (50 -CCTGGAGCAAAGGGAGAGAAAG-30 ) and C1qL2-R2 (50 -CTGTAAGACCACGCCACCTGAC-30 ) for mC1qL2. The 18S rRNA amplified using 18SF (50 ATGGTACTTTAGGCGCCTAC-30 ) and 18SR (50 -TATACGCTATTGGAGCTGG-30 ), was used as an internal control. The RNAs from two individual fish were used for RT-PCR. PCR was conducted according to the following program: 94 8C for 2 min, 29 cycles of 94 8C for 30 s, 55 8C for 30 s, 72 8C for 30 s, followed by a final extension at 72 8C for 10 min. PCR products were analyzed by electrophoresis on a 1.5% agarose gel. Northern blotting was also performed. RNA samples from three individual fish were mixed and electrophoresed according to the protocol of NorthernMax1-Gly (Ambion, USA). The antisense RNA probes were labeled by Dig RNA Labeling Kit (Roche, USA). Hybridization and immunological detection were performed using Dig Northern Starter Kit (Roche, USA). The signals were captured by GeneSnap chemical lighting assay system. The density of the 18S rRNA of every sample before transferred to membrane and the hybridization signals were scanned. The density ratios of the hybridization signals to the 18S rRNA were calculated as the relative expression level of each tissue.
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2.5. Expression of mC1qLs during ISKNV infection One hundred mandarin fish, 100 20 g, were bought and acclimated for 2 weeks before ISKNV challenge. The virus filtrates were prepared and injected into the fish intraperitoneally (i.p.) according to Zeng’s method (Zeng et al., 1999). Tissue samples were harvested before infection (0 day) and after infection at 1, 3, 5, 7, 9, 11, 13 and 15 days. Three parallel samples from three separate fish were collected at each time point for detection by PCR using ISKNV specific primers to confirm that the mandarin fish were surely infected with ISKNV. We sampled fish randomly at first 5 days when fish did not show visible clinical sign of infection. After 7 days of infection, we sampled the normal fish (no clinical sign) and moribund fish at each time point for detection. The total RNAs were extracted separately using SV total RNA Isolation Kit (Promega, USA) and quantified. The RTPCR was performed using the same primers and PCR conditions were the same as described above. We selected spleen and liver for the experiment because spleen was one of the main tissues of ISKNV infection (He et al., 1998) and liver was used as a control, which was not the main target tissue of ISKNV infection. The data analysis was carried out with Kodak Digital Science system. All statistics were performed using SPSS program to interpret the quantitative data after ISKNV injection. Significance of differences was analyzed by one-way ANOVA followed by Bonferroni’s post hoc adjustment. 3. Results and discussion 3.1. Cloning and sequence analysis of the full-length cDNAs encoding the mandarin fish C1qL1 and C1qL2 By 50 and 30 RACE amplification using degenerated primers, two full-length mandarin fish C1q-like cDNAs (mC1qL1 and mC1qL2) were obtained. mC1qL1 was 990 bp, containing a 71 bp 50 -UTR, a 723 bp ORF, and a 196 bp 30 -UTR with a putative polyadenylation signal and a poly(A) tail. The ORF of mC1qL1 encodes a protein of 240 amino acids, which includes a putative signal peptide of 23 amino acids (Fig. 1A). mC1qL2 was 1193 bp, containing a 100 bp 50 -UTR, followed by an ORF of 756 bp encoding a protein of 251 amino acid residues with a putative signal peptide of 22 amino acids, and a 30 -UTR of 337 bp with two putative polyadenyla-
c, Bos Taurus; cc, Carassius auratus color variety; ch, Gallus gallus; cp, Cyprinus carpio; d, Canis familiaris; f, Gallus gallus (red jungle fowl); gc, (Carassius auratus gibolio); h, Homo sapiens; l, Lethenteron japonicum; m, Siniperca chuatsi; mk, Macaca mulatta; ms, Mus musculus; p, Sus scrofa; Pt, Pan troglodyte; rb, Oryctolagus cuniculus; r, Rattus norvegicus; Tn, Tetraodon nigroviridis; z, Danio rerio.
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tion signals and a poly(A) tail (Fig. 1B). The mature peptides of both mC1qL1 and mC1qL2 are composed of three parts: a short amino-terminal region, a CLR region containing repeating triplet of Gly-X-Y, and a globular C1q domain. The domain organization of mC1qLs is characteristic of C1q proteins (Bohlson et al., 2007). In the C1q proteins, the Glycin residue in Gly-X-Y triplets is very conserved (Fig. 2). The number of Gly-X-Y repeats can range from 14 to well over 100 copies (Innamorati et al., 2006). In mC1qL1 and mC1qL2, the CLR included 24 and 22 Gly-X-Y repeats, respectively. Both mC1qL1 and mC1qL2 contain four cysteines, which are conserved in most C1q molecules (Fig. 2). The first cysteine participates in formation of inter-chain disulfide bridge in human C1q to yield A–B and C–C subunits. The second cysteine was free thiol group, which can be involved in the formation of disulphide bonds between C1q and IgG in immune complexes (Sellar et al., 1991). The third and fourth cysteine residues are considered to form an intrachain disulfide bond (Kishore et al., 2003). The Kozak sequence, (A/G)NNATGG, which is recognized by ribosomes as the translational start site and thus required for protein expression (Kozak, 1986), was also present within both mC1qL1 and mC1qL2 cDNA sequences. There were two N-linked glycosylation sites in mC1qL1, while mC1qL2 had a RGD motif, predicted by the SCAN PROSITE program (www.expasy.ch/prosite/). In mC1qL2 CLR, there was a motif, GEKGEP, which is also present in human mannose-binding lectin (MBL) and other defense collagens to stimulate phagocytic activity (Arora et al., 2001; Bohlson et al., 2007). mC1qL1 had a similar motif, GEKGDP, in which Glu(E) was substituted by an Asp(D) at the crucial position. C1q is a hexamer of trimers, each trimer contains three distinct polypeptide chains. It appears that only one such motif per trimer is required for induction of the function (Arora et al., 2001). We speculate that mandarin fish mC1qLs may possess the function to stimulate phagocytic activity. 3.2. Multiple sequence alignment and phylogenetic tree construction of C1q sequences mC1qL1 and mC1qL2 were similar to C1q sequences from other species by searching similarity regions in NCBI. The deduced amino acid sequences of mandarin fish mC1qL1 and mC1qL2 were aligned with the amino acid sequences of C1q from various species obtained from GenBank (Fig. 2). The identity between mC1qL1 and mC1qL2 was low (29%). mCqL1 exhibits highest similarity with Mus musculus C1qB (36.4% identity). mC1qL2 is highly identical to an unnamed protein from T. nigroviridis (CAF95737) (55.3%). The homology
