An i-type lysozyme from the Asiatic hard clam Meretrix meretrix potentially functioning in host immunity

Fish & Shellfish Immunology 30 (2011) 550e558

Contents lists available at ScienceDirect

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

An i-type lysozyme from the Asiatic hard clam Meretrix meretrix potentially functioning in host immunity Xin Yue a, b, Baozhong Liu a, *, Qinggang Xue c a

Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, 7 Nanhai Road, Qingdao 266071, China Graduate School of the Chinese Academy of Sciences, Beijing 100039, China c Department of Veterinary Science, Louisiana State University Agricultural Center, Baton Rouge, LA 70803, USA b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 11 August 2010 Received in revised form 31 October 2010 Accepted 28 November 2010 Available online 4 December 2010

Lysozymes function in animal immunity. Three types of lysozyme have been identified in animal kingdom and most lysozymes identified from bivalve molluscs belong to the invertebrate (i) type. In this research, we cloned and sequenced a new i-type lysozyme, named MmeLys, from the Asiatic hard clam Meretrix meretrix. MmeLys cDNA was constituted of 552 bp, with a 441 bp open reading frame encoding a 146 amino acid polypeptide. The encoded polypeptide was predicted to have a 15 amino acid signal peptide, and a 131 amino acid mature protein with a theoretical mass of 14601.44 Da and an isoelectric point (pI) of 7.14. MmeLys amino acid sequence bore 64% identity with the Manila clam (Venerupis philippinarum) i-type lysozyme and was grouped with other veneroid i-type lysozymes in a bivalve lysozyme phylogenetic tree predicted using Neighbor-Jointing method. Recombinantly expressed MmeLys showed lysozyme activity and strong antibacterial activity against Gram positive and Gram negative bacteria. MmeLys mRNA and protein were detected to be mainly produced in hepatopancreas and gill by the methods of semi-quantitative RT-PCR and western blotting. In addition, MmeLys gene expression increased following Vibrio parahaemolyticus challenge. Results of this research indicated that MmeLys represents a new i-type lysozyme that likely functions in M. meretrix immunity. Ó 2010 Elsevier Ltd. All rights reserved.

Keywords: Invertebrate lysozyme Meretrix meretrix Recombinant expression Antimicrobial activity Immunity

1. Introduction Lysozymes make up a large group of proteins that distribute ubiquitously among organisms ranging from virus to human [1]. Lysozymes share an enzymatic activity to cleave the b-(1, 4) linkage between N-acetylmuramic acid and N-acetylglucosamine of the peptidoglycan in bacterial cell walls [2e4], but differ in amino acid sequence, molecular mass, and biochemical properties. Depending on the amino acid sequence, lysozymes are classified into six types (i.e., phage type, bacterial type, plant type, chicken type, goose type, and invertebrate type); among them the chicken (c) type, goose (g) type and invertebrate (i) type are present in the animal kingdom [5]. The function of animal lysozymes is generally considered to be in host immunity via bacterial cell lysis [6], non-enzymatic antimicrobial activities [7e11], and immunomodulation [12,13]. For some species, lysozymes also function in digestion after adaptive evolution [14e18]. The presence of an i-type lysozyme in animals was proposed by Jollès and Jollès [19] based on the finding of differences in

* Corresponding author. Tel.: þ86 532 82898696; fax: þ86 532 82898578. E-mail address: [email protected] (B. Liu). 1050-4648/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2010.11.022

N-terminal sequence between the Starfish, Asterias rubens, lysozyme and c-type and g-type lysozymes. This hypothesis has been confirmed later by the identification of lysozymes from bivalve molluscs [18,20e23,26e28] and annelids [21,24,25] that share a similar N-terminus or conserved sequence region with the A. rubens lysozyme. In addition, multiple forms of i-type lysozyme have been found in some invertebrate species [18,23,26,27,29]. I-type lysozyme is considered to play an important role in the immunity and digestion in invertebrates. I-type lysozymes have been reported to have strong antimicrobial activity against Gram positive and Gram negative bacteria [22,26,30,31]. At the same time, they are found to express in multiple tissues and organs [28], and the expression level can be induced to increase by bacterial challenges [32,33]. On the other hand, high lysozyme activity has been detected in bivalve digestive tissues [14] and the associated species have the ability to use bacteria as nutrient source [34,35], suggesting that lysozyme may function as a digestive enzyme. Findings in the studies about eastern oyster (Crassostrea virgincia) lysozyme have supported the digestion function of i-type lysozymes [18]. Intriguingly, invertebrates could produce different lysozymes for immunity [28] and digestion [18], and digestive i-type lysozymes be evolved from immune lysozymes via positive selection [27].

