Structure of the ceramide moiety of GM1 ganglioside determines its occurrence in different detergent-resistant membrane domains in HL-60 cells

To investigate the effect of the ceramide moiety of GM1 ganglioside on its association with detergent resistant membrane domains (DRMs) in human leukemia HL-60 cells, [H-3] labeled GM1 molecular species (GM1s) with ceramides consisting of C18 sphingosine acetylated or acylated with C-8, C-12, C-14, C-16, C-18, C-22, C-24, C-18:(1), C-22:1, or C-24:1 fatty acids (FAs), or C20 sphingosine acetylated or acylated with C-8 or C-18 FA were prepared and added to culture media. GM1s uptake by HL-60 cells was affected by the structure of their ceramides. Resistance to removal with trypsin and the stoichiometry of [I-125] cholera toxin (CT) binding indicated that the added GM1s were incorporated into the membranes of the cells used for the isolation of DRMs in a manner resembling endogenous gangliosides. The ceramide moieties of the GM1s determined their occurrence in DRMs and the dependence of their recovery in this membrane fraction on the amount of Triton X-100 (TX) used for extraction as well as on cholesterol depletion. The GM1s with sphingosine acylated with C-14, C-16, C-18 C-22, or C-24 FAs were similarly abundant in DRMs. GMls acylated with C18:1, C22:1, or C24:1 were less abundant than those acylated with saturated FA of the same length. GMls acetylated or acylated with C-8 FA were detected in DRMs in the lowest proportion. Depletion of 73% of cell cholesterol with methyl-beta-cyclodextrin significantly affected the recovery in DRMs of GMls acetylated or acylated with C-8 or unsaturated FAs but not of GM1 acylated with Q(18), C-22, or C-24 FAs. After cross-linking with CT B subunit, all GMls were recovered in DRMs in a similarly high proportion irrespective of their ceramide structure or cholesterol depletion. DRMs prepared with low TX concentration at the TX/cell protein ratio of 0.3:1 were separated by multistep sucrose density gradient centrifugation into two fractions. The GMls with sphingosine acetylated or acylated with C-18 or C-18:1 FAs occurred in these fractions in different proportions.