Expression of nine-banded armadillo (Dasypus novemcinctus) interleukin-2 in E. coli

www.elsevier.com/locate/issn/10434666 Cytokine 32 (2005) 219e225

Expression of nine-banded armadillo (Dasypus novemcinctus) interleukin-2 in E. coli J.E. Adams, M.T. Pen˜a, T.P. Gillis, D.L. Williams, L.B. Adams, R.W. Truman* Laboratory Research Branch National Hansen’s Disease Programs, Louisiana State University, School of Veterinary Medicine, Skip Bertman Drive, Baton Rouge, LA 70803, USA Received 15 June 2005; received in revised form 26 August 2005; accepted 1 September 2005

Abstract The nine-banded armadillo (Dasypus novemcinctus) is the only immunologically intact animal that regularly develops lepromatous-type leprosy when inoculated with Mycobacterium leprae. However, the ability to exploit this model for understanding the pathogenesis of leprosy has been limited by a lack of suitable immunological reagents. Recently, efforts began to sequence the entire armadillo genome, and this sequence information will help make possible the development of a wide array of new immunological reagents suitable for use with armadillos. Using the available sequence data, a region of high homology to interleukin-2 of other mammals was identified. Primers were designed to amplify the coding region corresponding to the mature peptide and its exact sequence was confirmed. cDNA was made from ConA-stimulated armadillo PBMC. The amplified coding region was sub-cloned into a pET expression vector and transformed into Escherichia coli for over-expression. The subsequent product was characterized by SDS-PAGE and bioassays. Tritiated thymidine incorporation by CTLL-2 and armadillo lymphoblasts confirmed functionality of the recombinant product. The advent of the D. novemcinctus genome sequence and subsequent generation of immunological tools will assist in advancing the armadillo as a translational model for leprosy. Published by Elsevier Ltd. Keywords: Dasypus novemcinctus; Interleukin-2; Leprosy; Lymphocyte proliferation; Mycobacterium leprae; Nine-banded armadillo; Recombinant

1. Introduction Nine-banded armadillos, Dasypus novemcinctus, are the most abundant living members of the mammalian order Xenarthra. These exotic-looking cat-sized animals range throughout North and South America and are especially abundant in the southern United States. Armadillos in Texas and Louisiana are known to harbor Mycobacterium leprae, the etiological agent for leprosy [1e3]. This bacterium cannot be cultivated on artificial laboratory media and armadillos have developed into the hosts of choice for in vivo propagation of leprosy bacilli [4,5]. Nine-banded armadillos are the only immunologically intact animal species that exhibit high susceptibility to M. leprae. Like man, leprosy in the armadillo can manifest * Corresponding author. Tel.: þ1 225 578 9860; fax: þ1 225 578 9856. E-mail address: [email protected] (R.W. Truman). 1043-4666/$ - see front matter Published by Elsevier Ltd. doi:10.1016/j.cyto.2005.09.011

over a broad clinical spectrum of immunological and histopathological responses classifiable as Lepromatous or Tuberculoid and the associated borderline forms [6]. Owing to their unique susceptibility, armadillos have been considered potentially valuable models for leprosy pathogenesis, understanding factors underlying susceptibility and resistance to the infection, and for development of new diagnostics and vaccines [5]. Unfortunately, owing to their exotic nature and scant commercial value, relatively few armadillo-specific immunological reagents have been generated. As a result, few translational benefits have been realized from work with this model. Resistance to M. leprae is mediated through cellular immune responses and involves a complex interplay of cytokines and chemokines. It has not been possible to monitor the cytokine response of armadillos. IL-2 is an essential driver of any T-cell response [7] and the IL-2 of many mammals have already been cloned and overexpressed in Escherichia coli [8e13]. This protein often exhibits functional cross reactivity between

