Molecular cloning and characterisation of a carp ( Cyprinus carpio) cytokine-like cDNA that shares sequence similarity with IL-6 subfamily cytokines CNTF, OSM and LIF

Abstract

In the course of suppression subtractive hybridisation between sodium alginate-induced peritoneal cells (SA-PC) and normal head kidney cDNAs in common carp ( Cyprinus carpio), a cytokine-like cDNA clone was found. The clone, named M17, contains a 1600 bp nucleotide sequence that encodes a 215 amino acid putative protein that would have a pI of 9.01 and would include a 33 amino acid signal peptide. The 3′ untranslated region has seven ATTTA mRNA destabilising motifs that are common in cytokines and oncogenes. In a BLASTP search, M17 was most similar to chicken ciliary neurotrophic factor (CNTF) with 25% amino acid identity, followed by mammalian CNTF, cardiotrophin-1 and leukemia inhibitory factor (LIF) all of which belong to the IL-6 subfamily. However, M17 has some differences with CNTF in that CNTF has no signal sequence, the gene organisation of M17 is three exons and two introns, whereas that of CNTF is two exons and one intron, M17 has seven cysteines while CNTF has one cysteine, and M17 mRNA is detected in peripheral blood leukocytes as well as brain, whereas CNTF is expressed only in the nervous system. Compared to other members in the IL-6 subfamily cytokines, M17's cysteine positions and gene organisation are similar to those of oncostatin M and LIF, although amino acid identities are only 15–17%. Southern hybridisation suggested that M17 is a single copy gene. SA-PC showed significantly higher M17 mRNA levels than normal head kidney cells, which are considered to be a source of the SA-PC, indicating that M17 is inducible by inflammatory stimulation.