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Identification and characterization of the prepro-vasoactive intestinal peptide gene from the teleost Paralichthys olivaceus
Veterinary Immunology and Immunopathology 127 (2009) 249–258
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Veterinary Immunology and Immunopathology journal homepage: www.elsevier.com/locate/vetimm
Research paper
Identification and characterization of the prepro-vasoactive intestinal peptide gene from the teleost Paralichthys olivaceus Bo-Hye Nam a,*, Young-Ok Kim a, Hee Jeong Kong a, Woo-Jin Kim a, Sang-Jun Lee a, Tae-Jin Choi b a b
Biotechnology Research Institute, National Fisheries Research and Development Institute, 408-1, Sirang-ri, Gijang-eup, Gijang-gun, Busan 619-902, South Korea Department of Microbiology, Pukyong National University, 599-1, Daeyeon 3-dong, Nam-gu, Busan 608-737, South Korea
A R T I C L E I N F O
A B S T R A C T
Article history: Received 25 February 2008 Received in revised form 9 October 2008 Accepted 16 October 2008
The gene encoding the prepro form of a vasoactive intestinal peptide (VIP) was cloned, and its structural organization and expression profiles were determined in the teleost Paralichthys olivaceus. The prepro-VIP encodes both a VIP and a peptide-histidineisoleucine (PHI). The VIP gene comprises six exons with two distinct peptides encoded on separate exons, i.e., PHI on exon III and VIP on exon IV. Several elements involved in cytokine-mediated activation are highly conserved in an 806-bp segment of the 50 flanking region: cAMP responsive elements (CREs), binding sites for nuclear factor IL-6 (NF-IL-6), activating protein-1 (AP-1), stimulating protein-1 (Sp-1), two IL-6 responsive element binding proteins (IL-6 RE-BPs), and signal transducers and activators of transcription (STAT). The mRNA transcripts in normally conditioned fish are expressed in the brain, intestine, stomach, pyloric ceca, spleen, and heart, but not in the muscle, liver, head or trunk kidneys, and gill. However, prepro-VIP mRNA expression in the spleen and head kidney is significantly up- and down-regulated when exposed to an artificial bacterial challenge by Edwardsiella tarda. These results suggest that the typical features of neuropeptide VIPs are evolutionarily conserved from non-mammalian vertebrates to mammals and that the flounder VIP plays an important role in the immune system, especially inflammatory processes. ß 2008 Elsevier B.V. All rights reserved.
Keywords: Vasoactive intestinal peptide Prepro-VIP mRNA Paralichthys olivaceus
1. Introduction Vasoactive intestinal peptide (VIP) is structurally and functionally similar to pituitary adenylate cyclase-activating polypeptide (PACAP), and both peptides belong to the glucagon and secretin superfamily, which also includes glucagon, glucagon-like peptides (GLP)-1 and -2, peptidehistidine-methionine (PHM; in vertebrates other than humans, peptide-histidine-isoleucine [PHI] occurs in place of PHM), growth hormone-releasing hormone (GRF), PACAP-related peptide (PRP), and glucose-dependent
* Corresponding author. Tel.: +82 51 720 2452; fax: +82 51 720 2456. E-mail addresses: [email protected], [email protected] (B.-H. Nam). 0165-2427/$ – see front matter ß 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2008.10.320
insulinotropic peptide (GIP) in humans. These peptides show high levels of sequence identity at their N-termini, which are the bioactive cores of these peptides, and have similar receptor structures and signaling pathways (Sherwood et al., 2000). These features indicate that the glucagon and secretin superfamily originated from a common ancestral gene. The VIP, PACAP, and glucagon genes encode two or three bioactive peptides, whereas the GRF, secretin, and GIP genes encode only a single bioactive peptide. In the past, it was believed that the PACAP precursor gene was encoded with GRF in non-mammals and that the mammalian PRPs evolved from GRF in nonmammals. However, recent information on the evolution of PRP in non-mammalian vertebrates shows that the GRF peptides that encode PACAP in non-mammals are in fact the counterparts of mammalian PRPs (Lee et al., 2007). The
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PACAP precursor gene or cDNA has been sequenced in several fish species, including sockeye salmon, Coho salmon, Chinook salmon, rainbow trout, Atlantic salmon, catfish, and zebrafish (Parker et al., 1993, 1997; McRory et al., 1995; Fradinger and Sherwood, 2000; Cardoso et al., 2007), while a few VIP precursor cDNAs have been isolated and reported recently for Takifugu (NP_001106661), medaka (AM154517), zebrafish (EU031789 for VIP-I and EU031790 for VIP-II), and goldfish (Tse et al., 2002). VIP peptide fragments have been reported for several fish species, including the spotted dogfish (Dimaline et al., 1987), cod (Thwaites et al., 1989), goldfish (Uesaka et al., 1995), rainbow trout (Wang and Conlon, 1995), bowfin (Wang and Conlon, 1995), and pallid sturgeon (Kim et al., 2001). VIP is a 28-amino acid peptide that was originally isolated from porcine intestinal extracts as a vasodilator (Said and Mutt, 1970). VIP was later identified as a multifunctional neuropeptide in the central and peripheral nervous systems. Recently, this pleiotropic neuropeptide has been shown to play a key role in the maintenance of neuroendocrine-immune communication (reviewed by Delgado et al., 2004). Some of the neuropeptides are released from the central nervous system via the hypothalamic-pituitary axis as hormones or pro-hormones and arrive in the lymphoid organs via the circulation. Lymphocytes are the primary source of VIP in lymphoid organs, which express and secrete VIP upon activation by various stimuli (Gomariz et al., 1994; Martinez et al., 1999). Using immunohistochemistry, several investigators have demonstrated the presence of VIPergic nerve fibers in both central (thymus) and peripheral (spleen, lymph nodes, and mucosal-associated lymphoid tissue) lymphoid organs (Bellinger et al., 1996; Felten et al., 1985; Fink and Weihe, 1988). During the last decade, VIP has been shown to be a potent anti-inflammatory factor that acts by regulating the production of both anti- and pro-inflammatory mediators, and has been identified as a potential candidate in the treatment of inflammatory and autoimmune disorders in mammals (Gomariz et al., 2001; Pozo et al., 2007). The physiologic roles of VIP and PACAP in teleosts have been described (Matsuda et al., 2005). VIP and PACAP are distributed in the brains of teleosts and affect the release of growth hormone, gonadotropin, and prolactin from cultured teleost pituitary cells in vitro (Brinca et al., 2003). However, the functions of VIP in the nervous and immune systems of teleosts remain to be resolved. To investigate the structural and functional evolution of VIP, and to lay the foundation for developmental studies, we isolated and characterized the VIP gene of the flounder Paralichthys olivaceus. 2. Materials and methods Flounder (P. olivaceus) was supplied from the Genetic and Breeding Research Center of NFRDI on Geoje Island (southern South Korea). The procedure for the peripheral blood leukocytes (PBLs) preparation and stimulation has been described previously (Nam et al., 2007). Briefly, PBLs were prepared by density gradient centrifugation
over a percoll gradient (1.072 g/ml) at 400 g for 20 min. Following centrifugation, PBL were carefully removed and washed by phosphate buffered saline (PBS). And then, isolated PBLs were adjusted to a concentration of 107 cells per ml and cultured in RPMI1640 containing 500 mg/ml of LPS and normal fresh medium (RPMI1640 without LPS) at 20 8C for 1, 3, and 6 h. Following each incubation time, PBLs were harvested, washed twice with PBS and stored at 80 8C until use. Total RNA was isolated from the control and LPS-stimulated PBL cells with TRIzol Reagent (Invitrogen) according to the manufacturer’s instruction. Poly(A)+ RNAs were purified from the total RNAs of treated and untreated flounder PBLs using the PolyATtract mRNA Isolation System (Promega) according to the manufacturer’s instruction. The suppression subtractive hybridization (SSH) technique (Diatchenko et al., 1996) was used to characterize new genes involved in the flounder PBLs stimulated by LPS. Two micrograms of poly(A)+ RNA isolated from treated and untreated culture were used as tester and driver, respectively. SSH was performed using the PCR-Select cDNA subtraction kit (BD Biosciences Clontech) according to manufacturer’s instruction. The amplified cDNA fragments were subcloned into the pGEM-T vector (Promega) for sequencing. 2.1. cDNA cloning and sequencing A clone derived by SSH, LSPSL5-C-11, which carries an 885-bp insertion, showed significant sequence homology to the C-terminal portion of the Xenopus laevis Vip-A protein gene (AAH43792). To isolate the full-length cDNA of this SSH clone, 50 -RACE PCR was carried out using the SMART RACE cDNA Amplification Kit (BD Biosciences). Two gene-specific primers (VIP 50 -RACE-1 and VIP 50 RACE-2) were designed based on the sequence of the LSPSL5-C-11 clone (Table 1). First-strand cDNA was synthesized with the same poly(A)+ RNA used for SSH using the protocol provided with the Smart RACE cDNA amplification kit. The primer sets of VIP 50 -RACE-1, VIP 50 RACE-2, and a nested universal primer supplied with the kit were used for 50 -RACE. The PCR products generated were purified and cloned into the pGEM-T vector (Promega). Ten clones from the 50 -RACE PCR products were sequenced, and the longest PCR product was selected for determining the transcription start site. The complete cDNA of the flounder VIP gene was compiled by overlapping the sequences of the SSH clone and the 50 -RACE PCR products. Table 1 Sequence-specific primers used for PCR. Name
Sequence 0
Description 0
VIP-ORF-F VIP-ORF-R VIP-GW-1
5 -ATGTTACAACGGACCGGCCC-3 50 -CACCTCAAAGAGGCAGTTGA-30 50 -GTCTGCGTTTGTCTCTTCCTCC-30
VIP-GW-2 GAPDH-RT-F GAPDH-RT-R
50 -CCAGGGTTGACTCATCTGATC-30 50 -TCCCATGTTCGTCATGGGCGTGA-30 50 -ATTGAGCTCAGGGATGACCTTG-30
ppVIP RT-PCR ppVIP genomic walking GAPDH RT-PCR
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2.2. Genomic DNA cloning and sequencing
2.5. Artificial bacterial infection of fish
Genomic DNA was prepared from flounder whole blood using 4 M TNES-urea buffer (Asahida et al., 1996) and purified with phenol/chloroform (Sambrook and Russell, 2001). Two specific primers (VIP-ORF-F and VIP-ORF-R) were designed based on the 50 - and 30 -end sequences of the VIP open reading frame (ORF; Table 1). Ten nanograms of genomic DNA were used as a template. PCR was performed beginning at 95 8C for 5 min, followed by 30 cycles of 95 8C for 30 s, 55 8C for 30 s, and 72 8C for 3 min, with a final 5 min at 72 8C. The PCR product was purified and cloned into the T-Easy vector (Promega). The resultant positive clone was sequenced using an automatic sequencer (ABI3100; Applied Biosystems). Putative binding sites for transcription factors within the 50 flanking sequence were identified using the TESS (Transcription Element Search Software; Schung and Overton, 1997) software. The 50 -flanking region of the flounder VIP gene was amplified from flounder genomic DNA using a Universal GenomeWalkerTM kit following the manufacturer’s protocol (BD Biosciences). Briefly, the primary PCR reaction was carried out using the adaptor primer (AP-1) provided in the kit plus a gene-specific primer (VIP-GW-1). The primary PCR product was used as the template for a nested PCR reaction using the nested adaptor primer (AP-2) and a nested gene-specific primer (VIP-GW-2). The PCR product was cloned into the T-Easy vector (Promega) and sequenced (ABI3100; Applied Biosystems).
