Decay Accelerating Factor (daf) And Its Effect On Jurkat Cell Proliferation Signaling Mechanism

Decay-accelerating factor (DAF/CD55) is a glycosyl-phopatidylinositol anchored protein that widely distributed on hemopoietic and non-hemopoietic lineage. DAF can protect cells from complement-mediated lysis by either preventing the formation of or dissociating C3 and C5 convertases thus blocking both classical and alternative pathway of complement activation. DAF is a multifunctional molecule. In addition to its complement lysis inhibitory function, DAF plays an important role in T cells and monocytes activation and in bacterial, virus adhension events. Experiments have shown that DAF can take part in the activation of monocytes, leukocytes and T cells. Cross-linking of antibody to DAF synergy with trace amount PMA can induce the proliferation of T cells. The mechanism of signal transduction through GPI-anchored protein has been become a hot spot of research of both domestic and foreign countries. As is well known that the deficiency of GPI-anchored proteins affects the signal transduction and proliferation of T cells. Cross-linking of GPI-anchored proteins can activate many kinds of cells in vitro. Recent data confirmed that unlike transmembrane proteins, many GPI-anchored proteins distribute in special membrane microdomains known as "lipid rafts" which take part in cell signal transduction events. This indicated that the "lipid rafts"are important in GPI-protein mediating signal transduction. To research the effects and mechanism of DAF on the proliferation of Jurkat cells, the CD3 positive and negative Jurkat cells was used in our research. The proliferation activity of CD3 positive and negative Jurkat cells were measured with MTT method after cross-linking DAF with and without immobilized CD3 antibodies. The IL-2 dependent CTLL cells proliferation were used to measure the IL-2 production in the culture supernatant. The IL-2Rc~ chain expression of Jurkat cells were measured by cell ELISA. Cross-linking of DAF showed apparent effects on the proliferation and secretion of IL-2 of CD3 positive cells with the existence of immobilized CD3 antibodies. It could also induce the expression of IL-2Ra chain on CD3 positive cells, all these effects can be blocked by Src family PTK specific inhibitor PP2. But these effects don not exist in CD3 negative cells. So cross-linking of DAF may synergy with CD3 to facilitate CD3 positive Jurkat cell IL-2 secretion, promote IL-2Rc~ chain expression, thus induce its proliferation. All these effects are induced by Src family PTK. In order to identify the role of DAF in signal transduction of Jurkat cells. Antiphosphotyrosine immunoblot analysis and ECL(enhanced chemiluminescene) were used to assay the level of tyrosine phosphorylation of Jurkat cells after 5 minutes treated by cross-linking CD3 or DAF. Cross-linking of DAF or CD3 could induce tyrosine phosphorylation of CD3 positive cells, but the phosphorylation pattens were some kind of different,which indicated that the signal transduction pathway of DAF and CD3 were some kind of different. Co-immunoprecipitation analysis showed association between DAF and Src family kinase lck and fyn in both CD3 positive and CD3 negative cells, which indicated that DAF induce cell signal mainly through PTK. To identify the role of membrane microdomain in Jurkat cells signal transduction. In our exprements, mild detergent Triton X-l 00 was used to extract detergent resistant Jurkat cell membrane section under uncontinuous saccharu density gradient centriftigation con...