Molecular cloning and expression analysis of chymotrypsin-like serine protease from the Chinese shrimp, Fenneropenaeus chinensis

Fish & Shellfish Immunology 25 (2008) 589–597

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Fish & Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi

Molecular cloning and expression analysis of chymotrypsin-like serine protease from the Chinese shrimp, Fenneropenaeus chinensis Xiu-Zhen Shi, Xiao-Fan Zhao, Jin-Xing Wang* School of Life Sciences, Shandong University, Jinan, Shandong 250100, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 22 March 2008 Received in revised form 4 July 2008 Accepted 11 July 2008 Available online 24 July 2008

A new member of the serine protease (SP) chymotrypsin (designated Fc-chy) was isolated from the hepatopancreas of Chinese shrimp Fenneropenaeus chinensis. The full-length cDNA of Fc-chy contained 951 nucleotides with a polyadenylation sequence and a polyA tail. It encoded a peptide of 271 amino acids with a signal peptide of 17 amino acids and an activation peptide of 28 amino acids. The mature peptide concludes 226 amino acids. It contained the conserved catalytic triad (H, D, and S). Similarity analysis showed that Fc-chy shared high identity with chymotrypsins from the Pacific white shrimp, Litopenaeus vannamei. Northern blot, quantitative real-time PCR, in situ hybridization, and western blot analysis were carried out to analyze the expression pattern and distribution profiles of Fc-chy after bacteria and virus challenges. The results showed that Fc-chy transcription and Fc-chy protein levels were upregulated in the hepatopancreas after bacterial and viral infection. Fc-chy from the hepatopancreas was purified by affinity chromatography. It showed high hydrolytic activity toward the substrate N-succinyl-ala-ala-pro-phe p-nitroanilide (AAPF), and its activity was inhibited by Kazal-type SP inhibitor from Chinese shrimp. All of these may indicate that Fc-chy is involved in the innate immune reactions in Chinese shrimp. Ó 2008 Elsevier Ltd. All rights reserved.

Keywords: Chinese shrimp Fenneropenaeus chinensis Chymotrypsin Innate immunity Quantitative real-time PCR

1. Introduction Serine protease (SPs) constitutes one of the largest families of enzymes in the animal kingdom. They play important roles in dietary protein digestion, blood clotting, immune responses, signal transduction, hormone activation, inflammation, and development [1,2]. SPs are characterized by an active site termed the catalytic triad, which contains H, D, and S amino acids. The serine residue at the active site participates in the formation of a transient acylenzyme intermediate between the substrate and the protease [3]. SPs are the primary digestive enzymes of insects. Chymotrypsin is one of the SPs. In mammals, chymotrypsin participates in immune reactions and in many other physiological functions [4]. The chymotrypsins are primarily used as an aid of digestion and an antiinflammatory agent by preventing tissue damage and the fibrin clots. Also chymotrypsin formed the first line of defence against cancer by stripping away the proteins around the cancer cells. The chymotrypsins from crustaceans (United States Patent 6030612) have the activity of anti-cell–cell adhesion, so they are used for treating bacterial, viral, fungal, and parasitic infections in mammals. Studies on chymotrypsins in invertebrates have mainly been performed on insects since many insects are severe agriculture

* Corresponding author. Tel.: þ86 531 88364620; fax: þ86 531 88565610. E-mail address: [email protected] (J.-X. Wang). 1050-4648/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2008.07.011

pests and studying their digestive systems may aid in crop protection. These SPs are expressed in the midgut of larvae and participate in the digestion of dietary proteins. Plants employ protease inhibitors to block digestive proteases in the larval gut and thus protect themselves against insects [2]. Studies on Atlantic cod (Gadus morhua) have showed that chymotrypsin activity is involved in embryogenesis [5]. Chymotrypsin-like SPs from Manduca sexta also participate in the degradation of Cry toxinsda type of insecticidal crystal protein produced by Bacillus thuringiensis [6]. Scolexin, a new subfamily of chymotrypsin-like serine proteases in M. sexta, have been associated with response to bacteria, yeast, and baculovirus infection [7]. A chymotrypsin-like serine protease from the midgut of M. sexta interacts with the chitin synthesis and the formation of peritrophic matrix which is used to protect the digestive tract from the pathogen infection [8]. In Drosophila, a chymotrypsin-like serine protease is involved in immune defense reactions against bacteria [9]. Chymotrypsins had been cloned in the Pacific white shrimp, Litopenaeus vannamei [10,11]. Their polymorphism and evolution were analyzed. However, little is known about the roles of this protease in immune defense in shrimp. In this study, we cloned the cDNA of a chymotrypsin-like SP (Fc-chy) from the Chinese shrimp Fenneropenaeus chinensis. Northern blot, quantitative real-time PCR, and in situ hybridization were used to analyze the gene expression and distribution profiles. The expression level of proteins was studied by performing

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western blot with antiserum against recombinant mature chymotrypsin. The results indicated that this chymotrypsin from Chinese shrimp was upregulated after pathogen infection. Fc-chy was also purified from the hepatopancreas by affinity chromatography. It showed high activity toward the protease substrate N-succinyl-ala-ala-pro-phe p-nitroanilide (AAPF). Fc-chy activity was partially inhibited by the recombinant Kazal-type SP inhibitor from Chinese shrimp. All of these may suggest that Fc-chy is involved in the innate immune defence against pathogens.

