Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.24.69 (botulinum neurotoxin)
 1,901 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Native Clostridium botulinum gene coding for type A neurotoxin has been used to construct recombinant derivatives coding separately for L and H polypeptide chains of the toxin. The gene derivatives have been cloned into an expression vector pET28b in E. coli BL21 (DE3) cells. The recombinant L and H proteins seem to be the major individual proteins after IPTG induction of the recombinant cells. Each of the proteins has been accumulated only in inclusion bodies. The recombinant L chain (but not H chain) has been successfully resolubilized. Each of the proteins contains six His residues on the N terminus which allows purification on Ni-agarose columns with high yield. No toxic effect has been observed for both L and H chains after injection of 10 micrograms of recombinant preparations purified from inclusion bodies. Moreover, the injection resulted in an increase in the titer of specific antibodies which protected mice from 1 DLM of type A native botulinum neurotoxin. Hence, the recombinant neurotoxin protein derivatives which are present in E. coli inclusion bodies can be a source of material for producing diagnostic and therapeutic sera against type A botulinum neurotoxin.
Mol Gen Mikrobiol Virusol 2000
PMID:[Effective expression of fragments of a botulinum neurotoxin type A gene, coding for the L-chain and H-chain in E. coli, with formation of products causing protective immunity to administration of the toxin]. 1118 57

Neurotransmitter transporters are regulated through a variety of signal transduction mechanisms which appear to operate in order to maintain appropriate levels of transmitter in the synaptic cleft. One such mechanism is the trafficking of the transporter in association with synaptic vesicle release machinery. This report examines the specifics of trafficking regulation of the rat brain GABA transporter GAT1 by syntaxin 1A, a plasma membrane component of the SNARE complex which is involved in vesicle membrane fusion. In hippocampal neurons, botulinum neurotoxin 1C, which specifically cleaves syntaxin 1A, down-regulates plasma membrane GAT1 levels as assessed by surface biotinylation, suggesting that syntaxin 1A is a positive regulator of GAT1 surface expression. The up-regulation correlates with a decrease in intracellular GAT1 levels and is complete within several minutes. These data suggest that syntaxin 1A mediates the redistribution of GAT1 on a time scale important for the rapid regulation of extracellular GABA levels. Expression of different syntaxin 1A constructs in Xenopus oocytes suggests that several portions of the syntaxin 1A molecule are required for the trafficking of GAT1. These data suggest that the trafficking of GAT1 will be subject to regulatory control by the many molecules known to interact with various domains of syntaxin 1A.
Mol Membr Biol
PMID:Syntaxin 1A up-regulates GABA transporter expression by subcellular redistribution. 1139 10

As has been previously described, tetanus toxin (TeTx) and its H(C) fragment inhibit the sodium-dependent 5-hydroxytryptamine (5-HT) uptake in rat-brain synaptosomes, probably through a kinase mechanism affecting the 5-HT transporter. Now, the inhibition of 5-HT uptake in neurons in primary culture by TeTx in a dose-dependent manner is described in this work. This effect is also produced by the nontoxic C-terminal fragment of the TeTx heavy chain (H(C)-fragment), indicating that 5-HT uptake inhibition is a consequence of the toxin binding to the plasmatic membrane and not to its catalytic activity. This conclusion is supported by the fact that the 5-HT accumulation was not inhibited by the light chain of TeTx or the toxoid, and was even potentiated by botulinum neurotoxin A. These results correlate with the activation of phosphoinositide-phospholipase C activity in the cultures used in this study, this activity only being enhanced by TeTx and by its Hc-fragment. On the other hand, the use of tyrosine phosphorylation modulators indicates that both Na3VO4 and basic fibroblast growth factor (bFGF) produce an enhancement of 5-HT uptake in this system, which is also sensitive to TeTx inhibition. On the other hand, genistein alone is able to reduce the 5-HT transport in cultured neurons, and this effect did not appear to be additive to that elicited by TeTx. This result suggests that TeTx and genistein may share some events in their respective mechanisms of action. Furthermore, the incubation at different concentrations of 12-O-tetradecanoylphorbol 13-acetate (TPA) confirms the involvement of protein kinase C (PKC) in 5-HT transport modulation in rat-brain neuronal primary cultures. In summary, we shall demonstrate in this work that TeTx induces, through its Hc fragment, an inhibition of both basal and stimulated serotonin uptakes in primary neuronal cultures, in parallel to the activation of phosphoinositide-phospholipase C activity and PKC activation.
J Mol Neurosci 2001 Dec
PMID:Tetanus toxin modulates serotonin transport in rat-brain neuronal cultures. 1185 26

Nerve sprouts emerge from motor nerve terminals following blockade of exo-endocytosis for more than 3 days by botulinum neurotoxin (BoNT), and form functional synapses, albeit temporary. Upon restoration of synaptic activity to the parent terminal 7 and 90 days after exposure to BoNT/F or A respectively, a concomitant retraction of the outgrowths was observed. BoNT/E caused short-term neuroparalysis, and dramatically accelerated the recovery of BoNT/A-paralyzed muscle by further truncation of SNAP-25 and its replenishment with functional full-length SNARE. The removal of 9 C-terminal residues from SNAP-25 by BoNT/A leads to persistence of the inhibitory product due to the formation of a nonproductive SNARE complex(es) at release sites, whereas deletion of a further 17 amino acids permits replenishment and a speedy recovery.
Mol Cell Neurosci 2003 Apr
PMID:Dynamics of motor nerve terminal remodeling unveiled using SNARE-cleaving botulinum toxins: the extent and duration are dictated by the sites of SNAP-25 truncation. 1272 43

