Secretagogue Activity of Trachynilysin, a Neurotoxic Protein Isolated from Stonefish (Synanceia trachynis) Venom

Meunier, Frédéric A.; Ouanounou, Gilles; Mattei, César; Chameau, Pascal; Colasante, Cesare; Ushkaryov, Yuri A.; Dolly, J. Oliver; Kreger, Arnold S.; Molgó, Jordi

doi:10.1385/1-59259-132-9:595

Handbook of Neurotoxicology

DOI: 10.1385/1-59259-132-9:595

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Secretagogue Activity of Trachynilysin, a Neurotoxic Protein Isolated from Stonefish (Synanceia trachynis) Venom

Frédéric A. Meunier¹,

Gilles Ouanounou²,

César Mattei³

et al.

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Cited by 4 publications

(12 citation statements)

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“…1 Both authors contributed equally to this work. 2 National Health and Medical Research Council Research Fellows. 3 To whom correspondence should be addressed.…”

mentioning

confidence: 99%

Dynamin Inhibition Blocks Botulinum Neurotoxin Type A Endocytosis in Neurons and Delays Botulism

Harper

Martin

Nguyen

et al. 2011

Journal of Biological Chemistry

147

149

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The botulinum neurotoxins (BoNTs) are di-chain bacterial proteins responsible for the paralytic disease botulism. Following binding to the plasma membrane of cholinergic motor nerve terminals, BoNTs are internalized into an endocytic compartment. Although several endocytic pathways have been characterized in neurons, the molecular mechanism underpinning the uptake of BoNTs at the presynaptic nerve terminal is still unclear. Here, a recombinant BoNT/A heavy chain binding domain (Hc) was used to unravel the internalization pathway by fluorescence and electron microscopy. BoNT/A-Hc initially enters cultured hippocampal neurons in an activity-dependent manner into synaptic vesicles and clathrin-coated vesicles before also entering endosomal structures and multivesicular bodies. We found that inhibiting dynamin with the novel potent Dynasore analog, Dyngo-4a TM , was sufficient to abolish BoNT/ A-Hc internalization and BoNT/A-induced SNAP25 cleavage in hippocampal neurons. Dyngo-4a also interfered with BoNT/ A-Hc internalization into motor nerve terminals. Furthermore, Dyngo-4a afforded protection against BoNT/A-induced paralysis at the rat hemidiaphragm. A significant delay of >30% in the onset of botulism was observed in mice injected with Dyngo-4a. Dynamin inhibition therefore provides a therapeutic avenue for the treatment of botulism and other diseases caused by pathogens sharing dynamin-dependent uptake mechanisms.

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“…1 Both authors contributed equally to this work. 2 National Health and Medical Research Council Research Fellows. 3 To whom correspondence should be addressed.…”

mentioning

confidence: 99%

Dynamin Inhibition Blocks Botulinum Neurotoxin Type A Endocytosis in Neurons and Delays Botulism

Harper

Martin

Nguyen

et al. 2011

Journal of Biological Chemistry

147

149

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“…Interestingly, neither TLY (25) nor ␣-LTX (28) affects the number of large dense-core vesicles containing neuropeptides in motor nerve endings despite the depletion of small clear synaptic vesicles. TLY has also been reported to raise the intracellular Ca 2ϩ concentration in cultured mouse hippocampal neurons (29) and adrenal chromaffin cells (27). Simultaneous blockade of L, N, and P/Q voltagedependent Ca 2ϩ channels caused only a minor reduction of TLY-induced catecholamine secretion and little change in Ca 2ϩ signals (27), and removal of extracellular Ca 2ϩ and addition of EGTA or La 3ϩ completely abolished both secretion and Ca 2ϩ signals.…”

mentioning

confidence: 99%

“…Trachynilysin (TLY), a 159 kDa membrane-perturbing (hemolytic) toxic protein isolated from the venom of the stonefish Synanceia trachynis, also greatly increases quantal acetylcholine release from motor nerve endings (25,26) and catecholamine secretion from large dense-core vesicles of chromaffin cells (27). However, contrary to ␣-LTX, these two types of release are Ca 2ϩ -dependent.…”

mentioning

confidence: 99%

“…TLY has also been reported to raise the intracellular Ca 2ϩ concentration in cultured mouse hippocampal neurons (29) and adrenal chromaffin cells (27). Simultaneous blockade of L, N, and P/Q voltagedependent Ca 2ϩ channels caused only a minor reduction of TLY-induced catecholamine secretion and little change in Ca 2ϩ signals (27), and removal of extracellular Ca 2ϩ and addition of EGTA or La 3ϩ completely abolished both secretion and Ca 2ϩ signals. Moreover, depletion of intracellular Ca 2ϩ stores with caffeine inhibited TLY-induced catecholamine secretion by chromaffin cells.…”

mentioning

confidence: 99%

“…Moreover, depletion of intracellular Ca 2ϩ stores with caffeine inhibited TLY-induced catecholamine secretion by chromaffin cells. These results suggest that transmembrane Ca 2ϩ influx and the resulting mobilization of intracellular Ca 2ϩ stores are required for TLYinduced secretion (27). To determine the mechanism for the Ca 2ϩ influx, we decided to determine whether TLY activates existing ion channels or forms pores in the plasma membrane.…”

mentioning

confidence: 99%

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Trachynilysin, a Neurosecretory Protein Isolated from Stonefish (Synanceia trachynis) Venom, Forms Nonselective Pores in the Membrane of NG108-15 Cells

