Molecular characterization of LEAP-2 cDNA in common carp (Cyprinus carpio L.) and the differential expression upon a Vibrio anguillarum stimulus; indications for a significant immune role in skin.

LEAP-2 is a cysteine-rich cationic antimicrobial peptide (AMP) playing an important role in host innate immune system. LEAP-2 genes have been identified from higher vertebrates and several fish species. Here we report the cloning and identification of two LEAP-2 cDNA sequences from the liver of common carp (Cyprinus carpio L.). The LEAP-2A cDNA was 1325 bp long and contained an ORF of 279 bp encoding a protein of 92 amino acids. The LEAP-2B cDNA was 608 bp long and contained an ORF of 276 bp encoding a protein of 91 amino acids. Both LEAP-2 proteins consisted of 41 amino acid residues and shared four cysteines at the conserved positions in the predicted mature peptides, highly similar to LEAP-2 of other species. Sequence alignment showed that LEAP-2 amino acid sequences were well conserved in different species, and the phylogenetic relation of LEAP-2 was coincident with evolution of biological species. Expression analysis data revealed that LEAP-2A and LEAP-2B mRNAs were expressed in a wide range of common carp tissues including liver, spleen, head kidney, skin, gills, hindgut and foregut. When injected intraperitoneally with Vibrio anguillarum, the expression level of common carp LEAP-2A was quickly up-regulated in liver, spleen, head kidney, skin, gills, foregut and hindgut, however, the expression level of LEAP-2B was similarly up-regulated in spleen, skin, gills and hindgut but not in liver, head kidney and foregut. Our results showed that the LEAP-2A had a markedly high constitutive expression in skin, and the LEAP-2A and the LEAP-2B had a significantly high up-regulated expression after stimulus in skin. This differential expression of LEAP-2 in common carp suggests that it may play a key role in immune responses against invading pathogens and both LEAP-2 molecules may be involved in mucosal immunity.