Effect Of CD45 Glycosylation Of Galectin-3 Binding And Apoptosis Induced By Infection Route For HIV & Rhesus CD4 ~ + T Cells Expressing CD95 In

Galectin is a class of specific binding to the β-galactosidase of soluble glycoproteins. So far, there has been found fifteen different galectins. They are involved in adhesion between the cell-cell and cell-matrix, cell growth, differentiation and apoptosis via multivalent compound formed by a cell membrane glycans. Galectin-1and galectin-3are coexpressed in many cells and tissues, which play an important function and have been extensively studied. Above all, galectin is the most closely related to apoptosis. In galectin families, galectin-3is the only molecular own pro-apoptotic and anti-apoptotic effects. Since the importance in the regulation of biological activities in cells, galectin-3has become a target candidate for seeking new drugs for the therapy of diseases such as tumor, inflammatory diseases and acquired immune deficiency syndrome (AIDS).CD45is a type of I transmembrane protein and also is the main receptor of galectin-1and galectin-3. There are several splicing isoforms of CD45. CD45RABC is rich in O-glycans. CD45RO is a glycoprotein molecule containing only N-glycans. Galectin-1and galectin-3can bind to CD45thereby regulating the apoptosis of T cells. It has now been found that CD45is essential for galectin-3induced Jurkat cells apoptosis. O-glycans and N-glycans of CD45in galectin-3-induced apoptosis remains unknown.In this study, we identified that galectin-3-induced CD45+Jurkat cell apoptosis but not CD45-J45.01cell. Galectin-1can induce apoptosis of Jurkat and J45.01cell. The results suggest that CD45is necessary for galectin-3-induced apoptosis. CD45RABC own both O-glycans and N-glycans while CD45RO only own N-glycans. To further clarify the role of CD45O-glycans and N-glycans in galectin-3-induced Jurkat cells apoptosis, we successfully constructed CD45RO-J45.01cells and CD45RABC-J45.01cells. The gene of CD45RABC and CD45RO were transfected into J45.01via lentiviral system. Galectin-3can only bind to CD45RO-J45.01and CD45RABC-J45.01cell surface, but can not bind to J45.01cell surface. CD45RABC-J45.01cells are induced apoptosis by galectin-3but not CD45RO-J45.01cells. The experimental results show that the CD45O-glycans play an important role in galectin-3-mediated apoptosis of Jurkat cell.Then, what is the role of CD45N-glycans in galectin-3-induced Jurkat apoptosis? We mutated each11N-glycosylation sites on extracellular region of the CD45RO by site-directed mutagenesis. The extracellular region of the CD45RO N-glycosylation sites of asparagine (N) was mutated to glutamine (Q), respectively. By means of sequencing, each CD45RO gene with an N-glycosylation site depletion was transfected into J45.01. After flowcytometry sorting,11cell lines each of which expressed CD45RO with removal of one N-glycosylation site were successfully constructed. The amount of galectin-3binding to the the11glycosylation site mutation cell lines and apoptotic changes (compared with the wild-type CD45RO-J45.01cells) were tested. Binding assays showed that galectin-3binds to N36Q N71Q, N99Q, N109Q, N115Q, N217Q, N258Q, N307Q, N368Q matants were decreased, only binds to the N327Q mutant was increased. We took N36Q, N109Q, N217Q, N327Q and N368Q mutants as examples. Enhancement of apoptosis induction was detected in N36Q and N109Q mutant whereas no apoptosis was detected in N217Q, N327Q and N368Q mutant. These data demonstrated that site-specific removal of N-glycosylation affected galectin-3binding and galectin-3-induced apoptosis in CD45RO-J45.01cells.Finally, our study demonstrates that galectin-3-induced Jurkat cell death is regulated by both O-glycans and N-glycans presence in CD45. Moreover, our findings provide a better understanding on biological properties of CD45receptor and the mechanism associated with galectin-3-induced T cell apoptosis and would be helpful to the development of new therapeutic strategies for diseases. CD4+T lymphocyte number decrease via apoptosis is a clinical feature of HIV/SIV infection. One of the reasons for CD4+T cell apoptosis is the death receptor CD95, which plays an important role in apoptosis. Due to different infection routes, the amounts of the virus invaded the body, interaction between SIV and target cell, initiate the immune response patterns are different. The relationship between CD4+T cell count and the surface CD95changes by different routes of infection has not been clear. For the above shortcomings, the death receptor CD95was examinated with SIV/Rhesus AIDS model with infection of SIVmac239by intravenously and intrarectally, respectively. This is very important to further understand the mechanism of CD4+T cell apoptosis.28Chinese rhesus macaques were selected and the sera from the monkeys were detected to be negative for lentiviral SIV、STLV-1、SRV and B virus by indirect immunoenzymatic/fluorescence method detection. Their Mamu-A*01, Mamu-A*02, Mamu-B*08and Mamu-B*17gene related to SIV/HIV susceptibility were negative by PCR test.The PBMCs from rhesus monkeys were used for SIVmac239amplified and when P27antigen concentration is more than3ng/mL detected by ELISA, the viral supernatant was collected. The virulence of amplified viruses were titered by PBMCs and TZM-bl cells. Fourteen rhesus monkeys were challenged with1×105TCID50SIVmac239intrarectally or intravenously. Established infection was determined by monitoring viral loads, CD4+T lymphocyte numbers and the ratio of CD4+/CD8+. The bloods were collected at early stage of infection period at nine time points, and the CD4+T cell surface CD95expression amount was analysezed by flow cytometry.For intravenous group, CD95expression on the CD4+T cells began to increase after the infected following days, and reached the peak value on the seventh day, meanwhile CD4+T lymphocyte numbers were declined to the lowest point. Rectal group also began to rise on the following days after infection and CD95expression reached the peak value after10infected days, but CD4+T cells were reduced to the lowest point until to the17th day. Compared with intravenous infection, The peak value of CD95expression and the lowest point of CD4+T cells appeared later in rectal infection, and the peak value of CD95 expression was earlier to the lowest point of CD4+T cells. CD95expression of the CD4+T cells were increased and CD4+T cells were decreased in rhesus monkeys challenged with SIVmac239intrarectally or intravenously.Our results showed that CD95expressed on the surface of CD4+T cells varied from SIVmac239-infected rhesus monkeys with different infection routes. By detecting the relationship of expression of CD95on CD4+T cell and the number of CD4+T lymphocytes could be usefull for further understanding the pathogenesis for clinical AIDS infection pathway.