Peptides 32 (2011) 518–525
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Molecular cloning, characterization and expression analysis of cathepsin A gene in Chinese mitten crab, Eriocheir sinensis Wei-Wei Li, Lin He, Xing-Kun Jin, Hui Jiang, Li-Li Chen, Ying Wang, Qun Wang ∗ School of Life Science, East China Normal University, North Zhong-Shan Road, Shanghai, China
a r t i c l e
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Article history: Received 26 July 2010 Received in revised form 27 August 2010 Accepted 27 August 2010 Available online 15 September 2010 Keywords: Chinese mitten crab Eriocheir sinensis Cathepsin A Infection Quantitative real-time PCR
a b s t r a c t Cathepsins, a superfamily of hydrolytic enzymes produced and enclosed within lysosomes, function in immune response in vertebrates; however, their function within the innate immune system of invertebrates remains largely unknown. Therefore, we investigated the immune functionality of cathepsin A (catA) in Chinese mitten crab (Eriocheir sinensis), a commercially important and disease vulnerable aquaculture species. The full length catA cDNA (2200 bp) was cloned via PCR based upon an initial expressed sequence tag (EST) isolated from a hepatopancreatic cDNA library. The catA cDNA contained a 1398 bp open reading frame (ORF) that encoded a putative 465 amino acid (aa) protein. Comparisons with other reported vertebrate cathepsins sequences revealed percent identity range from 48 to 51%. CatA mRNA expression in E. sinensis was (a) tissue-speciﬁc, with the highest expression observed in gill and (b) responsive in hemocytes to a Vibrio anguillarum challenge, with peak exposure observed 12 h post-injection. Collectively, data demonstrate the successful isolation of catA from the Chinese mitten crab, and its involvement in the innate immune system of an invertebrate. © 2010 Elsevier Inc. All rights reserved.
1. Introduction A lysosome is an organelle with a cystic structure that contains a plethora of hydrolytic enzyme types that function in digestion , including phosphatase, ribonuclease, deoxyribonuclease, cathepsin, B-glucuronidase and acetyl-transferase enzymes. The lysosome has thus been likened to an “enzyme warehouse” and a cellular “digestive system” [7,25]. In marine invertebrates, lysosomes are contained within semi-granular and granular hemocytes and are released via hemocyte degranulation, which occurs during an immune response [39,48]. Once the lysosome enters the plasma, it releases proteolytic enzymes via membrane destabilization that then assist in the break down of foreign material [14,44,57]. Historically, cathepsins were described as a group of intracellular hydrolases that participate in lysosome-mediated protein turnover. The term “cathepsin”, ﬁrst introduced in 1920, is deﬁned as a “lysosomal proteolytic enzyme” regardless of enzyme class although the majority of cathepsins belong to the cysteine protease family . Therefore, in addition to cysteine proteases such as cathepsin L (catL), lysosome proteases also include serine proteases (cathepsins A and G) and aspartic proteases (cathepsins D and E). While several cathepsins are ubiquitously expressed, such as cathepsins B, L, H, and C, expression of recently identiﬁed cathepsins, such as cathepsins K, W, and X, are cell- or tissue-
