摘要：Our recent study showed the activation of ceramide (Cer) glycosylation and Cer-related genes in the mouse fibroblast under heat stress. We investigated whether the Cer level was affected or not when the Cer galactosylation was introduced into a fibroblast cell line Mop8 by transfection of Cer galactosyltransferase (GalT, alternatively called UGT8) gene. When cells were labeled with 14C-galactose, the parental Mop8 cells showed only glucosylceramide (GlcCer) in the monohexosylceramide region on 2D-high-performance thin layer chromatography (HPTLC), whereas the transfectant cells (namely MopGT) expressed a spot corresponding to GalCer in addition to GlcCer. MopGT cells showed a significant decrease in intracellular Cer. These results suggest contribution of Cer glycosylation to the cellular regulation of Cer, which is known as a pro-apoptotic factor. Incidentally, another novel spot (which we referred to as GTX) moving more slowly than GalCer was observed in MopGT by the metabolic labeling. Comparison with the previous report that, in CHO cells transfected with the brain GalT gene, galactosyl-alkylacyl glycerol was stably expressed in addition to GalCer and was easily hydrolyzed with alkaline treatment to produce galactosyl-alkyl glycerol as the lyso form [van der Bijl P et al. (1996) Biochem J 317: 589-597] suggested that our GTX corresponded to galactosyl-alkyl glycerol. Indeed, when GTX prepared by the HPTLC was analyzed by mass spectrometry (MS), m/z 501, 527 and 529 were predominantly observed as [M+Na]+, which were consistent with monogalactosyl-monoalkyl glycerol containing an alkyl chain of 16:0, 18:1 and 18:0, respectively. Galactosyl-alkylacyl glycerol (namely, GTXorg) was also prepared by preparative TLC without alkaline treatment. MS spectrum of GTXorg showed molecular ions corresponding to 16:0 alkyl and 16:0 acyl chains, 18:1 alkyl and 16:0 acyl chains, and 18:0 alkyl and 16:0 acyl chains, respectively. These findings support the view that substrate recognition of the ceramide-binding site of GalT is not very specific, accepting glycerolipids as substrates.
关键词：ceramide (Cer); galactosyl-alkylacyl glycerol; galactosylceramide (GalCer); galactosyltransferase (GalT) gene; collision-induced dissociation (CID); matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS)