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mammals, but the nucleic acid sequence appears to be only partially conserved. Monoclonal antibodies and primer sequences prepared for mouse or human proteins seldom react with armadillos (Promega, Madison, WI; R&D, Minneapolis, MN). Owing to the armadillo’s evolutionary and medical significance, The Human Genome Consortium recently began sequencing the Dasypus novemcinctus genome. Trace sequence reads for D. novemcinctus are already available (http:// www.ncbi.nlm.nih.gov/BLAST/tracemb.shtml) and annotation of the genome is now underway. We searched this sequence data for regions homologous to IL-2 and report here the identified sequence, cloning, over expression and biological activity of recombinant Dasypus novemcinctus IL-2. 2. Materials and methods 2.1. Database searches tBLASTn was employed by using the amino acid sequence of Mus musculus IL-2 (GI: 7110653) as a query sequence to search for homologous translated sequences in the high throughput genomic sequences (htgs) database. The putative genomic sequence of DnIL-2 (GI:33620804) was found at NISC (NIH Intramural Sequencing Center) comparative sequencing initiative (http://www.nisc.nih.gov/). The genomic sequence was submitted to FGENESH (http://www.softberry.com) to derive a putative cDNA and a corresponding translation for the putative amino acid sequence. The cDNA and the amino acid sequence were submitted to BLAST (http://www.ncbi.nlm.nih.gov/ BLAST/) to verify homology to other IL-2’s [14]. 2.2. Generation of D. novemcinctus cDNA Armadillo peripheral blood mononuclear cells (PBMC) were harvested from whole blood and treated with ConA (SigmaAldrich, St. Louis, MO). Aliquots of the ConA stimulated cells (5 mg/mL for 4 h) were washed 3
in cold PBS, harvested, snap frozen in liquid nitrogen, and stored at 70  C until RNA purification. Total RNA was purified using the FASTRNAÔ kit (Qbiogen, Carlsbad, CA) and the FASTPrep fpizo 120Ô instrument (Qbiogen, Carlsbad, CA) according to the manufacturer’s protocol. cDNA was generated from 1 mg RNA and the Advantage RT-for-PCR kit with random hexamers (BD Biosciences Clonetech, Palo Alto, CA) in a final volume of 50 mL according to the manufacturer’s recommendations. 2.3. Plasmid construction, expression and purification of DnIL-2 Primers designed to amplify the coding region corresponding to the mature peptide (the protein without the signal peptide) of DnIL-2 (MIL2cDNAf: 5#-TGCACCTACTTCAAGCTCTACAAA-3# and MIL2cDNAr: 5#-TAGCAAACCATACATCCAAAAATA-3#) (BIOMEDD, Baton Rouge, LA) were used with high fidelity polymerase, Pfu (Strategene, La Jolla, CA), to generate the fragment to be subcloned. This fragment was purified using QIAquick columns (QIAgen, Valencia, CA),

verified by automated DNA sequencing using an ABI prism 377 DNA sequencer (Applied Biosystems, Foster City, CA) (BIOMMED, Baton Rouge, LA) then reamplified using a primer containing the topo sequence (CACC) on the 5# terminus (MIL2-TOPO: 5#-CACCGCACCTACTTCAAGCTCTAC-3#). This topo fragment was subcloned into pET 200D/topo vector (Invitrogen, Carlsbad, CA) and transformed into E. coli BL21star (Invitrogen, Carlsbad, CA) according to manufacturer’s recommendations and verified by PCR and subsequent automated DNA sequencing. These cultures were grown to an OD600 of 0.6 and induced with a final concentration of 1 mM isopropyl b-D-thiogalactopyranoside (IPTG) (Sigma-Aldrich, St. Louis, MO). The overexpressed protein was then purified using a Ni-NTA Purification system using the recommended, denaturing regimen (Invitrogen Carlsbad, CA). The resulting product was concentrated using a Vivaspin 15 mL concentrator with a 10 KDD molecular weight cut-off (Vivascience, Hanover, Germany). The over expressed product was separated via SDS-PAGE (4% to 20% gradient polyacrylamide gel) and stained with coomassie brilliant blue (Biorad, Hercules, CA), compared against Kaleidoscope pre-stained standard (Biorad, Hercules, CA), and a western blot using the Anti-xpressÔ antibody (Invitrogen Carlsbad, CA) was used to verify the size and presence of the polyhistidine epitope of the recombinant product. 2.4. Lymphocyte isolation from D. novemcinctus and mitogen induction To avoid sacrificing animals and to permit re-examination of individual hosts, lymphocytes were isolated from armadillo peripheral blood. PBMC were purified from 14 mL peripheral blood collected in Vacutainer tubes containing 150 U of heparin. Blood was diluted in 2
volumes of cold Hanks’ Balanced Salt Solution (HBSS) and lymphocytes were isolated by density gradient centrifugation after layering over Ficoll-PaqueÔ Plus (Pharmacia Biotech, Piscataway, NJ) in a 50 mL tube and centrifuged at 400
g for 45 min at 18  C. The lymphocyte layer was removed using a sterile pipette and washed 3
with cold PBS. The suspension was centrifuged at 400
g for 10 min at 4  C and resuspended in 1 mL of RPMI 1640 media (RPMI) with 5% fetal bovine serum (FBS). Cell viability was determined using trypan blue exclusion. This cell suspension was seeded into 24 well culture plates (1.5 mL/well) at a concentration of 2
106 cells/mL. ConA was added to a final volume of 5 mg/mL. This concentration was found to be optimal by a titration assay where ConA was used at 5, 10 and 20 mg/mL (data not shown). The plates were incubated for 0, 4, 8, and 24 h [15] at 37  C, 5% CO2. After incubation, supernatants were harvested and clarified at 9300
g for 10 min and stored at 70  C until used. 2.5. CTLL-2 bioassay Briefly, the murine IL-2 dependent cell line CTLL-2 (ATCC# TIB-214) was obtained from the American Type Culture Collection (ATCC, Manassas, VA) and maintained in complete growth medium consisting of RPMI 1640 with 2 mM

