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Molecular identification of interleukin-2 in the lymphoid tissues of the common brushtail possum, Trichosurus vulpecula
Developmental and Comparative Immunology 36 (2012) 236–240
Contents lists available at ScienceDirect
Developmental and Comparative Immunology journal homepage: www.elsevier.com/locate/dci
Short communication
Molecular identification of interleukin-2 in the lymphoid tissues of the common brushtail possum, Trichosurus vulpecula L.J. Young a,c, M.L. Cross b, J.A. Duckworth b, S. Flenady c, K. Belov a,⇑ a
Faculty of Veterinary Science, The University of Sydney, NSW 2006, Australia Pest Control Technologies Team & NRC Possum Biocontrol at Landcare Research, Lincoln, New Zealand c Centre for Environmental Management, Central Queensland University, Rockhampton, Queensland, Australia b
a r t i c l e
i n f o
Article history: Received 24 March 2011 Revised 25 May 2011 Accepted 26 May 2011 Available online 12 June 2011 Keywords: Brushtail possum Cytokine Interleukin-2 Marsupial Trichosurus vulpecula
a b s t r a c t The common brushtail possum (Trichosurus vulpecula) is an Australian marsupial. Here we describe the identification of possum interleukin-2 in mitogen-stimulated lymph node cells. We used a strategy of Rapid amplification of cDNA ends using probes designed from recently-sequenced marsupial genomes to identify the IL2 gene and then confirmed that IL-2 expression in possum immune tissue occurs in a similar manner to that in their eutherian counterparts. The predictive possum IL-2 peptide showed 28% and 35% amino acid sequence homology with the mouse and human IL-2 molecules, respectively, consistent with the divergence found within this cytokine family. Despite this low sequence identity, possum IL-2 still possessed the characteristic hallmarks of mammalian IL-2, such as a predicted signal peptide and conserved family motifs. Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved.
1. Introduction The common brushtail possum (Trichosurus vulpecula) is a native Australian marsupial species. The immune system of the brushtail possum has been studied at the anatomical (Baker et al., 1999) and cellular level (Buddle et al., 1992), and reports to date suggest that the organisation and function of T and B cells (the effector cells of adaptive immunity) are similar to other mammals. Molecular investigations have revealed further insights into the workings of the possum immune system, and a number of key immunoglobulin molecules have been elucidated using molecular homology techniques (reviewed by Miller and Belov, 2000). Furthermore, important pro- and anti-inflammatory monokines such as interleukin-1b, tumor necrosis factor alpha and interleukin-10 (Wedlock et al., 1996, 1998, 1999) have been identified, with a view to utilising these molecules as potential adjuvants in possum vaccination programs. Notwithstanding the available information about immunity in this species, there are still important gaps in our knowledge about the canonical molecules associated with adaptive immunity and responses to infection. Interleukin-2 (IL-2) is a small (15 kDa) short-chain alpha helical cytokine that is involved in the regulation and differentiation of T cells and in the activation of NK cells (Gaffen and Liu, 2004). IL-2 is ⇑ Corresponding author. Tel.: +61 2 93513454; fax: +61 2 93513957. E-mail addresses: [email protected], [email protected] (K. Belov).
mainly produced by activated T helper cells (CD4+ and CD8+) after engagement of the T cell receptor (TCR), the constant regions of which have been cloned and sequenced in the brushtail possum (Zuccolotto et al., 2000). Despite repeated attempts to elucidate IL2 in possums, we and others (Harrison and Wedlock, 2000) have been unable to identify it until genome sequencing data from two marsupial species, the opossum (Monodelphis domestica) and the tammar wallaby (Macropus eugenii) recently became available. Using this sequence information to inform our molecular homology experimental approach, we renewed our efforts to identify this molecule in the brushtail possum, and report here the molecular characterisation of possum IL-2.
2. Materials and methods 2.1. Tissue and cell isolation Tissues containing resident and circulating lymphoid cells were collected from a 3.4 kg clinically healthy mature male possum that had been captured from the wild and acclimatised to captivity for four weeks prior to experimentation. The possum was euthanased humanely with appropriate approval from the Animal Ethics Committee of Landcare Research, Lincoln in accordance with the 1987 Animals Protection (Codes of Ethical Conduct) Regulations of New Zealand (Approval No. 10/02/01). Single cell mononuclear leukocyte suspensions were prepared in RPMI 1640 base medium
0145-305X/$ - see front matter Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.dci.2011.05.010
L.J. Young et al. / Developmental and Comparative Immunology 36 (2012) 236–240 Table 1 Stimulation indices for Con-A stimulated possum lymphoid cells. Mononuclear cells (105 in 200 lL) were cultured with/without Concanavalin A before being tritated for 16 h after periods of mitogen-stimulation of 24 or 72 h (six replicate wells for each sample). Data represent mean stimulation indices (proliferation value (cpm) with mitogen/proliferation value (cpm) without mitogen).
a
Tissue source
24 h time-point
72 h time-point
Retropharyngeal lymph nodes (LNRP)a Mesenteric lymph nodes (LNM) Blood Spleen
330 ± 17 205 ± 37 212 ± 16 192 ± 6
1035 ± 17 486 ± 59 410 ± 17 154 ± 6
LNRP tissue source used for RACE DNA construction.
