I-CELL DISEASE AND MENTAL RETARDATION

I-CELL 疾病和智力低下

• 批准号: 3394736
• 负责人: ARNOLD L MILLER
• 金额: $ 11.99万
• 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/3394736
• 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 是GlcNAc磷酸转移酶。 的 这种酶的活性没有严重降低， 负责两个常染色体遗传疾病，l-细胞 疾病（lCD）和假性Hurler多营养不良（PHP）。 虽然 对于这些疾病提出了单一酶缺乏， 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测基因 GlcNAc结构和功能突变 磷酸转移酶 淋巴母细胞，可以在 大量，证明GlcNAc的正常性质 磷酸转移酶，从而使其成为一个良好的系统， 纯化并研究该酶。 初步纯化结果 表明该酶是一种糖蛋白， 分子量的 我们的近期目标是净化正常的 酶的步骤，包括阴离子 交换层析，伴刀豆球蛋白A亲和 层析，凝胶过滤层析，亲和层析 层析和凝胶电泳。 表征 纯化酶的动力学、化学和物理性质 将产生关于其结构和功能的信息。 这些研究对于理解 负责不同互补作用的蛋白质水平 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 负责互补的突变是否 基团影响生物合成和/或翻译后加工 培养的lCD和PHP中GlcNAc磷酸转移酶的 成纤维细胞 多克隆抗体也将使其有可能用于 我们建立合作，以绘制和克隆基因的 酵素 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 磷酸化的替代识别系统或标记 甘露糖使酶靶向溶酶体。 这些研究将 利用密度梯度离心的亚细胞分级 溶酶体酶的胶体二氧化硅上 以前用3 H-甘露糖。