Subcellular biosynthesis and transport of gangliosides formed from exogenous lactosylceramide in rat liver

Trinchera, Marco; Ghidoni, Riccardo

doi:10.1042/bj2660363

Cited by 20 publications

(18 citation statements)

References 45 publications

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“…We conclude that the GSLs catabolism is decreased in CDG-Ia cells. This is supported by the finding that CDG-Ia fibroblasts displayed 30% less SM than normal controls, since the biosynthesis of SM occurs via a recycling route of the free sphingoid base released in the lysosomes (19).…”

Section: Gsl Degradation In Cdg-ia Fibroblasts After Labeling With ([supporting

confidence: 70%

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Increased Biosynthesis of Glycosphingolipids in Congenital Disorder of Glycosylation Ia (CDG-Ia) Fibroblasts

SALA

2002

Pediatric Research

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Congenital disorder of glycosylation Ia (CDG-Ia) is an autosomal recessive disease, characterized by the impaired biosynthesis of the N-linked oligosaccharide chains of proteins due to a deficiency of phosphomannomutase (PMM), the enzyme converting mannose-6-phosphate into mannose-1-phosphate. We investigated the consequences of the altered N-linked glycoprotein (GP) biosynthesis on the quantity and quality of glycosphingolipids (GSLs) in fibroblasts of CDG-Ia patients. First, we found that CDG-Ia fibroblasts contain an increased amount of total GSLs when compared with normal fibroblasts. Further, we assessed by metabolic labeling of CDG-Ia fibroblasts with radioactive sugar precursors, including galactose and N-acetylmannosamine, that a diminished biosynthesis of cellular GPs is antagonized by an increased biosynthesis of GSLs. An increased GSL biosynthesis was also observed by means of radiolabeled lipid precursors including sphingosine and lactosylceramide. Notably, also the degradation of GLSs is slowed down in CDG-Ia fibroblasts. Finally, when we labeled normal human fibroblasts and CHO cells with radioactive galactose in the presence and absence of deoxymannojirimycin (dMM), an inhibitor of N-glycan processing, we found that this cellular model mimics what occurs in CDG-Ia fibroblasts. Since an inverse relationship between GP expression and GSL content does exist, we assume that increased glycosphingolipid biosynthesis is secondary to protein hypoglycosylation. Altogether, our data suggest that the cell metabolic machinery may be able to partially re-equilibrate protein hypoglycosylation with increased biosynthesis of glycosphingolipids, possibly to preserve the overall physico-chemical equilibrium of the outer layer of the plasma membrane. Glycoproteins (GPs) and glycosphingolipids (GSLs) represent two major classes of glycoconjugates present in mammalian cells. The glycosylation of both classes of molecules shares some common features. Noteworthy, 1) the subcellular distribution of GP and GSL glycosylating enzymes is similar, with the Golgi apparatus being the compartment where the majority of glycosylations occurs (1-3); 2) some of the enzymes which glycosylate proteins and lipids are the same, e.g. those responsible for blood group antigenicity (4, 5); 3) the precursor pool of nucleotide-sugars of both GP and GSL glycosylation within the Golgi lumen is indistinguishable (6); 4) the GP and GSL transport through the Golgi complex displays biochemical and kinetical similarities (7); 5) a relevant portion of neosynthesized GPs and the totality of GSLs are delivered from the Golgi to the plasma membrane, the final site

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Section: Gsl Degradation In Cdg-ia Fibroblasts After Labeling With ([supporting

confidence: 70%

“…We used this molecule, since the labeling on the sphingoid base is conserved in degradation products originated from lactosylceramide itself (19). We detected a consistent impairment of glycohydrolases-assisted deglycosylation in CDG-Ia fibroblasts.…”

Section: Gsl Degradation In Cdg-ia Fibroblasts After Labeling With ([mentioning

confidence: 91%

Increased Biosynthesis of Glycosphingolipids in Congenital Disorder of Glycosylation Ia (CDG-Ia) Fibroblasts

SALA

2002

Pediatric Research

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“…Thus far, only limited data have been published on GlcT-1: successful solubilization of the enzyme from rat Golgi fraction (4), the different properties of liver and brain enzymes (5), and a specific inhibitor of the enzyme (6). Papers from several laboratories have demonstrated that the synthesis of GlcCer occurs at the cytosolic surface of the Golgi apparatus, whereas other glycosylation reactions of GSL synthesis take place at the lumenal side of the organelle (7)(8)(9). Although the topology of the lactosylceramide formation, the next step of the synthesis, remains unknown and controversial, recent studies suggest the lumenal orientation of the synthase (10,11).…”

Section: Introductionmentioning

confidence: 99%

Expression cloning of a cDNA for human ceramide glucosyltransferase that catalyzes the first glycosylation step of glycosphingolipid synthesis.