(Sigma, Australia) from the following tissues: Ficoll gradientseparated blood and spleen suspensions, homogenised tissues of the retropharyngeal lymph nodes (LNRP) and the mesenteric lymph nodes (LNM). Cell suspensions were adjusted to 106 viable cells/mL in RPMI (supplemented with 10% foetal calf serum and antibiotics) and stimulated in in vitro culture with and without 5 lg/mL of the T cell mitogen, Concanavalin A (Con A). Control and stimulated cells (viability of >90%) were harvested for molecular studies at 24 and 72 h (Table 1) when cells were in active blastogenesis. This activity was confirmed by visual inspection and by tritiated thymidine proliferation assay using standard techniques. Briefly, cellular proliferation of each cell preparation was confirmed by measuring beta emission following pulsing of the cells with 1 lCi/well of tritium-labelled thymidine over the last 18 h of stimulation. Stimulation indices were then determined by comparison of the stimulated cell cultures with their unstimulated control preparations.
237
2.2. Identification of the possum IL-2 gene A reverse-transcriptase polymerase chain reaction (RT-PCR) strategy followed by the rapid amplification of cDNA ends (RACE) technique was used to identify the expressed possum IL-2 gene. RNA was isolated from Con-A stimulated LNRP mononuclear cells using the GenElute Total RNA Miniprep Kit (Sigma–Aldrich) according to manufacturer’s instructions. Isolated RNA was treated with DNase 1 to remove residual genomic DNA. For RT-PCR, cDNA was synthesised using the superscript III RT system following the protocol recommended in the kit (Invitrogen). Primers were targeted to conserved regions of the IL-2 sequences predicted for the American gray short tailed opossum ( M. domestica) and the Australian tammar wallaby (M. eugenii) (accessed through ENSEMBL at http://www.ensembl.org/index.html). These primers (‘IL2FC1’ 50 -atgaa/gcaaggta/cccgctcc/ttgt-30 and ‘2Rex1’ 50 -acagtcacattaatgttg-30 ) were also subsequently used in RACE experiments to determine the entire IL-2 cDNA sequence. PCR using Taq DNA polymerase (Invitrogen) at an annealing temp of 50 °C for 35 cycles yielded a single DNA amplicon, which was gel-purified using the Wizard SV Gel and PCR Clean-up System (Promega, Australia) and directly sequenced at the Australian Genome Research Facility (AGRF; Brisbane, Australia). The returned sequence was edited to remove primer sequences and processed through Blastx (basic local alignment search tool; Altschul et al., 1997) for assignment of putative identity against known mammalian IL-2 gene sequences. Possum RACE DNA was performed using RNA obtained from Con-A stimulated LNRP in order to obtain the 50 - and 30 -untranslated regions of the IL-2 gene. RACE DNA was prepared
Fig. 1. Nucleotide and predicted amino acid sequence of possum IL-2. The nucleotide sequence comprises a coding domain of 426 bp to the stop codon, a 50 -UTR of 54 bp and a 30 UTR that contains one instability motif (attta in bold). A polyadenylation site (underlined), 19 bp upstream from the polyA tail (n = 24), is also evident. Putative N- and Oglycosylation sites are underlined and circled, respectively. The conserved region of the IL-2 family signature is shaded. GenBank Accession number for the possum nucleotide sequence is HQ717721.
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L.J. Young et al. / Developmental and Comparative Immunology 36 (2012) 236–240
following manufacturer’s instructions using the GeneRacer Kit (Invitrogen, Australia). Both 50 - and 30 - RACE amplicons were amplified directly using kit-supplied RACE primers (‘GeneRacer 50 nested primer’ 50 -ggacactgacatggactgaaggagta-30 and ‘GeneRacer 30 nested primer’ 50 -cgctacgtaacggcatgacagtg-30 ) matched to their 50 - or 30 -reverse and forward RT-PCR primers respectively. RACE PCR reaction volumes of 20 lL were performed in duplicate using 2 lL cDNA (1/10 dilution of original RACE cDNA template), 2.0 mM MgCl2, 0.5 units of Hi-Fidelity Platinum Taq Polymerase and remaining conditions as recommended by RACE kit instructions (Invitrogen, Australia). Gel-purified amplicons were directly sequenced by AGRF as described earlier.
Blast suite of programs (Altschul et al., 1997). ClustalW2 (accessed through http://www.ebi.ac.uk) was used to construct multiple sequence alignments. A phylogenetic tree was constructed using the program Geneious (Drummond et al. 2010) where sequences were aligned with MUSCLE and a neighbour joining tree was generated using 100 bootstrap replicates. A number of programs were accessed through the Expasy Tools website (http://au.expasy.org/ tools/) to identify family motifs, a predicted signal peptide and possible glycosylation sites.
