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 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.

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 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 Physicochemical modifications of lysosomal hydrolases during intracellular transport

1973 ◽  
Vol 132 (2) ◽  
pp. 267-282 ◽  
Author(s):  
Alfred Goldstone ◽  
Harold Koenig
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Lysosomal Enzymes ◽  
Intracellular Transport ◽  
Specific Activity ◽  
Rat Kidney ◽  
Basic Form ◽  
Acid Hydrolases ◽  
Lysosomal Hydrolases ◽  
Acetylneuraminic Acid

1. The following fractions were prepared from rat kidney and characterized ultrastructurally, biochemically and enzymically: (a) an ordinary rough microsomal (RM1) fraction; (b) a special rough microsomal (RM2) fraction enriched seven- to nine-fold in acid hydrolases over the homogenate; (c) a smooth microsomal (SM) fraction; (d) a Golgi (GM) fraction enriched 2.5-fold in acid hydrolases and 10-, 15- and 20-fold in sialyltransferase, N-acetyl-lactosamine synthetase and galactosyltransferase respectively; (e) a lysosomal (L) fraction enriched 15- to 23-fold in acid hydrolases. The frequency of Golgi sacs and tubules seen in the electron microscope and the specific activity of the three glycosyltransferases in these fractions increased in the order: RM2<RM1<SM<GM. 2. Five lysosomal hydrolases, acid phosphatase, β-N-acetyl-hexosaminidase, β-galactosidase, β-glucuronidase and arylsulphatase, were characterized in these fractions with respect to (a) solubility on freeze–thawing and (b) electrophoretic mobility in polyacrylamide gels. 3. In the RM2 fraction each of these hydrolases occurred largely or exclusively as a single bound basic form coincident with cationic glycoprotein bands in gels (Goldstone et al., 1973). 4. In the L fraction these hydrolases were present largely as soluble, acidic (anionic) forms. 5. The solubility, electrophoretic heterogeneity and anodic mobility of these hydrolases increased progressively in subcellular fractions in the order: RM2<RM1<SM<GM<L. 6. These findings, together with evidence cited in the text showing that N-acetylneuraminic acid residues are responsible for the solubility and electronegative charge of these acidic forms and incorporation of these residues into the Golgi apparatus, support the following scheme for the biosynthesis of lysosomal enzymes. Each hydrolase is synthesized as a bound basic glycoprotein enzyme in a restricted portion of the rough endoplasmic reticulum. The soluble, acidic forms are generated as the nascent glycoprotein enzymes migrate through the Golgi apparatus through the attachment of sugar sequences containing N-acetylneuraminic acid.

scholarly journals Lysosomal enzyme trafficking in mannose 6-phosphate receptor-positive mouse L-cells: demonstration of a steady state accumulation of phosphorylated acid hydrolases.

1986 ◽  
Vol 102 (3) ◽  
pp. 943-950 ◽  
Author(s):  
C A Gabel ◽  
S A Foster
Keyword(s):  
Steady State ◽  
Gradient Centrifugation ◽  
Human Fibroblasts ◽  
Absolute Number ◽  
Acid Hydrolases ◽  
Chase Period ◽  
L Cells ◽  
High Mannose ◽  
Mannose 6 Phosphate ◽  
Enzyme Deficient

During their transport from the endoplasmic reticulum to lysosomes, newly synthesized acid hydrolases are phosphorylated in the Golgi apparatus to generate a common recognition marker, mannose 6-phosphate (Man 6-P). The phosphorylated acid hydrolases then bind to the Man 6-P receptor and are transported by an unknown route to lysosomes. To learn more about the delivery pathway, we examined the fate of the phosphorylated oligosaccharides synthesized by a Man 6-P receptor-positive line of mouse L-cells. In contrast to the rapid degradation of the recognition marker previously observed in mouse lymphoma cells (Gabel, C. A., D. E. Goldberg, and S. Kornfield. 1982. J. Cell Biol., 95:536-542), the number of high mannose oligosaccharides phosphorylated by the L-cells after a 30-min pulse labeling with [2-3H]mannose increased continuously during a subsequent 4-h chase period to a maximum of 9.3% of the total cell-associated structures. After 19 h of chase the absolute number of phosphorylated oligosaccharides declined, but the loss was accompanied by a general loss of cellular oligosaccharides such that 7.4% of the cell-associated high mannose oligosaccharides remained phosphorylated. The longevity of the Man 6-P recognition marker in the L-cells was verified by analyzing the ability of an individual acid hydrolase, beta-glucuronidase, to serve as a ligand for the Man 6-P receptor. At least 60% of the steady state beta-glucuronidase molecules isolated from the L-cells could undergo receptor-mediated endocytosis into enzyme-deficient human fibroblasts. Dense lysosomal granules isolated by metrizamide gradient centrifugation from [3H]mannose-labeled L-cells were found to be highly enriched in their content of phosphomonoester-containing oligosaccharides. The data indicate that acid hydrolases may retain their Man 6-P recognition markers within lysosomes, and suggest the possibility that dephosphorylation occurs at a nonlysosomal location through which the newly synthesized enzymes pass en route to lysosomes.

scholarly journals Cathepsin D precursors in clathrin-coated organelles from human fibroblasts.

