The Mechanisms Of Soluble CD155 Production

Human CD155 was initially found as poliovirus receptor (PVR) in 1989, which could mediate the poliovirus infection, subsequently inducing poliomyelitis. CD155, a member of immunoglobulin superfamily (IgSF), belongs to nectin family of cell adhesion molecules, and plays important roles in cell adhesion, formation of adhesion junctions (AJs), and tumor cells migration. Because of the differences between CD155 and nectins, CD155 was designated as Nectin like molecule-5 (Necl-5) in 2003. This molecule is also important for the development of the CNS during embryogenesis. CD155 is broadly distributed on normal cells in many tissues at a relatively low level, while over-expressed on certain human tumor cells. Recently, CD155 was identified as the ligand of CD226, and the interaction of CD226 with CD155 was involved in the tumor cells lysis by NK cells which attracted more and more attention. Since CD155 is an adhesion molecule and immune function-regulating membrane molecule, the soluble form of CD155 (sCD155) may regulate functions in PV infection, cell adhesion, and immunological surveillance. Previous studies showed that many soluble adhesion molecules are related to the onset and development of certain diseases, and soluble forms are released into the extracellular space by the way of direct secreting from cells or proteolytic cleavage of membrane molecules by proteases. However, the production mechanism and function of sCD155 are still not well known.Firstly, we detected the distribution of CD155 on various human cell lines with indirect immunofluorescence staining and FCM analysis. The results indicated that CD155 was expressed on T cell lines and megakaryocytic cell lines but not B cell lines, NK cell lines, and monocytic cell lines. Particularly, CD155 was over-expressed on epithelial cell lines, endothelial cell lines, tumor cell lines derived form epithelial cells, glioma and melanoma cell lines. In order to investigate sCD155 levels in human sera or cell culture supernatants, 10 strains of monoclonal antibodies (mAb) to the ectodomain of CD155 were used for CD155 epitope determination and establishment of ELISA kit detecting sCD155. There were three epitope groups on the ectodomain of CD155 confirmed by the competitive ELISA. A sandwich ELISA for detecting sCD155 was established by using mAb FMU-CD155.2 as coating antibody and HRP-conjugated FMU-CD155.10 as detection antibody. This sandwich ELISA is reproducible with a detection limit of approximately 200ng/L rhCD155-Fc. sCD155 levels in sera of patients with certain tumors such as leukemia, lymphoma, pancreatic tumor, mammary tumor and pulmonary tumor were lower than that of healthy subjects, whereas sCD155 levels in sera from the patients with other tumors (stomach tumor, colorectal tumor, ovary tumor, and bone tumor) were not different.Finally, we investigated the mechanisms of sCD155 production using a model of CD155-expressing cell culture treated with protease inhibitors. K562 cells, which express a certain level of CD155 were stimulated by PMA for indicated time periods with or without serine protease inhibitor AEBSF and cysteine protease inhibitor NEM. The positive percentage of mCD155 on resting K562 cells was 15.7%, while the expression of mCD155 rised to 96.1% and 54.1% by adding of 5mM AEBSF and 0.2mM NEM respectively. Moreover, the inhibitors, AEBSF and NEM, regulated the mCD155 expression on resting K562 cells in a dose and time dependent pattern. The levels of mCD155 and sCD155 expression were both enhanced in PMA stimulated K562 cells. PMA plus AEBSF has more significant effect on upregulation of mCD155 expression than the effect by PMA plus NEM . Meanwhile, the sCD155 level in the supernatant of both PMA plus AEBSF-treated and PMA plus NEM-treated K562 cell culture decreased compared to the level of negative control groups. The other protease inhibitors had no effects on mCD155 expression and sCD155 secretion.In conclusion, we firstly confirmed that serine protease and cysteine protease are responsible for the proteolysis by which the soluble CD155 molecules are shedded from membrane bound CD155. The established sandwich ELISA kit for detecting sCD155 would be a useful tool for further investigation on the production mechanism and function of sCD155.