Comparative evolutionary analysis of IL6 in lagomorphs

Background and aims: Interleukin 6 (IL6), also known as interferon beta 2, is a class-I helical cytokine with a broad spectrum of biological activities in humoral and cellular defense. This class of cytokines has a gene structure conserved throughout vertebrates, with five coding exons. IL6 is involved in the immune response against rabbit hemorrhagic disease virus that causes a highly fatal disease in the European rabbit. Previously, IL6 from European rabbit samples belonging to the subspecies Oryctolagus cuniculus cuniculus, was shown to differ from the other mammals by extending for further 27 amino acids. This difference results from a mutation in the typical stop codon into a glutamate encoding codon. However, in other leporids (Sylvilagus spp. and Lepus spp.) that diverged from European rabbit approximately 12 million years ago this mutation was also not present. The purpose of this study was to confirm the mutation of the stop codon in other lagomorph specimens: Oryctolagus cuniculus algirus, Brachylagus idahoensis, Sylvilagus bachmanii, Lepus europaeus and Ochotona princeps. Methods: The IL6 gene was PCR-amplified and sequenced for the five lagomorph species. The obtained sequences were translated and compared with other mammalian IL6 sequences retrieved from public databases (GenBank, Ensembl and Uniprot). A maximum-likelihood (ML) tree was inferred in MEGA5 with the following options: HKY+G model, 500 bootstrap replicates and partial deletion to gaps/missing data treatment. Results: We confirmed the presence of the mutated stop codon in both O. c. cuniculus and O. c. algirus. In agreement with previous reports, we found that the stop codon is not mutated in S. bachmanii and L. europaeus. We further extended this observation to the leporid B. idahoensis and ochotonid O. princeps. In rabbits, sequence translation of IL6 continues into the exonic sequence and stops in the next STOP codon (81 nucleotides downstream). Typically, the IL6 protein has five cysteine residues that might be important to establish disulfide bonds. In rabbit, the 27 amino acid extension has four more cysteine residues. The inferred phylogeny for the IL6 gene is in agreement with what has been accepted for the mammals and lagomorphs.Conclusions: Our results indicate that in the ancestral of the Oryctolagusgenus, (approximately 2 million years ago), a single mutation at exon 5 occurred that made IL6 longer than for the other mammals. Biological implications of this extension remain to be assessed but the occurrence of the 4 extra cysteine residues might suggest some functional relevance