Demonstration of Direct Glycosylation of Nondegradable Glucosylceramide Analogs in Cultured Cells

Schwarzmann, Günter; Hofmann, Petra; Pütz, U.; Albrecht, Bernd

doi:10.1074/jbc.270.36.21271

Cited by 39 publications

(20 citation statements)

References 27 publications

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“…One possibility is that in normal cells, direct transport from the late endosomes to the Golgi complex occurs, but transport to the lysosomes is relatively minor. Consistent with this pathway are the observations that (i) a fraction of endocytosed Shiga toxin, a glycolipid-binding toxin, is transported to the trans-Golgi network (33)(34)(35)(36) during internalization and (ii) some exogenous glycosphingolipid analogs are transported in part to the Golgi complex during endocytosis (37,38). In this model, ''lipid sorting'' between the Golgi and the lysosomes would be altered in ML-IV cells, perhaps due to alteration of membrane properties or lysosomal accumulation of endogenous lipids.…”

Section: Resultssupporting

confidence: 49%

Abnormal transport along the lysosomal pathway in Mucolipidosis, type IV disease

Chen

Bach

Pagano

1998

Proc. Natl. Acad. Sci. U.S.A.

190

173

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Mucolipidosis, type IV (ML-IV) is an autosomal recessive storage disease that is characterized by lysosomal accumulation of sphingolipids, phospholipids, and acid mucopolysaccharides. Unlike most other storage diseases, the lysosomal hydrolases participating in the catabolism of the stored molecules appear to be normal. In the present study, we examined the hypothesis that the ML-IV phenotype might arise from abnormal transport along the lysosomal pathway. By using various markers for endocytosis, we found that plasma membrane internalization and recycling were nearly identical in ML-IV and normal fibroblasts. A f luorescent analog of lactosylceramide (LacCer) was used to study plasma membrane lipid internalization and subsequent transport. Lipid internalization at 19°C was similar in both cell types; however, 40-60 min after raising the temperature to 37°C, the f luorescent lipid accumulated in the lysosomes of ML-IV cells but was mainly concentrated at the Golgi complex of normal fibroblasts. Biochemical studies demonstrated that at these time points, hydrolysis of the lipid analog was minimal (ϳ7%) in both cell types. A f luorescence ratio imaging assay was developed to monitor accumulation of f luorescent LacCer in the lysosomes and showed that the apparent concentration of the lipid increased more rapidly and to a greater extent in ML-IV cells than in normal fibroblasts. By 60 min, LacCer apparently decreased in the lysosomes of normal fibroblasts but not in ML-IV cells, suggesting that lipid eff lux from the lysosomes was also impaired. These results demonstrate that there is a defect in ML-IV fibroblasts that affects membrane sorting and͞or late steps of endocytosis.

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Section: Resultssupporting

confidence: 49%

Abnormal transport along the lysosomal pathway in Mucolipidosis, type IV disease

Chen

Bach

Pagano

1998

Proc. Natl. Acad. Sci. U.S.A.

190

173

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“…A fraction of the internalized glucosylthioceramide is transported to the Golgi as indicated by its elongation to GM2 and GM3 by Golgi resident glycosyltransferases (Schwarzmann and Sandhoff, 1990;Schwarzmann et al, 1995;Schwarzmann, 2001). The glucosylthioceramide was taken up by control and LAMP-2-deficient hepatocytes.…”

Section: Trafficking Of Lipids Between the Plasma Membrane Lysosomesmentioning

confidence: 99%

“…This indicates mistargeting of a subset of lysosomal enzymes into the culture medium. 14 C]C 6 -GD1a with their relative mobilities denoted by standard abbreviations for GSL as explained in Schwarzmann et al (1995). In addition the mobilities of N-glycolyl neuraminic acid containing thiogangliosides GM3 and GM2 denoted as NeuGc-GM3 and NeuGc-GM2 are indicated, respectively.…”

