Identification And Functional Analysis Of Nile Tilapia NCC Activity Regulatory Proteins And Effectors In Fishes

Nonspecific cytotoxic cells(NCCs)are precursor cells of mammalian natural killer cells and play an important role in innate immunity of fish.The non-specific cytotoxic cell receptor type I(NCCRP-1)is one of its key cell membrane receptor protein and is the molecular basis for the function of NCC.However,the regulatory network and molecular mechanism of NCC activity mediated by NCCRP-1 is currently not clear.In this study,we used the main cultured species of Nile tilapia(Oreochromis niloticus)in South China to reveal the molecular network of NCC activity regulation in fishes.The research work was carried out in the following three aspects: I)Screening of the NCCRP-1 interaction proteins by yeast two-hybrid library,and verifying those interactions followed by point-to-point yeast two-hybridization;II)Through analyzing the regulation of natural cytotoxicity of NCC by the identified interacting proteins,we identified the NCC activation related signaling pathways mediated by NCCRP-1;III)A preliminary investigation was conducted on the function of the novel activated ligand molecule CD80/86 and the effector molecule NCC-lysin.The main results as follows:1.Characters of NCCRP-1 expression in tilapia and the identification of its interacted proteinsThe c DNA sequence and genomic sequence of NCCRP-1 in tilapia(designated by On-NCCRP-1)were obtained.It was distributed in the whole cell of NCC.q RT-PCR analysis found that On-NCCRP-1 had a higher distribution in the liver and head kidney of tilapia.After stimulated with different stimulants,On-NCCRP-1 expression was significantly up-regulated in many tissues and NCC(P < 0.05).The results indicated that On-NCCRP-1,act as an NCC receptor,plays an important signaling role in mediating immune responses.Based on the sequence of On-NCCRP-1,three yeast two-hybrid bait plasmids containing full length of On-NCCRP-1,extracellular binding region,and the plasmid with both intracellular transcriptional activation region and signal transduction region of On-NCCRP-1 were constructed,respectively.The yeast two-hybrid c DNA libraries were successfully constructed by using the c DNAs of head kidney and liver in tilapia.The potential interaction proteins were screened from liver and head kidney c DNA libraries using three bait vectors.Finally,11 gene fragments that potentially interact with NCCRP-1 were identified.Then,the 10 genes ORF were obtained.We performed the point-to-point yeast two-hybrid technology to verify the direct interactions of those 10 proteins with On-NCCRP-1.The results showed that On-NCCRP-1 could directly interact with On-C-type lectin and On-serotransferrin,indicating that two extracellular protein molecules had the ability to bind to On-NCCRP-1.2.Regulation of NCC activity by dependented-NCCRP-1 receptorOn-C-type lectin had a typical CRD domain.q RT-PCR analysis showed that On-C-type lectin was expressed in many tissues of tilapia with the highest expression in liver tissue.After stimulated with S.agalactiae,it was significantly up-regulated early in multiple tissues(P < 0.05),suggesting that this gene played an important role in immune response in the early stage of bacterial infection.Using the On-C-type lectin and On-serotransferrin recombinant proteins to stimulate NCCs isolated in vitro,it was found that,some of the toxic effectors of NCCs could be induced with high expression at 3 h and 6 h,suggesting NCCs be activated.At the same time,the toxicity test of FHM cells was performed with 3 h activated NCCs.We found that it could effectively induce the apoptosis signal of FHM cells.However,using mouse anti-NCCRP-1 antibody to block On-NCCRP-1,it could reduce the killing activity NCC to FHMs.These findings indicated that both On-C-type lectin and On-serotransferrin could mediate the killing activity of NCC through On-NCCRP-1.Yeast two-hybrid experiments revealed that On-NCCRP-1 interacts with On-SKP1,the main component of Skp1-Cul1-F-box protein(SCF)ubiquitin ligase complex that plays an important role in catalyzing the ubiquitination of proteins.This result suggests that On-NCCRP-1 may mediate ubiquitination.Moreover,dual luciferase reporter assays revealed that overexpression of On-NCCRP-1 activates NF-κB transcriptional activity.This suggests that On-NCCRP-1 may be involved in ubiquitination after being bound to On-SKP1,further promoting the degradation of NF-κB inhibitor IκB,thereby effectively promoting NF-κB signal transduction.3.Regulation of NCC activity by independented-NCCRP-1 receptorWe cloned and analyzed a CD28 gene(On-CD28)and a CD80/86 gene(On-CD80/86)from O.niloticus.q RT-PCR analysis showed that On-CD28 and On-CD80/86 transcripts were detected in all the examined tissues of tilapia,and the highest expression levels of On-CD28 and On-CD80/86 were detected in the brain and heart,respectively.Following a bacterial challenge using S.agalactiae in vivo,On-CD28 and On-CD80/86 were upregulated in some tissues.However,they showed different expression profiles in response to immunization with inactivated S.agalactiae in vitro.Yeast two-hybrid assay showed that On-CD28 could interacted with On-CD80/86 but not with On-NCCRP-1.Recombinantly expressed protein On-CD80/86 could induce the expression of NCC effector and enhance the killing effect of NCC on CIK cells independently to the On-NCCRP-1.These results suggest that the interaction between On-CD80/86 and On-CD28 may provide co-stimulatory signals for T cell activation in tilapia,while On-CD80/86 may be a novel NCC-activated ligand not mediated by On-NCCRP-1 receptors.It could provide important roles in the treatment of tilapia against pathogenic microorganisms.4.Sequence Analysis and Function Study of NCC Effector NK-lysinThis study cloned sphingolipid-like protein orthologs NK-lysin from tilapia(On-NKL).q RT-PCR analysis showed that On-NKL expression was observed in multiple tissues and head kidney leucocytes and nonspecific cytotoxic cells(NCCs)and is mostly abundant in gills.After bacterial challenge,On-NKL expression significantly varied in different tissues and NCCs.On-NKL-overexpressing fish featured significantly lower pathogen loads in tissues than control fish(P < 0.05).At the same time,it was found that the tilapia mortality rate of the On-NKL overexpression group was significantly lower than that of the control group within 20 days after challenge.The results showed that On-NKL could be a potential effector of fish NCC and play an important role in fish antibacterial infection.