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speciﬁc [20,54]. CatA is an important member of the cathepsin family that preferentially hydrolyzes various N-blocked peptides having aromatic and large hydrophobic amino acids at P1 and P2 positions both at acidic pH (carboxypeptidase activity) and neutral pH (amidase/esterase activity) [5,41]. As lysosomal protective protein, catA plays a structural role by forming a membrane-bound high molecular weight complex with b-galactosidase and neuraminidase in lysosomes of various cells and tissues [4,30]. CatA was also known as a multifunctional glycoprotein which activates lysosomal ␣-neuraminidase that protects ␣-galactosidase from proteolytic degradation through the formation of a multienzymic complex in lysosomes (protective function) and regulates the intracellular distribution of the glycosidases [10,17,22,38,50,51]. In general, CatA appears to prefer to release hydrophobic amino acid residues [26,32], and it has also been implicated in the process of autophagy, which occurs after the digestion of lysosome-associated membrane protein type2a (lamp2a) . Furthermore, defects in the enzymatic activity of catA have been reported to cause galactosialidosis, a lysosomal storage disorder in humans [10,42]. To investigate the potential immune functions of the lymphoid organ, Pongsomboon et al.  analyzed the expressed genes from the lymphoid organ of normal and Vibrio harveyi-infected Penaeus monodon using an expressed sequence tag (EST) approach. The result indicated that the transcripts of catA were found only in the V. harveyi-infected library. In the study of Litopenaeus vannamei , the transcript of catA was found in the hemocytes through Suppression Subtractive Hybridization (SSH) after the infection of WSSV indicating that the expression of catA is regulated during
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innate immune responses. These reports suggest that catA likely plays a major role in the lymphoid organ function and is probably implicated in degradation of foreign material that is sequestrated in the lymphoid organ spheroids and making it interesting candidate immune related genes. Chinese mitten crab, Eriocheir sinensis, is an economically important freshwater species for aquaculture in China. E. sinensis aquaculture is a rapidly developing industry in China, with a peak yield of 40 million tones in 2005 . However, bacterial- and viralborn disease have blossomed within booming E. sinensis cultures, incurring catastrophic losses to crab aquaculture [52,55]. Crab, as well as other invertebrates, do not possess an adaptive immune system and must rely on efﬁcient innate immune defenses ; therefore, obtaining a better understanding of the innate immune ability of crab and their defense mechanisms has become a research priority. In order to determine the participation of catA in innate immune responsiveness in crustaceans, and identify key mechanistic steps for improving immunity and associated mortality in aquaculture E. sinensis, we (1) cloned the full length catA cDNA using EST sequences identiﬁed from a E. sinensis hepatopancreatic cDNA library previously constructed by our laboratory, (2) examined mRNA tissue-dependent expression patterns, and (3) determined the temporal response of catA expression in response to an immune challenge via Vibrio anguillarum exposure.
Table 1 Sequences of primers. Primers
Primers for 5 RACE PCR 5 -CCTTCCACCCCATTCAAGTATCCGC-3 catA 5 ﬁrst GSP primer 5 -GGTGTGGCTCCTCCTTGTGACTCTGAC-3 catA 5 nested GSP primer Primers for 3 RACE PCR 5 -TGCTCAACTGGAGATGACGAGACTTCAC-3 catA 3 ﬁrst GSP primer 5 -GGGAGAGTCCTATGGAGGCATCTATGTG-3 catA 3 nested GSP primer ClontechTM Kit primers Universal primer A 5 -CTAATACGACTCACTATAGGGCAAGCmix AGTGGTATCAACGCAGAGT-3 5 -CTAATACGACTCACTATAGGGC-3 Nested universal 5 -AAGCAGTGGTATCAACGCAGAGT-3 primer A Primers for genomic DNA PCR 5 -CCTTGCGATCAAACTTCACTG-3 catA 5 primer 5 -AGAGGATGAGGCTGCCACAGGATAC-3 catA 3 primer Primers for RT-PCR and real-time PCR analysis 5 -GCCAGGGTAGGCACATAGA-3 catA 5 primer 5 -ATGGCAAGACACTGAGGAAGA-3 catA 3 primer 5 -CTCCTGCTTGCTGATCCACATC-3 ␤-actin 5 primer 5 -GCATCCACGAGACCACTTACA-3 ␤-actin 3 primer
Code A-R-5 -R A-R-5 -F
A-R-3 -R A-R-3 -F
A-G-R A-G-F A-RT-R A-RT-F ␤-1 ␤-2
