ATP-binding cassette transporters mediate differential biosynthesis of glycosphingolipid species

Monique Budani,Christiane Auray-Blais,Clifford Lingwood

doi:10.1016/j.jlr.2021.100128

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.

Highlights

Suppelementary key words ATP-binding cassette (ABC) transporter glucosylceramide flippase glycosphingolipid photoprobes metabolic labeling LacCer GlcCer pools metabolic channeling GSL anabolism GlcCer synthase
The first evidence of differential GSL anabolic regulation was our inhibition of LacCer and Gb3, but not ganglioside, biosynthesis in ABCB1-expressing cell lines by cyclosporin A [47], supporting an ABCB1 role as a flippase selectively involved in globo-series GSL biosynthesis
The probes were only used as a screen to identify potential GlcCer-binding proteins, while involvement in GSL biosynthesis would be confirmed through knockdown studies

Summary

Introduction

ABCB1 was first suggested as a potential mechanism for flipping GlcCer to the luminal leaflet of the Golgi for GSL biosynthesis when GSL levels were increased in ABCB1 retroviral cell transfection [45]. GCS overexpression in cancer has been shown to correlate with ABCB1 expression [48], and increased ABCB1 correlates with increased complex GSLs in drug-resistant cells [49]. Inhibiting GCS activity blocks ABCB1 overexpression [50], and vice versa [51], resensitizing drug-resistant cells to chemotherapeutic drugs. This can involve additional ABC transporters [52].

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Results

Conclusion