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Veterinary Immunology and Immunopathology 140 (2011) 335–340

Contents lists available at ScienceDirect

Veterinary Immunology and Immunopathology

journa l homepage: www.e lsev ier .com/ locate /vet imm

echnical report

xpressed sequence identification and characterization of the cDNAor Interleukin-4 from the mitogen-stimulated lymphoid tissue of a

arsupial, Macropus eugenii

auren Young ∗

arsupial Immunology Research Laboratory, Centre for Environmental Management, CQUniversity, Bruce Highway, Rockhampton,ueensland 4702, Australia

r t i c l e i n f o

rticle history:eceived 25 March 2010eceived in revised form6 November 2010ccepted 8 December 2010

eywords:ytokine

nterleukin-4acropus eugeniiarsupial

a b s t r a c t

Very few cytokines that are important to the understanding of T helper cell function arecharacterized in marsupials. Expression of a 645 bp cDNA product that codes for a pre-dicted Interleukin-4 peptide of 157 amino acids was detected in the lymph node tissues ofMacropus eugenii, the tammar wallaby. Using Rapid Amplification of cDNA Ends, both 5′-and 3′-untranslated regions were identified and a polyadenylation signal and three mRNAinstability motifs associated with secreted cytokine molecules were also present. The trans-lated cDNA sequence has a putative signal peptide of 24 amino acids, a predicted secondarystructure that is consistent with the short-chain alpha-helical cytokine family and 82% con-servation of residues associated with the Interleukin-4 family sequence motif. Comparisonsof wallaby nucleotide and predicted peptide sequences with the coding domains of other

allaby vertebrate species demonstrate the diversity within this gene family; with nucleotide andamino acid identities of 74% and 59% with opossum, 52% and 32% with human and 38%and 19% with chicken homologues respectively. Despite these differences in sequence con-servation, the putative Macropus eugenii Interleukin-4 mature peptide contains conservedstructural motifs and predicted receptor-binding residues that suggest that it may retain

ies asso

functional propert

Naïve CD4+ T helper (Th) cells may differentiate intone of at least four major Th cell populations; Th1, Th2,h17 and induced regulatory (iTreg) T cells (Zhu and Paul,008). Th1 and Th2 subsets produce immunoregulatoryolecules, the cytokines, which determine the nature of

he immune response to pathogens. IL-4, a classical Th2ytokine that influences B cell responses, is a memberf the short chain helical cytokine family. Members of

his immunoregulatory family are soluble, secreted pro-eins that possess four alpha helices (�A–D) folded in anp–up–down–down topology and which are joined by twoross-over connections of varying lengths (Hill et al., 2002).

∗ Tel.: +61 7 4923 2556; fax: +61 7 4930 6875.E-mail address: l.young@cqu.edu.au

165-2427/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.vetimm.2010.12.006

ciated with this important Th2 cytokine in other mammals.© 2010 Elsevier B.V. All rights reserved.

Originally identified as B cell growth factor (Paul, 1989),IL-4, together with IL-2 (a Th1 cytokine), plays an instruc-tional role in the activation of CD4 T cells to expressGATA-3, the master transcription factor for Th2 differenti-ation (Zhu and Paul, 2008). IL-4 upregulates expression ofantigen recognition and receptor molecules such as MajorHistocompatibility Class II (MHC Class II), IgM and CD23,the low affinity receptor for IgE (Alms et al., 1996), and isessential for the immunoglobulin class switch from IgG toIgE.

