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A novel tissue inhibitor of metalloproteinase in blood clam Tegillarca granosa: Molecular cloning, tissue distribution and expression analysis
Fish & Shellfish Immunology 33 (2012) 645e651
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A novel tissue inhibitor of metalloproteinase in blood clam Tegillarca granosa: Molecular cloning, tissue distribution and expression analysis Qing Wang a, b, Yongbo Bao a, *, Lihui Huo c, Hailong Gu a, b, Zhihua Lin a, * a
College of Biological & Environmental Sciences, Zhejiang Wanli University, 8 South Qianhu Road, Ningbo, Zhejiang 315100, PR China College of Fisheries and Life Sciences, Shanghai Ocean University, Shanghai 201306, China c College of Life Sciences and Bioengineering, Ningbo University, Ningbo 315211, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 February 2012 Received in revised form 1 June 2012 Accepted 26 June 2012 Available online 4 July 2012
Tissue inhibitor of metalloproteinases (TIMPs) were originally characterized as inhibitors of matrix metalloproteinases (MMPs), but their range of activities has been found to be broader as it includes the inhibition of several of the MMPs, etc. The cDNA encoding TIMP-4-like gene from blood clam Tegillarca granosa (designated as Tg-TIMP-4-like) which is the first tissue inhibitor of metalloproteinase identified in blood clams, was cloned and characterized. It was of 1164 bp, and an open reading frame (ORF) of 666 bp encoding a putative protein of 222 amino acids. The predicted amino acid sequence comprised all recognized functional domains found in other TIMP homologues and showed the highest (30.56%) identity to the TIMP-1.3 from Crassostrea gigas. Several highly conserved motifs including several TIMP signatures, amino acid residue Cys30 responsible for coordinating the metal ions, the Cys-X-Cys motif and the putative NTR (netrin) domain were almost completely conserved in the deduced amino acid of TgTIMP-4 like, which indicated that Tg-TIMP-4-like should be a member of the TIMP family. The mRNA expression of Tg-TIMP-4-like in the tissues of mantle, adductor muscle, foot, gill, hemocyte and hepatopancreas was examined by quantitative real-time PCR (qT-PCR) and mRNA transcripts of Tg-TIMP-4like were mainly detected in hemocyte, and weakly detected in the other tissues. We also observed that Tg-TIMP-4 like mRNA accumulated significantly during Vibrio parahaemolyticus, Peptidogylcan (PGN) and Lipopolysaccharide (LPS) challenge, whereas the timing and quantitative differences of mRNA expression against different challenge indicated that Tg-TIMP-4-like may play a pivotal role in mollusc defense mechanisms. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Tegillarca granosa Tissue inhibitors of metalloproteinases Immune response mRNA expression
1. Introduction Tissue inhibitors of metalloproteinases (TIMPs) are known widely as secreted multifunctional proteins that are vital to the regulation of ECM (extracellular matrix) metabolism. Variation in ECM composition is crucial for embryonic development, morphogenesis, and tissue remodeling and repair [1]. The major proteinases related to ECM catabolism are the matrix metalloproteinases (MMPs) [2], the ADAMs (a disintegrin and metalloproteinase) [3], and the ADAMTS [4] (a disintegrin and metalloproteinase with thrombospondin domains) metalloproteinases. The activities of these metalloproteinases are precisely regulated under physiologic conditions at the levels of transcription, zymogen activation, and inhibition by endogenous inhibitors. Thus, demolishing the balance * Corresponding authors. Tel./fax: þ86 574 88222015. E-mail addresses: [email protected] (Y. Bao), [email protected] (Z. Lin). 1050-4648/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fsi.2012.06.021
between the production of active enzymes and their inhibitiors may cause diseases which are associated with uncontrolled ECM turnover, inflammation, cell growth and migration, such as arthritis, cardiovascular disease, cancer, pulmonary disease, nephritis, neurological disorders and tissue ulceration [5]. TIMPs are endogenous inhibitors of these metalloproteinases and are accordingly important regulators of ECM turnover, tissue remodeling and cellular behaviors. The inhibitory activity of TIMP-1 was found in the early 1970s as a collagenase inhibitor in the media of cultured human skin fibroblasts [6], in bovine cartilage extracts, and aorta [7]. TIMPs are composed of two functional domains, an amino terminal (Nterminal) inhibitory domain and a carboxyl terminal (C-terminal) domain. The N-terminal domain is involved in metalloproteinase inhibition, whereas the C-terminal domain has sevral specific functions, including binding to MMPs and pro-MMP [8]. In human, the TIMP family consists of four members (TIMP-1 to -4) with high sequence identity and structural homology but with different
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tissue expression, regulation and inhibitory characteristics [9,10]. This restricted expression dictates the specific physiologic functions among the four TIMP members. In addition to their metalloproteinase inhibitory activity, TIMPs have other physiologic functions, including anti-angiogenesis [11], pro- and anti-apoptotic signaling [12], synaptic plasticity [13e15] and promoting cell proliferation [16], many of which are independent of the metalloproteinase inhibition pathway. TIMP genes are also found in invertebrates, such as Drosophila melanogaster [17] and Crassostrea gigas [18]. The first TIMP gene identified in mollusks was isolated in C. gigas, and the Cg-TIMPs are considered to be closely related to oyster ontogenesis, wound healing and defense mechanisms [18]. Because of the lack of adaptive immunity, invertebrates diminutively rely on innate immune responses to defend themselves efficiently against various pathogens [19,20]. The ECM components also function as docking sites responsible for microbial pathogen invasion [21]. Actually, many molecules that are produced by microorganisms have been identified through their intervention in ECM modifications. It is thought that these modifications are the necessary and primary events in the pathogenic mechanism of many infections. For example, bacterial proteases are reponsible for tissue damage and bacterial spread across tissue barriers, possibly as a result of the predicted function of latent MMP activation or protease inhibitor deactivation [22,23]. The extracellular proteases (ECP) produced by eastern oyster pathogen Perkinsus marinus could compromise the oyster’s immune response [24] and promote the protozoan’s ability to propagate during the pathogen infection [25,26]. TIMP research in mollusk helps reveal the mollusc immune mechanism. However, knowledge about TIMP in mollusks is still limited, and studies of molecular features and immune response of TIMP-4-like protein from commercial mollusk Tegillarca granosa are rare. TIMP-4 is the most recently identified and least studied member of the TIMP family: it was initially cloned in humans [27] and then in mice [28]. The main objectives of this study were: 1) to clone and characterize the full-length cDNA of the TIMP-4-like gene from bloody clam T. granosa; 2) to investigate the mRNA expression level of Tg-TIMP-4-like protein in different tissues; and 3) to examine the temporal expression profile of the Tg-TIMP-4-like transcript in hemocytes after live bacterial, LPS and PGN challenge. 2. Materials and methods 2.1. Animals, immune challenge Two-year to three-year old blood clams were collected from a commercial farm (Ningbo, China) and kept in seawater at 26 1.0 C in temperature and 30& in salinity. To minimize individual variability, at least 60 clams were used in each experiment. In Vibrio parahaemolyticus challenge group, for each individual, 20 ml of live V. parahaemolyticus suspended in PBS (2 108 cfu/ml, pH 7.2) was injected in the adductor muscle. Four individuals were randomly sampled at 1.5, 3, 6, 12, 24, and 48 h after injection. The LPS (Sigma, USA) and PGN (Sigma, USA) challenge group clams were injected with 20 ml 0.2 mg/ml drugs (diluted in 0.85% NaCl) per individual respectively following the procedures mentioned above. Unchallenged clams and 60 clams injected with 20 ml PBS buffer were used as the time 0 group (at 0 h) and the control group, respectively. 2.2. Tissues withdrawal, total RNA extraction and cDNA synthesis The 0.5 ml hemocyte pellets per individual from the blank, control and treated groups were collected, centrifuged (1000 g, 10 min, 4 C) immediately and were washed in sterile seawater. The healthy clam’s tissues (mantle, hepatopancreas, foot, adductor
