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)

Chinese hamster ovary cells transfected with human lysozyme cDNA encoding Asn instead of Gly22 synthesize a mutant lysozyme, [Asn22]lysozyme, with about 60% of the molecules bearing carbohydrate. This carbohydrate is predominantly of the complex type and contains a varied number of lactosamine repeats. In this study we show that the glycosylation of [Asn22] lysozyme fused to human cathepsin D is altered relative to [Asn22]lysozyme alone. The fusion protein is synthesized as a 66-kDa precursor that is cleaved to enzymatically active and antigenically positive cathepsin D and lysozyme. As compared with [Asn22]lysozyme the lysozyme moiety of the fusion protein shows an increased N-glycosylation and a decreased synthesis of lactosamine repeats. Cleavage of the precursor with cathepsin L has revealed that the lysozyme portion of the secreted fusion protein bears a complex type carbohydrate. The intracellularly released lysozyme portion of the fusion protein contains trimmed oligosaccharides. In the presence of NH4Cl the lysosomal targeting of the fusion protein is inhibited. The secreted protein is then enriched in molecules bearing phosphorylated high mannose oligosaccharides in their lysozyme moiety. Our results indicate that carbohydrate processing in [Asn22]lysozyme, including the synthesis of mannose 6-phosphate residues and of lactosamine repeats, is altered by the attached cathepsin D. The phosphorylation of the carbohydrate on the lysozyme portion results in a very efficient lysosomal targeting of the concerned fusion protein molecules.
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PMID:Synthesis of phosphorylated oligosaccharides in lysozyme is enhanced by fusion to cathepsin D. 836 10

B lymphocytes from patients with I-cell disease (ICD) maintain normal cellular levels of lysosomal enzymes despite a deficiency of the enzyme UDP-N-acetylglucosamine: lysosomal enzyme N-acetylglucosamine-1-phosphotransferase. We find that an ICD B lymphoblastoid cell line targets about 45% of the lysosomal protease cathepsin D to dense lysosomes. This targeting occurs in the absence of detectable mannose 6-phosphate residues on the cathepsin D and is not observed in ICD fibroblasts. The secretory protein pepsinogen, which is closely related to cathepsin D in both amino acid sequence and three-dimensional structure, is mostly excluded from dense lysosomes, indicating that the lymphoblast targeting pathway is specific. Carbohydrate residues are not required for lysosomal targeting, since a non-glycosylated mutant cathepsin D is sorted with comparable efficiency to the wild type protein. Analysis of a number of cathepsin D/pepsinogen chimeric proteins indicates that an extensive polypeptide determinant in the cathepsin D carboxyl lobe can confer efficient lysosomal sorting when introduced into the pepsinogen sequence. This determinant overlaps but is not identical to the recognition marker for phosphotransferase. These results indicate that a specific protein recognition event underlies Man-6-P-independent lysosomal sorting in ICD lymphoblasts.
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PMID:Mannose 6-phosphate-independent targeting of lysosomal enzymes in I-cell disease B lymphoblasts. 840 10

The transport of proteins from the secretory to the endocytic pathway is mediated by carrier vesicles coated with the AP-1 Golgi assembly proteins and clathrin. The mannose 6-phosphate receptors (MPHs) are two major transmembrane proteins segregated into these transport vesicles. Together with the GTPase ARF-1, these cargo proteins are essential components for the efficient translocation of the cytosolic AP-1 onto membranes of the trans-Golgi network, the first step of clathrin coat assembly, MPR-negative fibroblasts have a low capacity of recruiting AP-1 which can be restored by re-expressing the MPRs in these cells. This property was used to identify the protein motif of the cation-dependent mannose 6-phosphate receptor (CD-MPR) cytoplasmic domain that is essential for these interactions. Thus, the affinity of AP-1 for membranes and in vivo transport of cathepsin D were measured for MPR-negative cells re-expressing various CD-MPR mutants. The results indicate that the targeting of lysosomal enzymes requires the CD-PDR cytoplasmic domain that are different from tyrosine-based endocytosis motifs. The first is a casein kinase II phosphorylation site (ESEER) that is essential for high affinity binding of AP-1 and therefore probably acts as a dominant determinant controlling CD-MPR sorting in the trans-Golgi network. The second is the adjacent di-leucine motif (HLLPM), which, by itself, is not critical for AP-1 binding, but is absolutely required for a downstream sorting event.
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PMID:A casein kinase II phosphorylation site in the cytoplasmic domain of the cation-dependent mannose 6-phosphate receptor determines the high affinity interaction of the AP-1 Golgi assembly proteins with membranes. 856 75