2.3. Molecular characterisation of possum IL-2
3.1. Cell proliferation and molecular identification of possum IL-2
The nucleotide sequence for putative possum IL-2 was analysed for identity and similarity to other known IL-2 genes using the
Possum lymphocytes isolated from a number of immune tissue sources all responded to stimulation by the T-cell mitogen Con A (Table 1). In vitro mitogen stimulation of lymphocytes is known to lead to IL-2 secretion (Gaffen and Liu, 2004), and the high stimulation indices associated with our in vitro culture were thus optimal conditions for the cellular synthesis of possum IL-2. Preliminary RT-PCR experiments using marsupial-specific primers confirmed the presence of IL-2 amplicons of the correct size in cells optimised for the expression of this gene. Blastx analysis of the possum IL-2 RT-PCR product identified this amplicon as having approximately 45–58% similarity to known mammalian IL-2 peptide sequences. RACE PCR subsequently yielded a complete cDNA sequence of 764 bp that includes a 50 -UTR region of 54 bp and a 30 -UTR of 281 bp (see Fig. 1). The nucleotide sequence for possum IL-2 contains a single attaaa instability motif in the 30 -UTR and a polyadenylation signal 19 bp upstream from the polyA tail (see Fig. 1). This sequence has been deposited in GenBank and has been
Fig. 2A. Multiple sequence alignment of IL-2 peptide sequences. The predicted signal peptide for possum IL-2 is indicated above the alignment and the putative mature possum IL-2 peptide begins at residue 21. Residues that are completely conserved across mammalian species (upper case in the consensus line) have a black background. Residues that have a grey background are 80% conserved in this alignment (lower case in the consensus line). Dashes (–) represent gaps introduced by ClustalW2 to optimise the alignment. The predicted signal peptide for possum IL-2 is indicated above the alignment and the mature possum IL-2 peptide begins at residue 21. Conserved cysteine residues required for the formation of intrachain disulfide bonds essential to cytokine function are indicated by #. indicates conserved residues that are involved in IL-2 receptor interactions across a number of mammalian species. Accession numbers for these sequences are: possum Trichosurus vulpecula (this paper), opossum Monodelphis domestica (Wong et al., 2006), woodchuck Marmota monax gb|ABB84516.1, rabbit Oryctolagus cuniculus ref|NP_001156652.1, pig Sus scrofa ref|NP_999026.1, bat Rousettus leschenaultia dbj|BAH02558.1, cat Felis catus gb|AAX63392.1, horse Equus caballus gb|AAA20134.1, lama Lama glama sp|Q865X2.1, human Homo sapiens gb|AAH66254.1, camel Camelus dromedaries gb|ADH51719.1, macaca Macaca mulatta ref|NP_001040595.1, goat Capra hircus gb|AAG28783.1, dog Canis lupus familiaris ref|NP_001003305.1 and rat Rattus norvegicus ref|NP_446288.1.
3. Results and discussion
Fig. 2B. Phylogram of IL-2 amino acid sequences. The phylogram was constructed using an alignment that included a range of mammalian IL-2 sequences and an avian sequence for comparison. The phylogenetic tree was constructed using Geneious as outlined in the text. Internal branch reliability was assessed using 100 bootstrap replicates and branch support values over 50 are shown as percentages. Accession numbers for mammalian sequences are as for Fig. 2A. Accession number for the chicken Gallus gallus, an avian species, included for demonstration of grouping of the marsupial clade with other mammalian species, is gb|AAS00717.1.
L.J. Young et al. / Developmental and Comparative Immunology 36 (2012) 236–240
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Table 2 Sequence conservation of mammalian IL-2 peptides in comparison with predicted possum IL-2. Identity and similarity scores are recorded as reported using Blastp analysis against putative possum IL-2. %Gaps introduced by program defaults to optimise the alignments are also indicated. Animal and Accession number
%Identity
%Similarity
%Gaps
Opossum (Wong et al., 2006) Horse (gb|AAA20134.1) Lama (sp|Q865X2.1) Camel (gb|ADH51719.1) Pig (NP_999026.1) Whale (gb|AAD01426.1) Bat (dbj|BAH02558.1) Woodchuck (gb|ABB84516.1) Elephant seal(gb|AAC12258.1) Human (gb|AAH66254.1) Cat (gb|AAX63392.1) Dog (ref|NP_001003305.1) Macaca (ref|NP_001040595.1) Gerbil (emb|CAA48679.1) Mouse (gb|AAA39281.1) Rat (ref|NP_446288.1)
63 37 35 34 36 34 37 37 34 35 34 33 33 31 28 29
75 58 56 56 54 54 53 52 51 50 50 50 49 48 48 45
0 8 8 8 9 9 9 7 7 7 7 8 11 12 19 11
assigned accession number HQ717721. We also searched the publicly available possum EST database (http://www.possumbase.org.nz/) using our amplified RT-PCR sequence. No significant hits were obtained in this search, suggesting that the IL-2 gene sequence is not present in that database, explaining in part why this gene has not been identified in the possum genome until now. 3.2. Comparison of possum IL-2 with other mammalian IL-2 molecules Translation of the coding domain of the possum IL-2 nucleotide sequence (tIL-2) results in a predicted peptide length of 142aa to the stop codon. A calculated molecular weight of 15.8 kDa (13.8 kD for the mature transcript) is consistent with mammalian IL-2 gene products. Possum tIL-2 also shares many of the canonical features of other known mammalian IL-2 peptides (see Fig. 1). Both the 4helical cytokine (E = 2.53e15) motif and the IL-2 family signature [T-E-[LF]-x(2)-L-x-C-L-x(2)-E-L] are recognised (E = 1.19e08), although the IL-2 signature is not completely conserved (Fig. 2A). There is a predicted signal peptide of 20 aa residues from