I-CELL DISEASE AND MENTAL RETARDATION

I细胞疾病和假性Hurler多发性营养不良是由于缺乏 GlcNAc-1-磷酸转移酶(GlcNAcPTase)参与 6-磷酸甘露糖在溶酶体酶上的形成及其靶向性 到溶酶体。尽管一个单一的酶缺陷导致了这些 我们的生化和遗传学研究已经证明 几个互补群的存在表明不止一个 基因突变影响GlcNAcPTase的表达。这样做的一个主要目标是 实验室是为了解决每一种互补的分子基础 一群人。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。这个 目前提案的短期目标是获得高度提纯的或 利用新开发的Affigel 501纯化GlcNAcPTase， 子宫铁蛋白-亲和凝胶Ax和5-(3-烯丙基胺)-UDP-GlcNAc-Sepharose4B 亲和层析。随后使用一种独特的光亲和性 [~(32)P]4-SUDP结合制备十二烷基硫酸钠的标记技术 PAGE可用于鉴定和分离该蛋白的催化亚单位 酵素。在对亚基进行氨基酸测序后，多肽将被 合成到片段的序列和多克隆抗体 为这些序列做好准备。由此产生的阳性抗体可以用于 免疫亲和纯化GlcNAcPTase。氨基酸测序和 然后可以制备抗肽抗体来对抗 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基因是否 突变(S)负责各种互补性群的影响 GlcNAcPTase的生物合成和/或翻译后加工。在……里面 此外，使用这些特定抗体的合作研究将是 对控制病毒表达的基因(S)进行了克隆和定位 GlcNAcPTase。

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.