J.E. Adams et al. / Cytokine 32 (2005) 219e225 L-glutamine and supplemented with 10% rat T-STIM containing ConA (rat IL-2 culture supplement) (Becton-Dickson, Bedford, MA) and 10% fetal bovine serum (FBS). The biological activity of rDnIL-2 was determined in a cell proliferation assay [16]. The CTLL-2 cells were harvested in active log-phase and washed with RPMI media three times to remove residual rat IL2. Recombinant human IL-2 (rHuIL-2) (R&D Systems, Inc) was used a positive control for the assays. rHuIL-2 (1883 ng/ mL) recombinant DnIL-2 (rDnIL-2) (770 ng/mL), and supernatants were two-fold serially diluted in RPMI 10% FBS and 50 mL of each dilution was added to a 96-well microtiter plate. CTLL-2 cells were distributed in 50 mL RPMI medium at an optimal density (determined by titration) of 8
105 cells/mL to a 96-well microtiter plate containing 50 mL of the serially diluted recombinant IL-2’s or supernatants from the cells cultured for different time intervals. The cells were cultured for 20 h at 37  C with 5% CO2. After incubation, plates were pulsed with 10 mL tritiated thymidine (3[H]dTr) (Perkin-Elmer, Boston, MA) (10 mCi) per well and incubated for 16 h. The plates were harvested using a PHD Cell harvester (Cambridge Technologies, Inc., Cambridge, MA) and the incorporation of 3 [H]dTr was determined by liquid scintillation counting in cpm using a LS 6500 multi purpose scintillation counter (Beckman-Coulter, Fullerton, CA).

2.6. D. novemcinctus lymphocyte blasts We also assessed the ability of rDnIL-2 and D. novemcinctus PBMC ConA stimulated supernatants to maintain proliferation of D. novemcinctus lymphoblasts. Lymphoblasts were obtained after isolation of PBMCs from whole blood using Ficoll-Paque gradient. The cells were adjusted to 5
106 cells/ml in culture medium (DMEM with 10% FBS) and plated in 24 well tissue culture plates (1 ml aliquots) [12]. After the addition of Con A (5 mg/ml) the plates were incubated for 5 days. The cells were harvested and overlayed onto 10 ml of Ficoll-Paque and centrifuged at 700
g for 20 min. rDnIL-2, rHuIL-2, and supernatants were serially diluted with growth medium Dulbecco’s modified eagle medium (DMEM) and 50 mL was added to each well of a 96 well tissue culture plate. 50 mL of lymphoblast cells (5
105 cells/mL) was added and the plate was incubated for 24 h at 37  C or 33  C with 5% CO2. Proliferation of lymphoblasts was determined using thymidine incorporation as described with the CTLL-2 bioassay. 2.7. Protein quantification Protein concentrations were determined colorimetrically using the BCA protein assay kit (Pierce, Rockford, IL) on a Biorad plate reader (Hercules, CA) according to manufacturer’s recommendation. 2.8. Statistical analyses The means of the groups were compared using ANOVA and TukeyeKramer test was used to make multiple comparisons