(DIG RNA Labeling Kit, Roche) into antisense and sense DIG RNA probes. Northern blot analysis was performed according to the method of Kang et al. [15] in order to detect gene expression in different tissues (including hemocytes, heart, hepatopancreas, stomach, gills, intestine, and ovary) 24 h after the bacterial challenge. 2.5. In situ hybridization

2. Materials and methods

Hepatopancreas from bacteria-challenged and normal shrimp were used for in situ hybridization. Tissue preparation and hybridization was carried out according to the method of Kang et al. [15].

2.1. Immunity challenge of animals and hemocyte and tissue collection

2.6. Relative quantitative real-time PCR (qRT-PCR)

F. chinensis (10–20 g) was obtained from a shrimp farm in Qingdao, Shandong Province, China, and cultured in the laboratory in 500-L tanks filled with air-pumped fresh sea water. Vibrio anguillarum (3  107 cells) or white spot syndrome virus (WSSV, 200 mg total proteins extracted from the gills of natural heavily infected F. chinensis) was injected into the abdominal segment of each shrimp by a microliter syringe. Hemolymph was collected from the ventral sinus of challenged and control shrimp after culture for 24 h under a 1/10 volume of anticoagulant buffer (10% sodium citrate, pH 7) supplemented with 200 mM phenylthiourea as a melanization inhibitor, and then the hemolymph was centrifuged at 800g for 5 min (4  C) in order to collect the hemocytes. Tissues such as the heart, hepatopancreas, stomach, gills, intestine, and ovary were also collected from 3 shrimp for further studies. 2.2. Gene cloning Total RNA (5 mg) was isolated from the hepatopancreas of bacteria-challenged shrimp and then used to reverse transcribe first-strand cDNA by the methods of Du et al. [12]. Specific primer F1 (50 -CTCACCAACGACATTGCCCTCAT-30 ) and 30 anchor R primer (50 -GACCACGCGTATCGATGTCGAC-30 ) were used to amplify the 30 end of the chymotrypsin gene. The PCR conditions were as follows: 1 cycle at 94  C for 2 min; 35 cycles at 94  C for 30 s, 53  C for 45 s, and 72  C for 50 s; and 1 cycle at 72  C for 10 min. The 50 PCR primer (50 -TACGGCTGCGAGAAGACGACAGAA-30 ) and the specific primer R1 (50 -TCGTGAGTGAAGAAGTCGGTGGAGA CAATGGAG-30 ) were used to amplify the 50 end of the gene, with 1 cycle (94  C, 2 min), 35 cycles (94  C, 30 s; 53  C, 45 s; and 72  C, 50 s), and 1 cycle (72  C, 10 min) of PCR. 2.3. Phylogenetic and sequence analysis Homology analysis and cleavage site prediction was accomplished with BLASTP (http://www.ncbi.nlm.nih.gov/). Conceptual translation and characteristics of the protein were predicted using ExPASy (http://www.expasy.ch/). The motif was predicted with the help of SMART (http://smart.embl-heidelberg.de/). The signal peptide was searched with SignalP [13]. Sequence alignment was performed with ClustalW and GENEDOC softwares. The phylogenetic tree was prepared with MEGA 3.1 [14]. 2.4. Northern blot Specific primers F1 and R2 (50 -GTAGTGGACGCGGGTGAAGG-30 ) were used to amplify a fragment, which was then ligated into a pGEM-T-Easy vector. The recombinant pGEM-T-Easy-Fc-chy plasmid, with a negative-orientated insert of cDNA, was linearized by digestion with SpeI and SphI, separately. The 2 linear plasmids were then transcribed in vitro with T7 and SP6 RNA polymerase

