| Cathepsin is a lysosomal cysteine protease, but cystatins are natural tight-binding reversible inhibitors of cysteine protease. Because they exist in all living organisms and the regulation of cathepsin by cystatins, they are involved in various biological and pathological processes. In our preliminary studies, a spleen transcriptome library of large yellow croaker (Pseudosciana crocea) was contructed by induction with Aeromonas hydrophila. There are some sequences found in this library, which were characterized to the members of the cathepsin superfamily and the cystatin superfamily, after being sequenced and blasted, they are cathepsin B,L,S,cystatin B,F and a noval cystatin.In the present study, cathepsin B,L,S were cloned from the spleen cDNA library from large yellow croaker, with 993 nucleotides(nt),1011nt,1014nt encoding protein of 330 amino acids(aa),336aa,337aa. Then the recombinant Lyccathepsin B,L,S (rLyccathepsin B,L,S) were expressed in P.pastoris smd1168, and purified. The Km of cathepsin B,L,S to substrate Z-FR-AMC were detected ,which are 40.68μM,9.78μM and 52.49μM.Then we reported that a cloning from the spleen cDNA library from large yellow croaker is a cystatin B homologue (Lyccystatin B),with 590 nucleotides (nt) encoding a protein of 98 amino acids (aa),11KD. The deduced protein shared 50.51%-63.27%, 43.93%-48.98% sequence identity to the sequences found in other fishes and high vertebrates. The highest sequence identity of 63.27% was achieved by Lyccystatin B and a cystatin B from Japanese flounder. The Lyccystatin B contains typical conserved motifs of cystatin superfamily, a N-terminal Gly(4),QVVAG(46-50). The genomic DNA sequence of Lyccystatin B was cloned and sequenced, constituting of three exons and two introns, sharing the similar genomic structure with human cystatin B, house mouse cystatin B and zebrafish cystatin B. Tissue expression profile analysis with reverse transcription-PCR showed that Lyccystatin B gene was constitutively expressed in all eight tissues (intestine, gill, heart, muscle, spleen, kidney, liver, blood) examined, although at a different level. The highest level of Lyccystatin B mRNA was detected in kidney, and the lowest in gill. The transcript level was increased in kidney and spleen after induction with polyI:C or inactivated trivalent bacterial vaccine, analysis by relative quantitative real-time PCR. The recombinant Lyccystatin B (rLyccystatin B) was expressed in E.coli BL21, and puried. The inhibitory specific activity of rLyccystatin B against papain, legumain, rLyccathepsin B,L, S with the substrate Z-FR-AMC was detected. There are inhibitory activity of rLyccystatin B against papain, rLyccathepsin L,S, the Ki of which are 1.01×10-9M,1.82×10-11M,4.84×10-12M .There are no inhibitory activity of rLyccystatin B against legumain, rLyccathepsin B.We also reported that a cloning from the spleen cDNA library from large yellow croaker is a cystatin homologue (Lyccystatin), with 925 nucleotides (nt) encoding a protein of 147 amino acids (aa) with a 18aa signal peptide,17KD. The deduced protein shared 14%-17.33%,14.94%-26.88% sequence identity to the sequences of cystatin C found in other fishes and high vertebrates, it also shared only 16.33% sequence identity to the sequences of cystatin C which found in preliminary studies. The Lyccystatin contains typical conserved motifs of cystatin superfamily, a N-terminal Gly(35),QVTNV(79-83) and a C-terminal PR(125-126) known to interact with the active site of family C1 cysteine peptidases, it had the structural arrangement of four conserved cysteine residues with two disulphide bonds towards the carboxyl terminusas found in large known cystatins. But in other species, the middle typical conserved motif of the cystatin superfamily is QxVxG or QxxxG, which is QVTNV in large yellow croaker, Almost all of them are not the same. The sequence of Lyccystatin contains a N-linked carbohydrate recognition site. The phylogenetic tree based on the amino acid sequences of cystatin C,M,F from mammals and bony fishes shows that Lyccystatin is not together with the other cystatin C,M,F. The genomic DNA sequence of Lyccystatin was cloned and sequenced, constituting of three exons and two introns, sharing the similar genomic structure with human cystatin C, house mouse cystatin C and zebrafish cystatin C. However, the length of the exons from cystatin C of other species is almost the same, and Lyccystatin is not. But also in other species, the first intron is larger than the second intron, in contrast with other species, the first intron from Lyccystatin is smaller than the second intron. So we infer that Lyccystatin is a noval cystatin. Tissue expression profile analysis with reverse transcription-PCR showed that Lyccystatin gene was constitutively expressed in all eight tissues (intestine, gill, heart, muscle, spleen, kidney, liver, blood) examined, although at a different level. The highest level of Lyccystatin mRNA was detected in intestine, and the lowest in gill. The transcript level was increased in kidney and spleen after induction with polyI:C or inactivated trivalent bacterial vaccine, analysis by relative quantitative real-time PCR. The recombinant Lyccystatin (rLyccystatin) was expressed in P.pastoris smd1168, and puried. The inhibitory specific activity of rLyccystatin against papain, legumain, rLyccathepsin B,L,S with the substrate Z-FR-AMC was detected. There are inhibitory activity of rLyccystatin against papain, rLyccathepsin L,S, the Ki of (which are 2.90×10-8M,1.81×10-11M,2.02×10-11M.There are no inhibitory activity of rLyccystatin B against legumain, rLyccathepsin B.Finally we reported that a cloning from the spleen cDNA library from large yellow croaker is a cystatin F homologue (Lyccystatin F),with 935 nucleotides (nt) encoding a protein of 144 amino acids (aa) with a 26aa signal peptide,16KD. The deduced protein shared 40.69%-43.84%, 28.57%-36.73% sequence identity to the sequences found in other fishes and high vertebrates. The highest sequence identity of 43.84% was achieved by Lyccystatin F and a cystatin F from northern pike. It shared 28.57%, 29.76% sequence identity to the sequences of human cystatin F and house mouse cystatin F. The Lyccystatin F contains typical conserved motifs of cystatin superfamily, a N-terminal Gly(35),QVVRG (79-83) and a C-terminal PR(90-91) known to interact with the active site of family C1 cysteine peptidases, it had the structural arrangement of four conserved cysteine residues (C97,C108,C122,C142)with two disulphide bonds towards the carboxyl terminusas found in large known cystatins. There was a conserved S106N107 which may be the reactive site of legumain, a cysteine endopeptidase causing limited proteolysis of precursor proteins and protein splicing. The sequence of Lyccystatin F contains five N-linked carbohydrate recognition sites. Tissue expression profile analysis with reverse transcription-PCR showed that Lyccystatin gene was constitutively expressed in all seven tissues (intestine, heart, muscle, spleen, kidney, liver, blood) examined, although at a different level. The highest level of Lyccystatin F mRNA was detected in kidney, and not detectde in gill. The transcript level was increased in kidney and spleen after induction with polyI:C or inactivated trivalent bacterial vaccine, analysis by relative quantitative real-time PCR. The recombinant Lyccystatin F (rLyccystatin F) was expressed in P.pastoris smd1168, and puried. The inhibitory specific activity of rLyccystatin F against papain, legumain, rLyccathepsin B,L,S with the substrate Z-FR-AMC was detected. There are inhibitory activity of rLyccystatin against papain, legumain, rLyccathepsin B,L,S, the Ki of which are 1.42×10-8M,1.32×10-9M,2.34×10-8M,1.64×10-11M,9.39×10-11M.
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