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S Diment

Publications and source records attributed to S Diment.

9 recordsLinked to original sources
PubMed1992

Role of cathepsin D in antigen presentation of ovalbumin.

Modification of protein Ag by proteolysis is one of the principal steps in the presentation of Ag to Th cells. However, little is known about the enzymes participating in these events, their specificity or the characteristics of the natural fragments that they produce. Cathepsin D (CD) is an aspartyl protease identified in endosomes of APC. In this report, the role of CD in the processing of OVA has been investigated. OVA digested in vitro with purified CD was able to stimulate IL-2 secretion by three different OVA-specific I-Ad restricted Th cell hybridomas when it was presented by fixed APC. The digest of OVA was recognized in the context of I-Ad, but not by I-Ak-restricted OVA-specific Th cells. No difference was observed in the ability of OVA digested with CD to stimulate Th cells in the absence of FCS or in the presence of protease inhibitors indicating that extracellular proteases were not likely to contribute to processing of OVA. Taken together, these results suggest that CD is necessary and sufficient for the generation of an antigenic epitope from OVA. A fragment containing the epitope was isolated from the OVA digest by reverse phase HPLC. This fragment, which migrates in SDS-PAGE as a 10-kDa polypeptide, is a potent epitope. Its capacity to activate Th cells is compared to that of the tryptic peptide OVA323-339.

Animals↗
PubMed1990

Different roles for thiol and aspartyl proteases in antigen presentation of ovalbumin.

By using the model Ag, chicken OVA, the proteolytic events required for effective presentation of the antigenic epitope, OVA323-339 to H-2d-restricted Th cells were investigated. First, the ability of aspartyl and thiol proteases to generate antigenic fragments of Ova in vitro was determined. It was found that cathepsin D, an aspartyl protease, digested OVA to fragments that could be recognized by Th cells without further processing by APC. Cathepsin B, a thiol protease, was unable to generate antigenic fragments of OVA in vitro. These results provide evidence that APC do not require thiol protease activity for processing OVA. In contrast, APC were unable to present OVA to Th cells when thiol protease inhibitors were added to the incubation. Taken together, these observations indicate that thiol proteases may be important, not for processing, OVA, but for presentation of processed fragments by APC. This conclusion is supported by evidence obtained from experiments in which APC were treated with thiol protease inhibitors before addition of the antigenic peptide, OVA323-339. Under these conditions, the capacity of I-Ad at the cell surface to present OVA323-339 to Th cells was reduced. The results of these experiments provide evidence that Ag presentation of OVA may be achieved through the action of two different classes of proteases: aspartyl proteases such as cathepsin D, which process OVA to antigenic fragments, and thiol proteases such as cathepsin B, which are important for expression of functional MHC II molecules by APC.

Animals↗
PubMed1990

Immunolocalization of endosomal cathepsin D in rabbit alveolar macrophages.

Intravacuolar proteolysis appears to be an important component of antigen presentation, the activation of peptide hormones, and the conversion of biologically important mediators from inactive precursors. Cathepsin D has been identified in the endosomes of rabbit alveolar macrophages by biochemical analyses [Diment and Stahl, J. Biol. Chem. 260,15311, 1985; Diment et al., J. Biol. Chem. 263,6901, 1988]. Using affinity-purified goat antirabbit cathepsin D IgG, we have localized cathepsin D to the endosomes of rabbit alveolar macrophages. Immunofluorescent staining of frozen sections showed labeling in lysosomes and small vesicles in the periphery of the cell. Label was not seen on the plasma membrane. With immunoperoxidase labeling at the electron microscopic level on cells containing endocytosed mannose-BSA gold, we saw labeling in endosomes and classical lysosomes. When the results were quantitated using immunogold labeling of thin cryosections, we found that the majority of cathepsin D (62.2%) was present in lysosomes, 4.0% in large clear vacuoles, a surprisingly high percentage (29.3%) in small vesicles, 4.9% in endosomes, and none on the plasma membrane. We conclude from this study that, in addition to being present in lysosomes, cathepsin D is present in endosomes and in small peripheral vesicles.

Animals↗
PubMed1989

Cleavage of parathyroid hormone in macrophage endosomes illustrates a novel pathway for intracellular processing of proteins.

Most ligands which are taken up by macrophages are transported to lysosomes where they are degraded to their constituents by a concert of acid hydrolases. This process requires a number of intracellular events which result in the transport of ligands from light density endosomes to the more dense lysosomes. In contrast, our studies have shown that macrophages may process some incoming ligands in endosomes (Diment, S., and Stahl, P. D. (1985) J. Biol. Chem. 260, 15311-15317) and that cathepsin D, an aspartyl protease, is localized in these organelles (Diment, S., Leech, M. S., and Stahl, P. D. (1988) J. Biol. Chem. 263, 6901-6907). Using rabbit alveolar macrophages, which can be subjected to subcellular fractionation, we have traced the intracellular transport and processing of bovine parathyroid hormone (PTH-(1-84]. We present evidence that macrophages internalize PTH-(1-84). Once in endosomes the hormone is cleaved to fragments which include a bioactive peptide, PTH-(1-34), and then the fragments are returned to the extracellular medium, without delivery to lysosomes. The entire cycle from initial binding to release of PTH-(1-34) is achieved within 10-15 min, a time period consistent with findings in vivo. Our data provide evidence for a novel route for processing of an endocytosed ligand.

Animals↗
PubMed1988

Cathepsin D is membrane-associated in macrophage endosomes.