The induction of flounder VIP expression was examined in the spleen, kidney, and intestine using RT-PCR following an artificial bacterial infection with Edwardsiella tarda (strain KFE isolated from flounder in South Korea). For the artificial infection, healthy juvenile flounders (mean weight 100 g) obtained from the Genetic and Breeding Research Center were used. The fish were anesthetized with MS-222 (3-aminobenzoic acid ethyl ester; Sigma) and infected with E. tarda by intraperitoneal injection of a sublethal dose (1.2 106 cells) suspended in PBS buffer. Tissues were collected from three fish at 0, 1, 3, 6, 12, 24, and 72 h post-injection, and frozen at 80 8C for RNA extraction.
2.3. Alignment and phylogenetic tree analysis The relevant sequences were retrieved from GenBank for multiple sequence alignments using ClustalX (Thompson et al., 1997). Sequence homology was calculated by GENETYX version 8.0 (SDC Software Development, Japan). A phylogenetic tree was created based on the amino acid distances between the aligned sequences using the Neighbor-Joining method with 1000 bootstrap replications using the MEGA software (version 3.0; Kumar et al., 2004). For signal peptide prediction, the SignalP software (http:// www.cbs.dtu.dk/services/SignalP/) was used.
3. Results 3.1. Identification and characterization of the flounder prepro-VIP cDNA The SSH clone LSPSL5-C-11, which carries a 885-bp insertion, showed significant sequence homology to the Cterminal portion of the Xenopus Vip-A protein gene (AAH43792), lacking the N-terminal sequence that encodes the signal peptide and the PHI peptide coding sequence. The N-terminal portion of flounder VIP was amplified from the first-stranded cDNA of LPS-stimulated flounder PBLs using 50 -RACE with a primer based on the LSPSL5-C-11 sequence (Table 1). The 50 -RACE PCR product was approximately 350 bp in length and contained a 111bp 50 -untranslated region (UTR) and the N-terminal portion of prepro-VIP. The complete ORF obtained by joining the sequences of LSPSL5-C-11 and the 50 -RACE PCR product gave a predicted 148-aa prepro-VIP, including a 21-aa signal peptide (Fig. 1). The 30 -UTR consisted of 519 bp, excluding the poly(A) tail, and it harbored three ATTTA sequence elements, typically found in cytokine mRNAs, which mediate RNA instability. The overall sequence identities between the flounder prepro-VIP and its counterparts in Takifugu (GenBank accession no. NP_001106661) and zebrafish (GenBank accession no. EU031789) were approximately 93% and 78%, respectively.
2.4. Detection of prepro-VIP mRNA 3.2. Genomic organization Total RNA sample from the brain, muscle, liver, intestine, stomach, phyloric ceca, head kidney, trunk kidney, spleen, gill, heart, and skin of healthy flounders was subjected to DNase I treatment (Invitrogen) for avoiding genomic DNA contamination before being converted to cDNA. First-strand cDNA synthesis was performed using the Advantage RT-for-PCR Kit (BD Biosciences). The gene-specific primers (VIP-ORF-F and VIP-ORF-R) for flounder VIP amplification were designed based on the VIP cDNA sequence. As an internal control, the flounder GAPDH housekeeping gene was amplified using specific primers (GAPDH-RT-F and GAPDH-RT-R). The RTPCR reaction parameters included an initial 5-min step at 95 8C, followed by 30 cycles of 94 8C for 30 s, 60 8C for 30 s, and 72 8C for 1 min, and a final 5 min at 72 8C.
To determine the genomic structure of the flounder VIP gene, we amplified the genomic DNA of the coding region by PCR and the 50 -flanking region using the genome walking method. Sequence analysis showed that the genomic DNA sequence of the flounder VIP gene obtained in the present study contains 4884 bp and is composed of six exons and five introns (GenBank accession no. EU496523). The five introns are 871, 139, 1014, 638, and 339 bp in length, respectively. Typical intronic splice motifs are observed at the 50 (GT)- and 30 (AG)-ends of each intron. The first exon represents a 50 -UTR sequence of 101 bp. The second exon codes for a putative signal peptide of 21 amino acids and for 11 additional amino acids. The third and fourth exons encode PHI and VIP, respectively.
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Fig. 1. Nucleotide sequence of the flounder prepro-VIP mRNA and the deduced amino acid sequence. The numbering of nucleotides and amino acids begins with ATG and methionine (M), respectively, and is shown at the left and right margins, respectively. The putative peptides PHI and VIP are shaded. Three ATTTA sequence motifs in the 30 -untranslated region are shown in bold. The nucleotide sequence has GenBank accession no. EU496522.
The fifth exon contains 112 bp, which encodes a spacer protein sequence of unknown function. The sixth exon directs the synthesis of the two amino acid sequences, contains a stop codon, and a 30 -UTR (Fig. 2A). The 806-bp 50 sequences upstream from the transcriptional start site contain the major regulatory sequences of the VIP gene (Fig. 3). Major regulatory motifs for key transcription factors were identified in the flounder VIP promoter analysis. These cis-elements include a TATA box (nt 28 to 34, upstream from the putative flounder VIP transcriptional start site), two cAMP-responsive element (CRE) motifs (CGTCA; nt 90 to 94 and nt 100 to 104), and a putative cytokine-responsive element (CyRE), which includes binding sites for nuclear factor IL-6 (NF-IL-6; nt 138 to 144), activating protein-1 (AP-1; nt 225 to 234), stimulating protein-1 (Sp-1; nt 408 to 416), two IL-6 responsive element binding proteins (IL-6-RE-BP; nt 293 to 298 and nt 460 to 466), and signal transducers and activators of transcription (STAT; nt 658 to 663).
closer relationship between fish prepro-VIP and other nonmammalian VIP than between fish prepro-VIP and mammalian VIP. 3.4. Prepro-VIP mRNA expression To determine the tissue distribution of the prepro-VIP mRNA in adult flounder, RT-PCR was performed on several tissues (brain, muscle, liver, intestine, stomach, pyloric ceca, head kidney, trunk kidney, spleen, gill, and heart). A PCR primer set amplified a ca. 440-bp transcript from the brain, intestine, stomach, pyloric ceca, spleen, and heart, but not from the muscle, liver, head and trunk kidneys, and gill (Fig. 5A). Analysis of the expression of the prepro-VIP mRNA in several tissues (spleen, head kidney, and intestine) was also carried out using RT-PCR following infection with E. tarda. The flounder prepro-VIP mRNA of the intestinal tissue was expressed constitutively, while its expression was clearly up- and downregulated in the spleen and kidney after infection with E. tarda (Fig. 5B).
