Molecular cloning and characterization of a short type peptidoglycan recognition protein (CfPGRP-S1) cDNA from Zhikong scallop Chlamys farreri

Fish & Shellfish Immunology 23 (2007) 646e656 www.elsevier.com/locate/fsi

Molecular cloning and characterization of a short type peptidoglycan recognition protein (CfPGRP-S1) cDNA from Zhikong scallop Chlamys farreri Jianguo Su a,b,c, Duojiao Ni a,c, Linsheng Song a,*, Jianmin Zhao a, Limei Qiu a a

Institute of Oceanology, Chinese Academy of Sciences, 7 Nanhai Road, Qingdao 266071, PR China b Northwest A&F University, Yangling 712100, PR China c Graduate School, Chinese Academy of Sciences, Beijing 100039, PR China Received 6 October 2006; revised 19 January 2007; accepted 22 January 2007 Available online 20 February 2007

Abstract Peptidoglycan recognition protein (PGRP) specifically binds to peptidoglycan and plays a crucial role in the innate immune responses as a pattern recognition receptor (PRR). The cDNA of a short type PGRP was cloned from scallop Chlamys farreri (named CfPGRP-S1) by homology cloning with degenerate primers, and confirmed by virtual Northern blots. The full length of CfPGRP-S1 cDNA was 1073 bp in length, including a 50 untranslated region (UTR) of 59 bp, a 30 UTR of 255 bp, and an open reading frame (ORF) of 759 bp encoding a polypeptide of 252 amino acids with an estimated molecular mass of 27.88 kDa and a predicted isoelectric point of 8.69. BLAST analysis revealed that CfPGRP-S1 shared high identities with other known PGRPs. A conserved PGRP domain and three zinc-binding sites were present at its C-terminus. The temporal expression of CfPGRP-S1 gene in healthy, Vibrio anguillarum-challenged and Micrococcus lysodeikticus-challenged scallops was measured by RT-PCR analysis. The expression of CfPGRP-S1 was upregulated initially in the first 12 h or 24 h either by M. lysodeikticus or V. anguillarum challenge and reached the maximum level at 24 h or 36 h, then dropped progressively, and recovered to the original level as the stimulation decreased at 72 h. There was no significant difference between V. anguillarum and M. lysodeikticus challenge. The results indicated that the CfPGRP-S1 was a constitutive and inducible acute-phase protein which was involved in the immune response against bacterial infection. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Chlamys farreri Peptidoglycan recognition protein-S1 (PGRP-S1); Pattern recognition receptors (PRRs); Gene cloning; mRNA expression

1. Introduction Invertebrates lack a true adaptive immune system and rely on various innate immune responses against invading pathogens [1,2]. A prominent feature of the innate immune system is the rapid and massive response to invading microorganisms. The innate immune system develops effective strategies, termed pattern recognition to discriminate * Corresponding author. Tel.: þ86 532 82898552; fax: þ86 532 82880645. E-mail address: [email protected] (L. Song). 1050-4648/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2007.01.023

