Identification of interleukin-26 in the dromedary camel (Camelus dromedarius): Evidence of alternative splicing and isolation of novel splice variants

Abstract

Interleukin-26 (IL-26) is a member of the IL-10 family of cytokines. Though conserved across vertebrates, the IL-26 gene is functionally inactivated in a few mammals like rat, mouse and horse. We report here the identification, isolation and cloning of the cDNA of IL-26 from the dromedary camel. The camel cDNA contains a 516 bp open reading frame encoding a 171 amino acid precursor protein, including a 21 amino acid signal peptide. Sequence analysis revealed high similarity with other mammalian IL-26 homologs and the conservation of IL-10 cytokine family domain structure including key amino acid residues. We also report the identification and cloning of four novel transcript variants produced by alternative splicing at the Exon 3-Exon 4 regions of the gene. Three of the alternative splice variants had premature termination codons and are predicted to code for truncated proteins. The transcript variant 4 (Tv4) having an insertion of an extra 120 bp nucleotides in the ORF was predicted to encode a full length protein product with 40 extra amino acid residues. The mRNA transcripts of all the variants were identified in lymph node, where as fewer variants were observed in other tissues like blood, liver and kidney. The expression of Tv2 and Tv3 were found to be up regulated in mitogen induced camel peripheral blood mononuclear cells. IL-26-Tv2 expression was also induced in camel fibroblast cells infected with Camel pox virus in-vitro. The identification of the transcript variants of IL-26 from the dromedary camel is the first report of alternative splicing for IL-26 in a species in which the gene has not been inactivated.

Keywords: Alternative splicing; Camelids; Cytokine; IL-10; IL-26; Virus.

Fig. 1

Alignment of the deduced dromedary IL-26 amino acid sequence with that of other Camelids (predicted from the genome) and mammals. Predicted signal peptide cleavage site is marked by a downward arrow. The five conserved Cysteine residues are marked in highlight and annotated (C32, C79, C102, C121 and C124). The locations of the six alpha helices based on the SOPMA secondary structure prediction are also marked.

Fig. 2

Alignment of the Camel IL-26 (CdIL26) with Human IL26 (HsIL26), Camel IL10 (CdIL10) & Human IL10 (HsIL10) showing the structural conservation of the IL26 and IL10. Cd IL-26 has 24% amino acid identity and 54% similarity to Hs IL-10. The four cysteine residues and six alpha helices of IL-10 are shared by IL-26 are also marked. IL-26 has an additional Cysteine residue at 102 position, when compared to IL-10. Two signature motifs of IL- 10 family have also been identified and are marked. The residues in the human IL-26 predicted (Donnelly et al., 2010) to be involved in the IL20R1 & IL10R2 are marked by * and #, respectively.

Fig. 3

Unrooted phylogenetic tree of the dromedary camel IL-26 and other mammals. Phylogenetic relationships were reconstructed by the distance Neighbor Joining method using Clustal W multiple alignment, and bootstrapped 1000 times. The scale for the given branch length indicates 0.02 amino acid substitutions per site. XP prefix in the accession number denotes IL-26 sequences in the NCBI GenBank derived through prediction from the genome.

Fig. 4

Predicted exon intron usage of the dromedary IL-26 transcript variants. The cDNA sequence (start codon to stop codon) of the dromedary camel transcripts was aligned with the Camelus ferus shotgun sequence at the NCBI Genbank (LOCUS AGVR01041574) containing the IL-26 encoding region using the MGAlignIt program. Exonic regions are represented by solid boxes and intronic regions by slanting lines. The start and end positions of adjacent splicing exons are marked by numbers. The splice donor and acceptor sites are marked on all exon-intron boundaries. The thin bar below the mRNA sequence indicates the open reading frame.

Fig. 5

Multiple Alignment of the nucleotide sequence of the ORF of all the five IL-26 transcript variants in comparison with the predicted Camelus ferus IL-26 sequence (CFGB) in the NCBI Genbank. The predicted splice junctions in the transcript (corresponding to Tv1) are marked by ↓. The termination codons in the protein coding sequence of each transcript are boxed with the premature termination codons (PTC) in the dromedary camel transcript variants Tv2, Tv3 and Tv5 marked in red. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 6A

Alignment of the predicted amino acid sequences of the five transcript variants of dromedary IL-26. CfPrd refers to the Camelus ferus IL26 sequence predicted from the genome (NCBI GenBank Acc No XP 6189751.1). The IL-26 normal transcript variant, Tv1 has 171 amino acids like the predicted sequence from GenBank. Tv2 Tv3 & Tv5 result in truncated proteins due to premature termination of translation. Transcript variant Tv4 has additional 40 amino acids when compared to the Tv1 protein. IL-26 protein regions corresponding to the exon intron boundaries are marked by downward arrow. The second IL-10 signature motif sequence conserved in most mammalian IL-26 is marked. The truncated proteins encoded by the variants Tv2, Tv3 and Tv5 lack this motif.

Fig. 6B

Structural comparison of the human IL-26 and the predicted camel IL-26 proteins: Using the PHYRE automatic protein structure modeling server we created 3D homology models for the human IL-26 (i) and the predicted mature proteins of all the five alternative splice variants of camel IL-26-Tv1 to Tv5 (ii to vi)

Fig. 7

Expression of IL-26 alternative splice variants in different tissues of dromedary camel tissues and in-vitro mitogen stimulated PBMCs (A) Total mRNA was isolated from camel tissues were reverse transcribed and the cDNA was used as template for amplification with IL26XF1&IL26XR1 primers (B) Camel PBMCs were stimulated in vitro with PWM or Con A (10 μg/ml) for 16 hrs. Unstimulated PBMCs under identical culture conditions were also kept as control for 16 h. RNA isolated from stimulated samples and control (3 different camels) was used for cDNA synthesis. IL26 expression profile was analyzed with IL26XF1 and IL26XR1 primers on an Agilent 2100 Bioanalyzer using a DNA 1000 kit. Size standards for IL-26 transcript variants were made by PCR amplification of the previously cloned camel IL-26 Tv1 to Tv5 variants using IL26XF1 and IL26XR1. Equal mixture of these amplicons was used to create the size standards loaded in lane marked IL26Tv Mix.

Fig. 8

Analysis of Expression of camel IL-26 alternative splice variants in camel ovary fibroblasts cells infected with Camel pox virus: Camel ovary fibroblasts were infected with Camel Pox virus in-vitro and RNA isolated post 48 hrs after infection from virus infected and uninfected cells. IL-26 transcript variants were amplified by RT-PCR using IL26XF1 /IL26XR1 primers and analyzed on Agilent 2100 Bioanalyzer using a DNA 1000 kit. Size standards for IL-26 transcript variants were made by PCR amplification of the cloned camel IL-26 Tv1 to Tv5 variants using IL26XF1 and IL26XR1. Equal mixture of these amplicons was used to create the size standards and analyzed along with the samples. First panel in each set represents the virtual image for the fragments separated on the Agilent 2100 Bioanalyzer. The second and third panel represent the electropherograms of IL-26 transcript variants of control/infected (blue) overlaid with the IL-26Tv standard mix (red) for size comparison. The induction of IL-26 transcript variant 2 in virus infected samples is marked by an arrow. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)