Abstract: Objective To construct eleven cell lines each of which expressed CD45RO with removal of one N-glycosylation site and to examine its binding to galectin-3. Methods Individual site-directed N→Q mutagenesis of N-glycosylation site was performed for all eleven N-glycosylation sites in CD45RO and cloned into lentiviral vector pWPXL. Recombinant lentiviral was produced by 293T cells with co-transfection of pWPXL-mutated-CD45RO, the packaging plasmids psPAX2 and MD2.G. Human J45.01 cells were infected by the recombinant lentiviruses. The expression of CD45RO mutants was confirmed by RT-PCR and flow cytometry, and the binding of these cell lines to galectin-3 was evaluated by flow cytometry. Results Eleven cell lines each of which expressed CD45RO with removal of one N-glycosylation site were successfully constructed. RT-PCR, DNA sequencing and flow cytometry analysis revealed that the mutations were correct and CD45RO with removal of one N-glycosylation site was stably expressed on the surface of each cell lines. It was observed that galectin-3-binding to N327Q mutant was largely enhanced, to N36Q or N217Q mutant was largely suppressed. Conclusion Galectin-3 binding to CD45RO-J45.01 cells is regulated by N-glycosylation sites in CD45RO.

Key words: Galectin-3, binding, N-glycosylation site, CD45RO, Jurkat T cell