FUNCTIONAL/STRUCTURAL STUDIES OF MAMMALIAN NEURAMINIDASE

拟议的研究范围是研究哺乳动物溶酶体神经氨酸酶（neur），这是一种必需的水解酶，属于自然界中广泛分布的唾液酸酶超家族。 哺乳动物的神经氨酸酶，包括胞浆型、溶酶体型和质膜型，由于其明显的低丰度、不稳定性和膜结合特性而受到限制。 只是在最近几年才出现了关于这些酶的生理功能的重要线索。 溶酶体神经元通过去除末端唾液酸残基来启动唾液酸-糖缀合物的水解。该酶在唾液酸酶中是独特的，因为它需要与保护性蛋白/组织蛋白酶A（PPCA）结合用于细胞内路由和溶酶体活化，并且它与代谢的神经变性疾病相关。唾液酸沉积症是由neur基因座的结构性病变引起的，而半乳糖唾液酸沉积症（GS），neur和β-半乳糖苷酶（β-gal）的联合缺乏是由PPCA的缺乏引起的。 这两种疾病具有共同的临床和生化特征，可以归因于神经功能的丧失。 我们的总体目标是更广泛地了解溶酶体神经元功能，并将这些知识应用于唾液酸中毒和GS的病理生理学研究，从而有助于设计适当的治疗方法。 我们计划研究neur和PPCA之间的结构-功能关系，利用PPCA前体的3D结构对两种蛋白质之间的潜在接触位点进行靶向诱变，并利用在唾液酸沉积症患者中鉴定的天然neur突变。 通过这种方法，我们将确定neur/PPCA相互作用，细胞内运输和激活的关键残基/结构域。我们还将生化特性的一种新的神经氨酸酶亚型的想法与连接特定的生化特性与功能。 neur敲除小鼠的特征将有助于确定由neur缺乏引起的疾病的一般发病率，并将其与GS小鼠进行比较。 这些在体外和体内的研究将耦合到使用杆状病毒表达系统，以产生大量的neur蛋白的neur 3D结构的测定。最后，假设我们将成功地产生neur和PPCA的稳定复合物，我们将开始两种酶的复合物的结晶。 PI的实验室在开发这一研究领域处于独特的地位，因为它已经为拟议的研究建立了遗传和生物化学系统，并且可以依靠杰出的结构生物学家的专业知识来进行项目的晶体学部分。

The scope of the proposed research is to study mammalian lysosomal neuraminidase (neur), an essential hydrolase that belongs to the superfamily of sialidases found widely spread in nature. Information on the mammalian neuraminidases, including a cytosolic, a lysosomal and a plasma membrane form, has been limited by their apparent low abundance, instability and membrane-bound character. Only in recent years important clues have emerged as to the physiological function(s) of these enzymes. Lysosomal neur initiates the hydrolysis of sialo- glyconjugates by removing their terminal sialic acid residues. The enzyme is unique among sialidases in that it requires association with the protective protein/cathepsin A (PPCA) for intracellular routing and lysosomal activation, and it is associated with neurodegenerative diseases of metabolism. Sialidosis is caused by structural lesions at the neur locus, and galactosialidosis (GS), a combined deficiency of neur and beta- galactosidase (beta-gal) is caused by the absence of PPCA. The two diseases have common clinical and biochemical features that can be assigned to the loss of neur function. Our overall goal is to gain a broader understanding of lysosomal neur function(s), and to apply this knowledge to the study of the pathophysiology of sialidosis and GS which, in turn, could help in designing appropriate therapy. We plan to investigate the structure- function relationships between neur and PPCA, making use of the 3D structure of the PPCA precursor for targeted mutagenesis of potential contact sites between the two proteins, and of natural neur mutation(s) identified in patients with sialidosis. With this approach we will identify residues/domains crucial for neur/PPCA interaction, intracellular transport and activation. We will also biochemically characterize a novel neuraminidase isoform with the idea of linking specific biochemical properties with function. The characterization of neur knockout mice will be instrumental to determine the general penetrance of the disease that results from neur deficiency, and to compare it with the GS mouse. These in vitro and in vivo studies will be coupled to the determination of neur 3D structure using the baculovirus expression system to produce neur protein in large quantities. Finally, provided that we will be successful in generating stable complex of neur and PPCA we will initiate crystallization of the complex of the two enzymes. The PI's laboratory is in the unique position to develop this line of investigation as it has established genetic and biochemical systems for the proposed studies, and can rely on the expertise of an outstanding structural biologist for the crystallography part of the project.