Expression, purification, and characterization of Clostridium botulinum type B light chain

Gilsdorf, Janice; Gul, Nizamettin; Smith, Leonard A.

doi:10.1016/j.pep.2005.09.024

Cited by 28 publications

(19 citation statements)

References 43 publications

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“…2b, lanes 4 and 5). This is consistent with the results obtained by other researchers (6) who demonstrated that some dimers remain intact even under reducing conditions. Steric hindrance may make the substrate binding cleft and active site less accessible in the dimer; thus, the cleavage of VAMPtide by BoNT/B LC was slow without DTT (Fig.…”

Section: Vol 76 2010supporting

confidence: 93%

“…These results clearly show that the activities of different forms of BoNT/B are highly dependent on reaction conditions which affect the protein structure. This information is useful for understanding the present discrepancies in regard to the activity and inhibition of BoNT/B due to the different forms of active BoNT/B used in different studies (6,11). For example, it has been reported that the K m for BoNT/B LC without DTT treatment is about 0.08 mM and its k cat is about 40 s Ϫ1 (6,11).…”

Section: Vol 76 2010mentioning

confidence: 96%

“…This information is useful for understanding the present discrepancies in regard to the activity and inhibition of BoNT/B due to the different forms of active BoNT/B used in different studies (6,11). For example, it has been reported that the K m for BoNT/B LC without DTT treatment is about 0.08 mM and its k cat is about 40 s Ϫ1 (6,11). Nonnicked BoNT/B holotoxin has a K m of 0.3 mM and a k cat of 23.8 s Ϫ1 (16).…”

Section: Vol 76 2010mentioning

confidence: 99%

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Endopeptidase Activities of Botulinum Neurotoxin Type B Complex, Holotoxin, and Light Chain

Wang

Riding

Lindo

et al. 2010

Appl Environ Microbiol

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Botulinum neurotoxin (BoNT) serotype B (BoNT/B) is one of the serotypes of BoNT that causes deadly human botulism, though it is used clinically for treatment of many neuromuscular diseases. BoNT/B is produced by Clostridium botulinum, and it is secreted along with a group of neurotoxin-associated proteins (NAPs) in the form of a BoNT/B complex. The complex dissociates into a 150-kDa holotoxin and NAPs at alkaline pHs. The 150-kDa BoNT/B holotoxin can be nicked to produce a 50-kDa domain referred to as the light chain (LC) and a 100-kDa heavy chain, with the former possessing a unique endopeptidase activity. The two chains remain linked through a disulfide bond that can be reduced to separate the two chains. The endopeptidase activity is present in all three forms of the toxin (complex, purified BoNT/B holotoxin, and separated light chain), which are used by different researchers to develop detection methods and screen for inhibitors. In this research, the endopeptidase activities of the three forms, for the first time, were compared under the same conditions. The results show that enzyme activities of the three forms differ significantly and are largely dependent on nicking and disulfide reduction conditions. Under the conditions used, LC had the highest level of activity, and the complex had the lowest. The activity was enhanced by nicking of BoNT/B holotoxin and was enhanced even more by dithiothreitol (DTT) reduction after nicking. This information is useful for understanding the properties of BoNT endopeptidases and for comparing the efficacies of different inhibitors when they are tested with different forms of BoNT endopeptidase.

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Section: Vol 76 2010supporting

confidence: 93%

Section: Vol 76 2010mentioning

confidence: 96%

Section: Vol 76 2010mentioning

confidence: 99%

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Endopeptidase Activities of Botulinum Neurotoxin Type B Complex, Holotoxin, and Light Chain

Wang

Riding

Lindo

et al. 2010

Appl Environ Microbiol

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“…Materials-Recombinant BoNT LcA and light chain of serotype B (LcB) were purified as described (25)(26)(27), and similar purification of that of serotype D (LcD) will be published elsewhere. Recombinant LcE (BBtech, Inc., Dartmouth, MA) and LcF (LIST Biological Laboratories, Campbell, CA) were commercial products.…”

Section: Methodsmentioning

confidence: 99%

The C Terminus of the Catalytic Domain of Type A Botulinum Neurotoxin May Facilitate Product Release from the Active Site

Rahman

Frasca²,

Swaminathan

et al. 2013

Journal of Biological Chemistry

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Background:The function of C terminus of botulinum neurotoxin catalytic domain is unknown. Results: Synthetic C-terminal peptides competitively inhibited but at stoichiometric concentrations stimulated serotype A proteolytic activity. Conclusion: C terminus interacts with the active site and may function by removing a product. Significance: The inhibition and product removal appear to be a unique feature of type A botulinum neurotoxin among catalytic proteins.

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“…The heavy chain of BoNT binds to receptors on peripheral cholinergic neurons, leading to internalization of the toxin, and the light chain is a zinc metalloprotease. Both chains must be present in the disulfide-linked holotoxin form to cause botulism; i.e., individual chains are not toxic (1,15,29,39). Upon escape from endosomes into the neuronal cytosol, the light chain of each BoNT serotype cleaves only one peptide bond in its respective substrate.…”

mentioning

confidence: 99%

Use of a Recombinant Fluorescent Substrate with Cleavage Sites for All Botulinum Neurotoxins in High-Throughput Screening of Natural Product Extracts for Inhibitors of Serotypes A, B, and E

Hines

Kim

Stafford

et al. 2008

Appl Environ Microbiol

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The seven serotypes of botulinum neurotoxin (BoNTs) are zinc metalloproteases that cleave and inactivate proteins critical for neurotransmission. The synaptosomal protein of 25 kDa (SNAP-25) is cleaved by BoNTs A, C, and E, while vesicle-associated membrane protein (VAMP) is the substrate for BoNTs B, D, F, and G. BoNTs not only are medically useful drugs but also are potential bioterrorist and biowarfare threat agents. Because BoNT protease activity is required for toxicity, inhibitors of that activity might be effective for antibotulinum therapy. To expedite inhibitor discovery, we constructed a hybrid gene encoding (from the N terminus to the C terminus, with respect to the expressed product) green fluorescent protein, then a SNAP-25 fragment encompassing residues Met-127 to Gly-206, and then VAMP residues Met-1 to Lys-94. Cysteine was added as the C terminus. The expressed product, which contained the protease cleavage sites for all seven botulinum serotypes, was purified and coupled covalently through the C-terminal sulfhydryl group to maleimide-activated 96-well plates. The substrate was readily cleaved by BoNTs A, B, D, E, and F. Using this assay and an automated 96-well pipettor, we screened 528 natural product extracts for inhibitors of BoNT A, B, and E protease activities. Serotype-specific inhibition was found in 30 extracts, while 5 others inhibited two serotypes.

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Expression, purification, and characterization of Clostridium botulinum type B light chain

Cited by 28 publications

References 43 publications

Endopeptidase Activities of Botulinum Neurotoxin Type B Complex, Holotoxin, and Light Chain

Endopeptidase Activities of Botulinum Neurotoxin Type B Complex, Holotoxin, and Light Chain

The C Terminus of the Catalytic Domain of Type A Botulinum Neurotoxin May Facilitate Product Release from the Active Site

Use of a Recombinant Fluorescent Substrate with Cleavage Sites for All Botulinum Neurotoxins in High-Throughput Screening of Natural Product Extracts for Inhibitors of Serotypes A, B, and E

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