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Monoclonal Antibodies that Inhibit the Proteolytic Activity of Botulinum Neurotoxin Serotype/B.

Fan Y, Dong J, Lou J, Wen W, Conrad F, Geren IN, Garcia-Rodriguez C, Smith TJ, Smith LA, Ho M, Pires-Alves M, Wilson BA, Marks JD - Toxins (Basel) (2015)

Bottom Line: Eleven mAbs inhibited BoNT/B LC proteolytic activity.The fine epitopes of selected mAbs were identified by alanine-scanning mutagenesis, revealing that inhibitory mAbs bound near the active site, substrate-binding site or the extended substrate-binding site.The results provide mAbs that could prove useful for intracellular reversal of paralysis and identify epitopes that could be targeted by small molecules inhibitors.

View Article: PubMed Central - PubMed

Affiliation: Department of Anesthesia and Perioperative Care, University of California, San Francisco, San Francisco General Hospital, Room 3C-38, 1001 Potrero Avenue, San Francisco, CA 94110, USA. frank.fan@ucsf.edu.

ABSTRACT
Existing antibodies (Abs) used to treat botulism cannot enter the cytosol of neurons and bind to botulinum neurotoxin (BoNT) at its site of action, and thus cannot reverse paralysis. However, Abs targeting the proteolytic domain of the toxin could inhibit the proteolytic activity of the toxin intracellularly and potentially reverse intoxication, if they could be delivered intracellularly. As such, antibodies that neutralize toxin activity could serve as potent inhibitory cargos for therapeutic antitoxins against botulism. BoNT serotype B (BoNT/B) contains a zinc endopeptidase light chain (LC) domain that cleaves synaoptobrevin-2, a SNARE protein responsible for vesicle fusion and acetylcholine vesicle release. To generate monoclonal Abs (mAbs) that could reverse paralysis, we targeted the protease domain for Ab generation. Single-chain variable fragment (scFv) libraries from immunized mice or humans were displayed on yeast, and 19 unique BoNT/B LC-specific mAbs isolated by fluorescence-activated cell sorting (FACS). The equilibrium dissociation constants (KD) of these mAbs for BoNT/B LC ranged from 0.24 nM to 14.3 nM (mean KD 3.27 nM). Eleven mAbs inhibited BoNT/B LC proteolytic activity. The fine epitopes of selected mAbs were identified by alanine-scanning mutagenesis, revealing that inhibitory mAbs bound near the active site, substrate-binding site or the extended substrate-binding site. The results provide mAbs that could prove useful for intracellular reversal of paralysis and identify epitopes that could be targeted by small molecules inhibitors.

No MeSH data available.


Related in: MedlinePlus

Binding epitope of mAb 1B10.1 on BoNT/B surface. Shown are molecular models constructed with Pymol software based on the BoNT/B crystal structure (pdb ID: 1S0F). (A) Holotoxin LC structure with color-coding indicating the change in ΔΔG (kcal/mole) of binding of 1B10.1 using the scale shown at top left. The residues comprising the active site are shown in orange; (B) Expanded view of the 1B10.1 epitope on the surface of BoNT/B with color-coding as in (A). Substrate-binding S-pockets are shown in blue. The 1B10.1 epitope includes the S2 amino acid D244 and would cover the S4 and S6 binding sites of Syb-2.
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toxins-07-03405-f004: Binding epitope of mAb 1B10.1 on BoNT/B surface. Shown are molecular models constructed with Pymol software based on the BoNT/B crystal structure (pdb ID: 1S0F). (A) Holotoxin LC structure with color-coding indicating the change in ΔΔG (kcal/mole) of binding of 1B10.1 using the scale shown at top left. The residues comprising the active site are shown in orange; (B) Expanded view of the 1B10.1 epitope on the surface of BoNT/B with color-coding as in (A). Substrate-binding S-pockets are shown in blue. The 1B10.1 epitope includes the S2 amino acid D244 and would cover the S4 and S6 binding sites of Syb-2.

