scholarly journals Inhibition by cyanate of the processing of lysosomal enzymes

1983 ◽  
Vol 210 (3) ◽  
pp. 795-802 ◽  
Author(s):  
A Hasilik ◽  
R Pohlmann ◽  
K von Figura
Keyword(s):  
Golgi Apparatus ◽  
Lysosomal Enzyme ◽  
Cathepsin D ◽  
Lysosomal Enzymes ◽  
Intracellular Transport ◽  
Human Fibroblasts ◽  
Cultured Human Fibroblasts ◽  
High Mannose ◽  
In Cells

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.

scholarly journals Effect of monensin on intracellular transport and receptor-mediated endocytosis of lysosomal enzymes

1984 ◽  
Vol 217 (3) ◽  
pp. 649-658 ◽  
Author(s):  
R Pohlmann ◽  
S Krüger ◽  
A Hasilik ◽  
K von Figura
Keyword(s):  
Golgi Apparatus ◽  
Lysosomal Enzyme ◽  
Cathepsin D ◽  
Lysosomal Enzymes ◽  
Intracellular Transport ◽  
Human Fibroblasts ◽  
Free Medium ◽  
Intracellular Enzyme ◽  
Cultured Human Fibroblasts ◽  
Monensin A

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.

scholarly journals Biosynthesis and transport of cathepsin D in cultured human fibroblasts.

1983 ◽  
Vol 97 (1) ◽  
pp. 1-5 ◽  
Author(s):  
V Gieselmann ◽  
R Pohlmann ◽  
A Hasilik ◽  
K Von Figura
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Cathepsin D ◽  
Human Fibroblasts ◽  
Total Cell ◽  
Molecular Forms ◽  
Cell Extracts ◽  
Cultured Human Fibroblasts ◽  
Immune Precipitation ◽  
High Mannose

For study of the time order of glycosylation, formation of complex oligosaccharides and proteolytic maturation as well as the site of proteolytic maturation of cathepsin D, fibroblasts were subjected to pulse-chase labeling, and cathepsin D was isolated from either total cell extracts or subcellular fractions by immune precipitation and analyzed for its molecular forms and sensitivity to endo-beta-N-acetylglucosaminidase H. After a 10-min pulse, cathepsin D was detected in its glycosylated precursor form, indicating an early, probably a cotranslational, N-glycosylation of cathepsin D. Conversion of the high-mannose oligosaccharide side chains into forms resistant to endo-beta-N-acetylglucosaminidase H started after approximately 40 min, indicating that transport of cathepsin D from the endoplasmic reticulum to the trans-Golgi apparatus requires approximately 40 min. Processing of the 53-kdalton precursor polypeptide of cathepsin D to a 47-kdalton intermediate followed about 20 min after the formation of complex oligosaccharides, and, another 30 min later, 31-kdalton mature forms of cathepsin D were detected. Processing of cathepsin D was first observed in light membranes as a partial conversion of the 53-kdalton precursor into the 47-kdalton intermediate. Both the precursor and the intermediate are transferred into the high density-class lysosomes. After 8 h, the processing to the mature 31-kdalton form of cathepsin D is mostly completed.

scholarly journals Recognition and receptor-mediated uptake of phosphorylated high mannose-type oligosaccharides by cultured human fibroblasts.

1983 ◽  
Vol 96 (3) ◽  
pp. 915-919 ◽  
Author(s):  
M Natowicz ◽  
D W Hallett ◽  
C Frier ◽  
M Chi ◽  
P H Schlesinger ◽  
...  
Keyword(s):  
Lysosomal Enzymes ◽  
Intracellular Transport ◽  
Specific Activity ◽  
Human Fibroblasts ◽  
Lysosomal Hydrolases ◽  
Minimal Structure ◽  
Cultured Human Fibroblasts ◽  
Specific Uptake ◽  
High Mannose ◽  
Mannose 6 Phosphate

