Catalytic properties of botulinum neurotoxin subtypes A3 and A4

Abstract

Botulinum toxins (BoNT) are zinc proteases (serotypes A-G) which cause flaccid paralysis through the cleavage of SNARE proteins within motor neurons. BoNT/A was originally organized into two subtypes, BoNT/A1 and BoNT/A2, which are approximately 95% homologous and possess similar catalytic activities. Subsequently, two additional subtypes were identified, BoNT/A3 (Loch Maree) and BoNT/A4 (657Ba), which are 81 and 88% homologous with BoNT/A1, respectively. Alignment studies predicted that BoNT/A3 and BoNT/A4 were sufficiently different from BoNT/A1 to affect SNAP25 binding and cleavage. Recombinant light chain (LC) of BoNT/A3 (LC/A3) and BoNT/A4 (LC/A4) were subjected to biochemical analysis. LC/A3 cleaved SNAP25 at 50% of the rate of LC/A1 but cleaved SNAPtide at a faster rate than LC/A1, while LC/A4 cleaved SNAP25 and SNAPtide at slower rates than LC/A1. LC/A3 and LC/A4 had similar K(m) values for SNAP25 relative to LC/A1, while the k(cat) for LC/A4 was 10-fold slower than that for LC/A1, suggesting a defect in substrate cleavage. Neither LC/A3 nor LC/A4 possessed autocatalytic activity, a property of LC/A1 and LC/A2. Thus, the four subtypes of BoNT/A bind SNAP25 with similar affinity but have different catalytic capacities for SNAP25 cleavage, SNAPtide cleavage, and autocatalysis. The catalytic properties identified among the subtypes of LC/A may influence strategies for the development of small molecule or peptide inhibitors as therapies against botulism.

Figure 1. Purification of LC/As

LC/A subtype recombinant, purified protein (3μg) were subjected to 12% SDS-PAGE gel and visualized with Coomassie Brilliant Blue (shown).

Figure 2. Solubility of LC/A4

(A) Kyte and Doolittle mean hydrophobicity profile of LC/A1-4. Arrow indicates residues substitutions causing a calculated increase in hydrophobicity locally: LC/A1, LC/A2, LC/A3 (—), and LC/A4 (---). Alignment and hydrophobicity plot calculated using BioEdit. LC/A1-A3 were plotted as solid lines due to their overlapping sequence. (B) LC/A subtype alignment of residues within solubility pocket of LC/A4 (260, 264). Underlined lettering indicates residue substitution. (C) Quantification of LC/A4 and LC/A4-I264R solubility upon induction, lysis, and centrifugation as measured by densitometry of a Coomassie-stained, 12% SDS-PAGE gel.

Figure 3. Cleavage site identification of LC/A subtypes

GST-SNAP25 (141–206) (A) or GST-SNAP25(141-206-R198A) (B) (5 μM) was incubated with 0.2 μM of the indicated LC subtype (A1-A4) and a non-catalytic LC control (RYM, LC/A1-R363A, Y366F) for 1 hr at 37°C. Reaction were subjected to SDS-PAGE (shown) and the amount of cleaved GST-SNAP25(141–206) determined by densitometry of a Coomassie stained gel (C). Error bars represent the average of at least 3 independent experiments. In A and B, the SNAP25 (substrate) and cleaved product (product) are indicated with arrows, while “S” describes substrate and “P” describes the product band.

Figure 4. Structural folding of LC/A4

Circular dichroism (CD) spectroscopy of LC/A4 derivatives. LC/A1 (Unbroken line), LC/A4 (Dashed line), and LC/A4-I264R (Dotted line) were examined by CD at equimolar concentrations. Data represents an average of at least 10 independent scans

Figure 5. Auto-cleavage of LC/A subtypes

LC/A1-A4 or LC/A1-Y250A, Y251A (LC/A1 DYM) (6 μM) were incubated at 37°C for 6 h in 10 mM Tris (pH 7.6), 20 mM NaCl. Reactions were subjected to SDS-PAGE and auto-cleavage was observed following staining with Coomassie Brilliant Blue. Asterisk (*) designates purification background that remains constant from 0–16 hr incubation.