muscle, gill, and hemocytes) were collected from several clams to research on the tissue expression of Tg-TIMP-4-like. Tissues were washed by sterile seawater firstly, then cut into small pieces. Total RNA was extracted from tissues using Trizol Reagent (Invitrogen, USA) following the protocol and then treated with DNase I (Promega, USA). The treated total RNA was reverse transcribed using MMLV reverse transcriptase (Promega, USA) and Oligo(dT) primer. 2.3. Cloning of the full-length cDNA of Tg-TIMP-4-like gene One EST in heamocytes cDNA library was found to be homologous (identity ¼ 33%) to the TIMP gene from C. gigas (Genebank No. AAG42824). A forward primer (50 -AGAAACTTGGAGATGGTCG TTGT-30 ) and a reverse primer (50 -ATTCTTCCGCCATTCACATTC-30 ) were designed from the partial sequence of TIMP. The 30 end RACE PCR reaction was performed with the sense primer and universal vector primer T7. The PCR was carried out with the program of 35 cycles of 94 C for 40 s, 58 C for 40 s and 72 C for 50 s, and an extention of 72 C for 10 min. The 50 end RACE PCR reaction was performed with the reverse primer and vector primer T3. The PCR reaction conditions were the same as that described above, except for the Tm (57 C). The PCR products were cloned in pMD-18T vector (TAKARA) and sequenced in both directions with primers M13-47 and RV-M. The accessed sequences were verified and used to cluster analysis. 2.4. Analysis of nucleotide and amino acid sequences The nucleotide and deduced amino acid sequence of Tg-TIMP-4like cDNA were analyzed with DNAMAN 5.2.2. The deduced amino acid sequence was analysed to find signal peptide by signalP 3.0 software. Searches for homologies were realized with the amino acid sequence deduced from the TIMP cDNA cloned using the Blast program. N-glycosylating sites were predicted by NetNGlyc 1.0 Server software. The calculated molecular mass and theoretical isoelectric point was predicated by EMBOSS model of protein isoelectric point program. The protein domain features of Tg-TIMP4-like were predicted using Simple Modular Architecture Research Tool (http://smart.embl-heidelberg.de/). 2.5. Homologies of Tg-TIMP-4-like with invertebrate and vertebrate TIMPs A multiple sequence alignment was performed with ClustalW. An unrooted phylogenetic tree was constructed based on the amino sequences alignment by the neighbor-joining (NJ) algorithm embedded in Mega 4.1 program. The reliability of the branching was tested by bootstrap re-sampling (1000 pseudo-replicates). 2.6. Quantitative real-time PCR analysis of Tg-TIMP-4-like gene expression Quantitative real-time PCR analyses of the expression of TgTIMP-4-like transcript in tissues and the temporal expression profile in the hemocytes after V. parahaemolyticus, LPS and PGN challenge were performed using SYBR Green quantitative real-time Stratagene Mx3000P Real-time PCR System. The primer sequences, designed with primer 5 software, Tg-TIMP-4-like sense primer (50 -TGGAAATGGATGTAACAACCCTG-30 ) and antisense primer (50 -AGCGTAACAGTCGCCAGGTAATC-30 ) were used in qRT-PCR experiment. The forward primer (50 -CTTTCAAATGTCTGCCCTATCA ACT-30 ) and reverse prime (50 -TCCCGTATTGTTATTTTTCGTCACT-30 ) of Tg-18srRNA were used as an internal control to verify the successful reverse transcription and to calibrate the cDNA template. In a 96-well plate, each sample was run in triplicate along with the
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Fig. 1. Nucleotide and deduced amino acid sequences of Tg-TIMP-4-like. The letters in boxes are the start codon (ATG), the stop codon (TGA), and the polyadenylation signal sequence (ATTAAA) respectively. The asterisk (*) indicated the stop codon. The signal peptides were written on grey background. The motif Cys-X-Cys was marked in italic. NGlycosylation sites were highlighted in white on black background. Asn-Xaa-Ser/Thr sequons in the sequence output above are highlighted in blue. Asparagines predicted to be Nglycosylated are highlighted in red. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 2. Comparison of the TIMPs-4 between vertebrates and Tegillarca granosa. Amino acids conserved in all animal TIMP-4s are written in white on black background, and similar amino acids are shaded in grey. Gaps introduced to improve the alignment are shown as strigula; numbers refer to total amino acid of each mature protein.
internal control gene. The reaction component, thermal profile, and the data analysis were conducted as previously described [29]. All parameters are presented as means and standard deviations for each group data from triplications. The data from control and challenged groups were first checked for normality and transformed when necessary to meet the assumption of normal distribution.A two-way ANOVA was performed with SPSS 13.0 to detect statistical differences. Differences within the ANOVA were determined using a Dunnett t（2-sided）’s post-hoc test. P value of less than 0.05 was considered to be statistically significant. 3. Results and discussion 3.1. Molecular cloning and analysis of Tg-TIMP-4 like cDNA The cDNA sequence of Tg-TIMP-4 like was deposited in the GenBank database under accession number JN663889. This is the first tissue inhibitor of metalloproteinase gene identified in blood clams. The complete cDNA sequence of Tg-TIMP-4-like contains a 50 untranslated region (UTR) of 97 bp, a 30 UTR of 401 bp with a poly (A) tail. Canonical polyadenylation signal sequence AATAAA was
instead by ATTAAA in Tg-TIMP-4-like. The isolated cDNA also contains a ORF of 666 bp predicted to encode a 222-amino acid polypeptide. The nucleotide and deduced amino acid sequences of Tg-TIMP-4-like are shown in Fig. 1. The calculated molecular mass of the deduced mature Tg-TIMP-4-like protein was 22.67 kDa and the theoretical isoelectric point was 8.00. The three N-glycosylation sites of Tg-TIMP-4-like protein were predicted by NetNGlyc 1.0 Server software (Fig. 1). The amino acid sequence predicted to have an NTR domain which is TIMP subfamily by the SMART analysis indicated that it was a TIMP protein (Fig. 1). The analysis of the deduced amino acid sequence by SignalP 3.0 software revealed the presence of a 23-residue signal peptide with a predicted cleavage site located between residues 23 and 24, upstream of the Cys-TyrCys sequence (Fig. 1). 3.2. Homologous and phylogenetic analysis of Tg-TIMP-4-like We found that Tg-TIMP-4-like shares the highest similarities with C. gigas TIMP 1.3 (identity ¼ 30.56%) and the Homo sapiens TIMP-4 (identity 26.84%). Several structural features of TIMPs were almost completely conserved in the deduced amino acid of Tg-
