Molecular Composition and Extinction Coefficient of Native Botulinum Neurotoxin Complex Produced by Clostridium botulinum Hall A Strain

Bryant, Anne-Marie; Davis, Jenny; Cai, Shuowei; Singh, Bal Ram

doi:10.1007/s10930-013-9465-6

Cited by 15 publications

(14 citation statements)

References 38 publications

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“…Although their reducing SDS-PAGE results show 7 bands for NAPs, it is still unclear what the molecular composition is. NAPs purified in our lab is passed through a G-100 Sephadex column before eluting it through an anion exchange column to remove the additional impurities and also its exact molecular composition has been determined [20]. Also, there is variation in the buffer and salt composition used for CD analysis in our experiments.…”

Section: Discussionmentioning

confidence: 99%

“…It was demonstrated that the mechanism of toxin protection by HA-33 could be either due to the structural changes in the toxin by direct association with HA-33 or by potential blocking of the proteolytic cleavage sites on the toxin by HA-33, which renders the toxin protease-resistant in the GI tract. Based on these results and on further research, the exact molecular composition and a schematic model for BoNT/A complex was proposed showing the direct interaction of toxin with HA-33 [20]. Thus, the current research findings on the relevant roles of NTNH and Hn-33 in toxin protection strongly suggest that the stability of the toxin at acidic pH conditions of the stomach could be attributed to the protective action by NTNH, while HA-33 could be majorly involved in conferring proteolytic resistance to toxin in the protease-rich environment of the GI tract.…”

Section: Discussionmentioning

confidence: 99%

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Role of Neurotoxin Associated Proteins in the Low pH Induced Structural Changes in the Botulinum Neurotoxin Complex

Chellappan

Kumar²,

Cai

et al. 2014

Protein J

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BoNT (Botulinum Neurotoxin) produced by the bacterium Clostridium botulinum as a complex with NAPs causes botulism. It has been known that the NAPs protect the toxin from both extremes of pHs and proteases of the GI tract. In an attempt to emulate the physiological conditions encountered by the toxin, we examined BoNT/A, BoNT/A complex, and NAPs under different pH conditions and monitored their structural characteristics by far-UV CD and thermal denaturation analysis. BoNT/A complex showed the maximum CD signal with a molar ellipticity of −1.8 × 105 deg.cm2/dmol at 222 nm at both acidic and neutral pHs. Thermal denaturation analysis revealed NAPs to be the most stable amongst the three protein samples examined. Interestingly and quite uniquely, at pH 2.5, there was an increase in CD signal for BoNT complex as a function of temperature, which correlated with the NAPs profile, indicating a shielding effect of NAPs on BoNT complex at low pH. Calculation of the weighted mean of the ellipticities at the Tm for thermal unfolding of toxin and NAPs at neutral and acidic pHs showed variation with that of BoNT complex, suggesting structural reorganization in BoNT complex upon the association of NAPs and BoNT. In conclusion, this study reveals the structural behavior of BoNT complex and NAPs with pH changes substantially, which could be quite relevant for BoNT survival under extreme pH conditions in vivo.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Role of Neurotoxin Associated Proteins in the Low pH Induced Structural Changes in the Botulinum Neurotoxin Complex

Chellappan

Kumar²,

Cai

et al. 2014

Protein J

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“…Several studies have been performed to elucidate the biological and structural roles of the nontoxic botulinum complex proteins. Most of the studies have been performed on toxin complexes purified by chromatography or by in vitro reconstitution with recombinant BoNTs and nontoxic complex protein components expressed in Escherichia coli (45,(47)(48)(49). Structural studies of TCs have mainly used electron microscopy (EM), since high-quality crystals for X-ray diffraction of the LL and other complexes have not been obtained from native cultures.…”

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confidence: 99%

Immunoprecipitation of Native Botulinum Neurotoxin Complexes from Clostridium botulinum Subtype A Strains

