Identification of a protein binding to the phagocytosis activating protein (PAP) in immunized black tiger shrimp

Aquaculture 271 (2007) 112 – 120 www.elsevier.com/locate/aqua-online

Identification of a protein binding to the phagocytosis activating protein (PAP) in immunized black tiger shrimp Wilaiwan Chotigeat ⁎, Passanee Deachamag, Amornrat Phongdara Center for Genomics and Bioinformatics Research, Prince of Songkla University, Hat Yai, Songkhla, 90112, Thailand Received 2 June 2006; received in revised form 18 March 2007; accepted 19 March 2007

Abstract The phagocytosis activating protein (PAP) gene was isolated from Penaeus monodon infected with the white spot syndrome virus (WSSV). The endocytosis pathway of GST–PAP (glutathione-S-transferase–PAP) that caused activation of phagocytosis of shrimp haemocytes was investigated. In this study, a yeast two-hybrid screening assay of a WSSV-infected P. monodon cDNA library, was performed using PAP as bait to test for the interaction between PAP and other proteins in WSSV-infected shrimp. An alpha-2-macroglobulin (α2M) was one of the proteins that interacted with PAP. A GST pull-down assay showed that GST–PAP was capable of co-precipitating α2M whereas GST itself was not. An intramuscular injection with inactivated WSSV caused a significant increase in the expression of both the PAP and α2M gene in the haemolymph. The highest expression of both genes was detected at 24 h post-injection and remained constant for 1 week. Moreover, the uptake of GST–PAP by shrimp haemocytes was higher in the presence of α2M than in its absence. The results indicated that α2M may facilitate the entry of GST–PAP into phagocytic cells and increase the survival rate of the shrimp after being infected with WSSV. © 2007 Elsevier B.V. All rights reserved. Keywords: Alpha-2-macroglobulin; Phagocytosis activating protein; Penaeus monodon; Real-time PCR; Ribosomal protein L26; White spot syndrome virus; Yeast two-hybrid

1. Introduction Outbreaks of infectious diseases are causing significant economic losses for the shrimp farming industry, particularly that caused by white spot syndrome virus (WSSV) in Penaeus monodon. It is well known that crustaceans possess innate immune response systems including cellular and humoral mechanisms, that will rapidly and efficiently recognize and destroy non-self materials (Lee and SÖderhäll, 2002). Therefore, the development of basic knowledge of shrimp immunity is necessary to help establish strategies for prophylaxis ⁎ Corresponding author. Tel.: +66 74 288383; fax: +66 74 288117. E-mail address: [email protected] (W. Chotigeat). 0044-8486/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2007.03.019

and control of shrimp diseases in aquaculture (Bachere, 2000). In our previous study, we isolated a phagocytosis activating protein (PAP) gene (GenBank accession no. AY680836) from the haemolymph of a WSSV-infected P. monodon (Deachamag et al., 2006). This gene is highly homologous to the ribosomal protein L26 (RPL26) from Penaeus japonicus (98.6%) (Watanabe, 1998) and similar to RPL26 from Mus musculus (63.1%). Although this gene is highly homologous to RPL26, it was named after its phagocytic activation activity in shrimp (Deachamag et al., 2006). The percentage of phagocytosis of haemocytes increased when the haemocytes were incubated with the glutathione-S-transferase–PAP (GST–PAP). Furthermore, an intramuscular injection of formalin-inactivated

