A historical and proteomic analysis of botulinum neurotoxin type/G

Rebecca Terilli Veenhuis, Hercules Moura, Adrian R. Woolfitt, Jon Rees, David M. Schieltz, John R. Barr

Research output: Contribution to journalArticle

Abstract

Background: Clostridium botulinum is the taxonomic designation for at least six diverse species that produce botulinum neurotoxins (BoNTs). There are seven known serotypes of BoNTs (/A through/G), all of which are potent toxins classified as category A bioterrorism agents. BoNT/G is the least studied of the seven serotypes. In an effort to further characterize the holotoxin and neurotoxin-associated proteins (NAPs), we conducted an in silico and proteomic analysis of commercial BoNT/G complex. We describe the relative quantification of the proteins present in the/G complex and confirm our ability to detect the toxin activity in vitro. In addition, we review previous literature to provide a complete description of the BoNT/G complex. Results: An in-depth comparison of protein sequences indicated that BoNT/G shares the most sequence similarity with the/B serotype. A temperature-modified Endopep-MS activity assay was successful in the detection of BoNT/G activity. Gel electrophoresis and in gel digestions, followed by MS/MS analysis of/G complex, revealed the presence of four proteins in the complexes: neurotoxin (BoNT) and three NAPs - nontoxic-nonhemagglutinin (NTNH) and two hemagglutinins (HA70 and HA17). Rapid high-temperature in-solution tryptic digestions, coupled with MS/MS analysis, generated higher than previously reported sequence coverages for all proteins associated with the complex: BoNT 66%, NTNH 57%, HA70 91%, and HA17 99%. Label-free relative quantification determined that the complex contains 30% BoNT, 38% NTNH, 28% HA70, and 4% HA17 by weight comparison and 17% BoNT, 23% NTNH, 42% HA70, and 17% HA17 by molecular comparison. Conclusions: The in silico protein sequence comparisons established that the/G complex is phenetically related to the other six serotypes of C. botulinum. Proteomic analyses and Endopep-MS confirmed the presence of BoNT and NAPs, along with the activity of the commercial/G complex. The use of data-independent MSE data analysis, coupled to label-free quantification software, suggested that the weight ratio BoNT:NAPs is 1:3, whereas the molar ratio of BoNT:NTNH:HA70:HA17 is 1:1:2:1, within the BoNT/G progenitor toxin.

Original languageEnglish (US)
Article number232
JournalBMC Microbiology
Volume11
DOIs
StatePublished - Oct 19 2011
Externally publishedYes

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Neurotoxins
Proteomics
Proteins
Clostridium botulinum
Computer Simulation
botulinum toxin type G
Digestion
Gels
Biological Warfare Agents
Weights and Measures
Type A Botulinum Toxins
Temperature
Hemagglutinins
Electrophoresis
Software

ASJC Scopus subject areas

  • Microbiology
  • Microbiology (medical)

Cite this

A historical and proteomic analysis of botulinum neurotoxin type/G. / Terilli Veenhuis, Rebecca; Moura, Hercules; Woolfitt, Adrian R.; Rees, Jon; Schieltz, David M.; Barr, John R.

In: BMC Microbiology, Vol. 11, 232, 19.10.2011.

Research output: Contribution to journalArticle

Terilli Veenhuis, Rebecca ; Moura, Hercules ; Woolfitt, Adrian R. ; Rees, Jon ; Schieltz, David M. ; Barr, John R. / A historical and proteomic analysis of botulinum neurotoxin type/G. In: BMC Microbiology. 2011 ; Vol. 11.
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abstract = "Background: Clostridium botulinum is the taxonomic designation for at least six diverse species that produce botulinum neurotoxins (BoNTs). There are seven known serotypes of BoNTs (/A through/G), all of which are potent toxins classified as category A bioterrorism agents. BoNT/G is the least studied of the seven serotypes. In an effort to further characterize the holotoxin and neurotoxin-associated proteins (NAPs), we conducted an in silico and proteomic analysis of commercial BoNT/G complex. We describe the relative quantification of the proteins present in the/G complex and confirm our ability to detect the toxin activity in vitro. In addition, we review previous literature to provide a complete description of the BoNT/G complex. Results: An in-depth comparison of protein sequences indicated that BoNT/G shares the most sequence similarity with the/B serotype. A temperature-modified Endopep-MS activity assay was successful in the detection of BoNT/G activity. Gel electrophoresis and in gel digestions, followed by MS/MS analysis of/G complex, revealed the presence of four proteins in the complexes: neurotoxin (BoNT) and three NAPs - nontoxic-nonhemagglutinin (NTNH) and two hemagglutinins (HA70 and HA17). Rapid high-temperature in-solution tryptic digestions, coupled with MS/MS analysis, generated higher than previously reported sequence coverages for all proteins associated with the complex: BoNT 66{\%}, NTNH 57{\%}, HA70 91{\%}, and HA17 99{\%}. Label-free relative quantification determined that the complex contains 30{\%} BoNT, 38{\%} NTNH, 28{\%} HA70, and 4{\%} HA17 by weight comparison and 17{\%} BoNT, 23{\%} NTNH, 42{\%} HA70, and 17{\%} HA17 by molecular comparison. Conclusions: The in silico protein sequence comparisons established that the/G complex is phenetically related to the other six serotypes of C. botulinum. Proteomic analyses and Endopep-MS confirmed the presence of BoNT and NAPs, along with the activity of the commercial/G complex. The use of data-independent MSE data analysis, coupled to label-free quantification software, suggested that the weight ratio BoNT:NAPs is 1:3, whereas the molar ratio of BoNT:NTNH:HA70:HA17 is 1:1:2:1, within the BoNT/G progenitor toxin.",
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AU - Moura, Hercules

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AU - Schieltz, David M.

AU - Barr, John R.

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