Degradation of membrane-bound ganglioside GM2 by beta-hexosaminidase A - Stimulation by GM2 activator protein and lysosomal lipids

According to a recent hypothesis, glycosphingolipids originating from the p lasma membrane are degraded in the acidic compartments of the cell as compo nents of intraendosomal and intralysosomal vesicles and structures. Since m ost previous in vitro investigations used micellar ganglioside GM2 as subst rate, we studied the degradation of membrane-bound ganglioside GM2 by water -soluble P-hexosaminidase A in the presence of the GM2 activator protein in a detergent-free, liposomal assay system. Our results show that anionic li pids such as the lysosomal components bis(monoacylglycero)phosphate or phos phatidylinositol stimulate the degradation of GM2 by P-hexosaminidase A up to 180-fold in the presence of GM2 activator protein. In contrast, the degr adation rate of GM2 incorporated into liposomes composed of neutral lysosom al lipids such as dolichol, cholesterol, or phosphatidylcholine was signifi cantly lower than in negatively charged liposomes, This demonstrates that b oth, the GM2 activator protein and anionic lysosomal phospholipids, are nee ded to achieve a significant degradation of membrane-bound GM2 under physio logical conditions. The interaction of GM2 activator protein with immobiliz ed membranes was studied with surface plasmon resonance spectroscopy at an acidic pH value as it occurs in the lysosomes, Increasing the concentration of bis(monoacylglycero)phosphate in immobilized liposomes led to a signifi cant drop of the resonance signal in the presence of GM2 activator protein, This suggests that in the presence of bis(monoacylglycero)phosphate, which has been shown to occur in inner membranes of the acidic compartment, GM2 activator protein is able to solubilize lipids from the surface of immobili zed membrane structures.