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

Scotcher MC, McGarvey JA, Johnson EA, Stanker LH - PLoS ONE (2009)

Bottom Line: F1-40 was found to bind a peptide fragment of BoNT/A, designated L1-3, which spans from T125 to L200.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.

View Article: PubMed Central - PubMed

Affiliation: United States Department of Agriculture, Agricultural Research Service, Western Regional Research Center, Albany, California, United States of America.

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.

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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.
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pone-0004924-g002: 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.

Mentions: Seven recombinant peptide fragments of BoNT/A light chain (Lc, L1, L2, L1-1, L1-2, L1-3, L1-4) are shown in Figure 1. These recombinant peptides were expressed as fusions to glutathione-S-transferase (GST). The relative molecular weights (rMW) of these recombinant peptide-GST proteins, observed by SDS-PAGE and silver staining, are shown in Figure 2, panels A and C. In each case, the rMW corresponds to that predicted for the recombinant peptide-GST protein. Results from Western blot analyses are shown in panels B and D. Clearly, F1-40 bound to the light-chain fragment (Lc) and to subfragment L1, but binding to L2 was not detected (Figure 2, Panel B). The L1 peptide fragment corresponds to amino acids M1 to L200. This region was further subdivided by generating four additional recombinant peptide fragments L1-1 through L1-4 (Figure 1). In Western blotting experiments, F1-40 was observed to bind two of these recombinant peptide fragments, L1-3 and L1-4 (Figure 2, Panel D). In contrast, binding to L1-1 and L1-2 was not detected. These results, particularly antibody binding to L1-3, suggest that the epitope for F1-40 resides between amino acids T125 and L200.


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

Scotcher MC, McGarvey JA, Johnson EA, Stanker LH - PLoS ONE (2009)

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.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2654115&req=5

pone-0004924-g002: 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.
Mentions: Seven recombinant peptide fragments of BoNT/A light chain (Lc, L1, L2, L1-1, L1-2, L1-3, L1-4) are shown in Figure 1. These recombinant peptides were expressed as fusions to glutathione-S-transferase (GST). The relative molecular weights (rMW) of these recombinant peptide-GST proteins, observed by SDS-PAGE and silver staining, are shown in Figure 2, panels A and C. In each case, the rMW corresponds to that predicted for the recombinant peptide-GST protein. Results from Western blot analyses are shown in panels B and D. Clearly, F1-40 bound to the light-chain fragment (Lc) and to subfragment L1, but binding to L2 was not detected (Figure 2, Panel B). The L1 peptide fragment corresponds to amino acids M1 to L200. This region was further subdivided by generating four additional recombinant peptide fragments L1-1 through L1-4 (Figure 1). In Western blotting experiments, F1-40 was observed to bind two of these recombinant peptide fragments, L1-3 and L1-4 (Figure 2, Panel D). In contrast, binding to L1-1 and L1-2 was not detected. These results, particularly antibody binding to L1-3, suggest that the epitope for F1-40 resides between amino acids T125 and L200.

Bottom Line: F1-40 was found to bind a peptide fragment of BoNT/A, designated L1-3, which spans from T125 to L200.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.

View Article: PubMed Central - PubMed

Affiliation: United States Department of Agriculture, Agricultural Research Service, Western Regional Research Center, Albany, California, United States of America.

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.

Show MeSH
Related in: MedlinePlus