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)

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

In cultured human fibroblasts, maturation of the lysosomal enzymes beta-hexosaminidase and cathepsin D is inhibited by 10 mM-potassium cyanate. In cells treated with cyanate the two enzymes accumulate in precursor forms. The location of the accumulated precursor is probably non-lysosomal; in fractionation experiments the precursors separate from the bulk of the beta-hexosaminidase activity. The secretion of the precursor of cathepsin D, but not that of beta-hexosaminidase precursor, is enhanced in the presence of cyanate. The secreted cathepsin D, as well as that remaining within the cells, contains mostly high-mannose oligosaccharides cleavable with endo-beta-N-acetylglucosaminidase H. After removal of cyanate, the accumulated precursor forms of the lysosomal enzymes are largely released from the pretreated cells. It is concluded that cyanate interferes with the maturation of lysosomal-enzyme precursors by perturbing their intracellular transport. Most probably cyanate affects certain functions of the Golgi apparatus.
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PMID:Inhibition by cyanate of the processing of lysosomal enzymes. 622 28

In cultured human fibroblasts we observed that monensin, a Na+/H+-exchanging ionophore, (i) inhibits mannose 6-phosphate-sensitive endocytosis of a lysosomal enzyme, (ii) enhances secretion of the precursor of cathepsin D, while inhibiting secretion of the precursors of beta-hexosaminidase, (iii) induces secretion of mature beta-hexosaminidase and mature cathepsin D, and (iv) inhibits carbohydrate processing in and proteolytic maturation of the precursors remaining within the cells; this last effect appears to be secondary to an inhibition of the transport of the precursors. If the treated cells are transferred to a monensin-free medium, about half of the accumulated precursors are secreted, and the intracellular enzyme is converted into the mature form. Monensin blocks formation of complex oligosaccharides in lysosomal enzymes. In the presence of monensin, total phosphorylation of glycoproteins is partially inhibited, whereas the secreted glycoproteins are enriched in the phosphorylated species. The suggested inhibition by monensin of the transport within the Golgi apparatus [Tartakoff (1980) Int. Rev. Exp. Pathol. 22, 227-250] may be the cause of some of the effects observed in the present study (iv). Other effects (i, ii) are rather explained by interference by monensin with the acidification in the lysosomal and prelysosomal compartments, which appears to be necessary for the transport of endocytosed and of newly synthesized lysosomal enzymes.
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PMID:Effect of monensin on intracellular transport and receptor-mediated endocytosis of lysosomal enzymes. 623 17

Biosynthesis, transport, and maturation of cathepsin D and beta-hexosaminidase was examined in fibroblasts exposed to 1-deoxynojirimycin, a glucose analogue known to inhibit trimming glucosidases (Saunier, B., Kilker, R. D., Jr., Tkacz, J. S., Quaroni, A., and Herscovics, A. (1982) J. Biol. Chem. 257, 14155-14161; Hettkamp, H., Bause, E., and Legler, G. (1982) Biosci. Rep. 2, 899-906). Cells treated with 1-deoxynojirimycin contained precursors of cathepsin D and beta-hexosaminidase larger by about 1-2 kDa than control cells. The shift in molecular size was probably due to glucose residues that were rapidly removed from the precursors in the absence but not in the presence of 1-deoxynojirimycin. In addition, 1-deoxynojirimycin inhibited the glycosylation of the beta-chain precursor of beta-hexosaminidase and the synthesis of glycoproteins, including that of cathepsin D. The proteolytic processing of the larger precursors was retarded by several hours. The delay in proteolytic maturation was secondary to the accumulation of the larger precursors in organelles, which fractionated with membranes of the endoplasmic reticulum and Golgi complex. The accumulated cathepsin D precursor contained neither mannose 6-phosphate residues nor complex type oligosaccharides, which are formed in the cis and trans aspects of the Golgi complex. Cathepsin D precursors eventually released from the site of accumulation were apparently deglucosylated, acquired mannose 6-phosphate residues and complex type oligosaccharides, and were transferred into lysosomes as efficiently as in control cells. Our results suggest that transport of cathepsin D from the endoplasmic reticulum to the Golgi complex depends on removal of glucose residues from its carbohydrate.
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PMID:Cathepsin D and beta-hexosaminidase synthesized in the presence of 1-deoxynojirimycin accumulate in the endoplasmic reticulum. 623 13

