Direct quantitative determination of ceramide glycosylation in vivo: a new approach to evaluate cellular enzyme activity of glucosylceramide synthase

Gupta, Vineet; Patwardhan, Gauri A.; Zhang, Qianjin; Cabot, Myles C.; Jazwinski, S. Michal

doi:10.1194/jlr.d002949

Cited by 33 publications

(38 citation statements)

References 45 publications

(44 reference statements)

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“…Cellular Ceramide Glycosylation and GSL Analysis-Ceramide glycosylation that is catalyzed in cells was analyzed as described previously (41). Cells were grown 24 h in 35-mm dishes (5 ϫ 10 6 cells/dish) in 10% FBS RPMI 1640 medium and MBO-asGCS (100 nM) was introduced as described above.…”

Section: Methodsmentioning

confidence: 99%

“…After 12 h of growth in 10% FBS RPMI 1640 medium, the cells were switched to 1% BSA RPMI 1640 medium containing 5 M NBD C 6 -ceramide complexed to BSA (Invitrogen). After 2 h of incubation at 37°C, the lipids were extracted and resolved on Partisil high performance thin layer chromatography (HPTLC) plates with fluorescent indicator (number 4806-410; Whatman, Florham Park, NJ) in a solvent system containing chloroform, methanol, and 3.5 N ammonium hydroxide (85:15:1, v/v/v) as described previously (41). NBD C 6 -glucosylceramide and NBD C 6 -ceramide were identified using AlphaImager HP imaging system (Alpha Innotech, San Leandro, CA) and quantitated on a Synergy HT multi-detection microplate reader.…”

Section: Methodsmentioning

confidence: 99%

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Ceramide Glycosylation by Glucosylceramide Synthase Selectively Maintains the Properties of Breast Cancer Stem Cells

Gupta

Bhinge

Hosain

et al. 2012

Journal of Biological Chemistry

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Background: Glucosylceramide synthase catalyzes ceramide glycosylation that regulates the synthesis of glycosphingolipids. Results: Increased globo-series glycosphingolipids in breast cancer stem cells activate c-Src signaling and ␤-catenin-mediated transcription up-regulating stem cell factors. Conclusion: Ceramide glycosylation maintains the stemness of cancer stem cells. Significance: Glycosphingolipids in cell membrane actively participate in maintaining cancer stem cells.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Ceramide Glycosylation by Glucosylceramide Synthase Selectively Maintains the Properties of Breast Cancer Stem Cells

Gupta

Bhinge

Hosain

et al. 2012

Journal of Biological Chemistry

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“…It has been shown that glucosylceramide synthase localizes to the Golgi in HeLa cells ( 35 ) but it cannot be fully ruled out that some GlcCer synthase activity might also exist in the ER ( 36 ). To test if ceramide glycosylation is affected in the GPI anchor defi cient cells, we performed an in vivo ceramide glycosylation assay ( 28 ). As can be seen in Fig.…”

Section: Decrease In Hexcer Affects Downstream Glycosphingolipidsmentioning

confidence: 99%

“…Samples were resolved on partisil HPTLC plates with fl uorescent indicator (Whatman). To distinguish GalCer and GlcCer the HPTLC plates were impregnated with borate as described previously ( 28 ). After dipping the plates into a 1% aqueous sodium tetraborate solution, the HPTLC plates were activated at 120°C for 30 min.…”

Section: Cellular Ceramide Glycosylation Assaymentioning

confidence: 99%

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Glycosylphosphatidylinositol anchors regulate glycosphingolipid levels

