Fish & Shellfish Immunology 37 (2014) 115e121
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Molecular characterization, expression and function analysis of a five-domain Kazal-type serine proteinase inhibitor from pearl oyster Pinctada fucata Dianchang Zhang, Jianjun Ma, Shigui Jiang* Key Laboratory of South China Sea Fishery Resources Exploitation and Utilization, Ministry of Agriculture, South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou 510300, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 September 2013 Received in revised form 4 December 2013 Accepted 18 December 2013 Available online 27 December 2013
Serine proteinase inhibitors represent an expanding superfamily of endogenous inhibitors that are regulate proteolytic events and involved in a variety of physiological and immunological processes. A five-domain Kazal-type serine proteinase inhibitor (poKSPI) was identified and characterized from pearl oyster Pinctada fucata based on expressed sequence tag (EST) analysis. The full-length cDNA was 737 bp with an open reading frame (ORF) 660 bp encoding a 219 amino acid protein a theoretical molecular weight (Mw) of 23.3 kDa and an isoelectric point (pI) of 8.40. A putative signal peptide of 19 amino acid residues and five tandem Kazal domains were identified. Four of the Kazal domains had the highly conserved motif sequences with six cysteine residues responsible for the formation of disulfide bridges. The deduced amino acid sequence of the poKSPI shared high homology with KSPIs from Hirudo medicinalis. The poKSPI mRNA could be detected in all examined tissues, the expression level of the poKSPI mRNA was the highest in mantle and gonad, while the lowest in haemocyte and intestine. After LPS challenge, the expression level of the poKSPI mRNA in digestive gland was significantly up-regulated at 4 h post-challenge and reached the peak at 12 h post-challenge, which was 4.23-fold higher than control group; the expression level of the poKSPI mRNA in gill was also significantly up-regulated at 8 and 12 h post-challenge, which were 4.48 and 2.26-fold higher than control group. After Vibrio alginolyticus challenge, the expression levels of the poKSPI mRNA in digestive gland were significantly upregulated at 8, 12, 48 and 72 h post-challenge, which were 1.70, 1.79, 3.89 and 5.69-fold higher than control group, respectively; the expression level of the poKSPI mRNA in gill was significantly upregulated at 24 h post-challenge, which was 5.30-fold higher than control group. The recombinant poKSPI protein could inhibit chymotrypsin and trypsin activities in dose-dependent manner, when the ratios of rpoKSPI to chymotrypsin and trypsin were 36:1 and 72:1, respectively, the proteinase activities of chymotrypsin and trypsin could be almost completely inhibited, but the rpoKSPI could not inhibit subtilisin. Ó 2013 Published by Elsevier Ltd.
Keywords: Pinctada fucata Serine proteinase inhibitor Microbial challenge Quantitative real-time PCR Inhibitory activity
1. Introduction Serine proteinase inhibitors (SPIs) are found widely in all multicellular organisms and take part in controlling the various proteinase-mediated biological processes, such as digestion, complement system, blood coagulation, melanization, apoptosis, phagocytosis, cellular remodelling and reproductive processes [1e 4]. Some SPIs also show the potent bactericidal or bacteriostatic activities [5,6] and play the important roles as part of the humoural
* Corresponding author. Tel.: þ86 20 89108338; fax: þ86 20 84451442. E-mail address: [email protected] (S. Jiang). 1050-4648/$ e see front matter Ó 2013 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.fsi.2013.12.011
defence of the innate immune system against the invading pathogens [7]. Several microbial pathogens and parasites use the SPIs to counter the protective proteinases of hosts. For example, the intracellular parasite of human Toxoplasma gondii secretes a serine proteinase inhibitor to protect itself from the digestive enzymes during its residency in small intestine [8]. Rhodnius prolix, Dipetalogaster maximus and Triatoma infestans can produce the potent proteinase inhibitors to prevent blood coagulation and make them easier to suck blood from their hosts [9e11]. SPIs are classified into at least 63 families on the basis of amino acid sequence similarities (http://merops.sanger.ac.uk/). Kazal-type serine proteinase inhibitors (KSPIs) are well known and grouped into family Ⅰ1. All the KSPIs contain one or more Kazal domains and
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each domain consists of 50e60 amino acid residues and contains six well-conserved cysteine residues forming three intra-domain disulphide bridges, which are responsible for the stability of three-dimensional structure [12]. The reactive site (P1 residue) is located at the second position after the second cysteine residue of the domain and believed to contribute mainly to the inhibitory specificity [13], a few other adjacent amino acid residues also influence the binding specificity [14,15]. Recently, a variety of KSPIs are found in crustaceans [4,6,7,16e18]. They were believed to be involved in innate immune responses, because their expressions were up-regulated after bacterial challenge. Meanwhile, several of KSPIs also had bacteriostatic activity against some pathogens [6,7]. However, until now, the KSPIs were only studied in few mollusks [19e22], these results preliminarily demonstrated that KSPIs were involved in innate responses of mollusk. In contrast with the abundant knowledge on KSPIs in crustaceans, the information of KSPIs in bivalve mollusk is still poorly known. To further understand the molecular functions of KSPIs in the innate immune responses of bivalve mollusks, in this study, we cloned and characterized a Kazal-type serine proteinase inhibitor from pearl oyster Pinctada fucata (designated as poKSPI), which is the most important farmed bivalve mollusk for seawater pearl production in China [23,24], and investigated its tissue distribution and temporal expression profile after bacterial and lipopolysaccharide (LPS) challenge. This work would hopefully provide insight into the important functions in the innate immune responses of pearl oysters. 2. Materials and methods 2.1. EST and poKSPI cDNA analysis A cDNA library was constructed and analyzed in our previous studies [23]. These EST data were used to search for the Kazal-type serine proteinase inhibitors, and an EST (EST No. pmpca0_002189) of 453 bp was homologous to the KSPIs of Chlamys farreri (EU183309) and Argopecten irradians (AY830445), the plasmid of this EST was picked up and re-sequenced to get the full-length cDNA and ensure the correctness of the sequence. DNATool 6.0 software was used to deduce the poKSPI amino acid sequence. Searches for nucleotide and protein sequence similarities were conducted with BLAST algorithm at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/BLAST/). SignalP 4.0 software (http://www.cbs.dtu.dk/services/SignalP/) was used to predict the putative signal peptide cleavage site [25]. The simple modular architecture research tool (SMART) 4.0 program (http://www.smart.emblheidelberg.de/) and ScanProsite (http:// expasy.org/tools/) were used to predict the protein domain [26]. ClustalW (http://www.clustal.org/) [27] and MEGA 5.1 [28] were used to align poKSPI with other known KSPI sequences.
Munich, Germany, LPS 10 mg ml1) dissolved in phosphate-buffer saline (PBS) into the adductor muscles of each pearl oyster. The bacterial challenge experiment was carried out by injecting with 100 mL of Vibrio alginolyticus resuspended in PBS to OD600 ¼ 0.4 (1 OD ¼ 5 108 bacteria ml1) into the adductor muscles of each pearl oyster. While pearl oysters injected with 100 mL PBS were used as control group in the challenge experiments. The injected pearl oysters were returned into seawater tanks and 15 individuals were randomly sampled at 0, 2, 4, 8, 12, 24, 48 and 72 h in each group, respectively. The gill and digestive gland from the control and challenge groups were collected respectively and immediately stored in liquid nitrogen until used. For tissue distribution experiment, three healthy pearl oysters were dissected to sample haemocyte, gill, digestive gland, mantle, gonad, adductor muscle and intestine tissues, respectively, and then they were stored in liquid nitrogen until used. Total RNA samples were extracted using the TRIzol reagent (Invitrogen) according to the manufacture instructions. 2.3. Quantitative real-time RT-PCR analysis of poKSPI mRNA expression For the expression pattern of poKSPI in different tissues, total RNA was extracted from haemocyte, gill, digestive gland, mantle, gonad, intestine and adductor muscle of three healthy pearl oysters. For the temporal expression analysis, total RNA was extracted from gill and digestive gland sampled from the different time point in each group, respectively. The total RNA of each sample was treated with the DNase I (Promega) respectively, and then the single-strand cDNA was synthesized based on manufacture’s instruction of PrimerScriptÔ 1st Strand cDNA Synthesis Kit (TaKaRa) using random primer. cDNA mix was diluted to 1:10 and stored at 80 C for expression analysis. Two poKSPI gene-specific primers, poKSPI-F (50 -TGGAAGATGTCCCTGCATC-30 ) and poKSPI-R (50 -GTCCGCGCATTTAGCTGTA30 ), were used to amplify a product of 183 bp from cDNA template, and the PCR products were sequenced to verify the specificity of RTPCR. A 97 bp fragment was amplified using two primers of pearl oyster b-actin to calibrate the cDNA template as an internal control [23]. The SYBR Green RT-PCR assay was performed in the Eppendorf Realplex2 Detection System as described by Zhang et al. [23]. Dissociation curve analysis of amplification products was carried out at the end of each PCR reaction to confirm that only one PCR product was amplified and detected. After the PCR program, data were analyzed with the Realplex2 software (Eppendorf). The 2DDCT method was used to determine fold change for gene expression relative to controls [23]. All data were given in terms of relative mRNA expression as means S.D. the data were subjected to oneway analysis of variance (one-way ANOVA) followed by a multiple comparison with GraphPad Prism 5.0 Software, and the P value less than 0.05 were considered statistically significant.
