Physicochemical and immunological characterization of the type E botulinum neurotoxin binding protein purified fromClostridium botulinum

Abstract

Type E botulinum neurotoxin is produced by Clostridium botulinum along with a neurotoxin binding protein which helps protect the neurotoxin from adverse pH, temperature, and proteolytic conditions. The neurotoxin binding protein has been purified as a 118-kDa protein. Secondary structure content of the neurotoxin binding protein as revealed by far-UV circular dichroism spectroscopy was 19% alpha-helix, 50% beta-sheets, 28% random coils, and 3% beta-turns. This compared to 22% alpha-helix, 44% beta-sheets, 34% random coils, and no beta-turns of the type E botulinum neurotoxin. The complex of the two proteins revealed 25% alpha-helix, 45% beta-sheets, 27% random coils, and 3% beta-turns, suggesting a significant alteration at least in the alpha-helical folding of the two proteins upon their interaction. Tyrosine topography is altered considerably (28%) when the neurotoxin and its binding protein are separated, indicating strong interaction between the two proteins. Gel filtration results suggested that type E neurotoxin binding protein clearly complexes with type E neurotoxin. The interaction is favored at low pH as indicated by an initial binding rate of 8.4 min-1 at pH 5.7 compared to 4.0 min-1 at pH 7.5 as determined using a fiber optic-based biosensor. The neurotoxin and its binding protein apparently are of equivalent antigenicity, as both reacted equally on enzyme-linked immunosorbent assay to polyclonal antibodies raised against the toxoid of their complex.

Detection of Type A, B, E, and F Clostridium botulinum Neurotoxins in Foods by Using an Amplified Enzyme-Linked Immunosorbent Assay with Digoxigenin-Labeled Antibodies

“…The biological activity which is expressed in LD 50 or sometimes minimal mouse lethal dose is more relevant to the measurement of toxicity and is dependent on a variety of factors including the purification method for toxins and perhaps how the toxin is produced in culture and by what strain of a given type. Although BoNTs exhibit about 40% amino acid and 50 to 60% sequence homology among various serotypes, the immunological cross-reaction among serotypes is minimal (22,25,40). Less than 1% cross-reactivity among antibodies has been reported in previous ELISAs (31).…”

“…NAPs are known to protect the BoNTs from the acidic environment and proteases of the gastrointestinal tract (33,37,38,41). This protective complex is partly responsible for making BoNTs the most potent natural food poisoning agents known (26,29,40).…”

Llama Single Domain Antibodies Specific for the 7 Botulinum Neurotoxin Serotypes as Heptaplex Immunoreagents

“…The NAPs tend to be less conserved across serotypes, though homologies have been identified from recent genomic studies [26], [27] and cross-reactivities have been noted from immunological studies [28], [29], [30]. Within those subtypes so far studied, NAPS can also vary, with prominent components such as HA33 of subtype A1 being present in A4 but not A2 or A3 for example [27].…”

Botulinum neurotoxin structure, engineering, and novel cellular trafficking and targeting

“…It is notable that BoNT/E and /F, which have shortest duration of muscle paralysis are produced as a complex of two proteins -BoNT and the neurotoxin binding protein (NBP) (Singh et al, 1995) -whereas other serotypes are produced as a large complex of BoNT and five other neurotoxin associated proteins (Cai et al, 1999;Sharma and Singh, 2004). Another aspect that has not been examined closely to explain the longevity is a correlation between the native complex forms of BoNT and the longevity of the muscle paralysis.…”