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nonself invading pathogens from self-tissues. The innate pattern recognition recognizes the invading pathogens through a limited number of germ-line-encoded receptors, termed pattern recognition receptors (PRRs) or pattern recognition proteins (PRPs) which are highly conserved in evolution, such as lipopolysaccharide and beta-1,3-glucan binding protein (LGBP), peptidoglycan recognition protein (PGRP). The major targets of innate immune recognition are pathogen-associated molecular patterns (PAMPs) of microorganisms that are structurally distinct from anything produced by the host. The PAMPs include parts of the microorganism cell envelope, such as lipopolysaccharide (LPS), peptidoglycan (PGN), and beta-1,3-glucan (bG). PGRPs are a type of PRPs that recognize PGN which is the essential unique cell wall component of virtually all bacteria, but not present in eukaryotic cells [3,4]. PGN is an excellent target for recognition by the eukaryotic innate immune system, and can induce strong antibacterial responses in insects [5], and activates monocytes, macrophages, and B lymphocytes in mammals [6,7]. The first PGPR was characterized from haemolymph and cuticle of insect Bombyx mori in 1996 [8]. Subsequently, PGRP homologues have been identified in many species especially in insects and mammals. For example, insects have multiple PGRP genes that are classified into short (S) and long (L) transcripts and are often alternatively spliced into up to 19 different proteins [1e5]. There are seven short PGRPs (SA, SB1, SB2, SC1a, SC1b, SC2, and SD) and 10 long PGRPs in Drosophila. All Drosophila PGRP-S have short untranslated 50 regions, code for short peptides (w20 kDa) and have an N-terminus secretion signal peptide, suggesting they are extracellular proteins. On the other hand, PGRP-L have long untranslated 50 regions and code for longer peptides (30e90 kDa), some of which have predicted transmembrane domains, suggesting they are intracellular or membrane bound [9]. The short forms are present in the hemolymph, cuticle, and fat body cells, and sometimes in epidermal cells in the gut and hemocytes, whereas the long forms are mainly expressed in hemocytes [10]. Mammals have four PGRPs, PGLYRP1 is expressed primarily in polymorphonuclear leukocyte granules; PGLYRP2 is secreted from the liver into the blood and is also induced by bacteria in epithelial cells; PGLYRP3 and PGLYRP4 are expressed in the skin, eyes, salivary glands, throat, tongue, esophagus, stomach, and intestine [10,11]. The knowledge about the functions of different PGRPs is mainly from insects and mammals. PGRPs are well known to play central and diverse roles in activating insect immune reactions. The expression of insect PGRPs is often upregulated by exposure to bacteria. Insect PGRPs activate the Toll or IMD signal transduction pathways or induce proteolytic cascades that generate antimicrobial products, induce phagocytosis, hydrolyze peptidoglycan, and protect insects against infections [10]. Meanwhile, insect PGRPs have also been found to have other functions. For example, Drosophila PGRP-LB controls systemic immune responses as well as homeostasis at the barrier surfaces [12]. Drosophila PGRP-SC1/2 act in the larval gut to prevent activation of immune deficiency (IMD) pathway following bacterial ingestion to prevent bacteria induced developmental defects and larval death [13]. Mammalian PGRPs also play important roles in immune defense. PGLYRP2 is an N-acetylmuramoyl-L-alanine amidase that hydrolyzes bacterial peptidoglycan and reduces its proinflammatory activity. PGLYRP3 and PGLYRP4 are a new class of bactericidal and bacteriostatic proteins that have different structures, mechanism of actions, and expression patterns than antimicrobial peptides. These proteins kill bacteria by interacting with cell wall peptidoglycan, rather than permeabilizing bacterial membranes as other antibacterial peptides do [10,11]. In our previous study, the full-length cDNA of a short type PGRP (AiPGRP) has been cloned from scallop Argopecten irradians and its mRNA expression was examined after the stimulation of PGN and LPS in vitro [14]. In the present study, another short type PGRP (CfPGRP-S1) was cloned from another scallop Chlamys farreri by homology cloning. Its temporal expression after Micrococcus lysodeikticus and Vibrio anguillarum challenge in vivo was examined in order to evaluate the roles of CfPGRP-S1 in the immune process against different bacteria.

2. Materials and methods 2.1. Scallops The scallops C. farreri (weighing about 25  2 g) were purchased from Qingdao, Shandong Province, China, and maintained in aerated seawater at 15e18  C for a week before processing.