The intracellular transport of newly synthesized lysosomal hydrolases to lysosomes requires the presence of one or more phosphorylated high mannose-type oligosaccharides per enzyme. A receptor that mediates mannose-6-PO4-specific uptake of lysosomal enzymes is expressed on the surface of fibroblasts and presumably accounts for the intracellular transport of newly synthesized enzymes to the lysosome. In this study, we examined the internalization of lysosomal enzyme-derived phosphorylated oligosaccharides by cultured human fibroblasts. Oligosaccharides of known specific activity bearing a single phosphate in monoester linkage were internalized with Kuptake of 3.2 X 10(-7) M, whereas oligosaccharides bearing two phosphates in monoester linkage were internalized with a Kuptake of 3.9 X 10(-8) M. Thus, phosphorylated high mannose-type oligosaccharides appear to be the minimal structure required for recognition and uptake by the fibroblast receptor. The finding that the Kuptake for monophosphorylated oligosaccharides is 100-fold less than the reported Ki for mannose-6-phosphate indicates that the fibroblast phosphomannosyl receptor contains a binding site that recognizes features of the oligosaccharide in addition to mannose-6-phosphate.

scholarly journals Biosynthesis and transport of lysosomal enzymes in human monocytes and macrophages. Effects of ammonium chloride, zymosan and tunicamycin

1983 ◽  
Vol 214 (3) ◽  
pp. 671-678 ◽  
Author(s):  
M Imort ◽  
M Zühlsdorf ◽  
U Feige ◽  
A Hasilik ◽  
K von Figura
Keyword(s):  
Ammonium Chloride ◽  
Lysosomal Enzyme ◽  
Cathepsin D ◽  
Lysosomal Enzymes ◽  
Human Fibroblasts ◽  
Human Monocytes ◽  
Zymosan A ◽  
Polypeptide Patterns ◽  
Monocytes And Macrophages ◽  
Human Monocytes And Macrophages

Human monocytes and macrophages synthesize lysosomal enzymes as larger precursors. The polypeptide patterns of several lysosomal-enzyme precursors and their mature forms are similar to those observed in human fibroblasts. Like fibroblasts, the monocytes and macrophages release small amounts of lysosomal-enzyme precursors. The lysosomotropic NH4+ cation enhances this release. In contrast, zymosan, a degranulating agent, causes release of both the mature and the precursor forms of the lysosomal enzymes. Both NH4Cl and zymosan inhibit maturation of the precursors. The fractional amounts of mature cathepsin D and beta-hexosaminidase released in the presence of zymosan are strikingly different. Probably, in the macrophages several lysosomal organelles are packaged with different relative contents of lysosomal enzymes. The transport of the precursors of cathepsin D into lysosomes is inhibited by tunicamycin. Therefore oligosaccharide side chains are likely to function as signals in packaging of lysosomal enzymes in macrophages also.

scholarly journals Secretion of β-N-acetylglucosaminidase isoenzymes by normal human fibroblasts

1978 ◽  
Vol 173 (2) ◽  
pp. 433-439 ◽  
Author(s):  
P Willcox
Keyword(s):  
Lysosomal Enzyme ◽  
Human Fibroblast ◽  
Lysosomal Enzymes ◽  
Culture Medium ◽  
Human Fibroblasts ◽  
The Other ◽  
Normal Human Fibroblast ◽  
Acid Hydrolases ◽  
Human Fibroblast Cultures ◽  
Normal Human

1. Secretion of the lysosomal enzyme beta-N-acetylglucosaminidase (EC 3.2.1.30) by normal human fibroblast cultures was linear with respect to time up to 96h. 2. Two forms of the A isoenzyme of beta-N-acetylglucosaminidase were found in the culture medium. One form was similar to the isoenzyme found in other extracellular fluids, such as plasma and tears, the other resembled the intracellular (lysosomal) enzyme. The presence of the two isoenzymes in the culture medium appears to reflect two distinct secretory processes. 3. It is suggested that plasma acid hydrolases may be destined for incorporation into lysosomes in a manner analogous to that described for the packaging of lysosomal enzymes by fibroblasts.

scholarly journals The role of lysosomal enzymes in killing of mammalian cells by the lysosomotropic detergent N-dodecylimidazole.

