I-CELL DISEASE AND MENTAL RETARDATION

溶酶体酶具有独特的生物合成途径和 翻译后修饰。这笔赠款的长期目标 涉及酸性水解酶的结构信息研究，这是 对于它们的细胞内分选和随后的靶向是必要的 溶酶体或细胞外液。I-细胞病(ICD)与假性赫勒 多发性营养不良(PHP)，两种遗传性儿童疾病，将被用作 模型系统。导致这些疾病的主要缺陷是 关键酶GlcNAc磷酸转移酶缺失或严重降低 参与酸性水解酶的生物合成和靶向 溶酶体。存在遗传和生化异质性的证据 ICD和PHP。互补群在内部和之间的存在 这两种疾病提示不止一种基因突变。 这会影响转移酶的结构和功能。一 实验方法将从以下方面检查酶的选定性质 不同的互补基团，如酶在 各种条件，包括温度、添加酶抑制剂、 蔗糖负载，以及酶与各种凝集素的相互作用。这个 将从尸检的人肝中提纯转移酶 使用常规柱法和亲和柱法相结合的方法提取。这个 动力学、化学和物理性质的表征 来自正常来源和突变来源的酶将在 了解不同基因产物的功能(S) 酶活性。多克隆抗血清的生产将使我们能够 通过比较生物合成和基因突变来探索每个突变的基础 正常和突变细胞系中免疫沉淀物质的处理 加入标记氨基酸或~3H甘露糖后。淋巴母细胞的制备 正常对照组、ICD和PHP患者也将作为替代检查 来源：研究不同来源的GlcNAc磷酸转移酶蛋白 互补性小组。研究结构的另一种方法 酸性水解酶的胞内分离要求将采用 培养的正常、ICD和PHP皮肤成纤维细胞和淋巴母细胞 以前用~3H-甘露糖脉冲并在存在或不存在的情况下生长 已知的干扰正常糖基化的各种试剂 (衣霉素)、翻译后加工(苦马豆素)和细胞内 运输和分泌(莫能菌素)。亚细胞密度分级 Percoll梯度离心法和/或自由流电泳法 允许分离包含标记的酸性水解酶和 随后对存在的低聚糖类型进行分析。

Lysosomal enzymes share a unique pathway of biosynthesis and posttranslational modification. The long-term objectives of this grant involve studies of structural information on acid hydrolases which is necessary for their intracellular sorting and subsequent targeting to lysosomes or extracellular fluids. I-cell disease (ICD) and pseudoHurler polydystrophy (PHP), two inherited childhood disorders, will be used as model systems. The primary defect responsible for these disorders is an absence or severe reduction of glcNac phosphotransferase, a key enzyme involved in the biosynthesis and targeting of acid hydrolases to lysosomes. Evidence exists for genetic and biochemical heterogeneity in ICD and PHP. The existence of complementation groups within and between the two disorders suggest the involvement of more than one gene mutation which affects the structure and function of the transferase. One experimental approach will examine selected properties of the enzyme from the different complementation groups, such as the enzyme's stability under various conditions including temperature, addition of protease inhibitors, sucrose loading, and the enzyme's interaction with various lectins. The purification of the transferase from autopsied human liver will be carried out using a combination of conventional and affinity column methods. The characterization of kinetic, chemical, and physical properties of the enzyme from both the normal and mutant sources will be useful in understanding the functions of the different gene product(s) required for enzyme activity. The production of polyclonal antisera will allow us to probe the basis for each mutation by comparing the biosynthesis and processing of immunoprecipitable material in normal and mutant cell lines after adding labeled amino acids or 3H mannose. Lymphoblasts prepared from normal controls, ICD and PHP patients will also be examined as an alternate source to study the glcNac phosphotransferase protein from the various complementation groups. A second approach in studying the structural requirements for intracellular segregation of acid hydrolases will employ cultured normal, ICD, and PHP skin fibroblasts and lymphoblasts that have been previously pulsed with 3H-mannose and grown in the presence or absence of various agents known to interfere with normal glycosylation (tunicamycin), posttranslational processing (swainsonine) and intracellular transport and secretion (monensin). Subcellular fractionation by density gradient centrifugation on percoll and/or free flow electrophoresis will allow separation of the fractions containing labeled acid hydrolases and subsequent analysis of the types of oligosaccharides present.