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2.2. cDNA library construction and cloning the full-length cDNA of CfPGRP-S1 A cDNA library was constructed from the whole tissues of the scallop C. farreri challenged by V. anguillarum at 36 h after injection, using the ZAP-cDNA synthesis kit and ZAP-cDNA GigapackIII Gold cloning kit (Stratagene, USA). Two forward degenerate PCR primers SCF4 and SCF5 (Table 1) were designed based on the conserved region of different PGRPs to clone the corresponding fragment from scallop. The first round PCR reaction to get 30 -end was performed by using SCF4 and universal primer M13F with the cDNA library mixture as template. The PCR program was 94  C for 5 min, followed by 40 cycles of 94  C for 30 s, 55  C for 30 s, 72  C for 60 s and the final extension step at 72  C for 10 min. Using 1 ml of 1:10 dilution of the first round PCR product as nested PCR template, the 30 -end nested PCR reaction was performed under the same conditions except using SCF5 and universal primer T7. The generated PCR products were gel-purified and cloned into the pMD-18T vector (Takara, Japan). After transforming into the competent cells of Escherichia coli JM-109, the recombinants were identified through blue-white color selection in ampicillin-containing LB plates. Positive clones were sequenced in both directions. To get the 50 -end of the CfPGRP-S1 cDNA, a specific primer SCR7 was designed according to the sequence resulted from homology cloning. PCR was performed using cDNA library mixture as the template with the primer set of SCF7 and universal primer M13R. The PCR program was 94  C for 5 min, followed by 35 cycles of 94  C for 30 s, 58  C for 30 s, 72  C for 60 s and the final extension step at 72  C for 10 min. All the resulting sequences were verified and subjected to cluster analysis. 2.3. Sequence analysis The sequence was searched for similarity using BLAST program at web servers of NCBI (http://www.ncbi.nlm.nih. gov/BLAST/). The CfPGRP-S1 deduced amino acid sequence was analyzed with the Expert Protein Analysis System (http://www.expasy.org/). Multiple alignment of the CfPGRP-S1 with other PGRPs was performed with the ClustalW Multiple Alignment Program (http://www.ebi.ac.uk/clustalw) and Multiple Alignment show (http://www.bio-soft.net/ sms/index.html). Phylogenetic tree was constructed by a CLUSTALW alignment and MEGA2 Neighbor-Joining. 2.4. Bacterial challenge The scallops were challenged by injecting 50 ml of V. anguillarum or M. lysodeikticus suspension (resuspended in filtered seawater, OD600 ¼ 0.4) into the adductor muscles. The scallops in the control group received an injection of 50 ml filtered seawater instead of bacteria. The untreated scallops were used as blank. Hemolymph from blank, control and challenged scallops was collected using a 5-ml syringe from the adductor muscle at 0, 2, 6, 12, 24, 36, 48, and 72 h post-injection, and centrifuged at 800 g at 4  C for 10 min to harvest the hemocytes. For the blank group, the tissues including hemolymph, gill, mantle, adductor muscle, gonad and hepatopancreas were collected to investigate the Table 1 The major relevant primers used in these experiments Name M13F T7 M13R SCF4 SCF5 SCR7 SCF9 SCR10 OligodTSalIA OligodTSalIB CapFinderB1 CapFinderB2 AC3F AC3R

Sequence 0

Length (nt) 0

5 -CGCCAGGGTTTTCCCAGTCACGAC-3 50 -GTAATACGACTCACTATAGGGC-30 50 -AGCGGATAACAATTTCACACAGG-30 50 -TTYMTBRTBGGHGGWRA-30 50 -GGBMAVRTDTAYGARG-30 50 -GCCGCCAAACTCTTGTCGTTA-30 50 -CGTGGATGTAACGACAAGAGT-30 50 -GCTGATCTTGTATTTTGATGGA-30 50 -CTGCGCCAGAATTGGCAGGTCGAC(T)25V-30 50 -CTGCGCCAGAATTGGCAGGTCGAC-30 50 -GAGAGAACGCGTGACGAGAGACTGACAGGGGGGGGH-30 50 -GAGAGAACGCGTGACGAGAGACTGACAG-30 50 -GGAGAAGATGACACAGATCATG-30 50 -GCCAGACTCGTCGTATTCCT-30

Note: R ¼ AG; Y ¼ CT; M ¼ AC; K ¼ GT; S ¼ GC; W ¼AT; B ¼ GCT; H ¼ ACT; V ¼AGC; and D ¼ ATG.