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between the different groups (GraphPad InStat software, GraphPad Software, Inc, San Diego, CA). 3. Results 3.1. Sequences We found the nucleic acid sequence for the putative DnIL-2 (Fig. 1) at NISC using BLAST with the amino acid sequence of Mus musculus IL-2. The resulting 5979 bp genomic fragment was then submitted to FGNESH to map the coding region of DnIL-2 (Fig. 2). The genomic region contains four exons (Fig. 2) that compose a 468 bp predicted coding region. We verified this by sequencing cDNA from ConA stimulated PBMC. DnIL-2 showed the greatest homology after the first 20 amino acids when compared to other mammalian IL-2 amino acid sequences (Fig. 3) suggesting that these were part of the signal peptide. The entire DnIL-2 amino acid sequence (with signal peptide) was most homologous to that of Macaca mulatta (rhesus monkey) IL-2 (E-value: 1ee45 (84%)) and other mammalian IL-2’s: Cercocebus torquatus (red-crowned mangabey) (E-value: 1ee45 (83%)), Homo sapiens (E-value: 4ee45 (84%)), Papio hamadryas (hamadryas baboon) (E-value: 1ee45 (84%)), and Saimiri sciureus (common squirrel monkey) (E-value: 3ee45 (84%)). A ClustalW multiple alignment (http://searchlauncher.bcm.tmc.edu/multi-align/Options/clustalw. html) was done to examine similarities and differences between these IL-2’s and DnIL-2 (Fig. 4). Additionally, rat (Rattus norvegicus) and human (Homo sapiens) IL-2 was also examined for similarities and differences to DnIL-2 as rat T-STIM was used to maintain the CTLL-2 cells and rHuIL-2 was used as the positive control in bioassays. 3.2. Purification We found rDnIL-2 in the insoluble fraction (data not shown) of the E. coli lysate as has been reported with other mammalian recombinant IL-2’s [12,13]. The predicted rDnIL-2 fusion protein had a molecular weight of 19.7 KDD using ExPASY (http://us.expasy.org/tools/pi_tool.html). The high molecular weight was due to the inclusion of the histag and enterokinase recognition site (M R G S H H H H H H G M A S M T G G Q Q M G R D LY D D D D K D H P F T) added to the mature polypeptide sequence starting at residue A-21 (Fig. 4) in the recombinant product. We confirmed its size and purity by coomassie blue staining and a western blot using an antibody against the polyhistidine tag on the n-terminus of the recombinant product (Fig. 5). 3.3. Bioassays Multiple assays demonstrated rDnIL-2 and D. novemcinctus supernatants were capable of sustaining proliferation of CTLL-2 cells and D. novemcinctus lymphoblasts (Fig. 6a,b). Significant proliferation was seen when CTLL-2 cells or D. novemcinctus lymphoblasts were cultured with rDnIL-2

