Fish & Shellfish Immunology 18 (2005) 371e380

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Molecular cloning of leukocyte cell-derivedchemotaxin 2 in rainbow trout

Petros A. Kokkinos, Alexandra Kazantzi, Georgia Sfyroera1,Ioannis K. Zarkadis*

Department of Biology, School of Medicine, University of Patras, Rion 26500 Patra, Greece

Received 13 April 2004; revised 16 August 2004; accepted 21 September 2004

Available online 19 January 2005

Abstract

In humans, leukocyte cell-derived chemotaxin 2 (LECT2) is a 16 kDa chemotactic protein that consists of 133 amino

acids and three intramolecular disulphide bonds. Although it was originally demonstrated to have a chemotacticfunction in vitro, recent data sustain a further multifunctional role of LECT2 that extends from cell growth, dif-ferentiation, damage/repair process and carcinogenesis to autoimmune diseases. The in vivo function of LECT2 protein

still remains obscure. In order to study the phylogeny of LECT2, a full-length cDNA clone of LECT2 gene, 720 bp insize, was isolated in rainbow trout (Oncorhynchus mykiss). Its deduced amino acid sequence of 156 residues, presents 40,45 and 61% overall identity to human, mouse and carp LECT2 proteins, respectively. In contrast to mammalianLECT2 protein, trout LECT2 protein reveals two potential N-glycosylation sites. Phylogenetic analysis shows that

trout LECT2 is clustered with the known homologous proteins. Trout LECT2 mRNA is predominately expressed inliver and spleen, showing lower expression in kidney, intestine, heart and brain.� 2004 Elsevier Ltd. All rights reserved.

Keywords: Rainbow trout; LECT2; Cloning; Chemotaxis; Neutrophil; Chemotactic factor; Evolution; Phylogeny

1. Introduction

Chemotactic factors released from inflammatory sites play an important role in activating neutrophilfunctions [1]. Leukocyte cell-derived chemotaxin 2 (LECT2) was first purified from culture supernatants ofphytohaemagglutinin-activated human T-cell leukemia SKW-3 cells, as a new chemotactic factor to human

* Corresponding author. Tel.: C30 2610 997621; fax: C30 2610 991769.

E-mail address: zarkadis@med.upatras.gr (I.K. Zarkadis).1 Present address: Protein Chemistry Laboratory, Department of Pathology and Laboratory Medicine, University of Pennsylvania,

402 Stellar Chance Building, Philadelphia, PA 19104, USA.

1050-4648/$ - see front matter � 2004 Elsevier Ltd. All rights reserved.

doi:10.1016/j.fsi.2004.09.004

372 P.A. Kokkinos et al. / Fish & Shellfish Immunology 18 (2005) 371e380

neutrophils [2]. Human LECT2, a 16-kDa basic protein, consisting of 133 amino acid residues and threeintramolecular disulphide bonds, is specifically expressed in the adult and fetal livers [3]. Although LECT2was originally demonstrated to have a chemotactic function in vitro, recent data sustain a furthermultifunctional role extending from cell growth, differentiation, damage/repair process and carcinogenesisto autoimmune diseases [4e8]. Its in vivo function is still unknown.

LECT2 genes have been reported for various organisms in addition to humans, such as bovine (Bostaurus) [3], mouse (Mus musculus) [9], and carp (Cyprinus carpio) [10]. LECT2 sequences of rat (Rattusnorvegicus) and teleost tetraodon (Tetraodon nigroviridis) are deposited in databases with accessionnumbers XP_341487 and CAAE01014623.1, respectively. Bovine and mouse LECT2 seem to be identical tochondromodulin-II, a growth-promoting factor that stimulates the proliferation of chondrocytes andosteoblasts [11,12]. Moreover, LECT2 is homologous to the repeated units of mim-1 (myb-induced myeloidprotein-1), a chicken (Gallus gallus) protein whose biological function has not been identified [13,14],demonstrating their probable common origin. The presence of a hypothetical LECT2 gene in theCeanorhabditis elegans genome (accession number: AAA68720) suggests the ancient origin of LECT2 gene.

In this report, the molecular cloning of a full-length cDNA encoding the trout LECT2 protein, thepresence of the LECT2 gene in the trout genome as well as the expression of the corresponding mRNA introut tissues are described. Finally, the phylogenetic relations of trout LECT2 with the known homologousproteins from different species are examined.

