The Preliminary Study On The Interaction Of Cystatin B And Its Mutants Of G50E, Q71P With CLIC1

Objective To investigate the biological functions of cystatin B, inhibitor ofcathepsins, we constructed yeast expression vectors of cystatin B gene and its pointmutants of G50E, Q71P for yeast two-hybrid experiment and eukaryotic expressionvectors, tagged with FLAG, and of CLIC1gene tagged with GFP. The plasmid above was transiently transfected into mammalian cells to observe the localization ofexpressed protein respectively. The plasmid of cystatin B and its mutants weretransiently cotransfected with CLIC1into mammalian cells respectviely to definewhether two expressed proteins were colocalized. Finally, we confirmed the interactionof cystatin B and its mutants with CLIC1by co-immunoprecipitation assay.Methods pLenti6vector with full-length cystatin B was used as template foramplification. PCR product of cystatin B cDNA and vector were separately digested byappropriate restriction enzymes, and then ligated into pGADT7-cystatin B,pGBKT7-cystatin B, and pCDNA3-cystatin B-FLAG. We also put the full lengthCLIC1gene into vector pCDGFP in frame tagged with GFP, and mutants of cystatin B(G50E, Q71P) into pcDNA3tagged with FLAG. All plasmids were confirmed byrestriction enzyme analysis and DNA sequencing.Yeast expression vectors were divided into groups and co-transformed into AH109separately. Transformants were screened on SD/-Leu/-Trp/-His/3AT/X-α-gal plate.Clones exhibiting blue were transformants with active α-gal activity.Plasmid was transiently transfected into COS7cells to show the localization ofexpressed proteins. Cystatin B and its mutants were cotransfected with CLIC1intoCOS7cells and images were obtained by fluorescence microscope to investigatewhether each protein was colocalized with CLIC1. Finally, the interaction of cystatin Band its mutants with CLIC1were performed in293T cells by co-immunoprecipitationassay.Results We obtained plasmids in-framed with CLIC1gene, cystatin B gene and itsmutants. Yeast expressing couple of fusion proteins of cystatin B and CLIC1had α-galactosidase activity. The same results were obtained from cells expressing G50E withCLIC1and Q71P with CLIC1. CLIC1-GFP mainly distributed in nucleus, especiallyconcentrated in nuclear membrane, and less in cytoplasm. The distribution of cystatin B-FLAG was similar to that of CLIC1-GFP. Localization of G50E or Q71P mutants,however, was different to that of wild type. Cystatin B and CLIC1were colocalized inCOS7cells as well as its two mutants. Cystatin B and its two mutants could interactwith CLIC1in HEK293T cells.Conclusion Cystatin B and its two mutants of G50E and Q71P could interact withCLIC1in yeast cells. In COS7cells, cystatin B and its mutants of G50E or71P werecolocalized with CLIC1. Co-immunoprecipitation experiment suggested both cystatin Band its point mutants were interacted with CLIC1in HEK293T cells.