Meier E M, Schwarzmann G, Fürst W, Sandhoff K
Institute of Organic Chemistry and Biochemistry, University of Bonn, Federal Republic of Germany.
J Biol Chem. 1991 Jan 25;266(3):1879-87.
Ganglioside GD1a-GalNAc was isolated from Tay-Sachs brain, tritium-labeled in its sphingosine moiety, and its enzymic degradation studied in vitro and in cultured fibroblasts. When offered as micelles, GD1a-GalNAc was almost not hydrolyzed by Hex A or Hex B, while after incorporation of the ganglioside into the outer leaflet of liposomes, the terminal GalNAc residue was rapidly split off by Hex a. In striking contrast to ganglioside GM2, the major glycolipid substrate of Hex A, the enzymic hydrolysis of GD1a-GalNAc was not promoted by the GM2 activator protein, although the activator protein did bind GD1a-GalNAc to form a water-soluble complex. Pathobiochemical studies corroborate these results. After incorporation of [3H]GD1a-GalNAc into cultured skin fibroblasts from healthy subjects and from patients with different variants of GM2 gangliosidosis, its degradation was found to be strongly attenuated in mutant cells with Hex A deficiencies such as variant B (Tay-Sachs disease), variant B1 and variant 0 (Sandhoff disease), while in cells with variant AB (GM2 activator deficiency), its catabolism was blocked only at the level of GM2. In line with these metabolic studies, a normal content of GD1a-GalNAc was found in brains of patients who had succumbed to variant AB of GM2 gangliosidosis whereas in brains from variants B, B1, and 0, its concentration was considerably elevated (up to 19-fold). Together with studies on the enzymic degradation of GM2 derivatives with modifications in the ceramide portion, these results indicate that mainly steric hindrance by adjacent lipid molecules impedes the access of Hex A to membrane-bound GM2 (whose degradation therefore depends on solubilization by the GM2 activator) and in addition that the interaction between the GM2. GM2 activator complex and the enzyme must be highly specific.
神经节苷脂GD1a-GalNAc从患泰-萨克斯病的大脑中分离出来，在其鞘氨醇部分用氚标记，并在体外和培养的成纤维细胞中研究其酶促降解。当以胶束形式提供时，GD1a-GalNAc几乎不被己糖胺酶A或己糖胺酶B水解，而在神经节苷脂掺入脂质体的外层小叶后，末端的GalNAc残基被己糖胺酶A迅速裂解。与己糖胺酶A的主要糖脂底物神经节苷脂GM2形成鲜明对比的是，尽管GM2激活蛋白确实与GD1a-GalNAc结合形成水溶性复合物，但GD1a-GalNAc的酶促水解并未受到GM2激活蛋白的促进。病理生化研究证实了这些结果。将[3H]GD1a-GalNAc掺入健康受试者和患有不同类型GM2神经节苷脂沉积症患者的培养皮肤成纤维细胞后，发现在己糖胺酶A缺乏的突变细胞（如B型变异体（泰-萨克斯病）、B1型变异体和0型变异体（桑德霍夫病））中其降解明显减弱，而在AB型变异体（GM2激活蛋白缺乏）的细胞中，其分解代谢仅在GM2水平受阻。与这些代谢研究一致的是，在死于GM2神经节苷脂沉积症AB型变异体的患者大脑中发现GD1a-GalNAc含量正常，而在B型、B1型和0型变异体的大脑中，其浓度显著升高（高达19倍）。连同对神经酰胺部分有修饰的GM2衍生物的酶促降解研究，这些结果表明，相邻脂质分子的空间位阻主要阻碍己糖胺酶A接近膜结合的GM2（其降解因此依赖于GM2激活蛋白的增溶作用），此外，GM2-GM2激活蛋白复合物与酶之间的相互作用必须具有高度特异性。