Table 1 The homology between mC1qLs, other C1q and C1q-related proteins
mC1qL1 Mouse C1qB Human C1qB Zebrafish C1q domain Cattle C1qB Rat C1qB Dog C1qB Rat C1qC Tetraodon nigroviridis unnamed protein Fowl C1qC Rat C1qA Dog C1qA Cattle C1qC Zebrafish C1q domain protein Human C1qA Pig C1qA Monkey C1qA Cattle C1qA Mouse acrp30 Human clusterin Human collagen I-a1 Human precebellin Mouse clusterin
mC1qL1 (%)
mC1qL2 (%)
– 36.4 35.7 35.4 34.9 34.6 34.5 34.5 27.3
29.0 31.9 31.6 25.7 30.8 33.8 33.1 33.6 55.3
33.6 30.3 28.2 33.2 30.4 29.5 28.4 29.9 29.7 29.9 6.7 3.8 18 6.4
37.5 36.7 35.7 35.4 35.0 34.9 34.6 34.5 34.2 25.6 6.0 4.2 16.8 4.2
among mC1qLs and other C1q or C1q-related proteins is shown in Table 1. The homologies among mC1qLs and other C1q proteins were higher than those of mC1qLs and C1q-related proteins, including collagen, clusterin, Acrp30 and precebellin. mC1qLs should belong to C1q family although the homology between mC1qLs and other C1qs was low compared to the high identity (73%) of the three C1q subunits from human and rat. A phylogenetic tree was constructed using fulllength amino acid sequences of C1q and C1q-related proteins from various species by the NJ method (Fig. 3). The three subunits, A, B and C, of mammalian C1q clustered very well. mC1qL1 was clustered with two zebrafish hypothetical proteins containing C1q domain (NP_001003482 and NP_001018383) and further grouped with C1q proteins from other species. mC1qL2 was clustered with T. nigroviridis complement component C1q domain (CAF95737) and further clustered with C1qA proteins from other species. mC1qL2 may be the counterpart of C1qA of mammals. 3.3. Tissue distribution of mandarin fish mC1qL1 and mC1qL2 mRNAs In healthy mandarin fish, semi-quantitative RTPCR and Northern blotting showed that both mC1qL1
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Fig. 3. The phylogenetic tree of C1qs. The full-length amino acid sequences of C1qs, C1qLs and other proteins with C1q domain from different organisms were aligned using the ClusterX V1.83 program (Thompson et al., 1997). The phylogenetic tree was constructed using Bootstrap N-J method of MEGA3. Amino acid sequences are obtained from the GenBank as described in Fig. 2 legend. The added proteins (compared to Fig. 2) and their accession numbers were shown in Fig. 3.
and mC1qL2 mRNAs were expressed in all the tissues tested, including liver, spleen, head kidney, caudal kidney, intestine and gill (Fig. 4A and B). mC1qL1 mRNA expression level was the lowest in liver.
Compared with the expression level in liver, the expression level of mC1qL1 was high in head kidney (8.7 times), followed by intestine (6.7 times), gill (5.9 times), caudal kidney (3.7 times) and spleen (2.9
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Fig. 4. The expression of mandarin fish mC1qLs in tissues. The expression pattern of mC1qLs in tissues was detected by RT-PCR (A) and Northern blot (B). mC1qL1 and mC1qL2 were expressed in all tissues tested. (A) The RT-PCR results. (B) The Northern blot results. The numbers under (A) are the ratio of the expression of mC1qLs to 18S. The numbers under (B) are the results of density scanned. L: liver; S: spleen; HK: head kidney; CK: caudal kidney; I: intestine; G: gill.
times). mC1qL2 was also expressed at low level in liver, and its expression level was high in spleen (8.9 times), followed by head kidney (7.6 times), intestine (6.3 times), gill (4.6 times) and caudal kidney (2.7 times) compared to the expression level in liver. The expression level of mC1qL1 and mC1qL2 among different tissues are significantly different ( p < 0.05). Most complement proteins are produced in the liver and then released into the blood circulation. In contrast, C1q is not synthesized by hepatocytes (Gulati et al., 1993), but predominantly by macrophages (Lu et al., 2007). The mRNA expression of mC1qL1 and mC1qL2 in liver was different with Gulati’s result although the expression levels were relative low. The expression of C1q in many other tissues, including brain (Depboylu et al., 2005), monocyte (Moosig et al., 2006) and endothelial cells (Cao et al., 2003), suggests that the expression of mC1qLs in different tissues was normal. 3.4. Expression profiles of mandarin fish mC1qL1 and mC1qL2 after ISKNV challenge In order to test whether mandarin fish mC1qLs may participate in defense against virus infection, the mandarin fish were challenged with ISKNV, and expression of mC1qLs after virus infection was determined. Usually, the mandarin fish showed no visible clinical sign of infection 5 days post-infection. At 7 days after ISKNV infection, some infected fish began to die, at 11 days post-infection, most infected fish died and some fish started recovering from infection at 13 days post-infection. We used PCR to detect ISKNV infection in the randomly sampled fish. To determine the expression of mC1qLs, RT-PCR was performed with RNA samples using specific primers
