Characterization of the affinity of the G M2 activator protein for glycolipids by a fluorescence dequenching assay

Abstract

The G M2 activator protein is a substrate specific cofactor for degradation of G M2 ganglioside by lysosomal β-hexosaminidase A. Mutations in the gene encoding the activator result in the AB-variant form of G M2 gangliosidosis. The activator protein contains at least three functional elements; a hydrophobic binding pocket, an oligosaccharide binding site(s), and an area that interacts with hexosaminidase A. In this report a fluorescence dequenching assay specific for only the hydrophobic binding pocket is evaluated and optimized. It is shown that various glycolipids inhibit the transport between liposomes of a self-quenching fluorescent lipid probe, octadecylrhodamine, by the activator protein. The level of inhibition produced by each glycolipid is then used to characterize the oligosaccharide-binding specificity of the activator. The fluorescence dequenching assay is also used to evaluate the functionality of a truncated form of the activator protein. Our results indicate that this simple assay can be used to determine structure–function relationships within the normal or mutant forms of the activator. The data suggest that the C-terminus of the activator is required to produce a functional hydrophobic binding pocket.