The Expression And Characterization Of Recombinant Human Fc Chimeric Protein Containing Cysteine-rich (CR) And C-type Lectin Domain _4-7 (CTLD_4-7) Of Murine Mannose Receptor

The mannose receptor(MR, CD206) is a member of the MR family which is a subgroup of the C-type lectin superfamily. MR was the first MR family member to be discovered as a 175kDa endocytic receptor. MR expression is not MΦ-restricted; it is also expressed by hepatic and lymphatic endothelia and kidney mesangial cells. MR is a type-Ⅰmembrane protein with a single transmembrane domain and a cytoplasmic domain that mediates receptor internalisation and recycling. It contains three types of domains at its extracellular region, an N-terminal cysteine-rich(CR) domain capable of binding to sulphated sugar terminated in SO4-3-Gal or SO4-3/4-GalNAc, a fibronectin typeⅡ(FNⅡ) domain involved in collagen binding especially collagen typeⅠ,Ⅱ,Ⅲ,andⅣ, and eight CTLD responsible for binding to sugars terminated in mannose, fucose or N-acetyl glucosamine. MR mediates efficient clearance of endogenous molecules and could play a role in microbial recognization and antigen presentation and lymphocyte adhesion. In vitro studies have suggested that MR is abundantly present on both afferent and efferent lymphatic. Furthermore, both lymphocytes and some tumor cells bind to lymphatic tissues in an MR-dependent fashion. MR may serve as an adhesion molecule during cell migration to lymph nodes.L-selectin is a cell adhesion molecule expressed on the surface of most leukocytes and involved their rolling on inflamed vascular endothelium to promote to firm adhesion and transmigration. The study also showed that L-selectin can bind to MR expressed on efferent lymphatic sinuses, which mediates lymphocytes leaving the peripheral tissues via afferent lymphatic. It was suggested that the L-selectin positive tumor cells can interact with MR on the surface of lymphatic endothelial and contribute to tumor lymphatic metastasis.Previously studies in our lab showed that silencing the expression of L-selectin on P388D1 cells attenuated the P388D1 cell metastasis via lymphatic vessels. To understand the role of mannose receptor and L-selectin in lymphatic metastasis, the Plasmids encoding the Fc chimeric proteins containing CR or CTLD4-7 were constructed. Our preliminary data showed the CR-Fc and CTLD4-7-Fc could be expressed in COS-7 cells.Objective:This study is to optimize the condition for expression of the chimeric proteins containing CR or CTLD4-7 fused to the Fc region of human IgG (CR-Fc/CTLD4-7-Fc), to purify them from supernatants of transfected cells, to identify their interaction and to explore the role of mannose receptor and L-selectin in tumor lymphatic metastasis.Methods:Plasmids encoding the Fc chimeric proteins containing CR or CTLD4-7 were introduced into the 293T or COS-7 cell line by transient transfection with Lipofectamine 2000 following manufacturer's instruction. Immuno- cytochemical analysis and ELISA were used to detect the expression of the chimeric proteins and optimize the expression conditions. The recombinant CR-Fc chimeric protein was purified by Protein A chromatography from abundant culture supernatants and identified with SDS-PAGE and Western Blot analysis.The interaction between CR-Fc chimeric protein and L-selectin on P388D1 cells and Hepa1-6/L-selectin cells were analyzed by cell adhesion assays and flow cytometry analysis. IgG protein and Hepa1-6 cells were used as control.Results:1. The condition for more efficient expression of CR-Fc and CTLD4-7-Fc chimeric protein was optimized. CR-Fc chimeric protein was purified and exhibited as a single band with a molecular mass of 50 kDa on reducing SDS-PAGE. 2. Cell adhesion assay in vitro proved that Hepa1-6/L-selectin cells specifically attached CR-Fc chimeric protein coated at the bottom of 96 well plate, compared with Hepa1-6 cells (p<0.05).3. Flow cytometry analysis indicated that CR-Fc chimeric protein specifily recognized L-selectin on the surface of P388D1cells, compared with IgG protein (p<0.05).4. Immunofluorescence assay and Western blot analysis demonstrated that CTLD4-7-Fc chimeric protein was expressed abundantly in COS-7 cells, which provided a foundation for the future purification.Conclusions:The CR domain of mannose receptor could recognize and attach L-selectin on the cell surface, which may partly elucidate that the interaction between Mannose receptor and L-selectin is involved in the lymphatic metastasis.