2. Method and materials 2.1. Sample preparation Healthy E. sinensis (mean weight = 100 g) were collected from the Tongchuan aquatic product market in Shanghai, China. Crabs were placed in an ice bath for 1–2 min until lightly anesthetized prior to sacriﬁce. The following tissues were harvested, snap frozen in liquid nitrogen, and stored at −80 ◦ C until nucleic acid analysis, hepatopancreas, brain, gills, intestine, hemocytes, muscle, stomach, heart, and eyestalk. For cloning and expression analysis, tissues from 10 crabs were pooled and ground with a mortar and pestle prior to extraction. 2.2. Nucleic acid extraction Genomic DNA was extracted from E. sinensis hepatopancreas using the AxyprepTM Multisource Genomic DNA Miniprep Kit (Axygen, USA) according to the manufacturer’s protocol. Total RNA was extracted from E. sinensis using Trizol® reagent (RNA Extraction Kit, Invitrogen, CA, USA) according to the manufacturer’s protocol. The total RNA concentration and quality were estimated by spectrophotometry (absorbance at 260 nm) and agarose-gel electrophoresis, respectively. 2.3. First-strand cDNA synthesis Total RNA (5 g) isolated from hepatopancreas was reverse transcribed using the SMARTTM cDNA kit (Clonetech, USA) for cDNA cloning. For expression analysis, total RNA (4 g) was reverse transcribed using the PrimeScriptTM RT-PCR Kit (TaKaRa, Japan) for semi-quantitative RT-PCR analysis or the PrimeScriptTM Real-time PCR Kit (TaKaRa, Japan) for real-time quantitative RT-PCR (qRTPCR) analysis. 2.4. Rapid ampliﬁcation of cDNA ends (RACE) The E. sinensis catA cDNA sequence was extended using 5 and 3 RACE and a total of four gene-speciﬁc primers (Table 1) based upon the original EST sequence (as described above; GenBank accession no. FG360179). The initial 3 RACE PCR reaction was carried
out in a total volume of 50 l that contained 2.5 l of the ﬁrststrand cDNA reaction as template, 5 l of 10× Advantage 2 PCR buffer, 1 l of 10 mM dNTPs, 5 l of 10 M gene-speciﬁc primer (Table 1), 1 l of Universal Primer A Mix (UPM; Clonetech, USA), 34.5 l of sterile deionized water, and 1 U 50× Advantage 2 polymerase mix (Clonetech, USA). Diluted products of the ﬁrst PCR 3 RACE reaction (1:50 with Tricine–EDTA buffer) served as template for the 3 nested PCR reaction. With the exception of the template and nested primer (NUP), contents of the nested PCR reaction were identical to the initial reaction. For 5 RACE, SMARTTM cDNA kit UPM and NUP were used as forward primers in initial and nested PCR reactions in conjunction with the reverse genespeciﬁc primers GSP-5 -f and GSP-5 -n, respectively (see Table 1 for sequence). PCR ampliﬁcation conditions for initial 3 and 5 RACE were as follows: 5 cycles at 94 ◦ C for 30 s, 72 ◦ C for 3 min; 5 cycles at 94 ◦ C for 30 s, 72 ◦ C for 30 s, and 72 ◦ C for 3 min; 20 cycles at 94 ◦ C for 30 s, 68 ◦ C for 30 s, and 72 ◦ C for 3 min. Nested PCR ampliﬁcation conditions were as follows: 20 cycles at 94 ◦ C for 30 s, 68 ◦ C for 30 s, and 72 ◦ C for 3 min. PCR amplicons were size separated and visualized on an ethidium bromide stained 1.2% agarose gel. Amplicons of expected sizes were puriﬁed with E.Z.N.A® Gel Extraction Kit (Omiga BioTek, USA), and inserted into a pTZ57R® Vector (Promega, USA). Positive clones containing inserts of the expected size were sequenced using T7 and SP6 primers. 2.5. Cloning the catA gene Gene-speciﬁc primers (Table 1) corresponding to the cloned cDNA were used to obtain catA genomic DNA sequence. The ﬁnal PCR reaction was performed in a ﬁnal volume of 25 l and contained 2.5 l of 10× buffer for KOD-Plus-Ver.2, 2.5 l of 2 mM dNTPs, 1.5 l of 25 mM MgSO4 , 0.75 l of 10 M primer, 16 l of sterile deionized water, 0.5 l (1 U/l) KOD-Plus-PCR Cloning Enzyme (Toyobo, Japan), and 0.5 l genomic DNA isolated from hepatopancreas as template. PCR conditions were as follows: 94 ◦ C for 2 min; and 30 cycles of 98 ◦ C for 10 s and 68 ◦ C for 3 min. Amplicons of the expected size were size separated, puriﬁed, and cloned as described above.
