Fish & Shellfish Immunology 28 (2010) 504e509 Contents lists available at ScienceDirect Fish & Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi Short communication Sequence analysis, characterization and tissue distribution of channel catfish (Ictalurus punctatus Rafinesque, 1818) myeloperoxidase cDNA Hung-Yueh Yeh*, Phillip H. Klesius United States Department of Agriculture, Agricultural Research Service, Aquatic Animal Health Research Unit, 990 Wire Road, Auburn, AL 36832-4352, United States article info abstract Article history: Myeloperoxidase (EC 1.11.1.7), a heme-containing lysosomal glycoprotein, is found predominantly in Received 27 October 2009 azurophilic granules of neutrophils. This enzyme upon activation catalyzes hydrogen peroxide in the Received in revised form presence of various halide ions to form hypohalous acids. Subsequently, these reagents are able to kill 12 December 2009 the invading microorganisms. In this study, we report the identification, characterization and expression Accepted 12 December 2009 analysis of the channel catfish myeloperoxidase transcript. The full-length nucleotide sequence of channel Available online 24 December 2009 catfish myeloperoxidase cDNA had 3157 nucleotides, including an open reading frame, which appears to encode a putative peptide of 771 amino acid residues with a calculated molecular mass of 87.14 kDa. By Keywords: fi Channel catfish comparison with the human counterpart, the channel cat sh myeloperoxidase peptide can be divided into Ictalurus punctatus domains and has conservative features, including peroxidase catalytic sites, covalent linkage sites for Myeloperoxidase the heme group and all cysteine residues. The channel catfish myeloperoxidase transcript was detected by MPO RT-PCR in anterior kidneys, where the major leukocyte population is neutrophil precursors. Reagent Edwardsiella ictaluri development and the role of this enzyme in Edwardsiella ictaluri infection are under investigation. Published by Elsevier Ltd. 1. Introduction neutrophils, and in much lower amounts in monocytes and some tissue macrophages [6e11]. Upon activation, neutrophils produce Peroxidases, distributed ubiquitously in both prokaryotes and hydrogen peroxide and release myeloperoxidase from the granules eukaryotes, catalyze hydrogen peroxide into water and various organic that the latter catalyzes the former in the presence of chloride ions and inorganic substrates [1]. According to the recently published to form hypochlorous acid [7,12e15]. Subsequently, this reagent peroxidase databases [2,3],therearemorethan6000peroxidase is able to kill the invading microorganisms [6,16]. However, mye- sequences from over 940 organisms. The peroxidases can be classified loperoxidase has also been detected in microglia and macrophages into two large groups: heme-containing and non-heme-containing surrounding pathological lesions of multiple sclerosis and Alz- peroxidases [1e3]. The latter contains alkylhydro-, glutathione-, halo-, heimer's disease [17,18]. Recently, the studies of this enzyme have NADH-peroxidases, peroxiredoxins and manganese catalases. The been focused on the roles in chronic inflammation [see references former includes catalases, dyp-type peroxidases, di-heme cytochrome [11,19,20] for reviews]. C peroxidases, haloperoxidases, and animal and non-animal peroxi- In teleost fish, the myeloperoxidase activity has been detected in dases. The animal peroxidases are further classified and included channel catfish [21], black bullhead (Ameiurus melas Rafinesque,1820) “mammalian” and “non-mammalian vertebrate” peroxidases [2,3]. [22], Indian major carps (including Cirrhinus mrigala Hamilton, After we and others identified channel catfish peroxiredoxins 1822, Catla catla Hamilton,1822 and Labeo rohita Hamilton,1822) [23], (non-heme-containing peroxidases) [4,5], we found another perox- zebrafish (Danio rerio Hamilton-Buchanan, 1822) [24] and goldfish idasedmyeloperoxidasedESTwasup-regulatedintheearlystageof (Carassius auratus Linnaeus, 1758) [25].Thefish myeloperoxidase Edwardsiella ictaluri infection in catfish ovary cell line (CCO cells) [Yeh gene transcript has been cloned in zebrafish [26,27],andtheprotein and Klesius, unpublished data]. has been purified from turbot (Psetta maxima Linnaeus, 1758) anterior Myeloperoxidase (EC 1.11.1.7), a heme-containing lysosomal kidney neutrophils [28]. However, the roles of this enzyme in fish glycoprotein, is found predominantly in azurophilic granules of infected with various microorganisms have not been characterized. In this study, the channel catfish myeloperoxidase transcript was fi * Corresponding author. Tel.: þ1 334 887 3741; fax: þ1 334 887 2983. completely sequenced and characterized, and the expression pro le in E-mail address: [email protected] (H.