Novel Intrinsic and Extrinsic Approaches to Selectively Regulate Glycosphingolipid Metabolism

Glycosphingolipid (GSL) metabolism is a complex process involving proteins and enzymes at distinct locations within the cell. Mammalian GSLs are typically based on glucose or galactose, forming glucosylceramide (GlcCer) and galactosylceramide (GalCer). Most GSLs are derived from GlcCer, which is synthesized on the cytosolic leaflet of the Golgi, while all subsequent GSLs are synthesized on the lumenal side. We have utilized both pharamacological and genetic manipulation approaches to selectively regulate GSL metabolism and better understand its mechanistic details. We have developed analogues of GlcCer and GalCer by substituting the fatty acid moiety with an adamanatane frame. The resulting adamantylGSLs are more water-soluble than their natural counterparts. These analogues selectively interfere with GSL metabolism at particular points within the metabolic pathway. At 40 µM, adaGlcCer prevents synthesis of all GSLs downstream of GlcCer, while also elevating GlcCer levels, by inhibiting lactosylceramide (LacCer) synthase and glucocerebrosidase, respectively. AdaGalCer specifically reduces synthesis of globotriaosylceramide (Gb3) and downstream globo-series GSLs. AdaGalCer also increases Gaucher disease N370S glucocerebrosidase expression, lysosomal localization and activity. AdaGSLs, therefore, have potential as novel therapeutic agents in diseases characterized by GSL anomalies and as tools to study the effects of GSL modulation. Two predominant theories have been developed to explain how GlcCer accesses the Golgi lumen: one involving direct translocation from the cytosolic-to-lumenal leaflet of the Golgi by the ABC transporter P-glycoprotein (P-gp, ABCB1, MDR1), and the other involving retrograde transport of GlcCer by FAPP2 to the ER, followed by entry into the vesicular transport system for Golgi lumenal access. To examine the in vivo involvement of P-gp in GSL metabolism, we generated a knockout model by crossbreeding the Fabry disease mouse with the P-gp knockout mouse. HPLC analyses of tissue Gb3 levels revealed a tissue-specific reduction in MDR1/Fabry mice. TLC analyses, however, did not show such reduction. In addition, we performed a gene knockdown study using siRNA against P-gp and FAPP2. Results show these siRNA to have distinct effects on GSL levels that are cell-type specific. These results give rise to the prospect of unique therapeutic approaches by targeting P-gp or FAPP2 for synthesis inhibition of particular GSL pathways.

Keywords: Sphingolipids
Metabolism
Lysosomal storage disorders
adamantane
Glucosylceramide
Lactosylceramide
P-glycoprotein
MDR1
Gaucher disease
Fabry disease
siRNA
Gb2 galabiosylceramide
Glycolipids
Inhibitors
Knockout mouse
Crossbreeding
Glucosylceramide synthase
Lactosylceramide synthase
Ganglioside
Globo-series
Gb3 synthase
pharmacological chaperone
Glucocerebrosidase
Substrate reduction therapy
ATP8B1
Cholesterol
ABC transporters
P-type ATPase
Flippase
FAPP2
GLTP
Glycosyltransferase
Glycolipid analogues
ERAD
ABCB1
knockdown
GM3 and EGFR
GM3 and insulin receptor
Glycolipid synthesis
Glycolipid metabolism
enzyme enhancement therapy
Advisor: Lingwood, Clifford A.