Characterization of the affinity of the G(M2) activator protein for glycolipids by a fluorescence dequenching assay.
The G(M2) activator protein is a substrate specific cofactor for degradation of G(M2) ganglioside by lysosomal beta-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.
Smiljanic-Georgijev
N
N
Research Institute, The Hospital for Sick Children, University of Toronto, Ont., Canada.
Rigat
B
B
Xie
B
B
Wang
W
W
Mahuran
D J
DJ
D001665:Binding Sites; D002240:Carbohydrate Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005453:Fluorescence; D049289:G(M2) Activator Protein; D005678:G(M2) Ganglioside / Q000378:metabolism*; D006017:Glycolipids / Q000378:metabolism*; D054818:Hexosaminidase A; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry; D016133:Polymerase Chain Reaction; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D001619:beta-N-Acetylhexosaminidases / Q000378:metabolism
D049289:G(M2) Activator Protein; D006017:Glycolipids; D009844:Oligosaccharides; D011506:Proteins; D005678:G(M2) Ganglioside; D054818:Hexosaminidase A; D001619:beta-N-Acetylhexosaminidases