2.2. Pearl oysters and immune challenge 2.4. Recombinant expression and purification of poKSPI Pearl oysters P. fucata (shell length 6.15 0.32 cm, body weight 23.8 4.5 g) were obtained from pearl oyster culture base of South China Sea Fisheries Research Institute in Xincun village, Hainan province, China and maintained at 25e27 C with the recirculating seawater for one week before experiment. The pearl oysters were fed twice daily on Tetraselmis suecica and Isochrysis galbana. To take account of individual variability, 15 individual pearl oysters, which were divided into three replicates, were used for each time point (0, 2, 4, 8, 12, 24, 48 and 72 h) in the LPS and bacterial challenge experiments, respectively. The LPS challenge experiment was performed by injecting with 100 mL LPS (Escherichia coli 055:B5, #62326, SigmaeAldrich,
The fragment encoding mature protein of poKSPI was amplified with gene-specific primers poKSPI-F1 (50 - GGTGGATCCAGATCGCCACCCTACAAAC-30 ) and poKSPI-R1 (50 - GCGAAGCTTTTATTCATACTTCCTACATGGACC-30 ). For convenience of cloning, a Bam HI site was added to the 50 end of poKSPI-F1 and a Hind Ⅲ site was added to the 50 end of poKSPI-R1 before the stop codon. The PCR products was cloned into pMD18-T simple vector (Takara), digested by completely by restriction enzymes Bam HI and Hind Ⅲ, and then ligated into predigested expression vector pRSET. The recombinant plasmid pRSET-poKSPI was transformed into E. coli BL21 (Novagen). The positive transformant with pREST-poKSPI was
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picked up and sequenced, as well as, the transformant with only pRSET was used as the control group. Both of them were incubated at 37 C in liquid culture medium containing 100 mg ml1 ampicillin (QingDao MDBio Inc., QingDao City, Shandong, China) (LA) with shaking at 225 rpm. When the OD600 of bacteria concentration reached w0.6, isopropyl b-D-1-thiogalactopyranoside (IPTG) (MDBio) was added into LA medium at the final concentration of 0.8 mmol L1, then continued to incubate for 5 h. The recombinant poKSPI was purified and refolded by affinity chromatography on a resin with the chelated nickel ions according to His$Tag Purification Kit (MERCK, Jing’an District, Shanghai, China) as described by Cui et al. [29]. The resultant protein was separated by reducing 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDSPAGE), visualized with coomassie brilliant blue R250. The concentration of purified rpoKSPI was quantified by BCA method [29], and then the rpoKSPI was stored at 80 C for subsequent experiments. 2.5. Proteinase inhibition activity assays of rpoKSPI The inhibitory activities of rpoKSPI towards to chymotrypsin (bovine pancreas, Sigma), trypsin (bovine pancreas, Sigma) and subtilisin (Bacillus licheniformis, Sigma) were analyzed as described by Hergenhahn et al. [30]. The reaction mixture consisted of 100 mM TriseHCl, pH 8.0; 600 mM of N-benzoyl-Phe-Val-Arg-pnitroanilide as substrate for subtilin and trypsin and 300 mM Nsuccinyl-Ala-Ala-Pro-Phe-p-nitroanilide for chymotrypsin, the appropriate concentration of proteinase and proteinase inhibitors in a total volume of 100 mL. Proteinases (0.4 mM subtilisin, 0.4 mM trypsin or 0.2 mM chymotrypsin) were respectively incubated with the increasing concentration of inhibitor (3, 6, 9, 18, 36, 72-fold to each enzyme) in the buffer for 15 min at 30 C and then terminated by adding 50 mL of 50% acetic acid. The proteinase reaction product, p-nitroaniline, was measured at 405 nm using microplate reader. The percentage of remaining activity was calculated and plotted against the molar ratio of inhibitor to proteinase. 3. Results 3.1. Molecular characteristics of poKSPI A Kazal-type serine proteinase inhibitor with full-length cDNA sequence was identified from the cDNA library of P. fucata and deposited in GenBank under accession number GU361540. The complete cDNA sequence of poKSPI was 737 bp with an open reading frame (ORF) 660 bp encoding a 219 amino acid protein, a 50 -untranslated region (UTR) of 42 bp, a 30 UTR of 35 bp (Fig. 1). The deduced protein sequence contained a putative 19 aa signal peptide and five tandem Kazal domains, four of the Kazal domains had the highly conserved motif sequences C-X(6-9)-C-X(7)-C-X(10)-Y-X(3)C-X(2, 3)-C-X(9-16)-C with six cysteine residues responsible for the formation of disulfide bridges (C1eC5, C2eC4, C3eC6) (Supplemental data, Fig. 1). The poKSPI protein had a theoretical molecular weight (Mw) of 23.3 kDa and an isoelectric point (pI) of 8.40. The significant similarities were detected between the domains of poKSPI and other Kazal-type SPIs (Supplemental data, Fig. 1), the similarities among five domains of poKSPI were from 54.1% to 83.8%, and poKSPI-5 had a 64.9% similarity with Hirudo nipponia and 67.6% with Hirudo medicinalis [31]. In the P1 site of the poKSPI domains, there was Leu in 1st and 2nd, and Lys in 3rd, 4th and 5th domains (Fig. 1). 3.2. Spatial expression pattern of poKSPI mRNA in different tissues Quantitative real-time RT-PCR was performed to investigate the tissue expression pattern of the poKSPI mRNA with the b-actin as
Fig. 1. The nucleotide and deduced amino acid sequences of poKSPI (GenBank accession number GU361540). The putative signal peptide is underlined. The family signatures of Kazal-type domains are shaded. The potential N-glycosylation site (Asn47) is double underlined. The P1 sites (Leu32, Leu70, Lys108, Lys146 and Lys183) are boxed. The conserved cysteine residues are bolded. The stop codon is indicated by asterisk.
an internal control. The mRNA transcripts of poKSPI could be detected in all the examined tissues including gill, digestive gland, mantle, gonad, adductor muscle, intestine and haemocyte (Fig. 2). 3.3. Temporal expression profile of poKSPI mRNA after immune challenge The temporal expression profiles of the poKSPI mRNA in digestive gland and gill after LPS challenge were measured by quantitative real-time RT-PCR. LPS challenge induced the up-
Fig. 2. Expression levels of poKSPI mRNA were measured by quantitative real-time quantitative RT-PCR in different tissues. RNA samples were extracted from digestive gland (DG), mantle, intestine, gonad, adductor muscle (AM), gill and haemocyte of the adult tissues of pearl oyster. The b-actin gene was used as an internal control to calibrate the cDNA templates. Vertical bars represented the mean S.D (N ¼ 3).