While marsupials share major antibody isotypes with

eutherian mammals, there are differences in the onset andmagnitude of primary and secondary antibody responsesthat remain unexplained (Harrison and Wedlock, 2000).Since IL-4 plays a significant role in B cell activation andregulation, the study of this cytokine in metatherian

nd Imm

336 L. Young / Veterinary Immunology a

(marsupial) mammals in clearly warranted. To date, theexpressed cDNA sequence for IL-4 has not been reportedin marsupials, although the Th2 associated cytokines, IL5(Hawken et al., 1999) and IL10 (Wedlock et al., 1998)have both been characterized at the molecular level.Indeed, in a review of marsupial cytokines, Harrison andWedlock (2000) documented the difficulty in identifyingkey immunoregulatory molecules such as IL-4 and IL-2in marsupials. Since that time, information has come tolight concerning conserved regions in the structure of4-alpha helical cytokines (Hill et al., 2002), and we nowhave access to IL-4 nucleotide sequence data for phylo-genetically distant species (Li et al., 2007) and the modelmarsupial, Monodelphis domestica (Wong et al., 2006). Themore recent release of sequence data generated from a2× coverage of the tammar wallaby genome (Meug 1.0;http://www.ensembl.org/Macropus eugenii/Info/Index/)will further contribute to the availability of such data forfuture studies.

For the identification of cytokines that are known to beexpressed at low levels compared with other genes, cellculture conditions also need to be optimized to ensure thatthe RNA used in such studies is derived from T cells inactive blastogenesis (Young and Deane, 2007). Using anapproach that combined these factors, the cDNA expres-sion of Interleukin-4 was detected for the first time in anymarsupial species.

Single cell suspensions of mononuclear cells were iso-lated from the axillary lymph nodes of a tammar wallaby,stimulated with mitogens (25 �g/mL Con A or 25 �g/mLPHA) and assessed for proliferation as previously described(Young and Deane, 2007). RNA was extracted from culturedcells, unstimulated PBMC and spleen using TriReagent(Sigma–Aldrich, Australia) according to the manufac-turer’s directions. For initial RT-PCR experiments, cDNAwas oligo(dT)15 primed from total RNA using the A3500Reverse Transcription System (Promega, Australia). Con-sensus primers were designed to incorporate nucleotidescoding for the most conserved amino acids in known verte-brate species (Li et al., 2007; Ohtani et al., 2008), includingthe IL-4 predicted sequence of M. domestica (Wong et al.,2006). Primers were positioned in regions of the initiationcodon in exon 1 (4BNTbf: 5′-tgggtctcacmtyccaactga-3′) andwithin exon 4 (4BNTcr: 5′-tcttggnttcattcacaggaca-3′) todetect possible splice variants, which have been reportedfor some mammalian species (Gautherot et al., 2002;Waldvogel et al., 2004) (see Fig. 1). The reverse primer wasdesigned to amplify a region that coded for the structuralloop positioned within the short-chain helical cytokinefamily after helix �C (important for ligand binding toreceptor) and also included a highly conserved cysteineresidue (C99 in humans) considered essential for IL-4 fold-ing (Kruse et al., 1991).

PCR was performed using a reaction mix containing1 �L cDNA, 0.2 �M of each primer, 200 �M dNTPs, 1.5 mMMgCl2, 1.0 unit Platinum Taq DNA polymerase (Invitrogen,

Australia), buffer and nuclease-free water to 20 �L with thefollowing cycling conditions: 95 ◦C for 3 min followed by 35cycles of 95 ◦C for 30 s, 50 ◦C for 60 s and 72 ◦C for 60 s anda final extension step of 72 ◦C for 10 min. A 393 bp ampli-con was detected by agarose gel electrophoresis from the

unopathology 140 (2011) 335–340

PHA-stimulated lymphocyte cDNA, but not from unstimu-lated PBMC or spleen. This amplicon was gel-purified usingthe Wizard SV Gel and PCR Clean-up System (Promega,Australia) and directly sequenced at the Australian GenomeResearch Facility (AGRF; Brisbane, Australia). The returnedsequence was analysed by the basic local alignment searchtool algorithm package, blastx (Altschul et al., 1997) andreturned similarities of between 40% and 51% to knownmammalian IL-4 peptides. A Rapid Amplication of cDNAends (RACE) strategy (Frohman et al., 1988) was thenemployed to obtain the 5′- and 3′-UTR regions and theremainder of the coding sequence of the wallaby IL-4 cDNA.

