Expression And Functional Research Of Activating Receptor CD226on NK Cells

Innate immune system plays an important role within the skin as the second barrier defending pathogen invasion of the organism. As everyone knows, NK cell is the mainly effective cell in the innate immune. The functions of the NK cells include killing the tumor cell, killing the cells infected by the virus, bacteria or fungus.NK cells playing the role are mainly relying on the activated receptor or inhibitory receptors on its surface. The activated receptor mediating the NK cell cytotoxicity to the target cells, includes NKG2D, NKp30, NKp44CD226and so on. The inhibitory receptors such as CD94/NKG2A, KIR-L and TIGIT can inhibit the activation of the NK cells.In this study, we explored the function and recognition mechanisms of the CD226receptor. CD226can induce the adhesion and cytotoxicity of the NK cells to the target cell. It has been suggested that the CD226signal can induce the aggregation of the LFA-1to form the immune synapse. CD226is the type I transmembrane protein belonging to the immunoglobulin superfamily (IgSF). There are two immunoglobulin-like domains in its extracellular segment. The known ligands of the CD226are CD155and CD112molecules, both of them belonging to the type I transmembrane protein. CD112is also called nectin-2, belonging to the Nectin familiy. CD155is a poliovirus receptor (PVR). Both of the CD155and CD112is adhesion molecular. The mechanism of the CD226recognition to its ligands is not known. In this study, we constructed the vectors containing the two CD226extracellular domain and containing only the N terminal extracellular domain. We expressed the6His tag fusion protein in E.coli and got the two proteins. We purified the protein using Ni sepharose and identified its functions.The major results in our study were summarized as follows:1. Purified the CD226ECD1and CD226ECD proteins.We predicted the structures of the CD226protein. According to the results, we selected the19aa-129aa as the ECD1segment, and the19aa-243aa as the ECD segment. We constructed the vectors containing the ECD1and containing ECD. We expressed the6His tag fusion protein in E.coli and got the two proteins. Both of the proteins are inclusion bodies. We denatured and renatured the two inclusion bodies to get the soluble protein. And purify the protein using Ni sepharose. At last, we identified the two proteins via LC-MS and Western blot and got the protein at purity above95%.2. The function of the CD226ECD1and CD226ECD proteins.To identify whether there the two segments would be differences in their functions. We detected the protein-protein interaction via SPR, protein-cell interaction via FACS. The results showed that both of the ECD1and ECD proteins can bind to the CD155protein in the SPR experiments and both of the ECD1and ECD protein can bind to the K562and Hela cells with the CD226ligands. Then, we compared the blocking functions in cell-cell conjunction and immune synapse formation of the two proteins. The results showed that both of the two proteins can block the conjunction and immune synapse formation between the NK cells and target cells. Both of the two proteins can inhibit the target cell inducing NK cell activation, and activated signal (ERK) traducing in the NK cells3. Soluble CD226protein inhibit proliferation of the solid tumor cell and migration of the leukemic cells.In previous study, the researcher discovered that there is a level of soluble CD226in normal person serum. And the expression level of the soluble CD226usually up-regulated in the cancer patient’s serum. To confirm the function of this protein, we selected the19aa-248aa segments as the soluble extracellular CD226segments. We constructed the segments to the vectors and expressed soluble fusion proteins with6His and MBP tags on its N terminal in E.coli. We removed the6His tag and MBP tags using TEV protease. Then, purify the protein via Ni-sepharose and molecular sieve to get the protein solution at a purity above95%.We incubated the protein with the tumor cells for72h and counted the number of the tumor cells. The result suggested that compared with the BSA and blank control group, the cell number in the soluble CD226group was reduced dramatically. The reduction affection is dose dependent. We discovered the cell cycle was delayed via CFSE analysis. The phosphorylation level of Racl/Cdc42increased showed the cell migration inhibition. And, the phosphorylation level of Cyclin D1increased showed the cell was delayed in GO phase. The36h scratch test showed that the migration of the Hela cells was inhibited.Above all, CD226can play its role via its N terminal first extracellular domain. The CD226extracellular segments may be digested from the cell surface. The soluble CD226may play a role in inhibit the proliferation of the tumor cells.