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regulated expression of the poKSPI mRNA of digestive gland in time-dependent fashion. The expression level of the poKSPI mRNA was significantly up-regulated at 4 h post-challenge and reached the peak at 12 h post-challenge, which was 4.23-fold higher than control group, and then the expression level of the poKSPI mRNA dropped gradually (Fig. 3A). The expression level of the poKSPI mRNA in gill was also significantly up-regulated at 8 and 12 h postchallenge, which were 4.48 and 2.26-fold higher than control group, respectively, and then dropped dramatically at 24 h postchallenge (Fig. 3B). The expression levels of the poKSPI mRNA in digestive gland and gill were also significantly up-regulated after V. alginolyticus challenge (Fig. 4). In digestive gland, the expression levels of the poKSPI mRNA were significantly up-regulated at 8, 12, 48 and 72 h postchallenge, which were 1.70, 1.79, 3.89 and 5.69-fold higher than control group, respectively (Fig. 4A). In gill, the expression level of the poKSPI mRNA was significantly up-regulated at 24 h postchallenge, which was 5.30-fold higher than control group (Fig. 4B). 3.4. Expression and purification of recombinant poKSPI The recombinant plasmid pRSET-poKSPI was transformed into E. coli BL21 and then induced with IPTG to expression the
recombinant poKSPI protein as described above. After IPTG induction, the whole cell lysate of positive clone was separated by SDS-PAGE, and a distinct band with a molecular weight of about 24 kDa was detected, which was consistent with the predicted molecular mass (24.3 kDa), while negative clone with only pRSET plasmid lack this protein band (Fig. 5A). The purified and refolded rpoKSPI with only one target band was obtained by affinity chromatography and detected by SDS-PAGE (Fig. 5B). 3.5. Proteinase inhibition activity of rpoKSPI To investigate the inhibition activity of the recombinant poKSPI, tight-binding inhibition assay was carried out using increasing amount of the purified rpoKSPI against chymotrypsin, trypsin and subtilisin with corresponding artificial substrates. The dosee response curve of serine proteinase inhibition showed that rpoKSPI could inhibit chymotrypsin and trypsin activities in dosedependent manner, when the ratios of rpoKSPI to chymotrypsin and trypsin were 36:1 and 72:1, respectively, the proteinase activities of chymotrypsin and trypsin could be almost completely inhibited (Fig. 6). However, after incubation with different concentration of rpoKSPI, there was no evident alteration of subtilisin activity observed in tight-binding inhibition assay (Fig. 6). 4. Discussion
Fig. 3. Temporal expressions of the poKSPI transcript were detected by quantitative real-time RT-PCR after LPS challenge in digestive gland (A) and gill (B). b-Actin gene was used as an internal control to calibrate the cDNA template for all the samples. Vertical bars represented the mean S.D (N ¼ 3). Significant differences between the challenge group and the control group were indicated with the asterisk (* represented P < 0.05, ** represented P < 0.01).
Kazal-type serine proteinase inhibitors (KSPIs) existed widely in marine mollusks, and played the important roles in their innate immune processes. There were several types of KSPIs based on the number of Kazal domains. Recently, the KSPIs were identified and characterized from scallop A. with 6 Kazal domains [19], C. farreri with 12 Kazal domains [20], surf clam Mesodesma donacium with 2 Kazal domains [21] and abalone Haliotis discus discus with 2 Kazal domains [22]. In this study, a five Kazal-type serine proteinase inhibitor (poKSPI) was characterized from P. fucata (Fig. 1). A typical or canonical Kazal domain consisted of 40e60 amino acid residues including some spacer amino acids [15]. However, the Kazal domains of the poKSPI were only composed of 36e39 amino acid residues, which were shorter than other species, such as surf clam [21], swimming crab Portunus trituberculatus [16] and white shrimp Litopenaeus vannamei [32]. The five Kazal domains of the poKSPI showed highly homology to Kazal-type proteins of other species (Supplementary data, Fig. 1), most of them shared the characterized sequence motif of Kazal domain, described as C-X(1-7)-C-X(1-7)-CX(6)-Y-X3-C-X(2,3)-C-X(9-16)-C [33], and six conserved cysteine residues responsible for the formation of intra-domain disulfide bridges [13]. However, not all the domains of poKSPI were entirely consistent with the conserved sequence motif. The poKSPI-1 domain was short of C2 and C4. The ASPI-1 of A. irradians was short of C1 and C5 [19], and the CfKZSPI-1 and -4 of C. farreri were short of C1 and C5, C2 and C4, respectively [20]. So the poKSPI was also considered to be a non-classical inhibitor. The spatial expression pattern of the poKSPI in different tissues could offer useful cues to understand the biological roles of KSPIs in bivalve mollusks. In this study, the poKSPI mRNA was constitutively expressed in all examined tissues, although the expression levels varied between tissues. The expression levels of the poKSPI mRNA were the highest in mantle and gonad, and the lowest in haemocyte and intestine (Fig. 2). Similarly, the expression level of MdSPI-1 mRNA in surf clam was also higher in mantle than in other tissue [22]. The high expression level of KSPI mRNA in mantle was possibly due to the direct contact of this tissue with the external medium and consequently with contagious pathogens. The previous studies demonstrated that KSPI were involved in many immune processes [2e4] and some of them had
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Fig. 4. Temporal expressions of the poKSPI transcript were detected by quantitative real-time RT-PCR after V. alginolyticus challenge in digestive gland (A) and gill (B). b-Actin gene was used as an internal control to calibrate the cDNA template for all the samples. Vertical bars represented the mean S.D (N ¼ 3). Significant differences between the challenge group and the control group were indicated with the asterisk (* represented P < 0.05, ** represented P < 0.01).
antimicrobial ability [6,7], so the high expression level of the poKSPI mRNA in gonad could be considered to play an important role in resistance to pathogenic infection for reproductive cells. In addition, the wide tissue distribution of the poKSPI mRNA might suggest that it could be involved in multiplex biological functions in pearl oysters. As an important regulator of innate immune processes, the expression levels of the KSPI mRNAs have been demonstrated to be significantly up-regulated by immune challenge. The expression level of CfKZSPI mRNA in haemocyte of C. farreri was upregulated and reached 207.8-fold at 12 h after Vibrio anguillarum challenge [20]. MdSPI-1 and MdSPI-2 mRNA expressions in mantle
of surf clam were significantly up-regulated at 2 and 8 h after V. anguillarum challenge [21]. The Ab-KPI expression in haemocytes of abalone was increased 14-fold and 4-fold at 6 h postbacterial and -VHSV challenge, respectively [22]. In this study, to further understand the possible biological function of the poKSPI, its mRNA expression was examined at different time-point after LPS or V. alginolyticus challenge in gill and digestive gland. The gill represented one of the main interfaces between aquatic organisms and the external environment, bivalve mollusk gill was the first line of defencedefence against bacterial infection [34]; the digestive gland of mollusk was thought to be an important immune organ, which could secrete various enzymes to hydrolyze
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Acknowledgements This research was supported by National Infrastructure of Fishery Germplasm Resources Project (2013DKA-007), Major Science and Technology Projects of Guangdong (A201101A05, A201201A08), Central Institutes of Public Welfare Projects (2012TS05), and Institute of Science and Technology Cooperation Projects of Sanya City (2012YD84). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.fsi.2013.12.011. Fig. 5. The expression and purification of recombinant poKSPI. The cell cultures were grown for 3.5 h after IPTG induction and harvested. The recombinant protein was detected on 12% SDS-PAGE. A: lanes 1e3 were total protein of BL21 with pRSET-poKSPI, lane 4 was total protein of BL21 with pRSET; B: lane 1 was the purified recombinant poKSPI protein, lane 2 was total protein of BL21 with pRSET; M was protein marker.
microorganisms and involve in the digestive and defence functions [35]. So we selected the gill and digestive gland to research the temporal expression pattern of poKSPI after the immune challenge. The diverse expression profiles of the poKSPI mRNA after immune challenge suggested that the poKSPI could participate in various immune responses of pearl oysters triggered by different microorganisms. The inhibitory specificity primarily depends on P1 residue in the reactive site of Kazal domain [36]. Generally, the KSPIs with P1 Arg or Lys residues tend to inhibit trypsin and thrombin; the KSPIs with P1 Pro, Tyr, Phe, Leu and Met residues are considered to inhibit chymotrypsin and chymotrypsin-like enzymes; the KSPIs with P1 Ser and Met residues can inhibit elastase [1]. The several studies also demonstrated that the KSPIs with P1 Thr residue could inhibit subtilisin [6,7,37,38]. In this study, the rpoKSPI showed the strong inhibitory activities against chymotrypsin and trypsin, but could not inhibit subtilisin at all. These inhibitory activities could be speculated by the P1 sites of the poKSPI with two Leu and three Lys residues.
Fig. 6. The doseeresponse inhibitory curves of the recombinant poKSPI on subtilisin, trypsin and chymotrypsin. The reaction mixture consisted of 100 mM TriseHCl, pH 8.0; 600 mM of N-benzoyl-Phe-Val-Arg-p-nitroanilide as substrate for subtilin and trypsin and 300 mM N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide for chymotrypsin. Proteinases were respectively incubated with the increasing concentration of inhibitor (0e72 fold to each enzyme) for 15 min at 30 C and then terminated by adding 50 mL of 50% acetic acid. The increase in p-nitroaniline production was recorded by measuring the absorbance at 405 nm. The inhibitions of recombinant poKSPI were plotted as the residual activity of proteases against the concentrations of the inhibitor.
