Research Article
ATP-binding cassette transporters mediate differential biosynthesis of glycosphingolipid species

https://doi.org/10.1016/j.jlr.2021.100128Get rights and content
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Abstract

The cytosolic-oriented glucosylceramide (GlcCer) synthase is enigmatic, requiring nascent GlcCer translocation to the luminal Golgi membrane to access glycosphingolipid (GSL) anabolic glycosyltransferases. The mechanism by which GlcCer is flipped remains unclear. To investigate the role of GlcCer-binding partners in this process, we previously made cleavable, biotinylated, photoreactive GlcCer analogs in which the reactive nitrene was closely apposed to the GlcCer head group, while maintaining a C16-acyl chain. GlcCer-binding protein specificity was validated for both photoprobes. Using one probe, XLB, here we identified ATP-binding cassette (ABC) transporters ABCA3, ABCB4, and ABCB10 as unfractionated microsomal GlcCer-binding proteins in DU-145 prostate tumor cells. siRNA knockdown (KD) of these transporters differentially blocked GSL synthesis assessed in toto and via metabolic labeling. KD of ABCA3 reduced acid/neutral GSL levels, but increased those of LacCer, while KD of ABCB4 preferentially reduced neutral GSL levels, and KD of ABCB10 reduced levels of both neutral and acidic GSLs. Depletion of ABCA12, implicated in GlcCer transport, preferentially decreased neutral GSL levels, while ABCB1 KD preferentially reduced gangliosides, but increased neutral GSL Gb3. These results imply that multiple ABC transporters may provide distinct but overlapping GlcCer and LacCer pools within the Golgi lumen for anabolism of different GSL series by metabolic channeling. Differential ABC family member usage may fine-tune GSL biosynthesis depending on cell/tissue type. We conclude that ABC transporters provide a new tool for the regulation of GSL biosynthesis and serve as potential targets to reduce selected GSL species/subsets in diseases in which GSLs are dysregulated.

Supplementary key words

ABC transporter
glucosylceramide flippase
glycosphingolipid
photoprobes
metabolic labeling
LacCer
GlcCer pools
metabolic channeling
GSL anabolism
GlcCer synthase

Abbreviations

B3GALT1
β-1,3-galactosyltransferase 1
B4GALT6
β-1,4-galactosyltransferase 6
Cer
ceramide
FAPP2
phosphatidylinositol-four-phosphate adapter protein 2
GCS
GlcCer synthase
GlcCer
glucosylceramide
GLTP
glycolipid transfer protein
GSL
glycosphingolipid
KD
knockdown
LacCer
lactosylceramide
Lc3
lactotriaosylceramide
LCS
LacCer synthase
NBD
nitrobenzo-2-oxa-1,3-diazole
NC
negative control
siRNA
small interfering RNA
ST3GAL5
β-galactoside α-2,3-sialyltransferase 5, GM3 synthase
TMD
transmembrane domain

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