Qi Z et al. (2015), Cloning of interleukin-10 from African clawed f...

XB-ART-50355

J Immunol Res 2015 Jan 01;2015:462138. doi: 10.1155/2015/462138.

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Cloning of interleukin-10 from African clawed frog (Xenopus tropicalis), with the Finding of IL-19/20 homologue in the IL-10 locus.

Qi Z , Zhang Q , Wang Z , Zhao W , Gao Q .

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Interleukin-10 (IL-10) is a pleiotropic cytokine that plays an important role in immune system. In the present study, the IL-10 gene of African clawed frog (Xenopus tropicalis) was first cloned, and its expression pattern and 3D structure were also analyzed. The frog IL-10 mRNA encoded 172 amino acids which possessed several conserved features found in IL-10s from other species, including five-exon/four-intron genomic structure, conserved four cysteine residues, IL-10 family motif, and six α-helices. Real-time PCR showed that frog IL-10 mRNA was ubiquitous expressed in all examined tissues, highly in some immune related tissues including kidney, spleen, and intestine and lowly in heart, stomach, and liver. The frog IL-10 mRNA was upregulated at 24 h after LPS stimulation, indicating that it plays a part in the host immune response to bacterial infection. Another IL, termed as IL-20, was identified from the frog IL-10 locus, which might be the homologue of mammalian IL-19/20 according to the analysis results of the phylogenetic tree and the sequence identities.

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Species referenced: Xenopus tropicalis
Genes referenced: actl6a il10

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	Figure 1. Comparison of the gene organization of frog IL-10 gene with selected IL-10 molecules. The exons were indicated as black boxes and introns as black lines. The numbers above each box indicate the size (bp) of exons and the numbers below the lines indicate the size (bp) of introns. The gene organization of each gene was extracted from Ensembl Genome database.
	Figure 2. Multiple alignment of vertebrate IL-10. The multiple alignment was produced using Clustal W, and conserved amino acids were shaded using GeneDoc software. The signal peptides predicted by SignalP 4.1 server were underlined. The conserved IL-10 family signature motifs were boxed. The four conserved cysteine residues existing in IL-10 were indicated by black cycles below the alignment and the six conserved cysteine residues in IL-19/20 were indicated by black arrows above the alignment. The accession numbers for sequence used in this alignment were listed in Figure 6.
	Figure 3. Structure and molecular dynamics analysis of frog IL-10. (a) Superimposition of the frog IL-10 model with the human IL-10 (PDB: 2ILK). Frog IL-10 was shown in red color and human IL-10 was in white color; (b) RMSD of frog IL-10 postmolecular dynamics for 12 ns. The MD analysis was performed by Gromacs 4.0 package [28] on an Inspur, 12 GHz PC, and results were analyzed using Origin 6.0 software.
	Figure 4. Expression analysis of frog IL-10 in different tissues from healthy frog (a) and LPS stimulated frog (b). Six tissues including liver, spleen, kidney, intestine, heart, and stomach were sampled for real-time PCR analysis. The mean ± SD values were shown. The transcripts levels of IL-10 in healthy frog were relative to those of β-actin. The expression changes of IL-10 in LPS stimulated frog were expressed as fold change relative to the controls. P values generated by paired sample t-test between control and stimulated groups were shown above the bars. *P < 0.05.
	Figure 5. Gene synteny of IL-10 loci in vertebrates. The gene synteny of human, chicken, and zebrafish IL-10 locus was analyzed using Genomicus (v75.02) software and human IL-10 was set as a reference to compare the conserved synteny between different species. The gene synteny of frog IL-10 locus was based on the results of GenScan and BLAST search against NCBI database.
	Figure 6. Phylogenetic tree analysis of IL-10 family members from frog and other species. The tree was constructed by the “neighbor-joining” method using MEGA 6.0 software. Node values represent the percent of bootstrap confidence derived from 1,000 replicates. The accession number for each sequence followed the common species name.

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