2. Materials and methods

2.1. Chemicals

Restriction enzymes were obtained from New England Biolabs Inc. (Beverly, MA) and Minotech (Crete,Greece). The nylon membranes for hybridisation were provided by Biorad (Hercules, CA). Chemicals forautomated sequencing were provided by Amersham Biosciences (UK).

2.2. RNA isolation and cDNA library construction

Total liver cDNA was prepared from trout liver RNA extracted as previously described [15]. Using thepolyA Tract mRNA kit (Promega, Madisson, WI), polyadenylated RNA was obtained. Accordingly,double-stranded cDNA was synthesised from 1 mg of polyAC RNA, using cDNA synthesis kit(Amersham), and cDNAs over 500 bp were cloned in EcoRI digested lambda gt11 arms. The Y1090 strainEscherichia coli served as host for infection by lambda gt11 phages.

2.3. Cloning of trout LECT2

2.3.1. Trout LECT2 probe isolationDegenerated oligonucleotides were designed based on conserved regions of known LECT2 amino acid

sequences from different species, sense 20-mer: 50-GTN TAY GCN CCN GAR GAY GG-30 based onVYAPEDG amino acids and antisense 20-mer: 50-GT NGG YTC NSW NSW YTC YCA-30 based onCDSSDPT amino acids (the mixtures of nucleotides are represented by NZA, G, C and T; YZC and T;RZA and G; WZA and T; SZG and C). These primers were subsequently applied in a RT-PCRreaction using a Robus T I kit (Finnzymes), and as template 500 ng total trout liver RNA. The program wasas follows: 50 (C for 30 min, 95 (C for 15 min and 30 cycles of 95 (C for 1 min, 46 (C for 1 min and 72 (C

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for 30 s, followed by a final extension at 72 (C for 5 min. The PCR product of the expected size (260 bp)was gel purified, cloned into the pGEM-T vector (Promega) and sequenced.

2.3.2. Screening of a trout liver cDNA libraryThe 260 bp PCR fragment was labeled with 32P, using a random-primed DNA labeling kit (Amersham

Biosciences Corp). Lambda gt 11 recombinant phages, 1.5! 105, from a trout liver cDNA library werescreened under high-stringency conditions (65 (C). Positive plaques were cultured and the recombinantphages DNA were isolated. After digest with EcoRI, the longest insert was subcloned into the pGEM-3vector and sequenced.

2.4. Sequencing of LECT2

DNA sequence analysis of the trout LECT2 cDNA clone was performed by the dideoxy chain method(Amersham Sequenase kit) using the Vistra Sequencer 725 (Applied Biosystems) [16]. All sequences weredetermined at least twice for both strands.

2.5. Computer analysis

Analysis and assembly of data derived from DNA sequencing were performed using the Gene Tool Litesoftware. Image analysis was carried out with Kodak Digital Science (Electrophoresis Documentation andAnalysis System 120). Search for similarities with known genes was performed using BLAST software.Multiple alignments of LECT2 molecules from various species were carried out using the program ClustalW 1.5 [17]. Aligned sequences were used to create a phylogenetic tree in the program TreeView. The treewas constructed by the neighbour-joining method (MEGA) with pairwise deletion and 100 bootstrapreplicates [18].

2.6. Southern blot hybridisation

Trout genomic DNA (12 mg) was digested with BamHI, EcoRI, HindIII and PstI restriction endo-nucleases, electrophoresed on a 0.8% agarose gel, transferred onto a nylon membrane (Zeta-Probe, Biorad)and hybridized using as probe the 32P-labelled PCR fragment (260 bp) at 65 (C for 16 h. The membranewas washed twice with 40 mM sodium phosphate buffer (pH: 7.2) and 1 mM EDTA in 5% SDS, at roomtemperature for 15 min. The X-ray film was developed after 5 days of exposure.

2.7. Tissue mRNA expression of trout LECT2

2.7.1. Northern blot analysisTotal RNA was extracted from trout tissue samples (brain, intestine, kidney, liver, and spleen), using the

RNA extraction kit (Promega). Twelve micrograms of total RNA, was denatured with formamide,subjected to electrophoresis on a 1% agarose gel and transferred onto a nylon membrane (Zeta-Probe,Biorad). After prehybridisation at 43 (C for 30 min in a solution containing 50% formamide, 0.12 Msodium phosphate (pHZ 7.2), 0.25 M sodium chloride, 7% SDS and 1 mM EDTA, the membrane washybridized at 43 (C for 16 h, using the 32P radio-labelled probe described in the previous section. Finally,the membrane was washed twice, at room temperature, in 2! standard sodium citrate and 0.1% SDSbuffer for 15 min. The X-ray film was developed after 2 days of exposure.