Ouanounou

Malo

Stinnakre

et al. 2002

Journal of Biological Chemistry

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Trachynilysin, a protein toxin isolated from the venom of the stonefish Synanceia trachynis, has been reported to elicit massive acetylcholine release from motor nerve endings of isolated neuromuscular preparations and to increase both cytosolic Ca 2؉ and catecholamine release from chromaffin cells. In the present study, we used the patch clamp technique to investigate the effect of trachynilysin on the cytoplasmic membrane of differentiated NG108-15 cells in culture. Trachynilysin increased membrane conductance the most when the negativity of the cell holding membrane potential was reduced. The trachynilysin-induced current was carried by cations and reversed at about ؊3 mV in standard physiological solutions, which led to strong membrane depolarization and Ca 2؉ influx. La 3؉ blocked the trachynilysin current in a dose-, voltage-, and timedependent manner, and antibodies raised against the toxin antagonized its effect on the cell membrane. The inside-out configuration of the patch clamp technique allowed the recording of single channel activity from which various multiples of 22 pS elementary conductance were resolved. These results indicate that trachynilysin forms pores in the NG108-15 cell membrane, and they advance our understanding of the toxin's mode of action on motor nerve endings and neurosecretory cells.Neurotoxins have proved to be valuable tools for understanding molecular mechanisms involved in physiological processes, and their use has been instrumental in the purification and identification of key protein components of excitable membranes and synapses (1-3). Among the neurotoxins that promote neurotransmitter release, the most studied has been ␣-latrotoxin (␣-LTX) 1 (for recent reviews, see Refs. 4 and 5), a 120 kDa protein purified from the venom of the black widow spider (Lactrodectus mactans tredecimguttatus), which is known to trigger Ca 2ϩ -dependent and -independent release of a variety of neurotransmitters and hormones (6 -11). ␣-LTX forms nonselective cationic pores in planar lipid bilayers (12)(13)(14) and in the cytoplasmic membrane of differentiated PC12 (15) and neuroblastoma cells (16). In addition, two families of ␣-LTX receptors that facilitate pore formation in biological membranes (17, 18) have been identified, i.e. neurexins (19, 20) and latrophilins (21-24), and the pores are involved in the toxin's massive release of neurotransmitters (4,5,7,9,11).Trachynilysin (TLY), a 159 kDa membrane-perturbing (hemolytic) toxic protein isolated from the venom of the stonefish Synanceia trachynis, also greatly increases quantal acetylcholine release from motor nerve endings (25, 26) and catecholamine secretion from large dense-core vesicles of chromaffin cells (27). However, contrary to ␣-LTX, these two types of release are Ca 2ϩ -dependent. Interestingly, neither TLY (25) nor ␣-LTX (28) affects the number of large dense-core vesicles containing neuropeptides in motor nerve endings despite the depletion of small clear synaptic vesicles. TLY has also been reported to raise the intracellul...

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Glycerotoxin from Glycera convoluta stimulates neurosecretion by up-regulating N-type Ca2+ channel activity

et al. 2002

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We report here the puri®cation of glycerotoxin from the venom of Glycera convoluta, a novel 320 kDa protein capable of reversibly stimulating spontaneous and evoked neurotransmitter release at the frog neuromuscular junction. However, glycerotoxin is ineffective at the murine neuromuscular junction, which displays a different subtype of voltagedependent Ca 2+ channels. By sequential and selective inhibition of various types of Ca 2+ channels, we found that glycerotoxin was acting via Ca v 2.2 (N-type). In neuroendocrine cells, it elicits a robust, albeit transient, in¯ux of Ca 2+ sensitive to the Ca v 2.2 blockers w-conotoxin GVIA and MVIIA. Moreover, glycerotoxin triggers a Ca 2+ transient in human embryonic kidney (HEK) cells over-expressing Ca v 2.2 but not Ca v 2.1 (P/Q-type). Whole-cell patch±clamp analysis of Ca v 2.2 expressing HEK cells revealed an up-regulation of Ca 2+ currents due to a leftward shift of the activation peak upon glycerotoxin addition. A direct interaction between Ca v 2.2 and this neurotoxin was revealed by co-immunoprecipitation experiments. Therefore, glycerotoxin is a unique addition to the arsenal of tools available to unravel the mechanism controlling Ca 2+ -regulated exocytosis via the speci®c activation of Ca v 2.2.

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Secretagogue Activity of Trachynilysin, a Neurotoxic Protein Isolated from Stonefish (Synanceia trachynis) Venom

Cited by 4 publications

References 0 publications

Dynamin Inhibition Blocks Botulinum Neurotoxin Type A Endocytosis in Neurons and Delays Botulism

Dynamin Inhibition Blocks Botulinum Neurotoxin Type A Endocytosis in Neurons and Delays Botulism

Trachynilysin, a Neurosecretory Protein Isolated from Stonefish (Synanceia trachynis) Venom, Forms Nonselective Pores in the Membrane of NG108-15 Cells

Glycerotoxin from Glycera convoluta stimulates neurosecretion by up-regulating N-type Ca2+ channel activity

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