Studies of the action of ceramide-like substances (d- andl-PDMP) on sphingolipid glycosyltransferases and purified lactosylceramide synthase

Abstract

We have studied the effects of D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP) and its L-enantiomer on glycosphingolipids in cultured normal human kidney proximal tubular cells. We found that D-PDMP exerted a concentration-dependent reduction in the metabolic labelling and cellular levels of glucosylceramide (GlcCer), lactosylceramide (LacCer), and the globo-series glycosphingolipids, GbOSe3Cer and GbOse4Cer. It also directly inhibited the activity of UDP-glucose:ceramide beta 1--> 4-glucosyltransferase (GlcT-1) and UDP-galactose: GlcCer beta 1-->4 galactosyltransferase (GalT-2). In contrast, L-PDMP had opposite effects on the metabolic labelling of GlcCer, LacCer, and GbOse3Cer. The levels of GlcCer and LacCer were increased, while the labelling and level of GbOse4Cer were strongly reduced. Purified GalT-2 from human kidney was inhibited by D-PDMP and stimulated by L-PDMP. It appears likely that the different glycosphingolipid glycosyltransferases possess similar binding sites for the ceramide moiety, which are blocked by binding to D-PDMP and, in the case of GbOse4Cer synthase, by L-PDMP as well. The stimulatory effects of L-PDMP on GlcCer and LacCer synthases may be the result of binding to a modulatory site on the glycosyltransferases; in intact cells, the enzyme-analog complex may afford protection against the normal catabolic inactivation of the enzymes.

Novel Role of Lactosylceramide in Vascular Endothelial Growth Factor–Mediated Angiogenesis in Human Endothelial Cells

“…Because VEGF induced LacCer synthase activity, we first used D-PDMP, initially shown to be an inhibitor of GlcCer synthase 31 but later proven to be an inhibitor of purified LacCer synthase. 28,32 Our studies provided evidence that VEGF-induced LacCer/GlcCer synthesis, PECAM-1 gene/protein expression, and angiogenesis was inhibited by D-PDMP in a dose-dependent fashion. Moreover, this inhibitory effect was by passed by LacCer but not GlcCer, suggesting that VEGF targets the LacCer synthase to induce angiogenesis.…”

“…Because L-PDMP is a potent activator of LacCer synthase, 16,28 we examined whether this compound may alter PECAM-1 expression and angiogenesis. L-PDMP significantly induced PECAM-1 expression (supplemental Figure IIA) and angiogenesis (supplemental Figure IIB).…”

Lactosylceramide Mediates Shear-Induced Endothelial Superoxide Production and Intercellular Adhesion Molecule-1 Expression

“…D -PDMP was initially suggested to be a selective inhibitor of GlcT-1 with an IC 50 of ∼10 µ M [23], but subsequent studies revealed that it also inhibits GalT-2 (with equal potency), two of the globo-series synthases [15], and the sialyltransferase that converts LacCer to ganglioside GM3 [24]. Treatment of NIH 3T3 fibroblasts or kidney proximal tubular cells with D -PDMP reduced the cellular levels of GlcCer and LacCer and increased the levels of Cer in a concentration- and time-dependent fashion [15, 25]. The functional groups comprising D -PDMP (the acyl group in amide linkage, the phenyl group, and the morpholine) have provided sites for substitutions when attempting to design more selective GlcT-1 inhibitors.…”

“…NAC, gelatin, lucigenin, GlcCer, dimethylsulfoxide (DMSO) and phosphate-buffered saline (PBS) were from Sigma Chemical Company. D -PDMP, an inhibitor of GlcT-1 and GalT-2 activities [15, 16], was purchased from Matreya, Inc.…”

“…Exogenous LacCer was found to activate the plasma-membrane-associated NADPH oxidase [14]. HUVEC incubation with D -1-phenyl-2-decanoylamino-3-morpholino-1-propanol ( D -PDMP), an inhibitor of the GSL glycosyltransferases GlcT-1 and GalT-2 [15], blocked the TNF-α-induced ICAM-1 expression [14]. In sum, LacCer activated NADPH oxidase and the generated O 2 – · mediated the TNF-α-induced ICAM-1 expression [14].…”