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WSSV (white spot syndrome virus) produced a significantly increased expression (3-fold, p b 0.05) of the PAP gene in P. monodon 1 week post-injection (Deachamag et al., 2006). In addition, the RPL26 gene was activated in a mouse macrophage cell line when treated with silica, LPS and IFNγ (Segade et al., 1996). Generally, RPL26 is located at the ribosomal subunit interface of the 60S subunit inside the cell (Villrreal and Lee, 1998) and our previous study found that the phagocytic activity of haemocytes increased after incubation with GST–PAP (glutathione-S-transferase–PAP) (Deachamag et al., 2006). However, the pathway of the GST–PAP that was used to activate phagocytic cells of the shrimp, has not been clearly characterized. In present study, we have identified alpha-2-macroglobulin (α2M) as a PAP binding protein in the haemolymph of P. monodon by using the yeast twohybrid screening assay. A correlation between the expression of the PAP and α2M in the WSSV-infected shrimp was determined by using the Quantitative Realtime Polymerase Chain reaction (Q-PCR). Furthermore, we have shown that PAP protects the shrimp from WSSV infection after intramuscular injection of the GST–PAP into the shrimp before being challenged with WSSV. 2. Materials and methods 2.1. Yeast two-hybrid screening 2.1.1. Plasmid construction The PAP gene encoding a 144 amino acid protein (GenBank accession no. AY680836) was amplified by PCR from a PAP-pGEX-4T-1 plasmid produced in our previous study (Deachamag et al., 2006). The forward primer was flanked by a BamHI site (5′-GGG ATC CGG ATG AAG ATC A-3′) and the reverse primer flanked by a SalI site (5′-CCG TCG ACT TAA GAT GAG GTG-3′). PCR was performed in a final volume of 50 μl containing 200 ng DNA templates, 0.4 μM each of primer, 0.2 mM each of dNTP, 1.5 mM MgCl2, 10 mM Tris–HCl (pH 9.0), 50 mM KCl, 0.1% TritonX-100 and 2.5 U Taq DNA polymerase. Thirty cycles of PCR were carried out with 2 min of denaturation at 94 °C, 1 min of annealing at 50 °C followed by 1 min of extension and terminated by 10 min of incubation at 72 °C. All PCR experiments were performed using a thermocycler (Hybaid Limited, USA). The PCR product of 452 bp was subcloned in frame at the BamHI site and SalI sites into the pGBKT7 (CLONTECH), a bait vector that encodes the Gal4 DNA binding domain and the plasmid was named as PAP-BD. Plasmid DNA was prepared using the QIAprep Spin Miniprep Kit (QIAGEN).

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2.1.2. Protein interaction screening The yeast two-hybrid screening was carried out with the MATCHMAKER Gal4 Two-Hybrid System3 (CLONTECH). The bait PAP-BD (PAP-GAL4 DNA binding domain) was used to screen 105 independent recombinant clones of a haemocyte cDNA library from black tiger shrimp, obtained from a previous work in Saccharomyces cerevisiae strain AH109 (Tonganunt et al., 2005). The cDNA is expressed as a fusion to the GAL4 activation domain (AD). When bait and library fusion proteins interact in S. cerevisiae (AH109), the PAP-BD and AD-PAP binding proteins are brought into proximity, thus activating transcription of four reporter genes [ADE2, HIS3, MEL1 (encoding α-galactosidase) and lacZ (encoding α-galactosidase) while transformant markers for AH109 containing BD and AD plasmids are trp1 and leu2, respectively. Positive blue clones were selected for growth on a synthetic droupout (SD) plate lacking adenine, histidine, leucine and tryptophan (SD/− ade/−his/−leu/−trp) containing 5-bromo-4-chloro-3indolyl-α-D-galactopyranoside (X-Gal). Plasmid DNA isolated from those clones that activated all four yeast reporter genes [ADE2, HIS3, MEL1 (encoding αgalactosidase) and lacZ (encoding β-galactosidase) was transformed into E. coli Top10F′ to recover the plasmid and nucleic acid sequencing was carried out. Searching for gene database sequences was performed through the National Center for Biotechnology Information (NCBI) using BlastX. To confirm the screening result, β-galactosidase activity was activated when PAPBD interacted with AD-PAP binding protein. Therefore both of the PAP-BD and AD-PAP binding protein plasmids were retransformed into a host strain and plated on SD/−ade/−his/−leu/−trp. The transformants were tested for β-galactosidase activity by filter lift assay according to the manufacturer's instructions. Briefly, a single colony of the transformant was streaked on (SD/− ade/−his/−leu/−trp) plate and incubated at 30 °C, overnight. A sterile dry nitrocellulose filter was placed over the surface of the colonies to be assayed. The filter was lift and frozen in liquid nitrogen for 10 s. After thawing, the filter with colonies side up was placed on a presoaked filter with X-Gal solution to allow for the appearance of blue colonies. 2.2. In vitro pull-down assay 2.2.1. Expression of recombinant 6xHis-α2M As the clone identified by the yeast two-hybrid screening had homologies with α2M (GenBank accession no. AY826818) encoding 181 amino acid residues of the C-terminal. Therefore the α2M-pQE40 was

