Publication: Structural characterization of humanized nanobodies with neutralizing activity against the bordetella pertussis CyaA-hemolysin: Implications for a potential epitope of toxin-protective antigen
Issued Date
2016-04-01
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ISSN
20726651
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2-s2.0-84976328152
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Mahidol University
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SCOPUS
Bibliographic Citation
Toxins. Vol.8, No.4 (2016)
Suggested Citation
Aijaz Ahmad Malik, Chompounoot Imtong, Nitat Sookrung, Gerd Katzenmeier, Wanpen Chaicumpa, Chanan Angsuthanasombat Structural characterization of humanized nanobodies with neutralizing activity against the bordetella pertussis CyaA-hemolysin: Implications for a potential epitope of toxin-protective antigen. Toxins. Vol.8, No.4 (2016). doi:10.3390/toxins8040099 Retrieved from: https://repository.li.mahidol.ac.th/handle/20.500.14594/40662
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Title
Structural characterization of humanized nanobodies with neutralizing activity against the bordetella pertussis CyaA-hemolysin: Implications for a potential epitope of toxin-protective antigen
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Abstract
© 2016 by the authors; licensee MDPI, Basel, Switzerland. Previously, the 126-kDa CyaA-hemolysin (CyaA-Hly) fragment cloned from Bordetella pertussis—the causative agent of whooping cough—and functionally expressed in Escherichia coli was revealed as a key determinant for CyaA-mediated hemolysis against target erythrocytes. Here, phagemid-transfected E. coli clones producing nanobodies capable of binding to CyaA-Hly were selected from a humanized-camel VH/VHH phage-display library. Subsequently verified for binding activities by indirect ELISA and Western blotting, four CyaA-Hly-specific nanobodies were obtained and designated according to the presence/absence of VHH-hallmark amino acids as VHH2, VH5, VH18 and VHH37. In vitro neutralization assay revealed that all four ~17-kDa His-tagged VH/VHH nanobodies, in particular VHH37, which were over-expressed as inclusions and successfully unfolded-refolded, were able to effectively inhibit CyaA-Hly-mediated hemolysis. Phage-mimotope searching revealed that only peptides with sequence homologous to Linker 1 connecting Blocks I and II within the CyaA-RTX subdomain were able to bind to these four CyaA-Hly-specific nanobodies. Structural analysis of VHH37 via homology modeling and intermolecular docking confirmed that this humanized nanobody directly interacts with CyaA-RTX/Linker 1 through multiple hydrogen and ionic bonds. Altogether, our present data demonstrate that CyaA-RTX/Linker 1 could serve as a potential epitope of CyaA-protective antigen that may be useful for development of peptide-based pertussis vaccines. Additionally, such toxin-specific nanobodies have a potential for test-driven development of a ready-to-use therapeutic in passive immunization for mitigation of disease severity.