| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2003: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2002: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Research Abstract |
Lipid rafts are highly enriched in cholesterol and sphingolipids (glycosphingolipids (GSLs) and sphingomyelin (SM)). Among these lipid components, cholesterol has a critical function for maintaining raft structure. Cholesterol depletion from cell membranes using drugs, such as methyl-13-cyclodextrin (MβCD), generally results in disruption of raft-mediated cellular functions. Thus, despite some concerns about side effects of the drug on signaling events, this experimental approach has been widely used to verify the importance of cholesterol in raft organization and function.
In contrast to many studies manipulating the cholesterol level in lipid rafts, reports that address a role for sphingolipids in raft function are scarce. A previous study demonstrated that reduction of SM levels in Chinese hamster ovary (CHO) mutant cells induced decreased distribution of cholesterol in lipid rafts. Moreover, it was reported that addition of exogenous gangliosides to the culture of Madin-Darby canine
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kidney (MDCK) cells or CHO-K1 cells displaced glycosylphosphatidylinositol (GPI)-anchored proteins from lipid microdomains. All these studies suggested that changes in sphingolipid contents alter raft lipid and protein constituents. However, none of these studies contributed to our understanding of any functional change in membrane rafts by the manipulation of sphingolipid levels. Although an effect of GSL deficiency on functional raft formation has been suggested, whether sphingolipids are essential for raft-mediated signal transduction remains to be addressed. In the present study, we investig ated a role for sphingolipids in raft organization and function in lymphocytes, focusing particularly on a role for GSLs by the usage of a specific GSL synthesis inhibitor, D-PDMP. We demonstrated that the expression state of GPI-anchored proteins is affected by reduction of GSL levels. Further, our results showed that a reduced level of GSLs in membrane rafts modulates signaling through GPI-anchored proteins, but not through the TCR. By comparing the effect of D-PDMP with that of MMCD, we also identified that GSL and cholesterol depletion have distinctly different influences on GPI-anchored proteins in lipid rafts. Less
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