References [1] Laskowski Jr M, Kato I. Protein inhibitors of proteinases. Annu Rev Biochem 1980;49:593e626. [2] Jiravanichpaisal P, Lee BL, Söderhäll K. Cell-mediated immunity in arthropods: hematopoiesis, coagulation, melanization and opsonization. Immunobiology 2006;211(4):213e36. [3] Cao JX, Dai JQ, Dai ZM, Yin GL, Yang WJ. A male-reproduction related Kazal-type peptidase inhibitor gene in the prawn, Macrobrachium rosenbergii: molecular characterization and expression patterns. Mar Biotechnol 2007;9:45e55. [4] Visetnan S, Donpudsa S, Supungul P, Tassanakajon A, Rimphanitchayakit V. Kazal-type serine proteinase inhibitors from the black tiger shrimp Penaeus monodon and the inhibitory activities of SPIPm4 and 5. Fish Shellfish Immunol 2009;27(2):266e74. [5] Augustin R, Siebert S, Bosch TC. Identification of a Kazal-type serine protease inhibitor with potent anti-staphylococcal activity as part of Hydra’s innate immune system. Dev Comp Immunol 2009;33:830e7. [6] Donpudsa S, Tassanakajon A, Rimphanitchayakit V. Domain inhibitory and bacteriostatic activities of the five-domain Kazal-type serine proteinase inhibitor from black tiger shrimp Penaeus monodon. Dev Comp Immunol 2009;33(4):481e8. [7] Li XC, Zhang RR, Sun RR, Lan JF, Zhao XF, Wang JX. Three Kazal-type serine proteinase inhibitors from the red swamp crayfish Procambarus clarkii and the characterization, function analysis of hcPcSPI2. Fish Shellfish Immunol 2010;28(5e6):942e51. [8] Morris MT, Coppin A, Tomavo S, Carruthers VB. Functional analysis of Toxoplasma gondii protease inhibitor 1. J Biol Chem 2002;277:45259e66. [9] Friedrich T, Kroger B, Bialojan S, Lemaire HG, Hoffken HW, Reuschenbach P, et al. A Kazal-type inhibitor with thrombin specificity from Rhodnius prolixus. J Biol Chem 1993;268:16216e22. [10] Mende K, Petoukhova O, Koulitchkova V, Schaub GA, Lange U, Kaufmann R, et al. Dipetalogastin, a potent thrombin inhibitor from the blood-sucking insect Dipetalogaster maximus. Eur J Biochem 1999;266:583e90. [11] Campos IT, Amino R, Sampaio CA, Auerswald EA, Friedrich T, Lemaire HG, et al. Infestin, a thrombin inhibitor presents in Triatoma infestans midgut, a Chagas’ disease vector: gene cloning, expression and characterization of the inhibitor. Insect Biochem Mol Biol 2002;32:991e7. [12] van de Locht A, Lamba D, Bauer M, Huber R, Friedrich T, Kröger B, et al. Two heads are better than one: crystal structure of the insect derived double domain Kazal inhibitor Rhodniin in complex with thrombin. EMBO J 1995;14(21):5149e57. [13] Bode W, Huber R. Natural protein proteinase inhibitors and their interaction with proteinases. Eur J Biochem 1992;204:433e51. [14] Lu SM, Lu W, Qasim MA, Anderson S, Apostol I, Ardelt W, et al. Predicting the reactivity of proteins from their sequence alone: Kazal family of protein inhibitors of serine proteinases. Proc Natl Acad Sci USA 2001;98(4):1410e5. [15] Rimphanitchayakit V, Tassanakajon A. Structure and function of invertebrate Kazal-type serine proteinase inhibitors. Dev Comp Immunol 2010;34(4):377e86. [16] Wang SY, Cui ZX, Liu Y, Li QQ, Song CW. The first Kazal-type serine proteinase inhibitor in the swimming crab Portunus trituberculatus involved in immune response to bacteria and fungi. Aquaculture 2012;356e357:55e60. [17] Vargas-Albores F, Villalpando E. A new type of Kazal proteinase inhibitor related to shrimp Penaeus (Litopenaeus) vannamei immunity. Fish Shellfish Immunol 2012;33(1):134e7. [18] Qian YQ, Li Y, Yang F, Yu YQ, Yang JS, Yang WJ. Two Kazal-type protease inhibitors from Macrobrachium nipponense and Eriocheir sinensis: comparative analysis of structure and activities. Fish Shellfish Immunol 2012;32(3): 446e58. [19] Zhu L, Song LS, Chang YQ, Xu W, Wu LT. Molecular cloning, characterization and expression of a novel serine proteinase inhibitor gene in bay scallops (Argopecten irradians, Lamarck 1819). Fish Shellfish Immunol 2006;20(3): 320e31. [20] Wang B, Zhao J, Song L, Zhang H, Wang L, Li C, et al. Molecular cloning and expression of a novel Kazal-type serine proteinase inhibitor gene from
D. Zhang et al. / Fish & Shellfish Immunology 37 (2014) 115e121
[21]
[22]
[23]
[24]
[25]
[26] [27] [28]
[29]
Zhikong scallop Chlamys farreri, and the inhibitory activity of its recombinant domain. Fish Shellfish Immunol 2008;24(5):629e37. Maldonado-Aguayo W, Núñez-Acuña G, Valenzuela-Muñoz V, ChávezMardones J, Gallardo-Escárate C. Molecular characterization of two Kazal-type serine proteinase inhibitor genes in the surf clam Mesodesma donacium exposed to Vibrio anguillarum. Fish Shellfish Immunol 2013;34(6):1448e54. Wickramaarachchi WD, De Zoysa M, Whang I, Wan Q, Lee J. Kazal-type proteinase inhibitor from disk abalone (Haliotis discus discus): molecular characterization and transcriptional response upon immune stimulation. Fish Shellfish Immunol 2013;35:1039e43. Zhang DC, Jiang SG, Qiu LH, Su TF, Wu KC, Li YN, et al. Molecular characterization and expression analysis of the IkB gene from pearl oyster Pinctada fucata. Fish Shellfish Immunol 2009;26(1):84e90. Zhang DC, Jiang SG, Hu YT, Cui SG, Guo HY, Wu KC, et al. A multidomain galectin involved in innate immune response of pearl oyster Pinctada fucata. Dev Comp Immunol 2011;35(1):1e6. Petersen TN, Brunak S, von Heijne G, Nielsen H. SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods 2011;8(10): 785e6. Letunic I, Copley RP, Pils B, Pinkert S, Schultz J, Bork P. SMART 5: domains in the context of genomes and networks. Nucleic Acids Res 2006;34:257e60. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, et al. Clustal W and clustal X version 2.0. Bioinformatics 2007;23(21):2947e8. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 2011;28: 2731e9. Cui SG, Zhang DC, Jiang SG, Pu HL, Hu YT, Guo HY, et al. A macrophage migration inhibitory factor like oxidoreductase from pearl oyster Pinctada
[30]
[31]
[32] [33]
[34]
[35] [36]
[37]
[38]
121
fucata involved in innate immune responses. Fish Shellfish Immunol 2011;31(2):173e81. Hergenhahn HG, Aspan A, Söderhäll K. Purification and characterization of a high-Mr proteinase inhibitor of pro-phenol oxidase activation from crayfish plasma. Biochem J 1987;248:223e8. Sommerhoff CP, Söllner C, Mentele R, Piechottka GP, Auerswald EA, Fritz H. A Kazal-type inhibitor of human mast cell tryptase: isolation from the medical leech Hirudo medicinalis, characterization, and sequence analysis. Biol Chem Hoppe Seyler 1994;375(10):685e94. Jiménez-Vega F, Vargas-Albores F. A four-Kazal domain protein in Litopenaeus vannamei hemocytes. Dev Comp Immunol 2005;29:385e91. Stubbs MT, Morenweiser R, Sturzebecher J, Bauer M, Bode W, Huber R, et al. The three-dimensional structure of recombinant leech-derived tryptase inhibitor in complex with trypsin. Implications for the structure of human mast cell tryptase and its inhibition. J Biol Chem 1997;272:19931e7. Lichtenfels AJ, Lorenzi-Filho G, Guimaraes ET, Macchione M, Saldiva PH. Effects of water pollution on the gill apparatus of fish. J Comp Pathol 1996;115: 47e60. Tisca PG, Mosca F. Defense mechanisms in farmed marine mollusks. Vet Res Commun 2004;28:57e62. Kellenberger C, Boudier C, Bermudez I, Bieth JG, Luu B, Hietter H. Serine protease inhibition by insect peptides containing a cysteine knot and a triplestranded beta-sheet. J Biol Chem 1995;270:25514e9. Johansson MW, Keyser P, Söderhall K. Purification and cDNA cloning of a fourdomain Kazal proteinase inhibitor from crayfish blood cells. Eur J Biochem 1994;223(2):389e94. Somprasong N, Rimphanitchayakit V, Tassanakajon A. A fivedomain Kazaltype serine proteinase inhibitor from black tiger shrimp Penaeus monodon and its inhibitory activities. Dev Comp Immunol 2006;30:998e1008.