24 22 23 17 16 21 21 22 50 24 36 28 22 20

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tissue-specific expression of CfPGRP-S1. Total RNA was extracted from the samples using Trizol reagent (Invitrogen, USA) according to the manufacturer’s protocol. First strand cDNA was synthesized using oligodT primers (Invitrogen, USA). Reverse transcription was performed by the M-MLV reverse transcriptase (Promega, USA) with DNase I (Promega, USA) treated total RNA as template according to the manufacturer’s instructions. There were three replicates for each group. 2.5. Virtual Northern blot analysis The transcript of CfPGRP-S1 was detected by virtual Northern blots. Total RNAs were extracted from hemocytes of scallops challenged with M. lysodeikticus at 24 h post-injection and unchallenged scallops, and reversely transcribed into cDNA by using an anchored oligodT primer (OligodTSalIA) and CF primer (CapFinderB1) (Table 1) at elevated temperatures (48  C) for 1 h. The PCR reaction was performed in a PTC-100 Programmable Thermal Controller Cycler (Biorad, USA), using OligodTSalIB and CapFinderB2 (Table 1) as primers in a 25-ml reaction volume containing 1 U TaKaRa Ex Taq, 200 mM dNTPs, 2.5 mM MgCl2 and 20 pmol of each primer and 1 ml of reverse transcribed cDNA products. The LA-PCR program was 95  C, 1 min; 60  C, 1 min; 68  C, 12 min; seven cycles of 95  C, 30 s; 60  C, 30 s; 68  C, 12 min; seven additional cycles of 95  C, 30 s; 60  C, 30 s; 68  C, 14 min; and seven additional cycles of 95  C, 30 s; 60  C, 30 s; 68  C, 16 min. The PCR product (2e5 mg of amplified cDNA) was separated on a 0.7% agarose gel, denatured and subsequently blotted onto a nylon membrane in a conventional Southern transfer (downward with 10 standard SSC for 1e2 h) and UV-crosslinked. The probe was labeled with Digoxin by PCR amplification with primers SCF9 and SCR10 (Table 1). Prehybridization, hybridization, washing and detection were performed according to standard protocols [15,16]. 2.6. Quantification analysis of CfPGRP-S1 gene expression The mRNA expression levels of CfPGRP-S1 were measured by semi-quantitative RT-PCR. Two CfPGRP-S1 genespecific primers SCF9 and SCR10 (Table 1) were used to amplify a product of 345 bp. Two scallop b-actin primers AC3F and AC3R were used to amplify a 725-bp fragment as an internal control to verify the successful transcription and to calibrate the cDNA template for corresponding samples. Semi-quantitative RT-PCR was carried out in a PTC-100 Programmable Thermal Controller Cycler (Biorad, USA) in a 20 ml reaction volume containing 2 ml of 10 PCR buffer, 1.2 ml of MgCl2 (25 mmol L1), 1.6 ml of dNTP (2.5 mmol L1), 1 ml of each primer (10 pmol ml1), 14 ml of PCR-grade water, 0.2 ml (1 U) of Taq polymerase (Promega, USA), and 1 ml of cDNA mix. The PCR temperature profile for CfPGRP-S1 was 94  C for 3 min followed by 30 cycles of 94  C for 30 s, 58  C for 30 s, 72  C for 1 min. After the final cycle, samples were incubated for a further 10 min at 72  C then held at 4  C prior to analysis. The PCR temperature profile for actin was 94  C for 3 min

Fig. 1. Detection of PGRP-S1 mRNA by Virtual Northern Blots. Lane 1 contains 1 mg of total RNA from hemocytes challenged by M. lysoleikticus at 24 h post-injection, and Lane 2 contains 1 mg of total RNA from unchallenged hemocytes. The Marker indicates a 1.1 kb transcript in the hemocytes.

