Neobiosynthesis of Glycosphingolipids by Plasma Membrane-associated Glycosyltransferases*

Crespo, Pilar M.; Demichelis, Vanina Torres; Daniotti, José L.

doi:10.1074/jbc.m110.123422

Cited by 33 publications

(42 citation statements)

References 56 publications

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“…Sial-T2ϩ Cells-We recently demonstrated by biochemical approaches, combined with confocal microscopy and flow cytometric analysis, the expression and cis-activity of ectoSial-T2 at the cell surface of epithelial and melanoma cells (14). In this study, these results were further confirmed and validated by using the very sensitive and specific ELISA procedure.…”

Section: Gm3 Expressed At the Cell Surface Of Cho-k1 Wt Cells Is Catasupporting

confidence: 61%

“…Sial-T2ϩ Cells Sialylates GM3 Ganglioside Immobilized onto a Solid Surface-Many reports have described the significance of the regulation of the glycolipid metabolism at the plasma membrane (13,14,17,18). However, the possibility that ecto-Sial-T2 may catalyze the conversion of GM3, belonging to the surface of neighboring cells, to the ganglioside GD3 has not yet been described.…”

Section: Ecto-sial-t2 From Cho-k1mentioning

confidence: 99%

“…Under these experimental conditions, ecto-Sial-T2 expression was even observed at the cell surface (Fig. 2B, lower panels) (14). Next, P4-treated cells were washed and incubated at 37°C for 150 min with GM3 (50 pmol) absorbed on polystyrene microtitra- tion plates in a medium containing CMP-NeuAc, Mn ϩ2 , Mg ϩ2 , and P4 inhibitor.…”

Section: Ecto-sial-t2 From Cho-k1mentioning

confidence: 99%

“…3) (14,19). GD3 synthesis by ecto-Sial-T2 activity on the SK-Mel-28 cell surface was measured following essentially the same protocol as that described above for CHO-…”

Section: Sialylation Of Immobilized Gm3 Ganglioside By Ecto-sial-t2mentioning

confidence: 99%

“…However, regulation of glycosphingolipid expression has also been demonstrated to occur at the plasma membrane, with the existence of a plasma membrane-associated sialidase (Neu3), ␤-hexosaminidase, ␤-glucosidase, and ␤-galactosidase (1, 13) having been reported. In addition, we recently showed that both ectopically and endogenously expressed CMP-NeuAc:GM3 sialyltransferase (Sial-T2) 4 are able to sialylate GM3 at the plasma membrane (cis-catalytic activity) by using both the exogenous and endogenous donor (CMP-NeuAc) and acceptor (GM3) substrates (14). Thus, the current scenario shows the presence of both ganglioside glycosyltransferases and glycohydrolases at the plasma membrane, which locally modulate cellular glycolipid expression and, consequently, different signaling processes.…”

mentioning

confidence: 99%

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Trans-activity of Plasma Membrane-associated Ganglioside Sialyltransferase in Mammalian Cells

Vilcaes¹,

Demichelis²,

Daniotti³

2011

Journal of Biological Chemistry

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Gangliosides are acidic glycosphingolipids that contain sialic acid residues and are expressed in nearly all vertebrate cells. They are synthesized at the Golgi complex by a combination of glycosyltransferase activities followed by vesicular delivery to the plasma membrane, where they participate in a variety of physiological as well as pathological processes. Recently, a number of enzymes of ganglioside anabolism and catabolism have been shown to be associated with the plasma membrane. In particular, it was observed that CMP-NeuAc:GM3 sialyltransferase (Sial-T2) is able to sialylate GM3 at the plasma membrane (ciscatalytic activity). In this work, we demonstrated that plasma membrane-integrated ecto-Sial-T2 also displays a trans-catalytic activity at the cell surface of epithelial and melanoma cells. By using a highly sensitive enzyme-linked immunosorbent assay combined with confocal fluorescence microscopy, we observed that ecto-Sial-T2 was able to sialylate hydrophobically or covalently immobilized GM3 onto a solid surface. More interestingly, we observed that ecto-Sial-T2 was able to sialylate GM3 exposed on the membrane of neighboring cells by using both the exogenous and endogenous donor substrate (CMP-N-acetylneuraminic acid) available at the extracellular milieu. In addition, the trans-activity of ecto-Sial-T2 was considerably reduced when the expression of the acceptor substrate was inhibited by using a specific inhibitor of biosynthesis of glycolipids, indicating the lipidic nature of the acceptor. Our findings provide the first direct evidence that an ecto-sialyltransferase is able to trans-sialylate substrates exposed in the plasma membrane from mammalian cells, which represents a novel insight into the molecular events that regulate the local glycosphingolipid composition.Glycosphingolipids are amphipathic molecules consisting of a ceramide lipid moiety linked to a glycan chain of variable length and structure. Among these are found gangliosides, which are sialosylated glycosphingolipids mainly located in the outer layer of the plasma membrane of vertebrate cells (1, 2) and have been implicated in many physiological processes, including growth, differentiation, migration and apoptosis through modulating both cell signaling processes and cell-tocell and cell-to-matrix interactions (3-6). Furthermore, gangliosides have been associated with a wide range of pathological processes, being receptors for both viruses and antibodies (7,8).The biosynthesis of gangliosides is mainly carried out in the lumen of the Golgi cisternae by a complex system of membrane-bound glycolipid acceptors, nucleotide sugar donors, glycosyltransferases, and nucleotide sugar transporters (1, 9). These neosynthesized gangliosides move through the Golgi complex to the plasma membrane via the lumenal surface of transport vesicles (10 -12). It is very well documented that glycosphingolipid expression, including gangliosides, is mainly regulated at the transcriptional and posttranscriptional levels of glycolipid glycosyltransferases a...

