Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.23.5 (cathepsin D)
4,130 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Adherent cultures of rat peritoneal macrophages secrete lysozyme and the lysosomal marker enzymes beta-glucuronidase, beta-N-acetylglucosaminidase and acid phosphatase; the levels of secreted lysosomal cathepsin D, however, were found to be insignificant. Incubation of the cells at 4 degrees C for 15 min with yeast mannan or with 50 mM mannose, methyl alpha-glucopyranoside, or N-acetylglucosamine caused the concentration of cathepsin D in the culture medium to increase 30-40-fold; mannose-6-phosphate had no effect. 125I-labeled cathepsin D was prepared and the binding constant to the macrophage cell surface was determined to be KD = 27 nM. The data suggest that cathepsin D binds to the mannose receptor of macrophages and that binding to this receptor is not in equilibrium with the bulk medium.
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PMID:Binding of cathepsin D to the mannose receptor on rat peritoneal macrophages. 193 26

There are at least three stages in the targeting of soluble lysosomal enzymes: transfer of N-acetylglucosaminyl 1-phosphate to high-mannose oligosaccharide side chains, removal of N-acetylglucosamine and recognition of the "uncovered" mannose 6-phosphate residues. Defects in the transfer reaction cause mucolipidoses II and III. Those in the subsequent stages of the targeting may result in similar clinical disorders. To differentiate between possible defects of the targeting in cultured cells we have developed a procedure for a combined detection of the phosphorylation, uncovering of the transferred phosphate residues and the targeting of lysosomal enzymes. For this purpose cultured cells are metabolically labelled with [32P]phosphate and a lysosomal enzyme, such as cathepsin D, is isolated from the labelled cells and the medium by immunoprecipitation. The immunoprecipitates are dissolved with sodium dodecylsulphate and incubated in the presence and absence of calf intestine alkaline phosphatase. We show that the treatment of the denatured protein results in hydrolysis of phosphomonoester groups and that the phosphodiester and the peptide bonds remain intact. The initial and the residual radioactivity associated with the lysosomal enzyme which represent the total phosphate and the phosphodiester groups, respectively, are determined by gel-electrophoresis, fluorography and densitometry. This procedure extends one of the previously established methods for the diagnosis of mucolipidoses II and III.
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PMID:Determination of the phosphorylation, uncovering of mannose 6-phosphate groups and targeting of lysosomal enzymes. 207 12

Subcellular fractionation of human monocyte-macrophages (HMM) yielded a fraction rich in endosomes, lysosomes, and mitochondria. This pellet was further fractionated in a metrizamide gradient and the subcellular organelles were distributed among seven distinct bands. All of the bands contained lysosomal enzymes in similar amounts. However one band, poor in mitochondria, was markedly enriched in cathepsin D and cholesteryl ester hydrolase activities. A number of different ligands (low density lipoproteins (LDL), malondialdehyde-altered LDL, beta-migrating very low density lipoprotein, high density lipoprotein, reductively methylated LDL, mannose-bovine serum albumin, and transferrin) were presented to HMM at a concentration of 20 micrograms/ml at 4 degrees C. Three minutes after warming the cells at 37 degrees C all ligands except two were found predominantly in the cathepsin D- and cholesteryl ester hydrolase-rich fraction. Unlike the other ligands, LDL had distributed to other more dense fractions and reductively methylated LDL was found mainly in less dense fractions. At a lower concentration, 2 micrograms/ml, the distribution of LDL was identical to the other ligands. In vitro incubation of the fractions obtained from the gradient suggested that cathepsin D was largely responsible for the hydrolysis of the lipoproteins. We conclude that studies of LDL metabolism in HMM must take into account the different processing of this ligand at commonly used concentrations.
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PMID:Processing of lipoproteins in human monocyte-macrophages. 214 42

Lysosomal enzymes contain a common protein determinant that is recognized by UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase, the initial enzyme in the formation of mannose 6-phosphate residues. To identify this protein determinant, we constructed chimeric molecules between two aspartyl proteases: cathepsin D, a lysosomal enzyme, and pepsinogen, a secretory protein. When expressed in Xenopus oocytes, the oligosaccharides of cathepsin D were efficiently phosphorylated, whereas the oligosaccharides of a glycosylated form of pepsinogen were not phosphorylated. The combined substitution of two noncontinuous sequences of cathepsin D (lysine 203 and amino acids 265-292) into the analogous positions of glycopepsinogen resulted in phosphorylation of the oligosaccharides of the expressed chimeric molecule. These two sequences are in direct apposition on the surface of the molecule, indicating that amino acids from different regions come together in three-dimensional space to form this recognition domain. Other regions of cathepsin D were identified that may be components of a more extensive recognition marker.
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PMID:Generation of a lysosomal enzyme targeting signal in the secretory protein pepsinogen. 217 24

