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

A targeted delivery vehicle (DV) was developed for intracellular transport of emerging botulinum neurotoxin (BoNT) antagonists. The DV consisted of the isolated heavy chain (HC) of BoNT/A coupled to a 10-kDa amino dextran via the heterobifunctional linker 3-(2-pyridylthio)-propionyl hydrazide. The HC served to target BoNT-sensitive cells and promote internalization of the complex, while the dextran served as a platform to deliver model therapeutic molecules to the targeted cells. To determine the ability of this chimeric glycoprotein to enter neurons, dextran and HC were labeled independently with the fluorescent dyes Oregon green 488 and Cy3, respectively. Internalization of DV was monitored in primary cortical cells using laser confocal microscopy. Incubation of cells for 24 h with DV resulted in discrete punctate labeling of both soma and processes. The Cy3 and Oregon green 488 signals were generally co-localized, suggesting that the complex remained in the same intracellular compartment during the initial 24 h. The DV-associated fluorescence was reduced progressively by co-application of increasing concentrations of unlabeled BoNT/A holotoxin. The results suggest that the BoNT/A HC is able to mediate internalization of a coupled dextran, even though the latter bears no resemblance to the BoNT/A light chain (LC). The HC of BoNT/A thus offers promise as a selective carrier to deliver BoNT antagonists to the nerve terminal cytoplasm for inhibiting the proteolytic activity of internalized BoNT/A LC.
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PMID:Development of a delivery vehicle for intracellular transport of botulinum neurotoxin antagonists. 1190 43

Initial purification of two serotypic variants of recombinant botulinum neurotoxin toxin heavy chain fragment [rBoNT(Hc)], produced intracellularly in the yeast Pichia pastoris, using hydrophobic charge induction chromatography (HCIC) is reported. HCIC employs a matrix containing a weakly ionizable ligand that binds proteins through hydrophobic interactions at neutral pH and elutes the proteins by charge repulsion at acidic pH. HCIC optimization led to different purification conditions for each of the proteins even though they have 58% sequence similarity. The HCIC resin has a higher affinity for the fragment of serotype A than that of serotype B. The 10% dynamic breakthrough capacity for the serotype A fragment is >12.5 mg per ml of resin and is approximately 3.5 mg or the serotype B fragment per ml of resin. Stable elution conditions are also different for the two serotypes. The serotype A fragment is unstable when citrate is used to elute the product. However the serotype B fragment is stable when eluted with citrate buffer, and it is further purified by a overnight precipitation caused by the citrate buffer. This paper reports the development strategy, dynamic capacity breakthrough curves, resin and separation reproducibility, and preliminary scale-up data. The summation of the data demonstrates that HCIC is a scaleable process step for biopharmaceutical production of rBoNT(Hc) proteins.
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PMID:Initial purification of recombinant botulinum neurotoxin fragments for pharmaceutical production using hydrophobic charge induction chromatography. 1206 50

A cell permeabilization method for the release of intracellular proteins from microbial cells was developed. The method was applied to the recovery of recombinant botulinum neurotoxin fragments, expressed intracellularly in the yeast Pichia pastoris, by suspending the cells in an aqueous solution containing N,N-dimethyltetradecylamine. For the botulinum neurotoxin serotype B C-terminal heavy chain fragment, 1.8 mg g(-1) biomass were recovered. For the botulinum neurotoxin serotype A C-terminal heavy chain fragment, 3.7 mg g(-1) biomass were recovered. The concentration of recombinant protein in the cell extracts remained stable for up to 48 and 24 h for the serotype B and serotype A fragments, respectively. The permeabilization method was compared with high-pressure homogenization; the permeabilization method proved to be both more selective and more efficient.
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PMID:Recovery of intracellular recombinant proteins from the yeast Pichia pastoris by cell permeabilization. 1227 Jun 2

