A VHH That Neutralizes the Zinc Metalloproteinase Activity of Botulinum Neurotoxin Type A

Thanongsaksrikul, Jeeraphong; Srimanote, Potjanee; Maneewatch, Santi; Choowongkomon, Kiattawee; Tapchaisri, Pramuan; Makino, Sou‐ichi; Kurazono, Hisao; Chaicumpa, Wanpen

doi:10.1074/jbc.m109.073163

Cited by 57 publications

(85 citation statements)

References 42 publications

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“…In this study, the B-cell epitope of the Per a 1.0105 bound to the MAb was readily identified using a 12-mer random peptide phage display library to select phages that displayed peptides that mimic the original characteristic of the native epitope of the Per a 1.0105-specific MAb (epitope) 22. Validity of this technique has been demonstrated previously in several studies 17,23,24,25. The phage mimotopic peptide is REVLQKNFAKGV which is matched with the amino acids 99QDLLQNLRDKGV110 of the Per a 1.0105.…”

Section: Discussionmentioning

confidence: 76%

Specific B-cell Epitope of Per a 1: A Major Allergen of American Cockroach (Periplaneta americana) and Anatomical Localization

Sookrung

Khetsuphan

Chaisri

et al. 2014

Allergy Asthma Immunol Res

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PurposeCockroach (CR) is a common source of indoor allergens, and Per a 1 is a major American CR (Periplaneta americana) allergen; however, several attributes of this protein remain unknown. This study identifies a novel specific B cell epitope and anatomical locations of Per a 1.0105.MethodsRecombinant Per a 1.0105 (rPer a 1.0105) was used as BALB/c mouse immunogen for the production of monoclonal antibodies (MAb). The MAb specific B cell epitope was identified by determining phage mimotopic peptides and pair-wise alignment of the peptides with the rPer a 1.0105 amino acid sequence. Locations of the Per a 1.0105 in P. americana were investigated by immunohistochemical staining.ResultsThe rPer a 1.0105 (~13 kDa) had 100%, 98% and ≥90% identity to Per a 1.0105, Per a 1.0101, and Cr-PII, respectively. The B-cell epitope of the Per a 1.0105 specific-MAb was located at residues99 QDLLLQLRDKGV110 contained in all 5 Per a 1.01 isoforms and Per a 1.02. The epitope was analogous to the Bla g 1.02 epitope; however, this B-cell epitope was not an IgE inducer. Per a 1.0105 was found in the midgut and intestinal content of American CR but not in the other organs. The amount of the Per a 1 was ~544 ℃g per gram of feces.ConclusionsThe novel Per a 1 B-cell epitope described in this study is a useful target for allergen quantification in samples; however, the specific MAb can be used as an allergen detection reagent. The MAb based-affinity resin can be made for allergen purification, and the so-purified protein can serve as a standard and diagnostic allergen as well as a therapeutic vaccine component. The finding that the Per a 1 is contained in the midgut and feces is useful to increase yield and purity when preparing this allergen.

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Section: Discussionmentioning

confidence: 76%

Specific B-cell Epitope of Per a 1: A Major Allergen of American Cockroach (Periplaneta americana) and Anatomical Localization

Sookrung

Khetsuphan

Chaisri

et al. 2014

Allergy Asthma Immunol Res

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“…A llama was immunized simultaneously with all seven toxin toxoids and binders selected in solution towards biotinylated toxin. Other newly published reports have described the isolation of sdAb derived from naïve libraries that inhibit the catalytic activity of BoNT A Tx [25, 26]. In this paper, we report the development of a phage-display library prepared from a llama that had been immunized with only BoNT A Td, and on the use of bead-bound and plate-immobilized targets for selection.…”

Section: Introductionmentioning

confidence: 99%

Llama-derived single-domain antibodies for the detection of botulinum A neurotoxin

et al. 2010

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Single-domain antibodies (sdAb) specific for botulinum neurotoxin serotype A (BoNT A) were selected from an immune llama phage display library derived from a llama that was immunized with BoNT A toxoid. The constructed phage library was panned using two methods: panning on plates coated with BoNT A toxoid (BoNT A Td) and BoNT A complex toxoid (BoNT Ac Td) and panning on microspheres coupled to BoNT A Td and BoNT A toxin (BoNT A Tx). Both panning methods selected for binders that had identical sequences, suggesting that panning on toxoided material may be as effective as panning on bead-immobilized toxin for isolating specific binders.All of the isolated binders tested were observed to recognize bead-immobilized BoNT A Tx in direct binding assays, and showed very little cross-reactivity towards other BoNT serotypes and unrelated protein. Sandwich assays that incorporated selected sdAb as capture and tracer elements Correspondence to: Ellen R. Goldman, ellen.goldman@nrl.navy.mil. Electronic supplementary material The online version of this article

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“…Briefly, 0.4 mg/mL of lipid containing phosphatidylcholine (PC) [17], phosphatidylserine (PS) and cholesterol (7:1:2 molar ratio) was purged with N 2 to remove the organic solvent chloroform, resulting in the creation of a thin film. The lipid film was then vacuum dried for 2 h to remove the traces of organic solvent and was rehydrated with 10 mM sodium phosphate buffer, containing 200 mM NaCl, pH 7.2 or 4.5.…”

Section: Experimental Methodsmentioning

confidence: 99%

Structural and functional analysis of botulinum neurotoxin subunits for pH-dependent membrane channel formation and translocation

Chellappan¹,

Kumar²,

Santos³

et al. 2015

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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The structure–function relationship of Botulinum Neurotoxin (BoNT) proteins is greatly influenced by pH. While the low pH of endosome favors membrane interaction of the heavy chain (HC) for formation of a membrane channel and translocation of the light chain (LC), the catalytic activity of the LC requires neutral pH for cleavage of the soluble NSF attachment protein receptor (SNARE) complex in the cytosol. In this study, we monitored secondary structural characteristics of LC, HC and holotoxin at individual pHs 4.5 and 7.2 and at the transition pH 4.5 to 7.2 to identify the structural signatures underlying their function. The HC showed higher thermal stability at pH 4.5 with a melting temperature (Tm) of 60.4°C. The structural analysis of HC in the presence of liposomes showed no difference in ellipticity with that of HC at pH 7.2 at 208 and 222 nm but a 25.2 % decrease in ellipticity at 208 nm at acidic pH, indicating low pH-induced structural changes that might facilitate interaction with the membrane. Further, HC showed 18% release of K+ ions from liposomes at pH 4.5 as against 6% at neutral pH, reinforcing its role in membrane channel formation. LC on the other hand, showed maximum ellipticity at pH 7.2, a condition that is relevant to its endopeptidase activity in the cytosol of the neurons. Also, the similarity in the structures at pH 7.2 and transition pH 4.5 to 7.2 suggested that the flexibility acquired by the protein at low pH was reversible upon exposure to neutral pH for cleavage of SNARE proteins.

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A VHH That Neutralizes the Zinc Metalloproteinase Activity of Botulinum Neurotoxin Type A

Cited by 57 publications

References 42 publications

Specific B-cell Epitope of Per a 1: A Major Allergen of American Cockroach (Periplaneta americana) and Anatomical Localization

Specific B-cell Epitope of Per a 1: A Major Allergen of American Cockroach (Periplaneta americana) and Anatomical Localization

Llama-derived single-domain antibodies for the detection of botulinum A neurotoxin

Structural and functional analysis of botulinum neurotoxin subunits for pH-dependent membrane channel formation and translocation

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