Glucide And Lipid Recognition Mechanism Of LIP On Lipid Raft Of Tumor Cells

As a Cyclostomata animal,Lampreys are primitive jawless vertebrates,which located between invertebrates and vertebrates.They provide a wealth of genetic information about the origin and evolution of organisms.Previous studies have shown that Lamprey immune protein(LIP),which is isolated and purified from the serum of the jawless vertebrate,is a kind of protein who can electively kill the tumor cells.This paper focuses on the binding target identification of LIP in tumor cellsIn this study,we use immunofluorescence methods combined with(DeltaVision OMX)ultra-high resolution microscope,confocal microscope and other auxiliary equipmentse to interaction of recombined LIP with various types of molecules on the cell membrane were observed.It is inferred that the bingding target of tumor cells by LIP is located on the outside of the lipid raft.When the specific phosphatidylinositol-phospholipase C(PI-PLC)preincubated the cancer cells which could get rid of the GPI-APs of cell surfaces,it was found LIP could not conbind with the lipid raft anymore and inhibited the cytotoxic activity of LIP effectively at the same time.Sucrose gradient fractionation and other methods are used to enriching GPI-APs by Triton X-114 phase separation.PI-PLC and aqueous hydrogen fluoride treatment has been previously applied for isolating GPI-anchor from cell membrane.But after analyzing the structure,there is no difference between tumor cell and erythrocyte on GPI-anchor.So the bingding target of LIP to tumor cell is not GPI-anchor.100 N-Glycan miccoarray is used as a general test to help us determine binding characteristics of LIP to an array of 100 fundamental N-glycans.The result reveals that LIP are especially coated to be capable of immobilizing natural N-glycans with close-ring structure at their reducing end Neu5Gc,then we identified a glycan from GlycomeDB tumor glycan database which similar to the miccoarray.It is proved that LIP can be combined with N-Glycan chain containing Neu5Gc on tumor surface.Through the analysis of high-resolution liquid mass spectrometry,it was found that most of the components of LIP bound tumor were located in the high organic phase region,which may be the phospholipids on the surface of the lipid raft.After treated with SCDase,we found that the MCF-7 cells were no longer sensitive to LIP.It was also proved by MALDI-TOF-MS that both PI-PLC and SCDase could hydrolyze the sphingomyelin on the outside of the lipid raft,while phosphatidylcholine(PC)can not hydrolyzed by SCDase.So we can tell LIP can also bind to the sphingomyelin of lipid raft.We also found that cytotoxicity of LIP on MCF-7 cells was significantly lower than that of sphingomyelin on the surface of hydrolyzed cells.Therefore,we detect the relationship between sphingomyelin and LIP.By detecting,we found that the sphingomyelin numbers found on tumor cells was significantly higher than that of normal cells,then we use ORBITRAP to make a Lipidomic analyasis between MCF-7 cells and leukocyte.The result reveals there are no difference of sphingomyelin species between two cell lines.So this recognition of lip is related to the content of sphingomyelin in the lipid raft,but not to the species of sphingomyelin.Based on the experimental results,the Neu5Gc on N-glycan of GPI-APs and sphingomyelin on lipid raft are the recognition site of LIP,and the LIP could be positioned on the lipid raft,forming a membrane attack complex on the cell surface,pore-forming,eventually leading to the cell death.Neu5Gc and sphingomyelin are indispensable when LIP performs killer functions.The recognition mechanism will help us to better develop the potential of Neu5Gc and sphingomyelin in tumor recognition,killing and drug development,and provides a new idea for the research of anti-tumor.