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

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.

Mimtags: the use of phage display technology to produce novel protein-specific probes

In recent times the use of protein-specific probes in the field of proteomics has undergone evolutionary changes leading to the discovery of new probing techniques. Protein-specific probes serve two main purposes: epitope mapping and detection assays. One such technique is the use of phage display in the random selection of peptide mimotopes (mimtags) that can tag epitopes of proteins, replacing the use of monoclonal antibodies in detection systems. In this study, phage display technology was used to screen a random peptide library with a biologically active purified human interleukin-4 receptor (IL-4R) and interleukin-13 (IL-13) to identify mimtag candidates that interacted with these proteins. Once identified, the mimtags were commercially synthesised, biotinylated and used for in vitro immunoassays. We have used phage display to identify M13 phage clones that demonstrated specific binding to IL-4R and IL-13 cytokine. A consensus in binding sequences was observed and phage clones characterised had identical peptide sequence motifs. Only one was synthesised for use in further immunoassays, demonstrating significant binding to either IL-4R or IL-13. We have successfully shown the use of phage display to identify and characterise mimtags that specifically bind to their target epitope. Thus, this new method of probing proteins can be used in the future as a novel tool for immunoassay and detection technique, which is cheaper and more rapidly produced and therefore a better alternative to the use of monoclonal antibodies.