The molecular characterization of a cyclophilin A from Chinese mitten crab Eriocheir sinensis and the antifungal activity of its recombinant protein

Mengqiang Wang¹ · Daoxiang Zhang^1,2 · Lingling Wang¹ · Yunchao Gai¹ · Zhi Zhou¹ · Huan Zhang¹ · Linsheng Song*¹

¹Chinese Academy of Sciences, Institute of Oceanology, Key Laboratory of Experimental Marine Biology, Qingdao, China
²University of Chinese Academy of Sciences, Beijing, China

*Corresponding author: lshsong@ms.qdio.ac.cn

Financial support: This research was supported by National Basic Research Program of China (973 program, No. 2012CB114405 to Prof. Lingsheng Song), and Shandong Provincial Natural Science Foundation, China (No. JQ201110 to Prof. Lingling Wang).

Keywords: antifungal activity, cyclophilin A, Eriocheir sinensis, immune response, PPIase activity.

Background: Cyclophilin A (CypA), a receptor for the immunosuppressive agent cyclosporin A (CsA), is a cis-trans peptidyl-prolyl isomerase (PPIase) which accelerates the cis-trans isomerization of prolyl-peptide bonds and interacts with a variety of proteins to regulate their activities.

Results: The full-length cDNA of crab Eriocheir sinensis CypA (EsCypA) was cloned by EST and RACE technique. The complete sequence of EsCypA cDNA contained a 5' untranslated region (UTR) of 50 bp, a 3’ UTR of 233 bp with a polyA tail, and an open reading frame (ORF) of 495 bp encoding a polypeptide of 164 amino acids with the predicted molecular weight of 17.36 kDa. The deduced amino acid sequence of EsCypA contained two highly conserved signature sequences of peptidyl-prolyl cis-trans isomerase and a pro-isomerase domain. The mRNA transcripts of EsCypA were detectable in all the examined tissues, including haemocytes, gill, hepatopancreas, gonad, muscle and heart, with higher expression level in hepatopancreas and gonad. No significant difference in the relative mRNA expression level of EsCypA was observed during the whole course of bacteria challenge, whereas it was up-regulated during fungi challenge. The purified recombinant protein rEsCypA exhibited a significant PPIase activity and an antifungal activity.

Conclusions: All these results indicated that it was a typical CypA member and potentially involved in the innate immune responses of crab.

Cis-trans isomerization of the Xaa-Pro (Xaa stands for unspecified amino acid) bond is an important step in protein folding and a critical determinant of protein structure (Shen and Bax, 2010). Peptidyl-prolyl cis-trans isomerases (PPIases, EC 5.2.1.8) contain three unrelated subfamilies, including the cyclophilins (Cyps), FK binding proteins (FKBPs) and parvulins (Schiene-Fischer et al. 2012). According to their ability to bind the potent immunosuppressive drugs, the first two subfamilies are collectively known as immunophilins (Kang et al. 2008; Gauwerky et al. 2009). The Cyps was first identified from human as a cytosolic binding protein for the immunosuppressive drug cyclosporin A (CsA) (Handschumacher et al. 1984). Subsequently, various Cyps have been found in diverse organisms ranging from bacteria to human, and they are usually grouped based on their size and target location (Rutherford and Zuker, 1994). Five isoforms have been reported in yeast Cyps family, including CypA (Cyp1) of 17 kDa cytosolic protein, CypB (Cyp2) of 20 kDa secretory protein, CypC (Cyp3) of 18 kDa mitochondrial protein, CypD of 22.8 kDa endoplasmic reticulum protein, and Cyp4 of 33.4 kDa secretory protein (Frigerio and Pelham, 1993). Similarly, five members of Cyps family have also been identified from mammalian, CypA of 18 kDa soluble cytosolic proteins, CypB of 22 kDa proteins with signal sequences for ER, CypC of 23 kDa proteins with endoplasmic reticulum signal sequences, CypD of 22 kDa proteins with putative mitochondrial signal sequences, and Cyp40 of 40 kDa proteins with low affinities to CsA (Gething and Sambrook, 1992).

