Cloning, Expression And Characterization Of NMAAP1 And Trim59, Two Novel Membrane Proteins From Bcg-activated Macrophages

Macrophage is one of mononuclear cells in the body which widely exist in organizations; it is the first barrier of natural immune system. Macrophage also plays an important role in immune monitoring. Macrophages can secrete such as TNF, NO to kill cancer cells, on the other hand, macrophage could kill tumor cells directly through cell to cell contact,. The mechanism of this process is an important territory of tumor immunity. Up to date, lots of studies show that there must be some novel molecules on the membrane of macrophages, which mediate the cytotoxic process against tumor cells.In previous study, we found that BCG activated macrophages could kill MCA207 tumor cells through cell to cell contact. Membrane proteins from BCG activated macrophages and thioglycolate elicited macrophages were compared with a new method of SDS-page combine liquid chromatographytandem mass spectrometry. Comparisons resulted in a list of 454 proteins which were identified from BCG activated microphages only. From these proteins, we selected two novel proteins as our research targets.Part 1:We selected two novel proteins from our previous study using bioinformatics, named NMAAP1 and Trim59, and predicted the potential function of these two novel proteins.Result: NMAAP1 could combine with DAPK to mediate cell apoptosis; on the other hand, kiaa1754 (the homology member of NMAAP1) could combine to IP3R to mediate the Calcium Ion Channels, which might be the most important process in cell differentiation; meanwhile, NMAAP1 have a structure named Mab-21, which possibly mediate the organic evolution and phylogenetic development. Trim59 have several structures which might be involved in cell to cell contact.Part 2:We cloned and expressed the two proteins, and purified the proteins by affinity chromatography. Using these two proteins, we generated two polyclonal antibodies against these two proteins. After western-blotting detection, immunohistochemisty was processed.Result: In this study, we cloned the two novel proteins to generated fusion proteins in E. coil. The purified fusion proteins were applied for generation of polyclonal antibodies. Western-blotting detection showed that the polyclonal antibodies have high specificities to recognize target proteins. Using of the polyclonal antibodies, we detected the tissue distribution of these two novel proteins, and this process also showed our antibodies could combine to the nature protein. The results showed that NMAAP1 abundantly expressed in the thymus, which might be involved in T cell differentiation through IP3R combine to TCR. Trim59 abundantly expressed in the spleen, which indicate Trim59 is an important molecule in the immune system; on the other hand, Trim59 was detected from ovary, which indicate Trim59 might be involved in reproductive system.Part 3:We detected functions of the two novel proteins through antibodies blocked cell cytotoxicity. We cloned the full length of Trim59 and stably transfected it into Raw264.7 cell lines to investigate the function of Trim59.Result: Using the antibodies to block Trim59 on the membrane of BCG activated macrophages significantly reduced BCG activated macrophages'cytotoxicity against MCA207 tumor cells. This potentiated that Trim59 serves as an indispensable molecule in maintaining BCG activated macrophages'activity. However, blocking of NMAAP1 failed to down-regulate the cytotoxicity of BCG activated macrophages against MCA207 tumor cells. This result indicated that NMAAP1 is not the necessary molecule in BCG activated macrophages tumoricidal process.Overexpression of Trim59 in Raw264.7 cell line failed to lyse target MCA207 cells, which demonstrated Trim59 per se could not enhance macrophages cytotoxicity. Our results imply Trim59 might be an essential accessory molecule in mediating BCG activated macrophages tumoricidal functions. Interestingly, the phagocytosis of transfected Raw264.7 cells was enhanced notably; this might be related to the structure of Trim59, which have a ring finger domain, a B-box domain, and two coli-coli domains. We infer that Trim59 is an associated molecule, which promote macrophages to get in touch with tumor cells.Conclusion, NMAAP1 has a high expression in the thymus, and is not the necessary molecule in macrophages directly tumoricidal process, the structure of NMAAP1 indicate it might involved in the activate process. Trim59 has a high expression in spleen and ovary, which indicate Trim59 involved in immunization and reproduction. Trim59 is an indispensably molecule in macrophages tumoricidal process, and Trim59 act as an associated molecule in this process. On another hand, Trim59 could enhance the phagocytosis of macrophages.