Isolation And Characterization Of Two Antimicrobial Peptides And Midkine In Half-smooth Tongue Sole Cynoglossus Semilaevis

Half-smooth tongue sole (Cynoglossu semilaevis) is a kind of principal economicflatfish cultured in China. However, little information is available about the innateimmune mechanism of C. semilaevis. In this work, based on a normalized cDNAlibrary of C. semilaevis constructed by our laboratory, two antimicrobial peptides andone heparin binding growth factor have been isolated and characterized. The authorhas presented the gene organization, tissue and developmental expression patterns andthe activity of recombinant protein. The main results are reported as follows.Hepcidin, an antimicrobial peptide, has a dual function including innate immunityand iron regulation. Here, based on the sequence of an EST database, we have isolatedand characterized a hepcidin gene (referred to as CsHepcidin) from half-smoothtongue sole (Cynoglossus semilaevis). Analysis of the coding regions indicatedCsHepcidin gene comprised3exons and2introns. The putative CsHepcidin showed agreat similarity to other hepcidin orthologues, particularly with respect to its24aasignal peptide, typical RX(K/R)R motif and eight conserved cysteine residues in themature cationic peptide. Phylogenic analysis indicated that CsHepcidin was ahepcidin1-type peptide of acanthopterygians, with highly homologous with Soleasenegalensis hepcidin. In C. semilaevis ontogeny, the expression of CsHepcidinappeared to be developmentally regulated. CsHepcidin mRNA was detected at a lowlevel in unfertilized eggs, increased on6d after hatching, and decreased remarkably atmetamorphic stage. CsHepcidin transcripts showed a constitutive basal expression inmost of the tissues, especially in liver. Challenge with formalin-inactivated Vibrioanguillarum led to significantly up-regulations of CsHepcidin gene in liver, headkidney and spleen in time-dependent manners. Biological activity analysis showedthat recombinant CsHEP exhibited direct antimicrobial activity against bacterialpathogens in vitro, and particularly showed strong activity against the principal fishpathogens, Vibrio anguillarum and Edwardsiella tarda. All these results suggest that CsHepcidin may be involved in the initial response to invasion of microbial pathogens.Further exploration to elucidate the role of CsHepcidin in iron regulation andembryogenesis in C. semilaevis are needed.NK-lysin is an antimicrobial peptide that has powerful antibacterial property aswell as antitumor activity. To date, NK-lysin genes have been reported in a limitednumber of organisms. Here, the author identified a NK-lysin gene in C. semilaevis.The C. semilaevis NK-lysin gene is3541bp and consists of five exons and fourintrons. The CsNK-lysin cDNA consists of683bp, containing an open reading frame(ORF) of408bp, which encodes135amino acid residues. Comparison of its deducedamino acid sequence with other NK-lysins shows that CsNK-lysin has61.1%identityto Paralichthys olivaceus NK-lysin,49.6%to Salmo salar NK-lysin, while less than20%identity to other vertebrates NK-lysins. Multiple alignment of CsNK-lysin andother known saposin-like proteins reveals that the six cysteine residues which areimportant for structural folding are completely conserved. In this work, three types ofCsNK-lysin transcripts were identified because of the unproper splicing in intron4.The author examined the gene expression profile of CsNK-lysin with specific primersby real time PCR. Three types of CsNK-lysin transcripts appeared to have highlysimilar expression pattern, which were mainly detected in intestines and heart, anddetected at middle level in trunk kidney, spleen, brain and ovary. Challenge withformalin-inactivated V. anguillarum led to different regulations of CsNK-lysin gene inintestines, head kidney and spleen in a time-dependent manner. The results suggestthat CsNK-lysin probably be involved in the initial response of C. semilaevis toinvasion of microbial pathogens.Midkine is a kind of heparin-binding cytokine, and it promotes growth, survival,migration and other activities of target cells. Midkine plays important roles inreproduction, development and repair, and it is also involved in the onset andprogression of inflammatory diseases and malignancy. Full cDNA sequence ofMidkine gene in C. semilaevis has been isolated and characterized using RACE andreal-time PCR. Midkine gene consists of1098bp including5′-UTR of159bp,3′-UTR of504bp and an ORF of435bp. Putative amino acid sequence is144 residues long, including10conserved cystein residues, a highly conserved hingeregion, two clusters of basic residues, and a highly conserved Arg residue which isessential for binding midkine to its receptor. Midkine protein shared69%identity withOryzias latipes. Real-time PCR showed that Midkine was only detected in brain ofadult fish. Expression characterization of Midkine gene during embryogenesis, whichrevealed by real-time PCR, showed that it was first transcribed in gastrula embryos.The expression level of CsMidkine increased gradually to the highest level from15hpf (hours post fertilization) to19hpf, then dropped gradually at30hpf and increasedafter0.5d larva, then became consistent with a relatively stable expression level after6d larva. Whole mount in situ hybridization (WISH) revealed that Midkine washighly expressed in the future diencephalon, mid-hindbrain boundary (MHB) andhindbrain from19hpf to30hpf. These findings indicate that Midkine plays animportant role in the formation of brain during C. semilaevis embryogenesis. Thisresearch established the bases for further study of the detailed functions of Midkine inC. semilaevis. Based on the plasmid containing full length of CsMidkine cDNAsequence, the site-directed mutagenesis of predicted mature Midkine sequence wassuccessfully obtained by SOEing PCR method according to the E. coli codon bias.After being confirmed by sequencing, the resulting gene was cloned to the vectorpET32a (+) to yield an identified recombinant plasmid pET32a(+)-MK, which wasthen transformed into E. coli host strain BL21(DE3)pLysS. SDS-PAGE analysisrevealed that high level of fusion protein MK was induced by1mM IPTG at37℃and was mainly detected in sonicated supernatant. The yielded fusion protein MK wasapproximately33kD which was in accordance with the theoretical molecular weight.The successful expression of fusion protein MK will facilitate further studies for itsbiological functions in C. semilaevis.