X. Yue et al. / Fish & Shellfish Immunology 30 (2011) 550e558

During the annotation of an Asiatic hard clam, Meretrix meretrix, cDNA library [36], we identified an EST that potentially encodes a protein homologous to the Manila clam, Venerupis philippinarum (synonyms Tapes japonica), lysozyme and named it MmeLys. The objectives of this research were: (1) to clone and sequence the fulllength cDNA of MmeLys, (2) to determine the lysozyme activity and antibacterial activity of the encoded protein, (3) to analyze the tissue origin of the gene expression, and (4) to observe the changes in gene expression pattern in response to bacterial challenge. 2. Material and methods 2.1. Clams, Vibrio challenge and sample collection The Asiatic hard clams, M. meretrix (3.5e4.5 cm in shell length, 2e3 years old), used for the challenge were bought from a market in Qingdao, China and acclimated for 1e2 week (25  C, 30& salinity and under continuous aeration) in the laboratory. Before challenge, the clams were fed with the algae Isochrysis galbana. The Vibrio parahaemolyticus strain MM21 isolated from the moribund clams and characterized to pathogenic to M. meretrix [37] was used to challenge the clams. The challenge experiments were done according to the procedure reported by Yue et al. [37]. Briefly, the acclimated clams were split randomly into two groups; one group was injected with 100 ml per clam of MM21 suspension at the concentration of w5  106 CFU ml1 and the other group (control) received 100 ml per clam of phosphate buffered saline (PBS, 0.01 M, pH 7.2). Twenty four hours after challenge, tissues were sampled from hepatopancreas, gill and mantle of 4 clams per group. The dissected tissue blocks were reserved in liquid nitrogen before processing for RNA and protein extraction. 2.2. Full-length cDNA sequence determination The full-length cDNA sequence of MmeLys was determined by 30 and 50 rapid-amplification of cDNA ends (RACEs) using genespecific primers designed from the MmeLys EST sequence and adapter primers (T3 or T7) (Table 1). A plasmid DNA mixture that contained cDNAs of the entire M. meretrix cDNA library [36] was used as the template. The 50 RACE was performed in a total reaction volume of 20 ml containing 14.4 ml of PCR-grade water, 2 ml of 10PCR Buffer, 1.2 ml of MgCl2 (25 mM), 0.4 ml of dNTP mix (10 mM), 0.4 ml of each of the T3 primer and MmeLysR1 primer (Table 1) (10 mM), 0.2 ml (1U) of Taq polymerase (Promega) and 1 ml of

551

plasmid suspension. The PCR parameters were as followings: 1 cycle of 94  C for 4 min, 35 cycles of 94  C for 40 s, 57  C for 40 s, and 72  C for 40 s, followed by the final extension at 72  C for 10 min. The same PCR procedure was followed for the 30 RACE using the primers of T7 and MmeLysF1 (Table 1) except that an annealing temperature of 60  C instead of 57  C was used in the 35 cycles of amplification. PCR products were gel-purified and cloned into the pMD19-T simple vector (TaKaRa) and then sequenced at Shanghai Sangon Company (Shanghai, China). The predicted full-length MmeLys cDNA sequence was verified by PCR amplification using MmeLysF2 and MmeLysR2 (Table 1) and sequencing of the PCR product. 2.3. Computational sequence analysis MmeLys amino acid sequence was deduced from the cDNA sequence using the software BioEdit. Signal peptide was predicted by both neural networks and hidden Markov models on a Signal IP 3.0 Server [38]. Isoelectric points and molecular weight of the deduced protein were determined using the “Compute pI/MW” tool on the ExPASY Server (http://www.expasy.org/tools) [39]. Sequence similarity search was conducted using BLAST at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/ blast). Multiple sequence alignment was performed using the ClustalX [40]. 2.4. Phylogenetic analysis Phylogenetic analysis was done with amino acid sequence using the program Mega 3.0 [41]. Amino acid sequence of MmeLys and other i-type lysozymes identified from bivalve molluscs were aligned using ClustalX and a consensus tree was then constructed by Neighbor-Jointing (NJ) method. The robustness of each topology was checked by 1000 bootstrap replications. The i-type lysozymes included in this analysis were MmeLys, 3 sequences from V. philippinarum (ACU83237, BAC15553, BAB33389), 2 sequences from Calyptogena sp. (AAN16212, AAN16211), 2 sequences from Crassostrea gigas (BAF48045, Q6L6Q6), 2 sequences from Crassostrea virginica (B3A003, P83673), 2 sequences from Mytilus galloprovincialis (BAF63423, AAN16210), 1 sequence from Chlamys islandica (CAC34834), 1 sequence from Mytilus edulis (AAN16207), and 1 sequence from Ostrea edulis (Q6L6Q5). In addition, the lysozymes of Eisenia andrei (ABC68610) and Hirudo medicinalis (AAA96144) were included as outgroup. 2.5. MmeLys recombinant production

Table 1 Primers used in this study. Primer

Usage

Sequence

T7 T3 MmeLysF1 MmeLysR1 MmeLysF2 MmeLysR2 LYBamHI-F

30 -RACE PCR 50 -RACE PCR 30 -RACE PCR 50 -RACE PCR Sequence verify Sequence verify PCR for plasmid construction PCR for plasmid construction semi-quantitative RT-PCR semi-quantitative RT-PCR semi-quantitative RT-PCR semi-quantitative RT-PCR

50 -GTAATACGACTCACTATAGGGC-30 50 -AATTAACCCTCACTAAAGGG-30 50 -CCATACTGGACTGACTGTGGGAGA-30 50 -TGGCAACCACCTGATTCTAGC-30 50 -ACCGGCATGATCAGTTTAATTG-30 50 -ATGGTATAAACATAACATTCCT-30 50 -AGCGGATCCGCCAGCGTAGAGAAGAGAG-30

LYSal I- R lys-RT-F lys-RT-R actin-F actin-R

50 -CGCGTCGACTTAATGAACATTACTGCATC-30 50 -AGAATCAGGTGGTTGCCATC-30 50 -TCGGGCAGTGGTAGTAGGAG-30 50 -TTGTCTGGTGGTTCAACTATG-30 50 -GACTGATTTCTTACGGATG-30

A DNA fragment containing the predicted MmeLys coding region excluding signal peptide plus a BamH I restriction site at the 50 end and a Sal I restriction site at 30 end was generated by PCR using the primers of lysBamH IeF and lysSal I-R (Table 1). The PCR was done under the same conditions as that for verifying the predicted full-length MmeLys cDNA. After BamH I and Sal I cleavage, the DNA fragment was inserted into a pGEX-4T-1 plasmid (GE healthcare, containing N-terminal GST affinity tag) to construct a pGEX-MmeLys recombinant plasmid. The recombinant plasmid was then introduced into competent E. coli BL21 cells. The transformed E. coli was selected on the LB plate containing 100 mg/ml ampicillin. Plasmids were then extracted from selected cells and sequenced to confirm the in-frame insertion of the MmeLys coding sequence. After the confirmation, one colony of transformed E. coli was grown in 5 ml of LB broth containing 100 mg/ml ampicillin at 37  C overnight with shaking. An aliquot of 200 ml of this culture was inoculated into 200 ml of LB broth containing 100 mg/ml ampicillin and cultured at 37  C with shaking until the OD600 of