1987 ◽  
Vol 104 (5) ◽  
pp. 1223-1229 ◽  
Author(s):  
P D Wilson ◽  
R A Firestone ◽  
J Lenard
Keyword(s):  
Cell Viability ◽  
Ammonium Chloride ◽  
Lysosomal Enzyme ◽  
Lysosomal Enzymes ◽  
Human Fibroblasts ◽  
Cysteine Proteases ◽  
Cytotoxic Agents ◽  
Cysteine Cathepsins ◽  
Cathepsin Activity ◽  
Cysteine Cathepsin

The sensitivity of cultured human and hamster fibroblast cells to killing by the lysosomotropic detergent N-dodecylimidazole (C12-Im) was investigated as a function of cellular levels of general lysosomal hydrolase activity, and specifically of cysteine cathepsin activity. Fibroblasts from patients with mucolipidosis II (I-cell disease) lack mannose-6-phosphate-containing proteins, and therefore possess only 10-15% of the normal level of most lysosomal hydrolases. I-cell fibroblasts are about one-half as sensitive to killing by C12-Im as are normal human fibroblasts. Overall lysosomal enzyme levels of CHO cells were experimentally manipulated in several ways without affecting cell viability: Growth in the presence of 10 mM ammonium chloride resulted in a gradual decrease in lysosomal enzyme content to 10-20% of control values within 3 d. Subsequent removal of ammonium chloride from the growth medium resulted in an increase in lysosomal enzymes, to approximately 125% of control values within 24 h. Treatment with 80 mM sucrose caused extensive vacuolization within 2 h; lysosomal enzyme levels remained at control levels for at least 6 h, but increased 15-fold after 24 h of treatment. Treatment with concanavalin A (50 micrograms/ml) also caused rapid (within 2 h) vacuolation with a sevenfold rise in lysosomal enzyme levels occurring only after 24 h. The sensitivity of these experimentally manipulated cells to killing by C12-Im always paralleled the measured intracellular lysosomal enzyme levels: lower levels were associated with decreased sensitivity while higher levels were associated with increased sensitivity, regardless of the degree of vacuolization of the cells. The cytotoxicity of the cysteine proteases (chiefly cathepsin L in our cells) was tested by inactivating them with the irreversible inhibitor E-64 (100 micrograms/ml). Cell viability, protein levels, and other lysosomal enzymes were unaffected, but cysteine cathepsin activity was reduced to less than 20% of control values. E-64-treated cells were almost completely resistant to C12-Im treatment, although lysosomal disruption appeared normal by fluorescent visualization of Lucifer Yellow CH-loaded cells. It is concluded that cysteine cathepsins are the major or sole cytotoxic agents released from lysosomes by C12-Im. These observations also confirm the previous conclusions that C12-Im kills cells as a consequence of lysosomal disruption.

Glycosylation- and phosphorylation-dependent intracellular transport of lysosomal hydrolases

2009 ◽  
Vol 390 (7) ◽  
Author(s):  
Sandra Pohl ◽  
Katrin Marschner ◽  
Stephan Storch ◽  
Thomas Braulke
Keyword(s):  
Lysosomal Enzymes ◽  
Intracellular Transport ◽  
Dependent Manner ◽  
Lysosomal Storage ◽  
Lysosomal Hydrolases ◽  
Structural Requirements ◽  
Preferential Binding ◽  
High Mannose ◽  
Biosynthetic Studies ◽  
Storage Disorders

Abstract Lysosomes contain more than 50 soluble hydrolases that are targeted to lysosomes in a mannose 6-phosphate (Man6P)-dependent manner. The phosphorylation of man- nose residues on high mannose-type oligosaccharides of newly synthesized lysosomal enzymes is catalyzed by two multimeric enzymes, GlcNAc-1-phosphotransferase and GlcNAc-1-phosphodiester-α-N-acetylglucosaminidase, allowing the binding to two distinct Man6P receptors in the Golgi apparatus. Inherited defects in the GlcNAc-1-phosphotransferase complex result in missorting and cellular loss of lysosomal enzymes, and the subsequent lysosomal dysfunction causes the lysosomal storage disorders mucolipidosis types II and III. Biosynthetic studies and the availability of Man6P receptor-deficient mouse models have provided new insights into the structural requirements for preferential binding of subsets of lysosomal enzymes to Man6P receptors as well as the identification of alternative targeting pathways.

scholarly journals Effect of microtubule assembly status on the intracellular processing and surface expression of an integral protein of the plasma membrane.

