Epitope characterization and variable region sequence of f1-40, a high-affinity monoclonal antibody to botulinum neurotoxin type a (Hall strain)

Abstract

Background: Botulism, an often fatal neuroparalytic disease, is caused by botulinum neurotoxins (BoNT) which consist of a family of seven serotypes (A-H) produced by the anaerobic bacterium Clostridium botulinum. BoNT, considered the most potent biological toxin known, is a 150 kDa protein consisting of a 100 kDa heavy-chain (Hc) and a 50 kDa light-chain (Lc). F1-40 is a mouse-derived, IgG1 monoclonal antibody that binds the light chain of BoNT serotype A (BoNT/A) and is used in a sensitive immunoassay for toxin detection. We report the fine epitope mapping of F1-40 and the deduced amino acid sequence of the variable regions of the heavy and light chains of the antibody.

Methods and findings: To characterize the binding epitope of F1-40, three complementary experimental approaches were selected. Firstly, recombinant peptide fragments of BoNT/A light-chain were used in Western blots to identify the epitope domains. Secondly, a peptide phage-display library was used to identify the specific amino acid sequences. Thirdly, the three-dimensional structure of BoNT/A was examined in silico, and the amino acid sequences determined from the phage-display studies were mapped onto the three-dimensional structure in order to visualize the epitope. F1-40 was found to bind a peptide fragment of BoNT/A, designated L1-3, which spans from T125 to L200. The motif QPDRS was identified by phage-display, and was mapped to a region within L1-3. When the three amino acids Q138, P139 and D140 were all mutated to glycine, binding of F1-40 to the recombinant BoNT/A light chain peptide was abolished. Q-138, P-139 and D-140 form a loop on the external surface of BoNT/A, exposed to solvent and accessible to F1-40 binding.

Conclusions: The epitope of F1-40 was localized to a single exposed loop (ss4, ss5) on the Lc of BoNT. Furthermore amino acids Q138, P139 and D140 forming the tip of the loop appear critical for binding.

Figure 1. Peptide fragments of BoNT/A light chain.

Diagram is drawn to scale to facilitate size and location comparison between peptide fragments. Peptide fragments were expressed as fusions to GST at the N-terminal. N- and C-terminal amino acids of each peptide fragment are indicated.

Figure 2. Binding of F1-40 to peptide fragments of BoNT/A light chain.

A and C, Light chain fragment-GST fusion peptides separated by gel electrophoresis and stained with Silver Stain. B and D, Corresponding Western Blots using F1-40 for primary detection. Fusion peptide size is indicated in kDa.

Figure 3. Location of the QPDRS motif within the BoNT/A holotoxin.

A. Space-filled diagram of the three-dimensional model of the complete BoNT/A holotoxin showing the heavy (dark gray) and light (light gray) chains. QPDRS motif is the colored region within the light chain. B. Enlarged view of Q139 (red), P140 (blue), D141 (yellow), R145 (green) and S146 (pink) .

Figure 4. Binding of F1-40 to the Lc peptide fragment mutants.

A., Lc peptide fragment mutants separated by gel electrophoresis and stained with Silver Stain. B, Western Blot using rabbit polyclonal to Clostridium botulinum A toxoid # 20641 (Abcam Inc.) for primary detection. C, Western Blot using F1-40 for primary detection. Peptide size is indicated in kDa.

Figure 5. Competition ELISA for F1-40 by Lc peptide fragment mutants in solution.

The percent inhibition of binding of mAb F1-40 to immobilized Lc peptide fragment on a 96-well plate by various peptide fragments in solution (♦, Lc; ▵ Lc-Δ; ○ Lc-QPD; ▪ Lc-RS) was calculated by the formula (1-B/B₀)×100, where B = luminescent counts at each concentration of fusion peptide in solution, and B₀ = luminescent counts at 0 µg/mL fusion peptide in solution. Data is presented±1 standard error, n = 4.

Figure 6. Nucleotide and amino acid sequences for the variable regions of F1-40 k-light and heavy chains.

The nucleotide sequences for the entire cloned regions are shown, with the deduced amino acids from the leader sequence to the end of the fourth framework region. The leader sequence and three complementarity determining regions (CDRs) are shown highlighted in black, with the amino acids of the CDRs shown in bold. The four framework regions are shown highlighted in light gray, with the amino acids of the J-regions of both light and heavy chains shown underlined, within the fourth framework region. EMBL online Accession #s FM177889 & FM177890.