3.3. Sequence alignment and phylogenetic analyses 4. Discussion The deduced amino acid sequence of VIP is highly conserved between teleosts and humans. The flounder VIP contains five substitutions out of 28 residues compared to mammalian VIP (Table 2). While the amino acid sequence of the flounder PHI peptide shows complete identity (100%) with both the fish PHI and Xenopus PHI, the VIP has identical sequence among fishes (Table 2). A phylogenetic tree was constructed based on the deduced prepro-VIP amino acid sequences of reported species using the Neighbor-Joining method. As shown in Fig. 4, the flounder prepro-VIP formed a cluster with non-mammalian species, such as chicken, Xenopus, and other fishes. This finding was further supported by a high bootstrap value, indicating a
A clone that encodes the prepro-VIP was isolated from a subtracted cDNA library derived from flounder PBLs that were stimulated with LPS. The flounder prepro-VIP possesses features typical of neuropeptides. The precursor peptide (prepropeptide) always has three components: a signal sequence of approximately 16–30 amino acid residues at the N-terminus, one or more neuropeptide sequences, and one or several spacers regions (Holmgren and Jensen, 2001). The flounder prepro-VIP in the present study contains two related peptides, PHI and VIP, and a spacer sequence that follows a 21-aa signal peptide. Few VIP alternative splice transcripts have been described. In
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Fig. 2. Comparison of the gene organization and deduced amino acid sequences of the flounder VIP and those of other species. (A) Organization of the VIP genes of the flounder, zebrafish, chicken, and human. The lengths of the exons and introns are not proportional, so that exons can be aligned between genes. Exon numbering follows that used for the mammalian genes. Open boxes indicate untranslated regions, and closed and shaded boxes indicate the coding regions of exons for each gene. The accession numbers of the VIP genomic sequences used in the GenBank database are as follows: flounder, EU496523; zebrafish I, NW_001877362; zebrafish II, NW_001877173; chicken, NC_006090; and human, M33027. SP, signal peptide; CP, cryptic peptide; PHM, peptidehistidine-methionine; VIP, vasoactive intestinal peptide; PHI, peptide-histidine-isoleucine. (B) Multiple amino acid sequence alignment of VIP precursors. Each segment encoded by a different exon is boxed and annotated. Spaces introduced to help understand the aligned sequences are indicated by hyphens. Asterisks indicate the stop codons. The accession numbers of the VIP precursor used are as follows: flounder, EU496522; zebrafish I, NM_001114553; zebrafish II, NM_001114555; mouse, EDL03569; human, AAH09794; chicken, AAA87896; and frog, AAH43792.
Fig. 3. Schematic representation of the 50 -flanking region of the flounder VIP gene. Major regulatory motifs for key transcription factors are represented, including a TATA box (nt 28 to 34, upstream of the putative flounder VIP transcription start site), two cAMP-responsive element (CRE) motifs (CGTCA; nt 90 to 94 and nt 100 to 104), and a putative cytokine-responsive element (CyRE), which includes binding sites for nuclear factor IL-6 (NF-IL-6; nt 138 to 144), activating protein-1 (AP-1; nt 225 to 234), stimulating protein-1 (Sp-1; nt 408 to 416), two IL-6 responsive element binding proteins (IL-6RE-BP; nt 293 to 298 and nt 460 to 466), and signal transducers and activators of transcription (STAT; nt 658 to 663). The CRE shown as a shaded box contains two 5-bp CGTCA motifs, which are shown in bold and underlined.
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Table 2 The amino acid sequence identity of other forms of the PHI and VIP peptides in comparison to the floundera.
PHI (27aa)
VIP (28aa)
a
Species Flounder Takifugu Zebrafish I Zebrafish II Medaka Goldfish 2A Goldfish 11A Xenopus Chicken Cattle Rat Mouse Dog Monkey Chimpanzee Human Flounder Takifugu Zebrafish I Zebrafish II Medaka Goldfish 2A Goldfish 11A Sturgeon 1 Sturgeon 2 Bowfin Trout Cod Dogfish Xenopus Chicken Cattle Rat Mouse Dog Monkey Chimpanzee Human
Amino acid sequence HADGLFTSGYSKLLGQLSARRYLESLI ........................... ........................... ...................KEY....L ........................... ...................KEF....L ...................KEY....L ........................... .....I...V..H..AK.AVK...H.. ....V...D..R.......KK...... ....V...D..R....I..KK...... ....V...D..R....I..KK...... ....V...DF.R.......KK...... ....V...DF.........KK.....M ....V...DF.........KK.....M ....V...DF.........KK.....M HSDAIFTDNYSRFRKQMAVKKYLNSVLT ............................ ............................ ....V.......Y.....A........A ............................ ....V.......Y.....A........A ....V.......Y.....A........S ............................ ...S........................ ............................ ............................ ....V.............A........A ....V.......I.........I..L.A ....V....................... ....V....................... ....V.....T.L............I.N ....V.....T.L............I.N ....V.....T.L............I.N ....V.....T.L............I.N ....V.....T.L............I.N ....V.....T.L............I.N ....V.....T.L............I.N
Identity (%) 100 100 100 85.2 100 85.2 85.2 100 66.7 81.5 77.8 77.8 77.8 81.5 81.5 81.5 100 100 100 85.7 100 85.7 85.7 100 96.4 100 100 89.3 82.1 96.4 96.4 82.1 82.1 82.1 82.1 82.1 82.1 82.1
Dots indicate identical amino acids and the alternative amino acid is indicated when the sequence differ.
goldfish, two alternative VIP transcripts have been reported, one of which contains both VIP and PHI and the other has PHI only (Tse et al., 2002), while in the chicken, a shorter transcript that lacks the PHI coding region has also been described (Talbot et al., 1995). The flounder VIP gene spans 4884 bp and consists of six exons and five introns. The 101-bp exon I consists of the 50 UTR of the gene, 105-bp exon II encodes the signal peptide, 105-bp exon III encodes PHI, 126-bp exon IV encodes VIP, 112-bp exon V contains the spacer region (cryptic peptide), and 528-bp exon VI contains the termination codon of the prepro-VIP mRNA and the 30 -UTR of the gene. However, this structural organization of the flounder VIP gene differs from that of other species in several ways. For example, the human VIP gene consists of seven exons and six introns, and the cryptic peptide (spacer region) is encoded on exon III. Two teleost VIP genes have been sequenced from zebrafish: GenBank accession no. NW_001877362 for VIP
type I and NW_001877173 for VIP type II. The structural organization of the zebrafish VIP type I gene shows highly similarity to the flounder VIP gene. However, VIP type II has a genomic structure similar to that of the human VIP gene (Fig. 2A). Interestingly, the chicken VIP has two potential cryptic peptides at the N- and C-termini, which are encoded by exons III and VI, respectively (Fig. 2A and B). Previously, the function of cryptic peptide was considered simply to increase the length of the molecule and help the precursor to fold before cleavage (Holmgren and Jensen, 2001). Recently, the concept of ‘‘cryptic peptides’’ has been gaining popularity to address bioactive peptide hidden within larger precursors that are liberated by the action of proteases (Dylag et al., 2008). Therefore, we considered that the cryptic peptide region of VIP precursor supply more information in the aspect of VIP gene evolution, and applied to phylogenetic analysis with the amino acid sequences of cryptic peptide regions. In
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Fig. 4. Phylogenetic analysis of the prepropeptide form of VIP. The numbers indicate the frequencies with which the phylogram topology represented here is replicated for every 1000 bootstrap interactions. The accession numbers of the prepro-VIP amino acid sequences are as follows: human, AAH09794; monkey, XP_001096218; chimpanzee, XP_527541; mouse, EDL03569; rat, EDL92841; cattle, AF503910; dog, XP_541155; chicken, AAA87896; frog, AAH43792; trout, BX910000; medaka, AM154517; zebrafish 1, CK362621; zebrafish 2, XM_001337417; Takifugu, NP_001106661; goldfish 2A and 11A, obtained from Tse et al., 2002; and flounder, EU496522.
addition, using the cryptic peptide region for phylogenetic analysis could be useful to illuminate the close related species thanks to the high sequence variation unlike PHI (or PHM) and VIP region, that have a low phylogenetic resolution due to their low variability (see the Table 2). The cryptic peptide sequences were analyzed phylogenetically using the Neighbor-Joining method (Fig. 6). The results reveal that the cryptic peptide derived from zebrafish VIP type II (zebrafish II CP) forms a cluster with CP-1 of chicken and Xenopus and the CPs of mammals, while zebrafish I CP groups with CP-2s and that of flounder. This suggests that the VIP gene was duplicated into types I and II in zebrafish, and that the type II VIP gene subsequently evolved into the VIP genes of other vertebrates. Furthermore, a cryptic peptide should be an important clue to explaining the evolution of VIP genes. Vasoactive intestinal peptide is produced by lymphoid and neural cells, including the central (thymus) and peripheral (spleen, lymph nodes, and mucosal-associated lymphoid tissue) lymphoid organs. The flounder preproVIP mRNA was detected in the brain (including pituitary gland), intestine, stomach, pyloric ceca, spleen, and heart, but not in the muscle, liver, kidney, gill, and skin of
normally conditioned fish. When fish were subjected to an artificial bacterial challenge, to study the potential role of VIP in inflammatory processes, different expression patterns of the flounder prepro-VIP mRNA were observed depending on the specific tissue analyzed (Fig. 5B). The flounder prepro-VIP mRNA maintained a constitutive level of expression in intestinal tissue despite infection. However, the flounder prepro-VIP mRNA expression was significantly upregulated in the spleen and kidney after infection, which implies that VIP expression is tissuespecific. This also indicates that flounder VIP plays an important role in the inflammatory processes of fish, similar to its role observed in mammals. Additional evidence for the immunomodulatory role of flounder VIP is demonstrated by the presence of certain sequences in the 50 -flanking region of the flounder VIP gene. The flounder VIP gene contains consensus sequences for two cAMP response elements (CREs) as well as a CyRE in the promoter region. Most CREs contain one or multiple copies of the conserved sequence motif CGTCA or TGACG, and several have been shown to act as enhancers (Silver et al., 1987; Fink et al., 1988). In the CRE of the flounder VIP gene, two CGTCA motifs are present in a 25-bp sequence located
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Fig. 5. RT-PCR analysis of prepro-VIP mRNA in flounder tissues. (A) RT-PCR was conducted using cDNA from healthy flounder tissue. (B) Expression profiles of prepro-VIP mRNA in spleen, head kidney, and intestine tissues sampled at 0, 1, 3, 6, 12, 24, and 72 h after an artificial bacterial challenge. As a positive control for RT-PCR, the GAPDH gene was amplified to determine template concentration.
