Synthesis of novel NBD-GM1 and NBD-GM2 for the transfer activity of GM2-activator protein by a FRET-based assay system.
The ganglioside-activator protein is an essential cofactor for the lysosomal degradation of ganglioside GM2 (GM2) by beta-hexosaminidase A. It mediates the interaction between the water-soluble exohydrolase and its membrane-embedded glycolipid substrate at the lipid-water interphase. Mutations in the gene encoding this glycoprotein result in a fatal neurological storage disorder, the AB variant of GM2-gangliosidosis. In order to efficiently and sensitively probe the glycolipid binding and membrane activity of this cofactor, we synthesized two new fluorescent glycosphingolipid (GSL) probes, 2-NBD-GM1 and 2-NBD-GM2. Both compounds were synthesized in a convergent and multistep synthesis starting from the respective gangliosides isolated from natural sources. The added functionality of 2-aminogangliosides allowed us to introduce the chromophore into the region between the polar head group and the hydrophobic anchor of the lipid. Both fluorescent glycolipids exhibited an extremely low off-rate in model membranes and displayed very efficient resonance energy transfer to rhodamine-dioleoyl phosphoglycerol ethanolamine (rhodamine-PE) as acceptor. The binding to GM2-activator protein (GM2AP) and the degrading enzyme was shown to be unaltered compared to their natural analogues. A novel fluorescence-resonance energy transfer (FRET) assay was developed to monitor in real time the protein-mediated intervesicular transfer of these lipids from donor to acceptor liposomes. The data obtained indicate that this rapid and robust system presented here should serve as a valuable tool to probe quantitatively and comprehensively the membrane activity of GM2AP and other sphingolipid activator proteins and facilitate further structure-function studies aimed at delineating independently the lipid- and the enzyme-binding mode of these essential cofactors.
Schwarzmann
Günter
G
Kekulé-Institute für Organische Chemie und Biochemie, Gerhard-Domagk-Strasse 1, D-53121 Bonn, Germany. schwarzmann@uni-bonn.de
Wendeler
Michaela
M
Sandhoff
Konrad
K
D000818:Animals; D001921:Brain / Q000473:pathology; D002240:Carbohydrate Sequence; D002384:Catalysis; D002417:Cattle; D002855:Chromatography, Thin Layer; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry / Q000494:pharmacology; D005677:G(M1) Ganglioside / Q000737:chemistry*; D049289:G(M2) Activator Protein / Q000737:chemistry*; D005678:G(M2) Ganglioside / Q000737:chemistry*; D005732:Gangliosides / Q000737:chemistry; D005733:Gangliosidoses; D006017:Glycolipids / Q000737:chemistry; D006023:Glycoproteins / Q000737:chemistry; D006801:Humans; D008055:Lipids / Q000737:chemistry; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009154:Mutation; D013050:Spectrometry, Fluorescence; D049128:Sphingolipid Activator Proteins / Q000737:chemistry; D013107:Sphingolipids / Q000737:chemistry; D013329:Structure-Activity Relationship; D013661:Tay-Sachs Disease / Q000378:metabolism; D013997:Time Factors; D001619:beta-N-Acetylhexosaminidases / Q000737:chemistry
D005456:Fluorescent Dyes; D049289:G(M2) Activator Protein; D005732:Gangliosides; D006017:Glycolipids; D006023:Glycoproteins; D008055:Lipids; D049128:Sphingolipid Activator Proteins; D013107:Sphingolipids; D005678:G(M2) Ganglioside; D005677:G(M1) Ganglioside; D001619:beta-N-Acetylhexosaminidases