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TIMP-4-like. First, a signal sequence which is cleaved off upstream of the motif Cys-X-Cys, to produce the mature protein [30], and it is interesting that the X amino acid represented by Ser/Thr was conserved in vertebrates. However, this residue was replaced by a different amino acid (Arg/Met/Ser) in invertebrates, and the unique Tyr amino acid was observed in Tg-TIMP-4-like. Second, The calculated molecular mass of the deduced mature Tg-TIMP-4-like protein was 22.67 kDa, which is in close agreement with the molecular mass range of other TIMPs (20e23 kDa) [30]. However, the theoretical isoelectric point was 8.00, which is different from the acid pI of Cg-TIMPs (6.9 and 5.6) [31]. Third, residue Cys30, which binds the zinc and shares it with a metalloproteinase partner via amino nitrogen and carbonyl oxygen is conserved in vertebrates and in blood clams. Moreover, sequence alignment of the 9 proteins of TIMP-4 available in protein data banks indicates that 12 cysteines (as well as their location) and other 33 amino acids are highly conserved among all the proteins analyzed, though Tg-TIMP-4-like possess 4 addtional cysteines and these amino acids are represented in Fig. 2. These 12 cysteine residues were thought to form six disulfide bonds that divide the protein into two domains with each domain being folded into three loops stabilized by three disulfide bonds [32]. The remaining four cysteine residues in the carboxyterminal domain of the protein might form another two disulfide bonds. Finding all these conserved characteristics with the predicted NTR-domain supports the idea that Tg-TIMP-4-like is a TIMP gene in T. granosa. Because the taxonomy of TIMPs in invertebrates is not clear, and the TIMP gene from T. granosa is more closely related to H. sapiens TIMP-4 than to other TIMPs in vertebrates, we named the TIMP gene Tg-TIMP-4-like. The species and genebank accession number of TIMP sequences used in the homologous and phylogenetic analysis were listed in Table 1. Fifty three sequences of available animal TIMP proteins were used for the phylogenetic tree construction with neighbor-joining method by Mega 3.1. All the members were mainly clustered into
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five groups, Vertebrate TIMP-1, -2, -3, -4, and invertebrate TIMPs. Not surprisingly, all TIMPs from mollusk clustered together as a subgroup, TIMP genes from T. granosa and C. gigas firstly clustered a sister group, then clustered other invertebrate TIMPs into invertebrate TIMPs group distinct from other TIMP groups of vertebrates (Fig. 3). This might indicate that the TIMPs were classified preferentially according to function and not the traditional biotaxonomy in vertebrates; however, the grouping was not the same in invertebrates. This discrepancy might be because of inadequate research in invertevrates. 3.3. Tissue-specific expression of Tg-TIMP-4-like mRNA transcripts To investigate tissue expression of Tg-TIMP-4-like, real-time quantitative PCR (qRT-PCR) analyses were carried out using cDNA synthesized from total RNA of adductor muscle, mantle, gills, foot, digestive gland, and hemocytes. Similar to Cg-TIMP [18], Tg-TIMP4-like has a wide range of tissues distribution: it is in all the six tissues of T. granosa, and Tg-TIMP-4-like transcript was detected at highest levels in hemocytes (P < 0.01), and the expressions of TgTIMP-4-like in other five tissues were almost the same and observed in low level (Fig. 4). Presumably, mollusk hemocytes are phagocytic cells involved in many functions, including wound healing, shell repair, and internal defense [33]. Because organs of bivalve mollusks are bathed in hemolymph, hemocytes are free to circulate through the body cavity; thus, the signal detected in the five tissue types is contributed by hemocytes present there [18]. Furthermore, the five tissues of the blood clam might synthesize TIMP protein which needs to be lubricated afterwards. Considering that mollusks completely expose themselves to the aquatic environment with large numbers of viruses, bacteria, and protozoan parasites [34], the wide distribution of Tg-TIMP-4-like also suggests that it has important roles in immune defense. 3.4. Tg-TIMP-4-like expression was induced by bacterial challenge
Table 1 Species and Genebank accession number of TIMP sequences used for homologous and phylogenetic analysis of Tg-TIMP-4-like. Species
Genebank accession
Species
Genebank accession
Anolis carolinensis Bos taurus Bos taurus Bos taurus
XP_003217720 NP_776896 NP_776897 AAI42427
P25785 NP_035725 AAK62886 NP_001117980
Bos taurus
DAA16771
Caenorhabditis elegans Camponotus floridanus Canis lupus familiaris Cavia porcellus Crassostrea gigas Crassostrea gigas Culex quinquefasciatus Danio rerio Danio rerio Drosophila melanogaster Equus caballus
NP_505113 EFN65977 AB016817 NP_001166495 AAT73610 AAG42824 XP_001845008 NP_998461 NP_878294 CAA08989 NP_001075984
Equus caballus Gallus gallus Gallus gallus Harpegnathos saltator Homo sapiens H. sapiens H. sapiens H. sapiens Macaca mulatta
NP_001075339 AF004664 NP_990818 EFN82588 NP_003245 NP_003246 NP_000353 NP_003247 NP_001181776
Mus musculus Mus musculus Mus musculus Oncorhynchus mykiss Oryctolagus cuniculus Ovis aries Ovis aries Ovis aries Pagrus major Pan troglodytes Papio cynocephalus Papio cynocephalus Rattus norvegicus Rattus norvegicus Salmo salar Scyliorhinus torazame Sparus aurata Sparus aurata Takifugu rubripes Takifugu rubripes Takifugu rubripes Tegillarca granosa Xenopus laevis Xenopus laevis Xenopus (Silurana) tropicalis
AAB35920 NP_001009319 NP_001159658 NP_001159659 BAD52417 XP_516284 P49061 AAA99943 NP_001102863 NP_037018 ACI69648 AAD26150 CAP19942 CAP19941 NP_001033040 NP_001033041 AAO17737 JN663889 NP_001087748 NP_001079064 NP_001015760
The ECM is closely reported to pathogen and virus infection. As an inhibitor involved in ECM metabolism, TIMP is a predominant molecule against infection by pathogens [22,23]. qRT-PCR was used to monitor the expression of Tg-TIMP-4-like transcripts in adult animals stimulated by V. parahaemolyticus, LPS and PGN to investigate its involvement in the immune response (Fig. 5), the PBS group was used as a control. PBS injection induce a significant expression of Tg-TIMP-4-like mRNA after 6 h (3.26-fold, P < 0.01), and reached the highest point at 12 h post-injection (8.9-fold compared with the 0 h group, P < 0.01), then decreased to the origin level at 48 h. It is not unusual that PBS group was found a significant expression of Tg-TIMP-4-like mRNA, because the shell damage, from which the injections could be carried out, could induce the TIMP expression [18]. The variation of PBS group result was also caused by such factors as aquatic environment, etc. Although the shell damage or aquatic environment does induce the significant expression of TIMP, the two-way ANOVA analysis indicated that the drug or bacterial immune response also upregulated the Tg-TIMP-4-like expression significantly: The mRNA expression of Tg-TIMP-4 like was up-regulated significantly after the stimulation of PGN, and reached the highest point at 6 h postinjection (43.1-fold compared with the 0 h group, P < 0.01, Fig. 5), indicating that Tg-TIMP-4-like is acutely expressed against a Grampositive bacteria challenge. Okamoto et al. indicated that bacterial proteinases may participate in ECM destruction by activating the latent form of host MMP and inactivate protease inhibitors and metalloproteinases produced by the pathogen [22,35], which might increase the mRNA expression of Tg-TIMP-4-like as an effective
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Fig. 3. Neighbour-joining phylogenetic tree generated using the Mega 3.1 program based on multiple sequence alignment by ClustalW for TIMP amino acid sequences from NCBI. Tg-TIMP-4 like is marked by pound sign (#).
protease inhibitor. However, there was a sudden decrease to its original expression level at 12 h post-injection, indicating that it was also involved in the clearance of PNG after the acute response phase [36]. However, excessive protease upregulated Tg-TIMP-4-like expression again at 24 h and 48 h (5.5-fold, 5.3-fold compared with 0 h group, respectively), suggesting that the ECM-remolding process requires balance between the MMPs, ADMs, ADMTs and TIMPs [2]. Compared with PGN, LPS stimulation also induced significantly up-regulation of Tg-TIMP-4-like, and the mRNA expression of TgTIMP-4-like reached the highest point at 6 h post-injection (69.4-
Fig. 4. Expression of Tg-TIMP-4 like analyzed by real-time PCR mRNA in different clam tissues. Each bar are means S.E. (N ¼ 3) of four clams for different tissue. Significant difference is indicated by different letter (P < 0.05). 18SrRNA served as reference gene.
