The Discovery And Function Study Of BAP31, A Newly Defined Cancer/Testis Antigen

Tumor antigens refer to the neoantigens and/or the over-expressed antigens during the tumorigenesis, which can induce specific humoral and/or cellular immune response in human. Moreover, these antigens can be used as either markers of tumor or targets for immunotherapy. Cancer/testis antigens (CTA) consist a subpopulation of tumor antigens. Up to now 46 gene families with total 91 members of CTAs have been found. In general, the expression of CTAs is restricted to testis in normal tissue, while the expression of CTAs is significantly up-regulated in various cancer tissues. Some CTAs can induce specific humoral and/or cellular immune responses in human. Thus, CTAs have been thought as potential targets for the immunotherapy of cancer patients. Although more and more CTAs have been discovered, few of them can be used as ideal targets for cancer immunotherapy. There are limits in the methods established for identifying new CTAs. So, it's necessary for searching new technology for discovering unknown CTAs.Considering the unique expressing pattern of CTAs, that is, restricted to spermatogenic cells of human testis, we developed a new method to discovering unknown CTAs, named as spermatogenic cells specific monoclonal antibody-defined cancer/testis antigens (SADA). Firstly, we immunize BALA/c mice using human spermatogenic cells, then raise mAbs against spermatogenic cells using hybridomics. The expressing patterns of positive in testis and negative in other tissues were identified by newly developed mAbs and immunohistochemical staining. The mAbs which have CTA-like expressing patterns were selected and used for immunoprecipitation of the corresponding molecules. Finally, new candidates for new CTAs were determined based on the results from liquid chromatography-Mass spectrum/Mass spectrum (LC-MS/MS) assay.In our experiment, 19 candidates were discovered using the newly established SADA method and 5 of them had expression patterns similar with that of CTAs. In addition, we raised some mAbs that recognize cells in different stages of spermatogenesis. The molecules recognized by these mAbs may play important roles in spermatogenesis and these mAbs may be useful in the research on spermatogenesis.LC-MS/MS analysis is an important tool for identifying proteins. We choose 4 CTA-like mAbs, FM-1,FM-5,FM-6 and FM-18 for further identification. The results from immunocytochemistry staining showed that Hela cells expressed all the molecules recognized by these mAbs. So we performed immunoprecipitation using FM-1, FM-5, FM-6 or FM-18 and Hela cells'lysate and purifacation of the corresponding molecules recognized by these mAbs. FM-1 and FM-5 could be used in immunoprecipitation and LC-MS/MS assay showed that the molecule recognized by FM-1 is BAP31.BAP31, an evolutionarily conserved and ubiquitously expressed 28-kDa polytopic integral protein, was originally described as contributing to B-cell receptor activation. Its location in the ER, however, argued that its contribution to this activation might arise indirectly through an involvement in the egress of membrane-integrated protein complexes from this organelle. Consistent with this, BAP31 has been implicated in export of major histocompatibility complex classâ… (MHC-â… ) molecules from the ER and in the control of maturation or trafficking of cystic fibrosis transmembrane conductance regulator. In addition to its predicted role in regulating egress of membrane complexes from the ER, BAP31 is a BCL-2/BCL-XL-associated protein and plays an important role in apoptosis, both as a regulator of procaspase-8L processing and as a cleavage target of caspase-8. Using immunohistochemical staining, we found high-level expression of BAP31 in human testis, mainly localized in spermatogonia and primary spermatocyte, indicating that BAP31 may play a role in spermatogenesis. Moreover, expression of BAP31 is dramatically up-regulated in various carcinomas, including carcinomas of esophagus, colon, rectum, lung, breast, cervix, ovary and liver.The expression pattern of BAP31 is similar to that of CTAs, that is, the expression level of BAP31 is much lower in normal tissues than that in testis and tumor tissues. Considering that the chromosomal localization of BAP31 is Xq28, adjacent to various CTA genes including MAGE-A, NY-ESO-1, LAGE-1 and SAGE, we designate BAP31 as a new member of CTAs.Furthermore, we investigated the influence of BAP31 on cell cycle and biological functions in Hela cells. Flow cytometry analysis detected a constant increase in G1 DNA content in comparison with the control group. The percentage of cell DNA in replication phase S and division phase G2 are also decreased significantly. These data suggest that depletion of BAP31 could lead to an arrest of cells in G1 phase, a delayed transition into S phase, and eventually restrained proliferation of cells. Also, BAP31 depletion caused a significant decrease in the number of migrating cells relative to control transfected cells, indicating that BAP31 is essential for typical tumorigenic properties of cancerous cells. Moreover, down-regulation of BAP31 sensitizes cells to chemotherapeutic drugs-induced apoptosis. Acting as molecular chaperone, loss of BAP31 may cause retention of important proteins in the ER and in turn, cause changes in cell proliferation, cell cycle, etc. It has been known that BAP31 could interact with protein tyrosine phosphatase-like B (PTPLB), a novel member of the PTPL family, which may have a regulatory function in the aspects of coordinating ER dynamics and ER transitions during the cell cycle.CD molecules play central biological roles in hematopoietic cell differentiation, proliferation, activation and migration as well as host defense, inflammation, apoptosis, autoimmunity and organogenesis. With rapid development of genome project, the concept of CD is replaced by human cell differentiation molecule (HCDM). Because spermatogenesis is the differentiation progress of spermatogonia, some CD/HCDM may participate in the spermatogenesis. Studying the expression patterns of CD molecules in human testis may further our understanding of the mechanism of spermatogenesis, and even more, we may find new CTA candidates in CD molecules. We investigate the expression patterns of 29 CD molecules in human testis and found that 15 of them are positive in testis. Among them the expression pattern of CD326 is quite unique. CD326 is epithelial cell adhesion molecule (Ep-CAM), mainly expressed on epithelial cells and its expression is highly up-regulated in epithelial cell derived carcinomas. In human testis, CD326 mainly localized on the membrane of spermatogonia and some primary spermatocytes, indicating that it may play a role in spermatogenesis. The expression pattern of CD326 is much like the ubiquitously expressed CT molecules such as IL13Ra/CT19, TSP50/CT20, SPA-17/CT22, OY-TES-1/CT23 and PLU-1/CT31. It is resonable that CD326 may be a candidate for CTAs.