ABSTRACT
Human procathepsin D carries two N-linked glycosylation sites at asparagine residues 70 and 199, widely separated on the surface of the folded protein. We created monoglycosylated procathepsin D molecules by site-directed mutagenesis in vitro of the individual glycosylation sites. With only two exceptions, all 12 mutants of this type were expressed efficiently in mammalian cells. The expressed proteins were stable, targeted to the lysosome, and partially secreted into the medium. When both glycosylation sites were eliminated, however, the expressed proteins (9 different mutants) were stable but most were not secreted and targeted poorly to the lysosome. Mammalian fibroblasts appear to sort nascent procathepsin D efficiently only if it is N-glycosylated. Procathepsin D monoglycosylated at N70 is readily distinguished from the endogenous protein in transfected human cells and thus provides an excellent substrate for studying lysosomal targeting in an homologous system.
Subject(s)
Cathepsin D/metabolism , Enzyme Precursors/metabolism , Heat-Shock Proteins , Protein Processing, Post-Translational , Animals , CHO Cells , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cathepsin D/genetics , Cell Compartmentation , Cricetinae , Cricetulus , DNA, Complementary/genetics , Endoplasmic Reticulum Chaperone BiP , Enzyme Precursors/genetics , Fibroblasts/metabolism , Glycosylation , Humans , Lysosomes/metabolism , Molecular Chaperones/genetics , Molecular Chaperones/metabolism , Mutagenesis, Site-Directed , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/metabolism , Recombinant Fusion Proteins/metabolism , Species Specificity , TransfectionABSTRACT
A major pathway to the lysosome for soluble hydrolases involves the 6-phosphorylation of mannose residues. The initial step in this reaction is catalyzed by a phosphotransferase which recognizes lysosomal precursors. We constructed mutants of human procathepsin D whose targeting to the lysosome could be assayed directly in intact cells. Eight lysine residues were individually converted to glutamic acid on the surface of the carboxyl terminal lobe of the protein. Mutants with as many as four Lys to Glu mutations were normally targeted to the lysosome and processed to the mature form of the enzyme in transfected cells. We conclude that the C-terminal lobe of procathepsin D may not carry a determinant essential for lysosomal targeting in intact fibroblasts.
Subject(s)
Cathepsin D/metabolism , Enzyme Precursors/metabolism , Lysine , Lysosomes/metabolism , Protein Processing, Post-Translational , Protein Structure, Tertiary , Recombinant Fusion Proteins/metabolism , Animals , Biological Transport , CHO Cells , Cathepsin D/chemistry , Cathepsin D/genetics , Cell Line, Transformed , Chlorocebus aethiops , Cricetinae , Cricetulus , Enzyme Precursors/chemistry , Enzyme Precursors/genetics , Female , Glycosylation , Humans , Mannosephosphates/metabolism , Mutagenesis, Site-Directed , Phosphorylation , Phosphotransferases/metabolism , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/immunology , Renin/metabolism , Species SpecificityABSTRACT
When the 44-amino acid propeptide of human procathepsin D was deleted by mutagenesis in vitro, the mature protein was stably expressed and secreted from transfected mammalian cells. The secreted protein was correctly folded as judged by its binding to pepstatinylagarose. We were unable to detect lysosomal targeting of the propeptide-deleted protein, and targeting was not restored by the substitution of the propeptides from pepsin or renin. We conclude that its propeptide is not essential for the folding of nascent cathepsin D. Efficient lysosomal targeting in mammalian cells appears to require the precursor form of the molecule.