fold compared with the 0 h group, P < 0.01, Fig. 5), suggesting that it was also involved in the immune response against Gramnegative bacteria infection. But there is a slight decrease at 1.5 h, and after a sudden decrease at 12 h (6.3-fold), the Tg-TIMP-4-like transcripts were up-regulated to 26-fold at 24 h (26 fold, P < 0.01), then decreased suddenly to original level at 48 h. Although Tg-TIMP-4-like expression was more intense than in the PNG group, the result was almost accordance with the upregulation of expression in the PGN stimulation group. Saarialho-Kere indicated that the MMPs or other metalloproteinases could be activated by stimuli such as lipopolysaccharide, a potent inducer of metalloproteinase expression, which could caused Tg-TIMP-4-like transcript accumulation [37]. However, this result differed from the findings in C. gigas [38], where Montagnani reported that Cg-TIMP mRNA accumulation was induced by secretory/excretory molecules produced by Vibrio and probably not by bacterial cell wall compounds such as LPS. As to V. parahaemolyticus injection, the transcripts of Tg-TIMP-4like decreased extremely significant, by a factor 0.2 at 1.5 h compared with 0 h. Then transcripts of Tg-TIMP-4-like firstly exhibited a burst of increase at 3 h after Vibro injection, which were 17.0-fold (P < 0.01, Fig. 5) higher than that observed in 0 h group, and reached the highest point at 6 h (P < 0.01). The mRNA expression of Tg-TIMP-4-like decreased rapidly to 6.3-fold compared with 0 h at 12 h, and it was interesting that the TgTIMP-4-like transcripts increased significantly to 29.6-fold (P < 0.01) compared with 0 h at 48 h. However, the expression pattern of Vibro group differed from other groups slightly, the extremely significant depression of Tg-TIMP-4-like expression at
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Fig. 5. Temporal expression of Tg-TIMP-4 like mRNA relative to 18SrRNA analyzed by realtime PCR in clam hemocytes after PGN, V. Parahaemolyticus and LPS challenge. The values are shown as mean S.E. (N ¼ 4). Significant difference between challenged and 0 time point group is indicated with an asterisk at P < 0.05, and with two asterisks at P < 0.01.
1.5 h indicated that the pathogen might induce strong immunosuppression in clams (e.g., protease inhibitors inactivation and antimicrobial peptide degradation) [22,39,40]. The previous results in C. gigas together with the newly identified Tg-TIMP-4-like from T. granosa provided strong evidence that TIMPs might have a role in mollusks’ immune response. In the present study, the full-length cDNA of Tg-TIMP-4-like was cloned from the blood clam and analyzed. Interestingly, we found the hypothesized hallmark amino acid (Cys186) of invertebrates TIMPs in T. granosa. Tg-TIMP-4-like expression in adult tissues of the bloody clam were studied through qRT-PCR, and expression in hemocytes after V. parahaemolyticus, LPS, or PGN challenge was also examined. The results indicated that Tg-TIMP-4-like is an acute-phase, inducible protein that might play an important role in the immune responses against different invading microbes. Our future studies will examine the mechanisms of transcriptional control of Tg-TIMP-4-like and polymorphism of the Tg-TIMP-4-like and its association with disease susceptibility/resistance to V. parahaemolyticus. Acknowledgements This research was supported by National Science Foundation of China (31001097), Chinese Agriculture Research System (CARS-48), and Ningbo Science and Technology International Cooperation Research Projects (2010D10017). References [1] Brew K, Dinakarpandian D, Nagase H. Tissue inhibitors of metalloproteinases: evolution, structure and function. Biochim Biophys Acta 2000;1477(1e2):267e83. [2] Murphy G, Nagase H. Progress in matrix metalloproteinase research. Mol Aspects Med 2008;29(5):290e308. [3] Porter S, Clark IM, Kevorkian L, Edwards DR. The ADAMTS metalloproteinases. Biochem J 2005;386(Pt1):15e27. [4] Edwards DR, Handsley MM, Pennington CJ. The ADAM metalloproteinases. Mol Aspects Med 2008;29(5):258e89. [5] Brew K, Nagase H. The tissue inhibitors of metalloproteinases (TIMPs): an ancient family with structural and functional diversity. Biochim Biophys Acta 2010;1803(1):55e71. [6] Bauer EA, Stricklin GP, Jeffrey JJ, Eisen AZ. Collagenase production by human skin fibroblasts. Biochem Biophys Res Commun 1975;64(1):232e40. [7] Kuettner KE, Hiti J, Eisenstein R, Harper E. Collagenase inhibition by cationic proteins derived from cartilage and aorta. Biochem Biophys Res Commun 1976;72(1):40e6. [8] Langton KP, Barker MD, McKie N. Localization of the functional domains of human tissue inhibitor of metalloproteinases-3 and the effects of a Sorsby’s fundus dystrophy mutation. J Biol Chem 1998;273(27):16778e81. [9] Melendez-Zajgla J, Del Pozo L, Ceballos G, Maldonado V. Tissue Inhibitor of Metalloproteinases-4. The road less traveled. Mol Cancer 2008;7:85. [10] Nagase H, Murphy G. Tailoring TIMPs for selective metalloproteinase inhibition. In: Edwards D, Hoyer-Hansen G, Blasi F, Sloane BF, editors. The cancer degradome. New York: Springer Science; 2008. p. 787e810. [11] Seo DW, Li H, Guedez L, Wing field PT, Diaz T, Salloum R, et al. TIMP-2 mediated inhibition of angiogenesis: an MMP-independent mechanism. Cell 2003;114(2):171e80.
