We have measured the levels of glycosphingolipids and the activity of glycosphingolipid glycosyltransferases in human aortic intima and media from patients who died of atherosclerosis. The effects of lactosylceramide (LacCer) and glucosylceramide (GlcCer) from plaque intima on smooth muscle cell proliferation were assessed. When the GlcCer data was expressed as (micrograms GlcCer/mg cholesterol and/mg total phospholipid, a 28-fold and 7-fold increase in plaque intima compared to normal intima was observed. Similarly, the level of LacCer was elevated 5-fold and 4-fold, respectively, compared to unaffected intima. The activity of UDP-GlcCer: ceramide beta 1-->4 glucosyltransferase (GlcT-1) was similar in unaffected tissue, fatty streaks, and plaques. However, the activity of UDP-galactose: GlcCer, beta 1-->4 galactosyltransferase (GalT-2) activity was moderately higher in plaque than in unaffected tissue. LacCer, but not GlcCer derived from plaque intima exerted a approximately 2.8-fold increase in the proliferation of human aortic smooth muscle cells grown in tissue culture compared to control presumably due to a marked increase in LacCer molecular species containing C16:0, C22:1, and C24:0 fatty acids in plaque intima compared to control. In sum, our findings provide an interesting and novel pathogenic mechanism of lactosylceramide mediated plaque formation via stimulation of aortic smooth muscle cell proliferation.

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.

We have characterized a glycosphingolipid (GSL) receptor for Staphylococcus enterotoxin-B (SEB) in cultured human kidney proximal tubular (PT) cells. Solid-phase binding of [125I]SEB to the GSL receptor was concentration dependent and was not displaceable by two structurally related toxins, such as staphylococcal enterotoxin-A and toxic shock syndrome toxin-1. Rat kidney cells did not bind [125I]SEB. However, when the rat kidney cells were pre-incubated with digalactosylceramide, there was a concentration-dependent binding of [125I]SEB. Trimethylsilyl derivatization of methyl glycosides, followed by gas-liquid chromatography-mass spectrometry (GC-MS), revealed that galactose was the major sugar component of this putative receptor GSL. The sphingosines present in this GSL were d18:2, d22:2 and d23:0; the fatty acids present were palmitate, oleate and stearate. Permethylation of alditol acetates and GC-MS revealed two predominant sugars, namely 2, 3, 4 and 6 tetramethylgalactital and 2, 3 and 6 trimethylgalactital. The GSL receptor for SEB was sensitive to alpha-galactosidase, and resistant to beta-galactosidase and beta-glucosidase. Taken together, our studies reveal that the tentative structure of the receptor for SEB in human kidney PT cells is CerGal alpha 1-->4Gal. In summary, we have identified a GSL as one of the binding sites of SEB, a food-borne toxin. We believe that our finding may open up rational approaches for the therapy of SEB-induced glycopathology in man.