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Full length article
Molecular characterization, expression and function analysis of a five-domain Kazal-type serine proteinase inhibitor from pearl oyster Pinctada fucata Dianchang Zhang, Jianjun Ma, Shigui Jiang* Key Laboratory of South China Sea Fishery Resources Exploitation and Utilization, Ministry of Agriculture, South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou 510300, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 September 2013 Received in revised form 4 December 2013 Accepted 18 December 2013 Available online 27 December 2013
Serine proteinase inhibitors represent an expanding superfamily of endogenous inhibitors that are regulate proteolytic events and involved in a variety of physiological and immunological processes. A five-domain Kazal-type serine proteinase inhibitor (poKSPI) was identified and characterized from pearl oyster Pinctada fucata based on expressed sequence tag (EST) analysis. The full-length cDNA was 737 bp with an open reading frame (ORF) 660 bp encoding a 219 amino acid protein a theoretical molecular weight (Mw) of 23.3 kDa and an isoelectric point (pI) of 8.40. A putative signal peptide of 19 amino acid residues and five tandem Kazal domains were identified. Four of the Kazal domains had the highly conserved motif sequences with six cysteine residues responsible for the formation of disulfide bridges. The deduced amino acid sequence of the poKSPI shared high homology with KSPIs from Hirudo medicinalis. The poKSPI mRNA could be detected in all examined tissues, the expression level of the poKSPI mRNA was the highest in mantle and gonad, while the lowest in haemocyte and intestine. After LPS challenge, the expression level of the poKSPI mRNA in digestive gland was significantly up-regulated at 4 h post-challenge and reached the peak at 12 h post-challenge, which was 4.23-fold higher than control group; the expression level of the poKSPI mRNA in gill was also significantly up-regulated at 8 and 12 h post-challenge, which were 4.48 and 2.26-fold higher than control group. After Vibrio alginolyticus challenge, the expression levels of the poKSPI mRNA in digestive gland were significantly upregulated at 8, 12, 48 and 72 h post-challenge, which were 1.70, 1.79, 3.89 and 5.69-fold higher than control group, respectively; the expression level of the poKSPI mRNA in gill was significantly upregulated at 24 h post-challenge, which was 5.30-fold higher than control group. The recombinant poKSPI protein could inhibit chymotrypsin and trypsin activities in dose-dependent manner, when the ratios of rpoKSPI to chymotrypsin and trypsin were 36:1 and 72:1, respectively, the proteinase activities of chymotrypsin and trypsin could be almost completely inhibited, but the rpoKSPI could not inhibit subtilisin. Ó 2013 Published by Elsevier Ltd.
Keywords: Pinctada fucata Serine proteinase inhibitor Microbial challenge Quantitative real-time PCR Inhibitory activity
1. Introduction Serine proteinase inhibitors (SPIs) are found widely in all multicellular organisms and take part in controlling the various proteinase-mediated biological processes, such as digestion, complement system, blood coagulation, melanization, apoptosis, phagocytosis, cellular remodelling and reproductive processes [1e 4]. Some SPIs also show the potent bactericidal or bacteriostatic activities [5,6] and play the important roles as part of the humoural
* Corresponding author. Tel.: þ86 20 89108338; fax: þ86 20 84451442. E-mail address: [email protected] (S. Jiang). 1050-4648/$ e see front matter Ó 2013 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.fsi.2013.12.011
defence of the innate immune system against the invading pathogens [7]. Several microbial pathogens and parasites use the SPIs to counter the protective proteinases of hosts. For example, the intracellular parasite of human Toxoplasma gondii secretes a serine proteinase inhibitor to protect itself from the digestive enzymes during its residency in small intestine [8]. Rhodnius prolix, Dipetalogaster maximus and Triatoma infestans can produce the potent proteinase inhibitors to prevent blood coagulation and make them easier to suck blood from their hosts [9e11]. SPIs are classified into at least 63 families on the basis of amino acid sequence similarities (http://merops.sanger.ac.uk/). Kazal-type serine proteinase inhibitors (KSPIs) are well known and grouped into family Ⅰ1. All the KSPIs contain one or more Kazal domains and
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each domain consists of 50e60 amino acid residues and contains six well-conserved cysteine residues forming three intra-domain disulphide bridges, which are responsible for the stability of three-dimensional structure [12]. The reactive site (P1 residue) is located at the second position after the second cysteine residue of the domain and believed to contribute mainly to the inhibitory specificity [13], a few other adjacent amino acid residues also influence the binding specificity [14,15]. Recently, a variety of KSPIs are found in crustaceans [4,6,7,16e18]. They were believed to be involved in innate immune responses, because their expressions were up-regulated after bacterial challenge. Meanwhile, several of KSPIs also had bacteriostatic activity against some pathogens [6,7]. However, until now, the KSPIs were only studied in few mollusks [19e22], these results preliminarily demonstrated that KSPIs were involved in innate responses of mollusk. In contrast with the abundant knowledge on KSPIs in crustaceans, the information of KSPIs in bivalve mollusk is still poorly known. To further understand the molecular functions of KSPIs in the innate immune responses of bivalve mollusks, in this study, we cloned and characterized a Kazal-type serine proteinase inhibitor from pearl oyster Pinctada fucata (designated as poKSPI), which is the most important farmed bivalve mollusk for seawater pearl production in China [23,24], and investigated its tissue distribution and temporal expression profile after bacterial and lipopolysaccharide (LPS) challenge. This work would hopefully provide insight into the important functions in the innate immune responses of pearl oysters. 2. Materials and methods 2.1. EST and poKSPI cDNA analysis A cDNA library was constructed and analyzed in our previous studies [23]. These EST data were used to search for the Kazal-type serine proteinase inhibitors, and an EST (EST No. pmpca0_002189) of 453 bp was homologous to the KSPIs of Chlamys farreri (EU183309) and Argopecten irradians (AY830445), the plasmid of this EST was picked up and re-sequenced to get the full-length cDNA and ensure the correctness of the sequence. DNATool 6.0 software was used to deduce the poKSPI amino acid sequence. Searches for nucleotide and protein sequence similarities were conducted with BLAST algorithm at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/BLAST/). SignalP 4.0 software (http://www.cbs.dtu.dk/services/SignalP/) was used to predict the putative signal peptide cleavage site [25]. The simple modular architecture research tool (SMART) 4.0 program (http://www.smart.emblheidelberg.de/) and ScanProsite (http:// expasy.org/tools/) were used to predict the protein domain [26]. ClustalW (http://www.clustal.org/) [27] and MEGA 5.1 [28] were used to align poKSPI with other known KSPI sequences.
Munich, Germany, LPS 10 mg ml1) dissolved in phosphate-buffer saline (PBS) into the adductor muscles of each pearl oyster. The bacterial challenge experiment was carried out by injecting with 100 mL of Vibrio alginolyticus resuspended in PBS to OD600 ¼ 0.4 (1 OD ¼ 5 108 bacteria ml1) into the adductor muscles of each pearl oyster. While pearl oysters injected with 100 mL PBS were used as control group in the challenge experiments. The injected pearl oysters were returned into seawater tanks and 15 individuals were randomly sampled at 0, 2, 4, 8, 12, 24, 48 and 72 h in each group, respectively. The gill and digestive gland from the control and challenge groups were collected respectively and immediately stored in liquid nitrogen until used. For tissue distribution experiment, three healthy pearl oysters were dissected to sample haemocyte, gill, digestive gland, mantle, gonad, adductor muscle and intestine tissues, respectively, and then they were stored in liquid nitrogen until used. Total RNA samples were extracted using the TRIzol reagent (Invitrogen) according to the manufacture instructions. 2.3. Quantitative real-time RT-PCR analysis of poKSPI mRNA expression For the expression pattern of poKSPI in different tissues, total RNA was extracted from haemocyte, gill, digestive gland, mantle, gonad, intestine and adductor muscle of three healthy pearl oysters. For the temporal expression analysis, total RNA was extracted from gill and digestive gland sampled from the different time point in each group, respectively. The total RNA of each sample was treated with the DNase I (Promega) respectively, and then the single-strand cDNA was synthesized based on manufacture’s instruction of PrimerScriptÔ 1st Strand cDNA Synthesis Kit (TaKaRa) using random primer. cDNA mix was diluted to 1:10 and stored at 80 C for expression analysis. Two poKSPI gene-specific primers, poKSPI-F (50 -TGGAAGATGTCCCTGCATC-30 ) and poKSPI-R (50 -GTCCGCGCATTTAGCTGTA30 ), were used to amplify a product of 183 bp from cDNA template, and the PCR products were sequenced to verify the specificity of RTPCR. A 97 bp fragment was amplified using two primers of pearl oyster b-actin to calibrate the cDNA template as an internal control [23]. The SYBR Green RT-PCR assay was performed in the Eppendorf Realplex2 Detection System as described by Zhang et al. [23]. Dissociation curve analysis of amplification products was carried out at the end of each PCR reaction to confirm that only one PCR product was amplified and detected. After the PCR program, data were analyzed with the Realplex2 software (Eppendorf). The 2DDCT method was used to determine fold change for gene expression relative to controls [23]. All data were given in terms of relative mRNA expression as means S.D. the data were subjected to oneway analysis of variance (one-way ANOVA) followed by a multiple comparison with GraphPad Prism 5.0 Software, and the P value less than 0.05 were considered statistically significant.