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Mol Microbiol 1993;9(4):687e94. [22] Okamoto T, Akaike T, Suga M, Tanase S, Horie H, Miyajima S, et al. Activation of human matrix metalloproteinases by various bacterial proteinases. J Biol Chem 1997;272(9):6059e66. [23] Miyoshi S, Shinoda S. Microbial metalloproteases and pathogenesis. Microbes Infect 2000;2(1):91e8. [24] Garreis KG, La Peyre JF, Faisal M. The effect of Perkinsus marinus extracellular products and purified proteases on oyster defense parameters in vitro. Fish Shellfish Immuno 1996;6(8):581e97. [25] La Peyre JF, Yarnall HA, Faisal M. Contribution of Perkinsus marinus extracellular products in the infection of eastern oysters (Crassostrea gigas). J Invertebr Pathol 1996;68(3):312e3. [26] Muñoz P, Vance K, Gómez-Chiarri M. Protease activity in the plasma of American oysters, Crassostrea virginica, experimentally infected with the protozoan parasite Perkinsus marinus. J Parasitol 2003;89(5):941e51. [27] Greene J, Wang M, Liu YE, Raymond LA, Rosen C, Shi YE. Molecular cloning and characterization of human tissue inhibitor of metalloproteinase 4. J Biol Chem 1996;271(48):30375e80. [28] Leco KJ, Apte SS, Taniguchi GT, Hawkes SP, Khokha R, Schultz GA, et al. Murine tissue inhibitor of metalloproteinases-4 (Timp-4): cDNA isolation and expression in adult mouse tissues. FEBS Lett 1997;401(2e3):213e7. [29] Bao Y, Li L, Zhang G. The manganese superoxide dismutase gene in bay scallop Argopecten irradians: cloning, 3D modelling and mRNA expression. Fish Shellfish Immunol 2008;25:425e32. [30] Gomez DE, Alonso DF, Yoshiji H, Thorgeirsson UP. Tissue inhibitors of metalloproteinases: structure, regulation and biological functions. Eur J Cell Biol 1997;74(2):111e22. [31] Montagnani C, Tirape A, Boulo V, Escoubas JM. The two Cg-timp mRNAs expressed in oyster hemocytes are generated by two gene families and differentially expressed during ontogenesis. Dev Comp Immunol 2005;29(10):831e9. [32] Fernandez-Catalan C, Bode W, Huber R, Turk D, Calvete JJ, Lichte A, et al. Crystal structure of the complex formed by the membrane type 1-matrix metalloproteinase with the tissue inhibitor of metalloproteinases-2, the soluble progelatinase A receptor. EMBO J 1998;17(17):5238e48. [33] Cheng TC. In: Kennedy VS, Newell RIE, Eble AF, editors. The eastern oyster Crassostre virginica. College Park, MD: Maryland Sea Grant College; 1996. p. 299e333. [34] Berthe F. Report about mollusc diseaes. Mediterr Aquaculture Diagnostic Laboratories 2004;49:33e48. Open Access version, http://archimer.ifremer.fr/ doc/00000/3300/. [35] Lepore LS, Roelvink PR, Granados RR. Enhancin, the granulosis virus protein that facilitates nucleopolyhedrovirus (NPV) infections, is a metalloprotease. J Invertebr Pathol 1996;68(2):131e40. [36] Yang J, Qiu L, Wang L, Wei X, Zhang H, Zhang Y, et al. CfLGBP, a pattern recognition receptor in Chlamys farreri involved in the immune response against various bacteria. Fish Shellfish Immunol 2010;29(5):825e31. [37] Saarialho-Kere UK, Welgus HG, Parks WC. Distinct mechanisms regulate interstitial collagenase and 92-kDa gelatinase expression in human monocyticlike cells exposed to bacterial endotoxin. J Biol Chem 1993;268(23):17354e61. [38] Montagnani C, Avarre JC, de Lorgeril J, Quiquand M, Boulo V, Escoubas JM. First evidence of the activation of Cg-timp, an immune response component of pacific oysters, through a damage-associated molecular pattern pathway. Dev Comp Immunol 2007;31(1):1e11. [39] Cattaneo R. Four viruses, two bacteria, and one receptor: membrane cofactor protein (CD46) as pathogens’ magnet. J Virol 2004;78(9):4385e8. [40] Belas R, Manos J, Suvanasuthi R. Proteus mirabilis ZapA metalloproteinase degrades a broad spectrum of substrates, including antimicrobial peptides. Infect Immun 2004;72(9):5159e67.
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Short communication
A novel tissue inhibitor of metalloproteinase in blood clam Tegillarca granosa: Molecular cloning, tissue distribution and expression analysis Qing Wang a, b, Yongbo Bao a, *, Lihui Huo c, Hailong Gu a, b, Zhihua Lin a, * a
College of Biological & Environmental Sciences, Zhejiang Wanli University, 8 South Qianhu Road, Ningbo, Zhejiang 315100, PR China College of Fisheries and Life Sciences, Shanghai Ocean University, Shanghai 201306, China c College of Life Sciences and Bioengineering, Ningbo University, Ningbo 315211, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 February 2012 Received in revised form 1 June 2012 Accepted 26 June 2012 Available online 4 July 2012
Tissue inhibitor of metalloproteinases (TIMPs) were originally characterized as inhibitors of matrix metalloproteinases (MMPs), but their range of activities has been found to be broader as it includes the inhibition of several of the MMPs, etc. The cDNA encoding TIMP-4-like gene from blood clam Tegillarca granosa (designated as Tg-TIMP-4-like) which is the first tissue inhibitor of metalloproteinase identified in blood clams, was cloned and characterized. It was of 1164 bp, and an open reading frame (ORF) of 666 bp encoding a putative protein of 222 amino acids. The predicted amino acid sequence comprised all recognized functional domains found in other TIMP homologues and showed the highest (30.56%) identity to the TIMP-1.3 from Crassostrea gigas. Several highly conserved motifs including several TIMP signatures, amino acid residue Cys30 responsible for coordinating the metal ions, the Cys-X-Cys motif and the putative NTR (netrin) domain were almost completely conserved in the deduced amino acid of TgTIMP-4 like, which indicated that Tg-TIMP-4-like should be a member of the TIMP family. The mRNA expression of Tg-TIMP-4-like in the tissues of mantle, adductor muscle, foot, gill, hemocyte and hepatopancreas was examined by quantitative real-time PCR (qT-PCR) and mRNA transcripts of Tg-TIMP-4like were mainly detected in hemocyte, and weakly detected in the other tissues. We also observed that Tg-TIMP-4 like mRNA accumulated significantly during Vibrio parahaemolyticus, Peptidogylcan (PGN) and Lipopolysaccharide (LPS) challenge, whereas the timing and quantitative differences of mRNA expression against different challenge indicated that Tg-TIMP-4-like may play a pivotal role in mollusc defense mechanisms. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Tegillarca granosa Tissue inhibitors of metalloproteinases Immune response mRNA expression
1. Introduction Tissue inhibitors of metalloproteinases (TIMPs) are known widely as secreted multifunctional proteins that are vital to the regulation of ECM (extracellular matrix) metabolism. Variation in ECM composition is crucial for embryonic development, morphogenesis, and tissue remodeling and repair [1]. The major proteinases related to ECM catabolism are the matrix metalloproteinases (MMPs) [2], the ADAMs (a disintegrin and metalloproteinase) [3], and the ADAMTS [4] (a disintegrin and metalloproteinase with thrombospondin domains) metalloproteinases. The activities of these metalloproteinases are precisely regulated under physiologic conditions at the levels of transcription, zymogen activation, and inhibition by endogenous inhibitors. Thus, demolishing the balance * Corresponding authors. Tel./fax: þ86 574 88222015. E-mail addresses: [email protected] (Y. Bao), [email protected] (Z. Lin). 1050-4648/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fsi.2012.06.021
between the production of active enzymes and their inhibitiors may cause diseases which are associated with uncontrolled ECM turnover, inflammation, cell growth and migration, such as arthritis, cardiovascular disease, cancer, pulmonary disease, nephritis, neurological disorders and tissue ulceration [5]. TIMPs are endogenous inhibitors of these metalloproteinases and are accordingly important regulators of ECM turnover, tissue remodeling and cellular behaviors. The inhibitory activity of TIMP-1 was found in the early 1970s as a collagenase inhibitor in the media of cultured human skin fibroblasts [6], in bovine cartilage extracts, and aorta [7]. TIMPs are composed of two functional domains, an amino terminal (Nterminal) inhibitory domain and a carboxyl terminal (C-terminal) domain. The N-terminal domain is involved in metalloproteinase inhibition, whereas the C-terminal domain has sevral specific functions, including binding to MMPs and pro-MMP [8]. In human, the TIMP family consists of four members (TIMP-1 to -4) with high sequence identity and structural homology but with different