the start site (SignalP 3.0), consistent with the secretory properties of this mammalian cytokine. Two N-glycosylation and one O-glycosylation site are also predicted at residues 47–50, 62–65 and 23, respectively (Fig. 1). Cysteine residues, important for molecular folding and function of the mature protein (Zelus et al., 2000), are conserved by alignment with other known IL-2 peptide sequences (Fig. 2A) as are some amino acids essential for binding to the IL-2Rb and IL-2Rc chains of the high-affinity trimeric IL-2 receptor (Zelus et al., 2000). Many of the canonical features of mammalian IL-2 appear to be conserved in the possum gene and translated protein. Despite the conservation of canonical features, the overall level of amino acid identity between possum IL2 and IL2 of other species is low (Table 2). Phylogenetic analysis reveals that the putative possum IL-2 peptide forms a distinct marsupial clade with the predicted IL-2 opossum sequence when compared with other mammals (Fig. 2B). Nonetheless, when an avian IL-2 sequence is included in the analysis (chicken IL-2), the marsupial sequences cluster with members of the class Mammalia, which is consistent with the evolutionary distance between mammals and birds. The presence of a number of key features consistent with mammalian IL-2 molecules such as topology consistent with the 4-helical superfamily, a signal peptide (common to secreted immunoregulatory molecules) and a number of canonical features of the IL-2 family all suggest that possum IL-2 may play a similar immunological role in the modulation of T cell immune responses.
Fig. 3. Expression of the IL-2 gene in Con-A stimulated lymph node cells. An RT-PCR product of 311 bp was amplified in lymph node cells treated with Con A for 24hrs (Lane A – mesenteric lymph node and Lane B – retropharyngeal lymph node). Lane C contains an untreated control, demonstrating the lack of expression of this gene, and is representative of all control experiments. PCR conditions for this assay included an annealing temperature of 50 °C and amplification for 35 cycles. Lane D is a representative no-template control. IL-2 expression is shown in the top panel against molecular weight marker bands (M) of 500 and 250 bp in each panel. The bottom panel shows b-actin expression in the same tissues.
3.3. Expression of possum IL-2 under conditions of T cell stimulation Since IL-2 plays an essential role in T cell proliferation, regulation and memory functions associated with CMI (Malek, 2008), we chose to renew our efforts to characterise this cytokine gene in the brushtail possum and successfully identified this molecule in mitogen-stimulated lymph node tissue. Putative possum IL-2 was expressed in Con-A stimulated lymph node cells but was not readily detected in resting lymph node cells cultured under the same conditions (see Fig. 3) when normalised against the expression of the b-actin gene. Con A is a well-documented T cell mitogen used to assess proliferative responses in possums (Buddle et al., 1994), and the expression of the IL-2 gene in this study is consistent with the induction of this molecule under similar in vitro conditions to those used in studies of other mammalian species to date. This suggests, at least at the in vitro level, that the possum IL-2 gene may possess analogous immunoregulatory pathways to eutherian mammals. Until this report, the major regulators of adaptive immunity, specifically cytokines such as interleukin-2 that regulate important T helper cell responses, have not been successfully characterised in the possum (Harrison and Wedlock, 2000). This has proved a considerable drawback to studies of disease susceptibility in this species. Availability of genomic data has provided much needed information to progress our study of highly divergent and immunologically important regulatory molecules in the brushtail possum.
Acknowledgements L.J.Y. completed this work whilst on sabbatical at The University of Sydney. This research was funded by the NZ Foundation for Research, Science and Technology (Possum Biocontrol OBI C10X0501). References Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J.H., Zhang, Z., Miller, W., Lipman, D.J., 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402.
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Baker, M.L., Gemmell, E., Gemmell, R.T., 1999. Ontogeny of the immune system of the brushtail possum, Trichosurus vulpecula. Anat. Rec. 256, 354–365. Buddle, B.M., Aldwell, F.E., Jowett, G., Thomson, A., Jackson, R., Paterson, B.M., 1992. Influence of stress of capture on hematological values and cellular immuneresponses in the Australian brushtail possum (Trichosurus vulpecula). NZ Vet. J. 40, 155–159. Buddle, B.M., Aldwell, F.E., Pfeffer, A., Delisle, G.W., 1994. Experimental Mycobacterium bovis infection in the brushtail possum (Trichosurus vulpecula) – pathology, hematology and lymphocyte stimulation responses. Vet. Microbiol. 38, 241–254. Drummond, A.J., Ashton, B., Buxton, S., Cheung, M., Cooper, A., Heled, J., Kearse, M., Moir, R., Stones-Havas, S., Sturrock, S., Thierer, T., Wilson, A., 2010. Geneious v5.1, Available from :. Gaffen, S.L., Liu, K.D., 2004. Overview of interleukin-2 function, production and clinical applications. Cytokine 28, 109–123. Harrison, G.A., Wedlock, D.N., 2000. Marsupial cytokines. Structure, function and evolution. Dev. Comp. Immunol. 24, 473–484. Malek, T.R., 2008. The biology of interleukin-2. Annu. Rev. Immunol. 26, 453–479. Miller, R.D., Belov, K., 2000. Immunoglobulin genetics of marsupials. Dev. Comp. Immunol. 24, 485–490.
