Analysis The Structure Of Phospholipids In The Leaflet Of Lipid Raft And Non-raft Plasma Membrane

Objective: Lipid rafts are microdomains with unique structure and functions on the plasma membrane, which are riched in sphingolipids, cholesterol and GPI-anchored proteins. The functions of lipid rafts are involved in all the fields of life scicence. Especially in recent years, lipid rafts are considered as working platforms of signal transmembrane conduction, and has being attracting more and more attention. The molecular mechanism of rafts participating in cell transmembrane signal transduction is being important topics in molecular membrane biology and cell signal transduction areas. The structure is the basis of function. To understand the function of lipid rafts, the first thing to be done is making clear the structure of lipid rafts. Now, the strcture of outer leaflet of lipid rafts has been well understood. The sphingolipids, cholesterol and GPI-anchord proteins all are structural components of outer leaflet of lipid rafts, which is the structural basis for formation of lipid rafts. But, the strcture of inner leaflet of lipid rafts has not been well studied. A lot of signal moleculars (including lipids, acylated protein and membrane protein, etc.) cluster in the inner leaflet of lipid rafts, which is the structural basis for lipid rafts to exert its functions. What is the structural basis of signal transduction mediated by lipid rafts? How do these signal moleculars location on the rafts? What is the structural basis? What is the relationship between the inner and outer leaflet of lipid rafts? These all need to be studied furtherly.Methods: HcaF cells were cultured in the abdominal cavity of mice. After being harvested, the cells were homogenized with Teflon homogenizer, and then the plasma membrane was preparated with percoll gradient ultracentrifugation and lysed in 1%Tritonx-100. The lipid raft and non-raft plasma membrane were separated by sucrose gradient ultracentrifugation and identified by dot-blot with Cholera toxin B subunit (CTB). The total phospholipids in lipid raft and non-raft plasma membrane were extracted with Floch method and individual phospholipid was separated by high-performance thin-layer chromatography (HPTLC). Fatty acids in phospholipids were methyl esterated with 14% MeOH-BF3, and then the fatty acid methyl esters were extracted with petroleum ether. Fatty acid compositions were analyzed by gas chromatography.Results: Our results indicate that the major lipids in the lipid raft are phosphatidylcholine (PC), sphingomyelin (SM), phosphatidylethanolamine (PE) and phosphatidylserine (PS) and that in non-raft plasma membrane are PC and PE .It suggested that SM and PS mainly distributed in the lipid raft. Comparing the fatty acid compositions between the lipid raft and non-raft plasma membrane, we found that the fatty acid compositions of PE in the lipid raft are significantly different from that of non-raft plasma membrane: PE in the lipid raft contains more short and middle length fatty acyl side chain(C16,C18) whereas contains longer fatty acyl side chain (C18,C20,>C22) in the non-raft plasma membrane. The fatty acid compositions of PS are similar to that of PE in the lipid raft.As we known, the distribution of phospholipids on plasma membrane is asymmetric, PC and SM mainly distributed in the outer leaflet of membrane whereas PE and PS are most found in the inner leaflet. According to the distribution character of phospholipids and the results we obtained, we speculate that the phospholipids in outer leaflet of the lipid raft are mainly PC and SM, whereas that of non-raft plasma membrane are only PC; The phospholipids in the inner leaflet of the lipid raft are mainly PE and PS, whereas that of non-raft plasma membrane are only PE. To sum up, the major function of SM and PS in the plasma membrane is to refer to participate in the formation of lipid raft: SM distributes in the outer leaflet of lipid raft whereas PS mainly distributes in the inner leaflet of lipid raft. It is known that SM contains a long hydrocarbon chain (C24) in its ceramide part and like the phospholipids with asymmetric Acyl-chain. Phospholipids with asymmetric Acyl-chain often form complete cross-or part-cross lipid bilayers. SM mainly distributes in the outer leaflet of lipid raft. Thus, this requires the phospholipids in inner leaflet to be complementary at fatty acyl chain structure, or contain more asymmetric acyl chain or short length fatty acyl chain.Our results shown that PE in lipid raft contains more short chain fatty acids and the structure of fatty acids of PS is similar to that of PE in lipid raft. The structural characteristics maybe the immportent factors that promote these phospholipids to distribute in the lipid raft. It is known that PS is the activator of PKC and some trace phospholipids, such as inositophospholipids is enriched in lipid raft. The formation of lipid raft leads to these lipids involved in cell signaling to cluster in the inner leaflet of raft, witch form a microenvironment that is adapt to cell signal transduction. This may be one of the molecular mechanisms by witch lipid raft exerts its functions.