Precursors of cathepsin D and beta-hexosaminidase synthesized in the U937 monocyte line are processed to mature forms with similar kinetics as in fibroblasts. In U937 cells the processing of the precursor of the beta-chain of beta-hexosaminidase, however, results in a larger fragment that resembles a processing intermediate in fibroblasts. This difference is explained by differences in the equipment of the cells with proteinases, since cross-feeding of the precursors to the cells results in a processing characteristic for the recipient cell type. In sucrose gradients the precursors are found partly in a low- and partly in a high-density region. Mature polypeptides and activity of lysosomal enzymes fractionate mainly in the higher density region. In U937 cells the transport and maturation of endogenous lysosomal enzymes are less sensitive to bases (NH4Cl, chloroquine, tilorone) and to antibody against the mannose 6-phosphate specific receptors than in fibroblasts. A small portion of enzymes released from U937 cells contains the markers recognized by the mannose-6-phosphate specific receptors. U937 cells express these receptors and utilize them for transport of endogenous and exogenous lysosomal enzymes. It appears, however, that a fraction of lysosomal enzymes is transported in U937 cells independent of the mannose-6-phosphate-specific receptors.
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PMID:Processing and transport of lysosomal enzymes in human monocyte line U937. 623 73

During pulse-chase experiments in cultured porcine kidney cells, an early 75-kilodalton (kDa) form of beta-glucuronidase is converted to a late 72-kDa form. The relative molecular weight difference between the two forms is maintained on removal of high-mannose carbohydrate with endoglycosidase H. Both forms have the same partial NH2-terminal sequence, and both migrate as single polypeptide chains following reduction, alkylation, and electrophoresis under denaturing conditions. On treatment with carboxypeptidase Y, the early form released [35S]Met faster than the late form. Thus, the late form of beta-glucuronidase is generated by COOH-terminal proteolytic processing of the early form. During similar experiments, the mass of the 30-kDa heavy chain of porcine cathepsin D decreased by about 1 kDa. The heavy chain of the two-chain enzyme is derived from the COOH terminus of a 44-kDa single-chain enzyme. On treatment with carboxypeptidase Y, the early single-chain enzyme released COOH-terminal [35S]Met and [3H]Lys faster than the later 29-kDa heavy chain. Like beta-glucuronidase, cathepsin D evidently undergoes COOH-terminal proteolytic processing during biosynthesis.
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PMID:Carboxyl-terminal proteolytic processing during biosynthesis of the lysosomal enzymes beta-glucuronidase and cathepsin D. 636 Feb 5

Cathepsin D from porcine spleen contained mannose (3.3%), glucosamine (1.4%), and mannose 6-phosphate (0.08%). Essentially all of the oligosaccharides of cathepsin D could be released by endo-beta-N-acetylglucosaminidase H, pointing to oligomannoside types of structures. Three neutral oligosaccharide fractions, containing 5, 6, and 7 mannose residues, respectively, were isolated by gel permeation chromatography on Bio-Gel P-2. Studies using exoglycosidase digestions and 500-MHz 1H NMR spectroscopy revealed that their structures are [Man alpha 1 leads to 2]0 or 1 Man alpha 1 leads to 6[Man alpha 1 leads to 3]Man alpha 1 leads to 6[Man alpha 1 leads to 2)0 or 1 Man alpha 1 leads to 3]Man beta 1 leads to 4GlcNAc beta 1 leads to 4 GlcNAc. These structures are identical to what have recently been proposed by Takahashi et al. for the major oligosaccharide units of cathepsin D from the same source (T. Takahashi, P. G. Schimidt, and J. Tang (1983) J. Biol. Chem. 258, 2819-2930), except for the occurrence of two isomeric oligosaccharides containing six mannoses. Only a part (3.4%) of the oligosaccharides were acidic, containing phosphates in monoester linkage. The phosphorylated oligosaccharides also consisted of oligomannoside-type chains which were analogous to, but more heterogeneous in size than the neutral oligosaccharides. Cathepsin D was bound to a mannose- and N-acetylglucosamine-specific lectin (mannan-binding protein) isolated from rabbit liver with the Ki value of 5.4 X 10(-6) M.
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PMID:Oligosaccharides on cathepsin D from porcine spleen. 642 53