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obtained from previous work (Tonganunt et al., 2005). These recombinant plasmids (α2M-pQE40) as well as an insertless pQE40 were transformed into E. coli M15. The host harbouring the α2M-pQE40 plasmid and the pQE40 vector were used to produce the 6xHis-α2M fusion protein and 6xHis protein respectively. Both cultures were induced by 1 mM IPTG when the cell density at OD600 reached 0.5. After induction for 3 h, the cells were harvested by centrifugation at 4000 rpm and 4 °C for 10 min. The pellet was suspended in 300 μl of lysis buffer (100 mM Na2PO4, 10 mM Tris–Cl, 8 M Urea), sonicated ?6 × 10 s), and spun at 10,000 rpm at 4 °C for 20 min. The supernatant was used in the pulldown assay as the next step. 2.2.2. Expression of the recombinant GST–PAP Expression of GST–PAP was performed according to Deachamag et al. (2006). Briefly, the PCR fragment of 452 bp of PAP was ligated to pGEX-4T-1 (Amersham Biosciences, Sweden). The PAP-pGEX-4T-1 plasmid as well as the insertless pGEX-4T-1 was transformed into E. coli BL21 (Amersham Biosciences, Sweden). The PAP-pGEX-4T-1 plasmid was sequenced to confirm the presence of the insertion fragment sequence before fusion protein production. The host harbouring the PAPpGEX-4T-1 plasmid and the pGEX-4T-1 vector were used to produce the GST–PAP protein and the GST protein respectively. The GST–PAP protein and the GST protein production was accomplished by standard methods of bacterial growth in the presence of an antibiotic, followed by induction with 1 mM IPTG (isopropyl β-D-thiogalactopyronositol) as previously described above. 2.2.3. Pull-down assay The interaction between 6xHis-α2M and GST–PAP was examined by incubating the GST–PAP fusion protein (50 μl) with Glutathione Sepharose 4B resin (50 μl of 50% bead slurry, Amersham Biosciences) for 1 h at room temperature. The beads were washed 15 times with phosphate buffer saline (PBS), pH 7.4 to free them of unbound proteins. The recombinant 6xHis-α2M protein was added, and incubated for another 2 h at room temperature. After incubation, the beads were washed 15 times with PBS, pH 7.4 and the fusion protein was eluted with elution buffer (4% SDS, 120 mM Tris, pH 6.8, 0.2 M DTT). The protein was analyzed for interaction by 12% SDS-PAGE and transferred onto a nitrocellulose membrane. To confirm the presence of the GST-fusion protein, the blots were incubated with mouse anti-GST (Amersham Biosciences; 1:100,000) then blocked with 5% skim milk

in PBS/0.05% Tween 20 for 1 h and washed 3 times for 10 min each with PBS/0.05% Tween 20. A conjugated goat anti-mouse IgG-alkaline phosphatase (Pierce; diluted 1:100,000) was added and the alkaline phosphatase was detected with a developing solution {0.1 M NaHCO3, 1 mM MgCl2, pH 9.8, 0.23 mM of BCIP (Bromochloro indolyl phosphate) and 0.37 mM NBT (Nitroblue tetrazolium salt)} until the positive bands became sufficiently intense when the reaction was stopped by dipping the membrane in water. The membrane was then air-dried. To confirm the presence of the 6xHis-α2M protein, the blots were incubated with anti-His–HRP antibody (His–Probe conjugated horseradish peroxidase (Pierce; diluted 1:10,000) and any 6xHis-α2M protein was detected using Immuno Pure OPD (Pierce, USA). 2.3. Quantitation of the expression of the PAP and α2M genes by Real-time PCR 2.3.1. Inactivated WSSV vaccine (modified from Namikoshi et al., 2004) WSSV virus stock (Chotigeat et al., 2004) was treated with formalin (0.5% v/v) for 10 min at 25 °C. Formalin was removed by centrifuging twice at 20,000 ×g for 30 min at 4 °C. The pellet was resuspended in PBS to 1 × 10− 2 dilution of the original viral stock solution. 2.3.2. Intramuscular vaccination P. monodon shrimps (body weight 10–15 g) were divided into two groups, each with 25 shrimps. One group was injected intramuscularly with 100 μl of formalin-inactivated WSSV (1 × 10− 2 dilution of the virus preparation) per shrimp (Deachamag et al., 2006). The last group that served as a control was injected with 100 μl of PBS. After periods of 6 h, 24 h, 1 week, 2 weeks and 3 weeks the haemolymph was withdrawn to analyze for the expression of the PAP and α2M gene by Real-time PCR. 2.3.3. Real-time PCR Total RNA was extracted from the shrimp haemolymph using Trizol reagent according to the manufacturer's instruction (GIBCO BRL, USA). 12.5 μl of total RNA (4 μg) was incubated with 5 μl random primer (100 ng/μl) at 72 °C for 2 min and cooled on ice for 2 min, then 5 μl of 5× avian myeloblastosis virus (AMV) reverse transcriptase buffer, 1.5 μl of 10 mM dNTP and 1 μl of avian myeloblastosis virus (AMV) reverse transcriptase (5 U/μl) (Promega, USA) were added to make 25 μl reaction mixture for converting RNA to cDNA at 48 °C for 2 h.

