| The control of schistosomiasis mainly lies in the blocking of transmission and infection and alleviate pathogenesis.So far no vaccine against schistosomiasis has been available,the combination of chemical therapy and extinction of intermediate host of S.japonicum,oncomelania,is the main way to control schistosomiasis currently.Praziquantel(PZQ) leads the priority list of chemicals to treat schistosomiasis for the reasons of low price,less side effect,and good effect of annihilation.Thus,praziquantel has been used widely and exclusively in both clinic and epidemic areas for prevention from infection of S.japonicum.However,the exact mechanism by which PZQ kills the parasite remains unknown.The previous reports showed that PZQ has low effect on larvae of S.japonicum and progressive hepatic fibrosis of chronic schistosomiasis and strain of S.mansoni resistant against praziquantel has been developed except for PZQ.Therefore,identification of molecules,based on the understanding of the passway neurotransmitters such as catecholamine(CA) synthesis and metabolism,for novel targeting therapy and development of alternative chemicals against S.japonicum are urgently needed.The main causative agent of schistosomiasis is granunoma in liver from eggs deposists.Therefore,interfering and reduction of eggs is one of ways of anti-schistosomiasis.Catecholamine is one of the important neurotransmitters of Schistosoma,mainly synthesized in ganglion and nerve trunk of the parasite.Tyrosine Hydroxylase(TH) is the rate-limiting enzyme of catalyzing synthesis of catecholamine(CA) and regulate its production in a complicated pattern.It is reported that the signal protein human 14-3-3,through its binding motif,could interact with TH and up-regulate its activity.While in S.mansoni,serotonin with catecholamine affects its muscle contraction and rate of egg-laying.So we presume that the similar function might exist between Sj14-3-3 and SjTH and there may be one or more signal pathway to regulate the expression of SjTH,finally modulating the production of catecholamine and affecting the development and physiological function in S.japonicum.Therefore,experiments were designed to verify the interaction between Sj14-3-3 and SjTH and analyze the enzymatic activity of SjTH and the interference of catecholamine in order to find a molecular target to which the novel therapeutics of anti-schistosomiasis will be development.We have compared the homology of SjTH to THs of other different species such as human,bovine,rat,mouse,quail,drosophila,and S.mansoni,and a high homology was found in 3’-end.The specific primers were designed according to SmTH sequence in the conservative region.The fragment of SjTH was amplified with S.japoncum cDNA,with a size of 480bp and a homology of 78% with SmTH.Primers for RACE PCR were designed according to this partial fragment,and the 5’- and 3’-end sequence of SjTH were amplified with S.japonicum cDNA by RACE PCR respectively.The production of RACE PCR was subjected to sequence and spliced with 5’- and 3’-end sequence.The sequence overlapped was the same with the partial sequence cloned as above.Thus, 5’- and 3’-end RACE PCR was successful,and the open read fragments(ORF) of SjTH was screened with tool of ORF finder in NCBI.An ORF with 1392bp was identified and deemed as the coding gene of SjTH.The primers used to clone the complete cDNA fragment of SjTH gene were designed according to its ORF and subsequently PCR for complete sequence of SjTH was run and the product was sequenced again.Our results showed that complete sequence of SjTH has similarity with expected,with 1392bp in length.The complete sequence of SjTH gene was analyzed with vector NTI biosoftware.The coding sequence of SjTH was predicted to encode 463 amino acids with molecular mass at 53.97kDa and pI at 5.81.It was found that SjTH has 40% of homology with human TH and 75% with SmTH .As expected,a motif with specific amino acid sequence of RSXpSXP,which recognizes the binding domain of signal protein 14-3-3,was found in the C-terminal of SjTH,indicating that the SjTH would be able to interact with 14-3-3 through its motif.The prokaryotic fusion expression of SjTH was generated with pET28a/E.coli BL21 and purified by His tag using resin.The immune specificity of the recombinant SjTH was analyzed by Western blotting with monoclonal antibody against His.The enzymatic activity was test with tyrosing labeled with 3H.The result showed that the activity of SjTH depends on the existence of dihydrofolate reductase without dose-dependence of dihydrofolate reductase.Meanwhile,we prepared the polyclonal antibody against SjTH from rabbits.Immuno-localization of SjTH in S.japonicum showed that it is mainly distributed in the reproductive organs,ganglions and muscles of S.japonicum.Time phase of theSjTH expression in different development stages of adult,cercaria,and