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A single WAP domain-containing protein from Litopenaeus vannamei hemocytes
BBRC Biochemical and Biophysical Research Communications 314 (2004) 681–687 www.elsevier.com/locate/ybbrc
A single WAP domain-containing protein from Litopenaeus vannamei hemocytes Florinda Jimenez-Vega,a Gloria Yepiz-Plascencia,b Kenneth S€ oderh€ all,c b,* and Francisco Vargas-Albores a CIBNOR, PO Box 128, La Paz, BCS 23000, Mexico CIAD, PO Box 1735, Hermosillo, Sonora 83000, Mexico Department of Comparative Physiology, Uppsala University, Norbyv€agen 18 A, 752 36 Uppsala, Sweden b
c
Received 25 November 2003
Abstract A cDNA clone coding for a single WAP domain (SWD) protein was isolated from a hemocyte cDNA library of Litopenaeus vannamei. The full-length cDNA sequence is 0.4 kb long and encodes a 93-amino acid protein. Using this sequence as a probe a similar clone coding for a 92-amino acids protein was found in a cDNA library from Penaeus monodon hemocytes. The mRNA size was confirmed by Northern blot as well as that gene is expressed in hemocytes, but not in hepatopancreas. mRNA levels of the shrimp SWD protein were modified after injection of Vibrio alginolyticus, indicating the probable role of this protein in the immune response. Although amino acid sequence seems to be similar to those of other WAP domain-containing proteins, shrimp SWD protein does not have any other functional domain, similar to a mouse single WAP motif (SWAM) protein reported in mouse; however, the phylogenetic analysis shows that shrimp SWD is more related to other WAP proteins than to mouse SWAM. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Immunity; Shrimp; WAP domain; Penaeus vannamei; Penaeus monodon
Research on shrimp immunity has been stimulated by the growing problems of diseases in culture. For tropical countries, the shrimp culture is an economically important activity. However, diseases have a negative impact on the shrimp production throughout the world and consequently result in vast economic losses. Virus, bacterial, and fungal diseases are often the cause of massive mortalities during culture and the first larval stages of shrimps are particularly susceptible [1]. Such susceptibility to microbial infections could be related to their immunocompetence or capability to combat microorganisms. Thus, a better understanding of penaeid shrimp immunity would help us to design efficient strategies for disease control and insure the long-term survival of shrimp aquaculture [2]. In crustaceans, hemocytes are involved in the immediate defensive reactions such as nodulation, encapsulation, and phagocytosis [3]. These circulating cells are * Corresponding author. Fax: +52-62-280-00-55. E-mail address: [email protected] (F. Vargas-Albores).
0006-291X/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2003.12.145
also implicated in different immune responses such as melanization and coagulation, which are mediated by the release of hemocytic effectors such as the prophenoloxidase (ProPO)-activating system [4,5], trans-glutaminase [6] or antimicrobial peptides [7]. In addition, other immune-related proteins have been described in shrimp such as a clotting protein [8], lysozyme [9], LPSagglutinin [10], and a b-glucan binding protein [11]. Activation of the proPO systems process requires the participation of a protease [12] and its action needs to be precisely controlled to avoid tissue damage [4]. Universal proteinase inhibitors as a2MG [13], or more specific such as pacifastin, reported in crayfish [14] are implicated in this process. However, the full biochemical defensive battery of hemocytes is far to be described and cDNA methodology is being applied to describe the proteins synthesized by the hemocyte. Recently, expressed sequence tags (EST) from hemocyte cDNA library of shrimp have been published [15,16] and this is providing information about the genes expressed in these cells.
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Like in insects [17–19], antibacterial proteins, proteinases, and their inhibitors seem to be abundant in these cDNA libraries, revealing the importance of these proteins for shrimp immunity. Proteins containing the whey acidic protein (WAP) domain were initially associated with serine proteinase inhibitors [20–22], however this domain is present in other proteins as well and with a variety of functions. The WAP domain-containing proteins have been grouped into families based on function and tissue-specific location and include the antileukoproteinases (ALKI) [23], elastase inhibitor (elafin) proteins [24], as well as epididymal [25] and ovulatory [26] specific proteins. Although the WAP domain is 45–50 residues long, it can be found in larger proteins containing other active domains. For example, chelonianin have also a Kunitz domain [27], elafins have a cementoin domain [24], KAL-1 has a fibronectin domain [28], and the secretory leukoproteinase inhibitor which is formed by 2 WAP domains [29]. In this study, we report a cDNA sequence-deduced protein from Litopenaeus vannamei hemocytes that contain a WAP domain, but it does not have any other functional domains. This is the first report of a single WAP domain (SWD) containing protein in invertebrates and its primary structure is compared with those of the mouse SWAM (single WAP motif) proteins [22], as well as with other WAP-containing proteins. Additionally, the expression of the SWD protein is modified by inoculation of Vibrio alginolyticus indicating its possible participation in the shrimp immune response.