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Real-Time PCR was conducted in 50 μl reactions containing 25 μl of 2× iQ™SYBR®Green Supermix (BIO-RAD, USA), 20 pmol of each primer and 300 ng of cDNA. Deionized water was added to make 50 μl. The primers used to amplify the PAP gene were 5′ ATT GCA TCA TCC ACC ATG 3′ and 5′ GGG ACT TTG TCA TCT TCA 3′. The primers for the α2M gene were the forward primer: 5′ CGA GAT CTA CCA TTA ATG AGG ATA AC 3′ and reverse primer: 5′ CTA AGG TTT CAA TCG CAC CCT TCG A 3′. The β-actin gene was used as an internal standard. The primers for the βactin gene were 5′ CAG ATC ATG TTY GAG ACC TTC 3′ and 5′ GAT GTC CAC GTC RCA CTT CAT 3′. Thermal cycling and fluorescence detection were conducted using the COBAS Taq Man 48 (Roche, Germany). All samples were run in triplicate and a no template control was included for every primer. PCR reactions were conducted with the initial denaturation step at 95 °C for 5 min, followed by 40 cycles of 94 °C for 2 min, 50 °C for 1 min and 72 °C for 1 min. The standard curve for quantitation of PAP, α2M and βactin gene was prepared using serial dilutions of the linearized purified PCR products of PAP, α2M and βactin genes, respectively. The dynamic range of detection was determined by preparing 10-fold serial dilutions of the PAP, α2M and β-actin genes in the range of 1 × 107 to 1 × 103 copies. The copy number of each reacted product was calculated according to its molecular weight and then converted into the copy number based on Avogadro's number. The expression levels were compared statistically using a One Way ANOVA of SPSS program at a 95% confidence level (p b 0.05). 2.4. Survival rate of intramuscular GST–PAP vaccinated shrimp after being challenged by WSSV P. monodon shrimps weighing 10–15 g were divided into seven groups, each with 15 shrimps. Three groups were injected with either 4 μg/g body weight, 16 μg/g body weight or 32 μg/g body weight of GST–PAP, respectively. The remaining four groups served as controls. Three control groups were injected with either 4 μg/g body weight, 16 μg/g body weight or 32 μg/g body weight of GST alone; the last control group was injected with PBS. After 3 h, all groups were challenged by an intramuscular injection of 100 μl of 9 × 10− 6 dilution of the virus preparation. After being injected with WSSV, the mortality was recorded over a 15 day period. The relative percent survival (RPS) was calculated as (1 − infected group mortality / control group mortality) × 100 (Amend, 1981).

