I-CELL DISEASE AND MENTAL RETARDATION

I-CELL 疾病和智力低下

• 批准号: 3394744
• 负责人: ARNOLD L MILLER
• 金额: $ 16.52万
• 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 1990-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3394744
• 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.

与酶的生物合成和靶向性有关的酶 对于溶酶体细胞器来说，是GlcNAc磷酸转移酶。这个 这种酶的活性没有严重降低是 L-细胞对这两种常染色体遗传病负责 疾病(LCD)和假性Hurler多发性营养不良(PHP)。尽管一个 单酶缺乏症已被提出用于治疗这些疾病， 几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 突变(S)对GlcNAc结构和功能的影响 磷酸转移酶。淋巴母细胞，可以在 大量的，表现出GlcNAc的正常性质 因此，磷酸转移酶使其成为一个很好的系统，可以从 对该酶进行纯化和研究。初步纯化结果 揭示了该酶是一种超过30万的糖蛋白 分子量。我们的直接目标是净化正常的 酶通过包括阴离子的步骤达到表观均一性 刀豆蛋白A亲和交换层析 层析、凝胶过滤、亲和层析 层析和凝胶电泳法。刻画人物形象 纯化酶的动力学、化学和物理性质 将提供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 决定不同互补的蛋白质水平 组。这种方法的一个组成部分将需要多克隆 纯化的酶的抗血清。该抗体将用于 结合标记的氨基酸或甘露糖来确定 突变(S)是否导致了这种互补 基团影响生物合成和/或翻译后加工 LCD和PHP培养物中GlcNAc磷酸转移酶的研究 成纤维细胞。多克隆抗体也将使其有可能 美国将建立合作关系，绘制和克隆该基因 酵素。淋巴母细胞系统也将使我们能够研究 正常酸、液晶酸和苯丙氨酸的低聚糖结构 溶酶体中的水解酶，因为它们与 一种替代磷酸化的识别系统或标记 甘露糖以溶酶体为靶点的酶。这些研究将 利用密度梯度离心法进行亚细胞分离 溶酶体酶标记胶体硅胶的研究 前情提要：~3H-甘露糖。