In many mammalian cells, the transport of newly synthesized or externally added lysosomal enzymes to lysosomes is depend on their specific recognition by receptors for mannose 6-phosphate (Man-6-P). The physiological importance of this pathway was confirmed by the finding that fibroblasts from patients with mucolipidosis type II (ML-II ; I - cell disease) fail to phosphorylate mannose residues on their newly synthesized lysosomal enzymes, which results in the secretion of a large percentage of their acid hydrolases into the culture medium. However, lysosomal enzymes themselves do not contain the any consensus amino acid sequences for acquiring the Man-6-P recognition marker. Kornfeld et al revealed using cathepsin D-pepsinogen chimera proteins that UDP-N-acetylglucosamine: lysosomal enzyme N-acetylglucosamine-1-phosphotransferase recognizes not only oligosaccharides but also the three-dimensional structure of the lysosomal enzymes when transfers N-acetylglucosamine-1-phosphate to lysosomal acid hydrolases.
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PMID:[Lysosomal hydrolases have specific conformational domains for acquisition of mannose-6-phosphate]. 857 31

The membrane-association of early biosynthetic form of cathepsin D has been demonstrated in hepatoma cells, and this membrane-association is not mediated by mannose 6-phosphate residues, implying that a mannose 6-phosphate receptor-independent mechanism operates in the sorting of cathepsin D. In this paper, to demonstrate whether cathepsin D is associated with the lysosomal membranes, an in vitro binding experiment was carried out employing lysosomal cathepsin D or microsomal procathepsin D isolated from rat liver. Immunoblotting analysis revealed that an intermediate form of cathepsin D was associated with the lysosomal membranes; this lysosomal membrane-associated cathepsin D was released from the membranes by washing with Na2CO3 (pH 10.6) but not with solutions containing mannose 6-phosphate. This suggested that cathepsin D associates with the membranes by ionic-interaction, and that the membrane-associated cathepsin D resides as a peripheral membrane protein in the lysosomal membrane fraction. To confirm that the intermediate form of cathepsin D specifically interacts with the lysosomal integral membrane proteins, the lysosomal membrane fraction was treated with trypsin and the binding experiment was conducted. The result showed that the binding capacity of cathepsin D to the lysosomal membranes was apparently abolished and cathepsin D did not rebind to the membranes. These data suggest that the intermediate form of cathepsin D is preferentially recognized by the lysosomal membranous protein which complements the mannose 6-phosphate receptor-dependent intracellular sorting mechanism.
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PMID:Cathepsin D associates with lysosomal membranous protein. 859 33

We studied the biogenesis of the acrosome in sperm cells in immunogold-labeled ultrathin cryosections of rat testis, using a variety of antibodies against endosomal/lysosomal marker protein and acrosin, the major secretory protein of sperm cells. As expected, acrosomes and proacrosomal vesicles in the trans-Golgi region contained abundant acrosin. Rat lysosomal membrane glycoprotein (lgp) 120 and mouse lysosome-associated membrane protein-1 were not detectable in the acrosomal membrane. Similarly, the late endosomal markers cation-dependent and -independent mannose 6-phosphate receptors were absent from the acrosome and proacrosomal vesicles. Therefore, acrosomes do not exhibit these endosomal/lysosomal features. Apart from (pro) acrosomal vesicles, both spermatocytes and spermatids contained classical lysosomes (positive for rat lgp 120, mouse lysosome-associated membrane protein-1, and cathepsin D) that were negative for acrosin. Quantitative analysis of the immunogold labeling showed that spermatocytes express more mannose 6-phosphate receptors and lgp 120 than spermatids, whereas the opposite situation existed for acrosin. These data indicate differential synthetic activity of lysosomal and acrosomal constituents in different states of sperm differentiation. Together, our observations argue against a lysosomal /endosomal origin of the acrosome.
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PMID:Evidence for a nonlysosomal origin of the acrosome. 860 90

The localization and intracellular transport of major histocompatibility complex (MHC) class II molecules nd lysosomal hydrolases were studied in I-Cell Disease (ICD) B lymphoblasts, which possess a mannose 6-phosphate (Man-6-P)-independent targeting pathway for lysosomal enzymes. In the trans-Golgi network (TGN), MHC class II-invariant chain complexes colocalized with the lysosomal hydrolase cathepsin D in buds and vesicles that lacked markers of clathrin-coated vesicle-mediated transport. These vesicles fused with the endocytic pathway leading to the formation of "early" MHC class II-rich compartments (MIICs). Similar structures were observed in the TGN of normal beta lymphoblasts although they were less abundant. Metabolic labeling and subcellular fractionation experiments indicated that newly synthesized cathepsin D and MHC class II-invariant chain complexes enter a non-clathrin-coated vesicular structure after their passage through the TGN and segregation from the secretory pathway. These vesicles were also devoid of the cation-dependent mannose 6-phosphate (Man-6-P) receptor, a marker of early and late endosomes. These findings suggest that in ICD B lymphoblasts the majority of MHC class II molecules are transported directly from the TGN to "early" MIICs and that acid hydrolases cam be incorporated into MIICs simultaneously by a Man-6-P-independant process.
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PMID:The biogenesis of the MHC class II compartment in human I-cell disease B lymphoblasts. 860 11