for mC1qLs, and 18S rRNA cDNA fragment amplified from the same RNA samples was used as an internal control. The expression level of 18S rRNAs was not changed obviously after ISKNV injection (data not shown). The expression levels of mC1qL1 and mC1qL2 were also not changed obviously in liver (Fig. 5A and B). In spleen, the expression of mC1qL1 did not change obviously during the first 13 days after ISKNV challenge, but increased to a relative high level (1.62 times compared to 0 day, p > 0.05) at 15 days postchallenge (Fig. 5A). However, the expression level of mC1qL2 reached a peak (1.9 times compared to 0 day, p < 0.05) at 11 days post-challenge and decreased fast to a relative low level (0.73 times compared to 0 day, p > 0.05) at 15 days postchallenge (Fig. 5B). The expression level of mC1qL2 changed significantly ( p < 0.05) after ISKNV infection. At 15 days post-challenge, some fish survived and recovered from the ISKNV infection. The relatively high expression level of mC1qL2 at 11 days and mC1qL1 at 15 days post-infection in spleen indicated that there were some relationships between mC1qLs and ISKNV infection in mandarin fish. Very little is known about virus/host recognition and the mechanism involved in antivirus in fish. It was reported that neutralization of some fish viruses, e.g. VHSV and IHNV, is complement-independent, and depends solely on the presence of specific antibodies against the virus (Ellis, 2001). However, studies have also shown that the neutralizing ability of rainbow trout antibodies against rhabdoviruses is dependent on the presence of complement (Boshra et al., 2006). In addition, many animal retroviruses activate the classical complement pathway independent of spe-
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Acknowledgements This research was supported by National Natural Science Foundation of China under grant Nos. 30325035 and U0631008; National Basic Research Program of China under grand No. 2006CB101802; National High Technology Program of China under grand No. 2006AA100309; Guangdong Province Natural Science Foundation under grant No. 20023002; Science and Technology Bureau of Guangdong Province. The nucleotide sequences of mandarin fish mC1qL1 and mC1qL2 cDNAs have been submitted to NCBI database and the GenBank accession numbers are EU131872 and EU131873. References
Fig. 5. The expression pattern of mC1qL1 and mC1qL2 in spleen during ISKNV infection by RT-PCR. There are three parallel samples from three separate fish at every time point. The amount of PCR amplification of mC1qLs and 18S rRNA were quantified. The ratio of mC1qLs to 18S rRNA was used as Y-coordinate. The X-coordinate shows the times. (A) The expression pattern of mC1qL1 after ISKNV infection; (B) the expression pattern of mC1qL2 with ISKNV infection. At 7 days after ISKNV infection, some infected fish began to die, at 11 days post-infection, most infected fish died and some fish started recovering from infection at 13 days post-infection.
cific antibodies (Ikeda et al., 1998). In this study, the expression level of mC1qLs changed after ISKNV infection, suggesting that the complement system may participate in the antivirus response. We do not know whether the classical or all the three complement pathways are activated after virus infection. Future work is to investigate the role of mandarin fish complement system in defense against ISKNV. In conclusion, we have cloned two C1q-like cDNAs from mandarin fish (mC1qL1 and mC1qL2), and they shared common features with mammalian C1q. Expression of both mC1qL1 and mC1qL2 mRNAs in mandarin fish were constitutive and maybe related to the infection of ISKNV.
Altschul, S.F., Gish, W., Myers, E., Lipman, D.J., 1990. Basic local alignment search tool. J. Mol. Biol. 215, 403–410. Arora, M., Munoz, E., Tenner, A.J., 2001. Identification of a site on mannan-binding lectin critical for enhancement of phagocytosis. J. Biol. Chem. 276, 43087–43094. Aubert, B., Chesne, S., Arlaud, G.J., Colomb, M.G., 1985. Antibody independent interaction between the first component of human complement, C1, and the outer membrane of Escherichia coli D31 m4. Biochem. J. 232, 513–519. Bohlson, S.S., Fraser, D.A., Tenner, A.J., 2007. Complement proteins C1q and MBL are pattern recognition molecules that signal immediate and long-term protective immune functions. Mol. Immunol. 44, 33–43. Boshra, H., Li, J., Sunyer, J.O., 2006. Recent advances on the complement system of teleost fish. Fish Shellfish Immunol. 20, 239–262. Butko, P., Nicholson-Weller, A., Wessels, M.R., 1999. Role of complement component C1q in the IgG-independent opsonophagocytosis of group B streptococcus. J. Immunol. 163, 2761–2768. Cao, W., Bobryshev, Y.V., Lord, R.S., Oakley, R.E., Lee, S.H., Lu, J., 2003. Dendritic cells in the arterial wall express C1q: potential significance in atherogenesis. Cardiovasc. Res. 60, 175–186. Chen, B., Gui, J.F., 2004. Identification of a novel C1q family member in color crucian carp (Carassius auratus) ovary. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 138, 285–293. ¨ , L., 2000. Deng, M., He, J.G., Zuo, T., Weng, S.P., Zeng, K., LU Infectious spleen and kidney necrosis virus (ISKNV) from Siniperca chuatsi: development of a PCR detection method and the new evidence of iridovirus. Chin. J. Virol. 16, 365–369 (abstract in English). Depboylu, C., Scha¨fer, M.K., Schwaeble, W.J., Reinhart, T.A., Maeda, H., Mitsuya, H., Damadzic, R., Rausch, D.M., Eiden, L.E., Weihe, E., 2005. Increase of C1q biosynthesis in brain microglia and macrophages during lentivirus infection in the rhesus macaque is sensitive to antiretroviral treatment with 6-chloro-20 ,30 -dideoxyguanosine. Neurobiol. Dis. 20 (1), 12–26. Dierich, M.P., Ebenbichler, C.F., Marschang, P., Fust, G., Thielens, N.M., Arlaud, G.J., 1993. HIV and human complement: mechanisms of interaction and biological implication. Immunol. Today 14, 435–440. Dodds, A.W., Petry, F., 1993. The phylogeny and evolution of the first component of complement, C1. Behring Inst. Mitt. 93, 87–102.