Tetanus and botulinum neurotoxins selectively invade neurons following binding to complex gangliosides. Recent biochemical experiments demonstrate that two ganglioside binding sites within the tetanus neurotoxin HC-fragment, originally identified in crystallographic studies to bind lactose or sialic acid, are required for productive binding to target cells. Here, we determine by mass spectroscopy studies that the HC-fragment of botulinum neurotoxins A and B bind only one molecule of ganglioside GT1b. Mutations made in the presumed ganglioside binding site of botulinum neurotoxin A and B abolished the formation of these HC-fragment/ganglioside complexes, and drastically diminished binding to neuronal membranes and isolated GT1b. Furthermore, correspondingly mutated full-length neurotoxins exhibit significantly reduced neurotoxicity, thus identifying a single ganglioside binding site within the carboxyl-terminal half of the HC-fragment of botulinum neurotoxins A and B. These binding cavities are defined by the conserved peptide motif H...SXWY...G. The roles of tyrosine and histidine in botulinum neurotoxins A and B in ganglioside binding differ from those in the analogous tetanus neurotoxin lactose site. Hence, these findings provide valuable information for the rational design of potent botulinum neurotoxin binding inhibitors.
Mol Microbiol 2004 Feb
PMID:The HCC-domain of botulinum neurotoxins A and B exhibits a singular ganglioside binding site displaying serotype specific carbohydrate interaction. 1473 Dec 68

Clostridium botulinum and Clostridium tetani, respectively, produce potent toxins, botulinum neurotoxin (BoNT) and tetanus neurotoxin (TeTx), which are responsible for severe diseases, botulism and tetanus. Neurotoxin synthesis is a regulated process in Clostridium. The genes botR/A in C. botulinum A and tetR in C. tetani positively regulate expression of BoNT/A and associated non-toxic proteins (ANTPs), as well as TeTx respectively. The botR/A gene lies in close vicinity of the two operons which contain bont/A and antps genes in C. botulinum A, and tetR immediately precedes the tetX gene in C. tetani. We show that BotR/A and TetR function as specific alternative sigma factors rather than positive regulators based on the following results: (i) BotR/A and TetR associated with target DNAs only in the presence of the RNA polymerase core enzyme (Core), (ii) BotR/A and TetR directly bound with the core enzyme, (iii) BotR/A-Core recognized -35 and -10 regions of ntnh-bont/A promoter and (iv) BotR/A and TetR triggered in vitro transcription from the target promoters. In C. botulinum A, bont/A and antps genes are transcribed as bi- and tricistronic operons controlled by BotR/A. BotR/A and TetR are seemingly related to a new subgroup of the sigma70 family that includes TcdR and UviA, which, respectively, regulate production of toxins A and B in C. difficile and bacteriocin in C. perfringens. Sequences of -35 region are highly conserved in the promoter of target toxin genes in C. botulinum, C. tetani, C. difficile and C. perfringens. Overall, a common regulation mechanism probably controls toxin gene expression in these four toxigenic clostridial species.
Mol Microbiol 2005 Jan
PMID:BotR/A and TetR are alternative RNA polymerase sigma factors controlling the expression of the neurotoxin and associated protein genes in Clostridium botulinum type A and Clostridium tetani. 1561 31

Nuclear magnetic resonance (NMR) spectroscopy is a powerful technique for studying bimolecular interactions at the atomic scale. Our NMR laboratory is involved in the identification of small molecules, or ligands, that bind to target protein receptors such as tetanus neurotoxin (TeNT) and botulinum neurotoxin, anthrax proteins, and HLA-DR10 receptors on non-Hodgkin lymphoma cancer cells. Once low-affinity binders are identified, they can be linked together to produce multidentate synthetic high-affinity ligands (SHALs) that have very high specificity for their target protein receptors. An important nanotechnology application for SHALs is their use in the development of robust chemical sensors or biochips for the detection of pathogen proteins in environmental samples or body fluids. Here we describe a recently developed NMR competition assay based on transferred nuclear Overhauser effect spectroscopy that enables the identification of sets of ligands that bind to the same site, or a different site, on the surface of TeNT fragment C (TetC) than a known "marker" ligand, doxorubicin. Using this assay, one can identify the optimal pairs of ligands to be linked together for creating detection reagents, as well as estimate the relative binding constants for ligands competing for the same site.
Methods Mol Biol 2005
PMID:Application of NMR methods to identify detection reagents for use in development of robust nanosensors. 1565 83