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2.7.2. RT-PCRFifty nanograms of total RNA from brain, heart, intestine, kidney, liver and spleen were reverse-

transcribed using a Robus T I kit (Finnzymes) with primers TLECTF2: 50-TTGTCAGAGTGTGAGATGGT-30 and TLECTR2: 50-GAAGAACTTGGTGGGGTCAG-30. The program was as follows:50 (C for 30 min and 95 (C for 15 min followed by 30 cycles of 95 (C for 45 s, 56 (C for 45 s, 72 (C for45 s and a final extension at 72 (C for 10 min. An internal PCR control was performed using primersdesigned to amplify a fragment of the housekeeping beta-actin gene (beta-actin F: 50-CACCTTCTACAATGA GCTGC-30, beta-actin R: 50-AGGCAGCTCGTAGCTCTTCT-30).

3. Results

3.1. Isolation and sequence analysis of full-length cDNA clone of trout LECT2

Using the degenerated oligonucleotide primers mentioned above, a 260 bp product was amplified. Itsprimary structure of the deduced amino acid sequence showed 68 and 45% identity to carp and humanLECT2, respectively, and was therefore identified as trout LECT2. After screening of a trout liver cDNAlibrary with the PCR-amplified probe, the longest insert was isolated and sequenced. The full-length cDNAclone was 720 bp in size, encoding a potent protein of 156 amino acid residues with an open reading frameof 468 bp, comparable in size to that of carp, mouse and human LECT2 (160, 151 and 151 amino acidresidues, respectively). The trout LECT2 cDNA clone presents a Met initiating codon at nucleotide 34,followed by a hydrophobic sequence, characteristic of a putative signal peptide sequence (Met-1 to Cys-19).Moreover, two typical polyadenylation signals are present in a 210 bp 30 UTR region (Fig. 1). TroutLECT2 putative protein reveals sites for phosphorylation and myristylation, as well as two sites forpotential N-glycosylation (N-X-S/T). The first potential glycosylation site starts at position 31 (Asn-Ser-Ser) and the second one starts at position 97 (Asn-Leu-Ser).

3.2. Alignment of the trout LECT2 deduced amino acid sequence

The deduced amino acid sequence of trout LECT2 was aligned with the entire sequence of knownLECT2 proteins from other organisms, using the CLUSTAL W software. Trout LECT2 was also alignedwith chicken mim-1, a myb target gene that is expressed in normal granules of promyelocytes. The mim-1product consists of imperfect direct repeats and was divided into two domains a and b, so as to maximisethe alignment (Fig. 2).

Following alignment analysis, trout LECT2 showed the highest score of amino acid homology withtetraodon and carp LECT2, with 65 and 61% identity, respectively, followed by chicken MIMI-b andchicken MIMI-a with 49 and 40% identity, respectively. The overall identity of trout LECT2 to chickenmim-1 was 40% (data not shown). Accordingly, trout LECT2 showed 45, 41 and 40% identity to mouse,bovine and human LECT2, respectively, and the lowest score of homology (32%) was shown with theC. elegans hypothetical LECT2 protein. Based on amino acid identitiesesimilarities, a phylogenetic treeanalysis was constructed (Fig. 3).

3.3. Southern, Northern and RT-PCR analyses

In order to elucidate whether trout LECT2 gene exists in multiple copies in the trout genome, Southernblot analysis was carried out. More than one band in each lane was recognised after digestion of troutgenomic DNA with four restriction endonucleases: BamHI, EcoRI, HindIII and PstI (Fig. 4).

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Fig. 1. Complete nucleotide and deduced amino acid sequences of trout LECT2. The numbers of the nucleotide and amino acid

sequences are on the left side. Asterisk indicates stop codon; ‘‘ ’’ indicates a putative signal peptide of 19 residues; ‘‘ ’’

represents the putative poly-adenylation signal; ‘‘ ’’ represents the putativeN-glycosylation site; ‘‘ ’’, ‘‘ ’’ and ‘‘ ’’ represent

phosphorylation sites for serine, threonine and tyrosine, respectively; ‘‘ ’’ represents N-myristylation sites.