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2.6. Sequence analysis The putative signal peptide cleavage site was identiﬁed using the TargetP program (http://www.cbs.dtu.dk/services/TargetP/) [13,35]. E. sinensis full-length cDNA and deduced amino acid sequences were compared with other sequences reported in NCBI’s GenBank using the BLAST program (website). CatA cDNA and deduced amino acid sequences from E. sinensis and representative vertebrates were compared by multiple sequence alignment using ClustalX. Gene structure was identiﬁed via NCBI’s Spidey software (http://www.ncbi.nlm.nih.gov/IEB/Research/Ostell/Spidey/). An phylogenetic tree was constructed with MEGA4.0. E. sinensis catA cDNA and gene sequences were deposited under GenBank accession numbers GU593248 and GU593245, respectively. 2.7. RT-PCR analysis Tissue-dependent mRNA expression analysis was conducted via semi-quantitative RT-PCR. First-strand cDNA was prepared as described above. RT-PCR primers (Table 1) were designed based upon the cloned E. sinensis catA cDNA to produce a 250 bp amplicon. RT-PCR was performed in a ﬁnal volume of 25 l, and contained 2.5 l of 10× PCR buffer-II, 1 l of 10 mM dNTP mix, 0.25 l of 20 mM primer, 18.75 l of sterile deionized water, 0.25 U EX TaqTM HS DNA polymerase (TaKaRa, Japan) and 2 l of ﬁrst-strand cDNA as template. PCR conditions were as follows: 30 cycles at 94 ◦ C for 30 s, 60 ◦ C for 30 s, and 72 ◦ C for 1 min. Internal control PCR reactions for beta-actin were performed in a separate tube, as described above with the exception of an alternative gene-speciﬁc primer pair (Table 1), which was designed based upon a cloned E. sinensis ␤-actin cDNA fragment to produce a 276 bp amplicon. All RT-PCR reactions were completed in triplicate using independently extracted RNA. RT-PCR products were size separated on an ethidium bromide stained 1.5% agarose gel, visualized under ultraviolet light, and images were captured with a Gel Doc 2000 System (Tannon, China). 2.8. Real-time qRT-PCR analysis A real-time quantitative RT-PCR (qRT-PCR) assay was carried out using the CFX96TM Real-Time System (Bio-Rad, USA). Genespeciﬁc primers (Table 1) were designed based upon the cloned catA cDNA to produce a 250 bp amplicon. Samples were run in triplicate and normalized to the control gene ␤-actin, catA expression levels were calculated by the 2−Ct comparative CT method . Real-time qPCR ampliﬁcation reactions were carried out in a ﬁnal volume of 25 l, which contained 12.5 l 2× SYBR Premix Ex Taq (TaKaRa, Japan), 0.5 l diluted cDNA template, 11.0 l PCR-Grade water, and 0.5 l of each primer. PCR conditions were as follows, 95 ◦ C for 30 s; followed by 40 cycles of 95 ◦ C, and a 0.5 ◦ C/5 s incremental increase from 60 to 95 ◦ C that lasted 30 s per cycle. Resultant data was analyzed using the CFX ManagerTM software (Version 1.0). 2.9. Immune challenge and hemocyte isolation Chinese mitten crabs (n = 155; 70 ± 5 g wet weight) were acclimated for 1 week at 20–25 ◦ C in ﬁltered, aerated freshwater prior to