Loizides‐Mangold

David

Nesatyy

et al. 2012

Journal of Lipid Research

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This article is available online at http://www.jlr.org anchor has the core structure phosphatidylinositol (PI)-glucosamine (GlcN)-(Mannose) 3 -phosphoethanolamine (EtN-P), which is conserved among all species. After biosynthesis, the GPI anchor is attached posttranslationally to the newly generated C terminus of certain eukaryotic proteins destined for anchoring thereby tethering the protein to the membrane surface by the glycolipid moiety. GPI-anchored proteins can be released from the cell surface by phosphatidylinositol specifi c phospholipases and this cleavage event can induce major conformational changes on the GPIanchored protein itself ( 2 ).At least three organelles, the endoplasmic reticulum (ER), Golgi, and peroxisomes, are involved in the biosynthesis and remodeling of the GPI anchor. The biosynthesis is initiated on the outer side of the ER membrane. After the fi rst two reactions, the GPI anchor precursor is fl ipped and biosynthesis continues on the luminal side of the ER where the diacyl chains of phosphatidylinositol are then replaced by alkyl-acyl chains. This step is impaired in mutants of the peroxisomal alkyl phospholipid biosynthesis pathway ( 3 ).After protein attachment, the GPI anchor undergoes complex remodeling that begins in the ER with the removal of the inositol-linked acyl chain ( 4 ) and the remodeling of the GPI glycan part (5). Glycan remodeling is crucial for sorting GPI-anchored proteins into ER exit sites and their subsequent ER to Golgi transport ( 6 ). In mammalian cells, remodeling of the GPI anchor is then continued in the Golgi where the unsaturated fatty acid of the GPI anchor is replaced by a saturated fatty acid chain ( 7 ). Abstract Glycosylphosphatidylinositol (GPI) anchor biosynthesis takes place in the endoplasmic reticulum (ER).After protein attachment, the GPI anchor is transported to the Golgi where it undergoes fatty acid remodeling. The ER exit of GPI-anchored proteins is controlled by glycan remodeling and p24 complexes act as cargo receptors for GPI anchor sorting into COPII vesicles. In this study, we have characterized the lipid profi le of mammalian cell lines that have a defect in GPI anchor biosynthesis. Depending on which step of GPI anchor biosynthesis the cells were defective, we observed sphingolipid changes predominantly for very long chain monoglycosylated ceramides (HexCer). We found that the structure of the GPI anchor plays an important role in the control of HexCer levels. GPI anchordefi cient cells that generate short truncated GPI anchor intermediates showed a decrease in very long chain HexCer levels. Cells that synthesize GPI anchors but have a defect in GPI anchor remodeling in the ER have a general increase in HexCer levels. GPI-transamidase-defi cient cells that produce no GPI-anchored proteins but generate complete free GPI anchors had unchanged levels of HexCer. In contrast, sphingomyelin levels were mostly unaffected. We therefore propose a model in which the transport of very long chain ceramide from the ER to Golgi is regulated by...

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Regulation of lipid synthesis in myelin modulates neural activity and is required for motor learning

Kato

Aoyama

Nishida

et al. 2023

Glia

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Brain function relies on both rapid electrical communication in neural circuitry and appropriate patterns or synchrony of neural activity. Rapid communication between neurons is facilitated by wrapping nerve axons with insulation by a myelin sheath composed largely of different lipids. Recent evidence has indicated that the extent of myelination of nerve axons can adapt based on neural activity levels and this adaptive myelination is associated with improved learning of motor tasks, suggesting such plasticity may enhance effective learning. In this study, we examined whether another aspect of myelin plasticity—changes in myelin lipid synthesis and composition—may also be associated with motor learning. We combined a motor learning task in mice with in vivo two‐photon imaging of neural activity in the primary motor cortex (M1) to distinguish early and late stages of learning and then probed levels of some key myelin lipids using mass spectrometry analysis. Sphingomyelin levels were elevated in the early stage of motor learning while galactosylceramide levels were elevated in the middle and late stages of motor learning, and these changes were correlated across individual mice with both learning performance and neural activity changes. Targeted inhibition of oligodendrocyte‐specific galactosyltransferase expression, the enzyme that synthesizes myelin galactosylceramide, impaired motor learning. Our results suggest regulation of myelin lipid composition could be a novel facet of myelin adaptations associated with learning.

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Direct quantitative determination of ceramide glycosylation in vivo: a new approach to evaluate cellular enzyme activity of glucosylceramide synthase

Cited by 33 publications

References 45 publications

Ceramide Glycosylation by Glucosylceramide Synthase Selectively Maintains the Properties of Breast Cancer Stem Cells

Ceramide Glycosylation by Glucosylceramide Synthase Selectively Maintains the Properties of Breast Cancer Stem Cells

Glycosylphosphatidylinositol anchors regulate glycosphingolipid levels

Regulation of lipid synthesis in myelin modulates neural activity and is required for motor learning

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