Lazzarino D, Gabel C A
Department of Anatomy and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, New York 10032.
J Biol Chem. 1990 Jul 15;265(20):11864-71.
Cathepsin L, a lysosomal cysteine protease, is the major excreted protein of transformed mouse NIH 3T3 cells. Previous studies have shown that asparagine-linked oligosaccharides associated with the secreted hydrolase contain mannose 6-phosphate (Man 6-P), the recognition marker for transport of newly synthesized acid hydrolases to lysosomes. To investigate the mechanism by which cathepsin L evades targeting to lysosomes, we determined the structure of the enzyme's oligosaccharides and analyzed its interaction with the cation-independent mannose 6-phosphate (Man 6-PCl) receptor. Oligosaccharides associated with procathepsin L isolated from the medium of [3H]mannose-labeled J774 cells were remarkably homogeneous; all of the radiolabeled structures were high mannose-type units that contained two phosphomonoesters and 7 mannose residues. Both the alpha 1,3- and alpha 1,6-branches of the oligosaccharides were phosphorylated. Oligosaccharides released by endoglycosidase H from [3H]mannose-labeled procathepsin L bound to a Man 6-PCl receptor affinity column. Despite the high affinity binding of these oligosaccharides, the intact glycoprotein was not a good ligand for the Man 6-PCl receptor. Procathepsin L was internalized poorly by Man 6-P receptor-mediated endocytosis and the purified acid protease interacted weakly with a Man 6-PCl affinity column. In contrast, pro-beta-glucuronidase (another acid hydrolase produced by J774 cells) was an excellent ligand for the Man 6-PCl receptor as judged by the endocytosis and affinity chromatographic assays. Phosphorylated oligosaccharides associated with the J774-secreted pro-beta-glucuronidase were heterogeneous and contained both mono- and diphosphorylated species. Tryptic glycopeptides generated from [3H]mannose-labeled procathepsin L, unlike the intact protein, were excellent ligands for the Man 6-PCl receptor. The results indicate that oligosaccharides associated with procathepsin L are processed uniformly to diphosphorylated species that bind with high affinity to the Man 6-PCl receptor. Protein determinants inherent within the intact acid hydrolase, however, inhibit the high affinity binding of these oligosaccharides and, as a result, impair the interaction of procathepsin L with the receptor.
组织蛋白酶L是一种溶酶体半胱氨酸蛋白酶，是转化的小鼠NIH 3T3细胞的主要分泌蛋白。先前的研究表明，与分泌的水解酶相关的天冬酰胺连接的寡糖含有甘露糖6-磷酸（Man 6-P），这是新合成的酸性水解酶转运至溶酶体的识别标记。为了研究组织蛋白酶L逃避靶向溶酶体的机制，我们确定了该酶寡糖的结构，并分析了其与不依赖阳离子的甘露糖6-磷酸（Man 6-PCl）受体的相互作用。从[3H]甘露糖标记的J774细胞培养基中分离出的与组织蛋白酶L原相关的寡糖非常均一；所有放射性标记结构都是含有两个磷酸单酯和7个甘露糖残基的高甘露糖型单元。寡糖的α1,3-和α1,6-分支均被磷酸化。内切糖苷酶H从[3H]甘露糖标记的组织蛋白酶L原中释放出的寡糖与Man 6-PCl受体亲和柱结合。尽管这些寡糖具有高亲和力结合，但完整的糖蛋白并不是Man 6-PCl受体的良好配体。组织蛋白酶L原通过Man 6-P受体介导的内吞作用内化较差，并且纯化的酸性蛋白酶与Man 6-PCl亲和柱的相互作用较弱。相比之下，通过内吞作用和亲和色谱分析判断，β-葡萄糖醛酸酶原（J774细胞产生的另一种酸性水解酶）是Man 6-PCl受体的优良配体。与J774分泌的β-葡萄糖醛酸酶原相关的磷酸化寡糖是异质的，包含单磷酸化和双磷酸化物种。与完整蛋白不同，从[3H]甘露糖标记的组织蛋白酶L原产生的胰蛋白酶糖肽是Man 6-PCl受体的优良配体。结果表明，与组织蛋白酶L原相关的寡糖被均匀加工成双磷酸化物种，这些物种与Man 6-PCl受体具有高亲和力结合。然而，完整酸性水解酶中固有的蛋白质决定簇会抑制这些寡糖的高亲和力结合，结果损害了组织蛋白酶L原与受体的相互作用。