Mentions: Epitope location determined by the above method (Figure 4) correlated well with the classification of epitopes based on ability of mAbs to bind simultaneously to BoNT/B LC (Figure 1). The three mAbs displaying the most potent inhibition (16B3, 18E5 and 1B10.1, epitope cluster I, group 1) bound an epitope located near the catalytic site and the putative substrate-binding site and shared amino acids in the epitope (Figure 3A). The mAb 19D22, displaying the next most potent inhibition, also bound near the catalytic and substrate-binding sites, but at a structurally non-overlapping epitope with mAbs in the group 1 below the belt (Figure 3A). The remaining inhibitory mAbs in epitope cluster I, group 1 (2B25.1, 1B22 and 31A5) bound above and near the substrate-binding site but remote from the catalytic site (Figure 3B). The epitope of 31A5 includes an amino acid within the substrate-binding site, explaining why it is more inhibitory than 2B25.1, 1B22. In epitope cluster I, group 2, the weakly inhibitory mAbs 18A7, 18D10 and 31H3 shared amino acids in the epitope and bound an epitope below but near the substrate-binding site and more remote from the catalytic binding site than mAbs in epitope cluster I, group 1 (Figure 3C). In epitope cluster I, group 3, the non-inhibitory mAbs B6.1 and 18A6 bound far from the substrate-binding site (Figure 4D). Similarly, in epitope cluster II (mAbs 4B19, 19A9) and III (mAbs 18F2, 31G2 and 31E2), the minimally inhibitory or non-inhibitory mAbs bind remote from the substrate-binding site. The antibodies binding in these epitopes that displayed low levels of inhibition may be conformationally-specific, stabilizing catalytically inactive form of the enzyme [14]. Conformationally-specific antibodies have been identified for other proteases [33]. Residue K285 of the epitope of 18F2 is adjacent to one of the reported Ca2+-binding sites that includes R284 [34].


Monoclonal Antibodies that Inhibit the Proteolytic Activity of Botulinum Neurotoxin Serotype/B.

Fan Y, Dong J, Lou J, Wen W, Conrad F, Geren IN, Garcia-Rodriguez C, Smith TJ, Smith LA, Ho M, Pires-Alves M, Wilson BA, Marks JD - Toxins (Basel) (2015)

Binding epitope of mAb 1B10.1 on BoNT/B surface. Shown are molecular models constructed with Pymol software based on the BoNT/B crystal structure (pdb ID: 1S0F). (A) Holotoxin LC structure with color-coding indicating the change in ΔΔG (kcal/mole) of binding of 1B10.1 using the scale shown at top left. The residues comprising the active site are shown in orange; (B) Expanded view of the 1B10.1 epitope on the surface of BoNT/B with color-coding as in (A). Substrate-binding S-pockets are shown in blue. The 1B10.1 epitope includes the S2 amino acid D244 and would cover the S4 and S6 binding sites of Syb-2.
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Related In: Results  -  Collection