of nucleotides and amino acid residues were carried out using CLUSTAL as implemented by Megaline program of the Lasergene software (DNASTAR). A phylogenetic tree was constructed using the program MEGA2 [30]. Northern blot. Total RNA was isolated from L. vannamei (hemocytes and hepatopancreas) using Trizol LS reagent (GIBCO BRL) following the manufacturer’s instruction. Eight micrograms of total RNA was separated by electrophoresis in 1% agarose gel in the presence of formaldehyde with MOPS buffer [31] and then transferred to nylon Hybond-Nþ membrane (Amhersham) by capillarity blotting overnight. The membrane was hybridized at 65 °C using the radiolabeled L. vannamei probe. The membrane was exposed overnight at )80 °C and analyzed using a phosphoimager FujiX Bas2000 II (Fuji). Expression analysis. Juvenile shrimps (8–10 g) were inoculated with 20 ll heat killed V. alginolyticus (5 105 CFU/ll) in shrimp salt solution [32] and the hemolymph was withdrawn from the inoculated shrimp at different times (3, 6, 12, and 24 h), as previously described [32]. Hemocytes were recovered by centrifugation (800g, 10 min, 10 °C) and total RNA was isolated by Trizol as was described above. First strand cDNA synthesis was performed using SuperScript FirstStrand system Kit (Invitrogen) and (dT)12–18 primer following the product instruction manual. Specific primers, PvSWP-Fw (50 -GGT CAGCGTCAAGGAGGTTC-30 ) and PvSWP-Rv (50 -CTTCCGTTT CCGTAAGGAGG-30 ), were used for amplification with a 30-cycle PCR program (94 °C for 45 s; 60 °C for 1 min; and 72 °C for 2 min) in a Perkin Elmer 480 thermocycler. PCR products were separated by electrophoresis in a 2.0% agarose gel and visualized under UV light. The amplification of L. vannamei ribosomal protein L13/16 (primers: PvL13Fw, 50 -CAYCTTGMYMGGYCGCCTGG-30 and PvL13-Rv, 50 -CCA GCCKACYTCRTGMGASAGRCG-30 ) was used as internal control. The PCR products were quantified by densitometry measuring the relative intensity (RI) of the bands and using the ID Image Analysis Software (Kodak digital science).
Results Materials and methods A cDNA library from hemocytes of L. vannamei was constructed [9] using the ZAP Express cDNA synthesis kit (Stratagene). Isolated phagemids were obtained by in vivo excision using the ExAssist helper phage and the Escherichia coli XL1-Blue and XLOLR strains (Stratagene). Plasmid DNA was obtained by alkaline lysis and the purified DNA was used for sequencing. Similarly, a cDNA library was constructed from Penaeus monodon, but using UNI-ZAP XR cDNA synthesis kit (Stratagene). The full-length 433-bp fragment containing the WAP domain was purified and labeled with [a-32 P]dCTP using the Megaprimer DNA labeling system (Amersham) and used for initial screening of the P. monodon cDNA library. Recombinant lambda phages (50,000) were transferred to Hybond Nþ nylon membrane according to the instruction manual (Amersham). Hybridizations were carried out overnight at 59 °C in 6 SSC, 5 Denhardt’s solution (0.1% bovine serum albumin, 0.1% Ficoll, and 0.1 polyvinylpyrollidone), 0.5% SDS, and 20 mg/ml denatured salmon sperm DNA. The membranes were washed with 2 SSC for 15 min at 60 °C (temperature), then with 1 SSC for 15 min at 60 °C, followed by 0.5 SSC containing 0.1% SDS at 59 °C, followed by autoradiography. Secondary screenings were performed following the same procedure and the phagemids were obtained as described above. Sequencing and sequence analysis. Plasmid DNA from L. vannamei library was sequenced using T3 and T7 primers in an ALF sequencer (Amersham), and samples from P. monodon were sequenced using T3/T7 primers in an ABI Prism sequencer (Perkin Elmer). Alignments
A clone containing the complete cDNA sequence coding for a WAP domain-containing protein was isolated from a L. vannamei hemocyte cDNA library and both strands were thoroughly and completely sequenced (GenBank Accession No. AY465833). The full-length clone (433 bp) includes an open reading frame (ORF) of 282 nucleotides (93 amino acids) flanked by a 32-bp 50 -UTR and a 122-bp 30 -UTR containing a typical polyadenylation signal AATAAA (Fig. 1A). In a comparison of the nucleotide sequence against the GenBank EST database, the BLASTN program showed high similarity with sequences described as chelonianin in the penaeid shrimps: L. vannamei (BE188608), P. monodon (AW600774), and Marsupenaeus japonicus (AU176270). Furthermore, the comparison with the BLASTX algorithm showed high similarity with vertebrate proteins containing the WAP domain, such as chelonianin (P00993) from the turtle Careta careta, as well as with the human whey acidic protein (NP852479), antileukoproteinase or SPLI (P03973), and elastase inhibitor (2392716). To investigate the size of the mRNA, total RNA from hemocytes and hepatopancreas was transferred to a nylon membrane and hybridized with the radiolabeled
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Fig. 1. Penaeus WAP nucleotide and deduced protein sequences. (A) Nucleotide sequence of L. vannamei (GenBank Accession No. 465833). Initial and termination codons are in bold, specific primers used for expression studies are underlined, and polyadenylation signal is shown in italics. (B) Alignment of L. vannamei- and P. monodon (GenBank Accession No. 464465) -deduced amino acid sequences. Signal peptide cleavage is indicated by an arrow, conserved Cys are shadowed.
