Molecular characterization of hepcidin gene from mud loach (Misgurnus mizolepis; Cypriniformes)

Abstract

The gene encoding hepcidin, an antimicrobial peptide, was isolated and characterized in the mud loach Misgurnus mizolepis (Cypriniformes). Mud loach hepcidin shows a considerable degree of structural homology to other vertebrate hamp1 orthologues at both the gene and protein levels, particularly with respect to its tripartite genomic organization, typical transcription-factor-binding motifs in its promoter, and conserved cysteine residues in the mature cationic peptide. The mud loach possesses at least two allelic forms of hamp1, which are expected to be translated into the same hepcidin preproprotein. The two alleles are transmitted from parental fish to offspring with a Mendelian inheritance pattern, as demonstrated with polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) genotyping. Southern blot hybridization analysis showed a high degree of polymorphisms in the restriction patterns of individuals. Mud loach hamp1 mRNA is predominantly expressed in the liver, although many other tissues showed detectable levels of hamp1 transcripts in RT-PCR assay. Lipopolysaccharide and bacterial challenges induced significant hamp1 expression, whereas hamp1 was not clearly stimulated by polyinosinic:polycytidylic acid [poly(I:C)] injection. Iron overload and Cu exposure also elevated hamp1 transcripts in various tissues. The transcriptional activation of mud loach hamp1 in response to these stimuli varied among tissue types, and the liver appears predominantly involved in hepcidin-mediated iron regulation. However, hepcidin expression in the kidney and spleen was preferentially modulated by inflammation-mediated signals produced by immune challenges. Our results suggest that mud loach hepcidin has two basic functions, in iron regulation and antimicrobial activity, and that its transcription is also modulated by other environmental perturbations, including heavy metal exposure.