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.
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PMID:Immunolocalization of endosomal cathepsin D in rabbit alveolar macrophages. 219 19

Proteinase A, a yeast aspartyl protease that is highly homologous to the mammalian lysosomal aspartyl protease, cathepsin D, was expressed in Xenopus oocytes and its biosynthesis and post-translational modifications were characterized. While 29-45% of the proteinase A was secreted from oocytes, approximately 37% of the cell-associated proteinase A underwent proteolytic cleavage, characteristic of delivery to a lysosomal organelle. Although proteinase A is not targeted to the yeast vacuole by a mannose 6-phosphate receptor-dependent pathway, 2-5% of the proteinase A molecules expressed in oocytes bound to a Man-6-P receptor column. However, analysis of its [2-3H]mannose-labeled oligosaccharides revealed that 14-23% of these units contain phosphomannosyl residues. A hybrid molecule (H6), in which the propiece and first 12 amino acids of proteinase A were changed to the cathepsin D sequence, was also expressed in oocytes. The binding of H6 to the Man-6-P receptor was approximately 12-fold greater than observed for proteinase A. This increased level of receptor binding could be accounted for by three factors: 1) a small increase in the total amount of phosphorylated oligosaccharides, 2) an increase in the number of oligosaccharides which acquire two phosphomonoesters, and 3) the presence of a greater percentage of oligosaccharides with one phosphomonoester which exhibit high affinity binding to the Man-6-P receptor. These results demonstrate that proteinase A is recognized by UDP-GlcNAc:lysosomal enzyme N-acetylglucosaminylphosphotransferase. However, this interaction is altered by the addition of cathepsin D sequences, resulting in the generation of a higher affinity ligand for binding to the Man-6-P receptor.
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PMID:Expression of the yeast aspartyl protease, proteinase A. Phosphorylation and binding to the mannose 6-phosphate receptor are altered by addition of cathepsin D sequences. 252 Dec 20

Despite the clear differences between the amino acid sequence and enzymatic specificity of aspartic and cysteine endopeptidases, the biosynthetic processing of lysosomal members of these two families is very similar. With in vitro translation and pulse-chase analysis in tissue culture cells, the biosynthesis of cathepsin D, a aspartic protease, and cathepsins B, H and L, cysteine proteases, are compared. Both aspartic and cysteine endopeptidases undergo cotranslational cleavage of an amino-terminal signal peptide that mediates transport across the endoplasmic reticulum (ER) membrane. Addition of high-mannose carbohydrate also occurs cotranslationally in the lumen of the ER. Proteases of both enzyme classes are initially synthesized as inactive proenzymes possessing amino-terminal activation peptides. Removal of the propeptide generates an active single-chain enzyme. Whether the single-chain enzyme undergoes asymmetric cleavage into a light and a heavy chain appears to be cell type specific. Finally, late during their biosynthesis both classes of enzymes undergo amino acid trimming, losing a few amino acid residues at the cleavage site between the light and heavy chains and/or at their carboxyltermini. During biosynthesis these enzymes are also secreted to some extent. In most cells the secreted enzyme is the proenzyme bearing some complex carbohydrate. Under certain physiological conditions the inactive secreted enzymes may become activated as a result of a conformational change that may or may not result in autolysis. Analysis of the biochemical nature of the various processing steps helps define the cellular pathway followed by newly synthesized proteases targeted to the lysosome.
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PMID:Biosynthesis of lysosomal endopeptidases. 266 88

In human mammary cancer cells, pro-cathepsin D (pro-Cath-D) is induced by estrogens and 50% of it is secreted. To determine whether its secretion is characteristic of mammary cells or transformed cells, we compared its production, processing, and glycosylation in primary cultures of normal mammary epithelial cells to those found in breast cancer cell lines. The cytosolic concentration of total cathepsin D (precursor and mature enzyme) measured by enzyme-linked immunosorbent assay was 8 times higher in cancer cells. Its mRNA level estimated by Northern blot analysis was 8 to 50 times higher and its secretion was 30 times higher in cancer cells. Using pulse-chase labeling, the cellular processing of pro-Cath-D was altered in hormone-dependent and -independent breast cancer cells in comparison to normal cells. This alteration resulted in a lower accumulation of mature enzyme, while the secretion and cytoplasmic accumulation of pro-Cath-D was greater in breast cancer cells than in normal cells. NH4Cl increased secretion of the proenzyme in normal cells but not in cancer cells. The secreted proenzyme was markedly heterogeneous and had a more acidic pI in MCF7 cells than in normal mammary cells. These acidic forms disappeared following endo-beta-N-acetylglucosaminidase H treatment indicating that the structural difference between pro-Cath-D of normal and of cancer mammary cells was located on high mannose or hybrid N-linked oligosaccharides. This difference may be responsible for the altered routing of the pro-Cath-D in breast cancer cells.
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PMID:Increased secretion, altered processing, and glycosylation of pro-cathepsin D in human mammary cancer cells. 273 31