injection with approximately 70 l live V. anguillarum resuspended in 0.1 mol l−1 PBS (pH = 7.0, 109 CFU ml−1 ) or a vehicle control of PBS (pH = 7.0). Five crabs were randomly selected 2, 4, 6, 8, 12, 16, 32 and 48 h post-injection from experimental and control groups for hemolymph collection using a syringe (approximately 2.0 ml per crab). An equal volume of anticoagulant solution (glucose: 2.05 g, citrate: 0.8 g, NaCl: 0.42 g, double distilled water: add to 100 ml) was then added to each hemolymph sample, and centrifuged at 800 × g at 4 ◦ C to isolate hemocytes. Hemocytes were stored at −80 ◦ C after addition of 1 ml Trizol reagent (Invitrogen, CA, USA) for subsequent RNA extraction. 2.10. Statistical analysis Statistical analysis was performed using SPSS software (Ver11.0). Statistical signiﬁcance was determine using one-way ANOVA  and post hoc Duncan multiple range tests. Signiﬁcance was set at P < 0.05. 3. Results 3.1. Cloning and characterization of E. sinensis catA cDNA The full length catA cDNA sequence cloned from E. sinensis hepatopancreas was 2200 bp long and contained a 1398 bp ORF that encoded a 465 amino acid protein, a 17 bp 5 UTR, and a 785 bp 3 UTR. 3.2. Gene organization of catA The E. sinensis catA gene was comprised of four introns and ﬁve exons (Fig. 1). The six exons were 161, 163, 135, 212, 100 and 627 bp in length and were separated by ﬁve introns that were 415, 506, 169, 184 and 105 bp long (Fig. 1). All exon/intron boundary sites were consistent with the GT/AG rule . 3.3. Amino acid sequence alignment The homology of catA in E. sinensis and other species deduced amino acid catA sequences (Fig. 2) was explored via multiple sequence alignment using ClustalX (Fig. 3). Overall percent identify among catA and other reported sequences were as follows: with 51% identity to Salmo salar, 50% to Tetraodon nigroviridis, 51% to Monodelphis domestica, 48% to Macaca mulatta and 50% to Danio rerio. Alignment of the E. sinensis catA with those reported for other organisms revealed the conservation of the catalytic domain (serine carboxypeptidases). 3.4. Phylogenetic analysis of catA Phylogenetic analysis of catA, B, C, D, E, F, H, L and S from representative crustaceans, mammals, and pisces (Fig. 4) produced an NJ-phylogenetic tree, that contained three distinct branches, suggesting a phylogenetic relationship and shared common ancestor among catA, B, C, D, E, F, H, L and genes.
Fig. 1. The gene structure of catA in E. sinensis. Exons and introns are represented by red boxes and black solid lines, respectively. 5 and 3 UTRs are delineated by dotted lines. (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of the article.)
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Fig. 2. E. sinensis catA cDNA and deduced amino acid sequences. Amino acids corresponding to the start sites of the mature peptide are boxed. Putative signal peptide sequences are double underlined. The triad of conserved catalytic active sites are shaded, and their associated ﬂanking consensus motifs are single underlined.
3.5. Tissue distribution of catA expression in E. sinensis As determined by RT-PCR, detectable catA expression was widely observed in the hepatopancreas, hemocytes, gill, brain, muscle, heart, intestine, stomach and eye stalk of E. sinensis (Fig. 5). Expression was highest in gills, and comparable among hepatopancreas, hemocytes, brain, muscle, heart and stomach.
3.6. Temporal expression of catA in immune challenged hemocytes CatA expression in E. sinensis, as measured by real-time qRT-PCR, was induced in hemocytes following exposure to V. anguillarum (Fig. 6). CatA expression was signiﬁcantly greater than the vehicle control after 6, 8 and 12 h post V. anguillarum stimulation (P < 0.05).
CatA expression was up-regulated at 6 h post injection, and peaked to 7.9-fold that of the vehicle control after 12 h. CatA expression then decreased to levels not signiﬁcantly different from the vehicle 16 h post-injection.
