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 plasma membrane are degraded in the acidic compartments of the cell as components of intraendosomal and intralysosomal vesicles and structures. Since most previous in vitro investigations used micellar ganglioside GM2 as substrate, we studied the degradation of membrane-bound ganglioside GM2 by water-soluble beta-hexosaminidase A in the presence of the GM2 activator protein in a detergent-free, liposomal assay system. Our results show that anionic lipids such as the lysosomal components bis(monoacylglycero)phosphate or phosphatidylinositol stimulate the degradation of GM2 by beta-hexosaminidase A up to 180-fold in the presence of GM2 activator protein. In contrast, the degradation rate of GM2 incorporated into liposomes composed of neutral lysosomal lipids such as dolichol, cholesterol, or phosphatidylcholine was significantly lower than in negatively charged liposomes. This demonstrates that both, the GM2 activator protein and anionic lysosomal phospholipids, are needed to achieve a significant degradation of membrane-bound GM2 under physiological conditions. The interaction of GM2 activator protein with immobilized 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 significant 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 immobilized membrane structures.
Werth
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Kekulé-Institut für Organische Chemie und Biochemie, Universität Bonn, Gerhard-Domagk-Strasse 1, D-53121 Bonn, Germany.
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Wilkening
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Lemm
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Sandhoff
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D049289:G(M2) Activator Protein; D005678:G(M2) Ganglioside / Q000378:metabolism*; D054818:Hexosaminidase A; D006801:Humans; D007700:Kinetics; D008081:Liposomes; D008099:Liver / Q000201:enzymology; D008246:Lysophospholipids / Q000494:pharmacology; D008247:Lysosomes / Q000378:metabolism*; D008563:Membrane Lipids / Q000378:metabolism*; D050178:Monoglycerides; D010713:Phosphatidylcholines / Q000494:pharmacology; D011506:Proteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D001619:beta-N-Acetylhexosaminidases / Q000378:metabolism*
D049289:G(M2) Activator Protein; D008081:Liposomes; D008246:Lysophospholipids; D008563:Membrane Lipids; D050178:Monoglycerides; D010713:Phosphatidylcholines; D011506:Proteins; D011994:Recombinant Proteins; C012786:bis(monoacylglyceryl)phosphate; D005678:G(M2) Ganglioside; D054818:Hexosaminidase A; D001619:beta-N-Acetylhexosaminidases