I-CELL DISEASE AND MENTAL RETARDATION

I-CELL 疾病和智力低下

• 批准号: 3394745
• 负责人: ARNOLD L MILLER
• 金额: $ 17.53万
• 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 1992-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3394745
• 关键词: I cell disease; autosomal recessive trait; chromatography; electrophoresis; enzyme structure; fibroblasts; gene complementation; genetic mapping; genetic markers; glycoproteins; human tissue; immunochemistry; lymphoblast; lysosomes; mental retardation; molecular cloning; oligosaccharides; phosphotransferases; secretion; tissue /cell culture

The enzyme involved in the biosynthesis and targeting of enzymes to lysosomal organelles is the GlcNAc phosphotransferase. The absence of severe reduction of this enzyme's activity is responsible for the two autosomally inherited disorders, l-cell disease (lCD) and pseudo-Hurler polydystrophy (PHP). Although a single enzyme deficiency has been proposed for these disorders, the existence of several complementation groups suggests that more than one gene can regulate the expression of this enzyme. Our long range objective is to examine the effect of the gene mutation(s) on the structure and function of the GlcNAc phosphotransferase. Lymphoblast cells, which can be grown in large quantities, demonstrate normal properties for the GlcNAc phosphotransferase thus making it a good system from which to purify and study the enzyme. Preliminary purification results reveal that the enzyme is a glycoprotein of greater than 300,000 molecular weight. Our immediate goal is to purify the normal enzyme to apparent homogeneity by steps which include anion exchange chromatography, concanavalin A-affinity chromatography, gel filtration chromatography, affinity chromatography and gel electrophoresis. Characterization of the kinetic, chemical and physical properties of the purified enzyme will yield information concerning it's structure and function. These studies are necessary for understanding the alterations at the protein level responsible for the different complementation groups. An integral part of this approach will require polyclonal antisera to the purified enzyme. The antibody will be used in conjunction with labelled amino acids or mannose to determine whether the mutation(s) responsible for the complementation groups affect the biosynthesis and/or post-translational processing of the GlcNAc phosphotransferase in lCD and PHP cultured fibroblasts. The polyclonal antibody will also make it possible for us to establish collaborations to map and clone the gene for the enzyme. The lymphoblast system will also allow us to study the oligosaccharide structures on normal, lCD and PHP acid hydrolases within the lysosomes as they relate to the existence of an alternate recognition system or marker to phosphorylated mannose in targeting enzymes to lysosomes. These studies will utilize subcellular fractionation by density gradient centrifugation on colloiday silica of lysosomal enzymes that had been labeled previously with 3H-mannose.

参与酶的生物合成和靶向的酶 进入溶酶体细胞器的是 GlcNAc 磷酸转移酶。 这 这种酶的活性没有严重降低 导致两种常染色体遗传性疾病的原因是 L 细胞 疾病 (LCD) 和假性 Hurler 多营养不良症 (PHP)。 虽然一个 已提出单酶缺乏症可用于治疗这些疾病， 多个互补基团的存在表明 不止一个基因可以调节这种酶的表达。 我们的长期目标是检查基因的影响 GlcNAc 结构和功能的突变 磷酸转移酶。 淋巴细胞，可以在 大量，表现出 GlcNAc 的正常特性 磷酸转移酶，因此使其成为一个良好的系统 纯化并研究酶。 初步纯化结果 表明该酶是一种大于 300,000 的糖蛋白 分子量。 我们当前的目标是净化常态 通过包含阴离子的步骤使酶达到表观同质性 交换色谱法，刀豆球蛋白 A 亲和力 层析、凝胶过滤层析、亲和层析 色谱法和凝胶电泳。 的表征 纯化酶的动力学、化学和物理特性 将产生有关其结构和功能的信息。 这些研究对于理解这些变化是必要的 负责不同互补的蛋白质水平 组。 该方法的一个组成部分将需要多克隆 纯化酶的抗血清。 该抗体将用于 与标记的氨基酸或甘露糖结合以确定 突变是否负责互补 影响生物合成和/或翻译后加工的基团 LCD 和 PHP 培养物中 GlcNAc 磷酸转移酶的变化 成纤维细胞。 多克隆抗体也将使 我们建立合作来绘制和克隆基因 酶。 淋巴母细胞系统也将使我们能够研究 正常、LCD 和 PHP 酸上的寡糖结构 溶酶体内的水解酶，因为它们与以下物质的存在有关 磷酸化的替代识别系统或标记 甘露糖将酶靶向溶酶体。 这些研究将 通过密度梯度离心进行亚细胞分级 在已标记的溶酶体酶的胶体二氧化硅上 之前用3H-甘露糖。