| With the increased expansion of the scale for marine aquaculture and the worsening aquaculture environment in China,the frequent occurrence of diseases and drug abuse have seriously affected the healthy development of the breeding industry about China’s important economic fish species-large yellow croaker,and only the prevention and control of aquatic products by improving the autoimmunity of farmed animals can be considered to be an important way to solve this problem.Cathepsin(Cat)is a type of protease located on the lysosome,and its activity is reported to be involved in the regulation of antigen presentation pathway.Studies have shown that cathepsins interacts with a variety of cellular endogenous cathepsin inhibitors(Cystatins,Cys),and also plays an important role in mammalian disease resistance.In teleost,the related functions of cathepsins and cystatins in the immune response are still unclear.This study investigated the molecular and functional characteristic of Lc Cat F from the cathepsin family and LcCys-like in cystatins family.The Lc Cat F gene was cloned by large yellow croaker genome obtained earlier in this laboratory.The open reading frame(ORF)of Lc Cat F has 1,419 nucleotides in length totally and a putative protein contained 473 amino acids.The predicted molecular weight is almost43.3 k Da and p I is 6.23.Lc Cat F shares the sequence identity with fish from 80.14% to 92.09%,and with the highest relationship with Japanese killifish(92.09%).Real-Time q PCR analysis showed that Lc Cat F was constitutively expressed in 10 tissues or 4 cells tested,including muscle and liver or primary head kidney leukocytes(PKL),macrophages(PKM),granulocyte(PKG)and head kidney cell line LYCK cells,among which muscle,head kidney or LYCK cells shares the highest expression levels.After stimulation with LPS or poly(I:C),the expression of Lc Cat F in PKL cells or PKM cells were increased significantly.After injection of foreign antigen DLD or induction on PKM and PKL,the change of Lc Cat F in the gill and intestine or in PKM were also significantly increased,indicating that Lc Cat F can be induced by bacteria or viruses to participate in the specific immunity of large yellow croaker.After overexpression of pc DNA3.1-Lc Cat F in PKL,the expression levels of antigen presentation related genes(MHC-α,MHC-β,Ii,CD80,and CD83)were significantly upregulated,suggesting that Lc Cat F may be involved in the processing and presentation of foreign antigens.Lc Cat F participated in antigen presentation by regulating the expression of genes,and then participated in the specific immunity of large yellow croaker.In addition,LcCys-like gene was also cloned.LcCys-like shares an ORF with 414 bp in length and encodes a protein consisted of 138 amino acids.This putative protein has a predicted molecular weight of 15.4 k Da and has a p I at 7.68.LcCys-like contains typical cystatins family domains(Gly,Qx Vx G,PW,and C-terminally conserved cysteine),shares the sequence identity with fish at 13.2% to 59.4%.And the highest identity with caralfish cystatin-like at 59.4%.Real-time q PCR analysis showed that LcCys-like was constitutively expressed in the tested tissues or cells,with the highest expression levels in the spleen,gills and PKL.The expression pattern of LcCys-like in PKM,PKG and PKL is affected by LPS or poly(I:C).The stimulation of DLD can also significantly upregulate the change of LcCys-like in PKM and PKL as well as the gills and intestines of mucosal tissues,showing that LcCys-like has an immune response to bacterial or viral infections and effectively improve fish’s immunity.The recombinant LcCys-like(r LcCys-like)protein expressed by Pichia pastoris can inhibit the activity of a variety of proteases,among which the r Lc Cat F protein activity is strongly inhibited,suggesting that LcCys-like may participate in antigen presentation pathway through the regulation of Lc Cat F activity.After r LcCys-like stimulation in PKM,the change of IL-10 and TNF-α were significantly increased,the transcription of i NOS and the increase of NO content were promoted.Therefore,these results indicate that LcCys-like may be involved in the regulation of inflammatory cytokines,and participated in various physiological and pathological processes of large yellow croaker by inducing NO production.At the same time,the complete c-Jun cDNA of large yellow croaker Larimichthys crocea(Lcc-Jun)was cloned,its ORF is 984 bp long and encodes a protein of 327 amino acids(aa).Its transcripts were not only induced in spleen and head kidney by poly(I:C)or LPS,but also up-regulated in PKL,PKM,and PKG,suggesting that Lcc-Jun may be involved in immune responses induced by poly(I:C)and LPS.Overexpression of Lcc-Jun in PKL increased the expression of cytokines and transcription factors involved in T helper 1 and Th2 cell development and differentiation,suggesting that Lcc-Jun may play a role in regulation of Th1/Th2 cell response.In summary,this paper cloned Lc Cat F and LcCys-like,to clarify their expression patterns during virus or bacterial infection in large yellow croaker,and to clarify that Lc Cat F can be induced by bacteria or viruses,by regulating the expression of antigen presentation related genes to participate in antigen presentation,and then participate in the specific immunity of large yellow croaker.It revealed that LcCys-like may participate in the regulation of antigen presentation by inhibiting the activity of Lc Cat F.In addition,LcCys-like can promote the expression of inflammatory factors and i NOS in PKM,indicating that there may be a new relationship between LcCys-like and cytokines,inflammatory response and immune response in large yellow croaker.All results improve the understanding of the structure and function of Cathepsin F and cathepsin inhibitor Cystatin-like,and provide a theoretical basis for the study of Lc Cat F and LcCys-like in the anti-disease immune response,and show the great significance of disease prevention in large yellow croaker.What’s more,we also revealed that Lcc-Jun participates in the antibacterial and anti-disease immune process,regulates Th1/Th2 cell differentiation,and provides a theoretical basis for studying the role of c-Jun-related signaling pathways in the immune process of large yellow croaker. |