Total RNA was isolated from the hepatopancreas of shrimp at 2, 6, 12, and 24 h after bacteria and virus challenges. Subsequently, the DNase-treated (RNase-free DNase I, Takara, Japan) total RNA (5 mg) was reverse transcribed into first-strand cDNA, and diluted 100fold in nuclease-free water which was used as the template for qRTPCR. Primer RTF (50 -GAAGCCAGCCAGGTCTCCAT-30 ) and primer RTR (50 -GCAGTCTGACGGTCTTGATGTTT-30 ) were used as the primers. The specific primers actinF (50 -CATCAAGGAGAAACTGTGCTA-30 ) and actinR (50 -AGGAATGAGGGCTGGAAC-30 ) were used to amplify b-actin as the control. The qRT-PCR was performed following the manufacture’s instruction of SYBR Premix Ex Taq (Takara, Japan) using real-time thermal cycler (Bio-Rad, USA) in a total volume of 20 mL containing 10 mL of 2  Premix Ex Taq, 2 mL of the 1:100 diluted cDNA, 4 mL (1 mM) each of the forward and reverse primer. The amplification procedure consisted of an initial denaturation step at 95  C for 3 min and then 40 cycles of 95  C for 30 s, 62  C for 50 s followed by a melt from 60  C to 95  C. All samples were repeated in triplicate for real-time PCR analysis. The qRT-PCR data of the expression level of Fc-chy response to bacterial and viral challenge were calculated by 2DCT. The data obtained were subjected to the statistical analysis followed by an unpaired sample t-test. Significant difference was accepted at P < 0.05. 2.7. Expression, purification, and renaturation of recombinant Fc-chy Sequence coding of mature Fc-chy peptide was amplified with primer ExF (50 -TACTCAGAATTCAACCCCGCCGCGGGGAAGCCATG-30 ) and ExR (50 -TACTCACTCGAGCCTGCCGAAGGTGAGGCCGCTCA-30 ). Thereafter, the peptide was digested by EcoRI and XhoI and ligated into pET-30a(þ) vector. This recombinant plasmid was transformed into competent Escherichia coli BL21(DE3) cells to induce expression. Luria–Bertani broth (300 mL) with kanamycin (final concentration 100 mg ml1) was inoculated along with 3 mL of the overnight culture of pET-30a/Fc-chy at 37  C. When the OD600 reached 0.8, 300 mL isopropyl-1-thio-b-D-galactopyranoside (1 mM) was added, and the culture was continued at 37  C for another 4 h. The bacterial cells were precipitated by centrifugation at 6000g for 10 min, resuspended with 20 mL PBS containing 0.2% Triton X-100, and then sonicated. The soluble fraction was discarded. The precipitate was washed twice with Buffer A [50 mmol/L Tris–HCl (pH 8.0) and 5 mmol/L ethylenediaminetetraacetic acid (EDTA)] and Buffer B (50 mmol/L Tris–HCl (pH 8.0), 5 mmol/L EDTA, and 2 M urea), resuspended with 20 mL Buffer C (0.1 mol/L Tris–HCl (pH 8.0), 10 mmol/L DTT, and 8 mol/L urea), and shaken at 37  C at 200 rpm for 1 h. It was then dialyzed against a 1-L solution containing 0.1 mol/L Tris–HCl (pH 8.0), 5 mmol/L EDTA, and 5 mmol/L cysteine at 4  C for 16 h. Finally, it was centrifuged, and the soluble fraction was used for protein purification. Recombinant protein was purified by His-Bind resin chromatography (Novagen, USA), according to the manufacturer’s instructions.

X.-Z. Shi et al. / Fish & Shellfish Immunology 25 (2008) 589–597

2.8. Production of anti-Fc-chy immune serum Purified recombinant Fc-chy (rFc-chy) protein was used to produce polyclonal antiserum in a rabbit. For the first injection, 200 mg recombinant protein was emulsified in complete Freund’s adjuvant and injected subcutaneously into the back of the rabbit. After 3 weeks, another 200 mg protein was emulsified in incomplete Freund’s adjuvant and injected subcutaneously. After 2 weeks, 500 mg protein was injected directly into the muscle of the rabbit’s leg. For the last injection, 500 mg protein was injected into the parallel-ear vein of the rabbit. After 1 week, blood was collected by cardiac puncture. Serum was collected and stored at 20  C for use. 2.9. Western blot The heart, gills, stomach, intestine, ovary, and hepatopancreas of normal shrimp and the bacteria- and virus-challenged hepatopancreas at 2, 6, 12, and 24 h were homogenized on ice in a buffer containing 50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 3 mM EDTA. They were then centrifuged at 10,000g for 10 min. The supernatant was quantified according to the method of Bradford [16], with BSA as the standard. Further, 200 mg protein from these tissues was run on 12.5% SDS-PAGE, following Laemmli’s method [17]. Immunoblot analysis was performed according to a previously described method [18]. Western blot analysis was also performed using the antiserum against recombinant Chinese shrimp b-actin (from our laboratory) as control of the Fc-chy time course expression in the protein level. Western blot was repeated in triplicate. The western blot data were numerated using Quantity One (Bio-Rad) and statistical analysis was performed using Excel software.

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our laboratory), and 10 mM EDTA (Sangon, China) were used. The inhibitors were individually mixed with Fc-chy and incubated at 37  C for 20 min; subsequently, the substrate was added, and the solution was further incubated for 40 min. Inhibition was also measured at 405 nm.

