Substrates and Controls for the Quantitative Detection of Active Botulinum Neurotoxin in Protease-Containing Samples

Botulinum neurotoxins (BoNTs) are used in a wide variety of medical applications, but there is limited pharmacokinetic data on active BoNT. Monitoring BoNT activity in the circulation is challenging because BoNTs are highly toxic and are rapidly taken up by neurons and removed from the bloodstream. Previously we reported a sensitive BoNT “Assay with a Large Immunosorbent Surface Area” that uses conversion of fluorogenic peptide substrates to measure the intrinsic endopeptidase activity of bead-captured BoNT. However, in complex biological samples, protease contaminants can also cleave the substrates, reducing sensitivity and specificity of the assay. Here, we present a novel set of fluorogenic peptides that serve as BoNT-specific substrates and protease-sensitive controls. BoNT-cleavable substrates contain a C-terminal Nle, while BoNT-noncleavable controls contain its isomer ε-Ahx. The substrates are cleaved by BoNT subtypes A1-A3 and A5. Substrates and control peptides can be cleaved by non-BoNT proteases (e.g., trypsin, proteinase K, and thermolysin) while obeying Michaelis–Menten kinetics. Using this novel substrate/control set, we studied BoNT/A1 activity in two mouse models of botulism. We detected BoNT/A serum activities ranging from ∼3600 to 10 amol/L in blood of mice that had been intravenously injected 1 h prior with BoNT/A1 complex (100 to 4 pg/mouse). We also detected the endopeptidase activity of orally administered BoNT/A1 complex (1 μg) in blood 5 h after administration; activity was greatest 7 h after administration. Redistribution and elevation rates for active toxin were measured and are comparable to those reported for inactive toxin

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<p>Soleus muscles of mice treated with BoNT/C-wt and used for the analysis of EJPs of <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006567#ppat.1006567.g005" target="_blank">Fig 5B</a> were fixed immediately after the electrophysiological recordings and stained for <b>(A)</b> cleaved SNAP-25 (SNAP-25_c) or <b>(B)</b> Syntaxin-1A/1B (Stx-1A/1B), both shown in red. NMJs were spotted with α-Bungarotoxin (α-BTX, in green). The first row of panels represents the staining of a control muscle. Scale bar, 10 μm.</p

Botulinum neurotoxin C mutants reveal different effects of syntaxin or SNAP-25 proteolysis on neuromuscular transmission

<div><p>Botulinum neurotoxin serotype C (BoNT/C) is a neuroparalytic toxin associated with outbreaks of animal botulism, particularly in birds, and is the only BoNT known to cleave two different SNARE proteins, SNAP-25 and syntaxin. BoNT/C was shown to be a good substitute for BoNT/A1 in human dystonia therapy because of its long lasting effects and absence of neuromuscular damage. Two triple mutants of BoNT/C, namely BoNT/C <i>S51T/R52N/N53P</i> (BoNT/C α-51) and BoNT/C <i>L200W/M221W/I226W</i> (BoNT/C α-3W), were recently reported to selectively cleave syntaxin and have been used here to evaluate the individual contribution of SNAP-25 and syntaxin cleavage to the effect of BoNT/C <i>in vivo</i>. Although BoNT/C α-51 and BoNT/C α-3W toxins cleave syntaxin with similar efficiency, we unexpectedly found also cleavage of SNAP-25, although to a lesser extent than wild type BoNT/C. Interestingly, the BoNT/C mutants exhibit reduced lethality compared to wild type toxin, a result that correlated with their residual activity against SNAP-25. In spite of this, a local injection of BoNT/C α-51 persistently impairs neuromuscular junction activity. This is due to an initial phase in which SNAP-25 cleavage causes a complete blockade of neurotransmission, and to a second phase of incomplete impairment ascribable to syntaxin cleavage. Together, these results indicate that neuroparalysis of BoNT/C at the neuromuscular junction is due to SNAP-25 cleavage, while the proteolysis of syntaxin provides a substantial, but incomplete, neuromuscular impairment. In light of this evidence, we discuss a possible clinical use of BoNT/C α-51 as a botulinum neurotoxin endowed with a wide safety margin and a long lasting effect.</p></div

Time course of neurotransmission recovery in soleus muscles upon a local injection of a low dose of BoNT/C α-51.

<p><b>(A)</b> The black trace shows the analysis of evoked post synaptic junction potentials (EJP) on soleus muscles injected intramuscularly with 10 ng/kg of BoNT/C α-51. At indicated time points soleus muscles were collected and processed for the electrophysiological recordings of EJPs, as previously reported [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006567#ppat.1006567.ref027" target="_blank">27</a>]. Data are presented as a percentage of EJPs of control muscles. Each point represents an average EJP amplitude obtained from at least 45 muscle fibers from three different mice per condition. Error bars represent SEM. As a comparison, dotted trace shows the time course of EJP recovery obtained with 1 LD₅₀ of BoNT/C α-51. Statistical significance at each time point was determined by a Student's t-test comparing the mean values (**** p<0.0001, n.s. not significant). Error bars represent SEM. <b>(B and C)</b> Soleus muscles coming from the EJP analyses were fixed and stained for (B) cleaved SNAP-25 (SNAP-25_c) or (C) Syntaxin-1A/1B (Stx-1A/1B), both shown in red. NMJ were spotted with α-Bungarotoxin (α-BTX, in green). The first row of panels represents the staining of a control muscle.</p

Summary of the lethality of the different BoNT/C variants used in this study.