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followed by 22 cycles of 94  C for 30 s, 58  C for 30 s, 72  C for 1 min and then an additional extension at 72  C for 10 min. The PCR products were analyzed by electrophoresis on a 1.5% agarose gel containing ethidium bromide (EtBr). The electrophoresis photograph was analyzed with Band Leader Application Version 3.0. The validity of the results was assessed by an unpaired, two-tailed Student’s t-test to compare the CfPGRP-S1 gene expression levels in various tissues, in the healthy, control, and challenged scallops. 3. Results 3.1. cDNA cloning and sequencing of the CfPGRP-S1 gene The PCR product amplified by the degenerate primers was about 551 bp, and its nucleotide sequence was significantly homologous to other known PGRPs, especially to house mouse PRGP-L (E ¼ 6e19). Stop codon TAA,

Fig. 2. Nucleotide and predicted amino acid sequence of CfPGRP-S1. The two termination codons (taa) before start codon are under double line. The start codon (ATG) is boxed and the stop codon (TAA) is marked with an asterisk. The putative polyadenylation signal sequence (aataaa) is under bold line. The signal peptide is under dot line (1e22). PGRP domain is indicated as shaded residues (83e225). Ami_2 domain is indicated under wave line (100e231). Zn2þ binding sites (H112, H221, C229) are indicated in bold. The cysteines (C119, C125) forming disulfide bond are marked like C*.

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canonical polyadenylation signal sequence AATAAA and a poly(A) tail were identified in this fragment. CfPGRP-S1specific primer SCR7 was designed based on the sequence of the above 551 bp fragment, and used for the 50 -end of CfPGRP-S1 cloning. A fragment of 598 bp was amplified from the 50 -end of CfPGRP-S1 cDNA by using the primers of SCR7 and M13R. A 1073-bp nucleotide sequence representing the complete cDNA sequence of CfPGRP-S1 gene was obtained by cluster analysis of the above two fragments. Virtual Northern blot was used to confirm the length of this gene, and a single transcript of the approximate 1.1 kb was detected (Fig. 1). The nucleotide sequence and the deduced amino acid sequence of CfPGRP-S1 is shown in Fig. 2. The full-length cDNA sequence was of 1073 bp, contained a 50 untranslated region (UTR) of 59 bp with two termination codons, followed by an open reading frame (ORF) of 759 bp, a 30 UTR of 255 bp including a canonical polyadenylation signal sequence (AATAAA) at the position of 1014 bp to 1019 bp, and a poly(A) tail. Searching for sequence homology of the CfPGRP-S1 with other known PGRP sequences by BLASTn revealed that it was closely matched with human Homo sapiens PGLYRP1 (accession no. AF076483; E ¼ 0.26). The sequence of the CfPGRP-S1 gene was deposited in GenBank under accession no. AY987008. 3.2. Analysis of the PGRP-S1 amino acid sequence The ORF of CfPGRP-S1 was of 759 bp encoding a polypeptide of 252 amino acids (aa) with an estimated molecular mass (MM) of 27.88 kDa and a predicted isoelectric point (PI) of 8.69. There were a signal peptide (1e22 aa), a PGRP domain (83e225 aa; E ¼ 1.34e64), and an overlapping Ami_2 domain (100e231 aa; E ¼ 1.79e14). Searching for sequence similarities of the CfPGRP-S1 with known proteins by BLASTp revealed that it was closely matched with Euprymna scolopes peptidoglycan recognition protein 2 precursor (accession no. AAY27973; E ¼ 2e45, identities ¼ 45%, positives ¼ 67%, Gap ¼ 0%). Three Zn2þ binding sites (H112, H221, and C229) required for amidase activity were identified in CfPGRP-S1, which were corresponding to the Zn2þ binding sites (H42, H152, and C160) in Drosophila PGRP-LB [17] (Fig. 3). Another amino acid residue T227 in CfPGRP-S1 was predicted to corresponding to T158 in Drosophila PGRP-LB which was essential for amidase activity [17] (Fig. 3). There was a signal peptide but no transmembrane domain in CfPGRP-S1, it should belong to short PGRP based on its structure [18]. Based on overall amino acid sequences of C. farreri, A. irradians, D. melanogaster and H. sapiens PGRPs, a phylogenetic tree was constructed (Fig. 4). The results showed that CfPGRP-S1 was most similar to H. sapiens PGLYRP2, not AiPGRP or insect PGRPs. 3.3. The expression of CfPGRP-S1 Semi-quantitative RT-PCR was used to examine the expression of CfPGRP-S1 in different tissues in blank group (Fig. 5A) and the time-dependent expression pattern of CfPGRP-S1 in hemocytes of scallops challenged by V. anguillarum or M. lysodeikticus at 0, 2, 6, 12, 24, 36, 48, and 72 h after injection (Fig. 5B). The mRNA transcripts of CfPGRP-S1 could be detected in all tissues examined including hemolymph, gill, mantle, adductor muscle, gonad and hepatopancreas with no significant difference. After challenge by V. anguillarum, the expression of CfPGRP-S1 remained at a low level, and there was no significant difference (P > 0.05) among blank (data not shown), control and challenged groups in the first 12 h post-infection. It was upregulated at 24 h and this trend lasted until 36 h (P < 0.05), then decreased at 48 h (P < 0.05), and returned to normal levels at 72 h (P > 0.05). In the M. lysodeikticus-challenged group, there was no significant difference (P > 0.05) of CfPGRP-S1 expression in the first 6 h postinjection; the expression increased significantly at 12 h (P < 0.05), reached the highest at 24 h (P < 0.05), then decreased and dropped to the original level (P > 0.05) at 72 h. In the control and blank (data not shown) group, there was no significant difference among all the time points (P > 0.05). The mRNA expression profiles of CfPGRP-S1 stimulated by V. anguillarum and M. lysodeikticus were not significantly different (P > 0.05). 4. Discussion In the present study, the full-length cDNA of CfPGRP-S1 was cloned from scallop C. farreri and verified by virtual Northern blots. The full-length cDNA of CfPGRP-S1 was of 1073 bp, contained an ORF (nucleotides 60e818) encoding a protein of 252 aa with an estimated MM of 27.88 kDa and a predicted PI of 8.69 (alkaline). There was