between 90 and 114 nt from the transcription initiation site (Fig. 3). The spatial requirements for cooperation between the two CGTCA-containing domains of human VIP were tested by altering the orientation or distance between the two CGTCA motifs (Fink et al., 1988). The two CGTCA motifs in the human VIP CRE are located within a 15-bp sequence and have a palindromic structure. The flounder VIP CREs are encoded in a 25-bp sequence that includes the inverted orientation that results in 11–32% loss of CRE activity in the case of human VIP gene
expression. The presence of two adjacent functional CGTCA motifs within the VIP CRE is important for the proteinprotein interactions between two CGTCA-binding factors. The two domains of the VIP CRE may facilitate interaction between identical regulatory proteins, such as CRE binding proteins (CREBs). Further studies are necessary to determine the effects of orientation or distance between the two CGTCA motifs on the expression of the flounder VIP gene. The VIP CyRE is a complex regulatory element that is composed of binding sites for a variety of different
Fig. 6. Phylogenetic tree of the cryptic peptide of VIP. The tree was constructed using the Neighbor-Joining method with the MEGA 3.0 program. Bootstrap values were produced through an analysis of 1000 replications of the original dataset.
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transcription factors. Several studies have reported that there are functional STAT, AP-1, ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), IL-6-RE-BP, and NF-IL-6 sites within the 180-bp CyRE in the human VIP promoter region (Symes et al., 1993, 1994, 1995, 1997). We have identified a region of the flounder VIP CyRE that includes several putative transcription factor-binding sites for NF-IL-6, AP-1, two IL-6 RE-BPs, Sp-1, and STAT from 149 nt to 658 nt upstream of the transcriptional start site. This suggests that expression of the flounder VIP gene is regulated in a cytokine-dependent manner via CyRE. Further studies are needed to demonstrate unequivocally that the participation of cis-regulatory domains within the flounder VIP CyRE is required for full expression of the VIP gene. Acknowledgment This work is funded by a grant from the Nation Fisheries Research and Development Institute (RT-2008-BT-023). References Asahida, T., Kobayashi, T., Saitoh, K., Nakayama, I., 1996. Tissue preservation and total DNA extraction from fish stored at ambient temperature using buffers containing high concentration of urea. Fish. Sci. 62, 727–730. Bellinger, D.L., Lorton, D., Brouxhon, S., Felten, S., Felten, D.L., 1996. The significance of vasoactive intestinal polypeptide (VIP) in immunomodulation. Adv. Neuroimmunol. 6, 5–27. Brinca, L., Fuentes, J., Power, D.M., 2003. The regulatory action of estrogen and vasoactive intestinal peptide on prolactin secretion in sea bream (Sparus aurata L.). Gen. Comp. Endocrinol. 131, 117–125. Cardoso, J.C.R., Vieira, F.A., Gomes, A.S., Power, D.M., 2007. PACAP, VIP and their receptors in the metazoan: Insights about the origin and evolution of the ligand-receptor pair. Peptides 28, 1902–1919. Delgado, M., Pozo, D., Ganea, D., 2004. The significant of vasoactive intestinal peptide in immunomodulation. Pharmacol. Rev. 56, 249– 290. Diatchenko, L., Chris Lau, Y.-F., Campbell, A.P., Chenchik, A., Moqadam, F., Huang, B., Lukyanov, S., Lukyanov, K., Gurskaya, N., Sverdlov, E.D., Siebert, P.D., 1996. Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proc. Natl. Acad. Sci. U.S.A. 93, 6025–6030. Dimaline, R., Young, J., Thwaites, D.T., Lee, C.M., Shuttleworth, T.J., Thorndyke, M.C., 1987. A novel vasoactive intestinal peptide (VIP) from elasmobranch intestine has full affinity for mammalian pancreatic VIP receptors. Biochim. Biophys. Acta 930, 97–100. Dylag, T., Pachuta, A., Raoof, H., Kotlinska, J., Silberring, J., 2008. A novel cryptic peptide derived from the rat neuropeptide FF precursor reverses antinociception and conditioned place preference induced by morphine. Peptides 29, 473–478. Felten, D.L., Felten, S.Y., Carlson, S.L., Olschowka, J.A., Livnat, S., 1985. Noradrenergic and peptidergic innervation of lymphoid tissue. J. Immunol. 135, 755–765. Fink, J.S., Verhave, M., Kasper, S., Tsukada, T., Mandel, G., Goodman, R.H., 1988. The CGTCA sequence motif is essential for biological activity of the vasoactive intestinal peptide gene cAMP-regulated enhancer. Proc. Natl. Acad. Sci. U.S.A. 85, 6662–6666. Fink, T., Weihe, E., 1988. Multiple neuropeptides in nerve supplying mammalian lymph nodes: messenger candidates for sensory and autonomic neuroimmunomodulation. Neurosci. Lett. 90, 39–44. Fradinger, E.A., Sherwood, N.M., 2000. Characterization of the gene encoding both growth hormone-releasing hormone (GRF) and pituitary adenylate cyclase-activating polypeptide (PACAP) in the zebrafish. Mol. Cell. Endocrinol. 165, 211–219. Gomariz, R.P., Leceta, J., Garrido, E., Garrido, T., Delgado, M., 1994. Vasoactive intestinal peptide (VIP) mRNA expression in rat T and B lymphocytes. Regul. Pept. 50, 177–184. Gomariz, R.P., Martinez, C., Abad, C., Leceta, J., Delgado, M., 2001. Immunology of VIP: a review and therapeutical perspectives. Curr. Pharm. Des. 7, 89–111.
