STRUCTURE-FUNCTION RELATIONSHIPS OF LYSOSOMAL ENZYMES

I.我们已经表征了人β-糖基化位点 并测定了己糖氨酸酶B对个体的影响 低聚糖对酶的催化活性、转运和 酶的加工。糖基化是一个必不可少的步骤 溶酶体酶的最终表达，因为它只是 在构建甘露糖-6-磷酸识别标记后 这些酶被受体识别并传递到 溶酶体。五个潜在的糖基化位点(Asn-X-Ser/Thr) 氨基己糖苷酶的部分β链被单独修饰 定点突变及其在CoS中的表达 1细胞在SV-40晚期启动子的控制下。借此 分析，我们确定了五种可能性中的四种 通过添加低聚糖来修饰糖基化位点 锁链。任何一种低聚糖的缺乏都没有 极大地影响催化剂的活性或传递 酶转化为溶酶体。我们还演示了在 寡糖在氨基己糖苷酶B上的磷酸化；两 在四个寡糖链中，主要是 磷酸化。当这两个磷酸化位点被移除时 通过诱变，这种酶不能传递给溶酶体。这 这项工作还澄清了成熟酶的多肽结构。 II.从沙门氏菌中克隆了一条己糖氨酸酶α链基因 用一位患者的成纤维细胞mRNA制备文库 成人型泰-萨克斯病。我们目前正在对 克隆以确定导致这种形式的 疾病。

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.