433-bp cDNA WAP-containing probe from L. vannamei hemocytes. As shown in Fig. 2, a single 400 bp band, corresponding to the mRNA transcript, was detected in hemocytes, but not in hepatopancreas. Using the same probe, a cDNA library from P. monodon hemocytes was screened and a 452-nucleotide clone, encoding a 92amino acid protein, was found (GenBank Accession No. AY464465). The P. monodon cDNA clone was again similarly sequenced in both strands (100%) and the sequences were compared against GenBank data by BLASTN and BLASTX. By pairwise alignment, the deduce amino acid sequence showed high similarity
Fig. 2. Northern blot of L. vannamei SWD. Lane 1, RNA molecular markers; lane 2, 8 lg of total RNA from hemocytes; and lane 3, 8 lg of total RNA from hepatopancreas. Northern blot hybridization was performed as described in Materials and methods. The arrow indicates a 0.4 kb transcript present in the hemocytes.
(83%) with the WAP domain-containing protein from L. vannamei (Fig. 1B). Changes in gene expression by inoculation with V. alginolyticus were analyzed by RT-PCR. Specific oligonucleotides were designed based on the WAP domain-containing protein from L. vannamei. Total RNA was extracted from L. vannamei hemocytes several times after Vibrio inoculation. The RNA level increased at 3 and 6 h (2.28- and 3.12-fold, respectively), returning slowly to non-stimulated levels from about 12 to 24 h (Fig. 3). The deduced protein sequence of the L. vannamei WAP domain-containing clone corresponds to a 9.7kDa preprotein with a pI of 8.34. The signal peptide was predicted using the SignalP program [33] and predicts a cleavage between Ala24 and Val25, resulting in a 7.3kDa (69 residue) mature protein. Both, L. vannamei and P. monodon, proteins include eight cysteine residues probably forming a 4 disulfide core (4-DSC), typical for
Fig. 3. Time-course expression of SWD mRNA in L. vannamei hemocytes followed by RT-PCR. Specific primers were designed and used to amplify cDNA from inoculated animals at 3, 6, 12, and 24 h post-inoculation of V. alginolyticus: C is a non-inoculated shrimp used as control (top). Amplification of shrimp ribosomal protein L13 was used as internal control (bottom).
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Fig. 4. (A) Sequence alignment of the WAP domain, including: Homo sapiens elafin (P19957), Sus scrofa elafin (2501659), S. scrofa WAP-3 protein (2501660), H. sapiens epididymal protease inhibitor (10732863), Rattus norvegicus WDNM1 protein (P14730), C. careta chelonianin (P00993), Mus musculus SWAM1 protein (NP_619626), L. vannamei SWD (AY465833), and P. monodon SDW (AY464465). Conserved Cys residues and disulfide-bonding pattern are indicated. (B) Domain structure in WAP-proteins; (SS) signal sequence; (cem) cementoin domain; and (Fn3) fibronectin 3 domain.
Fig. 5. Phylogenetic relationship of shrimp SWD proteins to other WAP-containing proteins calculated by the Neighbor-Joining method. Model: amino acid, Poisson correction. Bootstrap 1000 replications.
WAP domain (Fig. 4). A phylogenetic tree was done with representative WAP-containing proteins and the consensus trees were confirmed by bootstrap (Fig. 5).