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Holmgren, S., Jensen, J., 2001. Evolution of vertebrate neuropeptides. Brain Res. Bull. 55, 723–735. Kim, J.B., Barton, B.A., Conlon, J.M., 2001. Characterization of two molecular forms of vasoactive intestinal polypeptide (VIP) from the pallid sturgeon, Scaphirhynchus albus (Acipenseriformes). Fish Physiol. Biochem. 25, 231–238. Kumar, S., Tamura, K., Nei, M., 2004. MEGA3: integrated software for molecular evolutionary genetic analysis and sequence alignment. Brief. Bioinform. 5, 150–163. Lee, L.T.O., Siu, F.K.Y., Tam, J.K.V., Lau, I.T.Y., Wong, A.O.L., Lin, M.C.M., Vaudry, H., Chow, B.K.C., 2007. Discovery of growth hormone-releasing hormones and receptors in nonmammalian vertebrates. Proc. Natl. Acad. Sci. U.S.A. 104, 2133–2138. Martinez, C., Delgado, M., Abad, C., Gomariz, R.P., Ganea, D., Leceta, J., 1999. Regulation of VIP production and secretion by murine lymphocytes. J. Neuroimmunol. 93, 126–138. Matsuda, K., Maruyama, K., Nakamachi, T., Miura, T., Uchiyama, M., Shioda, S., 2005. Inhibitory effects of pituitary adenylate cyclaseactivating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) on food intake in the goldfish, Carassius auratus. Peptides 26, 1611–1616. McRory, J.E., Parker, D.B., Ngamvongchon, S., Sherwood, N.M., 1995. Sequence and expression of cDNA for pituitary adenylate cyclase activating polypeptide (PACAP) and growth hormone-releasing hormone (GHRH)-like peptide in catfish. Mol. Cell. Endocrinol. 108, 169– 177. Nam, B.H., Byon, J.Y., Kim, Y.O., Park, E.M., Cho, Y.C., Cheng, J., 2007. Molecular cloning and characterization of the flounder (Paralichthys olivaceus) interleukin-6 gene. Fish Shellfish Immunol. 23, 231–236. Parker, D.B., Coe, I.M., Dixon, G.H., Sherwood, N.M., 1993. Two salmon neuropeptides encodes by one brain cDNA are structurally related to members of the glucagons superfamily. Eur. J. Biochem. 215, 439– 448. Parker, D.B., Power, M.E., River, J., Sherwood, N.M., 1997. Exon skipping in the gene encoding pituitary adenylate cyclase-activating polypeptide in salmon alters the expression of two hormones that stimulate growth release. Endocrinology 138, 414–423. Pozo, D., Gonzalez-Rey, E., Chorny, A., Anderson, P., Varela, N., Delgado, M., 2007. Turning immune tolerance with vasoactive intestinal peptide: a new therapeutic approach for immune disorder. Peptides 28, 1833– 1846. Said, S.I., Mutt, V., 1970. Polypeptide with broad biological activity: isolation from small intestine. Science 169, 1217–1218. Sambrook, J., Russell, D.W., 2001. Molecular cloning. A Laboratory Manual. Cold Spring Harbor Laboratory Press, New York. Schung, J., Overton, G.C., 1997. TESS: transcription element search system on the WWW. Technical Report CBIL-TR-1997-1001-v0.0, of the Computational Biology and Informatics Laboratory, School of Medicine, University of Pennsylvania. Sherwood, N.M., Krueckl, S.L., McRory, J.E., 2000. The origin and function of the pituitary adenylate cycles-activating polypeptide (PACAP)/ glucagons superfamily. Endocr. Rev. 21, 619–670. Silver, B.J., Bokar, J.A., Virgin, J.B., Vallen, E.A., Milsted, A., Nilson, J.H., 1987. Cyclic AMP regulation of the human glycoprotein hormone a-subunit gene is mediated by an 18-base-pair element. Proc. Natl. Acad. Sci. U.S.A. 84, 2198–2202. Symes, A.J., Rao, M.S., Lewis, S.E., Landis, S.C., Hyman, S.E., Fink, J.S., 1993. Ciliary neurotrophic factor coordinately activates transcription of neuropeptide genes in a neuroblastoma cell line. Proc. Natl. Acad. Sci. U.S.A. 90, 572–576. Symes, A.J., Lewis, S., Corpus, L., Rajan, P., Hyman, S.E., Fink, J.S., 1994. STAT proteins participate in the regulation of the vasoactive intestinal peptide gene by the ciliary neurotrophic factor family of cytokines. Mol. Endocrinol. 8, 1750–1763. Symes, A.J., Rajan, P., Corpus, L., Fink, J.S., 1995. C/EBP-related sites in addition to a Stat site are necessary for ciliary neurotrophic factorleukemia inhibitory factor-dependent transcriptional activation by the vasoactive intestinal peptide cytokine response element. J. Biol. Chem. 270, 8068–8075. Symes, A.J., Gearan, T., Eby, J., Fink, J.S., 1997. Integration of Jak-Stat and AP-1 signaling pathways at the vasoactive intestinal peptide cytokine response element regulates ciliary neurotrophic factor-dependent transcription. J. Biol. Chem. 272, 9648–9654. Talbot, R.T., Dunn, I.C., Wilson, P.W., Sang, H.M., Sharp, P.J., 1995. Evidence for alternative splicing of the chicken vasoactive intestinal polypeptide gene transcript. J. Mol. Endocrinol. 15, 81–91. Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., Higgins, D.G., 1997. The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 24, 4876–4882.
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Veterinary Immunology and Immunopathology journal homepage: www.elsevier.com/locate/vetimm
Research paper
Identification and characterization of the prepro-vasoactive intestinal peptide gene from the teleost Paralichthys olivaceus Bo-Hye Nam a,*, Young-Ok Kim a, Hee Jeong Kong a, Woo-Jin Kim a, Sang-Jun Lee a, Tae-Jin Choi b a b
Biotechnology Research Institute, National Fisheries Research and Development Institute, 408-1, Sirang-ri, Gijang-eup, Gijang-gun, Busan 619-902, South Korea Department of Microbiology, Pukyong National University, 599-1, Daeyeon 3-dong, Nam-gu, Busan 608-737, South Korea
A R T I C L E I N F O
A B S T R A C T
Article history: Received 25 February 2008 Received in revised form 9 October 2008 Accepted 16 October 2008
The gene encoding the prepro form of a vasoactive intestinal peptide (VIP) was cloned, and its structural organization and expression profiles were determined in the teleost Paralichthys olivaceus. The prepro-VIP encodes both a VIP and a peptide-histidineisoleucine (PHI). The VIP gene comprises six exons with two distinct peptides encoded on separate exons, i.e., PHI on exon III and VIP on exon IV. Several elements involved in cytokine-mediated activation are highly conserved in an 806-bp segment of the 50 flanking region: cAMP responsive elements (CREs), binding sites for nuclear factor IL-6 (NF-IL-6), activating protein-1 (AP-1), stimulating protein-1 (Sp-1), two IL-6 responsive element binding proteins (IL-6 RE-BPs), and signal transducers and activators of transcription (STAT). The mRNA transcripts in normally conditioned fish are expressed in the brain, intestine, stomach, pyloric ceca, spleen, and heart, but not in the muscle, liver, head or trunk kidneys, and gill. However, prepro-VIP mRNA expression in the spleen and head kidney is significantly up- and down-regulated when exposed to an artificial bacterial challenge by Edwardsiella tarda. These results suggest that the typical features of neuropeptide VIPs are evolutionarily conserved from non-mammalian vertebrates to mammals and that the flounder VIP plays an important role in the immune system, especially inflammatory processes. ß 2008 Elsevier B.V. All rights reserved.
Keywords: Vasoactive intestinal peptide Prepro-VIP mRNA Paralichthys olivaceus
1. Introduction Vasoactive intestinal peptide (VIP) is structurally and functionally similar to pituitary adenylate cyclase-activating polypeptide (PACAP), and both peptides belong to the glucagon and secretin superfamily, which also includes glucagon, glucagon-like peptides (GLP)-1 and -2, peptidehistidine-methionine (PHM; in vertebrates other than humans, peptide-histidine-isoleucine [PHI] occurs in place of PHM), growth hormone-releasing hormone (GRF), PACAP-related peptide (PRP), and glucose-dependent
* Corresponding author. Tel.: +82 51 720 2452; fax: +82 51 720 2456. E-mail addresses: [email protected], [email protected] (B.-H. Nam). 0165-2427/$ – see front matter ß 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2008.10.320
insulinotropic peptide (GIP) in humans. These peptides show high levels of sequence identity at their N-termini, which are the bioactive cores of these peptides, and have similar receptor structures and signaling pathways (Sherwood et al., 2000). These features indicate that the glucagon and secretin superfamily originated from a common ancestral gene. The VIP, PACAP, and glucagon genes encode two or three bioactive peptides, whereas the GRF, secretin, and GIP genes encode only a single bioactive peptide. In the past, it was believed that the PACAP precursor gene was encoded with GRF in non-mammals and that the mammalian PRPs evolved from GRF in nonmammals. However, recent information on the evolution of PRP in non-mammalian vertebrates shows that the GRF peptides that encode PACAP in non-mammals are in fact the counterparts of mammalian PRPs (Lee et al., 2007). The
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PACAP precursor gene or cDNA has been sequenced in several fish species, including sockeye salmon, Coho salmon, Chinook salmon, rainbow trout, Atlantic salmon, catfish, and zebrafish (Parker et al., 1993, 1997; McRory et al., 1995; Fradinger and Sherwood, 2000; Cardoso et al., 2007), while a few VIP precursor cDNAs have been isolated and reported recently for Takifugu (NP_001106661), medaka (AM154517), zebrafish (EU031789 for VIP-I and EU031790 for VIP-II), and goldfish (Tse et al., 2002). VIP peptide fragments have been reported for several fish species, including the spotted dogfish (Dimaline et al., 1987), cod (Thwaites et al., 1989), goldfish (Uesaka et al., 1995), rainbow trout (Wang and Conlon, 1995), bowfin (Wang and Conlon, 1995), and pallid sturgeon (Kim et al., 2001). VIP is a 28-amino acid peptide that was originally isolated from porcine intestinal extracts as a vasodilator (Said and Mutt, 1970). VIP was later identified as a multifunctional neuropeptide in the central and peripheral nervous systems. Recently, this pleiotropic neuropeptide has been shown to play a key role in the maintenance of neuroendocrine-immune communication (reviewed by Delgado et al., 2004). Some of the neuropeptides are released from the central nervous system via the hypothalamic-pituitary axis as hormones or pro-hormones and arrive in the lymphoid organs via the circulation. Lymphocytes are the primary source of VIP in lymphoid organs, which express and secrete VIP upon activation by various stimuli (Gomariz et al., 1994; Martinez et al., 1999). Using immunohistochemistry, several investigators have demonstrated the presence of VIPergic nerve fibers in both central (thymus) and peripheral (spleen, lymph nodes, and mucosal-associated lymphoid tissue) lymphoid organs (Bellinger et al., 1996; Felten et al., 1985; Fink and Weihe, 1988). During the last decade, VIP has been shown to be a potent anti-inflammatory factor that acts by regulating the production of both anti- and pro-inflammatory mediators, and has been identified as a potential candidate in the treatment of inflammatory and autoimmune disorders in mammals (Gomariz et al., 2001; Pozo et al., 2007). The physiologic roles of VIP and PACAP in teleosts have been described (Matsuda et al., 2005). VIP and PACAP are distributed in the brains of teleosts and affect the release of growth hormone, gonadotropin, and prolactin from cultured teleost pituitary cells in vitro (Brinca et al., 2003). However, the functions of VIP in the nervous and immune systems of teleosts remain to be resolved. To investigate the structural and functional evolution of VIP, and to lay the foundation for developmental studies, we isolated and characterized the VIP gene of the flounder Paralichthys olivaceus. 