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Capture assay for Botulinum Neurotoxin Type A utilizing the receptor protein SV2c
ARTICLE IN PRESS 2
Abstracts Toxins 2008 / Toxicon 51 (2008) 1–54
complexes in synaptosome lysates as detected by affinity chromatography techniques. SV2, synaptotagmin I, synaptophysin, VAMP2, and the vacuolar proton pump were identified as components of the BoNT–synaptic vesicle receptor complex by tandem LC-MS/MS. Density gradient analysis observed that the HCRs of BoNT/A and BoNT/B displayed unique interactions with synaptic vesicle proteins. Association of BoNTs to synaptic vesicle protein complexes, along with the ability to bind gangliosides, may explain the high affinity of BoNTs for peripheral neurons. Supported by a grant from the GLRCE-NIH-NIAID U54 AI057153. Keywords: Botulinum neurotoxins; Synaptic vesicles 10.1016/j.toxicon.2008.04.004
3. Botulinum neurotoxin interactions with substrate Axel Brunger Stanford University, Stanford, USA
The mechanism by which a CNT properly identifies and cleaves its target SNARE once inside the neuron involves one or more regions of enzyme–substrate interaction remote from the active site (so called exosites). We determined the structure of a CNT endopeptidase in complex with its target SNARE that illustrates the extensive enzyme–substrate interface and the basis of substrate selectivity. Our structure of BoNT/A, in complex with human SNAP-25, revealed multiple exosites, including several that are involved in substrate specificity, and one that likely functions as an allosteric activator of the toxin. Different crystal forms of wildtype BoNT/A revealed conformational variability of several loops near the active site. We also determined crystal structures of the apo form of the TeNT LC49 and that of the BoNT/C1 LC protease50. We found remarkable structural differences between the BoNT/C1 and BoNT/A LC proteases that may explain the dual substrate-binding ability of BoNT/C1. Keywords: Botulinum neurotoxin; SNARE; Protease
References Jin, R., Sikorra, S., Stegmann, C.M., Pich, A., Binz, T., Brunger, A.T., 2007. Biochemistry 46, 10685–10693. Burnett, J.C., Ruthel, G., Stegmann, C.M., Panchal, R.G., Nguyen, T.L., Hermone, A.R., Stafford, R.G., Lane, D.J., Kenny, T.A., McGrath, C.F., Wipf, P., Stahl, A.M., Schmidt, J.J., Gussio, R., Brunger, A.T., Bavari, S., 2007. J. Biol. Chem. 282, 5004–5014. Breidenbach, M.A., Brunger, A.T., 2004. Nature 432, 925–929. 10.1016/j.toxicon.2008.04.005
4. Capture assay for Botulinum Neurotoxin Type A utilizing the receptor protein SV2c Todd Christian, Nancy Shine List Biological Laboratories, Inc., Campbell, CA, USA
Recent biochemical and molecular genetic studies have established synaptic vesicle glycoprotein 2C (SV2c)
as the protein receptor for Botulinum Neurotoxin Type A (BoNT/A). The luminal domain loop of SV2c, between transmembrane domains 7 and 8, has been shown to be the location of BoNT/A binding. Utilizing this information, we have shown through GST pull-down experiments that we can detect binding of BoNT/A to SV2c using an immunoassay. This can be done with either the rat or the human forms of the SV2c protein. Upon specific binding of BoNT/A to its receptor protein, we can introduce our FRET peptide SNAPtides and detect enzymatic cleavage. We describe the reaction conditions and the detection limits using both rat and human forms of the SV2c binding region as well as multiple FRET substrates to determine the optimal conditions for this detection assay. There are three steps in the interruption of synaptic transmission by botulinum toxin. The first is binding to neuronal cells; the second is translocation of the enzymatic light chain out of the endosome; and finally cleavage of synaptosomal proteins to inhibit neurotransmitter release. With this assay we can monitor two of the three steps of toxin activity, binding and cleavage. In the future, we hope to establish this as a new functional assay for the detection and activity of BoNT/A and demonstrate a direct correlation to the gold standard, the mouse bioassay. Keywords: Receptor; SV2c; Detection; FRET 10.1016/j.toxicon.2008.04.006
5. C-terminal fragment of tetanus toxin heavy chain modulates sphingomyelinase activity in neuronal preparations Roger Cubı´ Pique´ , Mireia Herrando Grabulosa, Jose´ Aguilera A´vila , Carles Gil Giro´ Departament de Bioquı´mica i Biologia Molecular i Institut de Neurocie`ncies, `noma de Barcelona, Bellaterra (Cerdanyola del Valle´s), Spain Universitat Auto
Tetanus toxin (TeTx) reaches CNS by means of retrograde transport inside motor neuron axons. The C-terminal domain of TeTx (HC-TeTx) has been described as responsible for binding and internalization of the toxin into neurons. Analysis of this transport reveals common endocytic carriers for HC-TeTx and neurotrophin signalling (i.e. p75NTR, TrkB, NGF and BDNF). Moreover, in previous work from our group, the activation of neurotrophic signalling by HC-TeTx was described. On the basis of these observations, we decided to study the influence of HC-TeTx in p75NTR-dependent events. Since p75NTR lacks intrinsic enzymatic activity and has been described as a sphingomyelinase (SMase)-coupled receptor, the modulation of SMase activity after HC-TeTx treatment in cultured cerebellar granule neurons (CGN) has been determined and compared with the action of NGF. Neutral- and acid-sphingomyelinases are enzymes that catalyze the hydrolysis of sphingomyelin into ceramide and phosphorylcholine. Treatments of CGN at 7 DIV show a moderate yet consistent and significant enhancement of the nSMase activity after HC-TeTx, as assessed by means of enzymatic assays. HC-TeTx treatment of