The neurotoxin of Clostridium butyricum strain LCL155 (BuNT/LCL155) associated with type E food-borne botulism showed antigenic and biological properties different from those of C. botulinum type E (BoNT/E) andC. butyricum strain BL5262 (BuNT/BL5262). The specific toxicity of BuNT/LCL155 was found to be about 10% those of BoNT/E and BuNT/BL5262. Immunological analysis with monoclonal antibodies against BoNT/E showed that the heavy chain of BuNT/LCL155 differs partially from those of BoNT/E and BuNT/BL5262. Binding experiments with rat brain synaptic membrane revealed that BuNT/LCL155 possesses a binding activity lower than either BoNT/E or BuNT/BL5262. There was no difference in the catalytic activity of the three neurotoxins, which had been determined with a recombinant of the intracellular target protein SNAP-25. These data suggest that the BuNT/LCL155 shares the receptor-recognition site structurally different from BoNT/E and BuNT/BL5262, perhaps causing a decreased specific toxicity.
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PMID:Characterization of Clostridium butyricum neurotoxin associated with food-borne botulism. 1238 45

Clostridial botulinum neurotoxins (BoNTs) abort the process of neurotransmitter release at presynaptic motor nerve terminals, causing muscle paralysis. An enigmatic step in the intoxication process is the mechanism by which the neurotoxin heavy chain (HC) forms the conduit for the translocation of the light chain (LC) protease across the endosomal membrane into the cytosol, its site of action. Here we investigate the mechanism of LC translocation by using the combined detection of channel currents and substrate proteolysis, the two hallmark activities of BoNT. Our data are consistent with the translocation of the LC through the HC channel and show that the LC protease activity is retrieved in the trans compartment after translocation. We propose that the BoNT HC-LC complex embedded in the membrane is a transmembrane chaperone, a dynamic structural device that prevents aggregation and achieves translocation of the LC. In this regard, the complex is similar to the protein conducting/translocating channels of the endoplasmic reticulum, mitochondria and chloroplasts.
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PMID:Translocation of botulinum neurotoxin light chain protease through the heavy chain channel. 1249 Aug 83

Protein toxins are known to translocate through intracellular membranes to reach their cytosolic targets. Results from studies with botulinum neurotoxin suggest that the toxin heavy chain functions as both a channel and a chaperone for translocation of the catalytically active light chain.
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PMID:Moving across membranes. 1245 20

Previously, we demonstrated that the neurotoxin of strain 111 (111/NT) associated with type B infant botulism showed antigenic and biological properties different from that (Okra/NT) produced by a foodborne botulism-related strain, Okra. In this study, the neurotoxin genes of 111/NT and Okra/NT were amplified and the sequences determined. The nucleotide sequences of the genes for both neurotoxins possessed an open reading frame of 3873 bp that encoded 1291 amino acids. The identities of nucleotide sequences and amino acid sequences were 97.6% and 95.7%, respectively. The ratio of nonsynonymous to synonymous substitutions was 0.47. The amino acid substitutions between 111/NT and Okra/NT occurred mainly in the domain of the C-terminal half of heavy chain (H(C)) responsible for binding to its receptor complex of protein and ganglioside. To characterize the binding capability of the H(C), recombinant genes for the H(C) and two hybrid H(C) in which one half of Okra/NT was replaced by the homologous half of 111/NT were constructed and expressed in Escherichia coli. The binding activity of the recombinant H(C) of 111/NT to the protein receptor synaptotagmin II, in the presence of ganglioside GT1b, was 4.2-fold less than Okra/NT, consistent with the corresponding two NTs. The use of hybrid H(C) revealed that mutation of 23 residues in carboxy terminal half of H(C) (1029-1291) of Okra/NT could be attributed to the lower binding activity of 111/NT and thus the differences in binding affinity between the two BoNT/B.
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PMID:Sequence of the gene for Clostridium botulinum type B neurotoxin associated with infant botulism, expression of the C-terminal half of heavy chain and its binding activity. 1252 21