The hemagglutinating protein HA33 from Clostridium botulinum is associated with the large botulinum neurotoxin secreted complexes and is critical in toxin protection, internalization, and possibly activation. We report the crystal structure of serotype A HA33 (HA33/A) at 1.5 A resolution that contains a unique domain organization and a carbohydrate recognition site. In addition, sequence alignments of the other toxin complex components, including the neurotoxin BoNT/A, hemagglutinating protein HA17/A, and non-toxic non-hemagglutinating protein NTNHA/A, suggests that most of the toxin complex consists of a reoccurring beta-trefoil fold.
J Mol Biol 2005 Mar 04
PMID:The structure of the neurotoxin-associated protein HA33/A from Clostridium botulinum suggests a reoccurring beta-trefoil fold in the progenitor toxin complex. 1570 19

We have used a set of synthetic overlapping peptides encompassing the entire heavy (H) chain of botulinum neurotoxin serotype A (BoNT/A) to map, in two mouse strains (BALB/c, H2d, and SJL, H2S), the regions on the H-chain recognized by Abs in the last bleed of non-protective anti-BoNT/A antisera and in the bleed of protective antisera immediately following it in the bleeding schedule. Although the protective antisera bound slightly higher amounts of total (IgG+IgM) Abs, non-protective and protective BALB/c antisera showed similar peptide-binding profiles involving peptides N6/N7, N25, C2/C3, C9/C10/C11, C15, C18, C24, C30, and C31 and, at lower amounts of bound Abs, peptides N19, C6/C7, and C28. IgG+IgM antibodies of the protective SJL antisera recognized peptides N5, N22, and C21, and these peptides were only slightly recognized (N22, C21) or unrecognized (N5) by the non-protective antisera. Additionally, peptides N7/N8, N25, C11, C15, and less so N27/N28 bound two-fold or more Abs from the SJL protective antisera than the non-protective antisera. The Abs bound to peptides C4 and C29 were of relatively lower affinity. Peptides C2/C3, C7, C18/C19, C24, C30, and C31 bound higher amounts of Abs in the SJL protective versus the non-protective antisera, but the differences were less than double. We also mapped the binding profiles of the IgG Abs in these sera. BALB/c and SJL had 13-36-fold higher of IgG Abs that bound to BoNT/A in the protective antisera relative to non-protective antisera. The IgG Abs in the protective antisera of each mouse haplotype bound to the same peptides that bound total Abs in the correlate antiserum. But in both mouse strains, the non-protective Abs showed little or no IgG Abs that bound to these peptides. In the SJL haplotype, the IgG response to peptide N5 was transient, appearing strongly in early protective Abs and disappearing by day 70. It is not clear whether the response to region N5 plays a role in initiating and contributing to the protective activity of the toxin in the SJL strain in the early stages but is not needed in later hyperimmune stages of the Ab response. It is concluded that the switch in BALB/c and SJL mice from non-protective to protective Abs is not associated with major changes in the epitope-recognition profiles. Although some slight differences between non-protective and protective antisera appeared in their levels of Abs that were bound by some peptides, these differences are not sufficient to explain differences in the protection properties. Protection was mostly associated with the immunoglobulin class of the antibodies. IgM antibodies were non-protective, while IgG Abs produced after the switch were protective.
Mol Immunol 2005 Aug
PMID:Submolecular recognition profiles in two mouse strains of non-protective and protective antibodies against botulinum neurotoxin A. 1595 Jul 44

Botulism is caused by botulinum neurotoxin (BoNT), the most poisonous substance known. Potential use of BoNT as a biothreat agent has made development of sensitive assays for toxin detection and potent antitoxin for treatment of intoxication a high priority. To improve detection and treatment of botulism, molecular evolution and yeast display were used to increase the affinity of two neutralizing single chain Fv (scFv) antibodies binding BoNT serotype A (BoNT/A). Selection of yeast displayed scFv libraries was performed using methods to select for both increased association rate constant (k(on)) and decreased dissociation rate constants (k(off)). A single cycle of error prone mutagenesis increased the affinity of the 3D12 scFv 45-fold from a K(D) of 9.43x10(-10)M to a K(D) of 2.1x10(-11)M. Affinity of the HuC25 scFv was increased 37-fold from 8.44x10(-10)M to 2.26x10(-11)M using libraries constructed by both random and site directed mutagenesis. scFv variable region genes were used to construct IgG for use in detection assays and in vivo neutralization studies. While IgG had the same relative increases in affinity as scFv, (35-fold and 81-fold, respectively, for 3D12 and HuC25) higher solution equilibrium binding constants were observed for the IgG, with the 3D12 K(D) increasing from 6.07x10(-11)M to 1.71x10(-12)M and the HuC25 K(D) increasing from 4.51x10(-11)M to 5.54x10(-13)M. Affinity increased due to both an increase in k(on), as well as slowing of k(off). Higher affinity antibodies had increased sensitivity, allowing detection of BoNT/A at concentrations as low as 1x10(-13)M. The antibodies will also allow testing of the role of affinity in in vivo toxin neutralization and could lead to the generation of more potent antitoxin.
J Mol Biol 2005 Aug 05
PMID:Molecular evolution of antibody affinity for sensitive detection of botulinum neurotoxin type A. 1600 90


<< Previous 1 2 3 4 5 6 7 Next >>