Abstracts Toxins 2008 / Toxicon 51 (2008) 1–54
complexes in synaptosome lysates as detected by affinity chromatography techniques. SV2, synaptotagmin I, synaptophysin, VAMP2, and the vacuolar proton pump were identified as components of the BoNT–synaptic vesicle receptor complex by tandem LC-MS/MS. Density gradient analysis observed that the HCRs of BoNT/A and BoNT/B displayed unique interactions with synaptic vesicle proteins. Association of BoNTs to synaptic vesicle protein complexes, along with the ability to bind gangliosides, may explain the high affinity of BoNTs for peripheral neurons. Supported by a grant from the GLRCE-NIH-NIAID U54 AI057153. Keywords: Botulinum neurotoxins; Synaptic vesicles 10.1016/j.toxicon.2008.04.004
3. Botulinum neurotoxin interactions with substrate Axel Brunger Stanford University, Stanford, USA
The mechanism by which a CNT properly identifies and cleaves its target SNARE once inside the neuron involves one or more regions of enzyme–substrate interaction remote from the active site (so called exosites). We determined the structure of a CNT endopeptidase in complex with its target SNARE that illustrates the extensive enzyme–substrate interface and the basis of substrate selectivity. Our structure of BoNT/A, in complex with human SNAP-25, revealed multiple exosites, including several that are involved in substrate specificity, and one that likely functions as an allosteric activator of the toxin. Different crystal forms of wildtype BoNT/A revealed conformational variability of several loops near the active site. We also determined crystal structures of the apo form of the TeNT LC49 and that of the BoNT/C1 LC protease50. We found remarkable structural differences between the BoNT/C1 and BoNT/A LC proteases that may explain the dual substrate-binding ability of BoNT/C1. Keywords: Botulinum neurotoxin; SNARE; Protease
References Jin, R., Sikorra, S., Stegmann, C.M., Pich, A., Binz, T., Brunger, A.T., 2007. Biochemistry 46, 10685–10693. Burnett, J.C., Ruthel, G., Stegmann, C.M., Panchal, R.G., Nguyen, T.L., Hermone, A.R., Stafford, R.G., Lane, D.J., Kenny, T.A., McGrath, C.F., Wipf, P., Stahl, A.M., Schmidt, J.J., Gussio, R., Brunger, A.T., Bavari, S., 2007. J. Biol. Chem. 282, 5004–5014. Breidenbach, M.A., Brunger, A.T., 2004. Nature 432, 925–929. 10.1016/j.toxicon.2008.04.005
4. Capture assay for Botulinum Neurotoxin Type A utilizing the receptor protein SV2c Todd Christian, Nancy Shine List Biological Laboratories, Inc., Campbell, CA, USA
Recent biochemical and molecular genetic studies have established synaptic vesicle glycoprotein 2C (SV2c)
as the protein receptor for Botulinum Neurotoxin Type A (BoNT/A). The luminal domain loop of SV2c, between transmembrane domains 7 and 8, has been shown to be the location of BoNT/A binding. Utilizing this information, we have shown through GST pull-down experiments that we can detect binding of BoNT/A to SV2c using an immunoassay. This can be done with either the rat or the human forms of the SV2c protein. Upon specific binding of BoNT/A to its receptor protein, we can introduce our FRET peptide SNAPtides and detect enzymatic cleavage. We describe the reaction conditions and the detection limits using both rat and human forms of the SV2c binding region as well as multiple FRET substrates to determine the optimal conditions for this detection assay. There are three steps in the interruption of synaptic transmission by botulinum toxin. The first is binding to neuronal cells; the second is translocation of the enzymatic light chain out of the endosome; and finally cleavage of synaptosomal proteins to inhibit neurotransmitter release. With this assay we can monitor two of the three steps of toxin activity, binding and cleavage. In the future, we hope to establish this as a new functional assay for the detection and activity of BoNT/A and demonstrate a direct correlation to the gold standard, the mouse bioassay. Keywords: Receptor; SV2c; Detection; FRET 10.1016/j.toxicon.2008.04.006
5. C-terminal fragment of tetanus toxin heavy chain modulates sphingomyelinase activity in neuronal preparations Roger Cubı´ Pique´ , Mireia Herrando Grabulosa, Jose´ Aguilera A´vila , Carles Gil Giro´ Departament de Bioquı´mica i Biologia Molecular i Institut de Neurocie`ncies, `noma de Barcelona, Bellaterra (Cerdanyola del Valle´s), Spain Universitat Auto
Tetanus toxin (TeTx) reaches CNS by means of retrograde transport inside motor neuron axons. The C-terminal domain of TeTx (HC-TeTx) has been described as responsible for binding and internalization of the toxin into neurons. Analysis of this transport reveals common endocytic carriers for HC-TeTx and neurotrophin signalling (i.e. p75NTR, TrkB, NGF and BDNF). Moreover, in previous work from our group, the activation of neurotrophic signalling by HC-TeTx was described. On the basis of these observations, we decided to study the influence of HC-TeTx in p75NTR-dependent events. Since p75NTR lacks intrinsic enzymatic activity and has been described as a sphingomyelinase (SMase)-coupled receptor, the modulation of SMase activity after HC-TeTx treatment in cultured cerebellar granule neurons (CGN) has been determined and compared with the action of NGF. Neutral- and acid-sphingomyelinases are enzymes that catalyze the hydrolysis of sphingomyelin into ceramide and phosphorylcholine. Treatments of CGN at 7 DIV show a moderate yet consistent and significant enhancement of the nSMase activity after HC-TeTx, as assessed by means of enzymatic assays. HC-TeTx treatment of