Structural conversion of the serotype A recombinant botulinum neurotoxin heavy chain fragment (rBoNTA(Hc)) produced intracellularly in Pichia pastoris yeast was observed and characterized during purification development efforts. A pH screening study captured the transformation stages of the original recovered species into its derived counterpart and a number of analytical tools such as peptide mapping by LC/MS confirmed the formation of a disulfide bond, especially in samples of neutral to basic pH. A cation exchange chromatographic method proved useful in following the incidence of the reaction in various rBoNTA(Hc) samples. The disulfide formation kinetics were characterized using a one-quarter quadratic factorial design, following the investigation and development of controlled oxidation conditions using cysteine and cystamine as the redox pair. Temperature, pH and concentration of the redox pair had a significant effect on the yield and rate of the disulfide formation. This controlled reaction was eventually introduced as a functional unit operation in the purification process. The summation of preliminary scale-up and potency data showed scalability and robustness in the production of an active disulfide-bonded form of a recombinant botulism vaccine candidate. The presence of the disulfide bond did not effect the vaccine potency and it enhanced the molecule's thermal stability.
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PMID:Identifying and modulating disulfide formation in the biopharmaceutical production of a recombinant protein vaccine candidate. 1289 Jun 12

Several bacteria of the Clostridium genus (C. botulinum) produce 150 kDa di-chainal protein toxins referred as botulinum neurotoxins or BoNTs. They associate with non-toxic companion proteins and form a complex termed botulinum toxin or BoTx. The latter is used in clinic for therapeutic purpose. BoNTs affect cholinergic nerve terminals in periphery where they block acetylcholine release, thereby causing dysautonomia and motorparalysis (i.e. botulism). The cellular action of BoNTs can be depicted according to a three steps model: binding, internalisation and intraneuronal action. The toxins heavy chain mediates binding to specific receptors followed by endocytotic internalisation of BoNT/receptor complex. BoNT receptors may comprise gangliosides and synaptic vesicle-associated proteins as synaptotagmins. Vesicle recycling induces BoNT internalisation. Upon acidification of vesicles, the light chain of the neurotoxin is translocated into the cytosol. Here, this zinc-endopeptidase cleaves one or two among three synaptic proteins (VAMP-synaptobrevin, SNAP25, and syntaxin). As the three protein targets of BoNT play major role in fusion of synaptic vesicles at the release sites, their cleavage is followed by blockage of neurotransmitter exocytosis. The duration of the paralytic effect of the BoNTs is determined by 1) the turnover of their protein target; 2) the time-life of the toxin light chain in the cytosol, and 3) the sprouting of new nerve-endings that are retracted when the poisoned nerve terminal had recovered its full functionality.
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PMID:[Mode of action of botulinum neurotoxin: pathological, cellular and molecular aspect]. 1292 28

A recombinant heavy chain fragment C of botulinum neurotoxin serotype F (BoNTF(Hc)) has been expressed in Pichia pastoris for use as an antigen in a proposed human vaccine. P. pastoris cells were grown using glycerol batch, glycerol fed-batch, and methanol fed-batch methods to achieve high cell densities. The total cellular protein recovered after homogenization was 72 mg/g of cell paste. BoNTF(Hc) was purified from soluble Pichia cell lysate employing ion-exchange chromatographic (IEC) and hydrophobic interaction chromatographic (HIC) methods developed at the bench scale using 10-100 mL columns. The process was performed at the pilot scale using 1-4L columns for evaluation of scale up. The purification process resulted in greater than 98% pure product consisting of at least three forms of BoNTF(Hc) based on mass spectrometry and yielded up to 205 mg/kg cells at the bench scale and 170 mg/kg cells at the pilot scale. Full-length BoNTF(Hc) is present based on mass spectrometry and SDS-PAGE, however is postulated to be N-terminally blocked by acetylation. N-terminal sequencing showed that two of the three forms are missing the first 11 (80%) and 14 (20%) amino acids of the N-terminus from the full-length form. The ratios of the two clipped forms were consistent from the bench to pilot scales. Purified BoNTF(Hc) at the pilot scale was found to sufficiently protect mice against a high dose of BoNTF neurotoxin.
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PMID:Scale-up of the fermentation and purification of the recombinant heavy chain fragment C of botulinum neurotoxin serotype F, expressed in Pichia pastoris. 1468 Sep 33


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