4. Discussion Given their functional roles in intracellular protein degradation, it has been suggested that cathepsin cysteine proteases may be the most important group within the papain superfamily [28,44,45]. Despite the well documented role of catL, C and K in immune response among vertebrates [3,12,18,49], the functionality within the innate immune system of catA which belong to serine proteases family in invertebrates had yet to be explored in detail; therefore, we cloned the catA gene and full length cDNA, exam-
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Fig. 3. Multiple alignment of catA amino acid sequences in different species. Identical (*) and similar (. or:) amino acid residues are indicated. Gaps (−) were introduced to maximize the alignment. See Table 2 for GenBank accession numbers.
ined tissue-speciﬁc expression, and assessed responsiveness after an immune challenge in a commercially important aquaculture species, E. sinensis. In the present study, a catA gene with potential immune functions was identiﬁed from Chinese mitten crab, E. sinensis, designated as Es-catA. The sequence of the deduced protein shared similarity with other known serine proteases, such as catA from S. salar, T. nigroviridis, M. domestica, M. mulatta and D. rerio. Multiple sequence alignment revealed the conservation of the catalytic domain (serine carboxypeptidases) among catAs. The similarity, together with the conservation of characteristic domains, indicated that E. sinensis catA was a true member of the serine proteases family. In this experiment, we found that the catA intron sequences are remarkably short (Fig. 1). Introns are ubiquitous in eukaryotes, although their sizes vary considerably within a genome as well as between different species . Some of the largest introns are found in the human genome, where the total length of intron sequences in a gene often reaches tens of thousands of nucleotides such that transcription of a single gene requires several minutes and thousands of ATP molecules. Mutational preference for deletions may be the sole control on intron size [34,36]. Given the high
cost of transcription, however, one might expect that natural selection would favor shorter and fewer introns, especially in genes that, occasionally or constitutively, are expressed at a high level . However, whether the catA short intron sequences have any inﬂuence on its immune function were worth further studying. The papain superfamily of cysteine proteases arose early in evolution, possibly before the divergence of eukaryotes and prokaryotes . The high percent identity among residues buttressing the active site across taxa suggest that catA, D, E, S, L, F, H, C and B subfamilies evolved from gene duplication events . Phylogenic analysis of E. sinensis cathepsin proteins produced nine distinct clades, with proteins segregated based upon gene. An analysis of the tree revealed three main branches that contained catA and catB, C, F, H, L, S and catD, E, respectively. The topology of the tree was in accordance with protein classiﬁcations as CatB, C, F, H, L, K, O, S, V, W, and X are cysteine proteases of the papain family, and represent the largest and best-known cathepsin class, while catD and E are aspartic proteases and catA are serine proteases. Information from catA tissue-dependent mRNA expression may offer useful cues when speculating about function. In E. sinensis, catA transcripts were detected in all tissues examined, including
Table 2 Amino acid sequence percent identity of catA from E. sinensis compared to other cathepsins. Percent identity 1
51 66 65
48 64 63 79
50 79 72 65 62
1 2 3 4 5 6
E. sinensis S. salar T. nigroviridis M. domestica M. mulatta D. rerio
The sequences were downloaded from the GenBank and have the following accession numbers: Eriocheir sinensis (E. sinensis), GU593248; Salmo salar (S. salar), NP 001133654.1; Tetraodon nigroviridis (T. nigroviridis), CAF90164.1; Monodelphis domestica (M. domestica), XP 001374035.1; Macaca mulatta (M. mulatta), XP 001105880.1; Danio rerio (D. rerio), NP 956844.1.
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Fig. 4. Neighbor-joining phylogenetic tree of CatB, C, D, E, F, H, L and S amino acid sequences reported in representative taxa. Protein abbreviations and corresponding GenBank accession numbers are as follows: Cathepsin L: Eriocheir sinensis (GU593246), Homo sapiens (CAA77180.1); Cathepsin B: Gallus gallus (AAA87075.1), Homo sapiens (AAC37547.1); Cathepsin C: Eriocheir sinensis (GU593247), Homo sapiens (AAQ08887.1); Cathepsin D: Bos taurus (NP 001159993.1), Homo sapiens (AAA51922.1); Cathepsin E: Homo sapiens (AAA52300.1), Gallus gallus (XP 001235024.1); Cathepsin F: Homo sapiens (AAD41790.1), Bos taurus (NP 001068884.1); Cathepsin H: Homo sapiens (AAL23961.1), Bos taurus (AAI02387.1); Cathepsin S: Homo sapiens (AAC37592.1), Gallus gallus (NP 001026516.1); Cathepsin A: Eriocheir sinensis (GU593248), Salmo salar (NP 001133654.1), Tetraodon nigroviridis (CAF90164.1), Monodelphis domestica (XP 001374035.1), Macaca mulatta (XP 001105880.1), Danio rerio (NP 956844.1).