2. Materials and methods Flounder (P. olivaceus) was supplied from the Genetic and Breeding Research Center of NFRDI on Geoje Island (southern South Korea). The procedure for the peripheral blood leukocytes (PBLs) preparation and stimulation has been described previously (Nam et al., 2007). Briefly, PBLs were prepared by density gradient centrifugation
over a percoll gradient (1.072 g/ml) at 400 g for 20 min. Following centrifugation, PBL were carefully removed and washed by phosphate buffered saline (PBS). And then, isolated PBLs were adjusted to a concentration of 107 cells per ml and cultured in RPMI1640 containing 500 mg/ml of LPS and normal fresh medium (RPMI1640 without LPS) at 20 8C for 1, 3, and 6 h. Following each incubation time, PBLs were harvested, washed twice with PBS and stored at 80 8C until use. Total RNA was isolated from the control and LPS-stimulated PBL cells with TRIzol Reagent (Invitrogen) according to the manufacturer’s instruction. Poly(A)+ RNAs were purified from the total RNAs of treated and untreated flounder PBLs using the PolyATtract mRNA Isolation System (Promega) according to the manufacturer’s instruction. The suppression subtractive hybridization (SSH) technique (Diatchenko et al., 1996) was used to characterize new genes involved in the flounder PBLs stimulated by LPS. Two micrograms of poly(A)+ RNA isolated from treated and untreated culture were used as tester and driver, respectively. SSH was performed using the PCR-Select cDNA subtraction kit (BD Biosciences Clontech) according to manufacturer’s instruction. The amplified cDNA fragments were subcloned into the pGEM-T vector (Promega) for sequencing. 2.1. cDNA cloning and sequencing A clone derived by SSH, LSPSL5-C-11, which carries an 885-bp insertion, showed significant sequence homology to the C-terminal portion of the Xenopus laevis Vip-A protein gene (AAH43792). To isolate the full-length cDNA of this SSH clone, 50 -RACE PCR was carried out using the SMART RACE cDNA Amplification Kit (BD Biosciences). Two gene-specific primers (VIP 50 -RACE-1 and VIP 50 RACE-2) were designed based on the sequence of the LSPSL5-C-11 clone (Table 1). First-strand cDNA was synthesized with the same poly(A)+ RNA used for SSH using the protocol provided with the Smart RACE cDNA amplification kit. The primer sets of VIP 50 -RACE-1, VIP 50 RACE-2, and a nested universal primer supplied with the kit were used for 50 -RACE. The PCR products generated were purified and cloned into the pGEM-T vector (Promega). Ten clones from the 50 -RACE PCR products were sequenced, and the longest PCR product was selected for determining the transcription start site. The complete cDNA of the flounder VIP gene was compiled by overlapping the sequences of the SSH clone and the 50 -RACE PCR products. Table 1 Sequence-specific primers used for PCR. Name
Sequence 0
Description 0
VIP-ORF-F VIP-ORF-R VIP-GW-1
5 -ATGTTACAACGGACCGGCCC-3 50 -CACCTCAAAGAGGCAGTTGA-30 50 -GTCTGCGTTTGTCTCTTCCTCC-30
VIP-GW-2 GAPDH-RT-F GAPDH-RT-R
50 -CCAGGGTTGACTCATCTGATC-30 50 -TCCCATGTTCGTCATGGGCGTGA-30 50 -ATTGAGCTCAGGGATGACCTTG-30
ppVIP RT-PCR ppVIP genomic walking GAPDH RT-PCR
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2.2. Genomic DNA cloning and sequencing
2.5. Artificial bacterial infection of fish
Genomic DNA was prepared from flounder whole blood using 4 M TNES-urea buffer (Asahida et al., 1996) and purified with phenol/chloroform (Sambrook and Russell, 2001). Two specific primers (VIP-ORF-F and VIP-ORF-R) were designed based on the 50 - and 30 -end sequences of the VIP open reading frame (ORF; Table 1). Ten nanograms of genomic DNA were used as a template. PCR was performed beginning at 95 8C for 5 min, followed by 30 cycles of 95 8C for 30 s, 55 8C for 30 s, and 72 8C for 3 min, with a final 5 min at 72 8C. The PCR product was purified and cloned into the T-Easy vector (Promega). The resultant positive clone was sequenced using an automatic sequencer (ABI3100; Applied Biosystems). Putative binding sites for transcription factors within the 50 flanking sequence were identified using the TESS (Transcription Element Search Software; Schung and Overton, 1997) software. The 50 -flanking region of the flounder VIP gene was amplified from flounder genomic DNA using a Universal GenomeWalkerTM kit following the manufacturer’s protocol (BD Biosciences). Briefly, the primary PCR reaction was carried out using the adaptor primer (AP-1) provided in the kit plus a gene-specific primer (VIP-GW-1). The primary PCR product was used as the template for a nested PCR reaction using the nested adaptor primer (AP-2) and a nested gene-specific primer (VIP-GW-2). The PCR product was cloned into the T-Easy vector (Promega) and sequenced (ABI3100; Applied Biosystems).
The induction of flounder VIP expression was examined in the spleen, kidney, and intestine using RT-PCR following an artificial bacterial infection with Edwardsiella tarda (strain KFE isolated from flounder in South Korea). For the artificial infection, healthy juvenile flounders (mean weight 100 g) obtained from the Genetic and Breeding Research Center were used. The fish were anesthetized with MS-222 (3-aminobenzoic acid ethyl ester; Sigma) and infected with E. tarda by intraperitoneal injection of a sublethal dose (1.2 106 cells) suspended in PBS buffer. Tissues were collected from three fish at 0, 1, 3, 6, 12, 24, and 72 h post-injection, and frozen at 80 8C for RNA extraction.
2.3. Alignment and phylogenetic tree analysis The relevant sequences were retrieved from GenBank for multiple sequence alignments using ClustalX (Thompson et al., 1997). Sequence homology was calculated by GENETYX version 8.0 (SDC Software Development, Japan). A phylogenetic tree was created based on the amino acid distances between the aligned sequences using the Neighbor-Joining method with 1000 bootstrap replications using the MEGA software (version 3.0; Kumar et al., 2004). For signal peptide prediction, the SignalP software (http:// www.cbs.dtu.dk/services/SignalP/) was used.
3. Results 3.1. Identification and characterization of the flounder prepro-VIP cDNA The SSH clone LSPSL5-C-11, which carries a 885-bp insertion, showed significant sequence homology to the Cterminal portion of the Xenopus Vip-A protein gene (AAH43792), lacking the N-terminal sequence that encodes the signal peptide and the PHI peptide coding sequence. The N-terminal portion of flounder VIP was amplified from the first-stranded cDNA of LPS-stimulated flounder PBLs using 50 -RACE with a primer based on the LSPSL5-C-11 sequence (Table 1). The 50 -RACE PCR product was approximately 350 bp in length and contained a 111bp 50 -untranslated region (UTR) and the N-terminal portion of prepro-VIP. The complete ORF obtained by joining the sequences of LSPSL5-C-11 and the 50 -RACE PCR product gave a predicted 148-aa prepro-VIP, including a 21-aa signal peptide (Fig. 1). The 30 -UTR consisted of 519 bp, excluding the poly(A) tail, and it harbored three ATTTA sequence elements, typically found in cytokine mRNAs, which mediate RNA instability. The overall sequence identities between the flounder prepro-VIP and its counterparts in Takifugu (GenBank accession no. NP_001106661) and zebrafish (GenBank accession no. EU031789) were approximately 93% and 78%, respectively.
2.4. Detection of prepro-VIP mRNA 3.2. Genomic organization Total RNA sample from the brain, muscle, liver, intestine, stomach, phyloric ceca, head kidney, trunk kidney, spleen, gill, heart, and skin of healthy flounders was subjected to DNase I treatment (Invitrogen) for avoiding genomic DNA contamination before being converted to cDNA. First-strand cDNA synthesis was performed using the Advantage RT-for-PCR Kit (BD Biosciences). The gene-specific primers (VIP-ORF-F and VIP-ORF-R) for flounder VIP amplification were designed based on the VIP cDNA sequence. As an internal control, the flounder GAPDH housekeeping gene was amplified using specific primers (GAPDH-RT-F and GAPDH-RT-R). The RTPCR reaction parameters included an initial 5-min step at 95 8C, followed by 30 cycles of 94 8C for 30 s, 60 8C for 30 s, and 72 8C for 1 min, and a final 5 min at 72 8C.
To determine the genomic structure of the flounder VIP gene, we amplified the genomic DNA of the coding region by PCR and the 50 -flanking region using the genome walking method. Sequence analysis showed that the genomic DNA sequence of the flounder VIP gene obtained in the present study contains 4884 bp and is composed of six exons and five introns (GenBank accession no. EU496523). The five introns are 871, 139, 1014, 638, and 339 bp in length, respectively. Typical intronic splice motifs are observed at the 50 (GT)- and 30 (AG)-ends of each intron. The first exon represents a 50 -UTR sequence of 101 bp. The second exon codes for a putative signal peptide of 21 amino acids and for 11 additional amino acids. The third and fourth exons encode PHI and VIP, respectively.
