The GM2-activator
protein (GM2AP) is an essential cofactor for the lysosomal degradation of
ganglioside GM2 by
beta-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-beta-D-glucopyranoside (MUGS) indicated a weakened association with the
enzyme's alpha subunit. This identifies the
protein region affected by these mutations, the single short alpha 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.