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2.5. The uptake of GST–PAP by shrimp haemocyte in the presence of α2M 6xHis-α2M, GST–PAP and GST proteins were purified as described previously (Tonganunt et al., 2005; Deachamag et al., 2006). Purified PAP and α2M proteins were diluted in K199 to 100 μg/ml, 50 μg/ml and 25 μg/ml. Mixed protein solutions were prepared with 1:1 volumes of the above solutions. Haemocytes were collected from 15 shrimps. The haemocytes were prepared according to Itami et al. (1994). The haemocytes were divided into six groups and each group was performed in triplicate. The first group was incubated with 5 μl of GST–PAP and 6xHis-α2M while 5 μl of GST and 6xHis-α2M was incubated with the second group. Groups 3, 4 and 5 were incubated with either 5 μl of GST–PAP, GST and 6xHis-α2M, respectively. The last group (control group) had no added recombinant protein. After the mixtures of proteins or the single protein solutions were incubated at room temperature for 30 min, they were added to 50 μl of haemocyte suspension in K199. After incubation for 1 h, K199 and proteins not taken up were removed by centrifugation at 6500 ×g for 2 min at 4 °C and the haemocytes were washed twice with K199 to remove any soluble GST–PAP and 6xHis-α2M from the reaction. The haemocyte pellet was resuspended in 50 μl of PBS. The haemocytes were frozen and thawed 3 times, and the supernatant from lysed haemocytes was collected at 6500 ×g for 10 min. The GST–PAP taken up by the shrimp haemocytes was assayed by enzyme-linked immunosorbent assay (ELISA) as follows; 50 μl of the supernatants from the lysed haemocytes were each transferred to coat a well of a microtiter plate and incubated for 16–18 h at 16 °C. Five percent of non-fat milk in PBS was then added for 30 min to block any remaining attachment sites. After the plates were washed 3 times with PBS/0.2% Tween 20, 50 μl of mouse anti-GST (Amersham Biosciences; 1:2000) was added to each well and incubated for 1 h. Each well was washed 3 times for 10 min with PBS/ 0.2% Tween 20. A conjugated goat anti-mouse IgGalkaline phosphatase (Pierce; diluted 1:2000) was added. After 1 h incubation, wells were washed and any positive reaction was visualized by adding 100 μl of p-nitrophenyl phosphate (pNPP), (1 mg/ml of 10 mM diethanolamine, pH 9.5) for 30 min. The intensity of the color was quantified by measuring the absorbance at 405 nm using a spectrophotometer. The GST–PAP concentrations were compared statistically using a One Way ANOVA of SPSS program at a 95% confidence level (p b 0.05).

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3. Results 3.1. Identification of PAP binding proteins To identify haemocyte proteins that bind PAP, we performed a yeast two-hybrid screening assay with the full-length PAP as bait. The PAP gene encodes a 144 amino acid protein. Screening of the cDNA library obtained from WSSV-infected shrimps led to the isolation of four clones including an in frame α2M cDNA (AD312 and AD332), beta actin (AD331) and ribosomal protein L10 (AD350). The transformants were cultured onto selective media containing X-Gal. The blue colonies were used for confirmation by filter lift assay and showed blue patches caused by the cleavage of the substrate by αgalactosidase, that was expressed when the MEL1 promoter was activated (Fig. 1). Sequence analysis showed that clone AD332 consists of a 546 bp sequence (GenBank accession no. AY826818) that was 100% identical to the carboxyl-terminal of the α2M of P. monodon (Tonganunt et al., 2005; Lin et al. (2007) and P. japonicus (78% identity, 87% similarity, GenBank accession no. AB108542). The expression of PAP or α2M alone did not occur with SD medium and did not activate the lacZ receptor gene of the

Fig. 2. Coomassie stained 12% SDS-PAGE gel of GST pull-down assays. A GST pull-down was performed on the combined GST–PAP and 6xHis-α2M. The eluted proteins were loaded on SDS-PAGE gels. Lane M: low molecular weight standard marker; lane 1: cell lysate of GST–PAP (45 kDa); lane 2: the first elution of the purified GST–PAP; lane 3: the second elution of the purified GST–PAP; lane 4: the first elution of the combined GST–PAP and 6xHis-α2M and lane 5: the second elution of the combined GST–PAP and 6xHis-α2M; lane 6: cell lysate of 6xHis-α2M protein (46 kDa).