-Y. Yeh). varioustissueswasdetermined. 1050-4648/$ e see front matter Published by Elsevier Ltd. doi:10.1016/j.fsi.2009.12.007 H.-Y. Yeh, P.H. Klesius / Fish & Shellfish Immunology 28 (2010) 504e509 505 2. Materials and methods CA). Chromatograms were edited, trimmed and analyzed also at the USDA ARS Genomics and Bioinformatics Research Unit in Stone- 2.1. Fish ville, MS. The amino acid sequence of channel catfish myeloper- oxidase was deduced from nucleotide sequence by using Transeq Channel catfish (NWAC103 strain, weighed 25e30 g) were used [30], aligned with other myeloperoxidase amino acid sequences in this study. Prior to aseptical tissue excision, the catfish were deposited in GenBank using ClustalW2 [31]. ExPASy server [32] was euthanized by immersion in 300 mg/ml of tricaine methanesulfo- used to calculate the myeloperoxidase peptide molecular mass nate according to the Guidelines for the Use of Fishes in Research and pI, and to analyze the myeloperoxidase N-glycosylation sites. [29]. Spleen, anterior kidney, liver, intestine, skin and gill were The signal peptide site in the amino acid sequence was detected collected and immersed in 1 ml of TRI reagent (Molecular Research with SignalP 3.0 software [33]. Phylogenetic relationships of mye- Center, Inc., Cincinnati, OH). The Institutional Animal Care and Use loperoxidase amino acid sequences from various species were Committee of the USDA ARS Aquatic Animal Health Research Unit in analyzed with MEGA 4.0 software [34] based on the ClustalW2 Auburn, AL approved the experiment. alignment results. 2.2. RNA isolation and rapid amplification of cDNA ends (RACE) 2.5. RT-PCR Total RNA from channel catfish tissues was isolated by using a Tri Two-step RT-PCR assays were used to profile myeloperoxidase reagent (Molecular Research Center, Inc.) according to the manu- gene transcripts in various tissues as described previously [35e38]. facturer's instruction. The quality and quantity of total RNA were b-Actin was used as an internal control. The amplified products determined by using RNA 1200 chips on an Agilent Bioanalyzer were analyzed by 2% agarose gel electrophoresis and stained with (Agilent Technologies, Santa Clara, CA). A GeneRacer kit (Invitrogen, ethidium bromide. Images were recorded by a KODAK Gel Logic 440 Carlsbad, CA) was used to obtain full-length 50- and 30-end of cDNA Imaging System (version 4.0.3) (Eastman Kodak, Rochester, NY), according to the manufacturer's instruction. Total RNA (5 mg) from and processed with ImageJ software (version 1.41) [39]. three catfish anterior kidneys were used for this RACE construction. 3. Results and discussion 2.3. Myeloperoxidase gene amplification The full-length cDNA sequence of channel catfish myeloperox- 0 0 idase consists of 3157 nucleotides, including a 90-nucleotide Both specific full-length 5 - and 3 -RACE of the myeloperoxidase 50-untranslated region (UTR), an open reading frame and a 741- gene transcript were PCR amplified. The PCR reaction mixtures (50 ml nucleotide 30-UTR (GenBank accession no. GQ429001). The 30-UTR per reaction) contained the following reagents (in final concentra- has a mRNA instability sequence (attta) and a polyadenylation tions): 1Â Prime STAR PCR buffer (TaKaRa, Madison, WI), 200 mM signal sequence (tataaa) 168 and 86 nucleotides upstream of dNTP mix (TaKaRa), 300 mM each of gene specific primer and Gen- a 36-nucleotide polyadenylation tail, respectively. The human eRacer primer, 1.25 U Prime STAR HS DNA polymerase (TaKaRa) myeloperoxidase gene also uses the tataaa sequence as the poly- and 1 ml of cDNA template (equivalent to 250 ng RNA input). The adenylation signal [40]. The open reading frame appears to encode amplification was performed on a GeneAmp PCR System 9700 a 771 amino acid peptide with a calculated molecular mass of 87.14 thermocycler (Applied Biosystems, Foster City, CA) according to the kDa and a pI of 7.75 at pH 7.0. The peptide has five potential protocol described in the GeneRacer manual. The primers for PCR N-glycosylation sites at Asn111, Asn181, Asn379, Asn426 and Asn614 amplification are listed in the Table 1. The amplified PCR products (numbering after channel catfish), which may have a critical role were purified by agarose gel electrophoresis, and ligated into the pSC Ò in peroxidase activity. Castro et al. [28] reported that the turbot vector, followed by transformation of the vectors into the Solo Pack myeloperoxidase loses its peroxidase activity significantly, after competent Escherichia coli cells (Agilent Technologies) according to this enzyme is treated with peptide-N-glycosidase F to remove the manufacturer's instruction. At least six colonies per PCR product N-linked carbohydrates. This deglycosylation modification may were randomly selected for DNA