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aagtaatact ttttgccaca caggtagagc ctttgaaaat atgtgtaata tgtaaaaCAT Tttgtttgac acccccaTAA gtataaattg cttctcttgt tcaagagctt catatcaacc tctctaatca ctactcacag taacctcaac tcctgccaca GGCTTGCATT GCATTAAGTC TTGTACTCAT CACAAACAGT GCACCTACTT CAAGCTCTAC AAAGGAAACC CAGCAACAAC TTAAAGATGC TGTCCAAGAT GGTTAATgta agtatattcc ctttttaact aaaattatta cacttaattt ttctagctgg tgtgctattc tttctcagAA TAAGGATCTC AAACTCCCAA GGATGCTCAC ATTTAAATTT TACATGCCTA AGAGGgtaag ttatgtgtta aaattcaaag aaacatgaaa tttttaacat gttgacttaa gagcatctca tctgaaggaa aagaattagt tccaagttag ttaatagagt ttctattaaa atggcttggc ctatactgtt tagatttact tgggtgtatt ttcgggatca gaaaacaaga ataatattct ctttattgct gggacaattg taaaagaaga ttttgttgaa ttcctgcttt gtttaaaaat gaatttatta ttgtatcatg caacatgtca aaattgagtt cagaggagca cactttgaaa catcctttat tgtttgtgga agtttatttg aaccaaaatc ccagctctga catgtgctaa ctttcatcat ctttttatgt gaataataat tttatttact ttagtgagta aatgtagagt actcaaaaca gtgtctggca catggtaaac accaaataag ccttagcttt tattagtagt tgaggagtat gaagtaaaat tttggactta cacatctctt atgtcaatag acctggagat ttatggtgct atgtgattta tttgattgct tgggaggcca gatagttaga taagcacagc tcattcaagc tcctagaact acaacaaaga tgtaaagttt gcaaggatgg agcaaagctt tgatgctaga cagaacgtgg cccagggtga aaccaagtcc agaaaacaat gggatttaag ttgatttcct tctattagac tgagaatttt gtttcaaagt actgttttga aagacaaaaa gaaaatagct tagagactcc ataaccctta cctccaatca acctaagaga cactagactg tttttggagg aaaatagttc caagttgagc atgccgaaaa tagtaagttg aaagggcagt aaatggaaaa gtcaggagat gcataatgat tagtcctacc tttctctcta aaaattgtta gtgtataatt gggggtggag gctagagaat tctttgaaat tgaatagttt ctaaatgaca actcaaattc tatttgaaac ccattcactt gttcatttta ccaccattcc acatttgcaa aagattttag aaagtcatta aaaatttaaa gagatgctct taaaatttaa tcccggctct acctactcat gtggccttgg ggaaggcccc ttacttttga gattcagttt ccttatctgc gtactgccaa ccaccaaggg tggctgtgac acttaaaagg gcttagtgaa actataatgt accatagaag tacaagtatt atggctagtt atctgcagca tctagatcag gaggcctgag gaaaaaaaaa aatccttcaa atattggaac cttatatttg aatgcatctt aatgttgtaa aaacttaaga cacagcttta tgacttgctc cagactatat tgaatgtcat tattatagct atgttgggac tgtgataaat ggtgtccata gcagtcccta gcagtctaag attttcaaaa tcaaagctta gaattgcaga cattctggct gctctggttc taatgatttc tatgtaggtt agaagaaata gcctatatat ctgtgatttg tatttgggtg aaaggcaaac taacatgaat gagttcttag tcattaacag ttaatgctat ttatggttaa ttgatctcca gttttttcag cactggtcca tctttatagc aatgctgaaa acagggagaa aacaggtttc taaaatctat tgtagattaa tttctgattc attgagatga taaatattat cattattcta gGTCACAGAG TTGAAACATC TTCAGTGTCT AGTAGAAGAA CTCAAACCTT GCTCAAAGCC AAATGTCTCA ACTGGAACAT AATGGGGACT TAATCAGTAA TATCAATATA ACCGTTCTGG AGCTAAAGgt ctcctagaaa taaaataaat gcaaggggtg aaatattgta tttaaagttt tataatattt ttggtatttt gtaaaatatc attttaagcc cactgtaaac accaagtatt tttaatgtta gatgatatgt tattgagcta agaagtagaa gcttgaggtt ggacaagtcc cagcctctat atgaataatc accttcccct aatcagccac atctagtccc tcagcagtgg gagaaggcct gcagagtttt tcttcagatc atttaccagg atagttaata cagattacct gtgaggcttt ctaaatggca tttccctatc agaaatgatt tgtctcaaat aatggctgtc attaaataaa atggccaatg gaataaataa ctaactcttt atttagagga tatttttaag tgcacaggaa gtattaaaat atataaaggt ctcacatgat gtgaaagaga ttcaaggtgc tcttttatca aattcataag ttaataggta tcctacataa gcagtataat tttaactaca gtagaaccaa gcttctaaaa atacataaat ttattttagc ggaatttttt tcagcctaat ttaaaaaaaa caatacatca acattgagac cactgttatc tagagcccta ataaaatctt tgaccaatat cagaaaactc atgctaaatc tctgatgcaa ggttaaaata aagttatgtg aaagtcagca agactctttt taacttaaac aaacaaacaa aaacagagcc caagaacagg gaattaatgt ttacaatata tagagtttct tttcgtaatg aacaatggtg aattaaatat ttgaatggta aaaggggaaa ttttaggtat atatgttact ttaaggataa ggaacaacac agtgaaccct gttaaaccat gcactatagt tattagtaca attataaaaa tgtacttcta ttgattttaa aaggtgttca taacagggta gtatacagga acattgcatt ttatgcatga tttttctgta aacccactac tgtctaataa taaataattg tgtaagagga tacataatac tatagcagct ttttgtttgt ttgtttgttt gttttttcag ggaatacttt tgttcccaag ataggaatta cttcaaattg gaaaaagtta tcttttgtgt ttcactatta taaaaatatt tgtttcttag gtgaaatggc tcctatcatc aaaaaaagat taatttccaa aaaggcaaca gagaaaaatg gctaatgttt aacttttaaa tagatactgg gataaagggg atgggtatga gcagttatga agatgttcat ataaaccttc cccaaacaaa agcttgtcct tatcctcgga ttcactcaaa tagaatatta agtgtctctt aatgaaggac agaaaagaaa gtaaaatgtg catgccactg gtatatagat atacatacct agataaacac agagaggaaa agagatcaaa taacttcata tttgatgcag tatttaatga taaaatggtt acttgactac aatccaggaa aacatcttgt gttcattgag actgacagaa aaattaacag atgaagaaaa tcattgatat cataaatata attttgacac attttaatat tttgatcatt tcattttaga agatatgaat gactaatatg aggtcatgtg cctataaaac ttcaactgag aataattaca taaaaggcaa actaccctaa actaaaaaaa attaaaattt CAACATTCAT GTGTGACTAT GATGATGAGG CAGCAACCAT TGTAGAATTT CTGAACAAAT GGATTATCTT TTGTCAAAGC TAATTAAgtg ccttccattc aaaatacatc tatttattta aatatttaaa ttttataatt tatttttgga tgtatggttt tacttagatg atgaatatgg atcttttaag attctttttg taggccctag gggctctaaa atgctttcac tttaaattat atattgaatt gttacatata atgtctatat aggtcaatta ataaaattgt ttaataaact tgatgaatgg ttatttggga aaatattgtg aattatttat gtgaattcta agatggttaa aatgcttaac aaaagtcact tttcccatag agaggtatgt tcaaagctct tgctttctct ttccaagagt tgatcagatc cttgatatct tagttctggt ctgagaaaac tacctcataa tttgatcatg ccttaaattt aaacaaatgt gaaacatatt cttgaaggtc ttcatacaac cggtgtgcct ctctatccac acccaccacc cctcaggagg ctaagctata acaaaaccct cccttgtctt ttggctttgg ctttccattg ggtcttttga acttaaggga cacagaggag attctcctga ctccctccta gcaatattac atctggatgg cgatacctct tgaccaaag