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Fig. 1. Nucleotide sequence of the flounder prepro-VIP mRNA and the deduced amino acid sequence. The numbering of nucleotides and amino acids begins with ATG and methionine (M), respectively, and is shown at the left and right margins, respectively. The putative peptides PHI and VIP are shaded. Three ATTTA sequence motifs in the 30 -untranslated region are shown in bold. The nucleotide sequence has GenBank accession no. EU496522.
The fifth exon contains 112 bp, which encodes a spacer protein sequence of unknown function. The sixth exon directs the synthesis of the two amino acid sequences, contains a stop codon, and a 30 -UTR (Fig. 2A). The 806-bp 50 sequences upstream from the transcriptional start site contain the major regulatory sequences of the VIP gene (Fig. 3). Major regulatory motifs for key transcription factors were identified in the flounder VIP promoter analysis. These cis-elements include a TATA box (nt 28 to 34, upstream from the putative flounder VIP transcriptional start site), two cAMP-responsive element (CRE) motifs (CGTCA; nt 90 to 94 and nt 100 to 104), and a putative cytokine-responsive element (CyRE), which includes binding sites for nuclear factor IL-6 (NF-IL-6; nt 138 to 144), activating protein-1 (AP-1; nt 225 to 234), stimulating protein-1 (Sp-1; nt 408 to 416), two IL-6 responsive element binding proteins (IL-6-RE-BP; nt 293 to 298 and nt 460 to 466), and signal transducers and activators of transcription (STAT; nt 658 to 663).
closer relationship between fish prepro-VIP and other nonmammalian VIP than between fish prepro-VIP and mammalian VIP. 3.4. Prepro-VIP mRNA expression To determine the tissue distribution of the prepro-VIP mRNA in adult flounder, RT-PCR was performed on several tissues (brain, muscle, liver, intestine, stomach, pyloric ceca, head kidney, trunk kidney, spleen, gill, and heart). A PCR primer set amplified a ca. 440-bp transcript from the brain, intestine, stomach, pyloric ceca, spleen, and heart, but not from the muscle, liver, head and trunk kidneys, and gill (Fig. 5A). Analysis of the expression of the prepro-VIP mRNA in several tissues (spleen, head kidney, and intestine) was also carried out using RT-PCR following infection with E. tarda. The flounder prepro-VIP mRNA of the intestinal tissue was expressed constitutively, while its expression was clearly up- and downregulated in the spleen and kidney after infection with E. tarda (Fig. 5B).
3.3. Sequence alignment and phylogenetic analyses 4. Discussion The deduced amino acid sequence of VIP is highly conserved between teleosts and humans. The flounder VIP contains five substitutions out of 28 residues compared to mammalian VIP (Table 2). While the amino acid sequence of the flounder PHI peptide shows complete identity (100%) with both the fish PHI and Xenopus PHI, the VIP has identical sequence among fishes (Table 2). A phylogenetic tree was constructed based on the deduced prepro-VIP amino acid sequences of reported species using the Neighbor-Joining method. As shown in Fig. 4, the flounder prepro-VIP formed a cluster with non-mammalian species, such as chicken, Xenopus, and other fishes. This finding was further supported by a high bootstrap value, indicating a
A clone that encodes the prepro-VIP was isolated from a subtracted cDNA library derived from flounder PBLs that were stimulated with LPS. The flounder prepro-VIP possesses features typical of neuropeptides. The precursor peptide (prepropeptide) always has three components: a signal sequence of approximately 16–30 amino acid residues at the N-terminus, one or more neuropeptide sequences, and one or several spacers regions (Holmgren and Jensen, 2001). The flounder prepro-VIP in the present study contains two related peptides, PHI and VIP, and a spacer sequence that follows a 21-aa signal peptide. Few VIP alternative splice transcripts have been described. In
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Fig. 2. Comparison of the gene organization and deduced amino acid sequences of the flounder VIP and those of other species. (A) Organization of the VIP genes of the flounder, zebrafish, chicken, and human. The lengths of the exons and introns are not proportional, so that exons can be aligned between genes. Exon numbering follows that used for the mammalian genes. Open boxes indicate untranslated regions, and closed and shaded boxes indicate the coding regions of exons for each gene. The accession numbers of the VIP genomic sequences used in the GenBank database are as follows: flounder, EU496523; zebrafish I, NW_001877362; zebrafish II, NW_001877173; chicken, NC_006090; and human, M33027. SP, signal peptide; CP, cryptic peptide; PHM, peptidehistidine-methionine; VIP, vasoactive intestinal peptide; PHI, peptide-histidine-isoleucine. (B) Multiple amino acid sequence alignment of VIP precursors. Each segment encoded by a different exon is boxed and annotated. Spaces introduced to help understand the aligned sequences are indicated by hyphens. Asterisks indicate the stop codons. The accession numbers of the VIP precursor used are as follows: flounder, EU496522; zebrafish I, NM_001114553; zebrafish II, NM_001114555; mouse, EDL03569; human, AAH09794; chicken, AAA87896; and frog, AAH43792.
Fig. 3. Schematic representation of the 50 -flanking region of the flounder VIP gene. Major regulatory motifs for key transcription factors are represented, including a TATA box (nt 28 to 34, upstream of the putative flounder VIP transcription start site), two cAMP-responsive element (CRE) motifs (CGTCA; nt 90 to 94 and nt 100 to 104), and a putative cytokine-responsive element (CyRE), which includes binding sites for nuclear factor IL-6 (NF-IL-6; nt 138 to 144), activating protein-1 (AP-1; nt 225 to 234), stimulating protein-1 (Sp-1; nt 408 to 416), two IL-6 responsive element binding proteins (IL-6RE-BP; nt 293 to 298 and nt 460 to 466), and signal transducers and activators of transcription (STAT; nt 658 to 663). The CRE shown as a shaded box contains two 5-bp CGTCA motifs, which are shown in bold and underlined.
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Table 2 The amino acid sequence identity of other forms of the PHI and VIP peptides in comparison to the floundera.
PHI (27aa)
VIP (28aa)
a
Species Flounder Takifugu Zebrafish I Zebrafish II Medaka Goldfish 2A Goldfish 11A Xenopus Chicken Cattle Rat Mouse Dog Monkey Chimpanzee Human Flounder Takifugu Zebrafish I Zebrafish II Medaka Goldfish 2A Goldfish 11A Sturgeon 1 Sturgeon 2 Bowfin Trout Cod Dogfish Xenopus Chicken Cattle Rat Mouse Dog Monkey Chimpanzee Human
Amino acid sequence HADGLFTSGYSKLLGQLSARRYLESLI ........................... ........................... ...................KEY....L ........................... ...................KEF....L ...................KEY....L ........................... .....I...V..H..AK.AVK...H.. ....V...D..R.......KK...... ....V...D..R....I..KK...... ....V...D..R....I..KK...... ....V...DF.R.......KK...... ....V...DF.........KK.....M ....V...DF.........KK.....M ....V...DF.........KK.....M HSDAIFTDNYSRFRKQMAVKKYLNSVLT ............................ ............................ ....V.......Y.....A........A ............................ ....V.......Y.....A........A ....V.......Y.....A........S ............................ ...S........................ ............................ ............................ ....V.............A........A ....V.......I.........I..L.A ....V....................... ....V....................... ....V.....T.L............I.N ....V.....T.L............I.N ....V.....T.L............I.N ....V.....T.L............I.N ....V.....T.L............I.N ....V.....T.L............I.N ....V.....T.L............I.N
Identity (%) 100 100 100 85.2 100 85.2 85.2 100 66.7 81.5 77.8 77.8 77.8 81.5 81.5 81.5 100 100 100 85.7 100 85.7 85.7 100 96.4 100 100 89.3 82.1 96.4 96.4 82.1 82.1 82.1 82.1 82.1 82.1 82.1
Dots indicate identical amino acids and the alternative amino acid is indicated when the sequence differ.
goldfish, two alternative VIP transcripts have been reported, one of which contains both VIP and PHI and the other has PHI only (Tse et al., 2002), while in the chicken, a shorter transcript that lacks the PHI coding region has also been described (Talbot et al., 1995). The flounder VIP gene spans 4884 bp and consists of six exons and five introns. The 101-bp exon I consists of the 50 UTR of the gene, 105-bp exon II encodes the signal peptide, 105-bp exon III encodes PHI, 126-bp exon IV encodes VIP, 112-bp exon V contains the spacer region (cryptic peptide), and 528-bp exon VI contains the termination codon of the prepro-VIP mRNA and the 30 -UTR of the gene. However, this structural organization of the flounder VIP gene differs from that of other species in several ways. For example, the human VIP gene consists of seven exons and six introns, and the cryptic peptide (spacer region) is encoded on exon III. Two teleost VIP genes have been sequenced from zebrafish: GenBank accession no. NW_001877362 for VIP
type I and NW_001877173 for VIP type II. The structural organization of the zebrafish VIP type I gene shows highly similarity to the flounder VIP gene. However, VIP type II has a genomic structure similar to that of the human VIP gene (Fig. 2A). Interestingly, the chicken VIP has two potential cryptic peptides at the N- and C-termini, which are encoded by exons III and VI, respectively (Fig. 2A and B). Previously, the function of cryptic peptide was considered simply to increase the length of the molecule and help the precursor to fold before cleavage (Holmgren and Jensen, 2001). Recently, the concept of ‘‘cryptic peptides’’ has been gaining popularity to address bioactive peptide hidden within larger precursors that are liberated by the action of proteases (Dylag et al., 2008). Therefore, we considered that the cryptic peptide region of VIP precursor supply more information in the aspect of VIP gene evolution, and applied to phylogenetic analysis with the amino acid sequences of cryptic peptide regions. In
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Fig. 4. Phylogenetic analysis of the prepropeptide form of VIP. The numbers indicate the frequencies with which the phylogram topology represented here is replicated for every 1000 bootstrap interactions. The accession numbers of the prepro-VIP amino acid sequences are as follows: human, AAH09794; monkey, XP_001096218; chimpanzee, XP_527541; mouse, EDL03569; rat, EDL92841; cattle, AF503910; dog, XP_541155; chicken, AAA87896; frog, AAH43792; trout, BX910000; medaka, AM154517; zebrafish 1, CK362621; zebrafish 2, XM_001337417; Takifugu, NP_001106661; goldfish 2A and 11A, obtained from Tse et al., 2002; and flounder, EU496522.
