Stimulation of Glycosphingolipid Biosynthesis by L-Threo-1-Phenyl-2-Decanoylamino-1-Propanol and Its Homologs in B 16 Melanoma Cells

抄録

Previous studies have demonstrated that the ceramide analog D-Threo-1-phenyl-2-decanoylamino-3-morpholino-l-propanol (D-Threo-PDMP) inhibits glucosylceramide (GlcCer) synthase and thus leads to extensive depletion of glycosphingolipids (GSLs) biosynthesized from GlcCer [reviewed by Radin, N. S., Shayman, J. A., and Inokuchi, J. (1993) Adv. Lipid Res. 26, 183-213). In the present study, stereospecificity of PDMP activity was demonstrated with an enantiomeric pair, D-threo-PDMP and L-threo-PDMP. Treatment of B 16 melanoma cells with the D-threo or L-threo isomer produced contrasting changes of GSL biosynthesis, as monitored by metabolic labeling with [³H]Gal. D-PDMP markedly inhibited incorporation of radioactivity into GlcCer, LacCer, and GM3 as expected, whereas the L-threo isomer significantly increased it. Homologs of L-PDMP having different N-acyl chains were synthesized and also tested for their effects. Among them, the compounds having C₈-C₁₄ acyl chains increased incorporation of the radioactivity into GSLs to different degrees, demonstrating that the stimulatory effect of the L-threo homologs depends on acyl chain length. In order to elucidate the biochemical mechanisms of these PDMP effects, the activities of GlcCer synthase, LacCer synthase, and GM3 synthase in B 16 cell lysates were measured in the presence of PDMP. D-Threo-PDMP but not the L-threo isomer inhibited both LacCer and GM3 synthases as well as GleCer synthase, suggesting that the ceramide-like structure of the D-PDMP molecule interacted stereospecifically with these GSL-synthesizing enzymes. On the other hand, L-PDMP had no effect in the in vitro assays. However, LacCer synthase activity was found to be significantly elevated when the intact B 16 melanoma cells were preincubated with L-PDMP before measuring enzyme activities. Furthermore, the increase of LacCer synthase activity by L-PDMP was observed even when protein synthesis of the cells was blocked by cycloheximide, suggesting that L-PDMP affects post-translational modification of the enzyme. Thus, we were able to demonstrate distinctive and contrasting stereospecific actions of the two enantiomers on GSL biosynthesis.