Wedlock, D.N., Aldwell, F.E., Buddle, B.M., 1996. Molecular cloning and characterization of tumour necrosis factor alpha (TNF-alpha) from the Australian common brushtail possum, Trichosurus vulpecula. Immunol. Cell Biol. 74, 151–158. Wedlock, D.N., Aldwell, F.E., Buddle, B.M., 1998. Nucleotide sequence of a marsupial interleukin-10 cDNA from the Australian brushtail possum (Trichosurus vulpecula). DNA Seq. 9, 239–244. Wedlock, D.N., Goh, L.P., Parlane, N.A., Buddle, B.M., 1999. Molecular cloning and physiological effects of brushtail possum interleukin-1 beta. Vet. Immunol. Immunopathol. 67, 359–372. Wong, E.S., Young, L.J., Papenfuss, A.T., Belov, K., 2006. In silico identification of opossum cytokine genes suggests the complexity of the marsupial immune system rivals that of eutherian mammals. Immun. Res. 2, 4. Zelus, D., Robinson-Rechavi, M., Delacre, M., Auriault, C., Laudet, V., 2000. Fast evolution of interleukin-2 in mammals and positive selection in ruminants. J. Mol. Evol. 51, 234–244. Zuccolotto, P.D., Harrison, G.A., Deane, E.M., 2000. Cloning of marsupial T cell receptor alpha and beta constant region cDNAs. Immunol. Cell Biol. 78, 103–109.
Contents lists available at ScienceDirect
Developmental and Comparative Immunology journal homepage: www.elsevier.com/locate/dci
Short communication
Molecular identification of interleukin-2 in the lymphoid tissues of the common brushtail possum, Trichosurus vulpecula L.J. Young a,c, M.L. Cross b, J.A. Duckworth b, S. Flenady c, K. Belov a,⇑ a
Faculty of Veterinary Science, The University of Sydney, NSW 2006, Australia Pest Control Technologies Team & NRC Possum Biocontrol at Landcare Research, Lincoln, New Zealand c Centre for Environmental Management, Central Queensland University, Rockhampton, Queensland, Australia b
a r t i c l e
i n f o
Article history: Received 24 March 2011 Revised 25 May 2011 Accepted 26 May 2011 Available online 12 June 2011 Keywords: Brushtail possum Cytokine Interleukin-2 Marsupial Trichosurus vulpecula
a b s t r a c t The common brushtail possum (Trichosurus vulpecula) is an Australian marsupial. Here we describe the identification of possum interleukin-2 in mitogen-stimulated lymph node cells. We used a strategy of Rapid amplification of cDNA ends using probes designed from recently-sequenced marsupial genomes to identify the IL2 gene and then confirmed that IL-2 expression in possum immune tissue occurs in a similar manner to that in their eutherian counterparts. The predictive possum IL-2 peptide showed 28% and 35% amino acid sequence homology with the mouse and human IL-2 molecules, respectively, consistent with the divergence found within this cytokine family. Despite this low sequence identity, possum IL-2 still possessed the characteristic hallmarks of mammalian IL-2, such as a predicted signal peptide and conserved family motifs. Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved.
1. Introduction The common brushtail possum (Trichosurus vulpecula) is a native Australian marsupial species. The immune system of the brushtail possum has been studied at the anatomical (Baker et al., 1999) and cellular level (Buddle et al., 1992), and reports to date suggest that the organisation and function of T and B cells (the effector cells of adaptive immunity) are similar to other mammals. Molecular investigations have revealed further insights into the workings of the possum immune system, and a number of key immunoglobulin molecules have been elucidated using molecular homology techniques (reviewed by Miller and Belov, 2000). Furthermore, important pro- and anti-inflammatory monokines such as interleukin-1b, tumor necrosis factor alpha and interleukin-10 (Wedlock et al., 1996, 1998, 1999) have been identified, with a view to utilising these molecules as potential adjuvants in possum vaccination programs. Notwithstanding the available information about immunity in this species, there are still important gaps in our knowledge about the canonical molecules associated with adaptive immunity and responses to infection. Interleukin-2 (IL-2) is a small (15 kDa) short-chain alpha helical cytokine that is involved in the regulation and differentiation of T cells and in the activation of NK cells (Gaffen and Liu, 2004). IL-2 is ⇑ Corresponding author. Tel.: +61 2 93513454; fax: +61 2 93513957. E-mail addresses: [email protected], [email protected] (K. Belov).
mainly produced by activated T helper cells (CD4+ and CD8+) after engagement of the T cell receptor (TCR), the constant regions of which have been cloned and sequenced in the brushtail possum (Zuccolotto et al., 2000). Despite repeated attempts to elucidate IL2 in possums, we and others (Harrison and Wedlock, 2000) have been unable to identify it until genome sequencing data from two marsupial species, the opossum (Monodelphis domestica) and the tammar wallaby (Macropus eugenii) recently became available. Using this sequence information to inform our molecular homology experimental approach, we renewed our efforts to identify this molecule in the brushtail possum, and report here the molecular characterisation of possum IL-2.
