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. 2014 Feb;66(2):93-103.
doi: 10.1007/s00251-013-0747-0. Epub 2013 Nov 26.

Identification of a gene for an ancient cytokine, interleukin 15-like, in mammals; interleukins 2 and 15 co-evolved with this third family member, all sharing binding motifs for IL-15Rα

Johannes M Dijkstra  1 , Fumio Takizawa, Uwe Fischer, Maik Friedrich, Veronica Soto-Lampe, Christophe Lefèvre, Matthias Lenk, Axel Karger, Taei Matsui, Keiichiro Hashimoto
Affiliations

Identification of a gene for an ancient cytokine, interleukin 15-like, in mammals; interleukins 2 and 15 co-evolved with this third family member, all sharing binding motifs for IL-15Rα

Johannes M Dijkstra et al. Immunogenetics. 2014 Feb.

Abstract

Interleukins 2 and 15 (IL-2 and IL-15) are highly differentiated but related cytokines with overlapping, yet also distinct functions, and established benefits for medical drug use. The present study identified a gene for an ancient third IL-2/15 family member in reptiles and mammals, interleukin 15-like (IL-15L), which hitherto was only reported in fish. IL-15L genes with intact open reading frames (ORFs) and evidence of transcription, and a recent past of purifying selection, were found for cattle, horse, sheep, pig and rabbit. In human and mouse the IL-15L ORF is incapacitated. Although deduced IL-15L proteins share only ~21 % overall amino acid identity with IL-15, they share many of the IL-15 residues important for binding to receptor chain IL-15Rα, and recombinant bovine IL-15L was shown to interact with IL-15Rα indeed. Comparison of sequence motifs indicates that capacity for binding IL-15Rα is an ancestral characteristic of the IL-2/15/15L family, in accordance with a recent study which showed that in fish both IL-2 and IL-15 can bind IL-15Rα. Evidence reveals that the species lineage leading to mammals started out with three similar cytokines IL-2, IL-15 and IL-15L, and that later in evolution (1) IL-2 and IL-2Rα receptor chain acquired a new and specific binding mode and (2) IL-15L was lost in several but not all groups of mammals. The present study forms an important step forward in understanding this potent family of cytokines, and may help to improve future strategies for their application in veterinarian and human medicine.

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Figures

Fig. 1
Fig. 1
Phylogenetic distribution of IL-15L. White circles represent consensus intact IL-15L ORFs, gray circles represent non-typical but possibly intact IL-15L ORFs, and black circles represent incapacitated IL-15L ORFs; half circles refer to incomplete sequence information (for details, see Fig. S2). Mammals important for agriculture are highlighted in yellow. Branch knots within mammals are based on Meredith et al. (2011)
Fig. 2
Fig. 2
Schematic view of different types of mammalian IL-15L transcripts. a Transcript with consensus IL-15L ORF and additional out of frame AUGs in the 5′UTR (asterisks). Full-length transcripts including poly-A tail (AAAA) were determined for cattle and rabbit (Fig. S3). b In rabbits, however, most transcripts lack the exon3/4 sequence (ex3/4) and are unlikely to encode IL-15L protein (Figs. S3 C–E and S5B, middle picture). c In dog and bear, the consensus IL-15L AUG start codon has been replaced by GUG, and the region 5′ thereof is unlikely to encode the N-terminus of a functional cytokine (Figs. S2 and S4A–C). It was not determined whether these transcripts have a poly-A tail. d For human a hybrid IL-15Lψ-SUPT5H cDNA sequence was reported (GenBank DC400386), which is unlikely to encode modified IL-15L protein (Fig. S4 D and E). We did not investigate the 5′ and 3′ ends of this type of transcript
Fig. 3
Fig. 3
Alignment of deduced IL-15L, IL-15, IL-2, IL-21 and IL-4 amino acid sequences. *, for IL-15 only leader peptide fragments encoded by family consensus exon1 are shown. **, the sequence encoded by the last coding exon of IL-21 is not shown. Small font in the IL-4 sequence relates to a fragment deleted for lay-out reasons (see Fig. S6 A). Gaps, open spaces relate to exon borders, whereas hyphens connect residues encoded by the same exon. X shaded red, frame-shift. Known or predicted leader peptide and α-helix sequences are shaded gray and underlined, respectively (based on IPD accessions 2PSM, 2Z3R, 1Z92, 1ITL and 3TGX for murine IL-15 and human IL-15, IL-2, IL-4 and IL-21, respectively). Similar colored cysteines refer to known or expected disulfide bridges. Several residues, including a cysteine pair, are rather specific for IL-15L and are shaded green. Murine IL-15 and human IL-2 residues with "1" or "2" indications below contribute to patches 1 and 2 of their interface with IL-15Rα and IL-2Rα, respectively. The patch 1 and 2 contributing residues of mammalian IL-15 are shaded blue and pink, respectively, as are matching identical residues in the other compared cytokines; circles above the alignment indicate the mammalian IL-15 residues which are key for IL-15Rα binding (Olsen et al. 2007). The diamonds indicate positions important for binding of IL-2 and IL-15 to IL-2Rβ chain, and the open inverted triangle indicates the glutamine important for interaction with IL-2Rγ chain (Wang et al. ; Ring et al. 2012). The solid inverted triangles indicate positions important for binding of IL-4 and IL-21 to IL-4Rα and IL-21Rα, respectively (Hage et al. ; Bondensgaard et al. ; Hamming et al. 2012). For database accessions, see Figs. S2 and S6 A
Fig. 4
Fig. 4
Surface presentation of IL-15L by IL-15Rα. The expression of bovine IL-15L-FLAG, bovine IL-2Rα-Myc-His and bovine IL-15Rα-Myc-His in transfected HEK-293 cells was assayed by flow cytometry using anti-FLAG (left) and anti-Myc (right) mAbs. Surface binding was monitored using live cells (above) and transfection efficiencies were monitored using fixed/permeabilized cells (below). Only in the presence of IL-15Rα receptor chain, IL-15L-FLAG could be observed at the cell surface by using anti-FLAG (upper left histogram). Because the Myc-tags of IL-2Rα and IL-15Rα were connected to the intracellular cytoplasmic tails, anti-Myc fluorescence could only be observed in fixed/permeabilized cells. Percentages refer to the cells within the fluorescence range indicated by horizontal bar. The data shown are of a single experiment, representative of three independent experiments
Fig. 5
Fig. 5
Alignment of major parts of the sushi-domain of IL-15Rα and the first sushi-domain of IL-2Rα in representative species. All depicted fragments are encoded by a single exon. The numbers 1 and 2 above and below the alignment indicate the human IL-15Rα and IL-2Rα residues, which interact with IL-15 and IL-2 in patches 1 and 2, respectively, with the important key residues being underlined (Rickert et al. ; Olsen et al. 2007). To highlight the evolutionary conservation of the IL-15Rα residues contributing to patches 1 and 2, these residues are shaded blue and pink throughout the alignment, including in IL-2Rα. Other human IL-2Rα residues contributing to binding patches 1 and 2, or binding to IL-2 residues contributing to those patches, are shaded green (except the sushi-domain specific cysteine) and orange, as are identical matching residues in the other aligned sequences. In human IL-2Rα, the first and second sushi-domains engage in a beta strand exchange event in which the first 18 residues depicted here are swapped with a stretch of domain 2; this 1–18 region is thus not functionally comparable with the IL-15Rα fragments to which it is aligned here. The IL-2Rα residues indicated by pound symbols (#) interact with IL-2 residues that contribute to patch 1 (Rickert et al. 2005). For GenBank accession numbers, see Fig. S1 F
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