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)

The biosynthesis, processing, and intracellular transport of lysosomal acid phosphatase was studied using an in vitro cell-free translation system, pulse-chase experiments with primary cultured rat hepatocytes and subcellular fractionation techniques of rat liver after pulse-labeling with [35S]methionine in vivo. The single polypeptide of 45 kDa translated in the cell-free system from membrane-bound polysomal RNAs was converted to the 64 kDa form when the translation was carried out in the presence of microsomal vesicles. Pulse-chase experiments using cultured rat hepatocytes showed that acid phosphatase is initially synthesized as an endo-beta-N-acetylglucosaminidase H (Endo H)-sensitive form of 64 kDa, and processed via an Endo H-sensitive intermediate form of 62 kDa to an Endo H-resistant form with a 67 kDa mass. Phase separation with Triton X-114 showed that both the 64 and 67 kDa forms have hydrophobic properties. Treatment of the cells with chloroquine or tunicamycin, drugs which enhance the secretion of lysosomal hydrolases, had no effect on the normal transport of acid phosphatase to lysosomes. Acid phosphatase did not contain the phosphorylated high mannose type of oligosaccharide chains observed in cathepsin D. Subcellular fractionation experiments in conjunction with pulse-labeling in vivo showed that the acid phosphatase of the 67 kDa form was present in the Golgi heavy fraction (GF3) and the Golgi light fraction (GF1+2) enriched in cis and trans Golgi elements, respectively, at 30 min after the administration of [35S]methionine. Simultaneously, this polypeptide was also found in the lysosomal membrane fraction, thereby indicating that acid phosphatase is delivered to lysosomes in a membrane-bound form, immediately after reaching the trans-Golgi region.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Biosynthesis, processing, and intracellular transport of lysosomal acid phosphatase in rat hepatocytes. 169 35

In medium and in homogenates from baby-hamster kidney cells (BHK) transfected with human cathepsin D cDNA, an elevated activity of cathepsin D was found as compared to non-transfected cells. The elevated activity was removed by titrating the homogenates with an anti-(human cathepsin D) antibody. Metabolic labelling and immunoprecipitation revealed that, in the transfected cells, human cathepsin D was synthesized as a 53-kDa precursor indistinguishable from that found in human cells. A portion of the precursor was secreted and the remainder was processed to intermediate and mature chains within a few hours of synthesis. The precursor that was released from the transfected cells had a slightly smaller apparent size than that from cultured human fibroblasts. This difference was abrogated when the precursors were treated with glycopeptidase F. In the intracellular small chain a difference was observed in the size of carbohydrate chains that were cleavable with endo-beta-N-acetylglucosaminidase H. Sequence analysis of the N-termini of mature intracellular cathepsin D indicated a N-terminal trimming in both large and small chains from both human and transfected hamster cells. The proteolytic maturation of human cathepsin D in BHK cells closely resembles that in human cells, whereas a portion of the carbohydrate side chains is processed differently. The trimming of the N-termini in mature cathepsin D is proposed to be a part of the maturation and aging of this protein.
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PMID:Expression and maturation of human cathepsin D in baby-hamster kidney cells. 189 33

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

A significant elevation of cathepsin D activity was observed in six human hepatoma tissues as compared to 12 normal human livers. In isoelectric focusing experiments, cathepsin D purified from normal liver exhibited three different forms, with isoelectric points of 5.6, 6.1, and 6.7, while cathepsin D purified from hepatoma contained another five to six more acidic forms in addition to the forms observed in normal liver cathepsin D. When the tumor enzyme was treated with endo-beta-N-acetylglucosaminidase H followed by isoelectric focusing, the acidic components disappeared and were converted to forms identical to those of the normal liver cathepsin D. Determination of the mannose-6-phosphate content showed that hepatoma cathepsin D contains twice as much mannose-6-phosphate as normal liver cathepsin D. Peptide mapping and amino acid analysis showed that the protein moiety of cathepsin D from hepatoma is almost identical with that from normal liver. These findings indicate that the appearance of acidic variants in hepatoma cathepsin D is mainly due to changes in the oligosaccharide chains of the enzyme, which are closely associated with the increase of mannose-6-phosphate in the tumor enzyme.
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PMID:Elevated activity and increased mannose-6-phosphate in the carbohydrate moiety of cathepsin D from human hepatoma. 282 73