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Section: Gm3 Expressed At the Cell Surface Of Cho-k1 Wt Cells Is Catasupporting

confidence: 61%

Section: Ecto-sial-t2 From Cho-k1mentioning

confidence: 99%

Section: Ecto-sial-t2 From Cho-k1mentioning

confidence: 99%

“…3) (14,19). GD3 synthesis by ecto-Sial-T2 activity on the SK-Mel-28 cell surface was measured following essentially the same protocol as that described above for CHO-…”

Section: Sialylation Of Immobilized Gm3 Ganglioside By Ecto-sial-t2mentioning

confidence: 99%

mentioning

confidence: 99%

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Trans-activity of Plasma Membrane-associated Ganglioside Sialyltransferase in Mammalian Cells

Vilcaes¹,

Demichelis²,

Daniotti³

2011

Journal of Biological Chemistry

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Labeled gangliosides: their synthesis and use in biological studies

Schwarzmann

2018

FEBS Letters

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The unveiling of ganglioside metabolism and biological function in the last decades depended on the extensive study of inherited human disease, studies with cultured cells, and specifically designed mouse models. Nonetheless, only few of the accomplishments made so far would have been possible without the use of labeled gangliosides, such as their radiolabeled and/or fluorescent analogs, as well as other modified gangliosides bearing cross-linking, affinity, or paramagnetic groups. Over the years, chemists and biochemists have made tremendous progress toward developing labeled gangliosides for their application in biological systems. These labeled ganglioside probes of the ganglio series have been successfully incorporated in cultured vertebrate cells and in artificial model membranes. In this Review, I provide an overview over the different methods, mostly semisynthetic procedures, to obtain these labeled ganglioside probes that have been used to elucidate the molecular basis of ganglioside-related biology as well as the physicochemical properties of gangliosides.

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Metabolic pathways and intracellular trafficking of gangliosides

Daniotti

Iglesias-Bartolomé

2011

IUBMB Life

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Gangliosides constitute a large and heterogeneous family of acidic glycosphingolipids that contain one or more sialic acid residues and are expressed in nearly all vertebrate cells. Their de novo synthesis starts at the endoplasmic reticulum and is continued by a combination of glycosyltransferase activities at the Golgi complex, followed by vesicular delivery to the plasma membrane. At the cell surface, gangliosides participate in a variety of physiological as well as pathological processes. The cloning of genes for most of the glycosyltransferases responsible for ganglioside biosynthesis has produced a better understanding of the cellular and molecular basis of the ganglioside metabolism. In addition, the ability to delete groups of glycosphingolipid structures in mice has been enormously important in determining their physiological roles. Recently, a number of enzymes for ganglioside anabolism and catabolism have been shown to be associated with the plasma membrane, which might contribute to modulate local glycolipid composition, and consequently, the cell function.

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Neobiosynthesis of Glycosphingolipids by Plasma Membrane-associated Glycosyltransferases*

Cited by 33 publications

References 56 publications

Trans-activity of Plasma Membrane-associated Ganglioside Sialyltransferase in Mammalian Cells

Trans-activity of Plasma Membrane-associated Ganglioside Sialyltransferase in Mammalian Cells

Labeled gangliosides: their synthesis and use in biological studies

Metabolic pathways and intracellular trafficking of gangliosides

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