Ineraction of GM2 activator protein with glycosphingolipids

Abstract

GM2 activator protein is a protein cofactor that stimulates the hydrolysis of the GalNAc and the NeuAc in GM2 by β-hexosaminidase A and sialidase, respectively. To understand the mechanism of action of GM2 activator, the interaction of this protein with GM2 and/or β-hexosaminidase A has been the subject of interest since the purified GM2 activator became available. Numerous techniques including ultracentrifugation, isoelectric focusing, polyacrylamide gel electrophoresis, gel filtration, thin layer chromatogram overlay, and fluorescence dequenching assay have been used to investigate the binding and the affinity of GM2 activator to various glycosphingolipids. It has been generally accepted that GM2 activator must have a very weak binding with the enzyme, because they can be easily separated from each other by gel filtration. Therefore, the interaction of GM2 and GM2 activator has been the focus for most of the study. Although preferential association of GM2 activator with GM2 was detected by some methods, GM2 activator was found also to bind other glycosphingolipids. Isolation of the specific complex that consists of only GM2 activator and GM2 from an incubation mixture containing the activator protein and mixed glycosphingolipids has not been successfully carried out. Ultracentrifugation and gel-filtration are the mildest methods for the isolation of the complexes. However, these methods do not separate the complexes formed by specific interaction from that formed by non-specific association. In fluorescence dequenching assay, the attempt to isolate the complex of R18 lipid probe with GM2 activator was also not successful. Since GM2 activator and glycosphingolipids contain hydrophobic domains in their molecules, the non-specific hydrophobic interactions between the two components can greatly interfere with the isolation of true functional complexes. Among the reported methods, thin layer chromatography overlay and the assay based on the inhibition of fluorescence dequenching by various glycosphingolipids are more informative than the others on the binding between GM2 activator and the carbohydrate head groups of glycosphingolipids.