Cathepsin D was purified to apparently homogeneous form from normal human liver and hepatoma. The purified enzyme could not be distinguished between normal liver and hepatoma in terms of specific activity, subunit composition, antigenicity, amino acid composition and tryptic peptides. However, the hepatoma enzyme exhibited more charge heterogeneity to give multiple acidic variant forms which were devoid or much less in the normal liver enzyme. When the hepatoma enzyme was treated with endo-beta-N-acetylglucosaminidase H, the acidic variant forms disappeared and were converted into forms identical to those of normal liver. The content of mannose-6-phosphate in the hepatoma enzyme was twice as much as that in the normal liver enzyme. Thus, charge heterogeneity found in hepatoma cathepsin D is ascribed to increased phosphorylation on oligosaccharides bound to the enzyme, most probably due to cancer-associated, impaired processing in carbohydrate moiety. A significant elevation of cathepsin D activity per tissue proteins was observed in hepatoma as compared to normal liver. In contrast, true specific activity per cathepsin D protein in hepatoma was significantly lowered than that of normal liver. The lower true specific activity in hepatoma tissue may be attributed to an increased content in an inactive, large-molecular precursor form of the enzyme.
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PMID:[Tumor-associated impairment of the processing of hepatoma cathepsin D]. 283 81

The synthesis, transport and processing of lysosomal enzymes was examined in human hepatoma HepG2 cells and in human fibroblasts exposed to the Golgi alpha-mannosidase I inhibitor 1-deoxy-manno-nojirimycin. In HepG2 cells cathepsin D, beta-hexosaminidase and arylsulfatase B synthesized in the presence of 5 mM 1-deoxy-manno-nojirimycin contained exclusively endo-beta-N-acetylglucosaminidase H-cleavable oligosaccharides, indicating that alpha-mannosidase I had been inhibited efficiently. The proteolytic processing of intracellularly retained cathepsin D was retarded and the fraction of secreted cathepsin D was increased two-fold. In fibroblasts neither segregation nor maturation of cathepsin D were affected by 1-deoxy-manno-nojirimycin in spite of the inhibition of oligosaccharide processing. In the presence of the glucosidase I inhibitor 1-deoxynojirimycin, the precursor of cathepsin D (larger by about 1 kDa than the secreted form) accumulated transiently in light membranes in HepG2 cells. Release from the site of accumulation was accompanied by a decrease in size by about 1 kDa. This change was attributed to the removal of glucose residues. In fibroblasts the transient accumulation of larger precursors in the presence of 1-deoxynojirimycin was more pronounced than in HepG2 cells. The differential effects of alpha-mannosidase I and glucosidase I inhibitors on the transport of cathepsin D in HepG2 cells and fibroblasts may indicate that different intermediates in the biosynthetic pathway of asparagine-linked oligosaccharides participate in the transport of lysosomal enzymes in the two cell types.
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PMID:Cell type dependent inhibition of transport of cathepsin D in HepG2 cells and fibroblasts exposed to deoxy-manno-nojirimycin and deoxynojirimycin. 293 77

We have studied the posttranslational modifications of the 52-kD protein, an estrogen-regulated autocrine mitogen secreted by several human breast cancer cells in culture (Westley, B., and H. Rochefort, 1980, Cell, 20:353-362). The secreted 52-kD protein was found to be phosphorylated mostly (94%) on high-mannose N-linked oligosaccharide chains, and mannose-6-phosphate signals were identified. The phosphate signal was totally removed by alkaline phosphatase hydrolysis. The secreted 52-kD protein was partly taken up by MCF7 cells via mannose-6-phosphate receptors and processed into 48- and 34-kD protein moieties as with lysosomal hydrolases. By electron microscopy, immunoperoxidase staining revealed most of the reactive proteins in lysosomes. After complete purification by immunoaffinity chromatography, we identified both the secreted 52-kD protein and its processed cellular forms as aspartic and acidic proteinases specifically inhibited by pepstatin. The 52-kD protease is secreted in breast cancer cells under its inactive proenzyme form, which can be autoactivated at acidic pH with a slight decrease of molecular mass. The enzyme of breast cancer cells, when compared with cathepsin D(s) of normal tissue, was found to be similar in molecular weight, enzymatic activities (inhibitors, substrates, specific activities), and immunoreactivity. However, the 52-kD protein and its cellular processed forms of breast cancer cells were totally sensitive to endo-beta-N-acetylglucosaminidase H (Endo H), whereas several cellular cathepsin D(s) of normal tissue were partially Endo H-resistant. This difference, in addition to others concerning tissue distribution, mitogenic activity and hormonal regulation, strongly suggests that the 52-kD cathepsin D-like enzyme of breast cancer cells is different from previously described cathepsin D(s). The 52-kD estrogen-induced lysosomal proteinase may have important functions in facilitating the mammary cancer cells to proliferate, migrate, and metastasize.
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PMID:Phosphorylation, glycosylation, and proteolytic activity of the 52-kD estrogen-induced protein secreted by MCF7 cells. 354 22