Among all the Cyp members, CypA acts as a ubiquitously distributed intracellular protein and carries out a wide range of functions, including protein folding, receptor complex stabilization, apoptosis participation, and receptor signaling through its PPIase activity (Brazin et al. 2002; Lin and Lechleiter, 2002). CypA is also considered as a host cell receptor for the potent immunosuppressive drug CsA (Mikol et al. 1993). The immunosuppressive activity of CsA is mediated via Cyp-dependent calcineurin (Coto and Tavira, 2009), and calcineurin also plays a key role in initiating T lymphocyte activation by dephosphorylating the nuclear factor of activated T-cells (NFAT) (Li et al. 2012), which subsequently translocates from the cytoplasm to the nucleus. NFAT in nucleus is required for the induction of transcription of cytokines, such as Interleukin-2 (IL2), IL3, IL4, and interferon-γ (INFγ) (Yarilina et al. 2011). CypA-CsA complex can bind intracellular proteins and plays important roles in immune system of mammals (Angelucci et al. 2011). Recent studies revealed that CypA could activate innate immune system and inhibit the infectivity of wild type HIV-1 via interacting with HIV-1 gag protein (Towers et al. 2003; Sayah et al. 2004; Berthoux et al. 2005). Additionally, CypA was also suggested to play important roles in innate immune system of marine invertebrate. For examples, PmCypA from shrimp Penaeus monodon (Qiu et al. 2009), CfCypA from scallop Chlamys farreri (Song et al. 2009), and VpCypA2 from clams Venerupis philippinarum (Chen et al. 2011) were reported to be involved in the immune responses to pathogen infection. However, the activity of CypA protein and the detailed mechanism in invertebrates are still not quite clear.

The Chinese mitten crab Eriocheir sinensis is one of the most important aquaculture species in South-East Asia. With the development of intensive culture and environmental deterioration, various diseases caused by bacteria, viruses and rickettsia-like organisms had frequently occurred in cultured E. sinensis populations, which resulted in enormous losses to this aquaculture industry (Gai et al. 2009). A better understanding of the immune defence mechanisms of crab is necessary for the health management of crab farming. The main objectives of this study were (1) to clone the full-length cDNA of CypA from E. sinensis, (2) to investigate the tissue distribution of EsCypA transcript, and its temporal profile after microbe challenge, and (3) to validate the possible activity of recombinant EsCypA protein.

Crabs, microbe challenge and haemocyte collection

Healthy E. sinensis were collected from a local farm in Qingdao, China, and acclimated for two weeks before processing. Approximately two hundred crabs were employed for microbe challenge experiment, and they were divided into three groups. Seventy crabs in the first group were kept in tanks containing live Vibrio anguillarum strain M3 at final concentration of 8 x 10⁶ CFU mL^-1 and employed as bacteria challenge group. Other seventy crabs were transferred to the fungi-containing tanks with live Pichia pastoris strain GS115 at final concentration 8 x 10⁶ CFU mL^-1, and employed as fungi challenge group. Five individuals were randomly sampled at 1.5, 3, 6, 12, 24 and 48 hrs post challenge. The rest thirty unchallenged crabs were kept in the fresh seawater as blank group, and five individuals were randomly sampled at 0 hrs. The haemolymph was collected from cheliped using a syringe with an equal volume of anticoagulant (27 mmol L^-1 sodium citrate, 336 mmol L^-1 NaCl, 115 mmol L^-1 glucose, 9 mmol L^-1 EDTA, pH 7.0), and centrifuged at 800 g, at 4ºC for 10 min to harvest the haemocytes for RNA preparation. Hepatopancreas, muscle, gill, heart and gonad from five untreated crabs were collected to determine the tissue distribution of EsCypA mRNA transcript. Total RNA was immediately extracted using RNAiso plus reagent (TaKaRa, Japan) according to the manufacture’s protocol.

EST analysis and cloning the full-length EsCypA cDNA

One EST (CMCES_A_5626) of 473 bp from the cDNA library (Gai et al. 2009) was homologous to CypAs identified previously, and it was selected for further cloning of CypA cDNA from E. sinensis (designated as EsCypA). The clone corresponding to this EST was selected for completely sequencing with T7 promoter primer (Table 1). Two gene-specific primers, Es-CypA-Race-R1 and Es-CypA-Race-R2 (Table 1) were designed based on the certain EST to clone the 5’ sequence of EsCypA cDNA by rapid amplification of cDNA ends (RACE) technology. A pair of gene-specific primers Es-CypA-cDNA-F and Es-CypA-cDNA-R (Table 1) was designed to amplify and verify the cDNA sequences. All PCR amplification was performed in a PCR Thermal Cycler (Takara, Japan). And the PCR products were gel-purified and cloned into the pMD18-T simple vector (Takara, Japan). After being transformed into the competent cells Escherichia coli strain Top10 (TianGen, China), the positive recombinants were identified through anti-Amp selection and PCR screening with M13-47 and RV-M primers (Table 1). Three of the positive clones were sequenced on a PRISM 3730 automated sequencer (ABI, USA). The sequencing results were verified and subjected to cluster analysis.

