Identification of Glucosylceramide Flippases in Glycosphingolipid Biosynthesis

Glycosphingolipids (GSLs) participate in cell signaling, adhesion, differentiation, and inflammation. A variety of diseases have aberrant GSL metabolisms including GSL storage diseases, type II diabetes, drug resistant tumors, and Alzheimer’s disease. The major GSL precursor glucosylceramide (GlcCer) is synthesized on the cytosolic Golgi membrane leaflet, however GlcCer must be flipped to the luminal leaflet for subsequent complex GSL anabolism. The mechanism by which GlcCer is translocated is unclear. We have addressed this major 25-year knowledge gap in GSL biosynthesis in order to better understand GSL homeostasis and provide novel therapy targets. We synthesized and characterized photoreactive, biotinylated GlcCer analogue cross-linkers, XLA and XLB (diastereomers) to identify GlcCer interacting proteins. Using XLB and proteomics we identified microsomal GlcCer binding proteins, ABCA3, ABCB4 and ABCB10, which were depleted in DU145 cells by siRNA along with previously suggested GlcCer ATP-binding cassette (ABC) transporters (ABCA12 and ABCB1). Knockdown of ABC proteins differentially affected GSL biosynthesis, suggesting existence of Golgi and trans-Golgi network (TGN)-selective flippases. Putative flippase/LCS inhibitors 2A-AceGlcCer A/B, and 2A-AdaGlcCer A/B were synthesized. 2A-AdaGlcCer analogues appear to be LCS substrates. 2A-AdaGlcCer B is a LCS and flippase inhibitor whereas the A isomer is an activator of globo-series GSL synthesis. 2A-AceGlcCer analogues increased cellular GSLs. In summary, we demonstrated that ABC flippases facilitate metabolic channeling via formation of separate and overlapping Golgi pools of GlcCer for differential GSL-series biosynthesis, providing potential targets for drug therapy to reduce specific GSL species in GSL dysregulated diseases.Ph.D

The Dramatic Modulatory Role of the 2'N Substitution of the Terminal Amino Hexose of Globotetraosylceramide in Determining Binding by Members of the Verotoxin Family

Although globotetraosylceramide (Gb4) is only recognized by a single member of the verotoxin family namely, the pig edema disease toxin (VT2e), removal of the acetyl group from the terminal N-acetyl hexosamine of Gb4 to generate the free amino sugar containing species (aminoGb4) results in the generation of a glycolipid preferentially recognized by all members of the verotoxin family (i.e., VT1, VT2, VT2c, and VT2e). GT3, a site-specific mutant of VT2e, in which Gb4 recognition is lost but Gb3 binding is retained, also binds aminoGb4. We have now compared the binding of VT1, VT2, VT2e, and GT3 to a series of aminoGb4 derivatives using a TLC overlay technique. DimethylaminoGb4 is bound by VT1 and VT2 but not VT2e or GT3; formylaminoGb4 binds all toxins but poorly to VT2 and preferentially VT2e; trifluoroacetylaminoGb4 binds only VT2e and GT3; isopropylaminoGb4 binds VT1 and poorly to VT2; benzylaminoGb4 binds all four toxins. Thus, there is a marked distinction between the permissible amino substitutions for VT1 and VT2e binding. GT3 is a hybrid between these in that, according to the substitution, it behaves similarly either to VT1 or to VT2e. For each species, GT3 does not however, show a hybrid binding between that of VT1 and VT2e. Analysis of the binding as a function of pH shows opposite effects for VT1 and VT2e: decreased pH increases VT1, but decreases VT2e receptor glycolipid binding

The Dramatic Modulatory Role of the 2'N Substitution of the Terminal Amino Hexose of Globotetraosylceramide in Determining Binding by Members of the Verotoxin Family

Although globotetraosylceramide (Gb4) is only recognized by a single member of the verotoxin family namely, the pig edema disease toxin (VT2e), removal of the acetyl group from the terminal N-acetyl hexosamine of Gb4 to generate the free amino sugar containing species (aminoGb4) results in the generation of a glycolipid preferentially recognized by all members of the verotoxin family (i.e., VT1, VT2, VT2c, and VT2e). GT3, a site-specific mutant of VT2e, in which Gb4 recognition is lost but Gb3 binding is retained, also binds aminoGb4. We have now compared the binding of VT1, VT2, VT2e, and GT3 to a series of aminoGb4 derivatives using a TLC overlay technique. DimethylaminoGb4 is bound by VT1 and VT2 but not VT2e or GT3; formylaminoGb4 binds all toxins but poorly to VT2 and preferentially VT2e; trifluoroacetylaminoGb4 binds only VT2e and GT3; isopropylaminoGb4 binds VT1 and poorly to VT2; benzylaminoGb4 binds all four toxins. Thus, there is a marked distinction between the permissible amino substitutions for VT1 and VT2e binding. GT3 is a hybrid between these in that, according to the substitution, it behaves similarly either to VT1 or to VT2e. For each species, GT3 does not however, show a hybrid binding between that of VT1 and VT2e. Analysis of the binding as a function of pH shows opposite effects for VT1 and VT2e: decreased pH increases VT1, but decreases VT2e receptor glycolipid binding