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complexes: C1r-independent activation of C1s by cardiolipin vesicles. J. Immunol. 138, 1864–1870. Kozak, M., 1986. Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 44, 283–292. Kumar, S., Tamura, K., Nei, M., 2004. MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief. Bioinform. 5, 150–163. Letunic, I., Copley, R.R., Schmidt, S., Ciccarelli, F.D., Doerks, T., Schultz, J., Ponting, C.P., Bork, P., 2004. SMART 40: towards genomic data integration. Nucleic Acids Res. 1, D142–D144. Lu, J., Wu, X., Teh, B.K., 2007. The regulatory roles of C1q. Immunobiology 212 (4/5), 245–252. Moosig, F., Damm, F., Knorr-Spahr, A., Ritgen, M., Zeuner, R.A., Kneba, M., Ernst, M., Schro¨der, J.O., 2006. Reduced expression of C1q-mRNA in monocytes from patients with systemic lupus erythematosus. Clin. Exp. Immunol. 146 (3), 409–416. Nielsen, H., Engelbrecht, J., Brunak, S., vonHeijne, G., 1997. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng. 10, 1–6. Nonaka, M., Smith, S.L., 2000. Complement system of bony and cartilaginous fish. Fish Shellfish Immunol. 10, 215–228. Rimoldi, M.T., Tenner, A.J., Bobak, D.A., Joiner, K.A., 1989. Complement component C1q enhances invasion of human mononuclear phagocytes and fibroblasts by Trypanosoma cruzi trypomastigotes. J. Clin. Invest. 84, 1982–1989. Saitou, N., Nei, M., 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. 4, 406–425. Sellar, G.C., Blakes, D.J., Reid, K.B.M., 1991. Characterization and organization of the genes encoding the A-B- and C-chains of human complement subcomponent C1q. The complete derived amino acid sequence of human C1q. Biochem. J. 274, 481–490. Smith, S.L., 1998. Shark complement: an assessment. Immunol. Rev. 166, 67–78. Sunyer, J.O., Lambris, J.D., 1998. Evolution and diversity of the complement system of poikilothermic vertebrates. Immunol. Rev. 166, 39–57. Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., Higgins, D.G., 1997. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25, 4876–4882. Zeng, K., He, J.G., Weng, S.P., Huang, Z.J., Hou, K.T., Luo, J.R., Huang, W.P., Chen, J.H., 1999. Transmission, host range, temperature sensibility of infectious spleen and kidney necrosis (ISKNV) virus from Siniperca chuatsi. Virol. Sin. 14, 353–357.
Veterinary Immunology and Immunopathology 125 (2008) 37–46 www.elsevier.com/locate/vetimm
Molecular cloning of two C1q-like cDNAs in mandarin fish Siniperca chuatsi Hai-Hua Lao a,b, Ya-Nan Sun a, Zhi-Xin Yin a,c, Jing Wang a, Chao Chen a, Shao-Ping Weng a, Wei He a, Chang-Jun Guo a, Xian-De Huang a, Xiao-Qiang Yu d,*, Jian-Guo He a,** a
State Key Laboratory for Biocontrol, School of Life Sciences, Sun Yat-sen University, 135 Xingang Road West, Guangzhou 510275, PR China b Key Laboratory of Tropical & Subtropical Fish Breeding & Cultivation of CAFS, Pearl River Fisheries Research Institute, 1 Xingyu Road, Guangzhou 510380, PR China c Institute of Genetic Engineering, Southern Medical University, Guangzhou 510515, PR China d Division of Cell Biology and Biophysics, School of Biological Sciences, University of Missouri-Kansas City, Kansas City, MO 64110, USA Received 25 October 2007; received in revised form 30 April 2008; accepted 8 May 2008
Abstract C1q, a subunit of the C1 complex, plays a key role in the recognition of immune complexes to initiate the classical complement pathway. In this study, we reported two C1q-like cDNAs from mandarin fish (Siniperca chuatsi), mC1q-like-1 (mC1qL1) and mC1q-like-2 (mC1qL2). The full-length cDNA of mC1qL1was 990 bp, containing a 71 bp 50 -untranslated region (UTR), an open reading frame (ORF) of 723 bp, and a 196 bp long 30 -UTR. mC1qL2 cDNA was 1193 bp, containing a 100 bp 50 -UTR, followed by an ORF of 756 bp and a 30 -UTR of 337 bp. mC1qL1 and mC1qL2 share 29% identity in amino acid sequence. Both mC1qL1 and mC1qL2 contained three parts: a short amino-terminal region, a collagen-like region and a carboxyl-terminal globular C1q domain. The phylogenetic analysis showed that mC1qL1 clustered with two Danio rerio hypothetical proteins and further grouped with C1q proteins, while mC1qL2 clustered with C1qA proteins from other species. In healthy mandarin fish, mC1qL1 and mC1qL2 were expressed in all tissues tested, including liver, spleen, head kidney, caudal kidney, intestine and gill. mC1qL1 was highly expressed in head kidney, while mC1qL2 was mainly expressed in spleen. The expression level of mC1qL1 and mC1qL2 in liver were not changed obviously and mC1qL2 was significantly changed ( p < 0.05) in spleen after infectious spleen and kidney necrosis virus (ISKNV) infection. Mandarin fish C1q may play a role in response to ISKNV infection. # 2008 Elsevier B.V. All rights reserved. Keywords: mC1qL1; mC1qL2; Complement; Siniperca chuats; Tissue expression; ISKNV
1. Introduction
* Corresponding author. Tel.: +1 816 235 6379; fax: +1 816 235 1503. ** Corresponding author. Tel.: +86 20 84110976; fax: +86 20 84113819. E-mail addresses: [email protected] (X.-Q. Yu), [email protected] (J.-G. He). 0165-2427/$ – see front matter # 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2008.05.004
Mandarin fish, Siniperca chuatsi (Basilewsky), is a main economic cultured fish in China. With the rapid development of mandarin fish aquaculture, diseases, especially the viral disease, have become a major constraint and the most limiting factor in the mandarin fish culture industry. ISKNV (infectious spleen and
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kidney necrosis virus), a member of the Iridoviridae family, has caused high mortalities in mandarin fish and limited largely the mandarin fish cultures in China (He et al., 1998, 2000; Deng et al., 2000). The complement system, a family of more than 35 soluble serum proteins, plays essential roles in innate and adaptive immunity in host defense (Nonaka and Smith, 2000; Boshra et al., 2006). Activation of the complement system also contributes significantly to the orchestration and development of acquired immune responses (Boshra et al., 2006). The complement system can be activated through three pathways: the antibody-dependent classical pathway, the antibodyindependent alternative pathway, and the lectin pathway (Sunyer and Lambris, 1998). The classical complement pathway (CCP) is a major defense and clearance system in the blood circulation. It is activated at the level of C1q, a subcomponent of the C1 complex, by both immune complexes that react with the C1q globular region and nonimmune substances that react with the C1q collagen-like region (Kishore and Reid, 1999; Jiang et al., 1992). Fish possess complement pathways similar to those in mammals, and fish complement proteins identified thus far show highly homologies to their mammalian counterparts (Holland and Lambris, 2002). The human C1q molecule is composed of 18 polypeptide chains (six each of A-, B- and C-chains). Each of these chains has a short amino-terminal region, followed by a collagen-like region (CLR, repeating Gly-X-Y triplets, where X is often proline and Y is often hydroxylysine or hydroxyproline) and a carboxyterminal globular head region (Sellar et al., 1991). Every C1q chain also has four conserved cysteines, which can form inter- or intra-chain disulfide bridges that play an important role in formation of intact C1q molecules. C1q, the key component of the classical complement pathway in antimicrobial defense, can trigger rapid enhanced phagocytosis resulting in clearance of cellular debris, apoptotic cells and immune complexes, phagocytosis of bacteria, neutralization of retroviruses, and modulation of dendritic cells (DCs), B cells and fibroblasts (Bohlson et al., 2007; Kishore et al., 2004). Direct binding of C1q to a wide range of pathogens including viruses (Dierich et al., 1993; Kojouharova et al., 2003), parasites (Rimoldi et al., 1989), and both gram positive (Butko et al., 1999) and gram negative (Aubert et al., 1985; Kovacsovics et al., 1987) bacteria, has been reported. C1q is a versatile recognition protein and a major connecting link between classical pathway-driven innate immunity and acquired immunity (Kishore and Reid, 2000).