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tissue expression, regulation and inhibitory characteristics [9,10]. This restricted expression dictates the specific physiologic functions among the four TIMP members. In addition to their metalloproteinase inhibitory activity, TIMPs have other physiologic functions, including anti-angiogenesis [11], pro- and anti-apoptotic signaling [12], synaptic plasticity [13e15] and promoting cell proliferation [16], many of which are independent of the metalloproteinase inhibition pathway. TIMP genes are also found in invertebrates, such as Drosophila melanogaster [17] and Crassostrea gigas [18]. The first TIMP gene identified in mollusks was isolated in C. gigas, and the Cg-TIMPs are considered to be closely related to oyster ontogenesis, wound healing and defense mechanisms [18]. Because of the lack of adaptive immunity, invertebrates diminutively rely on innate immune responses to defend themselves efficiently against various pathogens [19,20]. The ECM components also function as docking sites responsible for microbial pathogen invasion [21]. Actually, many molecules that are produced by microorganisms have been identified through their intervention in ECM modifications. It is thought that these modifications are the necessary and primary events in the pathogenic mechanism of many infections. For example, bacterial proteases are reponsible for tissue damage and bacterial spread across tissue barriers, possibly as a result of the predicted function of latent MMP activation or protease inhibitor deactivation [22,23]. The extracellular proteases (ECP) produced by eastern oyster pathogen Perkinsus marinus could compromise the oyster’s immune response [24] and promote the protozoan’s ability to propagate during the pathogen infection [25,26]. TIMP research in mollusk helps reveal the mollusc immune mechanism. However, knowledge about TIMP in mollusks is still limited, and studies of molecular features and immune response of TIMP-4-like protein from commercial mollusk Tegillarca granosa are rare. TIMP-4 is the most recently identified and least studied member of the TIMP family: it was initially cloned in humans [27] and then in mice [28]. The main objectives of this study were: 1) to clone and characterize the full-length cDNA of the TIMP-4-like gene from bloody clam T. granosa; 2) to investigate the mRNA expression level of Tg-TIMP-4-like protein in different tissues; and 3) to examine the temporal expression profile of the Tg-TIMP-4-like transcript in hemocytes after live bacterial, LPS and PGN challenge. 2. Materials and methods 2.1. Animals, immune challenge Two-year to three-year old blood clams were collected from a commercial farm (Ningbo, China) and kept in seawater at 26 1.0 C in temperature and 30& in salinity. To minimize individual variability, at least 60 clams were used in each experiment. In Vibrio parahaemolyticus challenge group, for each individual, 20 ml of live V. parahaemolyticus suspended in PBS (2 108 cfu/ml, pH 7.2) was injected in the adductor muscle. Four individuals were randomly sampled at 1.5, 3, 6, 12, 24, and 48 h after injection. The LPS (Sigma, USA) and PGN (Sigma, USA) challenge group clams were injected with 20 ml 0.2 mg/ml drugs (diluted in 0.85% NaCl) per individual respectively following the procedures mentioned above. Unchallenged clams and 60 clams injected with 20 ml PBS buffer were used as the time 0 group (at 0 h) and the control group, respectively. 2.2. Tissues withdrawal, total RNA extraction and cDNA synthesis The 0.5 ml hemocyte pellets per individual from the blank, control and treated groups were collected, centrifuged (1000 g, 10 min, 4 C) immediately and were washed in sterile seawater. The healthy clam’s tissues (mantle, hepatopancreas, foot, adductor
muscle, gill, and hemocytes) were collected from several clams to research on the tissue expression of Tg-TIMP-4-like. Tissues were washed by sterile seawater firstly, then cut into small pieces. Total RNA was extracted from tissues using Trizol Reagent (Invitrogen, USA) following the protocol and then treated with DNase I (Promega, USA). The treated total RNA was reverse transcribed using MMLV reverse transcriptase (Promega, USA) and Oligo(dT) primer. 2.3. Cloning of the full-length cDNA of Tg-TIMP-4-like gene One EST in heamocytes cDNA library was found to be homologous (identity ¼ 33%) to the TIMP gene from C. gigas (Genebank No. AAG42824). A forward primer (50 -AGAAACTTGGAGATGGTCG TTGT-30 ) and a reverse primer (50 -ATTCTTCCGCCATTCACATTC-30 ) were designed from the partial sequence of TIMP. The 30 end RACE PCR reaction was performed with the sense primer and universal vector primer T7. The PCR was carried out with the program of 35 cycles of 94 C for 40 s, 58 C for 40 s and 72 C for 50 s, and an extention of 72 C for 10 min. The 50 end RACE PCR reaction was performed with the reverse primer and vector primer T3. The PCR reaction conditions were the same as that described above, except for the Tm (57 C). The PCR products were cloned in pMD-18T vector (TAKARA) and sequenced in both directions with primers M13-47 and RV-M. The accessed sequences were verified and used to cluster analysis. 2.4. Analysis of nucleotide and amino acid sequences The nucleotide and deduced amino acid sequence of Tg-TIMP-4like cDNA were analyzed with DNAMAN 5.2.2. The deduced amino acid sequence was analysed to find signal peptide by signalP 3.0 software. Searches for homologies were realized with the amino acid sequence deduced from the TIMP cDNA cloned using the Blast program. N-glycosylating sites were predicted by NetNGlyc 1.0 Server software. The calculated molecular mass and theoretical isoelectric point was predicated by EMBOSS model of protein isoelectric point program. The protein domain features of Tg-TIMP4-like were predicted using Simple Modular Architecture Research Tool (http://smart.embl-heidelberg.de/). 2.5. Homologies of Tg-TIMP-4-like with invertebrate and vertebrate TIMPs A multiple sequence alignment was performed with ClustalW. An unrooted phylogenetic tree was constructed based on the amino sequences alignment by the neighbor-joining (NJ) algorithm embedded in Mega 4.1 program. The reliability of the branching was tested by bootstrap re-sampling (1000 pseudo-replicates). 2.6. Quantitative real-time PCR analysis of Tg-TIMP-4-like gene expression Quantitative real-time PCR analyses of the expression of TgTIMP-4-like transcript in tissues and the temporal expression profile in the hemocytes after V. parahaemolyticus, LPS and PGN challenge were performed using SYBR Green quantitative real-time Stratagene Mx3000P Real-time PCR System. The primer sequences, designed with primer 5 software, Tg-TIMP-4-like sense primer (50 -TGGAAATGGATGTAACAACCCTG-30 ) and antisense primer (50 -AGCGTAACAGTCGCCAGGTAATC-30 ) were used in qRT-PCR experiment. The forward primer (50 -CTTTCAAATGTCTGCCCTATCA ACT-30 ) and reverse prime (50 -TCCCGTATTGTTATTTTTCGTCACT-30 ) of Tg-18srRNA were used as an internal control to verify the successful reverse transcription and to calibrate the cDNA template. In a 96-well plate, each sample was run in triplicate along with the
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Fig. 1. Nucleotide and deduced amino acid sequences of Tg-TIMP-4-like. The letters in boxes are the start codon (ATG), the stop codon (TGA), and the polyadenylation signal sequence (ATTAAA) respectively. The asterisk (*) indicated the stop codon. The signal peptides were written on grey background. The motif Cys-X-Cys was marked in italic. NGlycosylation sites were highlighted in white on black background. Asn-Xaa-Ser/Thr sequons in the sequence output above are highlighted in blue. Asparagines predicted to be Nglycosylated are highlighted in red. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 2. Comparison of the TIMPs-4 between vertebrates and Tegillarca granosa. Amino acids conserved in all animal TIMP-4s are written in white on black background, and similar amino acids are shaded in grey. Gaps introduced to improve the alignment are shown as strigula; numbers refer to total amino acid of each mature protein.
