Werth N, Schuette C G, Wilkening G, Lemm T, Sandhoff K
Kekulé-Institut für Organische Chemie und Biochemie, Universität Bonn, Gerhard-Domagk-Strasse 1, D-53121 Bonn, Germany.
J Biol Chem. 2001 Apr 20;276(16):12685-90. doi: 10.1074/jbc.M007970200. Epub 2001 Jan 16.
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.
根据最近的一种假说，源自质膜的糖鞘脂在细胞的酸性区室中作为内体和溶酶体内囊泡及结构的成分被降解。由于之前大多数体外研究使用胶束神经节苷脂GM2作为底物，我们在无去污剂的脂质体测定系统中，研究了在GM2激活蛋白存在的情况下，水溶性β-己糖胺酶A对膜结合神经节苷脂GM2的降解作用。我们的结果表明，在GM2激活蛋白存在的情况下，阴离子脂质如溶酶体成分双（单酰甘油）磷酸酯或磷脂酰肌醇可将β-己糖胺酶A对GM2的降解作用提高至180倍。相比之下，掺入由中性溶酶体脂质如多萜醇、胆固醇或磷脂酰胆碱组成的脂质体中的GM2的降解速率明显低于带负电荷的脂质体。这表明，在生理条件下，GM2激活蛋白和阴离子溶酶体磷脂对于实现膜结合GM2的显著降解都是必需的。在酸性pH值（如溶酶体中出现的pH值）下，利用表面等离子体共振光谱研究了GM2激活蛋白与固定化膜的相互作用。在固定化脂质体中增加双（单酰甘油）磷酸酯的浓度会导致在GM2激活蛋白存在时共振信号显著下降。这表明，在已证明存在于酸性区室内膜中的双（单酰甘油）磷酸酯存在的情况下，GM2激活蛋白能够从固定化膜结构表面溶解脂质。