Discussion Considering the participation of serine proteinases and its inhibitors in invertebrate immunity, we tried to
find clones coding for this kind of protein in a cDNA library of L. vannamei hemocytes. A cDNA sequence encoding a WAP domain-containing protein was found and both strands were fully sequenced. This clone has similarity with sequences described as chelonianin in the penaeid shrimps: L. vannamei (BE188608), P. monodon (AW600774), and M. japonicus (AU176270). However, chelonianin is a larger protein that contains also a Kunitz domain in the N-terminal part of the protein [27]. The cloned sequence contains a probable initial Met and a signal peptide with a conserved cleavage site and the size of the mature transcript was confirmed by Northern blot where a unique 400-bp band was detected (Fig. 2), confirming that the clone represents a mature message. Because this type of protein has not previously been described in invertebrates, a cDNA library from P. monodon hemocytes was screened and a clone for a similar protein was found. This P. monodon-deduced protein has also a unique WAP domain and 76 out of 92 amino acids are identical to the L. vannamei protein. In vertebrates, similar single WAP-proteins have been described as single WAP motif (SWAM) protein [22], but in the absence of homology evidences, we prefer to define the shrimp proteins here described as single WAP domain (SWD) protein. Since the sequences of the shrimp SWD proteins were almost identical to those described as penaeid chelonianin in the GenBank EST database, it is possible that such reported sequences correspond to SWD proteins, more than to chelonianin. The WAP motif (InterPro code IPR002221) is a 50residue protein with four disulfide bonds, conforming to a conserved tightly packed structure [34,35], named four disulfide core (4-DSC). WAP domain has been described in proteins with diverse functions, including: caltrin-like protein II, an inhibitor of calcium transport in the guinea pig [36]; SPAI-1, sodium/potassium ATPase inhibitor in the pig [37]; KAL1, the gene responsible for the Kallman syndrome [28]; WDNM1 that has been reported to be involved in cancer metastasis [38]; and SLPI and elafin. SLPI has been reported in human and mouse [29,39] as a protein that provides protection against a variety of pathogens [40–43] and prevents inflammation by protecting tissues against proteases released from neutrophils [44]. Elafin, with proteinase inhibitor spectrum different to SLPI [24], has also a protective role in various diseases [45]. In addition, independent of its anti-proteinase activity, SPLI also acts as a potent antimicrobial [42]. Recently, two members of WAP proteins (SWAM1 and SWAM2) with a potent antibacterial activity have been described in mouse [22]. Except for the conserved cysteine residues, the amino acids in the WAP domain are quite divergent which is reflected in the variety of functions for different WAP domain-containing proteins. Different distances between cysteines can also be observed, mainly in the amino extreme. The 4-DSC, characteristic structure of WAP
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domain, contains a high proportion of hydrophobic amino acids, which could fold as a b-sheet and provide the energy to stabilize the structure [20] and this structural conformation has been considered as a stable folding for a proteinase inhibitor and other functions [20]. The Penaeus SWD protein appears to be similar to the red turtle and human chelonianin, as well as to the human SLPI. However these proteins, in addition to the WAP domain, have also one Kunitz domain. As shown in Fig. 5, beside the number of domains, the Penaeus SWD protein seems to be phylogenetically more related to the WAP domain of these proteins than others, including the mouse SWAM1 and SWAM2 (NP_619626, NP_619625). Basically two types of proteins containing only a WAP domain have been described in vertebrates, differing in the presence of an N-terminal extension. This tail, in trappins, constitutes a trans-glutaminase substrate motif [21]. Thus, Penaeus SWD protein seems to be closely related to SWAM proteins [22], which have only a WAP domain and do not have TGase substrate N-terminal extension. Although elafin and SLPI are the most-studied WAP motif proteins with proteinase inhibitor activity [46], in other WAP proteins such activity has been proposed without any experimental evidence. Furthermore, some of them do not have the typical primary contact region, responsible for the proteinase inhibitor activity, described in SLPI and elafin [46]. Instead, the residues of the inhibitory sequence have been substituted by cationic and hydrophobic amino acids and an antibacterial activity has been proposed [22]. Appropriate positioning of cationic and hydrophobic amino acids makes the protein amphipathic [47], which permits insertion of the protein into the outer membrane of bacteria. penaeid SWD protein does not have the typical primary contact region characteristic for proteinase inhibitors (Elafin: LIRCAML), even though it does have hydrophobic and cationic residues. Besides the biochemical activity of the L. vannamei SWD protein remaining to be investigated, this protein appears to be involved in shrimp immunity. Changes in expression of L. vannamei SWD were detected by RT-PCR in hemocytes 3–6 h after inoculation of V. alginolyticus. A similar expression profile has been observed in Manduca sexta where the peptidoglycan recognition protein (PGRP) mRNA levels increase after 2–8 h of inoculation with bacteria [48]. On the other hand, modification in expression could be noted only after 8 h as occurred in Penaeus stylirostris proPO, finding a rise after 8 h and a decline, 12–32 h later. In the same shrimp, no changes in the LGBP expression were observed before 16 h; an increment was observed at 36 h [49]. In L. vannamei, the increment in penaeidin positive hemocytes is observed at 48 and 72 h, after a decrement at 6 and 12 h [50]. Also in vertebrates WAP domaincontaining proteins can be stimulated; mouse SLPI synthesis is observed from 60 min, obtaining the maxima
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expression in 24–36 h [51]. All these differences may indicate diversity in the mechanisms involving perhaps different transcription factors and/or a cascade of activators necessary for the modulation in gene expression. In summary, a single WAP domain protein, similar to mouse SWAM proteins, is described in penaeid shrimp (L. vannamei and P. monodon) and modification of its expression was observed after inoculation of V. alginolyticus. This is the first report of this type of protein in invertebrates.
Acknowledgments We thank Karin Johansson and Eakaphum Bangyeekhun for their help. The research was supported by Grants 41564-Z (to F.V.A.) and fellowship received (to F.J.V.) from CONACyT, Mexico, and from the Swedish Research Science Council and Formas to K.S.