addition, using the cryptic peptide region for phylogenetic analysis could be useful to illuminate the close related species thanks to the high sequence variation unlike PHI (or PHM) and VIP region, that have a low phylogenetic resolution due to their low variability (see the Table 2). The cryptic peptide sequences were analyzed phylogenetically using the Neighbor-Joining method (Fig. 6). The results reveal that the cryptic peptide derived from zebrafish VIP type II (zebrafish II CP) forms a cluster with CP-1 of chicken and Xenopus and the CPs of mammals, while zebrafish I CP groups with CP-2s and that of flounder. This suggests that the VIP gene was duplicated into types I and II in zebrafish, and that the type II VIP gene subsequently evolved into the VIP genes of other vertebrates. Furthermore, a cryptic peptide should be an important clue to explaining the evolution of VIP genes. Vasoactive intestinal peptide is produced by lymphoid and neural cells, including the central (thymus) and peripheral (spleen, lymph nodes, and mucosal-associated lymphoid tissue) lymphoid organs. The flounder preproVIP mRNA was detected in the brain (including pituitary gland), intestine, stomach, pyloric ceca, spleen, and heart, but not in the muscle, liver, kidney, gill, and skin of
normally conditioned fish. When fish were subjected to an artificial bacterial challenge, to study the potential role of VIP in inflammatory processes, different expression patterns of the flounder prepro-VIP mRNA were observed depending on the specific tissue analyzed (Fig. 5B). The flounder prepro-VIP mRNA maintained a constitutive level of expression in intestinal tissue despite infection. However, the flounder prepro-VIP mRNA expression was significantly upregulated in the spleen and kidney after infection, which implies that VIP expression is tissuespecific. This also indicates that flounder VIP plays an important role in the inflammatory processes of fish, similar to its role observed in mammals. Additional evidence for the immunomodulatory role of flounder VIP is demonstrated by the presence of certain sequences in the 50 -flanking region of the flounder VIP gene. The flounder VIP gene contains consensus sequences for two cAMP response elements (CREs) as well as a CyRE in the promoter region. Most CREs contain one or multiple copies of the conserved sequence motif CGTCA or TGACG, and several have been shown to act as enhancers (Silver et al., 1987; Fink et al., 1988). In the CRE of the flounder VIP gene, two CGTCA motifs are present in a 25-bp sequence located
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Fig. 5. RT-PCR analysis of prepro-VIP mRNA in flounder tissues. (A) RT-PCR was conducted using cDNA from healthy flounder tissue. (B) Expression profiles of prepro-VIP mRNA in spleen, head kidney, and intestine tissues sampled at 0, 1, 3, 6, 12, 24, and 72 h after an artificial bacterial challenge. As a positive control for RT-PCR, the GAPDH gene was amplified to determine template concentration.
between 90 and 114 nt from the transcription initiation site (Fig. 3). The spatial requirements for cooperation between the two CGTCA-containing domains of human VIP were tested by altering the orientation or distance between the two CGTCA motifs (Fink et al., 1988). The two CGTCA motifs in the human VIP CRE are located within a 15-bp sequence and have a palindromic structure. The flounder VIP CREs are encoded in a 25-bp sequence that includes the inverted orientation that results in 11–32% loss of CRE activity in the case of human VIP gene
expression. The presence of two adjacent functional CGTCA motifs within the VIP CRE is important for the proteinprotein interactions between two CGTCA-binding factors. The two domains of the VIP CRE may facilitate interaction between identical regulatory proteins, such as CRE binding proteins (CREBs). Further studies are necessary to determine the effects of orientation or distance between the two CGTCA motifs on the expression of the flounder VIP gene. The VIP CyRE is a complex regulatory element that is composed of binding sites for a variety of different
Fig. 6. Phylogenetic tree of the cryptic peptide of VIP. The tree was constructed using the Neighbor-Joining method with the MEGA 3.0 program. Bootstrap values were produced through an analysis of 1000 replications of the original dataset.
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transcription factors. Several studies have reported that there are functional STAT, AP-1, ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), IL-6-RE-BP, and NF-IL-6 sites within the 180-bp CyRE in the human VIP promoter region (Symes et al., 1993, 1994, 1995, 1997). We have identified a region of the flounder VIP CyRE that includes several putative transcription factor-binding sites for NF-IL-6, AP-1, two IL-6 RE-BPs, Sp-1, and STAT from 149 nt to 658 nt upstream of the transcriptional start site. This suggests that expression of the flounder VIP gene is regulated in a cytokine-dependent manner via CyRE. Further studies are needed to demonstrate unequivocally that the participation of cis-regulatory domains within the flounder VIP CyRE is required for full expression of the VIP gene. Acknowledgment This work is funded by a grant from the Nation Fisheries Research and Development Institute (RT-2008-BT-023). References Asahida, T., Kobayashi, T., Saitoh, K., Nakayama, I., 1996. Tissue preservation and total DNA extraction from fish stored at ambient temperature using buffers containing high concentration of urea. Fish. Sci. 62, 727–730. Bellinger, D.L., Lorton, D., Brouxhon, S., Felten, S., Felten, D.L., 1996. The significance of vasoactive intestinal polypeptide (VIP) in immunomodulation. Adv. Neuroimmunol. 6, 5–27. Brinca, L., Fuentes, J., Power, D.M., 2003. The regulatory action of estrogen and vasoactive intestinal peptide on prolactin secretion in sea bream (Sparus aurata L.). Gen. Comp. Endocrinol. 131, 117–125. Cardoso, J.C.R., Vieira, F.A., Gomes, A.S., Power, D.M., 2007. PACAP, VIP and their receptors in the metazoan: Insights about the origin and evolution of the ligand-receptor pair. Peptides 28, 1902–1919. Delgado, M., Pozo, D., Ganea, D., 2004. The significant of vasoactive intestinal peptide in immunomodulation. Pharmacol. Rev. 56, 249– 290. Diatchenko, L., Chris Lau, Y.-F., Campbell, A.P., Chenchik, A., Moqadam, F., Huang, B., Lukyanov, S., Lukyanov, K., Gurskaya, N., Sverdlov, E.D., Siebert, P.D., 1996. Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proc. Natl. Acad. Sci. U.S.A. 93, 6025–6030. Dimaline, R., Young, J., Thwaites, D.T., Lee, C.M., Shuttleworth, T.J., Thorndyke, M.C., 1987. A novel vasoactive intestinal peptide (VIP) from elasmobranch intestine has full affinity for mammalian pancreatic VIP receptors. Biochim. Biophys. Acta 930, 97–100. Dylag, T., Pachuta, A., Raoof, H., Kotlinska, J., Silberring, J., 2008. A novel cryptic peptide derived from the rat neuropeptide FF precursor reverses antinociception and conditioned place preference induced by morphine. Peptides 29, 473–478. Felten, D.L., Felten, S.Y., Carlson, S.L., Olschowka, J.A., Livnat, S., 1985. Noradrenergic and peptidergic innervation of lymphoid tissue. J. Immunol. 135, 755–765. Fink, J.S., Verhave, M., Kasper, S., Tsukada, T., Mandel, G., Goodman, R.H., 1988. The CGTCA sequence motif is essential for biological activity of the vasoactive intestinal peptide gene cAMP-regulated enhancer. Proc. Natl. Acad. Sci. U.S.A. 85, 6662–6666. Fink, T., Weihe, E., 1988. Multiple neuropeptides in nerve supplying mammalian lymph nodes: messenger candidates for sensory and autonomic neuroimmunomodulation. Neurosci. Lett. 90, 39–44. Fradinger, E.A., Sherwood, N.M., 2000. Characterization of the gene encoding both growth hormone-releasing hormone (GRF) and pituitary adenylate cyclase-activating polypeptide (PACAP) in the zebrafish. Mol. Cell. Endocrinol. 165, 211–219. Gomariz, R.P., Leceta, J., Garrido, E., Garrido, T., Delgado, M., 1994. Vasoactive intestinal peptide (VIP) mRNA expression in rat T and B lymphocytes. Regul. Pept. 50, 177–184. Gomariz, R.P., Martinez, C., Abad, C., Leceta, J., Delgado, M., 2001. Immunology of VIP: a review and therapeutical perspectives. Curr. Pharm. Des. 7, 89–111.
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