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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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9-O-acetyl GD3 is unable to induce mitochondrial damage. (A) GD3 and 9-O-acetyl GD3 (acGD3) were subjected to alkaline treatment, and run on TLC for 50 min, along with untreated GD3 and 9-O-acetyl GD3. (B) Isolated mitochondria were treated for 40 min with 200 μM Ca2+, 30 μM GD3, 30 μM 9-O-acetyl GD3 (acGD3), 30 μM de-acetylated 9-O-acetyl GD3 (de-acGD3), then supernatants subjected to Western blotting with anti-cytC mAb. (C) Isolated mitochondria were treated for 30 min with the indicated concentrations of GD3 and 9-O-acetyl GD3, then supernatants subjected to Western blotting with anti-caspase-9 p35 mAb. Data are representative of three independent experiments. (D) Isolated mitochondria were treated for 40 min with the indicated concentrations of GD3/9-O-acetyl GD3 mixes or GD3 alone, then supernatants subjected to Western blotting with anti-cytC mAb. Data are representative of two independent experiments.
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fig1: 9-O-acetyl GD3 is unable to induce mitochondrial damage. (A) GD3 and 9-O-acetyl GD3 (acGD3) were subjected to alkaline treatment, and run on TLC for 50 min, along with untreated GD3 and 9-O-acetyl GD3. (B) Isolated mitochondria were treated for 40 min with 200 μM Ca2+, 30 μM GD3, 30 μM 9-O-acetyl GD3 (acGD3), 30 μM de-acetylated 9-O-acetyl GD3 (de-acGD3), then supernatants subjected to Western blotting with anti-cytC mAb. (C) Isolated mitochondria were treated for 30 min with the indicated concentrations of GD3 and 9-O-acetyl GD3, then supernatants subjected to Western blotting with anti-caspase-9 p35 mAb. Data are representative of three independent experiments. (D) Isolated mitochondria were treated for 40 min with the indicated concentrations of GD3/9-O-acetyl GD3 mixes or GD3 alone, then supernatants subjected to Western blotting with anti-cytC mAb. Data are representative of two independent experiments.

Mentions: The interaction of GD3 with mitochondrial membranes causes cytochrome c release and caspase-9 activation, thereby triggering apoptosis (3–5). To verify whether 9-O-acetyl GD3 retains the proapoptotic functions of GD3, purified mitochondria were exposed to 9-O-acetyl GD3 and the amount of cytochrome c released in the supernatants quantitated by Western blot analysis and immunostaining. Fig. 1 B shows that, unlike GD3, purified 9-O-acetyl GD3 is completely unable to induce the release of cytochrome c from mitochondria. Accordingly, exposure of isolated mitochondria to purified 9-O-acetyl GD3 did not result in caspase-9 activation, unlike GD3, as revealed by the detection of the caspase-9 p35 fragment in the supernatants, by Western blot analysis and immunostaining (Fig. 1 C). To verify that the acetyl moiety is responsible for the loss of function of 9-O-acetyl GD3, we deacetylated 9-O-acetyl GD3 in vitro by alkaline treatment, in order to recover GD3 (Fig. 1 A). Deacetylated 9-O-acetyl GD3 is able to induce cytochrome c release from isolated mitochondria to the same extent as GD3 (Fig. 1 B).


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)

9-O-acetyl GD3 is unable to induce mitochondrial damage. (A) GD3 and 9-O-acetyl GD3 (acGD3) were subjected to alkaline treatment, and run on TLC for 50 min, along with untreated GD3 and 9-O-acetyl GD3. (B) Isolated mitochondria were treated for 40 min with 200 μM Ca2+, 30 μM GD3, 30 μM 9-O-acetyl GD3 (acGD3), 30 μM de-acetylated 9-O-acetyl GD3 (de-acGD3), then supernatants subjected to Western blotting with anti-cytC mAb. (C) Isolated mitochondria were treated for 30 min with the indicated concentrations of GD3 and 9-O-acetyl GD3, then supernatants subjected to Western blotting with anti-caspase-9 p35 mAb. Data are representative of three independent experiments. (D) Isolated mitochondria were treated for 40 min with the indicated concentrations of GD3/9-O-acetyl GD3 mixes or GD3 alone, then supernatants subjected to Western blotting with anti-cytC mAb. Data are representative of two independent experiments.
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Related In: Results  -  Collection

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

fig1: 9-O-acetyl GD3 is unable to induce mitochondrial damage. (A) GD3 and 9-O-acetyl GD3 (acGD3) were subjected to alkaline treatment, and run on TLC for 50 min, along with untreated GD3 and 9-O-acetyl GD3. (B) Isolated mitochondria were treated for 40 min with 200 μM Ca2+, 30 μM GD3, 30 μM 9-O-acetyl GD3 (acGD3), 30 μM de-acetylated 9-O-acetyl GD3 (de-acGD3), then supernatants subjected to Western blotting with anti-cytC mAb. (C) Isolated mitochondria were treated for 30 min with the indicated concentrations of GD3 and 9-O-acetyl GD3, then supernatants subjected to Western blotting with anti-caspase-9 p35 mAb. Data are representative of three independent experiments. (D) Isolated mitochondria were treated for 40 min with the indicated concentrations of GD3/9-O-acetyl GD3 mixes or GD3 alone, then supernatants subjected to Western blotting with anti-cytC mAb. Data are representative of two independent experiments.
Mentions: The interaction of GD3 with mitochondrial membranes causes cytochrome c release and caspase-9 activation, thereby triggering apoptosis (3–5). To verify whether 9-O-acetyl GD3 retains the proapoptotic functions of GD3, purified mitochondria were exposed to 9-O-acetyl GD3 and the amount of cytochrome c released in the supernatants quantitated by Western blot analysis and immunostaining. Fig. 1 B shows that, unlike GD3, purified 9-O-acetyl GD3 is completely unable to induce the release of cytochrome c from mitochondria. Accordingly, exposure of isolated mitochondria to purified 9-O-acetyl GD3 did not result in caspase-9 activation, unlike GD3, as revealed by the detection of the caspase-9 p35 fragment in the supernatants, by Western blot analysis and immunostaining (Fig. 1 C). To verify that the acetyl moiety is responsible for the loss of function of 9-O-acetyl GD3, we deacetylated 9-O-acetyl GD3 in vitro by alkaline treatment, in order to recover GD3 (Fig. 1 A). Deacetylated 9-O-acetyl GD3 is able to induce cytochrome c release from isolated mitochondria to the same extent as GD3 (Fig. 1 B).

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