The Interaction Between Dystroglycan And Sedlin Protein

Objective This study is to demonstrate if the interaction between DG and Sedlin occurs, and to further explore the specific domains of DG that interacting with Sedlin. Some experimental techniques were applied to the study in vivo( yeast cells and mamm-alian cells) and in vitro respectively, such as plasmid construction,yeast two-hybrid, we-stern blot(WB), immunofluorescence(IF),co-imumoprecipitation(CO-IP), laying the cy-tological foundation for exploring the functions of DG and Sedlin.Methods In yeast two-hybrid assay,we constructed the yeast expression plasmids( p GADT7- DAG1, p GBKT7- DAG1) of DG in turn, then they were cotransformed into yeast cells(AH109) with yeast expression plasmids(p GBKT7- SEDL, p GADT7-SEDL) of Sedlin respectively. Following the cell stainning on SD3- medium dish, positive cells were picked out, then determining the activities of X-α-gal of cells to demonstrate if there is interaction between DG and Sedlin by observing whether the color of cells turns blue;Then we continued to construct the eukaryotic expression plasmid(pc DNA3.1-DA G1-FLAG).In GST pull-down experiment pc DNA3.1-DAG1-FLAG was transfected into mammalian cells( HEK 293T), the total cellular proteins were extracted to suspend with fusion protein(GST-Sedlin) in vitro.and then the glutathione beads were collected by immunoblot to prove whether DG interact with Sedlin.In immunofluorescence experiment, pc DNA3.1-DAG1-FLAG and pc DGFP-SEDL were co-transfected into mammalian cells(COS7) respectively to observe if there was co-localized phenomenon between DG and Sedlin by fluorescence microscopy and confocal microscopy. In co-immunoprecipitation experiments pc DNA3.1-DAG1-FLAG and pc DGFP-SEDL were cotransfected into mammalian cells( HEK293T), and total cellular proteins wereextracted to incubate by FLAG antibody and precipitate by agarose beads,then the beads were collected for immumoblot to prove if DG interact with Sedlin.Next, we used the same experimental methods described above to study the interaction between the two domains of DG(α-DG,β-DG) and Sedlin further.Results The recombinant plasmids described above were proved to be constructed successfully by restriction digestion analysis and sequencing. Yeast two-hybrid experiment showed the yeast cells that had co-expressed the DG and Sedlin could grow normaly in SD3- medium, and the color of cells became blue in x-α-galactosidase activity assay, suggesting the existence of interaction between them. GST pull-down experiment showed that the DG could be detected in the experimental group(GST-Sedlin+DG-FLAG) after immunoblotting by FLAG antibody,then indicated the GST-Sedlin fusion protein could pull down the DG in vitro,suggesting that there was interaction between them. Immunofluorescence experiment showed that DG was mainly distributed in the cytoplasm in COS7 cells,and Sedlin was distributed in both cytoplasm and nucleus they had the obvious colocalization in the cytoplasm. Coimmunoprecipitati-on experiment also showed that Sedlin-GFP could be detected in the experimental group(Sedlin-GFP+DG-FLAG) after immunoblotting by GFP antibody, and indicated that Sedlin-GFP could be co-precipitated when DG-FLAG be precipitated by FLAG antibody, suggesting the existence of interaction between them. In further studies,the GST-Sedlin fusion protein could pull down the α-DG and β-DG in vitro too; then α-DG, β-DG and Sedlin also had the obvious co-localization in the cytoplasm of COS7 cells;and Sedlin could be coprecipitated when α-DG and β-DG were precipitated by antibody. This consistently showed that the existence of interaction between α-DG, β-DG and Sedlin too.Conclusion By Yeast two-hybrid, GST pull-down, IF and CO-IP, the interactionbetween DG and Sedlin was fully confirmed in vivo(yeast and mammalian cells) and in vitro respectively. And α-DG, β-DG, as the two domains of DG,both can interact with Sedlin, laying the foundation for studying the relationship between them in the future.