1984 ◽  
Vol 99 (3) ◽  
pp. 1101-1109 ◽  
Author(s):  
A A Rogalski ◽  
J E Bergmann ◽  
S J Singer
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Golgi Apparatus ◽  
G Protein ◽  
Rough Endoplasmic Reticulum ◽  
Intracellular Transport ◽  
Temperature Shift ◽  
Surface Expression ◽  
Microtubule Assembly ◽  
In Cells

We studied the effects of changes in microtubule assembly status upon the intracellular transport of an integral membrane protein from the rough endoplasmic reticulum to the plasma membrane. The protein was the G glycoprotein of vesicular stomatitis virus in cells infected with the Orsay-45 temperature-sensitive mutant of the virus; the synchronous intracellular transport of the G protein could be initiated by a temperature shift-down protocol. The intracellular and surface-expressed G protein were separately detected and localized in the same cells at different times after the temperature shift, by double-immunofluorescence microscopic measurements, and the extent of sialylation of the G protein at different times was quantitated by immunoprecipitation and SDS PAGE of [35S]methionine-labeled cell extracts. Neither complete disassembly of the cytoplasmic microtubules by nocodazole treatment, nor the radical reorganization of microtubules upon taxol treatment, led to any perceptible changes in the rate or extent of G protein sialylation, nor to any marked changes in the rate or extent of surface appearance of the G protein. However, whereas in control cells the surface expression of G was polarized, at membrane regions in juxtaposition to the perinuclear compact Golgi apparatus, in cells with disassembled microtubules the surface expression of the G protein was uniform, corresponding to the intracellular dispersal of the elements of the Golgi apparatus. The mechanisms of transfer of integral proteins from the rough endoplasmic reticulum to the Golgi apparatus, and from the Golgi apparatus to the plasma membrane, are discussed in the light of these observations, and compared with earlier studies of the intracellular transport of secretory proteins.

scholarly journals Expression of Mutant Dynamin Inhibits Toxicity and Transport of Endocytosed Ricin to the Golgi Apparatus

1998 ◽  
Vol 140 (3) ◽  
pp. 553-563 ◽  
Author(s):  
Alicia Llorente ◽  
Andrzej Rapak ◽  
Sandra L. Schmid ◽  
Bo van Deurs ◽  
Kirsten Sandvig
Keyword(s):  
Golgi Apparatus ◽  
Intracellular Transport ◽  
Trichostatin A ◽  
Selective Inhibition ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Tyrosine Sulfation ◽  
Cell Biol ◽  
The Difference ◽  
In Cells

Endocytosis and intracellular transport of ricin were studied in stable transfected HeLa cells where overexpression of wild-type (WT) or mutant dynamin is regulated by tetracycline. Overexpression of the temperature-sensitive mutant dynG273D at the nonpermissive temperature or the dynK44A mutant inhibits clathrin-dependent endocytosis (Damke, H., T. Baba, A.M. van der Blieck, and S.L. Schmid. 1995. J. Cell Biol. 131: 69–80; Damke, H., T. Baba, D.E. Warnock, and S.L. Schmid. 1994. J. Cell Biol. 127:915–934). Under these conditions, ricin was endocytosed at a normal level. Surprisingly, overexpression of both mutants made the cells less sensitive to ricin. Butyric acid and trichostatin A treatment enhanced dynamin overexpression and increased the difference in toxin sensitivity between cells with normal and mutant dynamin. Intoxication with ricin seems to require toxin transport to the Golgi apparatus (Sandirg, K., and B. van Deurs. 1996. Physiol. Rev. 76:949–966), and this process was monitored by measuring the incorporation of radioactive sulfate into a modified ricin molecule containing a tyrosine sulfation site. The sulfation of ricin was much greater in cells expressing dynWT than in cells expressing dynK44A. Ultrastructural analysis using a ricin-HRP conjugate confirmed that transport to the Golgi apparatus was severely inhibited in cells expressing dynK44A. In contrast, ricin transport to lysosomes as measured by degradation of 125I-ricin was essentially unchanged in cells expressing dynK44A. These data demonstrate that although ricin is internalized by clathrin-independent endocytosis in cells expressing mutant dynamin, there is a strong and apparently selective inhibition of ricin transport to the Golgi apparatus. Also, in cells with mutant dynamin, there is a redistribution of the mannose-6-phosphate receptor.

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