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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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Related in: MedlinePlus

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.
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pone-0004924-g001: 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.

Mentions: Total genomic DNA from Clostridium botulinum (Strain ATCC3502) was used as a template to amplify the fragments of the light chain (Lc, L1, L2) using the primers indicated (see Figure 1 and Table 1). Stop codons (TAA) were introduced when not present within the genomic DNA of the cloned region. All subsequent BoNT/A DNA fragments were cloned into plasmid pCR4-TOPO (Invitrogen) to allow sequencing using primers M13F and M13R. The pCR4-derived plasmids were then digested using BamHI and XhoI, the BoNT/A fragment was purified and ligated into BamHI- and XhoI-digested pGS-21a (Genscript) to yield the correspondently named pGS plasmid (e.g. pGS-L1 for fragment L1). All pGS-21a-derived plasmids were sequenced using primer pGS-F and pGS-R, to confirm the correct integration of the BoNT/A fragment into the vector. The BamHI and XhoI cloning sites of pGS-21a are located downstream of glutathione-S-transferase (GST), under the control of the T7 promoter.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0004924-g001: 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.
Mentions: Total genomic DNA from Clostridium botulinum (Strain ATCC3502) was used as a template to amplify the fragments of the light chain (Lc, L1, L2) using the primers indicated (see Figure 1 and Table 1). Stop codons (TAA) were introduced when not present within the genomic DNA of the cloned region. All subsequent BoNT/A DNA fragments were cloned into plasmid pCR4-TOPO (Invitrogen) to allow sequencing using primers M13F and M13R. The pCR4-derived plasmids were then digested using BamHI and XhoI, the BoNT/A fragment was purified and ligated into BamHI- and XhoI-digested pGS-21a (Genscript) to yield the correspondently named pGS plasmid (e.g. pGS-L1 for fragment L1). All pGS-21a-derived plasmids were sequenced using primer pGS-F and pGS-R, to confirm the correct integration of the BoNT/A fragment into the vector. The BamHI and XhoI cloning sites of pGS-21a are located downstream of glutathione-S-transferase (GST), under the control of the T7 promoter.

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