Membrane Binding Properties of the GM2 Activator Protein

GM2 激活蛋白的膜结合特性

• 批准号: 7075522
• 负责人: GAIL E FANUCCI
• 金额: $ 27.23万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-05 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7075522
• 关键词: binding proteins; conformation; electron spin resonance spectroscopy; gangliosides; glycosphingolipids; lipid bilayer membrane; lipid metabolism; lysosomes; membrane structure; protein binding; protein structure function

DESCRIPTION (provided by applicant): The GM2 Activator Protein (GM2AP) plays an essential role in the catabolism of GM2 in lysosomes. GM2 is a complex glycosphingolipid (GSL) that is a degradation intermediate in the breakdown of GM1 which is found in relatively high concentrations in the outer surface of plasma membranes in neuronal cells. GM2AP acts as a substrate specific co-factor by solubilizing GM2 from the intralysozomal vesicles and presenting the oligosaccharide moiety to beta-hexosaminidase A (HexA) for hydrolytic cleavage. Mutations in either HexA or GM2AP lead to the storage of GM2 within lysozomes and cell death. A well known example of this type of lysosomal storage disease is Tay-Sachs syndrome. The mechanism for how the GM2AP accessory protein regulates enzymatic degradation of GM2 as the first step in the GM2 lipid metabolism remains unclear. The proposed work will focus on elucidating the molecular details of this mechanism through site-directed spin labeling anomposition of late endosomes and intralysosomal vesicles are believed to alter the bilayer physical properties allowing GM2AP to extract GM2 from membranes. The current hypothesis is that in membranes of high curvature of neuronal lysozome composition, the binding is a transient event. Fluorescence based binding measurements will be used to test this hypothesis. Recently, crystal structures of the GM2AP bound to GM2, POPG and PAF were reported. The model of conformational changes predicted by X-ray crystallography data for specific and non-specific substrates will be tested by SDSL EPR experiments of GM2AP in solution where GM2 is extracted from micelles, as well as from lipid bilayers. Currently, the GM2AP has been expressed in E.coli, insect cells and in yeast. The effects of glycosylation on the membrane binding properties will also be examined. The general relevance of this research is that congenital mutations in GM2AP or HexA result in lysozomal storage diseases. The GM2-gangliosidoses (including Tay-Sachs disease) are a group of inherited disorders that result from defects in the catabolism of GM2. In fact, a variety of lysosomal storage diseases (Pompe, Fabry and the gangliosidoses) result from aberrations in lipid metabolism. A more detailed understanding of this critical first step in GM2 catabolism may lead to developments in drug or gene therapies for these fatal diseases.

描述（由申请方提供）：GM 2激活蛋白（GM 2AP）在溶酶体中的GM 2催化中起重要作用。GM 2是一种复合鞘糖脂（GSL），是GM 1分解的降解中间体，在神经元细胞的质膜外表面发现浓度相对较高。GM 2AP作为底物特异性辅因子，通过从溶酶体内囊泡中溶解GM 2并将寡糖部分呈递给β-氨基己糖苷酶A（HexA）进行水解裂解。HexA或GM 2AP中的突变导致GM 2在溶酶体内的储存和细胞死亡。这种类型的溶酶体贮积病的一个众所周知的例子是泰-萨二氏综合征。GM 2AP辅助蛋白如何调节GM 2的酶促降解作为GM 2脂质代谢的第一步的机制仍不清楚。拟议的工作将集中在阐明这一机制的分子细节，通过定点自旋标记的晚期内体和intrysosomal囊泡被认为是改变双层的物理性质，使GM 2AP提取GM 2膜。目前的假设是，在膜的高曲率的神经元溶酶体组成，结合是一个短暂的事件。将使用基于荧光的结合测量来检验该假设。近年来，与GM 2、POPG和PAF结合的GM 2AP的晶体结构被报道。将通过溶液中GM 2AP的SDSL EPR实验测试通过X射线晶体学数据预测的特异性和非特异性底物的构象变化模型，其中GM 2从胶束以及脂质双层中提取。目前，GM 2AP已经在大肠杆菌、昆虫细胞和酵母中表达。还将检查糖基化对膜结合性质的影响。这项研究的一般相关性是GM 2AP或HexA的先天性突变导致溶酶体贮积病。GM 2神经节苷脂沉积症（包括泰-萨二氏病）是一组遗传性疾病，由GM 2的catalysts缺陷引起。事实上，多种溶酶体贮积病（庞贝氏症、法布里病和神经节苷脂沉积症）是由脂质代谢异常引起的。更详细地了解GM 2催化剂的这一关键的第一步可能会导致这些致命疾病的药物或基因疗法的发展。