<p>Summary of the lethality of the different BoNT/C variants used in this study.</p

BoNT/C mutants are poorly lethal <i>in vivo</i>.

<p><b>(A)</b> Mouse bioassay for BoNT/C variants. CD1 female mice weighting 20–24 grams were injected intraperitoneally with the indicated doses of BoNT/C-wt (cyan) or BoNT/C α-51 (green) or BoNT/C α-3W (red). Survival after 96 hours is reported as the percentage of mice survived with respect to the total group treated with the same amount of toxin. <b>(B)</b> Animals of the mouse bioassay were monitored every 4 hours and their survival reported as a Kaplan-Meier plot. Top panel shows BoNT/C-wt, middle panel is for BoNT/C α-3W and bottom panel is for BoNT/C α-51.</p

Time course of neuroparalysis recovery upon a local injection of BoNT/C variants in the mouse hind limb.

<p><b>(A)</b> Digit Abduction Score (DAS) assay. 1 LD₅₀ of BoNT/C-wt (cyan), or BoNT/C α-51 (green) or BoNT/C α-3W (red) were injected intramuscularly in the mice hind limb and neuroparalysis was evaluated according to [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006567#ppat.1006567.ref049" target="_blank">49</a>]. The rescue from paralysis was monitored daily until complete recovery was attained. Traces are representative of three independent experiments with at least 5 mice per condition. Error bars represent SEM <b>(B)</b> Analysis of evoked post synaptic junction potentials (EJP) on injected soleus muscles. Mice were treated as in A and at indicated time points soleus muscles were collected and processed for recordings of EJPs, as previously reported [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006567#ppat.1006567.ref027" target="_blank">27</a>]. Data are presented as a percentage of EJPs of control muscles. Each point represents an average EJP amplitude obtained from at least 45 muscle fibers from three different mice per condition. Statistical significance at each time point was determined by a Student's t-test comparing the mean values between either BoNT/C α-51 (green) or BoNT/C α-3W (red) compared to BoNT/C-wt (cyan) (* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001, n.s. not significant). Error bars represent SEM.</p

BoNT/C mutants display noticeable lower potency than wild type BoNT/C.

<p><b>(A)</b> Activity of BoNT/C variants at the MPN hemidiaphragm assay. The black trace represents a dose-response calibration curve reporting the T₅₀ value obtained at indicated bath concentration of a reference wild type BoNT/C [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006567#ppat.1006567.ref045" target="_blank">45</a>]. Recombinant BoNT/C-wt (black diamond), tested at 100 pM displays a T₅₀ comparable to the previous BoNT/C-wt used at the same concentration. BoNT/C α-3W (white square) and BoNT/C α-51 (black square) need much higher concentrations to achieve a T₅₀ within the calibration curve. Error bars represent SD of n = 3–4 technical replicates. <b>(B)</b> Calculation of potency of BoNT/C mutants employing a power function fitted to the dose-response calibration curve in A. <b>(C)</b> Immunofluorescent analysis of hemidiaphragms derived from MPN assays. Hemidiaphragms treated with the indicated toxin and concentration were fixed immediately upon completion of paralysis and stained for cleaved SNAP-25 (SNAP-25_c, red). NMJs were spotted with α-Bungarotoxin (α-BTX, in green). Images shown are representative of at least three independent experiments. Scale bar, 10 μm.</p

Imaging of cleaved SNAP-25 and syntaxin-1 in muscles treated with BoNT/C-wt.

<p>Soleus muscles of mice treated with BoNT/C-wt and used for the analysis of EJPs of <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006567#ppat.1006567.g005" target="_blank">Fig 5B</a> were fixed immediately after the electrophysiological recordings and stained for <b>(A)</b> cleaved SNAP-25 (SNAP-25_c) or <b>(B)</b> Syntaxin-1A/1B (Stx-1A/1B), both shown in red. NMJs were spotted with α-Bungarotoxin (α-BTX, in green). The first row of panels represents the staining of a control muscle. Scale bar, 10 μm.</p

The HA complex interacts with carbohydrate receptors to cross epithelial cell monolayers.

<p>(A, B) TER of Caco-2 monolayers was measured when Alexa-488-labeled HA complex (HA*) pre-incubated with Lac, IPTG, α2,3-SiaLac, or α2,6-SiaLac was applied to the apical (A; 58 nM) or basolateral (B; 17 nM) chambers. Values are means ± SD (<i>n</i> = 4–12). (C) HA* (with or without carbohydrates) or the Alexa-488-labeled HA^33-DAFA complex (HA^33-DAFA *) was applied to the apical (at 58 nM) or basolateral (at 17 nM) chamber. The fluorescence signals in both chambers were quantified after 24 hours and the amount of transported HA*/HA^33-DAFA * was expressed as a percentage of the total HA*/HA^33-DAFA * used. Values are means ± SD (<i>n</i> = 3–22).</p