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分解的降解中间体，在神经元细胞的质膜外表面存在相对高浓度的GM1。GM2AP作为一种底物特异性辅助因子，可溶解溶胞内囊泡中的GM2，并将寡糖部分呈递给β -己糖氨酸酶a （HexA）进行水解裂解。HexA或GM2AP的突变都会导致GM2在溶酶体内的储存和细胞死亡。这类溶酶体贮积病的一个众所周知的例子是泰-萨克斯综合征。作为GM2脂质代谢的第一步，GM2AP辅助蛋白调控GM2酶降解的机制尚不清楚。我们的工作将集中于阐明这一机制的分子细节，通过位点定向自旋标记晚期核内体和溶酶体内囊泡的异常被认为改变了双分子层的物理性质，使GM2AP能够从膜中提取GM2。目前的假设是，在神经元溶酶体组成的高曲率膜中，结合是一个短暂的事件。基于荧光的结合测量将用于检验这一假设。近年来报道了GM2AP与GM2、POPG和PAF结合的晶体结构。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将通过SDSL EPR实验在GM2AP溶液中进行测试，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。糖基化对膜结合特性的影响也将被研究。本研究的一般相关性是GM2AP或HexA的先天性突变导致溶菌体储存病。GM2神经节脂质损伤（包括Tay-Sachs病）是一组由GM2分解代谢缺陷引起的遗传性疾病。事实上，多种溶酶体贮积病（Pompe， Fabry和神经节脂质中毒）是由脂质代谢异常引起的。更详细地了解GM2分解代谢的关键第一步，可能会导致这些致命疾病的药物或基因治疗的发展。

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.