Abstract
BackgroundBotulism, 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 FindingsTo 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.ConclusionsThe 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.
Highlights
Botulinum neurotoxins (BoNTs), the causative agents of botulism, are the most potent naturally-occurring toxins known [1]
The amino acids QPDRS, identified by phage display experiments mapped to this region, Q139, P140, and D141 form the tip of the loop and R145 and S146 are brought into close proximity by the tertiary structure of BoNT (Figure 3)
Replacement of R145 and S146 with glycine residues had no obvious effect on F1-40 binding as illustrated by Western blot, but competition ELISA experiments revealed a 7-fold decrease in the relative affinity of mutant Lc-RS for F1-40 compared to the recombinant Lc peptide
Summary
Botulinum neurotoxins (BoNTs), the causative agents of botulism, are the most potent naturally-occurring toxins known [1]. Seven different serotypes of BoNT, designated A to G, are produced predominantly by strains of Clostridium botulinum, and by some strains of C. butyricum and C. baratii [2]. 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
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