Limits...
Production of multiple brain-like ganglioside species is dispensable for fas-induced apoptosis of lymphoid cells.

Popa I, Therville N, Carpentier S, Levade T, Cuvillier O, Portoukalian J - PLoS ONE (2011)

Bottom Line: Activation of an acid sphingomyelinase (aSMase) leading to a biosynthesis of GD3 disialoganglioside has been associated with Fas-induced apoptosis of lymphoid cells.Hampering ganglioside production by inhibiting the key enzyme glucosylceramide synthase did not abrogate the apoptotic process.In addition, GM3 synthase-deficient lymphoid cells underwent Fas-induced apoptosis, suggesting that gangliosides are unlikely to play an indispensable role in transducing Fas-induced apoptosis of lymphoid cells.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Recherche Dermatologique, EA4169 Université de Lyon-1, Hôpital Edouard Herriot, Lyon, France.

ABSTRACT
Activation of an acid sphingomyelinase (aSMase) leading to a biosynthesis of GD3 disialoganglioside has been associated with Fas-induced apoptosis of lymphoid cells. The present study was undertaken to clarify the role of this enzyme in the generation of gangliosides during apoptosis triggered by Fas ligation. The issue was addressed by using aSMase-deficient and aSMase-corrected cell lines derived from Niemann-Pick disease (NPD) patients. Fas cross-linking elicited a rapid production of large amounts of complex a- and b-series species of gangliosides with a pattern and a chromatographic behavior as single bands reminiscent of brain gangliosides. The gangliosides were synthesized within the first ten minutes and completely disappeared within thirty minutes after stimulation. Noteworthy is the observation that GD3 was not the only ganglioside produced. The production of gangliosides and the onset of apoptotic hallmarks occurred similarly in both aSMase-deficient and aSMase-corrected NPD lymphoid cells, indicating that aSMase activation is not accountable for ganglioside generation. Hampering ganglioside production by inhibiting the key enzyme glucosylceramide synthase did not abrogate the apoptotic process. In addition, GM3 synthase-deficient lymphoid cells underwent Fas-induced apoptosis, suggesting that gangliosides are unlikely to play an indispensable role in transducing Fas-induced apoptosis of lymphoid cells.

Show MeSH

Related in: MedlinePlus

Characterization of the GM3 synthase-deficient lymphoid cell line.(a) Sequencing of the ST3GAL5 cDNA prepared from control (Con) and the RLS (GM3 synthase-deficient) cell lines. The arrow indicates the c.862C>T point mutation. (b) HPTLC analysis of gangliosides isolated from control (Con), the RLS and Elg (NPD type A) cell lines. GM3 was used as a standard lipid. (c) Cell surface expression of Fas/CD95. Intact control (Con), RLS and Tre (NPD type A) cell lines were incubated with an anti-CD95 (green line) or an irrelevant (black line) antibody. Labelling was evaluated by flow cytometry. (d) Cell surface expression of GM3 in control (Con) and RLS lymphoblasts, as evaluated by flow cytometry. Control murine melanoma (B16 cell line) and glucosylceramide synthase-deficient murine melanoma (GM95) cells were used as controls.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3101221&req=5

pone-0019974-g005: Characterization of the GM3 synthase-deficient lymphoid cell line.(a) Sequencing of the ST3GAL5 cDNA prepared from control (Con) and the RLS (GM3 synthase-deficient) cell lines. The arrow indicates the c.862C>T point mutation. (b) HPTLC analysis of gangliosides isolated from control (Con), the RLS and Elg (NPD type A) cell lines. GM3 was used as a standard lipid. (c) Cell surface expression of Fas/CD95. Intact control (Con), RLS and Tre (NPD type A) cell lines were incubated with an anti-CD95 (green line) or an irrelevant (black line) antibody. Labelling was evaluated by flow cytometry. (d) Cell surface expression of GM3 in control (Con) and RLS lymphoblasts, as evaluated by flow cytometry. Control murine melanoma (B16 cell line) and glucosylceramide synthase-deficient murine melanoma (GM95) cells were used as controls.

Mentions: Finally, the susceptibility of GM3 synthase-deficient cells to Fas-induced cell death was investigated. For this purpose, lymphoid cells from a patient with a genetic deficiency of ganglioside GM3 synthase activity [18] were used. Analysis of the cDNA encoding ST3 beta-galactoside alpha-2,3-sialyltransferase 5 gene (ST3GAL5 or SIAT9) showed homozygosity for the c.862C>T nonsense mutation which produces a premature termination codon at position R288 (Fig. 5a). This genetic defect was accompanied by the absence of detectable GM3 as evaluated by thin-layer chromatography of total cellular lipids (Fig. 5b); similar results were obtained by measuring the expression of GM3 at the cell surface using flow cytometry (Fig. 5d). Of note, the cell surface expression of the Fas/CD95 receptor remained unaltered (Fig. 5c).