Sequence analysis, multiple sequences alignment and phylogenetic analysis

The searches for nucleotide and protein sequence similarities were conducted with BLAST algorithm at the National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.gov/blast). The deduced amino acid sequence of EsCypA was analyzed with the Expert Protein Analysis System (Expasy, http://www.expasy.org/). SignalP 3.0 program was utilized to predict the presence and location of signal peptide (http://www.cbs.dtu.dk/services/SignalP/). The protein motif features were predicted by Simple Modular Architecture Research Tool (SMART) version 7.0 (http://smart.embl-heidelberg.de/). Multiple sequence alignment of EsCypA and other reported Cyps was performed with ClustalW multiple alignment program (http://www.ebi.ac.uk/Tools/clustalw2/) and multiple alignment show program (http://www.bioinformatics.org/sms/). An unrooted phylogenic tree was constructed based on the deduced amino acid sequences of EsCypA and other reported Cyps by the neighbour-joining (NJ) algorithm using the MEGA 5.05 software (http://www.megasoftware.net). To derive the confidence value for the phylogeny analysis, bootstrap trials were replicated 1000 times. The amino acid sequences information for homologous and phylogenetic analysis was shown in Table 2.

Tissue distribution of EsCypA mRNA and its temporal expression in haemocytes after microbechallenge

The mRNA transcripts of EsCypA in various tissues, including haemocytes, hepatopancreas, muscle, gill, heart and gonad of five untreated crabs, and its temporal expression in haemocytes of crabs challenged with V. anguillarum or P. pastoris were determined by real-time PCR. Total RNA from different tissues was isolated according to the protocol of RNAiso plus reagent (TaKaRa, Japan). The first-strand synthesis was carried out based on Promega M-MLV RT Usage information using the DNaseI (RQ1, Promega, USA)-treated total RNA as template and adaptor primer-oligo (dT) (Table 1) as primer. The reaction mixtures were incubated at 42ºC for 1 hr, terminated by heating at 95ºC for 5 min, and subsequently stored at -80ºC. Real-time PCR was performed with the SYBR premix Ex Taq II (Takara, Japan) on an ABI 7300 Real-Time Detection System (ABI, USA) to investigate the mRNA expression of EsCypA. The information of all primers used in this assay was shown in Table 1. The expression of EsCypA was normalized to the expression of β-actin gene for each sample. The comparative Ct method (2^-ΔΔCt method) was used to analyze the expression level of EsCypA. All data was given in terms of relative mRNA expression level as means ± S.D. (n = 5) and subjected to one-way analysis of variance (one-way ANOVA) followed by an unpaired, two-tailed student’s t-test. Differences were considered significant at p < 0.05.

Recombination, expression and purification of EsCypA in E. coli

The cDNA fragment encoding the mature peptide of EsCypA was amplified with two gene specific primers, Es-CypA-recombinant-F and Es-CypA-recombinant-R (Table 1), and then ligated to the expression vector pEASY-E1 (TransGen, China). The recombinant plasmid pEASY-E1/EsCypA was isolated via E.Z.N.A. Plasmid Mini Kit (Omega, USA) and transformed into E. coli strain BL21 (DE3) (Novagen, USA). The positive transformants E. coli BL21 (DE3)/pEASY-E1/EsCypA were incubated in LB medium containing 100 mg mL^-1 ampicillin at 37ºC with shaking at 220 rpm. When the culture mediums reached OD₆₀₀ of 0.5-0.7, the cells were incubated for 4 additional hrs with the induction of IPTG at the final concentration of 1 mmol L^-1. The recombinant protein (designed as rEsCypA) was purified by a Ni²⁺ chelating sepharose column under denatured condition (8 mol L^-1 urea), and refolded in gradient urea-TBS glycerol buffer (50 mmol L^-1 Tris-HCl, 50 mmol L^-1 NaCl, 5% glycerol, 2 mmol L^-1 reduced glutathione, 0.2 mmol L^-1 oxide glutathione, a gradient urea concentration of 6, 4, 3, 2, 0 mol L^-1 urea in each gradient, pH = 8.0, each gradient at 4ºC for 12 hrs). The resultant protein was separated by reducing 15% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and visualized with coomassie brilliant blue R-250.

