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Interaction between the two subdomains of the C‐terminal part of the botulinum neurotoxin A is essential for the generation of protective antibodies
Author(s) -
Tavallaie Mahmood,
Chenal Alexandre,
Gillet Daniel,
Pereira Yannik,
Manich Maria,
Gibert Maryse,
Raffestin Stephanie,
Popoff Michel R,
Marvaud Jean Christophe
Publication year - 2004
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/j.febslet.2004.06.094
Subject(s) - circular dichroism , chemistry , recombinant dna , antigenicity , toxin , neurotoxin , antibody , amino acid , escherichia coli , microbiology and biotechnology , stereochemistry , biochemistry , biology , immunology , gene
The botulinum neurotoxin A C‐terminal fragment (Hc), which mediates the binding of the toxin to neuronal cell surface receptors, comprises two subdomains, Hc‐N (amino acids 873–1095) and Hc‐C (amino acids 1096–1296). In order to define the minimal fragment of Hc carrying protective antigenic properties, Hc, Hc‐N and Hc‐C have been produced as recombinant proteins in Escherichia coli , and have been tested for their antigenicity in mouse protection assays. Hc, Hc‐N and Hc‐C induced similar antibody levels as shown by ELISA. However, a single immunization with Hc (10 μg) fully protected mice challenged with 10 3 mouse lethal dose 50 of toxin, whereas Hc‐N, Hc‐C, or Hc‐N plus Hc‐C did not give any protection. Triple immunizations with Hc‐N or Hc‐C were necessary to induce a higher level of protection. Circular dichroism and fluorescence studies showed that the isolated subdomains were folded and stable. However, an intense near‐UV dichroic signal was only observed in the Hc spectrum, revealing a highly structured interface between both subdomains. Taken together, the results show that the generation of protective antibodies requires the whole Hc domain and especially the native structure of the interfacial region between Hc‐N and Hc‐C.