The distribution of the different types of oligosaccharides in cathepsin D and in beta-hexosaminidase synthesized in cultured human fibroblasts was studied by using endo-beta-N-acetylglucosaminidase H as a probe for high-mannose oligosaccharides. The enzymes were specifically labelled in the protein or the carbohydrate moiety. In both enzymes, resistant and cleavable oligosaccharides were found. The resistant oligosaccharides prevailed in the secreted enzymes. Precursor molecules of cathepsin D contained two oligosaccharide side chains. Multiple forms of the precursor are synthesized with both, one or none of two oligosaccharides sensitive to the action of the endo-beta-N-acetylglucosaminidase H. In fibroblasts unable to phosphorylate lysosomal enzymes (mucolipidosis II) the excessively secreted lysosomal enzymes contained predominantly oligosaccharides resistant to endo-beta-N-acetylglucosaminidase H.
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PMID:Oligosaccharides in lysosomal enzymes. Distribution of high-mannose and complex oligosaccharides in cathepsin D and beta-hexosaminidase. 645 26

We have analyzed a soluble form of the glycoprotein (G) obtained from vesicular stomatitis virus (VSV) by treatment of intact virions with cathepsin D. This form lacks the carboxy-terminal and membrane-spanning domains and thus is analogous to the previously described secreted form of G, Gs. The molecular weight of the cathepsin D produced G, G(Cath D), measured by sedimentation equilibrium in the analytical ultracentrifuge is 57 600, indicating that it is a monomer. Intact G protein extracted from virions by octyl beta-D-glucoside also is monomeric, based on sedimentation equilibrium analysis. These results suggest that G may be monomeric in virions. The Stokes radii (Rs) of the two forms of G were obtained from their migration in nondenaturing polyacrylamide gradient gels. The Rs of G(Cath D) in the absence of nonionic detergent was 37 A; in the presence of nonionic detergent, it increased to 55 A. The Rs of detergent-extracted intact G was 63 A in nonionic detergent. From the molecular weight and Rs of G(Cath D), we calculated a sedimentation coefficient of 3.8 S; the value determined by centrifugation in a sucrose gradient was 3.7 S. Viruses such as VSV fuse with cell membranes at low pH [White, J., Matlin, K., & Helenius, A. (1981) J. Cell Biol. 89, 674-679]. We have used the fluorescent probe cis,trans,trans,cis-9,11,13,15-parinaric acid (cis-PnA) to detect a reversible conformational change in G(Cath D) when the protein was exposed to an acidic environment close to pH 5. cis-PnA binds to hydrophobic regions of protein, causing a quenching of the intrinsic tryptophan fluorescence and an increase in the fluorescence of the probe.
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PMID:Physical properties of a soluble form of the glycoprotein of vesicular stomatitis virus at neutral and acidic pH. 666 13

The amino acid sequences near the glycosylation sites and the oligosaccharide structures have been determined for the lysosomal protease cathepsin D from porcine spleen. Cathepsin D light and heavy chains were separately digested with proteases and the glycopeptides were purified. A single sequence was constructed from the amino acid sequence of the light chain glycopeptides which is: Tyr-Asn-Ser-Gly-Lys-Ser-Ser-Thr-Tyr-Val-Lys-Asn(CH2O)-Gly-Thr-Thr-Phe. A single glycopeptide sequence was also obtained for the heavy chain: Lys-Gly-Ser-Leu-Asp-Tyr-His-Asn(CH2O)-Val-Thr-Arg-Lys-Ala-Tyr. The light chain sequence is homologous with the sequence of porcine pepsin from residues 56 to 71. The heavy chain sequence is homologous with the pepsin sequence from residues 176 to 189. Thus, the 2 oligosaccharide-linked asparagines in cathepsin D correspond to residues 67 and 183 in pepsin and other homologous aspartyl proteases. These positions are located on the surface of the crystal structures of aspartyl proteases. Five oligosaccharides linked to Asn-67 were separated and their structures determined with proton NMR. Four major oligosaccharides are structural variants from the high mannose-type having 3, 5, 6, and 7 mannoses, respectively. A minor structure contained a third GlcNAc. Three oligosaccharide structures were found linked to Asn-183. Two major oligosaccharides are of the high mannose-type each with 5 mannose residues. One of the two contains a fucose linked to a GlcNAc. A third, very minor oligosaccharide contains galactose.
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PMID:Oligosaccharide units of lysosomal cathepsin D from porcine spleen. Amino acid sequence and carbohydrate structure of the glycopeptides. 682 41


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