TAttttttcc ATGTACAAGA TGGAGCAATT agtttgattg tacaattttc aaatgcacta gggggaaagc gcaaaactct tagccacaac ttatatagtt aattaataca tttccaggag cctagtagta ggtacagaga tccaatttaa cttaagcctg gaaagataac caaaagtaat gttaatgcta aaaatggata atcattatta gtttggacaa accattatct atgtgatttc cagaaatgct tcttataagg tgatcttcat TGGAGAATGT aagccattac catgtacttg tctaactaca tgaactgaat atttggtaga aatgcccact tcctccctgc tttagttgtt taactgttaa aaatagggtt atttggaatg agtatttttt caaatgtacc aaaaaaaaga acgctacctc ctagtcaggt agttgggata aactttctct catacatgta agtatttgtc tctcaggtta tgtaacatgc tcttttatag ATCATCTCAA gctacttttt tttatctcaa acagcacaga agaacaggga aactatactc cctttaccat ttggggagcc

agaattaaca TGCAACTCGT ACTACTGGAT taaataacaa tatgttccaa ttttctaaaa tgtaagaaaa agagatattt ttctagagaa cttgtgacaa gaattctcac taaaatttca gtgccagccc tataatcctg ccaacgctac gaactagtag atgagattat gtggtaaact taaagtgaat tagcaatact ataaaaatgc ataataaaga gatctgtaaa tttttttcca gactttttac taagctatta ttgagggtaa GCTAAATTTA tttatttgct ataagctaat gtctacccag ttcacagaaa aggactgatc gtcaggactg caaagtacaa tacaagttag gagatttgct caaatgccaa atggaaaagt aaaaattcat acaccaatgc ttataatact actttttttg agaggaagag cttgcttgca gggcaagttc acctacatag tagtattact tatgctagta ttgaaaataa GGATCTGAAA AAAGACTTGA gtaactatta agtatttatt ggaagtacta aatgggcttt ttttccttcc cttactgctt ctgttaagag

Fig. 1. The genomic sequence of DnIL-2 as obtained from NISC. The putative CATT box and TAATA box and coding region are indicated by uppercase bold letters.

or rHuIL-2. Supernatants derived from Con-A stimulated PBMC also drove proliferation of both CTLL-2 cells and D. novemcinctus lymphoblasts. Similar to what others have observed, greater proliferation was seen with supernatants that had been stimulated for 24 h than for shorter time periods [15]. Armadillo lymphoblasts incubated at 33  C showed a similar proliferative response (data not shown).

A representative dose response of CTLL-2 cells proliferation to different concentrations of rDnIL-2 and rHuIL-2 is shown in Fig. 7a. The 50% effective dose (ED 50) on CTLL2 proliferation was achieved at 0.023 pmol of rHuIL-2 and 0.19 pmol of our rDnIL-2 (Fig. 7a). Fig. 7b also shows a similar dose response on D. novemcinctus lymphoblasts proliferation with different concentrations of rDnIL-2 and rHuIL-2.

TATAA box

Polyadenylation

Exon I Exon II

Exon III

Signal

Exon IV

1 Kb

Fig. 2. Map of genomic DnIL-2. The 5979 bp region shown is predicted to contain the four exons that code for IL-2. Exon I (147 bp), Exon II (57 bp), Exon III (147 bp), and Exon IV (117 bp) are indicated by arrows. The putative TATAA box and putative polyadenylation signal are also indicated.

J.E. Adams et al. / Cytokine 32 (2005) 219e225

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atg tac aag atg caa ctc gtg gct tgc att gca tta agt ctt gta ctc atc aca M Y K M Q L V A C I A L S L V L I T aac agt gca cct act tca agc tct aca aag gaa acc cag caa caa ctg gag caa N S A P T S S S T K E T Q Q Q L E Q tta cta ctg gat tta aag atg ctg tcc aag atg gtt aat aat aag gat ctc aaa L L L D L K M L S K M V N N K D L K ctc cca agg atg ctc aca ttt aaa ttt tac atg cct aag agg gtc aca gag ttg L P R M L T F K F Y M P K R V T E L aaa cat ctt cag tgt cta gta gaa gaa ctc aaa cct ttg gag aat gtg cta aat K H L Q C L V E E L K P L E N V L N tta gct caa agc caa atg tct caa ctg gaa cat aat ggg gac tta atc agt aat L A Q S Q M S Q L E H N G D L I S N atc aat ata acc gtt ctg gag cta aag gga tct gaa aca aca ttc atg tgt gac I N I T V L E L K G S E T T F M C D tat gat gat gag gca gca acc att gta gaa ttt ctg aac aaa tgg att atc ttt Y D D E A A T I V E F L N K W I I F tgt caa agc atc atc tca aaa aga ctt gat aat taa C Q S I I S K R L D N stop

Fig. 3. The predicted coding region of 468 bp and resulting amino acid sequence of 155 aa of DnIL-2. The signal peptide is indicated in italics.