Lin

Tepp

Bradshaw

et al. 2015

Appl Environ Microbiol

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Botulinum neurotoxins (BoNTs) naturally exist as components of protein complexes containing nontoxic proteins. The nontoxic proteins impart stability of BoNTs in the gastrointestinal tract and during purification and handling. The two primary neurotoxin complexes (TCs) are (i) TC1, consisting of BoNT, nontoxin-nonhemagglutinin (NTNH), and hemagglutinins (HAs), and (ii) TC2, consisting of BoNT and NTNH (and possibly OrfX proteins). In this study, BoNT/A subtypes A1, A2, A3, and A5 were examined for the compositions of their TCs in culture extracts using immunoprecipitation (IP). IP analyses showed that BoNT/A1 and BoNT/A5 form TC1s, while BoNT/A2 and BoNT/A3 form TC2s. A Clostridium botulinum host strain expressing recombinant BoNT/A4 (normally present as a TC2) from an extrachromosomal plasmid formed a TC1 with complexing proteins from the host strain, indicating that the HAs and NTNH encoded on the chromosome associated with the plasmid-encoded BoNT/A4. Strain NCTC 2916 (A1/silent B1), which carries both an ha silent bont/b cluster and an orfX bont/a1 cluster, was also examined. IP analysis revealed that NCTC 2916 formed only a TC2 containing BoNT/A1 and its associated NTNH. No association between BoNT/A1 and the nontoxic proteins from the silent bont/b cluster was detected, although the HAs were expressed as determined by Western blotting analysis. Additionally, NTNH and HAs from the silent bont/b cluster did not form a complex in NCTC 2916. The stabilities of the two types of TC differed at various pHs and with addition of KCl and NaCl. TC1 complexes were more stable than TC2 complexes. Mouse serum stabilized TC2, while TC1 was unaffected. Botulinum neurotoxins (BoNTs) are produced by anaerobic, Gram-positive, spore-forming bacteria, including Clostridium botulinum and rare strains of Clostridium baratii and Clostridium butyricum. BoNTs are the most poisonous protein toxins known in nature, and they are classified as tier 1 category A select agents due to their potential use as bioterrorism agents (1). Purified BoNT is a protein with a molecular mass of ca. 150 kDa, consisting in its active form of a heavy chain (Hc) (ϳ100 kDa) and a light chain (Lc) (ϳ50 kDa) linked by a disulfide bond and noncovalent molecular interactions (2). The heavy chain contains two functional domains, a receptor binding domain and a translocation domain (3, 4), while the light chain is responsible for catalytic activity on SNARE substrates in the neuronal cell cytosol. BoNTs are distinguished immunologically into seven serotypes designated A to G based on toxin neutralization using homologous antitoxins (5). Recently, the existence of an eighth serotype, H, in a dual-toxin-producing strain of C. botulinum has been reported (5).Of the seven established serotypes, BoNT/A is of particular interest since it is the most potent BoNT, exhibits longer duration in humans than other serotypes, and is also implicated in most cases of human botulism in the continental United States (6, 7). Within serotype A, five subtypes of BoNT/A (A1 to A5) h...

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“…Clostridium species, however, naturally produce toxin complexes consisting of the core holotoxin in association with NAPs [62][63][64] . The NAPs protect the toxin from protease attack in the gastro-intestinal tract and are believed to facilitate toxin transport across the intestine epithelium 63,65 .…”

Section: Discussionmentioning

confidence: 99%

Isolation and Quantification of Botulinum Neurotoxin From Complex Matrices Using the BoTest Matrix Assays

Dunning

Piazza

Zeytin

et al. 2014

JoVE

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Accurate detection and quantification of botulinum neurotoxin (BoNT) in complex matrices is required for pharmaceutical, environmental, and food sample testing. Rapid BoNT testing of foodstuffs is needed during outbreak forensics, patient diagnosis, and food safety testing while accurate potency testing is required for BoNT-based drug product manufacturing and patient safety. The widely used mouse bioassay for BoNT testing is highly sensitive but lacks the precision and throughput needed for rapid and routine BoNT testing. Furthermore, the bioassay's use of animals has resulted in calls by drug product regulatory authorities and animal-rights proponents in the US and abroad to replace the mouse bioassay for BoNT testing. Several in vitro replacement assays have been developed that work well with purified BoNT in simple buffers, but most have not been shown to be applicable to testing in highly complex matrices. Here, a protocol for the detection of BoNT in complex matrices using the BoTest Matrix assays is presented. The assay consists of three parts: The first part involves preparation of the samples for testing, the second part is an immunoprecipitation step using anti-BoNT antibody-coated paramagnetic beads to purify BoNT from the matrix, and the third part quantifies the isolated BoNT's proteolytic activity using a fluorogenic reporter. The protocol is written for high throughput testing in 96-well plates using both liquid and solid matrices and requires about 2 hr of manual preparation with total assay times of 4-26 hr depending on the sample type, toxin load, and desired sensitivity. Data are presented for BoNT/A testing with phosphate-buffered saline, a drug product, culture supernatant, 2% milk, and fresh tomatoes and includes discussion of critical parameters for assay success. Video LinkThe video component of this article can be found at

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Molecular Composition and Extinction Coefficient of Native Botulinum Neurotoxin Complex Produced by Clostridium botulinum Hall A Strain

Cited by 15 publications

References 38 publications

Role of Neurotoxin Associated Proteins in the Low pH Induced Structural Changes in the Botulinum Neurotoxin Complex

Role of Neurotoxin Associated Proteins in the Low pH Induced Structural Changes in the Botulinum Neurotoxin Complex

Immunoprecipitation of Native Botulinum Neurotoxin Complexes from Clostridium botulinum Subtype A Strains

Isolation and Quantification of Botulinum Neurotoxin From Complex Matrices Using the BoTest Matrix Assays

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