552

X. Yue et al. / Fish & Shellfish Immunology 30 (2011) 550e558

culture reached 0.6 (approximately 2 h). Isopropyl-1-thio-b-D-galactopyranoside (IPTG) was then added into the culture to the final concentration of 1 mM and the culture was incubated for another 4 h at 37  C with shaking to induce the expression of the GSTMmeLys fusion protein. After incubation, bacterial cells were harvested by centrifugation at 5,000 rpm for 10 min at 4  C and resuspended in 40 ml of 0.01 M PBS, pH 7.2 containing 1% Triton X-100. The cells were lysed by sonication for 30 min in a combination of 1s sonication and 2s interval under 500 W power. The cell lysates were then centrifuged at 10,000 rpm for 10 min at 4  C and the precipitated pellets were collected for rMmeLys purification. To purify rMmeLys, the pellets were washed three times each with 10 ml of 50 mM TriseHCl, 5 mM EDTA, 0.1% Triton X-100, pH 8.0 and then three times each with 10 ml of 50 mM TriseHCl, 5 mM EDTA, 2 M urea, pH 8.0 by centrifugation at 10,000 rpm for 10 min at 4  C. The pellets were then dissolved and adjusted to the final concentration of 500 mg/ml in 0.1 M TriseHCl buffer, pH 8.0, containing 8 M urea and 10 mM DTT to denature the GST-MmeLys. After denaturation, GST-MmeLys was refolded by stepwise dialysis at 4  C against 8.0, 4.0, 2.0, 1.0 and 0 M urea solutions in 0.1 M TriseHCl containing 5 mM EDTA and 5 mM cysteine, pH 8.0, for 4 h each to completely remove urea and contaminants. The refolded recombinant GST-MmeLys was then purified by affinity chromatography using a GSTrap FF column according to the manufacture’s instruction (GE Healthcare). The GST tag was removed by hydrolyzing the GST-MmeLys bound to the affinity column with thrombin solution (GST removal buffer) according to the manufacture’s instruction (GE healthcare). Purified rMmeLys was compared with GST-MmeLys and GST tag by SDS-PAGE in 12% gel with Coomassie brilliant blue R250 (CBB-R250) staining to confirm the purity. 2.6. Identification of rMmeLys using mass spectrometry The amino acid sequence of rMmeLys was verified by mass spectrometry of SDS-PAGE separated GST-MmeLys. The GST-MmeLys bearing gel was excised manually after CBB-R250 staining and cut into 1 mm blocks. The gel blocks were washed twice in equal volume mixture of 100% methanol and 100 mM NH4HCO3 for 15 min to remove the CBB and then dehydrated with 100% acetonitrile for 5 min, followed by vacuum dry in a SPD 1010 SpeedVac system (ThermoSavant, Milford, MA, USA). The gel was then digested with 250 ng of sequencing-grade, modified trypsin (Promega, Madison, WI, USA) in 50 ml of 50 mM ammonium bicarbonate, pH 8.5 at 37  C for 16 h. After digestion, the aqueous phase of the reaction was collected and vacuum dried. The dried samples were then dissolved in 50% acetonitrile containing 0.1% formic acid and analyzed by LCESI-MS/MS in a LCQ DECA XPplus MS system (ThermoFinnigan) as reported by Jiang et al. [42]. The detected MS/MS spectra were analyzed against MmeLys and GST sequence using SEQUEST algorithm in the BioWorks 3.1 software package. 2.7. Lysozyme activity assay Lysozyme activity of rMmeLys was measured using a turbidimetric assay to monitor spectrophotometrically the degradation of Micrococcus lysodeikticus cell wall [43,44]. The assay were done using a commercial kit from Nanjing Jiancheng Bioengineering Institute (Nanjing, China) by mixing 5 ml M. lysodeikticus suspension prepared according to the manufacturer’s instruction with 500 ml of rMmeLys solution, 500 ml of hen egg white lysozyme (HEWL) solution at known units (reference), or 500 ml of deionized water (blank). After incubation at 37  C for 15 min, the suspension was measured for absorbance at 530 nm. The lysozyme unit of the

sample (rMmeLys solution) was then calculated according to the following formula:

