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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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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.
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pone-0004924-g006: 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.

Mentions: The cDNA sequences of the heavy and light chain variable regions for F1-40 are shown in Figure 6. The nucleotide sequence of the entire cloned regions is shown, with the corresponding amino acids shown from the start of the leader peptide to the end of the fourth framework region of each chain. The leader sequences, framework regions, complementarity determining regions (CDRs) and J-regions were identified by inspecting the alignment of the F1-40 heavy and light chains to other antibody sequences [26]–[30]. The nucleotide sequence reported was identical across the six individual colonies of E. coli harboring either pCR4-L-chain or pCR4-H-chain that were analyzed. EMBL Nucleotide Sequence Database accession numbers for these sequences are FM177889 and FM177890, for the light and heavy chains, respectively.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0004924-g006: 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.
Mentions: The cDNA sequences of the heavy and light chain variable regions for F1-40 are shown in Figure 6. The nucleotide sequence of the entire cloned regions is shown, with the corresponding amino acids shown from the start of the leader peptide to the end of the fourth framework region of each chain. The leader sequences, framework regions, complementarity determining regions (CDRs) and J-regions were identified by inspecting the alignment of the F1-40 heavy and light chains to other antibody sequences [26]–[30]. The nucleotide sequence reported was identical across the six individual colonies of E. coli harboring either pCR4-L-chain or pCR4-H-chain that were analyzed. EMBL Nucleotide Sequence Database accession numbers for these sequences are FM177889 and FM177890, for the light and heavy chains, respectively.

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