The ED 50 of D. novemcinctus lymphoblasts proliferation was achieved with 0.0013 pmol of rHuIL-2 and 0.028 pmol of rDnIL-2. 4. Discussion This report demonstrates that the coding region for the mature DnIL-2 was successfully amplified, subcloned, and overexpressed. DnIL-2 has high homology at the amino acid level to other mammalian IL-2’s and the recombinant protein appeared to be biologically adequate for sustaining proliferation of both CTLL-2 and armadillo lymphoblasts. The in silico predicted DnIL-2 had low Expect (E) values to other mammalian amino acid sequences of IL-2 (<4ee 45) indicating that the DnIL-2 was a close match [14]. The

deduced protein showed high amino acid sequence homology especially to the IL-2’s of Macaca mulatta as well as some other mammals (Fig. 4). It has been shown previously that the residue, D-40, in HuIL-2 is important for inducing proliferative activity of CTLL-2 cells and is responsible for high affinity receptor (IL-2R) binding [17]. Amino acid residues Y-64 and D-130 have been implicated in internal stabilization of HuIL-2 [17]. Residues 38e41 cannot be deleted without modifying the structure and function of murine IL-2 because they are related to the last turn of helix A and the first residue of the loop connecting a-helices A and B [18]. Residues K-55, R-58, F-62, and K-63 are located in the B a-helix of HuIL-2 and have been implicated as important residues for the binding of HuIL-2 to the low affinity (IL-2R) [19]. The translated cDNA sequence shows that all of these residues are conserved

Ph Ss Mm Ct Hs Dn Rn

10 MYRMQLLSCI MYRMQLLSCI MYRMQLLSCI MYRMQLLSCI MYRMQLLSCI MYKMQLVACI MYSMQLASCV ** *** :*:

20 ALSLALITNS ALSLALITNS ALSLALVTNS ALSLALVTNS ALSLALVTNS ALSLVLITNS ALTLVLLVNS **:*.*:.**

30 APTSSSTKKT APTSSSTKKT APTSSSTKKT APTSRSTKKT APTSSSTKKT APTSSSTKET APTSSPAKET **** .:*:*

40 QLQLEHLLLD QLQLEHLLLD QLQLEHLLLD QLQLEHLLLD QLQLEHLLLD QQQLEQLLLD QQHLEQLLLD * :**:****

50 LQMLLNGINN LQMLLNGINN LQMILNGINN LQMILNGINN LQMILNGINN LKMLSKMVNN LQVLLRGIDN *::: . ::

60 YKNPKLTRML YKNPKLTRML YKNPKLTRML YKNPKLTRML YKNPKLTRML -KDLKLPRML YKNLKLPMML *: **. **

Ph Ss Mm Ct Hs Dn Rn

70 TFKFYMPKKA TFKFYLPKKA TFKFYMPKKA TFKFYMPKKA TFKFYMPKKA TFKFYMPKRV TFKFYLPKQA *****:**:.

80 TELKHLQCLE TELKHLQCLE TELKHLQCLE TELKHLQCLE TELKHLQCLE TELKHLQCLV TELKHLQCLE *********

90 EELKPLEEVL EELKPLEEVL EELKPLEEVL EELKPLEEVL EELKPLEEVL EELKPLENVL NELGALQRVL :** .*:.**

100 NLAQSKNFHL NLAQSKNFHL NLAQSKNFHL NLAQSKNFHL NLAQSKNFHL NLAQSQMSQL DLTQSKSFHL :*:**: :*

110 RDTRDIISNI RDTRDIISNI RDTKDLISNI RDTKDLISNI RP-RDLISNI EHNGDLISNI EDAGNFISNI . ::****

120 NVLVLELKGS NVLVLELKGS NVIVLELKGS NVIVLELKGS NVIVLELKGS NITVLELKGS RVTVVKLKGS .: *::****

Ph Ss Mm Ct Hs Dn Rn

130 140 ETTFTCEYDD DTATIIEFLN ETTFTCEYDD DTATIIEFLN ETTLMCEYAD ETATIVEFLN ETTLMCEYAD ETATIVEFLN ETTFMCEYAD ETATIVEFLN ETTFMCDYDD EAATIVEFLN ENKFECQFDD EPATVVEFLR *..: *:: *:.**::***.

150 GWITFCQSII GWITFCQSII RWITFCQSII RWITFCQSII RWITFCQSII KWIIFCQSII RWIAICQSII ** :******

STLT-STLT-STLT-STLT-STLT-SKRLDN STMTQ.