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A single WAP domain-containing protein from Litopenaeus vannamei hemocytes Florinda Jimenez-Vega,a Gloria Yepiz-Plascencia,b Kenneth S€ oderh€ all,c b,* and Francisco Vargas-Albores a CIBNOR, PO Box 128, La Paz, BCS 23000, Mexico CIAD, PO Box 1735, Hermosillo, Sonora 83000, Mexico Department of Comparative Physiology, Uppsala University, Norbyv€agen 18 A, 752 36 Uppsala, Sweden b
c
Received 25 November 2003
Abstract A cDNA clone coding for a single WAP domain (SWD) protein was isolated from a hemocyte cDNA library of Litopenaeus vannamei. The full-length cDNA sequence is 0.4 kb long and encodes a 93-amino acid protein. Using this sequence as a probe a similar clone coding for a 92-amino acids protein was found in a cDNA library from Penaeus monodon hemocytes. The mRNA size was confirmed by Northern blot as well as that gene is expressed in hemocytes, but not in hepatopancreas. mRNA levels of the shrimp SWD protein were modified after injection of Vibrio alginolyticus, indicating the probable role of this protein in the immune response. Although amino acid sequence seems to be similar to those of other WAP domain-containing proteins, shrimp SWD protein does not have any other functional domain, similar to a mouse single WAP motif (SWAM) protein reported in mouse; however, the phylogenetic analysis shows that shrimp SWD is more related to other WAP proteins than to mouse SWAM. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Immunity; Shrimp; WAP domain; Penaeus vannamei; Penaeus monodon
Research on shrimp immunity has been stimulated by the growing problems of diseases in culture. For tropical countries, the shrimp culture is an economically important activity. However, diseases have a negative impact on the shrimp production throughout the world and consequently result in vast economic losses. Virus, bacterial, and fungal diseases are often the cause of massive mortalities during culture and the first larval stages of shrimps are particularly susceptible [1]. Such susceptibility to microbial infections could be related to their immunocompetence or capability to combat microorganisms. Thus, a better understanding of penaeid shrimp immunity would help us to design efficient strategies for disease control and insure the long-term survival of shrimp aquaculture [2]. In crustaceans, hemocytes are involved in the immediate defensive reactions such as nodulation, encapsulation, and phagocytosis [3]. These circulating cells are * Corresponding author. Fax: +52-62-280-00-55. E-mail address: [email protected] (F. Vargas-Albores).
0006-291X/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2003.12.145
also implicated in different immune responses such as melanization and coagulation, which are mediated by the release of hemocytic effectors such as the prophenoloxidase (ProPO)-activating system [4,5], trans-glutaminase [6] or antimicrobial peptides [7]. In addition, other immune-related proteins have been described in shrimp such as a clotting protein [8], lysozyme [9], LPSagglutinin [10], and a b-glucan binding protein [11]. Activation of the proPO systems process requires the participation of a protease [12] and its action needs to be precisely controlled to avoid tissue damage [4]. Universal proteinase inhibitors as a2MG [13], or more specific such as pacifastin, reported in crayfish [14] are implicated in this process. However, the full biochemical defensive battery of hemocytes is far to be described and cDNA methodology is being applied to describe the proteins synthesized by the hemocyte. Recently, expressed sequence tags (EST) from hemocyte cDNA library of shrimp have been published [15,16] and this is providing information about the genes expressed in these cells.
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Like in insects [17–19], antibacterial proteins, proteinases, and their inhibitors seem to be abundant in these cDNA libraries, revealing the importance of these proteins for shrimp immunity. Proteins containing the whey acidic protein (WAP) domain were initially associated with serine proteinase inhibitors [20–22], however this domain is present in other proteins as well and with a variety of functions. The WAP domain-containing proteins have been grouped into families based on function and tissue-specific location and include the antileukoproteinases (ALKI) [23], elastase inhibitor (elafin) proteins [24], as well as epididymal [25] and ovulatory [26] specific proteins. Although the WAP domain is 45–50 residues long, it can be found in larger proteins containing other active domains. For example, chelonianin have also a Kunitz domain [27], elafins have a cementoin domain [24], KAL-1 has a fibronectin domain [28], and the secretory leukoproteinase inhibitor which is formed by 2 WAP domains [29]. In this study, we report a cDNA sequence-deduced protein from Litopenaeus vannamei hemocytes that contain a WAP domain, but it does not have any other functional domains. This is the first report of a single WAP domain (SWD) containing protein in invertebrates and its primary structure is compared with those of the mouse SWAM (single WAP motif) proteins [22], as well as with other WAP-containing proteins. Additionally, the expression of the SWD protein is modified by inoculation of Vibrio alginolyticus indicating its possible participation in the shrimp immune response.
