The Expression Of Low Density Lipoprotein Receptor-related Protein 4 Intracellular Region And Its Interaction With Sorting Nexin 17 In Myasthenia Gravis Muscle Cells

BackgroundMyasthenia gravis(MG)is an autoimmune disease characterized by a synaptic transmission disorder in the neuromuscular junction(NMJ)that causes fluctuating myasthenia gravis.In about 80% of MG patients,the disease is mediated by antibodies directed against nicotinic acetylcholine receptors(nAChR).These antibodies increase AChR degradation and conversion,reduce the number of functional AChR on the fiber endplate,induce complement-mediated damage to muscle fibers,block the binding of acetylcholine to AChR.The results of the latest study show that low density lipoprotein receptor-related protein 4(LRP4)and muscle-specific receptor tyrosine kinase(Muscle specific kinase,MuSK),nicotinic acetylcholine receptor(nicotinic Acetylcholine receptor,nAChR)together constitute the NMJ end plate membrane-specific functional unit LRP4-MuSK-nAChR,the functional unit plays an important role in the process of neuromuscular excitatory chemical transfer.Previous study found that the content of Sorting nexin 17(SNX17)in MG patients was significantly higher than that in non-MG patients,and SNX17 and AChR were co-located at NMJ.SNX17 belongs to the family of cell sorting microtubule connexin,mainly through FERM-like primers and low density lipoprotein receptor(LDLR),low density lipoprotein receptor-related protein 1(LRP1),P-selectin and apolipoprotein 2(theta2 isoform of ApoE,ApoER2),and promote the efficient recycling of these membrane proteins to the cell membrane.The LRP4 and LRP1 belong to the LDLR family and have similar NPxY motifs.Therefore,the interaction between SNX17 and LRP4 may make LRP4 escape the lysosomes Degradation,and promote the recycling of LRP4 in the cell.In order to verify the above speculation,This study was performed by expressing LRP4 intracellular domain and SNX17,Co-immunoprecipitation(Co-IP)and Western blot were used to identify the interaction between them,to analysis of the role of SNX17 in the NMJ of MG patients.Methods1.Total RNA was extracted from muscle samples from MG patients,CDNA was reverse transcribed,and primers were designed using LRP4 intracellular domain as template to expand LRP4 intracellular domain gene.2.Construction of pET30a-LRP4 Intracellular Prokaryotic Expression Plasmid by Molecular Cloning Technique.3.Construction of recombinant expression plasmid pET30a-LRP4 intracellular domain by transformation of E.coli BL21(DE3)strain,using IPTG to induce expression of protein purification,and then by SDS-PAGE electrophoresis and Western blot identification.4.SNX17 gene was amplified from pET30a-SNX17 by using SNX17 gene as template.5.Construction of pEASY-Blunt M2-SNX17 Eukaryotic Expression Plasmid by Molecular Cloning Technique.6.The recombinant plasmid pEASY-Blunt M2-SNX17 was transfected into HEK293 cells and the total protein was extracted.SDS-PAGE and Western blot were used to identify HEK293 cells.7.Bi-directional Co-IP was performed by anti-His monoclonal antibody beads and anti-Myc monoclonal antibody beads.Results1.The recombinant plasmid pET30a-LRP4 and pEASY-Blunt M2-SNX17 were identified by restriction endonuclease and sequencing.2.The host protein of prokaryotic expression plasmid of pET30a-LRP4 was induced by SDS-PAGE and Western blot to induce the expression of His-LRP4 intracellular domain protein with molecular weight of about 17.8Kd.3.The Myc-SNX17 protein was transfected into the eukaryotic expression plasmid pEASY-Blunt M2-SNX17 by SDS-PAGE and Western blot.The molecular weight was about 53 Kd.4.Anti-His monoclonal antibody beads can precipitate Myc-SNX17 through His-LRP4 intracellular protein.5.Anti-Myc monoclonal antibody beads can precipitate His-LRP4 intracellular domain by Myc-SNX17.Conclusion1.LRP4 intracellular domain and SNX17 can interact.2.SNX17 maybe through the combination with LRP4,to induce the circulation of LRP4 in muscle cells,raise nAChR and repair damaged NMJ.