hepatopancreas, brain, gills, intestine, hemocytes, muscle, stomach, eyestalk and heart. It was notable that E. sinensis catA mRNA expression was highest in gills, with the possibility that the widely distributed presence of catA could be the result of the inﬁltration of hemocytes into different tissues. The crustacean gills directly contact the bacteria in culture water considering the potential role of catA in immunity, the high expression in gills might derive from its response against water bacteria. In human, CatA is mainly expressed in platelets , lymphokine-activated killer cells (LAK) , spleen cells, ﬁbroblasts, kidney, liver , melanoma cells , peripheral blood mononuclear cells (PBMC)  and human alveolar macrophages . CatA has also been demonstrated to be secreted from platelets and killer T lymphoid cells and then exerts putative extracellular functions [27,43] and also many of the other cathepsins like catL, C and K has been detected to have the function of immunity [3,12,18,49]. All the evidence suggested that catA may be involved in the process of immune reactions too. The innate immune system is the ﬁrst line of defense to protect the host in the ﬁrst hours to days of infection . Crustacean hemocytes are thought to be functionally analogous to vertebrate leukocytes and involved primarily with the recognition and removal of foreign materials, and the ﬁrst distinct phase of the immune response in crustaceans is approximately in the ﬁrst 12 h after challenge . We investigated the responsiveness of catA expression to an immune challenge in order to elucidate
Fig. 5. Tissue-dependent catA mRNA expression in E. sinensis. CatA and ␤-actin mRNA expression in gill (1), stomach (2), hepatopancreas (3), hemocytes (4), brain (5), heart (6), eye stalk (7), intestine (8), and muscle (9).
potential involvement in the invertebrate innate immune system. We observed a time-dependent upregulation in catA expression following V. anguillarum stimulation, with a signiﬁcant increase observed at 6 h post-injection (an 2.4-fold increase relative to the vehicle control), and we observed a total of three expression peaks in response to a single injection, after 6 h (2.4-fold), 8 h (4.1-fold) and 12 h (7.9-fold). Data suggest catA expression is immunoresponsive, and that catA may possess immune functionality in E. sinensis. However, in E. sinensis, the temporal response of the other immune-related serine protease gene expression is both geneand bacteria-dependent. The pathogen L. anguillarum, elicited two peaks in EscSP expression 2 and 12 h post-injection (4.96- and
Fig. 6. . Temporal catA mRNA expression in response to V. anguillarum challenge (black bars). Hemocytes collected from crabs injected with V. anguillarum (black bars) or vehicle (white bars) were compared with respect to catA mRNA expression (relative to ␤-actin) using Student’s t-tests. Bars represent mean ± S.E. (n = 6). Statistical signiﬁcance is indicated with an asterisk (P < 0.05).