Previously we identified an acid protease activity which was located in the endosomes of rabbit alveolar macrophages (Diment, S., and Stahl, P.D. (1985) J. Biol. Chem. 260, 15311-15317). In this study, the endosomal protease is identified as cathepsin D by immunoprecipitation with polyclonal antibodies raised against rabbit cathepsin D and by NH2-terminal sequence. In order to elucidate the mechanism for targeting of cathepsin D to endosomes, we first examined the membrane association of cathepsin D with light (rho = 1.05 g/ml) and heavy density (rho = 1.1 g/ml) vesicles from Percoll density gradients. After sequential washes, 8.4 and 21.9% of cathepsin D activity remained associated with heavy and light density vesicles, respectively. This membrane-associated cathepsin D could not be solubilized in either buffer at pH 5.0 containing mannose 6-phosphate and EDTA or in buffer at pH 10.6. Solubilization required the detergent Triton X-100. To determine whether membrane-associated cathepsin D was found in endosomes, the enzyme was radioiodinated within endosomes and lysosomes with internalized lactoperoxidase. The membrane-associated form was detected in endosomes, but much less in lysosomes. Biosynthetic studies combined with the same extraction procedure revealed that macrophage cathepsin D is first synthesized as an inactive membrane-associated precursor. The precursor is processed to an active, membrane-associated form and then to the active soluble form found in lysosomes. Our studies provide evidence that 1) cathepsin D is in endosomes of macrophages; 2) cathepsin D is transported to endosomes as a membrane-associated form; and 3) the membrane-associated form is a biosynthetic precursor for the soluble form found in endosomes and lysosomes.

Animals↗
PubMed1987

Generation of macrophage variants with 5-azacytidine: selection for mannose receptor expression.

Mannose receptors are expressed only in primary macrophages. Established macrophage-derived cell lines, although apparently possessing the potential to synthesize mannose receptors, do not express them on their plasma membranes. Using the drug 5-Azacytidine, mannose receptor expression was induced in the macrophage-derived mouse cell line J774. Receptor positive cells were sorted through a fluorescent activated cell sorter (FACS) prior to cloning. Clones were isolated which continuously express mannose receptors in culture. These macrophages were able to endocytose beta-glucuronidase and phagocytose yeast particles via mannose receptors. Secretion of the lysosomal enzyme beta-hexosaminidase was also reduced in proportion to the degree of mannose receptor expression.

Animals↗
PubMed1985

Macrophage endosomes contain proteases which degrade endocytosed protein ligands.

Rabbit alveolar macrophages rapidly internalize and degrade mannosylated bovine serum albumin (125I-mannose-BSA). Trichloroacetic acid-soluble degradation products appear in the cells as early as 6 min after uptake at 37 degrees C, and in the extracellular medium after 10 min. Incubation of endocytic vesicles containing this ligand in isotonic buffers at pH 7.4 + ATP resulted in intravesicular proteolysis, which was inhibited by monensin, nigericin, or ammonium chloride. At pH 5.0, degradation proceeded rapidly and was abolished by lysis of the vesicles with 0.1% Triton X-100. Readdition of lysosomes to the incubation mixture did not increase the rate of prelysosomal degradation. Proteolysis of 125I-mannose-BSA was optimal at pH 4.5, and inhibited by low concentrations of the cathepsin D inhibitor pepstatin A. After subcellular fractionation of the macrophages on Percoll gradients, 125I-mannose-BSA sedimented with prelysosomal vesicles and was not transported to secondary lysosomes. Addition of pepstatin A to extracellular medium during internalization of prebound 125I-mannose-BSA partially inhibited degradation of ligand, and resulted in transfer of undegraded 125I-mannose-BSA to lysosomes after 20 min. Using 125I-bovine serum albumin as a substrate for the protease in the presence of 0.1% Triton X-100, we have shown that as much as 36% of the total pepstatin A-sensitive activity sediments with nonlysosomal membranes. After intraendosomal iodination using lactoperoxidase, a labeled protease was isolated by affinity chromatography on pepstatin-agarose. The labeled protease, which had a subunit size of 46 kDa, was detected in endocytic vesicles after 5 min of internalization. These results suggest that a cathepsin D-like protease is responsible for the degradation of 125I-mannose-BSA in macrophages, and that this ligand is degraded in a prelysosomal vesicle.

Ammonium Chloride↗
PubMed1985

Iodinated fibroblast beta-glucuronidase as a ligand for receptor-mediated endocytosis.

Antibodies raised to human placental beta-glucuronidase were shown to cross-react with the beta-glucuronidase secreted by mouse 3T3 fibroblasts, but did not react with other lysosomal enzymes. The beta-glucuronidase secreted by 3T3 cells was purified 15000-fold by chromatography on an affinity column made from this antibody and resolved into a single component, of Mr 68000, by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Iodinated samples of purified enzyme were taken up into mouse peritoneal macrophages by receptor-mediated endocytosis at a rate similar to that calculated previously for unlabelled enzyme, and uptake was competitively inhibited by yeast mannan. Binding of beta-glucuronidase to macrophages was saturable, with a Kd of 7 X 10(-9)l/mol, an affinity comparable with that calculated for the binding of mannosylated bovine serum albumin (Kd 1.3 X 10(-9)l/mol), a ligand specific for mannose receptors. Four times as many molecules of mannosylated albumin (12000) as of beta-glucuronidase (3000), however, bound to each cell. This purification and iodination procedure did not therefore have any adverse effect on the uptake properties of secreted beta-glucuronidase, and provides a ligand with which to investigate binding and specific endocytosis into a range of different types of cell.

Animals↗