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 process was developed for production of a candidate vaccine antigen, recombinant C-terminal heavy chain fragment of the botulinum neurotoxin serotype E, rBoNTE(H(c)) in Pichia pastoris. P. pastoris strain GS115 was transformed with the rBoNTE(H(c)) gene inserted into pHILD4 Escherichia coli-P. pastoris shuttle plasmid. The clone was characterized for genetic stability, copy number, and BoNTE(H(c)) sequence. Expression of rBoNTE(H(c)) from the Mut(+) HIS4 clone was confirmed in the shake-flask, prior to developing a fed-batch fermentation process at 5 and 19 L scale. The fermentation process consists of a glycerol growth phase in batch and fed-batch mode using a defined medium followed by a glycerol/methanol transition phase for adaptation to growth on methanol and a methanol induction phase resulting in the production of rBoNTE(H(c)). Specific growth rate, ratio of growth to induction phase, and time of induction were critical for optimal rBoNTE(H(c)) production and minimal proteolytic degradation. A computer-controlled exponential growth model was used for process automation and off-gas analysis was used for process monitoring. The optimized process had an induction time of 9 h on methanol and produced up to 3 mg of rBoNTE(H(c)) per gram wet cell mass as determined by HPLC and Western blot analysis.
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PMID:Cell bank characterization and fermentation optimization for production of recombinant heavy chain C-terminal fragment of botulinum neurotoxin serotype E (rBoNTE(H(c)): antigen E) by Pichia pastoris. 1701 Apr 65

Clostridium botulinum type B neurotoxin was effectively bound to synaptotagmin 2 (Stg2) associated with ganglioside GT1b, however, the molecular interaction between the neurotoxin and the Stg2/GT1b complex has not been identified. Previously, we found that infant botulism-related strain 111 generated a low activity of the neurotoxin (111/NT), which differed in some amino acid residues, especially in the carboxyl terminal half of the heavy chain (H(C)), from the original neurotoxin of strain Okra (Okra/NT) associated with a food-borne botulism. In this study, we evaluated the binding capabilities of site-directed mutants of Okra/H(C) to the Stg2/GT1b complex and to GT1b alone, and investigated the relationship between the toxic action and receptor binding. Replacement of K1187 and E1190 with glutamic acid and lysine, respectively, which substituted for the 111/NT residues, caused a reduction of binding affinity to the Stg2/GT1b complex, suggesting that both these residues contribute to the different binding affinity between Okra/NT and 111/NT. Substitution of four residues, H1240, S1259, W1261 and Y1262, which form a ganglioside pocket, drastically decreased the binding of H(C) to the Stg2/GT1b complex and to GT1b. Mutation in the residues, K1186, E1189, K1191 and K1260 reduced the binding of H(C) to GT1b alone, but not to the Stg2/GT1b complex. Analyses of effects of mutant toxins on toxicity of BoNT/B to cerebellar granule cells suggest the association of cell toxicity with binding to Stg2/GT1b complex but not that to GT1b alone.
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PMID:Differential contribution of the residues in C-terminal half of the heavy chain of botulinum neurotoxin type B to its binding to the ganglioside GT1b and the synaptotagmin 2/GT1b complex. 1718 34

A unique strain of Clostridium botulinum serotype D 4947 produces toxin complexes that are composed of un-nicked components, including a neurotoxin (BoNT) and auxiliary proteins. This BoNT showed aberrant elution upon Superdex gel filtration, indicating a much lower molecular weight, due to hydrophobic interaction with the column. Limited trypsin proteolysis of BoNT produces two nicks; first nick yielded a BoNT 50 kDa light chain disulfide linked to a 100 kDa heavy chain (Hc), and a second nick arose in Hc C-terminal 10 kDa. The second nick occurred in the putative binding domain of the BoNT molecule and induced alterations in its secondary structure, leading to a significant reduction of mouse toxicity in comparison with that of the fully-activated singly nicked BoNT. These results help to clarify the role of the C-terminal half of the Hc in the oral toxicity of single-chain and more complex forms of BoNT.
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PMID:Effect of nicking the C-terminal region of the Clostridium botulinum serotype D neurotoxin heavy chain on its toxicity and molecular properties. 1720 Aug 83

