Stimulation of Glycosphingolipid Biosynthesis by L-Threo-1-Phenyl-2-Decanoylamino-1-Propanol and Its Homologs in B16 Melanoma Cells

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Abstract
Previous studies have demonstrated that the ceramide analog D-threo-1-phenyl-2-decano-ylamino-3-morpholino-1-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 B16 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 B16 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 GlcCer synthase, suggesting that the ceramide-like structure of the d-PDMP molecule interacted stereospecifically with these GSL-synthe-sizing 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 B16 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.

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