2. Materials and methods 2.1. Tissue and cell isolation Tissues containing resident and circulating lymphoid cells were collected from a 3.4 kg clinically healthy mature male possum that had been captured from the wild and acclimatised to captivity for four weeks prior to experimentation. The possum was euthanased humanely with appropriate approval from the Animal Ethics Committee of Landcare Research, Lincoln in accordance with the 1987 Animals Protection (Codes of Ethical Conduct) Regulations of New Zealand (Approval No. 10/02/01). Single cell mononuclear leukocyte suspensions were prepared in RPMI 1640 base medium
0145-305X/$ - see front matter Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.dci.2011.05.010
L.J. Young et al. / Developmental and Comparative Immunology 36 (2012) 236–240 Table 1 Stimulation indices for Con-A stimulated possum lymphoid cells. Mononuclear cells (105 in 200 lL) were cultured with/without Concanavalin A before being tritated for 16 h after periods of mitogen-stimulation of 24 or 72 h (six replicate wells for each sample). Data represent mean stimulation indices (proliferation value (cpm) with mitogen/proliferation value (cpm) without mitogen).
a
Tissue source
24 h time-point
72 h time-point
Retropharyngeal lymph nodes (LNRP)a Mesenteric lymph nodes (LNM) Blood Spleen
330 ± 17 205 ± 37 212 ± 16 192 ± 6
1035 ± 17 486 ± 59 410 ± 17 154 ± 6
LNRP tissue source used for RACE DNA construction.
(Sigma, Australia) from the following tissues: Ficoll gradientseparated blood and spleen suspensions, homogenised tissues of the retropharyngeal lymph nodes (LNRP) and the mesenteric lymph nodes (LNM). Cell suspensions were adjusted to 106 viable cells/mL in RPMI (supplemented with 10% foetal calf serum and antibiotics) and stimulated in in vitro culture with and without 5 lg/mL of the T cell mitogen, Concanavalin A (Con A). Control and stimulated cells (viability of >90%) were harvested for molecular studies at 24 and 72 h (Table 1) when cells were in active blastogenesis. This activity was confirmed by visual inspection and by tritiated thymidine proliferation assay using standard techniques. Briefly, cellular proliferation of each cell preparation was confirmed by measuring beta emission following pulsing of the cells with 1 lCi/well of tritium-labelled thymidine over the last 18 h of stimulation. Stimulation indices were then determined by comparison of the stimulated cell cultures with their unstimulated control preparations.
237
2.2. Identification of the possum IL-2 gene A reverse-transcriptase polymerase chain reaction (RT-PCR) strategy followed by the rapid amplification of cDNA ends (RACE) technique was used to identify the expressed possum IL-2 gene. RNA was isolated from Con-A stimulated LNRP mononuclear cells using the GenElute Total RNA Miniprep Kit (Sigma–Aldrich) according to manufacturer’s instructions. Isolated RNA was treated with DNase 1 to remove residual genomic DNA. For RT-PCR, cDNA was synthesised using the superscript III RT system following the protocol recommended in the kit (Invitrogen). Primers were targeted to conserved regions of the IL-2 sequences predicted for the American gray short tailed opossum ( M. domestica) and the Australian tammar wallaby (M. eugenii) (accessed through ENSEMBL at http://www.ensembl.org/index.html). These primers (‘IL2FC1’ 50 -atgaa/gcaaggta/cccgctcc/ttgt-30 and ‘2Rex1’ 50 -acagtcacattaatgttg-30 ) were also subsequently used in RACE experiments to determine the entire IL-2 cDNA sequence. PCR using Taq DNA polymerase (Invitrogen) at an annealing temp of 50 °C for 35 cycles yielded a single DNA amplicon, which was gel-purified using the Wizard SV Gel and PCR Clean-up System (Promega, Australia) and directly sequenced at the Australian Genome Research Facility (AGRF; Brisbane, Australia). The returned sequence was edited to remove primer sequences and processed through Blastx (basic local alignment search tool; Altschul et al., 1997) for assignment of putative identity against known mammalian IL-2 gene sequences. Possum RACE DNA was performed using RNA obtained from Con-A stimulated LNRP in order to obtain the 50 - and 30 -untranslated regions of the IL-2 gene. RACE DNA was prepared
Fig. 1. Nucleotide and predicted amino acid sequence of possum IL-2. The nucleotide sequence comprises a coding domain of 426 bp to the stop codon, a 50 -UTR of 54 bp and a 30 UTR that contains one instability motif (attta in bold). A polyadenylation site (underlined), 19 bp upstream from the polyA tail (n = 24), is also evident. Putative N- and Oglycosylation sites are underlined and circled, respectively. The conserved region of the IL-2 family signature is shaded. GenBank Accession number for the possum nucleotide sequence is HQ717721.