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To examine the tissue distribution profile of trout LECT2 mRNA, northern blot and RT-PCR analyseswere performed, using total RNA from trout brain, heart, intestine, kidney, liver, and spleen. The troutLECT2 mRNA is expressed highly in liver and weakly in spleen, identified as a single hybridising band of0.7 kb in size (Fig. 5). Although the results of expression in the other tissues were negative in northern blot,

Fig. 2. Alignment of the deduced amino acid sequence of trout LECT2 (TroutLECT2) with human LECT2 (HumanLECT2), bovine

LECT2 (BovineLECT2), mouse LECT2 (MouseLECT2), carp LECT2 (CarpLECT2), tetraodon nirigans unnamed protein

(TetraodonPROT), chicken MIMI a and b (MIMI-a and MIMI-b), and C. elegans hypothetical LECT2 protein (CElegLECT2).

Sequences were aligned with Clustal W program. ‘‘ ’’ shows the conserved cysteine residues, ‘‘*’’, the identity of amino acid residues,

‘‘:’’, the conserved substitutions, and ‘‘.’’, the semi-conserved substitutions observed. Dashes indicate gaps introduced for optimal

alignment.

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RT-PCR analysis, where a 420 bp fragment of LECT2 mRNA was amplified, showed that trout LECT2gene is also expressed at lower amounts in brain, heart, intestine and kidney (Fig. 6).

4. Discussion

In the present study, the nucleotide and deduced amino acid sequence of the full-length cDNA clone ofrainbow trout LECT2 with a predicted open reading frame of 156 amino acids, including in the N-terminus,a secretory signal sequence of 19 amino acids is reported. Trout LECT2 belongs to the LECT2 proteinfamily and shows several differences concerning certain motifs, from the other known chemotactic factors.The deduced trout LECT2 protein possesses sites for phosphorylation and myristylation, and as the carpand tetraodon counterparts, two sites for potential N-glycosylation (Asn-X-Ser/Thr), which are absent inmammalian LECT2. Surprisingly, trout and tetraodon LECT2 sequences possess 4 cysteine residues(Fig. 2), forming probably two intramolecular disulphide bonds, in contrast to carp and mammaliancounterparts (six cysteine residues and three disulphide bonds).

Phylogenetic tree analysis, based on the amino acid sequence alignment, showed that trout LECT2protein is clustered primarily with other teleost LECT2 molecules, and secondary with the counterpartsfrom different species. LECT2 and chicken mim-1 genes seem to have a common origin, evolving probablyfrom a common ancestor [13]. Moreover the presence of a homologe sequence in C. elegans genome

Fig. 3. Phylogenetic tree analysis of the aligned proteins with the Clustal W program. The relationships among the various components

were analysed by the neighbour-joining method. Numbers on the branches indicate percent bootstrap confidence values from 100

replicates.

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(137e307 residues of a 472 aa ORF), suggests the ancient origin of LECT2 gene, since its appearance seemsto have been established before the divergence of proteostomes and deuterostomes.

Southern blot analysis reveals two hybridising bands in each lane, suggesting either that the LECT2 geneis duplicated in trout genome, or LECT2 gene intron/s have recognition sites for the used enzymes. The first

Fig. 5. Northern blot analysis showing trout LECT2 mRNA expression in various trout tissues. The 28S and 18S rRNA subunits,

signed on the right, serve as internal weight markers. The molecular weight standards are shown on the left.

Fig. 4. Southern blot analysis of trout genomic DNA using a trout LECT2 specific probe. The molecular weight standards are shown

on the left.

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hypothesis could be sustained by the fact that trout is a tetraploid organism under diploidisation, and geneduplication is a common fact in its genome. Northern blot and RT-PCR analyses provided a tissuedistribution profile of trout LECT2. Trout LECT2 mRNA is expressed mainly in liver and spleen.Furthermore, RT-PCR analysis, as more sensitive method, showed lower expression of trout LECT2 genein heart, intestine, kidney and brain, tissues that were negative in Northern analysis. The LECT2 tissueexpression profile shows wider distribution in trout than is known in mammals [3].

The characterisation of LECT2 counterparts in invertebrates could enlighten the molecular evolution ofLECT2 gene. It seems that through millions of years, LECT2 was maintained in a quite conservative way,supporting a central and important role of its product. The functional properties of teleost LECT2 genesstill remain to be deciphered.

Acknowledgments

This work was supported by grants, ‘‘K.Karatheodori’’ (1959) from Res. Com. University of Patras, andEmpeirikeio Foundation (6351), Athens, Greece. GeneBank accession number of trout LECT2: AF363272.

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