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Glycobiology Advance Access published online on August 3, 2005
Glycobiology, doi:10.1093/glycob/cwj018
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© The Author 2005. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oupjournals.org
Received April 7, 2005
Revised July 6, 2005
Accepted July 18, 2005

Article

Synthesis of novel NBD-GM1 and NBD-GM2 for the Transfer-Activity of GM2-Activator Protein by a FRET-based Assay System

Günter Schwarzmann 1*, Michaela Wendeler 2, and Konrad Sandhoff 1*

1 Kekulé-Institute für Organische Chemie und Biochemie, Gerhard-Domagk-Strasse 1, D-53121 Bonn, Germany
2 Kekulé-Institute für Organische Chemie und Biochemie, Gerhard-Domagk-Strasse 1, D-53121 Bonn, Germany; Present Address: National Cancer Institute, Frederick, MD 21702, USA

* To whom correspondence should be addressed.
Günter Schwarzmann, E-mail: schwarzmann{at}uni-bonn.de
Konrad Sandhoff, E-mail: sandhoff{at}uni-bonn.de


  Abstract

The GM2-activator protein is an essential cofactor for the lysosomal degradation of ganglioside 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 probes, 2-NBD-GM1 and 2-NBD-GM2. Both compounds were synthesized in a convergent and multi-step synthesis starting from the respective gangliosides isolated from natural sources. The added functionality of 2-aminogangliosides allowed 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-PE as acceptor. The binding to 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.

Keywords: 2-amino-gangliosides/fluorescence resonance energy transfer/GM2-activator protein/2-NBD-GM1/2-NBD-GM2.
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