A historical and proteomic analysis of botulinum neurotoxin type/G

Details

• Alternative Title:
  BMC Microbiol
• Personal Author:
  Terilli, Rebecca R ; Moura, Hercules ; Woolfitt, Adrian R ; Rees, Jon ; Schieltz, David M ; Barr, John R
• Description:
  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.
  
  
• Subjects:
  Amino Acid Sequence Botulinum Toxins Clostridium Botulinum Hemagglutinins Molecular Sequence Data Proteomics Sequence Homology, Amino Acid Tandem Mass Spectrometry
• Source:
  BMC Microbiol. 2011; 11:232.
• Document Type:
  Journal Article
• Volume:
  11
• Collection(s):
  CDC Public Access
• Main Document Checksum:
  urn:sha256:ab44d09b86a726cc5f133f0f78fa8ee7792c29a547804023ee7df87c3ff16954
• Download URL:
  https://stacks.cdc.gov/view/cdc/3368/cdc_3368_DS1.pdf
• File Type:
  [PDF - 736.81 KB ]