3. Results 3.1. Gene cloning A 312-bp fragment was obtained from the SSH library [19] of hepatopancreas. The results of BLAST revealed that this fragment was highly homologous to the chymotrypsin of L. vannamei. A 561bp fragment with a polyA tail was obtained from the LD-PCR library by PCR with primers F1 and 30 anchor R. The 50 end (383 bp) was amplified using 50 PCR primer and R1. The full length of the cDNA encoding Fc-chy was 951 bp long and included an open reading frame of 816 bp, a 6-bp 50 untranslated region, and a 129-bp 30 untranslated region. This cDNA encoded a protein consisting of 271 amino acids with a signal peptide of 17 amino acids (Fig. 1). The expression primers ExF and ExR were used to amplify the full length of the mature gene and to verify the cDNA sequence from the 3 overlapping fragments. 3.2. Similarity and phylogenetic analysis BLASTP data suggested that Fc-chy shared high homology with chymotrypsins from Pacific white shrimp L. vannamei: chymotrypsin BI (GenBank accession no. CAA71672, 95%), chymotrypsin BI

2.10. Purification of Fc-chy from hepatopancreas Shrimp hepatopancreas at 24 h after bacterial challenge were homogenized on ice in 0.1 M Tris–HCl (pH 7.5). After centrifugation at 10,000g for 10 min, the supernatant was dialyzed in 0.1 M Tris– HCl (pH 7.5) at 4  C for at least 12 h. CNBr-activated Sepharose 4B (Amersham Biosciences, UK) was used for Fc-chy purification. Anti-Fc-chy immune serum was dialyzed overnight in the coupling buffer (0.1 M NaHCO3, pH 8.3 containing 0.5 M NaCl). Next, 0.5 g freeze-dried powder of CNBractivated Sepharose 4B was swelled and washed with 1 mM HCl, as described in the instructions. Sepharose 4B and serum were then mixed and gently rotated overnight at 4  C. Excess ligand was washed away with at least 5 medium volumes of the coupling buffer. Finally, 0.1 M Tris–HCl (pH 8.0) was used to block any remaining active groups. The homogenate was added to CNBr-activated Sepharose 4B coupled with the serum. The mixture was first washed with 0.1 M Tris–HCl (pH 7.5) and then eluted by 0.1 M Tris–HCl (pH 7.5) with 0.5 M NaCl and by 0.1 M Tris–HCl (pH 7.5) with 1 M NaCl. The purified Fc-chy was run on 12.5% SDS-PAGE. The N-terminal amino acids of the purified Fc-chy were sequenced by Genecore company. Western blot analysis was performed to validate the purified Fc-chy. 2.11. Fc-chy activity and inhibition assay For the activity analysis, 0.2 mM AAPF (Sigma, USA) was used per assay. The substrate was mixed with 100 mL purified Fc-chy (1 mg ml1) and incubated at 37  C for 40 min. Activity was measured with fluorescent microplate reader at 405 nm. For the inhibition assay, 5 inhibitors, namely, 0.1 mM N-tosylL-phenylalanine chloromethyl ketone (TPCK, Sigma), 1 mM soybean trypsin inhibitor (STI, Sigma), 10 mM PMSF (Sangon, China), 42 nM SPI (recombinant Kazal-type SP inhibitor from Chinese shrimp in

Fig. 1. Nucleotide and deduced amino acid sequences of the Fc-chy gene (GenBank accession no. EU433385). The nucleotides are numbered on the right, and the amino acids, on the left. The putative signal peptide is underlined. The trypsin-like SP domain is wave underlined. The catalytic triad (H, D, and S) is black shadowed. The letters in red indicate binding pocket residues. The boxed letters are the polyadenylation signal. The asterisk (*) indicates the stop codon.

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Fig. 2. Multiple alignment of trypsin-like serine protease domain amino acid sequences of chymotrypsin from Chinese shrimp and other animals, including B. taurus-CHYA (P00766), P. humanus corporis-CHY, T. molitor-CHY (ABC88746), A. grandis-CHY (AAT09847), M. sexta-CHY (2120321A), and trypsins from B. taurus protease, trypsin 2 (NM_174690), M. musculus trypsin (NP_035776), M. sexta trypsinogen-like protein 1 (AM690450), T. molitor trypsin-like protease (DQ356017), A. grandis trypsin-like SP (AY536264). The black shadowed letters indicate the differences in the binding pocket between chymotrypsin and trypsin.

precursor (Q00871, 95%), chymotrypsin BII precursor (P36178, 91%), and chymotrypsin BII (CAA71673, 81%). It also showed that Fc-chy had high similarity to serine collagenase 1 precursor from Atlantic sand fiddler crab Celuca pugilator (AAC47030, 74%), to collagenolytic serine protease from red king crab Paralithodes camtschaticus (AAL67441, 71%), and to chymotrypsin-like SP from Pediculus humanus corporis (AAV68346, 43%). Phylogenetic analysis (Fig. 3) showed that chymotrypsins could be divided into 3 groups: chymotrypsin from invertebrates, vertebrates, and urochordata. The invertebrate group contained 2 subgroups. Subgroup 1 included chymotrypsins from Pacific white shrimp (L. vannamei), Chinese shrimp (F. chinensis), human body louse (P. humanus corporis), mustard beetle (Phaedon cochleariae), yellow mealworm (Tenebrio molitor), tobacco hornworm (M. sexta), and boll weevil (Anthonomus grandis). Subgroup 2 consisted of 2 types of chymotrypsins from the African malaria mosquito (Anopheles gambiae). Fc-chy belonged to subgroup 1. Fc-chy showed higher homology to chymotrypsin B1 than to chymotrypsin B2 of Pacific white shrimp; hence, it may be a member of chymotrypsin B1. 3.3. Expression profiles after immune challenge In order to determine the expression profiles of Fc-chy in Chinese shrimp, total RNA was extracted from normal and bacteriachallenged shrimp. As shown in Fig. 4, a band of approximately 900 bp was detected in the hepatopancreas by northern blot analysis, and the Fc-chy expression level increased when shrimps were injected with the bacterium V. anguillarum. Northern blot analysis also revealed 2 other bands. Signals were not detected in other tissues in both normal and bacteria-challenged shrimp. The RT-PCR results of hepatopancreas from normal and saline-injected