2.2. Pearl oysters and immune challenge 2.4. Recombinant expression and purification of poKSPI Pearl oysters P. fucata (shell length 6.15 0.32 cm, body weight 23.8 4.5 g) were obtained from pearl oyster culture base of South China Sea Fisheries Research Institute in Xincun village, Hainan province, China and maintained at 25e27 C with the recirculating seawater for one week before experiment. The pearl oysters were fed twice daily on Tetraselmis suecica and Isochrysis galbana. To take account of individual variability, 15 individual pearl oysters, which were divided into three replicates, were used for each time point (0, 2, 4, 8, 12, 24, 48 and 72 h) in the LPS and bacterial challenge experiments, respectively. The LPS challenge experiment was performed by injecting with 100 mL LPS (Escherichia coli 055:B5, #62326, SigmaeAldrich,
The fragment encoding mature protein of poKSPI was amplified with gene-specific primers poKSPI-F1 (50 - GGTGGATCCAGATCGCCACCCTACAAAC-30 ) and poKSPI-R1 (50 - GCGAAGCTTTTATTCATACTTCCTACATGGACC-30 ). For convenience of cloning, a Bam HI site was added to the 50 end of poKSPI-F1 and a Hind Ⅲ site was added to the 50 end of poKSPI-R1 before the stop codon. The PCR products was cloned into pMD18-T simple vector (Takara), digested by completely by restriction enzymes Bam HI and Hind Ⅲ, and then ligated into predigested expression vector pRSET. The recombinant plasmid pRSET-poKSPI was transformed into E. coli BL21 (Novagen). The positive transformant with pREST-poKSPI was
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picked up and sequenced, as well as, the transformant with only pRSET was used as the control group. Both of them were incubated at 37 C in liquid culture medium containing 100 mg ml1 ampicillin (QingDao MDBio Inc., QingDao City, Shandong, China) (LA) with shaking at 225 rpm. When the OD600 of bacteria concentration reached w0.6, isopropyl b-D-1-thiogalactopyranoside (IPTG) (MDBio) was added into LA medium at the final concentration of 0.8 mmol L1, then continued to incubate for 5 h. The recombinant poKSPI was purified and refolded by affinity chromatography on a resin with the chelated nickel ions according to His$Tag Purification Kit (MERCK, Jing’an District, Shanghai, China) as described by Cui et al. [29]. The resultant protein was separated by reducing 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDSPAGE), visualized with coomassie brilliant blue R250. The concentration of purified rpoKSPI was quantified by BCA method [29], and then the rpoKSPI was stored at 80 C for subsequent experiments. 2.5. Proteinase inhibition activity assays of rpoKSPI The inhibitory activities of rpoKSPI towards to chymotrypsin (bovine pancreas, Sigma), trypsin (bovine pancreas, Sigma) and subtilisin (Bacillus licheniformis, Sigma) were analyzed as described by Hergenhahn et al. [30]. The reaction mixture consisted of 100 mM TriseHCl, pH 8.0; 600 mM of N-benzoyl-Phe-Val-Arg-pnitroanilide as substrate for subtilin and trypsin and 300 mM Nsuccinyl-Ala-Ala-Pro-Phe-p-nitroanilide for chymotrypsin, the appropriate concentration of proteinase and proteinase inhibitors in a total volume of 100 mL. Proteinases (0.4 mM subtilisin, 0.4 mM trypsin or 0.2 mM chymotrypsin) were respectively incubated with the increasing concentration of inhibitor (3, 6, 9, 18, 36, 72-fold to each enzyme) in the buffer for 15 min at 30 C and then terminated by adding 50 mL of 50% acetic acid. The proteinase reaction product, p-nitroaniline, was measured at 405 nm using microplate reader. The percentage of remaining activity was calculated and plotted against the molar ratio of inhibitor to proteinase. 3. Results 3.1. Molecular characteristics of poKSPI A Kazal-type serine proteinase inhibitor with full-length cDNA sequence was identified from the cDNA library of P. fucata and deposited in GenBank under accession number GU361540. The complete cDNA sequence of poKSPI was 737 bp with an open reading frame (ORF) 660 bp encoding a 219 amino acid protein, a 50 -untranslated region (UTR) of 42 bp, a 30 UTR of 35 bp (Fig. 1). The deduced protein sequence contained a putative 19 aa signal peptide and five tandem Kazal domains, four of the Kazal domains had the highly conserved motif sequences C-X(6-9)-C-X(7)-C-X(10)-Y-X(3)C-X(2, 3)-C-X(9-16)-C with six cysteine residues responsible for the formation of disulfide bridges (C1eC5, C2eC4, C3eC6) (Supplemental data, Fig. 1). The poKSPI protein had a theoretical molecular weight (Mw) of 23.3 kDa and an isoelectric point (pI) of 8.40. The significant similarities were detected between the domains of poKSPI and other Kazal-type SPIs (Supplemental data, Fig. 1), the similarities among five domains of poKSPI were from 54.1% to 83.8%, and poKSPI-5 had a 64.9% similarity with Hirudo nipponia and 67.6% with Hirudo medicinalis [31]. In the P1 site of the poKSPI domains, there was Leu in 1st and 2nd, and Lys in 3rd, 4th and 5th domains (Fig. 1). 3.2. Spatial expression pattern of poKSPI mRNA in different tissues Quantitative real-time RT-PCR was performed to investigate the tissue expression pattern of the poKSPI mRNA with the b-actin as
Fig. 1. The nucleotide and deduced amino acid sequences of poKSPI (GenBank accession number GU361540). The putative signal peptide is underlined. The family signatures of Kazal-type domains are shaded. The potential N-glycosylation site (Asn47) is double underlined. The P1 sites (Leu32, Leu70, Lys108, Lys146 and Lys183) are boxed. The conserved cysteine residues are bolded. The stop codon is indicated by asterisk.
an internal control. The mRNA transcripts of poKSPI could be detected in all the examined tissues including gill, digestive gland, mantle, gonad, adductor muscle, intestine and haemocyte (Fig. 2). 3.3. Temporal expression profile of poKSPI mRNA after immune challenge The temporal expression profiles of the poKSPI mRNA in digestive gland and gill after LPS challenge were measured by quantitative real-time RT-PCR. LPS challenge induced the up-
Fig. 2. Expression levels of poKSPI mRNA were measured by quantitative real-time quantitative RT-PCR in different tissues. RNA samples were extracted from digestive gland (DG), mantle, intestine, gonad, adductor muscle (AM), gill and haemocyte of the adult tissues of pearl oyster. The b-actin gene was used as an internal control to calibrate the cDNA templates. Vertical bars represented the mean S.D (N ¼ 3).