AH109 yeast cell, whereas when PAP and α2M were expressed together the lacZ receptor gene was activated (Fig. 1). The AD332 encodes 181 amino acids covering the 1316–1496 region of the C-terminal of α2M from P. monodon. The receptor binding region of the α2M is located between the amino acids at 1313–1440 (Lin et al., 2007). These results revealed that α2M was a PAP binding protein in the yeast two-hybrid assay and the binding region is located at the C-terminal of an α2Mlike protein. 3.2. GST pull-down assays

Fig. 1. Yeast two-hybrid assay. The filter containing the selected broken yeast cells and X-Gal solution was used to verify the activation of lacZ by interaction between two known proteins. S. cerevisiae AH109 cells co-transformed with pGBKT7-53 (P-AD) and pGADT7T (BD) as positive control (P-AD/BD); BD-PAP bound to AD-α2M (AD312 and AD332); BD-PAP bound to AD-other protein (AD311, AD313, AD331, AD333, AD 350). The negative control is a yeast cell that did not activate the MEL1.

To confirm the interaction between PAP and α2M, we carried out a GST pull-down experiment. GST–PAP is expected to be 45 kDa, consisting of the 29 kDa of GST and the 16 kDa of PAP. The GST–PAP was coupled to Glutathione Sepharose 4B beads and then incubated with the cell lysate containing the 6xHis-α2M (46 kDa). The 6xHis-α2M is 46 kDa, formed by the fusion of the 26 kDa 6xHis-DHFR (Dihydrofalate reductase) and the 20 kDa of α2M, had bound to the GST–PAP attached to the beads and was eluted and examined by SDS-PAGE. Lanes 4 and 5 of Fig. 2 show both the 6xHis-α2M (46 kDa) and the GST–PAP (45 kDa). Anti-His-Tag antibody and anti-GST antibody were used to visualize the specific protein in a Western blot analysis. As shown in Fig. 3, glutathione beads with attached GST–PAP effectively pulled down both GST (29 kDa) and GST–PAP. When the same membrane was probed with anti-His-Tag antibody, 6xHis-α2M was

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only present in the pull-down product (lane 3, Fig. 3). These data indicate that GST–PAP, not GST alone, was capable of co-precipitating with α2M. 3.3. Quantitation of the expression of PAP and α2M gene by Real-time PCR The expression of PAP and α2M in P. monodon haemolymph was activated by an intramuscular injection of formalin-inactivated WSSV and detected by Real-time Fig. 3. Western blot analysis of the eluted proteins from GST pulldown assays, separated on SDS-PAGE gels. The transferred proteins were detected by specific antibodies. The α2M band was detected with anti-His-Tag antibody. Lane 1: α2M positive control (46 kDa); lane 2: α2M combined with GST–PAP. GST band was detected with antiGST antibody. Lane 3: GST–PAP positive control (45 kDa); lane 4: GST (29 kDa); lane 5: GST–PAP combined with α2M. Lane M: prestained SDS-PAGE standards marker.

Fig. 4. The ratio of expression level of PAP (A) and α2M (B) in the haemolymph of P. monodon injected with formalin-inactivated WSSV. PBS was used as an injection control and β-actin used as an internal control (mean ± SD, n = 3 and p b 0.05).

Fig. 5. Percentage of survivor of shrimp weighing 10–15 g after being injected with recombinant protein GST–PAP 4 (A), 16 (B) and 32 (C) μg/g body weight and challenged by 9 × 10− 6 WSSV ( PBS, ▴ GST, × GST–PAP).

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Fig. 6. The uptake of GST–PAP by haemocytes after the haemocytes had been incubated with the proteins including GST–PAP ( ), GST ( ), α2M ( ), GST mixed α2M (□) and GST–PAP mixed with α2M ( ). The control group was the haemocytes that had not been incubated with recombinant protein. The amount of internal GST–PAP was estimated by subtracting the absorbance of the control group from the absorbance of the haemocytes incubated with the recombinant protein group.