PPIase activity analysis of recombinant EsCypA protein

PPIase activity was determined by an assay with α-chymotrypsin (Sigma, USA) that utilized N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (Sigma, USA) in 88% trans- and 12% cis- configuration as the substrate in TBS buffer (50 mmol L^-1 Tris-HCl, 50 mmol L^-1 NaCl, 5%, pH = 8.0) (Lee et al. 2007). The trans- form of the substrate is cleavable by α-chymotrypsin, which releases p-nitroanilide, while the cis- form cannot be cleaved. The p-nitroanilide was quantified spectrophotometrically at 405 nm, and PPIase activity was measured by the cis-trans isomerization of the proline alanine peptide bonnd in the peptide. For each assay, 45 μL of TBS buffer and 5 μL of 100 μg mL^-1 α-chymotrypsin were equilibrated in a well of the assay plate. Typically, 50 μL of 100 μg mL^-1 rEsCypA was then added, followed by 50 μL of 5 mmol L^-1 N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as substrate. Absorbance at 405 nm of each well was determined every 30 seconds by a precision micro plate reader (BioTek, USA). TBS buffer and 100 μg mL^-1 BSA were employed as blank control and negative control group, respectively. The PPIase activity is simply qualitatively represented by the change of OD₄₀₅.

Analysis of antifungal activity of recombinant EsCypA protein

Antifungal activity of the recombinant rEsCypA was assessed by a radial diffusion assay with the P. pastoris strain GS115 as substrate (Zhao et al. 2007a; Zhao et al. 2007b). The fungi concentration in medium was adjusted to 0.2 absorbance unit at 600 nm and poured onto 90 mm plates. Wells (diameter approximately 7.5 mm) were cut into freshly poured plates after the solidification of the agar. For radial diffusion assays, 100 μL of a gradient concentration of rEsCypA was pipetted into individual wells and the agar plates were incubated at 28ºC. After 48 hrs incubation, the radius of the clearing zone was measured. TBS buffer, 500 μg mL^-1 of zeocin (Invitrogen, USA), and a gradient concentration of BSA were employed as blank control, positive control and negative groups, respectively.

Nucleotide sequence

The complete cDNA sequence of EsCypA was deposited in GenBank under the accession number GQ240290.

Molecular features of EsCypA sequence

Sequencing of E. sinensis cDNA library yielded 7535 EST sequences, among which one EST CMCES_A_5626 was homologous to previously identified CypA. A 777 bp nucleotide sequence was obtained by overlapping the certain EST with the amplified fragments. It represented the complete cDNA sequence of EsCypA, and was deposited in GenBank under the accession number GQ240290. The complete sequence of EsCypA cDNA contained a 5’ untranslated region (UTR) of 50 bp, a 3’ UTR of 233 bp with a polyA tail, and an open reading frame (ORF) of 495 bp encoding a polypeptide of 164 amino acids with the predicted molecular weight of 17.36 kDa and theoretical isoelectric point of 8.482 (Figure 1). There was no availability of a signal peptide revealed by SignalP program analysis. Two highly conserved signature sequences of peptidyl-prolyl cis-trans isomerase (¹⁹RIVMELRADVVPKTAENF³⁶ and ⁴⁸YKGCAFHRVIPNFMCQGG⁶⁵) and a pro-isomerase domain (from Q⁴ to L¹⁶⁴) was identified in the amino acid sequence of EsCypA (Figure 1).

Multiple sequences alignment and the phylogeny of EsCypA

The deduced amino acid sequence of EsCypA shared significant homology with other reported CypAs, such as 85.9% identity with P. monodon, 81.7% with C. farreri, and 81.0% with both Ictalurus punctatus and Argopecten irradians (Figure 2). Fourteen amino acid residues critical for the fundamental structure and function of the CypA PPIase activity, including R⁵⁵, I⁵⁷, F⁶⁰, M⁶¹, Q⁶³, G⁷², A¹⁰¹, N¹⁰², A¹⁰³, Q¹¹¹, F¹¹³, W¹²¹, L¹²² and H¹²⁶ (Tu et al. 2003), were all conserved in EsCypA (Figure 2). A phylogenetic tree was constructed based on the amino acid sequences of 16 Cyps by the NJ method (Figure 3). EsCypA was first clustered with CypA from P. monodon, and then clustered with CypAs from Danio rerio and I. punctatus.