The complement system has been studied extensively in mammals, but considerably less known in lower vertebrates, particularly in teleost fish. The C1q just has been identified in zebrafish (Danio rerio) and Tetraodon nigroviridis because their full genomic DNA sequences are available. The full-length cDNA sequences of C1q in common carp (Cyprinus carpio) (BAD22535) and gibel carp (Carassius auratus gibelio) (AY662672) and an EST sequence (CV823415) similar to complement C1q subcomponent in killifish (Fundulus heteroclitus) can be found in the GenBank. Fish C1q has been reported in nurse shark (Ginglymostoma cirratum) (Smith, 1998) and channel catfish (Ictalurus punctatus) (Dodds and Petry, 1993), but the full-length cDNAs or genes are not available. In addition, full-length cDNA sequence containing the C1q globular domain but lacking the collagen domain has been cloned in color crucian carp (Carassius auratus color variety) (AY583317), and this C1q-like gene has unique functions in oogenesis, oocyte maturation and egg fertilization (Chen and Gui, 2004). To our knowledge, there is no relevant paper on fulllength fish C1q. Mandarin fish is an important economic fish in China. The breakouts of ISKNV do threaten the culture of mandarin fish and cause large loss every year. To understand the relationship between the complement system and ISKNV, here we report identification of two full-length C1q-like cDNAs in mandarin fish (S. chuatsi). Both mC1qL1 and mC1qL2 shared common features with mammalian C1q. The phylogenetic analysis showed that mC1qL2 were clustered with mammalian C1qAs and mC1qL1 clustered with zebrafish C1q-domain containing protein and further grouped with mammalian C1qs. mC1qL1 and mC1qL2 were expressed constitutively in all tissues tested. Both mC1qL1 and mC1qL2 were expressed at the lowest level 7 days after fish were challenged with ISKNV, while the ISKNV infected fish began to die at 7 days post-infection. The expression pattern of mC1qLs suggests that mandarin fish C1q may play a role in response to ISKNV infection. 2. Materials and methods 2.1. RNA extraction Three healthy mandarin fish, about 500 g, were obtained from fish farm in Nan Hai (Guangdong Province, China) and acclimated for 2 weeks. The fish were assured free of ISKNV infection by PCR (Deng et al., 2000). Total RNA was extracted from fresh liver
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of mandarin fish using SV Total RNA isolation Kit (Promega, USA). The quality and concentration were determined by agarose gel electrophoresis and spectrophotometer (Amersham, Sweden). 2.2. Cloning of C1q cDNA sequences from mandarin fish liver Degenerate oligonucleotide primers, C1qL1-R1 (50 -GTGATAACTGAAGAAAATAGACGCC-30 ) for mC1qL1 50 rapid amplification of cDNA ends (RACE), C1qL2-F1 (50 -TCAGGTGGCGTGGTCTTACAGC-30 ) and C1qL2-R1 (50 -ACAGACACATGCTGACCTTGGC-30 ) for the 30 and 50 RACE of mC1qL2, were designed based on two different EST sequences obtained previously from mandarin fish in our lab (AY909459, and another EST sequence has not been submitted in GenBank Database). The 30 and 50 end cDNA sequences of mandarin fish mC1qL1 and mC1qL2 were obtained by RACE using total RNA from mandarin fish liver as template. cDNA templates for the 50 and 30 RACE were synthesized using the BD SMARTTM RACE cDNA amplification kit (Clontech, USA) following the manufacture’s instruction. All the amplified fragments were cloned into pMD19-T vector (TaKaRa, Japan) and sequenced. The nucleotide sequences were determined from both strands by the dideoxy-chain termination method using a model 3730 sequencer. Then two fulllength cDNAs were obtained by aligning the overlapped fragments and the EST sequences. 2.3. Sequence analysis and alignment Homology searches and identity/similarity assessment were performed by BLAST at web servers of National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/blast) (Altschul et al., 1990). The signal peptide cleavage site was predicted with signalP 3.0 service (http://www.cbs.dtu.dk/ services/SignalP/) (Nielsen et al., 1997). SMART (http://smart.embl-heidelberg.de/) (Letunic et al., 2004) was used for protein domain determination and characterization. The predicted amino acid sequences of mC1qLs were multiple aligned with Fig. 1. Nucleotide and deduced amino acid sequences of mC1qL1 (A) and mC1qL2 (B) from Siniperca chuatsi. Amino acid sequence is shown with one-letter codes above the nucleotide sequence. The initiation codon (ATG) and the stop codon (TAG) are highlighted in bold. The predicted signal peptide is underlined (—). The Kozak sequence is marked by undee line ( ). The globular C1q domain is shown in gray. The two potential N-linked glycosylation sites in
mC1qL1 are shown in dot underlined ( ). The repeating triplet Glycin-X-Y is double underlined ( ). The conserved cysteine residues are boxed, a free one is in italic. The GE(K/Q/R)GEP motif in collagen-like region is shown in gray and double underlined ( ). The RGD sequence in collagen-like region of mC1qL2 is boxed. The residues for Ca2+ binding are undee lined ( ). The putative polyadenylation signals were shown in dotted underlined ( ).
