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The GM2-activator protein (GM2AP) is an essential cofactor for the lysosomal degradation of ganglioside GM2 by β-hexosaminidase A (HexA). It mediates the interaction between the water-soluble exohydrolase and its membrane-embedded glycolipid substrate at the lipid–water interface. Functional deficiencies in this protein result in a fatal neurological storage disorder, the AB variant of GM2 gangliosidosis. In order to elucidate this cofactor's mode of action and identify the surface region of GM2AP responsible for binding to HexA, we designed several variant forms of this protein and evaluated the consequences of these mutations for lipid- and enzyme-binding properties using a variety of biophysical and functional studies. The point mutants D113K, M117V and E123K showed a drastically decreased capacity to stimulate HexA-catalysed GM2 degradation. However, surface plasmon resonance (SPR) spectroscopy showed that the binding of these variants to immobilized lipid bilayers and their ability to solubilize lipids from anionic vesicles were the same as for the wild-type protein. In addition, a fluorescence resonance energy transfer (FRET)-based assay system showed that these variants had the same capacity as wild-type GM2AP for intervesicular lipid transfer from donor to acceptor liposomes. The concentration-dependent effect of these variants on hydrolysis of the synthetic substrate 4-methylumbelliferyl-2-acetamido-2-deoxy-6-sulfo-β-D-glucopyranoside (MUGS) indicated a weakened association with the enzyme's α subunit. This identifies the protein region affected by these mutations, the single short α helix of GM2AP, as the major determinant for the interaction with the enzyme. These results further confirm that the function of GM2AP is not restricted to a biological detergent that simply disrupts the membrane structure or lifts the substrate out of the lipid plane. In contrast, our data argue in favour of the critical importance of distinct activator–hexosaminidase interactions for GM2 degradation, and corroborate the view that the activator/lipid complex represents the true substrate for the degrading enzyme.