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Fig. 3. Alignment of C-terminal amino acid sequences containing conserved PGRP domains in C. farreri, D. melanogaster, and H. sapiens. Amino acid sequences are deduced from cDNA. Residues highlighted in black are identical and those in dark gray are similar. Percentage of sequences must agree for identity or similarity coloring to be added 60%. The numbers to the left indicate the amino acid position of start in the corresponding species. The right numbers represent the amino acid position in the corresponding species. E-value was determined by software. Sequence identities and similarities between the CfPGRP-S1 and homology were determined using the GAP alignment tool from seqweb. PGRP

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C.f.PGRP-S1

51 67

H.s.PGLYRP2 A.i.PGRP

16

D.m.PGRP-SA

85 63

D.m.PGRP-LE D.m.PGRP-LB D.M.PGRP-SD H.s.PGLYRP1

73 62

D.m.PGRP-SC2

83

D.m.PGRP-SC1A

99

99 D.m.PGRP-SC1B

47

D.m.PGRP-SB1 97

D.m.PGRP-SB2

22

H.s.PGLYRP3 99

H.s.PGLYRP4 D.m.PGRP-LCY

49

D.m.PGRP-LCX

98 41

99 D.m.PGRP-LCA

D.m.PGRP-LD D.m.PGRP-LAB 99 D.m.PGRP-LAC

D.m.PGRP-LF D.m.PGRP-LAA

0.2

Fig. 4. Phylogenetic tree analysis of CfPGRP-S1 with AiPGRP and all known PGRPs from D. melanogaster and H. sapiens. Phylogenetic tree was obtained from a CLUSTALW alignment and MEGA2 Neighbor-Joining of 23 sequences. The bar indicated the distance. The accession number of AiPGRP is AAR92030. Other abbreviations and accession numbers are the same as in Fig. 3.