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following manufacturer’s instructions using the GeneRacer Kit (Invitrogen, Australia). Both 50 - and 30 - RACE amplicons were amplified directly using kit-supplied RACE primers (‘GeneRacer 50 nested primer’ 50 -ggacactgacatggactgaaggagta-30 and ‘GeneRacer 30 nested primer’ 50 -cgctacgtaacggcatgacagtg-30 ) matched to their 50 - or 30 -reverse and forward RT-PCR primers respectively. RACE PCR reaction volumes of 20 lL were performed in duplicate using 2 lL cDNA (1/10 dilution of original RACE cDNA template), 2.0 mM MgCl2, 0.5 units of Hi-Fidelity Platinum Taq Polymerase and remaining conditions as recommended by RACE kit instructions (Invitrogen, Australia). Gel-purified amplicons were directly sequenced by AGRF as described earlier.
Blast suite of programs (Altschul et al., 1997). ClustalW2 (accessed through http://www.ebi.ac.uk) was used to construct multiple sequence alignments. A phylogenetic tree was constructed using the program Geneious (Drummond et al. 2010) where sequences were aligned with MUSCLE and a neighbour joining tree was generated using 100 bootstrap replicates. A number of programs were accessed through the Expasy Tools website (http://au.expasy.org/ tools/) to identify family motifs, a predicted signal peptide and possible glycosylation sites.
2.3. Molecular characterisation of possum IL-2
3.1. Cell proliferation and molecular identification of possum IL-2
The nucleotide sequence for putative possum IL-2 was analysed for identity and similarity to other known IL-2 genes using the
Possum lymphocytes isolated from a number of immune tissue sources all responded to stimulation by the T-cell mitogen Con A (Table 1). In vitro mitogen stimulation of lymphocytes is known to lead to IL-2 secretion (Gaffen and Liu, 2004), and the high stimulation indices associated with our in vitro culture were thus optimal conditions for the cellular synthesis of possum IL-2. Preliminary RT-PCR experiments using marsupial-specific primers confirmed the presence of IL-2 amplicons of the correct size in cells optimised for the expression of this gene. Blastx analysis of the possum IL-2 RT-PCR product identified this amplicon as having approximately 45–58% similarity to known mammalian IL-2 peptide sequences. RACE PCR subsequently yielded a complete cDNA sequence of 764 bp that includes a 50 -UTR region of 54 bp and a 30 -UTR of 281 bp (see Fig. 1). The nucleotide sequence for possum IL-2 contains a single attaaa instability motif in the 30 -UTR and a polyadenylation signal 19 bp upstream from the polyA tail (see Fig. 1). This sequence has been deposited in GenBank and has been
Fig. 2A. Multiple sequence alignment of IL-2 peptide sequences. The predicted signal peptide for possum IL-2 is indicated above the alignment and the putative mature possum IL-2 peptide begins at residue 21. Residues that are completely conserved across mammalian species (upper case in the consensus line) have a black background. Residues that have a grey background are 80% conserved in this alignment (lower case in the consensus line). Dashes (–) represent gaps introduced by ClustalW2 to optimise the alignment. The predicted signal peptide for possum IL-2 is indicated above the alignment and the mature possum IL-2 peptide begins at residue 21. Conserved cysteine residues required for the formation of intrachain disulfide bonds essential to cytokine function are indicated by #. indicates conserved residues that are involved in IL-2 receptor interactions across a number of mammalian species. Accession numbers for these sequences are: possum Trichosurus vulpecula (this paper), opossum Monodelphis domestica (Wong et al., 2006), woodchuck Marmota monax gb|ABB84516.1, rabbit Oryctolagus cuniculus ref|NP_001156652.1, pig Sus scrofa ref|NP_999026.1, bat Rousettus leschenaultia dbj|BAH02558.1, cat Felis catus gb|AAX63392.1, horse Equus caballus gb|AAA20134.1, lama Lama glama sp|Q865X2.1, human Homo sapiens gb|AAH66254.1, camel Camelus dromedaries gb|ADH51719.1, macaca Macaca mulatta ref|NP_001040595.1, goat Capra hircus gb|AAG28783.1, dog Canis lupus familiaris ref|NP_001003305.1 and rat Rattus norvegicus ref|NP_446288.1.
3. Results and discussion
Fig. 2B. Phylogram of IL-2 amino acid sequences. The phylogram was constructed using an alignment that included a range of mammalian IL-2 sequences and an avian sequence for comparison. The phylogenetic tree was constructed using Geneious as outlined in the text. Internal branch reliability was assessed using 100 bootstrap replicates and branch support values over 50 are shown as percentages. Accession numbers for mammalian sequences are as for Fig. 2A. Accession number for the chicken Gallus gallus, an avian species, included for demonstration of grouping of the marsupial clade with other mammalian species, is gb|AAS00717.1.
