TAY-SACHS AND RELATED GENETIC NEUROLOGICAL DISORDERS

泰萨克斯病和相关遗传神经系统疾病

• 批准号: 3415707
• 负责人: KUNIHIKO SUZUKI
• 金额: $ 19.01万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-01-01 至 1994-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3415707
• 关键词: Baculoviridae; Sandhoff disease; Tay Sachs disease; active sites; alleles; enzyme induction /repression; enzyme structure; enzyme substrate complex; fibroblasts; gangliosides; gangliosidosis; gene mutation; genetic markers; glycosidases; human genetic material tag; human tissue; lipid metabolism; molecular cloning; molecular genetics; molecular pathology; polymerase chain reaction; protein purification; protein structure; protein structure function; site directed mutagenesis; tissue /cell culture; transfection

DESCRIPTION: (Adapted from the applicants abstract) GM2 gangliosidosis is a heterogeneous group of genetic disorders due to defective catabolism of ganglioside GM2. This severe and nearly always fatal neurological disorder results when any one of the three tissue constituents required for normal degradation of GM2 ganglioside is genetically abnormal. Abnormality in the enzyme hexosaminidase alpha subunit leads to a disease clinically known as Tay Sachs disease, whereas abnormality in the subunit is identified as Sandhoff disease. A deficiency of the GM2 activator protein is termed "AB variant". cDNA's for all three proteins and genomic clones for the first two forms are now available for analyses of the genetic abnormalities at the nucleic acid level. During the past three years, the applicants' laboratory has identified seven of the thirteen known mutations in Tay Sachs disease, and the only known specific mutation in Sandhoff disease, and has successfully cloned cDNA's coding for the activator protein. This application proposes to extend their work to further characterize naturally occurring mutations in the three genes among patients with genetic GM2 gangliosidoses. Identification of naturally occurring mutations will be accomplished largely by standard molecular biological techniques, including cDNA and genomic library construction, screening, the polymerase chain reaction, sequencing, allele specific oligonucleotide screening, and transient functional expression in COS I cells. The second specific aim is to obtain and characterize full length cDNA's and the gene coding for the GM2 activator protein and to search for the structural requirement for the mannose phosphate recognition marker, taking advantage of the small size and only one glycosylation site of the protein. Characterization of the cDNA and the genomic structure of the GM2 activator proteins will also be done with standard recombinant DNA technologies, since they already have full length cDNA clones and are on their way to obtain genomic fragments. The third specific aim is to produce and purify milligram quantities of the three proteins using the new baculovirus system. The proteins will be used for subsequent experiments such as to produce antibodies. The fourth specific aim is to explore the structure/function relationship within the different domains of each of the constituents for the enzymatic reaction, the two subunits, the activator protein, and the substrate GM2 ganglioside, and interactions among the three proteins. These studies will extensively utilize sitedirected mutagenesis based on the knowledge gained from naturally occurring mutations and their enzymological properties coupled with information from computerized molecular modeling. Both naturally occurring and mutagenesis generated mutants will be evaluated functionally in the COS I cell and baculovirus systems. The new knowledge to be generated by this series of studies may contribute to eventual understanding of the molecular pathogenesis of these genetic neurological disorders and may be useful to future attempts at the gene therapy.

描述：（改编自申请人摘要）GM 2神经节苷脂沉积症是 一组异质的遗传性疾病， 神经节苷脂GM 2。 这种严重且几乎总是致命的神经系统疾病 当正常组织所需的三种组织成分中的任何一种 GM 2神经节苷脂的降解是遗传异常的。 异常 酶氨基己糖苷酶α亚基导致临床上称为 Tay Sachs病，而亚基的异常被鉴定为 山德霍夫病 GM 2激活蛋白的缺乏称为“AB 变量”。 所有三种蛋白质的cDNA和第一种蛋白质的基因组克隆 现时有两份表格可供分析基因异常， 核酸水平。 在过去的三年里，申请人 一个实验室已经确定了Tay中13个已知突变中的7个 萨克斯病，也是唯一已知的山德霍夫病的特异性突变， 并成功克隆了编码激活蛋白的cDNA。 这 应用程序建议扩展他们的工作，以进一步表征自然 遗传性GM 2患者的三个基因发生突变 神经节苷脂沉积 自然发生的突变的鉴定将是 主要通过标准分子生物学技术完成，包括 cDNA和基因组文库构建、筛选、聚合酶链 反应、测序、等位基因特异性寡核苷酸筛选，以及 COS I细胞中的瞬时功能性表达。 第二个具体目标是 为了获得和表征全长cDNA和编码该蛋白的基因， GM 2激活蛋白，并寻找其结构要求。 甘露糖磷酸识别标记，利用小尺寸 并且蛋白质只有一个糖基化位点。 表征 还将分析GM 2激活蛋白的cDNA和基因组结构。 用标准的重组DNA技术，因为他们已经有了 全长cDNA克隆，并正在获得基因组片段。 第三个具体目标是生产和纯化毫克量的 使用新的杆状病毒系统。 这些蛋白质将被用于 用于随后的实验，例如产生抗体。 第四 具体的目的是探索结构/功能的关系， 用于酶促反应的每种成分的不同区域， 两个亚基，激活蛋白和底物GM 2神经节苷脂， 以及三种蛋白质之间的相互作用。 这些研究将广泛 利用基于从以下获得的知识的定点突变 自然发生的突变及其酶学性质 与计算机分子模型的信息。 的天然 发生和诱变产生的突变体将在功能上进行评估 在COS I细胞和杆状病毒系统中。 新知识 这一系列研究产生的结果可能有助于 了解这些遗传性神经系统疾病的分子发病机制， 疾病，并可能是有用的基因治疗的未来尝试。