Sample lysozyme activity units ¼

OD530ðsampleÞ  OD530ðblankÞ  HEWL units OD530ðHEWLÞ  OD530ðblankÞ

Results were expressed as specific activity in the unit of lysozyme activity units per milligram rMmeLys protein. The GST removal buffer in which rMmeLys dissolved was also measured as sample for the lysozyme activity in the same procedure. All measurements were done in triplicate. 2.8. Antibacterial activity assay Antimicrobial activity was measured against one Gram-positive bacterium, M. luteus, and two Gram-negative bacteria, Pseudomonas aeruginosa and V. parahaemolyticus, using a solid phase assay modified from that of Lie et al. [45] and Minagawa et al. [46]. M. luteus, P. aeruginosa and V. parahaemolyticus were grown in the media of LB, TSB, and TSAYE (tryptic soy agar with 0.6% yeast extract) respectively to a density of 0.8 absorbance units at 600 nm (OD600). An aliquot of 6 ml per culture was then spread on a LB, TSB or TSAYE solid medium (9 cm in diameter) depending on the tested bacteria. Solutions of rMmeLys and HEWL were prepared in GST removal buffer and EDTA$2Na (40 mM) supplemented GST removal buffer. On each M. luteus or P. aeruginosa plate, 100 ml and 200 ml of rMmeLys solution at 250 mg/ml, 100 ml of HEWL at 250 mg/ml (positive control), and 100 ml of GST removal buffer (negative control) were spotted separately using Oxford cups. On a V. parahaemolyticus plate, 100 ml of rMmeLys solutions at 250 mg/ml to 750 mg/ml were spotted separately. The plates were then incubated at 30  C (V. parahaemolyticus) or 37  C (M. luteus and P. aeruginosa) for 24 h. After incubation, the diameter of the transparent circle around each spot cup was measured. 2.9. Detection of MmeLys mRNA using semi-quantitative RT-PCR MmeLys mRNA expression in hepatopancreas, gill and mantle was measured by semi-quantitative RT-PCR. Total RNAs were extracted from tissues and cDNA synthesized according to the techniques reported by Wang et al. [36]. Semi-quantitative RT-PCR was done using the gene specific primers, lys-RT-F and lys-RT-R (Table 1), and the synthesized cDNA as template. The gene specific primers lys-RT-F and lys-RT-R could generate a 202 bp PCR product. PCR conditions were as the followings: 1 cycle of 94  C for 4 min, 32 cycles of 94  C for 40 s, 55  C for 40 s, and 72  C for 40 s, and 1 cycle of 72  C for 10 min. Sample quantity used in the measurement was normalized with M. meretrix b-actin mRNA detected by PCR using the primers of actin-F and actin-R (Table 1) under the following amplification conditions: 1 cycle of 94  C for 4 min, 26 cycles of 94  C for 40 s, 53.5  C for 40 s and 72  C for 40 s, and 1 cycle of 72  C for 10 min. PCR products were analyzed in 1.5% agarose gel, stained with ethidium bromide and visualized under ultraviolet light. The detection specificity was confirmed by PCR product sequencing. 2.10. Preparation of anti-rMmeLys polyclonal antibody Anti-rMmeLys polyclonal antibodies were produced by immunizing 2 male rabbits with purified rMmeLys. The immunization was done by an initial multipoint subcutaneous injection of 0.5 mg per rabbit of purified rMmeLys emulsified in complete Freund’s adjuvant. Twenty days after the initial injection, the rabbits received 4 injections of same dose of antigen emulsified in incomplete Freund’s adjuvant, with a 10-day interval between the injections.

X. Yue et al. / Fish & Shellfish Immunology 30 (2011) 550e558

553

Fig. 1. Nucleotide sequence (upper) and deduced amino acid sequence (single letter code, below) of MmeLys cDNA. Numbering of the nucleotide and amino acid sequence is shown on the left and right, respectively. The underlined amino acid sequence indicates the predicted signal peptide; the poly-adenylation signal is bordered.

One month after the last injection, the immunized rabbits were bled and serum IgG purified by affinity chromatography using Protein A Sepharose column (GE healthcare). Antibody specificity was tested by western blot.

2.12. Statistical analysis MmeLys mRNA and protein expression results were presented as means  SD. Data were analyzed using one-way analysis of variance (ANOVA) followed by Turkey’s test when a significance (P < 0.05) was found using the SPSS 11.5 statistical software.

2.11. Detection of MmeLys protein using Western blot MmeLys protein was detected from hepatopancreas, gill and mantle tissue extracts using Western blot. Total proteins were extracted from tissues using a Total Protein Extraction Kit (BestBio, China) according to the manufacturer’s protocol. After determination of protein concentration by Bradford protein assay (Bio-Rad), equal quantity of total protein from each tissue was separated electrophoretically in a 12% SDS-polyacrylamide gels and transferred onto a PVDF membrane by electroblotting at 100 V for 1 h. The membrane was blocked with 5% (w/v) skimmed milk powder solution at 4  C overnight and then incubated with 1:3000 diluted rabbit anti-rMmeLys IgG in Tris buffered saline, pH8.0 (TBS) containing 0.1% skimmed milk powder at 37  C for 1 h. After incubation, the membrane was washed 3 times with TBS, 10 min each, followed by the incubation with a 1:1000 diluted horseradish peroxidase-conjugated goat anti-rabbit IgG solution (Huabio, China) in TBS containing 0.1% skimmed milk powder at 37  C for 1 h. After 3 washes of 10 min each in TBS, the MmeLys band on the membrane was visualized using a DAB assay kit (Solarbio).

3. Results 3.1. MmeLys cDNA sequence A 552 bp cDNA containing a 441 bp open reading frame (ORF) was determined by 50 and 30 RACEs (Fig. 1). Analysis in the 30 untranslated region (30 -UTR) revealed a canonical poly-adenylation signal (AATAAA) and a poly(A) tail. The ORF encoded a polypeptide of 146 amino acid residues with a theoretical molecular mass of 16082.35 and a pI of 6.99. Signal P prediction indicated that the 15 N-terminal amino acids formed a signal peptide. The predicted mature protein was thus constituted of 131 amino acids including 15 cysteine residues, with a theoretical mass of 14601.44 and a pI of 7.14. Search in the Conserved Domain Database detected a medicinal leech destabilase domain in the MmeLys amino acid sequence. The MmeLys cDNA and the deducted amino acid sequence has been deposited in GenBank under the accession number HM008915.

Fig. 2. Multiple amino acid sequence alignment of MmeLys and i-type lysozymes identified from some other bivalve organisms. Identical amino acid residues are indicated by asterisks (*). Possible active residues for lysozyme and isopeptidase activity are shadowed.