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Fig. 2. Multiple alignment of mC1qLs with other C1qs. The C1q and C1q-like (C1qL) proteins from different organisms were aligned using ClusterX V1.83. Identical or highly conserved residues are shaded in black, while similar residues are shaded in grey. Sequences for the alignment are obtained from GenBank: cC1qA (AAI05346), cC1qB (AAI12491), cC1qC (AAI14894), ccC1qL (AAS93680), gcC1qL (AAT76300), chC1q (NP_996874), cpC1qL (BAD22535), dC1qA (XP_535367), dC1qB (XP_544507), dC1qC (XP_544508), fC1qC (XP_417653), fC1qB (XP_425756), hC1qA (AAH71986), hC1qB (AAH08983), hC1qC (AAP97191), hC1qL2 (NP_872334), lC1qL (BAD22833), mkC1qA (XP_001101933), mkC1qB (XP_001110783), mkC1qC (XP_001102112), msC1qA (NP_031598), msC1qB (AAH67001), msC1qC (NP_031600), msC1qL1 (NP_035925), pC1qA (AAR20892), pC1qB (AAR20888), pC1qC (AAR20893), PtC1qA (XP_001166821), PtC1qC (XP_513188), rC1qA (AAH86605), rC1qB (CAA50440), rC1qC (AAH86409), rbC1qB (AAC61578), TnC1qD (CAF95737), zC1qD (NP_001003482), zC1qD-1 (NP_001018363), and zC1qD-2 (XP_697055). Abbreviations are A, alpha; B, beta; C, gamma; D, domain; L, like;
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C1qs from other organisms using the Clustal X V1.83 (Thompson et al., 1997). Phylogenetic tree was constructed based on the full-length amino acid sequences of C1q and C1q-related proteins using the neighbor-joining (NJ) algorithm (Saitou and Nei, 1987) within MEGA V3.1 (Kumar et al., 2004). The branch points of phylogenetic tree were validated by 1000 bootstrap replications. 2.4. Expression pattern of mC1qL1 and mC1qL2 by RT-PCR and Northern blotting analyses To determine the tissue expression profiles of mandarin fish mC1qL1 and mC1qL2, semi-quantitative RT-PCR and Northern blotting were performed using equal amounts of total RNA from various tissues of healthy mandarin fish, including liver, spleen, head kidney, caudal kidney, intestine and gill. The total RNAs were extracted using TRIzol reagent (Invitrogen, USA) and quantified. The first strand cDNAs were synthesized using RTase (promega, USA) and oligo-dT and random primer (9 bp) (Sangon, China) as primers. The PCR was carried out using C1qL1-F2 (50 -AACGGCATAGTCGGTCCCAAGG-30 ) and C1qL1-R1 for mC1qL1, C1qL2F4 (50 -CCTGGAGCAAAGGGAGAGAAAG-30 ) and C1qL2-R2 (50 -CTGTAAGACCACGCCACCTGAC-30 ) for mC1qL2. The 18S rRNA amplified using 18SF (50 ATGGTACTTTAGGCGCCTAC-30 ) and 18SR (50 -TATACGCTATTGGAGCTGG-30 ), was used as an internal control. The RNAs from two individual fish were used for RT-PCR. PCR was conducted according to the following program: 94 8C for 2 min, 29 cycles of 94 8C for 30 s, 55 8C for 30 s, 72 8C for 30 s, followed by a final extension at 72 8C for 10 min. PCR products were analyzed by electrophoresis on a 1.5% agarose gel. Northern blotting was also performed. RNA samples from three individual fish were mixed and electrophoresed according to the protocol of NorthernMax1-Gly (Ambion, USA). The antisense RNA probes were labeled by Dig RNA Labeling Kit (Roche, USA). Hybridization and immunological detection were performed using Dig Northern Starter Kit (Roche, USA). The signals were captured by GeneSnap chemical lighting assay system. The density of the 18S rRNA of every sample before transferred to membrane and the hybridization signals were scanned. The density ratios of the hybridization signals to the 18S rRNA were calculated as the relative expression level of each tissue.
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2.5. Expression of mC1qLs during ISKNV infection One hundred mandarin fish, 100 20 g, were bought and acclimated for 2 weeks before ISKNV challenge. The virus filtrates were prepared and injected into the fish intraperitoneally (i.p.) according to Zeng’s method (Zeng et al., 1999). Tissue samples were harvested before infection (0 day) and after infection at 1, 3, 5, 7, 9, 11, 13 and 15 days. Three parallel samples from three separate fish were collected at each time point for detection by PCR using ISKNV specific primers to confirm that the mandarin fish were surely infected with ISKNV. We sampled fish randomly at first 5 days when fish did not show visible clinical sign of infection. After 7 days of infection, we sampled the normal fish (no clinical sign) and moribund fish at each time point for detection. The total RNAs were extracted separately using SV total RNA Isolation Kit (Promega, USA) and quantified. The RTPCR was performed using the same primers and PCR conditions were the same as described above. We selected spleen and liver for the experiment because spleen was one of the main tissues of ISKNV infection (He et al., 1998) and liver was used as a control, which was not the main target tissue of ISKNV infection. The data analysis was carried out with Kodak Digital Science system. All statistics were performed using SPSS program to interpret the quantitative data after ISKNV injection. Significance of differences was analyzed by one-way ANOVA followed by Bonferroni’s post hoc adjustment. 3. Results and discussion 3.1. Cloning and sequence analysis of the full-length cDNAs encoding the mandarin fish C1qL1 and C1qL2 By 50 and 30 RACE amplification using degenerated primers, two full-length mandarin fish C1q-like cDNAs (mC1qL1 and mC1qL2) were obtained. mC1qL1 was 990 bp, containing a 71 bp 50 -UTR, a 723 bp ORF, and a 196 bp 30 -UTR with a putative polyadenylation signal and a poly(A) tail. The ORF of mC1qL1 encodes a protein of 240 amino acids, which includes a putative signal peptide of 23 amino acids (Fig. 1A). mC1qL2 was 1193 bp, containing a 100 bp 50 -UTR, followed by an ORF of 756 bp encoding a protein of 251 amino acid residues with a putative signal peptide of 22 amino acids, and a 30 -UTR of 337 bp with two putative polyadenyla-
c, Bos Taurus; cc, Carassius auratus color variety; ch, Gallus gallus; cp, Cyprinus carpio; d, Canis familiaris; f, Gallus gallus (red jungle fowl); gc, (Carassius auratus gibolio); h, Homo sapiens; l, Lethenteron japonicum; m, Siniperca chuatsi; mk, Macaca mulatta; ms, Mus musculus; p, Sus scrofa; Pt, Pan troglodyte; rb, Oryctolagus cuniculus; r, Rattus norvegicus; Tn, Tetraodon nigroviridis; z, Danio rerio.