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regulated expression of the poKSPI mRNA of digestive gland in time-dependent fashion. The expression level of the poKSPI mRNA was significantly up-regulated at 4 h post-challenge and reached the peak at 12 h post-challenge, which was 4.23-fold higher than control group, and then the expression level of the poKSPI mRNA dropped gradually (Fig. 3A). The expression level of the poKSPI mRNA in gill was also significantly up-regulated at 8 and 12 h postchallenge, which were 4.48 and 2.26-fold higher than control group, respectively, and then dropped dramatically at 24 h postchallenge (Fig. 3B). The expression levels of the poKSPI mRNA in digestive gland and gill were also significantly up-regulated after V. alginolyticus challenge (Fig. 4). In digestive gland, the expression levels of the poKSPI mRNA were significantly up-regulated at 8, 12, 48 and 72 h postchallenge, which were 1.70, 1.79, 3.89 and 5.69-fold higher than control group, respectively (Fig. 4A). In gill, the expression level of the poKSPI mRNA was significantly up-regulated at 24 h postchallenge, which was 5.30-fold higher than control group (Fig. 4B). 3.4. Expression and purification of recombinant poKSPI The recombinant plasmid pRSET-poKSPI was transformed into E. coli BL21 and then induced with IPTG to expression the
recombinant poKSPI protein as described above. After IPTG induction, the whole cell lysate of positive clone was separated by SDS-PAGE, and a distinct band with a molecular weight of about 24 kDa was detected, which was consistent with the predicted molecular mass (24.3 kDa), while negative clone with only pRSET plasmid lack this protein band (Fig. 5A). The purified and refolded rpoKSPI with only one target band was obtained by affinity chromatography and detected by SDS-PAGE (Fig. 5B). 3.5. Proteinase inhibition activity of rpoKSPI To investigate the inhibition activity of the recombinant poKSPI, tight-binding inhibition assay was carried out using increasing amount of the purified rpoKSPI against chymotrypsin, trypsin and subtilisin with corresponding artificial substrates. The dosee response curve of serine proteinase inhibition showed that rpoKSPI could inhibit chymotrypsin and trypsin activities in dosedependent manner, when the ratios of rpoKSPI to chymotrypsin and trypsin were 36:1 and 72:1, respectively, the proteinase activities of chymotrypsin and trypsin could be almost completely inhibited (Fig. 6). However, after incubation with different concentration of rpoKSPI, there was no evident alteration of subtilisin activity observed in tight-binding inhibition assay (Fig. 6). 4. Discussion
Fig. 3. Temporal expressions of the poKSPI transcript were detected by quantitative real-time RT-PCR after LPS challenge in digestive gland (A) and gill (B). b-Actin gene was used as an internal control to calibrate the cDNA template for all the samples. Vertical bars represented the mean S.D (N ¼ 3). Significant differences between the challenge group and the control group were indicated with the asterisk (* represented P < 0.05, ** represented P < 0.01).
Kazal-type serine proteinase inhibitors (KSPIs) existed widely in marine mollusks, and played the important roles in their innate immune processes. There were several types of KSPIs based on the number of Kazal domains. Recently, the KSPIs were identified and characterized from scallop A. with 6 Kazal domains [19], C. farreri with 12 Kazal domains [20], surf clam Mesodesma donacium with 2 Kazal domains [21] and abalone Haliotis discus discus with 2 Kazal domains [22]. In this study, a five Kazal-type serine proteinase inhibitor (poKSPI) was characterized from P. fucata (Fig. 1). A typical or canonical Kazal domain consisted of 40e60 amino acid residues including some spacer amino acids [15]. However, the Kazal domains of the poKSPI were only composed of 36e39 amino acid residues, which were shorter than other species, such as surf clam [21], swimming crab Portunus trituberculatus [16] and white shrimp Litopenaeus vannamei [32]. The five Kazal domains of the poKSPI showed highly homology to Kazal-type proteins of other species (Supplementary data, Fig. 1), most of them shared the characterized sequence motif of Kazal domain, described as C-X(1-7)-C-X(1-7)-CX(6)-Y-X3-C-X(2,3)-C-X(9-16)-C [33], and six conserved cysteine residues responsible for the formation of intra-domain disulfide bridges [13]. However, not all the domains of poKSPI were entirely consistent with the conserved sequence motif. The poKSPI-1 domain was short of C2 and C4. The ASPI-1 of A. irradians was short of C1 and C5 [19], and the CfKZSPI-1 and -4 of C. farreri were short of C1 and C5, C2 and C4, respectively [20]. So the poKSPI was also considered to be a non-classical inhibitor. The spatial expression pattern of the poKSPI in different tissues could offer useful cues to understand the biological roles of KSPIs in bivalve mollusks. In this study, the poKSPI mRNA was constitutively expressed in all examined tissues, although the expression levels varied between tissues. The expression levels of the poKSPI mRNA were the highest in mantle and gonad, and the lowest in haemocyte and intestine (Fig. 2). Similarly, the expression level of MdSPI-1 mRNA in surf clam was also higher in mantle than in other tissue [22]. The high expression level of KSPI mRNA in mantle was possibly due to the direct contact of this tissue with the external medium and consequently with contagious pathogens. The previous studies demonstrated that KSPI were involved in many immune processes [2e4] and some of them had
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Fig. 4. Temporal expressions of the poKSPI transcript were detected by quantitative real-time RT-PCR after V. alginolyticus challenge in digestive gland (A) and gill (B). b-Actin gene was used as an internal control to calibrate the cDNA template for all the samples. Vertical bars represented the mean S.D (N ¼ 3). Significant differences between the challenge group and the control group were indicated with the asterisk (* represented P < 0.05, ** represented P < 0.01).
antimicrobial ability [6,7], so the high expression level of the poKSPI mRNA in gonad could be considered to play an important role in resistance to pathogenic infection for reproductive cells. In addition, the wide tissue distribution of the poKSPI mRNA might suggest that it could be involved in multiplex biological functions in pearl oysters. As an important regulator of innate immune processes, the expression levels of the KSPI mRNAs have been demonstrated to be significantly up-regulated by immune challenge. The expression level of CfKZSPI mRNA in haemocyte of C. farreri was upregulated and reached 207.8-fold at 12 h after Vibrio anguillarum challenge [20]. MdSPI-1 and MdSPI-2 mRNA expressions in mantle
of surf clam were significantly up-regulated at 2 and 8 h after V. anguillarum challenge [21]. The Ab-KPI expression in haemocytes of abalone was increased 14-fold and 4-fold at 6 h postbacterial and -VHSV challenge, respectively [22]. In this study, to further understand the possible biological function of the poKSPI, its mRNA expression was examined at different time-point after LPS or V. alginolyticus challenge in gill and digestive gland. The gill represented one of the main interfaces between aquatic organisms and the external environment, bivalve mollusk gill was the first line of defencedefence against bacterial infection [34]; the digestive gland of mollusk was thought to be an important immune organ, which could secrete various enzymes to hydrolyze
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Acknowledgements This research was supported by National Infrastructure of Fishery Germplasm Resources Project (2013DKA-007), Major Science and Technology Projects of Guangdong (A201101A05, A201201A08), Central Institutes of Public Welfare Projects (2012TS05), and Institute of Science and Technology Cooperation Projects of Sanya City (2012YD84). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.fsi.2013.12.011. Fig. 5. The expression and purification of recombinant poKSPI. The cell cultures were grown for 3.5 h after IPTG induction and harvested. The recombinant protein was detected on 12% SDS-PAGE. A: lanes 1e3 were total protein of BL21 with pRSET-poKSPI, lane 4 was total protein of BL21 with pRSET; B: lane 1 was the purified recombinant poKSPI protein, lane 2 was total protein of BL21 with pRSET; M was protein marker.
microorganisms and involve in the digestive and defence functions [35]. So we selected the gill and digestive gland to research the temporal expression pattern of poKSPI after the immune challenge. The diverse expression profiles of the poKSPI mRNA after immune challenge suggested that the poKSPI could participate in various immune responses of pearl oysters triggered by different microorganisms. The inhibitory specificity primarily depends on P1 residue in the reactive site of Kazal domain [36]. Generally, the KSPIs with P1 Arg or Lys residues tend to inhibit trypsin and thrombin; the KSPIs with P1 Pro, Tyr, Phe, Leu and Met residues are considered to inhibit chymotrypsin and chymotrypsin-like enzymes; the KSPIs with P1 Ser and Met residues can inhibit elastase [1]. The several studies also demonstrated that the KSPIs with P1 Thr residue could inhibit subtilisin [6,7,37,38]. In this study, the rpoKSPI showed the strong inhibitory activities against chymotrypsin and trypsin, but could not inhibit subtilisin at all. These inhibitory activities could be speculated by the P1 sites of the poKSPI with two Leu and three Lys residues.
Fig. 6. The doseeresponse inhibitory curves of the recombinant poKSPI on subtilisin, trypsin and chymotrypsin. The reaction mixture consisted of 100 mM TriseHCl, pH 8.0; 600 mM of N-benzoyl-Phe-Val-Arg-p-nitroanilide as substrate for subtilin and trypsin and 300 mM N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide for chymotrypsin. Proteinases were respectively incubated with the increasing concentration of inhibitor (0e72 fold to each enzyme) for 15 min at 30 C and then terminated by adding 50 mL of 50% acetic acid. The increase in p-nitroaniline production was recorded by measuring the absorbance at 405 nm. The inhibitions of recombinant poKSPI were plotted as the residual activity of proteases against the concentrations of the inhibitor.