shrimp showed that when injected with saline, there were no changes of the Fc-chy expression level (data not shown) but it was upregulated in bacteria-challenged hepatopancreas. Fc-chy was upregulated at 24 h post-infection of either bacteria or viruses. In the time course qRT-PCR analysis (Fig. 5A) of gene expression in the hepatopancreas, the mRNA expression level of Fc-chy first decreased at 6 h after bacterial injection, and then considerably increased to about 2-fold at 24 h after injection. In the virus-challenged hepatopancreas, Fc-chy decreased 2 h after virus challenge and increased gradually from 6 to 24 h (Fig. 5B). The protein expression level of Fc-chy also increased to 2-fold at 24 h after bacterial or viral challenge in hepatopancreas (Fig. 8B and C). 3.4. Recombinant expression and purification After E. coli BL21(DE3)-pET-30a(þ)/Fc-chy was induced by 1 mM IPTG, the recombinant protein was highly expressed. The expected molecular mass of recombinant Fc-chy was approximately 32.7 kDa, which included 27.1 kDa Fc-chy and 5.6 kDa N-terminal expressed tag sequence (Fig. 7), the molecular weight of recombinant Fc-chy was as expected. Insoluble recombinant proteins were extracted from purified inclusion bodies by 8 M urea and renatured (see Materials and methods). They were purified by His-Bind resin chromatography. Purified rFc-chy was used for activity analysis using the specific substrate AAPF; no activity was detected. 3.5. Tissue distribution The results of northern blot tests (Fig. 4) revealed that Fc-chy was highly expressed in the hepatopancreas and that its expression increased after bacterial challenge. Hepatopancreas of normal and bacteria-challenged shrimp were selected for locating Fc-chy

X.-Z. Shi et al. / Fish & Shellfish Immunology 25 (2008) 589–597

100

100 Litopenaeus vannamei-CHYB1 Fenneropenaeus chinensis-CHY Litopenaeus vannamei-CHYB2

50 42

593

Pediculus humanus corporis-CHY Phaedon cochleariae-CHY

60

Tenebrio molitor-CHY

77 52

Manduca sexta-CHY

96

Anthonomus grandis-CHY Anopheles gambiae-CHY2 100

Anopheles gambiae-CHY1

Mus musculus-CHYA Bos taurus-CHYA

99

100

Bos taurus-CHYB 89

Gadus morhua-CHYB 100

Sparus aurata-CHYB Oikopleura dioica-CHYB

0.1 Fig. 3. Phylogenetic analysis of Fc-chy with other chymotrypsins. NJ tree was produced with the Mega 3.1 software. One thousand bootstraps carried out to check the repeatability of the result. L. vannamei-CHYB1, L. vannamei-CHYB2, P. humanus corporis-CHY, P. cochleariae-CHY (CAA76928), T. molitor-CHY, M. sexta-CHY, A. grandis-CHY, A. gambiae-CHY1 (CAA79325), A. gambiae-CHY2 (CAA79326), B. Taurus-CHYA, B. Taurus-CHYB (P00767), G. morhua-CHYB (P80646), S. aurata-CHYB (AAT45258), M. musculus-CHYA (AAL11034), and O. dioica-CHYB (AAT47850).

transcripts by in situ hybridization. As shown in Fig. 6, the gene was highly expressed in some cells of the hepatopancreatic tubules and the positive signal is much stronger in challenged shrimp than in normal shrimp. As shown in Fig. 8A, chymotrypsin was distributed in all tissues, including hemolymph, the heart, the hepatopancreas, the gills, the stomach, the intestine, and the ovary. The expression level in the hepatopancreas, stomach, and intestine was considerably higher than that in other tissues. 3.6. Characterization of Fc-chy Although we could purify rFc-chy from E. coli inclusion bodies (Fig. 7), we have not been able to refold the rFc-chy sufficiently to obtain the enzyme proteolytic activity. Therefore, we purified the Fc-chy from the hepatopancreas by affinity chromatography. The molecular mass of the purified protein was approximately 25 kDa,