554

X. Yue et al. / Fish & Shellfish Immunology 30 (2011) 550e558

3.2. Homology analysis of MmeLys Blastx searches in NCBI database revealed that the deduced protein of MmeLys shared 64% sequence identity with the lysozyme identified from the Manila clam V. philippinarum (Accession No. ACU83237). Multiple alignments of MmeLys with i-type lysozymes from other bivalves indicated that the amino acid residues critical for enzyme activities were conserved (i.e., the Glu26 and Asp38 in mature protein for lysozyme activity [47,48] and the His102 for isopeptidase activity (Fig. 2, shadowed) [49]). 3.3. Phylogenetic place of MmeLys in bivalve i-type lysozymes In the phylogenetic tree predicted on the basis of amino acid sequence alignment of MmeLys with other bivalve i-type lysozymes, M. meretix lysozyme was placed basally in a monophyletic clade formed by three veneroid lysozymes; MmeLys was sister to the group composed of Calyptogena sp. and V. philippinarum (Fig. 3). 3.4. rMmeLys production The GST removed rMmeLys was detected by SDS-PAGE and CBBR250 staining as a protein of w14.6 kDa (Fig. 4, lane 3), consistent with the mature protein predicted from the cDNA sequence. LC-ESIMS/MS analysis of the GST-MmeLys identified tryptic peptides encompassed in the MmeLys amino acid sequence (Fig. 5). One of the peptide fragments was identified to have the sequence of eQPGCSNVH, corresponding to the 123e131 of the mature MmeLys amino acid sequence (Fig. 5A). A peptide corresponding to the GST fragment (eLLLEYLEEK) was also identified (Fig. 5B). 3.5. Lysozyme activity of rMmeLys Purified rMmeLys was detected to have a specific lysozyme activity of 10.5  0.9 units/mg. No lysozyme activity was detected in the GST removal buffer, which demonstrated that the lysozyme activity detected was totally induced by purified rMmeLys.

Fig. 4. SDS-PAGE of the recombinant protein MmeLys. The gel was visualized by CBBR250 staining. Lane M: protein molecular standard (MBI); lane 1: purified fusion protein GST-MmeLys; lane 2: GST protein which was removed from fusion protein GSTMmeLys by the treatment of thrombin; lane 3: MmeLys. The target protein is indicated by an arrow.

rMmeLys produced a smaller transparent area on the P. aeruginosa plate (Fig. 6B) than on the M. luteus plate (Fig. 6A). Double loaded rMmeLys (i.e., 200 ml) did not show significant increase in bacterial inhibitory activity against either M. luteus or P. aeruginosa. On the other hand, rMmeLys and HEWL prepared in EDTA supplemented buffer resulted in a greater antibacterial activity compared to the same lysozyme prepared in the buffer without EDTA supplement (Fig. 6A and B). In addition, rMmeLys inhibited the growth of V. parahaemolyticus at the concentration of 375 mg/ml and above (Fig. 6C). 3.7. Tissue-specificity of MmeLys expression

3.6. Antibacterial activity of rMmeLys Purified rMmeLys showed strong antibacterial activity against M. luteus at the protein concentration of 250 mg/ml (Fig. 6A). It also inhibited the growth of P. aeruginosa, but the same amount of

Semi-quantitative RT-PCR detected MmeLys mRNA in hepatopancreas, gill and mantle (Fig. 7A). The mRNA expression level was higher in hepatopancreas and gill than in mantle. Western blot revealed the same expression pattern of the protein, with a more

Fig. 3. Phylogenetic analysis of i-type lysozymes of bivalve molluscs. The lysozymes of Eisenia andrei (ABC68610) and Hirudo medicinalis (AAA96144) were included as outgroup. The tree was constructed using Neighbor-Joining method. Bootstrap confidence was calculated from 1000 replications.

X. Yue et al. / Fish & Shellfish Immunology 30 (2011) 550e558

555

Fig. 5. Characteristic spectrum of matched peptide fragments. A: Spectrum of -QPGCSNVH for MmeLys; B: Spectrum of -LLLEYLEEK for GST.

intense band in hepatopancreas and gill extracts than in mantle extracts (Fig. 7B).

unchallenged individuals. No significant difference was detected in mantle between the challenged and unchallenged clams. 4. Discussion

3.8. MmeLys expression in response to Vibrio challenge The relative transcription level (Fig. 8A) and protein level (Fig. 8B) of MmeLys in hepatopancreas and gill were significantly higher (P < 0.05) in the Vibrio challenged clams than in

The cDNA encoding an i-type lysozyme was identified from the Asiatic hard clam (M. meretrix) was cloned and named MmeLys which was characterized using recombinantly expressed protein. Results of amino acid sequence analysis and lysozyme activity

Fig. 6. Antimicrobial activity of rMmeLys against different bacteria by the solid phase assay after 24 h incubation. A: M. luteus. (1) Positive control: HEWL (250 mg/ml, 100 ml). (2) HEWL (250 mg/ml, 100 ml) plus EDTA$2Na (40 mM). (3) rMmeLys (250 mg/ml, 100 ml). (4) rMmeLys (250 mg/ml, 100 ml) plus EDTA$2Na (40 mM). (5) rMmeLys (250 mg/ml, 200 ml). (6) Negative control: elution buffer containing thrombin (100 ml). B: P. aeruginosa. (1-5) same to those of plate A. C: V. parahaemolyticus. (1) rMmeLys (250 mg/ml, 100 ml). (2) rMmeLys (375 mg/ml, 100 ml). (3) rMmeLys (500 mg/ml, 100 ml). (4) rMmeLys (750 mg/ml, 100 ml).

556

X. Yue et al. / Fish & Shellfish Immunology 30 (2011) 550e558

Fig. 7. Tissue distribution of MmeLys analyzed by semi-quantitative RT-PCR and western blot. A: Tissue distribution of MmeLys transcript revealed by RT-PCR. b-actin served as a reference standard. B: Tissue distribution of MmeLys protein revealed by western blot.

