돌돔 (Oplegnathus fasciatus, Perciformes)으로부터 발굴된 항균 펩타이드 hepcidin의 유전자 구조 및 발현 기능에 관한 연구

Abstract

Hepcidin is a key peptide that plays an important role in the innate immune systems of both mammals and teleost fish. Multiple isoforms of hepcidin precursors (RbHEPC-1, -2, -3 and -4) were isolated from a marine teleost, rockbream (Oplegnathus fasciatus) at both RNA and genomic levels, and tissue-specific expression profiles under various stimulated conditions such as bacterial infection, iron overload and viral infection were characterized based on “electronic RT-PCR (redundancy analysis of RT-PCR clones)” and RT/real-time PCR. Rockbream hepcidin isoforms shared a high sequence identity each other and also shared conserved features with the orthologues from other vertebrate animals. The 5´-flanking regions of rockbream hepcidin genes represented typical binding sites for the transcription factors including C/EBP, HNF, AP and/or IL6/STAT. Genomic Southern blot hybridization analysis revealed the presence of multiple copies of hepcidin genes on the rockbream genome with highly polymorphic restriction patterns. Hepcidin precursors predominantly existed in liver and to a quite lesser extent, in other tissues with differential distribution of each isoform among tissues. Hepcidin mRNAs in liver, intestine, kidney and spleen of juvenile rockbreams were highly upregulated during challenges using various bacterial species (up to 7200 fold), iron overloading (up to more than 3400 fold) and viral (rockbream iridovirus) infection (up to more than 30 fold). Expression patterns of hepcidin isoforms during those treatments were largely governed by tissue types and also affected by kinds of stimulators. In overall, the significant induction of the isoform RbHEPC-2 was found in responsive to bacterial infection and iron overloading especially in kidney and spleen of juveniles, whereas the RbHEPC-1 responded sensitively to viral infection in liver and intestine of fingerlings. In addition, partially, improperly or alternatively spliced forms of hepcidin transcripts were identified and the partially processed RNA form was significantly induced by bacterial infection and iron overloading. Rockbream hepcidin genes (rbhepc-1, -2, -3 and -4) shared high similarity with each other in the cDNA and genomic sequence. However, it seems that small single nucleotide polymorphisms in the promoter and/or coding region may confer potentially unique function on each of hepcidin isoforms. Differential gene regulation of these multiple hepcidin isoforms were tissue-, stimulus- and isoform-specific during the biological stimulatory treatments. Furthermore, several variant hepcidin precursors including partially spliced RNA were identified for the first time in the present study. Knowledge obtained in this study provides a good basis to understand the innate immunity of fish, and consequently be useful to develop effective strategies for control of infectious diseases in aquaculture-relevant fish species. From the present advance, the next challenges may be the functional characterizations of various transcriptional elements and mature hepcidin peptides in order to better understand the complex cross-talk between various hepcidin isoforms in teleost species.