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Acetylation suppresses the proapoptotic activity of GD3 ganglioside.

Malisan F, Franchi L, Tomassini B, Ventura N, Condò I, Rippo MR, Rufini A, Liberati L, Nachtigall C, Kniep B, Testi R - J. Exp. Med. (2002)

Bottom Line: Moreover, cells which are resistant to the overexpression of the GD3 synthase, actively convert de novo synthesized GD3 to 9-O-acetyl-GD3.The coexpression of GD3 synthase with a viral 9-O-acetyl esterase, which prevents 9-O-acetyl-GD3 accumulation, reconstitutes GD3 responsiveness and apoptosis.Finally, the expression of the 9-O-acetyl esterase is sufficient to induce apoptosis of glioblastomas which express high levels of 9-O-acetyl-GD3.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Immunology and Signal Transduction, Department of Experimental Medicine and Biochemical Sciences, University of Rome Tor Vergata, 00133 Rome, Italy.

ABSTRACT
GD3 synthase is rapidly activated in different cell types after specific apoptotic stimuli. De novo synthesized GD3 accumulates and contributes to the apoptotic program by relocating to mitochondrial membranes and inducing the release of apoptogenic factors. We found that sialic acid acetylation suppresses the proapoptotic activity of GD3. In fact, unlike GD3, 9-O-acetyl-GD3 is completely ineffective in inducing cytochrome c release and caspase-9 activation on isolated mitochondria and fails to induce the collapse of mitochondrial transmembrane potential and cellular apoptosis. Moreover, cells which are resistant to the overexpression of the GD3 synthase, actively convert de novo synthesized GD3 to 9-O-acetyl-GD3. The coexpression of GD3 synthase with a viral 9-O-acetyl esterase, which prevents 9-O-acetyl-GD3 accumulation, reconstitutes GD3 responsiveness and apoptosis. Finally, the expression of the 9-O-acetyl esterase is sufficient to induce apoptosis of glioblastomas which express high levels of 9-O-acetyl-GD3. Thus, sialic acid acetylation critically controls the proapoptotic activity of GD3.

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Related in: MedlinePlus

HEK-293 cells are resistant to endogenous GD3 accumulation and synthesize 9-O-acetyl GD3. (A) The percentage of apoptotic HEK-293 nuclei was analyzed 40 h after transfection with empty vector (vector), wild-type GD3 synthase (wST8), dead mutant GD3 synthase (mST8), and caspase-8 as a positive control. Data represent the mean ± 1 SD from five independent experiments. (B) GD3 and 9-O-acetyl GD3 content of HEK-293 cells was analyzed by TLC and immunostaining 40 h after transfection with the indicated constructs. Data are representative of six independent experiments.
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fig3: HEK-293 cells are resistant to endogenous GD3 accumulation and synthesize 9-O-acetyl GD3. (A) The percentage of apoptotic HEK-293 nuclei was analyzed 40 h after transfection with empty vector (vector), wild-type GD3 synthase (wST8), dead mutant GD3 synthase (mST8), and caspase-8 as a positive control. Data represent the mean ± 1 SD from five independent experiments. (B) GD3 and 9-O-acetyl GD3 content of HEK-293 cells was analyzed by TLC and immunostaining 40 h after transfection with the indicated constructs. Data are representative of six independent experiments.

Mentions: Several cell lines undergo massive mitochondrial damage and apoptosis upon acute intracellular GD3 accumulation, a condition which can be experimentally achieved by GD3 synthase overexpression. Selected cell lines, however, while still sensitive to exogenous GD3 exposure, fail to undergo apoptosis upon GD3 synthase overexpression and endogenous GD3 accumulation. As shown in Fig. 3 A, HEK-293 cells, which undergo apoptosis upon exposure to exogenous GD3 (Fig. 2 B), are completely resistant to GD3 synthase overexpression. To verify that GD3 synthase overexpression was indeed inducing GD3 accumulation in HEK-293 cells, the amount of GD3 was assessed by TLC and immunostaining. Fig. 3 B shows that HEK-293 cells transfected with GD3 synthase, but not with a catalytically dead GD3 synthase mutant which lacks part of the catalytic site (1), accumulate GD3 efficiently. Interestingly, a simultaneous immunostaining for 9-O-acetyl GD3 revealed that GD3 synthase-transfected HEK-293 were also accumulating 9-O-acetyl GD3, indicating that part of the de novo synthesized GD3 was converted to 9-O-acetyl GD3, by the concomitant activation of endogenous sialic acid acetyltransferases.