Multiple biosynthetic forms of glucocerebrosidase were immunoprecipitated after synthesis in vitro using cell-free translation or in vivo using pulse-chase conditions in porcine kidney cells or human fibroblasts. The initial product in vitro was a 52-kDa polypeptide. When canine pancreatic microsomes were present during translation, the nascent polypeptide crossed the microsomal membrane and increased its mass to 60 kDa. Treatment of the 60-kDa polypeptide with endoglycosidase H to remove high mannose carbohydrate yielded a 51-kDa polypeptide. Thus, the membrane-translocated molecule was apparently a high mannose glycoprotein from which a signal peptide had been cleaved, as observed for the lysosomal protease cathepsin D (Erickson, A. H., and Blobel, G. (1979) J. Biol. Chem. 254, 11771-11774). Treatment of pancreatic microsomes or microsomes from porcine kidney cells with protease did not decrease the size of the polypeptide, which shows that this form is not a transmembrane protein bearing a cytoplasmic domain susceptible to digestion. The in vitro product synthesized in the presence of microsomal membranes was indistinguishable from the in vivo product synthesized during pulse-labeling of cultured porcine kidney cells. Following a 2-h chase period, the 60-kDa product was converted to a 59-kDa polypeptide. The major form of glucocerebrosidase detected after a 24-h chase period was a 56-kDa polypeptide, which in turn was converted to a 55-kDa polypeptide by 72 h. The same forms were precipitated from human fibroblasts but the rate of processing was accelerated in this cell type. Limited treatment of the 60-kDa form of glucocerebrosidase with endoglycosidase H suggested that high mannose carbohydrate is added to at least four sites on the polypeptide chain. By 24 h after synthesis, conversion to endoglycosidase H-resistant complex carbohydrate had occurred. Thus, both polypeptide and carbohydrate processing steps are involved in the biosynthesis of glucocerebrosidase.
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PMID:Biosynthesis of the lysosomal enzyme glucocerebrosidase. 393 53

We have investigated the basis for the specific recognition of lysosomal enzymes by UDP-GlcNAc:lysosomal enzyme N-acetylglucosaminylphosphotransferase. This enzyme is responsible for the selective phosphorylation of mannose residues on lysosomal enzymes. Two mammalian lysosomal enzymes, cathepsin D and uteroferrin, and two nonlysosomal glycoproteins were treated with endo-beta-N-acetylglucosaminidase H to remove those high mannose oligosaccharide units which are accessible on the native protein. These proteins were then tested as inhibitors of three different glycosyltransferases. The endo H-treated lysosomal enzymes were shown to be specific inhibitors of the phosphorylation of intact lysosomal enzymes. Proteolytic fragments of cathepsin D, including the entire light chain and heavy chain, did not retain the ability to be recognized by the N-acetylglucosaminylphosphotransferase. These findings indicate that the intact protein portion of lysosomal enzymes contains a specific recognition determinant which leads to high-affinity binding to the N-acetylglucosaminylphosphotransferase. The expression of this determinant appears to be dependent on the conformation of the protein.
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PMID:Lysosomal enzyme phosphorylation. Recognition of a protein-dependent determinant allows specific phosphorylation of oligosaccharides present on lysosomal enzymes. 609 68

Various biosynthetic forms of porcine spleen cathepsin D (Erickson, A. H. and Blobel, G. (1979) J. Biol. Chem. 254, 11771-11774), isolated by immunoprecipitation of in vivo- and in vitro-synthesized products, have been characterized by partial NH2-terminal sequence analysis. Two short lived and functionally distinct NH2-terminal sequence extensions, a "pre" sequence and a "pro" sequence, have been detected. Both sequence extensions are present in preprocathepsin D which is the primary translation product immunoprecipitated after translation of porcine spleen mRNA in a wheat germ cell-free system. Preprocathepsin D is not glycosylated and has an approximate Mr = 43,000. Its 20-residue pre sequence resembles the signal sequences of presecretory proteins in abundance of Leu residues (7 out of 20 residues). Addition of dog pancreatic microsomal vesicles to the translation system resulted in the cleavage of the pre sequence and yielded segregated and glycosylated procathepsin D (Mr = 46,000) that was indistinguishable from its in vivo-synthesized counterpart detected after pulse-labeling of cultured porcine kidney cells. Some of this in vivo-synthesized procathepsin D was secreted and persisted as such in the culture medium. The remainder was converted within a period of 15 min to 2 h to single chain cathepsin D (Mr = 44,000) by removal of a pro sequence which was estimated to be 44 residues. Its partial sequence showed considerable sequence homology to the 44-residue activation peptide of pepsinogen. It is possible, therefore, that the prosequence of procathepsin D serves as an activation peptide that keeps the enzyme inactive during intracellular transport to the lysosome. The enzymatically active single chain form of cathepsin D undergoes further cleavage into a light and a heavy chain (Mr = 15,000 and 30,000, respectively) over a period of 2-24 h after synthesis. The oligosaccharide moieties of procathepsin D and of the single chain and heavy chain forms of cathepsin D are cleaved by endoglycosidase H. Treatment of cells with tunicamycin arrests the biosynthetic pathway of cathepsin D at procathepsin D. The nonglycosylated procathepsin D is not proteolytically processed and its secretion is greatly inhibited.
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PMID:Biosynthesis of a lysosomal enzyme. Partial structure of two transient and functionally distinct NH2-terminal sequences in cathepsin D. 611 13


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