I-CELL DISEASE AND MENTAL RETARDATION

I-CELL 疾病和智力低下

• 批准号: 3394743
• 负责人: ARNOLD L MILLER
• 金额: $ 17.73万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 1978
• 资助国家: 美国
• 起止时间: 1978-06-01 至 1988-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3394743
• 关键词: I cell disease; autosomal recessive trait; chromatography; density gradient ultracentrifugation; electrophoresis; enzyme structure; extracellular; fibroblasts; gene complementation; gene expression; genetic translation; human tissue; hydrolase; immunochemistry; lymphoblast; lysosomes; mannose; mental retardation; mucopolysaccharidosis type I; oligosaccharides; radioassay; radioimmunoassay; secretion; spectrometry; tissue /cell culture; transferase

Lysosomal enzymes share a unique pathway of biosynthesis and posttranslational modification. The long-term objectives of this grant involve studies of structural information on acid hydrolases which is necessary for their intracellular sorting and subsequent targeting to lysosomes or extracellular fluids. I-cell disease (ICD) and pseudoHurler polydystrophy (PHP), two inherited childhood disorders, will be used as model systems. The primary defect responsible for these disorders is an absence or severe reduction of glcNac phosphotransferase, a key enzyme involved in the biosynthesis and targeting of acid hydrolases to lysosomes. Evidence exists for genetic and biochemical heterogeneity in ICD and PHP. The existence of complementation groups within and between the two disorders suggest the involvement of more than one gene mutation which affects the structure and function of the transferase. One experimental approach will examine selected properties of the enzyme from the different complementation groups, such as the enzyme's stability under various conditions including temperature, addition of protease inhibitors, sucrose loading, and the enzyme's interaction with various lectins. The purification of the transferase from autopsied human liver will be carried out using a combination of conventional and affinity column methods. The characterization of kinetic, chemical, and physical properties of the enzyme from both the normal and mutant sources will be useful in understanding the functions of the different gene product(s) required for enzyme activity. The production of polyclonal antisera will allow us to probe the basis for each mutation by comparing the biosynthesis and processing of immunoprecipitable material in normal and mutant cell lines after adding labeled amino acids or 3H mannose. Lymphoblasts prepared from normal controls, ICD and PHP patients will also be examined as an alternate source to study the glcNac phosphotransferase protein from the various complementation groups. A second approach in studying the structural requirements for intracellular segregation of acid hydrolases will employ cultured normal, ICD, and PHP skin fibroblasts and lymphoblasts that have been previously pulsed with 3H-mannose and grown in the presence or absence of various agents known to interfere with normal glycosylation (tunicamycin), posttranslational processing (swainsonine) and intracellular transport and secretion (monensin). Subcellular fractionation by density gradient centrifugation on percoll and/or free flow electrophoresis will allow separation of the fractions containing labeled acid hydrolases and subsequent analysis of the types of oligosaccharides present.

溶酶体酶具有独特的生物合成途径 翻译后修饰。 本次资助的长期目标 涉及酸性水解酶结构信息的研究 它们的细胞内分选和随后的靶向所必需的 溶酶体或细胞外液。 I 细胞病 (ICD) 和假Hurler 多营养不良（PHP）是两种遗传性儿童疾病，将被用作 模型系统。 造成这些疾病的主要缺陷是 glcNac 磷酸转移酶（一种关键酶）缺乏或严重减少 参与酸性水解酶的生物合成和靶向 溶酶体。 有证据表明遗传和生化异质性 ICD 和 PHP。 内部和之间存在互补基团 这两种疾病表明涉及不止一种基因突变 它影响转移酶的结构和功能。 一 实验方法将检查酶的选定特性 不同的互补基团，例如酶在不同条件下的稳定性 各种条件，包括温度、添加蛋白酶抑制剂、 蔗糖负载，以及酶与各种凝集素的相互作用。 这 将从尸检人肝脏中纯化转移酶 结合使用传统方法和亲和柱方法。 这 动力学、化学和物理性质的表征 正常来源和突变来源的酶都可用于 了解所需的不同基因产物的功能 酶活性。 多克隆抗血清的生产将使我们能够 通过比较生物合成和 正常和突变细胞系中免疫沉淀材料的加工 添加标记氨基酸或3H甘露糖后。 淋巴细胞制备自 正常对照、ICD 和 PHP 患者也将作为替代检查 研究不同来源的glcNac磷酸转移酶蛋白的来源 互补组。 结构研究的第二种方法 酸性水解酶细胞内分离的要求将采用 培养的正常、ICD 和 PHP 皮肤成纤维细胞和淋巴母细胞 之前已用 3H-甘露糖脉冲并在存在或不存在的情况下生长 已知干扰正常糖基化的各种药物 （衣霉素）、翻译后加工（苦马豆素）和细胞内 运输和分泌（莫能菌素）。 按密度进行亚细胞分级 Percoll 梯度离心和/或自由流动电泳将 允许分离含有标记的酸性水解酶的级分和 随后分析存在的寡糖类型。