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Show All Figures
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toxins-07-03405-f004: Binding epitope of mAb 1B10.1 on BoNT/B surface. Shown are molecular models constructed with Pymol software based on the BoNT/B crystal structure (pdb ID: 1S0F). (A) Holotoxin LC structure with color-coding indicating the change in ΔΔG (kcal/mole) of binding of 1B10.1 using the scale shown at top left. The residues comprising the active site are shown in orange; (B) Expanded view of the 1B10.1 epitope on the surface of BoNT/B with color-coding as in (A). Substrate-binding S-pockets are shown in blue. The 1B10.1 epitope includes the S2 amino acid D244 and would cover the S4 and S6 binding sites of Syb-2.
Mentions: Epitope location determined by the above method (Figure 4) correlated well with the classification of epitopes based on ability of mAbs to bind simultaneously to BoNT/B LC (Figure 1). The three mAbs displaying the most potent inhibition (16B3, 18E5 and 1B10.1, epitope cluster I, group 1) bound an epitope located near the catalytic site and the putative substrate-binding site and shared amino acids in the epitope (Figure 3A). The mAb 19D22, displaying the next most potent inhibition, also bound near the catalytic and substrate-binding sites, but at a structurally non-overlapping epitope with mAbs in the group 1 below the belt (Figure 3A). The remaining inhibitory mAbs in epitope cluster I, group 1 (2B25.1, 1B22 and 31A5) bound above and near the substrate-binding site but remote from the catalytic site (Figure 3B). The epitope of 31A5 includes an amino acid within the substrate-binding site, explaining why it is more inhibitory than 2B25.1, 1B22. In epitope cluster I, group 2, the weakly inhibitory mAbs 18A7, 18D10 and 31H3 shared amino acids in the epitope and bound an epitope below but near the substrate-binding site and more remote from the catalytic binding site than mAbs in epitope cluster I, group 1 (Figure 3C). In epitope cluster I, group 3, the non-inhibitory mAbs B6.1 and 18A6 bound far from the substrate-binding site (Figure 4D). Similarly, in epitope cluster II (mAbs 4B19, 19A9) and III (mAbs 18F2, 31G2 and 31E2), the minimally inhibitory or non-inhibitory mAbs bind remote from the substrate-binding site. The antibodies binding in these epitopes that displayed low levels of inhibition may be conformationally-specific, stabilizing catalytically inactive form of the enzyme [14]. Conformationally-specific antibodies have been identified for other proteases [33]. Residue K285 of the epitope of 18F2 is adjacent to one of the reported Ca2+-binding sites that includes R284 [34].

Bottom Line: Eleven mAbs inhibited BoNT/B LC proteolytic activity.The fine epitopes of selected mAbs were identified by alanine-scanning mutagenesis, revealing that inhibitory mAbs bound near the active site, substrate-binding site or the extended substrate-binding site.The results provide mAbs that could prove useful for intracellular reversal of paralysis and identify epitopes that could be targeted by small molecules inhibitors.

View Article: PubMed Central - PubMed

Affiliation: Department of Anesthesia and Perioperative Care, University of California, San Francisco, San Francisco General Hospital, Room 3C-38, 1001 Potrero Avenue, San Francisco, CA 94110, USA. frank.fan@ucsf.edu.

ABSTRACT
Existing antibodies (Abs) used to treat botulism cannot enter the cytosol of neurons and bind to botulinum neurotoxin (BoNT) at its site of action, and thus cannot reverse paralysis. However, Abs targeting the proteolytic domain of the toxin could inhibit the proteolytic activity of the toxin intracellularly and potentially reverse intoxication, if they could be delivered intracellularly. As such, antibodies that neutralize toxin activity could serve as potent inhibitory cargos for therapeutic antitoxins against botulism. BoNT serotype B (BoNT/B) contains a zinc endopeptidase light chain (LC) domain that cleaves synaoptobrevin-2, a SNARE protein responsible for vesicle fusion and acetylcholine vesicle release. To generate monoclonal Abs (mAbs) that could reverse paralysis, we targeted the protease domain for Ab generation. Single-chain variable fragment (scFv) libraries from immunized mice or humans were displayed on yeast, and 19 unique BoNT/B LC-specific mAbs isolated by fluorescence-activated cell sorting (FACS). The equilibrium dissociation constants (KD) of these mAbs for BoNT/B LC ranged from 0.24 nM to 14.3 nM (mean KD 3.27 nM). Eleven mAbs inhibited BoNT/B LC proteolytic activity. The fine epitopes of selected mAbs were identified by alanine-scanning mutagenesis, revealing that inhibitory mAbs bound near the active site, substrate-binding site or the extended substrate-binding site. The results provide mAbs that could prove useful for intracellular reversal of paralysis and identify epitopes that could be targeted by small molecules inhibitors.

No MeSH data available.


Related in: MedlinePlus