Research Of Functions And Molecular Characters Of Two Kind Of Cysteine Proteinases Inhibitors In Large Yellow Croaker(Pseudosciaena Crocea)

Cystatins are natuaL tight-binding reversibLe inhibitors of cysteine proteases. Because these cysteine proteases exist in aLL Living organisms and because they are invoLved in various bioLogicaL and pathoLogicaL processes, the controL of these protease functions by cystatins is of cardinaL importance. In the immune system, cystatins moduLate cathepsin activities and antigen presentation. They aLso induce tumor necrosis factor a and interLeukin 10 synthesis, and they stimuLate nitric oxide production by interferonγ-activated murine macrophages. A spLeen cDNA Library of Large yeLLow croaker was constructed by induction with poLyI:C. There were two reLative cysteine proteases inhibitors found in tow cLones respectivLy, which were characterized to the members of the cystatin superfamiLy, after being sequenced and bLasted. Three immune reLative gene homoLogues of Large yeLLow croaker were cLoned from the spLeen of Large yeLLow croaker, which were tumor necrosis factorα1(TNF-α1), tumor necrosis factorα2(TNF-α2) and interLeukin 10(IL-10) homoLogue respectiveLy.In the present study, it was reported that a cLoning from the spLeen cDNA Library from the Large yeLLow croaker is a cystatin homoLogue(Lyccys), with 688 nucLeotides (nt) encoding a protein of 118 amino acids (aa) with a 21aa signaL peptide, 13kDa. The deduced protein shared 27.1-45.9 % , 30.0-31.7 %å’Œ16.3-30.6% sequence identity to the sequences found in other fishes, some high vertebrates and some nematodes. The highest sequence identity of 45.9% was achieved by Lyccys and a cystatin from pufferfish. It shared 30.8%, 31.7% sequence identity to the sequences of human cystatin C and chicken egg white cystatin. The Lyccys contains typicaL conserved motifs of cystatin superfamiLy, a N-terminaL GLy-GLy (25, 26), QLVAG (69-73) and a C-terminaL PW (106-107) known to interact with the active site of famiLy Cl cysteine peptidases. There was a conserved S⁵²N⁵³ which may be the reactive site of Legumain, a cysteine endopeptidase causing Limited proteoLysis of precursor proteins and protein spLicing. It had the structuraL arrangement of four conserved cysteine residues (C⁵⁶, C⁸⁷, C⁹⁸ and C¹¹⁸) with two disuLphide bonds towards the carboxyL terminusas found in Large known cystatins, however there is a cysteine residue (C⁵⁶) in the different Location from the others, which was removed to N-terminus. This maybe affected the space structure and its bioLogicaL functions. The genomic DNA sequence of Lyccys was cLoned and sequenced, constituting of three exons and two introns, sharing the simiLar genomic structure with human cystatin C, mouse cystatin C and zebrafish cystatin. Tissue expression profiLe anaLysis with reverse transcription-PCR showed that Lyccys gene was constitutiveLy expressed in aLL eight tissues (intestine, giLL, heart, muscLe, spLeen, Liver, bLood and kidney) examined, aLthough at a different LeveL. The highest LeveL of Lycstefm mRNA was detected in spLeen and kidney, and the Lowest in giLL, muscLe and bLood. The transcript LeveL of Lyccys was increased in kidney and spLeen, but decreased in bLood after induction with poLyI:C or inactivated trivaLent bacteriaL vaccine, anaLysis by reLative quantitative reaL-time PCR. The recombinant Lyccys(GST) (rLyccys(GST)) was expressed in E.coLi BL-21, and purified. The inhibitory specific activity of rLyccys(GST) against papain with the substrate casein was detected to be 40 U/mg. The intraceLLuLar LocaLization of Lyccys in kidney and spLeen ceLLs with immune coLLoidaL goLd eLectron microscopy was suggested that Lyccys was onLy distributed in cytopLasm, mainLy attached to rough endopLasmic reticuLum(rER), Large in vesicLes (endosome or Lysosome), not found in nucLeues. The transcript LeveLs of TNF-α2 and IL-10 were increased in kidney and spLeen of Large yeLLow croaker by injection with rLyccys(GST) through reLative quantitative reaL-time PCR. It is specuLated that the cystatin homoLogue of Large yeLLow croaker probabLy pLay a cardinaL immunomoduLatory roLe simiLar to that of mammaLs.On the other hand, we reported the cLoning of a stefin gene homoLogue from the spLeen cDNA Library of Large yeLLow croaker (Pseudosciana crocea), an economicaLLy important marine fish (Lycstefm). The fuLL Length cDNA of Lycstefm is 556 nucLeotides (nt) encoding a protein of 99 amino acids (aa0, with a putative moLecuLar weight of 11 kDa. The deduced protein shares 47.5%,47.5%,46.1%, 42.7%,38.0%,38.0% sequence identity to the sequences found in pufferfish, cattLe, takifugu, pig, rainbow trout and zebrafish, respectiveLy; and 43.0%å’ŒP 38.0%, 38.4%å’Œ 32.0% sequence identity to the sequences of human stefin A, human stefin B, mouse stefin A and mouse stefin B respectiveLy. The deduced Lycstefin contains conserved activation LocaLization of N-terminaL GLy⁶-GLy⁷ ,a conserved QLVAG (48-51) motif, which were found in aLL stefins. The Lycstefin genomic DNA sequence was cLoned and sequenced, constituting of three exons and two introns, sharing the simiLar genomic structure with human stefin A, mouse stefin A and zebrafish cystatin B. Tissue expression profiLe anaLysis with reverse transcription-PCR (RT-PCR) showed that Lycstefin gene was constitutiveLy expressed in aLL eight tissues (intestine, giLL, heart, muscLe, spLeen, Liver, bLood and kidney) examined, aLthough at a different LeveL. The highest LeveL of Lycstefin mRNA was detected in bLood and kidney, and the Lowest in intestine. The transcript LeveL of Lycstefin was increased in kidney and spLeen , but decreased in bLood after induction with poLyI:C or inactivated trivaLent bacteriaL vaccine, anaLysis by reLative quantitative reaL-time PCR. The recombinant Lycstefin(GST) (rLycstefin(GST)) was expressed in E.coLi BL-21, and purified. The inhibitory specific activity of rLycstefin(GST) against papain with the substrate caseins was detected to be 42 U/mg. The intraceLLuLar LocaLization of Lycstefin in kidney and spLeen with immune coLLoidaL goLd eLectron microscopy was suggested that Lycstefin was mainLy distributed diffuseLy throughout the cytopLasm, and not attached to rough endopLasmic reticuLum(rER) . LittLe was found in nucLeues and vesicLes (endosome or Lysosome). The transcript LeveLs of TNF-α2 and IL-10 were not changed in kidney and spLeen of Large yeLLow croaker by injection with rLyccys(GST) through reLative quantitative reaL-time PCR. So it is suggested that the stefin homoLogue of Large yeLLow croaker couLdn't moduLate The transcript LeveLs of TNF-α2 and IL-10.