Functional analysis of glycosphingolipids by molecular biological approaches

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1. The promoter analysis of ceramide glucosyltransferaseIn several cell lines, ceramide are believed to serves as second messenger. In the case of stress or DNA damage, intracellular ceramide level elevates and induces apoptosis. In the other hand, ceramide glucosyltransferase, GlcT-1 inactivates ceramide and inhibits apoptosis. Thus, we constructed the chimera gene of the promoter and luciferase and introduced into several animal cell lines. Using these cell lines we examined a variety of genotoxic agents. In B16 cells 2μM camptothcine increase the activity twice and 10μM etoposide increased 3,2 times. We also examined 40 kinds of plant extracts and identified two kinds of plant extract that suppress the promoter activity.2. The functional analysis of GlcT-1 molecule by genetic engineeringGlcT-1 localizes in the Golgi membrane as well as its substrate ceramide. The enzyme is believed to binds to the membrane by the signal anchor sequence. To prove this hypothesis, we constructed cDNA lacking the sequence and introduced it to GlcT-1 deficient GM95 cells. The GlcT-1 lacking the signal anchor sequence did not show the activity, indicating the importance of the sequence. Localization of the mutant enzyme is now under examination.3. Expression cloning of proteins which inhibit apoptosisWe tried to isolate genes that can inhibit ceramide induced apotosis using retroviral-mediate expression cloning. In the course of the study, we happened to isolate genes that inhibit hydrogen peroxide induced apoptosis. We infected mouse 7-day cDNA library to NIH/3T3 cells and selected with hydrogen peroxide. The cDNAs were recovered by PCR from the genomic DNA of the survived cells. We have isolated 11 genes and two were functionally unknown.