The Interaction Mechanism Of CD226 With Its Ligands CD112/CD155 Molecules

Human platelet and T cell activation antigen 1 (PTA1) is a member of the immunoglobulin superfamily (IgSF) containing 2 Ig-V like domains in its extracellular region. It was designated as CD226 in the 7th Workshop and Conference on Human Leukocyte Differentiation Antigen (HLDA7) in 2000. In 1985, T lineage-specific activation antigen 1 (TLiSA1) was first discovered by a mAb LeoA1 which was prepared by immunizing mice with MLR generated activated lymphocytes. It was subsequently found TLiSA1 was involved in platelet activation and aggregation, hence renamed PTA1. In 1996, human CD226 was cloned by DNAX Research Institute designated as DNAM-1 (DNAX accessory molecule-1). The full length of CD226 cDNA has 2603bp, and its ORF encoding 336 amino acids is composed of 7 exons. There are some phosphorylation sites in its cytoplasmic region. At protein level, CD226 shows 22% homology with CD96 (Tactile) which is expressed on activated T cells and NK cells, and also shares certain homology to BGP-1, CD36, PRR, and drosophila nerve colloid protein-Neuroglian. CD226 is a 65kDaâ… type transmembrane glycoprotein expressed on cell surface of T cells, NK cells, NKT cells, monocytes/macrophages, megakaryocytes and platelets, and activated vascular endothelial cells, which is involved in a variety of immunological functions, including the differentiation of T cells and megakaryocytes, the cytotoxicity of NK cells against tumor cells and virus infected cells, the activation and aggregation of platelets, and the transmigration of monocytes/macrophages through vascular endothelial cells.In 2003, human poliovirus receptor (PVR/Necl-5/CD155) and its nectin/Necl (nectin-like) family member poliovirus receptor related 2 (PRR-2/ nectin-2/CD112) were identified as the ligands for CD226. CD112 also serves as the receptor for herpes simplex virus (HSV-1 and HSV-2). The CD226 ligands are expressed on many kinds of cells such as neuronal, epithelial, endothelial and fibroblastic cells. Interaction of CD226 with its ligands CD112 and CD155 induces intercellular adhesion, infiltration and cell signaling, participating in both innate and adaptive immunities. Until now however, the precise interaction mechanism between CD226 and its ligands is not fully understood. Although CD226, CD112 and CD155 share the similar three-dimensional structure, it is still unclear which domain(s) are involved in the activation and signal tranduction in this adhesion molecule. To explore the interaction of CD226 domain(s) with its ligands can help us to understand the function mechaniam of CD226 such as adhesion and cytotoxicity.At first, we cloned the full length of CD112 and CD155 from human colon cancer cell line SW480 by RT-PCR respectively. Primers were designed and synthesized according to the extracellular domain (ED) of CD112 and CD155 and the restriction enzyme sites in the vector. In order to obtain soluble Fc fusion protein, the ED of CD112 or CD155 was cloned into eukaryotic expression vector pCMD7 respectively, and the constructs were transfected into CHO cells. After G418 selection and limited dilution cloning, CHO cell lines which can stably secrete CD112ED-Fc and CD155ED-Fc (Fc tag in C-terminal) fusion protein were obtained, then the proteins were purified from the supernatants of the CHO cell culture by anti-Fc affinity chromatography column. Hybridomas secreting monoclonal antibodies (mAbs) to CD112 and CD155 were obtained via immunizing BALB/c mice with the purified fusion proteins and routine hybridoma technique. These mAbs can be used in the experiments of western blot, immunoprecipitation, immunohistochemistry (IHC), immunofluorescent staining, as well as functional study. Using these mAbs we investigated the expression pattern of CD155 in normal and tumor tissues, and the roles of CD112 or CD155 in anti-tumor immune responses.Furthermore, in order to overcome the shortcoming that the pCMD7 vector only has limited multiple cloning sites (MCS), pSecTag2B vector was reconstructed to express the fusion protein which contains 3C enzyme recognision site and C-terminal Fc tag. Using this vector, we obtained the CHO cell lines which can stably express CD226D1-Fc and CD226D2-Fc fusion proteins, with the human Fc tag at C terminal of the first or the second domain of CD226 extracellular region respectively. We analyzed the interaction domain(s) of CD226 and its ligands CD112 and CD155 with these fusion proteins. Moreover, the domain(s) of CD226 molecule participating in the NK cells killing to tumor cells and human PBMC adhesion to endothelial cells were identified.Using the model of soluble fusion proteins binding to coated CD226 molecule, we found that full length CD226 have much stronger ability to bind CD112 and CD155, while CD226D1 and D2 truncates only have low binding ability. In the flow-cytometric NK-cytotoxicity assay using CFSE/PI labeling, the presence of fusion protein CD226-Fc, CD112-Fc and CD155-Fc could inhibit the NK killing against K562 target cells obviously, and FMU-CD112.8 and FMU-CD155.12 mAbs could inhibit this killing activity to some extent, whereas CD226D1-Fc and CD226D2-Fc did not show any inhibitory effect on the cytotoxicity. These results demonstrated that the interactions of CD226-CD112 and CD226-CD155 both participate in the process of NK cell recognizing and killing target cells. In the experiment of fluorescein labeling PBMC adhere to endothelial cell line ECV304, we found the presence of full length CD226 could inhibit the adhesion between PBMC and ECV304 markedly, while CD226D1 and CD226D2 almost could not inhibit the adhesion.Finally, we constructed different vectors used for observation and analysis of the interaction by laser scanning confocal microscope (LSCM), which included all kinds of truncates of CD226 ED, chimeras of CD226/ICAM-1 fused to GFP, and CD112 or CD155 fused to RFP. We found that the chimeras which only have the first domain of CD226 (C-terminal linked to the fifth domain, transmembrane domain and cytoplamic region of ICAM-1) or the second domain of CD226 (N-terminal linked to the first domain of ICAM-1) could not bind CD112 or CD155 on the cell membrane effectively. Whereas the full length molecule containing CD226 D1 and D2 domains binded CD112 or CD155 effectively, and triggering polarization, aggregation, and capping of the receptor and its ligands were observed.In conclusion, the integrity of two Ig-V like domains of CD226 in extracellular region is necessary for its binding to its ligands which further mediated cell-cell adhesion, signal transduction and NK cell cytotoxicity. These results lay an important foundation for the further exploration of the molecular mechanism of activating receptor CD226 in the multiple immune responses.