Section: Activities and Trafficking Of Lysosomal Enzymesmentioning

confidence: 99%

Role of LAMP-2 in Lysosome Biogenesis and Autophagy

Eskelinen

Illert²,

Tanaka

et al. 2002

MBoC

Self Cite

321

260

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In LAMP-2–deficient mice autophagic vacuoles accumulate in many tissues, including liver, pancreas, muscle, and heart. Here we extend the phenotype analysis using cultured hepatocytes. In LAMP-2–deficient hepatocytes the half-life of both early and late autophagic vacuoles was prolonged as evaluated by quantitative electron microscopy. However, an endocytic tracer reached the autophagic vacuoles, indicating delivery of endo/lysosomal constituents to autophagic vacuoles. Enzyme activity measurements showed that the trafficking of some lysosomal enzymes to lysosomes was impaired. Immunoprecipitation of metabolically labeled cathepsin D indicated reduced intracellular retention and processing in the knockout cells. The steady-state level of 300-kDa mannose 6-phosphate receptor was slightly lower in LAMP-2–deficient hepatocytes, whereas that of 46-kDa mannose 6-phosphate receptor was decreased to 30% of controls due to a shorter half-life. Less receptor was found in the Golgi region and in vesicles and tubules surrounding multivesicular endosomes, suggesting impaired recycling from endosomes to the Golgi. More receptor was found in autophagic vacuoles, which may explain its shorter half-life. Our data indicate that in hepatocytes LAMP-2 deficiency either directly or indirectly leads to impaired recycling of 46-kDa mannose 6-phosphate receptors and partial mistargeting of a subset of lysosomal enzymes. Autophagic vacuoles may accumulate due to impaired capacity for lysosomal degradation

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“…Cholesterol and cholesterol esters were extracted as described (13), and the lower organic phase containing these lipids was collected and dried under N 2 . The extracted lipids were then separated by TLC using CHCl 3 For SL extraction and analysis (14,15), 900 l of cell suspension (see above) was mixed with 10 l of pyridine, followed by the addition of 4.5 ml of CH 3 OH and 4.5 ml of CHCl 3 , and stirred overnight at 40°C. The sample was then centrifuged for 10 min at 500 ϫ g to remove insoluble cellular debris.…”

Section: Methodsmentioning

confidence: 99%

Sphingolipid Storage Induces Accumulation of Intracellular Cholesterol by Stimulating SREBP-1 Cleavage

Puri

Jefferson²,

Singh

et al. 2003

Journal of Biological Chemistry

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We showed previously that the intracellular transport of sphingolipids (SLs) is altered in SL storage disease fibroblasts, due in part to the secondary accumulation of free cholesterol. In the present study we examined the mechanism of cholesterol elevation in normal human skin fibroblasts induced by treatment with SLs. When cells were incubated with various natural SLs for 44 h, cholesterol levels increased 25-35%, and cholesterol esterification was reduced. Catabolism of the exogenous SLs was not required for elevation of cholesterol because (i) a non-hydrolyzable and a degradable SL analog elevated cellular cholesterol to similar extents, and (ii) incubation of cells with various SL catabolites, including ceramide, had no effect on cholesterol levels. Elevated cholesterol was derived primarily from low density lipoproteins (LDL) and resulted from up-regulation of LDL receptors induced by cleavage of the sterol regulatory element-binding protein-1. Upon SL treatment, cholesterol accumulated with exogenous SLs in late endosomes and lysosomes. These results suggest a model in which excess SLs present in endocytic compartments serve as a "molecular trap" for cholesterol, leading to a reduction in cholesterol at the endoplasmic reticulum, induction of sterol regulatory element-binding protein-1 cleavage, and up-regulation of LDL receptors.Sphingolipid storage diseases (SLSDs) 1 are metabolic disorders that generally result from a defective lysosomal hydrolase or activator protein leading to accumulation of endogenous lipids in the lysosomes in many different cell types (1). We showed previously that a fluorescent glycosphingolipid (GSL) analog (BODIPY-lactosylceramide; LacCer) is internalized from the plasma membrane (PM) and transported to the Golgi complex in normal human skin fibroblasts (HSFs) but is mistargeted to endosomes and lysosomes in cells from multiple SLSDs, suggesting a common mechanism of cellular dysfunction in these biochemically distinct diseases (2). In a subsequent study we showed that these SLSD cells accumulate unesterified cholesterol in the late endosomes and lysosomes (3), and this cholesterol plays an important role in the altered sorting and targeting of the GSL analog in SLSD fibroblasts (3-6). Similarly, cholesterol has been reported to accumulate secondary to sphingomyelin storage in sphingomyelinase-deficient (Niemann-Pick A disease) human and mouse cells (7,8). The mechanism by which intracellular cholesterol is elevated and/or redistributes in SLSDs with primary defects in SL catabolism is unknown.In the current study, we show that incubation of normal HSFs with exogenous, naturally occurring SLs resulted in a dramatic increase and redistribution in cellular cholesterol reminiscent of that seen in SLSD cells. These observations led us to study further the relationship between SL accumulation and cellular cholesterol. We provide evidence that accumulation of SLs in endocytic vesicles serves as a molecular trap for cholesterol, binding and immobilizing the sterol as it circulates ...

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Demonstration of Direct Glycosylation of Nondegradable Glucosylceramide Analogs in Cultured Cells

Cited by 39 publications

References 27 publications

Abnormal transport along the lysosomal pathway in Mucolipidosis, type IV disease

Abnormal transport along the lysosomal pathway in Mucolipidosis, type IV disease

Role of LAMP-2 in Lysosome Biogenesis and Autophagy

Sphingolipid Storage Induces Accumulation of Intracellular Cholesterol by Stimulating SREBP-1 Cleavage

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