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tion signals and a poly(A) tail (Fig. 1B). The mature peptides of both mC1qL1 and mC1qL2 are composed of three parts: a short amino-terminal region, a CLR region containing repeating triplet of Gly-X-Y, and a globular C1q domain. The domain organization of mC1qLs is characteristic of C1q proteins (Bohlson et al., 2007). In the C1q proteins, the Glycin residue in Gly-X-Y triplets is very conserved (Fig. 2). The number of Gly-X-Y repeats can range from 14 to well over 100 copies (Innamorati et al., 2006). In mC1qL1 and mC1qL2, the CLR included 24 and 22 Gly-X-Y repeats, respectively. Both mC1qL1 and mC1qL2 contain four cysteines, which are conserved in most C1q molecules (Fig. 2). The first cysteine participates in formation of inter-chain disulfide bridge in human C1q to yield A–B and C–C subunits. The second cysteine was free thiol group, which can be involved in the formation of disulphide bonds between C1q and IgG in immune complexes (Sellar et al., 1991). The third and fourth cysteine residues are considered to form an intrachain disulfide bond (Kishore et al., 2003). The Kozak sequence, (A/G)NNATGG, which is recognized by ribosomes as the translational start site and thus required for protein expression (Kozak, 1986), was also present within both mC1qL1 and mC1qL2 cDNA sequences. There were two N-linked glycosylation sites in mC1qL1, while mC1qL2 had a RGD motif, predicted by the SCAN PROSITE program (www.expasy.ch/prosite/). In mC1qL2 CLR, there was a motif, GEKGEP, which is also present in human mannose-binding lectin (MBL) and other defense collagens to stimulate phagocytic activity (Arora et al., 2001; Bohlson et al., 2007). mC1qL1 had a similar motif, GEKGDP, in which Glu(E) was substituted by an Asp(D) at the crucial position. C1q is a hexamer of trimers, each trimer contains three distinct polypeptide chains. It appears that only one such motif per trimer is required for induction of the function (Arora et al., 2001). We speculate that mandarin fish mC1qLs may possess the function to stimulate phagocytic activity. 3.2. Multiple sequence alignment and phylogenetic tree construction of C1q sequences mC1qL1 and mC1qL2 were similar to C1q sequences from other species by searching similarity regions in NCBI. The deduced amino acid sequences of mandarin fish mC1qL1 and mC1qL2 were aligned with the amino acid sequences of C1q from various species obtained from GenBank (Fig. 2). The identity between mC1qL1 and mC1qL2 was low (29%). mCqL1 exhibits highest similarity with Mus musculus C1qB (36.4% identity). mC1qL2 is highly identical to an unnamed protein from T. nigroviridis (CAF95737) (55.3%). The homology
Table 1 The homology between mC1qLs, other C1q and C1q-related proteins
mC1qL1 Mouse C1qB Human C1qB Zebrafish C1q domain Cattle C1qB Rat C1qB Dog C1qB Rat C1qC Tetraodon nigroviridis unnamed protein Fowl C1qC Rat C1qA Dog C1qA Cattle C1qC Zebrafish C1q domain protein Human C1qA Pig C1qA Monkey C1qA Cattle C1qA Mouse acrp30 Human clusterin Human collagen I-a1 Human precebellin Mouse clusterin
mC1qL1 (%)
mC1qL2 (%)
– 36.4 35.7 35.4 34.9 34.6 34.5 34.5 27.3
29.0 31.9 31.6 25.7 30.8 33.8 33.1 33.6 55.3
33.6 30.3 28.2 33.2 30.4 29.5 28.4 29.9 29.7 29.9 6.7 3.8 18 6.4
37.5 36.7 35.7 35.4 35.0 34.9 34.6 34.5 34.2 25.6 6.0 4.2 16.8 4.2
among mC1qLs and other C1q or C1q-related proteins is shown in Table 1. The homologies among mC1qLs and other C1q proteins were higher than those of mC1qLs and C1q-related proteins, including collagen, clusterin, Acrp30 and precebellin. mC1qLs should belong to C1q family although the homology between mC1qLs and other C1qs was low compared to the high identity (73%) of the three C1q subunits from human and rat. A phylogenetic tree was constructed using fulllength amino acid sequences of C1q and C1q-related proteins from various species by the NJ method (Fig. 3). The three subunits, A, B and C, of mammalian C1q clustered very well. mC1qL1 was clustered with two zebrafish hypothetical proteins containing C1q domain (NP_001003482 and NP_001018383) and further grouped with C1q proteins from other species. mC1qL2 was clustered with T. nigroviridis complement component C1q domain (CAF95737) and further clustered with C1qA proteins from other species. mC1qL2 may be the counterpart of C1qA of mammals. 3.3. Tissue distribution of mandarin fish mC1qL1 and mC1qL2 mRNAs In healthy mandarin fish, semi-quantitative RTPCR and Northern blotting showed that both mC1qL1
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Fig. 3. The phylogenetic tree of C1qs. The full-length amino acid sequences of C1qs, C1qLs and other proteins with C1q domain from different organisms were aligned using the ClusterX V1.83 program (Thompson et al., 1997). The phylogenetic tree was constructed using Bootstrap N-J method of MEGA3. Amino acid sequences are obtained from the GenBank as described in Fig. 2 legend. The added proteins (compared to Fig. 2) and their accession numbers were shown in Fig. 3.
and mC1qL2 mRNAs were expressed in all the tissues tested, including liver, spleen, head kidney, caudal kidney, intestine and gill (Fig. 4A and B). mC1qL1 mRNA expression level was the lowest in liver.