L.J. Young et al. / Developmental and Comparative Immunology 36 (2012) 236–240
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Table 2 Sequence conservation of mammalian IL-2 peptides in comparison with predicted possum IL-2. Identity and similarity scores are recorded as reported using Blastp analysis against putative possum IL-2. %Gaps introduced by program defaults to optimise the alignments are also indicated. Animal and Accession number
%Identity
%Similarity
%Gaps
Opossum (Wong et al., 2006) Horse (gb|AAA20134.1) Lama (sp|Q865X2.1) Camel (gb|ADH51719.1) Pig (NP_999026.1) Whale (gb|AAD01426.1) Bat (dbj|BAH02558.1) Woodchuck (gb|ABB84516.1) Elephant seal(gb|AAC12258.1) Human (gb|AAH66254.1) Cat (gb|AAX63392.1) Dog (ref|NP_001003305.1) Macaca (ref|NP_001040595.1) Gerbil (emb|CAA48679.1) Mouse (gb|AAA39281.1) Rat (ref|NP_446288.1)
63 37 35 34 36 34 37 37 34 35 34 33 33 31 28 29
75 58 56 56 54 54 53 52 51 50 50 50 49 48 48 45
0 8 8 8 9 9 9 7 7 7 7 8 11 12 19 11
assigned accession number HQ717721. We also searched the publicly available possum EST database (http://www.possumbase.org.nz/) using our amplified RT-PCR sequence. No significant hits were obtained in this search, suggesting that the IL-2 gene sequence is not present in that database, explaining in part why this gene has not been identified in the possum genome until now. 3.2. Comparison of possum IL-2 with other mammalian IL-2 molecules Translation of the coding domain of the possum IL-2 nucleotide sequence (tIL-2) results in a predicted peptide length of 142aa to the stop codon. A calculated molecular weight of 15.8 kDa (13.8 kD for the mature transcript) is consistent with mammalian IL-2 gene products. Possum tIL-2 also shares many of the canonical features of other known mammalian IL-2 peptides (see Fig. 1). Both the 4helical cytokine (E = 2.53e15) motif and the IL-2 family signature [T-E-[LF]-x(2)-L-x-C-L-x(2)-E-L] are recognised (E = 1.19e08), although the IL-2 signature is not completely conserved (Fig. 2A). There is a predicted signal peptide of 20 aa residues from the start site (SignalP 3.0), consistent with the secretory properties of this mammalian cytokine. Two N-glycosylation and one O-glycosylation site are also predicted at residues 47–50, 62–65 and 23, respectively (Fig. 1). Cysteine residues, important for molecular folding and function of the mature protein (Zelus et al., 2000), are conserved by alignment with other known IL-2 peptide sequences (Fig. 2A) as are some amino acids essential for binding to the IL-2Rb and IL-2Rc chains of the high-affinity trimeric IL-2 receptor (Zelus et al., 2000). Many of the canonical features of mammalian IL-2 appear to be conserved in the possum gene and translated protein. Despite the conservation of canonical features, the overall level of amino acid identity between possum IL2 and IL2 of other species is low (Table 2). Phylogenetic analysis reveals that the putative possum IL-2 peptide forms a distinct marsupial clade with the predicted IL-2 opossum sequence when compared with other mammals (Fig. 2B). Nonetheless, when an avian IL-2 sequence is included in the analysis (chicken IL-2), the marsupial sequences cluster with members of the class Mammalia, which is consistent with the evolutionary distance between mammals and birds. The presence of a number of key features consistent with mammalian IL-2 molecules such as topology consistent with the 4-helical superfamily, a signal peptide (common to secreted immunoregulatory molecules) and a number of canonical features of the IL-2 family all suggest that possum IL-2 may play a similar immunological role in the modulation of T cell immune responses.
Fig. 3. Expression of the IL-2 gene in Con-A stimulated lymph node cells. An RT-PCR product of 311 bp was amplified in lymph node cells treated with Con A for 24hrs (Lane A – mesenteric lymph node and Lane B – retropharyngeal lymph node). Lane C contains an untreated control, demonstrating the lack of expression of this gene, and is representative of all control experiments. PCR conditions for this assay included an annealing temperature of 50 °C and amplification for 35 cycles. Lane D is a representative no-template control. IL-2 expression is shown in the top panel against molecular weight marker bands (M) of 500 and 250 bp in each panel. The bottom panel shows b-actin expression in the same tissues.
3.3. Expression of possum IL-2 under conditions of T cell stimulation Since IL-2 plays an essential role in T cell proliferation, regulation and memory functions associated with CMI (Malek, 2008), we chose to renew our efforts to characterise this cytokine gene in the brushtail possum and successfully identified this molecule in mitogen-stimulated lymph node tissue. Putative possum IL-2 was expressed in Con-A stimulated lymph node cells but was not readily detected in resting lymph node cells cultured under the same conditions (see Fig. 3) when normalised against the expression of the b-actin gene. Con A is a well-documented T cell mitogen used to assess proliferative responses in possums (Buddle et al., 1994), and the expression of the IL-2 gene in this study is consistent with the induction of this molecule under similar in vitro conditions to those used in studies of other mammalian species to date. This suggests, at least at the in vitro level, that the possum IL-2 gene may possess analogous immunoregulatory pathways to eutherian mammals. Until this report, the major regulators of adaptive immunity, specifically cytokines such as interleukin-2 that regulate important T helper cell responses, have not been successfully characterised in the possum (Harrison and Wedlock, 2000). This has proved a considerable drawback to studies of disease susceptibility in this species. Availability of genomic data has provided much needed information to progress our study of highly divergent and immunologically important regulatory molecules in the brushtail possum.
Acknowledgements L.J.Y. completed this work whilst on sabbatical at The University of Sydney. This research was funded by the NZ Foundation for Research, Science and Technology (Possum Biocontrol OBI C10X0501). References Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J.H., Zhang, Z., Miller, W., Lipman, D.J., 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402.
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