Renin is an aspartyl protease which is highly homologous to the lysosomal aspartyl protease cathepsin D. During its biosynthesis, cathepsin D acquires phosphomannosyl residues that enable it to bind to the mannose 6-phosphate (Man-6-P) receptor and to be targeted to lysosomes. The phosphorylation of lysosomal enzymes by UDP-GlcNAc:lysosomal enzyme N-acetylglucosaminylphosphotransferase (phosphotransferase) occurs by recognition of a protein domain that is thought to be present only on lysosomal enzymes. In order to determine whether renin, being structurally similar to cathepsin D, also acquires phosphomannosyl residues, human renin was expressed from cloned DNA in Xenopus oocytes and a mouse L cell line and its biosynthesis and posttranslational modifications were characterized. In Xenopus oocytes, the majority of the renin remained intracellular and underwent a proteolytic cleavage which removed the propiece. Most of the renin synthesized by oocytes was able to bind to a Man-6-P receptor affinity column (53%, 57%, and 90%, in different experiments), indicating the presence of phosphomannosyl residues. In the L cells, the majority of the renin was secreted but 5-6% of the renin molecules contained phosphomannosyl residues as demonstrated by binding of [35S]methionine-labeled renin to the Man-6-P receptor as well as direct analysis of [2-3H]mannose-labeled oligosaccharides. Although the level of renin phosphorylation differed greatly between the two cell types examined, these results demonstrate that renin is recognized by the phosphotransferase and suggest that renin contains at least part of the lysosomal protein recognition domain.
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PMID:Renin, a secretory glycoprotein, acquires phosphomannosyl residues. 296 Jun 82

Golgi-membrane-bound Gal beta 1-4GlcNAc alpha 2-6-sialyltransferase (CMP-N-acetylneuraminate:beta-galactoside alpha 2-6-sialyltransferase, EC 2.4.99.1) behaves as an acute-phase reactant increasing about 5-fold in serum in rats suffering from inflammation. The mechanism of release from the Golgi membrane is not understood. In the present study it was found that sialyltransferase could be released from the membrane by treatment with ultrasonic vibration (sonication) followed by incubation at reduced pH. Maximum release occurred at pH 5.6, and membranes from inflamed rats released more enzyme than did membranes from controls. Galactosyltransferase (UDP-galactose:N-acetylglucosamine galactosyltransferase; EC 2.4.1.38), another Golgi-located enzyme, which does not behave as an acute-phase reactant, remained bound to the membranes under the same conditions. Release of the alpha 2-6-sialyltransferase from Golgi membranes was substantially inhibited by pepstatin A, a potent inhibitor of cathepsin D-like proteinases. Inhibition of release of the sialyltransferase also occurred after preincubation of sonicated Golgi membranes with antiserum raised against rat liver lysosomal cathepsin D. Addition of bovine spleen cathepsin D to incubation mixtures of sonicated Golgi membranes caused enhanced release of the sialyltransferase. Intact Golgi membranes were incubated at lowered pH in presence of pepstatin A to inhibit any proteinase activity at the cytosolic face; subsequent sonication showed that the sialyltransferase had been released, suggesting that the proteinase was active at the luminal face of the Golgi. Golgi membranes contained a low level of cathepsin D activity (EC 3.4.23.5); the enzyme was mainly membrane-bound, since it could only be released by extraction with Triton X-100 or incubation of sonicated Golgi membranes with 5 mM-mannose 6-phosphate. Immunoblot analysis showed that the transferase released from sonicated Golgi membranes at lowered pH had an apparent Mr of about 42,000 compared with one of about 49,000 for the membrane-bound enzyme. Values of Km for the bound and released enzyme activities were comparable and were similar to values reported previously for liver and serum enzymes. The work suggests that a major portion of sialyltransferase containing the catalytic site is released from a membrane anchor by a cathepsin D-like proteinase located at the luminal face of the Golgi and that this explains the acute-phase behaviour of this enzyme.
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PMID:The role of a cathepsin D-like activity in the release of Gal beta 1-4GlcNAc alpha 2-6-sialyltransferase from rat liver Golgi membranes during the acute-phase response. 314 77


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