RACE DNA was synthesised according to the manufac-turer’s instructions using the GeneRacer Kit (Invitrogen,Australia) with RNA from PHA-stimulated cells previ-ously used to amplify the wallaby IL-4 amplicon. 5′- and3′-products were directly amplified from the mitogen-stimulated RACE template using the nested kit primers(Invitrogen, Australia) paired with each of the original con-sensus primers (5′-RACE primer with 4BNTbr and 4BNTfwith 3′-RACE primer). Buffered PCR reactions contained2 �L cDNA (1/20 dilution of RACE reaction), 0.4 �M eachprimer, 200 �M dNTPs, 2.0 mM MgCl2, 0.5 units of Hi-Fidelity Platinum Taq Polymerase (Invitrogen, Australia)and nuclease-free water to a volume of 20 �L. Cycling con-ditions were 94 ◦C for 2 min, 5 cycles of 94 ◦C for 30 s,55 ◦C for 50 s and 68 ◦C for 60 s, 35 cycles of 94 ◦C for 30 s,50 ◦C for 50 s and 68 ◦C for 70 s, with a final extensionstep at 68 ◦C for 10 min. Products were gel purified anddirectly sequenced by AGRF. An additional gene-specificprimer (5′-cagaggtctggcagatccttctctg-3′) was used to con-firm sequence data in the 5′-UTR region. The RACE reactionswere performed in triplicate and sequencing was per-formed in duplicate in both directions. A complete cDNAsequence of 645 bp was obtained, which has been assignedGenbank accession number HM011505.

The predicted coding sequence is 474 nucleotides inlength and has 65 bp of 5′-UTR sequence (see Fig. 1). The 3′-UTR extends 86 bp from the termination site to the polyAtail. A polyadenylation signal [attaaa] (Beaudoing et al.,2000) is 16 bp upstream from the polyA tail and three atttainstability motifs are present in the 3′-UTR. Consistent withother mammalian IL-4 molecules (Ekerfelt et al., 2002),expression of wallaby IL-4 was only readily detected inmitogen-stimulated but not resting lymph node cells. Ini-tial attempts to amplify this cytokine from a source of RACEDNA constructed from a M. eugenii mammary associatedlymph node (Harrison et al., 1999) failed to yield visibleDNA products upon electrophoresis as did a number of RT-PCR experiments on a range of unstimulated cells fromspleen and blood (not shown). Where IL-4 was detectedin mitogen-stimulated cells, no evidence of splice variants,reported in some other mammals (Gautherot et al., 2002),was visible.

Structural analyses of the 157 amino acid predictedtranslation product using the Simple Modular Architec-

ture Research Tool (Schultz et al., 1998; Letunic et al.,2009) revealed an IL4 13 domain (E = 2.25e−21), and aProsite scan (with similarity set at 80% to allow somemismatches) recognized the IL-4 and IL-13 family sig-nature; a signature that IL-4 and IL-13 share since they

L. Young / Veterinary Immunology and Immunopathology 140 (2011) 335–340 337

Fig. 1. Nucleotide and translated amino acid sequence of tammar wallaby Interleukin-4. The 474 bp coding domain (66–536) is flanked by both 5′- and3′-untranslated regions shown here in italics (nucleotides numbered on right of figure). The polyadenylation signal sequence is shown in dark relief and3 mino aca lycosyla

I the IL-4

asxm(42(sgpaChs4paei(

iaid

′-mRNA instability motifs (attta) are bold. The translated peptide is 157 a24 residue predicted signal peptide (bold font) and predicted N- and O-g

L-4 family signature is boxed . Primer sites used to successfully amplify

re distantly related (Combet et al., 2000). The IL-4ignature [LI]-x-E-[LIVM](2)-x(4,5)-[LIVM]-[TL]-x(5,7)-C-(4)-[IVA]-x-[DNS]-[LIVMA] is conserved across 82% of theotif in the N-terminal region of the M. eugenii sequence

see Fig. 1). A 24 residue signal peptide, common to IL-proteins, was predicted by SignalP 3.0 (Bendtsen et al.,