measurement indicated that MmeLys represented a new member of i-type lysozymes. MmeLys’ strong antibacterial activity, distribution in multiple tissues and increased expression following bacterial challenge suggested its function in M. meretrix host immunity. Amino acid sequence similarity between MmeLys and other i-type lysozymes qualified the M. meretrix lysozyme MmeLys as an i-type lysozyme. MmeLys contains a medicinal leech destabilase domain, which is conserved in all i-type lysozymes [24,49]. In addition, MmeLys bears 64% amino acid sequence identity with the Manila clam lysozyme, the first i-type lysozyme whose full amino acid sequence and structure were determined empirically [21,47,48]. Moreover, a phylogenetic analysis on MmeLys against different i-type lysozymes and the two other types (i.e., c-type and g-type) of lysozyme from invertebrates (e.g., arthropod c-type lysozymes and bivalve g-type lysozymes [50e52]) placed MmeLys in the i-type lysozyme group (data not shown). Indeed, several characteristics of i-type lysozymes are conserved in MmeLys. MmeLys sequence, for example, has high cysteine content (14 out of 131 residues), which is common in i-type lysozymes [18,26e28,53,54] and presumably important in maintaining the proteins’ stability in high-osmolarity seawater and in the digestive organs [21]. The lysozyme catalytic residues (i.e., Glu26 and Asp38) are also conserved in MmeLys. In addition, the MmeLys His102 appeared to correspond to the histidine residue that has been speculated to be responsible for the isopeptidase activity in destabilase [49] and the Manila clam lysozyme [47]. We have detected the lysozyme activity from MmeLys in this research. Other potential activities such as isopeptidase activity and perhaps chitinase activity that has been detected in both c-type lysozymes [1,5] and i-type lysozymes [18,47,48] should be analyzed in future studies. Results of our research also suggested the potential function of MmeLys in M. meretrix immunity. We detected strong antibacterial activity against Gram positive and Gram negative bacteria

from the recombinant MmeLys (rMmeLys). Notably, the M. meretrix lysozyme showed greater activity against Gram negative bacteria as compared to HEWL. Given the importance of Gram negative bacteria as pathogens or opportunistic pathogens in marine environment [37,55,56], this property of MmeLys can be particularly beneficial for the host immunity of M. meretrix. Interestingly, lysozymes identified from marine animals often show relatively stronger anti-Gram negative bacterium activity compared to lysozymes of land animals [26,30,52,57]. Also consistent with the immunity function is MmeLys’ expression in multiple M. meretrix tissues (i.e., hepatopancreas and gills). Remarkably, hepatopancreas and gills have been reported to represent important portal of the entry of pathogenic microorganisms in bivalve molluscs [30,58,59]; the presence of MmeLys in these tissue likely facilitate the lysozyme to function locally against invaded pathogens. Moreover, the expression of MmeLys was detected to increase in the clams after V. parahaemolyticus-challenge. We used a pathogenic V. parahaemolyticus strain to challenge M. meretrix [37]; the bacterium inducing MmeLys expression increase could be particularly protective to the host. However, it remains to be determined whether the induction is pathogen-specific. More studies will be done to analyze MmeLys expression patterns in response to challenges by different bacteria (Gram negative, Gram positive, pathogenic and nonpathogenic) so that the role of this M. meretrix lysozyme in host immunity can be better assessed. It should be noted that lysozyme presence in the hepatopancreas could also indicate the digestive function [14,18]. Unlike cvlysozyme 2 in the eastern oyster [18], however, MmeLys is not exclusively produced in hepatopancreas (i.e., digestive glands); its major expression sites also included gills. Thus, MmeLys is more likely to represent an evolutionary intermediate that retains the immune function, but is in the transitional process from functioning in immunity to functioning in digestion as in the case of cvlysozyme 3 [27]. Interestingly, MmeLys was predicted to have a pI significantly lower than that of the V. philippinarum lysozyme and Calyptogena lysozymes [27]. In addition, MmeLys was not placed to be sister to the V. philippinarum lysozyme in the phylogenetic tree of i-type bivalve lysozymes (Fig. 3) albeit the nearly 70% amino acid sequence identify between the two proteins and the close phylogenetic relationship between the two bivalve species. Studies using PCR or biochemical techniques to identify if M. meretrix has multiple lysozymes will help to justify this hypothesis. Lysozymes are antimicrobial proteins playing multiple roles in animals. Studies of these proteins have generated a great deal of

Fig. 8. A: The comparison of relative transcript level of MmeLys of different tissues between PBS-control clams and V. parahaemolyticus-challenge clams. Data was expressed as the ratio of MmeLys cDNA to the b-actin cDNA; B: The comparison of relative protein level of MmeLys of different tissues between PBS-control clams and V. parahaemolyticus-challenge clams. The concentration of total protein was determined using Bradford assay and adjusted to identical for the different tissues. Values were shown as mean  SD, which were calculated from the image using QuantityOne software (Bio-Rad, USA), n ¼ 3. Asterisks * indicated significant differences existed in expression level between V. parahaemolyticuschallenge and PBS-control at the level of 0.05 by one-way analysis of variance (one-way ANOVA).

