I-CELL DISEASE AND MENTAL RETARDATION

I-CELL 疾病和智力低下

• 批准号: 3394740
• 负责人: ARNOLD L MILLER
• 金额: $ 15.29万
• 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 1996-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3394740
• 关键词: I cell disease; SDS polyacrylamide gel electrophoresis; active sites; affinity chromatography; antiserum; gene complementation; gene expression; gene mutation; genetic mapping; human subject; inborn lysosomal enzyme disorder; mental retardation; molecular cloning; phosphotransferases; protein sequence; tissue /cell culture

I-cell disease and pseudo-Hurler polydystrophy result from a deficiency of GlcNAc-1-phosphate transferase (GlcNAcPTase) which is involved in the formation of mannose 6-phosphate on lysosomal enzymes for their targeting to lysosomes. Although a single enzyme defect is responsible for these disorders our biochemical and genetic studies have demonstrated the existence of several complementation groups indicating that more than one gene mutation affects the expression of GlcNAcPTase. A major goal of this laboratory is to resolve the molecular basis for each complementation group. An integral part of these studies requires the production of polyclonal antiserum to the purified enzyme or its synthetic peptides. The short term goal of the current proposal is to obtain highly purified or purified GlcNAcPTase by employing the newly developed Affigel 501, uteroferrin-Avidgel Ax, and 5(3-allylamine)-UDP-GlcNAc-Sepharose 4B affinity chromatographies. Subsequent use of a unique photoaffinity labeling technique with [32P]4-SUDP in conjunction with preparative SDS- PAGE can be used to identify and isolate the catalytic subunit of the enzyme. Following amino acid sequencing of the subunit, peptides will be synthesized to segments of the sequence, and polyclonal antibodies prepared to these sequences. The resulting positive antibodies can be used to immunoaffinity purify the GlcNAcPTase. Amino acid sequencing and antipeptide antibodies can then be prepared against other domains in a similar manner as for the catalytic domain. The resulting antibodies that cross-react with GlcNAcPTase will be used to determine whether the gene mutation(s) responsible for the various complementation groups affect the biosynthesis and/or post-translational processing of the GlcNAcPTase. In addition, using these specific antibodies collaborative studies will be carried out to clone and map the gene(s) controlling the expression of GlcNAcPTase.

I 细胞病和假性 Hurler 多发性营养不良是由于缺乏 GlcNAc-1-磷酸转移酶（GlcNAcPTase）参与 在溶酶体酶上形成甘露糖 6-磷酸用于靶向 到溶酶体。尽管单一酶的缺陷是造成这些的原因 我们的生化和遗传学研究已经证明 多个互补基团的存在表明不止一个 基因突变影响GlcNAcPTase的表达。此次活动的一个主要目标 实验室的目的是解决每种互补的分子基础 团体。这些研究的一个组成部分需要制作 针对纯化酶或其合成肽的多克隆抗血清。这 当前提案的短期目标是获得高度纯化或 采用新开发的Affigel 501纯化GlcNAcPTase， 子宫铁蛋白-Avidgel Ax 和 5(3-烯丙胺)-UDP-GlcNAc-Sepharose 4B 亲和色谱法。随后使用独特的光亲和力 [32P]4-SUDP 标记技术结合制备型 SDS- PAGE 可用于鉴定和分离催化亚基 酶。 对亚基进行氨基酸测序后，肽将被 合成序列片段和多克隆抗体 为这些序列做好准备。所得阳性抗体可用于 免疫亲和纯化 GlcNAcPTase。氨基酸测序和 然后可以针对其他结构域制备抗肽抗体 与催化结构域类似的方式。由此产生的抗体 与 GlcNAcPTase 的交叉反应将用于确定该基因是否 负责各种互补组的突变影响 GlcNAcPTase 的生物合成和/或翻译后加工。 在 此外，使用这些特定抗体的合作研究将 进行克隆和定位控制表达的基因 GlcNAcPT 酶。