STRUCTURE-FUNCTION RELATIONSHIPS OF LYSOSOMAL ENZYMES

溶酶体酶的结构-功能关系

• 批准号: 3918242
• 负责人: R L PROIA
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3918242
• 关键词: DNA; beta N acetylhexosaminidase; chromosome aberrations; cytogenetics; enzyme mechanism; gene expression; genetic mapping; genetic promoter element; glycoproteins; glycosylation; laboratory rabbit; lysosomes; messenger RNA; molecular cloning; nucleic acid probes; nucleic acid sequence; oligosaccharides; point mutation; protein engineering; protein sequence; simian virus 40; tissue /cell culture; transfection

I. We have characterized the glycosylation sites of human beta- hexosaminidase B and have determined the effect of individual oligosaccharides on the catalytic activity, transport and processing of the enzyme. Glycosylation is an essential step for the ultimate expression of lysosomal enzymes because it is only after the construction of a mannose-6-phosphate recognition marker that the enzymes are recognized by a receptor and delivered to lysosomes. The five potential glycosylation sites (Asn-X-Ser/Thr) of the hexosaminidase beta-chain were individually modified by site-directed mutagenesis and the constructs were expressed in Cos 1 cells under control of the SV-40 late promotor. By this analysis, we determined that four of the five potential glycosylation sites were modified by addition of oligosaccharide chains. The lack of any one of the oligosaccharides did not dramatically affect catalytic activity or the delivery of the enzyme to lysosomes. We also demonstrated a selectivity in the phosphorylation of the oligosaccharides on hexosaminidase B; two of the four oligosaccharide chains were predominantly phosphorylated. When these two phosphorylated sites were removed by mutagenesis, the enzyme was not delivered to lysosomes. This work has also clarified the peptide structure of the mature enzyme. II. A hexosaminidase alpha chain cDNA has been cloned from a library prepared with the fibroblast mRNA of a patient with the adult form of Tay-Sachs disease. We are currently sequencing the clone to identify the mutation responsible for this form of the disease.

I. 我们已经表征了人β-淀粉样蛋白的糖基化位点， 氨基己糖苷酶B，并确定了个体的影响 寡糖对催化活性、转运和 酶的加工。 糖基化是合成的重要步骤， 溶酶体酶的最终表达，因为它只是 在构建甘露糖-6-磷酸识别标记物后， 这些酶被受体识别并传递到 溶酶体 5个潜在的糖基化位点（Asn-X-Ser/Thr） 氨基己糖苷酶β链的每一个都被单独修饰， 定点诱变，并在Cos 1细胞中的SV-40晚期启动子的控制下。 受此 分析，我们确定五个潜在的四个， 糖基化位点通过加入寡糖进行修饰 店 缺乏任何一种低聚糖都不会 显着影响催化活性或交付的 酶转化为溶酶体。 我们还证明了选择性， 氨基己糖苷酶B上寡糖的磷酸化; 四种寡糖链中， 磷酸化 当这两个磷酸化位点被移除后 通过诱变，该酶不被递送到溶酶体。 这 工作还阐明了成熟酶的肽结构。 二. 已从一个具有氨基己糖苷酶α链cDNA的哺乳动物中克隆了氨基己糖苷酶α链cDNA。 用患有糖尿病的患者的成纤维细胞mRNA制备的文库 成人型泰-萨二氏病 我们目前正在对 克隆以确定负责这种形式的突变， 疾病