scholarly journals Evidence for lysosomal enzyme recognition by human fibroblasts via a phosphorylated carbohydrate moiety

1978 ◽  
Vol 170 (3) ◽  
pp. 643-650 ◽  
Author(s):  
Kurt Ullrich ◽  
Günther Mersmann ◽  
Ernst Weber ◽  
Kurt Von Figura
Keyword(s):  
Alkaline Phosphatase ◽  
Lysosomal Enzymes ◽  
General Scheme ◽  
Human Fibroblasts ◽  
High Uptake ◽  
Surface Receptors ◽  
Phosphatase Treatment ◽  
Mannose 6 Phosphate ◽  
Phosphorylated Sugars ◽  
Adsorptive Endocytosis

Adsorptive endocytosis of five different lysosomal enzymes from various human and non-human sources was susceptible to inhibition by mannose and l-fucose, methyl α-d-mannoside, α-anomeric p-nitrophenyl glycosides of mannose and l-fucose, mannose 6-phosphate and fructose 1-phosphate. A few exceptions from this general scheme were observed for particular enzymes, particularly for β-glucuronidase from human urine. The inhibition of α-N-acetylglucosaminidase endocytosis by mannose, p-nitrophenyl α-d-mannoside and mannose 6-phosphate was shown to be competitive. The loss of endocytosis after alkaline phosphatase treatment of lysosomal enzymes supports the hypothesis that the phosphorylated sugars compete with a phosphorylated carbohydrate on the enzymes for binding to the cell-surface receptors [Kaplan, Achord & Sly (1977) Proc. Natl. Acad. Sci. U.S.A.74, 2026–2030]. Endocytosis of ‘low-uptake’ forms of α-N-acetylglucosaminidase and α-mannosidase was likewise susceptible to inhibition by sugar phosphates and by alkaline phosphatase treatment, suggesting that ‘low-uptake’ forms are either contaminated with ‘high-uptake’ forms or are internalized via the same route as ‘high-uptake’ forms. The existence of an alternative route for adsorptive endocytosis of lysosomal enzymes is indicated by the unaffected adsorptive endocytosis of rat liver β-glucuronidase in the presence of phosphorylated sugars and after treatment with alkaline phosphatase.

scholarly journals Fibroblast receptor for lysosomal enzymes mediates pinocytosis of multivalent phosphomannan fragment

1980 ◽  
Vol 84 (1) ◽  
pp. 77-86 ◽  
Author(s):  
HD Fischer ◽  
M Natowicz ◽  
WS Sly ◽  
RK Bretthauer
Keyword(s):  
Molecular Weight ◽  
Alkaline Phosphatase ◽  
Lysosomal Enzymes ◽  
Human Fibroblasts ◽  
Recognition System ◽  
High Uptake ◽  
Acid Hydrolases ◽  
Phosphatase Treatment ◽  
Hydrolysis Of ◽  
Endoglycosidase H

Mild acid hydrolysis of phosphomannan secreted by the yeast hansenula holstii (NRRL Y- 2448) produces two phosphomannyl fragments which differ strikingly in their potency as inhibitors of pinocytosis of human β-glucuronidase by human fibroblasts. The larger molecular weight polyphosphomonoester fragment is 100,000-fold more potent an inhibitor of enzyme uptake than the smaller penta-mannosyl-monophosphate fragment. Binding to attached fibroblasts at 3 degrees C was much greater with the polyphosphomonoester fragment than with the pentamannosyl-monophosphate. The larger molecular weight fragment was also subject to adsorptive pinocytosis and was taken up by fibroblasts at a rate 30- fold greater than the rate of uptake of pentamannosyl-monophosphate. Evidence that the polyphosphomonoester fragment is taken up by the phosphomannosyl-recognition system that mediates uptake of lysosomal enzymes includes: (a) its pinocytosis is inhibited by the same compounds that competitively inhibit enzyme pinocytosis (mannose-6-phosphate and phosphomannan from saccharomyces cerevisiae mutant mnn-1); (b) alkaline phosphatase treatment greatly reduces its susceptibility to pinocytosis; (c) its pinocytosis is competitively inhibited by high-uptake human β-glucuronidase; and (d) this inhibition by high-uptake enzyme is dramatically reduced by prior treatment of the enzyme with alkaline phosphatase or endoglycosidase-H. Endoglycosidase-H treatment human β-glucuronidase dramatically reduced its susceptibility to pinocytosis by fibroblasts. The phosphomannosyl components of high- uptake enzyme released by endoglycosidase-H treatment were much less effective inhibitors of polyphosphomonoester pinocytosis than when present on the phosphomannyl-enzyme. These results suggest that high-uptake acid hydrolases may be polyvalent ligands analogous to the polyphosphomonoester mannan fragment whose pinocytosis depends on interaction of more than one phospho-mannosyl recognition marker with pinocytosis receptors on fibroblasts.

