Sorting of Non-Glycosylated Human Procathepsin S in Mammalian Cells

Abstract

Cathepsin S, a lysosomal cysteine protease, is synthesized as inactive precursor. It is activated in the lysosomes by a proteolytic cleavage of the propeptide. HEK 293-cells which do not express cathepsin S were transfected with cDNA of either wild type human procathepsin S or a mutant procathepsin S in which Asn of the only glycosylation site in the proregion was replaced by Gln. The cells expressed glycosylated and non-glycosylated procathepsin S, respectively. Large amounts of the precursors were secreted into the culture media by both transfectants. Secreted wild type procathepsin S contained Man-6-phosphate in the oligosaccharide chain. Wild type procathepsin S was activated in the cells but no maturation occurred in the culture media. In vitro processing of glycosylated as well as of non-glycosylated procathepsin S gave fully active enzymes thus indicating that the oligosaccharide chain was not necessary for proper folding. A reuptake of the glycosylated and non-glycosylated procathepsin S by HEK 293-cells could be observed. Small amounts of mature cathepsin S were detected in the lysosomes of the mutant transfectants. Subcellular fractionation showed non-glycosylated procathepsin S in the membrane fraction. Non-glycosylated procathepsin S was bound to the plasma membrane at 2 degrees C, suggesting an additional sorting motif in the cathepsin S molecule besides the Man-6-phosphate residue.

Mutations of the C-Terminal End of Cathepsin K Affect Proenzyme Secretion and Intracellular Maturation

“…The importance of glycosylation and its associated mannose-6-phosphate pathway on the cellular maturation of cathepsins B, L and S has already been observed in various studies using inhibitors or mutagenesis approaches (25)(26)(27). When the cathepsin K-expressing cells were incubated with tunicamycin, a strong inhibition of enzyme maturation and secretion of the proenzyme was detected, indicating that both processes require glycosylation.…”

Modulation of allergen-specific T-lymphocyte function by virus-like particles decorated with HLA class II molecules

“…The 293 cells (3 3 10 6 ) were transiently cotransfected with HLA-DRA*0101, HLA-DRB1*0101, CD54::GPI, CD80::GPI, CD86::GPI, CD58::GPI, cathepsin S, 19 Ii constructs, minigenes, GFP control vector, or empty control vector (30 mg DNA) by the calcium phosphate precipitation method in combinations as indicated and used 72 hours after transfection. VLP induction was achieved by cotransfection of MoMLV gag/pol.…”

Molecular and functional analysis of the antigen receptor of Art v 1–specific helper T lymphocytes

“…Generation of invariant chain constructs pcDNA1.1/Amp containing an invariant chain (Ii) with a SfuI-Eco47III cassette instead of class II-associated Ii peptide (CLIP) was kindly provided by J. vanBergen (Immunohematology and Blood Transfusion, Leiden, The Netherlands). 24 The double-stranded Art v 1 [25][26][27][28][29][30][31][32][33][34] oligonucleotides Art v 1 [25][26][27][28][29][30][31][32][33][34] upper 59-CGAAGTGCATCGAGTGGGAGAAGGCCCAGCACCAGGC-39 and Art v 1 [25][26][27][28][29][30][31][32][33][34] lower 59-GCCTGGTGCTGGGCCTTCTCCCACTCG ATGCACTT-39 were inserted into SfuI/Eco47III-digested pcDNA1.1/AmpIi. CLIP and HA 309-317 containing Iis were constructed as previously described.…”

“…20 More than 95% of patients with mugwort allergy are sensitized to Art v 1, the major allergen in mugwort pollen. Analyzing the T-cell response to Art v 1 by using a panel of specific T-cell lines and clones revealed that the presentation of the sole immunodominant epitope (Art v 1 [25][26][27][28][29][30][31][32][33][34][35][36] ) is highly associated with HLA-DRB1*01. 21,22 Thus allergy to Art v 1 is characterized by a uniform T-cell response.…”