004) (see Fig. 1). There are also three potential N-linkedCombet et al., 2000) and two potential O-linked glyco-ylation sites (Julenius et al., 2005) consistent with thelycoprotein nature of IL-4. Three-dimensional structuralrediction through PHYRE (Kelley and Sternberg, 2009)lso confirmed 29% identity (E = 7.6e−13) to human IL-4.ysteine residues C3, C24, C46 and C99 present in matureuman IL-4 are conserved by alignment in the wallabyequence (see Fig. 2); C46 and C99 are both essential for IL-biological function (Kruse et al., 1991). Also conserved areolar residues E9 and R88, both critical to effective inter-ctions with the high-affinity human IL-4R� chain (Kruset al., 1993; Zhang et al., 2002) and residue S125, involvedn interactions with the common gamma chain receptorGu et al., 2010).

When compared with other mammalian IL-4 sequences,t is not surprising that putative wallaby IL-4 nucleotidend amino acid coding domain sequences share mostdentity with other predicted marsupial sequences, M.omestica (59% identity when analysed using Blastx:

ids to the stop codon and is numbered from the left of the figure. There istion sites are underlined and circled respectively. The partially conserved

nucleotide product are also boxed .

align two sequences) and the more recently available, M.eugenii ENSEMBL sequence. IL-4 is predicted in ENSEMBL(EIL-4), although there are differences in the suggestedcDNA sequence, gene organisation and subsequent pep-tide length between the prediction and the cDNA sequence(cIL-4) reported in this study. According to the ENSEMBLwebsite, E-IL4 is predicted to be comprised of 6 exons andhas no obvious stop codon, yielding a peptide of 116 aminoacids. If cIL-4 is used to search ENSEMBL and the extractedsequence data is analysed using Genscan (accessed throughhttps://bm.angis.org.au), the result suggests a possible 5exon prediction for wallaby IL-4. Genscan predicts 3 exons(2 internal exons and one terminal exon) and two otherexons when suboptimal parameters are enabled; the fifthexon is poorly predicted with a weak score for the acceptorsplice site. Vertebrate IL-4 has a 4 exon/3 intron arrange-ment, so the predicted five exon structure for the wallabygene is novel for any mammalian species reported to date.Inspection by alignment with other species suggests thatthe highly variable exon 3 region of this gene (Perkins et al.,2000) may be replaced by two discrete exons in the wallaby

genome. Future studies to confirm splice sites and genearchitecture in the wallaby and other marsupial genomesare now required to determine the significance of this find-ing and what, if any, functional relevance this has to Th2immune responses in marsupials.

338 L. Young / Veterinary Immunology and Immunopathology 140 (2011) 335–340

Fig. 2. Multiple sequence alignment of mammalian IL-4 amino acid sequences. # Indicates the start of the putative IL-4 mature peptide after the 24 residuepredicted signal sequence. + Indicates conserved residues that are involved in high affinity receptor interactions in human IL-4. ˆ Indicates conservedcysteines involved in essential disulphide bridge formation in known IL-4 peptides. Black background indicates fully conserved residues across thesemammalian species (upper case in the consensus line), grey background indicates 80% conservation identity (lower case in the consensus line). Dots

or RefSlodytes N6346, bdicted o

indicate gaps introduced by ClustalW2 to optimise the alignment. Genbankeugenii ADG01643, human Homo sapiens NP 000580, chimpanzee Pan trogBAA78610, dog Canis lupus familiaris AAD11563, cattle Bos taurus NP 77Elephas maximus ABS28989 and mouse Mus musculus NP 067258. The pre