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PCR. The results in Fig. 4A show that the PAP expression increased as early as 24 h post-injection and continued to increase during week 1 until week 2 (7- and 8-fold, p b 0.05). Furthermore, the expression of the α2M gene was also activated by the intramuscular injection of WSSV and had increased 6 h post-injection, reached its highest expression by 24 h (175-fold, p b 0.05) and remained high until the end of 1 week Fig. 4B. 3.4. Survival rate of shrimp injected intramuscularly with GST–PAP followed by a challenge with WSSV The shrimps were injected intramuscularly with GST– PAP at either 4 μg/g body weight, 16 μg/g body weight or 32 μg/g body weight of shrimp. After 3 h of injection with the GST–PAP, the shrimps were challenged with a 9 × 10− 6 WSSV stock solution. Fifteen days after challenge, the relative percent survival (RPS) of the shrimps was 13%, 64% and 33%, respectively (Fig. 5). 3.5. The uptake of PAP by shrimp haemocytes in the presence of α2M To study the relationship between PAP and α2M in the immune response of P. monodon, purified α2M protein was tested for its ability to activate shrimp haemocytes to take up purified GST–PAP. GST–PAP at 100 μg/ml, 50 μg/ml and 25 μg/ml that were incubated with the haemocytes with or without α2M. The GST– PAP taken up by the haemocytes was determined by ELISA. Haemocytes mixed with α2M continuously absorbed GST–PAP (Fig. 6). The highest uptake GST– PAP occurred in at 50 μg/ml of GST–PAP (12-fold).

4. Discussion In our previous study, PAP was isolated from the haemolymph of P. monodon infected with the white spot syndrome virus (WSSV). The expression of PAP in the haemolymph of P. monodon was induced via the intramuscular injection of inactivated WSSV, inactivated Vibrio harveyi and fucoidan (Deachamag et al., 2006). Meanwhile, a yeast two-hybrid screening assay was performed to survey the endocytosis pathway of the injected GST–PAP that caused activation of the shrimp immune response. We have clearly demonstrated binding between PAP and α2M. This PAP binding protein: α2M, is a member of the α2-macroglobulin family, that includes several closely related proteinase inhibitors, C3, C4 and C5 of the complement system (Armstrong and Quigley, 1999). α2M from Litopenaeus vannamei has been shown to have a broad-spectrum inhibitory action against different proteinase types (Gollas-Galván et al., 2003). The proposed proteinase inhibitory mechanism of α2M is that the proteolytic cleavage site of the bait region of α2M activates the intrachain β-cysteinyl-γ-glutamyl thioester to bind to lysine side chains on the attacking proteinase (SottrupJensen, 1989; Sottrup-Jensen et al., 1990). Additionally, proteinase inhibitors like α2M can play an important role in controlling and regulating the proPO system to remove toxic intermediates from the system (Lee and SÖderhäll, 2002; Cerenius and SÖderhäll, 2004). Several lines of evidence also indicate that α2M plays a significant role in the immune system. α2M purified from crayfish blood has been found to act against pathogens. (Diéguez-Uribeondo and Cerenius, 1998).

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α2M:α-galactosidase conjugates, stimulate macrophages, via α2M receptors to induce a sixteen-fold higher T cell proliferative response than did unconjugated antigen (Osada et al., 1987). Moreover α2M:viral protein conjugates were effectively taken up by macrophages, and this was followed by an increase in the proliferation of immune T cells so that the production of antibodies was increased ten folds (Osada et al., 1988). The receptor binding domain was reported to be located at the C-terminal of α2M (Lin et al., 2007; Sottrup-Jensen, 1989) and may mediate endocytosis, then degradation of the α2M-proteinase complex (SottrupJensen, 1989). The GST–PAP also bound to the Cterminal of α2M, however it is not known if the GST– PAP occupies the whole or only part of the region of the receptor binding domain. Although GST–PAP was found in significant amounts in the cell lysates of the haemocytes incubated with the GST–PAP and α2M, this could mean that α2M binding enables GST–PAP to be taken up by haemocytes or to be bound at appropriate cell receptors. Expression of α2M has been detected in haemocytes but not detected in the hepatopancreas of the horseshoe crab (Iwaki et al., 1996), kuruma shrimp (Rattanachai et al., 2004), crayfish (Liang et al., 1992) and black tiger shrimp (Tonganunt et al., 2005). Lin et al. (2007) also reported that the full-length expression of α2M (GenBank accession no. DQ355145) in P. monodon was found only in haemocytes, but not in eyestalk, gill, muscle, hepatopancreas, and intestine. These data indicate that α2M is synthesized in haemocytes but not in the hepatopancreas while PAP was also synthesized in the haemocytes and was not found in the hepatopancreas or lymphoid organs (Deachamag et al., 2006). Moreover, Rattanachai et al. (2004) found that the highest expression of α2M mRNAwas observed 7 days after feeding kuruma shrimp with peptidoglycan (PG) and lipopolysaccharide (LPS) and induction of α2M was also detected (Gunnarsson et al., 2003). In addition, WSSV-infected P. monodon showed a significant up-regulation of the α2M gene (Tonganunt et al., 2005) while α2M itself increased significantly in haemocytes 12–48 h and returned to a normal level, 72 h post-peptidoglycan injection in P. monodon (Lin et al., 2007). In this study, injection of formalin-inactivated WSSV produced an increased expression of PAP and α2M in the haemolymph of the black tiger shrimp. A higher expression of both the PAP and α2M gene was detected as early as 24 h and remained constant until 1 week post-injection. Meanwhile, the intramuscular injection of shrimp with GST–PAP resulted in a better survival rate (64%) of shrimp after being infected with WSSV. In this study the dose of PAP