a putative 22 aa signal peptide in the deduced amino acid sequence of CfPGRP-S1, but no predicted transmembrane domain was found in the mature protein sequence, indicating that CfPGRP-S1 should belong to short PGRP family though it was bigger than other short PGRPs. Comparison of CfPGRP-S1 with another scallop PGRP, AiPGRP, indicated that these two PGRPs are both S-type PGRPs, but they only share about 33% identity at the protein level. Meanwhile, the full-length cDNA of AiPGRP was of 1018 bp, which was shorter than that of CfPGRP-S1. The ORF of AiPGRP was 618 bp in length encoding a protein of 205 aa residues with MM of 22.95 kDa and a PI of 6.54 (acidic). In the phylogenetic tree (Fig. 4), CfPGRP-S1 was clustered with H.s.PGLYRP2 firstly and then grouped with AiPGRP. The result demonstrated that CfPGRP-S1 had a nearer relation with H.s.PGLYRP2 than AiPGRP. Alignment analysis of the deduced amino acid sequence of CfPGRP-S1 with other PGRPs (Fig. 3) revealed that the C-terminal regions of all the PGRPs were conserved, high identities (23e46%) or similarities (46e63%) in the PGRP domains. Three PGRP domains (I, II, and III) [19] were identified in the C-terminal region of CfPGRP-S1. The conserved C-terminal regions of all the PGRPs were homologous to bacteriophage T7 lysozyme (w18%

domains I, II, and III were underlined with their names. The Zn2þ binding sites (H112, H221, C229) were marked with solid triangles (;), and the conserved cysteines (C119, C125) to form disulfide bond were marked with asterisks (*). The aligned sequences are as follows: C.f.PGRP-S1, C. farreri PGRP-S1 (AAY53765); H.s.PGLYRP2, H. sapiens PGLYRP2 (Q96PD5); D.m.PGRP-LB, D. melanogaster PGRP-LB (AAG23731); D.m.PGRP-SB1, D. melanogaster PGRP-SB1 (CAD89138); D.m.PGRP-SB2, D. melanogaster PGRP-SB2 (CAD89150); D.m.PGRP-SA, D. melanogaster PGRP-SA (CAD89125); D.m.PGRP-LE, D. melanogaster PGRP-LE (AAG32064); D.m.PGRP-SC1a, D. melanogaster PGRP-SC1a (CAD89163); D.m.PGRP-SC1b, D. melanogaster PGRP-SC1b (CAD89174); D.m.PGRP-SC2, D. melanogaster PGRP-SC2 (CAD89187); D.m.PGRP-LF, D. melanogaster PGRP-LF (AAF50301); H.s.PGLYRP3, H. sapiens PGLYRP3 (Q96LB9); H.s.PGLYRP4, H. sapiens PGLYRP4 (Q96LB8); H.s.PGLYRP1, H. sapiens PGLYRP1 (O75594); D.m.PGRP-SD, D. melanogaster PGRP-SD (CAD89198); D.m.PGRP-Lcx, D. melanogaster PGRP-Lcx (AAM18530); D.m.PGRP-Lca, D. melanogaster PGRP-Lca (AAF50302); D.m.PGRP-Lcy, D. melanogaster PGRP-Lcy (AAQ16306); D.m.PGRP-Lab, D. melanogaster PGRP-Lab (AAG23729); D.m.PGRP-Lac, D. melanogaster PGRP-Lac (AAG23730); D.m.PGRP-Laa, D. melanogaster PGRP-LAa (AAK00295); D.m.PGRP-LD, D. melanogaster PGRP-LD (AAG32062).

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A The expression level of CfPGRP-S1

0.65 0.64 0.63 0.62 0.61 0.60 0.59

pa nc re as

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na d

cl e

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he

pa

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0.58

B

The expression level of CfPGRP-S1

Tissues 0. 81 0. 79

control

0. 77

V.anguillarum

0. 75

M.lysoleikticus

0. 73 0. 71 0. 69 0. 67 0. 65 0. 63 0. 61 0. 59

0

2

6

12

24

36

48

72

Time (h) Fig. 5. Expression profile of CfPGRP-S1. Data plotted were mean  S.D. of three replicates. A: CfPGRP-S1 expression in different tissues in blank group; B: time-dependent expression pattern of CfPGRP-S1 in hemocytes.