X. Yue et al. / Fish & Shellfish Immunology 30 (2011) 550e558

knowledge in biochemistry, immunology and molecular evolution. In this study, we cloned and characterized an i-type lysozyme MmeLys, which likely functions in M. meretrix host immunity against bacterial infections. Further studies on its function will allow us to gain more insight about the role of this lysozyme superfamily played in bivalve immunity and could lead to the development of disease resistance markers for selective breeding of clam M. meretrix. Acknowledgements This work was financially supported by the Chinese National High-Tech R & D Program (2006AA10A410), the National Basic Research Program of China (2010CB126403) and the National Science & Technology Pillar Program (2006BAD09A02). References [1] Jollès P, Jollès J. What’s new in lysozyme research. Mol Cell Biochem 1984; 63:165e89. [2] Salton MRJ. The properties of lysozyme and its action on microorganisms. Bacteriol Rev 1957;21:82e99. [3] Jollès P. A chapter in molecular biology. Angew Chem 1969;8:227e94. [4] Chipman DM, Sharon N. Mechanism of lysozyme. Science 1969;165:454e65. [5] Callewaert L, Michiels CW. Lysozymes in the animal kingdom. J Biosci 2010;35:127e60. [6] Masschalck B, Michiels CW. Antimicrobial properties of lysozyme in relation to foodborne vegetative bacteria. Crit Rev Microbiol 2003;29:191e214. [7] During K, Porsch P, Mahn A, Brinkmann O, Gieffers W. The non-enzymatic microbicidal activity of lysozymes. FEBS Lett 1999;449:93e100. [8] Patrzykat A, Zhang L, Mendoza V, Iwama GK, Hancock RE. Synergy of histonederived peptides of coho salmon with lysozyme and flounder pleurocidin. Antimicrobial Agents Chemother 2001;45:1337e42. [9] Yan H, Hancock RE. Synergistic interactions between mammalian antimicrobial defense peptides. Antimicrobial Agents Chemother 2001;45:1558e60. [10] Lee-Huang S, Maiorov N, Huang PL, Lee HC, Chang Y, Kallenhach N, et al. Structural and functional modeling of human lysozyme reveals a unique nanopeptide, HL9, with anti-HIV activity. Biochemistry 2005;44:4648e55. [11] Baskova IP, Zavalova LL. Polyfunctionality of lysozyme destabilase from the medicinal leech. Russ J Bioorg Chem 2008;34:337e43. [12] Sava G, Ceschia V, Pacor S. Mechanism of the antineoplastic action of lysozyme: evidence for host mediated effects. Anticancer Res 1989;9:1175e80. [13] Park JW, Kim CH, Kim JH, Je BR, Roh KB, Kim SJ, et al. Clustering of peptidoglycan recognition protein-SA is required for sensing lysine-type peptidoglycan in insects. Proc Natl Acad Sci USA 2007;104:6602e7. [14] McHenery JG, Birkbeck TH. Lysozyme of the mussel, Mytilus edulis (L.). Mar Biol Lett 1979;1:111e9. [15] Dobson DE, Prager EM, Wilson AC. Stomach lysozymes of ruminants. I. Distribution and catalytic properties. J Biol Chem 1984;259:11607e16. [16] Lemos FJA, Ribeiro AF, Terra WR. A bacteria-digesting midgut-lysozyme from Musca domestica (diptera) larvae. Purification, properties and secretory mechanism. Insect Biochem Mol Biol 1993;23:533e41. [17] Kornegay JR, Schilling JW, Wilson AC. Molecular adaptation of a leaf-eating bird: stomach lysozyme of the hoatzin. Mol Biol Evol 1994;11:921e8. [18] Xue QG, Itoh N, Schey KL. A new lysozyme from the eastern oyster (Crassostrea virginica) indicates adaptive evolution of i-type lysozymes. Cell Mol Life Sci 2007;64:82e95. [19] Jollès J, Jollès P. The lysozyme from Asterias rubens. Eur J Biochem 1975; 54:19e23. [20] Jollès J, Fiala-Médioni A, Jollès P. The ruminant digestive model using bacteria already employed early in evolution by symbiotic mollusks. J Mol Evol 1996;43:525e7. [21] Ito Y, Yoshikawa A, Hotani T, Fukuda S, Sugimura K, Imoto T. Amino acid sequences of lysozymes newly purified from invertebrates imply wide distribution of a novel class in the lysozyme family. Eur J Biochem 1999;259:456e61. [22] Nilsen IW, Overbo K, Sandsdalen E, Sandaker E, Sletten K, Bjornar M. Protein purification and gene isolation of chlamysin, a cold active lysozyme-like enzyme with antibacterial activity. FEBS Lett 1999;464:153e8. [23] Nilsen IW, Myrnes B. The gene of chlamysin, a marine invertebrate-type lysozyme, is organized similar to vertebrate but different from invertebrate chicken-type lysozyme genes. Gene 2001;269:27e32. [24] Zavalova LL, Baskova IP, Lukyanov SA, Sass AV, Snezhkov EV, Akopov SA, et al. Destabilase from the medicinal leech is a representative of a novel family of lysozymes. Biochim Biophys Acta 2000;1478:69e77.  [25] Josková R, Silerová M, Procházková P, Bilej M. Identification and cloning of an invertebrate-type lysozyme from Eisenia andrei. Dev Comp Immunol 2009;33:932e8.