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that gave the best protection was 16 μg/g body weight. Additionally, the percentage of phagocytosis increased significantly (2-fold) after incubation of the GST–PAP with haemocytes as compared with non-activated haemocytes (Deachamag et al., 2006). These results indicate that the intramuscular injected GST–PAP or the incubated GST–PAP may enter the haemocytes via its protein partner α2M to activate the phagocytosis of shrimp haemocytes after infection. Acknowledgement This work was supported by Thailand Goverment Research Fund. We thank Dr. Brian Hodgson, faculty of Science, Prince of Songkla University, for checking the manuscript and for valuable comments. References Amend, D.F., 1981. Potency testing of fish vaccines. In: Anderson, D.P., Hennessen, W. (Eds.), Fish Biologics: Serodiagnostic and Vaccines. S. Karger, Basel, pp. 447–454. Armstrong, P.B., Quigley, J.P., 1999. α2-macroglobulin: an evolutionarily conserved arm of the innate immune system. Dev. Comp. Immunol. 23, 375–390. Bachere, E., 2000. Shrimp immunity and disease control. Aquaculture 191, 3–11. Cerenius, L., SÖderhäll, K., 2004. The prophenoloxidase activating system in invertebrates. Immunol. Rev. 198, 72–82. Chotigeat, W., Tongsupa, S., Supamataya, K., Phongdara, A., 2004. Effect of fucoidan on disease resistance of black tiger shrimp. Aquaculture 233, 23–30. Deachamag, P., Intaraphad, U., Phongdara, A., Chotigeat, W., 2006. Expression of a phagocytosis activating protein (PAP) gene in immunized black tiger shrimp. Aquaculture 255, 165–172. Diéguez-Uribeondo, J., Cerenius, J., 1998. The inhibition of extracellular proteinase from Aphanomyces spp. by three different proteinase inhibitors from crayfish blood. Mycol. Res. 102, 820–824. Gollas-Galván, T., Sotelo-Mundo, R.R., Yepiz-Plascencia, G., VargasRequena, C., Vargas-Albores, F., 2003. Purification and characterization of α2-macroglobulin from the white shrimp (Penaeus vannamei). Comp. Biochem. Physiol. C, Comp. Pharmacol. Toxicol. 134, 431–438. Gunnarsson, M., Frangsmyr, L., Stigbrard, T., Jensen, P.E.H., 2003. Stimulation of peripheral blood mononuclear cells with lipopolysaccharide induce expression of the plasma protein α2-macroglobulin. Protein Expr. Purif. 27, 238–243. Itami, T., Takahashi, Y., Tsuchihira, E., Igusa, H., Kondo, M., 1994. Enhancement of disease resistance of kuruma prawn Penaeus japonicus and increase in phagocytic activity of prawn hemocytes after oral administration of β 1,3-glucan Schizophyllan). In: Chou, L.M., Munro, A.D., Lam, T.J., Chen, T.W., Cheong, L.K.K., Ding, J.K., Hooi, K.K., Khoo, H.W., Phang, V.P.E., Shim, K.F., Tan, C.H. (Eds.), The 3rd Asian Fisheries Forum. Asian Fisheries Society, Manila, Philippines, pp. 375–378. Iwaki, D., Kawabata, S., Miura, Y., Kao, A., Armstrong, P.B., Quigley, J.P., et al., 1996. Molecular cloning of Limulus α2-macroglobulin. Eur. J. Biochem. 242, 822–831.

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