Tissue distribution of EsCypA mRNA

The mRNA expression level of EsCypA in different tissues of E. sinensis was quantified by real-time PCR with the β-actin gene as an internal control. For both EsCypA and β-actin genes, only one peak was observed at the corresponding melting temperature in the dissociation curve analysis, indicating that the PCR was specifically amplified (data not shown). The mRNA transcripts of EsCypA were detectable in all the examined tissues, including haemocytes, gill, hepatopancreas, gonad, muscle and heart (Figure 4). The expression level observed in muscle, heart, gill, hepatopancreas and gonad was 3.11-fold, 1.39-fold, 2.37-fold, 24.91-fold and 9.88-fold higher than that in haemocytes, respectively.

The temporal expression of EsCypA mRNA after microbe challenge

The temporal expression of EsCypA in haemocytes of E. sinensis after microbe challenge was detected via real-time PCR with β-actin gene as an internal control. No significant change in the relative expression level of EsCypA mRNA was observed during the whole course of bacteria challenge (Figure 5a). However, the relative expression level of EsCypA mRNA was up-regulated to 4.57-fold at 12 hrs (p < 0.05) and 2.61-fold at 24 hrs (p < 0.05) post P. pastoris challenge, and then down to the normal level at 48 hrs (Figure 5b).

The PPIase activity of rEsCypA

The rEsCypA protein was purified from the IPTG induced whole cell lysate and a signal sharp band was obtained with the exact molecular weight (Figure 6). Its PPIase activity was determined according to the described protocol. The rEsCypA is an active PPIase that can catalyze the cis- form of N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide to trans- form. The trans- form of the substrate can be cleavable by α-chymotrypsin, which releases p-nitroanilide, while the cis- form cannot be cleaved. The p-nitroanilide was quantified spectrophotometrically at 405 nm. The PPIase activity is simply qualitatively represented by the change of OD₄₀₅, and the results were compared with the spontaneous isomerization that occurs in the absence of rEsCypA in negative control and blank groups (Figure 7).

The antifungal activity of rEsCypA

Radial diffusion assay was employed to evaluate the antifungal activity of rEsCypA against P. pastoris (Zhao et al. 2007a; Zhao et al. 2007b). Based on the radius of the antifungal zone, rEsCypA was found to harbour remarkable in vitro inhibitive effect on fungi P. pastoris at the concentration of 1024 μg mL^-1, 512 μg mL^-1and 256 μg mL^-1, respectively, compared with the negative control groups (Table 3).

The first CypA, also called Cyp protein, was discovered in 1984 (Handschumacher et al. 1984) and its amino acid sequence was subsequently chartered in 1989 (Fischer et al. 1989; Takahashi et al. 1989). From then on, the CypAs have been identified in a wide range of species from prokaryotes and single cellular eukaryotes to higher plants and animals (Ribeiro et al. 2005). In the present study, the full length cDNA of a CypA was identified from Chinese mitten crab E. sinensis. The calculated molecular weight of EsCypA was of 17.36 kDa, which was consistent with the standard value of CypA proteins. Multiple sequence alignment revealed that two Cyp-type peptidl-prolyl cis-trans isomerase signature sequences existed in the deduced amino acid of EsCypA. They were located at sites 19-36 (RIVMELRADVVPKTAENF) and 48-65 (YKGCAFHRVIPNFMCQGG) (Figure 1). Fourteen amino acid residues critical for the fundamental structure and function of the CypA PPIase activity, including R⁵⁵, I⁵⁷, F⁶⁰, M⁶¹, Q⁶³, G⁷², A¹⁰¹, N¹⁰², A¹⁰³, Q¹¹¹, F¹¹³, W¹²¹, L¹²² and H¹²⁶ (Tu et al. 2003), were all conserved in EsCypA. Among them, the residues R⁵⁵, Q⁶³, and N¹⁰² composed of the enzyme active site, and the residues I⁵⁷, A¹⁰¹, F¹¹³, W¹²¹, and L¹²² constituted the binding site for CsA, while the other residues F⁶⁰, M⁶¹, G⁷², A¹⁰³, Q¹¹¹ and H¹²⁶ were involved in the biological functions of CypA (Eisenmesser et al. 2002; Dornan et al. 2003; Howard et al. 2003; Piotukh et al. 2005; Mark and Nilsson, 2007). Similarity analysis revealed that the deduced amino acid sequence of EsCypA shared significant homology with other reported CypA, such as 85.9% identity with P. monodon, 81.7% with C. farreri, and 81.0% with both I. punctatus and A. irradians. These results indicated that EsCypA protein was a PPIase and could act as an intracellular receptor for immunosuppressant drug CsA.