The design, synthesis, and initial inhibitory studies of di- and tetravalent glycoconjugates that target the heavy chain of botulinum neurotoxin A are reported.
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PMID:Synthesis of soluble multivalent glycoconjugates that target the Hc region of botulinum neurotoxin A. 1733 84

A plastic ELISA-on-a-chip (EOC) employing the concept of cross-flow immuno-chromatographic analysis was applied to the measurement of botulinum neurotoxin A (BoNT/A) as agent for bio-terrorism. Two monoclonal antibodies specific to the heavy chain of the toxin were raised and identified to form sandwich binding complexes as the pair with the analyte. For the construction of an immuno-strip, one was utilized as the capture antibody immobilized onto nitrocellulose membrane and the other as the detection coupled to an enzyme, horseradish peroxidase. The two plates of EOC used in this study were fabricated by injection molding of polycarbonate to improve the reproducibility of manufacture and, after inclusion of the immuno-strip, bonded using a UV-sensitive adhesive. Under optimal conditions of analysis, the chip produced a color signal in proportion to the analyte dose and the signal was quantified using a detector equipped with a digital camera. From the dose-response curve, the detection limit of BoNT/A was 2.0 ng mL(-1), approximately five times more sensitive than a commercial-version detection kit employing colloidal gold tracer.
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PMID:Plastic enzyme-linked immunosorbent assays (ELISA)-on-a-chip biosensor for botulinum neurotoxin A. 1738 46

The dynamics of Clostridium botulinum neurotoxins (BoNTs) protein-translocation across membranes was investigated by using a single molecule assay with millisecond resolution on excised patches of neuronal cells. Translocation of BoNT/A light chain (LC) by heavy chain (HC) was observed in real time as an increase of channel conductance: the HC channel is occluded by the LC during transit, then unoccluded after completion of translocation and release of LC-cargo. We identified an entirely unknown succession of intermediate conductance stages during LC translocation. For the single-chain BoNT/E, by contrast to the di-chain BoNT/A, we demonstrate that productive translocation requires proteolysis of the LC cargo from the HC chaperone. We propose a model for the set of protein-protein interactions between translocase and cargo at each step of translocation that supports the notion of an interdependent, tight interplay between the HC chaperone and the LC cargo preventing LC aggregation and dictating the outcome of translocation: productive passage of cargo or abortive channel occlusion by cargo.
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PMID:Single molecule detection of intermediates during botulinum neurotoxin translocation across membranes. 1756 59

Clostridial botulinum neurotoxins (BoNTs) exert their neuroparalytic action by arresting synaptic exocytosis. Intoxication requires the disulfide-linked, di-chain protein to undergo conformational changes in response to pH and redox gradients across the endosomal membrane with consequent formation of a protein-conducting channel by the heavy chain (HC) that translocates the light chain (LC) protease into the cytosol. Here, we investigate the role of the disulfide bridge in the dynamics of protein translocation. We utilize a single channel/single molecule assay to characterize in real time the BoNT channel and chaperone activities in Neuro 2A cells under conditions that emulate those prevalent across endosomes. We show that the disulfide bridge must remain intact throughout LC translocation; premature reduction of the disulfide bridge after channel formation arrests translocation. The disulfide bridge must be on the trans compartment to achieve productive translocation of LC; disulfide disruption on the cis compartment or within the bilayer during translocation aborts it. We demonstrate that a peptide linkage between LC and HC in place of a disulfide bridge is insufficient for productive LC translocation. The disulfide linkage, therefore, dictates the outcome of translocation: productive passage of cargo or abortive channel occlusion by cargo. Based on these and previous findings we suggest a sequence of events for BoNT LC translocation to be HC insertion, coupled LC unfolding, and protein conduction through the HC channel in an N to C terminus orientation and ultimate release of the LC from the HC by reduction of the disulfide bridge concomitant with LC refolding in the cytosol.
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PMID:Crucial role of the disulfide bridge between botulinum neurotoxin light and heavy chains in protease translocation across membranes. 1766 97