myracidium was examinted by RT-PCR and Western blotting and it showed that SjTH is reach in adult stage of female and male parasites and only slight flurescence was noted in cercariae.Summarily,we firstly cloned and characterized the coding gene of SjTH,and analyzed the enzymatic activity,localization,and exression in different stages of development.Interestingly,a binding motif of SjTH with a sequence of RSXpSXP,which recognizeds Sj14-3-3,was found in recombinant SjTH,suggensting that SjTH could interact with Sj14-3-3 and regulate the synthesis of catecholamine,a group of neurotransmitters critical to the survival and growth of S.japonicum in blood vessels of its host. Enzymatic activity of tyrosine hydroxylase of Schistosoma japonicum regulated by interaction between SjTH and Sj14-3-3 signaling protein Preziquantel(PZQ) is a shistosome-killing drug which has been used worldwide currently. The previous report indicated, however, PZQ has a approving therapeutic effect on adult worms than schistosomulae and a better parasiticide against matured eggs than unmatured eggs. Treatment of schistosomiasis with PZQ failed to inhibit the progress of hepatic fibrosis elicited by the soluble egg antigens from miracidia. Also elimination of adults from hosts will not be able to prevent the hosts from subsequent reinfection. Moreover, the near exclusive use of PZQ for treatment of human S.mansoni has raised concerns about the possible emergence of drug-resistant S.japonicum.It is stringent nowadays to develop a new drug and find new target molecules against schistosomes through approach of the signaling transduction or the pathways of neurotransmission. Soluble egg antigens (SEAs) secreted from miracidia stimulate host immune system to induce egg granuloma mainly in the liver leading to schistosomiasis. Therefore, it is a alternative strategy to suppress the maturation of schisosome and eggs-laying by interference with catecholamine metabolism. It is reported that catecholamine can reduce the tensity of muscle and repress the eggs of production in S.japonicum. Catecholamine is synthesized with tyrosine as substrate and catalyzed by a series of enzymes, including a rate-limiting enzyme, tyrosine hydroxylase(TH), which is the checkpoint for regulation of catecholamine synthesis . In the previous experiments, we cloned coding gene of SjTH successfully in adult S.japonicum and found that there was a motif which recognizes 14-3-3 protein in the C-terminal, comprised of a small peptide:YIRHHSRPMHTP. A novel phosphoserine containing motif initially identified in Raf was vital for interaction,and showed that target protein phosphorylation is important for 14-3-3 binding via a novel phosphoserine sequence motif.The peptide was according with RSXpSXP. Interaction between artificial phosphorylated peptide and Sj14-3-3 by BIAcore, which showed that the phosphorylated peptide could bind to Sj14-3-3.A perfect positive correlation of concentration of phosphorylated peptide was found to go with the combination of the Sj14-3-3 . So we believed that Sj14-3-3 could interact with phosphorylated SjTH. The published results suggested that human 14-3-3 could interact with phosporylated human TH and up-regulated activity of human TH. The present experiment was carried out to verify the hypothesis that the interaction of Sj14-3-3 with SjTH is similar to humans. Firstly,we observed the result of interaction between Sj14-3-3 and phosphorylated SjTH by BIAcore. Secondly, variation of enzymic activity was analysised with 3H tyrosine by scintillation in vitro.Finally,RNA interference(RNAi) with Sj14-3-3 or SjTH in vivo indicated the relation of the interaction between the two proteins which exerts influence on enzymic activity of SjTH.The SjTH was expressed in eukaryotic expression system.Primers were designed according to the coding gene of SjTH with restriction enzyme EcoR I and NotI. The PCR products were inserted into pcDNA3.1 and the recombinant plasmids were screened with antibiotics(G418)..The recombinant pcDNA3.1 carrying SjTH gene was purified with His tag by His binding purification kits.The purified fusion protein was phosporylated by CaM PKII in vitro and the interaction between Sj14-3-3 and phosphorylated SjTH was assayed by BIACORE(Co、BIOCORE3000). The enzymatic activity of SjTH was up-regulated during the limited range when different concentration of Sj14-3-3 was added,. The experiments above suggested that Sj14-3-3 could interact with phosphorylated SjTH through the motif with phosphoserine sites, activating SjTH and up-regulating the expression of catecholamines. The work set a basis for SjTH and/or Sj14-3-3 as the check point to regulate catecholamine synthesis in S.japonicum. Further approach for the effect of CA neurotransmitters on the adult development and egg-laying is in progress... |
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