1985 ◽  
Vol 101 (3) ◽  
pp. 824-829 ◽  
Author(s):  
E Schulze-Lohoff ◽  
A Hasilik ◽  
K von Figura
Keyword(s):  
Staphylococcus Aureus ◽  
Human Brain ◽  
Residence Time ◽  
Cathepsin D ◽  
Lysosomal Enzymes ◽  
Human Fibroblasts ◽  
Coated Membranes ◽  
The Mean ◽  
Mannose 6 Phosphate ◽  
And Staphylococcus Aureus

Coated vesicles were isolated from metabolically labeled human fibroblasts with the aid of affinity-purified antibodies against human brain clathrin and Staphylococcus aureus cells. The material adsorbed to the S. aureus cells was enriched in clathrin. When the S. aureus cells bearing the immunoadsorbed material were treated with 0.5% saponin, extracts containing the precursor form of cathepsin D were obtained. The extraction of the precursor was promoted in the presence of mannose 6-phosphate. Material adsorbed to S. aureus cells coated with control immunoglobulins was nearly free of clathrin and contained a small amount of the cathepsin D precursor (less than 20% of that adsorbed with anti-clathrin antibodies). The extraction of this cathepsin D precursor was independent of mannose 6-phosphate and was complete after a brief exposure to saponin. The amount of cathepsin D precursor in coated membranes varied between 0.4 and 2.5% of total precursor. Analysis of pulse chase-labeled fibroblasts revealed that cathepsin D was only transiently associated with coated membranes. The mean residence time of cathepsin D precursor in coated membranes was estimated to be 2 min. These observations support the view that coated membranes participate in the transfer of precursor forms of endogenous lysosomal enzymes to lysosomes.

scholarly journals Studies of the biosynthesis of the mannose 6-phosphate receptor in receptor-positive and -deficient cell lines.

1983 ◽  
Vol 97 (6) ◽  
pp. 1700-1706 ◽  
Author(s):  
D E Goldberg ◽  
C A Gabel ◽  
S Kornfeld
Keyword(s):  
Cell Lines ◽  
Lysosomal Enzymes ◽  
Cell Types ◽  
Receptor Protein ◽  
Complex Type ◽  
Cultured Cell ◽  
Selective Targeting ◽  
High Mannose ◽  
Mannose 6 Phosphate ◽  
Intracellular Pathway

Phosphomannosyl residues present on lysosomal enzymes are specifically recognized by the mannose 6-phosphate receptor protein. This interaction results in the selective targeting of lysosomal enzymes to lysosomes. While this pathway is operative in many cell types, we have found four cultured cell lines that are deficient in the ability to bind lysosomal enzymes containing phosphomannosyl residues to their intracellular or surface membranes (Gabel, C., D. Goldberg, and S. Kornfeld, 1983, Proc. Natl. Acad. Sci. USA, 80:775-779). These cells appear to segregate lysosomal enzymes by an alternate intracellular pathway. To determine the basis for the lack of mannose 6-phosphate receptor activity in these cell lines, we studied the biosynthesis of the receptor in receptor-positive (BW5147) and receptor-deficient (P388D1 and MOPC 315) cells. The cells were labeled with [2-3H]mannose or [35S]methionine and the receptor was immunoprecipitated with an antireceptor antiserum. BW5147 cells synthesize a receptor protein whose size increases after translation/glycosylation. MOPC 315 cells produce an apparently normal receptor and degrade it rapidly. P388D1 cells fail to synthesize any detectable receptor. The receptor from BW5147 and MOPC 315 cells is a glycoprotein with both high mannose and complex asparagine-linked oligosaccharides. The complex-type units become fully sialylated and remain so during long periods of chase.

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.

Regulation of Lysosomal Enzymes. II. Reversible Adaptation of Intestinal Acid Hydrolases During Starvation and Realimentation

1971 ◽  
Vol 49 (2) ◽  
pp. 170-176 ◽  
Author(s):  
I. D. Desai
Keyword(s):  
Lysosomal Enzymes ◽  
Specific Activity ◽  
The Body ◽  
Starvation Period ◽  
Small Intestinal Mucosa ◽  
Acid Hydrolases ◽  
Lysosomal Hydrolases ◽  
Intestinal Tissue ◽  
Body Tissues ◽  
Dietary Nutrients

The nutritional role of intestinal lysosomal enzymes in the regulation of reversible adaptation to starvation and realimentation by various dietary treatments is investigated. When rats were starved for periods ranging from 72 to 120 h, the specific activity of the representative lysosomal hydrolases, viz. acid phosphatase, β-glucuronidase, β-galactosidase, arylsulfatase, and cathepsin D of small intestinal mucosa, progressively increased reaching levels two to four times higher than the fed controls. During the starvation period of only 72 h, more than 60% of the intestinal weight and about 75% of the mucosal protein was lost. On realimentation with a complete diet such as laboratory rat chow, the specific activity of the intestinal lysosomal enzymes rapidly returned to normal and the lost weight of the intestinal tissue and of the body as a whole was restored. The regulatory effect of major dietary nutrients such as carbohydrates, proteins, and fats on the starvation-induced breakdown of the intestinal tissue reserves is presented and discussed. The findings of this study indicate that changes in the specific activity of the intestinal lysosomal enzymes are associated with adaptive processes requiring rapid turnover of body tissues for maintenance and survival of the animal during starvation and/or during conditions when dietary supply of certain nutrients is limited.

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