internal control gene. The reaction component, thermal profile, and the data analysis were conducted as previously described [29]. All parameters are presented as means and standard deviations for each group data from triplications. The data from control and challenged groups were first checked for normality and transformed when necessary to meet the assumption of normal distribution.A two-way ANOVA was performed with SPSS 13.0 to detect statistical differences. Differences within the ANOVA were determined using a Dunnett t（2-sided）’s post-hoc test. P value of less than 0.05 was considered to be statistically significant. 3. Results and discussion 3.1. Molecular cloning and analysis of Tg-TIMP-4 like cDNA The cDNA sequence of Tg-TIMP-4 like was deposited in the GenBank database under accession number JN663889. This is the first tissue inhibitor of metalloproteinase gene identified in blood clams. The complete cDNA sequence of Tg-TIMP-4-like contains a 50 untranslated region (UTR) of 97 bp, a 30 UTR of 401 bp with a poly (A) tail. Canonical polyadenylation signal sequence AATAAA was
instead by ATTAAA in Tg-TIMP-4-like. The isolated cDNA also contains a ORF of 666 bp predicted to encode a 222-amino acid polypeptide. The nucleotide and deduced amino acid sequences of Tg-TIMP-4-like are shown in Fig. 1. The calculated molecular mass of the deduced mature Tg-TIMP-4-like protein was 22.67 kDa and the theoretical isoelectric point was 8.00. The three N-glycosylation sites of Tg-TIMP-4-like protein were predicted by NetNGlyc 1.0 Server software (Fig. 1). The amino acid sequence predicted to have an NTR domain which is TIMP subfamily by the SMART analysis indicated that it was a TIMP protein (Fig. 1). The analysis of the deduced amino acid sequence by SignalP 3.0 software revealed the presence of a 23-residue signal peptide with a predicted cleavage site located between residues 23 and 24, upstream of the Cys-TyrCys sequence (Fig. 1). 3.2. Homologous and phylogenetic analysis of Tg-TIMP-4-like We found that Tg-TIMP-4-like shares the highest similarities with C. gigas TIMP 1.3 (identity ¼ 30.56%) and the Homo sapiens TIMP-4 (identity 26.84%). Several structural features of TIMPs were almost completely conserved in the deduced amino acid of Tg-
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TIMP-4-like. First, a signal sequence which is cleaved off upstream of the motif Cys-X-Cys, to produce the mature protein [30], and it is interesting that the X amino acid represented by Ser/Thr was conserved in vertebrates. However, this residue was replaced by a different amino acid (Arg/Met/Ser) in invertebrates, and the unique Tyr amino acid was observed in Tg-TIMP-4-like. Second, The calculated molecular mass of the deduced mature Tg-TIMP-4-like protein was 22.67 kDa, which is in close agreement with the molecular mass range of other TIMPs (20e23 kDa) [30]. However, the theoretical isoelectric point was 8.00, which is different from the acid pI of Cg-TIMPs (6.9 and 5.6) [31]. Third, residue Cys30, which binds the zinc and shares it with a metalloproteinase partner via amino nitrogen and carbonyl oxygen is conserved in vertebrates and in blood clams. Moreover, sequence alignment of the 9 proteins of TIMP-4 available in protein data banks indicates that 12 cysteines (as well as their location) and other 33 amino acids are highly conserved among all the proteins analyzed, though Tg-TIMP-4-like possess 4 addtional cysteines and these amino acids are represented in Fig. 2. These 12 cysteine residues were thought to form six disulfide bonds that divide the protein into two domains with each domain being folded into three loops stabilized by three disulfide bonds [32]. The remaining four cysteine residues in the carboxyterminal domain of the protein might form another two disulfide bonds. Finding all these conserved characteristics with the predicted NTR-domain supports the idea that Tg-TIMP-4-like is a TIMP gene in T. granosa. Because the taxonomy of TIMPs in invertebrates is not clear, and the TIMP gene from T. granosa is more closely related to H. sapiens TIMP-4 than to other TIMPs in vertebrates, we named the TIMP gene Tg-TIMP-4-like. The species and genebank accession number of TIMP sequences used in the homologous and phylogenetic analysis were listed in Table 1. Fifty three sequences of available animal TIMP proteins were used for the phylogenetic tree construction with neighbor-joining method by Mega 3.1. All the members were mainly clustered into
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five groups, Vertebrate TIMP-1, -2, -3, -4, and invertebrate TIMPs. Not surprisingly, all TIMPs from mollusk clustered together as a subgroup, TIMP genes from T. granosa and C. gigas firstly clustered a sister group, then clustered other invertebrate TIMPs into invertebrate TIMPs group distinct from other TIMP groups of vertebrates (Fig. 3). This might indicate that the TIMPs were classified preferentially according to function and not the traditional biotaxonomy in vertebrates; however, the grouping was not the same in invertebrates. This discrepancy might be because of inadequate research in invertevrates. 3.3. Tissue-specific expression of Tg-TIMP-4-like mRNA transcripts To investigate tissue expression of Tg-TIMP-4-like, real-time quantitative PCR (qRT-PCR) analyses were carried out using cDNA synthesized from total RNA of adductor muscle, mantle, gills, foot, digestive gland, and hemocytes. Similar to Cg-TIMP [18], Tg-TIMP4-like has a wide range of tissues distribution: it is in all the six tissues of T. granosa, and Tg-TIMP-4-like transcript was detected at highest levels in hemocytes (P < 0.01), and the expressions of TgTIMP-4-like in other five tissues were almost the same and observed in low level (Fig. 4). Presumably, mollusk hemocytes are phagocytic cells involved in many functions, including wound healing, shell repair, and internal defense [33]. Because organs of bivalve mollusks are bathed in hemolymph, hemocytes are free to circulate through the body cavity; thus, the signal detected in the five tissue types is contributed by hemocytes present there [18]. Furthermore, the five tissues of the blood clam might synthesize TIMP protein which needs to be lubricated afterwards. Considering that mollusks completely expose themselves to the aquatic environment with large numbers of viruses, bacteria, and protozoan parasites [34], the wide distribution of Tg-TIMP-4-like also suggests that it has important roles in immune defense. 3.4. Tg-TIMP-4-like expression was induced by bacterial challenge
Table 1 Species and Genebank accession number of TIMP sequences used for homologous and phylogenetic analysis of Tg-TIMP-4-like. Species
Genebank accession
Species
Genebank accession
Anolis carolinensis Bos taurus Bos taurus Bos taurus
XP_003217720 NP_776896 NP_776897 AAI42427
P25785 NP_035725 AAK62886 NP_001117980
Bos taurus
DAA16771
Caenorhabditis elegans Camponotus floridanus Canis lupus familiaris Cavia porcellus Crassostrea gigas Crassostrea gigas Culex quinquefasciatus Danio rerio Danio rerio Drosophila melanogaster Equus caballus
NP_505113 EFN65977 AB016817 NP_001166495 AAT73610 AAG42824 XP_001845008 NP_998461 NP_878294 CAA08989 NP_001075984
Equus caballus Gallus gallus Gallus gallus Harpegnathos saltator Homo sapiens H. sapiens H. sapiens H. sapiens Macaca mulatta
NP_001075339 AF004664 NP_990818 EFN82588 NP_003245 NP_003246 NP_000353 NP_003247 NP_001181776
Mus musculus Mus musculus Mus musculus Oncorhynchus mykiss Oryctolagus cuniculus Ovis aries Ovis aries Ovis aries Pagrus major Pan troglodytes Papio cynocephalus Papio cynocephalus Rattus norvegicus Rattus norvegicus Salmo salar Scyliorhinus torazame Sparus aurata Sparus aurata Takifugu rubripes Takifugu rubripes Takifugu rubripes Tegillarca granosa Xenopus laevis Xenopus laevis Xenopus (Silurana) tropicalis
AAB35920 NP_001009319 NP_001159658 NP_001159659 BAD52417 XP_516284 P49061 AAA99943 NP_001102863 NP_037018 ACI69648 AAD26150 CAP19942 CAP19941 NP_001033040 NP_001033041 AAO17737 JN663889 NP_001087748 NP_001079064 NP_001015760
The ECM is closely reported to pathogen and virus infection. As an inhibitor involved in ECM metabolism, TIMP is a predominant molecule against infection by pathogens [22,23]. qRT-PCR was used to monitor the expression of Tg-TIMP-4-like transcripts in adult animals stimulated by V. parahaemolyticus, LPS and PGN to investigate its involvement in the immune response (Fig. 5), the PBS group was used as a control. PBS injection induce a significant expression of Tg-TIMP-4-like mRNA after 6 h (3.26-fold, P < 0.01), and reached the highest point at 12 h post-injection (8.9-fold compared with the 0 h group, P < 0.01), then decreased to the origin level at 48 h. It is not unusual that PBS group was found a significant expression of Tg-TIMP-4-like mRNA, because the shell damage, from which the injections could be carried out, could induce the TIMP expression [18]. The variation of PBS group result was also caused by such factors as aquatic environment, etc. Although the shell damage or aquatic environment does induce the significant expression of TIMP, the two-way ANOVA analysis indicated that the drug or bacterial immune response also upregulated the Tg-TIMP-4-like expression significantly: The mRNA expression of Tg-TIMP-4 like was up-regulated significantly after the stimulation of PGN, and reached the highest point at 6 h postinjection (43.1-fold compared with the 0 h group, P < 0.01, Fig. 5), indicating that Tg-TIMP-4-like is acutely expressed against a Grampositive bacteria challenge. Okamoto et al. indicated that bacterial proteinases may participate in ECM destruction by activating the latent form of host MMP and inactivate protease inhibitors and metalloproteinases produced by the pathogen [22,35], which might increase the mRNA expression of Tg-TIMP-4-like as an effective
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Fig. 3. Neighbour-joining phylogenetic tree generated using the Mega 3.1 program based on multiple sequence alignment by ClustalW for TIMP amino acid sequences from NCBI. Tg-TIMP-4 like is marked by pound sign (#).