EsCypA mRNA transcript was found universally distributed in all the tested tissues, including haemolymph, gill, hepatopancreas, gonad, muscle and heart (Figure 4), which was also consistent with the opinion that Cyps were ubiquitous in animal tissues (Galat, 1993). The ubiquity of EsCypA transcripts in different tissues indicated that it could be potentially involved in some important physiological processes and played important roles in the basal metabolism of crab cells. In previous reports, the expression level of CypA was found to be highest expressed in hepatopancreas and haemolymph of shrimp P. monodon (Qiu et al. 2009), while in gonad and gill of scallop C. farreri (Song et al. 2009). In the present study, EsCypA was higher expressed in gonad, and it was suspected that EsCypA might be involved in the maturation of gonad for its molecular chaperones functions such as PPIase activity. The higher expression level of EsCypA in hepatopancreas also suggested that CypA might play an important role in the immune defence system of crab, as hepatopancreas was regarded as the main immune center in crustaceans and many immune-related genes were high expressed in this tissue (Gai et al. 2009; Lu et al. 2012). Haemocytes were demonstrated to play important roles in the immune response against the invasive pathogens in invertebrates, and they were always selected as candidate for investigating the immune response in crabs (Mu et al. 2009). The temporal expression of EsCypA mRNA in haemolymph of E. sinensis after microbe challenge was detected by real-time PCR, and its relative expression level exhibited no significant changes during the whole course of V. anguillarum challenge, while it was up-regulated after the challenge of fungi P. pastoris. In previous reports, the temporal expression profiles of CypA in various species were different for the tissue and stimulators. For example, the mRNA expression of PmCypA in the hepatopancreas of shrimp P. monodon was up-regulated after the stimulation of lipopolysaccharide (Qiu et al. 2009). The expression level of CfCypA in C. farreri was almost unchanged in haemocytes, but up-regulated in gonad post injection of V. anguillarum (Song et al. 2009). After challenged by L. anguillarum, the relative expression level of two CypA in V. philippinarum, VpCypA1 and VpCypA2, was opposite. VpCypA1 mRNA was down-regulated, while VpCypA2 mRNA in haemocytes was significantly up-regulated (Chen et al. 2011). These results collectively indicated that CypAs from marine invertebrate were not only a ubiquitous protein, but also an inducible protein and involved in the immune defence. However, the mRNA expression profiles of these CypAs were different from each other, indicating that CypA was a multifunctional molecule, and might be involved in different immune response in different species. Moreover, the specific response of EsCypA transcripts against fungi stimulation indicated the crab CypA might act as an antifungal protein.

The activities of EsCypA were further determined by examining its recombinant protein expressed in E. coli. Compared with the blank and control groups, the purified protein displayed significant PPIase activity and it could catalyze the cis-trans inter-conversion of Ala-Pro bond in the substrate. In the anti-fungi assay, rEsCypA was found to harbor remarkable in vitro inhibitive effect on fungi P. pastoris at the concentration of 1024 μg mL^-1, 512 μg mL^-1and 256 μg mL^-1, respectively. Similarly, the CypA isolated previously from Chinese cabbage B. campestris could inhibit the growth of several fungal pathogens (Lee et al. 2007). Moreover, some other proteins with PPIase activity also exhibited antifungal activity, such as FKBP from B. campestris (Park et al. 2007), Ess1 from Candida albicans (Li et al. 2005) and so on. These results indicated that the antifungal activity of rEsCypA might lie on its PPIase activity. However, compared with the authentic antimicrobial peptide (Zhao et al. 2007a; Zhao et al. 2007b), the inhibitive concentration of rEsCypA was rather high and the protein could not be so concentrated apparently in the normal physiological condition of crab. It was suspected EsCypA was mainly involved in immune defence via its PPIase activity, while its antifungal activity was of adjuvant.

In conclusion, the full-length cDNA encoding CypA was identified from Chinese mitten crab E. sinensis. It was constitutively expressed in all the tested tissues, and up-regulated in haemocytes after challenged by fungi. The purified rEsCypA protein showed significant PPIase activity to catalyze the cis-trans inter-conversion of Ala-Pro bond in the substrate and antifungal activity against P. pastoris. All these results indicated that it was a typical CypA member and was potentially involved in the innate immune responses of crabs.

We are grateful to all the laboratory members for their technical advices and helpful discussion. We also thank the anonymous reviewers for their comments and Prof. Zhaolan Mo (Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery) for her kindly providing V. anguillara strain M3.

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