of nucleotides and amino acid residues were carried out using CLUSTAL as implemented by Megaline program of the Lasergene software (DNASTAR). A phylogenetic tree was constructed using the program MEGA2 [30]. Northern blot. Total RNA was isolated from L. vannamei (hemocytes and hepatopancreas) using Trizol LS reagent (GIBCO BRL) following the manufacturer’s instruction. Eight micrograms of total RNA was separated by electrophoresis in 1% agarose gel in the presence of formaldehyde with MOPS buffer [31] and then transferred to nylon Hybond-Nþ membrane (Amhersham) by capillarity blotting overnight. The membrane was hybridized at 65 °C using the radiolabeled L. vannamei probe. The membrane was exposed overnight at )80 °C and analyzed using a phosphoimager FujiX Bas2000 II (Fuji). Expression analysis. Juvenile shrimps (8–10 g) were inoculated with 20 ll heat killed V. alginolyticus (5 105 CFU/ll) in shrimp salt solution [32] and the hemolymph was withdrawn from the inoculated shrimp at different times (3, 6, 12, and 24 h), as previously described [32]. Hemocytes were recovered by centrifugation (800g, 10 min, 10 °C) and total RNA was isolated by Trizol as was described above. First strand cDNA synthesis was performed using SuperScript FirstStrand system Kit (Invitrogen) and (dT)12–18 primer following the product instruction manual. Specific primers, PvSWP-Fw (50 -GGT CAGCGTCAAGGAGGTTC-30 ) and PvSWP-Rv (50 -CTTCCGTTT CCGTAAGGAGG-30 ), were used for amplification with a 30-cycle PCR program (94 °C for 45 s; 60 °C for 1 min; and 72 °C for 2 min) in a Perkin Elmer 480 thermocycler. PCR products were separated by electrophoresis in a 2.0% agarose gel and visualized under UV light. The amplification of L. vannamei ribosomal protein L13/16 (primers: PvL13Fw, 50 -CAYCTTGMYMGGYCGCCTGG-30 and PvL13-Rv, 50 -CCA GCCKACYTCRTGMGASAGRCG-30 ) was used as internal control. The PCR products were quantified by densitometry measuring the relative intensity (RI) of the bands and using the ID Image Analysis Software (Kodak digital science).
Results Materials and methods A cDNA library from hemocytes of L. vannamei was constructed [9] using the ZAP Express cDNA synthesis kit (Stratagene). Isolated phagemids were obtained by in vivo excision using the ExAssist helper phage and the Escherichia coli XL1-Blue and XLOLR strains (Stratagene). Plasmid DNA was obtained by alkaline lysis and the purified DNA was used for sequencing. Similarly, a cDNA library was constructed from Penaeus monodon, but using UNI-ZAP XR cDNA synthesis kit (Stratagene). The full-length 433-bp fragment containing the WAP domain was purified and labeled with [a-32 P]dCTP using the Megaprimer DNA labeling system (Amersham) and used for initial screening of the P. monodon cDNA library. Recombinant lambda phages (50,000) were transferred to Hybond Nþ nylon membrane according to the instruction manual (Amersham). Hybridizations were carried out overnight at 59 °C in 6 SSC, 5 Denhardt’s solution (0.1% bovine serum albumin, 0.1% Ficoll, and 0.1 polyvinylpyrollidone), 0.5% SDS, and 20 mg/ml denatured salmon sperm DNA. The membranes were washed with 2 SSC for 15 min at 60 °C (temperature), then with 1 SSC for 15 min at 60 °C, followed by 0.5 SSC containing 0.1% SDS at 59 °C, followed by autoradiography. Secondary screenings were performed following the same procedure and the phagemids were obtained as described above. Sequencing and sequence analysis. Plasmid DNA from L. vannamei library was sequenced using T3 and T7 primers in an ALF sequencer (Amersham), and samples from P. monodon were sequenced using T3/T7 primers in an ABI Prism sequencer (Perkin Elmer). Alignments
A clone containing the complete cDNA sequence coding for a WAP domain-containing protein was isolated from a L. vannamei hemocyte cDNA library and both strands were thoroughly and completely sequenced (GenBank Accession No. AY465833). The full-length clone (433 bp) includes an open reading frame (ORF) of 282 nucleotides (93 amino acids) flanked by a 32-bp 50 -UTR and a 122-bp 30 -UTR containing a typical polyadenylation signal AATAAA (Fig. 1A). In a comparison of the nucleotide sequence against the GenBank EST database, the BLASTN program showed high similarity with sequences described as chelonianin in the penaeid shrimps: L. vannamei (BE188608), P. monodon (AW600774), and Marsupenaeus japonicus (AU176270). Furthermore, the comparison with the BLASTX algorithm showed high similarity with vertebrate proteins containing the WAP domain, such as chelonianin (P00993) from the turtle Careta careta, as well as with the human whey acidic protein (NP852479), antileukoproteinase or SPLI (P03973), and elastase inhibitor (2392716). To investigate the size of the mRNA, total RNA from hemocytes and hepatopancreas was transferred to a nylon membrane and hybridized with the radiolabeled
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Fig. 1. Penaeus WAP nucleotide and deduced protein sequences. (A) Nucleotide sequence of L. vannamei (GenBank Accession No. 465833). Initial and termination codons are in bold, specific primers used for expression studies are underlined, and polyadenylation signal is shown in italics. (B) Alignment of L. vannamei- and P. monodon (GenBank Accession No. 464465) -deduced amino acid sequences. Signal peptide cleavage is indicated by an arrow, conserved Cys are shadowed.
