Synthesis of a novel photoactivatable glucosylceramide cross-linker

ATP-binding cassette transporters mediate differential biosynthesis of glycosphingolipid species

“…For identification of potential GlcCer flippases, GlcCer-based photoprobe stereoisomers XLA and XLB (see supplemental Fig. S1 in supplemental data for structure) were cross-linked in DU-145 cell unfractionated microsomes ± glycolipid transfer protein (GLTP) for cross-linker delivery and insertion into the microsomal membranes as previously ( 61 ). XLA and XLB were designed to mimic native GlcCer by maintaining the acyl group via native GlcCer fatty acid substitution with D, L-2-aminohexadecanoic acid, resulting in two diastereomers with different conformations at the stereogenic center circled in supplemental Fig.…”

“…As in ( 61 ), DU-145 cells detached by 0.05% trypsin/EDTA were collected in equal volume of FBS to inhibit trypsin. Cells were washed twice with ice-cold D-PBS (360 g , 4°C), and cell pellets were stored at –80°C.…”

“…To identify putative GlcCer flippases, XLA and XLB were delivered by GLTP and cross-linked to DU-145 unfractionated microsomal proteins as in ( 61 ). In dark conditions, 0.1 μg XLA or XLB was dried and suspended in water via sonication and stored at –20°C until use.…”

“…To identify putative GlcCer flippases, XLA and XLB were delivered by GLTP and cross-linked to DU-145 unfractionated microsomal proteins as in ( 61 ). Samples were solubilized with 1X radioimmunoprecipitation assay (RIPA) buffer (1X TBS, 1 mM EDTA, 1 mM EGTA, 0.1% SDS, 1% Triton X-100, 0.5% sodium deoxycholate) and centrifuged at 10,000 g for 10 min at room temperature.…”

“…Defining such flippases would close a major gap in understanding GSL biosynthesis, perhaps providing new opportunities for more selective drug therapy to treat diseases affected by aberrant GSL metabolism. In this study, we used novel GlcCer-based photoaffinity probes, XLA and XLB ( 61 ), to identify GlcCer-binding proteins as candidate GlcCer flippases and investigated their roles in GSL biosynthesis.…”