protease inhibitor. However, there was a sudden decrease to its original expression level at 12 h post-injection, indicating that it was also involved in the clearance of PNG after the acute response phase [36]. However, excessive protease upregulated Tg-TIMP-4-like expression again at 24 h and 48 h (5.5-fold, 5.3-fold compared with 0 h group, respectively), suggesting that the ECM-remolding process requires balance between the MMPs, ADMs, ADMTs and TIMPs [2]. Compared with PGN, LPS stimulation also induced significantly up-regulation of Tg-TIMP-4-like, and the mRNA expression of TgTIMP-4-like reached the highest point at 6 h post-injection (69.4-
Fig. 4. Expression of Tg-TIMP-4 like analyzed by real-time PCR mRNA in different clam tissues. Each bar are means S.E. (N ¼ 3) of four clams for different tissue. Significant difference is indicated by different letter (P < 0.05). 18SrRNA served as reference gene.
fold compared with the 0 h group, P < 0.01, Fig. 5), suggesting that it was also involved in the immune response against Gramnegative bacteria infection. But there is a slight decrease at 1.5 h, and after a sudden decrease at 12 h (6.3-fold), the Tg-TIMP-4-like transcripts were up-regulated to 26-fold at 24 h (26 fold, P < 0.01), then decreased suddenly to original level at 48 h. Although Tg-TIMP-4-like expression was more intense than in the PNG group, the result was almost accordance with the upregulation of expression in the PGN stimulation group. Saarialho-Kere indicated that the MMPs or other metalloproteinases could be activated by stimuli such as lipopolysaccharide, a potent inducer of metalloproteinase expression, which could caused Tg-TIMP-4-like transcript accumulation [37]. However, this result differed from the findings in C. gigas [38], where Montagnani reported that Cg-TIMP mRNA accumulation was induced by secretory/excretory molecules produced by Vibrio and probably not by bacterial cell wall compounds such as LPS. As to V. parahaemolyticus injection, the transcripts of Tg-TIMP-4like decreased extremely significant, by a factor 0.2 at 1.5 h compared with 0 h. Then transcripts of Tg-TIMP-4-like firstly exhibited a burst of increase at 3 h after Vibro injection, which were 17.0-fold (P < 0.01, Fig. 5) higher than that observed in 0 h group, and reached the highest point at 6 h (P < 0.01). The mRNA expression of Tg-TIMP-4-like decreased rapidly to 6.3-fold compared with 0 h at 12 h, and it was interesting that the TgTIMP-4-like transcripts increased significantly to 29.6-fold (P < 0.01) compared with 0 h at 48 h. However, the expression pattern of Vibro group differed from other groups slightly, the extremely significant depression of Tg-TIMP-4-like expression at
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Fig. 5. Temporal expression of Tg-TIMP-4 like mRNA relative to 18SrRNA analyzed by realtime PCR in clam hemocytes after PGN, V. Parahaemolyticus and LPS challenge. The values are shown as mean S.E. (N ¼ 4). Significant difference between challenged and 0 time point group is indicated with an asterisk at P < 0.05, and with two asterisks at P < 0.01.
1.5 h indicated that the pathogen might induce strong immunosuppression in clams (e.g., protease inhibitors inactivation and antimicrobial peptide degradation) [22,39,40]. The previous results in C. gigas together with the newly identified Tg-TIMP-4-like from T. granosa provided strong evidence that TIMPs might have a role in mollusks’ immune response. In the present study, the full-length cDNA of Tg-TIMP-4-like was cloned from the blood clam and analyzed. Interestingly, we found the hypothesized hallmark amino acid (Cys186) of invertebrates TIMPs in T. granosa. Tg-TIMP-4-like expression in adult tissues of the bloody clam were studied through qRT-PCR, and expression in hemocytes after V. parahaemolyticus, LPS, or PGN challenge was also examined. The results indicated that Tg-TIMP-4-like is an acute-phase, inducible protein that might play an important role in the immune responses against different invading microbes. Our future studies will examine the mechanisms of transcriptional control of Tg-TIMP-4-like and polymorphism of the Tg-TIMP-4-like and its association with disease susceptibility/resistance to V. parahaemolyticus. Acknowledgements This research was supported by National Science Foundation of China (31001097), Chinese Agriculture Research System (CARS-48), and Ningbo Science and Technology International Cooperation Research Projects (2010D10017). References [1] Brew K, Dinakarpandian D, Nagase H. Tissue inhibitors of metalloproteinases: evolution, structure and function. Biochim Biophys Acta 2000;1477(1e2):267e83. [2] Murphy G, Nagase H. Progress in matrix metalloproteinase research. Mol Aspects Med 2008;29(5):290e308. [3] Porter S, Clark IM, Kevorkian L, Edwards DR. The ADAMTS metalloproteinases. Biochem J 2005;386(Pt1):15e27. [4] Edwards DR, Handsley MM, Pennington CJ. The ADAM metalloproteinases. Mol Aspects Med 2008;29(5):258e89. [5] Brew K, Nagase H. The tissue inhibitors of metalloproteinases (TIMPs): an ancient family with structural and functional diversity. Biochim Biophys Acta 2010;1803(1):55e71. [6] Bauer EA, Stricklin GP, Jeffrey JJ, Eisen AZ. Collagenase production by human skin fibroblasts. Biochem Biophys Res Commun 1975;64(1):232e40. [7] Kuettner KE, Hiti J, Eisenstein R, Harper E. Collagenase inhibition by cationic proteins derived from cartilage and aorta. Biochem Biophys Res Commun 1976;72(1):40e6. [8] Langton KP, Barker MD, McKie N. Localization of the functional domains of human tissue inhibitor of metalloproteinases-3 and the effects of a Sorsby’s fundus dystrophy mutation. J Biol Chem 1998;273(27):16778e81. [9] Melendez-Zajgla J, Del Pozo L, Ceballos G, Maldonado V. Tissue Inhibitor of Metalloproteinases-4. The road less traveled. Mol Cancer 2008;7:85. [10] Nagase H, Murphy G. Tailoring TIMPs for selective metalloproteinase inhibition. In: Edwards D, Hoyer-Hansen G, Blasi F, Sloane BF, editors. The cancer degradome. New York: Springer Science; 2008. p. 787e810. [11] Seo DW, Li H, Guedez L, Wing field PT, Diaz T, Salloum R, et al. TIMP-2 mediated inhibition of angiogenesis: an MMP-independent mechanism. Cell 2003;114(2):171e80.
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