433-bp cDNA WAP-containing probe from L. vannamei hemocytes. As shown in Fig. 2, a single 400 bp band, corresponding to the mRNA transcript, was detected in hemocytes, but not in hepatopancreas. Using the same probe, a cDNA library from P. monodon hemocytes was screened and a 452-nucleotide clone, encoding a 92amino acid protein, was found (GenBank Accession No. AY464465). The P. monodon cDNA clone was again similarly sequenced in both strands (100%) and the sequences were compared against GenBank data by BLASTN and BLASTX. By pairwise alignment, the deduce amino acid sequence showed high similarity
Fig. 2. Northern blot of L. vannamei SWD. Lane 1, RNA molecular markers; lane 2, 8 lg of total RNA from hemocytes; and lane 3, 8 lg of total RNA from hepatopancreas. Northern blot hybridization was performed as described in Materials and methods. The arrow indicates a 0.4 kb transcript present in the hemocytes.
(83%) with the WAP domain-containing protein from L. vannamei (Fig. 1B). Changes in gene expression by inoculation with V. alginolyticus were analyzed by RT-PCR. Specific oligonucleotides were designed based on the WAP domain-containing protein from L. vannamei. Total RNA was extracted from L. vannamei hemocytes several times after Vibrio inoculation. The RNA level increased at 3 and 6 h (2.28- and 3.12-fold, respectively), returning slowly to non-stimulated levels from about 12 to 24 h (Fig. 3). The deduced protein sequence of the L. vannamei WAP domain-containing clone corresponds to a 9.7kDa preprotein with a pI of 8.34. The signal peptide was predicted using the SignalP program [33] and predicts a cleavage between Ala24 and Val25, resulting in a 7.3kDa (69 residue) mature protein. Both, L. vannamei and P. monodon, proteins include eight cysteine residues probably forming a 4 disulfide core (4-DSC), typical for
Fig. 3. Time-course expression of SWD mRNA in L. vannamei hemocytes followed by RT-PCR. Specific primers were designed and used to amplify cDNA from inoculated animals at 3, 6, 12, and 24 h post-inoculation of V. alginolyticus: C is a non-inoculated shrimp used as control (top). Amplification of shrimp ribosomal protein L13 was used as internal control (bottom).
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Fig. 4. (A) Sequence alignment of the WAP domain, including: Homo sapiens elafin (P19957), Sus scrofa elafin (2501659), S. scrofa WAP-3 protein (2501660), H. sapiens epididymal protease inhibitor (10732863), Rattus norvegicus WDNM1 protein (P14730), C. careta chelonianin (P00993), Mus musculus SWAM1 protein (NP_619626), L. vannamei SWD (AY465833), and P. monodon SDW (AY464465). Conserved Cys residues and disulfide-bonding pattern are indicated. (B) Domain structure in WAP-proteins; (SS) signal sequence; (cem) cementoin domain; and (Fn3) fibronectin 3 domain.
Fig. 5. Phylogenetic relationship of shrimp SWD proteins to other WAP-containing proteins calculated by the Neighbor-Joining method. Model: amino acid, Poisson correction. Bootstrap 1000 replications.
WAP domain (Fig. 4). A phylogenetic tree was done with representative WAP-containing proteins and the consensus trees were confirmed by bootstrap (Fig. 5).