References [1] Laskowski Jr M, Kato I. Protein inhibitors of proteinases. Annu Rev Biochem 1980;49:593e626. [2] Jiravanichpaisal P, Lee BL, Söderhäll K. Cell-mediated immunity in arthropods: hematopoiesis, coagulation, melanization and opsonization. Immunobiology 2006;211(4):213e36. [3] Cao JX, Dai JQ, Dai ZM, Yin GL, Yang WJ. A male-reproduction related Kazal-type peptidase inhibitor gene in the prawn, Macrobrachium rosenbergii: molecular characterization and expression patterns. Mar Biotechnol 2007;9:45e55. [4] Visetnan S, Donpudsa S, Supungul P, Tassanakajon A, Rimphanitchayakit V. Kazal-type serine proteinase inhibitors from the black tiger shrimp Penaeus monodon and the inhibitory activities of SPIPm4 and 5. Fish Shellfish Immunol 2009;27(2):266e74. [5] Augustin R, Siebert S, Bosch TC. Identification of a Kazal-type serine protease inhibitor with potent anti-staphylococcal activity as part of Hydra’s innate immune system. Dev Comp Immunol 2009;33:830e7. [6] Donpudsa S, Tassanakajon A, Rimphanitchayakit V. Domain inhibitory and bacteriostatic activities of the five-domain Kazal-type serine proteinase inhibitor from black tiger shrimp Penaeus monodon. Dev Comp Immunol 2009;33(4):481e8. [7] Li XC, Zhang RR, Sun RR, Lan JF, Zhao XF, Wang JX. Three Kazal-type serine proteinase inhibitors from the red swamp crayfish Procambarus clarkii and the characterization, function analysis of hcPcSPI2. Fish Shellfish Immunol 2010;28(5e6):942e51. [8] Morris MT, Coppin A, Tomavo S, Carruthers VB. Functional analysis of Toxoplasma gondii protease inhibitor 1. J Biol Chem 2002;277:45259e66. [9] Friedrich T, Kroger B, Bialojan S, Lemaire HG, Hoffken HW, Reuschenbach P, et al. A Kazal-type inhibitor with thrombin specificity from Rhodnius prolixus. J Biol Chem 1993;268:16216e22. [10] Mende K, Petoukhova O, Koulitchkova V, Schaub GA, Lange U, Kaufmann R, et al. Dipetalogastin, a potent thrombin inhibitor from the blood-sucking insect Dipetalogaster maximus. Eur J Biochem 1999;266:583e90. [11] Campos IT, Amino R, Sampaio CA, Auerswald EA, Friedrich T, Lemaire HG, et al. Infestin, a thrombin inhibitor presents in Triatoma infestans midgut, a Chagas’ disease vector: gene cloning, expression and characterization of the inhibitor. Insect Biochem Mol Biol 2002;32:991e7. [12] van de Locht A, Lamba D, Bauer M, Huber R, Friedrich T, Kröger B, et al. Two heads are better than one: crystal structure of the insect derived double domain Kazal inhibitor Rhodniin in complex with thrombin. EMBO J 1995;14(21):5149e57. [13] Bode W, Huber R. Natural protein proteinase inhibitors and their interaction with proteinases. Eur J Biochem 1992;204:433e51. [14] Lu SM, Lu W, Qasim MA, Anderson S, Apostol I, Ardelt W, et al. Predicting the reactivity of proteins from their sequence alone: Kazal family of protein inhibitors of serine proteinases. Proc Natl Acad Sci USA 2001;98(4):1410e5. [15] Rimphanitchayakit V, Tassanakajon A. Structure and function of invertebrate Kazal-type serine proteinase inhibitors. Dev Comp Immunol 2010;34(4):377e86. [16] Wang SY, Cui ZX, Liu Y, Li QQ, Song CW. The first Kazal-type serine proteinase inhibitor in the swimming crab Portunus trituberculatus involved in immune response to bacteria and fungi. Aquaculture 2012;356e357:55e60. [17] Vargas-Albores F, Villalpando E. A new type of Kazal proteinase inhibitor related to shrimp Penaeus (Litopenaeus) vannamei immunity. Fish Shellfish Immunol 2012;33(1):134e7. [18] Qian YQ, Li Y, Yang F, Yu YQ, Yang JS, Yang WJ. Two Kazal-type protease inhibitors from Macrobrachium nipponense and Eriocheir sinensis: comparative analysis of structure and activities. Fish Shellfish Immunol 2012;32(3): 446e58. [19] Zhu L, Song LS, Chang YQ, Xu W, Wu LT. Molecular cloning, characterization and expression of a novel serine proteinase inhibitor gene in bay scallops (Argopecten irradians, Lamarck 1819). Fish Shellfish Immunol 2006;20(3): 320e31. [20] Wang B, Zhao J, Song L, Zhang H, Wang L, Li C, et al. Molecular cloning and expression of a novel Kazal-type serine proteinase inhibitor gene from
D. Zhang et al. / Fish & Shellfish Immunology 37 (2014) 115e121
[21]
[22]
[23]
[24]
[25]
[26] [27] [28]
[29]
Zhikong scallop Chlamys farreri, and the inhibitory activity of its recombinant domain. Fish Shellfish Immunol 2008;24(5):629e37. Maldonado-Aguayo W, Núñez-Acuña G, Valenzuela-Muñoz V, ChávezMardones J, Gallardo-Escárate C. Molecular characterization of two Kazal-type serine proteinase inhibitor genes in the surf clam Mesodesma donacium exposed to Vibrio anguillarum. Fish Shellfish Immunol 2013;34(6):1448e54. Wickramaarachchi WD, De Zoysa M, Whang I, Wan Q, Lee J. Kazal-type proteinase inhibitor from disk abalone (Haliotis discus discus): molecular characterization and transcriptional response upon immune stimulation. Fish Shellfish Immunol 2013;35:1039e43. Zhang DC, Jiang SG, Qiu LH, Su TF, Wu KC, Li YN, et al. Molecular characterization and expression analysis of the IkB gene from pearl oyster Pinctada fucata. Fish Shellfish Immunol 2009;26(1):84e90. Zhang DC, Jiang SG, Hu YT, Cui SG, Guo HY, Wu KC, et al. A multidomain galectin involved in innate immune response of pearl oyster Pinctada fucata. Dev Comp Immunol 2011;35(1):1e6. Petersen TN, Brunak S, von Heijne G, Nielsen H. SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods 2011;8(10): 785e6. Letunic I, Copley RP, Pils B, Pinkert S, Schultz J, Bork P. SMART 5: domains in the context of genomes and networks. Nucleic Acids Res 2006;34:257e60. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, et al. Clustal W and clustal X version 2.0. Bioinformatics 2007;23(21):2947e8. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 2011;28: 2731e9. Cui SG, Zhang DC, Jiang SG, Pu HL, Hu YT, Guo HY, et al. A macrophage migration inhibitory factor like oxidoreductase from pearl oyster Pinctada
[30]
[31]
[32] [33]
[34]
[35] [36]
[37]
[38]
121
fucata involved in innate immune responses. Fish Shellfish Immunol 2011;31(2):173e81. Hergenhahn HG, Aspan A, Söderhäll K. Purification and characterization of a high-Mr proteinase inhibitor of pro-phenol oxidase activation from crayfish plasma. Biochem J 1987;248:223e8. Sommerhoff CP, Söllner C, Mentele R, Piechottka GP, Auerswald EA, Fritz H. A Kazal-type inhibitor of human mast cell tryptase: isolation from the medical leech Hirudo medicinalis, characterization, and sequence analysis. Biol Chem Hoppe Seyler 1994;375(10):685e94. Jiménez-Vega F, Vargas-Albores F. A four-Kazal domain protein in Litopenaeus vannamei hemocytes. Dev Comp Immunol 2005;29:385e91. Stubbs MT, Morenweiser R, Sturzebecher J, Bauer M, Bode W, Huber R, et al. The three-dimensional structure of recombinant leech-derived tryptase inhibitor in complex with trypsin. Implications for the structure of human mast cell tryptase and its inhibition. J Biol Chem 1997;272:19931e7. Lichtenfels AJ, Lorenzi-Filho G, Guimaraes ET, Macchione M, Saldiva PH. Effects of water pollution on the gill apparatus of fish. J Comp Pathol 1996;115: 47e60. Tisca PG, Mosca F. Defense mechanisms in farmed marine mollusks. Vet Res Commun 2004;28:57e62. Kellenberger C, Boudier C, Bermudez I, Bieth JG, Luu B, Hietter H. Serine protease inhibition by insect peptides containing a cysteine knot and a triplestranded beta-sheet. J Biol Chem 1995;270:25514e9. Johansson MW, Keyser P, Söderhall K. Purification and cDNA cloning of a fourdomain Kazal proteinase inhibitor from crayfish blood cells. Eur J Biochem 1994;223(2):389e94. Somprasong N, Rimphanitchayakit V, Tassanakajon A. A fivedomain Kazaltype serine proteinase inhibitor from black tiger shrimp Penaeus monodon and its inhibitory activities. Dev Comp Immunol 2006;30:998e1008.