Production of multiple brain-like ganglioside species is dispensable for fas-induced apoptosis of lymphoid cells.

Popa I, Therville N, Carpentier S, Levade T, Cuvillier O, Portoukalian J - PLoS ONE (2011)

Characterization of the GM3 synthase-deficient lymphoid cell line.(a) Sequencing of the ST3GAL5 cDNA prepared from control (Con) and the RLS (GM3 synthase-deficient) cell lines. The arrow indicates the c.862C>T point mutation. (b) HPTLC analysis of gangliosides isolated from control (Con), the RLS and Elg (NPD type A) cell lines. GM3 was used as a standard lipid. (c) Cell surface expression of Fas/CD95. Intact control (Con), RLS and Tre (NPD type A) cell lines were incubated with an anti-CD95 (green line) or an irrelevant (black line) antibody. Labelling was evaluated by flow cytometry. (d) Cell surface expression of GM3 in control (Con) and RLS lymphoblasts, as evaluated by flow cytometry. Control murine melanoma (B16 cell line) and glucosylceramide synthase-deficient murine melanoma (GM95) cells were used as controls.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3101221&req=5

pone-0019974-g005: Characterization of the GM3 synthase-deficient lymphoid cell line.(a) Sequencing of the ST3GAL5 cDNA prepared from control (Con) and the RLS (GM3 synthase-deficient) cell lines. The arrow indicates the c.862C>T point mutation. (b) HPTLC analysis of gangliosides isolated from control (Con), the RLS and Elg (NPD type A) cell lines. GM3 was used as a standard lipid. (c) Cell surface expression of Fas/CD95. Intact control (Con), RLS and Tre (NPD type A) cell lines were incubated with an anti-CD95 (green line) or an irrelevant (black line) antibody. Labelling was evaluated by flow cytometry. (d) Cell surface expression of GM3 in control (Con) and RLS lymphoblasts, as evaluated by flow cytometry. Control murine melanoma (B16 cell line) and glucosylceramide synthase-deficient murine melanoma (GM95) cells were used as controls.
Mentions: Finally, the susceptibility of GM3 synthase-deficient cells to Fas-induced cell death was investigated. For this purpose, lymphoid cells from a patient with a genetic deficiency of ganglioside GM3 synthase activity [18] were used. Analysis of the cDNA encoding ST3 beta-galactoside alpha-2,3-sialyltransferase 5 gene (ST3GAL5 or SIAT9) showed homozygosity for the c.862C>T nonsense mutation which produces a premature termination codon at position R288 (Fig. 5a). This genetic defect was accompanied by the absence of detectable GM3 as evaluated by thin-layer chromatography of total cellular lipids (Fig. 5b); similar results were obtained by measuring the expression of GM3 at the cell surface using flow cytometry (Fig. 5d). Of note, the cell surface expression of the Fas/CD95 receptor remained unaltered (Fig. 5c).

Bottom Line: Activation of an acid sphingomyelinase (aSMase) leading to a biosynthesis of GD3 disialoganglioside has been associated with Fas-induced apoptosis of lymphoid cells.Hampering ganglioside production by inhibiting the key enzyme glucosylceramide synthase did not abrogate the apoptotic process.In addition, GM3 synthase-deficient lymphoid cells underwent Fas-induced apoptosis, suggesting that gangliosides are unlikely to play an indispensable role in transducing Fas-induced apoptosis of lymphoid cells.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Recherche Dermatologique, EA4169 Université de Lyon-1, Hôpital Edouard Herriot, Lyon, France.

ABSTRACT
Activation of an acid sphingomyelinase (aSMase) leading to a biosynthesis of GD3 disialoganglioside has been associated with Fas-induced apoptosis of lymphoid cells. The present study was undertaken to clarify the role of this enzyme in the generation of gangliosides during apoptosis triggered by Fas ligation. The issue was addressed by using aSMase-deficient and aSMase-corrected cell lines derived from Niemann-Pick disease (NPD) patients. Fas cross-linking elicited a rapid production of large amounts of complex a- and b-series species of gangliosides with a pattern and a chromatographic behavior as single bands reminiscent of brain gangliosides. The gangliosides were synthesized within the first ten minutes and completely disappeared within thirty minutes after stimulation. Noteworthy is the observation that GD3 was not the only ganglioside produced. The production of gangliosides and the onset of apoptotic hallmarks occurred similarly in both aSMase-deficient and aSMase-corrected NPD lymphoid cells, indicating that aSMase activation is not accountable for ganglioside generation. Hampering ganglioside production by inhibiting the key enzyme glucosylceramide synthase did not abrogate the apoptotic process. In addition, GM3 synthase-deficient lymphoid cells underwent Fas-induced apoptosis, suggesting that gangliosides are unlikely to play an indispensable role in transducing Fas-induced apoptosis of lymphoid cells.

Show MeSH
Related in: MedlinePlus