Base 6583 4981 3638 2604 1908 1383 955 623 281

and the N-terminal amino acid sequence was IVGGVEATPH which was identical to the deduced amino acid sequence of the cloned cDNA. As we can see from Fig. 9A, there is a single band with same molecular mass in the western blot analysis of both the purified Fcchy (lane 3) and total proteins (200 mg) from the hepatopancreas (lane 4) with antibody against rFc-chy. All of these could confirm that the purified Fc-chy and the cloned cDNA are the same molecule. Analysis of the Fc-chy activity towards AAPF revealed high activity of the substrate (Fig. 9B). Several inhibitors were used to identify the enzyme. As seen in Fig. 9B, the activity could be inhibited by the active site inhibitor PMSF. The inhibitory effect of PMSF indicated that the enzyme was an SP. EDTA, a metalloprotease inhibitor, had no effect on its activity. TPCK, a short substrate-like chymotrypsin inhibitor, did not inhibit the enzyme, which indicated a chymotrypsin-like protease from the midgut of T. molitor larvae [20]. STI, a protease inhibitor from soybean seeds, showed high inhibitive activity. Inhibition by SPI suggested that the in vivo Kazal-type SP inhibitor of Chinese shrimp had inhibitive activity against chymotrypsin. 4. Discussion 4.1. Fc-chy is a chymotrypsin

18SRNA 2

1

2 1

2 1

2

lls st om ac h in te st in e O va ry

pa

he a

2 1

gi

2 1

rt nc re as

1

he pa

he m

2

to

1

oc yt es

M

Fig. 4. Northern blot analysis of Fc-chy transcripts with total RNA from different tissues of normal (control) and challenged shrimp. Lane M, RNA marker; lane 1, control tissues; lane 2, challenged tissues; 10 mg total RNA/lane. Tissues from left to right: hemocytes, heart, hepatopancreas, gills, stomach, intestine, ovary.

Purified Fc-chy showed high activity toward substrate AAPF; further, its activity was inhibited by PMSF and STI, similar to a chymotrypsin-like SP from the midgut of T. molitor larvae [20]. The inhibition characteristics imply that the purified Fc-chy was an SP. SPs differ in substrate specificity, and these differences are the result of small differences in their binding pockets. The difference between chymotrypsin and trypsin is that in trypsin, Ser189 (numbering as in bovine chymotrypsin) [21] is replaced by Asp at the bottom of the binding pocket; thus, the corresponding binding substrates change from those containing aromatic nonpolar side chains to those with a positively charged lysine or arginine side chain (Fig. 2). The binding pocket residues of SPs are variable and in chymotrypsins, usually consist of Ser189, Gly216, and Ala/Gly226

X.-Z. Shi et al. / Fish & Shellfish Immunology 25 (2008) 589–597

Relative expression level

A

B

Bacterial challenge 1.4

∗

1.2 1 0.8 0.6 0.4 0.2 0

0

2

6

12

24

Time (h)

Relative expression level

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Viral challenge 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0

∗

∗ 0

2

6

12

24

Time (h)

Fig. 5. The quantitative real-time PCR analysis of Fc-chy. (A) and (B) show the results of the quantitative real-time PCR analysis of Fc-chy expression with total RNA extracted from shrimp hepatopancreas at different time points after bacteria and virus challenges. Expression of the gene encoding b-actin was used as a control. The histograms show the statistical analysis of the quantitative real-time PCR results. The asterisks indicate significant differences (*: P < 0.05) from the control. Error bars represent  SD of 3 independent PCR amplifications and quantifications.

[22]. Identical to chymotrypsin from the Aedes aegypti midgut [23], the binding pocket residues of Fc-chy also contain Ser189 at the bottom of the pocket and have Gly216 lining the entrance of the pocket. Ala 226, a small amino acid found in vertebrate chymotrypsins also appears in Fc-chy. The activity of purified Fc-chy and its amino acid sequence character confirm it to be a chymotrypsinlike SP. Phylogenetic analysis revealed that Fc-chy showed high identity to chymotrypsin B1 from Pacific white shrimp L. vannamei; hence, it may belong to the chymotrypsin B1 subfamily. As a member of the SP family, it shows several typical structural features of SPs [24], such as the catalytic triad (H, D, and S), conserved serine catalytic site, and 6 cysteine residues. In the conserved trypsin-like SP domain, there are 6 cysteine residues common to all invertebrate chymotrypsins, and these are believed to form 3 disulfide bonds. In addition to the 3 pairs of conserved cysteine residues, there is another free cysteine residue in the signal peptide. In mammals, chymotrypsin has more than 6 cysteine residues, and 2 of the additional cysteine residues could form a disulfide bond to anchor the propeptide to the protein core [25]. Extra cysteine residues have been found in many insect SPs, but the extract role and pairing mode of these cysteine residues remain unclear [26]. 4.2. Tissue distribution and expression profiles of chymotrypsin SPs of the chymotrypsin (S1 peptidase) family constitute one of the largest enzyme families in the animal kingdom. There are 204 serine protease-like genes in Drosophila melanogaster [4], 305 in A. gambiae [27], and 57 in the honey bee Apis mellifera [28]. They are

