Protein determinants impair recognition of procathepsin L phosphorylated oligosaccharides by the cation-independent mannose 6-phosphate receptor.

摘要: 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 secreted hydrolase contain mannose 6-phosphate (Man 6-P), recognition marker for transport newly synthesized acid hydrolases to lysosomes. To investigate mechanism by which cathepsin L evades targeting lysosomes, we determined structure enzyme's and analyzed its interaction cation-independent 6-PCl) receptor. Oligosaccharides procathepsin isolated from medium [3H]mannose-labeled J774 cells were remarkably homogeneous; all radiolabeled structures high mannose-type units contained two phosphomonoesters 7 residues. Both alpha 1,3- 1,6-branches phosphorylated. released endoglycosidase H bound Man 6-PCl receptor affinity column. Despite binding these oligosaccharides, intact glycoprotein was not good ligand Procathepsin internalized poorly 6-P receptor-mediated endocytosis purified protease interacted weakly In contrast, pro-beta-glucuronidase (another produced cells) an excellent as judged chromatographic assays. Phosphorylated J774-secreted heterogeneous both mono- diphosphorylated species. Tryptic glycopeptides generated unlike protein, ligands The results indicate are processed uniformly species bind Protein determinants inherent within hydrolase, however, inhibit and, result, impair