STRUCTURE-FUNCTION RELATIONSHIPS OF LYSOSOMAL ENZYMES

溶酶体酶的结构-功能关系

• 批准号: 2573656
• 负责人: R L PROIA
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/2573656
• 关键词: Sandhoff disease; Tay Sachs disease; beta N acetylhexosaminidase; disease /disorder model; enzyme deficiency; enzyme mechanism; enzyme structure; gangliosides; gangliosidosis; gene targeting; genetically modified animals; laboratory mouse; model design /development; motor neurons; mucopolysaccharidosis; protein degradation; protein structure function

The GM2 gangliosidoses are severe neurodegenerative disorders characterized by impaired GM2 ganglioside degradation as a consequence of beta-hexosaminidase A deficiency. Mutations in the HEXA and HEXB genes, which encode the subunits of beta-hexosaminidase A, cause Tay- Sachs and Sandhoff disease, respectively. A third disease is caused by mutations in the GM2A gene encoding the GM2 activator protein. The GM2 activator protein forms a substrate-complex with the ganglioside enabling degradation by beta-hexosaminidase A. In humans, both the neurologic phenotype and neuropathology in the three diseases are very similar. Through targeted gene disruption in embryonic stem cells, we have previously created mouse models corresponding to Tay-Sachs and Sandhoff diseases. Unlike the human diseases, the mouse models showed dramatically different phenotypes as a result of differences in the ganglioside degradation pathway between mice and humans. We have now established mice with a disrupted Gm2alpha gene as a model for the third form of the GM2 gangliosidoses. The Gm2alpha gene disruption resulted in a null allele as evidenced by the absence of a Gm2alpha transcript in the knockout mice. Although the brain ganglioside level was elevated, ganglioside feeding experiments in fibroblasts indicated that the degradation pathway was not completely blocked in the Gm2alpha -/- mice. As of 18 weeks of age, there were no significant differences in motor function when mutant mice were compared to wild- type or heterozygous mice. At this age, the Sandhoff disease but not the Tay-Sachs disease mice were severely affected. The three gangliosidosis mice provide models for the human disorders allowing for a more detailed understanding of their pathobiology and for evaluation of potential therapies. We have also produced mice with both beta-hexosaminidase genes disrupted that are totally deficient in all three forms of the lysosomal enzyme (A, B and S). These totally beta-hexosaminidase deficient mice displayed facial and physical dysmorphia and severe movement difficulties, and had a short life span (1-4 months). In addition to gangliosidosis, the double knockout mice showed pathologic and biochemical features of mucopolysaccharidosis including dysostosis mutiplex. The novel phenotype of the totally beta-hexosaminidase deficient mice shows that GAGs like gangliosides are critical substrates for beta-hexosaminidase and that their lack of storage in Tay-Sachs and Sandhoff diseases is due to functional redundancy in the beta-hexosaminidase system.

GM 2神经节苷脂沉积症是严重的神经退行性疾病 其特征在于作为结果受损的GM 2神经节苷脂降解 β-氨基己糖苷酶A缺乏症 HEXA和HEXB突变 编码β-氨基己糖苷酶A亚基的基因导致Tay- 萨克斯和山德霍夫病。 第三种疾病是由 编码GM 2激活蛋白的GM 2A基因突变。 的 GM 2激活蛋白与神经节苷脂形成底物复合物 能够被β-氨基己糖苷酶A降解。 在人类中， 这三种疾病的神经表型和神经病理学非常相似， 相似 通过对胚胎干细胞进行靶向基因破坏， 之前已经建立了对应于Tay-Sachs的小鼠模型， 山德霍夫病 与人类疾病不同，小鼠模型显示， 不同的表型，由于不同的 神经节苷脂降解途径之间的小鼠和人类。 我们现在已经 建立了具有破坏的Gm 2 α基因的小鼠作为模型， GM 2神经节苷脂沉积症的第三种形式。 GM 2 α基因破坏 导致无效等位基因，如Gm 2 α的缺失所证明 基因敲除小鼠中的转录本。 虽然脑神经节苷脂水平 升高，成纤维细胞中神经节苷脂喂养实验表明 降解途径没有被完全阻断， GM 2 α-/-小鼠。 在18周龄时， 当突变小鼠与野生小鼠相比时， 型或杂合子小鼠。 在这个年龄，山德霍夫病，但不是 Tay-Sachs病小鼠受到严重影响。 三 神经节苷脂沉积症小鼠提供了人类疾病的模型， 为了更详细地了解它们的病理学， 评估潜在的治疗方法。 我们还培育了同时携带β-氨基己糖苷酶基因的小鼠 这三种形式都是完全缺乏的。 溶酶体酶（A、B和S）。 这些完全β-氨基己糖苷酶 缺陷小鼠表现出面部和身体畸形， 运动困难，寿命短（1-4个月）。在 除了神经节苷脂沉积症，双基因敲除小鼠还表现出病理性 包括成骨不全在内的粘多糖沉积症的生物化学特征 多重的 全β-氨基己糖苷酶的新表型 缺陷小鼠表明，像神经节苷脂这样的GAG是至关重要的， β-氨基己糖苷酶的底物，以及它们缺乏在 泰-萨二氏病和桑德霍夫病是由于大脑皮层的功能冗余所致。 β-氨基己糖苷酶系统。