typically synthesized as zymogens, which require proteolysis at a specific site for activation. In some cases, after an initiation protease becomes active upon stimulation, other downstream SP zymogens are sequentially activated in a cascade pathway, which eventually generates effector molecules by limited proteolysis. In the current study, the results of northern blot (Fig. 4) showed that Fc-chy mRNA was only expressed in the hepatopancreas; we observed 2 higher bands besides the approximately 900-bp band. It is probably because the antisense DIG RNA probe (base 385–777) is located in a fragment contained in the conserved trypsin-like SP domain and it also cross-reacted with mRNA of other serine proteases in the shrimp. In crustaceans, the hepatopancreas is reportedly the synthesis site of digestive enzymes [29]. Western blot analysis showed that chymotrypsin was distributed in all the tested tissues, but it was mainly distributed in the hepatopancreas, stomach, and intestine. Our results on transcription expression and western blot strongly suggested that Fc-chy was synthesized in the hepatopancreas and then secreted and transported to the stomach, intestine, and other tissues. The results of in situ hybridization showed that Fc-chy transcripts were located on some cells of the hepatopancreatic tubules. Lehnert et al. [29] had reported that digestive enzymes were synthesized in the hepatopancreas, and the F-cells were the synthesis sites of these enzymes. Thus, the positive signal detected by in situ hybridization may have originated from F-cells. The mRNA expression of Fc-chy in the hepatopancreas was upregulated after bacteria and virus challenge. The RT-PCR results (data not shown) showed that the expression level did not change after the same volume of saline injection. Hence, the upregulation

Fig. 6. In situ hybridization detection of the expression of chymotrypsin mRNA in the hepatopancreas of bacteria-challenged shrimp. (A) A hepatopancreatic specimen obtained from challenged shrimp and treated with sense Dig RNA probe. (B) A hepatopancreatic specimen obtained from normal shrimp and treated with antisense Dig RNA probe. (C) A hepatopancreatic specimen obtained from bacteria-challenged shrimp and treated with antisense Dig RNA probe. Bar ¼ 10 mm.

X.-Z. Shi et al. / Fish & Shellfish Immunology 25 (2008) 589–597

level at 24 h. Perhaps after bacteria or virus injection, translation and subsequent degradation of the mRNA stored in the hepatopancreas may have been initiated, followed by mRNA transcription in response to pathogen infection, which ultimately led to a gradual increase in the mRNA levels to the normal level or even to a higher level [30]. The expression level of Fc-chy in the hepatopancreas increased 24 h after bacteria and virus challenge. These results were consistent with the mRNA expression profiles. SPs can be divided into 2 types of molecules according to their domains: single-trypsin domain SPs (single-domain SPs) and cliptrypsin domain SPs (clip-domain SPs). In arthropods, clip-domain SPs mediate innate immunity and embryonic development [31,32]. Single-domain SPs are generally thought to be digestive proteases. Gorman et al. [33] reported 5 serine proteases in A. gambiae, including 3 clip-domain SPs and 2 single SPs. Expression of clipdomain SPs was induced by infection with malaria parasite. One of the single SPs was also induced by bacterial infection. Hence, they concluded that serine proteases played central roles in mosquito immune response by participating in processes, including hemolymph coagulation, melanization of pathogen surfaces, and antimicrobial peptide synthesis. Fc-chy is a single-domain SP. Chymotrypsin-like serine proteases conduct lots of physiological functions including digestive and degradative processes, blood clotting, humoral and cellular immunity, tissue remodeling, and embryonic development [34]. Some of the functions are mediated by the protease cascades in which the inactive protease is secreted and is activated by the upstream protease. The increase in Fc-chy mRNA and protein levels after bacteria and virus challenge suggested that it was likely to be related to the immune response in shrimp. In the study of phenoloxidase (PO) activity in Sydney rock oysters, proPO is activated to PO by the addition of chymotrypsin [35]. Perhaps Fc-chy is involved in a protease cascade for the PO activation. When the animals are immune challenged, Fc-chy could hydrolyze or modulate some proteins in the PO active system, and finally induce hemolymph coagulation, melanization of pathogen surfaces.