557

[26] Xue QG, Schey KL, Volety AK, Chu FL, La Peyre JF. Purification and characterization of lysozyme from plasma of the eastern oyster (Crassostrea virginica). Comp Biochem Physiol B Biochem Mol Biol 2004;139:11e25. [27] Xue QG, Hellberg ME, Schey KL, Itoh N, Eytan RI, Cooper RK, et al. A new lysozyme from the eastern oyster, Crassostrea virginica, and a possible evolutionary pathway for i-type lysozymes in bivalves from host defense to digestion. BMC Evol Biol 2010;10:213. [28] Itoh N, Xue QG, Li Y, Cooper RK, La-Peyre JF. cDNA cloning and tissue expression of plasma lysozyme in the estern oyster, Crassostrea virginica. Fish Shellfish Immunol 2007;23:957e68. [29] Olsen QM, Nilsen IW, Sletten K, Myrnes B. Multiple invertebrate lysozymes in blue mussel (Mytilus edulis). Comp Biochem Physiol B 2003;136:107e15. [30] Hermes-Lima M, Storey KB. Antioxidant defenses and metabolic depression in a pulmonate land snail. Am J Physiol Regul Integr Comp Physiol 1995; 268:1386e93. [31] Cong L, Yang X, Wang X, Tada M, Lu M, Liu H, et al. Characterization of an i-type lysozyme gene from the sea cucumber Stichopus japonicus, and enzymatic and nonenzymatic antimicrobial activities of its recombinant protein. J Biosci Bioeng 2009;107:583e8. [32] Li H, Parisi M, Toubiana M, Cammarata M, Roch P. Lysozyme gene expression and hemocyte behaviour in the Mediterranean mussel, Mytilus galloprovincialis, after injection of various bacteria or temperature stresses. Fish Shellfish Immunol 2008;25:143e52. [33] Zhao J, Qiu L, Ning X, Chen A, Wu H, Li C. Cloning and characterization of an invertebrate type lysozyme from Venerupis philippinarum. Comp Biochem Physiol B Biochem Mol Biol 2010;156:56e60. [34] Zobel CE, Felthons CB. Bacteria as food for certain marine invertebrates. J Mar Res 1938;1:312e27. [35] McHenery JG, Birkbeck TH. Uptake and processing of cultured microorganisms by bivalves. J Exp Mar Biol Ecol 1985;90:145e63. [36] Wang XM, Liu BZ, Wang GD, Tang BJ, Xiang JH. Molecular cloning and functional analysis of cathepsin B in nutrient metabolism during larval development in Meretrix meretrix. Aquaculture 2008;282:41e6. [37] Yue X, Liu BZ, Xiang JH, Jia JT. Identification and characterization of the pathogenic effect of a Vibrio parahaemolyticus-related bacterium isolated from clam Meretrix meretrix with mass mortality. J Invertebr Pathol 2010;103:109e15. [38] Bendtsen JD, Nielsen H, von Heijne G, Brunak S. Improved prediction of signal peptides:SignalP 3.0. J Mol Biol 2004;340:783e95. [39] Gasteiger E, Hoogland C, Gattiker A, Duvaud S, Wilkins MR, Appel RD, et al. Protein identification and analysis tools on the ExPASy server. In: Walker John M, editor. The proteomics protocols handbook. Humana Press; 2005 . p. 571e607. [40] Thompson JD, Gibsonl TJ, Plewniak F, Jeanmougin F, Desmon GH. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 1997;25:4876e82. [41] Kumar S, Tamura K, Nei M. MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinformatics 2004;5:150e63. [42] Jiang H, Li FH, Xie YS, Huang BX, Zhang JK, Zhang JQ, et al. Comparative proteomic profiles of the hepatopancreas in Fenneropenaeus chinensis response to hypoxic stress. Proteomics 2009;9:3353e67. [43] Shugar D. The measurement of lysozyme activity and the ultra-violet inactivation of interferon. Biochem Biophys Acta 1952;8:302e9. [44] Jollès P, Charlemagne D, Petit JF, Maire AC, Jollès J. Biochimie comparée des lysozymes. Bull Soc Chim Biol 1965;47:2241e57. [45] Lie O, Syed M, Solbu H. Improved agar plate assays of bovine lysozyme and haemolytic complement activity. Acta Vet Scand 1986;27:23e32. [46] Minagawa S, Hikima J, Hirono I, Aoki T. Expression of Japanese flounder cDNA in insect cells. Dev Comp Immunol 2001;25:439e45. [47] Takeshita K, Hashimoto Y, Ueda T, Imoto T. A small chimerically bifunctional monomeric protein: Tapes japonicus lysozyme. Cell Mol Life Sci 2003;60: 1944e51. [48] Goto T, Yoshito A, Kakuta Y, Takeshita K, Imoto T, Ueda T. Crystal structure of tapes japonica lysozyme with substrate analogue: structural basis of the catalytic mechanism and manifestation of its chitinase activity accompanied by quaternary structural change. J Biol Chem 2007;282:27459e67. [49] Zavalova LL, Lukyanov S, Baskova I, Snezhkov E, Akopov S, Berezhnoy S, et al. Genes from the medicinal leech (Hirudo medicinalis) coding for unusual enzymes that specifically cleave endoepsilon (gamma-Glu)-Lys isopeptide bonds and help to dissolve blood clots. Mol Gen Genet 1996;253:20e5. [50] Moreira-Ferro CK, Daffre S, James AA, Marinotti O. A lysozyme in the salivary glands of the malaria vector Anopheles darlingi. Insect Mol Biol 1998;7:257e64. [51] Zhao JM, Song LS, Li CH, Zou H, Ni DJ, Wang W, et al. Molecular cloning of an invertebrate goose-type lysozyme gene from Chlamys farreri, and lytic activity of the recombinant protein. Mol Immunol 2007;107:583e8. [52] Mai W, Hu C. cDNA cloning, expression and antibacterial activity of lysozyme C in the blue shrimp (Litopenaeus stylirostris). Prog Nat Sci 2009;19:837e44. [53] Matsumoto T, Nakamura AM, Takahashi KG. Cloning of cDNAs and hybridization analysis of lysozymes from two oyster species, Crassostrea gigas and Ostrea edulis. Comp Biochem Physiol 2006;B 145:325e30. [54] Itoh N, Takahashi KG. cDNA cloning and in situ hybridization of a novel lysozyme in the Pacific oyster, Crassostrea gigas. Comp Biochem Physiol 2007;B 148:160e6. [55] Paillard C, Roux FL, Borrego JJ. Bacterial disease in marine bivalves, a review of recent studies: trends and evolution. Aquat Living Resour 2004;17:477e98.

558

X. Yue et al. / Fish & Shellfish Immunology 30 (2011) 550e558

[56] Beaz-Hidalgo R, Balboa S, Romalde JL, Figueras MJ. Diversity and pathogenecity of Vibrio species in cultured bivalve mollusks. Environ Microbiol Rep 2010;2:34e43. [57] Mai W, Hu C. Molecular cloning, characterization, expression and antibacterial analysis of a lysozyme homologue from Fenneropenaeus. Mol Biol Rep 2009;36:1587e95.

[58] Soldatov AA, Gostyukhina OL, Golovina IV. Antioxidant enzyme complex of tissues of the bivalve Mytilus galloprovincialis Lam. Under normal and oxidative-stress conditions: a review. Appl Biochem Microbiol 2007;43:556e62. [59] Zhang H, Wang H, Wang L, Song L, Song X, Zhao J, et al. Cflec-4. a multidomain C-type lectin involved in immune defense of Zhikong scallop Chlamys farreri. Dev Comp Immunol 2009;33:780e8.