identities, w33% similarities) which hydrolyzed the bonds between the N-acetylmuramic acids and the peptides of bacterial PGN. Several other amino acid residues in the C-terminal regions of PGRPs were also highly conserved. For example, there were one asparagine, one glycine, one isoleucine, one phenylalanine, and one proline identical in PGRP domains I and II; two cysteines, one arginine, one glutamine, and one histidine identical in PGRP domains II and III (Fig. 3). These residues were likely to be important for the tertiary structure, cellular location, or function of these PGRPs [19]. The remaining N-terminal portions of PGRP molecules were highly diversified and had no homology to each other. A conserved cysteine (C119, C125) pair engaged in the formation of the disulfide bond that was required for proper tertiary structure [17,20] was identified in the deduced amino acid sequence of CfPGRP-S1 (Fig. 3). These cysteines seemed to be essential for the structure and function of PGRPs. A mutation in one of these cysteines in Drosophila PGRP-SA (C80) corresponding to C125 in CfPGRP-S1 abolished the ability of PGRP-SA to activate the Toll pathway and induce protective responses against Gram-positive bacteria, but the immunity to Gram-negative bacteria and fungi was not affected [21]. Moreover, a mutation in one of these cysteines in human PGLYRP2 (C419) corresponding to C119 in CfPGRP-S1 abolished the amidase activity of PGLYRP2 [22]. Another cysteine (C168 in Drosophila PGRP-SC1B and the C530 in Homo PGLYRP2) which corresponds to C229 in C. farreri (Fig. 3) was also essential for the amidase activity of these PGRPs [22,23].

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In invertebrates, several families of PRPs have been identified in insects, including beta-GRPs, GNBPs, PGRPs, C-type lectins, hemolin, scavenger receptor, and complement-like proteins [24e26]. But very few PRRs were reported in mollusk. In the present study, we cloned the first PGRP in C. farreri by homology cloning. As a PRP, PGRPs are expressed in response to the corresponding PAMPs. Human PGRP family members express selectively in various organs and tissues [19]. Most of the insect PGRPs are expressed in immune organs, consistent with their role in insect immunity [27]. In C. farreri, CfPGRP-S1 was ubiquitously distributed in the tested tissues. The wide tissues distribution may contribute to the open circulatory system in scallop which is different from insects and mammals. The expression of several insect PGRPs is upregulated by exposure to bacteria or purified bacterial PGN [20,27e 30]. In the present study, CfPGRP-S1 was upregulated by bacteria (both Gram-negative and Gram-positive). For V. anguillarum challenge, the expression of CfPGRP-S1 increased significantly at 24 h and lasted to 48 h post-injection (P < 0.05). In M. lysodeikticus-challenged group, the expression of CfPGRP-S1 was upregulated significantly at 12 h and lasted to 48 h post-injection (P < 0.05). The expression of CfPGRP-S1 was induced more by M. lysodeikticus challenge than by V. anguillarum challenge, and the up-regulation of PGRP gene was later in V. anguillarumchallenged groups than that in M. lysodeikticus-challenged groups, but no significant difference of CfPGRP-S1 expression was observed between the two groups (P > 0.05). In our previous study, the PGRP in bay scallop (AiPGRP) was found to be upregulated only by the treatment with PGN (purified from Micrococcus luteus, Fluka, USA) in primary mixed cultured hemocytes. LPS (purified from Escherichia coli 026:B6, sigma, USA) did not induce the expression of AiPGRP. The up-regulation of CfPGRP-S1 resulted from V. anguillarum-challenge may due to the PGN in V. anguillarum cell wall. Acknowledgements The authors are grateful to Dr. Hongyue Dang, Mr. Hao Wang, Mr. Chenghua Li, Dr. Limei Qiu and other laboratory members for technical advice and helpful discussions. This research was supported by 973 National Key Fundamental Research Program (No. 2006CB101806) and 863 High Technology Project (No. 20060110A4013) from the Chinese Ministry of Science and Technology, and a grant (No. 30671597) from NSFC to Prof. Linsheng Song. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17]

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