Dynamic Redistribution Of "Don’t Eat Me" Signal CD47during Epithelial Cell Apopotosis

Apoptosis is one of the most important mechanisms during body’s natural processes in metazoans. To eliminate the apoptotic cells timely and accurately. On one hand, the apoptotic cells exhibit some special molecules as "eat-me" signals, which can interact with the molecules and counter-receptors present on phagocytes surface; on the other hand, apoptotic cells lose in exhibiting the functional "don’t-eat-me" signals normally present on healthy cells to the phagocytes according to the fated apoptosis programme.CD47, the ligand of SIRPα on another cell. Apart from the diverse function studies of CD47-SIRPα interaction, the cell-surface CD47on viable cells and its engagement of SIRPa on phagocyte, has been characterized as a key don’t-eat-me signal.However, the CD47-SIRPα interaction is disabled when cells undergo apoptosis. The detailed molecular mechanism(s) of such don’t eat me signals inactivating, especially in epithelial cells remain unknown. The objective of this study was to investigate the changes happened to CD47that disabled CD47-SIRPα don’t eat me signal during epithelial cells apoptosis.Using immunofluorescence and Flow cytometry, we find the expression level of CD47on epithelial lines HT-29, T-47D and MCF-7is not decreased during apoptosis, but the cells could not adhere to SIRPa coated wells as viable ones. Biochemistry assay:in healthy epithelial cells CD47locate in some kind(s) of macrodomain complex on the cell plasma but diffuse to other part of the plasma membrane during apoptosis.With different methods, we found that the complex most of CD47locate in was not lipid rafts or proteins polymer. However, according to our recent progress, CD47may interact with some other proteins indirectly through its glycans to form this kind of complex which probably to be CD47-lectin lattice. As a results when these aggregated CD47interact with SIRP a on another phagocyte, they will show a strong "don’t eat me" signal which can inhibit phagocyte swallow healthy cell. Once apoptosis happens, the complex disassemble. In this case, when a phagocye meet the apoptotic cell, there would not be a threshold "don’t eat me" signal that can inhibit phagocytosis and the apoptotic cell would be phagocytosed.In our study, we used PNGase F and NaIO4to cut or destroy the polysaccharide on CD47. This led to the diffusion of CD47on the cell surface, and the treated cell failed to adhere to SIRP α-Ex coated wells. Further, we constructed CD47mutants which with one or more N-glycans deletion, and some of these CD47mutants showed a diffused CD47expression on plasma membrane of transfected CaCO2cells, which just liked apoptotic ones. And we will continue to assay the function of these CD47mutants and try to prove this model on epithelial cells.