Adamantyl Glycosphingolipids Provide a New Approach to the Selective Regulation of Cellular Glycosphingolipid Metabolism

Abstract

Mammalian glycosphingolipid (GSL) precursor monohexosylceramides are either glucosyl-or galactosylceramide (GlcCer or GalCer). Most GSLs derive from GlcCer. Substitution of the GSL fatty acid with adamantane generates amphipathic mimics of increased water solubility, retaining receptor function. We have synthesized adamantyl GlcCer (adaGlcCer) and adamantyl GalCer (adaGalCer). AdaGlcCer and adaGalCer partition into cells to alter GSL metabolism. At low dose, adaGlcCer increased cellular GSLs by inhibition of glucocerebrosidase (GCC). Recombinant GCC was inhibited at pH 7 but not pH 5. In contrast, adaGalCer stimulated GCC at pH 5 but not pH 7 and, like adaGlcCer, corrected N370S mutant GCC traffic from the endoplasmic reticulum to lysosomes. AdaGalCer reduced GlcCer levels in normal and lysosomal storage disease (LSD) cells. At 40 M adaGlcCer, lactosylceramide (LacCer) synthase inhibition depleted LacCer (and more complex GSLs), such that only GlcCer remained. In Vero cell microsomes, 40 M adaGlcCer was converted to adaLacCer, and LacCer synthesis was inhibited. AdaGlcCer is the first cell LacCer synthase inhibitor. At 40 M adaGalCer, cell synthesis of only Gb 3 and Gb 4 was significantly reduced, and a novel product, adamantyl digalactosylceramide (adaGb 2 ), was generated, indicating substrate competition for Gb 3 synthase. AdaGalCer also inhibited cell sulfatide synthesis. Microsomal Gb 3 synthesis was inhibited by adaGalCer. Metabolic labeling of Gb 3 in Fabry LSD cells was selectively reduced by adaGalCer, and adaGb 2 was produced. AdaGb 2 in cells was 10-fold more effectively shed into the medium than the more polar Gb 3 , providing an easily eliminated "safety valve" alternative to Gb 3 accumulation. Adamantyl monohexosyl ceramides thus provide new tools to selectively manipulate normal cellular GSL metabolism and reduce GSL accumulation in cells from LSD patients.

Structure-dependent Pseudoreceptor Intracellular Traffic of Adamantyl Globotriaosyl Ceramide Mimics

“…This could explain the dominant trafficking effect of adaGb 3 in Gb 3 -expressing cells and the ability of adamantyl monohexosyl ceramides to modulate GSL metabolism (72). The lack of OHEtadaGb 3 efficacy could be consistent with this mechanism.…”

“…The amphipathic GSL analogues we have made, which in part retain the receptor function of membrane GSLs (71), provide new insight into these processes and the means to alter cellular GSL metabolism selectively (72). We now show that adaGSLs have an immediate effect on plasma membrane GSL receptor function and intracellular traffic.…”

Verotoxin Receptor-Based Pathology and Therapies

“…Although the fatty acid is important in VT-Gb 3 binding (Kiarash et al, 1994;Pezeshkian et al, 2015), these species (unlike the free sugar) retained the biological activity of the membrane embedded GSL in solution (Lingwood et al, 2006). AdaGlcCer and adaGalCer proved effective inhibitors of cellular GSL biosynthesis (Kamani et al, 2011), while adaSGC was an effective mimic of SGC (3-sulfogalactosyl ceramide), binding to Hsp70 (Mamelak and Lingwood, 2001), thereby inhibiting its ATPase activity (Whetstone and Lingwood, 2003) and chaperone action in cells (Park et al, 2009). Hsp70-SGC binding promotes aggregation of Hsp70 for high affinity peptide binding and blocks ATP-mediated peptide release (Harada et al, 2015).…”

Inhibition of Glycosphingolipid Synthesis Ameliorates Atherosclerosis and Arterial Stiffness in Apolipoprotein E −/− Mice and Rabbits Fed a High-Fat and -Cholesterol Diet

“…mice, rats, rabbits up to 10 times the effective dose. In mice, the effective dose of D-PDMP is 10 mg per kg (mpk) body weight when given orally compared to the use of 100mpk of various adamanate derivatives used by other investigators 8, 9 . Since D-PDMP turnover time is a short ~52 min in mice 10 , it is rapidly detoxified and excreted with little or no side effects.…”