Protein determinants impair recognition of procathepsin L phosphorylated oligosaccharides by the cation-independent mannose 6-phosphate receptor.
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.
Lazzarino
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Department of Anatomy and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, New York 10032.
Gabel
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CA
D000818:Animals; D002241:Carbohydrates / Q000032:analysis; D056668:Cathepsin L; D002403:Cathepsins / Q000302:isolation & purification / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D003546:Cysteine Endopeptidases; D004705:Endocytosis; D010450:Endopeptidases; D004792:Enzyme Precursors / Q000302:isolation & purification / Q000378:metabolism*; D005966:Glucuronidase / Q000302:isolation & purification / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D008358:Mannose / Q000378:metabolism; D008360:Mannosephosphates / Q000378:metabolism; D051379:Mice; D009844:Oligosaccharides / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface / Q000378:metabolism*
D002241:Carbohydrates; D004792:Enzyme Precursors; D008360:Mannosephosphates; D009844:Oligosaccharides; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface; D005966:Glucuronidase; D002403:Cathepsins; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D056668:Cathepsin L; C534278:Ctsl protein, mouse; D008358:Mannose