Expression,localization And Functional Analysis Of Cystathionine ?-lyase Cgl1 From Tetrahymena Thermophila

Sulfur-containing amino acids have important regulatory functions in different organisms.The specific amino acids participate in the cellular functional regulation and play a role in DNA and protein modification,immune cell response,and maintenance of the cellular redox reaction balance.The sulfur transfer pathway is the core pathway for the metabolism of sulfur-containing amino acids in cells.Therefore,the control of the transsulfur pathway involved in the synthesis of sulfur-containing amino acids is essential for maintaining normal cellular function.Tetrahymena is an important model organism which has sensitive environmental response mechanisms and fine cellular metabolic pathway.However,the sulfur transfer pathway is less clear in the model organism.In this study,cystathionine ?-lyase from the transgenic sulfur pathway in Tetrahymena thermophila was characterized for the first time.The main results were as following:1.Identification of cystathionine ?-lyase in Tetrahymena thermophilaT.thermophila contains evolutionarily conserved cystathionine ?-lyase?CGL1,TTHERM₀0052400?,open reading frame 1230 bp encoding 409 amino acids.CGL1 showed higher expression levels in vegetative growth stage,while maintained a low level of expression during starvation and sexual reproduction.The CGL1 gene was synthesized and the recombinant expression plasmid p GST-CGL1 was constructed.The plasmid was transformed into E.coli BL21?DE3?,and GST-Cgl1 was expressed and purified by affinity chromatography.Enzyme activity analysis showed that GST-Cgl1 cleave cystathionine to produce cysteine,while cleavage of cysteine and homocysteine to produce H2 S.The GST-Cgl1 maintain high stability under 45?,the relative enzyme activity is above 70%;after 45?,the enzyme activity drops sharply.The optimum p H is 8.0.2.Localization of Cgl1 in the cytoplasm and nucleus The recombinant plasmid p NEO4-3HA-CGL1 and p XS75-CGL1 were constructed and transformed into Tetrahymena.The mutant strains were screened with paromomycin and identified by PCR.Immunofluorescence staining showed that the HA-Cgl1 localized in the parental macronucleus in vegetatively growing stage;localized in the cytoplasm during starvation stage;and localized in the parental macronuclear cytoplasm during sexual reproduction.Furthermore,the overexpressed HA-Cgl1 localized in the micronucleus and the macronucleus during the vegetative stage,and enriched around the nuclear membrane.HA-Cgl1 located in macronucleus and cytoplasm during early anlagen stage and localized in the new micronucleus and the parental macronucleus during late anlagen stage.3.Overexpression of CGL1 inhibits cell proliferation and sexual reproductionOverexpressed CGL1 inhibits cellular proliferation in vegetatively growing stage.Furthermore,overexpressed CGL1 aborted the sexual development.The micronucleus failed to complete the division which implied that Cgl1 could be involved in DNA replication.4.The knockdown of CGL1 affects stability of the nucleus during sexual reproductionThe plasmid p NEO4-KO-CGL1 was constructed and transformed into Tetrahymena cells.The stable CGL1 knockout strain failed to obtain which imply that CGL1 is essential for cellular survival.Furthermore,p SMC1 hp NEO-CGL1 was constructed and transformed into Tetrahymena cell.Expression of CGL1 was interfered by small RNA under Cd ions induction.CGL1 knockdown affected cell proliferation in vegetatively growing stage.During sexual development stage,CGL1 knockdown mutant cells failed to form zygotic nucleus and the developing micronucleus degenerate abnormally.The results indicated that Cgl1 is involved in the regulation of the formation of functional nuclei during Tetrahymena sexual reproduction.In summary,this study identified a cystathionine ?-lyase gene from T.thermophila.The GST-Cgl1 was expressed in E.coli BL21?DE3?and purified by affinity chromotography.GST-Cgl1 cleaved cystathionine to produce cysteine,and cleaved cysteine and homocysteine to produce H2 S.HA-Cgl1 localized in the cytoplasm and nucleus in Tetrahymena.Both knockdown and overexpression of CGL1 affect the proliferation of Tetrahymena in vegetatively growing stage and the stability of the nucleus during sexual reproduction.The results showed that cystathionine ?-lyase has enzymatic activity for cystathionine,cysteine and homocysteine,and involved in the proliferation of cells,maintained the stability of nuclear development during sexual reproduction in Tetrahymena.