Expression, Subcellular Localization And Function Of P9-ZFD Protein In Pathogenesis Of MG

Myasthenia gravis(MS)is a T-cell-dependent and antibody-mediated organ-specific autoimmune disease of neuromuscular junction. In 85 % of patients, this disease is mediated by autoantibodies directed against the nicotinic acetylcholine receptor(nAChR), leading to nAChR loss at neuromuscular junctions. The pathogenicity of anti-nAChR antibodies has been clearly demonstrated. However, there is no absolute correlation between the severity of the disease and antibody titer. Above all, about 15 % of patients with typical generalized MG do not have detectable anti-nAChR antibody(SNMG). Although these patients do not differ from seropositive MG patients(SPMG)in clinical characteristics or respose to plasmapheresis and immunosuppressive therapy. In addition, Plasmapheresis is beneficial in SNMG, and MG can be transferred to experimental animals even by injection of sera from SNMG patients. Actually the pathogenetic mechanisms underlying the initiation and progression of MG are not completely understood. Some patients with MG should have autoAbs to unknown components of target organs. The production of autoantibodies is mediated by cytokines produced by T helper(Th)cells. Cytokines exert an important role in the pathogenesis of MG by modulating autoantibody production and interfering with cell-mediated immunity. A number of recent studies in both EAMG and human MG have shown that significant skeletal muscle tissue destruction has already occurred and many new autoantigens are liberated during the autoimmunity of MG, leading to the presentation of these cryptic epitopes to antigen-presenting cells(APC)surfaces and subsequent de novo activation of autoreactive T and/or B lymphocytes involving cytokine release. Especially in genetically susceptible MG individuals who may be defective in the regulatory controls on autoreactive lymphocytesprimed during tissue damage, the expression of many cryptic epitopes or peptide molecules including Titin and TNFR are obviously up-regulated. TNF is possibly involved in the autoimmune pathogenesis of MG by acting in concert with other cytokines within the TNF-TNFR1 signaling pathway. So we consider that MG should require the existence of more than one trigger condition, with the cumulative effects of several mechanisms and the influences of both genetic and environmental factors required to initiate clinical disease. Previous report from our laboratory demonstrated that the differential gene P9 had very low homology to known gene sequence and was mainly expressed by skeletal muscle and thymus. Furthermore, we had confirmed that P9 mRNA level was significantly higher in the skeletal muscle and thymus tissues of MG patients with thymoma or thymic hyperplasia than in that of controls. which clearly indicated that P9 gene should be a new MG-related gene . P9 gene consists of 3442 base pair and codes 709 amino acid. It is a difficulty task to express so big a protein. So we analyze P9 gene by InterPro database and find that P9 gene includes two function domains(P9-ZFD and F-box)and three candidate membrane-spanning segments. P9-ZFD is an important adaptor protein which TRAF2 region is expressed as a part of TNF-induced formation of a multiprotein receptor complex(MPRC)at the cell membrane. It could recruit cytokines and key pathway-specific enzymes to TNF-TNFR1 signaling pathways and cause MG by modulating autoantibody production and interfering with cell-mediated immunity. Our present works is to express and purify the protein coded by P9-ZFD fragment and to prepare P9-ZFD polyclonal antibody for investigating the function of P9-ZFD protein in the autoimmune pathogenesis of MG by studing 1)the expression in situ and subcellular distribution of P9-ZFD protein in the skeletal muscles of MG and Non-MG. 2)the expression level of P9-ZFD protein in different human tissues and its expression difference in the skeletal muscles of MG patients withdifferent clinical and pathological classification, Non-MG and patients with other muscle disease(OMD...