The Interaction Between Depp And Sedlin Protein And A Pilot Study On The Mechanism Of Spondyloepiphyseal Dysplasia Tarda Caused By SEDL Gene Mutation

Objective To investigate the interaction region of Depp for Sedlin by yeast two-hybrid assay; Construction the eukaryocyte expression vectors of Depp protein and protein N-C terminal deletion mutants and Sedlin protein, co-transfected into COS7 cells to observe in mammals Co-localization of cells; Construction of the four point mutants D47Y, S73L, F83S, V130D of Sedlin protein to pcDGFP eukaryocyte expression vector,transfected into COS7 cells to observe them single localization difference with the wild type, then,co-transfected with Depp protein with Flag tag into COS7 cells together to observe in mammals co-localization of cells,respectively; co-transfect Depp protein tagged with GFP and Flag-tagged Sedlin protein into HEK293T cells together for Immunoprecipitation to detect Depp protein can interact with Sedlin.protein in mammalian cells.Methods PCR method was used to amplify the N terminal 300bp and C terminal 339bp DNA fragments of Depp from plasmid pACT2-Depp,then the amplified fragments were inserted into pGADT7 vector respectively to construct lack mutants pGADT7-Depp-N300 and pGADT7-Depp-C339.Each mutant was cotransformed into AH 109 yeast competent cells with pGBKT7-SEDL,then the yeast cells were cultured on (-Trp/-Leu/-His/+3-AT)/X-α-gal plate, The blue clones were X-a-gal activity positive clone;Amplify BD-SEDL-D47Y, S73L, F83S, V130D as the template with pGBKT7-SEDL with site-directed mutagenesis, respectively, then co-transformed with pGADT7-Depp into AH 109 yeast competent cells, screened blue clones. The whole sequence of Sedlin was amplified frompGBKT7-SEDL by PCR method, then the amplified fragments were recycled after by BamH I and EcoR I digested, the recovery was cloned into the vector with GFP tagged, After successfully constructed pcDGFP-SEDL with its four points mutants, then transfected into COS7 cells and observed their localizatione in cells with wild-type difference under a fluorescence microscop, and then with Flag-Depp protein cotransfected into COS7 cells to observe their localization under a fluorescence microscope. Amplify Sedlin coding sequence by PCR method. The amplified fragments were inserted into pCDNA3.1 to construct eukaryocyte expression vector pCDNA-FLAG-SEDL. Identified by restriction enzyme digestion and sequencing, co-transfected the plasmid pCDGFP-Depp and pCDNA-FLAG-SEDL into COS7 cells to observe Sedlin protein and Depp protein intracellular localization by immunofluorescence;co-transfected into HEK293T cells, extracted cell lysatefor immunoprecipitation, then detected whether Sedlin protein can interact with Depp protein in mammalian cells by Western blot.Results Each recombined plasmid was constructed correctly confirmed by restriction enzymatic analysis and DNA sequencing. Nutrition Screening and a-galactosidase activity tests indicated Depp protein N- terminal can interact with Sedlin protein also as C-terminal, only D47Y of Sedlin mutant proteins was positive, the other three point mutations was negative. Immunofluorescence microscopy experiments observed Depp protein and Sedlin protein co-localized at a concentrated region around the nucleus similar as N-terminal in COS7 cells. Co-transfected four point mutants of Sedlin protein with Flag-Depp protein into COS7 cells, indicating that only GFP-SEDL-D47Y colocalization similar to wild type,both distributed in the perinuclear and nucleus, while the S73L, F83S, V130D were mainly localized in the perinuclear. Western blot analysis showed that both Depp protein and its N- terminal can express,but C- terminal can not;immunoprecipitation and Western blot proved that Sedlin protein can interact with Depp protein in HEK293T cells.Conclusion The critical region of Depp for its interaction with Sedlin located in N-terminal 100 amino acid residues; Immunofluorescence assay showed that protein Depp have a colocalization with Sedlin at a concentrated region around perinuclear in COS7 cells,which indicated the interaction between Depp and Sedlin in mammalian cells; Sedlin mutant proteins single and dual Transfection indicated D47Y cellular localization similar as and wild-type,while S73L, F83S, V130D were mainly distributed in the perinuclear. Co-immunoprecipitation experiments show that Depp protein and Sedlin protein an interact in mammalian cytoplasmic.