Acetylation suppresses the proapoptotic activity of GD3 ganglioside.

Malisan F, Franchi L, Tomassini B, Ventura N, Condò I, Rippo MR, Rufini A, Liberati L, Nachtigall C, Kniep B, Testi R - J. Exp. Med. (2002)

HEK-293 cells are resistant to endogenous GD3 accumulation and synthesize 9-O-acetyl GD3. (A) The percentage of apoptotic HEK-293 nuclei was analyzed 40 h after transfection with empty vector (vector), wild-type GD3 synthase (wST8), dead mutant GD3 synthase (mST8), and caspase-8 as a positive control. Data represent the mean ± 1 SD from five independent experiments. (B) GD3 and 9-O-acetyl GD3 content of HEK-293 cells was analyzed by TLC and immunostaining 40 h after transfection with the indicated constructs. Data are representative of six independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196066&req=5

fig3: HEK-293 cells are resistant to endogenous GD3 accumulation and synthesize 9-O-acetyl GD3. (A) The percentage of apoptotic HEK-293 nuclei was analyzed 40 h after transfection with empty vector (vector), wild-type GD3 synthase (wST8), dead mutant GD3 synthase (mST8), and caspase-8 as a positive control. Data represent the mean ± 1 SD from five independent experiments. (B) GD3 and 9-O-acetyl GD3 content of HEK-293 cells was analyzed by TLC and immunostaining 40 h after transfection with the indicated constructs. Data are representative of six independent experiments.
Mentions: Several cell lines undergo massive mitochondrial damage and apoptosis upon acute intracellular GD3 accumulation, a condition which can be experimentally achieved by GD3 synthase overexpression. Selected cell lines, however, while still sensitive to exogenous GD3 exposure, fail to undergo apoptosis upon GD3 synthase overexpression and endogenous GD3 accumulation. As shown in Fig. 3 A, HEK-293 cells, which undergo apoptosis upon exposure to exogenous GD3 (Fig. 2 B), are completely resistant to GD3 synthase overexpression. To verify that GD3 synthase overexpression was indeed inducing GD3 accumulation in HEK-293 cells, the amount of GD3 was assessed by TLC and immunostaining. Fig. 3 B shows that HEK-293 cells transfected with GD3 synthase, but not with a catalytically dead GD3 synthase mutant which lacks part of the catalytic site (1), accumulate GD3 efficiently. Interestingly, a simultaneous immunostaining for 9-O-acetyl GD3 revealed that GD3 synthase-transfected HEK-293 were also accumulating 9-O-acetyl GD3, indicating that part of the de novo synthesized GD3 was converted to 9-O-acetyl GD3, by the concomitant activation of endogenous sialic acid acetyltransferases.

Bottom Line: Moreover, cells which are resistant to the overexpression of the GD3 synthase, actively convert de novo synthesized GD3 to 9-O-acetyl-GD3.The coexpression of GD3 synthase with a viral 9-O-acetyl esterase, which prevents 9-O-acetyl-GD3 accumulation, reconstitutes GD3 responsiveness and apoptosis.Finally, the expression of the 9-O-acetyl esterase is sufficient to induce apoptosis of glioblastomas which express high levels of 9-O-acetyl-GD3.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Immunology and Signal Transduction, Department of Experimental Medicine and Biochemical Sciences, University of Rome Tor Vergata, 00133 Rome, Italy.

ABSTRACT
GD3 synthase is rapidly activated in different cell types after specific apoptotic stimuli. De novo synthesized GD3 accumulates and contributes to the apoptotic program by relocating to mitochondrial membranes and inducing the release of apoptogenic factors. We found that sialic acid acetylation suppresses the proapoptotic activity of GD3. In fact, unlike GD3, 9-O-acetyl-GD3 is completely ineffective in inducing cytochrome c release and caspase-9 activation on isolated mitochondria and fails to induce the collapse of mitochondrial transmembrane potential and cellular apoptosis. Moreover, cells which are resistant to the overexpression of the GD3 synthase, actively convert de novo synthesized GD3 to 9-O-acetyl-GD3. The coexpression of GD3 synthase with a viral 9-O-acetyl esterase, which prevents 9-O-acetyl-GD3 accumulation, reconstitutes GD3 responsiveness and apoptosis. Finally, the expression of the 9-O-acetyl esterase is sufficient to induce apoptosis of glioblastomas which express high levels of 9-O-acetyl-GD3. Thus, sialic acid acetylation critically controls the proapoptotic activity of GD3.

Show MeSH
Related in: MedlinePlus