ClustalW2 (http://www.ebi.ac.uk) multiple sequencealignments processed through GeneDoc (www.psc.edu/biomed/genedoc) and phylogenetic analysis per-formed through the Phylogeny.fr platform (throughhttp://au.expasy.org/tools/) confirm that when compared

with other species, M. eugenii IL-4 (cIL-4) is most similarto M. domestica and that there is significant variabilityin both nucleotide and amino acid conservation of thecoding domain of this gene and its translated product(see Table 1 and Fig. 3). Sequence variability within exon

eq accession numbers for these sequences are: tammar wallaby MacropusP 001011714, horse Equus caballus ABZ91978, dolphin Tursiops truncates

at Rousettus leschenaultii BAH02559, pig Sus scrofa CAA48407, elephantpossum Monodelphis domestica sequence is from Wong et al. (2006).

3 of mammals accounts for some of this variation (seearea in Fig. 2 bounded by wallaby amino acids 64–121),although differences in and around this area (which codesfor residues before and after the structurally significant �Bhelix) are not unusual in mammalian IL-4 genes (Perkins

et al., 2000; Gautherot et al., 2002). The variability inClustalW2 identity scores between the marsupial and theeutherian mammal sequences (a measure of the diver-gence in this gene family; see Table 1) and between themammals and the phylogenetically distant species such as

L. Young / Veterinary Immunology and Immunopathology 140 (2011) 335–340 339

F geneticm s assessa througp bers for

cuioe

TCdcifi2m(nTEbmar

ig. 3. Phylogram of mammalian IL-4 amino acid sequences. The phyloented in the PhyML program (v3.0 aLRT). Internal branch reliability wa

s percentages. The analysis was performed on the Phylogeny.fr platformhylogenetic tree was generated using TreeDyn (v198.3). Accession num

hicken and pufferfish, demonstrates the difficulties whensing stand-alone sequence homology approaches to

dentify cytokines such as IL-4; one of the most divergentf mammalian proteins (Makalowski et al., 1996; Conklint al., 2005).

able 1lustalW2 multiple sequence alignment identity scores for the codingomain of known vertebrate IL-4 nucleotide and amino acid sequencesompared with the same region of Tammar Wallaby IL-4. Sequences usedn this alignment were extracted from Genbank in addition to the puffer-sh (Li et al., 2007) and the predicted opossum sequence (Wong et al.,006). Default conditions for ClustalW2 were used to construct the align-ent and identity scores were obtained after processing through GeneDoc

under default conditions). Genbank and RefSeq Accession numbers forucleotide sequences are: elephant Elephas maximus EU000424, dolphinursiops truncatus AB020732, pig Sus scrofa X68330, horse Equus caballusU438769, cattle Bos taurus NM 173921, dog Canis familiaris AF054833,at Rousettus leschenaultii AB472359, human Homo sapiens NM 000589,ouse Mus musculus NM 021283, chicken Gallus gallus NM 001007079

nd frog Xenopus tropicalis NM 001113807. See Fig. 2 for IL-4 amino acideference numbers.

Species % Identity

Nucleotide Amino acid

Opossum 74 59Elephant 48 37Pig 49 36Dolphin 47 36Horse 47 34Cattle 46 34Dog 48 33Human 52 32Bat 46 31Mouse 43 25Chicken 38 19African clawed-frog 33 15Pufferfish 31 10

tree was reconstructed using the maximum likelihood method imple-ed using 500 bootstrap replicates and branch support values are shown

h the Expasy website (http://au.expasy.org/tools/) and the graphic of thethese sequences are as for Fig. 2.

Short-chain helical cytokine protein structures are moreconserved than their sequences (Hill et al., 2002), a factorthat has prevented the elucidation of marsupial IL-4 untilnow. Despite expected differences in nucleotide sequenceand predicted amino acid composition, many of the aminoacid residues that confer functionality on IL-4 appear to beconserved in the putative IL-4 wallaby protein.

Acknowledgements

This work was completed during a period of study leaveto LJY supported by Central Queensland University. Fund-ing was also provided by the Centre for EnvironmentalManagement (CQUniversity Australia).

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