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9.90-fold, respectively) ; while V. anguillarum injection induced three EsproPO peaks 2 h (3.68-fold), 12 h (32.1-fold), and 48 h (18.6fold) post-injection . Collective results indicate that E. sinensis catA is a constitutive and inducible acute-phase protein involved in defense response against bacterial infection. 5. Conclusions In the present study, we report the cloning, sequence analysis, tissue-speciﬁc distribution, and immune responsiveness of a catA gene and associated cDNA in E. sinensis. The conservation of key amino acids and motifs among taxa and cathepsin subgroups support a common evolutionary origin and functionality. Further study concerning catA’s mechanism of action within the innate immune system of E. sinensis may elucidate its exact immunological and provide a vehicle for the prevention of viral and bacterial infections among aquaculture stocks. Acknowledgments This research was supported by the National Natural Science Foundation of China (No. 30671607, 30972241), Program of Shanghai Education Commission (No. 08SG24). References  Berti PJ, Storer AC. Alignment/phylogeny of the papain superfamily of cysteine proteases. J Mol Biol 1995;246:273–83.  Birkus G, Wang R, Liu X, Kutty N, MacArthur H, Cihlar T, et al. A is the major hydrolase catalyzing the intracellular hydrolysis of the antiretroviral nucleotide phosphonoamidate prodrugs GS-7340 and GS-9131. Antimicrob Agents Chemother 2007;51:543–50.  Biroc SL, Gay S, Hummel K, Magill CT, Palmer J, Spencer DR. Cysteine protease activity is up-regulated in inﬂamed ankle joints of rats with adjuvant-induced arthritis and decreases with in vivo administration of a vinyl sulfone cysteine protease inhibitor. Arthritis Rheum 1997;44:703–11.  Bonten D, Wang JN, Toy L, Mann A, Mignardot G, Yogalingam, et al. Targeting macrophages with baculovirus-produced lysosomal enzymes: implications for enzyme replacement therapy of the glycoprotein storage disorder galactosialidosis. FEBS Lett 2004;9:971–3.  Bonten E, Spoel A, Fornerod M, Grosveld G, d’Azzo A. Characterization of human lysosomal neuraminidase deﬁnes the molecular basis of the metabolic storage disorder sialidosis. Genes Dev 1996;10:3156–69.  Breathnach R, Chambon P. Organization and expression of eukaryotic split genes coding for proteins. Annu Rev Biochem 1981;50:349–83.  Bright NA, Reaves BJ, Mullock BM, Luzio JP. Dense core lysosomes can fuse with late endosomes and are re-formed from the resultant hybrid organelles. J Cell Sci 1997;110:2027–40.  Castillo-Davis CI, Mekhedov SL, Hartl DL, Koonin EV, Kondrashov FA. Selection for short introns in highly expressed genes. Nat Genet 2002;31:415–8.  Cuervo AM, Mann L, Bonten EJ, d’Azzo A, Dice JF. Cathepsin A regulate chaperone-mediated autophagy through cleavage of the lysosomal receptor. J EMBO 2003;22:47–59.  D’Azzo A, Hoogeveen A, Reuser AJ, Robinson D, Galjaard H. Molecular defect combined beta-galactosidase and neuraminidase deﬁciency in man. Proc Natl Acad Sci USA 1982;79:4535–9.  Deutsch M, Long M. Intron–exon structures of eukaryotic model organisms. Nucleic Acids Res 1999;27:3219–28.  Drake FH, Dodds RA, James IE, Connor JR, Debouck C, Richardson S. Cathepsin K, but not cathepsins B, L, or S, is abundantly expressed in human osteoclasts. J Biol Chem 1996;271:12511–6.  Emanuelsson O, Nielsen H, Brunak S, Von Heijne G. Predicting subcellular localization of proteins based on their N-terminal amino acids sequence. J Mol Biol 2000;300:1005–16.  Faurschou M, Borregaard N. Neutrophil granules and secretory vesicles in inﬂammation. Microb Infect 2003;5:1317–27.  Gai YC, Qiu LM, Wang LI, Song LS, Mu CK, Zhao JM. A clip domain serine protease (cSP) from the Chinese mitten crab Eriocheir sinensis: cDNA characterization and mRNA expression. Fish Shellﬁsh Immunol 2009;27: 670–7.  Gai YC, Zhao JM, Song LS, Li CH, Zheng PL, Qiu LM. A prophenoloxidase from the Chinese mitten crab Eriocheir sinensis: gene cloning, expression and activity analysis. Fish Shellﬁsh Immunol 2008;24:156–67.  Galjart HJ, Gillemans N, Harris A, van der Horst GT, Verheijen FW, Galjaard H, et al. Expression of cDNA encoding the human ‘protective protein’ associated with lysosomal␤-galactosidase and neuraminidase: homology to yeast proteases. Cell 1988;54:755–64.
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