Membrane Binding Properties of the GM2 Activator Protein

描述(由申请人提供)：GM2激活蛋白(GM2AP)在溶酶体中GM2的分解代谢中起重要作用。GM2是一种复杂的鞘糖脂(GSL)，是GM1降解的中间产物，GM1存在于神经细胞质膜外表面，浓度相对较高。GM2AP作为底物特异性辅助因子，通过溶解溶菌体内小泡中的GM2，并将寡糖部分呈递给β-己糖氨酸酶A(HexA)进行水解性切割。HexA或GM2AP中的任何一个突变都会导致GM2储存在溶菌体中并导致细胞死亡。这种溶酶体储积症的一个著名例子是泰-萨克斯综合征。作为GM2脂代谢的第一步，GM2AP辅助蛋白如何调节GM2的酶降解的机制尚不清楚。拟议的工作将集中于通过定点自旋标记晚期内小体和溶酶体内小泡的异常位置来阐明这一机制的分子细节，这些异常被认为改变了允许GM2AP从膜中提取GM2的双层物理性质。目前的假设是，在神经元溶菌体组成的高曲率的膜上，结合是一个瞬时事件。基于荧光的结合测量将被用来检验这一假设。最近，人们报道了GM2、POPG和PAF结合的GM2AP的晶体结构。通过X射线结晶学数据预测的特定和非特定底物的构象变化模型将通过GM2AP在溶液中的SDSL EPR实验进行验证，其中GM2从胶束中提取，以及从脂质双层中提取。目前，GM2AP已在大肠杆菌、昆虫细胞和酵母中表达。糖基化对膜结合性能的影响也将被检测。这项研究的总体相关性是，GM2AP或HEXA的先天性突变会导致溶菌体储存疾病。GM2神经节苷脂增多症(包括Tay-Sachs病)是一组遗传性疾病，由GM2分解代谢缺陷引起。事实上，各种溶酶体储存性疾病(Pompe、Fabry和神经节苷脂增多症)都是由脂代谢异常引起的。更详细地了解GM2分解代谢的这一关键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。

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.