Fig. 4. ClustalW alignment of closest matches in NCBI nr database using BLAST where Ph- Papio hamadryas; Ss- Saimiri sciureus; Mm- Macaca mulatta; CtCercocebus torquatus; Hs- Homo sapiens; Dn- Dasypus novemcinctus (bold-faced); and Rn- Rattus norvegicus. The signal peptide is indicated in italics. ‘‘*’’ indicates positions which have a single, fully conserved residue. ‘‘:’’ indicates that one of the following ‘‘strong’’ groups is fully conserved: STA NEQK NHQK NDEQ QHRK MILV MILF HY FYW. ‘‘.’’ indicates that one of the following ‘‘weaker’’ groups is fully conserved: CSA ATV SAG STNK STPA SGND SNDEQK NDEQHK NEQHRK FVLIH FYM.

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Fig. 5. Purification of rDnIL-2. (A) A coomassie stained gel with uninduced E. coli transformed with pET D topo þ DnIL-2 plasmid (U), E. coli transformed with pET D topo þ DnIL-2 plasmid induced with IPTG (I), and the purified rDnIL-2 (P); and, (B) a western blot using Anti-xpressÔ showing uninduced E. coli transformed with pET D topo þ DnIL-2 plasmid (U), E. coli transformed with pET D topo þ DnIL-2 plasmid induced with IPTG (I), and the purified rDnIL-2 (P). rDnIL-2 is indicated by arrows.

in DnIL-2, and it possesses similar functionality. The two ahelices on the N-terminus of the protein that are responsible for binding to the IL-2 high affinity and low affinity receptors may be preferred targets to DnIL-2 for future attempts to raise monoclonal antibodies.

Fig. 7. (a) Relative efficacy of recombinant IL-2’s to induce CTLL-2 proliferation on a per pmol basis. (C) represents the recombinant human IL-2 (rHuIL-2) and (:) represents recombinant D. novemcinctus IL-2 (rDnIL-2). Each point represents the mean  SD of triplicate wells. (b) Relative efficacy of recombinant IL-2’s to induce armadillo blasts on a per pmol basis. (C) represents the recombinant human IL-2 (rHuIL-2) and (:) represents recombinant D. novemcinctus IL-2 (rDnIL-2). Each point represents the mean  SD of triplicate wells.

Fig. 6. (a) CTLL-2 proliferation (in cpm) after incubation with media, supernatants generated by induction of D. novemcinctus PBMC with ConA at 0 h, 4 h, 8 h, and 24 h, and recombinants (recombinant D. novemcinctus IL-2 (rDn) and recombinant human IL-2 (rHu)). Media is cells with no recombinant IL-2 or supernatant and used as a negative control. Error bars represent mean  SD of triplicate wells. (b) Armadillo blast proliferation (in cpm) after incubation with media, supernatants generated by induction of D. novemcinctus PBMC with ConA at 0 h, 4 h, 8 h, and 24 h, and recombinants (recombinant D. novemcinctus IL-2 (rDn) and recombinant human IL-2 (rHu)). Media indicates cells with no recombinant IL-2 or supernatant and used as a negative control. Error bars represent mean  SD of triplicate wells.

We left the his-tag intact on the N-terminus of rDnIL-2 and this should not have had any significant untoward effect. It has been found that the presence of the six histidine residues from the his-tag of glutathione S-transferase fusion proteins on the amino terminus of the ovine IL-2 fusion protein did not affect the proliferative stimulus of recombinant IL-2 [12]. The lower activity of rDnIL-2 when compared to that of rHuIL-2 may be due to a portion of the rDnIL-2 having been irreversibly denatured and biologically inactive after purification from inclusion bodies leading to some loss of activity; however, rDnIL-2 was clearly biologically active and sustained proliferation in CTLL-2 cells and armadillo lymphoblasts. This is the first published report of a protein from D. novemcinctus to be overexpressed and bioassayed. rDnIL2 shows properties similar to other mammalian IL-2’s. This protein plays an essential role in the immune response and can be used effectively in model studies of the pathogenesis of leprosy. With the D. novemcinctus genome nearly complete, a vast array of armadillo cell markers, receptors, and cytokines will likely evolve in the near future and these animals will advance as important translational models for leprosy research.

J.E. Adams et al. / Cytokine 32 (2005) 219e225

Acknowledgements The authors would like to thank Kyle Andrews, Tana Pittman, Naoko Robbins, and Heidi Zhang for their technical assistance and express our appreciation to Dr. Jean Chang at the Broad Institute for information regarding the Dasypus novemcinctus sequencing project. Financial support: This study was supported in part by the National Hansen’s Disease Program and funds from the National Institute for Allergy and Infectious Disease, contract number YI-AI-2646-01.

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