Ichikawa

Sakiyama

Suzuki

et al. 1996

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

297

242

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We have isolated a cDNA encoding human ceramide glucosyltransferase (glucosylceramide synthase, UDP-glucose:N-acylsphingosine D-glucosyltransferase, EC 2.4.1.80) by expression cloning using as a recipient GM-95 cells lacking the enzyme. The enzyme catalyzes the first glycosylation step of glycosphingolipid synthesis and the product, glucosylceramide, serves as the core of more than 300 glycosphingolipids. The cDNA has a G+C-rich 5' untranslated region of 290 nucleotides and the open reading frame encodes 394 amino acids (44.9 kDa). A hydrophobic segment was found near the N terminus that is the potential signal-anchor sequence. In addition, considerable hydrophobicity was detected in the regions close to the C terminus, which may interact with the membrane. A catalytically active enzyme was produced from Escherichia coli transfected with the cDNA. Northern blot analysis revealed a single transcript of 3.5 kb, and the mRNA was widely expressed in organs. The amino acid sequence of ceramide glucosyltransferase shows no significant homology to ceramide galactosyltransferase, which indicates different evolutionary origins of these enzymes.

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“…GlcT-1 is a type III membrane protein. Its catalytic domain is located at the cytosolic surface of the Golgi apparatus (17)(18)(19)(20); thus, GlcT-1-mediated reactions occur on the cytosolic surface. Other glycosylation reactions involved in GSL synthesis, however, take place on the luminal surface of the Golgi (21,22).…”

mentioning

confidence: 99%

Drosophila Glucosylceramide Synthase

Kohyama-Koganeya

Sasamura

Oshima

et al. 2004

Journal of Biological Chemistry

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Glucosylceramide synthase (GlcT-1) catalyzes the formation of glucosylceramide (GlcCer), the core structure of major glycosphingolipids (GSLs), from ceramide and UDP-glucose. Ceramide and its metabolites, such as sphingosine-1-phosphate, are now known to be important mediators of apoptosis and cell survival. Recently, we have shown that GlcT-1 functions to regulate intracellular ceramide levels via glycosylation of ceramide. In this study, we employ the fruit fly Drosophila melanogaster as a model system for understanding the in vivo roles of GlcT-1. We isolated and characterized a GlcT-1 homologue (DGlcT-1) from Drosophila. When DGlcT-1 was expressed in GM-95 cells deficient in GSLs (because of the absence of GlcT-1 activity), these cells regained the ability to synthesize GSLs. Northern blot and in situ hybridization analyses revealed that the expression of DGlcT-1 mRNA was ubiquitous throughout development, suggesting that DGlcT-1 is important for development and differentiation. Indeed, RNA interference experiments demonstrated that the loss of GlcT-1 function enhances apoptotic cell death. Conversely, targeted expression of GlcT-1 partially rescued cell death caused by the proapoptotic factors Reaper and Grim, suggesting that ceramide generation might be one signal pathway that executes the cell death program. We also found that GlcT-1 localized not only in the Golgi apparatus but also in the perinuclear endoplasmic reticulum, providing the first visual evidence of GlcT-1 in membranes. These results indicate that GlcT-1 might down-regulate ceramide generated in these membranes.Ceramide and its metabolites, such as sphingosine-1-phosphate, have emerged as novel second messengers for intracellular signaling pathways that are involved in regulation of diverse cellular responses to exogenous stimuli (1-3).

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Subcellular biosynthesis and transport of gangliosides formed from exogenous lactosylceramide in rat liver

Cited by 20 publications

References 45 publications

Increased Biosynthesis of Glycosphingolipids in Congenital Disorder of Glycosylation Ia (CDG-Ia) Fibroblasts

Increased Biosynthesis of Glycosphingolipids in Congenital Disorder of Glycosylation Ia (CDG-Ia) Fibroblasts

Expression cloning of a cDNA for human ceramide glucosyltransferase that catalyzes the first glycosylation step of glycosphingolipid synthesis.

Drosophila Glucosylceramide Synthase

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