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Fig. 7. SDS-PAGE analysis of the expression of recombinant Fc-chy. Lane 1, total protein of E. coli with pET-30a(þ)/Fc-chy; lane 2, total protein of E. coli with pET-30a(þ)/Fc-chy induced with 1 mM IPTG; lane 3, purified recombinant protein; and lane 4, standard marker protein.

was the result of the bacteria or virus injection and not the injury. Time course expression analysis (Fig. 5) showed that after bacterial or virus injection, the mRNA expression level of Fc-chy declined in the first 2 h and then gradually increased to a considerably higher

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Fig. 8. Western blot analysis of Fc-Chy. A. Expression of Fc-Chy in different tissues obtained from normal shrimp. Lanes left to right: total protein (200 mg) from hemolymph, heart, hepatopancreas, gills, stomach, intestine, and ovary. B. Expression of Fc-chy in bacterial challenged hepatopancreas. Lanes: total protein (200 mg) from the hepatopancreas of normal shrimp and shrimp injected with bacteria and observed at 2, 6, 12, and 24 h. C. Expression of Fc-Chy in viral challenged hepatopancreas. Lanes: total protein (200 mg) from the hepatopancreas of normal shrimp and shrimp injected with virus and observed at 2, 6, 12, and 24 h. Histogram below was the statistical analysis of western blot result. Data was expressed as the ratio of Fc-chy to the b-actin. Asterisks indicate significant differences (*: P < 0.05) comparing to that of the control. Error bars represent the  SD. of 3 independent PCR amplification and quantification.

X.-Z. Shi et al. / Fish & Shellfish Immunology 25 (2008) 589–597

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Fig. 9. Fc-chy purification and characterization. A. SDS-PAGE and western blot analysis of Fc-chy from the hepatopancreas purified by affinity chromatography. Lane 1: purified Fcchy from the hepatopancreas, lane 2: protein marker, lane 3: western blot analysis of purified Fc-chy, with antibody against recombinant Fc-chy; lane 4: western blot analysis of total protein (200 mg) from hepatopancreas, with antibody against recombinant Fc-chy; B. Fc-chy activity towards substrate AAPF (control), inhibition analysis with inhibitors such as PMSF (active site inhibitor), STI (a protease inhibitor from soybean seeds), SPI (recombinant Kazal-type SP inhibitor from Chinese shrimp), TPCK (a short substrate-like chymotrypsin inhibitor), and EDTA (metalloprotease inhibitor). Histogram was the statistical analysis of inhibition. Data were expressed as the ratio of inhibitors to the control at OD405.

The hepatopancreas is a crucial organ of the immune system in shrimp. It initiates the humoral immune response and participates in the cellular immune response by some highly specialized cells and phagocytes [36]. Many immune-related genes are identified from the hepatopancreas after bacterial and viral infections in shrimp [37,38]. In conclusion, a chymotrypsin-like SP was identified in Chinese shrimp. The entire length of this serine protease was first cloned in this study, and its expression pattern after bacterial and viral injection was also described for the first time in shrimp. Further studies are required to detect the mechanism underlying chymotrypsin involvement in immune reactions. Acknowledgements This work was supported by grants from the National Natural Science Foundation of China (No. 30770282) and the National High Technology Research and Development Program of China (863 Program) (No. 2007AA09Z425 and 2006AA100311). References [1] O’Connell AR, Lee BW, Stenson-Cox C. Caspase-dependant activation of chymotrypsin-like proteases mediates nuclear events during Jurkat T cell apoptosis. Biochem Biophys Res Commun 2006;345:608–16. [2] Herrero S, Combes E, Oers MMV, Vlak JM, RAde Maagd, Beekwilder J. Identification and recombinant expression of a novel chymotrypsin from Spodoptera exigua. Insect Biochem Mol Biol 2005;35:1073–82. [3] Rawlings ND, Barrett AJ. Families of serine peptidases. Methods Enzymol 1994;244:19–61. [4] Ross J, Jiang H, Kanost MR, Wang Y. Serine proteases and their homologs in the Drosophila melanogaster genome: an initial analysis of sequence conservation and phylogenetic relationships. Gene 2003;304:117–31. [5] Sveinsdo´ttir H, Thorarensen H, Gudmundsdo´ttir A´. Involvement of trypsin and chymotrypsin activities in Atlantic cod (Gadus morhua) embryogenesis. Aquaculture 2006;260:307–14. [6] Miranda R, Zamudio FZ, Bravo A. Processing of Cry1Ab endotoxin from Bacillus thuringiensis by Manduca sexta and Spodoptera frugiperda midgut proteases: role in protoxin activation and toxin inactivation. Insect Biochem Mol Biol 2001;31:1155–63. [7] Finnerty CM, Karplus PA, Granados RR. The insect immune protein scolexin is a novel serine proteinase homolog. Protein Sci 1999;8:242–8. [8] Broehan G, Zimoch L, Wessels A, Ertas B, Merzendorfer H. A chymotrypsin-like serine protease interacts with the chitin synthase from the midgut of the tobacco hornworm. J Exp Biol 2007;210:3636–43. [9] de Morais Guedes S, Vitorino R, Domingues R, Tomer K, Correia AJF, Amado F, et al. Proteomics of immune-challenged Drosophila melanogaster larvae hemolymph. Biochem Biophys Res Commun 2005;328:106–15. [10] Sellos D, Van Wormhoudt A. Molecular cloning of a cDNA that encodes a serine protease with chymotryptic and collagenolytic activities in the hepatopancreas of the shrimp Penaeus vanameii (Crustacea, Decapoda). FEBS Lett 1992;309(3):219–24.

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