Effects of D‐threo‐PDMP, an inhibitor of glucosylceramide synthetase, on expression of cell surface glycolipid antigen and binding to adhesive proteins by B16 melanoma cells

Abstract

Incubating B16 melanoma cells with an inhibitor of glucosylceramide (GlcCer) synthetase, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-threo-PDMP), led to a considerable decrease in the levels of GlcCer and lactosylceramide (LacCer). The content of ganglioside GM3 was little affected, but the ability to bind a monoclonal antibody against the ganglioside (M2590) was greatly reduced, suggesting that the reduction in the simple glycolipids led to encryption of the membrane antigen. This interpretation is supported by the observation that permeabilization of the treated cells with Triton X-100 restored immunological reactivity. Specificity of the PDMP effect was shown by its lack of effect on the reactivity of two other surface antigens to anti-melanoma monoclonal antibodies M562 and M622, and of the major histocompatibility antigens to anti-H-2KbDb monoclonal antibody. The ability of the treated cells to attach to laminin or type IV collagen was lost but that to fibronectin was not. The effects of the enzyme inhibitor were counteracted by including GlcCer in the culture medium. This indicates that the lipid was absorbed by the cells and utilized like endogenously-formed GlcCer. Cells preattached to laminin or collagen could be induced to round up by addition of inhibitor. In contrast, L-threo-PDMP (which does not block the synthesis of GlcCer) had no effect on the immunologic reactivity of GM3 or the adhesion properties of the cells. However, it did produce considerable accumulation of LacCer. These data suggest that the simple glycolipid, GlcCer, is an essential factor for antigenic expression of the more complex glycolipids on cell surfaces and that there is a close association and interaction between glycolipids and adhesive receptors on the cell surface.

Reduction of Glycosphingolipid Levels in Lipid Rafts Affects the Expression State and Function of Glycosylphosphatidylinositol-anchored Proteins but Does Not Impair Signal Transduction via the T Cell Receptor

“…With regard to the concentration of D-PDMP, we were able to inhibit GM1 expression in Jurkat cells at a concentration of 10 M without detriment to cell viability and cell growth (data not shown). As a control for D-PDMP, we used L-PDMP, the stereoisomer of D-PDMP, which has been shown to be inactive in inhibiting GlcCer synthase (27). As previously reported, we did not observe any reduction in GM1 expression by L-PDMP treatment (Fig.…”

Ganglioside GM3 Participates in the Pathological Conditions of Insulin Resistance

“…Ganglioside Analysis-The total lipids were extracted from the cells with chloroform/methanol/water (4:4:0.3, 2:4:0.3, v/v/v), successively, and purified to obtain acidic glycolipid fraction as described (18). Ganglioside composition was determined by TLC plate with chloroform/ methanol/water (65:25:4).…”

Insulin Resistance as a Membrane Microdomain Disorder

“…One approach to depleting cellular gangliosides is the use of inhibitor of glucosylceramide synthase, D-PDMP, a wellknown tool for studying various functional roles of endogenous glycosphingolipids including gangliosides. 12,16) D-PDMP lowered GM3 content in 3T3-L1 adipocytes treated without or with TNFa (Fig. 1B), with a concomitant increase of tyrosine phosphorylation of IRS-1 in response to insulin stimulation (Fig.…”