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 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 An alternative hypothesis of cellular transport of lysosomal enzymes in fibroblasts. Effect of inhibitors of lysosomal enzyme endocytosis on intra- and extra-cellular lysosomal enzyme activities

1978 ◽  
Vol 176 (3) ◽  
pp. 943-950 ◽  
Author(s):  
Kurt Von Figura ◽  
Ernst Weber
Keyword(s):  
Cell Surface ◽  
Lysosomal Enzyme ◽  
Lysosomal Enzymes ◽  
Alternative Hypothesis ◽  
Fold Increase ◽  
Cellular Transport ◽  
Intracellular Enzyme ◽  
Surface Receptors ◽  
Mannose 6 Phosphate ◽  
A Minor

Recapture of lysosomal enzymes secreted by fibroblasts was inhibited by growing the cells in the presence of either free or immobilized antibodies against lysosomal enzymes or in the presence of phosphorylated carbohydrates known to interact with the cell-surface receptors for lysosomal enzymes. The following results were obtained. 1. Conditions that prevent recapture of released lysosomal enzymes increase the rate of extracellular accumulation of these enzymes up to twice that of controls. 2. Growing cells for 12 days in the presence of 0.5mm-mannose 6-phosphate, which decreases β-N-acetylglucosaminidase endocytosis to less than 10% of that of controls, has no effect on the intracellular activity of this and four other lysosomal enzymes. 3. Growing cells for 4 days in the presence of 50mm-mannose 6-phosphate, which is a 1000-fold higher concentration than that required for 50% inhibition of lysosomal enzyme endocytosis, leads to a 4-fold increase in extracellular β-N-acetylglucosaminidase accumulation and a decrease in intracellular enzyme. These results give evidence that, in fibroblasts, transfer of lysosomal enzymes into lysosomes does not require secretion before a receptor-mediated recapture [Hickman & Neufeld (1972) Biochem. Biophys. Res. Commun.49, 992–999]. We propose that (a) lysosomal enzymes are present in a receptor-bound form in those vesicles that fuse with the cell membrane, (b) the major part of the lysosomal enzyme cycles via the cell surface in a receptor-bound form and (c) only a minor part of the lysosomal enzyme is released into the extracellular space during its life cycle.

scholarly journals Is movement of mannose 6-phosphate-specific receptor triggered by binding of lysosomal enzymes?

1987 ◽  
Vol 104 (6) ◽  
pp. 1735-1742 ◽  
Author(s):  
T Braulke ◽  
C Gartung ◽  
A Hasilik ◽  
K von Figura
Keyword(s):  
Cell Surface ◽  
Lysosomal Enzymes ◽  
Free Cell ◽  
Cell Surface Receptors ◽  
Receptor Ligands ◽  
Receptor Ligand ◽  
Surface Receptors ◽  
Weak Bases ◽  
Specific Receptors ◽  
Mannose 6 Phosphate

Mannose 6-phosphate-specific receptors with an apparent molecular mass of 215,000 are present in fibroblasts at the cell surface and in intracellular membranes. The cell surface receptors mediate endocytosis of exogenous lysosomal enzymes and exchange with the intracellular receptors, which function in the sorting of endogenous lysosomal enzymes. In the present study, several methods independent of receptor ligands were designed in order to examine the exchange of receptors under conditions where receptor-ligand complexes do not dissociate (weak bases and monensin) or where receptor-ligand complexes are not formed due to absence of endogenous ligands as a result of inhibition of protein synthesis. Weak bases and monensin reduce the concentration of receptors at the cell surface by 20-30% and free cell surface receptors were replaced by occupied receptors. The latter continued to be exchanged with internal ligand-occupied receptors and the rates of the exchange were similar to the control values. The exchange of receptors between the cell surface and internal membranes was also not affected when the receptor ligands were depleted from the transport compartments by treating the cells with cycloheximide for up to 10 h. We conclude from these results that movement of mannose 6-phosphate-specific receptors along the endocytosis and sorting pathways is constitutive and not triggered by binding or dissociation of ligands.

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