Discussion Considering the participation of serine proteinases and its inhibitors in invertebrate immunity, we tried to
find clones coding for this kind of protein in a cDNA library of L. vannamei hemocytes. A cDNA sequence encoding a WAP domain-containing protein was found and both strands were fully sequenced. This clone has similarity with sequences described as chelonianin in the penaeid shrimps: L. vannamei (BE188608), P. monodon (AW600774), and M. japonicus (AU176270). However, chelonianin is a larger protein that contains also a Kunitz domain in the N-terminal part of the protein [27]. The cloned sequence contains a probable initial Met and a signal peptide with a conserved cleavage site and the size of the mature transcript was confirmed by Northern blot where a unique 400-bp band was detected (Fig. 2), confirming that the clone represents a mature message. Because this type of protein has not previously been described in invertebrates, a cDNA library from P. monodon hemocytes was screened and a clone for a similar protein was found. This P. monodon-deduced protein has also a unique WAP domain and 76 out of 92 amino acids are identical to the L. vannamei protein. In vertebrates, similar single WAP-proteins have been described as single WAP motif (SWAM) protein [22], but in the absence of homology evidences, we prefer to define the shrimp proteins here described as single WAP domain (SWD) protein. Since the sequences of the shrimp SWD proteins were almost identical to those described as penaeid chelonianin in the GenBank EST database, it is possible that such reported sequences correspond to SWD proteins, more than to chelonianin. The WAP motif (InterPro code IPR002221) is a 50residue protein with four disulfide bonds, conforming to a conserved tightly packed structure [34,35], named four disulfide core (4-DSC). WAP domain has been described in proteins with diverse functions, including: caltrin-like protein II, an inhibitor of calcium transport in the guinea pig [36]; SPAI-1, sodium/potassium ATPase inhibitor in the pig [37]; KAL1, the gene responsible for the Kallman syndrome [28]; WDNM1 that has been reported to be involved in cancer metastasis [38]; and SLPI and elafin. SLPI has been reported in human and mouse [29,39] as a protein that provides protection against a variety of pathogens [40–43] and prevents inflammation by protecting tissues against proteases released from neutrophils [44]. Elafin, with proteinase inhibitor spectrum different to SLPI [24], has also a protective role in various diseases [45]. In addition, independent of its anti-proteinase activity, SPLI also acts as a potent antimicrobial [42]. Recently, two members of WAP proteins (SWAM1 and SWAM2) with a potent antibacterial activity have been described in mouse [22]. Except for the conserved cysteine residues, the amino acids in the WAP domain are quite divergent which is reflected in the variety of functions for different WAP domain-containing proteins. Different distances between cysteines can also be observed, mainly in the amino extreme. The 4-DSC, characteristic structure of WAP
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domain, contains a high proportion of hydrophobic amino acids, which could fold as a b-sheet and provide the energy to stabilize the structure [20] and this structural conformation has been considered as a stable folding for a proteinase inhibitor and other functions [20]. The Penaeus SWD protein appears to be similar to the red turtle and human chelonianin, as well as to the human SLPI. However these proteins, in addition to the WAP domain, have also one Kunitz domain. As shown in Fig. 5, beside the number of domains, the Penaeus SWD protein seems to be phylogenetically more related to the WAP domain of these proteins than others, including the mouse SWAM1 and SWAM2 (NP_619626, NP_619625). Basically two types of proteins containing only a WAP domain have been described in vertebrates, differing in the presence of an N-terminal extension. This tail, in trappins, constitutes a trans-glutaminase substrate motif [21]. Thus, Penaeus SWD protein seems to be closely related to SWAM proteins [22], which have only a WAP domain and do not have TGase substrate N-terminal extension. Although elafin and SLPI are the most-studied WAP motif proteins with proteinase inhibitor activity [46], in other WAP proteins such activity has been proposed without any experimental evidence. Furthermore, some of them do not have the typical primary contact region, responsible for the proteinase inhibitor activity, described in SLPI and elafin [46]. Instead, the residues of the inhibitory sequence have been substituted by cationic and hydrophobic amino acids and an antibacterial activity has been proposed [22]. Appropriate positioning of cationic and hydrophobic amino acids makes the protein amphipathic [47], which permits insertion of the protein into the outer membrane of bacteria. penaeid SWD protein does not have the typical primary contact region characteristic for proteinase inhibitors (Elafin: LIRCAML), even though it does have hydrophobic and cationic residues. Besides the biochemical activity of the L. vannamei SWD protein remaining to be investigated, this protein appears to be involved in shrimp immunity. Changes in expression of L. vannamei SWD were detected by RT-PCR in hemocytes 3–6 h after inoculation of V. alginolyticus. A similar expression profile has been observed in Manduca sexta where the peptidoglycan recognition protein (PGRP) mRNA levels increase after 2–8 h of inoculation with bacteria [48]. On the other hand, modification in expression could be noted only after 8 h as occurred in Penaeus stylirostris proPO, finding a rise after 8 h and a decline, 12–32 h later. In the same shrimp, no changes in the LGBP expression were observed before 16 h; an increment was observed at 36 h [49]. In L. vannamei, the increment in penaeidin positive hemocytes is observed at 48 and 72 h, after a decrement at 6 and 12 h [50]. Also in vertebrates WAP domaincontaining proteins can be stimulated; mouse SLPI synthesis is observed from 60 min, obtaining the maxima
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expression in 24–36 h [51]. All these differences may indicate diversity in the mechanisms involving perhaps different transcription factors and/or a cascade of activators necessary for the modulation in gene expression. In summary, a single WAP domain protein, similar to mouse SWAM proteins, is described in penaeid shrimp (L. vannamei and P. monodon) and modification of its expression was observed after inoculation of V. alginolyticus. This is the first report of this type of protein in invertebrates.
Acknowledgments We thank Karin Johansson and Eakaphum Bangyeekhun for their help. The research was supported by Grants 41564-Z (to F.V.A.) and fellowship received (to F.J.V.) from CONACyT, Mexico, and from the Swedish Research Science Council and Formas to K.S.
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