Compared with the expression level in liver, the expression level of mC1qL1 was high in head kidney (8.7 times), followed by intestine (6.7 times), gill (5.9 times), caudal kidney (3.7 times) and spleen (2.9
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Fig. 4. The expression of mandarin fish mC1qLs in tissues. The expression pattern of mC1qLs in tissues was detected by RT-PCR (A) and Northern blot (B). mC1qL1 and mC1qL2 were expressed in all tissues tested. (A) The RT-PCR results. (B) The Northern blot results. The numbers under (A) are the ratio of the expression of mC1qLs to 18S. The numbers under (B) are the results of density scanned. L: liver; S: spleen; HK: head kidney; CK: caudal kidney; I: intestine; G: gill.
times). mC1qL2 was also expressed at low level in liver, and its expression level was high in spleen (8.9 times), followed by head kidney (7.6 times), intestine (6.3 times), gill (4.6 times) and caudal kidney (2.7 times) compared to the expression level in liver. The expression level of mC1qL1 and mC1qL2 among different tissues are significantly different ( p < 0.05). Most complement proteins are produced in the liver and then released into the blood circulation. In contrast, C1q is not synthesized by hepatocytes (Gulati et al., 1993), but predominantly by macrophages (Lu et al., 2007). The mRNA expression of mC1qL1 and mC1qL2 in liver was different with Gulati’s result although the expression levels were relative low. The expression of C1q in many other tissues, including brain (Depboylu et al., 2005), monocyte (Moosig et al., 2006) and endothelial cells (Cao et al., 2003), suggests that the expression of mC1qLs in different tissues was normal. 3.4. Expression profiles of mandarin fish mC1qL1 and mC1qL2 after ISKNV challenge In order to test whether mandarin fish mC1qLs may participate in defense against virus infection, the mandarin fish were challenged with ISKNV, and expression of mC1qLs after virus infection was determined. Usually, the mandarin fish showed no visible clinical sign of infection 5 days post-infection. At 7 days after ISKNV infection, some infected fish began to die, at 11 days post-infection, most infected fish died and some fish started recovering from infection at 13 days post-infection. We used PCR to detect ISKNV infection in the randomly sampled fish. To determine the expression of mC1qLs, RT-PCR was performed with RNA samples using specific primers
for mC1qLs, and 18S rRNA cDNA fragment amplified from the same RNA samples was used as an internal control. The expression level of 18S rRNAs was not changed obviously after ISKNV injection (data not shown). The expression levels of mC1qL1 and mC1qL2 were also not changed obviously in liver (Fig. 5A and B). In spleen, the expression of mC1qL1 did not change obviously during the first 13 days after ISKNV challenge, but increased to a relative high level (1.62 times compared to 0 day, p > 0.05) at 15 days postchallenge (Fig. 5A). However, the expression level of mC1qL2 reached a peak (1.9 times compared to 0 day, p < 0.05) at 11 days post-challenge and decreased fast to a relative low level (0.73 times compared to 0 day, p > 0.05) at 15 days postchallenge (Fig. 5B). The expression level of mC1qL2 changed significantly ( p < 0.05) after ISKNV infection. At 15 days post-challenge, some fish survived and recovered from the ISKNV infection. The relatively high expression level of mC1qL2 at 11 days and mC1qL1 at 15 days post-infection in spleen indicated that there were some relationships between mC1qLs and ISKNV infection in mandarin fish. Very little is known about virus/host recognition and the mechanism involved in antivirus in fish. It was reported that neutralization of some fish viruses, e.g. VHSV and IHNV, is complement-independent, and depends solely on the presence of specific antibodies against the virus (Ellis, 2001). However, studies have also shown that the neutralizing ability of rainbow trout antibodies against rhabdoviruses is dependent on the presence of complement (Boshra et al., 2006). In addition, many animal retroviruses activate the classical complement pathway independent of spe-
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Acknowledgements This research was supported by National Natural Science Foundation of China under grant Nos. 30325035 and U0631008; National Basic Research Program of China under grand No. 2006CB101802; National High Technology Program of China under grand No. 2006AA100309; Guangdong Province Natural Science Foundation under grant No. 20023002; Science and Technology Bureau of Guangdong Province. The nucleotide sequences of mandarin fish mC1qL1 and mC1qL2 cDNAs have been submitted to NCBI database and the GenBank accession numbers are EU131872 and EU131873. References
Fig. 5. The expression pattern of mC1qL1 and mC1qL2 in spleen during ISKNV infection by RT-PCR. There are three parallel samples from three separate fish at every time point. The amount of PCR amplification of mC1qLs and 18S rRNA were quantified. The ratio of mC1qLs to 18S rRNA was used as Y-coordinate. The X-coordinate shows the times. (A) The expression pattern of mC1qL1 after ISKNV infection; (B) the expression pattern of mC1qL2 with ISKNV infection. At 7 days after ISKNV infection, some infected fish began to die, at 11 days post-infection, most infected fish died and some fish started recovering from infection at 13 days post-infection.
cific antibodies (Ikeda et al., 1998). In this study, the expression level of mC1qLs changed after ISKNV infection, suggesting that the complement system may participate in the antivirus response. We do not know whether the classical or all the three complement pathways are activated after virus infection. Future work is to investigate the role of mandarin fish complement system in defense against ISKNV. In conclusion, we have cloned two C1q-like cDNAs from mandarin fish (mC1qL1 and mC1qL2), and they shared common features with mammalian C1q. Expression of both mC1qL1 and mC1qL2 mRNAs in mandarin fish were constitutive and maybe related to the infection of ISKNV.
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