| C-type lectins are key players in the immune system, mediating clearance of pathogens and cell-cell interactions in the innate and adaptive immunity. Recently we have cloned a novel C-type lectin LSECtin, which is a member of a family comprising CD23, DC-SIGN and DC-SIGNR. LSECtin is a type II integral membrane protein and expressed in liver and lymph node. It possesses a typical carbohydrate recognition domain(CRD) and binds to mannose, N-acetylglucosamine (GlcNAc) and fucose in a Ca2+-dependent manner but not to galactose(Liu et al., J Biol Chem. 2004,279:18748-58).In this study, we performed immnohistochemistry analysis and double immunofluorescein staining, indicating that LSECtin was expressed in Kupffer cells and liver sinusoidal endothelial cells. To verify the hypothesis that LSECtin is a cell adhesion molecule, sLSECtin-Fc fusion protein was used in the screening a series of cell lines to identify the LSECtin ligand by flow cytometry. Among the cell lines tested, Jurkat, a human T lymphocyte leukaemia cell line, had maximum expression of LSECtin ligand after treatment with PMA in combined with ionomycin. Then we sought to determine whether LSECtin ligand naturally expressed on T cells in vivo, both purified sLSECtin-Fc protein and LSECtin expressed in MCF7 cells bound activated peripheral blood T cells. These results suggest that LSECtin is a novel cell adhesion molecule in liver and can recognize activated T cells.To identify LSECtin ligand on activated T cells, we performed an immunoblot analysis on lysates of surface-biotinylated activated Jurkat cells with sLSECtin-Fc. A 140-kD and an 80-kD ligand specifically interacted with sLSECtin-Fc. Mass spectrometry analysis suggests that CD44 is a potential LSECtin ligand. On the basis of these results, we confirmed the interaction between LSECtin and CD44. Firstly, blocking anti-CD44 antibody inhibited LSECtin binding to activated T cells. Secondly, purified sLSECtin-Fc protein bound to CD44 transfectants and purified sCD44-Fc protein bound to LSECtin transfectants.CD44 exists in different isoforms due to alternative splicing and post-translational modifications such as O- and N-glycosylations and chondroitin and heparan sulphate chains, which contributes to the complexity and diversity of its function.We isolated... |