Two immunoassay platforms were developed for either the sensitive or rapid detection of botulinum neurotoxin A (BoNT/A), using high-affinity recombinant monoclonal antibodies against the receptor binding domain of the heavy chain of BoNT/A. These antibodies also bind the same epitopes of the receptor binding domain present on a nontoxic recombinant heavy chain fragment used for assay development and testing in the current study. An enzyme-linked immunosorbent assay (ELISA) microarray using tyramide amplification for localized labeling was developed for the specific and sensitive detection of BoNT. This assay has the sensitivity to detect BoNT in buffer and blood plasma samples down to 14fM (1.4 pg mL(-1)). Three capture antibodies and one antibody combination were compared in the development of this assay. Using a selected pair from the same set of recombinant monoclonal antibodies, a renewable surface microcolumn sensor was developed for the rapid detection of BoNT/A in an automated fluidic system. The ELISA microarray assay, because of its sensitivity, offers a screening test with detection limits comparable to the mouse bioassay, with results available in hours instead of days. The renewable surface assay is less sensitive but much faster, providing results in less than 10 min.
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PMID:Enzyme-amplified protein microarray and a fluidic renewable surface fluorescence immunoassay for botulinum neurotoxin detection using high-affinity recombinant antibodies. 1772 91

The purpose of this work was to map the continuous regions recognized by human, horse and mouse anti-botulinum neurotoxin B (BoNT/B) antibodies (Abs). We synthesized a panel of sixty 19-residue peptides (peptide C31 was 24 residues) that overlapped consecutively by 5 residues and together encompassed the entire heavy chain of BoNT/B (H/B, residues 442-1291). Abs from the three host species recognized similar, but not identical, peptides. There were also peptides recognized by two or only by one host species. Where a peptide was recognized by Abs of more than one host species, these Abs were at different levels among the species. Human, horse and mouse Abs bound, although in different amounts, to regions within peptides 736-754, 778-796, 848-866, 932-950, 974-992, 1058-1076 and 1128-1146. Human and horse Abs bound to peptides 890-908 and 1170-1188. Human and mouse Abs recognized peptides 470-488/484-502 overlap, 638-656, 722-740, 862-880, 1030-1048, 1072-1090, 1240-1258 and 1268-1291. We concluded that the antigenic regions localized with the three antisera are quite similar, exhibiting in some cases a small shift to the left or to the right. This is consistent with what is known about protein immune recognition. In the three-dimensional structure, the regions recognized on H/B by anti-BoNT/B Abs occupied surface locations and analysis revealed no correlation between these surface locations and surface electrostatic potential, hydrophilicity, hydrophobicity, or temperature factor. A region that bound mouse Abs overlapped with a recently defined site on BoNT/B that binds to mouse and rat synaptotagmin II, thus providing a molecular explanation for the blocking (protecting) activity of these Abs. The regions thus localized afford candidates for incorporation into a synthetic vaccine design.
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PMID:Immune recognition of botulinum neurotoxin B: antibody-binding regions on the heavy chain of the toxin. 1789 17

Botulinum neurotoxins cause botulism, a neuroparalytic disease in humans and animals. We constructed a replication-incompetent adenovirus encoding a synthesized codon-optimized gene for expression of the heavy chain C-fragment (H(C)50) of botulinum neurotoxin type C (BoNT/C). This recombinant human serotype 5 adenoviral vector (Ad5) was evaluated as a genetic vaccine candidate against botulism caused by BoNT/C in a mouse model. A one-time intramuscular injection with 10(5) to 2 x 10(7)pfu of adenoviral vectors elicited robust serum antibody responses against H(C)50 of BoNT/C as assessed by ELISA. Immune sera showed high potency in neutralizing the active BoNT/C in vitro. After a single dose of 2 x 10(7)pfu adenoviral vectors, the animals were completely protected against intraperitoneal challenge with 100 x MLD(50) of active BoNT/C. The protective immunity appeared to be vaccine dose-dependent. The anti-toxin protective immunity could last for at least 7 months without a booster injection. In addition, we observed that pre-existing immunity to the wild-type Ad5 in the host had no significant influence on the protective efficacy of vaccination. The data suggest that an adenovirus-vectored genetic vaccine is a highly efficient prophylaxis candidate against botulism.
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PMID:Protective immunity against botulism provided by a single dose vaccination with an adenovirus-vectored vaccine. 1789 56


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