I-CELL DISEASE AND MENTAL RETARDATION

I-CELL 疾病和智力低下

• 批准号: 2262392
• 负责人: ARNOLD L MILLER
• 金额: $ 14.58万
• 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/2262392
• 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多营养不良是由于缺乏 GlcNAc-1-磷酸转移酶（GlcNAc-1-phosphate transferase，GlcNAc-1-phosphate transferase，GlcNAc-1-phosphate transferase），它参与 在溶酶体酶上形成甘露糖6-磷酸用于其靶向 到溶酶体。虽然一个单一的酶缺陷是负责这些 我们的生物化学和遗传学研究已经证明， 存在几个互补组，表明不止一个 基因突变影响GlcNAcPT酶的表达。一个主要目标是 实验室的任务是解决每个互补的分子基础 组这些研究的一个组成部分需要制作 纯化的酶或其合成肽的多克隆抗血清。的 本发明的短期目标是获得高度纯化或 通过使用新开发的Affigel 501， 子宫铁蛋白-Avidgel Ax和5（3-烯丙基胺）-UDP-GlcNAc-Sepharose 4 B 亲和层析。随后使用独特的光亲和性 [32 P]4-SUDP结合制备型SDS-标记技术 PAGE可用于鉴定和分离蛋白质的催化亚基。 酵素 在亚基的氨基酸测序之后，将肽与亚基的氨基酸序列进行比较。 合成的序列片段和多克隆抗体 准备好这些序列。产生的阳性抗体可用于 免疫亲和纯化GlcNAcPT酶。氨基酸测序和 然后可以制备抗多肽中其它结构域的抗肽抗体， 类似于催化域的方式。产生的抗体 与GlcNAcPT酶交叉反应将用于确定基因是否 负责各种互补基团的突变影响 在一些实施方案中，GlcNAcPT酶是GlcNAcPT酶的生物合成和/或翻译后加工的酶。 在 此外，使用这些特异性抗体的合作研究将是 进行克隆和定位控制以下表达的基因： GlcNAcPT酶。