A previous observation that insulin-like growth factor II (IGF-II) inhibits the cellular uptake of a lysosomal enzyme by inhibiting binding to the IGF-II/mannose 6-phosphate receptor led to the proposal that, in a cell producing IGF-II, the routing of lysosomal enzymes might be altered. To test this hypothesis MCF-7 breast cancer cells were transfected with pRc/CMV vector only (CMV) or vector containing IGF-II complementary DNA encoding either mature (M-II) or precursor (P-II) IGF-II, and the routing of cathepsin D, a predominant lysosomal enzyme in this cell line, was examined. The concentration of IGF-II in media conditioned by P-II clones (11.2 +/- 4.3 micrograms/ml) was much higher than in media conditioned by M-II clones (1.3 +/- 1.5 micrograms/ml). Metabolic labeling experiments were performed with 10 mM mannose 6-phosphate present in the medium to block reuptake of lysosomal enzymes. Cell extracts (C) and media (M) were immuno-precipitated with a cathepsin D antiserum, and immunoprecipitates were analyzed by SDS-PAGE. The mean of the C/M ratio of cathepsin D for the seven P-II clones (1.60 +/- 0.13) was significantly lower than for the six CMV clones (3.47 +/- 0.48). Similar results were obtained when conditioned M and C were examined by immunoblotting after a 48-h incubation. The mean of the C/M ratio for the seven P-II clones (11.4 +/- 1.6) was significantly lower than for the six CMV clones (24.9 +/- 5.2). There was also a strong negative correlation between the ratio of intracellular cathepsin D to extracellular cathepsin D and relative cathepsin D synthesis (r = 0.843), consistent with increased cathepsin D production in cells overexpressing IGF-II. It is concluded that endogenous IGF-II modulates the routing of cathepsin D in MCF-7 cells.
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PMID:Insulin-like growth factor II modulates the routing of cathepsin D in MCF-7 breast cancer cells. 861 24

In the human carcinoma cell line HEp-2, endosomes are multivesicular bodies (MVBs) which gradually mature and eventually fuse with other mature endosomes and/or with preexisting lysosomes. Selective inhibition of the vacuolar H(+)-ATPase with bafilomycin A1 (Baf) did not influence endocytic uptake and recycling of the protein toxin ricin. Moreover, quantification of immunogold labeling on ultracryosections revealed that the frequency by which MVBs containing internalized cationized gold (Cat.Au) also labeled for the cation-independent mannose-phosphate receptor (MPR) was the same with and without Baf, suggesting that formation and maturation of MVBs were unchanged in the presence of Baf. However, degradation of ricin was strongly reduced by bafilomycin, and this reduction was not only due to increased pH in lysosomes. Thus, quantitative lmmunogold labeling showed that the Baf-induced alkalinization reduced the transfer of internalized Cat.Au to lysosomes, as defined by their content of human lysosome-associated membrane protein 1 (h-lamp-1) and cathepsin D. Accordingly, although low vacuolar pH does not seem to be required for transport to MPR-containing endosomes, it seems to be important for a late fusion step along the endocytic pathway.
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PMID:Inhibition of the vacuolar H(+)-ATPase with bafilomycin reduces delivery of internalized molecules from mature multivesicular endosomes to lysosomes in HEp-2 cells. 874 Dec 16

Cathepsin D carries a mannose 6-phosphate sorting signal which is recognized by a specific mannose 6-phosphate receptor, presumably at the site of the trans Golgi network, which segregates cathepsin D from the secretory proteins, and results in targeting of the enzyme to the acidic prelysosomal compartments and lysosomes in mammalian cells. Recent evidence implies that another sorting signal resides within the polypeptide backbone of the precursor cathepsin D. To evaluate the role of the propeptide region of cathepsin D in mannose 6-phosphate receptor-independent targeting to lysosomes, we prepared a deletion mutant of rat cathepsin D lacking the propeptide portion and analyzed its intracellular targeting mechanism after transfection of the mutant cDNA as well as the wild-type cDNA into COS cells. The glycosylated mutant protein was retained intracellularly, and extracellular release of mutant protein was not observed after a 48 h chase. A cell fractionation experiment demonstrated that in the cells expressing the wild-type cathepsin D, the processed form of 44 kDa cathepsin D was recovered in the dense lysosomal fraction. In contrast, in the cells expressing the mutant protein, virtually all of the cell-associated cathepsin D was present in the light fraction which was enriched in the marker enzyme NADPH cytochrome c reductase, and this molecular form of cathepsin D was not observed in the dense lysosomal fraction. An immunofluorescence study revealed that the deletion mutant protein was accumulated within the endoplasmic reticulum, unlike the wild-type protein. These results suggest that the mutant cathepsin D is not correctly recognized by the intracellular sorting system in the endoplasmic reticulum, implying that the propeptide region of cathepsin D is essential for the export of cathepsin D from the endoplasmic reticulum.
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PMID:Intracellular targeting of lysosomal cathepsin D in COS cells. 874 16


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