미꾸라지(Misgurnus mizolepis)로부터 항균 단백질 유전자 hamp, leap2 및 wap65의 분리 및 발현 특징 분석

Abstract

In teleosts, the innate immunity is a pivotal protective system against invading pathogens and the antimicrobial proteins including antimicrobial peptide and iron sequestrating protein play the crucial roles in the first defense line of the innate system. To get a deeper insight into the coordinated cross-talks between liver-expressed antimicrobial proteins in the mud loach Misgurnus mizolepis (Cypriniformes), this study was aimed to isolate and characterize the genetic determinants of three antimicrobial proteins, hepcidin (HAMP), liver expressed antimicrobial peptide 2 (LEAP2) and warm-temperature acclimation associated 65-kDa protein (WAP65), and scrutinize their basal and stimulated mRNA expression patterns in response to various experimental immune challenges. 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 hamp 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 hamp, 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. Full-length cDNAs of leap2 isoform genes (leap2a and leap2b) were cloned from mud loach. Both mud loach leap2 isoform genes displayed a three-exon structure that is common to most teleostean leap2 genes. Several consensus binding sites for transcription factors including C/EBP, HNF, HIF-1, MRE, AP-1, NF-AT, IL6-REBP, USF, Ahr, and/or STAT were found in their 5re found in theHNF. Both LEAP2 mature peptides consisted of 41 amino acid residues and represented four cysteines at the conserved positions to form two disulfide bonds, which is also common to other teleostean LEAP2 orthologues. Genomic organizations including the structural characteristics of 5and/or STAT ygions of two warm-temperature-acclimation-associated 65 kDa protein (wap65-1 and wap65-2) isoform genes were characterized. Both mud loach wap65 isoform genes displayed a ten-exon structure that is common to most teleostean wap65 genes. The two mud loach wap65 isoforms were predicted to possess various stress/immune-related transcription factor binding sites in their regulatory regions; however the predicted motif profiles were different between the two isoforms, in which the inflammation-related transcription factor binding motifs such as NF-κB and CREBP sites are more highlighted in the wap65-2 isoform than in wap65-1 isoform. The deduced amino acid sequences of the two mud loach WAP65 isoforms (WAP65-1 and WAP65-2) share moderate levels of sequence homology with their corresponding orthologues from teleosts and with human hemopexin, a possible mammalian homologue. Both isoforms display conserved features, including essential motifs and/or residues that are important for the protein structure of hemopexin. In overall, WAP65-2 is more homologous to human hemopexin than is WAP65-1. From various gene expression analyses, mud loach hamp mRNA is predominantly expressed in the liver under basal, non-stimulated conditions, although many other tissues showed detectable levels of hamp transcripts in RT-PCR assay. Hamp transcripts were developmentally regulated where mRNA expression level of hamp was sharply elevated at post-gastrulation stage, decreased at 3-4 somites stage and rebounded at hatched larvae. Lipopolysaccharide (LPS) and bacterial challenges induced significantly hamp expression, whereas hamp was not clearly stimulated by polyinosinic:polycytidylic acid [poly(I:C)] injection. Iron overload and Cu exposure also elevated hamp transcripts in various tissues, suggesting that hamp is associated with various cellular pathways involved in host defense mechanism. The transcriptional activation of mud loach hamp 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. Expression assays revealed that leap2 gene reveals an isoform-specific tissue distribution pattern in which leap2a mRNAs were expressed in a wide range of mud loach tissues including liver, ovary, intestine, skin, muscle, testis and kidney, while leap2b mRNAs were largely predominant in the liver. The onset mRNA expressions of leap2a and leap2b during development were different from each other and varied depending upon embryonic and larval development stages. Leap2a and leap2b mRNA transcripts were differentially modulated by various stimuli, including immune challenge (LPS, polyI:C or bacterial injection), iron overload and heavy metal exposure (cadmium, chromium, copper, iron, manganese, nickel or zinc). Leap2a and leap2b displayed up- and down-regulated expression patterns in response to various stimuli and the modulation patterns were also apparently variable among tissues. Both mud loach wap65 transcripts are predominantly expressed in liver, although the transcripts are ubiquitously detectable in most tissues with variable basal expression levels. Both wap65 isoforms are also differentially regulated during embryonic development, and the changes in transcript levels during embryogenesis are greater for wap65-2 than for wap65-1. The transcription of the wap65-1 genes is differentially modulated by various stimuli, including thermal changes, immune challenge (lipopolysaccharide injection or bacterial infection), and heavy metal exposure (cadmium, copper, or nickel). The isoform wap65-1 is more responsive to warm temperature treatments than wap65-2, whereas wap65-2 is much more strongly stimulated by immune and heavy metal challenges than is wap65-1. Taken together, the results of this study suggest that mud loach WAP65 isoforms are potentially involved in multiple cellular pathways and that the two mud loach wap65 isoforms undergo functional partitioning or subfunctionalization. Taken together, the three liver-expressed antimicrobial proteins characterized in this study turned out to play important roles in mud loach innate immunity particularly during acute phase of bacterial invasion. Of the three proteins, HAMP might be the central component for the cross talks between iron regulation and bactericidal action. LEAP2 might display supportive roles for HAMP action, while WAP65-2 would be closely associated with the iron sequestration defense mechanism in the innate immunity. Data from this study could be a useful and fundamental basis for future strategy to perform molecular genetic breeding for the development of bacterial disease-resistant fish models.