Role of glycosylation in the expression of human procathepsin D.
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.
Fortenberry
S C
SC
Research Service, Audie L. Murphy Memorial Veterans Administration Hospital, San Antonio, Texas, USA.
Schorey
J S
JS
Chirgwin
J M
JM
D000818:Animals; D016466:CHO Cells; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002402:Cathepsin D / Q000235:genetics / Q000378:metabolism*; D002451:Cell Compartmentation; D006224:Cricetinae; D003412:Cricetulus; D018076:DNA, Complementary / Q000235:genetics; D000091342:Endoplasmic Reticulum Chaperone BiP; D004792:Enzyme Precursors / Q000235:genetics / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism; D006031:Glycosylation; D006360:Heat-Shock Proteins; D006801:Humans; D008247:Lysosomes / Q000378:metabolism; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011499:Protein Processing, Post-Translational; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013045:Species Specificity; D014162:Transfection
D002352:Carrier Proteins; D018076:DNA, Complementary; D000091342:Endoplasmic Reticulum Chaperone BiP; D004792:Enzyme Precursors; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D016271:Proto-Oncogene Proteins c-myc; D011993:Recombinant Fusion Proteins; C058345:procathepsin D; D002402:Cathepsin D