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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: 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.The coexpression of GD3 synthase with a viral 9-O-acetyl esterase, which prevents 9-O-acetyl-GD3 accumulation, reconstitutes GD3 responsiveness and apoptosis.

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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Deacetylation of de novo synthesized 9-O-acetyl GD3 triggers apoptosis of HEK-293 cells. (A) GD3 and 9-O-acetyl GD3 content of HEK-293 cells was analyzed by TLC and immunostaining 40 h after transfection with wild-type GD3 synthase (ST8), wild-type 9-O-acetylesterase (Est), dead mutant GD3 synthase (mST8), and dead mutant 9-O-acetylesterase (mEst). Data are representative of three independent experiments. (B) The percentage of apoptotic HEK-293 nuclei was analyzed 40 h after transfection with the indicated constructs. Data represent the mean ± 1 SD from three independent experiments.
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fig4: Deacetylation of de novo synthesized 9-O-acetyl GD3 triggers apoptosis of HEK-293 cells. (A) GD3 and 9-O-acetyl GD3 content of HEK-293 cells was analyzed by TLC and immunostaining 40 h after transfection with wild-type GD3 synthase (ST8), wild-type 9-O-acetylesterase (Est), dead mutant GD3 synthase (mST8), and dead mutant 9-O-acetylesterase (mEst). Data are representative of three independent experiments. (B) The percentage of apoptotic HEK-293 nuclei was analyzed 40 h after transfection with the indicated constructs. Data represent the mean ± 1 SD from three independent experiments.

Mentions: The above mentioned findings suggested that GD3 to 9-O-acetyl GD3 conversion, mediated by endogenous sialic acid acetyltransferases, might represent an efficient cell survival strategy in response to stress-induced acute GD3 accumulation. To test this hypothesis, we first decided to force the in vivo deacetylation of de novo–synthesized 9-O-acetyl GD3 by coexpressing a specific sialic acid acetylesterase. We used the influenza type C viral 9-O-acetylesterase, known to efficiently de-acetylate 9-O-acetyl GD3 in mammalian cells (26). We also prepared an enzymatically dead version of the same viral acetylesterase by substituting a critical Ser57 residue with Ala, based on structural informations (27). We transiently coexpressed the wild-type 9-O-acetylesterase, or the dead 9-O-acetylesterase, together with the wild-type GD3 synthase, or the dead GD3 synthase in HEK-293 cells. The accumulation of GD3 and 9-O-acetyl GD3 in cotransfected cell cultures was monitored along with the induction of apoptosis. As shown in Fig. 4 A, the coexpression of both dead mutants, or the coexpression of the dead GD3 synthase together with the wild-type 9-O-acetylesterase, did not result in any GD3 or 9-O-acetyl GD3 accumulation. Moreover, the coexpression of the wild-type GD3 synthase together with the dead 9-O-acetylesterase, resulted in the accumulation of both GD3 and 9-O-acetyl GD3. This last condition parallels what already shown in Fig. 3, and no specific apoptosis can be induced in HEK-293 cells (Fig. 4 B). However, the coexpression of the wild-type forms of both enzymes resulted in enhanced accumulation of GD3 and in the disappearance of 9-O-acetyl GD3, due to the action of the 9-O-acetylesterase. Importantly, in this last condition, extensive apoptosis of HEK-293 could be induced (Fig. 4 B). Apoptosis is strictly dependent on specific deacetylation of 9-O-acetyl GD3, since the dead 9-O-acetylesterase is ineffective, and it does not occur in the absence of de novo 9-O-acetyl GD3 synthesis. These results strongly suggest that when the attempt to transform part of the accumulating GD3 into 9-O-acetyl GD3 is reversed by the action of the 9-O-acetylesterase, a critical GD3 threshold is exceeded and HEK-293 cells undergo apoptosis.


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)

Deacetylation of de novo synthesized 9-O-acetyl GD3 triggers apoptosis of HEK-293 cells. (A) GD3 and 9-O-acetyl GD3 content of HEK-293 cells was analyzed by TLC and immunostaining 40 h after transfection with wild-type GD3 synthase (ST8), wild-type 9-O-acetylesterase (Est), dead mutant GD3 synthase (mST8), and dead mutant 9-O-acetylesterase (mEst). Data are representative of three independent experiments. (B) The percentage of apoptotic HEK-293 nuclei was analyzed 40 h after transfection with the indicated constructs. Data represent the mean ± 1 SD from three independent experiments.
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Related In: Results  -  Collection

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fig4: Deacetylation of de novo synthesized 9-O-acetyl GD3 triggers apoptosis of HEK-293 cells. (A) GD3 and 9-O-acetyl GD3 content of HEK-293 cells was analyzed by TLC and immunostaining 40 h after transfection with wild-type GD3 synthase (ST8), wild-type 9-O-acetylesterase (Est), dead mutant GD3 synthase (mST8), and dead mutant 9-O-acetylesterase (mEst). Data are representative of three independent experiments. (B) The percentage of apoptotic HEK-293 nuclei was analyzed 40 h after transfection with the indicated constructs. Data represent the mean ± 1 SD from three independent experiments.
Mentions: The above mentioned findings suggested that GD3 to 9-O-acetyl GD3 conversion, mediated by endogenous sialic acid acetyltransferases, might represent an efficient cell survival strategy in response to stress-induced acute GD3 accumulation. To test this hypothesis, we first decided to force the in vivo deacetylation of de novo–synthesized 9-O-acetyl GD3 by coexpressing a specific sialic acid acetylesterase. We used the influenza type C viral 9-O-acetylesterase, known to efficiently de-acetylate 9-O-acetyl GD3 in mammalian cells (26). We also prepared an enzymatically dead version of the same viral acetylesterase by substituting a critical Ser57 residue with Ala, based on structural informations (27). We transiently coexpressed the wild-type 9-O-acetylesterase, or the dead 9-O-acetylesterase, together with the wild-type GD3 synthase, or the dead GD3 synthase in HEK-293 cells. The accumulation of GD3 and 9-O-acetyl GD3 in cotransfected cell cultures was monitored along with the induction of apoptosis. As shown in Fig. 4 A, the coexpression of both dead mutants, or the coexpression of the dead GD3 synthase together with the wild-type 9-O-acetylesterase, did not result in any GD3 or 9-O-acetyl GD3 accumulation. Moreover, the coexpression of the wild-type GD3 synthase together with the dead 9-O-acetylesterase, resulted in the accumulation of both GD3 and 9-O-acetyl GD3. This last condition parallels what already shown in Fig. 3, and no specific apoptosis can be induced in HEK-293 cells (Fig. 4 B). However, the coexpression of the wild-type forms of both enzymes resulted in enhanced accumulation of GD3 and in the disappearance of 9-O-acetyl GD3, due to the action of the 9-O-acetylesterase. Importantly, in this last condition, extensive apoptosis of HEK-293 could be induced (Fig. 4 B). Apoptosis is strictly dependent on specific deacetylation of 9-O-acetyl GD3, since the dead 9-O-acetylesterase is ineffective, and it does not occur in the absence of de novo 9-O-acetyl GD3 synthesis. These results strongly suggest that when the attempt to transform part of the accumulating GD3 into 9-O-acetyl GD3 is reversed by the action of the 9-O-acetylesterase, a critical GD3 threshold is exceeded and HEK-293 cells undergo apoptosis.

Bottom Line: 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.The coexpression of GD3 synthase with a viral 9-O-acetyl esterase, which prevents 9-O-acetyl-GD3 accumulation, reconstitutes GD3 responsiveness and apoptosis.

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